Expanded diversity of Asgard archaea and their relationships with eukaryotes.

Asgard is a recently discovered superphylum of archaea that appears to include the closest archaeal relatives of eukaryotes1-5. Debate continues as to whether the archaeal ancestor of eukaryotes belongs within the Asgard superphylum or whether this ancestor is a sister group to all other archaea (that is, a two-domain versus a three-domain tree of life)6-8. Here we present a comparative analysis of 162 complete or nearly complete genomes of Asgard archaea, including 75 metagenome-assembled genomes that-to our knowledge-have not previously been reported. Our results substantially expand the phylogenetic diversity of Asgard and lead us to propose six additional phyla that include a deep branch that we have provisionally named Wukongarchaeota. Our phylogenomic analysis does not resolve unequivocally the evolutionary relationship between eukaryotes and Asgard archaea, but instead-depending on the choice of species and conserved genes used to build the phylogeny-supports either the origin of eukaryotes from within Asgard (as a sister group to the expanded Heimdallarchaeota-Wukongarchaeota branch) or a deeper branch for the eukaryote ancestor within archaea. Our comprehensive protein domain analysis using the 162 Asgard genomes results in a major expansion of the set of eukaryotic signature proteins. The Asgard eukaryotic signature proteins show variable phyletic distributions and domain architectures, which is suggestive of dynamic evolution through horizontal gene transfer, gene loss, gene duplication and domain shuffling. The phylogenomics of the Asgard archaea points to the accumulation of the components of the mobile archaeal 'eukaryome' in the archaeal ancestor of eukaryotes (within or outside Asgard) through extensive horizontal gene transfer.

Ergone Derivatives from the Deep-Sea-Derived Fungus Aspergillus terreus YPGA10 and 25,28-Dihydroxyergone-Induced Apoptosis in Human Colon Cancer SW620 Cells.

Ten new ergone derivatives (1-10) and five known analogues (11-15) were isolated from the deep-sea-derived fungus Aspergillus terreus YPGA10. The structures including the absolute configurations were established by detailed analysis of the NMR spectroscopic data, HRESIMS, ECD calculation, and coupling constant calculation. All the structures are characterized by a highly conjugated 25-hydroxyergosta-4,6,8(14),22-tetraen-3-one nucleus. Structurally, compound 2 bearing a 15-carbonyl group and compounds 5-7 possessing a 15ß-OH/OCH3 group are rarely encountered in ergone derivatives. Bioassay results showed that compounds 1 and 11 demonstrated cytotoxic effects on human colon cancer SW620 cells with IC50 values of 8.4 and 3.1 µM, respectively. Notably, both compounds exhibited negligible cytotoxicity on the human normal lung epithelial cell BEAS-2B. Compound 11 was selected for preliminary mechanistic study and was found to inhibit cell proliferation and induce apoptosis in human colon cancer SW620 cells. In addition, compound 1 displayed cytotoxic activity against five human leukemia cell lines with IC50 values ranging from 5.7 to 8.9 µM. Our study demonstrated that compound 11 may serve as a potential candidate for the development of anticolorectal cancer agents.

Phenylspirodrimane with Moderate Reversal Effect of Multidrug Resistance Isolated from the Deep-Sea Fungus Stachybotrys sp. 3A00409.

Two new phenylspirodrimanes, stachybotrins K and L (1 and 2), together with eight known analogues (3-10), were isolated from deep-sea-derived Stachybotrys sp. MCCC 3A00409. Their structures were determined by extensive NMR data and mass spectroscopic analysis. Absolute configurations of new compounds were determined through a comparison of their circular dichroism (CD) spectra with other reported compounds. The possible reversal effects of all compounds were assayed in the resistant cancer cell lines. Stachybotrysin B (8) can reverse multidrug resistance (MDR) in ABCB1-overexpression cells (KBv200, Hela/VCR) at the non-cytotoxic concentration. Doxorubicin accumulation assay and molecular-docking analysis reveal that the mechanism of its reversal MDR effect may be related to the increase in the intracellular concentration of substrate anticancer drugs.

An Ancient Respiratory System in the Widespread Sedimentary Archaea Thermoprofundales.

Thermoprofundales, formerly Marine Benthic Group D (MBG-D), is a ubiquitous archaeal lineage found in sedimentary environments worldwide. However, its taxonomic classification, metabolic pathways, and evolutionary history are largely unexplored because of its uncultivability and limited number of sequenced genomes. In this study, phylogenomic analysis and average amino acid identity values of a collection of 146 Thermoprofundales genomes revealed five Thermoprofundales subgroups (A-E) with distinct habitat preferences. Most of the microorganisms from Subgroups B and D were thermophiles inhabiting hydrothermal vents and hot spring sediments, whereas those from Subgroup E were adapted to surface environments where sunlight is available. H2 production may be featured in Thermoprofundales as evidenced by a gene cluster encoding the ancient membrane-bound hydrogenase (MBH) complex. Interestingly, a unique structure separating the MBH gene cluster into two modular units was observed exclusively in the genomes of Subgroup E, which included a peripheral arm encoding the [NiFe] hydrogenase domain and a membrane arm encoding the Na+/H+ antiporter domain. These two modular structures were confirmed to function independently by detecting the H2-evolving activity in vitro and salt tolerance to 0.2 M NaCl in vivo, respectively. The peripheral arm of Subgroup E resembles the proposed common ancestral respiratory complex of modern respiratory systems, which plays a key role in the early evolution of life. In addition, molecular dating analysis revealed that Thermoprofundales is an early emerging archaeal lineage among the extant MBH-containing microorganisms, indicating new insights into the evolution of this ubiquitous archaea lineage.

Impact of environmental factors on diversity of fungi in sediments from the Shenzhen River Estuary.

In this study, to explore the relationship between environmental factors and fungal diversity in the Shenzhen River ecosystem, multiple methods including chemical analysis, culture isolation, qPCR analysis of fungal ITS region and ITS-based Illumina next-generation-sequencing were integrated. A total of 115 isolates were finally isolated and could be classified into 23 genera. Top three abundant genera isolated were Meyerozyma (18 strains), Aspergillus (17 strains) and Penicillium (14 strains). Based on the Illumina sequencing approach, 829 OTUs were affiliated to seven phyla, 17 known classes, and 162 genera, indicating the Shenzhen estuary sediments are rich in fungal diversity. The major fungal genera were Meyerozyma, Trichoderma and Talaromyces. Environmental factors showed a gradient change in Shenzhen estuary, and fungal abundance was only significantly correlated with NH4+. Shannon index was significantly correlated with pH and IC (P < 0.05). Principal coordinate analysis based on OTU level grouped into three clusters among sampling sites along with the IC and pH gradient. Functional guilds analysis suggests most of the fungi in this studying area were almost all saprotrophs, suggesting a large number of saprophytic fungi may play a significant role in the organic matter decomposition and nutrient cycling process. In summary, this study will deepen our understanding of fungi community in Shenzhen River ecosystem and their distribution and potential function shaped by environmental factors.

Ion Mobility-Derived Collision Cross-Sections Add Extra Capability in Distinguishing Isomers and Compounds with Similar Retention Times: The Case of Aphidicolanes.

The hyphenation of ion mobility spectrometry with high-resolution mass spectrometry has been widely used in the characterization of various metabolites. Nevertheless, such a powerful tool remains largely unexplored in natural products research, possibly mainly due to the lack of available compounds. To evaluate the ability of collision cross-sections (CCSs) in characterizing compounds, especially isomeric natural products, here we measured and compared the traveling-wave IMS-derived nitrogen CCS values for 75 marine-derived aphidicolanes. We established a CCS database for these compounds which contained 227 CCS values of different adducts. When comparing the CCS differences, 36 of 57 pairs (over 60%) of chromatographically neighboring compounds showed a ΔCCS over 2%. What is more, 64 of 104 isomeric pairs (over 60%) of aphidicolanes can be distinguished by their CCS values, and 13 of 18 pairs (over 70%) of chromatographically indistinguishable isomers can be differentiated from the mobility dimension. Our results strongly supported CCS as an important parameter with good orthogonality and complementarity with retention time. CCS is expected to play an important role in distinguishing complex and diverse marine natural products.

Asperochratides A-J, Ten new polyketides from the deep-sea-derived Aspergillus ochraceus.

Ten new C9 polyketides (asperochratides A-J, 1-10) and 14 known miscellaneous compounds (11-24) were isolated from the deep-sea-derived fungus Aspergillus ochraceus. Structures of the new compounds were elucidated by extensive spectroscopic analyses, modified Mosher's method, Mo2(OAc)4 induced circular dichroism (ICD) experiments, and ECD calculations. Structurally, compounds 1-11 and 16-18 share the same polyketide origin of the skeleton and belong to aspyrone co-metabolites. All isolates were tested for cytotoxic, anti-food allergic, anti-H1N1 virus, anti-microbe, and anti-inflammatory activities in vitro. Results showed that compounds 5-8 and 13-17 exerted significant cytotoxic effects on BV-2 cell line, and compound 16 showed the potential of anti-inflammatory activities.

Terpenoids from the Deep-Sea-Derived Fungus Penicillium thomii YPGA3 and Their Bioactivities.

A chemical study of the ethyl acetate (EtOAc) extract from the deep-sea-derived fungus Penicillium thomii YPGA3 led to the isolation of a new austalide meroterpenoid (1) and seven known analogues (28), two new labdane-type diterpenoids (9 and 10) and a known derivative (11). The structures of new compounds 1, 9, and 10 were determined by comprehensive analyses via nuclear magnetic resonance (NMR) and mass spectroscopy (MS) data. The absolute configurations of 1, 9, and 10 were determined by comparisons of experimental electronic circular dichroism (ECD) with the calculated ECD spectra. Compound 1 represented the third example of austalides bearing a hydroxyl group at C-5 instead of the conserved methoxy in other known analogues. To our knowledge, diterpenoids belonging to the labdane-type were discovered from species of Penicillium for the first time. Compound 1 showed cytotoxicity toward MDA-MB-468 cells with an IC50 value of 38.9 M. Compounds 2 and 11 exhibited inhibition against α-glucosidase with IC50 values of 910 and 525 M, respectively, being more active than the positive control acarbose (1.33 mM).

Periodic density as an endpoint of customized plankton community responses to petroleum hydrocarbons: A level of toxic effect should be matched with a suitable time scale.

As an endpoint of community response to contaminants, average periodic density of populations (APDP) has been introduced to model species interactions in a community with 4 planktonic species. An ecological model for the community was developed by means of interspecific relationship including competition and predation to calculate the APDP. As a case study, we reported here the ecotoxicological effects of petroleum hydrocarbons (PHC) collected from Bohai oil field on densities of two algae, Platymonas subcordiformis and Isochrysis galbana, a rotifer, Brachionus plicatilis, and of a cladocera, Penilia avirostris, in single species and a microcosm experiment. Time scales expressing toxic effect increased with increasing levels of toxic effect from molecule to community. Remarkable periodic changes in densities were found during the tests in microcosm experiment, revealing a strong species reaction. The minimum time scale characterizing toxic effect at a community level should be the common cycle of population densities of the microcosm. In addition, the cycles of plankton densities shortened in general with increasing PHC, showing an evident toxic effect on the microcosm. Using APDP as the endpoint, a threshold concentration for the modeled microcosm was calculated to be 0.404 mg-PHC L-1. The APDP was found to be more sensitive and reliable than the standing crops of populations as the endpoint. This indicated that the APDP, an endpoint at the community level, could be quantitatively related to the endpoints at the population level, and led to the quantitative concentration-toxic effect relationship at the community level.

Protection against Pseudomonas plecoglossicida in Epinephelus coioides immunized with a cspA1-knock-down live attenuated vaccine.

Pseudomonas plecoglossicida is well-known as the cause of viscera granulomas disease in fish. In this study, a cspA1 knock-down strain was constructed and tested in Epinephelus coioides to observe the changes in virulence and evaluate its potential as an attenuated live vaccine. The results showed that the cspA1 knock-down strain caused a significant reduction in the ability of biofilm formation, motility, adhesion and virulence. E. coioides vaccinated with cspA1 knock-down strain were more tolerant of the infection by wild-type P. plecoglossicida. The relative percent survival value of E. coioides vaccinated with cspA1 knock-down strain reached 80% after challenging with wild-type P. plecoglossicida. In the meanwhile, the expression level of genes associated with immunity, including IL-1ß, IgM, MHC-I and MHC-II, was up-regulated after vaccination, indicating that the cspA1 knock-down strain can induce effective and durable immune response in E. coioides and it may be an effective attenuated live vaccine candidate for the prevention of infections by P. plecoglossicida.

Phenotypic characterization, virulence, and immunogenicity of Pseudomonas plecoglossicida rpoE knock-down strain.

Pseudomonas plecoglossicida, a temperature dependent bacterial pathogen in fish, expresses rpoE gene that is sensitive to temperature and probably critical for pathogen virulence and disease development. In this study, the rpoE silence strain rpoE-RNAi-1 was constructed by gene knock-down. The rpoE-RNAi-1 displayed significant changes in biofilm formation, swarming motility, adhesion and virulence. Meanwhile, vaccination of grouper with rpoE-RNAi-1 led to a relative percent survival (RPS) value of 85% after challenged with the wild-type P. plecoglossicida. qRT-PCR assays showed that vaccination with rpoE-RNAi-1 enhanced the expression of immune-related genes, including MHC-I, MHC-II, IgM, and IL-1ß, indicating that it was able to induce humoral and cell-mediated immune response in grouper. These results validated the possibility of rpoE as a potential target for constructing P. plecoglossicida live attenuated vaccine.

Sarocladione, a unique 5,10:8,9-diseco-steroid from the deep-sea-derived fungus Sarocladium kiliense.

A novel ergostane, sarocladione (1), was isolated from the deep-sea-derived fungus Sarocladium kiliense, along with 20 known compounds. The structure of 1 was determined mainly by a detailed analysis of its experimental and calculated NMR spectroscopic data. It is worth noting that 1 was the first steroid bearing a 5,10:8,9-diseco moiety. All 21 compounds were tested for in vitro antitumor activities against five cancer cell lines. ß-Sitostenone (7) and 4,6-dihydroxyeudesmane (20) showed significant effects on HeLa-S3 cells with the IC50 values of 9.2 µM and 9.3 µM, respectively.

Aphidicolin Chemistry of the Deep-Sea-Derived Fungus Botryotinia fuckeliana MCCC 3A00494.

Aphidicolin, a potent DNA polymerase α inhibitor, has been explored in clinical trials for the treatment of cancer. So far, about 300 modified aphidicolins have been discovered. However, none have shown a stronger effect. Herein, we report 71 new (aphidicolins A1-A71, 1-71) and eight known (72-79) aphidicolin congeners from Botryotinia fuckeliana MCCC 3A00494, a fungus isolated from the western Pacific Ocean (-5572 m). The structures of 1-71 were determined through extensive spectroscopic analysis, X-ray crystallography, chemical derivatization, modified Mosher's method, and the ECD exciton chirality method. Compounds 54-57 and 58-64 are novel 6/6/5/6/5 pentacyclic aphidicolins featuring tetrahydrofuran and dihydrofuran rings, respectively, while compounds 65-71 are rare noraphidicolins. Aphidicolin A8 (8) significantly induced apoptosis in T24 (IC50 = 2.5 µM) and HL-60 (IC50 = 6.1 µM) cancer cells by causing DNA damage. By docking its structure to the human DNA polymerase α binding pocket, 8 was found to form tight intermolecular contacts, elaborating aphidicolin A8 as a potently cytotoxic lead compound.

Three new cyclopiane-type diterpenes from a deep-sea derived fungus Penicillium sp. YPGA11 and their effects against human esophageal carcinoma cells.

Cyclopianes, featuring a highly rigid 6/5/5/5-fused tetracyclic framework, are structurally unique and biologically significant and belong to a rarely reported diterpenoid family. Chemical investigation of an EtOAc extract of a deep-sea-derived Penicillium sp. led to the isolation of three new cyclopiane diterpenes, namely, conidiogenols C-D (1-2) and conidiogenone L (3). The structures were determined by extensive analyses of the spectroscopic data in association with ECD calculations and chemical conversion for configurational assignments. Compound 1 represents the second example of cyclopianes bearing a hydroxyl group at C-13. Compound 2, the third example of conidiogenols, possesses a distinct α-oriented 1-hydroxy group relative to other analogues. The bioassay study demonstrated that compounds 2 and 4-6 exhibited moderate inhibitory effects against five esophageal cancer cell lines with IC50 values ranging from 25 to 55 µM. The cytotoxicities of all compounds toward esophageal cancer cell lines were evaluated for the first time.

Butenolide Derivatives with α-Glucosidase Inhibitions from the Deep-Sea-Derived Fungus Aspergillus terreus YPGA10.

Three new butenolide derivatives, namely aspernolides N-P (1-3), together with six known analogues (4-9), were isolated from the ethyl acetate (EtOAc) extract of the deep sea-derived fungus Aspergillus terreus YPGA10. The structures of compounds 1-3 were determined on the basis of comprehensive analyses of the nuclear magnetic resonance (NMR) and mass spectroscopy (MS) data, and the absolute configurations of 1 and 2 were determined by comparisons of experimental electronic circular dichroism (ECD) with calculated ECD spectra. Compound 1 represents the rare example of Aspergillus-derived butenolide derivatives featured by a monosubstituted benzene ring. Compounds 6-9 exhibited remarkable inhibitory effects against α-glucosidase with IC50 values of 3.87, 1.37, 6.98, and 8.06 µM, respectively, being much more active than the positive control acarbose (190.2 µM).

Graphostromols A-K, Eleven New Chained Polyketides from the Deep-Sea-Derived Graphostroma sp.

Chromatographic separation of the AcOEt extract of the fermented cultures of the deep-sea-derived fungus Graphostroma sp. resulted in the isolation of 11 new chained polyketides, namely graphostromols A-K (1-11). The structures of the new compounds were established by the extensive analyses of their NMR and HR-ESI-MS data. The absolute configuration at C-11 in 1 was determined on the basis of the modified Mosher's method. Compounds 1-5, bearing a 2,2,10,10-tetramethyldodecane skeleton, were discovered for the first time from nature. All isolates were evaluated for their cytotoxicities against five different cancer cell lines (HeLa, Eca-109, Biu-87, Bel-7402, and PANC-1). However, none showed positive effects.

Peniginsengins B⁻E, New Farnesylcyclohexenones from the Deep Sea-Derived Fungus Penicillium sp. YPGA11.

Chemical examination of the EtOAc extract of the deep sea-derived fungus Penicillium sp. YPGA11 resulted in the isolation of four new farnesylcyclohexenones, peniginsengins B⁻E (1⁻4), and a known analog peniginsengin A (5). The structures of compounds 1⁻4 were determined on the basis of comprehensive analyses of the nuclear magnetic resonance (NMR) and mass spectroscopy (MS) data, and the absolute configurations of 1, 2, and 4 were determined by comparisons of experimental electronic circular dichroism (ECD) with calculated ECD spectra. Compounds 1⁻5, characterized by a highly oxygenated 1-methylcyclohexene unit and a (4E,8E)-4,8-dimethyldeca-4,8-dienoic acid side chain, are rarely found in nature. Compounds 2⁻4 exhibited antibacterial activity against Staphylococcus aureus.

Discovery, gene modification, and optimization of fermentation of an enduracidin-producing strain.

Enduracidin significantly inhibits Gram-positive bacteria and had been widely used in many fields. However, as the poor technology for production of enduracidin and its scarcity, identification of novel strategies for production of enduracidin is important. Our group developed two methods to improve the yield of the production of enduracidin. The yield of enduracidin was increased by three- to fivefold. The highest yields of enduracidin A and enduracidin B achieved were 63.7 and 82.13 mg/ml. Thus, our results might provide a new reference method for the industrial production of enduracidin.

Spirograterpene A, a Tetracyclic Spiro-Diterpene with a Fused 5/5/5/5 Ring System from the Deep-Sea-Derived Fungus Penicillium granulatum MCCC 3A00475.

A novel spiro-tetracyclic diterpene, spirograterpene A (1), was isolated from the deep-sea-derived fungus Penicillium granulatum MCCC 3A00475, together with two biosynthetically related cyclopianes, conidiogenone I (2) and conidiogenone C (3). The relative configuration of 1 was elucidated by extensive spectroscopic analyses, and the absolute structure was established by the modified Mosher's method. Compound 1 is the second example of a diterpene featuring a 5/5/5/5 spirocyclic carbon skeleton. It showed modest antiallergic activity.

Cytotoxic and Antibacterial Compounds from the Coral-Derived Fungus Aspergillus tritici SP2-8-1.

Three novel compounds, 4-methyl-candidusin A (1), aspetritone A (2) and aspetritone B (3), were obtained from the culture of a coral-derived fungus Aspergillus tritici SP2-8-1, together with fifteen known compounds (4-18). Their structures, including absolute configurations, were assigned based on NMR, MS, and time-dependent density functional theory (TD-DFT) ECD calculations. Compounds 2 and 5 exhibited better activities against methicillin-resistant strains of S. aureus (MRSA) ATCC 43300 and MRSA CGMCC 1.12409 than the positive control chloramphenicol. Compound 5 displayed stronger anti-MRSA and lower cytotoxic activities than 2, and showed stronger antibacterial activities against strains of Vibrio vulnificus, Vibrio rotiferianus, and Vibrio campbellii than the other compounds. Compounds 2 and 10 exhibited significantly stronger cytotoxic activities against human cancer cell lines HeLa, A549, and Hep G2 than the other compounds. Preliminary structure-activity relationship studies indicated that prenylation of terphenyllin or candidusin and the tetrahydrobenzene moiety in anthraquinone derivatives may influence their bioactivity.