Aureane-type sesquiterpene tetraketides as a novel class of immunomodulators with interleukin-17A inhibitory activity.

Interleukin (IL)-17A, a pro-inflammatory cytokine, is a fundamental function in the onset and advancement of multiple immune diseases. To uncover the primary compounds with IL-17A inhibitory activity, a large-scale screening of the library of traditional Chinese medicine constituents and microbial secondary metabolites was conducted using splenic cells from IL-17A-GFP reporter mice cultured under Th17-priming conditions. Our results indicated that some aureane-type sesquiterpene tetraketides isolated from a wetland mud-derived fungus, Myrothecium gramineum, showed remarkable IL-17A inhibitory activity. Nine new aureane-type sesquiterpene tetraketides, myrogramins A-I (1, 4-11), and two known ones (2 and 3) were isolated and identified from the strain. Compounds 1, 3, 4, 10, and 11 exhibited significant IL-17A inhibitory activity. Among them, compound 3, with a high fermentation yield dose-dependently inhibited the generation of IL-17A and suppressed glycolysis in splenic cells under Th17-priming conditions. Strikingly, compound 3 suppressed immunopathology in both IL-17A-mediated animal models of experimental autoimmune encephalomyelitis and pulmonary hypertension. Our results revealed that aureane-type sesquiterpene tetraketides are a novel class of immunomodulators with IL-17A inhibitory activity, and hold great promise applications in treating IL-17A-mediated immune diseases.

Peniterpenoids A-C, new sesquiterpenoid metabolites from a wheat cyst nematode Penicillium janthinellum.

Three new sesquiterpenoids, peniterpenoids A - C (1-3), together with six known metabolites (4-9) were isolated from an entomogenous fungus Penicillium janthinellum (LB1.20090001) collected from a wheat cyst nematode. The structures of the new compounds were elucidated based on NMR and HRESIMS spectroscopic analyses. The absolute configuration of the C-8 secondary alcohol of peniterpenoid B (2) was determined by [Rh2(OCOCF3)4]-induced ECD experiment. Subsequently, the antimicrobial and DPPH scavenging activities were determined. Compounds 6-8 exhibited moderate antibacterial activities against Staphylococcus aureus (CGMCC1.2465) with MIC values of 25.0, 50.0 and 12.5 µg/mL, respectively.

Cordycepamides A-E and cordyglycoside A, new alkaloidal and glycoside metabolites from the entomopathogenic fungus Cordyceps sp.

Five new alkaloidal metabolites cordycepamides A-E (1-5), and one glycoside metabolite cordyglycoside A (6), together with six known compounds (7-12) were isolated from the entomopathogenic fungus Cordyceps sp. (LB1.18060004) from unidentified insect collected in Baoshan City, Yunnan Province, People's Republic of China. The structures were characterized by NMR and HRESIMS spectroscopic analyses. Cordycepamides A and B (1 and 2) were mixtures of two isomers in 5:4 ratio by integration of 1H NMR spectra. In additional, the structure of cordycepamide A (1) was further confirmed by X-ray crystallography as a pair of enantiomers. Absolute configurations of sugar moiety of cordyglycoside A (6) was confirmed by the acid hydrolysis and subsequent HPLC analysis. The isolated metabolites were evaluated for antimicrobial, cytotoxicity, and the DPPH scavenging assay, only 4 showed modest antioxidant effects in the DPPH scavenging assay (IC50 = 51.42 ± 3.08 µM).

A breakthrough in the artificial cultivation of Chinese cordyceps on a large-scale and its impact on science, the economy, and industry.

Chinese cordyceps, an entity of the Chinese caterpillar fungus (Ophiocordyceps sinensis, syn. Cordyceps sinensis) that parasitizes ghost moth larvae, is one of the best known traditional Chinese medicines and is found exclusively on the Tibetan Plateau with limited natural resources. Although the fungus O. sinensis can grow on artificial substrates and the ghost moth has been successfully reared, the large-scale artificial cultivation of Chinese cordyceps has only recently been accomplished after several decades of efforts and attempts. In this article, research progress related to this breakthrough from living habitats, the life history of the fungus, its host insect, fungal isolation and culture, host larvae rearing, infection cycle of the fungus to the host, primordium induction, and fruiting body development have been reviewed. An understanding of the basic biology of O. sinensis, its host insect and the simulation of the Tibetan alpine environment resulted in the success of artificial cultivation on a large scale. Practical workshop production has reached annual yields of 2.5, 5, and 10 tons in 2014, 2015, and 2016, respectively. There was no difference in the chemical components detected between the cultivated and natural Chinese cordyceps. However, the artificial cultivation system can be controlled to avoid heavy metal contamination and results in high-quality products. Although omics studies, including genomic, transcriptomic, proteomic, and metabolomic studies, have helped to understand the biology of the fungus, the success of the artificial cultivation of the Chinese cordyceps is clearly a milestone and provides the possibility for research on the in-depth mechanisms of the interaction between the fungus and host insects and their adaptation to the harsh habitats. This cultivation will not only result in a large industry to alleviate the pressure of human demand but also protect the limited natural resources for sustainable utilization.

The Microbiome and Metabolites in Fermented Pu-erh Tea as Revealed by High-Throughput Sequencing and Quantitative Multiplex Metabolite Analysis.

Pu-erh is a tea produced in Yunnan, China by microbial fermentation of fresh Camellia sinensis leaves by two processes, the traditional raw fermentation and the faster, ripened fermentation. We characterized fungal and bacterial communities in leaves and both Pu-erhs by high-throughput, rDNA-amplicon sequencing and we characterized the profile of bioactive extrolite mycotoxins in Pu-erh teas by quantitative liquid chromatography-tandem mass spectrometry. We identified 390 fungal and 629 bacterial OTUs from leaves and both Pu-erhs. Major findings are: 1) fungal diversity drops and bacterial diversity rises due to raw or ripened fermentation, 2) fungal and bacterial community composition changes significantly between fresh leaves and both raw and ripened Pu-erh, 3) aging causes significant changes in the microbial community of raw, but not ripened, Pu-erh, and, 4) ripened and well-aged raw Pu-erh have similar microbial communities that are distinct from those of young, raw Ph-erh tea. Twenty-five toxic metabolites, mainly of fungal origin, were detected, with patulin and asperglaucide dominating and at levels supporting the Chinese custom of discarding the first preparation of Pu-erh and using the wet tea to then brew a pot for consumption.

Development of Ling-zhi industry in China - emanated from the artificial cultivation in the Institute of Microbiology, Chinese Academy of Sciences (IMCAS).

Ling-zhi is a medicinal herb that generally refers to a fungus in the genus Ganoderma. It has been used as a medicinal mushroom in traditional Chinese medicine for more than 2000 years. Mycologists at the Institute of Microbiology, Chinese Academy of Sciences (IMCAS) first artificially cultivated the Ling-zhi fruiting body in the late 1960s (X.J. Liu's team). In IMCAS, different research teams have extensively studied Ling-zhi in the aspects of national resource surveys, systematic taxonomy, chemical analysis, and processing for medicinal and health applications. The research results from IMCAS have provided essential support and prompted the development of the Ling-zhi industry in China to some extent. This review aims to summarize the history of research on Ling-zhi in IMCAS and its role in the development of the Ling-zhi economy.

Stachybisbins A and B, the first cases of seco-bisabosquals from Stachybotrys bisbyi.

Stachybisbins A (1) and B (2), two new meroterpenoids with unprecedented seco-bisabosqual skeleton, together with three biogenetically related metabolites (3-5), were isolated from a wetland fungal strain of Stachybotrys bisbyi. The structures of the new compounds were determined by spectroscopic analyses, modified Mosher's method, and quantum chemical CD method. The cytotoxic activities of all compounds were tested against HL-60, SMMC-7721, A-549, MCF-7, and SW480 human cancer cell lines.

Two new ramulosin derivatives from the entomogenous fungus Truncatella angustata.

Two new ramulosin derivatives, 7α-hydroxy-8-dihydroramulosin (1) and 7-ketoramulosin (2), along with three known metabolites, (+)-ramulosin (3), 6-hydroxyramulosin (4), and 8-dihydroramulosin (5), were isolated from the crude extract of Truncatella angustata, an entomogenous fungus isolated from the Septobasidium-infected insect Aspidiotus sp. The structures of 1 and 2 were elucidated by nuclear magnetic resonance experiments, and 1 was further confirmed by X-ray crystallography. The absolute configuration of 1 was assigned by single-crystal X-ray diffraction analysis using Cu Kα radiation, whereas that of 2 was determined by electronic circular dichroism (ECD) calculations. Compounds 1-5 were tested for cytotoxicity against four human carcinoma cell lines, HeLa, A549, MCF-7, and T24. Compound 4 showed weak cytotoxic effects against A549 and T24.

Farming of a defensive fungal mutualist by an attelabid weevil.

The mutualism between fungus-growing animals and fungi is a classic example of a complex interspecies association. A handful of insects, notably the well-recognized fungus-farming ants, termites and beetles, have developed advanced agriculture, which includes seeding new gardens with crop propagules, improving growth conditions and protecting and harvesting the fungal crop. More examples, which could be called 'proto-fungiculture', involve fewer adaptations, as exemplified by marine snails that farm intertidal fungi on marsh grass. Recent work has indicated that the solitary leaf-rolling weevil Euops chinensis (family Attelabidae) has a protofarming symbiosis with the mycangial fungus Penicillium herquei (family Trichocomaceae). In this study, we investigated how the weevils create cradles (leaf-rolls) for their offspring and protect the fungal garden. We describe new specialized structures and behaviors that E. chinensis females use for leaf-rolling and fungus inoculation. The fungus P. herquei produces the antibiotic (+)-scleroderolide in laboratory culture and in leaf-rolls, which can serve to inhibit microbial 'weeds' and pests, thus protecting the fungal garden against potential infection. The fungiculture of E. chinensis differs from other advanced insect fungiculture systems because female weevils do not continuously tend the inoculated microbe and do not depend nutritionally on the fungus. The defensive role of the cultivated fungus makes the attelabid weevils exceptional in 'proto-fungiculture' animals.

[Two new polyesters from wetland soil-derived fungus Talaromyces flavus].

Two new polyesters, talapolyesters G-H (1-2) were isolated from the wetland soil-derived fungus Talaromyces flavus BYD07-13, and their structures were determined by NMR and MS spectroscopic data. The absolute configurations of the residues were determined by alkaline hydrolysis. The cytotoxicity against five tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480) of 1-2 was examined.

Cordyceps industry in China.

Cordyceps, as a general term, describes a group of ascomycetous fungi growing on arthropods and other related fungi. Some cordyceps have been used in traditional Chinese medicine for centuries and cordyceps-derived products are currently a big industry in China. A number of medicinal and health products have been developed and extensively commercialized from natural Chinese cordyceps, its anamorphic fungus (Hirsutella sinensis), and other fungi known as Chinese cordyceps. The lack of a defined classification system for medicinal cordyceps fungi is a source of confusion in the industry and the public, and even among pharmaceutical scientists. This review summarizes the cordyceps fungi currently used in the industry in China with a special reference to clarify Chinese cordyceps and associated fungi. Cordyceps militaris, Cordyceps guangdongensis and Isaria cicadae are well recognized and commercialized cordyceps fungi in China. Except the natural Chinese cordyceps and its anamorphic fungus, Paecilomyces hepiali, Mortierella hepiali, Cephalosporium sinensis and Clonostachys rosea isolated from natural Chinese cordyceps are classified as Chinese cordyceps-associated fungi. P. hepiali is a cordyceps fungus based on current phylogenetic analysis of Hypocreales, while M. hepiali is a fungus in the Zygomycetes and should only be treated as associated fungus of Chinese cordyceps. C. sinensis and C. rosea belong to the Hypocreales and their relationship to cordyceps fungi should be further studied. The exploitation of the resources of cordyceps fungi and their quality control in the industry should be major topics for future studies. Cooperation between the industry and the research community will enhance the whole cordyceps industry.

Hawaiinolides E-G, cytotoxic cassane and cleistanthane diterpenoids from the entomogenous fungus Paraconiothyrium hawaiiense.

Hawaiinolides E-G (1-3), three additional new secondary metabolites including two cassane (1 and 2) types of diterpene lactones and one cleistanthane (3) diterpenoid, were isolated from the scale-up fermentation extract of Paraconiothyrium hawaiiense, an entomogenous fungus isolated from the Septobasidium-infected insect Diaspidiotus sp. The structures of 1-3 were elucidated by nuclear magnetic resonance experiments, and 1 and 3 were further confirmed by X-ray crystallography. The absolute configurations of 1 and 3 were assigned by single-crystal X-ray diffraction analysis using Cu Kα radiation, whereas that of 2 was deduced via the circular dichroism data. Compound 1 showed significant cytotoxic effects against the human tumor cell line T24, with an IC50 value (9.32 µM) comparable to that of the positive control cisplatin.

Phylogeography and evolution of a fungal-insect association on the Tibetan Plateau.

Parasitoidism refers to a major form of interspecies interactions where parasitoids sterilize and/or kill their hosts typically before hosts reach reproductive age. However, relatively little is known about the evolutionary dynamics of parasitoidism. Here, we investigate the spatial patterns of genetic variation of Chinese cordyceps, including both the parasitoidal fungus Ophiocordyceps sinensis and its host insects. We sampled broadly from alpine regions on the Tibetan Plateau and obtained sequences on seven fungal and three insect DNA fragments from each of the 125 samples. Seven and five divergent lineages/cryptic species were identified within the fungus and host insects, respectively. Our analyses suggested that O. sinensis and host insects originated at similar geographic regions in southern Tibet/Yunnan, followed by range expansion to their current distributions. Cophylogenetic analyses revealed a complex evolutionary relationship between O. sinensis and its host insects. Significant congruence was found between host and parasite phylogenies and the time estimates of divergence were similar, raising the possibility of the occurrence of cospeciation events, but the incongruences suggested that host shifts were also prevalent. Interestingly, one fungal genotype was broadly distributed, consistent with recent gene flow. In contrast, the high-frequency insect genotypes showed limited geographic distributions. The dominant genotypes from both the fungus and the insect hosts may represent ideal materials from which to develop artificial cultivation of this important Chinese traditional medicine. Our results demonstrate that both historical and contemporary events have played important roles in the phylogeography and evolution of the O. sinensis-ghost moth parasitoidism on the Tibetan Plateau.

Trichocladinols I-K, oxatricyclic and oxabicyclic polyketides from Trichocladium opacum.

Three new polyketides, trichocladinols I-K (1-3) with oxabicyclic (1 and 3) and oxatricyclic (2) skeletons, together with three knovwnl metabolites, massarilactone G (4), massarigenin D (5), and rosigenin (6), were isolated from the solid-substrate fermentation cultures of the ascomycete fungus Trichocladium opacum. The structures of the new compounds were elucidated primarily by analysis of NMR data. The absolute configurations of 1-3 were deduced from circular dichroism (CD) data. Compounds 1-3 were tested for cytotoxicity against five human carcinoma cell lines, HeLa, A549, MCF-7, HCT116, and T24. Compound I showed a weak cytotoxic effect against HeLa cells, but 2 and 3 were inactive.

Stereochemical determination of new cytochalasans from the plant endophytic fungus Trichoderma gamsii.

Three new cytochalasans, trichalasins E (1), F (2) and H (7), together with four known analogues, trichalasin C (3), aspochalasin K (4), trichalasin G (5) and aspergillin PZ (8), were isolated from one endophytic fungus Trichoderma gamsii inhabiting in the traditional medicinal plant Panax notoginseng (BurK.) F.H. Chen. Trichalasins E (1) contains a unique hydroperoxyl group, which is the first report in all known analogues, whereas trichalasin H (7) possesses the rare 6/5/6/6/5 pentacyclic skeleton with 12-oxatricyclo [6.3.1.0(2,7)] moiety as that of aspergillin PZ (8). The relative configurations of the new compounds were characterized by analysis of coupling constants and ROESY correlations, and the absolute configurations of trichalasins E (1), H (7) and aspergillin PZ (8) were determined by modified Mosher's reaction. In addition, compounds 1-5, 7 and 8 were tested cytotoxic activities against several cancer cell lines.

Guanacastane diterpenoids from the plant endophytic fungus Cercospora sp.

Cercosporenes A-F (1-6, respectively), six new guanacastane diterpenes, including a homodimer (5) and a heterodimer (6), were isolated from the crude extract of the fungus Cercospora sp., endophytic to the medicinal plant Fallopia japonica. The structures of 1-6 were elucidated by nuclear magnetic resonance experiments, and 4 and 5 were further confirmed by X-ray crystallography. The absolute configuration of 1 and 3 was assigned by electronic circular dichroism calculations, whereas that of 6 was deduced by the application of the circular dichroism exciton chirality method. In addition to its cytotoxicity against a panel of five human tumor cell lines, HeLa, A549, MCF-7, HCT116, and T24, heterodimer 6 also induced autophagy in HCT116 cells.

Bacterial and fungal communities in Pu'er tea samples of different ages.

Pu'er is a major kind of postfermented tea and is made with a "large leaf" variety of Camellia sinensis (C. sinensis assamica), whose distribution is limited to the mountains of southern Yunnan, China. The quality of Pu'er tea is believed to increase with storage (aging, maturing) because of postfermentation by microbes. The effect of storage period (from < 1 to 192 mo) on the bacteria and fungi in Pu'er tea was investigated by a culture-dependent and a PCR-DGGE method. The individual numbers of fungi and bacteria decreased with increasing storage time and were significantly greater in ripened tea than in raw Pu'er tea. Both methods indicated that yeast, Aspergillus spp., and Penicillium spp. were the dominant fungi in almost all the samples. However, the common bacteria detected by the culture-dependent method were species of Pseudomonas, Achromobacter, Alcaligenes, Sporosarcina, and Bacillus, whereas those detected by PCR-DGGE were species of Staphylococcus, Arthrobacter, and Streptomyces. According to ordination analysis, bacterial community structure differed between ripened and raw Pu'er tea. Bacterial diversity was positively correlated with aging time, while fungal diversity in both raw and ripened tea increased during the first 60 mo of aging and then decreased. Changes in polyphenol content were correlated with the changes in fungal diversity. These results suggest that the relationship between storage time and the quality of Pu'er tea is complex and involves changes in polyphenol content and microbial abundance and diversity.