Functional Properties, Rheological Characteristics, Simulated Digestion, and Fermentation by Human Fecal Microbiota of Polysaccharide from Morchella importuna.

Morchella importuna polysaccharide (MIP) has been proven to have obvious hypoglycemic effects on mice with type 2 diabetes (T2DM). This study looked at the functional and rheological characteristics of MIP, and investigated the effects of MIP on the human fecal microbiota through in vitro fermentation experiments. The outcomes demonstrate the excellent oil-holding capacity, emulsifying, foaming, and rheological characteristics of MIP. After salivary gastrointestinal digestion, the Mw of MIP decreased from 398.2 kDa and 21.5 kDa to 21.9 kDa and 11.7 kDa. By 16S rRNA sequencing of bacteria fermented in vitro, it was found that MIP did not improve the richness and diversity of intestinal microorganisms, but it may exert an anti-T2DM function by significantly increasing the relative abundance of Firmicutes and promoting Ruminococcaceae_UCG_014, Bacteroides, and Blautia proliferation. Escherichia-Shigella could also be inhibited to improve the intestinal microenvironment. In addition, the fermentation of MIP increased the total short-chain fatty acid (SCFA) concentration from 3.23 mmol/L to 39.12 mmol/L, and the propionic acid content increased significantly. In summary, MIP has excellent processing performance and is expected to exert potential anti-T2DM activity through the human intestinal microbiota, which has broad market prospects.

The effect of sequential extraction on the physicochemical and rheological properties of Naematelia aurantialba polysaccharides.

To overcome the difficulty of separation and low rate of extraction caused by highly viscous polysaccharides from Naematelia aurantialba (NA), four N. aurantialba polysaccharides (NAPs) were sequentially extracted using water (enzyme-/ultrasound-assisted extraction), alkali (0.1 mol/L NaOH), and acid (0.1 mol/L HCl), and named E-NAP, U-NAP, Al-NAP, and Ac-NAP. The properties of four NAPs were different. The yields of NAPs were 26.05 % (Ac-NAP) > 20.33 % (Al-NAP) > 17.99 % (U-NAP) > 12.77 % (E-NAP), respectively. The monosaccharide composition of NAPs was composed primarily of mannose, xylose, glucose, glucuronic acid, and galactose. Sequential extraction improved the purity and solubility of NAPs, but decreased the particle size, thermal stability, water retention, and crystallinity. Two polysaccharides, U-NAP and Al-NAP, had a triple helix structure. All the NAPs were pseudoplastic fluids with concentration/frequency-dependent entangled structure. Al-NAP with the highest viscosity exhibited an elastic gel, while Ac-NAP with the lowest viscosity was a viscous gel. The behavior of NAPs differed from that predicted using the Cox-Merz rule, and in particular, E-NAP and U-NAP more significantly deviated from the rule. In this study, four NAPs with different properties were extracted sequentially, which provided a theoretical basis for the down-stream processing with high added-value and utilization of NA and NAP.

Function of Transcription Factors PoMYB12, PoMYB15, and PoMYB20 in Heat Stress and Growth of Pleurotus ostreatus.

MYB transcription factors (TFs) have been extensively studied in plant abiotic stress responses and growth and development. However, the role of MYB TFs in the heat stress response and growth and development of Pleurotus ostreatus remains unclear. To investigate the function of PoMYB12, PoMYB15, and PoMYB20 TFs in P. ostreatus, mutant strains of PoMYB12, PoMYB15, and PoMYB20 were generated using RNA interference (RNAi) and overexpression (OE) techniques. The results indicated that the mycelia of OE-PoMYB12, OE-PoMYB20, and RNAi-PoMYB15 mutant strains exhibited positive effects under heat stress at 32 °C, 36 °C, and 40 °C. Compared to wild-type strains, the OE-PoMYB12, OE-PoMYB20, and RNAi-PoMYB15 mutant strains promoted the growth and development of P. ostreatus. These mutant strains also facilitated the recovery of growth and development of P. ostreatus after 24 h of 36 °C heat stress. In conclusion, the expression of PoMYB12 and PoMYB20 supports the mycelium's response to heat stress and enhances the growth and development of P. ostreatus, whereas PoMYB15 produces the opposite effect.

Transcriptome Analysis Revealed That Hydrogen Peroxide-Regulated Oxidative Phosphorylation Plays an Important Role in the Formation of Pleurotus ostreatus Cap Color.

Pleurotus ostreatus is widely cultivated in China. H2O2, as a signaling molecule, can regulate the formation of cap color, but its regulatory pathway is still unclear, severely inhibiting the breeding of dark-colored strains. In this study, 614 DEGs specifically regulated by H2O2 were identified by RNA-seq analysis. GO-enrichment analysis shows that DEGs can be significantly enriched in multiple pathways related to ATP synthesis, mainly including proton-transporting ATP synthesis complex, coupling factor F(o), ATP biosynthetic process, nucleoside triphosphate metabolic processes, ATP metabolic process, purine nucleoside triphosphate biosynthetic and metabolic processes, and purine ribonuclease triphosphate biosynthetic metabolic processes. Further KEGG analysis revealed that 23 DEGs were involved in cap color formation through the oxidative phosphorylation pathway. They were enriched in Complexes I, III, IV, and V in the respiratory chain. Further addition of exogenous uncoupling agents and ATP synthase inhibitors clarifies the important role of ATP synthesis in color formation. In summary, H2O2 may upregulate the expression of complex-encoding genes in the respiratory chain and promote ATP synthesis, thereby affecting the formation of cap color. The results of this study lay the foundation for the breeding of dark-colored strains of P. ostreatus and provide a basis for the color-formation mechanism of edible fungi.

MAC Family Transcription Factors Enhance the Tolerance of Mycelia to Heat Stress and Promote the Primordial Formation Rate of Pleurotus ostreatus.

Pleurotus ostreatus is a typical tetrapolar heterologous edible mushroom, and its growth and development regulatory mechanism has become a research hotspot in recent years. The MAC1 protein is a transcription factor that perceives copper and can regulate the expression of multiple genes, thereby affecting the growth and development of organisms. However, its function in edible mushrooms is still unknown. In this study, two transcription factor genes, PoMCA1a and PoMAC1b, were identified. Afterwards, PoMAC1 overexpression (OE) and RNA interference (RNAi) strains were constructed to further explore gene function. The results showed that the PoMAC1 mutation had no significant effect on the growth rate of mycelia. Further research has shown that OE-PoMAC1a strains and RNAi-PoMAC1b strains exhibit strong tolerance under 32 °C heat stress. However, under 40 °C heat stress, the OE of PoMAC1a and PoMAC1b promoted the recovery of mycelial growth after heat stress. Second, the OE of PoMAC1a can promote the rapid formation of primordia and shorten the cultivation cycle. In summary, this study indicated that there are functional differences between PoMAC1a and PoMAC1b under different heat stresses during the vegetative growth stage, and PoMAC1a has a positive regulatory effect on the formation of primordia during the reproductive growth stage.

Nitric Oxide Negatively Regulates the Rapid Formation of Pleurotus ostreatus Primordia by Inhibiting the Mitochondrial aco Gene.

Nitric oxide (NO) is as a signaling molecule that participates in the regulation of plant development and in a number of physiological processes. However, the function and regulatory pathway of NO in the growth and development of edible mushrooms are still unknown. This study found that NO played a negative role in the transformation of Pleurotus ostreatus from vegetative growth to reproductive growth by the exogenous addition of NO donors and scavengers. Further studies showed that NO can inhibit the gene expression and enzyme activity of aconitase (ACO). Moreover, the overexpression (OE) of mitochondrial aco and RNA interference (RNAi) confirmed that ACO participates in the regulation of the primordia formation rate. The effects of aco OE and RNAi on the tricarboxylic acid (TCA) cycle and energy metabolism were further measured. The results showed that RNAi-aco mutant strains can affect the enzyme activities of isocitrate dehydrogenase of mitochondria (ICDHm) and α-ketoglutarate dehydrogenase (α-KGDH) in the TCA cycle, thereby reducing the production of nicotinamide adenine dinucleotide (NADH) in the TCA cycle, decreasing the contents of adenosine triphosphate (ATP) and hydrogen peroxide (H2O2), and negatively regulating the rapid formation of primordia. In addition, H2O2 was significantly increased during the transformation from vegetative growth to reproductive growth of P. ostreatus. Additionally, the exogenous addition of H2O2 and its scavengers further confirmed the positive regulation by H2O2 in primordia formation. This study shows that during the growth and development of P. ostreatus, NO can inhibit the expression of the mitochondrial aco gene and ACO protein in the TCA cycle, reduce the production of ATP and H2O2 in the respiratory chain, and negatively regulate the rate of primordia formation.

Mnsod1 promotes the development of Pleurotus ostreatus and enhances the tolerance of mycelia to heat stress.

BACKGROUND: Superoxide dismutases (SODs, EC 1.15.1.1) are defense proteins that can be used as sweepers to clear reactive oxygen species (ROS). They have been widely studied in the plant. Intensive research demonstrates that SOD plays an essential role in plants. However, in Pleurotus ostreatus, the function and regulatory pathway of SOD in the growth and development and the abiotic stress response have not been clear. RESULTS: In this study, three MnSOD-encoding genes of the P. ostreatus CCMSSC00389 strain were cloned and identified. Mnsod1, Mnsod2, and Mnsod3 were interrupted by 3, 7, and 2 introns, and encoded proteins of 204, 220, and 344 amino acids, respectively. By comparing the relative expression of three MnSOD-encoding genes in mycelia, the results showed that the gene with the highest primary expression was Mnsod1. Subsequently, the function of P. ostreatus Mnsod1 was explored by overexpression (OE) and RNA interference (RNAi). The results showed that during the growth and development of P. ostreatus, MnSOD1 protein increased gradually from mycelia to the fruiting body, but decreased in spores. The change of Mnsod1 transcription level was not consistent with the changing trend of MnSOD1 protein. Further studies showed that during primordia formation, the expression of Mnsod1 gradually increased, reaching a peak at 48 h, and the transcription level was 2.05-folds compared to control. H2O2 content progressively accumulated during the formation of primordia, and its change trend was similar to that of Mnsod1 transcription. OE-Mnsod1-1 and OE-Mnsod1-21 strains accelerated the formation of primordia. The results suggested that Mnsod1 may participate in the formation rate of P. ostreatus primordium by regulating the signal molecule H2O2. In addition, OE-Mnsod1-1 and OE-Mnsod1-21 strains shortened the mycelial recovery time after heat stress and improved the tolerance of the strains to 2.5 mM and 5 mM H2O2, which showed that Mnsod1 was involved in the response of P. ostreatus mycelium to heat stress. CONCLUSIONS: This study indicates that Mnsod1 plays an active role in the formation of P. ostreatus primordia and the response to abiotic stress.

Alternative oxidase gene induced by nitric oxide is involved in the regulation of ROS and enhances the resistance of Pleurotus ostreatus to heat stress.

BACKGROUND: In China, during the cultivation process of Pleurotus ostreatus, the yield and quality of fruiting bodies are easily affected by high temperatures in summer. Nitric oxide (NO) plays an important regulatory role in the response to abiotic stress, and previous studies have found that NO can induce alternative oxidase (aox) experssion in response to heat stress (HS) by regulating aconitase. However, the regulatory pathway of NO is complex, and the function and regulation of the aox gene in the response to HS remain unclear. RESULTS: In this study, we found that NO affected nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP) levels, reduced hydrogen peroxide (H2O2) and superoxide anion (O2-) contents, and slowed O2- production. Further RNA-Seq results showed that NO regulated the oxidation-reduction process and oxidoreductase activity, affected the cellular respiration pathway and activated aox gene expression. The function of aox was determined by constructing overexpression (OE) and RNA interference (RNAi) strains. The results showed that the OE-aox strains exhibited obviously improved growth recovery after exposure to HS. During exposure to HS, the OE-aox strains exhibited reduced levels of NADH, the product of the tricarboxylic acid (TCA) cycle, and decreased synthesis of ATP, which reduced the production and accumulation of reactive oxygen species (ROS), whereas the RNAi-aox strains exhibited the opposite result. In addition, aox mediated the expression of antioxidant enzyme genes in the mycelia of P. ostreatus under HS through the retrograde signaling pathway. CONCLUSIONS: This study shows that the expression of the aox gene in P. ostreatus mycelia can be induced by NO under HS, that it regulates the TCA cycle and cell respiration to reduce the production of ROS, and that it can mediate the retrograde signaling pathway involved in the mycelial response to HS.

Cell metabolomics to study the cytotoxicity of carbon black nanoparticles on A549 cells using UHPLC-Q/TOF-MS and multivariate data analysis.

Carbon black nanoparticles (CBNPs) are core component of fine particulate matter (PM2.5) in the atmosphere. It was reported that the particle in the atmosphere with smaller size and the larger the specific surface area are easier to reach the deep respiratory tract or even the alveoli through the respiratory barrier and cause lung injury. Therefore, it has been believed that ultrafine or nanometer particles with more toxic than those with larger particle sizes. Moreover, it was confirmed that CBNPs could induce inflammation, oxidative stress and changes in cell signaling and gene expression in mammalian cells and organs. However, the cytotoxicity mechanism of them has been uncertain so far. The aim of the present study was to explore the underlying mechanism of cytotoxicity induced by CBNPs on A549 cells. In the current research, the viabilities of A549 cells were detected by Cell Counting Kit-8 (CCK-8) assay. The further metabolomics studies were conducted to detect the cytotoxic effect of CBNPs on A549 cells with an IC50 value of 70 µg/mL for 48 h. Potential differential compounds were identified and quantified using a novel on-line acquisition method based on ultra-liquid chromatography quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF/MS). The cytotoxicity mechanism of CBNPs on A549 cells was evaluated by multivariate data analysis and statistics. As a result, a total of 32 differential compounds were identified between CBNPs exposure and control groups. In addition, pathway analysis showed the metabolic changes were involved in the tricarboxylic acid (TCA) cycle, alanine, aspartate and glutamate metabolism, histidine metabolism and so on. It is also suggested that CBNPs may induce cytotoxicity by affecting the normal process of energy metabolism and disturbing several vital signaling pathways and finally induce cell apoptosis.

Nitric Oxide Improves the Tolerance of Pleurotus ostreatus to Heat Stress by Inhibiting Mitochondrial Aconitase.

Pleurotus ostreatus is widely cultivated in China. However, its cultivation is strongly affected by seasonal temperature changes, especially the high temperatures of summer. Nitric oxide (NO) was previously reported to alleviate oxidative damage to mycelia by regulating trehalose. In this study, we found that NO alleviated oxidative damage to P. ostreatus mycelia by inhibiting the protein and gene expression of aconitase (ACO), and additional studies found that the overexpression and interference of aco could affect the content of citric acid (CA). Furthermore, the addition of exogenous CA can induce alternative oxidase (aox) gene expression under heat stress, reduce the content of H2O2 in mycelium, and consequently protect the mycelia under heat stress. An additional analysis focused on the function of the aox gene in the heat stress response of mycelia. The results show that the colony diameter of the aox overexpression (OE-aox) strains was significantly larger than that of the wild-type (WT) strain under heat stress (32°C). In addition, the mycelia of OE-aox strains showed significantly enhanced tolerance to H2O2 In conclusion, this study demonstrates that NO can affect CA accumulation by regulating aco gene and ACO protein expression and that CA can induce aox gene expression and thereby be a response to heat stress.IMPORTANCE Heat stress is one of the abiotic stresses that affect the growth and development of edible fungi. Our previous study found that exogenous NO had a protective effect on mycelia under heat stress. However, its regulatory mechanism had not been elucidated. In this study, we found that NO altered the respiratory pathway of mycelia under heat stress by regulating aco The results have enhanced our understanding of NO signaling pathways in P. ostreatus.

Expression patterns of two pal genes of Pleurotus ostreatus across developmental stages and under heat stress.

BACKGROUND: Phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) is the first key enzyme in the phenylpropanoid pathway. The pal gene has been widely studied in plants and participates in plant growth, development and defense systems. However, in Pleurotus ostreatus, the biological functions of pal during organismal development and exposure to abiotic stress have not been reported. RESULTS: In this study, we cloned and characterized the pal1 (2232 bp) and pal2 (2244 bp) genes from the basidiomycete P. ostreatus CCMSSC 00389. The pal1 and pal2 genes are interrupted by 6 and 10 introns, respectively, and encode proteins of 743 and 747 amino acids, respectively. Furthermore, prokaryotic expression experiments showed that PAL enzymes catalyzed the conversion of L-phenylalanine to trans-cinnamic acid. The function of pal1 and pal2 was determined by constructing overexpression (OE) and RNA interference (RNAi) strains. The results showed that the two pal genes had similar expression patterns during different developmental stages. The expression of pal genes was higher in the reproductive growth stage than in the vegetative growth stage. And the interference of pal1 and pal2 delayed the formation of primordia. The results of heat stress assays showed that the RNAi-pal1 strains had enhanced mycelial tolerance to high temperature, while the RNAi-pal2 strains had enhanced mycelial resistance to H2O2. CONCLUSIONS: These results indicate that two pal genes may play a similar role in the development of P. ostreatus fruiting bodies, but may alleviate stress through different regulatory pathways under heat stress.

Characterization of serum metabolites as biomarkers of carbon black nanoparticles-induced subchronic toxicity in rats by hybrid triple quadrupole time-of-flight mass spectrometry with non-targeted metabolomics strategy.

Carbon black nanoparticles (CBNPs) are one of atmospheric particles components and have been closely related with a series of lung diseases. It can reach the depths of the respiratory tract or even alveolar more easily than those micro-particles. Although some of its toxicities have been confirmed in animals or human bodies, the subchronic toxicity mechanism of CBNPs has been uncertain so far. Therefore, it is very necessary to establish a novel method and clarify the mechanism of subchronic toxicity caused by concentration adjustments of small molecule metabolites in vivo. In animal experiments, CB exposure, recovery and control group were set up. The concentration of CBNPs in chamber was 30.06 ±â€¯4.42 mg/m3. We developed a UHPLC-Q-TOF-MS/MS-based non-targeted metabolomic analysis strategy to analyze serum samples of rats. Then, differential metabolites in serum were found by multivariate data analysis and 39 potential biomarkers were identified. It was showed that main metabolic pathways associated with CBNPs exposure were hormones metabolism, amino acid metabolism, nucleotide metabolism and lipid metabolism. It is worth noting that long-term exposure to CBNPs had the greatest impact on steroid hormones biosynthesis so that the risk of infertility could increase. The results provided a new mechanistic insight into the metabolic alterations owing to CBNPs induced subchronic toxicity.

Metabolites identification of (+)-usnic acid in vivo by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

(+)-usnic acid (UA) is an active natural phenolic acid ingredient originating from Chinese traditional Tibetan herb. Usnea acid is expected to become a new agent for anticancer and remarkable antitumor. To reveal its metabolic profile, metabolites identification of UA in vivo was studied using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) in this present study. The chromatographic separation was performed on a C18 column with a mobile phase consisted of methanol and water with a flow rate of 0.4 ml/min. The mass spectral analysis conducted in a negative electrospray ionization mode combined with information-dependent acquirement technology (IDA) was used to trace all the potential UA metabolites. Several sensitive and specific multiple data-mining techniques especially key product ions (KPIs) filter were applied to hunt and identify metabolites rapidly. As a result, a total of 36 metabolites were detected after oral administration of UA, including 33, 8 and 16 in rat urine, plasma and bile, respectively. These results showed that the probable metabolite pathways of UA were oxidation, reduction, dihydroxylation, glycine conjugation, glucuronide conjugation, N-acetylcysteine conjugation and methylation. It is the first time to elucidate the profile of UA in vivo. These results not only provided the basis of UA pharmacological properties, but also gave the guidance in clinical medication. Moreover, the analysis strategy and methodology proposed in this paper could be widely used in characterization of other phenolic acids metabolites.

Chemical profiling and total quality assessment of Isodon japonica using data-independent acquisition mode combined with superimposed multiple product ion UHPLC-Q-TOF-MS and chemometric analysis.

In this paper, an analytical strategy combined data acquisition with a practical mining strategy aimed at rapid characterization and quantitation of ent-kaurane diterpenoids in Isodon japonica using ultra high-performance liquid chromatography-triple time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS). First, an effective self-built filter template based on drug phase I/II metabolic reaction theory and a components library data set were established. Second, the mass spectra of ent-kaurane diterpenoid standards were studied and their mass spectrum cleavage pathways were summarized. Next, the methanol extract of this herb was studied by data-independent acquisition mode (DIA). With the aid of a self-built filter template, the peaks of ent-kaurane diterpenoids were easily picked out and rapidly classified as ent-kaurane diterpenoids from a complex matrix. A total of 24 ent-kaurane diterpenoids were structurally identified. Meanwhile, the self-built filter template provided a convenient and fast method for the structural characterization and Isodon japonica was used to illustrate this approach for the first time. Furthermore, eight major bioactive diterpenoids were simultaneously quantified by a newly developed superimposed multiple product ion (SMPI) with UPLC-Q-TOF-MS/MS method. Principal component analysis (PCA) revealed significant differences in different batches of samples. These combined qualitative and quantitative methods were used to provide a potential approach for the holistic quality evaluation of traditional Chinese medicine (TCM) and its preparations.

UHPLC-Q-TOF-MS/MS-oriented characteristic components dataset and multivariate statistical techniques for the holistic quality control of Usnea.

The holistic quality evaluation of Traditional Chinese Medicine (TCM) is confronted with significant challenges due to its extreme chemical complexity. In this study, a sensitive strategy based on ultra-high-performance liquid chromatography-triple/time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) and chemometric analysis was established and validated for the qualitative and semi-quantitative analyses of characteristic components in Usnea. First, three mass spectrometry fragmentation patterns of phenolic acid standards were studied and summarized. Then, an extract of this herb was analyzed by the full-scan MS spectra and identified by extracted ion chromatography (XIC). Based on the abovementioned methods, a total of 38 compounds (8 dibenzofurans, 11 didepsides, 13 depsidones, and 6 mono-substituted phenyl rings) were identified. Subsequently, the qualities of Usnea samples from different regions were evaluated by the semi-quantitative analysis based on their relative peak areas. Furthermore, principal component analysis (PCA) was performed to compare the Usnea herbs and to find possible diagnostic chemical components. This novel and powerful strategy could provide a potential approach for the holistic quality control of TCM.

A practical strategy for the characterization of ponicidin metabolites in vivo and in vitro by UHPLC-Q-TOF-MS based on nontargeted SWATH data acquisition.

Ponicidin is an active natural ent-kaurane diterpenoid ingredient originating from many Isondon herbs and is expected to become a new anticancer agent. In this study, a practical strategy was developed for the identification of ponicidin metabolites in vivo and in vitro utilizing ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). The analytical strategy was as follows: potential ponicidin metabolites were detected by a novel on-line data acquisition approach, i.e., sequential window acquisition of all theoretical fragment-ion spectra (SWATH™). Compared to the traditional information-dependent acquisition (IDA) method, SWATH™ significantly improved the hit rate of low-level or trace metabolites because it could obtain all MS/MS spectra. Moreover, many data post-processing methods were used to deduce the metabolites structures. As a result, a total of 20 metabolites were characterized in vivo and in vitro. The results showed that ponicidin could undergo general metabolic reactions, such as oxidation, reduction, hydrolysis, methylation and glucuronidation. Furthermore, there was an obvious difference in the ponicidin metabolites among four species in vitro. This is the first time that the SWATH™ data acquisition mode has been used to characterize ponicidin metabolites in trace amounts or in a biological matrix. These results not only provided a better understanding of the safety and efficacy of ponicidin but also showed a valuable methodology for the identification of other ent-kaurane diterpenoid metabolites.