Phylogenomics of Neogastropoda: the backbone hidden in the bush.

The molluscan order Neogastropoda encompasses over 15,000 almost exclusively marine species playing important roles in benthic communities and in the economies of coastal countries. Neogastropoda underwent intensive cladogenesis in early stages of diversification, generating a 'bush' at the base of their evolutionary tree, that has been hard to resolve even with high throughput molecular data. In the present study to resolve the bush, we use a variety of phylogenetic inference methods and a comprehensive exon capture dataset of 1,817 loci (79.6% data occupancy) comprising 112 taxa of 48 out of 60 Neogastropoda families. Our results show consistent topologies and high support in all analyses at (super)family level, supporting monophyly of Muricoidea, Mitroidea, Conoidea, and, with some reservations, Olivoidea and Buccinoidea. Volutoidea and Turbinelloidea as currently circumscribed are clearly paraphyletic. Despite our analyses consistently resolving most backbone nodes, three prove problematic: First, uncertain placement of Cancellariidae, as the sister group to either a Ficoidea-Tonnoidea clade, or to the rest of Neogastropoda, leaves monophyly of Neogastropoda unresolved. Second, relationships are contradictory at the base of the major 'core Neogastropoda' grouping. Third, coalescence-based analyses reject monophyly of the Buccinoidea in relation to Vasidae. We analysed phylogenetic signal of targeted loci in relation to potential biases, and we propose most probable resolutions in the latter two recalcitrant nodes. The uncertain placement of Cancellariidae may be explained by orthology violations due to differential paralog loss shortly after the whole genome duplication, which should be resolved with a curated set of longer loci.

Targeting Dysregulated Ion Channels in Liver Tumors with Venom Peptides.

The regulation of cellular processes by ion channels has become central to the study of cancer mechanisms. Designing molecules that can modify ion channels specific to tumor cells is a promising area of targeted drug delivery and therapy. Despite their potential in drug discovery, venom peptides-a group of natural products-have largely remained understudied and under-characterized. In general, venom peptides display high specificity and selectivity for their target ion channels. Therefore, they may represent an effective strategy for selectively targeting the dysregulation of ion channels in tumor cells. This review examines existing venom peptide therapies for different cancer types and focuses on the application of snail venom peptides in hepatocellular carcinoma (HCC), the most common form of primary liver cancer worldwide. We provide insights into the mode of action of venom peptides that have been shown to target tumors. We also explore the benefit of using new computational methods like de novo protein structure prediction to screen venom peptides and identify potential druggable candidates. Finally, we summarize the role of cell culture, animal, and organoid models in developing effective therapies against HCC and highlight the need for creating models that represent the most disproportionately affected ethnicities in HCC.

Comparative mucomic analysis of three functionally distinct Cornu aspersum Secretions.

Every animal secretes mucus, placing them among the most diverse biological materials. Mucus hydrogels are complex mixtures of water, ions, carbohydrates, and proteins. Uncertainty surrounding their composition and how interactions between components contribute to mucus function complicates efforts to exploit their properties. There is substantial interest in commercializing mucus from the garden snail, Cornu aspersum, for skincare, drug delivery, tissue engineering, and composite materials. C. aspersum secretes three mucus-one shielding the animal from environmental threats, one adhesive mucus from the pedal surface of the foot, and another pedal mucus that is lubricating. It remains a mystery how compositional differences account for their substantially different properties. Here, we characterize mucus proteins, glycosylation, ion content, and mechanical properties that could be used to provide insight into structure-function relationships through an integrative "mucomics" approach. We identify macromolecular components of these hydrogels, including a previously unreported protein class termed Conserved Anterior Mollusk Proteins (CAMPs). Revealing differences between C. aspersum mucus shows how considering structure at all levels can inform the design of mucus-inspired materials.

Discovering Venom-Derived Drug Candidates Using Differential Gene Expression.

Venoms are a diverse and complex group of natural toxins that have been adapted to treat many types of human disease, but rigorous computational approaches for discovering new therapeutic activities are scarce. We have designed and validated a new platform-named VenomSeq-to systematically identify putative associations between venoms and drugs/diseases via high-throughput transcriptomics and perturbational differential gene expression analysis. In this study, we describe the architecture of VenomSeq and its evaluation using the crude venoms from 25 diverse animal species and 9 purified teretoxin peptides. By integrating comparisons to public repositories of differential expression, associations between regulatory networks and disease, and existing knowledge of venom activity, we provide a number of new therapeutic hypotheses linking venoms to human diseases supported by multiple layers of preliminary evidence.

VenomFlow: An Automated Bioinformatic Pipeline for Identification of Disulfide-Rich Peptides from Venom Arsenals.

Animal venoms are among the most complex natural secretions known, comprising a mixture of bioactive compounds often referred to as toxins. Venom arsenals are predominately made up of cysteine-rich peptide toxins that manipulate molecular targets, such as ion channels and receptors, making these venom peptides attractive candidates for the development of therapeutics to benefit human health. With the rise of omic strategies that utilize transcriptomic, proteomic, and bioinformatic methods, we are able to identify more venom proteins and peptides than ever before. However, identification and characterization of bioactive venom peptides remains a significant challenge due to the unique chemical structure and enormous number of peptides found in each venom arsenal (upward of 200 per organism). Here, we introduce a rapid and user-friendly in silico bioinformatic pipeline for the de novo identification and characterization of raw RNAseq reads from venom glands to elucidate cysteine-rich peptides from the arsenal of venomous organisms.Implementation: This project develops a user-friendly automated bioinformatics pipeline via a Galaxy workflow to identify novel venom peptides from raw RNAseq reads of terebrid snails. While designed for venomous terebrid snails, with minor adjustments, this pipeline can be made universal to identify secreted disulfide-rich peptide toxins from any venomous organism.

From Flatland to Jupiter: Searching for Rules of Interaction Across Biological Scales.

In this future-spanning perspective, we examine how an agent-based model could be used to define general rules for interactions across biological systems and evolutionary time. To date, there have been a number of attempts to simulate the emergence of ecological communities using agent-based models of individuals that have evolving traits. Here we speculate whether it is possible to use this computational modeling to simulate self-organizing systems and, importantly, to decipher universal principles that govern biological interactions. This perspective is a thought exercise, meant to extrapolate from current knowledge to how we may make Jupiter-shot leaps to further advance the biosciences in the 21st century.

Advancing Discovery of Snail Mucins Function and Application.

Mucins are a highly glycosylated protein family that are secreted by animals for adhesion, hydration, lubrication, and other functions. Despite their ubiquity, animal mucins are largely uncharacterized. Snails produce mucin proteins in their mucous for a wide array of biological functions, including microbial protection, adhesion and lubrication. Recently, snail mucins have also become a lucrative source of innovation with wide ranging applications across chemistry, biology, biotechnology, and biomedicine. Specifically, snail mucuses have been applied as skin care products, wound healing agents, surgical glues, and to combat gastric ulcers. Recent advances in integrated omics (genomic, transcriptomic, proteomic, glycomic) technologies have improved the characterization of gastropod mucins, increasing the generation of novel biomaterials. This perspective describes the current research on secreted snail mucus, highlighting the potential of this biopolymer, and also outlines a research strategy to fulfill the unmet need of examining the hierarchical structures that lead to the enormous biological and chemical diversity of snail mucus genes.

Diet Diversity in Carnivorous Terebrid Snails Is Tied to the Presence and Absence of a Venom Gland.

Predator-prey interactions are thought to play a driving role in animal evolution, especially for groups that have developed venom as their predatory strategy. However, how the diet of venomous animals influences the composition of venom arsenals remains uncertain. Two prevailing hypotheses to explain the relationship between diet and venom composition focus on prey preference and the types of compounds in venom, and a positive correlation between dietary breadth and the number of compounds in venom. Here, we examined venom complexity, phylogenetic relationship, collection depth, and biogeography of the Terebridae (auger snails) to determine if repeated innovations in terebrid foregut anatomy and venom composition correspond to diet variation. We performed the first molecular study of the diet of terebrid marine snails by metabarcoding the gut content of 71 terebrid specimens from 17 species. Our results suggest that the presence or absence of a venom gland is strongly correlated with dietary breadth. Specifically, terebrid species without a venom gland displayed greater diversity in their diet. Additionally, we propose a revision of the definition of venom complexity in conoidean snails to more accurately capture the breadth of ecological influences. These findings suggest that prey diet is an important factor in terebrid venom evolution and diversification and further investigations of other understudied organisms, like terebrids, are needed to develop robust hypotheses in this area.

Induced Disassembly of a Virus-like Particle under Physiological Conditions for Venom Peptide Delivery.

Virus-like particles (VLPs) show considerable promise for the in vivo delivery of therapeutic compounds such as bioactive venom peptides. While loading and targeting protocols have been developed for numerous VLP prototypes, induced disassembly under physiological conditions of neutral pH, moderate temperature, and aqueous medium remain a challenge. Here, we implement and evaluate a general mechanism, based on ring-opening metathesis polymerization (ROMP), for controllable VLP disassembly. This mechanism is independent of cell-specific factors or the manipulation of environmental conditions such as pH and temperature that cannot be readily controlled in vivo. The ROMP substrate norbornene is covalently conjugated to surface-exposed lysine residues of a P22 bacteriophage-derived VLP, and ROMP is induced by treatment with the water-soluble ruthenium catalyst AquaMet. Disruption of the P22 shell and release of a GFP reporter is confirmed via native agarose electrophoresis, TEM, and dynamic light scattering (DLS) analyses. Our ROMP disassembly strategy does not depend on the particular structure or morphology of the P22 nanocontainer and is adaptable to other VLP prototypes for the potential delivery of venom peptides for pharmacological applications.

Comparative Animal Mucomics: Inspiration for Functional Materials from Ubiquitous and Understudied Biopolymers.

The functions of secreted animal mucuses are remarkably diverse and include lubricants, wet adhesives, protective barriers, and mineralizing agents. Although present in all animals, many open questions related to the hierarchical architectures, material properties, and genetics of mucus remain. Here, we summarize what is known about secreted mucus structure, describe the work of research groups throughout the world who are investigating various animal mucuses, and relate how these studies are revealing new mucus properties and the relationships between mucus hierarchical structure and hydrogel function. Finally, we call for a more systematic approach to studying animal mucuses so that data sets can be compared, omics-style, to address unanswered questions in the emerging field of mucomics. One major result that we anticipate from these efforts is design rules for creating new materials that are inspired by the structures and functions of animal mucuses.

Macroevolutionary Analyses Suggest That Environmental Factors, Not Venom Apparatus, Play Key Role in Terebridae Marine Snail Diversification.

How species diversification occurs remains an unanswered question in predatory marine invertebrates, such as sea snails of the family Terebridae. However, the anatomical disparity found throughput the Terebridae provides a unique perspective for investigating diversification patterns in venomous predators. In this study, a new dated molecular phylogeny of the Terebridae is used as a framework for investigating diversification of the family through time, and for testing the putative role of intrinsic and extrinsic traits, such as shell size, larval ecology, bathymetric distribution, and anatomical features of the venom apparatus, as drivers of terebrid species diversification. Macroevolutionary analysis revealed that when diversification rates do not vary across Terebridae clades, the whole family has been increasing its global diversification rate since 25 Ma. We recovered evidence for a concurrent increase in diversification of depth ranges, while shell size appeared to have undergone a fast divergence early in terebrid evolutionary history. Our data also confirm that planktotrophy is the ancestral larval ecology in terebrids, and evolutionary modeling highlighted that shell size is linked to larval ecology of the Terebridae, with species with long-living pelagic larvae tending to be larger and have a broader size range than lecithotrophic species. Although we recovered patterns of size and depth trait diversification through time and across clades, the presence or absence of a venom gland (VG) did not appear to have impacted Terebridae diversification. Terebrids have lost their venom apparatus several times and we confirm that the loss of a VG happened in phylogenetically clustered terminal taxa and that reversal is extremely unlikely. Our findings suggest that environmental factors, and not venom, have had more influence on terebrid evolution.

Selective Inhibition of Liver Cancer Cells Using Venom Peptide.

Increasingly cancer is being viewed as a channelopathy because the passage of ions via ion channels and transporters mediate the regulation of tumor cell survival, death, and motility. As a result, a potential targeted therapy for cancer is to use venom peptides that are selective for ion channels and transporters overexpressed in tumor cells. Here we describe the selectivity and mechanism of action of terebrid snail venom peptide, Tv1, for treating the most common type of liver cancer, hepatocellular carcinoma (HCC). Tv1 inhibited the proliferation of murine HCC cells and significantly reduced tumor size in Tv1-treated syngeneic tumor-bearing mice. Tv1's mechanism of action involves binding to overexpressed transient receptor potential (TRP) channels leading to calcium dependent apoptosis resulting from down-regulation of cyclooxygenase-2 (COX-2). Our findings demonstrate the importance of modulating ion channels and the unique potential of venom peptides as tumor specific ligands in the quest for targeted cancer therapies.

The chemical basis of a signal of individual identity: shell pigment concentrations track the unique appearance of Common Murre eggs.

In group-living species with parental care, the accurate recognition of one's own young is critical to fitness. Because discriminating offspring within a large colonial group may be challenging, progeny of colonial breeders often display familial or individual identity signals to elicit and receive parental provisions from their own parents. For instance, the common murre (or common guillemot: Uria aalge) is a colonially breeding seabird that does not build a nest and lays and incubates an egg with an individually unique appearance. How the shell's physical and chemical properties generate this individual variability in coloration and maculation has not been studied in detail. Here, we quantified two characteristics of the avian-visible appearance of murre eggshells collected from the wild: background coloration spectra and maculation density. As predicted by the individual identity hypothesis, there was no statistical relationship between avian-perceivable shell background coloration and maculation density within the same eggs. In turn, variation in both sets of traits was statistically related to some of their physico-chemical properties, including shell thickness and concentrations of the eggshell pigments biliverdin and protoporphyrin IX. These results illustrate how individually unique eggshell appearances, suitable for identity signalling, can be generated by a small number of structural mechanisms.

Building on Success: A Bright Future for Peptide Therapeutics.

The primary aim of this review article is to highlight current exciting and future looking areas of research in peptide science as applied to the discovery and development of novel therapeutics. Among the strengths of peptides as drug candidates are their high potency, specificity, and good safety profile. These positive attributes of peptides along with advances in drug delivery technologies have generated renewed interest in the discovery, optimization, and development of peptides as therapeutics. The intent of this review is to demonstrate that peptides have broad applicability in many therapeutic areas by examining some of the most compelling indications and targets for peptide therapeutics. For example, target selection for peptide therapeutics is challenging due to the inherent properties of peptides; therefore, identifying a clear differentiation strategy for a new peptide program over a small molecule or antibody program from the outset is critical for successful navigation of drug development hurdles. In this review, some of the latest techniques that accentuate the advantages and overcome the druggability limitations of peptides will be covered. Emerging technologies for enhancing the pharmacokinetics of peptides to achieve sufficient in vivo half-lives will be described and evaluated, as well as novel technologies for getting peptides across cell membranes to reach intracellular targets and across the blood-brain-barrier to reach central nervous system targets.

Breakthroughs in Venom Peptide Screening Methods to Advance Future Drug Discovery.

BACKGROUND: Venom peptides are a proven resource for identifying novel drugs, however the process of identifying bioactive venom peptides is currently labor intensive, costly, and rarely results in pharmaceutical success. As venom peptides are modulators of ion channels and receptors their potential for manipulating cell signals in diseased states are unique and offer an untapped resource for finding new medicines. Recent advances in -omic technologies, and microfluidic biosensing systems have transformed how venom peptides are discovered and characterized. CONCLUSION: This review will cover past, present and future approaches for screening venom peptides for drug discovery and development. Specifically, we will highlight online high-resolution microfluidic biosensing systems and new fluorescence detection methods that can be adapted to expand the discovery and characterization of venom peptide drugs.

Using Drosophila behavioral assays to characterize terebrid venom-peptide bioactivity.

The number of newly discovered peptides from the transcriptomes and proteomes of animal venom arsenals is rapidly increasing, resulting in an abundance of uncharacterized peptides. There is a pressing need for a systematic, cost effective, and scalable approach to identify physiological effects of venom peptides. To address this discovery-to-function gap, we developed a sequence driven:activity-based hybrid approach for screening venom peptides that is amenable to large-venom peptide libraries with minimal amounts of peptide. Using this approach, we characterized the physiological and behavioral phenotypes of two peptides from the venom of predatory terebrid marine snails, teretoxins Tv1 from Terebra variegata and Tsu1.1 from Terebra subulata. Our results indicate that Tv1 and Tsu1.1 have distinct bioactivity. Tv1 (100 µM) had an antinociceptive effect in adult Drosophila using a thermal nociception assay to measure heat avoidance. Alternatively, Tsu1.1 (100 µM) increased food intake. These findings describe the first functional bioactivity of terebrid venom peptides in relation to pain and diet and indicate that Tv1 and Tsu1.1 may, respectively, act as antinociceptive and orexigenic agents. Tv1 and Tsu1.1 are distinct from previously identified venom peptides, expanding the toolkit of peptides that can potentially be used to investigate the physiological mechanisms of pain and diet.

Beach to Bench to Bedside: Marine Invertebrate Biochemical Adaptations and Their Applications in Biotechnology and Biomedicine.

The ocean covers more than 70% of the surface of the planet and harbors very diverse ecosystems ranging from tropical coral reefs to the deepest ocean trenches, with some of the most extreme conditions of pressure, temperature, and light. Organisms living in these environments have been subjected to strong selective pressures through millions of years of evolution, resulting in a plethora of remarkable adaptations that serve a variety of vital functions. Some of these adaptations, including venomous secretions and light-emitting compounds or ink, represent biochemical innovations in which marine invertebrates have developed novel and unique bioactive compounds with enormous potential for basic and applied research. Marine biotechnology, defined as the application of science and technology to marine organisms for the production of knowledge, goods, and services, can harness the enormous possibilities of these unique bioactive compounds acting as a bridge between biological knowledge and applications. This chapter highlights some of the most exceptional biochemical adaptions found specifically in marine invertebrates and describes the biotechnological and biomedical applications derived from them to improve the quality of human life.

How to Make a Mimic? Brood Parasitic Striped Cuckoo Eggs Match Host Shell Color but Not Pigment Concentrations.

Hosts of avian brood parasites often use visual cues to reject foreign eggs, and several lineages of brood parasites have evolved mimetic eggshell coloration and patterning to circumvent host recognition. What is the mechanism of parasitic egg color mimicry at the chemical level? Mimetic egg coloration by Common Cuckoos Cuculus canorus is achieved by depositing similar concentrations of colorful pigments into their shells as their hosts. The mechanism of parasitic egg color mimicry at the chemical level in other lineages of brood parasites remains unexplored. Here we report on the chemical basis of egg color mimicry in an evolutionarily independent, and poorly studied, host-parasite system: the Neotropical Striped Cuckoo Tapera naevia and one of its hosts, the Rufous-and-white Wren Thryophilus rufalbus. In most of South America, Striped Cuckoos lay white eggs that are identical to those of local host species. In Central America, however, Striped Cuckoos lay blue eggs that match those of the Rufous-and-white Wren, suggesting that blue egg color in these cuckoo populations is an adaptation to mimic host egg appearance. Here we confirm that Striped Cuckoo eggs are spectrally similar to those of their hosts and consistently contain the same major eggshell pigment, biliverdin. However, wren eggshells lacked protoporphyrin, which was present in the parasitic cuckoo eggshells. Furthermore, biliverdin concentrations were significantly lower in cuckoo eggshells than in host eggshells. Similarity of host-parasite eggshell appearance, therefore, need not always be paralleled by a quantitative chemical match to generate effective visual mimicry in birds.