Legacy and Emerging Plasticizers and Stabilizers in PVC Floorings and Implications for Recycling.

Hazardous chemicals in building and construction plastics can lead to health risks due to indoor exposure and may contaminate recycled materials. We systematically sampled new polyvinyl chloride floorings on the Swiss market (n = 151). We performed elemental analysis by X-ray fluorescence, targeted and suspect gas chromatography-mass spectrometry analysis of ortho-phthalates and alternative plasticizers, and bioassay tests for cytotoxicity and oxidative stress, and endocrine, mutagenic, and genotoxic activities (for selected samples). Surprisingly, 16% of the samples contained regulated chemicals above 0.1 wt %, mainly lead and bis(2-ethylhexyl) phthalate (DEHP). Their presence is likely related to the use of recycled PVC in new flooring, highlighting that uncontrolled recycling can delay the phase-out of hazardous chemicals. Besides DEHP, 29% of the samples contained other ortho-phthalates (mainly diisononyl and diisodecyl phthalates, DiNP and DiDP) above 0.1 wt %, and 17% of the samples indicated a potential to cause biological effects. Considering some overlap between these groups, they together make up an additional 35% of the samples of potential concern. Moreover, both suspect screening and bioassay results indicate the presence of additional potentially hazardous substances. Overall, our study highlights the urgent need to accelerate the phase-out of hazardous substances, increase the transparency of chemical compositions in plastics to protect human and ecosystem health, and enable the transition to a safe and sustainable circular economy.

Pharmacological characterization of potent and selective NaV1.7 inhibitors engineered from Chilobrachys jingzhao tarantula venom peptide JzTx-V.

Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers.

Time-resolved monitoring of polycyclic aromatic hydrocarbons adsorbed on atmospheric particles.

Real-time monitoring of individual particles from atmospheric aerosols was performed by means of a specifically developed single-particle fluorescence spectrometer (SPFS). The observed fluorescence was assigned to particles bearing polycyclic aromatic hydrocarbons (PAH). This assignment was supported by an intercomparison with classical speciation on filters followed by gas chromatography-mass spectrometry (GC-MS) analysis. As compared with daily averaged data, our time-resolved approach provided information about the physicochemical dynamics of the particles. In particular, distinctions were made between background emissions related to heating, and traffic peaks during rush hours. Also, the evolution of the peak fluorescence wavelength provided an indication of the aging of the particles during the day.

Multianalyte profiling of per- and polyfluoroalkyl substances (PFASs) in liquid commercial products.

The chemical properties of poly- and perfluoroalkyl substances (PFASs) make them widespread for use in a number of industrial and commercial products to confer water and oil-repellency characteristics and to reduce surface tension e.g. in aqueous film-forming foams (AFFFs). Some PFASs, especially perfluoroctane sulfonate, and several perfluoroalkyl carboxylic acids, are known to cause significant human and environmental negative impact. Our knowledge on the content of PFASs in products remains scarce due to limited information available, thus impeding any precise assessment of human exposure and environmental release upon use. This study aimed at analyzing a wide variety of liquid products (n = 194) likely to contain PFASs, including impregnating agents, lubricants, cleansers, polishes, AFFFs and other industrial products. By means of LC- and GC-MS/MS analytical techniques, 24 PFASs (from 41 targeted PFASs) were detected and quantified in 55% of samples. PFAS quantification and profiling was found to be consumer product specific. PFASs were mostly detected in AFFF (90%) and impregnating agents (60%) with mainly ionic and neutral species, respectively. In particular, the fluorotelomer alcohols 6:2, 8:2 and 10:2 FTOHs were detected in 40-50% of impregnating agents. Further investigation by Fast Atom Bombardment Mass Spectrometry (FAB-MS) on a set of AFFF samples allowed the characterization of 8 different PFAS classes as major components in these formulations. Results demonstrated that numerous and diversified PFAS are currently used in specific commercial products, implying significant human exposure and environmental release that necessitate further research concerning their toxicological impact.

Discrimination of hexabromocyclododecane from new polymeric brominated flame retardant in polystyrene foam by nuclear magnetic resonance.

Hexabromocyclododecane (HBCDD) is a brominated flame retardant (BFR) and major additive to polystyrene foam thermal insulation that has recently been listed as a persistent organic pollutant by the Stockholm Convention. During a 2013/2014 field analytical survey, we measured HBCDD content ranging from 0.2 to 2.4% by weight in 98 polystyrene samples. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses indicated that expandable (EPS) and extruded (XPS) polystyrene foams significantly differed in the α/γ HBCDD isomer ratio, with a majority of α and γ isomers in XPS and EPS, respectively. Interestingly, this technique indicated that some recent materials did not contain HBCDD, but demonstrated bromine content when analysed with X-ray fluorescence (XRF). Further investigation by Nuclear Magnetic Resonance (NMR) was able to discriminate between the BFRs present. In addition to confirming the absence or presence of HBCDD in polystyrene samples, high-field NMR spectroscopy provided evidence of the use of brominated butadiene styrene (BBS) as copolymer in the production of polystyrene. Use of this alternative flame retardant is expected to cause fewer health and environmental concerns. Our results highlight a trend towards the use of copolymerized BFRs as an alternative to HBCDD in polystyrene foam boards. In addition to providing a rapid NMR method to identify polymeric BFR, our analytical approach is a simple method to discriminate between flame-retardants in polystyrene foam insulating materials.

Engineering potent and selective analogues of GpTx-1, a tarantula venom peptide antagonist of the Na(V)1.7 sodium channel.

NaV1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residue peptide, termed GpTx-1, with potent activity on NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20× and 1000× over NaV1.4 and NaV1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp(29), Lys(31), and Phe(34) near the C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against NaV1.4. A structure-guided campaign afforded additive improvements in potency and NaV subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a NaV1.7 IC50 value of 1.6 nM and >1000× selectivity against NaV1.4 and NaV1.5.

Uncovering intense protein diversification in a cone snail venom gland using an integrative venomics approach.

Marine cone snail venoms are highly complex mixtures of peptides and proteins. They have been studied in-depth over the past 3 decades, but the modus operandi of the venomous apparatus still remains unclear. Using the fish-hunting Conus consors as a model, we present an integrative venomics approach, based on new proteomic results from the venom gland and data previously obtained from the transcriptome and the injectable venom. We describe here the complete peptide content of the dissected venom by the identification of numerous new peptides using nanospray tandem mass spectrometry in combination with transcriptomic data. Results reveal extensive mature peptide diversification mechanisms at work in the venom gland. In addition, by integrating data from three different venom stages, transcriptome, dissected, and injectable venoms, from a single species, we obtain a global overview of the venom processing that occurs from the venom gland tissue to the venom delivery step. In the light of the successive steps in this venom production system, we demonstrate that each venom compartment is highly specific in terms of peptide and protein content. Moreover, the integrated investigative approach discussed here could become an essential part of pharmaceutical development, as it provides new potential drug candidates and opens the door to numerous analogues generated by the very mechanisms used by nature to diversify its peptide and protein arsenal.

δ-Conotoxins synthesized using an acid-cleavable solubility tag approach reveal key structural determinants for NaV subtype selectivity.

Conotoxins are venom peptides from cone snails with multiple disulfide bridges that provide a rigid structural scaffold. Typically acting on ion channels implicated in neurotransmission, conotoxins are of interest both as tools for pharmacological studies and as potential new medicines. δ-Conotoxins act by inhibiting inactivation of voltage-gated sodium channels (Nav). Their pharmacology has not been extensively studied because their highly hydrophobic character makes them difficult targets for chemical synthesis. Here we adopted an acid-cleavable solubility tag strategy that facilitated synthesis, purification, and directed disulfide bridge formation. Using this approach we readily produced three native δ-conotoxins from Conus consors plus two rationally designed hybrid peptides. We observed striking differences in Nav subtype selectivity across this group of compounds, which differ in primary structure at only three positions: 12, 23, and 25. Our results provide new insights into the structure-activity relationships underlying the Nav subtype selectivity of δ-conotoxins. Use of the acid-cleavable solubility tag strategy should facilitate synthesis of other hydrophobic peptides with complex disulfide bridge patterns.

When everything converges: integrative taxonomy with shell, DNA and venomic data reveals Conus conco, a new species of cone snails (Gastropoda: Conoidea).

Cone snails have long been studied both by taxonomists for the diversity of their shells and by biochemists for the potential therapeutic applications of their toxins. Phylogenetic approaches have revealed that different lineages of Conus evolved divergent venoms, a property that is exploited to enhance the discovery of new conotoxins, but is rarely used in taxonomy. Specimens belonging to the Indo-West Pacific Conus lividus species complex were analyzed using phenetic and phylogenetic methods based on shell morphology, COI and 28S rRNA gene sequences and venom mRNA expression and protein composition. All methods converged to reveal a new species, C. conco n. sp. (described in Supplementary data), restricted to the Marquesas Islands, where it diverged recently (∼3mya) from C. lividus. The geographical distribution of C. conco and C. lividus and their phylogenetic relationships suggest that the two species diverged in allopatry. Furthermore, the diversity of the transcript sequences and toxin molecular masses suggest that C. conco evolved unique toxins, presumably in response to new selective pressure, such as the availability of new preys and ecological niches. Furthermore, this new species evolved new transcripts giving rise to original toxin structures, probably each carrying specific biological activity.

Performance evaluation of currently used portable X ray fluorescence instruments for measuring the lead content of paint in field samples.

Field-portable X-ray fluorescence (FP-XRF) instruments are important for non-destructive, rapid and convenient measurements of lead in paint, in view of potential remediation. Using real-life paint samples, we compared measurements from three FP-XRF instruments currently used in Switzerland with laboratory measurements using inductively coupled plasma mass spectrometry after complete sample dissolution. Two FP-XRF devices that functioned by lead L shell excitation frequently underestimated the lead concentration of samples. Lack of accuracy correlated with lead depth and/or the presence of additional metal elements (Zn, Ba or Ti). A radioactive source emitter XRF that enabled the additional K shell excitation showed higher accuracy and precision, regardless of the depth of the lead layer in the sample or the presence of other elements. Inspection of samples by light and electron microscopy revealed the diversity of real-life samples, with multi-layered paints showing various depths of lead and other metals. We conclude that the most accurate measurements of lead in paint are currently obtained with instruments that provide at least sufficient energy for lead K shell excitation.

Chemical camouflage--a frog's strategy to co-exist with aggressive ants.

Whereas interspecific associations receive considerable attention in evolutionary, behavioural and ecological literature, the proximate bases for these associations are usually unknown. This in particular applies to associations between vertebrates with invertebrates. The West-African savanna frog Phrynomantis microps lives in the underground nest of ponerine ants (Paltothyreus tarsatus). The ants usually react highly aggressively when disturbed by fiercely stinging, but the frog is not attacked and lives unharmed among the ants. Herein we examined the proximate mechanisms for this unusual association. Experiments with termites and mealworms covered with the skin secretion of the frog revealed that specific chemical compounds seem to prevent the ants from stinging. By HPLC-fractionation of an aqueous solution of the frogs' skin secretion, two peptides of 1,029 and 1,143 Da were isolated and found to inhibit the aggressive behaviour of the ants. By de novo sequencing using tandem mass spectrometry, the amino acid sequence of both peptides consisting of a chain of 9 and 11 residues, respectively, was elucidated. Both peptides were synthesized and tested, and exhibited the same inhibitory properties as the original frog secretions. These novel peptides most likely act as an appeasement allomone and may serve as models for taming insect aggression.

Predicting function from sequence in a large multifunctional toxin family.

Venoms contain active substances with highly specific physiological effects and are increasingly being used as sources of novel diagnostic, research and treatment tools for human disease. Experimental characterisation of individual toxin activities is a severe rate-limiting step in the discovery process, and in-silico tools which allow function to be predicted from sequence information are essential. Toxins are typically members of large multifunctional families of structurally similar proteins that can have different biological activities, and minor sequence divergence can have significant consequences. Thus, existing predictive tools tend to have low accuracy. We investigated a classification model based on physico-chemical attributes that can easily be calculated from amino-acid sequences, using over 250 (mostly novel) viperid phospholipase A2 toxins. We also clustered proteins by sequence profiles, and carried out in-vitro tests for four major activities on a selection of isolated novel toxins, or crude venoms known to contain them. The majority of detected activities were consistent with predictions, in contrast to poor performance of a number of tested existing predictive methods. Our results provide a framework for comparison of active sites among different functional sub-groups of toxins that will allow a more targeted approach for identification of potential drug leads in the future.

Position-specific scoring matrix and hidden Markov model complement each other for the prediction of conopeptide superfamilies.

Classified into 16 superfamilies, conopeptides are the main component of cone snail venoms that attract growing interest in pharmacology and drug discovery. The conventional approach to assigning a conopeptide to a superfamily is based on a consensus signal peptide of the precursor sequence. While this information is available at the genomic or transcriptomic levels, it is not present in amino acid sequences of mature bioactives generated by proteomic studies. As the number of conopeptide sequences is increasing exponentially with the improvement in sequencing techniques, there is a growing need for automating superfamily elucidation. To face this challenge we have defined distinct models of the signal sequence, propeptide region and mature peptides for each of the superfamilies containing more than 5 members (14 out of 16). These models rely on two robust techniques namely, Position-Specific Scoring Matrices (PSSM, also named generalized profiles) and hidden Markov models (HMM). A total of 50 PSSMs and 47 HMM profiles were generated. We confirm that propeptide and mature regions can be used to efficiently classify conopeptides lacking a signal sequence. Furthermore, the combination of all three-region models demonstrated improvement in the classification rates and results emphasise how PSSM and HMM approaches complement each other for superfamily determination. The 97 models were validated and offer a straightforward method applicable to large sequence datasets.

Conus consors snail venom proteomics proposes functions, pathways, and novel families involved in its venomic system.

For some decades, cone snail venoms have been providing peptides, generally termed conopeptides, that exhibit a large diversity of pharmacological properties. However, little attention has been devoted to the high molecular mass (HMM) proteins in venoms of mollusks. In order to shed more light on cone snail venom HMM components, the proteins of dissected and injected venom of a fish-hunting cone snail, Conus consors, were extensively assessed. HMM venom proteins were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by mass spectrometry (MS). The MS data were interpreted using UniProt database, EST libraries from C. consors venom duct and salivary gland, and their genomic information. Numerous protein families were discovered in the lumen of the venom duct and assigned a biological function, thus pointing to their potential role in venom production and maturation. Interestingly, the study also revealed original proteins defining new families of unknown function. Only two groups of HMM proteins passing the venom selection process, echotoxins and hyaluronidases, were clearly present in the injected venom. They are suggested to contribute to the envenomation process. This newly devised integrated HMM proteomic analysis is a big step toward identification of the protein arsenal used in a cone snail venom apparatus for venom production, maturation, and function.

Large-scale discovery of conopeptides and conoproteins in the injectable venom of a fish-hunting cone snail using a combined proteomic and transcriptomic approach.

Predatory marine snails of the genus Conus use venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable venom from Conus consors, an indo-pacific fish-hunting cone snail. By matching MS/MS data against an extensive set of venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone snail venom to date, and delineate the major protein families present in the delivered venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.

Peak capacity optimisation for high resolution peptide profiling in complex mixtures by liquid chromatography coupled to time-of-flight mass spectrometry: application to the Conus consors cone snail venom.

The high resolution profiling of complex mixtures is indispensable for obtaining online structural information on the highest possible number of the analytes present. This is particularly relevant for natural extracts, as for the venom of the predatory marine snail Conus consors, which contains numerous bioactive peptides with molecular masses ranging between 1000 and 5000 Da. The goal of the present work was to maximise peak capacity of peptides separations by LC-MS while maintaining a reasonable analysis time. The best gradient performance using the C. consors venom as a real sample was obtained with a mobile phase flow rate as high as possible to maximise performance in the gradient mode, and gradient time comprised between 75 and 350 min when using a 150 mm column length. The present study also confirmed that an elevated temperature (up to 90 °C) improves performance under ultra-high pressure liquid chromatography (UHPLC) conditions. However, the thermal stability of the analytes had to be critically evaluated. For the profiling of C. consors, analyte degradation was not clearly observable at 90 °C with analysis times of approximately 100 min. Finally, the MS source was found to cause significant additional band broadening in the UHPLC mode (σ(ext)(2) was 10-24 times higher using TOF-MS vs. UV detection). Thus, if the MS contributes strongly to the peak capacity loss, classical 2.1mm I.D. columns can be replaced by 3.0mm I.D. to mitigate this problem. Based on these considerations, the optimal generic profiling conditions applied to the C. consors venom provided a peak capacity higher than 1100 for a gradient time of around 100 min, doubling the values reached by classical HPLC separation. UHPLC-QTOF-MS/MS experiments carried out in these conditions provided exploitable data that matched with peptides present in the C. consors venom. These optimal LC conditions are thus compatible with online peptide deconvolution and matching against transcriptomic data and, to some extent, de novo sequencing in such complex mixtures.

ConoDictor: a tool for prediction of conopeptide superfamilies.

ConoDictor is a tool that enables fast and accurate classification of conopeptides into superfamilies based on their amino acid sequence. ConoDictor combines predictions from two complementary approaches-profile hidden Markov models and generalized profiles. Results appear in a browser as tables that can be downloaded in various formats. This application is particularly valuable in view of the exponentially increasing number of conopeptides that are being identified. ConoDictor was written in Perl using the common gateway interface module with a php submission page. Sequence matching is performed with hmmsearch from HMMER 3 and ps_scan.pl from the pftools 2.3 package. ConoDictor is freely accessible at http://conco.ebc.ee.

Recruitment of glycosyl hydrolase proteins in a cone snail venomous arsenal: further insights into biomolecular features of Conus venoms.

Cone snail venoms are considered an untapped reservoir of extremely diverse peptides, named conopeptides, displaying a wide array of pharmacological activities. We report here for the first time, the presence of high molecular weight compounds that participate in the envenomation cocktail used by these marine snails. Using a combination of proteomic and transcriptomic approaches, we identified glycosyl hydrolase proteins, of the hyaluronidase type (Hyal), from the dissected and injectable venoms ("injectable venom" stands for the venom variety obtained by milking of the snails. This is in contrast to the "dissected venom", which was obtained from dissected snails by extraction of the venom glands) of a fish-hunting cone snail, Conus consors (Pionoconus clade). The major Hyal isoform, Conohyal-Cn1, is expressed as a mixture of numerous glycosylated proteins in the 50 kDa molecular mass range, as observed in 2D gel and mass spectrometry analyses. Further proteomic analysis and venom duct mRNA sequencing allowed full sequence determination. Additionally, unambiguous segment location of at least three glycosylation sites could be determined, with glycans corresponding to multiple hexose (Hex) and N-acetylhexosamine (HexNAc) moieties. With respect to other known Hyals, Conohyal-Cn1 clearly belongs to the hydrolase-type of Hyals, with strictly conserved consensus catalytic donor and positioning residues. Potent biological activity of the native Conohyals could be confirmed in degrading hyaluronic acid. A similar Hyal sequence was also found in the venom duct transcriptome of C. adamsonii (Textilia clade), implying a possible widespread recruitment of this enzyme family in fish-hunting cone snail venoms. These results provide the first detailed Hyal sequence characterized from a cone snail venom, and to a larger extent in the Mollusca phylum, thus extending our knowledge on this protein family and its evolutionary selection in marine snail venoms.

A novel µ-conopeptide, CnIIIC, exerts potent and preferential inhibition of NaV1.2/1.4 channels and blocks neuronal nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: The µ-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new µ-conopeptide (µ-CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant. EXPERIMENTAL APPROACH: µ-CnIIIC was sequenced, synthesized and characterized by its direct block of elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. µ-CnIIIC was also studied on HEK-293 cells expressing various rodent VGSCs and also on voltage-gated potassium channels and nicotinic acetylcholine receptors (nAChRs) to assess cross-interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data. KEY RESULTS: Synthetic µ-CnIIIC decreased twitch tension in mouse hemidiaphragms (IC(50) = 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), compared with µ-SIIIA, µ-SmIIIA and µ-PIIIA. µ-CnIIIC blocked Na(V)1.4 (IC(50) = 1.3 nM) and Na(V)1.2 channels in a long-lasting manner. Cardiac Na(V)1.5 and DRG-specific Na(V)1.8 channels were not blocked at 1 µM. µ-CnIIIC also blocked the α3ß2 nAChR subtype (IC(50) = 450 nM) and, to a lesser extent, on the α7 and α4ß2 subtypes. Structure determination of µ-CnIIIC revealed some similarities to α-conotoxins acting on nAChRs. CONCLUSION AND IMPLICATIONS: µ-CnIIIC potently blocked VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels.

High-resolution picture of a venom gland transcriptome: case study with the marine snail Conus consors.

Although cone snail venoms have been intensively investigated in the past few decades, little is known about the whole conopeptide and protein content in venom ducts, especially at the transcriptomic level. If most of the previous studies focusing on a limited number of sequences have contributed to a better understanding of conopeptide superfamilies, they did not give access to a complete panorama of a whole venom duct. Additionally, rare transcripts were usually not identified due to sampling effect. This work presents the data and analysis of a large number of sequences obtained from high throughput 454 sequencing technology using venom ducts of Conus consors, an Indo-Pacific living piscivorous cone snail. A total of 213,561 Expressed Sequence Tags (ESTs) with an average read length of 218 base pairs (bp) have been obtained. These reads were assembled into 65,536 contiguous DNA sequences (contigs) then into 5039 clusters. The data revealed 11 conopeptide superfamilies representing a total of 53 new isoforms (full length or nearly full-length sequences). Considerable isoform diversity and major differences in transcription level could be noted between superfamilies. A, O and M superfamilies are the most diverse. The A family isoforms account for more than 70% of the conopeptide cocktail (considering all ESTs before clustering step). In addition to traditional superfamilies and families, minor transcripts including both cysteine free and cysteine-rich peptides could be detected, some of them figuring new clades of conopeptides. Finally, several sets of transcripts corresponding to proteins commonly recruited in venom function could be identified for the first time in cone snail venom duct. This work provides one of the first large-scale EST project for a cone snail venom duct using next-generation sequencing, allowing a detailed overview of the venom duct transcripts. This leads to an expanded definition of the overall cone snail venom duct transcriptomic activity, which goes beyond the cysteine-rich conopeptides. For instance, this study enabled to detect proteins involved in common post-translational maturation and folding, and to reveal compounds classically involved in hemolysis and mechanical penetration of the venom into the prey. Further comparison with proteomic and genomic data will lead to a better understanding of conopeptides diversity and the underlying mechanisms involved in conopeptide evolution.