Homogeneous Polyporus polysaccharide exerts anti-bladder cancer effects via autophagy induction.

CONTEXT: Polyporus polysaccharide (PPS), the leading bioactive ingredient extracted from Polyporus umbellatus (Pers.) Fr. (Polyporaceae), has been demonstrated to exert anti-bladder cancer and immunomodulatory functions in macrophages. OBJECTIVE: To explore the effects of homogeneous Polyporus polysaccharide (HPP) on the proliferation and autophagy of bladder cancer cells co-cultured with macrophages. MATERIALS AND METHODS: MB49 bladder cancer cells and RAW264.7 macrophages were co-cultured with or without HPP intervention (50, 100, or 200 µg/mL) for 24 h. The cell counting kit-8 (CCK-8) assay and 5-ethynyl-2″-deoxyuridine (EdU) staining evaluated MB49 cell proliferation. Monodansylcadaverine (MDC) staining and transmission electron microscopy (TEM) observed autophagosomes. Western blotting detected the expression levels of autophagy-related proteins and PI3K/Akt/mTOR pathway proteins. RESULTS: HPP inhibited the proliferation of MB49 cells co-cultured with RAW264.7 cells but not MB49 cells alone. HPP altered the expression of autophagy-related proteins and promoted the formation of autophagosomes in MB49 cells in the co-culture system. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) not only antagonized HPP-induced autophagy but also attenuated the inhibitory effects of HPP on MB49 cell proliferation in the co-culture system. HPP or RAW264.7 alone was not sufficient to induce autophagy in MB49 cells. In addition, HPP suppressed the protein expression of the PI3K/Akt/mTOR pathway in MB49 cells in the co-culture system. DISCUSSION AND CONCLUSIONS: HPP induced bladder cancer cell autophagy by regulating macrophages in the co-culture system, resulting in the inhibition of cancer cell proliferation. The PI3K/Akt/mTOR pathway was involved in HPP-induced autophagy in the co-culture system.

Homogeneous Polyporus Polysaccharide Inhibit Bladder Cancer by Resetting Tumor-Associated Macrophages Toward M1 Through NF-κB/NLRP3 Signaling.

Bladder cancer(BC)is one of the most common urinary system tumors, which characterized by a high incidence. Polyporus polysaccharide is the main active component of polyporus, which is clinically used in the treatment of bladder cancer, but the mechanism is not clear. In previous study, we isolated homogeneous polyporus polysaccharide(HPP) with high purity from polyporus. The goal of this study was to assess the polarization of macrophages induced by HPP in the bladder tumor microenvironment and explored its anti-bladder cancer mechanism through BBN bladder cancer rat model and Tumor associated macrophages(TAM). The results suggested that HPP regulates TAM polarization to improve the tumor inflammatory microenvironment, possibly through the NF-κB/NLRP3 signaling pathway. Our results suggested that HPP may be a potential therapeutic agent for bladder tumors.

Regiospecific 7-hydroxylation of ten-carbon monoterpenes by detoxifying CYP5035S7 monooxygenase of the white-rot fungus Polyporus arcularius.

In a previous study, we identified CYP5035S7 of the white-rot fungus Polyporus arcularius with a broad activity towards monoterpenes such as p-cymene. Therefore, in this study we aimed at further exploring the substrate scope of detoxifying CYP5035S7 towards terpenes and semi-preparatively isolating some of the products via whole-cell biotransformation, in order to obtain information about the enzyme's reactivity. We noticed a clear preference for the monoterpene skeleton and elucidated a distinct regioselectivity pattern based on key structural and electronic features of its substrates. This study illustrates how minimal characterisation effort may already suffice to provide vital information on enzymatic reactivity by the comparison of structural derivatives.

Use of transcriptomic profiling to identify candidate genes involved in Polyporus umbellatus sclerotial formation affected by oxalic acid.

Polyporus umbellatus is a precious medicinal fungus. Oxalic acid was observed to affect sclerotial formation and sclerotia possessed more medicinal compounds than mycelia. In this study, the transcriptome of P. umbellatus was analysed after the fungus was exposed to various concentrations of oxalic acid. The differentially expressed genes (DEGs) encoding a series of oxidases were upregulated, and reductases were downregulated, in the low-oxalic-acid (Low OA) group compared to the control (No OA) group, while the opposite phenomenon was observed in the high-oxalic-acid (High OA) group. The detection of reactive oxygen species (ROS) in P. umbellatus mycelia was performed visually, and Ca2+ and H2O2 fluxes were measured using non-invasive micro-test technology (NMT). The sclerotial biomass in the Low OA group increased by 66%, however, no sclerotia formed in the High OA group. The ROS fluorescence intensity increased significantly in the Low OA group but decreased considerably in the High OA group. Ca2+ and H2O2 influx significantly increased in the Low OA group, while H2O2 exhibited efflux in the High OA group. A higher level of oxidative stress formed in the Low OA group. Different concentrations of oxalic acid were determined to affect P. umbellatus sclerotial formation in different ways.

Do the Main Components of the Sclerotia of Umbrella Polypore Mushroom, Polyporus umbellatus (Agaricomycetes), Correlate with Armillaria Associates?

Polyporus umbellatus is a traditional Chinese medicinal mushroom. The growth of P. umbellatus sclerotia requires the rhizomorphs of Armillaria spp. to supply nutrition. Whether the main components (MC) of sclerotia of P. umbellatus are related to the phylogeny of Armillaria associates or other environmental factors is largely unknown. In this study, we collected 17 sclerotia and soil samples from northeast to southwest China. In total, 17 Armillaria associates were isolated, and sclerotial MC contents and soil characteristics (total N, P, K, and organic matter) were determined. The analysis revealed that the MC content of P. umbellatus did not resemble a Brownian motion process in phylogeny of Armillaria associates, but were significantly influenced by the total N content of the soil. These results provide clear evidence that sclerotia of P. umbellatus associating with phylogenetic related Armillaria associates possess differing MC content. The mechanisms of nutrient exchange in P. umbellatus-Armillaria associations now require further elucidation.

Green seaweeds (Ulva fasciata sp.) as nitrogen source for fungal cellulase production.

In developing countries, local enzyme production can help decrease the dependency of imported enzymes for bioconversion of e.g. cellulosic feedstocks, but the use of conventional nitrogen sources contributes significantly to such enzyme production cost. Use of local resources is therefore important to consider. Green seaweeds are marine macroalgae that are rich in nitrogen, but not exploited for their nitrogen content. Cellulase production was accomplished by using cocoa pod husk (CPH) and green seaweed (GS) (Ulva fasciata sp.) as growth substrates for Polyporus ciliatus CBS 366.74 in submerged cultivation. The nitrogen concentration of GS was comparable to that of CPH with 0.6% w/v peptone at 4% w/v substrate concentration. A decline of cellulase activity in peptone supplemented GS growth media indicated nitrogen sufficiency of GS to serve as a potential nitrogen source for the fungal growth and cellulase production. Comparison of enzyme production on CPH growth media supplemented with either GS or peptone based on equivalent carbon to nitrogen ratios was done for two Polyporus strains namely; P. ciliatus CBS 366.74 and P. brumalis CBS 470.77. Peptone could be substituted by up to 0.6% w/v with GS at inclusion levels of 50-100% of substrate concentration to attain satisfactory cellulase productivity. However, the cellulase productivity response varied among the two Polyporus species. This study demonstrated that green seaweeds may be used as alternative nitrogen sources for fungal cellulase production.

Three exopolysaccharides from the liquid fermentation of Polyporus umbellatus and their bioactivities.

The exopolysaccharides were extracted and separated from the broth of the liquid fermentation of P. umbellatus, and the antioxidant activities and other relative bioactivities were investigated, aiming to find clues for a wider use in the future. Three novel exopolysaccharides of PPS1, PPS2 and PPS3 with molecular weight of 3.7×104-6.9×104Da were obtained. Monosaccharide analysis showed that they were mainly composed of mannose, along with galactose and glucose with different molar ratio, and their structural features were also investigated by FT-IR, NMR and SEM. The antioxidant activity assay in vitro showed these exopolysaccharides exhibited a significant scavenging effect on DPPH· and other free radicals in a dose-dependent manner. Significantly, the stimulate nitric oxide production and phagocytic activity implied that the polysaccharides could enhance the immunity of RAW 264.7 macrophages. Other assays revealed that they have obvious cellular aging delaying activity and the DNA damage protecting activity. In conclusion, these three exopolysaccharides might have potential applications in the fields of pharmaceuticals, cosmetics, and food products.

Biological wheat straw valorization: Multicriteria optimization of Polyporus brumalis pretreatment in packed bed bioreactor.

The purpose of this work was to optimize the pretreatment process of wheat straw by Polyporus brumalis_BRFM985 in order to improve carbohydrate accessibility for more efficient bioconversion. Indeed, there is growing demands to develop sustainable routes for lignocellulosic feedstocks valorization into value-added products in energy, chemicals, materials, and animal feed fields. To be achieved, implementation of cheap and ecofriendly biomass pretreatment processes is necessary. In this frame, white rot basidiomycetes, well known for their ability to degrade lignin efficiently and selectively, are of great interest. The pretreatment of wheat straw by Polyporus brumalis_BRFM985 was performed in packed bed bioreactor and optimized using response surface methodology. The four pretreatment parameters optimized were metals addition (Cu, Mn, and Fe), time of culture, initial water content, and temperature. Multicriteria optimization highlighted that wheat straw pretreatment by Polyporus brumalis_BRFM985 in the presence of metals with high initial water content of 3.6 g H2 O/g at 27°C for 15-16 days led to an improvement of carbohydrate accessibility with minimal matter loss.

SWATH label-free proteomics analyses revealed the roles of oxidative stress and antioxidant defensing system in sclerotia formation of Polyporus umbellatus.

Understanding the initiation and maturing mechanisms is important for rational manipulating sclerotia differentiation and growth from hypha of Polyporus umbellatus. Proteomes in P. umbellatus sclerotia and hyphae at initial, developmental and mature phases were studied. 1391 proteins were identified by nano-liquid chromatograph-mass spectrometry (LC-MS) in Data Dependant Acquisition mode, and 1234 proteins were quantified successfully by Sequential Window Acquisition of all THeoretical fragment ion spectra-MS (SWATH-MS) technology. There were 347 differentially expressed proteins (DEPs) in sclerotia at initial phase compared with those in hypha, and the DEP profiles were dynamically changing with sclerotia growth. Oxidative stress (OS) in sclerotia at initial phase was indicated by the repressed proteins of respiratory chain, tricarboxylic acid cycle and the activation of glycolysis/gluconeogenesis pathways were determined based on DEPs. The impact of glycolysis/gluconeogenesis on sclerotium induction was further verified by glycerol addition assays, in which 5% glycerol significantly increased sclerotial differentiation rate and biomass. It can be speculated that OS played essential roles in triggering sclerotia differentiation from hypha of P. umbellatus, whereas antioxidant activity associated with glycolysis is critical for sclerotia growth. These findings reveal a mechanism for sclerotial differentiation in P. umbellatus, which may also be applicable for other fungi.

Polyporus squamosus Lectin 1a (PSL1a) Exhibits Cytotoxicity in Mammalian Cells by Disruption of Focal Adhesions, Inhibition of Protein Synthesis and Induction of Apoptosis.

PSL1a is a lectin from the mushroom Polyporus squamosus that binds to sialylated glycans and glycoconjugates with high specificity and selectivity. In addition to its N-terminal carbohydrate-binding domain, PSL1a possesses a Ca2+-dependent proteolytic activity in the C-terminal domain. In the present study, we demonstrate that PSL1a has cytotoxic effects on mammalian cancer cells, and we show that the cytotoxicity is dependent on the cysteine protease activity. PSL1a treatment leads to cell rounding and detachment from the substratum, concomitant with disruption of vinculin complexes in focal adhesions. We also demonstrate that PSL1a inhibits protein synthesis and induces apoptosis in HeLa cells, in a time- and concentration-dependent manner.

Ligninolytic fungus Polyporus sp. S133 mediated metabolic degradation of fluorene

ABSTRACT This study aimed to investigate the impact of nonionic surfactants on the efficacy of fluorine degradation by Polyporus sp. S133 in a liquid culture. Fluorene was observed to be degraded in its entirety by Polyporus sp. S133 subsequent to a 23-day incubation period. The fastest cell growth rate was observed in the initial 7 days in the culture that was supplemented with Tween 80. The degradation process was primarily modulated by the activity of two ligninolytic enzymes, laccase and MnP. The highest laccase activity was stimulated by the addition of Tween 80 (2443 U/L) followed by mixed surfactant (1766 U/L) and Brij 35 (1655 U/L). UV-vis spectroscopy, TLC analysis and mass spectrum analysis of samples subsequent to the degradation process in the culture medium confirmed the biotransformation of fluorene. Two metabolites, 9-fluorenol (λmax 270, tR 8.0 min and m/z 254) and protocatechuic acid (λmax 260, tR 11.3 min and m/z 370), were identified in the treated medium.

Ligninolytic fungus Polyporus sp. S133 mediated metabolic degradation of fluorene.

This study aimed to investigate the impact of nonionic surfactants on the efficacy of fluorine degradation by Polyporus sp. S133 in a liquid culture. Fluorene was observed to be degraded in its entirety by Polyporus sp. S133 subsequent to a 23-day incubation period. The fastest cell growth rate was observed in the initial 7 days in the culture that was supplemented with Tween 80. The degradation process was primarily modulated by the activity of two ligninolytic enzymes, laccase and MnP. The highest laccase activity was stimulated by the addition of Tween 80 (2443U/L) followed by mixed surfactant (1766U/L) and Brij 35 (1655U/L). UV-vis spectroscopy, TLC analysis and mass spectrum analysis of samples subsequent to the degradation process in the culture medium confirmed the biotransformation of fluorene. Two metabolites, 9-fluorenol (λmax 270, tR 8.0min and m/z 254) and protocatechuic acid (λmax 260, tR 11.3min and m/z 370), were identified in the treated medium.

Transcriptomic analysis of the white rot fungus Polyporus brumalis provides insight into sesquiterpene biosynthesis.

Object of this study was to identify genes and enzymes that are involved in sesquiterpene biosynthesis in the wood rotting fungus, Polyporus brumalis. Sesquiterpenes, ß-eudesmane and ß-eudesmol, were produced by the mycelium of P. brumalis cultured in modified medium. However, theses final products were not observed when the fungus was grown in potato dextrose medium. We used next generation sequencing (NGS) to identify differentially expressed genes (DEGs) related to terpene metabolism. This approach generated 25,000 unigenes and 127 metabolic pathways that were assigned to Kyoto Encyclopedia Genes Groups (KEGG). Further analysis of samples from modified medium indicated significant upregulation of 8 unigenes involved in the mevalonate (MVA) and methylerythritol phosphate (MEP) biosynthetic pathways. These pathways generate isopentenyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP), which are precursors for the synthesis of sesquiterpenes. Furthermore, genes encoding germacrene A synthase, which facilitate the cyclization of FPP, were only differentially expressed in mycelium from fungi grown in modified medium. Our data provide a resource for studying the molecular mechanisms underpinning sesquiterpene biosynthesis and terpene metabolism.

Transcriptome analysis of genes involved in defence response in Polyporus umbellatus with Armillaria mellea infection.

Polyporus umbellatus, a species symbiotic with Armillaria mellea and it also exhibits substantial defence response to Armillaria mellea infection. There are no genomics resources databases for understanding the molecular mechanism underlying the infection stress of P. umbellatus. Therefore, we performed a large-scale transcriptome sequencing of this fungus with A. mellea infection using Illumina sequencing technology. The assembly of the clean reads resulted in 120,576 transcripts, including 38,444 unigenes. Additionally, we performed a gene expression profiling analysis upon infection treatment. The results indicated significant differences in the gene expression profiles between the control and the infection group. In total, 10933 genes were identified between the two groups. Based on the differentially expressed genes, a Gene Ontology annotation analysis showed many defence-relevant categories. Meanwhile, the Kyoto Encyclopedia of Genes and Genomes pathway analysis uncovered some important pathways. Furthermore, the expression patterns of 13 putative genes that are involved in defence response resulting from quantitative real-time PCR were consistent with their transcript abundance changes as identified by RNA-seq. The sequenced genes covered a considerable proportion of the P. umbellatus transcriptome, and the expression results may be useful to strengthen the knowledge on the defence response of this fungus defend against Armillaria mellea invasion.

Oxalic acid and sclerotial differentiation of Polyporus umbellatus.

The present investigation aimed to uncover the effects of exogenous oxalic acid during the sclerotial formation of Polyporus umbellatus, with an emphasis on determining the content of the endogenic oxalic acid in the fungus. To this end, the oxalic acid content of the vegetative mycelia, sclerotia, culture mediums and sclerotial exudate were measured using High Performance Liquid Chromatography (HPLC). Furthermore, the lipid peroxidation was estimated by detecting thiobarbituric bituric acid reactive substances (TBARS). The results showed that the exogenous oxalic acid caused a delay in sclerotial differentiation (of up to 9 or more days), suppressed the sclerotial biomass and decreased the lipid peroxidation significantly in a concentration-dependent manner. Oxalic acid was found at very low levels in the mycelia and the maltose medium, whereas it was found at high levels in the mycelia and sucrose medium. After sclerotial differentiation, oxalic acid accumulated at high levels in both the sclerotia and the sclerotial exudate. Oxalic acid was therefore found to inhibit P. umbellatus sclerotial formation.

Biotransformation of (-)-α-pinene and geraniol to α-terpineol and p-menthane-3,8-diol by the white rot fungus, Polyporus brumalis.

In this study, the monoterpenes, α-pinene and geraniol, were biotransformed to synthesize monoterpene alcohol compounds. Polyporus brumalis which is classified as a white rot fungus was used as a biocatalyst. Consequently α-terpineol was synthesized from α-pinene by P. brumalis mycelium, after three days. Moreover, another substrate, the acyclic monoterpenoids geraniol was transformed into the cyclic compound, p-menthane-3, 8-diol (PMD). The main metabolites, i.e., α-terpineol and PMD, are known to be bioactive monoterpene alcohol compounds. This study highlights the potential of fungal biocatalysts for monoterpene transformation.

[Physicochemical properties of medicinal fungus Polyporus umbellatus sclerotial exudate].

This study was conducted to investigate the physicochemical properties of Polyporus umbellatus sclerotial exudate. Morphological characteristics of the sclerotia and its exudate were observed during different stages of sclerotial formation. The pH of the exudate was detected at different time during cultivation. A phenol-sulfuric acid method was employed to determine the polysaccharide content of P. umbellatus sclerotial exudate during cultivating time. Additionally, the protein content was measured by means of BCA protein assay. Furthermore, CAT content was detected using ultraviolet absorption method. That the protein content of the exudate and CAT specific activity rose gradually during the passage of the cultivating time indicated a high level of oxidative stress during P. umbellatus sclerotial exudate formation. The results showed that the pH of the exudate increased gradually and then dropped down during sclerotial formation. That the pH of the exudate maintained the acidity state during the cultivation indirectly indicated that acidic environment would help sclerotial formation. The exudate produced gradually and was absorbed by the sclerotia itself.

Decolorization and degradation mechanism of Amaranth by Polyporus sp. S133.

Polyporus sp. S133 decolorized the Amaranth in 72 h (30 mg L(-1)) under static and shaking conditions. Liquid medium containing glucose has shown the highest decolorization of Amaranth by Polyporus sp. S133. When the effect of increasing inoculum concentration on decolorization of Amaranth was studied, maximum decolorization was observed with 15 % inoculum concentration. Significant increase in the enzyme production of laccase (102.2 U L(-1)) was observed over the period of Amaranth decolorization compared to lignin peroxidase and manganese peroxidase. Germination rate of Sorghum vulgare and Triticum aestivum was less with Amaranth treatment as compared to metabolites obtained after its decolorization. Based on the metabolites detected by GC-MS, it was proposed that Amaranth was bio-transformed into two intermediates, 1-hydroxy-2-naphthoic acid and 1,4-naphthaquinone. Overall findings suggested the ability of Polyporus sp. S133 for the decolorization of azo dye and ensured the ecofriendly degradation of Amaranth.

Identification of differentially expressed genes involved in laccase production in tropical white-rot fungus Polyporus sp. PG15.

Carbon sources and copper ion are the main influencing factors on the production of fungal laccase. To investigate the regulation of carbon source and copper ion in laccase production on the molecular level in tropical white-rot fungus PG15, a comparative analysis of gene expression patterns was performed by cDNA-amplified fragment length polymorphism (AFLP) technique. Selective amplifications with 120 primer combinations allowed the identification of 92 differentially expressed transcript-derived fragments (TDFs), ranging from 200 to 750 bp in size. The TDFs were from PG15 supplemented with different carbon sources and copper ion concentrations, majority of which downregulated laccase production. Twenty-one fragments that matched the database were functionally annotated and analyzed according to the up- and downregulation patterns identified by cDNA-AFLP. These fragments were probably involved in laccase production at the metabolism, signal transduction, transcription, or post-translation levels. This study provides the first catalog of genes involved in laccase production, together with their putatively functional annotations. These data provide potential candidates for improving laccase production in fungi by marker-assisted selection or genetic engineering.

Enhanced lignin biodegradation by a laccase-overexpressed white-rot fungus Polyporus brumalis in the pretreatment of wood chips.

The laccase gene of Polyporus brumalis was genetically transformed to overexpress its laccase. The transformants exhibited increased laccase activity and effective decolorization of the dye Remazol Brilliant Blue R than the wild type. When the transformants were pretreated with wood chips from a red pine (softwood) and a tulip tree (hardwood) for 15 and 45 days, they showed higher lignin-degradation activity as well as higher wood-chip weight loss than the wild type. When the wood chips treated with the transformant were enzymatically saccharified, the highest sugar yields were found to be 32.5 % for the red pine wood and 29.5 % for the tulip tree wood, on the basis of the dried wood weights, which were 1.6-folds higher than those for the wild type. These results suggested that overexpression of the laccase gene from P. brumalis significantly contributed to the pretreatment of lignocellulose for increasing sugar yields.