Red-on-Yellow Queen: Bio-Layer Interferometry Reveals Functional Diversity Within Micrurus Venoms and Toxin Resistance in Prey Species.

Snakes in the family Elapidae largely produce venoms rich in three-finger toxins (3FTx) that bind to the α 1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specific mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specificity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan → serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

Monkeying around with venom: an increased resistance to α-neurotoxins supports an evolutionary arms race between Afro-Asian primates and sympatric cobras.

BACKGROUND: Snakes and primates have a multi-layered coevolutionary history as predators, prey, and competitors with each other. Previous work has explored the Snake Detection Theory (SDT), which focuses on the role of snakes as predators of primates and argues that snakes have exerted a selection pressure for the origin of primates' visual systems, a trait that sets primates apart from other mammals. However, primates also attack and kill snakes and so snakes must simultaneously avoid primates. This factor has been recently highlighted in regard to the movement of hominins into new geographic ranges potentially exerting a selection pressure leading to the evolution of spitting in cobras on three independent occasions. RESULTS: Here, we provide further evidence of coevolution between primates and snakes, whereby through frequent encounters and reciprocal antagonism with large, diurnally active neurotoxic elapid snakes, Afro-Asian primates have evolved an increased resistance to α-neurotoxins, which are toxins that target the nicotinic acetylcholine receptors. In contrast, such resistance is not found in Lemuriformes in Madagascar, where venomous snakes are absent, or in Platyrrhini in the Americas, where encounters with neurotoxic elapids are unlikely since they are relatively small, fossorial, and nocturnal. Within the Afro-Asian primates, the increased resistance toward the neurotoxins was significantly amplified in the last common ancestor of chimpanzees, gorillas, and humans (clade Homininae). Comparative testing of venoms from Afro-Asian and American elapid snakes revealed an increase in α-neurotoxin resistance across Afro-Asian primates, which was likely selected against cobra venoms. Through structure-activity studies using native and mutant mimotopes of the α-1 nAChR receptor orthosteric site (loop C), we identified the specific amino acids responsible for conferring this increased level of resistance in hominine primates to the α-neurotoxins in cobra venom. CONCLUSION: We have discovered a pattern of primate susceptibility toward α-neurotoxins that supports the theory of a reciprocal coevolutionary arms-race between venomous snakes and primates.

Electrostatic resistance to alpha-neurotoxins conferred by charge reversal mutations in nicotinic acetylcholine receptors.

The evolution of venom resistance through coevolutionary chemical arms races has arisen multiple times throughout animalia. Prior documentation of resistance to snake venom α-neurotoxins consists of the N-glycosylation motif or the hypothesized introduction of arginine at positions 187 at the α-1 nicotinic acetylcholine receptor orthosteric site. However, no further studies have investigated the possibility of other potential forms of resistance. Using a biolayer interferometry assay, we first confirm that the previously hypothesized resistance conferred by arginine at position 187 in the honey badger does reduce binding to α-neurotoxins, which has never been functionally tested. We further discovered a novel form of α-neurotoxin resistance conferred by charge reversal mutations, whereby a negatively charged amino acid is replaced by the positively charged amino acid lysine. As venom α-neurotoxins have evolved strong positive charges on their surface to facilitate binding to the negatively charged α-1 orthosteric site, these mutations result in a positive charge/positive charge interaction electrostatically repelling the α-neurotoxins. Such a novel mechanism for resistance has gone completely undiscovered, yet this form of resistance has convergently evolved at least 10 times within snakes. These coevolutionary innovations seem to have arisen through convergent phenotypes to ultimately evolve a similar biophysical mechanism of resistance across snakes.

A single-step, high throughput, and highly reproducible method for measuring IgM quantity and avidity directly from fish serum via biolayer interferometry (BLI).

Quantification of specific antibody responses is critical in determining activation of MHCII-dependent immune memory and is generally performed by enzyme-linked immunosorbent assay (ELISA). Antibody avidity for a particular antigen is also informative of the quality of the adaptive immune response following vaccination. Avidity can be determined by chaotropic elution ELISA, pre-absorption ELISA, or surface plasmon resonance (SPR), although multimeric antibodies such as IgM are problematic for SPR. ELISA-based assays are very time consuming, require secondary antibody reagents, and are poorly repeatable. Here we demonstrate that biolayer interferometry (BLI) using an Octet HTX instrument can robustly and reproducibly quantify and determine avidity of specific IgM for an antigen directly from fish serum in a single step. We collected sera from giant grouper (Epinephelus lanceolatus) that had been vaccinated with the hapten 2,4-dinitrophenol conjugated to keyhole limpet hemocyanin (DNP-KLH) and from control fish injected with phosphate buffered saline. The specific IgM in the serum and its avidity for DNP were quantified via ELISA and BLI. BLI was precise and highly repeatable for determination of the quantity and avidity of antibody in the serum compared to ELISA. The wet-lab preparation and machine running time for BLI was 3-5 times faster than ELISA to generate the same amount of data. The ELISA inter-plate variation significantly affected reproducibility while BLI was consistent and repeatable between samples and plates. Indeed, the consistency of BLI data indicated that technical triplicates were redundant. Biological replication alone was sufficient to elucidate the effect of treatments. However, BLI required a lower serum dilution than ELISA for similar sensitivity, and thus more serum was required to produce high resolution data. BLI is an extremely high-throughput assay, providing teleost serum IgM quantification and avidity data as a single-step, agile alternative to ELISA.

Symmetrical Viewpoint Representations in Face-Selective Regions Convey an Advantage in the Perception and Recognition of Faces.

Learning new identities is crucial for effective social interaction. A critical aspect of this process is the integration of different images from the same face into a view-invariant representation that can be used for recognition. The representation of symmetrical viewpoints has been proposed to be a key computational step in achieving view-invariance. The aim of this study was to determine whether the representation of symmetrical viewpoints in face-selective regions is directly linked to the perception and recognition of face identity. In Experiment 1, we measured fMRI responses while male and female human participants viewed images of real faces from different viewpoints (-90, -45, 0, 45, and 90° from full-face view). Within the face regions, patterns of neural response to symmetrical views (-45 and 45° or -90 and 90°) were more similar than responses to nonsymmetrical views in the fusiform face area and superior temporal sulcus, but not in the occipital face area. In Experiment 2, participants made perceptual similarity judgements to pairs of face images. Images with symmetrical viewpoints were reported as being more similar than nonsymmetric views. In Experiment 3, we asked whether symmetrical views also convey an advantage when learning new faces. We found that recognition was best when participants were tested with novel face images that were symmetrical to the learning viewpoint. Critically, the pattern of perceptual similarity and recognition across different viewpoints predicted the pattern of neural response in face-selective regions. Together, our results provide support for the functional value of symmetry as an intermediate step in generating view-invariant representations.SIGNIFICANCE STATEMENT The recognition of identity from faces is crucial for successful social interactions. A critical step in this process is the integration of different views into a unified, view-invariant representation. The representation of symmetrical views (e.g., left profile and right profile) has been proposed as an important intermediate step in computing view-invariant representations. We found view symmetric representations were specific to some face-selective regions, but not others. We also show that these neural representations influence the perception of faces. Symmetric views were perceived to be more similar and were recognized more accurately than nonsymmetric views. Moreover, the perception and recognition of faces at different viewpoints predicted patterns of response in those face regions with view symmetric representations.

Assessing the Binding of Venoms from Aquatic Elapids to the Nicotinic Acetylcholine Receptor Orthosteric Site of Different Prey Models.

The evolution of an aquatic lifestyle from land dwelling venomous elapids is a radical ecological modification, bringing about many evolutionary changes from morphology to diet. Diet is an important ecological facet which can play a key role in regulating functional traits such as venom composition and prey-specific targeting of venom. In addition to predating upon novel prey (e.g., fish, fish eggs and invertebrates), the venoms of aquatic elapids also face the challenge of increased prey-escape potential in the aquatic environment. Thus, despite the independent radiation into an aquatic niche on four separate occasions, the venoms of aquatic elapids are evolving under convergent selection pressures. Utilising a biolayer interferometry binding assay, this study set out to elucidate whether crude venoms from representative aquatic elapids were target-specific to the orthosteric site of postsynaptic nicotinic acetylcholine receptor mimotopes of fish compared to other terrestrial prey types. Representatives of the four aquatic lineages were: aquatic coral snakes representative was Micrurus surinamensis;, sea kraits representative was Laticauda colubrina; sea snakes representatives were two Aipysurus spp. and eight Hydrophis spp; and water cobras representative was Naja annulata. No prey-specific differences in crude venom binding were observed from any species tested, except for Aipysurus laevis, which showed slight evidence of prey-potency differences. For Hydrophis caerulescens, H. peronii, H. schistosus and M. surinamensis, there was a lack of binding to the orthosteric site of any target lineage. Subsequent testing on the in vitro chick-biventer cervicis muscle preparation suggested that, while the venoms of these species bound postsynaptically, they bound to allosteric sites rather than orthosteric. Allosteric binding is potentially a weaker but faster-acting form of neurotoxicity and we hypothesise that the switch to allosteric binding is likely due to selection pressures related to prey-escape potential. This research has potentially opened up the possibility of a new functional class of toxins which have never been assessed previously while shedding light on the selection pressures shaping venom evolution.

Determinants of broadband access and affordability: An analysis of a community survey on the digital divide.

Broadband access in the home is a necessity, especially since the COVID-19 pandemic. Increasingly, connectivity is of vital importance for school, work, family, and friends. Existing international research on the implementation of broadband has studied its adoption patterns with a focus on the rural/urban digital divide. This paper explores the digital divide in a case study of the seventh largest city, by population, in the United States; San Antonio is a majority-minority city where over half of the people are Hispanic. This paper focuses on the five key affordability factors that drive broadband adoption. Researchers test social exclusion theory, the structural facets of poverty and social marginality to ascertain its potential impact on broadband access. The authors conducted a survey in both English and Spanish to learn more about the affordability factors that influence the broadband digital divide. Through our analysis, we found evidence that four of the factors (geographical disparities, profit-based discrimination, technology deployment cost, and socio-economic factors) played a role in the digital divide in this case study. The results of this study demonstrate that the digital divide is not exclusively a rural/urban digital divide, but can also occur in an intra-city context. This is especially evident in low-income areas within the city because they have substantially lower broadband adoption rates. The results of this study demonstrate the importance of looking closely at issues of social exclusion of marginalized groups and the affordability of broadband access intra-city.

Distinct but Overlapping Patterns of Response to Words and Faces in the Fusiform Gyrus.

Converging evidence suggests that the fusiform gyrus is involved in the processing of both faces and words. We used fMRI to investigate the extent to which the representation of words and faces in this region of the brain is based on a common neural representation. In Experiment 1, a univariate analysis revealed regions in the fusiform gyrus that were only selective for faces and other regions that were only selective for words. However, we also found regions that showed both word-selective and face-selective responses, particularly in the left hemisphere. We then used a multivariate analysis to measure the pattern of response to faces and words. Despite the overlap in regional responses, we found distinct patterns of response to both faces and words in the left and right fusiform gyrus. In Experiment 2, fMR adaptation was used to determine whether information about familiar faces and names is integrated in the fusiform gyrus. Distinct regions of the fusiform gyrus showed adaptation to either familiar faces or familiar names. However, there was no adaptation to sequences of faces and names with the same identity. Taken together, these results provide evidence for distinct, but overlapping, neural representations for words and faces in the fusiform gyrus.

Neural responses to expression and gaze in the posterior superior temporal sulcus interact with facial identity.

Neural models of human face perception propose parallel pathways. One pathway (including posterior superior temporal sulcus, pSTS) is responsible for processing changeable aspects of faces such as gaze and expression, and the other pathway (including the fusiform face area, FFA) is responsible for relatively invariant aspects such as identity. However, to be socially meaningful, changes in expression and gaze must be tracked across an individual face. Our aim was to investigate how this is achieved. Using functional magnetic resonance imaging, we found a region in pSTS that responded more to sequences of faces varying in gaze and expression in which the identity was constant compared with sequences in which the identity varied. To determine whether this preferential response to same identity faces was due to the processing of identity in the pSTS or was a result of interactions between pSTS and other regions thought to code face identity, we measured the functional connectivity between face-selective regions. We found increased functional connectivity between the pSTS and FFA when participants viewed same identity faces compared with different identity faces. Together, these results suggest that distinct neural pathways involved in expression and identity interact to process the changeable features of the face in a socially meaningful way.

Morphing between expressions dissociates continuous from categorical representations of facial expression in the human brain.

Whether the brain represents facial expressions as perceptual continua or as emotion categories remains controversial. Here, we measured the neural response to morphed images to directly address how facial expressions of emotion are represented in the brain. We found that face-selective regions in the posterior superior temporal sulcus and the amygdala responded selectively to changes in facial expression, independent of changes in identity. We then asked whether the responses in these regions reflected categorical or continuous neural representations of facial expression. Participants viewed images from continua generated by morphing between faces posing different expressions such that the expression could be the same, could involve a physical change but convey the same emotion, or could differ by the same physical amount but be perceived as two different emotions. We found that the posterior superior temporal sulcus was equally sensitive to all changes in facial expression, consistent with a continuous representation. In contrast, the amygdala was only sensitive to changes in expression that altered the perceived emotion, demonstrating a more categorical representation. These results offer a resolution to the controversy about how facial expression is processed in the brain by showing that both continuous and categorical representations underlie our ability to extract this important social cue.

Brain regions involved in processing facial identity and expression are differentially selective for surface and edge information.

Although different brain regions are widely considered to be involved in the recognition of facial identity and expression, it remains unclear how these regions process different properties of the visual image. Here, we ask how surface-based reflectance information and edge-based shape cues contribute to the perception and neural representation of facial identity and expression. Contrast-reversal was used to generate images in which normal contrast relationships across the surface of the image were disrupted, but edge information was preserved. In a behavioural experiment, contrast-reversal significantly attenuated judgements of facial identity, but only had a marginal effect on judgements of expression. An fMR-adaptation paradigm was then used to ask how brain regions involved in the processing of identity and expression responded to blocks comprising all normal, all contrast-reversed, or a mixture of normal and contrast-reversed faces. Adaptation in the posterior superior temporal sulcus--a region directly linked with processing facial expression--was relatively unaffected by mixing normal with contrast-reversed faces. In contrast, the response of the fusiform face area--a region linked with processing facial identity--was significantly affected by contrast-reversal. These results offer a new perspective on the reasons underlying the neural segregation of facial identity and expression in which brain regions involved in processing invariant aspects of faces, such as identity, are very sensitive to surface-based cues, whereas regions involved in processing changes in faces, such as expression, are relatively dependent on edge-based cues.

Verbal redundancy aids memory for filmed entertainment dialogue.

Three studies investigated the effects of presentation modality and redundancy of verbal content on recognition memory for entertainment film dialogue. U.S. participants watched two brief movie clips and afterward answered multiple-choice questions about information from the dialogue. Experiment 1 compared recognition memory for spoken dialogue in the native language (English) with subtitles in English, French, or no subtitles. Experiment 2 compared memory for material in English subtitles with spoken dialogue in English, French, or no sound. Experiment 3 examined three control conditions with no spoken or captioned material in the native language. All participants watched the same video clips and answered the same questions. Performance was consistently good whenever English dialogue appeared in either the subtitles or sound, and best of all when it appeared in both, supporting the facilitation of verbal redundancy. Performance was also better when English was only in the subtitles than when it was only spoken. Unexpectedly, sound or subtitles in an unfamiliar language (French) modestly improved performance, as long as there was also a familiar channel. Results extend multimedia research on verbal redundancy for expository material to verbal information in entertainment media.

The first deep rebreather dive using hydrogen: case report.

Bounce diving with rapid descents to very deep depths may provoke the high-pressure neurological syndrome (HPNS). The strategy of including small fractions of nitrogen in the respired gas to produce an anti-HPNS narcotic effect increases the gas density which may exceed recommended guidelines. In 2020 the 'Wetmules' dive team explored the Pearse Resurgence cave (New Zealand) to 245 m breathing trimix (approximately 4% oxygen, 91% helium and 5% nitrogen). Despite the presence of nitrogen, one diver experienced HPNS tremors beyond 200 m. The use of hydrogen (a light yet slightly narcotic gas) has been suggested as a solution to this problem but there are concerns, including the potential for ignition and explosion of hydrogen-containing gases, and accelerated heat loss. In February 2023 a single dive to 230 m was conducted in the Pearse Resurgence to experience hydrogen as a breathing gas in a deep bounce dive. Using an electronic closed-circuit rebreather, helihydrox (approximately 3% oxygen, 59% helium and 38% hydrogen) was breathed between 200 and 230 m. This was associated with amelioration of HPNS symptoms in the vulnerable diver and no obvious adverse effects. The use of hydrogen is a potential means of progressing deeper with effective HPNS amelioration while maintaining respired gas density within advised guidelines.

Venom biotechnology: casting light on nature's deadliest weapons using synthetic biology.

Venoms are complex chemical arsenals that have evolved independently many times in the animal kingdom. Venoms have attracted the interest of researchers because they are an important innovation that has contributed greatly to the evolutionary success of many animals, and their medical relevance offers significant potential for drug discovery. During the last decade, venom research has been revolutionized by the application of systems biology, giving rise to a novel field known as venomics. More recently, biotechnology has also made an increasing impact in this field. Its methods provide the means to disentangle and study venom systems across all levels of biological organization and, given their tremendous impact on the life sciences, these pivotal tools greatly facilitate the coherent understanding of venom system organization, development, biochemistry, and therapeutic activity. Even so, we lack a comprehensive overview of major advances achieved by applying biotechnology to venom systems. This review therefore considers the methods, insights, and potential future developments of biotechnological applications in the field of venom research. We follow the levels of biological organization and structure, starting with the methods used to study the genomic blueprint and genetic machinery of venoms, followed gene products and their functional phenotypes. We argue that biotechnology can answer some of the most urgent questions in venom research, particularly when multiple approaches are combined together, and with other venomics technologies.

The Target Selects the Toxin: Specific Amino Acids in Snake-Prey Nicotinic Acetylcholine Receptors That Are Selectively Bound by King Cobra Venoms.

Snake venom is an adaptive ecological trait that has evolved primarily as a form of prey subjugation. Thus, the selection pressure for toxin diversification is exerted by the prey's physiological targets, with this pressure being particularly acute for specialist feeders, such as the King Cobra species, all of which are snake-prey specialists. However, while extensive research has been undertaken to elucidate key amino acids that guide toxin structure-activity relationships, reciprocal investigations into the specific sites guiding prey-lineage selective effects have been lacking. This has largely been due to the lack of assay systems amenable to systematic amino acid replacements of targeted proteins in the prey's physiological pathways. To fill this knowledge gap, we used a recently described approach based upon mimotope peptides corresponding to the orthosteric site of nicotinic acetylcholine receptor alpha-1 subunits, a major binding site for snake venom neurotoxins that cause flaccid paralysis. We investigated the venoms of four different types of King Cobra (Cambodian, Javan, Malaysian, and Thai). This approach allowed for the determination of the key amino acid positions in King Cobra snake prey that are selectively bound by the toxins, whereby replacing these amino acids in the snake-prey orthosteric site with those from lizards or rats resulted in a significantly lower level of binding by the venoms, while conversely replacing the lizard or rat amino acids with those from the snake at that position increased the binding. By doing such, we identified three negatively charged amino acids in the snake orthosteric site that are strongly bound by the positively charged neurotoxic three-finger toxins found in King Cobra venom. This study, thus, sheds light on the selection pressures exerted by a specialist prey item for the evolution of lineage-selective toxins.

Differential coagulotoxic and neurotoxic venom activity from species of the arboreal viperid snake genus Bothriechis (palm-pitvipers).

The viperid snake genus Bothriechis consists of eleven species distributed among Central and South America, living across low and high-altitude habitats. Despite Bothriechis envenomations being prominent across the Central and South American region, the functional effects of Bothriechis venoms are poorly understood. Thus, the aim of this study was to investigate the coagulotoxic and neurotoxic activities of Bothriechis venoms to fill this knowledge gap. Coagulotoxic investigations revealed Bothriechis nigroviridis and B. schlegelii to have pseudo-procoagulant venom activity, forming weak clots that rapidly break down, thereby depleting fibrinogen levels and thus contributing to a net anticoagulant state. While one sample of B. lateralis also showed weaker pseudo-procoagulant activity, directly clotting fibrinogen, two samples of B. lateralis venom were anticoagulant through the inhibition of thrombin and factor Xa activity. Differential efficacy of PoliVal-ICP antivenom was also observed, with the pseudo-procoagulant effect of B. nigroviridis venom poorly neutralised, despite this same activity in the venom of B. schlegelii being effectively neutralised. Significant specificity of these fibrinogen cleaving toxins was also observed, with no activity upon model amphibian, avian, lizard or rodent plasma observed. However, upon avian plasma the venom of B. nigroviridis exerted a complete anticoagulant effect, in contrast to the pseudo-procoagulant effect seen on human plasma. Neurotoxic investigations revealed B. schlegelii to be unique among the genus in having potent binding to the orthosteric site of the alpha-1 postsynaptic nicotinic acetylcholine receptor (with B. lateralis having a weaker but still discernible effect). This represents the first identification of postsynaptic nAChR neurotoxic activity for Bothriechis. In conclusion this study identifies notable differential activity within the coagulotoxic and postsynaptic neurotoxic activity of Bothriechis venoms, supporting previous research, and highlights the need for further studies with respect to antivenom efficacy as well as coagulotoxin specificity for Bothriechis venoms.

Convergent evolution of toxin resistance in animals.

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.

Radiating pain: venom has contributed to the diversification of the largest radiations of vertebrate and invertebrate animals.

BACKGROUND: Understanding drivers of animal biodiversity has been a longstanding aim in evolutionary biology. Insects and fishes represent the largest lineages of invertebrates and vertebrates respectively, and consequently many ideas have been proposed to explain this diversity. Natural enemy interactions are often important in diversification dynamics, and key traits that mediate such interactions may therefore have an important role in explaining organismal diversity. Venom is one such trait which is intricately bound in antagonistic coevolution and has recently been shown to be associated with increased diversification rates in tetrapods. Despite ~ 10% of fish families and ~ 16% of insect families containing venomous species, the role that venom may play in these two superradiations remains unknown. RESULTS: In this paper we take a broad family-level phylogenetic perspective and show that variation in diversification rates are the main cause of variations in species richness in both insects and fishes, and that venomous families have diversification rates twice as high as non-venomous families. Furthermore, we estimate that venom was present in ~ 10% and ~ 14% of the evolutionary history of fishes and insects respectively. CONCLUSIONS: Consequently, we provide evidence that venom has played a role in generating the remarkable diversity in the largest vertebrate and invertebrate radiations.

Not Goanna Get Me: Mutations in the Savannah Monitor Lizard (Varanus exanthematicus) Nicotinic Acetylcholine Receptor Confer Reduced Susceptibility to Sympatric Cobra Venoms.

Antagonistic coevolutionary relationships provide intense selection pressure which drive changes in the genotype. Predator-prey interactions have caused some venomous snakes and their predators/prey to evolve α-neurotoxin resistance through changes at the orthosteric site of nicotinic acetylcholine receptors. The presence of negatively charged amino acids at orthosteric site positions 191 and 195 is the ancestral state. These negatively charged amino acids have exerted a selection pressure for snake venom α-neurotoxins to evolve with strong positive charges on their molecular surface, with the opposite-charge attraction facilitating the binding by the neurotoxins. We aimed to test the effects of a series of mutations whereby one or both negatively charged amino acids are replaced by uncharged residues to ascertain if this was a novel form of reduced venom susceptibility in the varanid species. Using a biolayer interferometry assay, we tested the relative binding of α-neurotoxin-rich snake venoms against the orthosteric sites of V. giganteus (Perentie) and V. komodoensis (Komodo dragon), which both possess the negatively charged aspartic acid at position 191; V. mertensi (Merten's water monitor), which also has aspartic acid at position 195; and Varanus exanthematicus (savannah monitor), which lacks negatively charged amino acids at both positions 191 and 195. The orthosteric sites of these species are otherwise identical. In order to complete the structure-function relationship examination, we also tested a mutant version with the negatively charged aspartic acid at both positions 191 and 195. It was demonstrated that the presence of a negatively charged amino acid at either position 191 or 195 is crucial for the successful binding of snake venom α-neurotoxins, with V. giganteus, V. komodoensis and V. mertensi all strongly bound. The mutant version containing a negatively charged amino acid at both positions was bound equipotently to the native forms of V. giganteus, V. komodoensis and V. mertensi. Thus, the presence of a negatively charged amino acid at both positions does not increase binding affinity. In contrast, Varanus exanthematicus, lacking a negatively charged amino acid at either position, displayed dramatically less sensitivity to neurotoxins compared with the other species. V. exanthematicus is distinguished from the other species examined in this study by being a small, terrestrial, slow-moving species living sympatrically with a high density of large cobra species that have neurotoxin-rich venoms. Thus, this vulnerable prey item seems to have evolved a novel form of reduced susceptibility to snake venom neurotoxins under a strong selection pressures from these neurotoxic predators. These results therefore contribute to the body of knowledge of predator/prey chemical arm races while providing novel insights into the structure-activity relationships of the orthosteric site of the nicotinic acetylcholine receptor alpha-subunit.

Human Intestinal Macrophages Are Involved in the Pathology of Both Ulcerative Colitis and Crohn Disease.

BACKGROUND: Intestinal macrophages are key immune cells in the maintenance of intestinal immune homeostasis and have a role in the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms by which macrophages exert a pathological influence in both ulcerative colitis (UC) and Crohn disease (CD) are not yet well understood. METHODS: We purified intestinal macrophages from gastrointestinal mucosal biopsies (patients with UC, patients with CD, and healthy donors) and analyzed their transcriptome by RNA sequencing and bioinformatics, confirming results with quantitative polymerase chain reaction and immunohistochemistry. RESULTS: Compared with those of healthy donors, intestinal macrophages in patients with UC and with CD showed cellular reprograming of 1287 and 840 dysregulated genes, respectively (false discovery rate ≤ 0.1). The UC and CD intestinal macrophages showed an activated M1 inflammatory phenotype and the downregulation of genes engaged in drug/xenobiotic metabolism. Only macrophages from CD showed, concomitant to an M1 phenotype, a significant enrichment in the expression of M2 and fibrotic and granuloma-related genes. For the first time, we showed (and validated by quantitative polymerase chain reaction and immunohistochemistry) that intestinal macrophages in patients with IBD present both M1 and M2 features, as recently described for tumor-associated macrophages, that affect key pathways for IBD pathology, represented by key markers such as MMP12 (fibrosis), CXCL9 (T-cell attraction), and CD40 (T-cell activation). CONCLUSIONS: Our data support the therapeutic targeting of macrophages to maintain remission in IBD but also indicate that a shift toward an M2 program-as proposed by some reports-may not limit the recruitment and activation of T cells because M2 features do not preclude M1 activation in patients with UC or CD and could exacerbate M2-related CD-specific features such as fibrosis and the formation of granulomas.