Control of lipolysis by a population of oxytocinergic sympathetic neurons.

Oxytocin (OXT), a nine-amino-acid peptide produced in the hypothalamus and released by the posterior pituitary, has well-known actions in parturition, lactation and social behaviour1, and has become an intriguing therapeutic target for conditions such as autism and schizophrenia2. Exogenous OXT has also been shown to have effects on body weight, lipid levels and glucose homeostasis1,3, suggesting that it may also have therapeutic potential for metabolic disease1,4. It is unclear, however, whether endogenous OXT participates in metabolic homeostasis. Here we show that OXT is a critical regulator of adipose tissue lipolysis in both mice and humans. In addition, OXT serves to facilitate the ability of ß-adrenergic agonists to fully promote lipolysis. Most surprisingly, the relevant source of OXT in these metabolic actions is a previously unidentified subpopulation of tyrosine hydroxylase-positive sympathetic neurons. Our data reveal that OXT from the peripheral nervous system is an endogenous regulator of adipose and systemic metabolism.

Polyene Carboxylic Acids from a Streptomyces sp. Isolated from Tibet Soil.

Six new polyene carboxylic acids named serpentemycins E-J (1-6), together with three known analogs (7-9), were isolated from the fermentation medium of Streptomyces sp. TB060207, which was isolated from arid soil collected from Tibet, China. The structures of the new compounds were elucidated mainly on the basis of HR-ESI-MS and NMR spectroscopic analyses. The inhibitory activities of compounds 1-9 against NO production in LPS-activated RAW264.7 cells were evaluated. Compound 9 has an inhibition rate of 87.09% to 60.53% at concentrations ranging from 5.0 to 40.0 µM.

The unique Akt inhibitor SC66 suppressed AMPK activity and abolished autophagy through the EGFR-p62 pathway.

Akt is usually considered to be a negative regulator of both autophagy and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling. In the present study, we found that SC66, a pyridine-based allosteric Akt inhibitor, suppressed basal and H2 O2 -induced autophagy concurrent with decreased phosphorylation and activity of AMPK. SC66 treatment led to the formation of a high molecular weight (HMW) form of SQSTM1/p62 (p62), which is an autophagic substrate and is essential for selective autophagy. Moreover, we observed that SC66 inhibited the binding of p62 and microtubule-associated protein light chain 3 (LC3). The immunoprecipitation results revealed the interaction between p62 and epidermal growth factor receptor (EGFR), and knockdown of EGFR reversed SC66-mediated autophagy inhibition without affecting the phosphorylation of acetyl-CoA carboxylase (ACC), a well-known substrate of AMPK. SC66 increased the interaction between EGFR and Beclin 1 and markedly decreased the association of EGFR with VPS34, a critical protein for autophagy induction. Collectively, the data presented here indicate that EGFR-p62 pathway plays a critical role in Akt-mediated positive regulation of autophagy.

Genomics-driven discovery of a new cyclodepsipeptide from the guanophilic fungus Amphichorda guana.

Two potential non-ribosomal peptide synthetases (NRPSs) were identified in the genome of a guanophilic fungus Amphichorda guana by bioinformatics analysis and gene knockout experiments. Liquid chromatography coupled with mass spectrometry (LC-MS) guided isolation led to the discovery of a new cyclodepsipeptide isaridin H (1) and seven known analogs, desmethylisaridin E (2), isaridin E (3), isariin A (4), iso-isariin B (5), iso-isariin D (6), isariin E (7), and nodupetide (8). The absolute configuration of isaridin H (1) was achieved by Marfey's method. Isaridin H (1) showed significant antifungal activity against Botrytis cinerea and Alternaria solani.

The CREB coactivator CRTC2 controls hepatic lipid metabolism by regulating SREBP1.

Abnormal accumulation of triglycerides in the liver, caused in part by increased de novo lipogenesis, results in non-alcoholic fatty liver disease and insulin resistance. Sterol regulatory element-binding protein 1 (SREBP1), an important transcriptional regulator of lipogenesis, is synthesized as an inactive precursor that binds to the endoplasmic reticulum (ER). In response to insulin signalling, SREBP1 is transported from the ER to the Golgi in a COPII-dependent manner, processed by proteases in the Golgi, and then shuttled to the nucleus to induce lipogenic gene expression; however, the mechanisms underlying enhanced SREBP1 activity in insulin-resistant obesity and diabetes remain unclear. Here we show in mice that CREB regulated transcription coactivator 2 (CRTC2) functions as a mediator of mTOR signalling to modulate COPII-dependent SREBP1 processing. CRTC2 competes with Sec23A, a subunit of the COPII complex, to interact with Sec31A, another COPII subunit, thus disrupting SREBP1 transport. During feeding, mTOR phosphorylates CRTC2 and attenuates its inhibitory effect on COPII-dependent SREBP1 maturation. As hepatic overexpression of an mTOR-defective CRTC2 mutant in obese mice improved the lipogenic program and insulin sensitivity, these results demonstrate how the transcriptional coactivator CRTC2 regulates mTOR-mediated lipid homeostasis in the fed state and in obesity.

New Diterpenoids and Isocoumarin Derivatives from the Mangrove-Derived Fungus Hypoxylon sp.

Two new diterpenoids, hypoxyterpoids A (1) and B (2), and four new isocoumarin derivatives, hypoxymarins A-D (4-7), together, with seven known metabolites (3 and 8-13) were obtained from the crude extract of the mangrove-derived fungus Hypoxylon sp. The structures of the new compounds were elucidated on the basis of 1- and 2-dimensional (1D/2D) nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric analysis. The absolute configurations of compounds 1, 2, 4, 5, and 7 were determined by comparison of experimental and calculated electronic circular dichroism (ECD) spectra, and the absolute configurations of C-4' in 6 and C-9 in 7 were determined by [Rh2(OCOCF3)4]-induced ECD spectra. Compound 1 showed moderate α-glucosidase inhibitory activities with IC50 values of 741.5 ± 2.83 µM. Compounds 6 and 11 exhibited DPPH scavenging activities with IC50 values of 15.36 ± 0.24 and 3.69 ± 0.07 µM, respectively.

Genetic dereplication driven discovery of a tricinoloniol acid biosynthetic pathway in Trichoderma hypoxylon.

A genetic dereplication approach in combination with differential gene expression led to the discovery of three new sesquiterpenes, tricinoloniol acids (TRAs) A-C (1-3) and the known fusidilactone A (4) from T. hypoxylon. Comparative transcriptomic analysis and targeted deletion identified the biosynthetic route for TRAs. Our results demonstrate an alternative application of the genetic dereplication method for exploring the biosynthesis of cryptic secondary metabolites (SMs), which utilizes the coordinated expression of trichothecene (tri) and tra cluster genes.

Silencing of lncRNA UCA1 curbs proliferation and accelerates apoptosis by repressing SIRT1 signals by targeting miR-204 in pediatric AML.

The long noncoding RNA urothelial carcinoma-associated 1 (UCA1) has been reported to sustain the proliferation of acute myeloid leukemia (AML) cells through downregulating cell cycle regulators p27kip1 . Yet, the foundational mechanism of UCA1 in AML pathologies remains unclear. Herein, we found an escalation of UCA1 expression and suppression of miR-204 expression in pediatric AML patients and cells. UCA1 silencing suppressed cell proliferative abilities, promoted apoptotic rates, decreased Ki67, and increased cleaved caspase-3 in AML cells. Moreover, UCA1 sponged miR-204 and suppressed its expression. UCA1 overexpression inversed the miR-204 suppressed proliferation and promoted apoptosis. UCA1 also boosted the expression of SIRT1, a miR-204 target, via the sponging interaction. Furthermore, miR-204 inhibited inducible nitric oxide synthase and cyclooxygenase-2 expression, while UCA1 overexpression inversed the inhibitory effects in AML cells. Our findings concluded that UCA1 downregulation repressed cell proliferation and promoted apoptosis through inactivating SIRT1 signals by upregulating miR-204 in pediatric AML.

Disulfide Cleavage in a Dimeric Epipolythiodioxopiperazine Natural Product Diminishes Its Apoptosis-Inducing Effect but Enhances Autophagy in Tumor Cells.

Gliocladicillin C (3) is a cytotoxic epipolythiodioxopiperazine (ETP) isolated from the Ophiocordyceps-associated fungus Clonostachys rogersoniana. Although the disulfides/polysulfides in ETPs are believed to account for their cytotoxicity, and 11'-deoxyverticillin A was demonstrated to induce apoptosis and autophagy, how they mediate apoptosis and autophagy remained unknown. Here, we revealed that 3 activated caspase-dependent apoptosis and autophagy in human tumor cells, while the prepared disulfide-cleavage product failed to induce reactive oxygen species production and PARP cleavage, but further enhanced the autophagic flux compared to 3. Gliocladicillin C and its derivative also increased the phosphorylation of AMP-activated protein kinase and stimulated autophagy by affecting the glycolytic pathway. These results demonstrated that the disulfides played an essential role in inducing apoptosis, but not autophagy.

Different Role of Raptor and Rictor in Regulating Rasfonin-Induced Autophagy and Apoptosis in Renal Carcinoma Cells.

Both Raptor and Rictor are the key components in the complexes of mammalian target of rapamycin (mTOR), which play a vital role in mediating autophagy. Unlike mTOR, the regulatory role of either Raptor or Rictor in the regulation of autophagic process is relatively less explored. In present study, we found that rasfonin, which isolated from Talaromyces sp. 3656-A1 and was a fungal natural product, activated both caspase-dependent apoptosis and autophagy in ACHN, a renal carcinoma cell line. Knockdown of Raptor decreased both rasfonin-induced autophagic flux and PARP-1 cleavage, and in contrast, Rictor silencing increased apoptosis concomitantly enhancing rasfonin-induced autophagy. Unexpectedly, API-2, which was widely used as an inhibitor of Akt, promoted rasfonin-dependent autophagy in Raptor-depleted but not Rictor-deprived cells. Collectively, these results demonstrated that Raptor and Rictor could play a distinctly regulatory role in rasfonin-enhanced autophagy and apoptosis.

Density functional theory (DFT) studies of vanadium-titanium based selective catalytic reduction (SCR) catalysts.

Based on density functional theory (DFT) and basic structure models, the chemical reactions on the surface of vanadium-titanium based selective catalytic reduction (SCR) denitrification catalysts were summarized. Reasonable structural models (non-periodic and periodic structural models) are the basis of density functional calculations. A periodic structure model was more appropriate to represent the catalyst surface, and its theoretical calculation results were more comparable with the experimental results than a non-periodic model. It is generally believed that the SCR mechanism where NH3 and NO react to produce N2 and H2O follows an Eley-Rideal type mechanism. NH2NO was found to be an important intermediate in the SCR reaction, with multiple production routes. Simultaneously, the effects of H2O, SO2 and metal on SCR catalysts were also summarized.

Genetic dereplication of Trichoderma hypoxylon reveals two novel polycyclic lactones.

Previous study demonstrated large scale production of trichochecenes which limited the discovery of novel metabolites in Trichoderma hypoxylon. By genetic deletion of trichothecene synthase encoding gene thtri5, we created the dereplication mutant which eliminated the production of trichothecenes. Through chemical isolation, we characterized a couple of rare new polycyclic lactones tricholactones A and B from the thtri5 deletion strain. The structures of these two compounds were well determined by NMR, HR-ESI-MS and IECD analysis.

Cytotoxic, Anti-Migration, and Anti-Invasion Activities on Breast Cancer Cells of Angucycline Glycosides Isolated from a Marine-Derived Streptomyces sp.

Four angucycline glycosides were previously characterized from marine-derived Streptomyces sp. OC1610.4. Further investigation of this strain cultured on different fermentation media from that used previously resulted in the isolation of two new angucycline glycosides, vineomycins E and F (1-2), and five known homologues, grincamycin L (3), vineomycinone B2 (4), fridamycin D (5), moromycin B (7), and saquayamycin B1 (8). Vineomycin F (2) contains an unusual ring-cleavage deoxy sugar. All the angucycline glycosides isolated from Streptomyces sp. OC1610.4 were evaluated for their cytotoxic activity against breast cancer cells MCF-7, MDA-MB-231, and BT-474. Moromycin B (7), saquayamycin B1 (8), and saquayamycin B (9) displayed potent anti-proliferation against the tested cell lines, with IC50 values ranging from 0.16 to 0.67 µM. Saquayamycin B (9) inhibited the migration and invasion of MDA-MB-231 cells in a dose-dependent manner, as detected by Transwell and wound-healing assays.

A highly efficient genetic system for the identification of a harzianum B biosynthetic gene cluster in Trichoderma hypoxylon.

Trichoderma hypoxylon is a fungicolous species which produces rich secondary metabolites. However, no genetic transformation method is available for further studies. Here, we developed a marker-less transformation system based on the complementation of an uridine/uracil biosynthetic gene by protoplast transformation. An uridine/uracil auxotrophic mutant of Δthpyr4 was obtained by using a positive screening protocol with 5'-fluoroorotic acid as a selective reagent. To improve the homologous integration rates, the orthologues of ku70 and lig4 which play critical roles in non-homologous end-joining recombination were disrupted. The resulting thlig4 mutant showed remarkable transformation rates of 89 %, while no change was found in the thku70 deletion mutant compared with the WT strain. This suggests that thlig4 play a key role in the non-homologous recombination in this strain. Using this system, the biosynthetic gene cluster of trichothecene (tri) harzianum B was identified by deletion of the thtri5 in T. hypoxylon. Comparative genome analysis revealed that the trichothecene biosynthetic gene cluster in T. hypoxylon shared similar organizations with T. arundinaceum and T. brevicompactum, even though their encoded products are different in structures. Taken together, the highly efficient genetic system provides a convenient tool for studying the biosynthetic diversity and mining the novel natural product from the fungi.

Botryane Sesquiterpenoids, Cyclopentadepsipeptides, Xanthones, and Trichothecenes from Trichoderma oligosporum.

Five new botryane sesquiterpenes (1: -5: ), one new cyclopentadepsipeptide (9: ), and two new xanthones (11:  - 12: ), together with 11 known compounds, were isolated from Trichoderma oligosporum. The structures of the new compounds were identified by comprehensive spectroscopic methods including nuclear magnetic resonance and mass spectrometry. The cytotoxicity of 1: -19: was evaluated against K562, A549, and ASPC cell lines. Compounds 5, 8, 11, 17: , and 18: showed cytotoxicity against the K562 cell line with more than 50% inhibition at 12.5 µM. As to A549 cell line, compound 8: showed the strongest cytotoxicity with approximately 50% inhibition at 25.0 µM. No compounds showed cytotoxicity against the ASPC cell line.

Angucycline Glycosides from an Intertidal Sediments Strain Streptomyces sp. and Their Cytotoxic Activity against Hepatoma Carcinoma Cells.

Four angucycline glycosides including three new compounds landomycin N (1), galtamycin C (2) and vineomycin D (3), and a known homologue saquayamycin B (4), along with two alkaloids 1-acetyl-ß-carboline (5) and indole-3-acetic acid (6), were isolated from the fermentation broth of an intertidal sediments-derived Streptomyces sp. Their structures were established by IR, HR-ESI-MS, 1D and 2D NMR techniques. Among the isolated angucyclines, saquayamycin B (4) displayed potent cytotoxic activity against hepatoma carcinoma cells HepG-2, SMMC-7721 and plc-prf-5, with IC50 values 0.135, 0.033 and 0.244 µM respectively, superior to doxorubicin. Saquayamycin B (4) also induced apoptosis in SMMC-7721 cells as detected by its morphological characteristics in 4',6-diamidino-2-phenylindole (DAPI) staining experiment.

Decalin-Containing Tetramic Acids and 4-Hydroxy-2-pyridones with Antimicrobial and Cytotoxic Activity from the Fungus Coniochaeta cephalothecoides Collected in Tibetan Plateau (Medog).

New tetramic acid derivatives, (±)-conipyridoins A-D (1-4), conipyridoins E (5) and F (6), and new 4-hydroxy-2-pyridone alkaloids (±)-didymellamide E (7), (+)-didymellamide B (8), (+)-N-hydroxyapiosporamide (9), and didymellamides F-H (10-12) were isolated and identified from the solid culture of the fungus Coniochaeta cephalothecoides. Chiral resolution of 1, 2, 3, 4, and 7 gave five pairs of enantiomers: 1a/1b, 2a/2b, 3a/3b, 4a/4b, and 7a/7b, respectively. Stereochemistry of 1a and 1b, and 2a and 2b was established and confirmed by the single-crystal X-ray diffraction and electronic circular dichroism (ECD) methods. Absolute configuration in 3a, 3b, 4a, 4b, 7a, and 7b was assigned by ECD calculations. Compounds 1-6 possess an unprecedented chemical skeleton featuring a decalin ring and a tetramic acid moiety. Compound 11 significantly inhibited the growth of Candida albicans and Aspergillus fumigatus with minimum inhibitory concentration (MIC) of 3.13 and 1.56 µM, respectively, and was further confirmed to be a new chitin synthesis inhibitor. Compound 5 exhibited the strongest activity against the growth of both Staphylococcus aureus and MRSA with MIC value of 0.97 µM. In the light of a co-occurrence of 3-acyl tetramic acids and biogenetically related pyridine alkaloids, the biosynthetic pathway for 1-12 was postulated.

Characterization and mechanism of anti-Aeromonas salmonicida activity of a marine probiotic strain, Bacillus velezensis V4.

The bacterium Aeromonas salmonicida is the causative agent of furunculosis, a systemic, ubiquitous disease of fish in the salmon family, characterized by high mortality and morbidity. Probiotics are a promising approach for prevention of furunculosis in aquaculture. A bacterial strain with anti-A. salmonicida properties, Bacillus velezensis V4, was isolated and the mechanisms underlying these properties were investigated. Anti-A. salmonicida compounds present in cell-free supernatant of V4 were purified and structurally identified as members of the iturin, macrolactin, and difficidin groups. The compounds contributed jointly to inhibition of A. salmonicida, and the diversity of the compounds was related to the versatility of their mode of action. Addition of the compounds to A. salmonicida cell suspensions reduced cell density. Analyses by confocal microscopy and scanning electron microscopy revealed cell membrane disruption, deletion of cellular content, and cell lysis of A. salmonicida. The V4 genome was sequenced, and gene clusters involved in synthesis of anti-Aeromonas compounds were detected and identified. A possible probiotic effect on growth performance of Oncorhynchus mykiss (rainbow trout) was investigated by addition of 0, 1, and 3 % (v/w) V4. Relative to control, mortality was reduced 27.25 % in the 1 % addition group and 81.86 % in the 3 % addition group. Feed coefficient ratio was reduced 19.49 % and weight gain ratio was increased 71.22 % in the 1 % addition group. Our findings demonstrate that V4 is an effective probiotic strain in O. mykiss and has clear potential for both control of furunculosis and growth promotion of aquaculture animals.

A novel high-throughput nematicidal assay using embryo cells and larvae of Caenorhabditis elegans.

Human health safety and environmental concerns have resulted in the widespread deregistration of several agronomic important nematicides. New and safer nematicides are urgently needed. However, a high-throughput bioassay for screening potential nematicides has not been established. We developed a two-step high-throughput nematicidal screening method to combine a cell-based MTS colorimetric assay with Caenorhabditis elegans embryo cells for preliminary cytotoxicity screening (step 1) followed by in vitro larval assay for nematicidal activity (step 2). Based on three conventional nematicides' test, high correlations were obtained between cell viability and larval viability and "r" values were 0.78 for Avermectin, 0.95 for Fosthiazate, and 0.65 for Formaldehyde solution. Further assays with 60 fungal secondary metabolites (extracts, fractions and pure compounds) also demonstrated the high correlation between cell viability and larval viability (r=0.60) and between the C. elegans cell viability and the juvenile viability of soybean cyst nematode Heterodera glycines (r=0.48) and pine wood nematode Bursaphelenchus xylophilus (r=0.56). Six metabolites with high cytotoxicity have performed high larval mortality with a LC50 range of 6.8-500µg/ml. These results indicate that the proposed two-step screening assay represents an efficient and labor-saving method for screening natural nematicidal products.

Arundinols A-C and arundinones A and B from the plant endophytic fungus Microsphaeropsis arundinis.

Three new ent-eudesmane sesquiterpenoids, arundinols A-C (1-3), one isochroman-1-one, arundinone A (4), and a polyoxygenated benzofuran-3(2H)-one dimer, arundinone B (5), were isolated from the extract of a plant endophytic fungus, Microsphaeropsis arundinis. Their structures were elucidated primarily by NMR experiments, and 1 was confirmed by X-ray crystallography. The absolute configuration of 1 was assigned by X-ray crystallography using Cu Kα radiation, whereas those of the C-11 tertiary alcohols in 2 and 3 were deduced via the circular dichroism data of the in situ formed [Rh(2)(OCOCF(3))(4)] complexes. Arundinone B (5) represents the first dimeric benzofuran-3(2H)-one, showing cytotoxicity against T24 and A549 cells. The co-isolated known compound 6 showed a modest inhibitory effect against Staphylococcus aureus.