Inducing secondary metabolite production of Aspergillus sydowii through microbial co-culture with Bacillus subtilis.

BACKGROUND: The co-culture strategy which mimics natural ecology by constructing an artificial microbial community is a useful tool to activate the biosynthetic gene clusters to generate new metabolites. However, the conventional method to study the co-culture is to isolate and purify compounds separated by HPLC, which is inefficient and time-consuming. Furthermore, the overall changes in the metabolite profile cannot be well characterized. RESULTS: A new approach which integrates computational programs, MS-DIAL, MS-FINDER and web-based tools including GNPS and MetaboAnalyst, was developed to analyze and identify the metabolites of the co-culture of Aspergillus sydowii and Bacillus subtilis. A total of 25 newly biosynthesized metabolites were detected only in co-culture. The structures of the newly synthesized metabolites were elucidated, four of which were identified as novel compounds by the new approach. The accuracy of the new approach was confirmed by purification and NMR data analysis of 7 newly biosynthesized metabolites. The bioassay of newly synthesized metabolites showed that four of the compounds exhibited different degrees of PTP1b inhibitory activity, and compound N2 had the strongest inhibition activity with an IC50 value of 7.967 µM. CONCLUSIONS: Co-culture led to global changes of the metabolite profile and is an effective way to induce the biosynthesis of novel natural products. The new approach in this study is one of the effective and relatively accurate methods to characterize the changes of metabolite profiles and to identify novel compounds in co-culture systems.

Naphthacemycins from a Streptomyces sp. as Protein-Tyrosine Phosphatase Inhibitors.

Nine new naphthacemycins (1-9), along with one known naphthacemycin (10) were isolated from the culture of Streptomyces sp. N12W1565. Their structures were elucidated on the basis of spectroscopic analysis, including UV, NMR, and HRESIMS. All the compounds showed significant activity, with IC50 values less than 10 µM against protein-tyrosine phosphatase 1B (PTP1B). The anti-PTP1B structure-activity relationship of naphthacemycins (1-10) is discussed. These findings provide a promising starting point for the development of naphthacemycins as potential anti-PTP1B agents.

Azaphilones with protein tyrosine phosphatase inhibitory activity isolated from the fungus Aspergillus deflectus.

Six undescribed azaphilones, deflectins C1-C3, deflectins D1-D2, and deflectin E, along with five known azaphilones were obtained from a solid culture of the wild fungus Aspergillus deflectus NCC0415. Their structures were determined by HRESIMS, NMR and ECD analyses, together with the GIAO 13C NMR calculation method. All compounds displayed strong or moderate inhibitory activity against protein tyrosine phosphatases SHP2 and PTP1B. Structure-activity relationship analysis of these azaphilones suggested that the length of the ketone aliphatic side chain would affect their SHP2 and PTP1B inhibitory activity. In addition, the presence of a Δ8(12) double bond on γ-lactone ring and the presence of CH3-2' in fatty chains may increase their inhibitory activity.

Indole-Diterpenoids with Protein Tyrosine Phosphatase Inhibitory Activities from the Marine-Derived Fungus Penicillium sp. KFD28.

Five new indole-terpenoids named penerpenes E-I (1-5), along with seven known ones (6-12), were isolated from the marine-derived fungus Penicillium sp. KFD28 from a bivalve mollusk, Meretrix lusoria. The structures of the new compounds were elucidated from spectroscopic data and ECD spectroscopic analyses. Compound 1 was assigned as an indole-diterpenoid with a unique 6/5/5/6/6/5/5 heptacyclic ring system. Compound 2 represents an indole-diterpenoid with a new carbon skeleton derived from paxilline by the loss of three carbons (C-23/24/25). Compound 3 contains an additional oxygen atom between C-21 and C-22 compared to paxilline to form an unusual 6/5/5/6/6/7 hexacyclic ring system bearing a 1,3-dioxepane ring, which is rarely encountered in natural products. Compounds 1, 2, 4, and 6 showed inhibitory activities against protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 14, 27, 23, and 13 µM, respectively.

Penerpenes A-D, Four Indole Terpenoids with Potent Protein Tyrosine Phosphatase Inhibitory Activity from the Marine-Derived Fungus Penicillium sp. KFD28.

Four unusual indole-terpenoids, penerpenes A-D (1-4), along with two known ones paxilline (5) and emindole SB (6), were isolated from the marine-derived fungus Penicillium sp. KFD28. The absolute structures of 1-4 were elucidated on the basis of spectroscopic data and ECD spectra analysis along with quantum ECD calculations. Compounds 1 and 2 showed potent inhibitory activity toward protein tyrosine phosphatases (PTP1B and TCPTP). Plausible biosynthetic pathways of compounds 1-4 are proposed.

Ubiquitin-Specific Peptidase 10 (USP10) Inhibits Hepatic Steatosis, Insulin Resistance, and Inflammation Through Sirt6.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, insulin resistance and inflammation, and the pathogenic mechanism of NAFLD is poorly understood. Ubiquitin-specific peptidase 10 (USP10), a member of the ubiquitin-specific protease family, is involved in environmental stress responses, tumor growth, inflammation, and cellular metabolism. However, the role of USP10 in hepatic steatosis, insulin resistance, and inflammation remains largely unexplored. USP10 expression was detected in livers of patients with NAFLD, mice with high-fat diet (HFD)-induced obesity, and genetically obese (ob/ob) mice, as well as in palmitate-induced hepatocytes. The function of USP10 in hepatic steatosis, insulin resistance, and inflammation was investigated using hepatocyte-specific USP10 deficiency or overexpression in mice induced by HFD treatment or genetic defect. The molecular mechanisms underlying USP10-regulated hepatic steatosis were further investigated in HFD-treated mice. USP10 expression was significantly decreased in the fatty livers of NAFLD patients and obese mice and in palmitate-treated hepatocytes. USP10 deficiency exacerbated the metabolic dysfunction induced by HFD treatment for 12 weeks. Conversely, USP10 overexpression significantly suppressed metabolic dysfunction in mice after HFD treatment and inhibited the development of NAFLD in ob/ob mice. Further investigation indicated that USP10 regulates hepatic steatosis by interacting with Sirt6 and inhibiting its ubiquitination and degradation. Sirt6 overexpression markedly ameliorated the effects of USP10 deficiency in hepatic steatosis, insulin resistance, and inflammation. Conversely, Sirt6 deficiency decreased the ameliorative effects of USP10 overexpression in response to HFD treatment. Conclusion: USP10 inhibits hepatic steatosis, insulin resistance, and inflammation through Sirt6.

Tisp40 deficiency attenuates renal ischemia reperfusion injury induced apoptosis of tubular epithelial cells.

Renal ischemia reperfusion (IR) is a major cause of acute kidney injury (AKI) and no effective treatments have been established. Tisp40 is a transcription factor of the CREB/ATF family and involves in cell apoptosis, proliferation and differentiation, but its role in renal IR remains unknown. Here, we investigated the role of Tisp40 in renal IR injury. In vivo, Tisp40 knockout (KO) and wild-type (WT) mice were subjected to thirty minutes of bilateral renal ischemia and 48h reperfusion, the blood and kidneys were harvested for analysis. In vitro, Tisp40 overexpression and vector cells were subjected to hypoxia/reoxygenation (HR), the apoptosis rate and the expressions of related proteins were measured. Following IR, the expressions of Tisp40 protein, serum creatinine (sCr), blood urea nitrogen (BUN) and apoptosis of tubular cells were significantly increased in WT mice. However, Tisp40 deficiency significantly attenuated the increase of sCr, BUN and apoptosis of tubular cells. Following HR, apoptosis of tubular cells was increased in Tisp40 overexpression cells compared with vector cells. Mechanistically, Tisp40 promoted the expressions of C/EBP homologous protein (CHOP), Bax and Cleaved caspase3 and suppressed the expression of Bcl-2 in renal IR injury. In conclusion, Tisp40 aggravates tubular cells apoptosis in renal IR injury.

Identification of Tisp40 as an Essential Regulator of Renal Tubulointerstitial Fibrosis via TGF-ß/Smads Pathway.

BACKGROUND: Tisp40, a transcription factor of the CREB/CREM family, is involved in cell proliferation, differentiation and other biological functions, but its role in renal tubulointerstitial fibrosis is unknown. METHODS: In our study, we investigated the effects of Tisp40 on extracellular matrix (ECM) accumulation, epithelial-mesenchymal transition (EMT) and the underlying molecular mechanisms in transforming growth factor-ß (TGF-ß)-stimulated TCMK-1 cells by quantitative real-time polymerase chain reaction (qPCR), Western blot analysis and immunofluorescence in vitro, and further explored the role of Tisp40 on renal fibrosis induced by ischemia-reperfusion (I/R) by qPCR, Western blot analysis, hydroxyproline analysis, Masson trichrome staining and immunohistochemistry staining in vivo. RESULTS: The data showed that Tisp40 was upregulated in a model of renal fibrosis induced by I/R injury (IRI). Upon IRI, Tisp40-deficient mice showed attenuated renal fibrosis compared with wild-type mice. Furthermore, the expression of α-smooth muscle actin, E-cadherin, fibronectin, and collagen I was suppressed. Tisp40 overexpression aggravated ECM accumulation and EMT in the TGF-ß-stimulated TCMK-1 cell line, whereas the opposite occurred in cells treated with small interfering RNA (siRNA) targeting Tisp40. Importantly, it is changes in the Smad pathway that attenuate renal fibrosis. CONCLUSION: These findings suggest that Tisp40 plays a critical role in the TGF-ß/ Smads pathway involved in this process. Hence, Tisp40 could be a useful therapeutic target in the fight against renal tubulointerstitial fibrosis.

Trivaric acid, a new inhibitor of PTP1b with potent beneficial effect on diabetes.

AIM: To screen a potential PTP1b inhibitor from the microbial origin-based compound library and to investigate the potential anti-diabetic effects of the inhibitor in vivo and determine its primary anti-diabetic mechanism in vitro and in silico. METHODS: PTP1b inhibitory activity was measured using recombination protein as the enzyme and p-NPP as the substrate. The binding of the inhibitor to PTP1b was analysed by docking in silico and confirmed by ITC experiments. The intracellular signalling pathway was detected by Western blot analysis in HepG2 cells. The anti-diabetic effects were evaluated using a diabetic mice model in vivo. RESULTS: Among 545 microbial origin-based pure compounds tested, trivaric acid, a tridepside, was selected as a PTP1B inhibitor exhibiting strong inhibitory activity with an IC50 of 173nM. Docking and ITC studies showed that trivaric acid was able to spontaneously bind to PTP1b and may inhibit PTP1b by blocking the catalytic domain of the phosphatase. Trivaric acid also enhanced the ability of insulin to stimulate the IR/IRS/Akt/GLUT2 pathway and increase the glucose consumption in HepG2 cells. In diabetic mice, trivaric acid that had been encapsulated into Eudrgit L100-5.5 showed significant anti-diabetic effects, improving insulin resistance, leptin resistance and lipid profile and weight control at doses of 5mg/kg and 50mg/kg. SIGNIFICANCE: Trivaric acid is a potential lead compound in the search for anti-diabetic agents targeting PTP1b.

Discovery of a new structural class of competitive hDHODH inhibitors with in vitro and in vivo anti-inflammatory, immunosuppressive effects.

Human dihydroorotate dehydrogenase (hDHODH) is an inner mitochondrial membrane enzyme that involves in the fourth step of the biosynthesis of pyrimidine base. Inhibitors of hDHODH have been proven efficacy for the treatments of inflammation, rheumatoid arthritis, multiple sclerosis and cancer. In the present study, ascochlorin (ASC) and its derivatives, natural compounds from fungal metabolites, were discovered as hDHODH inhibitors by high-throughput screening. Enzyme kinetics studies showed that ASC competitively binds to hDHODH at the site of coenzyme Q substrate. In ex vivo study, ASC significantly inhibited the ConA-stimulated T lymphocytes proliferation and interleukin-2, interferon-γ production. Furthermore, ASC showed significant in vivo anti-inflammatory and immunosuppressive effects on the mice ears swelling, allogenic skin grafts and rat collagen-induced arthritis animal disease models. ASC significantly reduced ears edema level of mice, increased the survival time of allogenic skin implanted on the mice and attenuated arthritis severity of rat model. In conclusion, ASC was identified as a new structural class of hDHODH inhibitors with efficient anti-inflammatory, immunosuppressive activity, and may be a promising candidate for the development of new therapy in the treatment of autoimmune diseases.

[Study on the expression of angiogenesis and spontaneous apoptosis and their relevance in laryngeal squamous cell carcinoma].

OBJECTIVE: To investigate the relationship among angiogenesis, spontaneous apoptosis and clinicopathological parameters in laryngeal squamous cell carcinoma (LSCC). METHOD: The intratumor microvessel density (IMVD), apoptotic index (AI) and vascular endothelial growth factor (VEGF) expression were detected by immunohistochemistry SABC and terminal uridine deoxynucleotidyl transferase mediated nick end labeling(TUNEL) methods in 34 LSCC patients. RESULT: The average IMVD was (21.50 +/- 8.87), and median of AI was 1.15%. The average IMVD in positive and negative cervical lymphatic metastasis was (26.33 +/- 9.70) and (17.68 +/- 6.06) respectively, and the IMVD with positive lymphatic metastasis tumors was statistical significantly higher than those with negative cervical lymphatic metastasis tumors (P < 0.01). The average IMVD had statistical difference in histological grading (P < 0.01), and analysis by one to one, the average IMVD had statistical difference between high and median grading. Expression of VEGF had a significantly positive correlation with IMVD (r = 0.51, P < 0.01). Statistical analysis revealed a significantly inverse correlation between AI and IMVD (r = -0.53, P < 0.01). We failed to find the statistical difference between IMVD and tumor T-stage in LSCC. CONCLUSION: IMVD may be an important indicator to predict cervical lymphatic metastasis in LSCC. VEGF might be an important angiogenic factor, and could promote tumor angiogenesis in LSCC. Tumor angiogenesis might contribute to tumor malignant progression by inhibiting spontaneous apoptosis in LSCC.

[Cloning and expression of gB gene of infectious laryngotracheitis virus in M. smegmatis].

Firstly, the complete gB gene of a domestic isolation stain were amplified by PCR, and the 2.6 kb gene fragment was obtained. Then the recombinant plasmid pY-alpha-gB was constructed by cloning PCR product into pY-alpha vector, and the shuttle expression plasmid pR-alpha-gB was constructed by cloning the hsp-alpha-gB gene into the downstream sequences of pRR3 vector. The recombinant plasmid was identified by restriction enzyme digestion and the sequence analysis, which was electrophoreted into M. smegmatis mc2 155. At last, the expressed gB proteins were successfully detected by ELISA and Western blot, which seems to be immunogenic crucially. The recombinant bacterial stain M. smegmatis mc2 155 (pR-alpha-gB)could protect SPF chick embryo from one lethal dose of ILTV.

[Study on the relationship between angiogenesis and cervical lymphatic metastasis in head and neck squamous cell carcinomas].

OBJECTIVE: To investigate the relationship between tumor angiogenesis and cervical lymphatic metastasis and the expression of vascular endothelial growth factor in head and neck squamous cell carcinomas. METHOD: Fifty-eight head and neck carcinoma tissue slides were immunohistochemically stained for anti-CD34 monoclonal antibody and anti-VEGF polyclonal antibody by SABC method. The intratumor microvessel density (IMVD) and the expression of vascular endothelial growth factor were measured. RESULT: 1. The mean IMVD with cervical lymphatic metastasis was significantly higher than those without cervical lymphatic metastasis in head and neck squamous cell carcinomas(27.92 +/- 9.11 and 20.69 +/- 7.08, P < 0.01); 2. The expression of vascular endothelial growth factor had a positive correlation with IMVD in head and neck carcinoma tissues (rs = 0.487, P < 0.01). CONCLUSION: IMVD may be an important predictor to cervical lymphatic metastasis in head and neck squamous cell carcinomas. Vascular endothelial growth factor may stimulate tumor angiogenesis in head and neck squamous cell carcinomas.