Medicinal Mushrooms: Past, Present and Future.

The survival of Homo sapiens is continually under threat from agencies capable of inflicting calamitous damage to the overall health and well-being of humankind. One strategy aimed at combatting this threat is focused on medicinal mushrooms and derivatives thereof. Mushrooms themselves have been consumed as part of the human diet for centuries, whereas 'mushroom nutriceuticals' is a more recently adopted term describing mushroom-derived products taken as dietary supplements to enhance general health and fitness. Among the most extensively studied pharmacologically active components of mushrooms are polysaccharides and polysaccharide-protein complexes, triterpenes, lectins, and fungal immunomodulatory proteins. Medicinal mushrooms have been credited with a wide range of therapeutic properties including antitumour/anti-cancer, antioxidant, hepatoprotective, anti-diabetic, antimicrobial, cholesterol-lowering and genoprotective activities as well as protection against atherosclerosis, cardiovascular, chronic inflammatory and autoimmune diseases, and neurodegenerative conditions. This review examines the past, present and future of medicinal mushroom development including the two legs concept for the mushroom industry and the pyramid model summarizing the various human applications of mushrooms. It considers numerous issues the industry needs to address to exploit fully the opportunities presented by the continued increasing demand for medicinal mushrooms, and by the future overall expansion of the medicinal mushroom movement.

Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing.

Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional direct RNA nanopore sequencing, the 5' and 3' ends of poly(A) RNA cannot be identified unambiguously. This is due in part to RNA degradation in vivo and in vitro that can obscure transcription start and end sites. In this study, we aimed to identify individual full-length human RNA isoforms among ∼4 million nanopore poly(A)-selected RNA reads. First, to identify RNA strands bearing 5' m7G caps, we exchanged the biological cap for a modified cap attached to a 45-nt oligomer. This oligomer adaptation method improved 5' end sequencing and ensured correct identification of the 5' m7G capped ends. Second, among these 5'-capped nanopore reads, we screened for features consistent with a 3' polyadenylation site. Combining these two steps, we identified 294,107 individual high-confidence full-length RNA scaffolds from human GM12878 cells, most of which (257,721) aligned to protein-coding genes. Of these, 4876 scaffolds indicated unannotated isoforms that were often internal to longer, previously identified RNA isoforms. Orthogonal data for m7G caps and open chromatin, such as CAGE and DNase-HS seq, confirmed the validity of these high-confidence RNA scaffolds.

Ligno(hemi)cellulolytic Enzyme Profiles during the Developmental Cycle of the Royal Oyster Medicinal Mushroom Pleurotus eryngii (Agaricomycetes) Grown on Supplemented Agri-Wastes.

We have determined the production profiles of major ligno(hemi)cellulolytic enzymes at different stages of the mushroom development cycle during industrial scale cultivation of Pleurotus eryngii on supplemented agri-wastes. Endo-1,4-ß-glucanase, cellobiohydrolase and endoxylanase levels remained relatively low during substrate colonization, increased sharply when small fruit bodies appeared, and peaked at maturation. ß-Glucosidase and ß-xylosidase levels decreased when substrate colonization was complete, increased with the appearance of small fruit bodies and primordia, respectively, and reached maxima at maturation. Laccase peaked along with substrate colonization but, after falling sharply in the upper substrate layers, remained relatively low until postinduction. Levels increased slightly when primordia appeared, fell to minimal values during the small and mature fruit body stages, and increased again postharvest. Manganese peroxidase (Mn-P) exhibited a similar pattern initially but high enzyme levels also coincided with primordia formation. Laccase and Mn-P activity patterns were compatible with a lignin-degradation function associated with substrate colonization and, in the former case, a putative role in fruit body morphogenesis. Based on the relatively low levels of polysaccharidases recorded during the initial stages of substrate colonization, we conclude that reducing sugar levels in noncolonized substrate were adequate for sustainable vegetative growth at that stage. We further conclude that the increase in enzyme production later in the developmental cycle was consistent with the replenishment of depleted reducing sugar from cellulose in the growth substrate to levels required for fruit body formation. These data provide new information describing combined temporal and spatial enzyme production profiles throughout the mushroom development cycle under a set of conditions used in industrial scale production.

The yeast scavenger decapping enzyme DcpS and its application for in vitro RNA recapping.

Eukaryotic mRNAs are modified at their 5' end early during transcription by the addition of N7-methylguanosine (m7G), which forms the "cap" on the first 5' nucleotide. Identification of the 5' nucleotide on mRNA is necessary for determination of the Transcription Start Site (TSS). We explored the effect of various reaction conditions on the activity of the yeast scavenger mRNA decapping enzyme DcpS and examined decapping of 30 chemically distinct cap structures varying the state of methylation, sugar, phosphate linkage, and base composition on 25mer RNA oligonucleotides. Contrary to the generally accepted belief that DcpS enzymes only decap short oligonucleotides, we found that the yeast scavenger decapping enzyme decaps RNA transcripts as long as 1400 nucleotides. Further, we validated the application of yDcpS for enriching capped RNA using a strategy of specifically tagging the 5' end of capped RNA by first decapping and then recapping it with an affinity-tagged guanosine nucleotide.

A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome.

BACKGROUND: The initiating nucleotide found at the 5' end of primary transcripts has a distinctive triphosphorylated end that distinguishes these transcripts from all other RNA species. Recognizing this distinction is key to deconvoluting the primary transcriptome from the plethora of processed transcripts that confound analysis of the transcriptome. The currently available methods do not use targeted enrichment for the 5'end of primary transcripts, but rather attempt to deplete non-targeted RNA. RESULTS: We developed a method, Cappable-seq, for directly enriching for the 5' end of primary transcripts and enabling determination of transcription start sites at single base resolution. This is achieved by enzymatically modifying the 5' triphosphorylated end of RNA with a selectable tag. We first applied Cappable-seq to E. coli, achieving up to 50 fold enrichment of primary transcripts and identifying an unprecedented 16539 transcription start sites (TSS) genome-wide at single base resolution. We also applied Cappable-seq to a mouse cecum sample and identified TSS in a microbiome. CONCLUSIONS: Cappable-seq allows for the first time the capture of the 5' end of primary transcripts. This enables a unique robust TSS determination in bacteria and microbiomes.  In addition to and beyond TSS determination, Cappable-seq depletes ribosomal RNA and reduces the complexity of the transcriptome to a single quantifiable tag per transcript enabling digital profiling of gene expression in any microbiome.

Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging.

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged ß-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.

Site-specific protein labeling by intein-mediated protein ligation.

Intein-mediated protein ligation (IPL) employs an intein to create a protein possessing a C-terminal thioester that can be ligated to a protein or peptide with an amino-terminal cysteine via a native peptide bond. Here we present a procedure to conduct isolation and labeling of recombinant proteins expressed in E. coli using synthetic short peptides possessing a fluorescent moiety. This approach can be readily utilized for site-specific conjugation of a fluorophore to the C-terminus of a protein of interest, without the drawback of non-specific chemical labeling. This chapter also gives a general review of the critical parameters of intein-mediated cleavage and ligation reactions.

Fluorescent site-specific labeling of Escherichia coli expressed proteins with Sfp phosphopantetheinyl transferase.

Fluorescent tagging of proteins has become a critical step in optical analysis of protein function in vitro and in living cells. Here we describe a two-tag system for expression and isolation of a protein of interest from Escherichia coli and subsequent site-specific fluorescent labeling with Sfp phosphopantetheinyl transferase (Sfp synthase). In the example presented, adenoviral protein E3-14.7 K (E14.7) was expressed as a tripartite fusion protein with a fluorophore-targeting peptide tag and a chitin-binding domain. This system allows for rapid isolation of the recombinant fusion protein from crude bacterial cell lysate via a single chitin column. Sfp synthase-mediated labeling with fluorophore conjugated to coenzyme A-SH (CoA-SH) resulted in covalent attachment of a fluorescent dye to a specific residue of the peptide tag via a phosphopantetheinyl linker. The fluorescently labeled E14.7 fusion protein was analyzed with a fluorescence imager and subsequently transfected into mammalian cells for imaging with a fluorescence microscope.

Comparison of endoglucanase-1 (EG1) induction in the edible straw mushroom Volvariella volvacea by lactose and/or cellobiose with or without added sorbose.

We have compared the induction of an endoglucanase (EG1) by α-lactose and/or cellobiose, with or without added L-sorbose, in submerged cultures of Volvariella volvacea, to better understand the mechanism whereby cellulase formation is triggered by these soluble disaccharides. EG1 levels induced by α-lactose and cellobiose were 28.6% and 6.7%, respectively of the highest levels recorded with crystalline cellulose. Sorbose did not induce EG1 and strongly repressed enzyme levels when added to α-lactose but not cellobiose-containing cultures. EG1 levels in cultures containing all three saccharides were similar to those recorded with sorbose and cellobiose although enzyme induction was delayed by 12 h. When V. volvacea was pre-grown for 24 h in medium containing sorbose as the sole carbon source, followed by addition of α-lactose or cellobiose or a mixture of the two, EG1 levels recorded in the α-lactose-supplemented cultures were again markedly lower compared with cultures containing only α-lactose. Maximal enzyme levels in cultures with added cellobiose or cellobiose and α-lactose were not affected although appearance of EG1 in culture supernatants was again delayed by 12 h. Semi-quantitative RT-PCR revealed that higher, more prolonged, levels of eg1 transcription occurred in V. volvacea cultures induced with α-lactose compared with cellobiose- or α-lactose + cellobiose-induced cultures. However, eg1 transcription levels in cultures induced with cellobiose or with cellobiose + lactose, and the corresponding cultures with added sorbose, were not markedly different.

Molecular cloning and transcriptional expression analysis of an intracellular beta-glucosidase, a family 3 glycosyl hydrolase, from the edible straw mushroom, Volvariella volvacea.

A beta-glucosidase, with a molecular mass of 95 kDa, was isolated from extracts of Volvariella volvacea mycelium grown on crystalline cellulose. Degenerate primers based on the N-terminal sequences of purified beta-glucosidase and two protease-generated peptides were used to generate cDNA fragments encoding a portion of the beta-glucosidase gene (bgl), and rapid amplification of cDNA ends was used to obtain full-length cDNA clones. The cDNA of bgl contained an ORF of 2586 bp coding for 862 amino acids. Alignment of the deduced amino-acid sequence of beta-glucosidase with deduced amino acid sequences of other microbial beta-glucosidases showed the highest overall homology with glycoside hydrolase family 3 beta-glucosidases from fungi. Transcripts of bgl were detected in total RNA extracted from mycelium grown on cellulose and cellobiose, and from mycelium pre-grown for 72 h in basal medium containing 1% (w/v) sorbitol following addition of alpha-lactose, beta-lactose, cellobiose, d- xylose, l-sorbose, beta-gentiobiose, sophorose or d-galactose. Addition of l-sorbose and d-glucosamine to mycelium grown on 1% (w/v) crystalline cellulose greatly increased the level of bgl expression. bgl Was expressed at various stages of the mushroom developmental cycle (substrate colonization to mature fruit body), although the number of bgl transcripts in pinhead and button stages was slightly smaller.

Cloning of multiple cellulase cDNAs from Volvariella volvacea and their differential expression during substrate colonization and fruiting.

We used PCR-based methods to clone and sequence four previously unidentified cellulase cDNAs: cbhI-I, cbhI-II, cbhII-I and egII. CbhI-I, cbhI-II and cbhII-I consist of 1710, 1610 and 1453 bp, respectively, and encode for 512, 458 and 442 amino acids, respectively. EgII consists of 1180 bp encoding for 310 amino acids, and belongs to family 61 of the glycosyl hydrolases. CbhI-I, cbhII-I and egII all have a modular structure, with the catalytic domain (CD) and cellulose-binding domain (CBD) located at the C-terminus in cbhI-I and egII, and at the N-terminus in cbhII-I. CbhI-II shows high homology to cbhI-I but lacks a CBD. Northern blotting revealed that cbhI-I, cbhI-II and cbhII-I were coordinately expressed at various stages of the mushroom developmental cycle (substrate colonization to mature fruit body), although the number of cbhI-I transcripts was much smaller. No egII expression was detectable during the substrate colonization phase but transcription levels increased as fruit body morphogenesis progressed.

Molecular cloning of a new laccase from the edible straw mushroom Volvariella volvacea: possible involvement in fruit body development.

Cloning of a laccase-encoding cDNA from the edible straw mushroom, Volvariella volvacea, was performed using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The cDNA of the putative laccase gene (lac4) consisted of 1689 bp, including an open reading frame encoding a 23-amino acid signal peptide at the N-terminal end and a 540-amino acid mature protein with a predicted molecular mass of 58173 Da and a pI value of 6.1. The 10 histidine residues and one cysteine residue required to co-ordinate the four copper atoms at the active site of the protein were all conserved. The amino acid sequence of V. volvacea lac4 has a high degree of identity with other basidiomycete laccases. Transcription of the laccase gene was analysed by RT-PCR and, unlike many other laccase genes, shown to be regulated independently of either copper or aromatic compounds under the test conditions. However, the laccase gene is strongly expressed during that part of the mushroom developmental cycle involving fruit body morphogenesis.

Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvacea.

We have isolated a laccase (lac1) from culture fluid of Volvariella volvacea, grown in a defined medium containing 150 micro m CuSO4, by ion-exchange and gel filtration chromatography. Lac1 has a molecular mass of 58 kDa as determined by SDS/PAGE and an isoelectric point of 3.7. Degenerate primers based on the N-terminal sequence of purified lac1 and a conserved copper-binding domain were used to generate cDNA fragments encoding a portion of the lac1 protein and RACE was used to obtain full-length cDNA clones. The cDNA of lac1 contained an ORF of 1557 bp encoding 519 amino acids. The amino acid sequence from Ala25 to Asp41 corresponded to the N-terminal sequence of the purified protein. The first 24 amino acids are presumed to be a signal peptide. The expression of lac1 is regulated at the transcription level by copper and various aromatic compounds. RT-PCR analysis of gene transcription in fungal mycelia grown on rice-straw revealed that, apart from during the early stages of substrate colonization, lac1 was expressed at every stage of the mushroom developmental cycle defined in this study, although the levels of transcription varied considerably depending upon the developmental phase. Transcription of lac1 increased sharply during the latter phase of substrate colonization and reached maximum levels during the very early stages (primordium formation, pinhead stage) of fruit body morphogenesis. Gene expression then declined to approximately 20-30% of peak levels throughout the subsequent stages of sporophore development.

Induction of laccase activity in the edible straw mushroom, Volvariella volvacea.

Volvariella volvacea, strain V14, produces multiple forms of extracellular laccase when grown in submerged culture in a defined medium with glucose as sole carbon source, and on cotton waste 'compost' representative of the conditions used for industrial-scale mushroom cultivation. In liquid culture, enzyme synthesis is associated with the onset of secondary growth, and is positively regulated by copper (up to 200 microM CuSO(4)) and by various aromatic compounds. In solid-state systems, only low levels of laccase are detectable during the vegetative growth phase but enzyme activity increases sharply at the onset of fruiting and during sporophore development.

L-DOPA oxidation products prevent H2O2-induced oxidative damage to cellular DNA.

Using the single-cell gel electrophoresis ("Comet") assay, we show that tyrosinase-generated L-DOPA oxidation products prevent H2O2-induced oxidative DNA damage in cultured tissue cells. We propose that these oxidation products trigger cellular processes that up-regulate the overall antioxidant status of the cell, and could be incorporated into treatments of pathological conditions associated with elevated oxidative DNA damage and other manifestations of increased oxidative stress.

Role of tyrosinase in the genoprotective effect of the edible mushroom, Agaricus bisporus.

A heat-labile protein has been identified in fruit bodies of the edible mushroom, Agaricus bisporus, which protects Raji cells (a human lymphoma cell line) against H2O2-induced oxidative damage to cellular DNA. This protein has been purified following salt fractionation, combined with ion-exchange, hydrophobic interaction and adsorption chromatography. Based on catalytic and electrophoretic properties, and inhibition studies using tropolone, the protein was identified as tyrosinase. The genoprotective effect of A. bisporus tyrosinase, determined using the single-cell gel electrophoresis met") assay, has been shown to be dependent upon the enzymic hydroxylation of tyrosine to L-DOPA and subsequent conversion of this metabolite to dopaquinone. The possible role of dopaquinone, and other L-DOPA oxidation products, in enhancing the cellular antioxidant defence mechanisms is discussed.

Mushroom-derived preparations in the prevention of H2O2-induced oxidative damage to cellular DNA.

Aqueous extracts of the sporophores of eight mushroom species were assessed for their ability to prevent H2O2-induced oxidative damage to cellular DNA using the single-cell gel electrophoresis ("Comet") assay. The highest genoprotective effects were obtained with cold (20 degrees C) and hot (100 degrees C) water extracts of Agaricus bisporus and Ganoderma lucidum fruit bodies, respectively. No protective effects were observed with Mushroom Derived Preparations (MDPs) from Flammulina velutipes, Auricularia auricula, Hypsizygus marmoreus, Lentinula edodes, Pleurotus sajor-caju, and Volvariella volvacea. These findings indicate that some edible mushrooms represent a valuable source of biologically active compounds with potential for protecting cellular DNA from oxidative damage.