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Bitterlings are carp-like teleost fish (Cypriniformes: Acheilanathidae) known for their specialized brood parasitic lifestyle. Bitterling embryos, in fact, develop inside the gill chamber of their freshwater mussel hosts. However, little is known about how their parasitic lifestyle affects brain development in comparison to nonparasitic species. Here, we document the development of the brain of the rosy bitterling, Rhodeus ocellatus, at four embryonic stages of 165, 185, 210, 235 hours postfertilization (hpf) using micro-computed tomography (microCT). Focusing on developmental regionalization and brain ventricular organization, we relate the development of the brain divisions to those described for zebrafish using the prosomeric model as a reference paradigm. Segmentation and three-dimensional visualization of the ventricular system allowed us to identify changes in the longitudinal brain axis as a result of cephalic flexure during development. The results show that during early embryonic and larval development, histological differentiation, tissue boundaries, periventricular proliferation zones, and ventricular spaces are all detectable by microCT. The results of this study visualized with differential CT profiles are broadly consistent with comparable histological studies, and with the genoarchitecture of teleosts like the zebrafish. Compared to the zebrafish, our study identifies distinct developmental heterochronies in the rosy bitterling, such as a precocious development of the inferior lobe.

Convergence is the phenomenon whereby similar phenotypes evolve independently in different lineages. One example is resistance to toxins in animals. Toxins have evolved many times throughout the tree of life. They disrupt molecular and physiological pathways in target species, thereby incapacitating prey or deterring a predator. In response, molecular resistance has evolved in many species exposed to toxins to counteract their harmful effects. Here, we review current knowledge on the convergence of toxin resistance using examples from a wide range of toxin families. We explore the evolutionary processes and molecular adaptations driving toxin resistance. However, resistance adaptations may carry a fitness cost if they disrupt the normal physiology of the resistant animal. Therefore, there is a trade-off between maintaining a functional molecular target and reducing toxin susceptibility. There are relatively few solutions that satisfy this trade-off. As a result, we see a small set of molecular adaptations appearing repeatedly in diverse animal lineages, a phenomenon that is consistent with models of deterministic evolution. Convergence may also explain what has been called ‘autoresistance’. This is often thought to have evolved for self-protection, but we argue instead that it may be a consequence of poisonous animals feeding on toxic prey. Toxin resistance provides a unique and compelling model system for studying the interplay between trophic interactions, selection pressures and the molecular mechanisms underlying evolutionary novelties.

Standardization and reduction of variation is key to behavioural screening of animal models in toxicological and pharmacological studies. However, individual variation in behavioural and physiological phenotypes remains in each laboratory population and can undermine the understanding of toxicological and pharmaceutical effects and their underlying mechanisms. Here, we used zebrafish (ABTL-strain) larvae to explore individual consistency in activity level and emergence time, across subsequent days of early development (6–8 dpf). We also explored the correlation between these two behavioural parameters. We found inter-individual consistency over time in activity level and emergence time, but we did not find a consistent correlation between these parameters. Subsequently, we investigated the impact of variation in activity level on the effect of a 1% ethanol treatment, suitable for our proof-of-concept case study about whether impact from pharmacological treatments might be affected by inter-individual variation in basal locomotion. The inter-individual consistency over time in activity level did not persist in this test. This was due to the velocity change from before to after exposure, which turned out to be a dynamic individual trait related to basal activity level: low-activity individuals raised their swimming velocity, while high-activity individuals slowed down, yielding diametrically opposite response patterns to ethanol exposure. We therefore argue that inter-individual consistency in basal activity level, already from 6 dpf, is an important factor to take into account and provides a practical measure to improve the power of statistical analyses and the scope for data interpretation from behavioural screening studies.

Paraffin histology is one of the most important and commonly-used laboratory techniques in diagnostic histopathology. The discovery of paraffin embedding is often attributed to the pathologist Edwin Klebs. Klebs was following the lead of Stricker, who embedded embryos in a mixture of hot stearin and white beeswax. We show that Klebs experimented with paraffin wax for embedding tumour tissue. But he quickly rejected it as unsuitable because paraffin wax did not infiltrate the tissue. One of Klebs' correspondents, embryologist Wilhelm His, Sr., learned of Klebs' experiments and decided to try paraffin embedding. His dehydrated chicken embryos in alcohol, cleared them in lavender oil, and dripped hot paraffin wax onto them. This process allowed His to cut good sections. Here, we have replicated His's paraffin embedding protocol in order to determine whether His had indeed made the landmark discovery of infiltration embedding with paraffin wax. We followed the protocol that he gives in his 1868 monograph on the early development of the chicken. The protocol described by His failed, in our hands, to yield sections of the quality that he illustrates in his monograph. Typically, the tissue disintegrated when sectioned due to poor infiltration of the wax. Usable sections could only be obtained if His's protocol was modified by melting the embedded embryos in fresh paraffin wax. One explanation for our findings is that we failed to faithfully replicate His's protocol. Another is that his protocol was incomplete. We suggest that His is likely to have discovered and perfected infiltration embedding with paraffin wax but did not publish a complete protocol.Die paraffinbasierte histologische Untersuchung ist eines der wichtigsten angewendeten Laborverfahren in der diagnostischen Histopathologie. Die Entwicklung der Paraffineinbettung wird oft dem Pathologen Edwin Klebs zugeschrieben. Klebs orientierte sich an den Arbeiten von Stricker, der Embryonen in eine Mischung aus heißem Tristearin und weißem Bienenwachs einbettete, und experimentierte mit Paraffin, um Tumorgewebe einzubetten. Er verwarf den Ansatz aber rasch, da Paraffin das Gewebe nicht infiltrierte. Klebs stand im Briefwechsel mit dem Embryologen Wilhelm His Senior, der von dessen Experimenten erfuhr und beschloss, sich an der Paraffineinbettung zu versuchen. His entwässerte Hühnerembryonen in Alkohol, klärte sie mit Lavendelöl und träufelte heißes Paraffin darüber. Dieses Verfahren ermöglichte ihm die Anfertigung guter Schnitte. In der vorliegenden Arbeit haben wir His’ Protokoll der Paraffineinbettung nachvollzogen, um zu ermitteln, ob His tatsächlich die Infiltrationseinbettung mit Paraffin gelungen ist. Wir befolgten das Protokoll aus seiner 1868 erschienenen Monografie. Anhand der Anweisungen von His gelang es uns nicht, Schnitte mit der von ihm illustrierten Qualität herzustellen. In der Regel zerfiel das Gewebe beim Schneiden wegen schlechter Wachsinfiltration. Brauchbare Schnitte wurden nur bei Abwandlung von His’ Protokoll durch das Wiedereinschmelzen der eingebetteten Embryos in frischem Paraffin erreicht. Möglicherweise ist es uns nicht gelungen ist, das Protokoll von His detailgetreu zu replizieren, oder das Protokoll war unvollständig. Nach unserer Einschätzung hat His wahrscheinlich die Infiltrationseinbettung mit Paraffin erfunden und perfektioniert, aber kein vollständiges Protokoll veröffentlicht.

Paraffin histology is one of the most important and commonly used laboratory techniques in diagnostic histopathology. The discovery of paraffin embedding is often attributed to the pathologist Edwin Klebs. Klebs was following the lead of Stricker, who embedded embryos in a mixture of hot stearin and white beeswax. We show that Klebs experimented with paraffin wax for embedding tumour tissue. But he quickly rejected it as unsuitable because paraffin wax did not infiltrate the tissue. One of Klebs' correspondents, embryologist Wilhelm His, Sr., learned of Klebs' experiments and decided to try paraffin embedding. His dehydrated chicken embryos in alcohol, cleared them in lavender oil, and dripped hot paraffin wax onto them. This process allowed His to cut good sections. Here, we have replicated His's paraffin embedding protocol in order to determine whether His had indeed made the landmark discovery of infiltration embedding with paraffin wax. We followed the protocol that he gives in his 1868 monograph on the early development of the chicken. The protocol described by His failed, in our hands, to yield sections of the quality that he illustrates in his monograph. Typically, the tissue disintegrated when sectioned due to poor infiltration of the wax. Usable sections could only be obtained if His's protocol was modified by melting the embedded embryos in fresh paraffin wax. One explanation for our findings is that we failed to faithfully replicate His's protocol. Another is that his protocol was incomplete. We suggest that His is likely to have discovered and perfected infiltration embedding with paraffin wax but did not publish a complete protocol.Die paraffinbasierte histologische Untersuchung ist eines der wichtigsten angewendeten Laborverfahren in der diagnostischen Histopathologie. Die Entwicklung der Paraffineinbettung wird oft dem Pathologen Edwin Klebs zugeschrieben. Klebs orientierte sich an den Arbeiten von Stricker, der Embryonen in eine Mischung aus heißem Tristearin und weißem Bienenwachs einbettete, und experimentierte mit Paraffin, um Tumorgewebe einzubetten. Er verwarf den Ansatz aber rasch, da Paraffin das Gewebe nicht infiltrierte. Klebs stand im Briefwechsel mit dem Embryologen Wilhelm His Senior, der von dessen Experimenten erfuhr und beschloss, sich an der Paraffineinbettung zu versuchen. His entwässerte Hühnerembryonen in Alkohol, klärte sie mit Lavendelöl und träufelte heißes Paraffin darüber. Dieses Verfahren ermöglichte ihm die Anfertigung guter Schnitte. In der vorliegenden Arbeit haben wir His’ Protokoll der Paraffineinbettung nachvollzogen, um zu ermitteln, ob His tatsächlich die Infiltrationseinbettung mit Paraffin gelungen ist. Wir befolgten das Protokoll aus seiner 1868 erschienenen Monografie. Anhand der Anweisungen von His gelang es uns nicht, Schnitte mit der von ihm illustrierten Qualität herzustellen. In der Regel zerfiel das Gewebe beim Schneiden wegen schlechter Wachsinfiltration. Brauchbare Schnitte wurden nur bei Abwandlung von His’ Protokoll durch das Wiedereinschmelzen der eingebetteten Embryos in frischem Paraffin erreicht. Möglicherweise ist es uns nicht gelungen ist, das Protokoll von His detailgetreu zu replizieren, oder das Protokoll war unvollständig. Nach unserer Einschätzung hat His wahrscheinlich die Infiltrationseinbettung mit Paraffin erfunden und perfektioniert, aber kein vollständiges Protokoll veröffentlicht.

More than 400,000 people each year suffer adverse effects following bites from venomous snakes. However, snake venom is also a rich source of bioactive molecules with known or potential therapeutic applications. Manually ‘milking’ snakes is the most common method to obtain venom. Safer alternative methods to produce venom would facilitate the production of both antivenom and novel therapeutics. This protocol describes the generation, maintenance and selected applications of snake venom gland organoids. Snake venom gland organoids are 3D culture models that can be derived within days from embryonic or adult venom gland tissues from several snake species and can be maintained long-term (we have cultured some organoids for more than 2 years). We have successfully used the protocol with glands from late-stage embryos and recently deceased adult snakes. The cellular heterogeneity of the venom gland is maintained in the organoids, and cell type composition can be controlled through changes in media composition. We describe in detail how to derive and grow the organoids, how to dissociate them into single cells, and how to cryopreserve and differentiate them into toxin-producing organoids. We also provide guidance on useful downstream assays, specifically quantitative real-time PCR, bulk and single-cell RNA sequencing, immunofluorescence, immunohistochemistry, fluorescence in situ hybridization, scanning and transmission electron microscopy and genetic engineering. This stepwise protocol can be performed in any laboratory with tissue culture equipment and enables studies of venom production, differentiation and cellular heterogeneity.

Molecular genetic data have recently been incorporated in attempts to reconstruct the ecology of the ancestral snake, though this has been limited by a paucity of data for one of the two main extant snake taxa, the highly fossorial Scolecophidia. Here we present and analyze vision genes from the first eye-transcriptomic and genome-wide data for Scolecophidia, for Anilios bicolor, and A. bituberculatus, respectively. We also present immunohistochemistry data for retinal anatomy and visual opsin-gene expression in Anilios. Analyzed in the context of 19 lepidosaurian genomes and 12 eye transcriptomes, the new genome-wide and transcriptomic data provide evidence for a much more reduced visual system in Anilios than in non-scolecophidian (=alethinophidian) snakes and in lizards. In Anilios, there is no evidence of the presence of 7 of the 12 genes associated with alethinophidian photopic (cone) phototransduction. This indicates extensive gene loss and many of these candidate gene losses occur also in highly fossorial mammals with reduced vision. Although recent phylogenetic studies have found evidence for scolecophidian paraphyly, the loss in Anilios of visual genes that are present in alethinophidians implies that the ancestral snake had a better-developed visual system than is known for any extant scolecophidian.

Background: The outflow tract of crocodilians resembles that of birds and mammals as ventricular septation is complete. The arterial anatomy, however, presents with a pulmonary trunk originating from the right ventricular cavum, and two aortas originating from either the right or left ventricular cavity. Mixing of blood in crocodilians cannot occur at the ventricular level as in other reptiles but instead takes place at the aortic root level by a shunt, the foramen of Panizza, the opening of which is guarded by two facing semilunar leaflets of both bicuspid aortic valves. Methods: Developmental stages of Alligator mississipiensis, Crocodilus niloticus and Caiman latirostris were studied histologically. Results and Conclusions: The outflow tract septation complex can be divided into two components. The aorto-pulmonary septum divides the pulmonary trunk from both aortas, whereas the interaortic septum divides the systemic from the visceral aorta. Neural crest cells are most likely involved in the formation of both components. Remodeling of the endocardial cushions and both septa results in the formation of bicuspid valves in all three arterial trunks. The foramen of Panizza originates intracardially as a channel in the septal endocardial cushion.

Evolutionary developmental biology (evo-devo) is the study of the evolution of developmental mechanisms. Here, I review some of the theories, models, and laws in evo-devo, past and present. Nineteenth-century evo-devo was dominated by recapitulation theory and archetypes. It also gave us germ layer theory, the vertebral theory of the skull, floral organs as modified leaves, and the "inverted invertebrate" theory, among others. Newer theories and models include the frameshift theory, the genetic toolkit for development, the ABC model of flower development, the developmental hourglass, the zootype, Urbilateria, and the hox code. Some of these new theories show the influence of archetypes and recapitulation. Interestingly, recent studies support the old "primordial leaf," "inverted invertebrate," and "segmented head" theories. Furthermore, von Baer's first three laws may now need to be rehabilitated, and the hourglass model modified, in view of what Abzhanov has pointed out about the maternal-zygotic transition. There are many supposed "laws" of evo-devo but I argue that these are merely generalizations about trends in particular lineages. I argue that the "body plan" is an archetype, and is often used in such a way that it lacks any scientific meaning. Looking to the future, one challenge for evo-devo will be to develop new theories and models to accommodate the wealth of new data from high-throughput sequencing, including single-cell sequencing. One step in this direction is the use of sophisticated in silico analyses, as in the "transcriptomic hourglass" models.

Bitterlings, a group of freshwater teleosts, provide a fascinating example among vertebrates of the evolution of brood parasitism. Their eggs are laid inside the gill chamber of their freshwater mussel hosts where they develop as brood parasites. Studies of the embryonic development of bitterlings are crucial in deciphering the evolution of their distinct early life‐history. Here, we have studied 255 embryos and larvae of the rosy bitterling (Rhodeus ocellatus) using in vitro fertilization and X‐ray microtomography (microCT). We describe 11 pre‐hatching and 13 post‐hatching developmental stages spanning the first 14 days of development, from fertilization to the free‐swimming stage. In contrast to previous developmental studies of various bitterling species, the staging system we describe is character‐based and therefore more compatible with the widely‐used stages described for zebrafish. Our bitterling data provide new insights into to the polarity of the chorion, and into notochord vacuolization and yolk sac extension in relation to body straightening. This study represents the first application of microCT scanning to bitterling development and provides one of the most detailed systematic descriptions of development in any teleost. Our staging series will be an important tool for heterochrony analysis and other comparative studies of teleost development, and may provide insight into the co‐evolution of brood parasitism., A selection of microCT images of rosy bitterling embryos.

6 páginas, 3 figuras., Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A2 (PLA2) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense., This work was funded from a studentship supported by Elizabeth Artin Kazandjian to T.D.K.; grant PE 2600/1 from the German Research Foundation (DFG) to D.P.; grant OPUS 1354156 from the U.S. National Science Foundation to H.W.G.; grants FAPESP 2017/18922-2 and 2019/05026-4 from the São Paulo Research Foundation to R.R.d.S.; grants RPG-2012-627 and RFG-10193 from the Leverhulme Trust to R.A.H. and W.W.; grant MR/L01839X/1 from the U.K. Medical Research Council to J.M.G., R.A.H., J.J.C., and N.R.C.; Career Development Fellowships APP1162503 and DP190103787 from the Australian National Health and Medical Research Council to I.V.; fellowship DE160101142 and grant DP160104025 from the Australian Research Council, and fellowship FRIPRO-YRT #287462 from the Research Council of Norway to E.A.B.U.; and a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by the Wellcome Trust and Royal Society to N.R.C.

More than 400,000 people each year suffer adverse effects following bites from venomous snakes. However, snake venom is also a rich source of bioactive molecules with known or potential therapeutic applications. Manually 'milking' snakes is the most common method to obtain venom. Safer alternative methods to produce venom would facilitate the production of both antivenom and novel therapeutics. This protocol describes the generation, maintenance and selected applications of snake venom gland organoids. Snake venom gland organoids are 3D culture models that can be derived within days from embryonic or adult venom gland tissues from several snake species and can be maintained long-term (we have cultured some organoids for more than 2 years). We have successfully used the protocol with glands from late-stage embryos and recently deceased adult snakes. The cellular heterogeneity of the venom gland is maintained in the organoids, and cell type composition can be controlled through changes in media composition. We describe in detail how to derive and grow the organoids, how to dissociate them into single cells, and how to cryopreserve and differentiate them into toxin-producing organoids. We also provide guidance on useful downstream assays, specifically quantitative real-time PCR, bulk and single-cell RNA sequencing, immunofluorescence, immunohistochemistry, fluorescence in situ hybridization, scanning and transmission electron microscopy and genetic engineering. This stepwise protocol can be performed in any laboratory with tissue culture equipment and enables studies of venom production, differentiation and cellular heterogeneity.

© King Abdulaziz City for Science and Technology 2020, The emergence of bacterial resistance due to the indiscriminate use of antibiotics warrants the need for developing new bioactive agents. In this context, antimicrobial peptides are highly useful for managing resistant microbial strains. In this study, we report the isolation and characterization of peptides obtained from the venom of the toadfish Thalassophryne nattereri. These peptides were active against Gram-positive and Gram-negative bacteria and fungi. The primary amino acid sequences showed similarity to Cocaine and Amphetamine Regulated Transcript peptides, and two peptide analogs—Tn CRT2 and Tn CRT3—were designed using the AMPA algorithm based on these sequences. The analogs were subjected to physicochemical analysis and antimicrobial screening and were biologically active at concentrations ranging from 2.1 to 13 μM. Zeta potential analysis showed that the peptide analogs increased the positive charge on the cell surface of Grampositive and Gram-negative bacteria. The toxicity of Tn CRT2 and Tn CRT3 were analyzed in vitro using a hemolytic assay and tetrazolium salt reduction in fibroblasts and was found to be significant only at high concentrations (up to 40 μM). These results suggest that this methodological approach is appropriate to design novel antimicrobial peptides to fight bacterial infections and represents a new and promising discovery in fish venom., This research was supported by FAPESP grant No. 2017/00032-0.

This study describes the application of at-line nanofractionation to the screening of snake venoms for antibacterial activity against Gram-positive and Gram-negative bacteria, the detection of proteins of interest, and their partial or full identification. A method was developed to identify bioactive peptides in crude snake venoms based on reversed-phase liquid chromatography (LC), with parallel nanofractionation onto 384-well plates and mass spectrometry (MS). Bioactivity assays were based on a resazurin-reduction assay. Accurate masses of the bioactive peptides were determined, and peptides were then identified via nanoLC–MS/MS analysis of tryptic digests, allowing full or partial identification of the bioactive proteins. Crude venoms from 41 species were screened for their antibacterial bioactivity. Venoms showing the highest activity were further screened using at-line nanofractionation, which resulted in the elucidation of 28 bioactive proteins.

Background Venom has evolved in parallel in multiple animals for the purpose of self-defense, prey capture or both. These venoms typically consist of highly complex mixtures of toxins: diverse bioactive peptides and/or proteins each with a specific pharmacological activity. Because of their specificity, they can be used as experimental tools to study cell mechanisms and develop novel medicines and drugs. It is therefore potentially valuable to explore the venoms of various animals to characterize their toxins and identify novel toxin-families. This study focuses on the annotation and exploration of the transcriptomes of six scorpion species from three different families. The transcriptomes were annotated with a custom-built automated pipeline, primarily consisting of Basic Local Alignment Search Tool searches against UniProt databases and filter steps based on transcript coverage. Results We annotated the transcriptomes of four scorpions from the family Buthidae, one from Iuridae and one from Diplocentridae using our annotation pipeline. We found that the four buthid scorpions primarily produce disulfide-bridged ion-channel targeting toxins, while the non-buthid scorpions have a higher abundance of non-disulfide-bridged toxins. Furthermore, analysis of the “unidentified” transcripts resulted in the discovery of six novel putative toxin families containing a total of 37 novel putative toxins. Additionally, 33 novel toxins in existing toxin-families were found. Lastly, 19 novel putative secreted proteins without toxin-like disulfide bonds were found. Conclusions We were able to assign most transcripts to a toxin family and classify the venom composition for all six scorpions. In addition to advancing our fundamental knowledge of scorpion venomics, this study may serve as a starting point for future research by facilitating the identification of the venom composition of scorpions and identifying novel putative toxin families. Electronic supplementary material The online version of this article (10.1186/s12864-019-6013-6) contains supplementary material, which is available to authorized users.

Rationale The endocannabinoid system (ECS) comprises the cannabinoids anandamide and 2-arachidonoylglycerol and the cannabinoid receptors 1 and 2 (Cnr1 and Cnr2). The function of these receptors in relation to zebrafish larval behavior is poorly understood, even though the zebrafish larva has become a versatile animal model in biomedical research. Objectives The objective of the present study is to characterize the function of Cnr1 and Cnr2 in relation to behavior in zebrafish. Methods Behavioral analysis of zebrafish larvae was performed using a visual motor response (VMR) test, which allows locomotor activity to be determined under basal conditions and upon a dark challenge. Results Treatment with the non-specific Cnr agonists WIN55,212-2 and CP55,940 resulted in a decrease in locomotion. This was observed for both basal and challenge-induced locomotion, although the potency for these two effects was different, which suggests different mechanisms of action. In addition, WIN55,212-2 increased the reaction time of the startle response after the dark challenge. Using the Cnr1 antagonist AM251 and a cnr1−/− mutant line, it was shown that the effects were mediated by Cnr1 and not Cnr2. Interestingly, administration of the antagonist AM251 alone does not have an effect on locomotion, which indicates that endogenous cannabinoid activity does not affect locomotor activity of zebrafish larvae. Upon repeated dark challenges, the WIN55,212-2 effect on the locomotor activity decreased, probably due to desensitization of Cnr1. Conclusions Taken together, these results show that Cnr1 activation by exogenous endocannabinoids modulates both basal and challenge-induced locomotor activity in zebrafish larvae and that these behavioral effects can be used as a readout to monitor the Cnr1 responsiveness in the zebrafish larva model system.

Background: The venom of Phoneutria nigriventer spider is a source of numerous bioactive substances, including some toxins active in insects. An example is PnTx4(5-5) that shows a high insecticidal activity and no apparent toxicity to mice, although it inhibited NMDA-evoked currents in rat hippocampal neurons. In this work the analgesic activity of PnTx4(5-5) (renamed Γ-ctenitoxin-Pn1a) was investigated. Methods: The antinociceptive activity was evaluated using the paw pressure test in rats, after hyperalgesia induction with intraplantar injection of carrageenan or prostaglandin E2 (PGE2). Results: PnTx4(5-5), subcutaneously injected, was able to reduce the hyperalgesia induced by PGE2 in rat paw, demonstrating a systemic effect. PnTx4(5-5) administered in the plantar surface of the paw caused a peripheral and dose-dependent antinociceptive effect on hyperalgesia induced by carrageenan or PGE2. The hyperalgesic effect observed in these two pain models was completely reversed with 5 µg of PnTx4(5-5). Intraplantar administration of L-glutamate induced hyperalgesic effect that was significantly reverted by 5 μg of PnTx4(5-5) injection in rat paw. Conclusion: The antinociceptive effect for PnTx4(5-5) was demonstrated against different rat pain models, i.e. induced by PGE2, carrageenan or glutamate. We suggest that the antinociceptive effect of PnTx4(5-5) may be related to an inhibitory activity on the glutamatergic system.

The mean primary diameter (d) of nanoparticles (NPs) is commonly used as the best basis to assess the dose metric for expressing the toxicity of spherical NPs. However, d bears no relevance for nonspherical NPs. In the present study, the mean surface area to volume ratio, applicable to both spherical and nonspherical NPs, was used to replace d given the aim of obtaining the best dose metric (volume, surface area, or number) of differently shaped metallic NPs in vivo (9 organisms) and in vitro (6 mammalian cell lines). The slope of the curves obtained by relating the total particle number of NPs at various effect concentrations to the mean surface area to volume ratio was subsequently used to deduce the best dose metric. For the majority of the organisms studied, it was found that NP volume is the most appropriate dose metric, independent of the composition of the NPs tested. For 3 organisms exposed to Ag NPs, however, surface area was found to be the best dose metric. It is therefore concluded that the optimum dose metric depends on organism and NP properties. Environ Toxicol Chem 2016;35:2466-2473. © 2016 SETAC.

Initial association of platelets after vascular injury is mediated by glycoprotein (GP)Ib-IX-V binding to von Willebrand factor (vWf) immobilized on exposed collagens and eventually leads to thrombus formation. This article provides data about a new P-I class snake venom metalloproteinase (SVMP), barnettlysin-I (Bar-I), purified from the venom of Bothrops barnetti. This Data in Brief manuscript complements the main research article by providing additional data of the biochemical characterization of Bar-I 10.1016/j.bbagen.2015.12.021 [1].

The venom of the Brazilian armed spider Phoneutria nigriventer is a rich source of biologically active peptides that have potential as analgesic drugs. In this study, we investigated the analgesic and adverse effects of peptide 3-5 (Tx3-5), purified from P. nigriventer venom, in several mouse models of pain. Tx3-5 was administered by intrathecal injection to mice selected as models of postoperative (plantar incision), neuropathic (partial sciatic nerve ligation) and cancer-related pain (inoculation with melanoma cells) in animals that were either sensitive or tolerant to morphine. Intrathecal administration of Tx3-5 (3-300 fmol/site) in mice could either prevent or reverse postoperative nociception, with a 50 % inhibitory dose (ID50) of 16.6 (3.2-87.2) fmol/site and a maximum inhibition of 87 ± 10 % at a dose of 30 fmol/site. Its effect was prevented by the selective activator of L-type calcium channel Bay-K8644 (10 μg/site). Tx3-5 (30 fmol/site) also produced a partial antinociceptive effect in a neuropathic pain model (inhibition of 67 ± 10 %). Additionally, treatment with Tx3-5 (30 fmol/site) nearly abolished cancer-related nociception with similar efficacy in both morphine-sensitive and morphine-tolerant mice (96 ± 7 and 100 % inhibition, respectively). Notably, Tx3-5 did not produce visible adverse effects at doses that produced antinociception and presented a TD50 of 1125 (893-1418) fmol/site. Finally, Tx3-5 did not alter the normal mechanical or thermal sensitivity of the animals or cause immunogenicity. Our results suggest that Tx3-5 is a strong drug candidate for the treatment of painful conditions.

Venomous snakes are important subjects of study in evolution, ecology, and biomedicine. Many venomous snakes have alpha-neurotoxins (&alpha, neurotoxins) in their venom. These toxins bind the alpha-1 nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction, causing paralysis and asphyxia. Several venomous snakes and their predators have evolved resistance to &alpha, neurotoxins. The resistance is conferred by steric hindrance from N-glycosylated asparagines at amino acids 187 or 189, by an arginine at position 187 that has been hypothesized to either electrostatically repulse positively charged neurotoxins or sterically interfere with &alpha, neurotoxin binding, or proline replacements at positions 194 or 197 of the nAChR ligand-binding domain to inhibit &alpha, neurotoxin binding through structural changes in the receptor. Here, we analyzed this domain in 148 vertebrate species, and assessed its amino acid sequences for resistance-associated mutations. Of these sequences, 89 were sequenced de novo. We find widespread convergent evolution of the N-glycosylation form of resistance in several taxa including venomous snakes and their lizard prey, but not in the snake-eating birds studied. We also document new lineages with the arginine form of inhibition. Using an in vivo assay in four species, we provide further evidence that N-glycosylation mutations reduce the toxicity of cobra venom. The nAChR is of crucial importance for normal neuromuscular function and is highly conserved throughout the vertebrates as a result. Our research shows that the evolution of &alpha, neurotoxins in snakes may well have prompted arms races and mutations to this ancient receptor across a wide range of sympatric vertebrates. These findings underscore the inter-connectedness of the biosphere and the ripple effects that one adaption can have across global ecosystems.

The ability to culture complex organs is currently an important goal in biomedical research. It is possible to grow organoids (3D organ-like structures) in vitro; however, a major limitation of organoids, and other 3D culture systems, is the lack of a vascular network. Protocols developed for establishing in vitro vascular networks typically use human or rodent cells. A major technical challenge is the culture of functional (perfused) networks. In this rapidly advancing field, some microfluidic devices are now getting close to the goal of an artificially perfused vascular network. Another development is the emergence of the zebrafish as a complementary model to mammals. In this review, we discuss the culture of endothelial cells and vascular networks from mammalian cells, and examine the prospects for using zebrafish cells for this objective. We also look into the future and consider how vascular networks in vitro might be successfully perfused using microfluidic technology.

Venom systems have evolved on multiple occasions across the animal kingdom, and they can act as key adaptations to protect animals from predators [1]. Consequently, venomous animals serve as models for a rich source of mimicry types, as non-venomous species benefit from reductions in predation risk by mimicking the coloration, body shape, and/or movement of toxic counterparts [2, 3, 4, 5]. The frequent evolution of such deceitful imitations provides notable examples of phenotypic convergence and are often invoked as classic exemplars of evolution by natural selection. Here, we investigate the evolution of fangs, venom, and mimetic relationships in reef fishes from the tribe Nemophini (fangblennies). Comparative morphological analyses reveal that enlarged canine teeth (fangs) originated at the base of the Nemophini radiation and have enabled a micropredatory feeding strategy in non-venomous Plagiotremus spp. Subsequently, the evolution of deep anterior grooves and their coupling to venom secretory tissue provide Meiacanthus spp. with toxic venom that they effectively employ for defense. We find that fangblenny venom contains a number of toxic components that have been independently recruited into other animal venoms, some of which cause toxicity via interactions with opioid receptors, and result in a multifunctional biochemical phenotype that exerts potent hypotensive effects. The evolution of fangblenny venom has seemingly led to phenotypic convergence via the formation of a diverse array of mimetic relationships that provide protective (Batesian mimicry) and predatory (aggressive mimicry) benefits to other fishes [2, 6]. Our results further our understanding of how novel morphological and biochemical adaptations stimulate ecological interactions in the natural world.

A general approach is proposed that allows for quantifying the relative toxic contribution of ions released from metallic nanoparticles and of the particles themselves, as exemplified for the case of differently shaped zinc oxide (ZnO) nanoparticles (NPs) exposed to zebrafish embryos. First of all, the toxicity of suspensions of ZnO nanoparticles (NP(total))--nanospheres, nanosticks, cuboidal submicron particles (SMPs), and Zn(NO3)2--to the embryos was assessed. The observed toxicity of ZnO NP(total) is assumed to result from the combined effect of the particles present in the suspensions (NP(particle)) and of the dissolved Zn(2+) ions released from the particles (NP(ion)). Different addition models were used to explicitly account for the toxicity of NP(particle). The median lethal concentrations (LC50) of NP(particle) of nanospheres, nanosticks, and SMPs were found to range between 7.1 mg Zn/L and 11.9 mg Zn/L (i.e., to differ by a factor of 1.7). Behavioral performance showed no significant differences among all types of the NP(particle). The median effective concentrations (EC50) of the particles were found to range between 1.0 mg Zn/L and 2.2 mg Zn/L. At the LC50 of each particle suspension, the main contribution to lethality to zebrafish embryos was from the NP(particle) (52%-72%). For hatching inhibition, the NP(particle) was responsible for 38% to 83% of the adverse effects observed. The ZnO nanosticks were more toxic than any of the other NPs with regard to the endpoints mortality and hatching inhibition. The main contribution to toxicity to zebrafish embryos was from the NP(particle) at the LC50 and EC50 of each particle suspension.

Three sizes of copper nanoparticles (Cu NPs; 25 nm, 50 nm, and 100 nm), 1 submicron-sized particle, and Cu(NO3 )2 were added to the culture buffer of zebrafish embryos from 24 h postfertilization to 120 h postfertilization. In suspensions of Cu NPs and the Cu submicron-sized particle, the main contribution to the toxicity to zebrafish embryos was from the particle form of Cu particles (Cu NPparticle , >71%) rather than from dissolved Cu from the Cu particles (Cu NPion ). All particles tested as well as copper nitrate inhibited hatching, altered behavioral responses, and increased the incidence of malformations. Different kinds of abnormalities were observed in the morphology and behavior of the zebrafish embryos, depending on the particle size of the Cu suspensions tested. The median lethal concentrations of Cu NPparticle (25 nm, 50 nm, and 100 nm), the submicron-sized particle, and copper nitrate were 0.58 mg/L, 1.65 mg/L, 1.90 mg/L, 0.35 mg/L, and 0.70 mg/L, respectively. Submicron-sized particles and copper nitrate were more toxic than Cu NPs, and smaller Cu NPs were more toxic than larger Cu NPs. Dissolution of Cu NPs and the subsequent ion toxicity was not the primary mechanism of Cu NP toxicity in zebrafish embryos.

Snakes possess many extreme morphological and physiological adaptations. Identification of the molecular basis of these traits can provide novel understanding for vertebrate biology and medicine. Here, we study snake biology using the genome sequence of the Burmese python (Python molurus bivittatus), a model of extreme physiological and metabolic adaptation. We compare the python and king cobra genomes along with genomic samples from other snakes and perform transcriptome analysis to gain insights into the extreme phenotypes of the python. We discovered rapid and massive transcriptional responses in multiple organ systems that occur on feeding and coordinate major changes in organ size and function. Intriguingly, the homologs of these genes in humans are associated with metabolism, development, and pathology. We also found that many snake metabolic genes have undergone positive selection, which together with the rapid evolution of mitochondrial proteins, provides evidence for extensive adaptive redesign of snake metabolic pathways. Additional evidence for molecular adaptation and gene family expansions and contractions is associated with major physiological and phenotypic adaptations in snakes; genes involved are related to cell cycle, development, lungs, eyes, heart, intestine, and skeletal structure, including GRB2-associated binding protein 1, SSH, WNT16, and bone morphogenetic protein 7. Finally, changes in repetitive DNA content, guanine-cytosine isochore structure, and nucleotide substitution rates indicate major shifts in the structure and evolution of snake genomes compared with other amniotes. Phenotypic and physiological novelty in snakes seems to be driven by system-wide coordination of protein adaptation, gene expression, and changes in the structure of the genome.

Phα1β toxin is a peptide purified from the venom of the armed spider Phoneutria nigriventer , with markedly antinociceptive action in models of acute and persistent pain in rats. Similarly to ziconotide, its analgesic action is related to inhibition of high voltage activated calcium channels with more selectivity for N-type. In this study we evaluated the effect of Phα1β when injected peripherally or intrathecally in a rat model of spontaneous pain induced by capsaicin. We also investigated the effect of Phα1β on Ca 2+ transients in cultured dorsal root ganglia (DRG) neurons and HEK293 cells expressing the TRPV1 receptor. Intraplantar or intrathecal administered Phα1β reduced both nocifensive behavior and mechanical hypersensitivity induced by capsaicin similarly to that observed with SB366791, a specific TRPV1 antagonist. Peripheral nifedipine and mibefradil did also decrease nociceptive behavior induced by intraplantar capsaicin. In contrast, ω-conotoxin MVIIA (a selective N-type Ca 2+ channel blocker) was effective only when administered intrathecally. Phα1β, MVIIA and SB366791 inhibited, with similar potency, the capsaicin-induced Ca 2+ transients in DRG neurons. The simultaneous administration of Phα1β and SB366791 inhibited the capsaicin-induced Ca 2+ transients that were additive suggesting that they act through different targets. Moreover, Phα1β did not inhibit capsaicin-activated currents in patch-clamp recordings of HEK293 cells that expressed TRPV1 receptors. Our results show that Phα1β may be effective as a therapeutic strategy for pain and this effect is not related to the inhibition of TRPV1 receptors.

The marine snail peptide ziconotide (ω-conotoxin MVIIA) is used as an analgesic in cancer patients refractory to opioids, but may induce severe adverse effects. Animal venoms represent a rich source of novel drugs, so we investigated the analgesic effects and the side-effects of spider peptide Phα1β in a model of cancer pain in mice with or without tolerance to morphine analgesia. Cancer pain was induced by the inoculation of melanoma B16-F10 cells into the hind paw of C57BL/6 mice. After 14 days, painful hypersensitivity was detected and Phα1β or ω-conotoxin MVIIA (10-100 pmol/site) was intrathecally injected to evaluate the development of antinociception and side-effects in control and morphine-tolerant mice. The treatment with Phα1β or ω-conotoxin MVIIA fully reversed cancer-related painful hypersensitivity, with long-lasting results, at effective doses 50% of 48 (32-72) or 33 (21-53) pmol/site, respectively. Phα1β produced only mild adverse effects, whereas ω-conotoxin MVIIA induced dose-related side-effects in mice at analgesic doses (estimated toxic dose 50% of 30 pmol/site). In addition, we observed that Phα1β was capable of controlling cancer-related pain even in mice tolerant to morphine antinociception (100% of inhibition) and was able to partially restore morphine analgesia in such animals (56 ± 5% of inhibition). In this study, Phα1β was as efficacious as ω-conotoxin MVIIA in inducing analgesia in a model of cancer pain without producing severe adverse effects or losing efficacy in opioid-tolerant mice, indicating that Phα1β has a good profile for the treatment of cancer pain in patients.

In the present work, we report the isolation and partial biochemical characterization of BpLec, a C-type lectin purified from Bothrops pauloensis venom by one chromatographic step on an affinity agarose column immobilized with d-galactose. This protein was homogeneous by SDS-PAGE under reducing and nonreducing conditions, and was shown to be a 33.6 kDa homodimer by MALDI TOF analysis. BpLec presented an isoeletric point of 5.36. Its partial sequence of 132 amino acids for each subunit, determined by Edman degradation, revealed high identity (between 86% and 95%) when aligned with sequences of other related proteins. BpLec was capable of agglutinating native dog and cat erythrocytes and this activity was inhibited by β-galactosides and EDTA. Its hemagglutinating activity was abolished at high temperatures and stable in any pH range. BpLec was effective in inhibiting Gram-positive but not Gram-negative bacteria. In addition, BpLec agglutinated promastigote forms of Leishmania (Leishmania) amazonensis.

The humerus of fossorial moles has a highly derived anatomy, reflecting the ecological specialization of these animals for digging. It is short and broad, with enlarged muscle attachment sites and pronounced articulations compared to non-fossorial sister taxa and other mammals. Both condyles are rotated in opposite directions, resulting in a torsion which is unique among eutherian mammals. The development of this exceptional bone was studied in embryonic stages of the fossorial Iberian mole (Talpa occidentalis) from mesenchymal condensation to incipient ossification based on histological serial sections using 3D reconstruction methods. For comparison, embryonic stages of the semi-fossorial Japanese shrew mole (Urotrichus talpoides) as well as a sister taxon of moles, the terrestrial North American least shrew (Cryptotis parva), were studied. Results show that the humerus of Talpa already shows its derived anatomy with broadened muscle attachment sites and distinct articulations at early cartilaginous stages, when ossification has just started in the mid-diaphyseal region. The torsion takes place simultaneously with the medial rotation of the forelimbs. The supracondylar foramen is closed in all studied Talpa embryos, but patent in Cryptotis and Urotrichus. This is an example of developmental penetrance, suggesting that variation of adult elements can be found at early stages as well.

Venomous snakes have evolved their efficient venomous arsenals mainly to immobilize prey. The highly variable toxic peptides in these venoms target a myriad of neurotoxic and haemotoxic receptors and enzymes and comprise highly interesting candidates for drug discovery. Discovery of bioactive compounds from snake venoms, however, is a challenge to achieve. We have developed and applied a methodology to rapidly assess bioactives in a snake venom proteome. Our microfluidic platform opens up efficient and rapid profiling of venomous anti-cholinergic receptor compounds. The key advantages of our methodology are: (i) nano amounts of venom needed; and (ii) a direct correlation of selected bioaffinities with accurate mass. To achieve this, we have for the first time successfully constructed a functional post nano-LC split to MS and bioaffinity profiling. In our method, comprehensive venom profiles with accurate masses and corresponding bioaffinities are obtained in one analytical run and will subsequently allow immediate purification of bioactive peptides with LC-MS, guided by accurate masses of the bioactives only. We profiled several neurotoxic Elapidae snake venoms using our methodology in combination with the acetylcholine binding protein (AChBP) as biological target protein. The latter is a homologue of nicotinic acetylcholine receptors (nAChRs), a drug target in neurodegenerative diseases and cognitive decline such as Parkinson's and Alzheimer's, and in pain related diseases. Our methodology was evaluated and validated with high-affinity α-bungarotoxin and haemotoxic/proteolytic Vipera ammodytes venom spiked with α-bungarotoxin. Thereafter, the methodology was applied to profile the venom proteomes of Dendroaspis jamesoni kaimosae, Naja annulifera and Naja nivea. Gathering comprehensive profiling data took less than 2 h per snake venom measured. The data yielded 20 AChBP ligands of which the corresponding accurate masses were used to retrieve information from literature regarding their function and targeting specificity. We found that from these 20 ligands, 11 were previously reported on, while information on the others could not be found. From these 11 peptides, five have been reported to have nAChR affinity, while the others are reported as cytotoxic, cardiotoxic or as orphan toxin. Our methodology has the potential to aid the field of profiling complex animal venoms for drug discovery. © 2012 Elsevier Ltd.