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Harmful cyanobacterial blooms (cyanoHABs) cause recurrent toxic events in global watersheds. Although public health agencies monitor the causal toxins of most cyanoHABs and scientists in the field continue developing precise detection and prediction tools, the potent anticholinesterase neurotoxin, guanitoxin, is not presently environmentally monitored. This is largely due to its incompatibility with widely employed analytical methods and instability in the environment, despite guanitoxin being among the most lethal cyanotoxins. Here, we describe the guanitoxin biosynthesis gene cluster and its rigorously characterized nine-step metabolic pathway from l-arginine in the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. Through environmental sequencing data sets, guanitoxin ( gnt ) biosynthetic genes are repeatedly detected and expressed in municipal freshwater bodies that have undergone past toxic events. Knowledge of the genetic basis of guanitoxin biosynthesis now allows for environmental, biosynthetic gene monitoring to establish the global scope of this neurotoxic organophosphate.

The l-DOPA dioxygenase of Amanita muscaria (AmDODA) participates in the biosynthesis of betalain- and hygroaurin-type natural pigments. AmDODA is encoded by the dodA gene, whose DNA sequence was inferred from cDNA and gDNA libraries almost 30 years ago. However, reports on its heterologous expression rely on either the original 5'-truncated cDNA plasmid or artificial gene synthesis. We provide unequivocal evidence that the heterologous expression of AmDODA from A. muscaria specimens is not possible by using the coding sequence previously inferred for dodA . Here, we rectify and reannotate the full-length coding sequence for AmDODA and express a 205-aa His-tagged active enzyme, which was used to produce the l-DOPA hygroaurin, a rare fungal pigment. Moreover, AmDODA and other isozymes from bacteria were submitted to de novo folding using deep learning algorithms, and their putative active sites were inferred and compared. The wide catalytic pocket of AmDODA and the presence of the His-His-His and His-His-Asp motifs can provide insight into the dual cleavage of l-DOPA at positions 2,3 and 4,5 as per the mechanism proposed for nonheme dioxygenases., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Toxic cyanobacteria blooms are a frequent problem in subtropical reservoirs and freshwater systems. The purpose of this study was to investigate the occurrence of potentially toxic cyanobacteria and the environmental conditions associated with the presence of cyanotoxins in a Brazilian subtropical reservoir. Five collections were carried out at seven sampling locations in the reservoir, during the rainy and dry seasons, between the years 2016 and 2017. There was permanent occurrence of Raphidiopsis raciborskii (Woloszynska) Aguilera, Berrendero Gómez, Kastovsky, Echenique & Salerno (Phycologia 57(2):130-146, 2018), ranging between dominant and abundant, with an average biomass of 38.8 ± 29.9 mg L -1 . Also abundant were Dolichospermum solitarium, D. planctonicum, Planktothrix isothrix, and Aphanizomenon gracile. Saxitoxin (STX) was detected in all the collected samples (0.11 ± 0.05 µg L -1 ). Microcystin (MC) was also detected, but at lower concentrations (0.01 ± 0.0 µg L -1 ). Low availability of NO 3 - and phosphorus limitation had significant effects on the R. raciborskii biomass and the levels of STX and MC. It was observed that R. raciborskii was sensitive to thermal stratification, at the same time that STX levels were higher. This suggested that STX was produced under conditions that restricted the growth of R. raciborskii. These are important findings, because they add information about the permanent occurrence of STX and R. raciborskii in an aquatic ecosystem limited by phosphorus, vulnerable to climatic variations, and polluted by domestic effluents., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Nitrones derived from natural antioxidants are emerging as highly specific therapeutics against various human diseases, including stroke, neurodegenerative pathologies, and cancer. However, the development of useful pseudo-natural nitrones requires the judicious choice of a secondary metabolite as the precursor. Betalains are nitrogen-containing natural pigments that exhibit marked antioxidant capacity and pharmacological properties and, hence, are ideal candidates for designing multifunctional nitrones. In this work, we describe the semisynthesis and properties of a biocompatible and antioxidant betalain-nitrone called OxiBeet. This bio-based compound is a better radical scavenger than ascorbic acid, gallic acid, and most non-phenolic antioxidants and undergoes concerted proton-coupled electron transfer. The autoxidation of OxiBeet produces a persistent nitroxide radical, which, herein, is studied via electron paramagnetic resonance spectroscopy. In addition, femtosecond transient absorption spectroscopy reveals that excited state formation is not required for the oxidation of OxiBeet. The results are compared with those obtained using betanin, a natural betalain, and pBeet, the imine analog of OxiBeet. The findings of this study will enable the development of antioxidant and spin-trap nitrones based on the novel N-oxide 1,7-diazaheptamethinium scaffold and betalain dyes with enhanced hydrolytic stability in aqueous alkaline media., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Ibuprofen soft gelatin capsules were subjected to degradation under acidic, basic, oxidation, photolytic, thermal, humidity, and metal ions conditions. To analyse the degradation products, a reversed-phase liquid chromatography (RP-LC) indicative stability method was successfully developed. Chromatographic separation was achieved using a Poroshell HPH-C18 150 x 4.6 mm, 4 μm, column at 25 °C, with a mobile phase constituted by 0.1% phosphoric acid and acetonitrile in gradient at a flow rate of 1.0 mL• min -1 , using ultraviolet detection at 220 nm and injection volume of 20 μL. In total, eight unknown impurities were found. The peaks RRt 0.49, RRt 0.75, and RRt 0.95 were above 0.17%, corresponding to the identification threshold. Those were identified and characterized by LC-MS-QTOF, with the same chromatographic conditions, except for the exchange of 0.1% phosphoric acid for 0.1% formic acid. The impurities originated from the interaction of ibuprofen with excipients: esterification with PEG, with sorbitol/sorbitan, and with glycerol by-products, which has not yet been reported in the literature. The developed method can be used in several pharmaceutical areas as quality control of impurities, studies of forced degradation, and for the development of future formulations.

Several studies have indicated the presence of the neonicotinoid insecticide imidacloprid (IMI) in aquatic ecosystems in concentrations up to 320.0 µg L -1 . In the present study, we evaluated the effects of the highest IMI concentration detected in surface water (320.0 µg L -1 ) on the survival of Chironomus sancticaroli, Daphnia similis, and Danio rerio in three different scenarios of water contamination. The enzymatic activities of glutathione S-transferase (GST), catalase (CAT), and ascorbate peroxidase (APX) in D. rerio also were determined. For this evaluation, we have simulated a lotic environment using an indoor system of artificial channels developed for the present study. In this system, three scenarios of contamination by IMI (320.0 µg L -1 ) were reproduced: one using reconstituted water (RW) and the other two using water samples collected in unpolluted (UW) and polluted (DW) areas of a river. The results indicated that the tested concentration was not able to cause mortality in D. similis and D. rerio in any proposed treatment (RW, UW, and DW). However, C. sancticaroli showed 100% of mortality in the presence of IMI in the three proposed treatments, demonstrating its potential to impact the community of aquatic nontarget insects negatively. Low IMI concentrations did not offer risks to D. rerio survival. However, we observed alterations in GST, CAT, and APX activities in treatments that used IMI and water with no evidence of pollution (i.e., RW and UW). These last results demonstrated that fish are more susceptible to the effects of IMI in unpolluted environments.

While thermal treatment of paper can lead to the formation of aromatic structures via hydrothermal treatment (low temperature) or pyrolysis (high temperature), neither of these approaches allow patterning the substrates. Somewhere in between these two extremes, a handful of research groups have used CO2 lasers to pattern paper and induce carbonization. However, none of the previously reported papers have focused on the possibility to form fluorescent derivatives via laser-thermal engraving. Exploring this possibility, this article describes the possibility of using a CO2 laser engraver to selectively treat paper, resulting in the formation of fluorescent compounds, similar to those present on the surface of carbon dots. To determine the most relevant variables controlling this process, 3 MM chromatography paper was treated using a standard 30 W CO2 laser engraver. Under selected experimental conditions, a blue fluorescent pattern was observed when the substrate was irradiated with UV light (365 nm). The effect of various experimental conditions (engraving speed, engraving power, and number of engraving steps) was investigated to maximize the fluorescence intensity. Through a comprehensive characterization effort, it was determined that 5-(hydroxymethyl)furfural and a handful of related compounds were formed (varying in amount) under all selected experimental conditions. To illustrate the potential advantages of this strategy, that could complement those applications traditionally developed from carbon dots (sensors, currency marking, etc.), a redox-based optical sensor for sodium hypochlorite was developed.

The term cylindrospermopsins (CYNs) refers to a structurally related class of cyanobacterial metabolites comprised of a tricyclic guanidine group and a hydroxymethyluracil moiety. Most reports in environmental aquatic samples refer to cylindrospermopsin (CYN), and reports on other CYN alkaloids are scarce, due, in part, to a lack of versatile isolation protocols. Thus, using commercially available solid phase extraction (SPE) cartridges, we optimized an isolation protocol for the complete recovery of CYN, 7-deoxy-cylindrospermopsin (7D-CYN) and 7-deoxy-desulfo-cylindrospermopsin (7D-desulfo-CYN) from the same aliquot. The isolation protocol was adaptable depending on the nature of the sample (solid biomass, culture broth or environmental water sample) and tolerates up to 4 L of dense culture broth or 400 mg of lyophilized biomass. To quantitate the CYN alkaloids, we validated an LC-DAD-MS 2 method, which takes advantage of the UV absorption of the uracil group (λ 262 nm). Using electrospray ionization (ESI) in a positive ion mode, the high-resolution MS 1 data confirms the presence of the protonated alkaloids, and the MS 2 fragment assignment is reported as complementary proof of the molecular structure of the CYNs. We isolated three CYN alkaloids with different water solubility using the same lyophilized sample, with a purity that ranged from 95% to 99%. The biological activity of the purified CYNs, along with a synthetic degradation product of CYN (desulfo-cylindrospermopsin), was evaluated by assessing necrosis and apoptosis in vitro using flow cytometry. CYN's lethal potency in HepG2 cells was greater than the other analogs, due to the presence of all four functional groups: guanidine, uracil, C-7 hydroxyl and the sulfate residue.

Blue natural pigments are rare, especially among plants. However, flowering species that evolved to attract Hymenoptera pollinators are colored by blue anthocyanin-metal complexes. Plants lacking anthocyanins are pigmented by betalains but are unable to produce blue hues. By extending the π-system of betalains, we designed a photostable and metal-free blue dye named BeetBlue that did not show toxicity to human hepatic and retinal pigment epithelial cells and does not affect zebrafish embryonal development. This chiral dye can be conveniently synthesized from betalamic acid obtained from hydrolyzed red beetroot juice or by enzymatic oxidation of l-dopa. BeetBlue is blue in the solid form and in solution of acidified polar molecular solvents, including water. Its capacity to dye natural matrices makes BeetBlue the prototype of a new class of low-cost bioinspired chromophores suitable for a myriad of applications requiring a blue hue., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The bloom-forming cyanobacterium Nodularia spumigena CENA596 encodes the biosynthetic gene clusters (BGCs) of the known natural products nodularins, spumigins, anabaenopeptins/namalides, aeruginosins, mycosporin-like amino acids, and scytonemin, along with the terpenoid geosmin. Targeted metabolomics confirmed the production of these metabolic compounds, except for the alkaloid scytonemin. Genome mining of N. spumigena CENA596 and its three closely related Nodularia strains-two planktonic strains from the Baltic Sea and one benthic strain from Japanese marine sediment-revealed that the number of BGCs in planktonic strains was higher than in benthic one. Geosmin-a volatile compound with unpleasant taste and odor-was unique to the Brazilian strain CENA596. Automatic annotation of the genomes using subsystems technology revealed a related number of coding sequences and functional roles. Orthologs from the Nodularia genomes are involved in the primary and secondary metabolisms. Phylogenomic analysis of N. spumigena CENA596 based on 120 conserved protein sequences positioned this strain close to the Baltic Nodularia . Phylogeny of the 16S rRNA genes separated the Brazilian CENA596 strain from those of the Baltic Sea, despite their high sequence identities (99% identity, 100% coverage). The comparative analysis among planktic Nodularia strains showed that their genomes were considerably similar despite their geographically distant origin.

Microcystins (MCs) are serine/threonine phosphatase inhibitors synthesized by several members of the phylum Cyanobacteria. Mining the draft genome sequence of the nostocalean MC-producing Fischerella sp. strain CENA161 led to the identification of three contigs containing mcy genes. Subsequent PCR and Sanger sequencing allowed the assembling of its complete biosynthetic mcy gene cluster with 55,016 bases in length. The cluster encoding ten genes (mcyA-J) with a central bidirectional promoter was organized in a similar manner as found in other genera of nostocalean cyanobacteria. However, the nucleotide sequence of the mcy gene cluster of Fischerella sp. CENA161 showed significant differences from all the other MC-producing cyanobacterial genera, sharing only 85.2 to 74.1% identities. Potential MC variants produced by Fischerella sp. CENA161 were predicted by the analysis of the adenylation domain binding pockets and further investigated by LC-MS/MS analysis. To our knowledge, this study presents the first complete mcy cluster characterization from a strain of the genus Fischerella, providing new insight into the distribution and evolution of MCs in the phylum Cyanobacteria., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We previously showed that luminous fungi share a common mechanism in bioluminescence, and identified hispidin as a luciferin precursor in Neonothopanus nambi mycelium. Here we showed the presence of hispidin as a bioluminescent active compound at 25-1000 pmol g -1 in the fruiting bodies of Mycena chlorophos, Omphalotus japonicus, and Neonothopanus gardneri. These results suggest that luminous mushrooms contain hispidin as a luciferin precursor. We also found that non-luminous "young" fruiting bodies exhibited luminescence by hispidin treatment. Furthermore, we observed a gradual luminescence enhancement of the cell-free fruiting body extract by the addition of hispidin biosynthetic components, namely caffeic acid, ATP and malonyl-CoA. These findings suggest that continuous weak glow of luminous mushrooms is regulated by slow recycling biosynthesis of hispidin.

The bioavailability and metabolism of anthocyanins and ellagitannins following acute intake of grumixama fruit, native Brazilian cherry, by humans, and its in vitro antiproliferative activity against breast cancer cells (MDA-MB-231) were investigated. A single dose of grumixama juice was administered to healthy women (n = 10) and polyphenol metabolites were analyzed in urine and plasma samples collected over 24 h. The majority of the metabolites circulating and excreted in urine were phenolic acids and urolithin conjugates, the gut microbiota catabolites of both classes of polyphenols, respectively. According to pharmacokinetic parameters, the subjects were divided into two distinct groups, high and low urinary metabolite excretors. The pool of polyphenol metabolites found in urine samples showed a significant inhibition of cell proliferation and G2/M cell cycle arrest in MDA-MB-231 cells. Our findings demonstrate the large interindividual variability concerning the polyphenol metabolism, which possibly could reflect in health promotion.

Bioluminescent fungi are spread throughout the globe, but details on their mechanism of light emission are still scarce. Usually, the process involves three key components: an oxidizable luciferin substrate, a luciferase enzyme, and a light emitter, typically oxidized luciferin, and called oxyluciferin. We report the structure of fungal oxyluciferin, investigate the mechanism of fungal bioluminescence, and describe the use of simple synthetic α-pyrones as luciferins to produce multicolor enzymatic chemiluminescence. A high-energy endoperoxide is proposed as an intermediate of the oxidation of the native luciferin to the oxyluciferin, which is a pyruvic acid adduct of caffeic acid. Luciferase promiscuity allows the use of simple α-pyrones as chemiluminescent substrates.

Cyanobacteria produce a broad range of natural products, many of which are potent protease inhibitors. Biosynthetic gene clusters encoding the production of novel protease inhibitors belonging to the spumigin and anabaenopeptin family of nonribosomal peptides were identified in the genome of the bloom-forming cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. The genetic architecture and gene organization of both nonribosomal peptide biosynthetic clusters were compared in parallel with their chemical structure variations obtained by liquid chromatography (LC-MS/MS). The spumigin (spu) and anabaenopeptin (apt) gene clusters are colocated in the genomes of S. torques-reginae ITEP-024 and Nodularia spumigena CCY9414 and separated by a 12 kb region containing genes encoding a patatin-like phospholipase, l-homophenylalanine (l-Hph) biosynthetic enzymes, and four hypothetical proteins. hphABCD gene cluster encoding the production of l-Hph was linked to all eight apt gene clusters investigated here. We suggest that while the HphABCD enzymes are an integral part of the anabaenopeptin biosynthetic pathway, they provide substrates for the biosynthesis of both anabaenopeptins and spumigins. The organization of the spu and apt suggests a plausible model for the biosynthesis of the 4-(4-hydroxyphenyl)-2-acid (Hpoba) precursor of spumigin variants in S. torques-reginae ITEP-024 based on the acceptable substrates of HphABCD enzymes.

Polyphenols from diverse sources have shown anti-inflammatory activity. In the context of atherosclerosis, macrophages play important roles including matrix metalloproteinases synthesis involved in degradation of matrix extracellular components affecting the atherosclerotic plaque stability. We prepared a propolis extract and pinocembrin in ethanol solution. Propolis extract was chemically characterized using LC-MS. The effect of treatments on gene expression and proteolytic activity was measured in vitro using murine macrophages activated with LPS. Cellular toxicity associated with both treatments and the vehicle was determined using MTT and apoptosis/necrosis detection assays. MMP-9 gene expression and proteolytic activity were measured using qPCR and zymography, respectively. Thirty-two compounds were identified in the propolis extract, including pinocembrin among its major components. Treatment with either ethanolic extract of propolis or pinocembrin inhibits MMP-9 gene expression in a dose-dependent manner. Similarly, an inhibitory effect was observed in proteolytic activity. However, the effect showed by ethanolic extract of propolis was higher than the effect of pinocembrin, suggesting that MMP-9 inhibition results from a joint contribution between the components of the extract. These data suggest a potential role of polyphenols from Chilean propolis in the control of extracellular matrix degradation in atherosclerotic plaques.

High resolution mass spectrometry investigation of an extract of the toxic cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 led to the discovery of four new spumigin congeners. The structures for these peptides were postulated on the basis of accurate mass data and isotopic pattern information of both full scan and product ion spectra. This is the first reported evidence of spumigins on Sphaerospermopsis species., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

There is a long-standing concern that creatine supplementation could be associated with cancer, possibly by facilitating the formation of carcinogenic heterocyclic amines (HCAs). This study provides compelling evidence that both low and high doses of creatine supplementation, given either acutely or chronically, does not cause a significant increase in HCA formation. HCAs detection was unrelated to creatine supplementation. Diet was likely to be the main factor responsible for HCAs formation after either placebo (n = 6) or creatine supplementation (n = 3). These results directly challenge the recently suggested biological plausibility for the association between creatine use and risk of testicular germ cell cancer. Creatine supplementation has been associated with increased cancer risk. In fact, there is evidence indicating that creatine and/or creatinine are important precursors of carcinogenic heterocyclic amines (HCAs). The present study aimed to investigate the acute and chronic effects of low- and high-dose creatine supplementation on the production of HCAs in healthy humans (i.e. 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-(1,6-dimethylfuro[3,2-e]imidazo[4,5-b])pyridine (IFP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx)). This was a non-counterbalanced single-blind crossover study divided into two phases, in which low- and high-dose creatine protocols were tested. After acute (1 day) and chronic supplementation (30 days), the HCAs PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx were assessed through a newly developed HPLC-MS/MS method. Dietary HCA intake and blood and urinary creatinine were also evaluated. Out of 576 assessments performed (from 149 urine samples), only nine (3 from creatine and 6 from placebo) showed quantifiable levels of HCAs (8-MeIQx: n = 3; 4,8-DiMeIQx: n = 2; PhIP: n = 4). Individual analyses revealed that diet rather than creatine supplementation was the main responsible factor for HCA formation in these cases. This study provides compelling evidence that both low and high doses of creatine supplementation, given either acutely or chronically, did not cause increases in the carcinogenic HCAs PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx in healthy subjects. These findings challenge the long-existing notion that creatine supplementation could potentially increase the risk of cancer by stimulating the formation of these mutagens., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Cyanobacteria from underexplored and extreme habitats are attracting increasing attention in the search for new bioactive substances. However, cyanobacterial communities from tropical and subtropical regions are still largely unknown, especially with respect to metabolite production. Among the structurally diverse secondary metabolites produced by these organisms, peptides are by far the most frequently described structures. In this work, liquid chromatography/electrospray ionization coupled to high resolution quadrupole time-of-flight tandem mass spectrometry with positive ion detection was applied to study the peptide profile of a group of cyanobacteria isolated from the Southeastern Brazilian coastal forest. A total of 38 peptides belonging to three different families (anabaenopeptins, aeruginosins, and cyanopeptolins) were detected in the extracts. Of the 38 peptides, 37 were detected here for the first time. New structural features were proposed based on mass accuracy data and isotopic patterns derived from full scan and MS/MS spectra. Interestingly, of the 40 surveyed strains only nine were confirmed to be peptide producers; all of these strains belonged to the order Nostocales (three Nostoc sp., two Desmonostoc sp. and four Brasilonema sp.).

Tryptophan (TRP) is essential for many physiological processes, and its metabolism changes in some diseases such as infection and cancer. The most studied aspects of TRP metabolism are the kynurenine and serotonin pathways. A minor metabolic route, tryptamine and N,N-dimethyltryptamine (DMT) biosynthesis, has received far less attention, probably because of the very low amounts of these compounds detected only in some tissues, which has led them to be collectively considered as trace amines. In a previous study, we showed a metabolic interrelationship for TRP in melanoma cell lines. Here, we identified DMT and N,N-dimethyl-N-formyl-kynuramine (DMFK) in the supernatant of cultured SK-Mel-147 cells. Furthermore, when we added DMT to the cell culture, we found hydroxy-DMT (OH-DMT) and indole acetic acid (IAA) in the cell supernatant at 24 h. We found that SK-Mel-147 cells expressed mRNA for myeloperoxidase (MPO) and also had peroxidase activity. We further found that DMT oxidation was catalyzed by peroxidases. DMT oxidation by horseradish peroxidase, H2O2 and MPO from PMA-activated neutrophils produced DMFK, N,N-dimethyl-kynuramine (DMK) and OH-DMT. Oxidation of DMT by peroxidases apparently uses the common peroxidase cycle involving the native enzyme, compound I and compound II. In conclusion, this study describes a possible alternative metabolic pathway for DMT involving peroxidases that has not previously been described in humans and identifies DMT and metabolites in a melanoma cell line. The extension of these findings to other cell types and the biological effects of DMT and its metabolites on cell proliferation and function are key questions for future studies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX), while cylindrospermopsin (CYN), which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins.

Indoleamine 2,3-dioxygenase (IDO) is an interferon-γ (IFN-γ)-induced tryptophan-degrading enzyme, producing kynurenine (KYN) that participates in the mechanism of tumor immune tolerance. Thus, IDO inhibition has been considered a strategy for anticancer therapy. The aim of this study was to identify whether the metabolites originated from the competitive routes of tryptophan metabolism, such as the serotonergic or N, N-dimethyltryptamine (DMT) pathways, have inhibitory effects on recombinant human IDO (rhIDO) activity. Serotonin and melatonin had no effect; on the other hand, tryptamine (TRY) and DMT modulated the activity of rhIDO as classical non-competitive inhibitors, with Ki values of 156 and 506 μM, respectively. This inhibitory effect was also observed on constitutively expressed or IFN-γ-induced IDO in the A172 human glioma cell line. TRY and DMT increased the cytotoxic activity of peripheral blood mononuclear cells (PBMCs) in co-culture assays. We conclude that the IDO inhibition by TRY and DMT contributed to a more effective tumor-reactive response by the PBMCs., (Copyright © 2013 John Wiley & Sons, Ltd.)

Objective: The goal of this study was to monitor imatinib mesylate therapeutically in the Tumor Biology Laboratory, Department of Hematology and Hemotherapy, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (USP). A simple and sensitive method to quantify imatinib and its metabolite (CGP74588) in human serum was developed and fully validated in order to monitor treatment compliance., Methods: The method used to quantify these compounds in serum included protein precipitation extraction followed by instrumental analysis using high performance liquid chromatography coupled with mass spectrometry. The method was validated for several parameters, including selectivity, precision, accuracy, recovery and linearity., Results: The parameters evaluated during the validation stage exhibited satisfactory results based on the Food and Drug Administration and the Brazilian Health Surveillance Agency (ANVISA) guidelines for validating bioanalytical methods. These parameters also showed a linear correlation greater than 0.99 for the concentration range between 0.500 µg/mL and 10.0 µg/mL and a total analysis time of 13 minutes per sample. This study includes results (imatinib serum concentrations) for 308 samples from patients being treated with imatinib mesylate., Conclusion: The method developed in this study was successfully validated and is being efficiently used to measure imatinib concentrations in samples from chronic myeloid leukemia patients to check treatment compliance. The imatinib serum levels of patients achieving a major molecular response were significantly higher than those of patients who did not achieve this result. These results are thus consistent with published reports concerning other populations.

Species of Microcystis are the most common bloom-forming cyanobacteria in several countries. Despite extensive studies regarding the production of bioactive cyanopeptides in this genus, there are limited data on isolated strains from Brazil. Three Microcystis sp. strains were isolated from the Salto Grande Reservoir (LTPNA01, 08 and 09) and investigated for the presence of mcy genes, microcystins and other cyanopeptides. Microcystin and microginin production was confirmed in two isolates using high-resolution tandem mass spectrometry after electrospray ionization (ESI-Q-TOF), and the structures of two new microginin congeners were proposed (MG756 Ahda-Val-Leu-Hty-Tyr and MG770 MeAhda-Val-Leu-Hty-Tyr). The biosynthesis profile of the identified cyanopeptides was evaluated at different growth phases via a newly developed HPLC-UV method. Results demonstrated no substantial differences in the production of microcystins and microginins after data normalization to cell quota, suggesting a constitutive biosynthesis. This study represents the first confirmed co-production of microginins and microcystins in Brazilian strains of Microcystis sp. and highlights the potential of Brazilian cyanobacteria as a source of natural compounds with pharmaceutical interest., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Propolis is a polyphenol-rich resinous substance extensively used to improve health and prevent diseases. The effects of polyphenols from different sources of propolis on atherosclerotic lesions and inflammatory and angiogenic factors were investigated in LDL receptor gene (LDLr-/-) knockout mice. The animals received a cholesterol-enriched diet to induce the initial atherosclerotic lesions (IALs) or advanced atherosclerotic lesions (AALs). The IAL or AAL animals were divided into three groups, each receiving polyphenols from either the green, red or brown propolis (250 mg/kg per day) by gavage. After 4 weeks of polyphenol treatment, the animals were sacrificed and their blood was collected for lipid profile analysis. The atheromatous lesions at the aortic root were also analyzed for gene expression of inflammatory and angiogenic factors by quantitative real-time polymerase chain reaction and immunohistochemistry. All three polyphenol extracts improved the lipid profile and decreased the atherosclerotic lesion area in IAL animals. However, only polyphenols from the red propolis induced favorable changes in the lipid profiles and reduced the lesion areas in AAL mice. In IAL groups, VCAM, MCP-1, FGF, PDGF, VEGF, PECAM and MMP-9 gene expression was down-regulated, while the metalloproteinase inhibitor TIMP-1 gene was up-regulated by all polyphenol extracts. In contrast, for advanced lesions, only the polyphenols from red propolis induced the down-regulation of CD36 and the up-regulation of HO-1 and TIMP-1 when compared to polyphenols from the other two types of propolis. In conclusion, polyphenols from propolis, particularly red propolis, are able to reduce atherosclerotic lesions through mechanisms including the modulation of inflammatory and angiogenic factors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the unusual gas-phase dissociation behavior of two epimer pairs of protonated gonyautoxins (GTX) following electrospray ionization in comparison to their deprotonated counterparts. The chemical structures of the investigated GTX1-4 variants vary in their substitution pattern at N-1 and the stereochemical orientation of the hydroxysulfate group at C-11 (11α for GTX1/2 versus 11β for GTX3/4). The direct comparison of mass spectra in positive and negative ion modes illustrated two distinct features: first, an intriguing difference between protonated 11α and 11β species, where 11α conformations exhibited almost complete dissociation of [M + H](+) ions via facile SO(3) elimination, while 11β species remained mostly intact as [M + H](+); and second, the lack of such differences for the deprotonated counterparts. In this study, we propose an acid-catalyzed elimination mechanism from density functional theory calculations, initiated by a proton transfer of a guanidinium proton to the hydroxysulfate group with simultaneous SO(3) release, which is only possible for the 11α conformation based on intramolecular distances. The same mechanism explains the lack of a comparable SO(3) loss in the negative ion mode. CID experiments supported this proposed mechanism for GTX1 and GTX2. Computational modeling of product ions seen in the CID spectra of GTX3 and GTX4 established that the lowest energy dissociation pathway for the 11β epimers is elimination of water with the possibility for further SO(3) release from the intermediate product. Experimental data for structurally analogous decarbamoyl gonyautoxins confirmed the evidence for the GTX compounds as well as the proposed elimination mechanisms.

Microcystins (MC) are a family of hepatotoxic cyclic heptapeptides produced by a number of different cyanobacterial species. Considering the recent advances in the characterization of deprotonated peptides by mass spectrometry, the fragmentation behavior of four structurally related microcystin compounds was investigated using collision-induced dissociation (CID) experiments on an orbitrap mass spectrometer. It is demonstrated in this study that significant structural information can be obtained from the CID spectra of deprotonated microcystins. A predominant ring-opening reaction at the isoMeAsp residue, as well as two major complementary fragmentation pathways, was observed, reducing the complexity of the product ion spectra in comparison with spectra observed from protonated species. This proposed fragmentation behavior was applied to characterize [Leu(1)]MC-LR from a cyanobacterial cell extract. In conclusion, CID spectra of microcystins in the negative ion mode provide rich structurally informative mass spectra which greatly enhance confidence in structural assignments, in particular when combined with complementary positive ion CID spectra., (Copyright © 2011 John Wiley & Sons, Ltd.)

The acute poisoning of chronic renal patients during hemodialysis sessions in 1996 in Caruaru City (Pernambuco State, Brazil) stimulated an intensive search for the cause of this severe complication. This search culminated in the identification of microcystins (MC), hepatotoxic cyclic heptapeptides produced by cyanobacteria, as the causative agents. More than ten years later, additional research data provides us with a better understanding of the factors related to cyanobacterial bloom occurrence and production of MC in Brazil and other South American countries. The contamination of water bodies and formation of toxic blooms remains a very serious concern, especially in countries in which surface water is used as the main source for human consumption. The purpose of this review is to highlight the discoveries of the past 15 years that have brought South American researchers to their current level of understanding of toxic cyanobacteria species and that have contributed to their knowledge of factors related to MC production, mechanisms of action and consequences for human health and the environment., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Anatoxin-a(s) is a potent irreversible inhibitor of the enzyme acetylcholinesterase with a unique N-hydroxyguanidine methylphosphate ester chemical structure. Determination of this toxin in environmental samples is hampered by the lack of specific methods for its detection. Using the toxic strain of Anabaena lemmermani PH-160 B as positive control, the fragmentation characteristics of anatoxin-a(s) under collision-induced dissociation conditions have been investigated and new LC-MS/MS methods proposed. Recommended ion transitions for correct detection of this toxin are 253>58, 253>159, 235>98 and 235>96. Chromatographic separation is better achieved under HILIC conditions employing a ZIC-HILIC column. This method was used to confirm for the first time the production of anatoxin-a(s) by strains of Anabaena oumiana ITEP-025 and ITEP-026. Considering no standard solutions are commercially available, our results will be of significant use for the correct identification of this toxin by LC-MS/MS.

Introduction: Ayahuasca is obtained by infusing the pounded stems of Banisteriopsis caapi in combination with the leaves of Psychotria viridis. P. viridis is rich in the psychedelic indole N,N-dimethyltryptamine, whereas B. caapi contains substantial amounts of beta-carboline alkaloids, mainly harmine, harmaline and tetrahydroharmine, which are monoamine-oxidase inhibitors. Because of differences in composition in ayahuasca preparations, a method to measure their main active constituents is needed., Objective: To develop a gas chromatographic method for the simultaneous determination of dimethyltryptamine and the main beta-carbolines found in ayahuasca preparations., Methodology: The alkaloids were extracted by means of solid phase extraction (C(18)) and detected by gas chromatography with nitrogen/phosphorous detector., Results: The lower limit of quantification (LLOQ) was 0.02 mg/mL for all analytes. The calibration curves were linear over a concentration range of 0.02-4.0 mg/mL (r(2 )> 0.99). The method was also precise (RSD < 10%)., Conclusion: A simple gas chromatographic method to determine the main alkaloids found in ayahuasca was developed and validated. The method can be useful to estimate administered doses in animals and humans for further pharmacological and toxicological investigations of ayahuasca., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Cylindrospermopsin (CYN) belongs to a group of toxins produced by several strains of freshwater cyanobacteria. It is a compact zwitterionic molecule composed of a uracil section and a tricyclic guanidinium portion with a primarily hepatotoxic effect. Using low multi-stage and high-resolution mass spectrometry, the gas-phase reactions of this toxin have been investigated. Our data show that collision-induced dissociation (CID) spectra of CYN are dominated by neutral losses, and three major initial fragmentation pathways are clearly distinguishable. Interestingly, comparative analysis of protonated and cationizated molecules showed a significant difference in the balance of the SO3 and terminal ring elimination. These data indicate that the differential ion mobility of H+, Li+, Na+ and K+ leads to different fragmentation pathways, giving rise to mass spectra with different profiles.

The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4 degrees C and the supernatants were used in enzymatic assays at 30 degrees C, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 +/- 0.15 absorbance variation min(-1) mg protein(-1), at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 +/- 0.02 mmol p-nitroaniline min(-1) mg protein(-1). The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50 degrees C, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.