The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells.

BACKGROUND: Triptans are anti-migraine drugs with a potential central site of action. However, it is not known to what extent triptans cross the blood-brain barrier (BBB). The aim of this study was therefore to determine if triptans pass the brain capillary endothelium and investigate the possible underlying mechanisms with focus on the involvement of the putative proton-coupled organic cation (H+/OC) antiporter. Additionally, we evaluated whether triptans interacted with the efflux transporter, P-glycoprotein (P-gp). METHODS: We investigated the cellular uptake characteristics of the prototypical H+/OC antiporter substrates, pyrilamine and oxycodone, and seven different triptans in the human brain microvascular endothelial cell line, hCMEC/D3. Triptan interactions with P-gp were studied using the IPEC-J2 MDR1 cell line. Lastly, in vivo neuropharmacokinetic assessment of the unbound brain-to-plasma disposition of eletriptan was conducted in wild type and mdr1a/1b knockout mice. RESULTS: We demonstrated that most triptans were able to inhibit uptake of the H+/OC antiporter substrate, pyrilamine, with eletriptan emerging as the strongest inhibitor. Eletriptan, almotriptan, and sumatriptan exhibited a pH-dependent uptake into hCMEC/D3 cells. Eletriptan demonstrated saturable uptake kinetics with an apparent Km of 89 ± 38 µM and a Jmax of 2.2 ± 0.7 nmol·min-1·mg protein-1 (n = 3). Bidirectional transport experiments across IPEC-J2 MDR1 monolayers showed that eletriptan is transported by P-gp, thus indicating that eletriptan is both a substrate of the H+/OC antiporter and P-gp. This was further confirmed in vivo, where the unbound brain-to-unbound plasma concentration ratio (Kp,uu) was 0.04 in wild type mice while the ratio rose to 1.32 in mdr1a/1b knockout mice. CONCLUSIONS: We have demonstrated that the triptan family of compounds possesses affinity for the H+/OC antiporter proposing that the putative H+/OC antiporter plays a role in the BBB transport of triptans, particularly eletriptan. Our in vivo studies indicate that eletriptan is subjected to simultaneous brain uptake and efflux, possibly facilitated by the putative H+/OC antiporter and P-gp, respectively. Our findings offer novel insights into the potential central site of action involved in migraine treatment with triptans and highlight the significance of potential transporter related drug-drug interactions.

Nontargeted metabolomic insights into the behavioral effects of 5-MeO-MiPT in zebrafish (Danio rerio).

5-Methoxy-N-methyl-N-isopropyltryptamine (5-MeO-MiPT) is a novel psychoactive substance exhibiting a tryptamine structure. Despite its increasing prevalence, the environmental impact of 5-MeO-MiPT remains unexplored. Our prior investigation revealed that 5-MeO-MiPT induced inhibited spontaneous movement and prompted anxiety-like behavior in adult zebrafish-a validated toxicological model. To elucidate this phenomenon and establish a correlation between metabolomics and behavioral changes induced by 5-MeO-MiPT, zebrafish were administered varying drug concentrations. Zebrafishes were subjected to injections of different 5-MeO-MiPT concentrations. Subsequent metabolomic analysis of endogenous metabolites affected by the drug unveiled substantial variations in metabolic levels between the control group and the drug-injected cohorts. A total of 22 distinct metabolites emerged as potential biomarkers. Further scrutiny identified seven pathways significantly influenced by 5-MeO-MiPT. A focused exploration into amino acid metabolism, lipid metabolism, and energy metabolism unveiled that the metabolic repercussions of 5-MeO-MiPT on zebrafish resulted in observable brain damage. Notably, the study identified a consequential disruption in the liver-brain pathway. The comprehensive metabolomic approach employed herein effectively discerned the impact of 5-MeO-MiPT on zebrafish metabolism. This approach also shed light on the mechanism underpinning the anxiety-like behavior observed in zebrafish post-drug injection. Specifically, our findings indicate that 5-MeO-MiPT induces brain damage, particularly within the liver-brain pathway.

A journey with psychedelic mushrooms: From historical relevance to biology, cultivation, medicinal uses, biotechnology, and beyond.

Psychedelic mushrooms containing psilocybin and related tryptamines have long been used for ethnomycological purposes, but emerging evidence points to the potential therapeutic value of these mushrooms to address modern neurological, psychiatric health, and related disorders. As a result, psilocybin containing mushrooms represent a re-emerging frontier for mycological, biochemical, neuroscience, and pharmacology research. This work presents crucial information related to traditional use of psychedelic mushrooms, as well as research trends and knowledge gaps related to their diversity and distribution, technologies for quantification of tryptamines and other tryptophan-derived metabolites, as well as biosynthetic mechanisms for their production within mushrooms. In addition, we explore the current state of knowledge for how psilocybin and related tryptamines are metabolized in humans and their pharmacological effects, including beneficial and hazardous human health implications. Finally, we describe opportunities and challenges for investigating the production of psychedelic mushrooms and metabolic engineering approaches to alter secondary metabolite profiles using biotechnology integrated with machine learning. Ultimately, this critical review of all aspects related to psychedelic mushrooms represents a roadmap for future research efforts that will pave the way to new applications and refined protocols.

Spatial localization of monoterpenoid indole alkaloids in Rauvolfia tetraphylla by high resolution mass spectrometry imaging.

Monoterpenoid indole alkaloids (MIAs) are a large group of biosynthetic compounds, which have pharmacological properties. One of these MIAs, reserpine, was discovered in the 1950s and has shown properties as an anti-hypertension and anti-microbial agent. Reserpine was found to be produced in various plant species within the genus of Rauvolfia. However, even though its presence is well known, it is still unknown in which tissues Rauvolfia produce reserpine and where the individual steps in the biosynthetic pathway take place. In this study, we explore how matrix assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI) mass spectrometry imaging (MSI) can be used in the investigation of a proposed biosynthetic pathway by localizing reserpine and the theoretical intermediates of it. The results show that ions corresponding to intermediates of reserpine were localized in several of the major parts of Rauvolfia tetraphylla when analyzed by MALDI- and DESI-MSI. In stem tissue, reserpine and many of the intermediates were found compartmentalized in the xylem. For most samples, reserpine itself was mainly found in the outer layers of the sample, suggesting it may function as a defense compound. To further confirm the place of the different metabolites in the reserpine biosynthetic pathway, roots and leaves of R. tetraphylla were fed a stable-isotope labelled version of the precursor tryptamine. Subsequently, several of the proposed intermediates were detected in the normal version as well as in the isotope labelled versions, confirming that they were synthesized in planta from tryptamine. In this experiment, a potential novel dimeric MIA was discovered in leaf tissue of R. tetraphylla. The study constitutes to date the most comprehensive spatial mapping of metabolites in the R. tetraphylla plant. In addition, the article also contains new illustrations of the anatomy of R. tetraphylla.

Engineering Saccharomyces cerevisiae for the de novo Production of Halogenated Tryptophan and Tryptamine Derivatives.

The indole scaffold is a recurring structure in multiple bioactive heterocycles and natural products. Substituted indoles like the amino acid tryptophan serve as a precursor for a wide range of natural products with pharmaceutical or agrochemical applications. Inspired by the versatility of these compounds, medicinal chemists have for decades exploited indole as a core structure in the drug discovery process. With the aim of tuning the properties of lead drug candidates, regioselective halogenation of the indole scaffold is a common strategy. However, chemical halogenation is generally expensive, has a poor atom economy, lacks regioselectivity, and generates hazardous waste streams. As an alternative, in this work we engineer the industrial workhorse Saccharomyces cerevisiae for the de novo production of halogenated tryptophan and tryptamine derivatives. Functional expression of bacterial tryptophan halogenases together with a partner flavin reductase and a tryptophan decarboxylase resulted in the production of halogenated tryptophan and tryptamine with chlorine or bromine. Furthermore, by combining tryptophan halogenases, production of di-halogenated molecules was also achieved. Overall, this works paves the road for the production of new-to-nature halogenated natural products in yeast.

In silico characterization of the psilocybin biosynthesis pathway.

Nearly all mushrooms of the Psilocybe genus contain the natural product psilocybin, which is a psychoactive alkaloid derived from l-tryptophan. Considering their use in ancient times, as well as their psychedelic properties, these mushrooms have re-emerged with psychotherapeutic potential for treating depression, which has triggered increased pharmaceutical interest. However, the psilocybin biosynthesis pathway was only recently defined and, as such, little exists in the way of structural data. Accordingly, the aim of this study was to structurally characterize this pathway by generating homology models for the four Psilocybe cubensis enzymes involved in psilocybin biosynthesis (PsiD, a decarboxylase; PsiH, a monooxygenase; PsiK, a phosphotransferase; PsiM, a methyltransferase). Following initial model generation and alignment with the identified structural templates, repeated refinement of the models was carried out using secondary structure prediction, geometry evaluation, energy minimization, and molecular dynamics simulations in water. The final models were then evaluated using molecular docking interactions with their substrates, i.e., psilocybin precursors (l-tryptophan, tryptamine, 4-hydroxytryptamine, and norbaeocystin/baeocystin), all of which generated feasible binding modes for the expected biotransformation. Further plausibility of the psilocybin → aeruginascin, 4-hydroxytryptamine → norpsilocin, and tryptamine → N,N-dimethyltryptamine conversions, all mediated by the generated model for PsiM, suggests valid routes of formation for these key secondary metabolites. The structural characterization of these enzymes and their binding modes which emerged from this study can lead to a better understanding of psilocybin synthesis, thereby paving the way for the development of novel substrates and selective inhibitors, as well as improved biotechnological manipulation and production of psilocybin in vitro.

Evaluation of Human Performance Aiding Live Synthetically Engineered Bacteria in a Gut-on-a-Chip.

Synbiotics are a new class of live therapeutics employing engineered genetic circuits. The rapid adoption of genetic editing tools has catalyzed the expansion of possible synbiotics, exceeding traditional testing paradigms in terms of both throughput and model complexity. Herein, we present a simplistic gut-chip model using common Caco2 and HT-29 cell lines to establish a dynamic human screening platform for a cortisol sensing tryptamine producing synbiotic for cognitive performance sustainment. The synbiotic, SYN, was engineered from the common probiotic E. coli Nissle 1917 strain. It had the ability to sense cortisol at physiological concentrations, resulting in the activation of a genetic circuit that produces tryptophan decarboxylase and converts bioavailable tryptophan to tryptamine. SYN was successfully cultivated within the gut-chip showing log-phase growth comparable to the wild-type strain. Tryptophan metabolism occurred quickly in the gut compartment when exposed to 5 µM cortisol, resulting in the complete conversion of bioavailable tryptophan into tryptamine. The flux of tryptophan and tryptamine from the gut to the vascular compartment of the chip was delayed by 12 h, as indicated by the detectable tryptamine in the vascular compartment. The gut-chip provided a stable environment to characterize the sensitivity of the cortisol sensor and dynamic range by altering cortisol and tryptophan dosimetry. Collectively, the human gut-chip provided human relevant apparent permeability to assess tryptophan and tryptamine metabolism, production, and transport, enabled host analyses of cellular viability and pro-inflammatory cytokine secretion, and succeeded in providing an efficacy test of a novel synbiotic. Organ-on-a-chip technology holds promise in aiding traditional therapeutic pipelines to more rapidly down select high potential compounds that reduce the failure rate and accelerate the opportunity for clinical intervention.

DNA Authentication and Chemical Analysis of Psilocybe Mushrooms Reveal Widespread Misdeterminations in Fungaria and Inconsistencies in Metabolites.

The mushroom genus Psilocybe is best known as the core group of psychoactive mushrooms, yet basic information on their diversity, taxonomy, chemistry, and general biology is still largely lacking. In this study, we reexamined 94 Psilocybe fungarium specimens, representing 18 species, by DNA barcoding, evaluated the stability of psilocybin, psilocin, and their related tryptamine alkaloids in 25 specimens across the most commonly vouchered species (Psilocybe cubensis, Psilocybe cyanescens, and Psilocybe semilanceata), and explored the metabolome of cultivated P. cubensis. Our data show that, apart from a few well-known species, the taxonomic accuracy of specimen determinations is largely unreliable, even at the genus level. A substantial quantity of poor-quality and mislabeled sequence data in public repositories, as well as a paucity of sequences derived from types, further exacerbates the problem. Our data also support taxon- and time-dependent decay of psilocybin and psilocin, with some specimens having no detectable quantities of them. We also show that the P. cubensis metabolome possibly contains thousands of uncharacterized compounds, at least some of which may be bioactive. Taken together, our study undermines commonly held assumptions about the accuracy of names and presence of controlled substances in fungarium specimens identified as Psilocybe spp. and reveals that our understanding of the chemical diversity of these mushrooms is largely incomplete. These results have broader implications for regulatory policies pertaining to the storage and sharing of fungarium specimens as well as the use of psychoactive mushrooms for recreation and therapy. IMPORTANCE The therapeutic use of psilocybin, the active ingredient in "magic mushrooms," is revolutionizing mental health care for a number of conditions, including depression, posttraumatic stress disorder (PTSD), and end-of-life care. This has spotlighted the current state of knowledge of psilocybin, including the organisms that endogenously produce it. However, because of international regulation of psilocybin as a controlled substance (often included on the same list as cocaine and heroin), basic research has lagged far behind. Our study highlights how the poor state of knowledge of even the most fundamental scientific information can impact the use of psilocybin-containing mushrooms for recreational or therapeutic applications and undermines critical assumptions that underpin their regulation by legal authorities. Our study shows that currently available chemical studies are mainly inaccurate, irreproducible, and inconsistent, that there exists a high rate of misidentification in museum collections and public databases rendering even names unreliable, and that the concentration of psilocybin and its tryptamine derivatives in three of the most commonly collected Psilocybe species (P. cubensis, P. cyanescens, and P. semilanceata) is highly variable and unstable in museum specimens spanning multiple decades, and our study generates the first-ever insight into the highly complex and largely uncharacterized metabolomic profile for the most commonly cultivated magic mushroom, P. cubensis.

Tryptamine, a Microbial Metabolite in Fermented Rice Bran Suppressed Lipopolysaccharide-Induced Inflammation in a Murine Macrophage Model.

Fermentation is thought to alter the composition and bioavailability of bioactive compounds in rice bran. However, how this process affects the anti-inflammatory effects of rice bran and the bioactive compounds that might participate in this function is yet to be elucidated. This study aimed to isolate bioactive compounds in fermented rice bran that play a key role in its anti-inflammatory function. The fermented rice bran was fractionated using a succession of solvent and solid-phase extractions. The fermented rice bran fractions were then applied to lipopolysaccharide (LPS)-activated murine macrophages to evaluate their anti-inflammatory activity. The hot water fractions (FRBA), 50% ethanol fractions (FRBB), and n-hexane fractions (FRBC) were all shown to be able to suppress the pro-inflammatory cytokine expression from LPS-stimulated RAW 264.7 cells. Subsequent fractions from the hot water fraction (FRBF and FRBE) were also able to reduce the inflammatory response of these cells to LPS. Further investigation revealed that tryptamine, a bacterial metabolite of tryptophan, was abundantly present in these extracts. These results indicate that tryptamine may play an important role in the anti-inflammatory effects of fermented rice bran. Furthermore, the anti-inflammatory effects of FRBE and tryptamine may depend on the activity of the aryl hydrocarbon receptor.

Single mutations toggle the substrate selectivity of multifunctional Camptotheca secologanic acid synthases.

Terpene indole alkaloids (TIAs) are plant-derived specialized metabolites with widespread use in medicine. Species-specific pathways derive various TIAs from common intermediates, strictosidine or strictosidinic acid, produced by coupling tryptamine with secologanin or secologanic acid. The penultimate reaction in this pathway is catalyzed by either secologanin synthase (SLS) or secologanic acid synthase (SLAS) according to whether plants produce secologanin from loganin or secologanic acid from loganic acid. Previous work has identified SLSs and SLASs from different species, but the determinants of selectivity remain unclear. Here, combining molecular modeling, ancestral sequence reconstruction, and biochemical methodologies, we identified key residues that toggle SLS and SLAS selectivity in two CYP72A (cytochrome P450) subfamily enzymes from Camptotheca acuminata. We found that the positions of foremost importance are in substrate recognition sequence 1 (SRS1), where mutations to either of two adjacent histidine residues switched selectivity; His131Phe selects for and increases secologanin production whereas His132Asp selects for secologanic acid production. Furthermore, a change in SRS3 in the predicted substrate entry channel (Arg/Lys270Thr) and another in SRS4 at the start of the I-helix (Ser324Glu) decreased enzyme activity toward either substrate. We propose that the Camptotheca SLASs have maintained the broadened activities found in a common asterid ancestor, even as the Camptotheca lineage lost its ability to produce loganin while the campanulid and lamiid lineages specialized to produce secologanin by acquiring mutations in SRS1. The identification here of the residues essential for the broad substrate scope of SLASs presents opportunities for more tailored heterologous production of TIAs.

Tentative identification of in vitro metabolites of O-acetylpsilocin (psilacetin, 4-AcO-DMT) by UHPLC-Q-Orbitrap MS.

4-Acetoxy-N,N-dimethyltryptamine (4-AcO-DMT, psilacetin, O-acetylpsilocin) is a synthetic tryptamine with psychedelic properties. Psilacetin may also act as precursor drug of psilocin, similar to psilocybin, but little is known about its metabolism. In this study, the phase I and phase II in vitro metabolism of 4-AcO-DMT was investigated with pooled human liver microsomes, and the reaction mixture was analyzed using liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry. Fifteen metabolites were formed after incubation of pooled human liver microsomes with 4-AcO-DMT (12 phase I metabolites and 3 phase II metabolites). The proposed metabolite structures were based on accurate mass analysis and MS/MS fragmentation patterns. The biotransformations included hydrolysis, hydroxylation, N-demethylation, oxidation, and conjugation with glucuronic acid. The hydrolysis metabolite was the most abundant compound. For the development of new methods for the identification of 4-AcO-DMT consumption, the beta-hydroxylation metabolite of 4-AcO-DMT (M2-1) is recommended as a biomarker. The data reported in this work might be applicable to metabolic transformation of 4-AcO-DMT in vivo and also forensically helpful.

ß-Methyltryptamine Provoking the Crucial Role of Strictosidine Synthase Tyr151-OH for Its Stereoselective Pictet-Spengler Reactions to Tryptoline-type Alkaloids.

Strictosidine synthase (STR), the gate enzyme for monoterpenoid indole alkaloid biosynthesis, catalyzes the Pictet-Spengler reaction (PSR) of various tryptamine derivatives with secologanin assisted by "indole sandwich" stabilization. Continuous exploration with ß-methyltryptamine (IPA) stereoselectively delivered the C6-methylstrictosidines and C6-methylvincosides by enzymatic and nonenzymatic PSR, respectively. Unexpectedly, the first "nonindole sandwich" binding mode was witnessed by the X-ray structures of STR1-ligand complexes. Site-directed mutagenesis revealed the critical cryptic role of the hydroxyl group of Tyr151 in IPA biotransformation. Further computational calculations demonstrated the adjustable IPA position in STR1 upon the binding of secologanin, and Tyr151-OH facilitates the productive PSR binding mode via an advantageous hydrogen-bond network. Further chemo-enzymatic manipulation of C6-methylvincosides successfully resulted in the discovered antimalarial framework (IC50 = 0.92 µM).

Effect of Chemical Penetration Enhancer-Adhesive Interaction on Drug Release from Transdermal Patch: Mechanism Study Based on FT-IR Spectroscopy, 13C NMR Spectroscopy, and Molecular Simulation.

Chemical penetration enhancers (CPEs) are commonly added into transdermal patches to impart improved skin permeation of drug. However, significant unexplained variability in drug release kinetics in transdermal patches is possible as a result of the addition of CPEs; investigations into the underlying mechanisms are still limited. In the present study, a diverse set of CPEs was employed to draw broad conclusions. Solubility parameters of CPEs and acrylate pressure-sensitive adhesive were calculated by molecular dynamics simulation and Fedors group contribution method to evaluate drug-adhesive miscibility. CPE-adhesive interaction was characterized by FT-IR study, 13C NMR spectroscopy, and molecular docking simulation. Results showed that release enhancement ratio (ERR) of CPEs for zolmitriptan was rank ordered as isopropyl myristate > azone > Plurol Oleique® CC497 > Span® 80 > N-methylpyrrolidone > Transcutol® P. It was found that solubility parameter difference (Δδ) between CPE and adhesive was negatively related with ERR. It was proved that hydrogen bonding between CPE and adhesive would increase drug release rate, but only if the CPE showed good miscibility with adhesive. CPE like isopropyl myristate, which had good miscibility with adhesive, could decrease drug-adhesive interaction leading to the release of drug from adhesive.

Participating mechanism of a major contributing gene ysnE for auxin biosynthesis in Bacillus amyloliquefaciens SQR9.

The phytohormone indole-3-acetic acid (IAA) has been demonstrated to contribute to the plant growth-promoting effect of rhizobacteria, but the IAA biosynthesis pathway in rhizobacteria remains unclear. The ysnE gene, encoding a putative tryptophan acetyltransferase, has been demonstrated to be involved in and strongly contribute to IAA production in Bacillus, but the mechanism is unknown. In this study, to investigate how ysnE participates in IAA biosynthesis in the plant growth-promoting rhizobacterium Bacillus amyloliquefaciens SQR9, differences in the produced IAA biosynthesis intermediates between wild-type SQR9 and ΔysnE were analyzed and compared, and the effects of different intermediate compounds on the production of IAA and the accumulation of other intermediates were also investigated. The results showed that the mutant ΔysnE produced more indole-3-lactic acid (ILA) and tryptamine (TAM) than the SQR9 wild-type strain (nearly 1.6- and 2.1-fold), while the production of tryptophol (TOL) was significantly decreased by 46%. When indole-3-pyruvic acid (IPA) served as the substrate, the concentration of ILA in the ΔysnE fermentation broth was much higher than that of the wild type, while IAA and TOL were significantly lower, and ΔysnE was lower than SQR9 in IAA and TOL with the addition of TAM. The TOL content in the ΔysnE fermentation broth was much lower than that in the wild-type SQR9 with the addition of ILA. We suggest that ysnE may be involved in the IPA and TAM pathways and play roles in indole acetaldehyde (IAAld) synthesis from IPA and TAM and in the conversion of ILA to TOL.

Transfer of triptans into human breast milk and estimation of infant drug exposure through breastfeeding.

Clinical data on the transfer of triptans into human breast milk remain scarce. In a lactation study including 19 breastfeeding women with migraine, we examined the excretion of six different triptans into milk. Following intake of a single dose, each participant collected seven breast milk samples at predefined intervals up to 24 hours after dose. Triptan concentrations in milk were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Infant drug exposure was estimated by calculating the relative infant dose (RID). Twenty-two breast milk sample sets were obtained for sumatriptan (n = 8), rizatriptan (n = 5), zolmitriptan (n = 4), eletriptan (n = 3), almotriptan (n = 1) and naratriptan (n = 1). Based on the average concentration in milk throughout the day, estimated mean RIDs (with range in parenthesis) were as follows: eletriptan 0.6% (0.3%-0.8%), sumatriptan 0.7% (0.2%-1.8%), rizatriptan 0.9% (0.3%-1.4%), almotriptan 1.8% (-), zolmitriptan 2.1% (0.7%-5.3%) and naratriptan 5.0% (-). Infant drug exposure through breastfeeding appears to be low and indicates that use of the triptans in this study is compatible with breastfeeding. Naratriptan may not be first choice in breastfeeding mothers initiating triptans during the neonatal period.

Melatonin- and Ferulic Acid-Based HDAC6 Selective Inhibitors Exhibit Pronounced Immunomodulatory Effects In Vitro and Neuroprotective Effects in a Pharmacological Alzheimer's Disease Mouse Model.

The structures of melatonin and ferulic acid were merged into tertiary amide-based histone deacetylase 6 (HDAC6) inhibitors to develop multi-target-directed inhibitors for neurodegenerative diseases to incorporate antioxidant effects without losing affinity and selectivity at HDAC6. Structure-activity relationships led to compound 10b as a hybrid molecule showing pronounced and selective inhibition of HDAC6 (IC50 = 30.7 nM, > 25-fold selectivity over other subtypes). This compound shows comparable DPPH radical scavenging ability to ferulic acid, comparable ORAC value to melatonin and comparable Cu2+ chelating ability to EDTA. It also lacks neurotoxicity on HT-22 cells, exhibits a pronounced immunomodulatory effect, and is active in vivo showing significantly higher efficacy in an AD mouse model to prevent both Aß25-35-induced spatial working and long-term memory dysfunction at lower dose (0.3 mg/kg) compared to positive control HDAC6 inhibitor ACY1215 and an equimolar mixture of the three entities ACY1215, melatonin and ferulic acid, suggesting potentially disease-modifying properties.

Hypoxia Routes Tryptophan Homeostasis Towards Increased Tryptamine Production.

The liver is the central hub for processing and maintaining homeostatic levels of dietary nutrients especially essential amino acids such as tryptophan (Trp). Trp is required not only to sustain protein synthesis but also as a precursor for the production of NAD, neurotransmitters and immunosuppressive metabolites. In light of these roles of Trp and its metabolic products, maintaining homeostatic levels of Trp is essential for health and well-being. The liver regulates global Trp supply by the immunosuppressive enzyme tryptophan-2,3-dioxygenase (TDO2), which degrades Trp down the kynurenine pathway (KP). In the current study, we show that isolated primary hepatocytes when exposed to hypoxic environments, extensively rewire their Trp metabolism by reducing constitutive Tdo2 expression and differentially regulating other Trp pathway enzymes and transporters. Mathematical modelling of Trp metabolism in liver cells under hypoxia predicted decreased flux through the KP while metabolic flux through the tryptamine branch significantly increased. In line, the model also revealed an increased accumulation of tryptamines under hypoxia, at the expense of kynurenines. Metabolic measurements in hypoxic hepatocytes confirmed the predicted reduction in KP metabolites as well as accumulation of tryptamine. Tdo2 expression in cultured primary hepatocytes was reduced upon hypoxia inducible factor (HIF) stabilisation by dimethyloxalylglycine (DMOG), demonstrating that HIFs are involved in the hypoxic downregulation of hepatic Tdo2. DMOG abrogated hepatic luciferase signals in Tdo2 reporter mice, indicating that HIF stability also recapitulates hypoxic rewiring of Trp metabolism in vivo. Also in WT mice HIF stabilization drove homeostatic Trp metabolism away from the KP towards enhanced tryptamine production, leading to enhanced levels of tryptamine in liver, serum and brain. As tryptamines are the most potent hallucinogens known, the observed upregulation of tryptamine in response to hypoxic exposure of hepatocytes may be involved in the generation of hallucinations occurring at high altitude. KP metabolites are known to activate the aryl hydrocarbon receptor (AHR). The AHR-activating properties of tryptamines may explain why immunosuppressive AHR activity is maintained under hypoxia despite downregulation of the KP. In summary our results identify hypoxia as an important factor controlling Trp metabolism in the liver with possible implications for immunosuppressive AHR activation and mental disturbances.

Molecular and physiological analysis of indole-3-acetic acid degradation in Bradyrhizobium japonicum E109.

Bradyrhizobium japonicum E109 is a bacterium widely used for inoculants production in Argentina. It is known for its ability to produce several phytohormones and degrade indole-3-acetic acid (IAA). The genome sequence of B. japonicum E109 was recently analyzed and it showed the presence of genes related to the synthesis of IAA by indole-3-acetonitrile, indole-3-acetamide and tryptamine pathways. Nevertheless, B. japonicum E109 is not able to produce IAA and instead has the ability to degrade this hormone under saprophytic culture conditions. This work aimed to study the molecular and physiological features of IAA degradation and identify the genes responsible of this activity. In B. japonicum E109 we identified two sequences coding for a putative 3-phenylpropionate dioxygenase (subunits α and ß) responsible for the IAA degradation that were homologous to the canonical cluster of iacC and iacD of Pseudomonas putida 1290. These genes form a separate cluster together with three additional genes with unknown functions. The degradation activity was found to be constitutively expressed in B. japonicum E109. As products of IAA degradation, we identified two compounds, 3-indoleacetic acid 2,3-oxide and 2-(2-hydroperoxy-3-hydroxyindolin-3-yl) acetic acid. Our report proposes, for the first time, a model for IAA degradation in Bradyrhizobium.

Determination of six underivatized biogenic amines by LC-MS/MS and study of biogenic amine production during trout (Salmo trutta) storage in ice.

Biogenic amines (BAs) are natural components of food produced mainly during metabolism in animals and plants. The determination of BAs is important because of their potential toxicity and their potential use as food spoilage indicators. In the present study, a method for the determination of six BAs (putrescine, cadaverine, histamine, ß-phenylethylamine, tyramine, and tryptamine) by Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) with Atmospheric Pressure Chemical Ionisation (APCI) source has been used on trout samples (Salmo trutta) stored in ice for 15 days. The results showed that on day 15 quite large amounts of putrescine (76.530 mg/kg), cadaverine (85.530 mg/kg), tryptamine (25.210 mg/kg), and histamine (15.975mg/kg) were detected, while the other BAs remained low (ß-phenylethylamine: 3.230 mg/kg, tyramine: 0.165mg/kg). Furthermore, microbiological data (Total Vial Count- TVC, Pseudomonas spp, and Shewanella putrefaciens) showed that trout samples became organoleptically unacceptable on day 12, while volatile compound analysis showed a significant increase in total amounts of alcohols, aldehydes, and ketones on days 12 and 15.

Integrated metabolo-transcriptomics and functional characterization reveals that the wheat auxin receptor TIR1 negatively regulates defense against Fusarium graminearum.

Fusarium head blight (FHB) caused by Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schw.) Perch) results in large yield losses in annual global wheat production. Although studies have identified a number of wheat FHB resistance genes, a deeper understanding of the mechanisms underlying host plant resistance to F. graminearum is required for the control of FHB. Here, an integrated metabolomics and transcriptomics analysis of infected wheat plants (Triticum aestivum L.) enabled identification of 789 differentially accumulated metabolites, including flavonoids, phenolamides, tryptamine derivatives, and phytohormones, and revealed altered expression of more than 100 genes that function in the biosynthesis or regulation of these pathways. Our data regarding the effects of F. graminearum infection on flavonoids and auxin signaling led to follow-up experiments that showed that exogenous kaempferide and apigenin application on spikes increased wheat resistance to FHB, while exogenous auxin treatment increased FHB susceptibility. RNAi-mediated knockdown of the gene encoding the auxin receptor, TaTIR1, increased FHB resistance. Our data supported the use of TaTIR1 knockdown in controlling FHB. Our study provides insights on the wheat response to F. graminearum infection and its FHB resistance mechanisms while illustrating the potential of TaTIR1 knockdown in increasing FHB resistance during crop improvement programs.