A Cardiolipin from Muribaculum intestinale Induces Antigen-Specific Cytokine Responses.

An systematic phenotypic screen of the mouse gut microbiome for metabolites with an immunomodulatory effect identified Muribaculum intestinale as one of only two members with an oversized effect on T-cell populations. Here we report the identification and characterization of a lipid, MiCL-1, as the responsible metabolite. MiCL-1 is an 18:1-16:0 cardiolipin, whose close relatives are found on concave lipid surfaces of both mammals and bacteria. MiCL-1 was synthesized to confirm the structural analysis and functionally characterized in cell-based assays. It has a highly restrictive structure-activity profile, as its chain-switched analog fails to induce responses in any of our assays. MiCL-1 robustly induces the production of pro-inflammatory cytokines like TNF-α, IL-6, and IL-23, but has no detectable effect on the anti-inflammatory cytokine IL-10. As is the case with other recently discovered immunomodulatory lipids, MiCL-1 requires functional TLR2 and TLR1 but not TLR6 in cell-based assays.

Revisiting Coley's Toxins: Immunogenic Cardiolipins from Streptococcus pyogenes.

Coley's toxins, an early and enigmatic form of cancer (immuno)therapy, were based on preparations of Streptococcus pyogenes. As part of a program to explore bacterial metabolites with immunomodulatory potential, S. pyogenes metabolites were assayed in a cell-based immune assay, and a single membrane lipid, 18:1/18:0/18:1/18:0 cardiolipin, was identified. Its activity was profiled in additional cellular assays, which showed it to be an agonist of a TLR2-TLR1 signaling pathway with a 6 µM EC50 and robust TNF-α induction. A synthetic analog with switched acyl chains had no measurable activity in immune assays. The identification of a single immunogenic cardiolipin with a restricted structure-activity profile has implications for immune regulation, cancer immunotherapy, and poststreptococcal autoimmune diseases.

New Anti-Inflammatory ß-Resorcylic Acid Lactones Derived from an Endophytic Fungus, Colletotrichum sp.

The endophytic fungus Colletotrichum gloeosprioides JS0419, isolated from the leaves of the halophyte Suaeda japonica, produced four new ß-resorcylic acid derivatives, colletogloeopyrones A and B (1 and 2) and colletogloeolactones A and B (3 and 4), and seven known ß-resorcylic acid lactones (RALs). The structures of these compounds were elucidated via analysis of the high-resolution mass spectrometry and nuclear magnetic resonance data. Compounds 1 and 2 showed a dihydrobenzopyranone ring with a linear C9 side chain, which is rarely observed in RALs. All isolated compounds were evaluated for their anti-inflammatory activities. Colletogloeopyrone A (1), monocillin II (5), and monocillin II glycoside (6) were effective in reducing nitric oxide production without cytotoxicity. They also inhibited the secretion of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), as demonstrated by the expression of mRNA corresponding to IL-6 and TNF-α. Mechanistically, compounds 5 and 6 significantly inhibited the protein expression of nuclear factor-κB, IκBα, IKKα/ß, inducible nitric oxide synthase, and cyclooxygenase (COX)-2, whereas compound 1 only inhibited COX-2 expression. This study indicated that RAL-type compounds 1, 5, and 6 demonstrated potential anti-inflammatory activity by inhibiting the synthesis of pro-inflammatory cytokines.

Rare ß-Resorcylic Acid Derivatives from a Halophyte-Associated Fungus Colletotrichum gloeosporioides JS0419 and Their Antifungal Activities.

Six new ß-resorcylic acid derivatives (1-5 and 7) were isolated from a halophyte-associated fungus, Colletotrichum gloeosporioides JS0419, together with four previously reported ß-resorcylic acid lactones (RALs). The relative and absolute stereochemistry of 1 was completely established by a combination of spectroscopic data and chemical reactions. The structures of the isolated compounds were elucidated by analysis of HRMS and NMR data. Notably, compounds 1-3 had a ß-resorcylic acid harboring a long unesterified aliphatic side chain, whereas the long aliphatic chains were esterified to form macrolactones in 4-9. Among the isolated compounds, monocillin I and radicicol showed potent antifungal activities against Cryptococcus neoformans, comparable to clinically available antifungal agents and radicicol showed weak antifungal activity against Candida albicans. These findings provide insight into the chemical diversity of fungal RAL-type compounds and their pharmacological potential.

Adiponectin-Secretion-Promoting Cyclic Peptide-Polyketide Hybrids from a Halophyte-Associated Fungus, Colletotrichum gloeosporioides JS0417.

Three new cyclic peptide-polyketide hybrids (1-3) and two new chaetiacandin-type polyketides (4 and 5) along with nine known compounds were isolated from cultures of a halophyte-associated fungus, Colletotrichum gloeosporioides JS0417. Spectroscopic analysis revealed that 1-3 were cyclic depsipeptides where 3,5,11-trihydroxy-2,6-dimethyldodecanoic acid was linked to two amino acids through amide and ester bonds to form a 12-membered ring. Relative and absolute configurations for the peptides were determined with spectroscopic analysis and chemical reactions. The cyclic depsipeptides 2 and 6 were determined to act as strong adiponectin-secretion-promoting modulators with potential to treat metabolic diseases associated with hypoadiponectinemia. Notably, a known compound, tryptophol, significantly inhibited PGE2 synthesis and also promoted adiponectin secretion, exhibiting a similar biological activity profile to aspirin, but with greater potency. The presence of an isoleucine moiety and non-glycosylation may be important for biological activity of the cyclic peptide-polyketide hybrids, and non-methoxylation of the side chain may influence activity of the indole derivatives.

Lyme Disease, Borrelia burgdorferi, and Lipid Immunogens.

The human immune system detects potentially pathogenic microbes with receptors that respond to microbial metabolites. While the overall immune signaling pathway is known in considerable detail, the initial molecular signals, the microbially produced immunogens, for important diseases like Lyme disease (LD) are often not well-defined. The immunogens for LD are produced by the spirochete Borrelia burgdorferi, and a galactoglycerolipid (1) has been identified as a key trigger for the inflammatory immune response that characterizes LD. This report corrects the original structural assignment of 1 to 3, a change of an α-galactopyranose to an α-galactofuranose headgroup. The seemingly small change has important implications for the diagnosis, prevention, and treatment of LD.

Azaphilones from an Endophytic Penicillium sp. Prevent Neuronal Cell Death via Inhibition of MAPKs and Reduction of Bax/Bcl-2 Ratio.

Fourteen azaphilone-type polyketides (1-14), including nine new ones (1-6 and 8-10), were isolated from cultures of Vitex rotundifolia-associated Penicillium sp. JVF17, and their structures were determined by spectroscopic analysis together with computational methods and chemical reactions. Neuroprotective effects of the isolated compounds were evaluated against glutamate-induced neurotoxicity. Treatment with compounds 3, 6, 7, and 11-14 increased cell viabilities of hippocampal neuronal cells damaged by glutamate, with compound 12 being the most potent. Compound 12 markedly decreased intracellular Ca2+ and nuclear condensation levels. Mechanistically, molecular markers of apoptosis induced by treatment with glutamate, i.e., phosphorylation of MAPKs and elevated Bax/Bcl-2 expression ratio, were significantly lowered by compound 12. The azaphilones with an isoquinoline core structure were more active than those with pyranoquinones, but N-substitution decreased the activity. This study, including the structure-activity relationship, indicates that the azaphilone scaffold is a promising lead toward the development of novel neuroprotective agents.

Colletotrichalactones A-Ca, unusual 5/6/10-fused tricyclic polyketides produced by an endophytic fungus, Colletotrichum sp. JS-0361.

Three unusual polyketides with a 5/6/10-fused ring system, named colletotrichalactones A-Ca (1-3a), were isolated from cultures of the endophytic fungus, Colletotrichum sp. JS-0361, which was isolated from a leaf of Morus alba. Their structures, including their absolute stereochemistries, were completely established using extensive spectroscopic methods together with a chemical reaction utilizing competing enantioselective acylation coupled with LC/MS. Compounds possessing this ring skeleton were previously reported in three studies. Our rigorous chemical investigation revealed the complete configuration of this skeleton, which agreed with the results for glabramycin B with this ring skeleton established by computational chemistry and enantioselective synthesis in previous reports. 1 and 2 had unstable aldehyde groups that were easily converted to acetal groups in the presence of solvents. Meanwhile, compound 3a, with terminal acetal functionality, was deduced to be an artefact originating from compound 3 with a terminal aldehyde group. Compounds 1 and 3a displayed moderate-to-potent cytotoxic activities against MCF7 cells with IC50s of 35.06 and 25.20 µM, respectively.

Beta resorcylic acid lactones (RALs) from fungi: chemistry, biology, and biosynthesis.

ß-Resorcylic acid lactones (RALs) are one of the major polyketides produced by fungi, and some of them have a diverse array of biological activities. Most RALs feature a 14-membered macrocyclic ring fused to ß-resorcylic acid (2,4-dihydroxybenzoic acid). In this review, more than 100 RAL-type of compounds are structurally classified into three groups; 14-membered RALs with 17R configuration, 14-membered RALs with 17S configuration, and benzopyranones/benzofuranones, and they are reviewed comprehensively in terms of chemistry, biological activities, and biosynthetic pathways.

Unusual bridged angucyclinones and potent anticancer compounds from Streptomyces bulli GJA1.

Two new unique angucyclinones (1 and 2) with unprecedented ether bridges connecting carbons 5 and 7 were isolated from the cultures of Streptomyces bulli GJA1, an endophyte of Gardenia jasminoides, together with two known ones (3 and 4). The MS2-based molecular networking system facilitated the isolation of compounds with target functionalities. The stereochemistry of 1 was completely established by ROESY and ECD experiments. Compound 3 showed antivirulence activities by inhibiting the peptide toxin (PSMs) production and the biofilm formation of MRSA. Compounds 3 and 4 showed very potent anti-proliferative effects against OV1 and ES2 ovarian cancer cells.

Secondary Metabolites of The Endophytic Fungus Alternaria alternata JS0515 Isolated from Vitex rotundifolia and Their Effects on Pyruvate Dehydrogenase Activity.

The fungal strain Alternaria alternata JS0515 was isolated from Vitex rotundifolia (beach vitex). Twelve secondary metabolites, including one new altenusin derivative (1), were isolated. The isolated metabolites included seven known altenusin derivatives (2-8), two isochromanones (9, 10), one perylenequinone (11), and one benzocycloalkanone (12). Their structures were determined via 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and computational electronic circular dichroism (ECD) analysis. Compounds 3 and 11 increased pyruvate dehydrogenase (PDH) activity in AD-293 human embryonic kidney cells and significantly inhibited PDH phosphorylation. The IC50 values of 3 and 11 were 32.58 and 27.82 µM, respectively.

Neuroprotective Glycosylated Cyclic Lipodepsipeptides, Colletotrichamides A-E, from a Halophyte-Associated Fungus, Colletotrichum gloeosporioides JS419.

Glutamate neurotoxicity has been implicated in neuronal death in both acute CNS injury and in chronic neurodegenerative diseases. Five unique cyclic depsipeptides with neuroprotective activity, colletotrichamides A-E (1-5), were isolated from cultures of a halophyte Suaeda japonica-associated fungus, Colletotrichum gloeosporioides JS419. Spectroscopic analysis revealed that they were glycosylated cyclic lipodepsipeptides. Their relative configurations were determined by ROESY and J-based configuration analysis, and absolute configurations were established by chemical reactions including modified Mosher's method, advanced Marfey's method, and sugar derivatization. This is the first report of a glycosylated dimethyl-trioxygenated dodecanoyl moiety, and the relative as well as absolute stereochemistry was elucidated herein for the first time. Colletotrichamide C exhibited strong neuroprotective activity against glutamate in hippocampal HT22 cells.

Neuroprotective Secondary Metabolite Produced by an Endophytic Fungus, Neosartorya fischeri JS0553, Isolated from Glehnia littoralis.

Roots of Glehnia littoralis have been used to heal stroke as a traditional medicine. Even though many studies on this plant have been conducted, the secondary metabolites produced by its endophytes and their bioactivities have not been investigated thus far. Therefore, a new meroditerpenoid named sartorypyrone E (1) and eight known compounds (2-9) were isolated from extracts of cultured Neosartorya fischeri JS0553, an endophyte of G. littoralis. The isolated metabolites were identified using spectroscopic methods and chemical reaction, based on a comparison to literature data. Relative and absolute stereochemistries of compound 1 were also elucidated. To identify the protective effects of isolated compounds (1-9) in HT22 cells against glutamate-induced cytotoxicity, we assessed inhibition of cell death, intracellular reactive oxygen species (ROS) accumulation, and calcium ion (Ca2+) influx. Among the isolates, compound 8, identified as fischerin, showed significant neuroprotective activity on glutamate-mediated HT22 cell death through inhibition of ROS, Ca2+ influx, and phosphorylation of mitogen-activated protein kinase, including c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38. The results suggested that the metabolites produced by the endophyte N. fischeri JS0553 might be related to the neuroprotective activity of its host plant, G. littoralis.

Simultaneous determination of the bioactive compounds from Sparassis crispa (Wulf.) by HPLC-DAD and their inhibitory effects on LPS-stimulated cytokine production in bone marrow-derived dendritic cell.

Sparassis crispa (Wulf.) belonging to the family of Sparassidaceae, has been widely used as an edible mushroom due to its unique flavor and functions to improve health. In this study, the compounds isolated from the extract of this mushroom were simultaneously quantified by the developed HPLC-DAD method and evaluated for the inhibitory activities on the production of the LPS-stimulated cytokines (IL-12p40, IL-6, and TNF-α) in bone marrow-derived dendritic cells (BMDCs). The contents of this compounds were 0.1928 ± 0.0118, 4.4137 ± 0.0240, 0.5237 ± 0.0005, 2.7303 ± 0.0206 mg/g for sparoside A (1), methyl 2,4-dihydroxy-3-methoxy-6-methylbenzoate (2), sparalide A (3), and 5'-deoxy-5'-methylthioadenosine (4), respectively, which demonstrated that they are the major constituents of this mushroom. Thus, our results were found that the sparoside A (1), 5-hydroxy-7-methoxyphthalide (6), 5-methoxy-7-hydroxyphthalide (7), nicotinamide (10), and adenosine (11) inhibit LPS-stimualted cytokine production, compound 6 is the most potent inhibitory activities on the production of IL-12p40, IL-6, and TNF-α with IC50 values of 0.19, 0.18, and 0.91 µM, respectively.

Neuroprotective Compound from an Endophytic Fungus, Colletotrichum sp. JS-0367.

Colletotrichum sp. JS-0367 was isolated from Morus alba (mulberry), identified, and cultured on a large scale for chemical investigation. One new anthraquinone (1) and three known anthraquinones (2-4) were isolated and identified using spectroscopic methods including 1D/2D-NMR and HRESIMS. Although the neuroprotective effects of some anthraquinones have been reported, the biological activities of the four anthraquinones isolated in this study have not been reported. Therefore, the neuroprotective effects of these compounds were determined against murine hippocampal HT22 cell death induced by glutamate. Compound 4, evariquinone, showed strong protective effects against HT22 cell death induced by glutamate by the inhibition of intracellular ROS accumulation and Ca2+ influx triggered by glutamate. Immunoblot analysis revealed that compound 4 reduced the phosphorylation of MAPKs (JNK, ERK1/2, and p38) induced by glutamate. Furthermore, compound 4 strongly attenuated glutamate-mediated apoptotic cell death.

Anti-influenza effect of the major flavonoids from Salvia plebeia R.Br. via inhibition of influenza H1N1 virus neuraminidase.

To determine the compounds responsible for its anti-influenza activities, we isolated the three flavonoids, 6-hydroxyluteolin 7-O-ß-d-glucoside (1), nepitrin (2), homoplantaginin (3) from the MeOH extract of Salvia plebeia R.Br. and identified them by comparing the spectroscopic data with that reported in the literature. The contents of the three flavonoids in the whole extract were 108.74 ± 0.95, 46.26 ± 2.19, and 69.35 ± 1.22 mg/g for 6-hydroxyluteolin 7-O-ß-d-glucoside, nepitrin, and homoplantaginin, respectively, which demonstrates that they are the major constituents of this plant. The three flavonoids were evaluated for their inhibitory activities against influenza virus H1N1 A/PR/9/34 neuraminidase and H1N1-induced cytopathic effect (CPE) on Madin-Darby canine kidney (MDCK) cells. Our results demonstrated the following arrangement for their anti-influenza activities: nepitrin (2) > 6-hydroxyluteolin 7-O-ß-d-glucoside (1) > homoplantaginin (3). The potent inhibitory activities of these flavonoids against influenza suggested their potential to be developed as novel anti-influenza drugs in the future.

New Aromatic Compounds from the Fruiting Body of Sparassis crispa (Wulf.) and Their Inhibitory Activities on Proprotein Convertase Subtilisin/Kexin Type 9 mRNA Expression.

Successive chromatography of EtOAc-soluble extracts of the fruiting body of Sparassis crispa (Wulf.) resulted in isolation of four new aromatic compounds, sparoside A (1) and sparalides A-C (3-5), two new naturally occurring compounds, 2 and 6, and eight known compounds, 7-14. The chemical structures were determined by interpretation of nuclear magnetic resonance and mass spectrometry spectroscopic data. Extract, solvent-soluble fractions of the extract, and all of the pure compounds isolated from the fractions were subjected to the mRNA expression assay for proprotein convertase subtilisin/kexin type 9 (PCSK9). Among them, sparoside A (1), hanabiratakelide A (8), adenosine (11), and 5α,6α-epoxy-(22E,24R)-ergosta-8(14),22-diene-3ß,7ß-diol (14) exhibited potent inhibitory activities on PCSK9 mRNA expression, with IC50 values of 20.07, 7.18, 18.46, and 8.23 µM, respectively (berberine, positive control, IC50 = 8.04 µM), suggesting that compounds 1, 8, 11, and 14 are suitable for use in supplements to the statins for hyperlipidemia treatments.

Carrier frequency of Wilson's disease in the Korean population: a DNA-based approach.

Wilson's disease (WD) is an autosomal recessive disorder caused by ATP7B gene mutation. The frequency of WD is about 1 in 30 000 worldwide. In the present study, we screened 14 835 dried blood spots (DBSs) from asymptomatic Korean neonates and retrospectively reviewed massively parallel sequencing of 1090 control individuals to estimate carrier frequency. TaqMan real-time PCR assays were conducted to detect six mutations that account for 58.3% of mutations in Korean WD patients: c.2333G>T (p.Arg778Leu), c.2621C>T (p.Ala874Val), c.3086C>T (p.Thr1029Ile), c.3247C>T (p.Leu1083Phe), c.3556G>A (p.Gly1186Ser) and c.3809A>G (p.Asn1270Ser). We also retrospectively reviewed data from 1090 individuals with various indications other than WD for whom whole-exome or panel sequencing data were available. Mutant allele frequency based on the six most common mutations was 0.0067 among the total of 14 835 DBSs screened. Given that these six mutations account for 58.3% of mutations in Korean WD patients, the corrected mutant allele frequency is 0.0115 (95% confidence interval (CI): 0.0103-0.0128). Corresponding incidence (q2) and carrier frequency (2pq) were estimated to be 1:7561 and 1:44, respectively. In retrospective data analysis of 1090 control individuals, allele frequency of pathogenic or likely pathogenic variants was 0.0096 (95% CI: 0.0063-0.0146). Corresponding carrier frequency was estimated to be 1:53. Estimated allele and carrier frequencies based on DNA screening were relatively higher than those reported previously based on clinical ascertainment.

Bioactive secondary metabolites produced by an endophytic fungus Gaeumannomyces sp. JS0464 from a maritime halophyte Phragmites communis.

Endophytes, important plant-associated mycobionts, have attracted a great deal of attention because of their bioactive secondary metabolites. Even though halophytes have been reported to overcome salt stress via associations with their endophytes, few studies have investigated the metabolites produced by the endophytes from halophytes. In this study, a dark septate endophytic fungal strain (JS0464), identified as Gaeumannomyces sp. by ITS sequencing, was isolated from the rhizome of a halophyte, Phragmites communis, in Suncheon bay, South Korea. This strain was cultured on a large scale and extracted with ethyl acetate. Chemical investigations of extracts of JS0464 led to the isolation of two glycosylated dialkylresorcinol derivatives (1-2), an anthraquinone derivative (3) and eight known compounds (4-11), which were identified by spectroscopic analyses incorporating one-dimensional/2D NMR and MS. Nine compounds showed significant nitric oxide reduction activity in lipopolysaccharide-stimulated microglia BV-2 cells, seven of which did not impair cell viability. The results suggest that endophytes from the halophytes could be potential resources for bioactive natural products.

Antiviral activities of compounds from aerial parts of Salvia plebeia R. Br.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia plebeia R. Br. is an edible plant widely spread in many countries. It has been used as a traditional medicine to treat common cold, flu, cough, hepatitis, hemorrhoids, etc. The purpose of the study is to explicate antiviral compounds responsible for its traditional use for the common cold or flu. MATERIALS AND METHODS: The methanolic extract of the aerial parts of S. plebeia was extracted with CHCl3, EtOAc, and n-BuOH, successively. The EtOAc and CHCl3 fractions were subjected to a successive of chromatographic method, which led to the isolation of fourteen compounds. Inhibition activities of the isolated compounds were evaluated against influenza A (H1N1) neuraminidase. RESULTS: Chemical investigation of the methanolic extracts of S. plebeia resulted in the isolation of two novel benzoylated monoterpene glycosides, named as plebeiosides A (1) and B (2), together with twelve known compounds including four flavonoids (4-5, 7, 10), two sesquiterpenoids (8, 12), four phenolics (9-10, 13-14), a steroid (6), and a triterpenoid (3). Their chemical structures were elucidated based on spectroscopic data and absolute stereochemistries of 1 and 2 were determined by comparison of optical rotations of their hydrolysates with literature values. Compounds 5, 7, 9, and 11 exhibited potent enzymatic inhibition against H1N1 neuraminidase (IC50 values ranging from 11.18±1.73 to 19.83±2.28µM). Furthermore, two flavonoids (5 and 7) and one rosmarinic acid methyl ester (9) reduced cytopathic effects of the H1N1 virus during replication. CONCLUSIONS: The antiviral activities of the flavonoids and phenolics isolated from the extracts of S. plebeia supported the traditional application of this medicine on common cold or flu. In this study, benzoylated monoterpene glycosides were first found to exist in this species. Moreover, the present study suggested potential of three compounds (5, 7, and 9) to be new lead structures for the development of new neuraminidase inhibitors in the future.