Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and fermentation optimization.

Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

Aggravated hepatic fibrosis induced by phenylalanine and tyrosine was ameliorated by chitooligosaccharides supplementation.

Hepatic fibrosis is a classic pathological manifestation of metabolic chronic hepatopathy. The pathological process might either gradually deteriorate into cirrhosis and ultimately liver cancer with inappropriate nutrition supply, or be slowed down by several multifunctional nutrients, alternatively. Herein, we found diet with excessive phenylalanine (Phe) and tyrosine (Tyr) exacerbated hepatic fibrosis symptoms of liver dysfunction and gut microflora dysbiosis in mice. Chitooligosaccharides (COS) could ameliorate hepatic fibrosis with the regulation of amino acid metabolism by downregulating the mTORC1 pathway, especially that of Phe and Tyr, and also with the alleviation of the dysbiosis of gut microbiota, simultaneously. Conclusively, this work presents new insight into the role of Phe and Tyr in the pathologic process of hepatic fibrosis, while revealing the effectiveness and molecular mechanism of COS in improving hepatic fibrosis from the perspective of metabolites.

Characterization of Cinnamomum kanehirae Extract-Stimulated Triterpenoids Synthesis in Submerged Fermentation of Antrodia camphorata via Untargeted Metabolomics.

The underlying mechanisms of Cinnamomum kanehirae-stimulated growth and metabolism of Antrodia camphorata remain unknown. Herein, we first observed that the methanol extract of C. kanehirae trunk (MECK) (2 g/L) showed a potent stimulatory effect on A. camphorata triterpenoids production (115.6 mg/L). Second, MECK treatment considerably increased the category and abundance of many secondary metabolites in the mycelia. We identified 93 terpenoids (8 newly formed and 49 upregulated) in the MECK-treated mycelia, wherein 21 terpenoids were the same as those in the fruiting bodies. Third, 42 out of the 93 terpenoids were annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, mainly involving monoterpenoids and diterpenoids syntheses. Finally, 27 monoterpenes and 16 sesquiterpenes were detected in the MECK, and the two terpenoids with the highest abundance (linalool and α-pinene) were selected for verification and found to considerably increase the terpenoids production of A. camphorata and demonstrate the regulation of mRNA expression levels of nine key genes in the mevalonate pathway via RT-qPCR. This study is beneficial for elucidating the terpenoids synthesis mechanism in A. camphorata.

Phospholipids (PLs) know-how: exploring and exploiting phospholipase D for its industrial dissemination.

Owing to their numerous nutritional and bioactive functions, phospholipids (PLs), which are major components of biological membranes in all living organisms, have been widely applied as nutraceuticals, food supplements, and cosmetic ingredients. To date, PLs are extracted solely from soybean or egg yolk, despite the diverse market demands and high cost, owing to a tedious and inefficient manufacturing process. A microbial-based manufacturing process, specifically phospholipase D (PLD)-based biocatalysis and biotransformation process for PLs, has the potential to address several challenges associated with the soybean- or egg yolk-based supply chain. However, poor enzyme properties and inefficient microbial expression systems for PLD limit their wide industrial dissemination. Therefore, sourcing new enzyme variants with improved properties and developing advanced PLD expression systems are important. In the present review, we systematically summarize recent achievements and trends in the discovery, their structural properties, catalytic mechanisms, expression strategies for enhancing PLD production, and its multiple applications in the context of PLs. This review is expected to assist researchers to understand current advances in this field and provide insights for further molecular engineering efforts toward PLD-mediated bioprocessing.

Influence of Short-Term Consumption of Hericium erinaceus on Serum Biochemical Markers and the Changes of the Gut Microbiota: A Pilot Study.

Hericium erinaceus (H. erinaceus) is widely studied as a medicinal and edible fungus. Recent studies have shown that H. erinaceus has protective effects for diseases, such as inflammatory bowel disease and cancer, which are related to gut microbiota. To investigate the benefits of H. erinaceus intake on gut microbiota and blood indices in adulthood, we recruited 13 healthy adults to consume H. erinaceus powder as a dietary supplement. Blood changes due to H. erinaceus consumption were determined by routine hematological examination and characterized by serum biochemical markers. Microbiota composition was profiled by 16S ribosomal RNA gene sequencing. Results showed that daily H. erinaceus supplementation increased the alpha diversity within the gut microbiota community, upregulated the relative abundance of some short-chain fatty acid (SCFA) producing bacteria (Kineothrix alysoides, Gemmiger formicilis, Fusicatenibacter saccharivorans, Eubacterium rectale, Faecalibacterium prausnitzii), and downregulated some pathobionts (Streptococcus thermophilus, Bacteroides caccae, Romboutsia timonensis). Changes within the gut microbiota were correlated with blood chemical indices including alkaline phosphatase (ALP), low-density lipoprotein (LDL), uric acid (UA), and creatinine (CREA). Thus, we found that the gut microbiota alterations may be part of physiological adaptations to a seven-day H. erinaceus supplementation, potentially influencing beneficial health effects.

Hepatoprotective Effect of Cereal Vinegar Sediment in Acute Liver Injury Mice and Its Influence on Gut Microbiota.

Cereal vinegar sediment (CVS) is a natural precipitate formed during the aging process of traditional grain vinegar. It has been used as Chinese traditional medicine, while its composition and function are reported minimally. In this study, we measured CVS in terms of saccharide, protein, fat and water content, and polyphenol and flavonoid content. Furthermore, we determined the amino acids, organic acids, and other soluble metabolites in CVS using reverse-phase high-performance liquid chromatography (RP-HPLC), HPLC, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) platforms. The hepatoprotective effect of CVS was evaluated in acute CCl4-induced liver injury mice. Administration of CVS for 7 days prior to the CCl4 treatment can significantly decrease liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and reactive oxygen species (ROS) levels, compared with those in the hepatic injury model group. The gut microbiota was changed by CCl4 administration and was partly shifted by the pretreatment of CVS, particularly the Muribaculaceae family, which was increased in CVS-treated groups compared with that in the CCl4 administration group. Moreover, the abundances of Alistipes genus and Muribaculaceae family were correlated with the liver ALT, AST, and malondialdehyde (MDA) levels. Our results illustrated the composition of CVS and its hepatoprotective effect in mice, suggested that CVS could be developed as functional food to prevent acute liver injury.

Edgeworthia gardneri (Wall.) Meisn. water extract improves diabetes and modulates gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: In Chinese folk medicine, the flower of Edgeworthia gardneri (Wall.) Meisn. is used to treat various metabolic diseases, such as hyperglycemia, hypertension, and hyperlipidemia. AIM OF THE STUDY: This study aimed to explore the antidiabetes potential of the flower of E. gardneri and investigate whether it can benefit the entire gut bacteria community. MATERIALS AND METHODS: Chemical constituents of the extract were analyzed by UHPLC-Q Exactive Mass Spectrometer (UHPLC-QE-MS). The antidiabetes effect of the water extract (WAE) of the flower of E. gardneri was evaluated in diabetic mice induced by high-fat diet (HFD) and streptozotocin (STZ) (six groups, n = 8) daily at doses of 1, 2, and 3 g/kg for 4 weeks. The gut microbiota was analyzed using high-throughput 16S rRNA gene sequencing. Short-chain fatty acids (SCFAs) in the fecal were also investigated. RESULTS: UHPLC-QE-MS analysis identified 29 compounds, including five alkaloids, six coumarins, four flavonoids, 11 organic acids, and three additional compounds, in the WAE. Results showed that the high dose of WAE considerably decreased the blood glucose level by 30.0%. Furthermore, E. gardneri significantly ameliorated insulin resistance and lipid metabolism dysfunction and repaired islet, hepatic, and white fat and colon histology in diabetic mice. Diabetic mice treated with WAE showed apparent changes in the structure and composition of the gut microbiota. WAE reversed the changes in Clostridiales, Lachnospiraceae, S24-7, Rikenellaceae, and Dorea in diabetic mice. The correlation analysis indicated that key OTUs were related to diabetes indices. The amounts of SCFAs, including acetic, propionic, and valeric acids, were significantly high in WAE-treated diabetic groups. CONCLUSIONS: E. gardneri treatment improved the glucose metabolism and reshaped the unbalanced gut microbiota of diabetic mice. Our study provides evidence for application of E. gardneri to treatment of diabetes mellitus.

Therapeutic Effect and Potential Mechanisms of Lion's Mane Medicinal Mushroom, Hericium erinaceus (Agaricomycetes), Mycelia in Submerged Culture on Ethanol-Induced Chronic Gastric Injury.

Hericium erinaceus (HE) is an edible and medicinal mushroom traditionally used for the treatment of gastric injury in clinical practice. However, scientific evidence of its pharmacological activities has not yet been revealed. This study was designed to investigate the therapeutic effect of HE mycelia in submerged culture on ethanol-induced chronic gastric injury (ECGI) in mice. Gastric injury model was induced by ethanol with chronic and binge ethanol feeding in mice, and then mice were treated with HE mycelia. The stomachs were removed for histopathological examination and inflammatory cytokines measurement. Meanwhile, total proteins of gastric tissue were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling analysis to quantitatively identify differentially expressed proteins (DEPs) in three groups of animals. Bioinformatics analysis of DEPs was conducted through clustering analysis, Venn analysis, Gene Ontology (GO) annotation enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment. The histopathologic characteristics and biochemical data showed that HE mycelia (0.5 and 1.0 g/kg) exhibited therapeutic effects on the ECGI mice. Based on the results of iTRAQ analysis, a total of 308 proteins were differentially expressed in the ethanol group when compared with the control group; 205 DEPs in the high dose of HE (HEH) group when compared with control group; and 230 DEPs in HE group (1.0 g/kg) when compared with ethanol group. KEGG analysis showed that the p53 signaling pathway was closely related to the therapeutic effect of HE mycelia on ECGI. Furthermore, the expression levels of several DEPs, including keratin (KRT) 16, KRT6b and transglutaminase E (TGE), were verified by quantitative real-time polymerase chain reaction (qRT-PCR). In conclusion, H. erinaceus mycelia could relieve ethanol-induced chronic gastric injury in mice by ameliorating inflammation as well as regulating epidermal differentiation.

Depolymerized konjac glucomannan: preparation and application in health care.

Konjac glucomannan (KGM) is a water-soluble polysaccharide obtained from the roots and tubers of konjac plants. Recently, a degraded product of KGM, depolymerized KGM (DKGM), has attracted attention because of its low viscosity, improved hydrophily, and favorable physiological functions. In this review, we describe the preparation of DKGM and its prebiotic effects. Other health benefits of DKGM, covering antioxidant and immune activity, are also discussed, as well as its safety. DKGM could be a candidate for use as a tool for the treatment of various diseases, including intestinal flora imbalance, and oxidative- and immune-related disorders.

Mining of a phospholipase D and its application in enzymatic preparation of phosphatidylserine.

Phosphatidylserine (PS) is useful as the additive in industries for memory improvement, mood enhancement and drug delivery. Conventionally, PS was extracted from soybeans, vegetable oils, egg yolk, and biomass; however, their low availability and high extraction cost were limiting factors. Phospholipase D (PLD) is a promising tool for enzymatic synthesis of PS due to its transphosphatidylation activity. In this contribution, a new and uncharacterized PLD was first obtained from GenBank database via genome mining strategy. The open reading frame consisted of 1614 bp and potentially encoded a protein of 538-amino-acid with a theoretical molecular mass of 60 kDa. The gene was successfully cloned and expressed in Escherichia coli. Its enzymatic properties were experimentally characterized. The temperature and pH optima of PLD were determined to be 60°C and 7.5, respectively. Its hydrolytic activity was improved by addition of Ca2+ at 5 mM as compared with the control. The enzyme displayed suitable transphosphatidylation activity and PS could be synthesized with L-serine and soybean lecithin as substrates under the catalysis of PLD. This PLD enzyme might be a potential candidate for industrial applications in PS production. To the best of our knowledge, this is the first report on genome mining of PLDs from GenBank database.

Bioassay-guided fractionation of ethyl acetate extract from Armillaria mellea attenuates inflammatory response in lipopolysaccharide (LPS) stimulated BV-2 microglia.

BACKGROUND: Armillaria mellea (A. mellea) is a traditional Chinese medicinal and edible mushroom, which is proved to possess a lot of biological activities, including anti-oxidation, immunopotentiation, anti-vertigo and anti-aging activities. However, little information is available in regard to its neuroprotection activity in inflammation-mediated neurodegenerative diseases. PURPOSE: We have found that A. mellea has an anti-inflammatory activity in LPS-induced RAW264.7 cells in our previous study. The objective of this study is to investigate the anti-neuroinflammatory mechanism of a bioassay-guided fractionation (Fr.2) and its active components/compounds. METHODS: Compounds were isolated by preparative high performance liquid chromatography (pre-HPLC) and their structures were established by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopic analyses. The anti-neuroinflammatory effect of Fr.2 and each compounds were investigated in lipopolysaccharide (LPS)-stimulated murine microglia cell lineBV-2. RESULTS: We demonstrated that Fr.2 significantly decreased the production of inflammation mediator nitric oxide (NO) and inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1beta (IL-1ß) in a dose-dependent manner (10, 30, 100µg/ml). In addition, Fr.2 markedly down-regulated the phosphorylation levels of nuclear factor kappa B p65 (NF-κB p65), inhibitory κB-α (IκB-α) and c-Jun N-terminal kinases (JNKs) pathways. Sevens compounds were isolated from Fr.2, among them, three compounds, 5-hydroxymethylfurfural (CP1), vanillic acid (CP4) and syringate (CP5) were reported for the first time in A. mellea. NO and inflammatory cytokines (TNF-α, IL-6, IL-1ß) secretion indicated that daidzein (CP6) and genistein (CP7) showed a more outstanding anti-inflammation potential at non-toxic concentrations (10, 30, 100µM) than the other five compounds. CONCLUSIONS: In conclusion, Fr.2 may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and suppress inflammation pathway in activated microglia. Daidzein and genistein may serve as the effective anti-inflammation compounds of Fr.2.

The common dietary flavonoid myricetin attenuates liver fibrosis in carbon tetrachloride treated mice.

SCOPE: Myricetin is found in most berries, vegetables, and various medicinal herbs, which has been reported to possess various bio-activities. However, the role of myricetin on liver fibrosis remains to be elucidated. METHODS AND RESULTS: Hepatic stellate cell (HSC) line CFSC-8B was stimulated by transforming growth factor ß1 (TGF-ß1) or platelet-derived growth factor BB (PDGF-BB) to induce liver fibrosis in vitro. The results showed that myricetin significantly ameliorated TGF-ß1- or PDGF-BB-induced HSCs activation, cell migration, and extracellular matrix production; blocked TGF-ß1-induced phosphorylation of Smad2, P38, extracellular signal-regulated kinase (ERK), and protein kinase B (Akt); and downregulated PDGF-BB stimulated phosphorylation of extracellular signal-regulated kinase and Akt in HSCs in a dose-dependent manner. Meanwhile, the carbon tetrachloride (CCl4 ) induced mouse model has been used to study antifibrosis role of myricetin in vivo. Our data demonstrated that myricetin suppressed α-smooth muscle actin and collagen type I deposition and blocked phosphorylation of Smad2, mitogen-activated protein kinases, and Akt in CCl4 -treated mice. CONCLUSION: Myricetin inhibits the activation of HSCs and ameliorates CCl4 -induced liver fibrosis in mice and may serve as a potential therapeutic agent in the treatment of liver fibrosis.

Anti-Inflammatory Effects of Ethanol Extract of Lion's Mane Medicinal Mushroom, Hericium erinaceus (Agaricomycetes), in Mice with Ulcerative Colitis.

This study investigated the anti-inflammatory activity of ethanol extracts of Hericium erinaceus in the inflammatory bowel disease (IBD) model. Twenty C57BL/6 mice were exposed to 2% (w/v) dextran sulfate sodium (DSS) in their drinking water for 7 d to induce acute intestinal inflammation. Orally administrated ethanol extract of H. erinaceus (HEEE) (250 mg/kg/d and 500 mg/kg/d body weight) could significantly (P < 0.05) improve body weight and colon length and decreased the intestinal bleeding of DSS-treated mice compared with DSS-treated mice not given HEEE. HEEE markedly reduced DSS-induced myeloperoxidase accumulation in colon tissues, attenuated histological change in the neutrophils and lymphocyte infiltration, and protected the mucosal epithelium. Mechanistically, HEEE ameliorated colitis not only by suppressing the production of inflammatory mediators including tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 in colon tissues but also by adjusting the production of nitric oxide, malondialdehyde, and superoxide dismutase in serum to suppress the oxidative stress. These results suggest that HEEE can be applied as a protective agent in the treatment of IBDs.

Antiplatelet Aggregation and Antithrombosis Efficiency of Peptides in the Snake Venom of Deinagkistrodon acutus: Isolation, Identification, and Evaluation.

Two peptides of Pt-A (Glu-Asn-Trp 429 Da) and Pt-B (Glu-Gln-Trp 443 Da) were isolated from venom liquor of Deinagkistrodon acutus. Their antiplatelet aggregation effects were evaluated with platelet-rich human plasma in vitro; the respective IC50 of Pt-A and Pt-B was 66 µM and 203 µM. Both peptides exhibited protection effects on ADP-induced paralysis in mice. After ADP administration, the paralysis time of different concentration of Pt-A and Pt-B lasted as the following: 80 mg/kg Pt-B (152.8 ± 57.8 s) < 40 mg/kg Pt-A (163.5 ± 59.8 s) < 20 mg/kg Pt-A (253.5 ± 74.5 s) < 4 mg/kg clopidogrel (a positive control, 254.5 ± 41.97 s) < 40 mg/kg Pt-B (400.8 ± 35.9 s) < 10 mg/kg Pt-A (422.8 ± 55.4 s), all of which were statistically shorter than the saline treatment (666 ± 28 s). Pulmonary tissue biopsy confirmed that Pt-A and Pt-B prevented the formation of thrombi in the lung. Unlike ADP injection alone, which caused significant reduction of peripheral platelet count, Pt-A treatment prevented the drop of peripheral platelet counts; interestingly, Pt-B could not, even though the same amount of Pt-B also showed protection effects on ADP-induced paralysis and thrombosis. More importantly, intravenous injection of Pt-A and Pt-B did not significantly increase the hemorrhage risks as clopidogrel.

Screening and isolation for anti-hepatofibrotic components from medicinal mushrooms using TGF-(ß1-induced live fibrosis in hepatic stellate cells.

Liver fibrosis is a wound-healing response to chronic liver injury that could lead to liver failure, but treatment remains ineffective. In this study, we investigated anti-hepatic fibrosis activity of n-hexane, chloroform, ethyl acetate, and methanol extracts of mycelia from six commercially available medicinal mushrooms in submerged culture, namely Antrodia camphorata, Cephalosporium sinensis, Cordyceps mortierella, Hericium erinaceus, Ganoderma lucidum, and Armillaria mellea. Their anti-fibrotic activities were evaluated via inhibition against accumulation of TGF-ß1-induced collagen deposition in CFSC-8B cells. Hex, Chl, and MeOH extracts of A. camphorata and Hex extract of A. mellea significantly decreased collagen production. Bioactivity-guided fractionation led to the identification of seven compounds using UPLC-Q-TOF-MS from the Hex Fr.2 of A. camphorata. At the molecular level, Hex Fr.2 of A. camphorata suppressed α-SMA, Collagen I, Collagen III, and Fibronectin expression induced by TGF-ß1 in CFSC-8B cells as indicated by qRT-PCR analysis. They also inhibited α-SMA and Collagen I protein expression according to western blot analyses. Mechanistically, Hex Fr.2 of A. camphorata negatively regulates TGF-ß1/Smad2/3 signaling. Our studies demonstrate that A. camphorata has in vitro anti-hepatofibrotic activity and that there is great potential for the discovery of new drugs for the treatment of liver fibrosis by screening more medicinal mushrooms.

Anti-inflammatory activity of mycelial extracts from medicinal mushrooms.

Medicinal mushrooms have been essential components of traditional Chinese herbal medicines for thousands of years, and they protect against diverse health-related conditions. The components responsible for their anti-inflammatory activity have yet to be fully studied. This study investigates the anti-inflammatory activity of n-hexane, chloroform, ethyl acetate, and methanol extracts of mycelia in submerged culture from 5 commercially available medicinal mushrooms, namely Cephalosporium sinensis, Cordyceps mortierella, Hericium erinaceus, Ganoderma lucidum, and Armillaria mellea. MTT colorimetric assay was applied to measure the cytotoxic effects of different extracts. Their anti-inflammatory activities were evaluated via inhibition against production of lipopolysaccharide (LPS)-induced nitric oxide (NO) in murine macrophage-like cell line RAW264.7 cells. Of the 20 extracts, n-hexane, chloroform, ethyl acetate, and methanol extracts from C. sinensis, C. mortierella, and G. lucidum; chloroform extracts from H. erinaceus and A. mellea; and ethyl acetate extracts from A. mellea at nontoxic concentrations (<300 µg/mL) dose-dependently inhibited LPS-induced NO production. Among them, the chloroform extract from G. lucidum was the most effective inhibitor, with the lowest half maximal inhibitory concentration (64.09 ± 6.29 µg/mL) of the LPS-induced NO production. These results indicate that extracts from medicinal mushrooms exhibited anti-inflammatory activity that might be attributable to the inhibition of NO generation and can therefore be considered a useful therapeutic and preventive approach to various inflammation-related diseases.

Enhanced biotransformation of dehydroepiandrosterone to 3ß,7α,15α-trihydroxy-5-androsten-17-one with Gibberella intermedia CA3-1 by natural oils addition.

Dihydroxylation of dehydroepiandrosterone (DHEA) is an essential step in the synthesis of many important pharmaceutical intermediates. However, the solution to the problem of low biohydroxylation conversion in the biotransformation of DHEA has yet to be found. The effects of natural oils on the course of dihydroxylation of DHEA to 3ß,7α,15α-trihydroxy-5-androsten-17-one (7α,15α-diOH-DHEA) were studied. With rapeseed oil (2 %, v/v) addition, the bioconversion efficiency was improved, and the 7α,15α-diOH-DHEA yield was increased by 40.8 % compared with that of the control at DHEA concentration of 8.0 g/L. Meantime, the ratio of 7α,15α-diOH-DHEA to 7α-OH-DHEA was also increased by 4.5 times in the rapeseed oil-containing system. To explain the mechanism underlying the increase of 7α,15α-diOH-DHEA yield, the effects of rapeseed oil on the pH of the bioconversion system, the cell growth and integrity of Gibberella intermedia CA3-1, as well as the membrane composition were systematically studied. The addition of rapeseed oil enhanced the substrate dispersion and maintained the pH of the system during bioconversion. Cells grew better with favorable integrity. The fatty acid profile of G. intermedia cells revealed that rapeseed oil changed the cell membrane composition and improved cell membrane permeability for lipophilic substrates.

Alpha-terpineol promotes triterpenoid production of Antrodia cinnamomea in submerged culture.

Antrodia cinnamomea is a medicinal mushroom producing potent bioactive triterpenoids. However, triterpenoids of A. cinnamomea in submerged culture are much less than those in fruiting bodies. Here we evaluated effects of different extracts from a host-related species, Cinnamomum camphora, on the mycelial growth and triterpenoid production of A. cinnamomea in submerged culture. The hot water extract of the stem showed the strongest promotion of the mycelial growth. The petroleum ether extract of the stem (PES) (0.05 g L(-1)) showed the greatest stimulatory effect on content and production of triterpenoids. A total of 39 compounds including terpenoids, phenolic and aromatic compounds were identified in the PES by GC-MS analysis. Furthermore, the effects of seven compounds contained in the PES on the mycelial growth and triterpenoid production of A. cinnamomea were evaluated. Among them, α-terpineol (0.5 mg L(-1)) showed the greatest stimulatory effect on the triterpenoid content (23.31 mg g(-1)) and triterpenoid production (91.33 mg L(-1)) of A. cinnamomea. Results of LC-MS analysis showed that α-terpineol (0.5 mg L(-1)) stimulated the syntheses of six triterpenoids in the mycelia of A. cinnamomea. This indicates that α-terpineol can act as an elicitor for triterpenoid biosynthesis in A. cinnamomea.

Stimulated production of steroids in Inonotus obliquus by host factors from birch.

Steroids was considered as one of the bioactive components in Inonotus obliquus, while this kind of secondary metabolites are less accumulated in cultured mycelia. In this study, effect of extracts from bark and core of host-related species, birch (Betula platyphylla Suk.), on steroid production of I. obliquus in submerged culture were evaluated. The results showed that all dosages (0.01 and 0.1 g/L) of aqueous extracts and methanol extracts from birch bark and birch core possessed significantly stimulatory effect on steroid production of I. obliquus (P < 0.05). Among the eight extracts, the aqueous extract (0.01 g/L) from birch bark gave the highest steroid production (225.5 ± 8.7 mg/L), which is 97.3% higher than that of the control group. The aqueous extract (0.01 and 0.1 g/L) from birch bark could simultaneously stimulated mycelial growth and steroid content, while the methanol extract from birch bark only elevated the steroid content. High performance liquid chromatography analysis showed that productions of betulin, ergosterol, cholesterol, lanosterol, stigmasterol, and sitosterol in I. obliquus simultaneously increased in the presence of aqueous extract and methanol extract from birch bark. The results presented herein indicate that extracts from birch bark could act as an inducer for steroid biosynthesis of I. obliquus.

Nanoparticle-delivered transforming growth factor-ß siRNA enhances vaccination against advanced melanoma by modifying tumor microenvironment.

Achievement of potent immunoresponses against self/tumor antigens and effective therapeutic outcome against advanced tumors remain major challenges in cancer immunotherapy. The specificity and efficiency of two nanoparticle-based delivery systems, lipid-calcium-phosphate (LCP) nanoparticle (NP) and liposome-protamine-hyaluronic acid (LPH) NP, provide us an opportunity to address both challenges. A mannose-modified LCP NP delivered both tumor antigen (Trp 2 peptide) and adjuvant (CpG oligonucleotide) to the dendritic cells and elicited a potent, systemic immune response regardless of the existence or the stage of tumors in the host. This vaccine was less effective, however, against later stage B16F10 melanoma in a subcutaneous syngeneic model. Mechanistic follow-up studies suggest that elevated levels of immune-suppressive cytokines within the tumor microenvironment, such as TGF-ß, might be responsible. We strategically augment the efficacy of LCP vaccine on an advanced tumor by silencing TGF-ß in tumor cells. The delivery of siRNA using LPH NP resulted in about 50% knockdown of TGF-ß in the late stage tumor microenvironment. TGF-ß down-regulation boosted the vaccine efficacy and inhibited tumor growth by 52% compared with vaccine treatment alone, as a result of increased levels of tumor infiltrating CD8+ T cells and decreased level of regulatory T cells. Combination of systemic induction of antigen-specific immune response with LCP vaccine and targeted modification of tumor microenvironment with LPH NP offers a flexible and powerful platform for both mechanism study and immunotherapeutic strategy development.