An Aspergillus nidulans Platform for the Complete Cluster Refactoring and Total Biosynthesis of Fungal Natural Products.

Fungal natural products (NPs) comprise a vast number of bioactive molecules with diverse activities, and among them are many important drugs. However, the yields of fungal NPs from native producers are usually low, and total synthesis of structurally complex NPs is challenging. As such, downstream derivatization and optimization of lead fungal NPs can be impeded by the high cost of obtaining sufficient starting material. In recent years, reconstitution of NP biosynthetic pathways in heterologous hosts has become an attractive alternative approach to produce complex NPs. Here, we present an efficient, cloning-free strategy for the cluster refactoring and total biosynthesis of fungal NPs in Aspergillus nidulans. Our platform places our genes of interest (GOIs) under the regulation of the robust asperfuranone afo biosynthesis gene machinery, allowing for their concerted activation upon induction. We demonstrated the utility of our system by creating strains that can synthesize high-value NPs, citreoviridin (1), mutilin (2), and pleuromutilin (3), with good to high yield and purity. This platform can be used not only for producing NPs of interests (i.e., total biosynthesis) but also for elucidating cryptic biosynthesis pathways.

Combination of Exercise and Vegetarian Diet: Relationship with High Density-Lipoprotein Cholesterol in Taiwanese Adults Based on MTHFR rs1801133 Polymorphism.

We examined the association between high-density lipoprotein cholesterol (HDL-C), and exercise and vegetarian diets, in Taiwanese adults, based on the Methylenetetrahydrofolate reductase (MTHFR) rs1801133 polymorphism. Using regression models, we analyzed historical data collected from 9255 Taiwan Biobank (TWB) participants from 2008 through 2015. Exposure to exercise was associated with higher HDL-C (ß = 1.0508 and 1.4011 for GG and GA + AA individuals, respectively), whereas a vegetarian diet was associated with lower HDL-C (ß = -6.2793 and -4.6359 for those with GG and GA + AA genotype, respectively). We found an interaction between exercise and diet among GG individuals (p = 0.0101). Compared with no exercise/no vegetarian diet, vegetarian diet/no exercise was associated with a 5.1514 mg/dl reduction in HDL-C among those with GG genotype (ß = -5.1514, p < 0.0001) and a 4.8426 mg/dl reduction (ß = -4.8426, p < 0.0001) among those with GA + AA genotype. Vegetarian diets in combination with exercise predicted a 6.5552 mg/dl reduction in HDL-C among GG individuals (ß = -6.5552) and a 2.8668 mg/dl reduction among GA + AA individuals (p < 0.05). These findings demonstrated that vegetarian diet alone was associated with lower HDL-C, no matter the rs1801133 genotype. However, the inclusion of regular exercise predicted much lower levels among GG individuals, whereas levels among GA + AA individuals were relatively higher.

Vegetarian Diets along with Regular Exercise: Impact on High-Density Lipoprotein Cholesterol Levels among Taiwanese Adults.

Background and objectives: High-density lipoprotein cholesterol (HDL-C) is important for improving risk estimates of atherosclerotic cardiovascular disease. We investigated the effect of omnivore and diverse vegetarian diets in connection with exercise on HDL-C. Materials and Methods: Historical data of 9588 biobank participants (4025 exercisers and 5563 non-exercisers) aged 30-70 years were categorized as omnivores (n = 8589), former vegetarians (n = 544), lacto-ovo vegetarians (n = 417), and strict vegetarians (n = 38). We used multiple linear regression for analyses. Results: HDL-C levels were higher in exercisers compared to non-exercisers. Compared with omnivores, strict vegetarians had decreased levels of HDL-C (ß = -5.705; p = 0.001) followed by lacto-ovo vegetarians (ß = -3.900; p < 0.001) and former vegetarians (ß = -0.329; p = 0.475). The test for trend was significant (p < 0.001). After categorization by exercise modalities, the ß-value was -13.984 for strict vegetarians, -4.419 for lacto-ovo vegetarians, and -1.864 for former vegetarians, respectively (p < 0.05). There was an interaction between diet and exercise (p = 0.009). Omnivores who exercised regularly had significantly higher HDL-C, whereas strict vegetarians who exercised regularly had significantly lower HDL-C. Conclusions: In summary, strict vegetarian diets in conjunction with regular exercise might not serve as healthful behaviors to be implemented in everyday life considering the negative impact on HDL-C.

Overexpression of a three-gene conidial pigment biosynthetic pathway in Aspergillus nidulans reveals the first NRPS known to acetylate tryptophan.

Fungal nonribosomal peptide synthetases (NRPSs) are megasynthetases that produce cyclic and acyclic peptides. In Aspergillus nidulans, the NRPS ivoA (AN10576) has been associated with the biosynthesis of grey-brown conidiophore pigments. Another gene, ivoB (AN0231), has been demonstrated to be an N-acetyl-6-hydroxytryptophan oxidase that putatively acts downstream of IvoA. A third gene, ivoC, has also been predicted to be involved in pigment biosynthesis based on publicly available genomic and transcriptomic information. In this paper, we report the replacement of the promoters of the ivoA, ivoB, and ivoC genes with the inducible promoter alcA in a single cotransformation. Co-overexpression of the three genes resulted in the production of a dark-brown pigment in hyphae. In addition, overexpression of each of the Ivo genes, ivoA-C, individually or in combination, allowed us to isolate intermediates and confirm the function of each gene. IvoA was found to be the first known NRPS to carry out the acetylation of the amino acid, tryptophan.

Mechanistic Study of the Phytocompound, 2- ß -D-Glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne in Human T-Cell Acute Lymphocytic Leukemia Cells by Using Combined Differential Proteomics and Bioinformatics Approaches.

Bidens pilosa, a medicinal herb worldwide, is rich in bioactive polyynes. In this study, by using high resolution 2-dimensional gel electrophoresis coupled with mass spectrometry analysis, as many as 2000 protein spots could be detected and those whose expression was specifically up- or downregulated in Jurkat T cells responsive to the treatment with 2-ß-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne (GHTT) can be identified. GHTT treatment can upregulate thirteen proteins involved in signal transduction, detoxification, metabolism, energy pathways, and channel transport in Jurkat cells. Nine proteins, that is, thioredoxin-like proteins, BH3 interacting domain death agonist (BID protein involving apoptosis), methylcrotonoyl-CoA carboxylase beta chain, and NADH-ubiquinone oxidoreductase, were downregulated in GHTT-treated Jurkat cells. Further, bioinformatics tool, Ingenuity software, was used to predict signaling pathways based on the data obtained from the differential proteomics approach. Two matched pathways, relevant to mitochondrial dysfunction and apoptosis, in Jurkat cells were inferred from the proteomics data. Biochemical analysis further verified both pathways involving GHTT in Jurkat cells. These findings do not merely prove the feasibility of combining proteomics and bioinformatics methods to identify cellular proteins as key players in response to the phytocompound in Jurkat cells but also establish the pathways of the proteins as the potential therapeutic targets of leukemia.

An efficient system for heterologous expression of secondary metabolite genes in Aspergillus nidulans.

Fungal secondary metabolites (SMs) are an important source of medically valuable compounds. Genome projects have revealed that fungi have many SM biosynthetic gene clusters that are not normally expressed. To access these potentially valuable, cryptic clusters, we have developed a heterologous expression system in Aspergillus nidulans . We have developed an efficient system for amplifying genes from a target fungus, placing them under control of a regulatable promoter, transferring them into A. nidulans , and expressing them. We have validated this system by expressing nonreducing polyketide synthases of Aspergillus terreus and additional genes required for compound production and release. We have obtained compound production and release from six of these nonreducing polyketide synthases and have identified the products. To demonstrate that the procedure allows transfer and expression of entire secondary metabolite biosynthetic pathways, we have expressed all the genes of a silent A. terreus cluster and demonstrate that it produces asperfuranone. Further, by expressing the genes of this pathway in various combinations, we have clarified the asperfuranone biosynthetic pathway. We have also developed procedures for deleting entire A. nidulans SM clusters. This allows us to remove clusters that might interfere with analyses of heterologously expressed genes and to eliminate unwanted toxins.

Engineering fungal nonreducing polyketide synthase by heterologous expression and domain swapping.

We reannotated the A. niger NR-PKS gene, e_gw1_19.204, and its downstream R domain gene, est_GWPlus_C_190476, as a single gene which we named dtbA. Heterologous expression of dtbA in A. nidulans demonstrated that DtbA protein produces two polyketides, 2,4-dihydroxy-3,5,6-trimethylbenzaldehyde (1) and 6-ethyl-2,4-dihydroxy-3,5-dimethylbenzaldehyde (2). Generation of DtbAΔR+TE chimeric PKSs by swapping the DtbA R domain with the AusA (austinol biosynthesis) or ANID_06448 TE domain enabled the production of two metabolites with carboxylic acids replacing the corresponding aldehydes.

Reconstitution of the early steps of gliotoxin biosynthesis in Aspergillus nidulans reveals the role of the monooxygenase GliC.

The gliotoxin, a member of the epipolythiodioxopiperazine (ETP), has received considerable attention from the scientific community for its wide range of biological activity. Despite the identification of gliotoxin cluster, however, the sequence of steps in the gliotoxin biosynthesis has remained elusive. As an alternative to the gene knock-out and biochemical approaches used so far, here we report using a heterologous expression approach to determine the sequence of the early steps of gliotoxin biosynthesis in Aspergillus nidulans. We identified the GliC, a monooxygenases that involved in the second step of gliotoxin biosynthesis pathway through the catalyzing the hydroxylation at the α-position of L-Phe.

Illuminating the diversity of aromatic polyketide synthases in Aspergillus nidulans.

Genome sequencing has revealed that fungi have the ability to synthesize many more natural products (NPs) than are currently known, but methods for obtaining suitable expression of NPs have been inadequate. We have developed a successful strategy that bypasses normal regulatory mechanisms. By efficient gene targeting, we have replaced, en masse, the promoters of nonreducing polyketide synthase (NR-PKS) genes, key genes in NP biosynthetic pathways, and other genes necessary for NR-PKS product formation or release. This has allowed us to determine the products of eight NR-PKSs of Aspergillus nidulans, including seven novel compounds, as well as the NR-PKS genes required for the synthesis of the toxins alternariol (8) and cichorine (19).

Recent advances in awakening silent biosynthetic gene clusters and linking orphan clusters to natural products in microorganisms.

Secondary metabolites from microorganisms have a broad spectrum of applications, particularly in therapeutics. The growing number of sequenced microbial genomes has revealed a remarkably large number of natural product biosynthetic clusters for which the products are still unknown. These cryptic clusters are potentially a treasure house of medically useful compounds. The recent development of new methodologies has made it possible to begin unlock this treasure house, to discover new natural products and to determine their biosynthesis pathways. This review will highlight some of the most recent strategies to activate silent biosynthetic gene clusters and to elucidate their corresponding products and pathways.

Role of Cybr, a cytohesin binder and regulator, in CD4(+) T-cell function and host immunity.

Cytohesin binder and regulator (Cybr) is known to regulate leukocyte adhesion and migration. However, its function in T-cells is poorly understood. Here, we investigated the role of Cybr in CD4(+) T-cell function and host immunity. Cybr inhibited p38 phosphorylation following CD4(+) T-cell stimulation. Since p38 regulates the expression of T-box expressed in T-cells (T-bet) but not GATA binding protein 3 (GATA-3) in T-cells, Cybr decreased the expression of T-bet and IFN-gamma in CD4(+) T-cells. Moreover, we found that host immunity against Listeria infection and IFN-gamma production in blood were significantly compromised in Cybr-overexpressing transgenic mice. In summary, our data suggest that Cybr represses the expression of T-bet and IFN-gamma via an inhibition of p38 in T-cells and consequently reduces host resistance to bacterial infection in mice.

Major determinants and long-term outcomes of successful balloon dilatation for the pediatric patients with isolated native valvular pulmonary stenosis: a 10-year institutional experience.

PURPOSE: We report herein major determinants and long-term outcomes of balloon dilatation (BD) for 27 pediatric patients with isolated native valvular pulmonary stenosis (VPS). MATERIALS AND METHODS: From May 1997 to May 2003, 27 pediatric patients with VPS (pressure gradients >or= 40 mmHg) were enrolled in this retrospective study. Single-balloon maneuver was applied in 26 patients, and double-balloon maneuver in 1. After BD, the pressure gradients were documented simultaneously by pullback maneuver by cardiac catheterization and echocardiography within 24 hours, at 1-month, 3-month, 1-year, and 4-to-10-year follow-ups. RESULTS: Before BD, the echocardiographic gradients ranged from 40 to 101 mmHg (61+/-19, 55), and from 40 to 144 mmHg (69+/-32, 60) by pressure recordings. After BD, the gradients ranged from 12 to 70 mmHg (29+/-13, 27) by pressure recording (p<0.001), and from 11 to 64 mmHg (27+/-12, 26) by echocardiography within 24 hrs (p<0.001). The ratios of the systolic pressure of the right ventricle to those of the left ventricle were 55 to 157% (89+/-28, 79%) before BD, and 30 to 79% (47 +/- 13, 42%) after BD (p<0.001). Follow-up (7.7+/-5.7, 4.5 years) echocardiographic gradients ranged from 11 to 61 mmHg (25+/-11, 24). Two patients did not have immediate success owing to infundibular spasm. Improved right ventricular compliance could be accounted for the ultimate success in these 2 patients. The ultimate successful rate was 100%. CONCLUSION: BD can achieve excellent long-term outcomes in the pediatric patients with isolated native VPS.

Cytopiloyne, a polyacetylenic glucoside, prevents type 1 diabetes in nonobese diabetic mice.

Some polyacetylenes from the plant Bidens pilosa have been reported to treat diabetes. In this study, we report that the cytopiloyne from B. pilosa, which is structurally different from the above-mentioned polyacetylenes and inhibits CD4(+) T cell proliferation, effectively prevents the development of diabetes in nonobese diabetic mice as evidenced by a normal level of blood glucose and insulin and normal pancreatic islet architecture. Cytopiloyne also suppresses the differentiation of type 1 Th cells but promotes that of type 2 Th cells, which is consistent with it enhancing GATA-3 transcription. Also, long-term application of cytopiloyne significantly decreases the level of CD4(+) T cells inside pancreatic lymph nodes and spleens but does not compromise total Ab responses mediated by T cells. Coculture assays imply that this decrease in CD4(+) T cells involves the Fas ligand/Fas pathway. Overall, our results suggest that cytopiloyne prevents type 1 diabetes mainly via T cell regulation.

Flavonoids, centaurein and centaureidin, from Bidens pilosa, stimulate IFN-gamma expression.

Bidens pilosa is used as an ethnical medicine for bacterial infection or immune modulation in Asia, America and Africa. Here, we employed an IFN-gamma promoter-driven luciferase reporter construct and T cells to characterize immunomodulatory compounds from this plant based on a bioactivity-guided isolation principle. We found that PHA, a positive control, caused a six-fold increase in IFN-gamma promoter activity. In contrast, hot water crude extracts from Bidens pilosa and its butanol subfraction increased IFN-gamma promoter activity to two- and six-fold, respectively. Finally, centaurein (EC(50)=75 microg/ml) and its aglycone, centaureidin (EC(50)=0.9 microg/ml), isolated from this butanol subfraction, augmented IFN-gamma promoter activity by approximately four-fold. Consistent with the role of centaurein or its aglycone in IFN-gamma regulation, we showed that centaurein induced the activity of NFAT and NFkappaB enhancers, located within the IFN-gamma promoter, in Jurkat cells. Overall, our results showed that centaurein regulated IFN-gamma transcription, probably via NFAT and NFkappaB in T cells.

The effect of centaurein on interferon-gamma expression and Listeria infection in mice.

We previously found that centaurein enhanced IFN-gamma transcription in T cells. Here, we demonstrate that centaurein increased the IFN-gamma expression in T and NK cells and the serum IFN-gamma level in mice. Centaurein elevated the transcription of T-bet but not GATA-3, which is consistent with its effect on that of IFN-gamma but not IL-4. Additionally, centaurein effectively protected mice against Listeria infection. Moreover, centaurein per se or in combination with antibiotics could treat Listeria infection. Our mechanistic studies suggest that centaurein augments IFN-gamma expression via a transcriptional up-regulation of T-bet and that centaurein protects against or treats Listeria infection via a modulation of IFN-gamma expression.

Cytopiloyne, a novel polyacetylenic glucoside from Bidens pilosa, functions as a T helper cell modulator.

An extract of Bidens pilosa, an anti-diabetic Asteraceae plant, has recently been reported to modulate T cell differentiation and prevent the development of non-obese diabetes (NOD) in NOD mice. In this paper, a novel bioactive polyacetylenic glucoside, cytopiloyne (1), was identified from the Bidens pilosa extract using ex vivo T cell differentiation assays based on a bioactivity-guided fractionation and isolation procedure. Its structure was elucidated as 2beta-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne by various spectroscopic methods. Functional studies showed that cytopiloyne was able to inhibit the differentiation of naïve T helper (Th0) cells into type I T helper (Th1) cells but to promote the differentiation of Th0 cells into type II T helper (Th2) cell. Accordingly, cytopiloyne also suppressed IFN-gamma expression and promoted IL-4 expression in mouse splenocytes ex vivo. These results suggest that cytopiloyne functions as a T cell modulator that may directly contribute to the ethnopharmacological effect of Bidens pilosa extract on preventing diabetes. Moreover, cytopiloyne can serve as an index compound for quality control of lot-to-lot extract preparations of Bidens pilosa.

The distinct effects of a butanol fraction of Bidens pilosa plant extract on the development of Th1-mediated diabetes and Th2-mediated airway inflammation in mice.

Bidens pilosa is claimed to be useful for immune or anti-inflammatory disorders; however, little scientific evidence has been published concerning its function. In this paper, immune disease mouse models were used to study the function of a butanol fraction of B.pilosa. We demonstrated treatment with the butanol fraction of B.pilosa ameliorated Th1 cell-mediated autoimmune diabetes in nonobese diabetic (NOD) mice but caused deterioration of Th2 cell-mediated airway inflammation induced by ovalbumin (OVA) in BALB/c mice. We next showed that Th2 cytokines (IL-4 and/or IL-5) increased but Th1 cytokine (IFN-gamma) decreased following injections with the butanol fraction of B.pilosa in both mouse strains. Accordingly, Th2 cytokine-regulated IgE production in mouse serum increased following treatment with this fraction. Finally, we found that the butanol fraction of B.pilosa inhibited Th1 cell differentiation but promoted Th2 cell differentiation. Taken together, the butanol fraction of B.pilosa has a dichotomous effect on helper T cell-mediated immune disorders, plausibly via modulation of T cell differentiation.

Polyacetylenic compounds and butanol fraction from Bidens pilosa can modulate the differentiation of helper T cells and prevent autoimmune diabetes in non-obese diabetic mice.

Compelling evidence suggests that infiltrating CD4+ type I helper T (Th1) cells in the pancreatic islets play a pivotal role in the progression of diabetes in non-obese diabetic (NOD) mice. We demonstrate in the present report that a butanol fraction of B. pilosa suppressed the development of diabetes, helped maintain levels of blood sugar and insulin in NOD mice in a dose-dependent manner and elevated the serum IgE levels regulated by Th2 cytokines in NOD mice. Moreover, the butanol fraction inhibited the differentiation of naive helper T (Th0) cells into Th1 cells but enhanced their transition into type II helper T (Th2) cells using an in vitro T cell differentiation assay. Two polyacetylenic compounds, 2-beta-D-glucopyranosyloxy-1-hydroxy-5(E)-tridecene-7,9,11-triyne and 3-beta-D-glucopyranosyloxy-1-hydroxy-6(E)-tetradecene-8,10,12-triyne, identified from the butanol fraction also prevented the onset of diabetes like the butanol fraction. The latter compound showed a stronger activity for T cell differentiation than the former. In summary, the butanol fraction of B. pilosa and its polyacetylenes can prevent diabetes plausibly via suppressing the differentiation of Th0 cells into Th1 cells and promoting that of Th0 cells into Th2 cells.

A novel p53 mutant retained functional activity in lung carcinomas.

The gene p53 is a critical tumor suppressor that can respond to multiple signals of cellular gatekeepers for growth and division. The mdm2 gene is one of the downstream target genes for transcriptional activation by the product of p53 tumor suppressor gene. Transactivation of mdm2 gene is represented by the presence of a functional P53 protein. To understand the biological function of mutant p53 in tumorigenesis, we constructed a number of p53 mutants by site-directed mutagenesis (H179Y, L194R, S240R, R249S, A276D, E286Q), followed by characterization of each P53 mutant's ability to transactivate mdm2, bax and p21waf. The transactivation properties of p53 mutants were compared by co-transfection with pGL-3-mdm2, pGL-3-bax and pGL-3-p21waf into the P53 null cell line H1299 derived from a non-small cell lung carcinoma. Among them mt p53 S240R and E286Q were shown to have enhanced transactivating activity of pGL3-mdm2, at about 43.2 and 28.2% of the wt p53 vector, respectively, while the remaining four had nearly the same level of activity as the negative control did. Furthermore, data indicated that mt p53 S240R had as high an ability to suppress the growth of the p53 null cell line H1299 as wild type p53. Therefore, mutant p53 alone is an insufficient indicator of poor prognosis. Instead, functional p53 may affect lung cancer prognosis.