Formaldehyde as a Chemical Defence Agent of Fruiting Bodies of Mycena rosea and its Role in the Generation of the Alkaloid Mycenarubin†C.

Mycenarubin C, a previously unknown red pyrroloquinoline alkaloid, was isolated from fruiting bodies of the mushroom Mycena rosea and its structure was elucidated mainly by NMR spectroscopy and mass spectrometry. Unlike mycenarubin A, the major pyrroloquinoline alkaloid in fruiting bodies of M. rosea, mycenarubin C, contains an eight-membered ring with an additional C1 unit that is hitherto unprecedented for pyrroloquinoline alkaloids known in nature. Incubation of mycenarubin A with an excess of formaldehyde revealed that mycenarubin C was generated nearly quantitatively from mycenarubin A. An investigation into the formaldehyde content of fresh fruiting bodies of M. rosea showed the presence of considerable amounts of formaldehyde, with values of 5 µg per gram of fresh weight in fresh fruiting bodies. Although mycenarubin C did not show bioactivity against selected bacteria and fungi, formaldehyde inhibits the growth of the mycoparasite Spinellus fusiger at concentrations present in fruiting bodies of M. rosea. Therefore, formaldehyde might play an ecological role in the chemical defence of M. rosea against S. fusiger. In turn, S. fusiger produces gallic acid-presumably to detoxify formaldehyde by reaction of this aldehyde with amino acids and gallic acid to Mannich adducts.

The pathogen Moniliophthora perniciosa promotes differential proteomic modulation of cacao genotypes with contrasting resistance to witches´ broom disease.

BACKGROUND: Witches' broom disease (WBD) of cacao (Theobroma cacao L.), caused by Moniliophthora perniciosa, is the most important limiting factor for the cacao production in Brazil. Hence, the development of cacao genotypes with durable resistance is the key challenge for control the disease. Proteomic methods are often used to study the interactions between hosts and pathogens, therefore helping classical plant breeding projects on the development of resistant genotypes. The present study compared the proteomic alterations between two cacao genotypes standard for WBD resistance and susceptibility, in response to M. perniciosa infection at 72 h and 45 days post-inoculation; respectively the very early stages of the biotrophic and necrotrophic stages of the cacao x M. perniciosa interaction. RESULTS: A total of 554 proteins were identified, being 246 in the susceptible Catongo and 308 in the resistant TSH1188 genotypes. The identified proteins were involved mainly in metabolism, energy, defense and oxidative stress. The resistant genotype showed more expressed proteins with more variability associated with stress and defense, while the susceptible genotype exhibited more repressed proteins. Among these proteins, stand out pathogenesis related proteins (PRs), oxidative stress regulation related proteins, and trypsin inhibitors. Interaction networks were predicted, and a complex protein-protein interaction was observed. Some proteins showed a high number of interactions, suggesting that those proteins may function as cross-talkers between these biological functions. CONCLUSIONS: We present the first study reporting the proteomic alterations of resistant and susceptible genotypes in the T. cacao x M. perniciosa pathosystem. The important altered proteins identified in the present study are related to key biologic functions in resistance, such as oxidative stress, especially in the resistant genotype TSH1188, that showed a strong mechanism of detoxification. Also, the positive regulation of defense and stress proteins were more evident in this genotype. Proteins with significant roles against fungal plant pathogens, such as chitinases, trypsin inhibitors and PR 5 were also identified, and they may be good resistance markers. Finally, important biological functions, such as stress and defense, photosynthesis, oxidative stress and carbohydrate metabolism were differentially impacted with M. perniciosa infection in each genotype.

Crude polysaccharide from a wild mushroom enhances immune response in murine macrophage cells by TLR/NF-κB pathway.

OBJECTIVE: Mushroom crude polysaccharides offer a complete package of various medicinal activities. In this context, the present study aimed to unveil structural and biomedical properties of crude polysaccharide (MLHWP) obtained from an edible wild mushroom Macrocybe lobayensis (R. Heim) Pegler & Lodge. METHOD: Chemical characterization was accomplished with the help of spectrophotometry, Fourier-transform infrared spectroscopy, HPTLC and GC-MS. Immunomodulatory activity of the crude polysaccharide and its signalling mechanism was assessed using RAW 264.7 cells. Furthermore, antioxidant activity was analysed based on radical scavenging, metal ion chelating and reducing effect. KEY FINDINGS: Compositional study revealed that MLHWP possessed triple helical structure and its backbone consisted of ß-linked glucan along with xylose, rhamnose, mannose and galactose. Investigation on bioactive potency revealed that MLHWP augmented macrophage activity in terms of viability, phagocytosis, NO and ROS generation. Gene expression studies indicated that MLHWP signalled through TLR and modulated expression of immunomodulation-related genes including NF-κB, COX-2, IFN-γ, TNF-α, iNOS and Iκ-ßα. Besides, MLHWP displayed noticeable antioxidant potential as reflected in all investigating assays. CONCLUSIONS: Overall, the results portrayed possibility of MLHWP as pharmaceutical agent with multidimensional application.

Combining microfluidics and RNA-sequencing to assess the inducible defensome of a mushroom against nematodes.

BACKGROUND: Fungi are an attractive source of nutrients for predators. As part of their defense, some fungi are able to induce the production of anti-predator protein toxins in response to predation. A previous study on the interaction of the model mushroom Coprinopsis cinerea by the fungivorous nematode Aphelenchus avenae on agar plates has shown that the this fungal defense response is most pronounced in the part of the mycelium that is in direct contact with the nematode. Hence, we hypothesized that, for a comprehensive characterization of this defense response, an experimental setup that maximizes the zone of direct interaction between the fungal mycelium and the nematode, was needed. RESULTS: In this study, we conducted a transcriptome analysis of C. cinerea vegetative mycelium upon challenge with A. avenae using a tailor-made microfluidic device. The device was designed such that the interaction between the fungus and the nematode was confined to a specific area and that the mycelium could be retrieved from this area for analysis. We took samples from the confrontation area after different time periods and extracted and sequenced the poly(A)+ RNA thereof. The identification of 1229 differentially expressed genes (DEGs) shows that this setup profoundly improved sensitivity over co-cultivation on agar plates where only 37 DEGs had been identified. The product of one of the most highly upregulated genes shows structural homology to bacterial pore-forming toxins, and revealed strong toxicity to various bacterivorous nematodes. In addition, bacteria associated with the fungivorous nematode A. avenae were profiled with 16S rRNA deep sequencing. Similar to the bacterivorous and plant-feeding nematodes, Proteobacteria and Bacteroidetes were the most dominant phyla in A. avenae. CONCLUSIONS: The use of a novel experimental setup for the investigation of the defense response of a fungal mycelium to predation by fungivorous nematodes resulted in the identification of a comprehensive set of DEGs and the discovery of a novel type of fungal defense protein against nematodes. The bacteria found to be associated with the fungivorous nematode are a possible explanation for the induction of some antibacterial defense proteins upon nematode challenge.

Lepista sordida Water Extract Enhances the Maturation of Mouse Dendritic Cells in Vitro and in Vivo.

BACKGROUND: Lepista sordida (LS) extract has been shown to possess anti-oxidant, anti-aging, and anti-tumor activities. However, the immunostimulatory effect of LS extract has not been elucidated. OBJECTIVE: To characterize the impact of a water extract of LS (WE-LS) on the maturation and function of mouse dendritic cell (DC) in vitro and in vivo. METHODS: Mouse bone marrow-derived DCs (BMDCs) were generated. Next, DC maturation was determined by flow cytometry, and cytokine production was measured by ELISA after WE-LS treatment. In addition, DC-induced OVA-specific T cell activation was assayed by [3H]-thymidine incorporation assay. Furthermore, the in vivo effects of WE-LS on DC maturation and Th1 responses in the spleens of mice were assessed by flow cytometry. RESULTS: WE-LS treatment up-regulated co-stimulatory (CD40 and CD80) and MHC class II molecules, increased the production of tumor necrosis factor-alpha (TNF-α), IL-6 and IL-12, and enhanced both the proliferation and IFN-γ secretion of allogenic T cells in BMDCs, partially mediated by the TLR2 and TLR4 signaling pathways. Moreover, the in vivo administration of WE-LS to mice enhanced the up-regulation of CD40, CD80 and MHC class II molecules in spleen DCs. WE-LS also increased the generation of T helper type 1 (Th1) cells in vivo. CONCLUSION: These results suggest that WE-LS might have the potential to promote immunity against infection and cancer or to serve as an adjuvant in vaccines and immunotherapies.

An unusual case of wheat dependent exercise induced anaphylaxis (WDEIA) triggered by Tri a 14 in a pediatric patient: a case report.

Summary: Anakinra, one of the novel biological agents, is a recombinant human IL-1 receptor antagonist. It is preferred as an alternative drug for familial Mediterranean fever cases where colchicine is not sufficient or cannot be used due to its side effects. Like all other biologics, hypersensitivity reactions to anakinra are quite rare. This is the first case which was successfully desensitized with anakinra after a severe immediate-type hypersensitivity reaction. We report a case of WDEIA in an asthmatic boy admitted to our Unit with suspected mushroom acute toxicity. The symptoms occurred during a gym session, approximately 2 hours after the ingestion of a meal based on pasta and cooked mushroom found in the family's garden. Acute toxicity due to mushroom ingestion was then excluded. Triptase serum levels resulted elevated in acute phase and normal after 24 hours. Food specific IgE showed a sensitization to lipid transfer protein Pru p 3 and to Tri a 14. This case highlights that WDEIA is underdiagnosed, especially when patients are firstly visited in Emergency Unit. Moreover, Tri a 14 is seldom described as responsible for WDEIA, compared to omega 5 gliadin.

Immunomodulatory Properties of Plants and Mushrooms.

Plants and mushrooms are used for medicinal purposes and the screening of molecules possessing biological activities. A single plant or mushroom may produce both stimulatory and inhibitory effects on immune cells, depending on experimental conditions, but the reason behind this dichotomy remains obscure. We present here a large body of experimental data showing that water extracts of plants and mushrooms usually activate immune cells, whereas ethanol extracts inhibit immune cells. The mode of extraction of plants and mushrooms may thus determine the effects produced on immune cells, possibly due to differential solubility and potency of stimulatory and inhibitory compounds. We also examine the possibility of using such plant and mushroom extracts to treat immune system disorders.

Immunomodulatory properties of medicinal mushrooms: differential effects of water and ethanol extracts on NK cell-mediated cytotoxicity.

Medicinal mushrooms have been used for centuries in Asian countries owing to their beneficial effects on health and longevity. Previous studies have reported that a single medicinal mushroom may produce both stimulatory and inhibitory effects on immune cells, depending on conditions, but the factors responsible for this apparent dichotomy remain obscure. We show here that water and ethanol extracts of cultured mycelium from various species (Agaricus blazei Murrill, Antrodia cinnamomea, Ganoderma lucidum and Hirsutella sinensis) produce opposite effects on NK cells. Water extracts enhance NK cell cytotoxic activity against cancer cells, whereas ethanol extracts inhibit cytotoxicity. Water extracts stimulate the expression and production of cytolytic proteins (perforin and granulysin) and NKG2D/NCR cell surface receptors, and activate intracellular signaling kinases (ERK, JNK and p38). In contrast, ethanol extracts inhibit expression of cytolytic and cell surface receptors. Our results suggest that the mode of extraction of medicinal mushrooms may determine the nature of the immunomodulatory effects produced on immune cells, presumably owing to the differential solubility of stimulatory and inhibitory mediators. These findings have important implications for the preparation of medicinal mushrooms to prevent and treat human diseases.

Characterisation of a New Fungal Immunomodulatory Protein from Tiger Milk mushroom, Lignosus rhinocerotis.

Lignosus rhinocerotis (Tiger milk mushroom) is an important folk medicine for indigenous peoples in Southeast Asia. We previously reported its de novo assembled 34.3 Mb genome encoding a repertoire of proteins including a putative bioactive fungal immunomodulatory protein. Here we report the cDNA of this new member (FIP-Lrh) with a homology range of 54-64% to FIPs from other mushroom species, the closest is with FIP-glu (LZ-8) (64%) from Ganoderma lucidum. The FIP-Lrh of 112 amino acids (12.59 kDa) has a relatively hydrophobic N-terminal. Its predicted 3-dimensional model has identical folding patterns to FIP-fve and contains a partially conserved and more positively charged carbohydrates binding pocket. Docking predictions of FIP-Lrh on 14 glycans commonly found on cellular surfaces showed the best binding energy of -3.98 kcal/mol to N-acetylgalactosamine and N-acetylglucosamine. Overexpression of a 14.9 kDa soluble 6xHisFIP-Lrh was achieved in pET-28a(+)/BL21 and the purified recombinant protein was sequence verified by LC-MS/MS (QTOF) analysis. The ability to haemagglutinate both mouse and human blood at concentration ≥0.34 µM, further demonstrated its lectin nature. In addition, the cytotoxic effect of 6xHisFIP-Lrh on MCF-7, HeLa and A549 cancer cell lines was detected at IC50 of 0.34 µM, 0.58 µM and 0.60 µM, respectively.

Modulation of T cell response by Phellinus linteus.

Phellinus linteus, a species of mushroom, has been shown to contribute to health benefits, such as anti-inflammatory activity and immunomodulatory efficacy. The aim of this study was to analyze the most effective constituents of P. linteus fermented broths, polysaccharides, and to evaluate their immunoregulatory effects on T cells. Four fermented broths (PL1-4) and the dialyzate medium (MD) were prepared from P. linteus mycelia, and the polysaccharide contents of each were analyzed. The P. linteus samples were tested for biological activity in the regulation of T cell activation. In T cells, the production of mitogen-induced interleukin (IL)-2 and cell cycle progression were dose-responsively inhibited by PL3 and MD, primarily through cell-cycle arrest in S phase. PL3 broth, which contained large quantities of polysaccharides, significantly decreased the ratio of interferon-gamma (IFN-γ) to interleukin 4 (IL-4) in T cells. Thus, P. linteus fermented broths produced additive effects on the regulation of the Th1/Th2 balance and show promise for the development of immunomodulatory therapeutics.

Selected wild strains of Agaricus bisporus produce high yields of mushrooms at 25 °C / Cepas silvestres seleccionadas de Agaricus bisporus con capacidad para producir un alto rendimiento a temperaturas de 25°C

Background. To cultivate the button mushroom Agaricus bisporus in warm countries or during summer in temperate countries, while saving energy, is a challenge that could be addressed by using the biological diversity of the species. Aims. The objective was to evaluate the yield potential of eight wild strains previously selected in small scale experiments for their ability to produce mature fruiting bodies at 25 °C and above. Methods. Culture units of 8 kg of compost were used. The yield expressed as weight or number per surface unit and earliness of fruiting were recorded during cultivation in climatic rooms at 17, 25 or 30 °C. Results. Only strains of A. bisporus var. burnettii were able to fruit at 30 °C. At 25 °C they produced the highest yields (27 kg m−2) and had best earliness. The yields at 25 °C for the strains of A. bisporus var. bisporus ranged from 12 to 16 kg m−2. The yield ratios 25 °C/17 °C ranged from 0.8 to 1.2. Conclusions. The variety burnettii originated in the Sonoran Desert in California showed adaptation for quickly producing fruiting bodies at high temperature when humidity conditions were favorable. Strains of the variety bisporus showed interesting potentials for their ability to produce mature fruiting bodies at higher temperature than present cultivars and might be used in breeding programs (AU)

Antecedentes. El cultivo del champiñón (Agaricus bisporus) en países de clima tropical, o durante el verano en países de clima templado, además del ahorro energético que supone, es un reto que podría abordarse con el uso adecuado de la diversidad biológica de la especie. Objetivos. El objetivo del presente estudio fue examinar el rendimiento potencial de ocho cepas silvestres, previamente seleccionadas en experimentos a pequeña escala, para determinar su capacidad para producir frutos maduros a temperaturas ≥25 °C. Métodos. Se utilizaron unidades experimentales de cultivo con 8 kg de compost. El rendimiento se expresó como peso o número de frutos producidos por unidad de superficie; en naves climatizadas, a una temperatura de 17, 25 o 30 °C, durante todo el cultivo, se registró el momento de fructificación. Resultados. Sólo las cepas de A. bisporus var. burnettii fueron capaces de fructificar a 30 °C. Éstas produjeron un alto rendimiento a 25 °C (27 kg/m2) y la fructificación aconteció más temprano. El rendimiento a 25 °C para las cepas de A. bisporus var. bisporus varió de 12 a 16 kg/m2, con una proporción 25 °C/17 °C de 0,8 a 1,2. Conclusiones. La variedad burnettii, originaria del desierto de Sonora en California, está adaptada para producir cuerpos fructíferos a temperaturas elevadas cuando las condiciones de humedad son favorables. Para las cepas de la variedad bisporus se demostró un interesante potencial para producir cuerpos fructíferos maduros a una mayor temperatura que la utilizada en los cultivares actuales y puede ser utilizada en los programas de mejora genética (AU)

Methylated glycans as conserved targets of animal and fungal innate defense.

Effector proteins of innate immune systems recognize specific non-self epitopes. Tectonins are a family of ß-propeller lectins conserved from bacteria to mammals that have been shown to bind bacterial lipopolysaccharide (LPS). We present experimental evidence that two Tectonins of fungal and animal origin have a specificity for O-methylated glycans. We show that Tectonin 2 of the mushroom Laccaria bicolor (Lb-Tec2) agglutinates Gram-negative bacteria and exerts toxicity toward the model nematode Caenorhabditis elegans, suggesting a role in fungal defense against bacteria and nematodes. Biochemical and genetic analysis of these interactions revealed that both bacterial agglutination and nematotoxicity of Lb-Tec2 depend on the recognition of methylated glycans, namely O-methylated mannose and fucose residues, as part of bacterial LPS and nematode cell-surface glycans. In addition, a C. elegans gene, termed samt-1, coding for a candidate membrane transport protein for the presumptive donor substrate of glycan methylation, S-adenosyl-methionine, from the cytoplasm to the Golgi was identified. Intriguingly, limulus lectin L6, a structurally related antibacterial protein of the Japanese horseshoe crab Tachypleus tridentatus, showed properties identical to the mushroom lectin. These results suggest that O-methylated glycans constitute a conserved target of the fungal and animal innate immune system. The broad phylogenetic distribution of O-methylated glycans increases the spectrum of potential antagonists recognized by Tectonins, rendering this conserved protein family a universal defense armor.

Adaptabilidad de cepas brasileñas de Agaricus subrufescens Peck a la fructificación sobre diferentes capas de cobertura en cultivo comercial / Adaptability of Brazilian strains of Agaricus subrufescens Peck to fruiting on various casing materials in commercial crops

Antecedentes. Agaricus subrufescens Peck es un hongo cuyo cultivo ha despertado gran interés en todo el mundo en los últimos años, adquiriendo gran popularidad. Sus propiedades medicinales y culinarias hacen prever una rápida expansión del cultivo en todo el mundo. Objetivos. El trabajo plantea como objetivo la evaluación del efecto sobre los principales parámetros de producción de 3 cepas de Agaricus subrufescens que se han hecho fructificar sobre 5 capas de cobertura diferentes. Métodos. Se ha llevado a cabo un ciclo de cultivo de Agaricus subrufescens en condiciones controladas en el que se han evaluado los principales parámetros de producción. Resultados. Los mejores resultados han sido proporcionados por la cepa ABL 99/30. Las coberturas basadas en turba presentan mejor comportamiento que las basadas en suelo mineral. El mayor rendimiento (6,75 kg/m−2, eficiencia biológica 27,57 kg/dt) ha sido proporcionado por la combinación ABL 99/30-Euroveen. Conclusiones. Nuestros resultados evidencian que la combinación de la cepa ABL 99/30 utilizando una capa de cobertura basada en turba (Euroveen) ofrece un alto potencial para ser utilizada a escala comercial por el sector productor de hongos. La disponibilidad de alternativas a las especies de hongos comestibles cultivadas habitualmente puede suponer un mejor aprovechamiento de recursos y una mayor rentabilidad económica de la actividad (AU)

Background. Agaricus subrufescens Peck is a mushroom whose cultivation has aroused great interest worldwide in recent years, and is becoming increasingly popular. A rapid expansion of culture throughout the world is foreseen because of its medicinal and culinary properties. Aims. This work assesses the effect of 5 different casing layers on the production of 3 strains of Agaricus subrufescens. Methods. A growth cycle of Agaricus subrufescens under controlled conditions has been carried out. The main production parameters were evaluated. Results. The best results were provided by the ABL 99/30 strain. Peat-based casings have a better yield than those based on mineral soil. The highest yield (6.75 kg/m2, biological efficiency 27.57 kg/dt) was provided by the combination ABL 99/30-Euroveen. Conclusions. Our results suggest that the combination of the strain ABL 99/30 using a peat-based casing layer (Euroveen) offers a high potential for use on a commercial scale by the edible mushroom production sector. The availability of alternatives to the usually cultivated species can make better use of resources, and increase the profitability of this activity (AU)

Different immunology mechanisms of Phellinus igniarius in inhibiting growth of liver cancer and melanoma cells.

To assess inhibition mechanisms of a Phellinus igniarius (PI) extract on cancer, C57BL/6 mice were orally treated with PI extractive after or before implanting H22 (hepatocellular carcinoma ) or B16 (melanoma) cells. Mice were orally gavaged with different doses of PI for 36 days 24h after introduction of H22 or B16 cells. Mice in another group were orally treated as above daily for 42 days and implanted with H22 cells on day 7. Then the T lymphocyte, antibody, cytokine, LAK, NK cell activity in spleen, tumor cell apoptosis status and tumor inhibition in related organs, as well as the expression of iNOS and PCNA in tumor tissue were examined. The PI extract could improve animal immunity as well as inhibit cancer cell growth and metastasis with a dose-response relationship. Notably, PI's regulation with the two kinds of tumor appeared to occur in different ways, since the antibody profile and tumor metastasis demonstrated variation between animals implanted with hepatocellular carcinoma and melanoma cells.

Evaluation of fungal extracts to determine immunomodulatory properties.

BACKGROUND: Immunoglobulin E-mediated allergies have doubled in prevalence during recent decades in developed countries.This increase has been attributed, in part, to high hygiene standards, which have reduced exposure to microbes. The capacity of microbes to induce type 1 helper T cell (TH1) responses may imply suppression of TH2 responses. However, little research has been performed with fungal extracts. OBJECTIVES: To evaluate the TH1-inducing properties of fungal extracts. METHODS: A total of 24 fungal extracts, including Cetavlon-precipitated polysaccharides from different yeasts, molds, and mushrooms were prepared.The extracts were screened for production of interferon (IFN)gamma in human peripheral blood mononuclear cells. The active compounds were further purified by mild acid hydrolysis and by column chromatography and studied in human peripheral blood mononuclear cells. RESULTS: Expression of IFN-gamma was induced by several extracts. The strongest expression of IFN-gamma was induced by Candida albicans. The Cetavlon-precipitated mannans of fungi induced cytokine responses that were similar or superior to those induced by whole extracts, C albicans being the most potent inducer of IFN-gamma. Column chromatography-fractionated mild acid hydrolysis of Calbicans mannan was performed. Fractions containing oligosaccharides of 12-16 mannoses induced production of tumor necrosis factor. CONCLUSIONS: Several fungal extracts induce IFN-gamma. The most promising preparations were yeast-derived oligosaccharides. Further research should be focused on purification and eventual synthesis of the extracts.