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The biosynthesis of the potent neurotoxin tetrodotoxin (TTX, 1) is still unresolved. We used MS-guided screening and nuclear magnetic resonance analyses including long-range HSQMBC to characterize ...

Natural products containing the highly unusual phosphotriester ring are known to be potent serine hydrolase inhibitors. The long-chain bicyclic enol-phosphotriester salinipostins (SPTs) from the marine actinomycete Salinispora have been identified as selective antimalarial agents. A potential regulatory function has been suggested for phosphotriesters based on their structural relationship with actinomycete signaling molecules and the prevalence of spt-like biosynthetic gene clusters across actinomycetes. In this study, we established a mass spectrometry–guided screening method for phosphotriesters focusing on their characteristic fragment ions. Applying this screening method to the SPT producer Salinispora tropica CNB-440, new SPT analogs (4-6) were discovered and their structures were elucidated by spectroscopic analyses. Previously known and herein-identified SPT analogs inhibited the activity of human monoacylglycerol lipase (MAGL), a key serine hydrolase in the endocannabinoid system, in the nanomolar range. Our method could be applied to the screening of phosphotriesters, potential serine hydrolase inhibitors and signaling molecules.

Bacterial hormones, such as the iconic gamma-butyrolactone A-factor, are essential signaling molecules that regulate diverse physiological processes, including specialized metabolism. These low molecular weight compounds are common in Streptomyces species and display species–specific structural differences. Recently, unusual gamma-butyrolactone natural products called salinipostins were isolated from the marine actinomycete genus Salinispora based on their antimalarial properties. As the salinipostins possess a rare phosphotriester motif of unknown biosynthetic origin, we set out to explore its construction by the widely conserved 9-gene spt operon in Salinispora species. We show through a series of in vivo and in vitro studies that the spt gene cluster dually encodes the salinipostins and newly identified natural A-factor-like gamma-butyrolactones (Sal-GBLs). Remarkably, homologous biosynthetic gene clusters are widely distributed among many actinomycete genera, including Streptomyces, suggesting the significance of this operon in bacteria.

The biosynthesis of tetrodotoxin (TTX, 1), a potent neurotoxin widely distributed in marine and terrestrial metazoans, remains unresolved. A significant issue has been identifying intermediates and...

The North American newt genera Taricha and Notophthalmus (order Caudata) are well known for the combination of potent toxicity, aposematic coloration, and striking defense postures that protects these animals from predation. This suite of traits is centered around the neurotoxin tetrodotoxin, which causes paralysis and death in metazoans by disrupting the initiation and propagation of electrical signals in the nerves and muscles. Tetrodotoxin defends newts from predation across multiple life history stages and its role in generating arms-race coevolution between Taricha newts and garter snake (genus Thamnophis) predators is well studied. However, understanding the broader picture of chemical defenses in Taricha and Notophthalmus requires an expanded comprehension of the defensive chemical ecology of tetrodotoxin that includes possible coevolutionary interactions with insect egg predators, protection against parasites, as well as mimicry complexes associated with tetrodotoxin and aposematic coloration in both genera. Herein the authors review what is known about the structure, function, and pharmacology of tetrodotoxin to explore its evolution and chemical ecology in the North American newt. Focus is made specifically on the origin and possible biosynthesis of tetrodotoxin in these taxa as well as providing an expanded picture of the web of interactions that contribute to landscape level patterns of toxicity and defense in Taricha and Notophthalmus.

Saxitoxin and its analogues, paralytic shellfish toxins (PSTs), are potent and specific voltage-gated sodium channel blockers. These toxins are produced by some species of freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates of PSTs, as well as new analogues, from such organisms and proposed the biosynthetic and metabolic pathways of PSTs. In this study, 12β-deoxygonyautoxin 5 (12α-gonyautoxinol 5 = gonyautoxin 5-12(

Streptococcus mutans is a common constituent of dental plaque and a major etiologic agent of dental caries (tooth decay). In this study, we elucidated the biosynthetic pathway encoded by muc, a hybrid polyketide synthase and nonribosomal peptide synthetase (PKS/NRPS) biosynthetic gene cluster (BGC), present in a number of globally distributed S. mutans strains. The natural products synthesized by muc included three N-acyl tetramic acid compounds (reutericyclin and two novel analogues) and an unacylated tetramic acid (mutanocyclin). Furthermore, the enzyme encoded by mucF was identified as a novel class of membrane-associated aminoacylases and was responsible for the deacylation of reutericyclin to mutanocyclin. A large number of hypothetical proteins across a broad diversity of bacteria were homologous to MucF, suggesting that this may represent a large family of unexplored acylases. Finally, S. mutans utilized the reutericyclin produced by muc to impair the growth of neighboring oral commensal bacteria. Since S. mutans must be able to out-compete these health-associated organisms to persist in the oral microbiota and cause disease, the competitive advantage conferred by muc suggests that this BGC is likely to be involved in S. mutans ecology and therefore dental plaque dysbiosis and the resulting caries pathogenesis.

The scallop, Patinopecten yessoensis, was screened for new saxitoxin analogues to study the metabolism of paralytic shellfish toxins (PSTs), and this resulted in the discovery of two new analogues: M5-hemiaminal (HA) and M6-HA. M5-HA was isolated and its structure was determined by using NMR spectroscopy. It contains hydrogen at C-4 with opposite stereochemistry to that in saxitoxin, and a hemiaminal was formed between 9-NH2 and the hydrated ketone at C-12 in α-orientation. This is the first reported structural feature in a natural saxitoxin analogue, whereas the same ring system has previously been reported in a synthetic saxitoxin analogue, FD-saxitoxin. Acid hydrolysis of the carbamoyl N-sulfate in M5-HA produced M6-HA which was also identified in P. yessoensis by using LC-MSMS. M5-HA was not synthetically produced from M1 (11-hydroxy gonyautoxin-5) and M3 (11,11-dihydroxy gonyautoxin-5) through incubation in aqueous buffers. Furthermore, PSTs in the hepatopancreas of P. yessoensis, cultured in a bay located in northeastern Japan, were chronologically analyzed in 2018. The highest concentrations of M1/M3/M5-HA were observed two weeks after C-toxins had reached their highest concentrations, which provides evidence that M1/M3/M5-HA are metabolites of C-toxins. The voltage-gated sodium channel blockage activity of M6-HA was not detected at the concentration of 140 nM by using the Neuro-2A veratridine/ouabain assay.

Bacteria communicate by small-molecule chemicals that facilitate intra- and inter-species interactions. These extracellular signaling molecules mediate diverse processes including virulence, bioluminescence, biofilm formation, motility, and specialized metabolism. The signaling molecules produced by members of the phylum Actinobacteria are generally comprised of γ-butyrolactones, γ-butenolides, and furans. The best known actinomycete γ-butyrolactone is A-factor, which triggers specialized metabolism and morphological differentiation in the genus Streptomyces. Salinipostins A-K are unique γ-butyrolactone molecules with rare phosphotriester moieties that were recently characterized from the marine actinomycete genus Salinispora. The production of these compounds has been linked to the 9-gene biosynthetic gene cluster spt. Critical to salinipostin assembly is the γ-butyrolactone synthase encoded by spt9. Here, we report the global distribution of spt9 among sequenced bacterial genomes, revealing a surprising diversity of gene homologs across 12 bacterial phyla, the majority of which are not known to produce γ-butyrolactones. Further analyses uncovered a large group of spt-like gene clusters outside of the genus Salinispora, suggesting the production of new salinipostin-like diversity. These gene clusters show evidence of horizontal transfer between many bacterial taxa and location specific homologous recombination exchange among Salinispora strains. The results suggest that γ-butyrolactone production may be more widespread than previously recognized. The identification of new γ-butyrolactone biosynthetic gene clusters is the first step towards understanding the regulatory roles of the encoded small molecules in Actinobacteria.ImportanceSignaling molecules orchestrate a wide variety of bacterial behaviors. Among Actinobacteria, γ-butyrolactones mediate morphological changes and regulate specialized metabolism. Despite their importance, few γ-butyrolactones have been linked to their cognate biosynthetic gene clusters. A new series of γ-butyrolactones called the salinipostins was recently identified from the marine actinomycete genus Salinispora and linked to the spt biosynthetic gene cluster. Here we report the detection of spt-like gene clusters in diverse bacterial families not known for the production of this class of compounds. This finding expands the taxonomic range of bacteria that may employ this class of compounds and provides opportunities to discover new compounds associated with chemical communication.

Bacterial hormones, such as the iconic gamma-butyrolactone A-factor, are essential signaling molecules that regulate diverse physiological processes, including specialized metabolism. These low molecular weight compounds are common in Streptomyces species and display species-specific structural differences. Recently, unusual gamma-butyrolactone natural products called salinipostins were isolated from the marine actinomycete genus Salinispora based on their anti-malarial properties. As the salinipostins possess a rare phosphotriester motif of unknown biosynthetic origin, we set out to explore its construction by the widely conserved 9-gene spt operon in Salinispora species. We show through a series of in vivo and in vitro studies that the spt gene cluster dually encodes the saliniphostins and newly identified A-factor-like gamma-butyrolactones (Sal-GBLs). Remarkably, homologous biosynthetic gene clusters are widely distributed amongst many actinomycete genera, including Streptomyces, suggesting the significance of this operon in bacteria.

The biosynthesis of tetrodotoxin (TTX

The fundamental building blocks of the proton, quarks and gluons, have been known for decades. However, we still have an incomplete theoretical and experimental understanding of how these particles and their dynamics give rise to the quantum bound state of the proton and its physical properties, such as for example its spin. The two up and the single down quarks that comprise the proton in the simplest picture account only for a few percent of the proton mass, the bulk of which is in the form of quark kinetic and potential energy and gluon energy from the strong force. An essential feature of this force, as described by quantum chromodynamics, is its ability to create matter-antimatter quark pairs inside the proton that exist only for a very short time. Their fleeting existence makes the antimatter quarks within protons difficult to study, but their existence is discernible in reactions where a matter-antimatter quark pair annihilates. In this picture of quark-antiquark creation by the strong force, the probability distributions as a function of momentum for the presence of up and down antimatter quarks should be nearly identical, since their masses are quite similar and small compared to the mass of the proton. In the present manuscript, we show evidence from muon pair production measurements that these distributions are significantly different, with more abundant down antimatter quarks than up antimatter quarks over a wide range of momentum. These results revive interest in several proposed mechanisms as the origin of this antimatter asymmetry in the proton that had been disfavored by the previous results and point to the future measurements that can distinguish between these mechanisms., 10 pages, 5 figures, 5 tables; Matches published version

Total syntheses of Cep-212 and Cep-210, predicted biosynthetic intermediates of tetrodotoxin isolated from the Japanese toxic newt, have been accomplished from geraniol by an intramolecular hetero Diels-Alder reaction as a key step in a highly stereoselective manner. The success of these syntheses enabled us to determine their absolute configurations by using a chiral normal-phase HPLC/MS analysis of the bis-dinitrobenzene derivative of natural Cep-212 and reference derivatives prepared from chemically synthesized enantiomers.

The analysis of volatiles released by marine Salinispora bacteria uncovered a new class of natural compounds displaying an unusual bicyclic [3.1.0]-lactone skeleton. Although only sub-μg quantities of the compounds were available, the combination of analytical methods, computational spectroscopy, and synthesis allowed unambiguous structural identification of the compounds, called salinilactones, without the need for isolation. Orthogonal hyphenated methods, GC/MS and solid-phase GC/IR allowed to propose a small set of structures consistent with the data. A candidate structure was selected by comparison of DFT-calculated IR spectra and the experimental IR-spectrum. Synthesis confirmed the structure and absolute configuration of three bicyclic lactones, salinilactones A–C. The salinilactones are structurally closely related to the A-factor class of compounds, autoregulators from streptomycete bacteria. They exhibited inhibitory activity against Salinispora and Streptomyces strains.

This work reports on the creation of an electroencephalogram (EEG) index and the discovery of a power ratio related to alpha band frequencies (8–13 Hz) that were experimentally found to have a high correlation with human mental fatigue. The EEG was measured from occipital lobe of the human brain by microfabricated dry candle-like microneedle electrodes (CMEs) that can avoid hairs and penetrate through the high-impedance stratum corneum. The newly identified EEG index exhibited higher correlations to mental fatigue states, which were rated subjectively via the visual analogue scale (VAS), than did heart rate variance, which is often used as the objective measure of fatigue levels. Interestingly, it was found that the index trend changed 10 min before the VAS did, which implies that the index could assess the onset of mental fatigue even before the subjects recognised it themselves. The identification of this EEG index, along with the ease of obtaining EEG measurements via CME, which is due to the fact that the CME does not require any skin preparation or conductive gels and can acquire EEGs from hair-covered parts of the scalp, can be expected to promote practical fatigue assessment applications that use EEGs.

This paper reports the EEG-Hat, the hat-type EEG device with candle-like dry microneedle electrodes. Current wearable EEG devices have two major problems; 1) no adjustability to every participant and 2) no ability to measure EEG at hairy part. If these problems are solved, health care technologies in daily life will be developed drastically. The EEG-Hat potentially solves these problems. This device can adjust the electrodes to different size and shape of human head. This device has shutter-like structures to separate hair, so using this device, it is possible to measure EEG without separating hair by hand. First, we quantified the force to press the electrodes and the amount of separating hair. Then, the EEG-Hat fabricated based on the findings successfully measured EEG from 3 hairy parts without manual separation.

We investigate a possible relation between frustration and phase-transition points in spin glasses. The relation is represented as a condition of the number of frustrated plaquettes in the lattice at phase-transition points at zero temperature and was reported to provide very close points to the phase-transition points for several lattices. Although there has been no proof of the relation, the good correspondence in several lattices suggests the validity of the relation and some important role of frustration in the phase transitions. To examine the relation further, we present a natural extension of the relation to diluted lattices and verify its effectiveness for bond-diluted square lattices. We then confirm that the resulting points are in good agreement with the phase-transition points in a wide range of dilution rate. Our result supports the suggestion from the previous work for non-diluted lattices on the importance of frustration to the phase transition of spin glasses., 6 pages, 4 figures

Streptococcus mutans is a common constituent of dental plaque and a major etiologic agent of dental caries (tooth decay). In this study, we elucidated the biosynthetic pathway encoded by muc, a hybrid polyketide synthase and nonribosomal peptide synthetase (PKS/NRPS) biosynthetic gene cluster (BGC), present in a number of globally distributed S. mutans strains. The natural products synthesized by muc included three N-acyl tetramic acid compounds (reutericyclin and two novel analogues) and an unacylated tetramic acid (mutanocyclin). Furthermore, the enzyme encoded by mucF was identified as a novel class of membrane-associated aminoacylases and was responsible for the deacylation of reutericyclin to mutanocyclin. A large number of hypothetical proteins across a broad diversity of bacteria were homologous to MucF, suggesting that this may represent a large family of unexplored acylases. Finally, S. mutans utilized the reutericyclin produced by muc to impair the growth of neighboring oral commensal bacteria. Since S. mutans must be able to out-compete these health-associated organisms to persist in the oral microbiota and cause disease, the competitive advantage conferred by muc suggests that this BGC is likely to be involved in S. mutans ecology and therefore dental plaque dysbiosis and the resulting caries pathogenesis.

Streptococcus mutansis a common constituent of dental plaque and an etiologic agent of dental caries (tooth decay). Here we elucidate a biosynthetic pathway, encoded by globally distributed strains ofS. mutans, which produces a series of bioactive small molecules including reutericyclin and twoN-acyl tetramic acid analogues active against oral commensal bacteria. This pathway may provideS. mutanswith a competitive advantage, promoting dysbiosis and caries pathogenesis.

Author(s): Aidala, CA; Arrington, JR; Ayuso, C; Bowen, BM; Bowen, ML; Bowling, KL; Brown, AW; Brown, CN; Byrd, R; Carlisle, RE; Chang, T; Chang, WC; Chen, A; Chen, JY; Christian, DC; Chu, X; Dannowitz, BP; Daugherity, M; Diefenthaler, M; Dove, J; Durandet, C; El Fassi, L; Erdos, E; Fox, DM; Geesaman, DF; Gilman, R; Goto, Y; Guo, L; Guo, R; Hague, T; Hicks, CR; Holt, RJ; Isenhower, D; Jiang, X; Katich, JM; Kerns, BM; Kinney, ER; Kitts, ND; Klein, A; Kleinjan, D; Kras, J; Kudo, Y; Lin, PJ; Liu, D; Liu, K; Liu, MX; Lorenzon, W; Makins, NCR; Martinez, JD; McClellan, RE; McDonald, B; McGaughey, PL; McNease, SE; Medeiros, MM; Miller, B; Miller, AJ; Miyasaka, S; Miyachi, Y; Mooney, IA; Morton, DH; Nadim, B; Nagai, K; Nakahara, K; Nakano, K; Nara, S; Obata, S; Peng, JC; Prasad, S; Puckett, AJR; Ramson, BJ; Raymond, RS; Reimer, PE; Rubin, JG; Salinas, R; Sanftl, F; Sawada, S; Sawada, T; Scott, MBC; Selensky, LE; Shibata, TA; Shiu, S; Su, D; Tadepalli, AS; Teo, M; Tice, BG | Abstract: The SeaQuest spectrometer at Fermilab was designed to detect oppositely-charged pairs of muons (dimuons) produced by interactions between a 120 GeV proton beam and liquid hydrogen, liquid deuterium and solid nuclear targets. The primary physics program uses the Drell–Yan process to probe antiquark distributions in the target nucleon. The spectrometer consists of a target system, two dipole magnets and four detector stations. The upstream magnet is a closed-aperture solid iron magnet which also serves as the beam dump, while the second magnet is an open aperture magnet. Each of the detector stations consists of scintillator hodoscopes and a high-resolution tracking device. The FPGA-based trigger compares the hodoscope signals to a set of pre-programmed roads to determine if the event contains oppositely-signed, high-mass muon pairs.

Tetrodotoxin (TTX, 1), a potent neurotoxin, has been found in various animal species in both marine and terrestrial environments. In this study, a new TTX analogue, 8- epiTTX (2), and a possible biosynthetic shunt compound of TTX, Cep-226A (3), were isolated from the newt Cynops ensicauda popei. The voltage-gated sodium ion channel (Nav) blocking activity of 2 and 6- epiTTX (4), a known analogue, were investigated by a colorimetric cell-based assay and compared with that of 1. The EC50 values for 2 and 4 were determined to be 110 ± 40 and 33 ± 11 nM, respectively, which were larger than that of 1 (1.9 ± 0.7 nM). The results indicated that the equatorial hydroxy group at C-8 in TTX significantly contributes to its Nav blocking activity, whereas the 6-epimer of TTX retains substantial activity, consistent with its previously reported toxicity in mice and binding affinity to rat brain membrane preparations. The presence of these epimers of TTX (2 and 4) and Cep-226A (3) in newts supports our hypothesis that TTX is derived from a monoterpene in terrestrial environments.

This paper reports successful measurement of even-related potential (ERP) using candle-like dry microneedle electrodes, which can acquire high-quality electroencephalogram (EEG) from hairy parts without any pretreatment. In our previous work, we successfully measured spontaneous EEG activity and its application to assess the stress state of the subjects. ERPs originate from electrophysiological response to stimulus and are one of the most important indices to capture the cognitive and sensory activities. In this work, using the candle-like dry microelectrodes, we demonstrate successful measurement of ERPs elicited by oddball tasks. Two oddball tasks using pure tone stimuli and speech stimuli were assigned to the subjects, where EEG was acquired from the parietal region (Cz in international 10-20 system). Note that no pretreatment, such as removal of hairs and abrasion of the scalp, was applied. As a result, P300 and mismatch negativity (MMN) were successfully measured in the both oddball tasks from the averaged EEG after the stimuli. Based on these results and given the attractive natures of the candle-like dry microneedle electrodes; they do not need any skin treatment and conductive gels and they can measure EEG from the hairy parts, the developed electrodes will accelerate cognitive neuroscience research using ERPs.