KMT5A Knockdown Suppresses Osteosarcoma Cell Proliferation and Metastasis Through Ꞵ-Catenin Signalling.

PURPOSE: Osteosarcoma (OS) is the most common malignant solid bone tumor in children and young adults. We aimed to investigate the effects and cellular mechanisms of KMT5A on OS cell activity. METHODS: The protein expression was evaluated in the clinical normal, adjacent and OS osteogenic tissues. Knockdown of KMT5A was achieved by KMT5A siRNAs in a human OS cell line, MG63, to detect cell proliferation and metastasis. RESULTS: KMT5A expression was upregulated in clinical OS tissues. Knockdown of KMT5A inhibited cell proliferation but enhanced cell death, with significantly reduced cyclinD1 and Bcl2 and increased cleaved-caspase9 levels. KMT5A knockdown also suppressed OS cell migration and invasion capacity and deceased MMP3 and vimentin expression. ß-catenin levels were upregulated in OS tissues and blocking KMT5A resulted in a significant decline in ß-catenin expression in the OS cells. Further administration of ß-catenin activator remarkably increased protein levels of KMT5A, cyclinD1, Bcl2, MMP3, and vimentin, which showed reversed effects of KMT5A knockdown on OS cell activity. CONCLUSION: KMT5A knockdown plays an inhibitory role in OS cell proliferation and metastasis through ß-catenin signalling, which provides basic evidence and suggests potential targets for OS therapeutic research.

Enzymatic Hydrolysis Lignin-Derived Porous Carbons through Ammonia Activation: Activation Mechanism and Charge Storage Mechanism.

The low energy density and low cost performance of electrochemical capacitors (ECs) are the principal factors that limit the wide applications of ECs. In this work, we used enzymatic hydrolysis lignin as the carbon source and an ammonia activation methodology to prepare nitrogen-doped lignin-derived porous carbon (NLPC) electrode materials with high specific surface areas. We elucidated the free radical mechanism of ammonia activation and the relationship between nitrogen doping configurations, doping levels, and preparation temperatures. Furthermore, we assembled NLPC∥NLPC symmetric ECs and NLPC∥Zn asymmetric ECs using aqueous sulfate electrolytes. Compared with the ECs using KOH aqueous electrolyte, the energy densities of NLPC∥NLPC and NLPC∥Zn ECs were significantly improved. The divergence of charge storage characteristics in KOH, Na2SO4, and ZnSO4 electrolytes were compared by analyzing their area surface capacitance. This work provides a strategy for the sustainable preparation of lignin-derived porous carbons toward ECs with high energy densities.

Physicochemical characteristics and functional properties of high methoxyl pectin with different degree of esterification.

Moderate alkali de-esterification can change the physicochemical characteristics and thus the functional properties of high methoxyl pectin (HMP). The results revealed that de-esterification could increase negative charges (Zeta potential from -21 to -31 mV), decrease molecular weight (from 448 to 136 kDa) and apparent viscosity of HMP. Homogalacturonan (HG) content decreased (from 62% to 49%) while rhamnogalacturonan Ⅰ (RG-Ⅰ) content increased (from 32% to 46%) after de-esterification. The group characteristics of HMP with different degree of esterification (DE) were similar and no obvious impact was made on degree of crystallinity by alkali de-esterification. A conformation transition of HMP molecule implied by Congo red test were occurred as the DE decreased. With the decrease of DE, the molecular structure of HMP became shorter and smaller, and the entanglement was weaker. The de-esterification caused slight decrease of thermal stability. Alkali de-esterification would weaken the gel property and the emulsifying ability of HMP.

Meta-Analysis of Hepatic Arterial Infusion for Liver Metastases From Colorectal Cancer.

The aim of the present study was to evaluate the potential benefits of hepatic arterial infusion chemotherapy (HAIC) in the management of colorectal liver metastases (CRLM). Electronic databases, including PubMed, EMBASE, Medline, Web of Science, and Cochrane Library, were comprehensively searched from inception to November 2020. Prospective randomized trials with HAIC vs. systemic chemotherapy (SC) were selected. The overall survival (OS), tumor response rates (RRs), progression-free survival (PFS), and corresponding 95% confidence intervals (CIs) were assessed in the meta-analysis. Subsequently, the heterogeneity between studies, sensitivity, publication bias, and meta-regression analyses were performed. Finally, 18 studies, which contained 1,766 participants (922 in the HAIC group and 844 in the SC group) were included. There was a significantly higher OS rate in the HAIC as palliative treatment group (HR, 0.17; 95% CI, 0.08-0.26; P = 0.000) and HAIC as adjuvant treatment group compared with SC group (HR, 0.63; 95% CI, 0.38-0.87; P = 0.000). The complete and partial tumor RRs were also increased significantly in the HAIC as palliative treatment group (RR = 2.09; 95% CI, 1.36-3.22; P = 0.001) and as adjuvant treatment group compared with SC group (RR = 2.14; 95% CI, 1.40-3.26; P = 0.000). However, PFS did not differ significantly between the HAIC and SC groups (P > 0.05). Meta-regression analysis showed potential covariates did not influence on the association between HAIC and OS outcomes (P > 0.05). The results of the present study suggested that HAIC may be a potential therapeutic regimen that may improve the outcomes of patients with CRLM. The present meta-analysis has been registered in PROSPERO (no. CRD 42019145719).

Pulmonary Delivery of Biological Drugs.

In the last decade, biological drugs have rapidly proliferated and have now become an important therapeutic modality. This is because of their high potency, high specificity and desirable safety profile. The majority of biological drugs are peptide- and protein-based therapeutics with poor oral bioavailability. They are normally administered by parenteral injection (with a very few exceptions). Pulmonary delivery is an attractive non-invasive alternative route of administration for local and systemic delivery of biologics with immense potential to treat various diseases, including diabetes, cystic fibrosis, respiratory viral infection and asthma, etc. The massive surface area and extensive vascularisation in the lungs enable rapid absorption and fast onset of action. Despite the benefits of pulmonary delivery, development of inhalable biological drug is a challenging task. There are various anatomical, physiological and immunological barriers that affect the therapeutic efficacy of inhaled formulations. This review assesses the characteristics of biological drugs and the barriers to pulmonary drug delivery. The main challenges in the formulation and inhalation devices are discussed, together with the possible strategies that can be applied to address these challenges. Current clinical developments in inhaled biological drugs for both local and systemic applications are also discussed to provide an insight for further research.

MiR-181c-5p Promotes Inflammatory Response during Hypoxia/Reoxygenation Injury by Downregulating Protein Tyrosine Phosphatase Nonreceptor Type 4 in H9C2 Cardiomyocytes.

BACKGROUND: Constitutive nuclear factor kappa B (NFκB) activation has been shown to exacerbate during myocardial ischemia/reperfusion (I/R) injury. We recently showed that miR-181c-5p exacerbated cardiomyocytes injury and apoptosis by directly targeting the 3'-untranslated region of protein tyrosine phosphatase nonreceptor type 4 (PTPN4). However, whether miR-181c-5p mediates cardiac I/R injury through NFκB-mediated inflammation is unknown. Thus, the present study aimed to investigate the role of miR-181c-5p during myocardial I/R injury and explore its mechanism in relation to inflammation in H9C2 cardiomyocytes. METHODS AND RESULTS: In hypoxia/reoxygenation (H/R, 6 h hypoxia followed by 6 h reoxygenation)-stimulated H9C2 cardiomyocytes or postischemic myocardium of rat, the expression of miR-181c-5p was significantly upregulated, which was concomitant increased NFκB activity when compared to the nonhypoxic or nonischemic control groups. This is indicative that miR-181c-5p may be involved in NFκB-mediated inflammation during myocardial I/R injury. To investigate the potential role of miR-181c-5p in H/R-induced cell inflammation and injury, H9C2 cardiomyocytes were transfected with the miR-181c-5p agomir. Overexpression of miR-181c-5p significantly aggravated H/R-induced cell injury (increased lactate dehydrogenase (LDH) level) and exacerbated NFκB-mediated inflammation (greater phosphorylation and degradation of IκBα, phosphorylation of p65, and increased levels of proinflammatory cytokines tumor necrosis factor α (TNFα), interleukin (IL)-6, and IL-1ß). In contrast, inhibition of miR-181c-5p by its antagomir transfection in vitro had the opposite effect. Furthermore, overexpression of miR-181c-5p significantly enhanced lipopolysaccharide-induced NFκB signalling. Additionally, knockdown of PTPN4, the direct target of miR-181c-5p, significantly aggravated H/R-induced phosphorylation and degradation of IκBα, phosphorylation of p65, and the levels of proinflammatory cytokines. PTPN4 knockdown also cancelled miR-181c-5p antagomir mediated anti-inflammatory effects in H9C2 cardiomyocytes during H/R injury. CONCLUSIONS: It is concluded that miR-181c-5p may exacerbate myocardial I/R injury and NFκB-mediated inflammation via PTPN4, and that targeting miR-181c-5p/PTPN4/NFκB signalling may represent a novel strategy to combat myocardial I/R injury.

Structural characterization of pectin-bismuth complexes and their aggregation in acidic conditions.

Bismuth-contained therapies are effective in treating gastric ulcer and eliminating Helicobacter pylori (Hp). Anion polysaccharides ligand could reduce the intake of bismuth, and enhance drug efficacy of bismuth compounds. In this study, pectin-bismuth (PB) was prepared and the changes of PB structure in acidic environment were reported for the first time. The structure of PB was characterized by FT-IR, XRD, and TGA, which suggested that combined with bismuth could alter the crystal structure of pectin. XPS confirmed the ionic binding of Bi3+ with carboxyl groups of pectin. The aggregating of PB with different pH level were also investigated, and the influence of pH on PB structure were observed by SEM. Results showed that PB has much larger volume of flocculation in acidic environment compared with bismuth nitrate. Additionally, apparent shear stress (τa) of PB suspension was evaluated. These results revealed the structural characteristics and acid-induced aggregation of pectin-bismuth, and bismuth could aggregate in acidic solution with the gelation of pectin.

Ursolic Acid Regulates Cell Cycle and Proliferation in Colon Adenocarcinoma by Suppressing Cyclin B1.

Aims: The biological functions of cyclin B1 (CCNB1) in colon adenocarcinoma (COAD) will be explored in this study. Furthermore, the therapeutic effects and potential molecular mechanisms of ursolic acid (UA) in COAD cells will also be investigated in vitro. Methods: COAD data were obtained from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Differentially expressed genes (DEGs) were determined with differential analysis. The biological functions of CCNB1 were analyzed through the GeneCards, the Search Tool for the Retrieval of Interacting Genes (STRING), and the Database for Annotation, Visualization, and Integrated Discovery (DAVID) databases. Therapeutic effects of UA on COAD cell lines HCT-116 and SW-480 were analyzed by CCK-8 and high-content screening (HCS) imaging assay. Flow cytometry was utilized to detect cell cycle changes of SW-480 and HCT-116 cells. Levels of mRNA and expression proteins of HCT-116, SW-480, and normal colon epithelial cells NCM-460 were determined by qRT-PCR and western blot. Results: CCNB1 was highly expressed and acted as an oncogene in COAD patients. CCNB1 and its interacting genes were significantly enriched in the cell cycle pathway. UA effectively inhibited the proliferation and injured COAD cells. In addition, UA arrested cell cycle of COAD cells in S phase. With regard to the molecular mechanisms of UA, we demonstrated that UA can significantly downregulate CCNB1 and its interacting genes and proteins, including CDK1, CDC20, CCND1, and CCNA2, which contributed to cell cycle blocking and COAD treatment. Conclusion: Results from this study revealed that UA possesses therapeutic effects on COAD. The anti-COAD activities of UA are tightly related to suppression of CCNB1 and its interacting targets, which is crucial in abnormal cell cycle process.

Using two-fluid nozzle for spray freeze drying to produce porous powder formulation of naked siRNA for inhalation.

Spray freeze drying is an attractive technology to produce powder formulation for inhalation. It can be used to generate large porous particles which tend to aerosolize efficiently and do not aggregate readily. It also avoids material to be exposed to elevated temperature. In this study, we reported the use of two-fluid nozzle to produce spray freeze dried powder of small interfering RNA (siRNA). The effect of atomization gas flow rate and liquid feed rate were inspected initially using herring sperm DNA (hsDNA) as nucleic acid model. The atomization gas flow rate was found to have a major impact on the aerosol properties. The higher the atomization gas flow rate, the smaller the particle size, the higher the fine particle fraction (FPF). In contrast, the liquid feed rate had very minor effect. Subsequently, spray freeze dried siRNA powder was produced at various atomization gas flow rates. The particles produced were highly porous as examined with the scanning electron microscopy, and the structural integrity of the siRNA was demonstrated with gel electrophoresis. The gene-silencing effect of the siRNA was also successfully preserved in vitro. The best performing siRNA formulation was prepared at the highest atomization gas flow rate investigated with a moderate FPF of 30%. However, this was significantly lower than that obtained with the corresponding hsDNA counterparts (FPF ∼57%). A direct comparison between the hsDNA and siRNA formulations revealed that the former exhibited a lower density, hence a smaller aerodynamic diameter despite similar geometric size.

Synthesis, Structure⁻Activity Relationships and In Vitro Toxicity Profile of Lactose-Based Fatty Acid Monoesters as Possible Drug Permeability Enhancers.

Permeability enhancers are receiving increased attention arising from their ability to increase transepithelial permeability and thus, bioavailability of orally or pulmonary administered biopharmaceutics. Here we present the synthesis and the in vitro assaying of a series of lactose-based non-ionic surfactants, highlighting the relationship between their structure and biological effect. Using tensiometric measurements the critical micelle concentrations (CMCs) of the surfactants were determined and demonstrate that increasing hydrophobic chain length reduces surfactant CMC. In vitro testing on Caco-2 intestinal and Calu-3 airway epithelia revealed that cytotoxicity, assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays, is presented for most of the surfactants at concentrations greater than their CMCs. Further biological study demonstrates that application of cytotoxic concentrations of the surfactants is associated with depolarizing mitochondrial membrane potential, increasing nuclear membrane permeability and activation of effector caspases. It is, therefore, proposed that when applied at cytotoxic levels, the surfactants are inducing apoptosis in both cell lines tested. Importantly, through the culture of epithelial monolayers on Transwell® supports, the surfactants demonstrate the ability to reversibly modulate transepithelial electrical resistance (TEER), and thus open tight junctions, at non-toxic concentrations, emphasizing their potential application as safe permeability enhancers in vivo.

Spray freeze drying of small nucleic acids as inhaled powder for pulmonary delivery.

The therapeutic potential of small nucleic acids such as small interfering RNA (siRNA) to treat lung diseases has been successfully demonstrated in many in vivo studies. A major barrier to their clinical application is the lack of a safe and efficient inhaled formulation. In this study, spray freeze drying was employed to prepare dry powder of small nucleic acids. Mannitol and herring sperm DNA were used as bulking agent and model of small nucleic acid therapeutics, respectively. Formulations containing different solute concentration and DNA concentration were produced. The scanning electron microscope (SEM) images showed that the porosity of the particles increased as the solute concentration decreased. Powders prepared with solute concentration of 5% w/v were found to maintain a balance between porosity and robustness. Increasing concentration of DNA improved the aerosol performance of the formulation. The dry powder formulation containing 2% w/w DNA had a median diameter of 12.5 µm, and the aerosol performance study using next generation impactor (NGI) showed an emitted fraction (EF) and fine particle fraction (FPF) of 91% and 28% respectively. This formulation (5% w/v solute concentration and 2% w/w nucleic acid) was adopted subsequently to produce siRNA powder. The gel retardation and liquid chromatography assays showed that the siRNA remained intact after spray freeze drying even in the absence of delivery vector. The siRNA powder formulation exhibited a high EF of 92.4% and a modest FPF of around 20%. Further exploration of this technology to optimise inhaled siRNA powder formulation is warranted.

From Pulmonary Surfactant, Synthetic KL4 Peptide as Effective siRNA Delivery Vector for Pulmonary Delivery.

Pulmonary delivery of small interfering RNA (siRNA) has huge potential for the treatment of a wide range of respiratory diseases. The ability of naked siRNA to transfect cells in the lungs without a delivery vector has prompted the investigation of whether an endogenous component is at least partially responsible for the cellular uptake of siRNA, and whether a safe and efficient delivery system could be developed from this component to further improve the transfection efficiency. Surfactant protein B (SP-B), a positively charged protein molecule found in lung surfactant, is one of the possible candidates. While the role of SP-B in siRNA transfection remains to be determined, the SP-B mimic, synthetic KL4 peptide, was investigated in this study as a potential siRNA carrier. KL4 is a 21-residue cationic peptide that was able to bind to siRNA to form nanosized complexes. It mediated siRNA transfection effectively in vitro on human lung epithelial cells, A549 cells, and BEAS-2B cells, which was comparable to Lipofectamine 2000. When commercial pulmonary surfactant (Infasurf) was added in the transfection medium, the gene silencing effect of siRNA in cells transfected with Lipofectamine 2000 was completely abolished, whereas those transfected with KL4 remained unaffected. At 4 °C, KL4 failed to deliver siRNA into the cells, indicating that an energy-dependent process was involved in the uptake of the complexes. Chlorpromazine (inhibitor of chathrin-mediated endocytosis), but not nystatin (inhibitor of caveolae-mediated endocytosis), inhibited the uptake of KL4/siRNA complexes, suggesting that they entered cells through clathrin-mediated endocytosis. There was no sign of cytotoxicity or immune response caused by KL4 and KL4/siRNA complexes. Overall, this study demonstrated that synthetic KL4 peptide is a promising candidate for siRNA carrier for pulmonary delivery and could be a potential platform for delivering other types of nucleic acid therapeutics.

Delivery of RNAi Therapeutics to the Airways-From Bench to Bedside.

RNA interference (RNAi) is a potent and specific post-transcriptional gene silencing process. Since its discovery, tremendous efforts have been made to translate RNAi technology into therapeutic applications for the treatment of different human diseases including respiratory diseases, by manipulating the expression of disease-associated gene(s). Similar to other nucleic acid-based therapeutics, the major hurdle of RNAi therapy is delivery. Pulmonary delivery is a promising approach of delivering RNAi therapeutics directly to the airways for treating local conditions and minimizing systemic side effects. It is a non-invasive route of administration that is generally well accepted by patients. However, pulmonary drug delivery is a challenge as the lungs pose a series of anatomical, physiological and immunological barriers to drug delivery. Understanding these barriers is essential for the development an effective RNA delivery system. In this review, the different barriers to pulmonary drug delivery are introduced. The potential of RNAi molecules as new class of therapeutics, and the latest preclinical and clinical studies of using RNAi therapeutics in different respiratory conditions are discussed in details. We hope this review can provide some useful insights for moving inhaled RNAi therapeutics from bench to bedside.

Incorporation of a Nuclear Localization Signal in pH Responsive LAH4-L1 Peptide Enhances Transfection and Nuclear Uptake of Plasmid DNA.

The major intracellular barriers associated with DNA delivery using nonviral vectors are inefficient endosomal/lysosomal escape and poor nuclear uptake. LAH4-L1, a pH responsive cationic amphipathic peptide, is an efficient DNA delivery vector that promotes the release of nucleic acid into cytoplasm through endosomal escape. Here we further enhance the DNA transfection efficiency of LAH4-L1 by incorporating nuclear localizing signal (NLS) to promote nuclear importation. Four NLSs were investigated: Simian virus 40 (SV40) large T-antigen derived NLS, nucleoplasmin targeting signal, M9 sequence, and the reverse SV40 derived NLS. All peptides tested were able to form positively charged nanosized complexes with DNA. Significant improvement in DNA transfection was observed in slow-dividing epithelial cancer cells (Calu-3), macrophages (RAW264.7), dendritic cells (JAWSII), and thymidine-induced growth-arrested cells, but not in rapidly dividing cells (A549). Among the four NLS-modified peptides, PK1 (modified with SV40 derived NLS) and PK2 (modified with reverse SV40 derived NLS) were the most consistent in improving DNA transfection; up to a 10-fold increase in gene expression was observed for PK1 and PK2 over the unmodified LAH4-L1. Additionally PK1 and PK2 were shown to enhance cellular uptake as well as nuclear entry of DNA. Overall, we show that the incorporation of SV40 derived NLS, in particular, to LAH4-L1 is a promising strategy to improve DNA delivery efficiency in slow-dividing cells and dendritic cells, with development potential for in vivo applications and as a DNA vaccine carrier.

Five New Cytotoxic Metabolites from the Marine Fungus Neosartorya pseudofischeri.

The marine fungus Neosartorya pseudofischeri was isolated from Acanthaster planci from the South China Sea. In a preliminary bioactivity screening, the crude methanol extract of the fungal mycelia showed significant inhibitory activity against the Sf9 cell line from the fall armyworm Spodoptera frugiperda. Five novel compounds, including 5-olefin phenylpyropene A (1), 13-dehydroxylpyripyropene A (4), deacetylsesquiterpene (7), 5-formyl-6-hydroxy-8-isopropyl-2- naphthoic acid (9) and 6,8-dihydroxy-3-((1E,3E)-penta-1,3-dien-1-yl)isochroman-1-one (10), together with eleven known compounds, phenylpyropene A (2) and C (3), pyripyropene A (5), 7-deacetylpyripyropene A (6), (1S,2R,4aR,5R,8R,8aR)-1,8a-dihydroxy-2-acetoxy-3,8-dimethyl-5- (prop-1-en-2-yl)-1,2,4a, 5,6,7,8,8a-octahydronaphthalene (8), isochaetominine C (11), trichodermamide A (12), indolyl-3-acetic acid methyl ester (13), 1-acetyl-ß-carboline (14), 1,2,3,4-tetrahydro-6-hydroxyl-2-methyl-l,3,4-trioxopyrazino[l,2-a]-indole (15) and fumiquinazoline F (16), were obtained. The structures of these compounds were determined mainly by MS and NMR data. The absolute configuration of 9 was assigned by the single-crystal X-ray diffraction studies. Compounds 1-11 and 15 showed significant cytotoxicity against the Sf9 cells from S. frugiperda.

Gliotoxin Inhibits Proliferation and Induces Apoptosis in Colorectal Cancer Cells.

The discovery of new bioactive compounds from marine natural sources is very important in pharmacological research. Here we developed a Wnt responsive luciferase reporter assay to screen small molecule inhibitors of cancer associated constitutive Wnt signaling pathway. We identified that gliotoxin (GTX) and some of its analogues, the secondary metabolites from marine fungus Neosartorya pseufofischeri, acted as inhibitors of the Wnt signaling pathway. In addition, we found that GTX downregulated the ß-catenin levels in colorectal cancer cells with inactivating mutations of adenomatous polyposis coli (APC) or activating mutations of ß-catenin. Furthermore, we demonstrated that GTX induced growth inhibition and apoptosis in multiple colorectal cancer cell lines with mutations of the Wnt signaling pathway. Together, we illustrated a practical approach to identify small-molecule inhibitors of the Wnt signaling pathway and our study indicated that GTX has therapeutic potential for the prevention or treatment of Wnt dependent cancers and other Wnt related diseases.

Inhalable dry powder formulations of siRNA and pH-responsive peptides with antiviral activity against H1N1 influenza virus.

Pulmonary delivery of siRNA has considerable therapeutic potential for treating viral respiratory infectious diseases including influenza. By introducing siRNA that targets the conserved region of viral genes encoding nucleocapsid protein (NP), viral mRNAs can be degraded and viral replication can be inhibited in mammalian cells. To enable siRNA to be used as an antiviral agent, the nucleic acid delivery barrier must be overcome. Effective local delivery of siRNA to lung tissues is required to reduce the therapeutic dose and minimize systemic adverse effects. To develop a formulation suited for clinical application, complexes of pH-responsive peptides, containing either histidine or 2,3-diaminopropionic acid (Dap), and siRNA were prepared into dry powders by spray drying with mannitol, which was used as a bulking agent. The spray-dried (SD) powders were characterized and found to be suitable for inhalation with good stability, preserving the integrity of the siRNA as well as the biological and antiviral activities. The formulations mediated highly effective in vitro delivery of antiviral siRNA into mammalian lung epithelial cells, leading to significant inhibition of viral replication when the transfected cells were subsequently challenged with H1N1 influenza virus. SD siRNA powders containing pH-responsive peptides are a promising inhalable formulation to deliver antiviral siRNA against influenza and are readily adapted for the treatment of other respiratory diseases.

Exploring the chemodiversity and biological activities of the secondary metabolites from the marine fungus Neosartorya pseudofischeri.

The production of fungal metabolites can be remarkably influenced by various cultivation parameters. To explore the biosynthetic potentials of the marine fungus, Neosartorya pseudofischeri, which was isolated from the inner tissue of starfish Acanthaster planci, glycerol-peptone-yeast extract (GlyPY) and glucose-peptone-yeast extract (GluPY) media were used to culture this fungus. When cultured in GlyPY medium, this fungus produced two novel diketopiperazines, neosartins A and B (1 and 2), together with six biogenetically-related known diketopiperazines,1,2,3,4-tetrahydro-2, 3-dimethyl-1,4-dioxopyrazino[1,2-a]indole (3), 1,2,3,4-tetrahydro-2-methyl-3-methylen e-1,4-dioxopyrazino[1,2-a]indole (4), 1,2,3,4-tetrahydro-2-methyl-1,3,4-trioxopyrazino[1,2-a] indole (5), 6-acetylbis(methylthio)gliotoxin (10), bisdethiobis(methylthio)gliotoxin (11), didehydrobisdethiobis(methylthio)gliotoxin (12) and N-methyl-1H-indole-2-carboxamide (6). However, a novel tetracyclic-fused alkaloid, neosartin C (14), a meroterpenoid, pyripyropene A (15), gliotoxin (7) and five known gliotoxin analogues, acetylgliotoxin (8), reduced gliotoxin (9), 6-acetylbis(methylthio)gliotoxin (10), bisdethiobis(methylthio) gliotoxin (11) and bis-N-norgliovictin (13), were obtained when grown in glucose-containing medium (GluPY medium). This is the first report of compounds 3, 4, 6, 9, 10 and 12 as naturally occurring. Their structures were determined mainly by MS, 1D and 2D NMR data. The possible biosynthetic pathways of gliotoxin-related analogues and neosartin C were proposed. The antibacterial activity of compounds 2-14 and the cytotoxic activity of compounds 4, 5 and 7-13 were evaluated. Their structure-activity relationships are also preliminarily discussed.

Pseudaboydins A and B: novel isobenzofuranone derivatives from marine fungus Pseudallescheria boydii associated with starfish Acanthaster planci.

Two novel isobenzofuranone derivatives, pseudaboydins A (1) and B (2), along with five known compounds, including (R)-2-(2-hydroxypropan-2-yl)-2,3-dihydro-5-hydroxybenzofuran (3), (R)-2-(2-hydroxypropan-2-yl)-2,3-dihydro-5-methoxybenzofuran (4), 3,3'-dihydroxy-5,5'-dimethyldiphenyl ether (5), 3-(3-methoxy-5-methylphenoxy)-5-methylphenol (6) and (-)-regiolone (7), were isolated from the culture broth of the marine fungus, Pseudallescheria boydii, associated with the starfish, Acanthaster planci. Their structures were elucidated primarily based on NMR and MS data. The absolute configurations of 1-4 were determined by CD spectroscopy and single-crystal X-ray diffraction studies. The cytotoxic and antibacterial activities of 1-4 were evaluated. Pseudaboydin A (1) showed moderate cytotoxic activity against human nasopharyngeal carcinoma cell line HONE1, human nasopharyngeal carcinoma cell line SUNE1 and human glandular lung cancer cell line GLC82 with IC50 values of 37.1, 46.5 and 87.2 µM, respectively.