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Abnormal blood coagulation is a major health problem and natural anticoagulants from blood-feeding organisms have been investigated as novel therapeutics. NAPc2, a potent nematode-derived inhibitor of coagulation, has an unusual mode of action that requires coagulation factor Xa but does not inhibit it. Molecular dynamics simulations of NAPc2 and factor Xa were generated to better understand NAPc2. The simulations suggest that parts of NAPc2 become more rigid upon binding factor Xa and reveal that two highly conserved residues form an internal salt bridge that stabilises the bound conformation. Clotting time assays with mutants confirmed the utility of the salt bridge and suggested that it is a conserved mechanism for stabilising the bound conformation of secondary structure-poor protease inhibitors.

Antimicrobial resistance has emerged as a global public health threat, and development of novel therapeutics for treating infections caused by multi-drug resistant bacteria is urgent. Staphylococcus aureus is a major human and animal pathogen, responsible for high levels of morbidity and mortality worldwide. The intracellular survival of S. aureus in macrophages contributes to immune evasion, dissemination, and resilience to antibiotic treatment. Here, we present a confocal fluorescence imaging assay for monitoring macrophage infection by green fluorescent protein (GFP)-tagged S. aureus as a front-line tool to identify antibiotic leads. The assay was employed in combination with nanoscaled chemical analyses to facilitate the discovery of a new, active rifamycin analogue. Our findings indicate a promising new approach for the identification of antimicrobial compounds with macrophage intracellular activity. The antibiotic identified here may represent a useful addition to our armory in tackling the silent pandemic of antimicrobial resistance.

As one of the first families of marine natural products to undergo clinical trials, the didemnin depsipeptides have played a significant role in inspiring the discovery of marine drugs. Originally developed as anticancer therapeutics, the recent re-evaluation of these compounds including synthetically derived dehydrodidemnin B or Aplidine, has led to their advancement towards antiviral applications. While conventionally associated with production in colonial tunicates of the family Didemnidae , recent studies have identified their biosynthetic gene clusters from the marine-derived bacteria Tistrella mobilis . While these studies confirm the production of didemnin X/Y, the low titer and general lack of understanding of their biosynthesis in Tistrella currently prevents the development of effective microbial or synthetic biological approaches for their production. To this end, we conducted a survey of known species of Tistrella and report on their ability to produce the didemnin depsipeptides. These data were used to develop conditions to produce didemnin B at titers over 15 mg/L.

The oxytosis/ferroptosis regulated cell death pathway recapitulates many features of mitochondrial dysfunction associated with the aging brain and has emerged as a potential key mediator of neurodegeneration. It has thus been proposed that the oxytosis/ferroptosis pathway can be used to identify novel drug candidates for the treatment of age-associated neurodegenerative diseases that act by preserving mitochondrial function. Previously, we identified cannabinol (CBN) as a potent neuroprotector. Here, we demonstrate that not only does CBN protect nerve cells from oxytosis/ferroptosis in a manner that is dependent on mitochondria and it does so independently of cannabinoid receptors. Specifically, CBN directly targets mitochondria and preserves key mitochondrial functions including redox regulation, calcium uptake, membrane potential, bioenergetics, biogenesis, and modulation of fusion/fission dynamics that are disrupted following induction of oxytosis/ferroptosis. These protective effects of CBN are at least partly mediated by the promotion of endogenous antioxidant defenses and the activation of AMP-activated protein kinase (AMPK) signaling. Together, our data highlight the potential of mitochondrially-targeted compounds such as CBN as novel oxytotic/ferroptotic inhibitors to rescue mitochondrial dysfunction as well as opportunities for the discovery and development of future neurotherapeutics., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Next-generation sequencing studies of saliva and dental plaque from subjects in both healthy and diseased states have identified bacteria belonging to the Rothia genus as ubiquitous members of the oral microbiota. To gain a deeper understanding of molecular mechanisms underlying the chemical ecology of this unexplored group, we applied a genome mining approach that targets functionally important biosynthetic gene clusters (BGCs). All 45 genomes that were mined, representing Rothia mucilaginosa , Rothia dentocariosa , and Rothia aeria , harbored a catechol-siderophore-like BGC. To explore siderophore production further, we grew the previously characterized R. mucilaginosa ATCC 25296 in liquid cultures, amended with glycerol, which led to the identification of the archetype siderophore enterobactin by using tandem liquid chromatography-mass spectrometry (LC-MS/MS), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) spectroscopy. Normally attributed to pathogenic gut bacteria, R. mucilaginosa is the first commensal oral bacterium found to produce enterobactin. Cocultivation studies including R. mucilaginosa or purified enterobactin revealed that enterobactin reduced growth of certain strains of cariogenic Streptococcus mutans and pathogenic strains of Staphylococcus aureus Commensal oral bacteria were either unaffected, reduced in growth, or induced to grow adjacent to enterobactin-producing R. mucilaginosa or the pure compound. Taken together with Rothia 's known capacity to ferment a variety of carbohydrates and amino acids, our findings of enterobactin production add an additional level of explanation to R. mucilaginosa 's prevalence in the oral cavity. Enterobactin is the strongest Fe(III) binding siderophore known, and its role in oral health requires further investigation. IMPORTANCE The communication language of the human oral microbiota is vastly underexplored. However, a few studies have shown that specialized small molecules encoded by BGCs have critical roles such as in colonization resistance against pathogens and quorum sensing. Here, by using a genome mining approach in combination with compound screening of growth cultures, we identified that the commensal oral community member R. mucilaginosa harbors a catecholate-siderophore BGC, which is responsible for the biosynthesis of enterobactin. The iron-scavenging role of enterobactin is known to have positive effects on the host's iron pool and negative effects on host immune function; however, its role in oral health remains unexplored. R. mucilaginosa was previously identified as an abundant community member in cystic fibrosis, where bacterial iron cycling plays a major role in virulence development. With respect to iron's broad biological importance, iron-chelating enterobactin may explain R. mucilaginosa 's colonization success in both health and disease., (Copyright © 2020 Uranga et al.)

Marine cyanobacteria (blue-green algae) have been shown to possess an enormous capacity to produce structurally diverse natural products that exhibit a broad spectrum of potent biological activities, including cytotoxic, antifungal, antiparasitic, antiviral, and antibacterial activities. Using mass-spectrometry-guided fractionation together with molecular networking, cyanobacterial field collections from American Samoa and Palmyra Atoll yielded three new cyclic peptides, tutuilamides A-C. Their structures were established by spectroscopic techniques including 1D and 2D NMR, HR-MS, and chemical derivatization. Structure elucidation was facilitated by employing advanced NMR techniques including nonuniform sampling in combination with the 1,1-ADEQUATE experiment. These cyclic peptides are characterized by the presence of several unusual residues including 3-amino-6-hydroxy-2-piperidone and 2-amino-2-butenoic acid, together with a novel vinyl chloride-containing residue. Tutuilamides A-C show potent elastase inhibitory activity together with moderate potency in H-460 lung cancer cell cytotoxicity assays. The binding mode to elastase was analyzed by X-ray crystallography revealing a reversible binding mode similar to the natural product lyngbyastatin 7. The presence of an additional hydrogen bond with the amino acid backbone of the flexible side chain of tutuilamide A, compared to lyngbyastatin 7, facilitates its stabilization in the elastase binding pocket and possibly explains its enhanced inhibitory potency.

This report describes the first application of the novel NMR-based machine learning tool "Small Molecule Accurate Recognition Technology" (SMART 2.0) for mixture analysis and subsequent accelerated discovery and characterization of new natural products. The concept was applied to the extract of a filamentous marine cyanobacterium known to be a prolific producer of cytotoxic natural products. This environmental Symploca extract was roughly fractionated, and then prioritized and guided by cancer cell cytotoxicity, NMR-based SMART 2.0, and MS 2 -based molecular networking. This led to the isolation and rapid identification of a new chimeric swinholide-like macrolide, symplocolide A, as well as the annotation of swinholide A, samholides A-I, and several new derivatives. The planar structure of symplocolide A was confirmed to be a structural hybrid between swinholide A and luminaolide B by 1D/2D NMR and LC-MS 2 analysis. A second example applies SMART 2.0 to the characterization of structurally novel cyclic peptides, and compares this approach to the recently appearing "atomic sort" method. This study exemplifies the revolutionary potential of combined traditional and deep learning-assisted analytical approaches to overcome longstanding challenges in natural products drug discovery.

The discovery of biologically active small molecules requires sifting through large amounts of data to identify unique or unusual arrangements of atoms. Here, we develop, test and evaluate an atom-based sort to identify novel features of secondary metabolites and demonstrate its use to evaluate novelty in marine microbial and sponge extracts. This study outlines an important ongoing advance towards the translation of autonomous systems to identify, and ultimately elucidate, atomic novelty within a complex mixture of small molecules., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A lack of selectivity of classical agonists for the nicotinic acetylcholine receptors (nAChR) has prompted us to identify and develop a distinct scaffold of α7 nAChR-selective ligands. Noncanonical 2,4,6-substituted pyrimidine analogues were framed around compound 40 for a structure-activity relationship study. The new lead compounds activate selectively the α7 nAChRs with EC 50 's between 30 and 140 nM in a PNU-120596-dependent, cell-based calcium influx assay. After characterizing the expanded lead landscape, we ranked the compounds for rapid activation using Xenopus oocytes expressing human α7 nAChR with a two-electrode voltage clamp. This approach enabled us to define the molecular determinants governing rapid activation, agonist potency, and desensitization of α7 nAChRs after exposure to pyrimidine analogues, thereby distinguishing this subclass of noncanonical agonists from previously defined types of agonists (agonists, partial agonists, silent agonists, and ago-PAMs). By NMR, we analyzed p K a values for ionization of lead candidates, demonstrating distinctive modes of interaction for this landscape of ligands.

Three decahydroisoquinoline alkaloids, lepadins I-K, were isolated from a specimen of Didemnum sp. collected in the Bahamas. The structures of the new compounds were assigned by an integrated analysis of MS, IR, and 1 H, 13 C, and 2D NMR spectra. Like previously reported lepadins, the structures of the new compounds contain a decahydroquinoline heterocyclic core in lepadin I, and a new variation, an octahydroquinoline in lepadin J, but differ from earlier reported compounds by acylation of the 3-hydroxyl group by a rare 3'-methylthioacrylate. The absolute configuration of lepadin I was solved by interpretation of NOE measurements, and exciton coupled circular dichroism (ECCD) of the corresponding N- p-bromobenzoyl derivative. The latter constitutes a general method for determination of absolute configuration of the entire lepadin family. The configuration of the remote side-chain secondary carbinol was solved by the modified Mosher's esters method. Lepadin I inhibited butyrylcholineesterase (BuChE, IC 50 3.1 μM), but only weakly inhibited acetylcholineesterase (AChE) (10% at 100 μM).

Viruses are underrepresented as targets in pharmacological screening efforts, given the difficulties of devising suitable cell-based and biochemical assays. In this study we found that a pre-fractionated organic extract of the Red Sea sponge Amphimedon chloros was able to inhibit the West Nile Virus NS3 protease (WNV NS3). Using liquid chromatography⁻mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy, the identity of the bioactive compound was determined as a 3-alkylpyridinium with m / z = 190.16. Diffusion Ordered Spectroscopy (DOSY) NMR and NMR relaxation rate analysis suggest that the bioactive compound forms oligomers of up to 35 kDa. We observed that at 9.4 μg/mL there was up to 40⁻70% inhibitory activity on WNV NS3 protease in orthogonal biochemical assays for solid phase extracts (SPE) of A. chloros . However, the LC-MS purified fragment was effective at inhibiting the protease up to 95% at an approximate amount of 2 µg/mL with negligible cytotoxicity to HeLa cells based on a High-Content Screening (HCS) cytological profiling strategy. To date, 3-alkylpyridinium type natural products have not been reported to show antiviral activity since the first characterization of halitoxin, or 3-alkylpyridinium, in 1978. This study provides the first account of a 3-alkylpyridinium complex that exhibits a proposed antiviral activity by inhibiting the NS3 protease. We suggest that the here-described compound can be further modified to increase its stability and tested in a cell-based assay to explore its full potential as a potential novel antiviral capable of inhibiting WNV replication.

Various algorithms comparing 2D NMR spectra have been explored for their ability to dereplicate natural products as well as determine molecular structures. However, spectroscopic artefacts, solvent effects, and the interactive effect of functional group(s) on chemical shifts combine to hinder their effectiveness. Here, we leveraged Non-Uniform Sampling (NUS) 2D NMR techniques and deep Convolutional Neural Networks (CNNs) to create a tool, SMART, that can assist in natural products discovery efforts. First, an NUS heteronuclear single quantum coherence (HSQC) NMR pulse sequence was adapted to a state-of-the-art nuclear magnetic resonance (NMR) instrument, and data reconstruction methods were optimized, and second, a deep CNN with contrastive loss was trained on a database containing over 2,054 HSQC spectra as the training set. To demonstrate the utility of SMART, several newly isolated compounds were automatically located with their known analogues in the embedded clustering space, thereby streamlining the discovery pipeline for new natural products.

Natural product screening programs have uncovered molecules from diverse natural sources with various biological activities and unique structures. However, much is yet underexplored and additional information is hidden in these exceptional collections. We applied untargeted mass spectrometry approaches to capture the chemical space and dispersal patterns of metabolites from an in-house library of marine cyanobacterial and algal collections. Remarkably, 86% of the metabolomics signals detected were not found in other available datasets of similar nature, supporting the hypothesis that marine cyanobacteria and algae possess distinctive metabolomes. The data were plotted onto a world map representing eight major sampling sites, and revealed potential geographic locations with high chemical diversity. We demonstrate the use of these inventories as a tool to explore the diversity and distribution of natural products. Finally, we utilized this tool to guide the isolation of a new cyclic lipopeptide, yuvalamide A, from a marine cyanobacterium.

Background: Genetic progress in selection for greater body mass and meat yield in poultry has been associated with an increase in gait problems which are detrimental to productivity and welfare. The incidence of suboptimal gait in breeding flocks is controlled through the use of a visual gait score, which is a subjective assessment of walking ability of each bird. The subjective nature of the visual gait score has led to concerns over its effectiveness in reducing the incidence of suboptimal gait in poultry through breeding. The aims of this study were to assess the reliability of the current visual gait scoring system in ducks and to develop a more objective method to select for better gait., Results: Experienced gait scorers assessed short video clips of walking ducks to estimate the reliability of the current visual gait scoring system. Kendall's coefficients of concordance between and within observers were estimated at 0.49 and 0.75, respectively. In order to develop a more objective scoring system, gait components were visually scored on more than 4000 pedigreed Pekin ducks and genetic parameters were estimated for these components. Gait components, which are a more objective measure, had heritabilities that were as good as, or better than, those of the overall visual gait score., Conclusions: Measurement of gait components is simpler and therefore more objective than the standard visual gait score. The recording of gait components can potentially be automated, which may increase accuracy further and may improve heritability estimates. Genetic correlations were generally low, which suggests that it is possible to use gait components to select for an overall improvement in both economic traits and gait as part of a balanced breeding programme.

Ultra-high resolution band-selective HSQC (bsHSQC) has been employed for detection of 35 Cl- 37 Cl isotope shifted 13 C NMR signals for assignment of regioisomerism in bromo-chloro natural products. Optimum pulse sequence and instrumental parameters for maximization of detection of the isotope shifts were explored. The chlorine isotope shifts (Δδ) were detected within crosspeaks and were shown to vary with hybridization of 13 C, substitution of 13 C, presence of β-chloro substituents, and their relative configuration. Deconvolution of Cl-substituted CH bsHSQC crosspeaks may provide other useful information, including a potentially MS-independent method for quantitating 37 Cl/ 35 C isotopic fractionation during the biosynthesis of halogenated natural products. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Pseudomonads are cosmopolitan microorganisms able to produce a wide array of specialized metabolites. These molecules allow Pseudomonas to scavenge nutrients, sense population density and enhance or inhibit growth of competing microorganisms. However, these valuable metabolites are typically characterized one-molecule-one-microbe at a time, instead of being inventoried in large numbers. To index and map the diversity of molecules detected from these organisms, 260 strains of ecologically diverse origins were subjected to mass-spectrometry-based molecular networking. Molecular networking not only enables dereplication of molecules, but also sheds light on their structural relationships. Moreover, it accelerates the discovery of new molecules. Here, by indexing the Pseudomonas specialized metabolome, we report the molecular-networking-based discovery of four molecules and their evolutionary relationships: a poaeamide analogue and a molecular subfamily of cyclic lipopeptides, bananamides 1, 2 and 3. Analysis of their biosynthetic gene cluster shows that it constitutes a distinct evolutionary branch of the Pseudomonas cyclic lipopeptides. Through analysis of an additional 370 extracts of wheat-associated Pseudomonas, we demonstrate how the detailed knowledge from our reference index can be efficiently propagated to annotate complex metabolomic data from other studies, akin to the way in which newly generated genomic information can be compared to data from public databases.

Colibactin is an as-yet-uncharacterized genotoxic secondary metabolite produced by human gut bacteria. Here we report the biosynthetic discovery of two new precolibactin molecules from Escherichia coli, including precolibactin-886, which uniquely incorporates the highly sought genotoxicity-associated aminomalonate building block into its unprecedented macrocyclic structure. This work provides new insights into the biosynthetic logic and mode of action of this colorectal-cancer-linked microbial chemical.

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry (MS) techniques are well-suited to high-throughput characterization of NP, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social Molecular Networking (GNPS; http://gnps.ucsd.edu), an open-access knowledge base for community-wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS, crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of 'living data' through continuous reanalysis of deposited data.

Many halogenases interchangeably incorporate chlorine and bromine into organic molecules. On the basis of an unsubstantiated report that the alga Ochromonas danica, a prodigious producer of chlorosulfolipids, was able to produce bromosulfolipids, we have investigated the promiscuity of its halogenases toward bromine incorporation. We have found that bromosulfolipids are produced with the exact positional and stereochemical selectivity as in the chlorosulfolipid danicalipin A when this alga is grown under modified conditions containing excess bromide ion.

We introduce a means of efficiently photo-uncaging active compounds from amino-1,4-benzoquinone in aqueous environments. Aqueous photochemistry of this photocage with one-photon red light is typically not efficient unless the photocaged molecules are allowed to assemble into nanoparticles. A variety of biologically active molecules were functionalized with the photocage and subsequently formulated into water-dispersible nanoparticles. Red light irradiation through various mammalian tissues achieved efficient photo-uncaging. Co-encapsulation of NIR fluorescent dyes and subsequent photomodulation provides a NIR fluorescent tool to assess both particle location and successful photorelease.

Background: Poultry account for the most numerous species farmed for meat and have been subject to intense selection over approximately 60 generations. To assess morphological changes which have occurred in the avian leg due to selection for rapid growth and high meat yields, divergent lines of chicken (Gallus gallus) and duck (Anas platyrhynchos) were studied between 3 and 7 weeks of age. For each line, femoral and tibiotarsal morphology was recorded using computed tomography scanning and tibiotarsal bone quality measures (stiffness, bending stress and porosity) were assessed., Results: In chicken and duck, divergence in hindlimb morphology has occurred in the commercial meat lines compared to their lighter conspecifics. As expected, the differences were largest between species. Leg development nears completion much earlier in ducks than in chickens. Duck tibiotarsi showed a large degree of lateral curvature, which is expected to affect foot position during swimming and walking, and thus to influence gait. All lines have adapted their tibiotarsal morphology to suit the loading forces they experience; however bone quality was found to be poorer in chickens., Conclusions: We demonstrate that intensive selection for growth rate in both chickens and ducks has resulted in leg morphology changes, which are likely to influence gait. Ducks represent an interesting compromise of adaptation for efficient locomotion in two media-on land and in water. Some aspects of bone morphology in the duck, such as lateral curvature of the tibiotarsus, may result from adaptation to swimming, which potentially imposes limitations on terrestrial locomotion.

Four new chlorobromohydrins, mollenynes B-E, were isolated from the marine sponge Spirastrella mollis collected from Hogsty Reef, Bahamas. Their structures were elucidated by integrated analysis of NMR, MS, and computational methods. A high-resolution band-selective HSQC experiment was developed to identify (13)C NMR signals in samples at the nanomole-scale that arise from Cl-substituted (13)C by exploiting the (35)Cl/(37)Cl isotope shift.

An innovative approach was developed for the discovery of new natural products by combining mass spectrometric metabolic profiling with genomic analysis and resulted in the discovery of the columbamides, a new class of di- and trichlorinated acyl amides with cannabinomimetic activity. Three species of cultured marine cyanobacteria, Moorea producens 3L, Moorea producens JHB, and Moorea bouillonii PNG, were subjected to genome sequencing and analysis for their recognizable biosynthetic pathways, and this information was then compared with their respective metabolomes as detected by MS profiling. By genome analysis, a presumed regulatory domain was identified upstream of several previously described biosynthetic gene clusters in two of these cyanobacteria, M. producens 3L and M. producens JHB. A similar regulatory domain was identified in the M. bouillonii PNG genome, and a corresponding downstream biosynthetic gene cluster was located and carefully analyzed. Subsequently, MS-based molecular networking identified a series of candidate products, and these were isolated and their structures rigorously established. On the basis of their distinctive acyl amide structure, the most prevalent metabolite was evaluated for cannabinomimetic properties and found to be moderate affinity ligands for CB1.

A small library of synthetic (-)-palmyrolide A diastereomers, analogues, and acyclic precursors have been examined with respect to their interaction with voltage-gated sodium channels (VGSCs). Toward this goal, the ability of (-)-palmyrolide A and analogues to antagonize veratridine-stimulated Na(+) influx in primary cultures of mouse cerebrocortical neurons was assessed. We found that synthetic (-)-palmyrolide A and its enantiomer functioned as VGSC antagonists to block veratridine-induced sodium influx. A detailed NMR and computational analysis of four diastereomers revealed that none had the same combination of shape and electrostatic potential as exhibited by natural (-)-palmyrolide A. These data indicate that the relative configuration about the tert-butyl and methyl substituents appears to be a prerequisite for biological function. Additional testing revealed that the enamide double bond was not necessary for blocking veratridine-induced sodium influx, whereas the acyclic analogues and other macrolide diastereomers tested were inactive as inhibitors of VGSCs, suggesting that the intact macrolide was required.

Most (75%) of the anti-infectives that save countless lives and enormously improve quality of life originate from microbes found in nature. Herein, we described a global visualization of the detectable molecules produced from a single microorganism, which we define as the 'molecular network' of that organism, followed by studies to characterize the cellular effects of antibacterial molecules. We demonstrate that Streptomyces roseosporus produces at least four non-ribosomal peptide synthetase-derived molecular families and their gene subnetworks (daptomycin, arylomycin, napsamycin and stenothricin) were identified with different modes of action. A number of previously unreported analogs involving truncation, glycosylation, hydrolysis and biosynthetic intermediates and/or shunt products were also captured and visualized by creation of a map through MS/MS networking. The diversity of antibacterial compounds produced by S. roseosporus highlights the importance of developing new approaches to characterize the molecular capacity of an organism in a more global manner. This allows one to more deeply interrogate the biosynthetic capacities of microorganisms with the goal to streamline the discovery pipeline for biotechnological applications in agriculture and medicine. This is a contribution to a special issue to honor Chris Walsh's amazing career.

Glycosyl groups are an essential mediator of molecular interactions in cells and on cellular surfaces. There are very few methods that directly relate sugar-containing molecules to their biosynthetic machineries. Here, we introduce glycogenomics as an experiment-guided genome-mining approach for fast characterization of glycosylated natural products (GNPs) and their biosynthetic pathways from genome-sequenced microbes by targeting glycosyl groups in microbial metabolomes. Microbial GNPs consist of aglycone and glycosyl structure groups in which the sugar unit(s) are often critical for the GNP's bioactivity, e.g., by promoting binding to a target biomolecule. GNPs are a structurally diverse class of molecules with important pharmaceutical and agrochemical applications. Herein, O- and N-glycosyl groups are characterized in their sugar monomers by tandem mass spectrometry (MS) and matched to corresponding glycosylation genes in secondary metabolic pathways by a MS-glycogenetic code. The associated aglycone biosynthetic genes of the GNP genotype then classify the natural product to further guide structure elucidation. We highlight the glycogenomic strategy by the characterization of several bioactive glycosylated molecules and their gene clusters, including the anticancer agent cinerubin B from Streptomyces sp. SPB74 and an antibiotic, arenimycin B, from Salinispora arenicola CNB-527.

Acyl carrier proteins (ACPs) are a group of highly conserved and abundant proteins in bacteria. ACPs play a central role as the acyl group carriers in bacterial fatty acid biosynthesis, providing building blocks for membrane biogenesis and the production of secondary metabolites. In the versatile human pathogen Pseudomonas aeruginosa, three ACP homologs have been identified. One homolog, AcpP, exhibits the strongest sequence homology to the canonical Escherichia coli ACP. Here we report the (1)H, (13)C and (15)N assignments of the holo-AcpP of P. aeruginosa.

Arginine-rich motifs (ARMs) capable of binding diverse RNA structures play critical roles in transcription, translation, RNA trafficking, and RNA packaging. The regulatory HIV-1 protein Rev is essential for viral replication and belongs to the ARM family of RNA-binding proteins. During the early stages of the HIV-1 life cycle, incompletely spliced and full-length viral mRNAs are very inefficiently recognized by the splicing machinery of the host cell and are subject to degradation in the cell nucleus. These transcripts harbor the Rev Response Element (RRE), which orchestrates the interaction with the Rev ARM and the successive Rev-dependent mRNA export pathway. Based on established criteria for predicting intrinsic disorder, such as hydropathy, combined with significant net charge, the very basic primary sequences of ARMs are expected to adopt coil-like structures. Thus, we initiated this study to investigate the conformational changes of the Rev ARM associated with RNA binding. We used multidimensional NMR and circular dichroism spectroscopy to monitor the observed structural transitions, and described the conformational landscapes using statistical ensemble and molecular-dynamics simulations. The combined spectroscopic and simulated results imply that the Rev ARM is intrinsically disordered not only as an isolated peptide but also when it is embedded into an oligomerization-deficient Rev mutant. RRE recognition triggers a crucial coil-to-helix transition employing an induced-fit mechanism., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Correlated networks of amino acids have been proposed to play a fundamental role in allostery and enzyme catalysis. These networks of amino acids can be traced from surface-exposed residues all the way into the active site, and disruption of these networks can decrease enzyme activity. Substitution of the distal Gly121 residue in Escherichia coli dihydrofolate reductase results in an up to 200-fold decrease in the hydride transfer rate despite the fact that the residue is located 15 Å from the active-site center. In this study, nuclear magnetic resonance relaxation experiments are used to demonstrate that dynamics on the picosecond to nanosecond and microsecond to millisecond time scales are changed significantly in the G121V mutant of dihydrofolate reductase. In particular, picosecond to nanosecond time scale dynamics are decreased in the FG loop (containing the mutated residue at position 121) and the neighboring active-site loop (the Met20 loop) in the mutant compared to those of the wild-type enzyme, suggesting that these loops are dynamically coupled. Changes in methyl order parameters reveal a pathway by which dynamic perturbations can be propagated more than 25 Å across the protein from the site of mutation. All of the enzyme complexes, including the model Michaelis complex with folate and nicotinamide adenine dinucleotide phosphate bound, assume an occluded ground-state conformation, and we do not observe sampling of a higher-energy closed conformation by (15)N R2 relaxation dispersion experiments. This is highly significant, because it is only in the closed conformation that the cofactor and substrate reactive centers are positioned for reaction. The mutation also impairs microsecond to millisecond time scale fluctuations that have been implicated in the release of product from the wild-type enzyme. Our results are consistent with an important role for Gly121 in controlling protein dynamics critical for enzyme function and further validate the dynamic energy landscape hypothesis of enzyme catalysis.

The ability to correlate the production of specialized metabolites to the genetic capacity of the organism that produces such molecules has become an invaluable tool in aiding the discovery of biotechnologically applicable molecules. Here, we accomplish this task by matching molecular families with gene cluster families, making these correlations to 60 microbes at one time instead of connecting one molecule to one organism at a time, such as how it is traditionally done. We can correlate these families through the use of nanospray desorption electrospray ionization MS/MS, an ambient pressure MS technique, in conjunction with MS/MS networking and peptidogenomics. We matched the molecular families of peptide natural products produced by 42 bacilli and 18 pseudomonads through the generation of amino acid sequence tags from MS/MS data of specific clusters found in the MS/MS network. These sequence tags were then linked to biosynthetic gene clusters in publicly accessible genomes, providing us with the ability to link particular molecules with the genes that produced them. As an example of its use, this approach was applied to two unsequenced Pseudoalteromonas species, leading to the discovery of the gene cluster for a molecular family, the bromoalterochromides, in the previously sequenced strain P. piscicida JCM 20779(T). The approach itself is not limited to 60 related strains, because spectral networking can be readily adopted to look at molecular family-gene cluster families of hundreds or more diverse organisms in one single MS/MS network.

The use of genome sequences has become routine in guiding the discovery and identification of microbial natural products and their biosynthetic pathways. In silico prediction of molecular features, such as metabolic building blocks, physico-chemical properties or biological functions, from orphan gene clusters has opened up the characterization of many new chemo- and genotypes in genome mining approaches. Here, we guided our genome mining of two predicted enediyne pathways in Salinispora tropica CNB-440 by a DNA interference bioassay to isolate DNA-targeting enediyne polyketides. An organic extract of S. tropica showed DNA-interference activity that surprisingly was not abolished in genetic mutants of the targeted enediyne pathways, ST_pks1 and spo. Instead we showed that the product of the orphan type II polyketide synthase pathway, ST_pks2, is solely responsible for the DNA-interfering activity of the parent strain. Subsequent comparative metabolic profiling revealed the lomaiviticins, glycosylated diazofluorene polyketides, as the ST_pks2 products. This study marks the first report of the 59 open reading frame lomaiviticin gene cluster (lom) and supports the biochemical logic of their dimeric construction through a pathway related to the kinamycin monomer., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mono- and multispecies microbial populations alter the chemistry of their surrounding environments during colony development thereby influencing multicellular behavior and interspecies interactions of neighboring microbes. Here we present a methodology that enables the creation of three-dimensional (3D) models of a microbial chemotype that can be correlated to the colony phenotype through multimodal imaging analysis. These models are generated by performing matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) imaging mass spectrometry (IMS) on serial cross-sections of microbial colonies grown on 8 mm deep agar, registering data sets of each serial section in MATLAB to create a model, and then superimposing the model with a photograph of the colonies themselves. As proof-of-principle, 3D models were used to visualize metabolic exchange during microbial interactions between Bacillus subtilis and Streptomyces coelicolor, as well as, Candida albicans and Pseudomonas aeruginosa. The resulting models were able to capture the depth profile of secreted metabolites within the agar medium and revealed properties of certain mass signals that were previously not observable using two-dimensional MALDI-TOF IMS. Most significantly, the 3D models were capable of mapping previously unobserved chemical distributions within the array of sub-surface hyphae of C. albicans and how this chemistry is altered by the presence of P. aeruginosa, an opportunistic pathogen known to alter virulence of C. albicans. It was determined that the presence of C. albicans triggered increased rhamnolipid production by P. aeruginosa, which in turn was capable of inhibiting embedded hyphal growth produced beneath the C. albicans colony at ambient temperature.

In the present paper we show a comprehensive in vitro, ex vivo and in vivo study on hydrolytic detoxification of nerve agent and pesticide OPs (organophosphates) catalysed by purified hBChE (human butyrylcholinesterase) in combination with novel non-pyridinium oxime reactivators. We identified TAB2OH (2-trimethylammonio-6-hydroxybenzaldehyde oxime) as an efficient reactivator of OP-hBChE conjugates formed by the nerve agents VX and cyclosarin, and the pesticide paraoxon. It was also functional in reactivation of sarin- and tabun-inhibited hBChE. A 3-5-fold enhancement of in vitro reactivation of VX-, cyclosarin- and paraoxon-inhibited hBChE was observed when compared with the commonly used N-methylpyridinium aldoxime reactivator, 2PAM (2-pyridinealdoxime methiodide). Kinetic analysis showed that the enhancement resulted from improved molecular recognition of corresponding OP-hBChE conjugates by TAB2OH. The unique features of TAB2OH stem from an exocyclic quaternary nitrogen and a hydroxy group, both ortho to an oxime group on a benzene ring. pH-dependences reveal participation of the hydroxy group (pKa=7.6) forming an additional ionizing nucleophile to potentiate the oxime (pKa=10) at physiological pH. The TAB2OH protective indices in therapy of sarin- and paraoxon-exposed mice were enhanced by 30-60% when they were treated with a combination of TAB2OH and sub-stoichiometric hBChE. The results of the present study establish that oxime-assisted catalysis is feasible for OP bioscavenging.

In polymicrobial infections, microbes can interact with both the host immune system and one another through direct contact or the secretion of metabolites, affecting disease progression and treatment options. The thick mucus in the lungs of patients with cystic fibrosis is highly susceptible to polymicrobial infections by opportunistic pathogens, including the bacterium Pseudomonas aeruginosa and the fungus Aspergillus fumigatus. Unravelling the hidden molecular interactions within such polymicrobial communities and their metabolic exchange processes will require effective enabling technologies applied to model systems. In the present study, MALDI-TOF and MALDI-FT-ICR imaging mass spectrometry (MALDI-IMS) combined with MS/MS networking were used to provide insight into the interkingdom interaction between P. aeruginosa and A. fumigatus at the molecular level. The combination of these technologies enabled the visualization and identification of metabolites secreted by these microorganisms grown on agar. A complex molecular interplay was revealed involving suppression, increased production, and biotransformation of a range of metabolites. Of particular interest is the observation that P. aeruginosa phenazine metabolites were converted by A. fumigatus into other chemical entities with alternative properties, including enhanced toxicities and the ability to induce fungal siderophores. This work highlights the capabilities of MALDI-IMS and MS/MS network analysis to study interkingdom interactions and provides insight into the complex nature of polymicrobial metabolic exchange and biotransformations.

Stairway climbing provides a ubiquitous and inconspicuous method of burning calories. While typically two strategies are employed for climbing stairs, climbing one stair step per stride or two steps per stride, research to date has not clarified if there are any differences in energy expenditure between them. Fourteen participants took part in two stair climbing trials whereby measures of heart rate were used to estimate energy expenditure during stairway ascent at speeds chosen by the participants. The relationship between rate of oxygen consumption ([Formula: see text]) and heart rate was calibrated for each participant using an inclined treadmill. The trials involved climbing up and down a 14.05 m high stairway, either ascending one step per stride or ascending two stair steps per stride. Single-step climbing used 8.5±0.1 kcal min(-1), whereas double step climbing used 9.2±0.1 kcal min(-1). These estimations are similar to equivalent measures in all previous studies, which have all directly measured [Formula: see text] The present study findings indicate that (1) treadmill-calibrated heart rate recordings can be used as a valid alternative to respirometry to ascertain rate of energy expenditure during stair climbing; (2) two step climbing invokes a higher rate of energy expenditure; however, one step climbing is energetically more expensive in total over the entirety of a stairway. Therefore to expend the maximum number of calories when climbing a set of stairs the single-step strategy is better.

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35-40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all (1)H, (13)C, and (15)N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes.

Microarrays have been used extensively to identify differential gene expression at the level of transcriptional control in oncogenesis. However, increasing evidence indicates that changes in translational control are critical to oncogenic transformation. This study identifies mRNA transcripts that are differentially regulated, primarily at the level of translation, in the immortalized human embryonic prostate epithelial cell line 267B1 and the v-Ki-ras-transformed counterpart by comparing total mRNA to polysome-bound mRNA by using Affymetrix oligonucleotide microarrays. Among the transcripts that were identified were those encoding proteins involved in DNA replication, cell cycle control, cell-to-cell interactions, electron transport, G protein signaling, and translation. Many of these proteins are known to contribute to oncogenesis or have the potential to contribute to oncogenesis. Differential expression of RNA-binding proteins and the presence of highly conserved motifs in the 5' and 3' untranslated regions of the mRNAs are consistent with multiple pathways and mechanisms governing the changes in translational control. Although Alu sequences were found to be associated with increased translation in transformed cells, an evolutionarily conserved motif was identified in the 3' untranslated regions of ephrinB1, calreticulin, integrin alpha3, and mucin3B that was associated with decreased polysome association in 267B1/Ki-ras.