MrCreC, a carbon catabolite repression gene, is required for the growth, conidiation, stress tolerance and virulence of Metarhizium robertsii.

As a key component of carbon source metabolism in fungi, CreC WD40 repeat protein is regulated by carbon catabolite repression (CCR). However, the understanding of the functions of CreC in entomopathogenic fungi is currently limited. Here, CreC in Metarhizium robertsii (MrCreC) was identified, and its roles in fungal development, conidiation, environmental stress response, and insecticidal virulence were explored. MrCreC is localized to cytoplasm, and MrCreC deletion affects fungal growth on various nutrients. Compared to the wild type, the sporulation of ΔMrCreC strain was significantly decreased by 60.3%. Further qPCR analysis found that deletion of MrCreC resulted in repression of sporulation-related genes such as AbaA, FlbA, Flbc, MedA, FlbD, FluG, and wetA. In addition, MrCreC loss did not alter heat stress tolerance but resulted in enhanced tolerance to UV-B. Interestingly, bioassays showed that the virulence following exposures to topical applications or injection of conidial suspensions of both infection and injection was impaired compared with that of the wild type. Further analysis showed that the adhesion and cuticle penetration genes in ΔMrCreC was down-regulated during infection, and the appressorial formation rate was significantly reduced. A deletion of MrCreC significantly also reduced immune escape and nutrient utilization genes in insect hemocoel. In conclusion, MrCreC is involved in the growth, development and virulence of M. robertsii. These findings advance our understanding of the function of CCR pathway-related genes.

Optimisation of enzyme-assisted extraction of Erythronium sibiricum bulb polysaccharide and its effects on immunomodulation.

In this study, polysaccharides of Erythronium sibiricum bulb were extracted using enzyme-assisted extraction technology and then optimised by response surface methodology. The characteristics and immunomodulatory activities of the polysaccharide (E1P) were investigated. Setting the yield of polysaccharides as the index, the effects of amylase content, zymolytic time, extraction pH and zymolytic temperature were investigated. The optimal extraction conditions for polysaccharides were as follows: amylase content, 1% weight of pre-treated powder; zymolytic time, 2 h; extraction pH, 7.5; and zymolytic temperature, 55 °C. The yield was predicted to be 61.10%, which agreed with the value obtained in confirmatory experiments (59.71% ± 2.72%). Further research indicated that the primary component of E1P is glucose; however, it also contains a small quantity of galactose and arabinose. In vitro assays showed that E1P and ESBP (another kind of E. sibiricum bulb polysaccharide extracted by water decoction in our previous study) could significantly promote the cellular viability and phagocytosis of RAW264.7 cells without cytotoxicity. Moreover, they could enhance the ability to secrete nitric oxide and cytokines such as TNF-α and IL-1ß. However, the immunomodulatory activities of E1P were better than those of ESBP. According to the results of this study, enzyme-assisted extraction represents a new strategy for extracting E. sibiricum bulb polysaccharides with higher yield and better immune activity.

Optimization of the composite enzyme extraction of polysaccharide from Erythronium sibiricum bulb and its immunoregulatory activities.

An efficient compound enzyme extraction process was developed and optimized to extract the polysaccharide from Erythronium sibiricum bulb via response surface methodology. The polysaccharide E2P was obtained. Then, the preliminary characteristics of E2P were determined via colorimetry and chromatography. Additionally, the immunoregulatory activities of E2P and another polysaccharide (ESBP, extracted using the hot water method) were compared. The optimized extraction results were as follows: temperature (54.56 °C), time (2.52 h), pH (6.53), and enzyme concentration ratio (0.5% cellulase:1.5% amylase). The yield (64.18% ± 2.91%) obtained under the aforementioned conditions was considerably higher than the yield of ESBP (37.25% ± 0.17%). The total sugar, uronic acid, starch, and protein contents of E2P were 81.77% ± 2.84%, 3.31% ± 0.45%, 3.29% ± 0.01%, and 0.24% ± 0.02%, respectively. The HPLC result suggested that the predominant monosaccharides of E2P included glucose, galactose, and arabinose, with a molar ratio of 543.2:1:1.8. The in vitro tests in RAW264.7 cells indicated that ESBP exhibited better immunomodulatory activities than E2P. In particular, ESBP can promote the proliferation, phagocytosis, and cytokine secretion abilities of cytokines, such as nitric oxide, tumor necrosis factor-α, and interleukin (IL)-1ß of RAW264.6 cells. By contrast, E2P can only promote phagocytosis ability and the secretion of IL-1ß.

Immunomodulatory effects of four polysaccharides purified from Erythronium sibiricum bulb on macrophages.

Four neutral polysaccharides (ESBP1-1, ESBP1-2, ESBP2-1 and ESBP3-1) were successfully purified from the water extracted crude polysaccharides of Erythronium sibiricum bulbs through the combination of DEAE Sepharose CL-6B and Sephadex G-100 chromatography; their average molecular weights were 1.3 × 104, 1.7 × 104, 9.4 × 105 and 4.1 × 105 Da, respectively. Monosaccharide component analysis indicated that ESBP1-1 and ESBP1-2 were mainly composed of glucose (Glc). ESBP2-1 was composed of Glc, galactose (Gal) and arabinose, with a molar ratio of 24.3:1.1:1, whereas ESBP3-1 comprised Glc and Gal at a molar ratio of 14.8:1. In-vitro study showed that all of the four polysaccharides were able to considerably promote the proliferation and neutral red phagocytosis of RAW 264.7 macrophage cell. They could also stimulate the production of the cell lines' secretory molecules [nitric oxide, tumour necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß)] in a dose-dependent manner. However, ESBP1-2 was not included in IL-1ß. Overall, these results suggested that polysaccharides from E. sibiricum bulbs can be developed as immunomodulatory ingredients for complementary medicines or functional foods. However, further animal or clinical studies are required.

DNM1, a Dynamin-Related Protein That Contributes to Endocytosis and Peroxisome Fission, Is Required for the Vegetative Growth, Sporulation, and Virulence of Metarhizium robertsii.

Although dynamins and dynamin-related proteins (DRPs), a large GTPase superfamily, are involved in the budding of transport vesicles and division of organelles in eukaryotic cells, the function of these proteins in entomopathogenic fungi has not been reported to date. Here, DNM1, a DRP in Metarhizium robertsii, was characterized using gene disruption and complementation strategies. Mutant phenotype assays showed that the ΔDnm1 strain displayed increased defects in radial growth (∼24%) and conidial production (∼42%) compared to those of the wild type (WT), and reduced conidiation levels were accompanied by the repression of several key conidiation-related genes, including flbA, wetA, and flbD Additionally, mutant bioassays revealed that disruption of Dnm1 impaired the virulence (both topical inoculation and injection) of M. robertsii in the insect Galleria mellonella Further analysis demonstrated that deleting Dnm1 in fungi suppressed the transcriptional levels of several virulence genes in the insect hemocoel. Moreover, we found that DNM1 colocalized with peroxisomes and mitochondria. Importantly, disruption of Dnm1 abolished normal fungal endocytosis, resulting in significantly decreased numbers of, as well as morphological changes in, peroxisomes. These findings indicate that deletion of Dnm1 causes significant changes in the vegetative growth, sporulation, and virulence of M. robertsii due to changes in cell function and peroxisomes.IMPORTANCEDnm1 was found to be involved in fungal development and virulence, mediated peroxisomal fission, and normal endocytosis. This finding provides new insights into the cellular processes and pathogenicity in entomopathogenic fungi.

The Intermediates in Branched-Chain Amino Acid Biosynthesis Are Indispensable for Conidial Germination of the Insect-Pathogenic Fungus Metarhizium robertsii.

Metarhizium spp. are well-known biocontrol agents used worldwide to control different insect pests. Keto-acid reductoisomerase (ILVC) is a key enzyme for branched-chain amino acid (BCAA) biosynthesis, and it regulates many physiological activities. However, its functions in insect-pathogenic fungi are poorly understood. In this work, we identified MrilvC in M. robertsii and dissected its roles in fungal growth, conidiation, germination, destruxin biosynthesis, environmental stress response, and insecticidal virulence. BCAA metabolism affects conidial yields and germination. However, BCAAs cannot recover the conidial germination of an MrilvC-deficient strain. Further feeding assays with intermediates showed that some conidia of the ΔMrilvC mutant start to germinate. Therefore, it is the germination defect that causes the complete failures of conidial penetration and pathogenicity in the ΔMrilvC mutant. In conclusion, we found intermediates in BCAA biosynthesis are indispensable for Metarhizium robertsii conidial germination. This study will advance our understanding of the fungal germination mechanism.IMPORTANCE Branched-chain amino acid (BCAA) metabolism plays a significant role in many biological activities beyond protein synthesis. Spore germination initiates the first stage of vegetative growth, which is critical for the virulence of pathogenic fungi. In this study, we demonstrated that the keto-acid reductoisomerase MrILVC, a key enzyme for BCAA biosynthesis, from the insect-pathogenic fungus Metarhizium robertsii is associated with conidial germination and fungal pathogenicity. Surprisingly, the germination of the ΔMrilvC mutant was restored when supplemented with the intermediates of BCAA metabolism rather than three BCAAs. The result was significantly different from that of plant-pathogenic fungi. Therefore, this report highlights that the intermediates in BCAA biosynthesis are indispensable for conidial germination of M. robertsii.

Genome-wide study of saprotrophy-related genes in the basal fungus Conidiobolus heterosporus.

Conidiobolus spp. are important saprophytic basal fungi. However, to date, no genomic-level data for decaying plant materials in the genus Conidiobolus has been reported. Here, we report that the 33.4-Mb genome of Conidiobolus heterosporus encodes 10,857 predicted genes. Conidiobolus heterosporus harbors 394 CAZyme-encoding genes belonging to 4 major modules but does not encode a polysaccharide lyase (PL). Many carbohydrate esterases (CEs) belonging to the family CE12 play crucial roles as pectin acetylesterases, and 14 genes were upregulated in the IM (fungus grown on inducing medium) among 17 expressed CE12 family genes. In addition, most of the genes in the GH132 CAZyme family showed a greater than 5-fold increase in expression in the IM compared with that in the wild type. Furthermore, 122 P450-encoding genes grouped into 11 families were detected in the fungal genome, most of which belonged to the CYP547 family (36 genes) followed by CYP548 (27 genes) and CYP5856 (25 genes). Interestingly, members of the families CYP5014 and CYP5136 were identified, the first time such enzymes have been described in a fungus. Our findings provide new insights into the genomics and genomic features of the saprophytic basal fungus C. heterosporus.Key Points• Genome of the saprobiotic basal fungus C. heterosporus was sequenced and analyzed.• 394 CAZymes but no PL family genes were found and expression levels were determined.• CE12 and GH132 proteins may play roles in the pectin and plant material degradation.• A large number of P450s but few P450 families existed in the fungus.

Degradation of Fungal MicroRNAs Triggered by Short Tandem Target Mimics Is via the Small-RNA-Degrading Nuclease.

MicroRNAs (miRNAs) have been recognized as sequence-specific regulators of the genome, transcriptome, and proteome in eukaryotes. However, the functions and working mechanisms of hundreds of fungal miRNA-like (miR-like) RNAs are obscure. Here, we report that a short tandem target mimic (STTM) triggered the degradation of several fungal miR-like RNAs in two different fungal species, Metarhizium robertsii and Aspergillus flavus, and that small-RNA-degrading nucleases (SDNs) were indispensable for such degradation. STTMs were most effective when the fungal polymerase II (Pol II) promoter was used for their expression, while the Pol III promoter was less effective. The length of the STTM spacer, approximately 48 to 96 nucleotides, and the number of miR-like RNA binding sites, from 2 to 4 copies, showed no significant difference in the degradation of miR-like RNAs. STTMs modulated the miR-like RNA expression levels in at least two different fungal species, which further impacted fungal asexual growth and sporulation. Further analysis showed that the degraded miR-like RNAs in STTM mutants led to the upregulation of potential target genes involved in fungal development and conidial production, which result in different phenotypes in these mutants. The STTM technology developed in this study is an effective and powerful tool for the functional dissection of fungal miR-like RNAs.IMPORTANCE The development and application of STTM technology to block miR-like RNAs in M. robertsii and A. flavus may allow for efficient generation of miR-like RNA mutants in various fungi, providing a powerful tool for functional genomics of small RNA molecules in fungi.

An efficient method for rapid screening of triterpenoid saponins in three Glycyrrhiza species using rapid resolution liquid chromatography quadrupole time-of-flight mass spectrometry combined with mass defect filtering.

Triterpenoid saponins, a major bioactive component of liquorice, possess high hydrophilicity and often co-occur with other impurities of similar polarity. Additionally, subtle structural differences of some triterpenoid saponins bring challenges to comprehensive characterisation. In this study, triterpenoid saponins of three Glycyrrhiza species were systematically analysed using rapid resolution liquid chromatography quadrupole time-of-flight mass spectrometry (RRLC-Q-TOF-MS) coupled with mass defect filtering (MDF). Firstly, comprehensive date acquisition was achieved using RRLC-Q-TOF-MS. Secondly, a polygonal MDF method was established by summarizing known and speculated substituents and modifications based on the core structure to rapidly screen potential triterpenoid saponins. Thirdly, based on the fragmentation patterns of reference compounds, an identification strategy for characterisation of triterpenoid saponins was proposed. The strategy divided triterpenoid saponins into three distinct classes. By this strategy, 98 triterpenoid saponins including 10 potential new ones were tentatively characterised. Finally, triterpenoid saponins of three Glycyrrhiza species were further analysed using principle component analysis (PCA) and orthogonality partial least squares discriminant analysis (OPLS-DA). Among these, 18 compounds with variable importance in projections (VIP) > 1.0 and P values < 0.05 were selected to distinguish three Glycyrrhiza species. Overall, our study provided a reference for quality control and rational use of the three species.

Structural identification and comprehensive comparison of saponin-related impurities present in the three different compound glycyrrhizin tablets.

Compound Glycyrrhizin Tablet (CGT) is a glycyrrhizin-containing (monoammonium glycyrrhizate, MAG) preparation, which has been widely used in clinical treatment of chronic liver diseases, eczema, atopic dermatitis and other conditions. However, the impurity profile of CGT has not yet been completely elucidated. In this study, eight main saponin-related impurity compounds were initially isolated and identified. Thereafter, based on the characteristic MS/MS fragmentation pathways analysis of the isolated compounds, a novel strategy for characterization and identification of saponin-related impurities was proposed. Then, a total of 41 saponin-related impurities were identified or tentatively characterized in CGTs. Furthermore, principal component analysis (PCA), Wayne diagram and heatmap analysis revealed that the process-related impurity profile in CGTs from three different manufacturers was significantly different. Overall, our findings provided additional technological support for evaluating saponin-related impurities, thereby laying a solid foundation to develop strategies for future product quality improvement.

Integration of dsRNA against host immune response genes augments the virulence of transgenic Metarhizium robertsii strains in insect pest species.

The slow lethality of fungal biopesticides to insects restrains their widespread application as a strategy of pest control. In this study, unary, binary and ternary transgenic Metarhizium robertsii were created by integrating genes that encode the scorpion neurotoxin BjαIT, the cuticle-degrading protease Pr1A, and a double-stranded RNA (dsRNA) that targets host gnbp3, individually or collectively under a constitutive promoter to enhance virulence. Compared with the parental wild type, all unary transgenic strains had increased virulence against four insect species, Tenebrio molitor, Locusta migratoria, Plutella xylostella and Galleria mellonella, whereas the binary transgenic strain expressing both pr1A and BjαIT had increased virulence to T. molitor and L. migratoria, with no change in virulence against P. xylostella and G. mellonella. Importantly, all ternary transgenic strains simultaneously expressing pr1A, BjαIT, and the dsRNA specific to host gnbp3 exhibited the highest increase in insect-specific virulence. This finding highlights a novel strategy for genetic engineering of dsRNAs that target genes associated with the host immune response alongside virulence genes to maximize fungal virulence and lethality against insect pests.

Isolation, purification, and characterization of antioxidant peptides from fresh mare's milk.

In this study, the whey protein of fresh mare's milk was used as raw material. The antioxidant peptide liquid XMNDT was extracted from fresh mare's milk solution and purified. The peptide had a molecular weight of 1594.89 kDa and was mainly composed of VAPFPQPVVPYPQR. Antioxidant peptide XMNDT could inhibit the proliferation of A549 lung cancer cells in the G1 phase, accelerate cell apoptosis, increase the activity of SOD and the amount of GSH, and reduce the secretion of MDA. It also exhibited certain antioxidant capacity and free radical scavenging. These data can provide a basis for research on new antioxidant properties by reducing the inflammation caused by aging.

Functional Analysis of Keto-Acid Reductoisomerase ILVC in the Entomopathogenic Fungus Metarhizium robertsii.

Ketol-acid reductoisomerase (ILVC) is the second enzyme in the branched-chain amino acid (BCAA) biosynthesis, which regulates many physiological activities in a variety of organisms from bacteria to fungi and plants. In this work, function mechanisms of ILVC in Metarhizium robertsii Metchnikoff (Hypocreales: Clavicipitaceae) were explored with site-directed mutagenesis, reductase activity assays and transcriptomics analysis. The reductase activity assays showed that ILVC from phytopathogenic fungi exhibited significantly higher activities than those from entomopathogenic fungi but lower than those from yeast. Site-directed mutagenesis and enzymatic activities of MrILVC with different active-site mutants (Arg-113, Ser-118, Asp-152, Asp-260, and Glu-264) confirmed that active sites of MrILVC are conserved with plant and bacterial ILVCs. Deleting MrilvC causes the complete failures of vegetative growth and conidial germination, feeding with branched-chain amino acids (BCAAs) recovers the fungal growth but not conidial germination, while both characteristics are restored when supplemented with yeast extract. Compared to ΔMrilvC cultured in czapek agar (CZA), plenty of genes involved in the biosynthesis of antibiotics and amino acids were up- or down-regulated in the wild type or ΔMrilvC feeding with either BCAAs or yeast extract. Further analysis showed some genes, such as catalase A, participate in mycelial growth and conidial germination was down-regulated in ΔMrilvC from CZA, revealing that MrILVC might control the fungal development by gene regulation and BCAAs or yeast extract could play partial roles of MrILVC. This study will advance our understanding of ILVC function mechanisms in fungi.

Synthesis and properties of a novel photochromic compound with modulated fluorescence in the solid state.

A new photochromic compound, 1,3-diphenyl-4-(3-bromobenzal)-5-hydroxypyrazole 4-phenylsemicarbazone (DP3BrBP-PSC), has been prepared. Its photochromic and thermobleaching behavior, crystal structure, and fluorescent property have been investigated in detail. The results show that the compound exhibits reversible enol-keto photoisomerization due to the intermolecular double-proton transfer, excellent photostability, high fatigue resistance, and remarkable fluorescence.

Genome-Wide Identification and Functional Prediction of Long Non-coding RNAs Involved in the Heat Stress Response in Metarhizium robertsii.

Long non-coding RNAs (lncRNAs) play a significant role in stress responses. To date, only a few studies have reported the role of lncRNAs in insect-pathogenic fungi. Here, we report a genome-wide transcriptional analysis of lncRNAs produced in response to heat stress in Metarhizium robertsii, a model insect-pathogenic fungus, using strand-specific RNA sequencing. A total of 1655 lncRNAs with 1742 isoforms were identified, of which 1081 differentially expressed (DE) lncRNAs were characterized as being heat responsive. By characterizing their genomic structures and expression patterns, we found that the lncRNAs possessed shorter transcripts, fewer exons, and lower expression levels than the protein-coding genes in M. robertsii. Furthermore, target prediction analysis of the lncRNAs revealed thousands of potential DE lncRNA-messenger RNA (mRNA) pairs, among which 5381 pairs function in the cis-regulatory mode. Further pathway enrichment analysis of the corresponding cis-regulated target genes showed that the targets were significantly enriched in the following biological pathways: the Hippo signaling pathway and cell cycle. This finding suggested that these DE lncRNAs control the expression of their corresponding neighboring genes primarily through environmental information processing and cellular processes. Moreover, only 26 trans-regulated lncRNA-mRNA pairs were determined. In addition, among the targets of heat-responsive lncRNAs, two classic genes that may be involved in the response to heat stress were also identified, including hsp70 (XM_007821830 and XM_007825705). These findings expand our knowledge of lncRNAs as important regulators of the response to heat stress in filamentous fungi, including M. robertsii.

Water-soluble polysaccharide from Erythronium sibiricum bulb: Structural characterisation and immunomodulating activity.

A water-soluble polysaccharide named ESBP2-1 was isolated from Erythronium sibiricum bulb through anion-exchange and size-exclusion chromatography. ESBP2-1 is a neutral polysaccharide fraction with an average molecular weight of 9.4×105Da and is composed of glucose, galactose and arabinose in a ratio of 24.3:1.1:1. Methylation analysis, partial hydrolysis and NMR studies revealed that the backbone of ESBP2-1 primarily consists of repeating →1)-α-d-Glcp-(4→ units where the disaccharide side chains of Arap-(1→6)-α-d-Galp-(1→ residue are attached to the O-6 position of Glc. An in vitro assay showed that ESBP2-1 significantly promoted the proliferation and neutral red phagocytosis of RAW 264.7 macrophage cells. In addition, ESBP2-1 stimulated the production of secretory molecules (nitric oxide, TNF-α and IL-1ß) of RAW264.7 cells in a dose-dependent manner. These results suggest that ESBP2-1 is a potential immunostimulator.

Optimised extraction of Erythronium sibiricum bulb polysaccharides and evaluation of their bioactivities.

In this study, the extraction of Erythronium sibiricum bulb polysaccharides (ESBP) through hot water decoction was optimised using response surface methodology (RSM) and a three-level, four-factor Box-Behnken design. The optimum extraction conditions were as follows: extraction time of 4.28h, extraction temperature of 90°C, ratio of liquid to raw material of 37 mL/g and extraction cycle number of three. The experimental yield (37.25%±0.17%) agreed with the predicted value of the RSM model (37.465%). Preliminary ESBP characterisation was conducted through physicochemical analysis. Biological activity test results showed that ESBP exhibited antioxidant activities and excellent anti-inflammatory and analgesic activities, indicating its potential as an anti-inflammatory and analgesic agent.

A New Type of Liquid Silymarin Proliposome Containing Bile Salts: Its Preparation and Improved Hepatoprotective Effects.

Silymarin, a known extract, is used in the treatment of liver diseases with various origins, but its current administration form cannot target the liver because of its poor oral bioavailability. A new type of oral silymarin proliposome aimed at improving silymarin's poor bioavailability and hepatoprotective effects, is introduced in this work. Silymarin-loaded liquid proliposome were prepared using a simple dissolving process. The morphology, particle size, zeta potential, and entrapment efficiency of the silymarin liposomes were analysed. The everted gut sac transport model was used to measure the intestinal transport of liposomes. The liposomal hepatoprotective activity was evaluated in three types of experimental hepatitis animal models. After staining with haematoxylin and eosin, the livers were microscopically examined to analyse any pathological changes. The prepared silymarin proliposome formed silymarin liposomes with a multilayer liposome structure and improved intestinal transport. In an injured liver, the silymarin liposomes produced a stronger hepatoprotective effect through a significant decrease in both the aminotransferase and MDA levels and a significant increase in the SOD and GSH-PX levels compared to orally administered silymarin tablets. This effect was also confirmed histopathologically. In a word, incorporation of silymarin into a liposomal carrier system increased intestinal absorption and showed better hepatoprotective effects compared to silymarin tablets.