Chaetoxylariones A-G: undescribed chromone-derived polyketides from co-culture of Chaetomium virescens and Xylaria grammica enabled via the molecular networking strategy.

By co-culturing two endophytic fungi (Chaetomium virescens and Xylaria grammica) collected from the medicinal and edible plant Smilax glabra Roxb. and analyzing them with MolNetEnhancer module on GNPS platform, seven undescribed chromone-derived polyketides (chaetoxylariones A-G), including three pairs of enantiomer ones (2a/2b, 4a/4b and 6a/6b) and four optical pure ones (1, 3, 5 and 7), as well as five known structural analogues (8-12), were obtained. The structures of these new compounds were characterized by NMR spectroscopy, single-crystal X-ray diffraction, 13C NMR calculation and DP4+ probability analyses, as well as the comparison of the experimental electronic circular dichroism (ECD) data. Structurally, compound 1 featured an unprecedented chromone-derived sulfonamide tailored by two isoleucine-derived δ-hydroxy-3-methylpentenoic acids via the acylamide and NO bonds, respectively; compound 2 represented the first example of enantiomeric chromone derivative bearing a unique spiro-[3.3]alkane ring system; compound 3 featured a decane alkyl side chain that formed an undescribed five-membered lactone ring between C-7' and C-10'; compound 4 contained an unexpected highly oxidized five-membered carbocyclic system featuring rare adjacent keto groups; compound 7 featured a rare methylsulfonyl moiety. In addition, compound 10 showed a significant inhibition towards SW620/AD300 cells with an IC50 value of PTX significantly decreased from 4.09 µM to 120 nM, and a further study uncovered that compound 10 could obviously reverse the MDR of SW620/AD300 cells.

Discovery of two ent-atisane diterpenoid lactones with AChE inhibitory activity from the roots of Euphorbia fischeriana.

Euphorlactone A (1), a rare rearranged ent-atisane norditerpenoid with an undescribed 3-nor-2,4-olide-ent-atisane scaffold, and euphorlactone B (2), a new ent-atisane diterpenoid with an unprecedented seven-membered lactone ring C, were isolated from the roots of Euphorbia fischeriana. Their planar structures with absolute configurations were extensively elucidated by analysis of 1D and 2D NMR data, electronic circular dichroism (ECD) calculations, Rh2(OCOCF3)4-induced ECD curves, and single-crystal X-ray diffraction. Euphorlactone A (ELA) showed a remarkable AChE (acetylcholinesterase) inhibitory activity (IC50 = 2.13 ± 0.06 µM and Ki = 0.058 µM), which was five times stronger than that of the positive control (rivastigmine, IC50 = 12.46 ± 0.82 µM), and further in vitro enzyme inhibition kinetic analysis and molecular docking studies were performed to investigate the AChE inhibitory mechanism.

Indoloquinazoline alkaloids with cardiomyocyte protective activity against cold ischemic injury from Aspergillus clavatonanicus.

The arthropod-associated fungi have been demonstrated to be a remarkable producer of structurally captivating and bioactive secondary metabolites for drug discovery. In this study, eleven new indoloquinazoline alkaloids, namely aspergilloids A-K (1-11), along with five known congeners (12-16), were obtained from fungus Aspergillus clavatonanicus, which was isolated from the gut of a centipede collected in our Tongji campus. All these compounds were rarely defined by a 6/5/5 indolone ring system in conjugation with a five-membered spiral ring (1-5 and 10-16) or an opening five-membered spiral ring (6-9). Their structures were elucidated by widespread spectroscopic analyses, mainly including HRESIMS and 1D and 2D NMR data, single-crystal X-ray diffraction, and electronic circular dichroism (ECD) data analyses. The cardiomyocyte protective activity assay revealed that compounds 1, 2, 5, 12-14, and 16 ameliorated cold ischemic injury at 48 h post cold ischemia (CI), and compounds 1, 5, and 14 prevented cold ischemia induced Ser9 dephosphorylation of GSK3ß at 12 h post CI. Our current study highlights indoloquinazoline alkaloids as the first class of natural cardiomyocyte protective agents against cold ischemic injury, which furnishes promising lead molecules for the development of new cardioprotectants in heart transplantation medicine.

Coregulation of dimorphism and symbiosis by cyclic AMP signaling in the lichenized fungus Umbilicaria muhlenbergii.

Umbilicaria muhlenbergii is the only known dimorphic lichenized fungus that grows in the hyphal form in lichen thalli but as yeast cells in axenic cultures. However, the regulation of yeast-to-hypha transition and its relationship to the establishment of symbiosis are not clear. In this study, we show that nutrient limitation and hyperosmotic stress trigger the dimorphic change in U. muhlenbergii Contact with algal cells of its photobiont Trebouxia jamesii induced pseudohyphal growth. Treatments with the cAMP diphosphoesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) induced pseudohyphal/hyphal growth and resulted in the differentiation of heavily melanized, lichen cortex-like structures in culture, indicating the role of cAMP signaling in regulating dimorphism. To confirm this observation, we identified and characterized two Gα subunits UmGPA2 and UmGPA3 Whereas deletion of UmGPA2 had only a minor effect on pseudohyphal growth, the ΔUmgpa3 mutant was defective in yeast-to-pseudohypha transition induced by hyperosmotic stress or T. jamesii cells. IBMX treatment suppressed the defect of ΔUmgpa3 in pseudohyphal growth. Transformants expressing the UmGPA3G45V or UmGPA3Q208L dominant active allele were enhanced in the yeast-to-pseudohypha transition and developed pseudohyphae under conditions noninducible to the wild type. Interestingly, T. jamesii cells in close contact with pseudohyphae of UmGPA3G45V and UmGPA3Q208L transformants often collapsed and died after coincubation for over 72 h, indicating that improperly regulated pseudohyphal growth due to dominant active mutations may disrupt the initial establishment of symbiotic interaction between the photobiont and mycobiont. Taken together, these results show that the cAMP-PKA pathway plays a critical role in regulating dimorphism and symbiosis in U. muhlenbergii.

Two novel polyketones from the leaves and twigs of Clerodendrum trichotomum.

Two novel polyketones, along with four known compounds, were isolated from the leaves and twigs of Clerodendrum trichotomum. Their structures were determined to be clerodendruketone A (1), clerodendruketone B (2), ecdysanols E (3), ecdysanols D (4), 5,5'-dimethoxy-7-oxolariciresinol (5), and (-)-(7'S,8S,8'R)-4,4'-dihydroxy-3,3',5,5'-tetramethoxy-7',9-epoxy-lignan-9'-ol-7-one (6) through the methods of NMR, HRESIMS and ECD data analyses. The antioxidant effects against free radical were tested by DPPH assay. The antibacterial activity against Escherichia coli and Staphylococcus aureus were tested by turbidimetry assay. The results demonstrated that compounds 1 and 2 had significative antibacterial activity, and compound 3 had moderate antioxidant activity.

[A new norsesquiterpenoid from Schisandra chinensis].

This study investigated the chemical components from the leaves and stems of Schisandra chinensis. Three norsesquiterpenoids were isolated from S. chinensis by various column chromatographies(silica gel, Sephadex LH-20, and MCI), reversed-phase medium-pressure preparative, and semi-preparative high-performance liquid chromatography(HPLC). Their structures were identified based on physicochemical properties, mass spectrometry(MS), nuclear magnetic resonance(NMR), ultraviolet(UV), and electro-nic circular dichroism(ECD) as(3R,4R,5R,6S,7E)-3,4,5,6-tetrahydroxy-7-megastigmen-9-one(1),(3S,5R,6R,7E)-3,5,6-trihydroxy-7-megastigmen-9-one(2), and(3S,4R,9R)-3,4,9-trihydroxymegastigman-5-ene(3). Compound 1 was a new compound, and its absolute configuration was determined by ECD. Compounds 2 and 3 were isolated from the Schisandra plant for the first time.

Euphorfistrines A-G, cytotoxic and AChE inhibiting triterpenoids from the roots of Euphorbia fischeriana.

Seven new triterpenoids including two cycloartanes (1-2), a lanostane (3), a tirucallane (4), a dammarane (5), an ursane (6), and an oleanane (7), along with nineteen known triterpenoids (8-26), have been obtained from the roots of Euphorbia fischeriana. Their structures were established by NMR, HRESIMS, single-crystal X-ray diffraction analysis, Mosher's method, NMR calculations, ECD analysis, and comparison with structurally related known analogues. Among them, compounds 1 and 8 were a pair of cycloartane-type triterpenoids epimers. Our bioassays have established that compounds 1-5 and 10 displayed moderate cytotoxic effects, and the structure-activity relationships of cycloartane-type triterpenoids (CTTs) were further examined. Notably, some triterpenoids displayed moderate inhibitory effects against AChE by an in vitro screened experiment. Triterpenoid 7 (Euphorfistrine G, ETG) displayed the potent inhibitory effect with IC50 = 2.45 and Ki = 2.30 µM (inhibition kinetic). And, in silico docking analyses have been performed to investigate the inhibitory mechanism of compound 7.

Chemical characteristics of the fungus Ganoderma lucidum and their inhibitory effects on acetylcholinesterase.

The chemical characteristic of a well-known folk medicine Ganoderma lucidum has been investigated by a series of chromatographic technologies, which displayed the presences of 45 lanostane type triterpenoids, including two new nor-lanostane triterpenoids (40, 41). Their structures were identified on the basis of spectroscopic data analysis (UV, IR, HRESIMS, 1D, and 2D NMR). Notably, some triterpenoids displayed moderate inhibitory effects against AChE (acetylcholinesterase) by an in vitro screened experiment. Triterpenoid 2 displayed the potent inhibitory effect with IC50 10.8 and Ki 14.95 µM (inhibition kinetic). The preliminary SAR has been discussed by the docking analyses between ganoderic acids (1, 2) and AChE.

Comparative Analysis of Chemical Composition, Anti-Inflammatory Activity and Antitumor Activity in Essential Oils from Siegesbeckiaorientalis, S. glabrescens and S. pubescens with an ITS Sequence Analysis.

Herba Siegesbeckiae (HS), derived from the aerial parts of three plants, Siegesbeckia orientalis (SO), S. glabrescens (SG), and S. pubescens (SP), has been used for the treatment of inflammatory diseases in China for centuries. In the present study, hydrodistillation was applied to extract essential oils from dried SO, SG, and SP aerial parts, and chemical composition analysis by gas chromatography⁻mass spectrometry (GC-MS) led to the identification of a total of 148 compounds (56 in SO, 62 in SG, and 59 in SP). The main components in the essential oils of SO, SG, and SP differed significantly. In vitro anti-inflammatory activity assays showed that SP essential oils (IC50, 0.97 µg/mL) significantly reduced the ability of lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages to release NO, and the SO essential oil (IC50, 14.99 µg/mL) was better than the others at inhibiting the LPS-induced release of cytokine IL-6. Furthermore, the essential oils exhibited antitumor activities (IC50, 37.72⁻123.16 µg/mL) against Hep3B (liver) and Hela (cervical) cells. Linear regression analysis showed that, caryophyllene oxide peak area percentages showed remarkably high negative correlation coefficients with IC50 values of Hep3B and Hela cytotoxicity, which suggested the contribution of this compound on the cancer cell cytotoxicity of three essential oils. Finally, the ITS1-5.8S-ITS2 region was amplified and sequenced in order to generate genomic reference sequences for each plant. These can be used to identify the origins of the plants, and will assist other research studies related to these three plants.

The calcium-binding protein EpANN from the lichenized fungus Endocarpon pusillum enhances stress tolerance in yeast and plants.

Annexins are calcium-phospholipid binding proteins that play a significant role in the Ca2+signaling pathway. These proteins are essential for plants to effectively respond to abiotic stresses. However, their functions and mechanisms remain largely unknown in fungi. In this study, an annexin gene, Epann, was cloned from the lichenized fungus Endocarpon pusillum, a drought resistant organism. Our results showed that Epann was induced by several abiotic stresses in E. pusillum. Heterologous expression of the Epann gene enhanced the stress tolerance of Saccharomyces cerevisiae. Under heat-shock conditions, the EpANN proteins were significantly aggregated and the aggregation sites were located on peroxisomes. In heat-shocked cells, Epann reduced the reactive oxygen species level mainly through its intracellular peroxidase activity and regulation of stress-related genes. Transgenic Arabidopsis plants overexpressing Epann exhibited a higher germination rate under oxidative stress and stronger drought tolerance. Our results provide a mechanistic understanding of the role of annexins in abiotic stress responses and suggest that this lichenized fungal gene could be a promising resource to generate stress-tolerant transgenic organisms.

[Diversity of Bacillus species inhabiting on the surface and endophyte of lichens collected from Wuyi Mountain].

OBJECTIVE: The present work reported the isolation, identification and diversity of Bacillus species colonizing on the surface and endophyte in lichens collected from Wuyi Mountain. METHODS: Nine lichen samples of Evernia, Stereocaulon, Menegazzia and other 6 genera belonging to 7 families were collected from Wuyi mountain nature reserve. The bacillus-like species colonizing on the surface and endophyte in these lichens were isolated and identified by 16S rRNA gene sequence analysis. RESULTS: There was no bacillus-like species isolated from Evernia, Ramalina and Lecarona. A total of 34 bacillus-like bacteria were isolated from another 6 lichen samples. These bacteria were identified as 24 species and were classified into Bacillus, Paenibacillus, Brevibacillus, Lysinibacillus and Viridiibacillus. Paenibacillus and Bacillus are the dominant genera, and accounting for 41. 2% and 35. 3% of all isolated bacteria respectively. Brevibacillus, Lysinibacillus and Viridiibacillu were first reported being isolated from lichens. There were different species and quantity of bacillus colonizing on the surface and endophyte in different lichens. The quantity of bacillus colonizing on the surface of Physcia was more than 3.85 x 10(6) cfu/g and was the largest in the isolated bacteria, while the species of bacillus colonizing on the surface and endophyte in Stereocaulon was the most abundant. Most of the isolated bacteria were colonizing on (in) one lichen genera, but Paenibacillus taichungensis, Paenibacillus odorifer, Brevibacillus agri, Lysinibacillus xylanilyticus was respectively colonizing on (in) 2-3 lichen genera and Bacillus mycoides was colonizing on (in) Menegazzia, Cladonia Physcia, and Stereocaulon. CONCLUSION: There are species and quantity diversity of bacillus colonizing on (in) lichens.

Genome characteristics reveal the impact of lichenization on lichen-forming fungus Endocarpon pusillum Hedwig (Verrucariales, Ascomycota).

BACKGROUND: Lichen is a classic mutualistic organism and the lichenization is one of the fungal symbioses. The lichen-forming fungus Endocarpon pusillum is living in symbiosis with the green alga Diplosphaera chodatii Bialsuknia as a lichen in the arid regions. RESULTS: 454 and Illumina technologies were used to sequence the genome of E. pusillum. A total of 9,285 genes were annotated in the 37.5 Mb genome of E. pusillum. Analyses of the genes provided direct molecular evidence for certain natural characteristics, such as homothallic reproduction and drought-tolerance. Comparative genomics analysis indicated that the expansion and contraction of some protein families in the E. pusillum genome reflect the specific relationship with its photosynthetic partner (D. chodatii). Co-culture experiments using the lichen-forming fungus E. pusillum and its algal partner allowed the functional identification of genes involved in the nitrogen and carbon transfer between both symbionts, and three lectins without signal peptide domains were found to be essential for the symbiotic recognition in the lichen; interestingly, the ratio of the biomass of both lichen-forming fungus and its photosynthetic partner and their contact time were found to be important for the interaction between these two symbionts. CONCLUSIONS: The present study lays a genomic analysis of the lichen-forming fungus E. pusillum for demonstrating its general biological features and the traits of the interaction between this fungus and its photosynthetic partner D. chodatii, and will provide research basis for investigating the nature of its drought resistance and symbiosis.

Adaptive and blind audio watermarking algorithm based on dither modulation and butterfly optimization algorithm.

How to improve the robustness to resist attacks and how to adaptively match the key parameters of the watermarking algorithm with the performance requirements to achieve the best performance in different applications are two hot issues in the research of audio watermarking algorithms. An adaptive and blind audio watermarking algorithm based on dither modulation and butterfly optimization algorithm (BOA) is proposed. Based on the convolution operation, a stable feature is designed to carry the watermark, which will improve the robustness by means of the stability of this feature to prevent the watermark loss. Blind extraction will be achieved only by comparing the feature value and the quantized value without the original audio. The BOA is used to optimize the key parameters of the algorithm which can be matched with the performance requirements by coding the population and constructing the fitness function. Experimental results confirm that this proposed algorithm can adaptively search for the optimal key parameters that match the performance requirements. Compared with other related algorithms in recent years, it exhibits strong robustness against various signal processing attacks and synchronization attacks.

Erratum to: Developmental recording of the ghost-moth larvae after ex situ infection by Ophiocordyceps sinensis.

This paper contains errors in the authors' affiliations. Wenjia Li, is a Ph.D. candidate in the University of Chinese Academy of Sciences, Jiangchun Wei and Xingzhong Liu are professors in the University of Chinese Academy of Sciences, which is missing in the original version. The affiliations have been corrected.

A novel colchicine-myricetin heterozygous molecule: design, synthesis, and effective evaluations on the pathological models of acute lung injury in vitro and in vivo.

Acute lung injury (ALI) is an inflammatory condition and there are no effective treatments. A novel new compound----colchicine-myricetin hybrid (CMyrH) was herein designed and synthesized. To evaluate the activity of CMyrH in ALI, we used a bleomycin (BLM) induced BEAS-2B injury model in vitro and established a well-recognized rat model of BLM-induced lung injury in vivo. The results demonstrated that colchicine-myricetin hybrid protected BEAS-2B cells against BLM-induced cell injury in an increased dose manner, and reduced wet/dry weight ratio, histological scoring, and inflammation cytokines IL-1ß, IL-6, IL-18, and TNF-α levels of lung tissue of the rats. Furthermore, we found colchicine-myricetin hybrid inhibited caspase-1, ASC, GSDMD, and NLRP-3 expression in vivo. Meanwhile, we used molecular docking to analyze the binding mode of colchicine-myricetin hybrid and human neutrophil elastase (HNE), it revealed that colchicine-myricetin hybrid showed strong binding affinity toward human neutrophil elastase when compared to its parent molecules. In conclusion, It is suggested that colchicine-myricetin hybrid antagonized acute lung injury by focusing on multi-targets via multi-mechanisms, and might be served as a potential therapeutic agent for acute lung injury.

Antiproliferative ent-abietane diterpenoids from Euphorbia fischeriana.

Euphorfinoids M and N (1 and 2), two previously undescribed ent-abietane diterpenoids, together with seven known analogues (3-9), were isolated from the roots of wild Euphorbia fischeriana. Their structures were elucidated by spectroscopic analysis, including extensive NMR, HR-ESIMS, ECD, and comparison with structurally related known analogues. Bioassays against proliferative effects of HeLa cell line showed that compound 1 was the most active with IC50 3.62 ± 0.31 µM.

Regulation of symbiotic interactions and primitive lichen differentiation by UMP1 MAP kinase in Umbilicaria muhlenbergii.

Lichens are of great ecological importance but mechanisms regulating lichen symbiosis are not clear. Umbilicaria muhlenbergii is a lichen-forming fungus amenable to molecular manipulations and dimorphic. Here, we established conditions conducive to symbiotic interactions and lichen differentiation and showed the importance of UMP1 MAP kinase in lichen development. In the initial biofilm-like symbiotic complexes, algal cells were interwoven with pseudohyphae covered with extracellular matrix. After longer incubation, fungal-algal complexes further differentiated into primitive lichen thalli with a melanized cortex-like and pseudoparenchyma-like tissues containing photoactive algal cells. Mutants deleted of UMP1 were blocked in pseudohyphal growth and development of biofilm-like complexes and primitive lichens. Invasion of dividing mother cells that contributes to algal layer organization in lichens was not observed in the ump1 mutant. Overall, these results showed regulatory roles of UMP1 in symbiotic interactions and lichen development and suitability of U. muhlenbergii as a model for studying lichen symbiosis.

Euphorfinoids A and B, a pair of ent-atisane diterpenoid epimers from the roots of Euphorbia fischeriana, and their bioactivities.

Euphorfinoids A and B (1 and 2), a pair of ent-atisane diterpenoid epimers with a vicinal 2,3-diol moiety, together with four known analogues (3-6), were isolated from the roots of wild Euphorbia fischeriana. Their structures were elucidated by spectroscopic analysis, including extensive NMR, HR-ESIMS, NMR calculations, X-ray diffraction, and comparison with structurally related known analogues. Our bioassays have established that compound 1 displayed moderate anti-proliferative effects on Hcc1806 cell line with IC50 15.53 ± 0.21 µM, and compound 5 showed remarkable inhibitory effects against AChE with IC50 32.56 ± 2.74 µM by an in vitro screened experiment.

The Jurassic epiphytic macrolichen Daohugouthallus reveals the oldest lichen-plant interaction in a Mesozoic forest ecosystem.

Lichens are well known as pioneer organisms or stress-tolerant extremophiles, potentially playing a core role in the early formation of terrestrial ecosystems. Epiphytic macrolichens are known to contribute to the water- and nutrient cycles in forest ecosystem. But due to the scarcity of fossil record, the evolutionary history of epiphytic macrolichens is poorly documented. Based on new fossil of Jurassic Daohugouthallus ciliiferus, we demonstrate the hitherto oldest known macrolichen inhabited a gymnosperm branch. We applied energy dispersive X-ray spectroscopy and geometric morphometric analysis to complementarily verify lichen affinity of D. ciliiferus and quantitatively assess the potential relationships with extant lichenized lineages, providing new approaches for study of this lichen adpression fossil. Considering the results, and the inferred age of D. ciliiferus, a new family, Daohugouthallaceae, is established. This work updates current knowledge to the early evolution of epiphytic macrolichens and reveals more complex lichen-plant interactions in a Jurassic forest ecosystem.

Morphological and Phylogenetic Characterizations Reveal Five New Species of Astrothelium (Trypetheliales, Ascomycota) from China.

The lichenized fungal genus Astrothelium is an important element of crustose lichen communities in tropical to subtropical forests. Morphological and molecular phylogenetic approaches to investigate species diversity of Astrothelium (Trypetheliaceae) from Southern China were carried out in this study. Bayesian and maximum-likelihood (ML) analyses were generated based on the combined data set of internal transcribed spacer (ITS), partial regions of the nuclear ribosomal large subunit (LSU), and the largest subunit of RNA polymerase II gene sequences (RPB1). The morphological comparison with the known Astrothelium taxa and molecular phylogeny support five new species: Astrothelium jiangxiense sp. nov., A. luminothallinum sp. nov., A. pseudocrassum sp. nov., A. subeustominspersum sp. nov., and A. subrufescens sp. nov. All these species are described and illustrated in detail.