Flavanone-rich citrus beverages counteract the transient decline in postprandial endothelial function in humans: a randomised, controlled, double-masked, cross-over intervention study.

Specific flavonoid-rich foods/beverages are reported to exert positive effects on vascular function; however, data relating to effects in the postprandial state are limited. The present study investigated the postprandial, time-dependent (0-7 h) impact of citrus flavanone intake on vascular function. An acute, randomised, controlled, double-masked, cross-over intervention study was conducted by including middle-aged healthy men (30-65 years, n 28) to assess the impact of flavanone intake (orange juice: 128·9 mg; flavanone-rich orange juice: 272·1 mg; homogenised whole orange: 452·8 mg; isoenergetic control: 0 mg flavanones) on postprandial (double meal delivering a total of 81 g of fat) endothelial function. Endothelial function was assessed by flow-mediated dilatation (FMD) of the brachial artery at 0, 2, 5 and 7 h. Plasma levels of naringenin/hesperetin metabolites (sulphates and glucuronides) and nitric oxide species were also measured. All flavanone interventions were effective at attenuating transient impairments in FMD induced by the double meal (7 h post intake; P<0·05), but no dose-response effects were observed. The effects on FMD coincided with the peak of naringenin/hesperetin metabolites in circulation (7 h) and sustained levels of plasma nitrite. In summary, citrus flavanones are effective at counteracting the negative impact of a sequential double meal on human vascular function, potentially through the actions of flavanone metabolites on nitric oxide.

Forazoline†A: marine-derived polyketide with antifungal in†vivo efficacy.

Forazoline A, a novel antifungal polyketide with in vivo efficacy against Candida albicans, was discovered using LCMS-based metabolomics to investigate marine-invertebrate-associated bacteria. Forazoline A had a highly unusual and unprecedented skeleton. Acquisition of (13)C-(13)C gCOSY and (13)C-(15)N HMQC NMR data provided the direct carbon-carbon and carbon-nitrogen connectivity, respectively. This approach represents the first example of determining direct (13)C-(15)N connectivity for a natural product. Using yeast chemical genomics, we propose that forazoline A operated through a new mechanism of action with a phenotypic outcome of disrupting membrane integrity.

Activation of the nuclear factor E2-related factor 2 pathway by novel natural products halomadurones A-D and a synthetic analogue.

Two novel chlorinated pyrones, halomadurones A and B, and two novel brominated analogues, halomadurones C and D, were isolated from a marine Actinomadura sp. cultivated from the ascidian Ecteinascidia turbinata. Additionally, a non-halogenated analogue, 2-methyl-6-((E)-3-methyl-1,3-hexadiene)-γ-pyrone, was synthesized to understand the role of the halogens for activity. Halomadurones C and D demonstrated potent nuclear factor E2-related factor antioxidant response element (Nrf2-ARE) activation, which is an important therapeutic approach for treatment of neurodegenerative diseases.

Baeyer-Villiger C-C bond cleavage reaction in gilvocarcin and jadomycin biosynthesis.

GilOII has been unambiguously identified as the key enzyme performing the crucial C-C bond cleavage reaction responsible for the unique rearrangement of a benz[a]anthracene skeleton to the benzo[d]naphthopyranone backbone typical of the gilvocarcin-type natural anticancer antibiotics. Further investigations of this enzyme led to the isolation of a hydroxyoxepinone intermediate, leading to important conclusions regarding the cleavage mechanism.

Microbial strain prioritization using metabolomics tools for the discovery of natural products.

Natural products profoundly impact many research areas, including medicine, organic chemistry, and cell biology. However, discovery of new natural products suffers from a lack of high throughput analytical techniques capable of identifying structural novelty in the face of a high degree of chemical redundancy. Methods to select bacterial strains for drug discovery have historically been based on phenotypic qualities or genetic differences and have not been based on laboratory production of secondary metabolites. Therefore, untargeted LC/MS-based secondary metabolomics was evaluated to rapidly and efficiently analyze marine-derived bacterial natural products using LC/MS-principal component analysis (PCA). A major goal of this work was to demonstrate that LC/MS-PCA was effective for strain prioritization in a drug discovery program. As proof of concept, we evaluated LC/MS-PCA for strain selection to support drug discovery, for the discovery of unique natural products, and for rapid assessment of regulation of natural product production.

Genome-based cluster deletion reveals an endocrocin biosynthetic pathway in Aspergillus fumigatus.

Endocrocin is a simple anthraquinone frequently identified in extracts of numerous fungi. Several biosynthetic schemes for endocrocin synthesis have been hypothesized, but to date, no dedicated secondary metabolite gene cluster that produces this polyketide as its major metabolite has been identified. Here we describe our biosynthetic and regulatory characterization of the endocrocin gene cluster in Aspergillus fumigatus. This is the first report of this anthraquinone in this species. The biosynthetic genes required for endocrocin production are regulated by the global regulator of secondary metabolism, LaeA, and encode an iterative nonreducing polyketide synthase (encA), a physically discrete metallo-ß-lactamase type thioesterase (encB), and a monooxygenase (encC). Interestingly, the deletion of a gene immediately adjacent to encC, termed encD and encoding a putative 2-oxoglutarate-Fe(II) type oxidoreductase, resulted in higher levels of endocrocin production than in the wild-type strain, whereas overexpression of encD eliminated endocrocin accumulation. We found that overexpression of the encA transcript resulted in higher transcript levels of encA-D and higher production of endocrocin. We discuss a model of the enc cluster as one evolutionary origin of fungal anthraquinones derived from a nonreducing polyketide synthase and a discrete metallo-ß-lactamase-type thioesterase.

Peptidolipins B-F, antibacterial lipopeptides from an ascidian-derived Nocardia sp.

A marine Nocardia sp. isolated from the ascidian Trididemnum orbiculatum was found to produce five new lipopeptides, peptidolipins B-F (1-5), which show distinct similarities to the previously reported L-Val(6) analog of peptidolipin NA. Synthetic modification of peptidolipin E (4) was used to determine the location of an olefin within the lipid chain. The advanced Marfey's method was used to determine the absolute configurations of the amino acids. Peptidolipins B (1) and E (4) demonstrated moderate antibacterial activity against methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus.

First natural analogs of the cytotoxic thiodepsipeptide thiocoraline A from a marine Verrucosispora sp.

A marine Verrucosispora sp. isolated from the sponge Chondrilla caribensis f. caribensis was found to produce thiocoraline, a potent cytotoxic compound. Five new analogs of thiocoraline were isolated and represent the first analogs of thiocoraline. 22'-Deoxythiocoraline (2), thiochondrilline C (5), and 12'-sulfoxythiocoraline (6) demonstrated significant cytotoxicity against the A549 human cancer cell line with EC(50) values of 0.13, 2.86, and 1.26 µM, respectively. The analogs provide insight into the SAR and biosynthesis of thiocoraline. The DP4 probability method was used to analyze ab initio NMR calculations to confirm stereochemical assignments.

Antiproliferative compounds of Cyphostemma greveana from a Madagascar dry forest.

Bioassay-guided fractionation of the EtOH extracts obtained from a plant identified as Cyphostemma greveana Desc. (Vitaceae) led to the identification of one macrolide, lasiodiplodin (1), three sesquiterpenoids, 12-hydroxy-15-oxoselina-4,11-diene (2), 1ß,6α-dihydroxyeudesm-4(15)-ene (3), and (7R*)-opposit-4(15)-ene-1ß,7-diol (5), and a new diterpenoid, 16,18-dihydroxykolavenic acid lactone (4). All the isolates were tested against the A2780 human ovarian cancer cell line, and compound 4 and a fraction containing 5 as the major constituent showed antiproliferative activities with IC(50) values of 0.44 µM (0.14 µg/ml) and 0.045 µg/ml, respectively. A partial synthesis of compound 5 was carried out, but the pure synthetic compound was inactive, indicating that the activity of the fraction containing it must be due to a very minor and as yet unidentified substance.

Antiproliferative cardenolide glycosides of Elaeodendron alluaudianum from the Madagascar Rainforest.

Bioassay-guided fractionation of an ethanol extract of a Madagascar collection of Elaeodendron alluaudianum led to the isolation of two new cardenolide glycosides (1 and 2). The (1)H and (13)C NMR spectra of both compounds were fully assigned using a combination of 2D NMR experiments, including (1)H-(1)H COSY, HSQC, HMBC, and ROESY sequences. Both compounds 1 and 2 were tested against the A2780 human ovarian cancer cell line and the U937 human histiocytic lymphoma cell line assays, and showed significant antiproliferative activity with IC(50) values of 0.12 and 0.07 microM against the A2780 human ovarian cancer cell line, and 0.15 and 0.08 microM against the U937 human histiocytic lymphoma cell line, respectively.

Antiproliferative and antimalarial anthraquinones of Scutia myrtina from the Madagascar forest.

Bioassay-guided fractionation of an ethanol extract of a Madagascar collection of the bark of Scutia myrtina led to the isolation of three new anthrone-anthraquinones, scutianthraquinones A, B and C (1-3), one new bisanthrone-anthraquinone, scutianthraquinone D (4), and the known anthraquinone, aloesaponarin I (5). The structures of all compounds were determined using a combination of 1D and 2D NMR experiments, including COSY, TOCSY, HSQC, HMBC, and ROESY sequences, and mass spectrometry. All the isolated compounds were tested against the A2780 human ovarian cancer cell line for antiproliferative activities, and against the chloroquine-resistant Plasmodium falciparum strains Dd2 and FCM29 for antiplasmodial activities. Compounds 1, 2 and 4 showed weak antiproliferative activities against the A2780 ovarian cancer cell line, while compounds 1-4 exhibited moderate antiplasmodial activities against P. falciparum Dd2 and compounds 1, 2, and 4 exhibited moderate antiplasmodial activities against P. falciparum FCM29.

Tauroursodeoxycholic acid attenuates endoplasmic reticulum stress and protects the liver from chronic intermittent hypoxia induced injury.

Obstructive sleep apnea that characterized by chronic intermittent hypoxia (CIH) has been reported to associate with chronic liver injury. Tauroursodeoxycholic acid (TUDCA) exerts liver-protective effects in various liver diseases. The purpose of this study was to test the hypothesis that TUDCA could protect liver against CIH injury. C57BL/6 mice were subjected to intermittent hypoxia for eight weeks and applied with TUDCA by intraperitoneal injection. The effect of TUDCA on liver histological changes, liver function, oxidative stress, inflammatory response, hepatocyte apoptosis and endoplasmic reticulum (ER) stress were investigated. The results showed that administration of TUDCA attenuated liver pathological changes, reduced serum alanine aminotransferase and aspartate aminotransferase level, suppressed reactive oxygen species activity, decreased tumor necrosis factor-α and interleukin-1ß level and inhibited hepatocyte apoptosis induced by CIH. TUDCA also inhibited CIH-induced ER stress in liver as evidenced by decreased expression of ER chaperone 78 kDa glucose-related protein, unfolded protein response transducers and ER proapoptotic proteins. Altogether, the present study described a liver-protective effect of TUDCA in CIH mice model, and this effect seems at least partly through the inhibition of ER stress.

Natural Product Discovery Using Planes of Principal Component Analysis in R (PoPCAR).

Rediscovery of known natural products hinders the discovery of new, unique scaffolds. Efforts have mostly focused on streamlining the determination of what compounds are known vs. unknown (dereplication), but an alternative strategy is to focus on what is different. Utilizing statistics and assuming that common actinobacterial metabolites are likely known, focus can be shifted away from dereplication and towards discovery. LC-MS-based principal component analysis (PCA) provides a perfect tool to distinguish unique vs. common metabolites, but the variability inherent within natural products leads to datasets that do not fit ideal standards. To simplify the analysis of PCA models, we developed a script that identifies only those masses or molecules that are unique to each strain within a group, thereby greatly reducing the number of data points to be inspected manually. Since the script is written in R, it facilitates integration with other metabolomics workflows and supports automated mass matching to databases such as Antibase.

Macrotermycins A-D, Glycosylated Macrolactams from a Termite-Associated Amycolatopsis sp. M39.

Bioassay-guided metabolomic analyses led to the characterization of four new 20-membered glycosylated polyketide macrolactams, macrotermycins A-D, from a termite-associated actinomycete, Amycolatopsis sp. M39. M39's sequenced genome revealed the macrotermycin's putative biosynthetic gene cluster. Macrotermycins A and C had antibacterial activity against human-pathogenic Staphylococcus aureus and, of greater ecological relevance, they also had selective antifungal activity against a fungal parasite of the termite fungal garden.

Chemical Genomics, Structure Elucidation, and in Vivo Studies of the Marine-Derived Anticlostridial Ecteinamycin.

A polyether antibiotic, ecteinamycin (1), was isolated from a marine Actinomadura sp., cultivated from the ascidian Ecteinascidia turbinata. 13C enrichment, high resolution NMR spectroscopy, and molecular modeling enabled elucidation of the structure of 1, which was validated on the basis of comparisons with its recently reported crystal structure. Importantly, ecteinamycin demonstrated potent activity against the toxigenic strain of Clostridium difficile NAP1/B1/027 (MIC = 59 ng/µL), as well as other toxigenic and nontoxigenic C. difficile isolates both in vitro and in vivo. Additionally, chemical genomics studies using Escherichia coli barcoded deletion mutants led to the identification of sensitive mutants such as trkA and kdpD involved in potassium cation transport and homeostasis supporting a mechanistic proposal that ecteinamycin acts as an ionophore antibiotic. This is the first antibacterial agent whose mechanism of action has been studied using E. coli chemical genomics. On the basis of these data, we propose ecteinamycin as an ionophore antibiotic that causes C. difficile detoxification and cell death via potassium transport dysregulation.

Probing the regiospecificity of enzyme-catalyzed steroid glycosylation.

The potential of a uniquely permissive engineered glycosyltransferase (OleD ASP) as a catalyst for steroid glycosylation is highlighted. The ability of OleD ASP to glucosylate a range of cardenolides and bufadienolides was assessed using a rapid LC-UV/MS-SPE-NMR analytical platform. While a bias toward OleD-catalyzed C3 monoglucosylation was observed, subtle alterations of the steroidal architecture, in some cases, invoked diglucosylation or, in one case (digoxigenin), C12 glucosylation. This latter case represents the first, and highly efficient, synthesis of digoxigenin 12-O-ß-D-glucoside.

Microtermolides A and B from termite-associated Streptomyces sp. and structural revision of vinylamycin.

Microtermolides A (1) and B (2) were isolated from a Streptomyces sp. strain associated with fungus-growing termites. The structures of 1 and 2 were determined by 1D- and 2D-NMR spectroscopy and high-resolution mass spectrometry. Structural elucidation of 1 led to the re-examination of the structure originally proposed for vinylamycin (3). Based on a comparison of predicted and experimental (1)H and (13)C NMR chemical shifts, we propose that vinylamycin's structure be revised from 3 to 4.

Structure and biosynthesis of the antibiotic bottromycin D.

Drug resistant infectious diseases are quickly becoming a global health crisis. While Streptomyces spp. have been a major source of antibiotics over the past 50 years, efficient methods are needed to identify new antibiotics and greatly improve the rate of discovery. LCMS-based metabolomics were applied to analyze extracts of 50 Streptomyes spp. Using this methodology, we discovered bottromycin D and used whole genome sequencing to determine its biosynthesis by a ribosomal pathway.