Study of IsCT analogue peptide against Candida albicans and toxicity/teratogenicity in zebrafish embryos (Danio rerio).

Aim: An IsCT analogue peptide (PepM3) was designed based on structural studies of wasp mastoparans and tested against Candida albicans. Its effects on fungal cell membranes and toxicity were evaluated. Materials & methods: Antifungal activity was analyzed using a microdilution susceptibility test. Toxicity was assessed using human skin keratinocytes (HaCaT) and zebrafish embryos. Results: PepM3 demonstrated activity against C. albicans and a synergistic effect with amphotericin B. The peptide presented fungicidal action with damage to the fungal cell membrane, low toxicity in HaCat cells and was nonteratogenic in zebrafish embryos. Conclusion: Evaluating structural modifications is essential for the development of new agents with potential activity against fungal pathogens and for the reduction of toxic and teratogenic effects.

Study of mastoparan analog peptides against Candida albicans and safety in zebrafish embryos (Danio rerio).

Aim: In this work, mastoparan analog peptides from wasp venom were tested against Candida albicans and safety assays were performed using cell culture and model zebrafish. Materials & methods: Minimal inhibitory concentration was determined and toxicity was performed using human skin keratinocyte and embryo zebrafish. Also, permeation of peptides through embryo chorion was performed. Results: The peptides demonstrated anti-C. albicans activity, with low cytotoxicity and nonteratogenicity in Danio rerio. The compounds had different permeation through chorion, suggesting that this occurs due to modifications in their amino acid sequence. Conclusion: The results showed that the studied peptides can be used as structural study models for novel potential antifungal agents.

Simultaneous determination of anionic and nonionic surfactants in commercial laundry wastewater and anaerobic fluidized bed reactor effluent by online column-switching liquid chromatography/tandem mass spectrometry.

This study presents a new method developed for the simultaneous determination of anionic surfactant (linear alkylbenzene sulfonate - LAS, 4 homologs) and nonionic surfactant (linear alcohol ethoxylate - LAE) in commercial laundry wastewater. The surfactants were identified and quantified using online column-switching solid-phase extraction (SPE) coupled with liquid chromatography/tandem mass spectrometry (LC-MS/MS). Ten and three transitions (m/z) were identified for LAS and LAE, respectively. The detection and quantification limits were 75 and 200µg/L for LAS, respectively, and 75µg/L for LAE. This method was applied to the determination of the surfactants in the influent and effluent of an anaerobic fluidized bed reactor that was used for the treatment of commercial laundry wastewater. After 480days of operation with a hydraulic retention time (HRT) of 18h, the removal of 45.9±5.6% LAS and 99.2±4.3% LAE from an influent with surfactant concentrations of 26.1±12.9mg/L and 23.8±6.8mg/L, respectively, was obtained. Under these conditions, the breakage of longer-chain LAS homologs with the release of carbon units was observed with an increase in the number of shorter homolog chains. This SPE online sample treatment method is simple, fast and effective for the analysis of both surfactants. This technique is pioneering in its simultaneous measurement of two surfactant categories in anaerobic fluidized bed reactors.

Sulfamethoxazole and ciprofloxacin removal using a horizontal-flow anaerobic immobilized biomass reactor.

The antibiotics sulfamethoxazole (SMTX) and ciprofloxacin (CIP) are commonly used in human and veterinary medicine, which explains their occurrence in wastewater. Anaerobic reactors are low-cost, simple and suitable technology to wastewater treatment, but there is a lack of studies related to the removal efficiency of antibiotics. To overcome this knowledge gap, the objective of this study was to evaluate the removal kinetics of SMTX and CIP using a horizontal-flow anaerobic immobilized biomass reactor. Two different concentrations were evaluated, for SMTX 20 and 40 µg L(-1); for CIP 2.0 and 5.0 µg L(-1). The affluent and effluent analysis was carried out in liquid chromatography/tandem mass spectrometry (LC-MS/MS) with the sample preparation procedure using an off-line solid-phase extraction. This method was developed, validated and successfully applied for monitoring the affluent and effluent samples. The removal efficiency found for both antibiotics at the two concentrations studied was 97%. Chemical oxygen demand (COD) exhibited kinetic constants that were different from that observed for the antibiotics, indicating the absence of co-metabolism. Also, though the antibiotic concentration was increased, there was no inhibitory effect in the removal of COD and antibiotics.

Rapid determination of 12 antibiotics and caffeine in sewage and bioreactor effluent by online column-switching liquid chromatography/tandem mass spectrometry.

This study presents a column-switching solid-phase extraction online-coupled to a liquid chromatography/tandem mass spectrometry (SPE-LC-MS/MS) method for simultaneous analysis of 12 antibiotics (7 sulfonamides and 5 fluoroquinolones) and caffeine detected in the sewage and effluent of a pilot anaerobic reactor used in sewage treatment. After acidification and filtration, the samples were directly injected into a simple and conventional LC system. Backflush and foreflush modes were compared based on the theoretical plates and peak asymmetry observed. The method was tested in terms of detection (MDL) and quantification limit (MQL), linearity, relative recovery, and precision intra- and inter-day in lab-made sewage samples. The method presented suitable figures of merit in terms of detection, varying from 8.00 × 10(-5) to 6.00 × 10(-2) ng (0.800 up to 600 ng L(-1); caffeine) with direct injection volume of only 100 µL and 13 min of total analysis time (sample preparation and chromatographic run). When the method was applied in the analysis of sewage and effluent of the anaerobic reactor (n = 15), six antibiotics and caffeine were detected in concentrations ranging from 0.018 to 1097 µg L(-1). To guarantee a reliable quantification, standard addition was used to overcome the matrix effect.

The effects of the C-terminal amidation of mastoparans on their biological actions and interactions with membrane-mimetic systems.

Polycationic peptides may present their C-termini in either amidated or acidic form; however, the effects of these conformations on the mechanisms of interaction with the membranes in general were not properly investigated up to now. Protonectarina-MP mastoparan with an either amidated or acidic C-terminus was utilized to study their interactions with anionic and zwitterionic vesicles, using measurements of dye leakage and a combination of H/D exchange and mass spectrometry to monitor peptide-membrane interactions. Mast cell degranulation, hemolysis and antibiosis assays were also performed using these peptides, and the results were correlated with the structural properties of the peptides. The C-terminal amidation promotes the stabilization of the secondary structure of the peptide, with a relatively high content of helical conformations, permitting a deeper interaction with the phospholipid constituents of animal and bacterial cell membranes. The results suggested that at low concentrations Protonectarina-MP interacts with the membranes in a way that both terminal regions remain positioned outside the external surface of the membrane, while the α-carbon backbone becomes partially embedded in the membrane core and changing constantly the conformation, and causing membrane destabilization. The amidation of the C-terminal residue appears to be responsible for the stabilization of the peptide conformation in a secondary structure that is richer in α-helix content than its acidic congener. The helical, amphipathic conformation, in turn, allows a deeper peptide-membrane interaction, favoring both biological activities that depend on peptide structure recognition by the GPCRs (such as exocytosis) and those activities dependent on membrane perturbation (such as hemolysis and antibiosis).

Determination of steroids, caffeine and methylparaben in water using solid phase microextraction-comprehensive two dimensional gas chromatography-time of flight mass spectrometry.

Analysis of several emerging contaminants (steroids, caffeine and methylparaben) in water using automated solid-phase microextraction with comprehensive two dimensional gas chromatography coupled to time of flight mass spectrometry (SPME-GCxGC-ToF/MS) is presented. Experimental design was used to determine the best SPME extraction conditions and the steroids were not derivatized prior to injection. SPME-GCxGC-ToF/MS provided linear ranges from 0.6 to 1200µgL(-1) and limits of detection and quantitation from 0.02 to 100µgL(-1). A series of river water samples obtained locally were subjected to analysis. SPME-GCxGC-ToF/MS is readily automated, straightforward and competitive with other methods for low level analysis of emerging contaminants.

Larvicidal activity against Aedes aegypti of essential oils from northeast Brazil.

The larvicidal activities of essential oils from the leaves of Artemisia vulgaris L., Cymbopogon flexuosus (Nees ex Steud.) Wats. and Piper tuberculatum Jacq. were evaluated using third-instar larvae of Aedes aegypti. The essential oils were obtained by hydrodistillation and analyzed by GC and GC-MS. The essential oil of P. tuberculqatum had the lowest LC50 value (106.3 +/- 2.2 microg/mL), followed by that of A. vulgaris (114.1 +/- 1.7 microg/mL) and C. flexuosus (121.6 +/- 0.8 micro/mL). The results show that these essential oils may be potent sources of natural larvicides.

Development of a new stir bar sorptive extraction coating and its application for the determination of six pesticides in sugarcane juice.

In this article, a novel polydimethylsiloxane/activated carbon (PDMS-ACB) material is proposed as a new polymeric phase for stir bar sorptive extraction (SBSE). The PDMS-ACB stir bar, assembled using a simple Teflon(®)/glass capillary mold, demonstrated remarkable stability and resistance to organic solvents for more than 150 extractions. The SBSE bar has a diameter of 2.36 mm and a length of 2.2 cm and is prepared to contain 92 µL of polymer coating. This new PDMS-ACB bar was evaluated for its ability to determine the quantity of pesticides in sugarcane juice samples by performing liquid desorption (LD) in 200 µL of ethyl acetate and analyzing the solvent through gas chromatography coupled with mass spectrometry (GC-MS). A fractional factorial design was used to evaluate the main parameters involved in the extraction procedure. Then, a central composite design with a star configuration was used to optimize the significant extraction parameters. The method used demonstrated a limit of quantification (LOQ) of 0.5-40 µg/L, depending on the analyte detected; the amount of recovery varied from 0.18 to 49.50%, and the intraday precision ranged from 0.072 to 8.40%. The method was used in the analysis of real sugarcane juice samples commercially available in local markets.

Nigriventrine: a low molecular mass neuroactive compound from the venom of the spider Phoneutria nigriventer.

Nigriventrine was isolated from the "armed" spider Phoneutria nigriventer, in which it constitutes about 0.4% of the total venom content. Its structure was determined to be [1,1'-(1-hydroxyhydrazine-1,2-diyl)bis(oxy)bis(4-hydroxy-2,6-dioxopiperidine-4 carboxylic acid)] by NMR, HR-ES/IMS and MS/MS methods. The intracerebroventricular application of nigriventrine in rat brain, followed by the detection of c-Fos protein expression, indicated that the compound was neuroactive in the motor cortex, sensory cortex, piriform cortex, median preoptic nucleus, dorsal endopiriform nucleus, lateral septal nucleus and hippocampus of rat brain. Nigriventrine causes convulsions in rats, even when peripherally applied.

Monoamine oxidase inhibitory activities of indolylalkaloid toxins from the venom of the colonial spider Parawixia bistriata: functional characterization of PwTX-I.

Colonial spiders evolved a differential prey-capture behaviour in concert with their venom chemistry, which may be a source of novel drugs. Some highly active tetrahydro-beta-carboline (THbetaC) toxins were recently isolated from the venom of the colonial spider Parawixia bistriata; the spiders use these toxins as part of their chemical arsenal to kill and/or paralyze preys. The major THbetaC compound isolated from this venom was identified as 6-hydroxytrypargine, also known as PwTX-I. Most natural compounds of animal origin occur in low abundance, and the natural abundance of PwTX-I is insufficient for complete functional characterization. Thus, PwTx-I was synthesized using a Pictet-Spengler condensation strategy, and the stereoisomers of the synthetic toxin were separated by chiral chromatography. The fraction of venom containing a mixture of three natural THbetaC toxins and enantiomers of PwTx-I were analyzed for inhibition of monoamine oxidase (MAO)-A and -B and for toxicity to insects. We reveal that the mixture of the natural THbetaC toxins, as well as the enantiomers of PwTx-I, were non-competitive inhibitors of MAO-A and MAO-B and caused potent paralysis of honeybees. The (-)-PwTX-I enantiomer is 2-fold more potent than the (+)-PwTX-I enantiomer in the assays performed.

Electrospray ionization quadrupole time-of-flight and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometric analyses to solve micro-heterogeneity in post-translationally modified peptides from Phoneutria nigriventer (Aranea, Ctenidae) venom.

Previous studies of the fractionated venom of the Brazilian armed spider Phoneutria nigriventer, obtained by gel filtration, have demonstrated the presence of a fraction PhM, a pool of small peptides (up to 2000 Da) that provoke contractions in smooth muscle of guinea pig ileum. Initial attempts to sequence these peptides were largely unsuccessful because of the low purification yield and the fact that the majority seemed to be blocked at their N-termini. In the present work, analysis of this venom fraction by mass spectrometry has revealed the existence of a highly complex mixture of peptides with molecular weights corresponding to those observed for the muscle-active peptides previously described (800-1800 Da). These peptides appear to be a family of isoforms with some particular features. The amino acid sequences of 15 isoforms have been determined by tandem mass spectrometry (MS/MS) using both electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q/ToFMS) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-ToF/ToFMS). These molecules contain post-translational modifications such as proteolysis and C-terminal amidation, which combine to generate additional isoforms. All the isoforms sequenced in this study possess an N-terminal pyroglutamic acid residue. A search for sequence similarities with other peptides in databanks revealed that these peptides are structurally related to the tachykinins, a family of neuro-hormone peptides. The data obtained in this study will be essential for the subsequent steps of this research, the synthesis of these peptides and pharmacological characterization of their biological activity.