Hepatoprotective Effect of Wheat-Based Solid-State Fermented Antrodia cinnamomea in Carbon Tetrachloride-Induced Liver Injury in Rat

Antrodia cinnamomea (A. cinnamomea) is an indigenous medical fungus in Taiwan and has multiple biological functions, including hepatoprotective and immune-modulatory effects. Currently, the commercially available A. cinnamomea are mainly liquid- and solid-state fermented A. cinnamomea. However, the hepatoprotective effect of solid-state fermented A. cinnamomea has never been reported. Here we evaluate the ability of air-dried, ground and non-extracted wheat-based solid-state fermented A. cinnamomea (WFAC) to protect against carbon tetrachloride (CCl₄)-induced hepatic injury in vivo. The results showed that oral administration of WFAC dose dependently (180, 540 and 1080 mg/kg) ameliorated the increase in plasma aspartate aminotransferase and alanine aminotransferase levels caused by chronic repeated CCl₄ intoxication in rats. WFAC significantly reduced the CCl₄-induced increase in hepatic lipid peroxidation levels and hydroxyproline contents, as well as reducing the spleen weight and water content of the liver. WFAC also restored the hepatic soluble protein synthesis and plasma albumin concentration in CCl₄-intoxicated rats, but it did not affect the activities of superoxide dismutase, catalase, or glutathione peroxidase. In addition, a hepatic morphological analysis showed that the hepatic fibrosis and necrosis induced by CCl₄ were significantly ameliorated by WFAC. Furthermore, the body weights of control rats and WFAC-administered rats were not significantly different, and no adverse effects were observed in WFAC-administered rats. These results indicate that WFAC is a nontoxic hepatoprotective agent against chronic CCl₄-induced hepatic injury.     

Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.     

Enhanced antioxidant and antitumor activities of Antrodia cinnamomea cultured with cereal substrates in solid state fermentation

Growing exclusively on stout camphor trees in Taiwan, Antrodia cinnamomea is known for its extraordinary antioxidant and antitumor activities. As an alternative to the limited supply of natural source, cultured A. cinnamomea from solid state or submerged liquid fermentation still offers many of its medicinal effects. To further enhance the production of functional compounds and corresponding activities, oat, wheat, buckwheat and pearl barley were used as substrates for solid state fermentation of A. cinnamomea in this study. Among these cereal-based culturing, the methanol extract of A. cinnamomea mycelia grown on oats showed stronger overall antioxidant properties. EC₅₀ for the antioxidant activity (conjugated diene method), the DPPH radicals scavenging ability and reducing power were estimated to be around 0.57 mg/mL, 1.07 mg/mL and 0.31 mg/mL, respectively. Incubating cultured cells with 150 ppm of the oat-cultured mycelial extract for 24 h greatly reduced the viability of MCF-7 breast cancer cells and HepG2 hepatocellular carcinoma cells to 29% and 76%, while 3T3 normal fibroblasts were virtually unaffected. In general, cereal-based solid state fermentation of A. cinnamomea produced more of the secondary metabolites and their methanolic extracts showed stronger antioxidant and anti-tumor activities than extracts obtained from liquid fermentation at the same concentration.     

MicroRNA-Like Small RNAs Prediction in the Development of Antrodia cinnamomea

Antrodia cinnamomea, a precious, host-specific brown-rot fungus that has been used as a folk medicine in Taiwan for centuries is known to have diverse bioactive compounds with potent pharmaceutical activity. In this study, different fermentation states of A. cinnamomea (wild-type fruiting bodies and liquid cultured mycelium) were sequenced using the next-generation sequencing (NGS) technique. A 45.58 Mb genome encoding 6,522 predicted genes was obtained. High quality reads were assembled into a total of 13,109 unigenes. Using a previously constructed pipeline to search for microRNAs (miRNAs), we then identified 4 predicted conserved miRNA and 63 novel predicted miRNA-like small RNA (milRNA) candidates. Target prediction revealed several interesting proteins involved in tri-terpenoid synthesis, mating type recognition, chemical or physical sensory protein and transporters predicted to be regulated by the miRNAs and milRNAs.     

Production of bioactive metabolites by submerged fermentation of the medicinal mushroom Antrodia cinnamomea: recent advances and future development

Edible and medicinal mushrooms have usually been considered as a sustainable source of unique bioactive metabolites, which are valued as promising provisions for human health. Antrodia cinnamomea is a unique edible and medicinal fungus widespread in Taiwan, which has attracted much attention in recent years for its high value in both scientific research and commercial applications owing to its potent therapeutic effects, especially for its hepatic protection and anticancer activity. Due to the scarcity of the fruiting bodies, the cultivation of A. cinnamomea by submerged fermentation appears to be a promising substitute which possesses some unique advantages, such as short culture time period and its high feasibility for scale-up production. However, the amount of fungal bioactive metabolites derived from the cultured mycelia of A. cinnamomea grown by submerged fermentation is much less than those obtained from the wild fruiting bodies. Hence, there is an urgent need to bridge such a discrepancy on bioactive metabolites between the wild fruiting bodies and the cultured mycelia. The objective of this article is to review recent advances and the future development of the mycelial submerged fermentation of A. cinnamomea in terms of enhancement for the production of fungal bioactive components by the optimization of culture conditions and the regulation of fungal metabolism. This review provides valuable information for further biotechnological applications of A. cinnamomea as well as other mushrooms being the source of bioactive ingredients by submerged fermentation.     

Chloroplast Genome Evolution in Early Diverged Leptosporangiate Ferns

In this study, the chloroplast (cp) genome sequences from three early diverged leptosporangiate ferns were completed and analyzed in order to understand the evolution of the genome of the fern lineages. The complete cp genome sequence of Osmunda cinnamomea (Osmundales) was 142,812 base pairs (bp). The cp genome structure was similar to that of eusporangiate ferns. The gene/intron losses that frequently occurred in the cp genome of leptosporangiate ferns were not found in the cp genome of O. cinnamomea. In addition, putative RNA editing sites in the cp genome were rare in O. cinnamomea, even though the sites were frequently predicted to be present in leptosporangiate ferns. The complete cp genome sequence of Diplopterygium glaucum (Gleicheniales) was 151,007 bp and has a 9.7 kb inversion between the trnL-CAA and trnV-GCA genes when compared to O. cinnamomea. Several repeated sequences were detected around the inversion break points. The complete cp genome sequence of Lygodium japonicum (Schizaeales) was 157,142 bp and a deletion of the rpoC1 intron was detected. This intron loss was shared by all of the studied species of the genus Lygodium. The GC contents and the effective numbers of co-dons (ENCs) in ferns varied significantly when compared to seed plants. The ENC values of the early diverged leptosporangiate ferns showed intermediate levels between eusporangiate and core leptosporangiate ferns. However, our phylogenetic tree based on all of the cp gene sequences clearly indicated that the cp genome similarity between O. cinnamomea (Osmundales) and eusporangiate ferns are symplesiomorphies, rather than synapomorphies. Therefore, our data is in agreement with the view that Osmundales is a distinct early diverged lineage in the leptosporangiate ferns.     

Comparative Cellular Analysis of Pathogenic Fungi with a Disease Incidence in Brachypodium distachyon and Miscanthus x giganteus

Based on a growing demand on renewable energy, fast growing perennial grasses have been identified as energy crops with a high capacity in sustainable biomass production. Among these grasses, the giant reed Miscanthus x giganteus delivers one of the highest biomass yields. Despite its potential for an extended cultivation, only little is known about putative fungal pathogens that might cause biomass losses. Molecular targets that are related to fungal resistance have not been identified because cellular and molecular tools have not been established in this energy crop. Therefore, our study was aimed to evaluate a method to compare the penetration process of fungal plant pathogens in the model grass Brachypodium distachyon and M. giganteus. In a screening with 13 different fungal species on detached leaves, we identified four filamentous fungi that infected both B. distachyon and M. giganteus and have not been previously described as M. giganteus pathogens. Spray inoculations with these four fungi on intact M. giganteus leaves of whole plants confirmed their pathogenicity. Microscopic analysis of the fungal infections and the hyphal propagation within the leaf tissue revealed that the four newly identified fungi used very similar strategies for penetration and colonization in B. distachyon and M. giganteus. This suggests that B.?distachyon could be suitable to establish model pathosystems for these fungal pathogens that colonize M. giganteus. The already existing genetic tools for B. distachyon might improve the identification of defense-related targets and mechanisms supporting fungal resistance in M. giganteus.     

Processing characteristics of submerged fermentation of Antrodia cinnamomea in airlift bioreactor

Three 5-L airlift bioreactors including airlift reactor with solid draft tube (ALs), airlift reactor with net draft tube (ALn) and bubble column reactor (BC) were investigated for their suitability for cultivating Antrodia cinnamomea, and a stirred tank reactor (ST) was used for comparison. Results indicated that after 7 days fermentation, ALs yielded the highest mycelium content (313 mg/100 mL) and had the lowest dissolved oxygen in the broth. Among different aeration rates (0.025, 0.05, 0.1, 0.5, 1 vvm) used during cultivation of A. cinnamomea in ALs, the aeration rate 0.1 vvm resulted in a volumetric oxygen transfer coefficient of 10.8 h⁻¹ and produced the highest mycelium content. When the optimal conditions were used for the fermentation of A. cinnamomea in an industrial 500-L ALs, the mycelium content in the broth reached 542 mg/100 mL in 28 days. The IC₅₀ values of the ethanol extracts of A. cinnamomea mycelium cultivated in 5-L and 500-L ALs for 28 days were 23 and 17 μg/mL, respectively, for hepatocellular carcinoma cells HepG2. And after 42 days cultivation in 500-L ALs, the IC₅₀ value of the mycelium ethanol extract was reduced to 10 μg/mL.     

Glycosylation of Candida albicans Cell Wall Proteins Is Critical for Induction of Innate Immune Responses and Apoptosis of Epithelial Cells

C. albicans is one of the most common fungal pathogen of humans, causing local and superficial mucosal infections in immunocompromised individuals. Given that the key structure mediating host-C. albicans interactions is the fungal cell wall, we aimed to identify features of the cell wall inducing epithelial responses and be associated with fungal pathogenesis. We demonstrate here the importance of cell wall protein glycosylation in epithelial immune activation with a predominant role for the highly branched N-glycosylation residues. Moreover, these glycan moieties induce growth arrest and apoptosis of epithelial cells. Using an in vitro model of oral candidosis we demonstrate, that apoptosis induction by C. albicans wild-type occurs in early stage of infection and strongly depends on intact cell wall protein glycosylation. These novel findings demonstrate that glycosylation of the C. albicans cell wall proteins appears essential for modulation of epithelial immunity and apoptosis induction, both of which may promote fungal pathogenesis in vivo.     

Enhanced production of triterpenoid in submerged cultures of Antrodia cinnamomea with the addition of citrus peel extract

In recent years, Antrodia cinnamomea has become a well-known medicinal mushroom in Taiwan. Triterpenoids are considered one of the most biologically active components found in A. cinnamomea. The aim of this research is to investigate the feasibility of enhancing triterpenoid production in shake flask cultures of A. cinnamomea by adding citrus peel extract. As a result of its containing essential oils, citrus peel extract is inhibitory to mycelial growth. In the experiments, the appropriate adding time is determined to be on day 7. Of the various citrus peel extracts tested, tangerine proves to be the most effective in enhancing polyphenol and triterpenoid production. With an addition of 2 % (v/v), the content and production of total polyphenols rises from 5.95 mg/g DW of the control and 56.73 mg/L to 23.52 mg/g DW and 224.39 mg/L, respectively, on day 28. The production of triterpenoids also increases from 99.93 to 1,028.02 mg/L, for more than a tenfold increase. An optimal level of tangerine peel additive is determined to be around 4 %. Furthermore, when compared with the mycelia of the control culture, the profiles of the HPLC analysis show that the mycelia cultured with the tangerine-peel addition contain more kinds of triterpenoids. This study demonstrates that the addition of citrus peel extract effectively enhances the production of bioactive metabolites in the submerged cultures of A. cinnamomea.     

Silibinin triggers yeast apoptosis related to mitochondrial Ca2+ influx in Candida albicans

Candida albicans is a common yeast that resides in the human body, but can occasionally cause systemic fungal infection, namely candidiasis. As this infection rate is gradually increasing, it is becoming a major problem to public health. Accordingly, we for the first time investigated the antifungal activity and mode of action of silibinin, a natural product extracted from Silybum marianum (milk thistle), against C. albicans. On treatment with 100 μM silibinin, generation of reactive oxygen species (ROS) from mitochondria, which can cause yeast apoptosis via oxidative stress, was increased by 24.17% compared to that in untreated cells. Subsequently, we found disturbances in ion homeostasis such as release of intracellular K⁺ and accumulation of cytoplasmic and mitochondrial Ca²⁺. Among these phenomena, mitochondrial Ca²⁺ overload particularly plays a crucial role in the process of apoptosis, promoting the activation of pro-apoptotic factors. Therefore, we investigated the significance of mitochondrial Ca²⁺ in apoptosis by employing 20 mM ruthenium red (RR). Additional apoptosis hallmarks such as mitochondrial membrane depolarization, cytochrome c release, caspase activation, phosphatidylserine (PS) exposure, and DNA damage were observed in response to silibinin treatment, whereas RR pre-treatment seemed to block these responses. In summary, our results suggest that silibinin induces yeast apoptosis mediated by mitochondrial Ca²⁺ signaling in C. albicans.     

Novel Introner-Like Elements in fungi Are Involved in Parallel Gains of Spliceosomal Introns

Spliceosomal introns are key components of the eukaryotic gene structure. Although they contributed to the emergence of eukaryotes, their origin remains elusive. In fungi, they might originate from the multiplication of invasive introns named Introner-Like Elements (ILEs). However, so far ILEs have been observed in six fungal species only, including Fulvia fulva and Dothistroma septosporum (Dothideomycetes), arguing against ILE insertion as a general mechanism for intron gain. Here, we identified novel ILEs in eight additional fungal species that are phylogenetically related to F. fulva and D. septosporum using PCR amplification with primers derived from previously identified ILEs. The ILE content appeared unique to each species, suggesting independent multiplication events. Interestingly, we identified four genes each containing two gained ILEs. By analysing intron positions in orthologues of these four genes in Ascomycota, we found that three ILEs had inserted within a 15 bp window that contains regular spliceosomal introns in other fungal species. These three positions are not the result of intron sliding because ILEs are newly gained introns. Furthermore, the alternative hypothesis of an inferred ancestral gain followed by independent losses contradicts the observed degeneration of ILEs. These observations clearly indicate three parallel intron gains in four genes that were randomly identified. Our findings suggest that parallel intron gain is a phenomenon that has been highly underestimated in ILE-containing fungi, and likely in the whole fungal kingdom.     

Diversity and function of fungi associated with the fungivorous millipede,Brachycybe lecontii

Fungivorous millipedes (subterclass Colobognatha) likely represent some of the earliest known mycophagous terrestrial arthropods, yet their fungal associates remain elusive. Here we describe relationships between fungi and the fungivorous millipede, Brachycybe lecontii. Their fungal community is surprisingly diverse, including 176 genera, 39 orders, four phyla, and several undescribed species. Of particular interest are twelve genera conserved across wood substrates and millipede clades that comprise the core fungal community of B. lecontii. Wood decay fungi, long speculated to serve as the primary food source for Brachycybe species, were absent from this core assemblage and proved lethal to millipedes in pathogenicity assays while entomopathogenic Hypocreales were more common in the core but had little effect on millipede health. This study represents the first survey of fungal communities associated with any colobognath millipede, and these results offer a glimpse into the complexity of millipede fungal communities.     

The MEK/ERK pathway is the primary conduit for Borrelia burgdorferi-induced inflammation and P53-mediated apoptosis in oligodendrocytes

Lyme neuroborreliosis (LNB) affects both the central and peripheral nervous systems. In a rhesus macaque model of LNB we had previously shown that brains of rhesus macaques inoculated with Borrelia burgdorferi release inflammatory mediators, and undergo oligodendrocyte and neuronal cell death. In vitro analysis of this phenomenon indicated that while B. burgdorferi can induce inflammation and apoptosis of oligodendrocytes per se, microglia are required for neuronal apoptosis. We hypothesized that the inflammatory milieu elicited by the bacterium in microglia or oligodendrocytes contributes to the apoptosis of neurons and glial cells, respectively, and that downstream signaling events in NFkB and/or MAPK pathways play a role in these phenotypes. To test these hypotheses in oligodendrocytes, several pathway inhibitors were used to determine their effect on inflammation and apoptosis, as induced by B. burgdorferi. In a human oligodendrocyte cell line (MO3.13), inhibition of the ERK pathway in the presence of B. burgdorferi markedly reduced inflammation, followed by the JNK, p38 and NFkB pathway inhibition. In addition to eliciting inflammation, B. burgdorferi also increased total p53 protein levels, and suppression of the ERK pathway mitigated this effect. While inhibition of p53 had a minimal effect in reducing inflammation, suppression of the ERK pathway or p53 reduced apoptosis as measured by active caspase-3 activity and the TUNEL assay. A similar result was seen in primary human oligodendrocytes wherein suppression of ERK or p53 reduced apoptosis. It is possible that inflammation and apoptosis in oligodendrocytes are divergent arms of MAPK pathways, particularly the MEK/ERK pathway.     

Label-Free Quantitative Proteomic Analysis of Puccinia psidii Uredospores Reveals Differences of Fungal Populations Infecting Eucalyptus and Guava

Puccinia psidii sensu lato (s.l.) is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to P. psidii dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava) and eucalyptus leaves (PpEucalyptus). NanoUPLC-MS^E was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database) resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.     

Development of Snake Fungal Disease after Experimental Challenge with Ophidiomyces ophiodiicola in Cottonmouths (Agkistrodon piscivorous)

Snake fungal disease (SFD) is a clinical syndrome associated with dermatitis, myositis, osteomyelitis, and pneumonia in several species of free-ranging snakes in the US. The causative agent has been suggested as Ophidiomyces ophiodiicola, but other agents may contribute to the syndrome and the pathogenesis is not understood. To understand the role of O. ophiodiicola in SFD, a cottonmouth snake model of SFD was designed. Five cottonmouths (Agkistrodon piscivorous) were experimentally challenged by nasolabial pit inoculation with a pure culture of O. ophiodiicola. Development of skin lesions or facial swelling at the site of inoculation was observed in all snakes. Twice weekly swabs of the inoculation site revealed variable presence of O. ophiodiicola DNA by qPCR in all five inoculated snakes for 3 to 58 days post-inoculation; nasolabial flushes were not a useful sampling method for detection. Inoculated snakes had a 40% mortality rate. All inoculated snakes had microscopic lesions unilaterally on the side of the swabbed nasolabial pit, including erosions to ulcerations and heterophilic dermatitis. All signs were consistent with SFD; however, the severity of lesions varied in individual snakes, and fungal hyphae were only observed in 3 of 5 inoculated snakes. These three snakes correlated with post-mortem tissue qPCR evidence of O. ophiodiicola. The findings of this study conclude that O. ophiodiicola inoculation in a cottonmouth snake model leads to disease similar to SFD, although lesion severity and the fungal load are quite variable within the model. Future studies may utilize this model to further understand the pathogenesis of this disease and develop management strategies that mitigate disease effects, but investigation of other models with less variability may be warranted.     

Ectomycorrhizal fungal communities associated to Nothofagus species in Northern Patagonia

Ectomycorrhizal fungi constitute an important component of soil biota in Nothofagus forests in Patagonia. However, ectomycorrhizal fungal community is poorly known in this region. Here, we assess biodiversity and community compositions of ectomycorrhizal fungal species associated with Nothofagus dombeyi, N. obliqua and N. alpina. We selected three monospecific Nothofagus forest sites for each species within the boundaries of the Lanin National Park in Northern Patagonia. Ectomycorrhizal fungal species were identified based on morphotyping and rDNA (ITS and 28S rDNA) sequence analysis using both universal and taxon-specific primers. Contrary to previous studies on congeneric host trees, our results showed no significant differences among Nothofagus forest types in terms of fungal biodiversity and community composition. However, altitude had a strong effect on the structure of the ectomycorrhizal fungal community associated with Nothofagus spp.