An integrative omics perspective for the analysis of chemical signals in ecological interactions.

All living organisms emit, detect, and respond to chemical stimuli, thus creating an almost limitless number of interactions by means of chemical signals. Technological and intellectual advances in the last two decades have enabled chemical signals analyses at several molecular levels, including gene expression, molecular diversity, and receptor affinity. These advances have also deepened our understanding of nature to encompass interactions at multiple organism levels across different taxa. This tutorial review describes the most recent analytical developments in 'omics' technologies (i.e., genomics, transcriptomics, proteomics, and metabolomics) and provide recent examples of its application in studies of chemical signals. We highlight how studies have integrated an enormous amount of information generated from different omics disciplines into one publicly available platform. In addition, we stress the importance of considering different signal modalities and an evolutionary perspective to establish a comprehensive understanding of chemical communication.

Development and validation of a UHPLC-MS/MS bioanalytical method to quantify in plasma the analgesic candidate PT-31 following a preliminary pharmacokinetic study in rats.

A selective and sensitive UHPLC-MS/MS bioanalytical method to determine PT-31, an analgesic drug candidate, in rat plasma was developed and validated. Analyses were performed using a UHPLC-MS/MS system equipped with an electrospray ionization interface operating in the positive ionization mode using a C18 reversed-phase column with a mobile phase of water:acetonitrile (68:31, v/v) containing 0.1% acetic acid eluting in a gradient mode with a flow rate of 0.3 mL/min. Plasma samples were deproteinized with cold acetonitrile containing 0.01% TFA (1:2, v/v) and 50 µL of the supernatant were injected into the system. PT-31 and phenytoin (internal standard) retention times were roughly 1.0 and 1.5 min, respectively. Linear standard curves were plotted for the 0.01-10 µg/mL concentration range, with a coefficient of determination > 0.99. The method's precision was over 88%. Maximum intra- and inter-day relative standard deviations were 14.6% and 11.6%, respectively. Interfering substances were not detected in the chromatogram, indicating that the method was specific. PT-31 stability was assessed under different temperature and storage settings. The method was used to characterize PT-31 plasma pharmacokinetics following administration of 5 mg/kg i.v. to Wistar rats. Therefore, the method described is sensitive, linear, precise and specific enough to determine PT-31 in preclinical pharmacokinetic investigations. Copyright © 2015 John Wiley & Sons, Ltd.

Analysis of insect cuticular compounds by non-lethal solid phase micro extraction with styrene-divinylbenzene copolymers.

Insect cuticular hydrocarbons including relatively non-volatile chemicals play important roles in cuticle protection and chemical communication. The conventional procedures for extracting cuticular compounds from insects require toxic solvents, or non-destructive techniques that do not allow storage of subsequent samples, such as the use of SPME fibers. In this study, we describe and tested a non-lethal process for extracting cuticular hydrocarbons with styrene-divinylbenzene copolymers, and illustrate the method with two species of bees and one species of beetle. The results demonstrate that these compounds can be efficiently trapped by Chromosorb® (SUPELCO) and that this method can be used as an alternative to existing methods.

Citizen science in marine litter research: A review.

Citizen science (CS) can help to tackle the emerging and worldwide problem of marine litter (ML), from collecting data to engaging different stakeholders. We reviewed what and how the scientific literature is reporting CS on ML to identify possible gaps to be improved. The 92 search results (separate occasions when 48 different CS initiatives were discussed across 85 publication records) revealed an under-representation of studies in developing regions. Most search results focused on the science of ML, whilst information regarding citizen scientists was commonly vague or missing, preventing critical analysis of good practices on this aspect. The studies concentrated on the shoreline and did not harmonize types and sizes of items collected, thus precluding data meta-analyses. The standardisation of CS methods and approaches and the detailed report of aspects related to citizen scientists are essential to support the science we need for the advances in CS efforts to face ML.

Cuticular hydrocarbons in the stingless bee Schwarziana quadripunctata (Hymenoptera, Apidae, Meliponini): differences between colonies, castes and age.

Chemical communication is of fundamental importance to maintain the integration of insect colonies. In honey bees, cuticular lipids differ in their composition between queens, workers and drones. Little is known, however, about cuticular hydrocarbons in stingless bees. We investigated chemical differences in cuticular hydrocarbons between different colonies, castes and individuals of different ages in Schwarziana quadripunctata. The epicuticle of the bees was extracted using the non-polar solvent hexane, and was analyzed by means of a gas chromatograph coupled with a mass spectrometer. The identified compounds were alkanes, branched-alkanes and alkenes with chains of 19 to 33 carbon atoms. Discriminant analyses showed clear differences between all the groups analyzed. There were significant differences between bees from different colonies, workers of different age and between workers and virgin queens.

Characterisation of the in vivo interactions between detomidine and methadone in horses: Pharmacokinetic and pharmacodynamic modelling.

BACKGROUND: Pharmacokinetic (PK)/pharmacodynamic (PD) modelling offers new insights to design protocols for sedation and analgesia in standing horses. OBJECTIVES: To evaluate the parameters and interactions between detomidine and methadone when given alone or combined in standing horses. STUDY DESIGN: Randomised, placebo-controlled, blinded, crossover. METHODS: Eight adult healthy horses were given six treatments intravenously: saline (SAL); detomidine (5 µg/kg bwt; DET); methadone (0.2 mg/kg bwt; MET) alone or combined with detomidine (2.5 [MLD], 5 [MMD] or 10 [MHD] µg/kg bwt). Venous blood samples were obtained at predetermined times between 0 and 360 min after drug administration. Plasma detomidine and methadone were measured using a single, liquid/liquid extraction technique by liquid chromatography coupled with a triple quadrupole mass spectrometer (LC-MS/MS). Sequential PK/PD modelling compared rival models, with and without PK and PD interaction between drugs, to fit the PD data including height of the head above the ground (HHAG), a visual analogue scale for sedation (VAS), electrical (ET), thermal (TT) and mechanical (MT) nociceptive thresholds and gastrointestinal motility (GIM) [1]. RESULTS: Two and three compartment models best described the PK of detomidine and methadone, respectively. Detomidine decreased its own clearance as well as the clearance of methadone. The interaction of methadone on the effect of detomidine revealed an infra-additive (partial antagonism) effect for HHAG (α = -1.33), VAS (α = -0.98) and GIM (α = -1.05), a positive potentiation for ET (pot = 0.0041) and TT (pot = 0.133) and a synergistic to additive effect for MT (α = 0.78). MAIN LIMITATIONS: This is a small experimental study. CONCLUSIONS: Different PK/PD interactions were demonstrated for each PD parameter and could be modelled in vivo. The modelling of our data will allow us to simulate and predict the effect of constant rate infusions of both drugs for future investigations.

Acid diterpenes from Copaiba oleoresin (Copaifera langsdorffii): Chemical and plasma stability and intestinal permeability using Caco-2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Copaiba oleoresin has been used in folk medicine in the treatment of bronchitis, syphilis, skin diseases and ulcers due to its anti-inflammatory and antiseptic activities, but there is no information about major compounds oral absorption to support the traditional use. AIM OF STUDY: Considering the potential of copalic (CA) and kaurenoic acid (KA) - major biological activity (in vitro) diterpenes found in the oleoresin, this study aimed to evaluate the intestinal permeability of CA and KA using Caco-2 cells model as predictive test for oral drug absorption. MATERIALS AND METHODS: Chemical stability at pH 1.2 and 7.4 and plasma stability were evaluated to mimic physiological conditions of the gastrointestinal tract. The intestinal permeability of CA and KA was evaluated in Caco-2 cells in the presence and absence of the P-glycoprotein inhibitor verapamil. RESULTS: CA and KA were rapidly degraded at pH 1.2 (0.2 M Clark-Lubs buffer). At pH 7.4 (0.1 M phosphate buffer), CA was stable for up to 24 h and KA for up to 6 h. In human plasma, CA and KA can be considered stable for 24 h and 12 h at 37 °C, respectively. Caco-2 cells were considered viable when incubated with CA or KA in the range of 3.9-250 µM for 24 h. CA and KA exhibited moderate apparent permeability (Papp) of 4.67 (±0.08) × 10-6 cm/s and 4.66 (±0.04) × 10-6 cm/s, respectively. Simultaneous incubation with verapamil showed that P-glycoprotein does not play a relevant role on CA and KA oral absorption, with Papp of 4.48 (±0.26) × 10-6 cm/s and 5.37 (±0.72) × 10-6 cm/s observed for CA and KA, respectively. CONCLUSION: The oral absorption of both CA and KA is driven by mainly passive permeability, is not limited by p-glycoprotein, but enteric-coated dosage forms should be used to avoid chemical instability in the gastric pH.

Fragmentation pattern of amides by EI and HRESI: study of protonation sites using DFT-3LYP data.

Amides are important natural products which occur in a few plant families. Piplartine and piperine, major amides in Piper tuberculatum and P. nigrum, respectively, have shown a typical N-CO cleavage when analyzed by EI-MS or HRESI-MS. In this study several synthetic analogs of piplartine and piperine were subjected to both types of mass spectrometric analysis in order to identify structural features influencing fragmentation. Most of the amides showed an intense signal of the protonated molecule [M + H]+ when subjected to both HRESI-MS and EI-MS conditions, with a common outcome being the cleavage of the amide bond (N-CO). This results in the loss of the neutral amine or lactam and the formation of aryl acylium cations. The mechanism of N-CO bond cleavage persists in α,ß-unsaturated amides because of the stability caused by extended conjugation. Computational methods determined that the protonation of the piperamides and their derivatives takes place preferentially at the amide nitrogen supporting the dominant the N-CO bond cleavage.

Chemical profiling of two congeneric sea mat corals along the Brazilian coast: adaptive and functional patterns.

Metabolomic profiles were explored to understand environmental and taxonomic influences on the metabolism of two congeneric zoanthids, Palythoa caribaeorum and P. variabilis, collected across distinct geographical ranges. Integrated mass spectrometry data suggested the major influence of geographical location on chemical divergence when compared to species differentiation.

Inhibition of acute nociceptive responses in rats after i.c.v. injection of Thr6-bradykinin, isolated from the venom of the social wasp, Polybia occidentalis.

BACKGROUND AND PURPOSE: In this work, a neuroactive peptide from the venom of the neotropical wasp Polybia occidentalis was isolated and its anti-nociceptive effects were characterized in well-established pain induction models. EXPERIMENTAL APPROACH: Wasp venom was analysed by reverse-phase HPLC and fractions screened for anti-nociceptive activity. The structure of the most active fraction was identified by electron-spray mass spectrometry (ESI-MS/MS) and it was further assessed in two tests of anti-nociceptive activity in rats: the hot plate and tail flick tests. KEY RESULTS: The most active fraction contained a peptide whose structure was Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH, which corresponds to that of Thr(6)-BK, a bradykinin analogue. This peptide was given by i.c.v. injection to rats. In the tail flick test, Thr(6)-BK induced anti-nociceptive effects, approximately twice as potent as either morphine or bradykinin also given i.c.v. The anti-nociceptive activity of Thr(6)-BK peaked at 30 min after injection and persisted for 2 h, longer than bradykinin. The primary mode of action of Thr(6)-BK involved the activation of B(2) bradykinin receptors, as anti-nociceptive effects of Thr(6)-BK were antagonized by a selective B(2) receptor antagonist. CONCLUSIONS AND IMPLICATIONS: Our data indicate that Thr(6)-BK acts through B(2) bradykinin receptors in the mammalian CNS, evoking antinociceptive behaviour. This activity is remarkably different from that of bradykinin, despite the structural similarities between both peptides. In addition, due to the increased metabolic stability of Thr(6)-BK, relative to that of bradykinin, this peptide could provide a novel tool in the investigation of kinin pathways involved with pain.

Colony membership is reflected by variations in cuticular hydrocarbon profile in a Neotropical paper wasp, Polistes satan (Hymenoptera, Vespidae).

Nestmate recognition is one the most important features in social insect colonies. Although epicuticular lipids or cuticular hydrocarbons have both structural and defensive functions in insects, they also seem to be involved in several aspects of communication in wasps, bees and ants. We analyzed and described for the first time the cuticular hydrocarbons of a Neotropical paper wasp, Polistes satan, and found that variation in hydrocarbon profile was sufficiently strong to discriminate individuals according to their colony membership. Therefore, it seems that small differences in the proportion of these compounds can be detected and used as a chemical-based cue by nestmates to detect invaders and avoid usurpation.

Gas-phase dissociation study of erythrinian alkaloids by electrospray ionization mass spectrometry and computational methods.

Alkaloids from plants of the genus Erythrina display important biological activities, including anxiolytic action. Characterization of these alkaloids by mass spectrometry (MS) has contributed to the construction of a spectral library, has improved understanding of their structures and has supported the proposal of fragmentation mechanisms in light of density functional calculations. In this study, we have used low-resolution and high-resolution MSn analyses to investigate the fragmentation patterns of erythrinian alkaloids; we have employed the B3LYP/6-31+G(d,p) model to obtain their reactive sites. To suggest the fragmentation mechanism of these alkaloids, we have studied their protonation sites by density functional calculation, and we have obtained their molecular electrostatic potential map and their gas-phase basicity values. These analyses have indicated the most basic sites on the basis of the proton affinities of the nitrogen and oxygen atoms. The protonated molecules were generated by two major fragmentations, namely, neutral loss of CH3 OH followed by elimination of H2 O. High-resolution analysis confirmed elimination of NH3 by comparison with the losses of H2 and •CH3 . NH3 was eliminated from compounds that did not bear a substituent on ring C. The benzylic carbocation initiated the dissociation mechanism, and the first reaction involved charge transfer from a lone pair of electrons in the oxygen atoms. The second reaction consisted of ring contraction with loss of a CO molecule. The presence of hydroxy and epoxy groups could change the intensity or the occurrence of the fragmentation pathways. Given that erythrinian alkaloids are applied in therapeutics and are promising leads for the development of new drugs, the present results could aid identification of several analogues of these alkaloids in biological samples and advance pharmacokinetic studies of new plant derivatives based on MSn and MS/MS analyses. Copyright © 2017 John Wiley & Sons, Ltd.

Gas-phase fragmentation of protonated piplartine and its fungal metabolites using tandem mass spectrometry and computational chemistry.

Piplartine, an alkaloid produced by plants in the genus Piper, displays promising anticancer activity. Understanding the gas-phase fragmentation of piplartine by electrospray ionization tandem mass spectrometry can be a useful tool to characterize biotransformed compounds produced by in vitro and in vivo metabolism studies. As part of our efforts to understand natural product fragmentation in electrospray ionization tandem mass spectrometry, the gas-phase fragmentation of piplartine and its two metabolites 3,4-dihydropiplartine and 8,9-dihydropiplartine, produced by the endophytic fungus Penicillium crustosum VR4 biotransformation, were systematically investigated. Proposed fragmentation reactions were supported by ESI-MS/MS data and computational thermochemistry. Cleavage of the C-7 and N-amide bond, followed by the formation of an acylium ion, were characteristic fragmentation reactions of piplartine and its analogs. The production of the acylium ion was followed by three consecutive and competitive reactions that involved methyl and methoxyl radical eliminations and neutral CO elimination, followed by the formation of a four-member ring with a stabilized tertiary carbocation. The absence of a double bond between carbons C-8 and C-9 in 8,9-dihydropiplartine destabilized the acylium ion and resulted in a fragmentation pathway not observed for piplartine and 3,4-dihydropiplartine. These results contribute to the further understanding of alkaloid gas-phase fragmentation and the future identification of piplartine metabolites and analogs using tandem mass spectrometry techniques. Copyright © 2017 John Wiley & Sons, Ltd.

Metabolic adjustments in two Amazonian cichlids exposed to hypoxia and anoxia.

The effects of graded hypoxia on the physiological and biochemical responses were examined in two closely related species of cichlids of the Amazon: Astronotus crassipinnis and Symphysodon aequifasciatus. Ten fish of each species were exposed to graded hypoxia for 8 h in seven oxygen concentrations (5.92, 3.15, 1.54, 0.79, 0.60, 0.34, and 0.06 mg O(2) L(-)(1)), with the aim to evaluate hypoxia tolerance and metabolic adjustments, where plasma glucose and lactate levels, hepatic and muscle glycogen contents, and maximum enzyme activities (PK, LDH, MDH and CS) in skeletal and cardiac muscles were measured. Another experimental set was done to quantify oxygen consumption (MO(2)) and opercular movements in two oxygen concentrations. Hypoxia tolerance differed between the two species. Astronotus crassipinnis was able to tolerate anoxia for 178 min while S. aequifasciatus was able to withstand 222 min exposure in deep hypoxia (0.75 mg O(2) L(-)(1)). Suppressed MO(2) was observed during exposure to 0.34 (A. crassipinnis) and 0.79 mg O(2) L(-)(1) (S. aequifasciatus), while opercular movements increased in both species exposed to hypoxia. Higher levels of muscle and liver glycogen and larger hypoxia-induced increases in plasma glucose and lactate were observed in A. crassipinnis, which showed a higher degree of hypoxia tolerance. Changes in enzyme levels were tissue-specific and differed between species suggesting differential abilities in down-regulating oxidative pathways and increasing anaerobic metabolism. Based on the present data, we conclude that these animals are good anaerobes and highly adapted to their environment, which is allowed by their abilities to regulate metabolic pathways and adjust their enzyme levels.

Analgesic activity of the lignans from Lychnophora ericoides.

Lychnophora ericoides is a Brazilian medicinal plant that is commercially available as an analgesic and anti-inflammatory agent. The extract from roots, which yielded 10 lignans, showed analgesic activity in the mouse writhing test and the lignan, cubebin, was one of the most active. Anti-inflammatory and anti-pyretic activities from cubebin (10 mg/kg) revealed no significant effects. In addition two previously unknown methyl clusin derivatives are reported.

Phenylpropanoid derivatives and biflavones at different stages of differentiation and development of Araucaria angustifolia.

Chemical investigations carried out with tissues at different developmental stages of Araucaria angustifolia established the presence of E and Z isomers of octadecyl p-coumarate and octadecyl ferulate in undifferentiated callus; in the seedling stems, the source of explants, three biflavones of the amentoflavone-type were isolated, whereas the diterpene, trans-communic acid, was obtained from the seedling roots. Adult stems accumulated the benzaldehydes, vanillin, p-hydroxybenzaldehyde and coniferaldehyde; the lignans, pinoresinol, eudesmin and lariciresinol; and the isoflavones, cabreuvine and irisolidone.

Antimalarial use of volatile oil from leaves of Virola surinamensis (Rol.) Warb. by Waiãpi Amazon Indians.

The Amazon Indians Waiãpi living in the West of Amapá State of Brazil, treat malaria with an inhalation of vapor obtained from leaves of Viola surinamensis. The essential oil obtained from adult and plantlet leaves was analyzed by GC/MS and 11 monoterpenes, 11 sesquiterpenes and three phenylpropanoids were identified. Plantlet essential oil caused 100% of growth inhibition after 48 h in the development of the young trophozoite to schizont stage and the sesquiterpene nerolidol (100 microg/ml) was identified as one of the active constituents (100% of growth inhibition was obtained). In addition, examination of [U14C]-glucose incorporation showed that activity of nerolidol is related to the inhibition of glycoprotein biosynthesis.

The potential of secondary metabolites from plants as drugs or leads against protozoan neglected diseases - part II.

Infections with protozoan parasites are a major cause of disease and mortality in many tropical countries of the world. Diseases caused by species of the genera Trypanosoma (Human African Trypanosomiasis and Chagas Disease) and Leishmania (various forms of Leishmaniasis) are among the seventeen "Neglected Tropical Diseases" (NTDs) defined by the WHO. Furthermore, malaria (caused by various Plasmodium species) can be considered a neglected disease in certain countries and with regard to availability and affordability of the antimalarials. Living organisms, especially plants, provide an innumerable number of molecules with potential for the treatment of many serious diseases. The current review attempts to give an overview on the potential of such plant-derived natural products as antiprotozoal leads and/or drugs in the fight against NTDs. In part I, a general description of the diseases, the current state of therapy and need for new therapeuticals, assay methods and strategies applied in the search for new plant derived natural products against these diseases and an overview on natural products of terpenoid origin with antiprotozoal potential were given. The present part II compiles the current knowledge on natural products with antiprotozoal activity that are derived from the shikimate pathway (lignans, coumarins, caffeic acid derivatives), quinones of various structural classes, compounds formed via the polyketide pathways (flavonoids and related compounds, chromenes and related benzopyrans and benzofurans, xanthones, acetogenins from Annonaceae and polyacetylenes) as well as the diverse classes of alkaloids. In total, both parts compile the literature on almost 900 different plant-derived natural products and their activity data, taken from over 800 references. These data, as the result of enormous efforts of numerous research groups world-wide, illustrate that plant secondary metabolites represent an immensely rich source of chemical diversity with an extremely high potential to yield a wealth of lead structures towards new therapies for NTDs. Only a small percentage, however, of the roughly 200,000 plant species on earth have been studied chemically and only a small percentage of these plants or their constituents has been investigated for antiprotozoal activity. The repository of plant-derived natural products hence deserves to be investigated even more intensely than it has been up to present.

The potential of secondary metabolites from plants as drugs or leads against protozoan neglected diseases - part I.

Infections with protozoan parasites are a major cause of disease and mortality in many tropical countries of the world. Diseases caused by species of the genera Trypanosoma (Human African Trypanosomiasis and Chagas Disease) and Leishmania (various forms of Leishmaniasis) are among the seventeen "Neglected Tropical Diseases" (NTDs) defined as such by WHO due to the neglect of financial investment into research and development of new drugs by a large part of pharmaceutical industry and neglect of public awareness in high income countries. Another major tropical protozoan disease is malaria (caused by various Plasmodium species), which -although not mentioned currently by the WHO as a neglected disease- still represents a major problem, especially to people living under poor circumstances in tropical countries. Malaria causes by far the highest number of deaths of all protozoan infections and is often (as in this review) included in the NTDs. The mentioned diseases threaten many millions of lives world-wide and they are mostly associated with poor socioeconomic and hygienic environment. Existing therapies suffer from various shortcomings, namely, a high degree of toxicity and unwanted effects, lack of availability and/or problematic application under the life conditions of affected populations. Development of new, safe and affordable drugs is therefore an urgent need. Nature has provided an innumerable number of drugs for the treatment of many serious diseases. Among the natural sources for new bioactive chemicals, plants are still predominant. Their secondary metabolism yields an immeasurable wealth of chemical structures which has been and will continue to be a source of new drugs, directly in their native form and after optimization by synthetic medicinal chemistry. The current review, published in two parts, attempts to give an overview on the potential of such plant-derived natural products as antiprotozoal leads and/or drugs in the fight against NTDs.

Effects of chlorogenic acid on neutrophil locomotion functions in response to inflammatory stimulus.

AIM OF THE STUDY: Species of Lychnophora are used in Brazilian folk medicine as analgesic and anti-inflammatory agents. Chlorogenic acid (CGA) and their analogues are important components of polar extracts of these species, as well in several European and Asian medicinal plants. Some of these phenolic compounds display anti-inflammatory effects. In this paper we report the isolation of CGA from Lychnophora salicifolia and its effects on functions involved in neutrophils locomotion. MATERIALS AND METHODS: LC-MS(n) data confirmed the presence of CGA in the plant. Actions of CGA were investigated on neutrophils obtained from peritoneal cavity of Wistar rats (4h after 1% oyster glycogen solution injection; 10 ml), and incubated with vehicle or with 50, 100 or 1000 µM CGA in presence of lipopolysaccharide from Escherichia coli (LPS, 5 µg/ml). Nitric oxide (NO; Griess reaction); prostaglandin E(2) (PGE(2)), interleukin-1ß (IL-1ß) and tumor necrosis factor-α [TNF-α; enzyme-linked immunosorbent assay (EIA)]; protein (flow cytometry) and gene (RT-PCR) expression of L-selectin, ß(2)integrin and platelet-endothelial cell adhesion molecule-1 (PECAM-1) were quantified. In vitro neutrophil adhesion to primary culture of microvascular endothelial cell (PMEC) and neutrophil migration in response to formyl-methionil-leucil-phenilalanine (fMLP, 10(-8)M, Boyden chamber) was determined. RESULTS: CGA treatment did not modify the secretion of inflammatory mediators, but inhibited L-selectin cleavage and reduced ß(2) integrin, independently from its mRNA synthesis, and reduced membrane PECAM-1 expression; inhibited neutrophil adhesion and neutrophil migration induced by fMLP. CONCLUSIONS: Based on these findings, we highlight the direct inhibitory actions of CGA on adhesive and locomotion properties of neutrophils, which may contribute to its anti-inflammatory effects and help to explain the use of Lychnophora salicifolia as an anti-inflammatory agent.