Integrating high-throughput analysis to create an atlas of replication origins in Trypanosoma cruzi in the context of genome structure and variability

Trypanosoma cruzi is the etiologic agent of the most prevalent human parasitic disease in Latin America, Chagas disease. Its genome is rich in multigenic families that code for virulent antigens and are present in the rapidly evolving genomic compartment named Disruptive. DNA replication is a meticulous biological process in which flaws can generate mutations and changes in chromosomal and gene copy numbers. Here, integrating high-throughput and single-molecule analyses, we were able to identify Predominant, Flexible, and Dormant Orc1Cdc6-dependent origins as well as Orc1Cdc6-independent origins. Orc1Cdc6-dependent origins were found in multigenic family loci, while independent origins were found in the Core compartment that contains conserved and hypothetical protein-coding genes, in addition to multigenic families. In addition, we found that Orc1Cdc6 density is related to the firing of origins and that Orc1Cdc6-binding sites within fired origins are depleted of a specific class of nucleosomes that we previously categorized as dynamic. Together, these data suggest that Orc1Cdc6-dependent origins may contribute to the rapid evolution of the Disruptive compartment and, therefore, to the success of T. cruzi infection and that the local epigenome landscape is also involved in this process.

Venom composition of neglected Bothropoid snakes from the Amazon Rainforest: ecological and toxinological implications

Snake venoms have evolved in several families of Caenophidae, and their toxins have been assumed to be biochemical weapons with a role as a trophic adaptation. However, it remains unclear how venom contributes to the success of venomous species for adaptation to different environments. Here we compared the venoms from Bothrocophias hyoprora, Bothrops taeniatus, Bothrops bilineatus smaragdinus, Bothrops brazili, and Bothrops atrox collected in the Amazon Rainforest, aiming to understand the ecological and toxinological consequences of venom composition. Transcriptomic and proteomic analyses indicated that the venoms presented the same toxin groups characteristic from bothropoids, but with distinct isoforms with variable qualitative and quantitative abundances, contributing to distinct enzymatic and toxic effects. Despite the particularities of each venom, commercial Bothrops antivenom recognized the venom components and neutralized the lethality of all species. No clear features could be observed between venoms from arboreal and terrestrial habitats, nor in the dispersion of the species throughout the Amazon habitats, supporting the notion that venom composition may not shape the ecological or toxinological characteristics of these snake species and that other factors influence their foraging or dispersal in different ecological niches.

ToxCodAn-Genome: an automated pipeline for toxin-gene annotation in genome assembly of venomous lineages

Background: The rapid development of sequencing technologies resulted in a wide expansion of genomics studies using venomous lineages. This facilitated research focusing on understanding the evolution of adaptive traits and the search for novel compounds that can be applied in agriculture and medicine. However, the toxin annotation of genomes is a laborious and time-consuming task, and no consensus pipeline is currently available. No computational tool currently exists to address the challenges specific to toxin annotation and to ensure the reproducibility of the process. Results: Here, we present ToxCodAn-Genome, the first software designed to perform automated toxin annotation in genomes of venomous lineages. This pipeline was designed to retrieve the full-length coding sequences of toxins and to allow the detection of novel truncated paralogs and pseudogenes. We tested ToxCodAn-Genome using 12 genomes of venomous lineages and achieved high performance on recovering their current toxin annotations. This tool can be easily customized to allow improvements in the final toxin annotation set and can be expanded to virtually any venomous lineage. ToxCodAn-Genome is fast, allowing it to run on any personal computer, but it can also be executed in multicore mode, taking advantage of large high-performance servers. In addition, we provide a guide to direct future research in the venomics field to ensure a confident toxin annotation in the genome being studied. As a case study, we sequenced and annotated the toxin repertoire of Bothrops alternatus, which may facilitate future evolutionary and biomedical studies using vipers as models. Conclusions: ToxCodAn-Genome is suitable to perform toxin annotation in the genome of venomous species and may help to improve the reproducibility of further studies. ToxCodAn-Genome and the guide are freely available at https://github.com/pedronachtigall/T oxCodAn-Genome.

A novel metalloproteinase-derived cryptide from Bothrops cotiara venom inhibits angiotensin-converting enzyme activity

Snake venoms are primarily composed of proteins and peptides, which selectively interact with specific molecular targets, disrupting prey homeostasis. Identifying toxins and the mechanisms involved in envenoming can lead to the discovery of new drugs based on natural peptide scaffolds. In this study, we used mass spectrometry-based peptidomics to sequence 197 peptides in the venom of Bothrops cotiara, including a novel 7-residue peptide derived from a snake venom metalloproteinase. This peptide, named Bc-7a, features a pyroglutamic acid at the N-terminal and a PFR motif at the C-terminal, homologous to bradykinin. Using FRET (fluorescence resonance energy transfer) substrate assays, we demonstrated that Bc-7a strongly inhibits the two domains of angiotensin converting enzyme (Ki < 1 μM). Our findings contribute to the repertoire of biologically active peptides from snake venoms capable of inhibiting angiotensin-converting enzyme (ACE), beyond current known structural motifs and precursors. In summary, we report a novel snake venom peptide with ACE inhibitory activity, suggesting its potential contribution to the hypotensive effect observed in envenomation.

Comparing morphological and secretory aspects of cephalic glands among the New World coral snakes brings novel insights on their biological roles

Oral and other cephalic glands have been surveyed by several studies with distinct purposes. Despite the wide diversity and medical relevance of the New World coral snakes, studies focusing on understanding the biological roles of the glands within this group are still scarce. Specifically, the venom glands of some coral snakes were previously investigated but all other cephalic glands remain uncharacterized. In this sense, performing morphological and molecular analysis of these glands may help better understand their biological role. Here, we studied the morphology of the venom, infralabial, rictal, and harderian glands of thirteen species of Micrurus and Micruroides euryxanthus. We also performed a molecular characterization of these glands from selected species of Micrurus using transcriptomic and proteomic approaches. We described substantial morphological variation in the cephalic glands of New World coral snakes and structural evidence for protein-secreting cells in the inferior rictal glands. Our molecular analysis revealed that the venom glands, as expected, are majorly devoted to toxin production, however, the infralabial and inferior rictal glands also expressed some toxin genes at low to medium levels, despite the marked morphological differences. On the other hand, the harderian glands were dominated by the expression of lipocalins, but do not produce toxins. Our integrative analysis, including the prediction of biological processes and pathways, helped decipher some important traits of cephalic glands and better understand their biology.

Role of ErbB and IL-1 signaling pathways in the dermonecrotic lesion induced by Loxosceles sphingomyelinases D

Sphingomyelinase D (SMase D), the main toxic component of Loxosceles venom, has a well-documented role on dermonecrotic lesion triggered by envenomation with these species; however, the intracellular mechanisms involved in this event are still poorly known. Through differential transcriptomics of human keratinocytes treated with L. laeta or L. intermedia SMases D, we identified 323 DEGs, common to both treatments, as well as upregulation of molecules involved in the IL-1 and ErbB signaling. Since these pathways are related to inflammation and wound healing, respectively, we investigated the relative expression of some molecules related to these pathways by RT-qPCR and observed different expression profiles over time. Although, after 24 h of treatment, both SMases D induced similar modulation of these pathways in keratinocytes, L. intermedia SMase D induced earlier modulation compared to L. laeta SMase D treatment. Positive expression correlations of the molecules involved in the IL-1 signaling were also observed after SMases D treatment, confirming their inflammatory action. In addition, we detected higher relative expression of the inhibitor of the ErbB signaling pathway, ERRFI1, and positive correlations between this molecule and pro-inflammatory mediators after SMases D treatment. Thus, herein, we describe the cell pathways related to the exacerbation of inflammation and to the failure of the wound healing, highlighting the contribution of the IL-1 signaling pathway and the ERRFI1 for the development of cutaneous loxoscelism.

The venom composition of the snake tribe Philodryadini: ‘Omic’ techniques reveal intergeneric variability among South American racers

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas, Chlorosoma and Xenoxybelis. The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis, respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

Independent Recruitment of Different Types of Phospholipases A2 to the Venoms of Caenophidian Snakes: The Rise of PLA2-IIE within Pseudoboini (Dipsadidae)

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

Independent recruitment of different types of Phospholipases A2 to the venoms of Caenophidian snakes: the rise of PLA2-IIE within Pseudoboini (Dipsadidae)

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

Clues on the dynamics of DNA replication in Giardia lamblia Icon for the Forest of Biologists

Genomic replication is a critical, regulated process that ensures accurate genetic information duplication. In eukaryotic cells, strategies have evolved to prevent conflicts between replication and transcription. Giardia lamblia, a binucleated protozoan, alternates between tetraploid and octoploid genomes during its cell cycle. Using single-molecule techniques like DNA combing and nanopore-based sequencing, we investigated the spatio-temporal organization of DNA replication, replication fork progression, and potential head-on replication-transcription collisions in Giardia trophozoites. Our findings indicate that Giardia chromosomes are replicated from few active origins, which are widely spaced and exhibit faster replication rates compared to other protozoan parasites. Immunofluorescence assays revealed that around 20% of trophozoites show asynchronous replication between nuclei. Forksense and gene ontology analyses disclosed that genes in regions with potential head-on collisions are linked to chromatin dynamics, cell cycle regulation, and DNA replication/repair pathways, possibly explaining the observed asynchronous replication in part of the population. This study offers the first comprehensive view of replication dynamics in Giardia, the cause of giardiasis, a diarrheal disease impacting millions worldwide.

Venom phenotype conservation suggests integrated specialization in a lizard-eating snake

Biological specialization reduces the size of niche space while increasing efficiency in the use of available resources. Specialization often leads to phenotypic changes via natural selection aligning with niche space constraints. Commonly observed changes are in size, shape, behavior, and traits associated with feeding. One often selected trait for dietary specialization is venom, which, in snakes, often shows variation dependent on diet across and within species. The Neotropical Blunt-headed Treesnake (Imantodes cenchoa) is a highly specialized, rear-fanged, arboreal, lizard hunter that displays a long thin body, enlarged eyes, and a large Duvernoy's gland. However, toxin characterization of I. cenchoa has never been completed. Here, we use RNA-seq and mass spectrometry to assemble, annotate, and analyze the venom gland transcriptomes of four I. cenchoa from across their range. We find a lack of significant venom variation at the sequence and expression levels, suggesting venom conservation across the species. We propose this conservation provides evidence of a specialized venom repertoire, adapted to maximize efficiency of capturing and processing lizards. Importantly, this study provides the most complete venom gland transcriptomes of I. cenchoa and evidence of venom specialization in a rear-fanged snake, giving insight into selective pressures of venom across all snake species.

Caracterização de adaptações do repertório molecular de serpentes das tribos Xenodontini e Pseudoboini relacionadas à sua ecologia de predação

Among the wide spectrum of food ecologies, some groups of snakes have become ophiophagous, feeding mainly on other snakes. Due to the relationship between diet and venom composition, these ophiophagous groups probably needed to develop specific toxins targeting snakes and some type of resistance to the venoms of the snakes they preyed on, associating these characteristics with the development of ophiophagy. In the Neotropics, more precisely in the Dipsadidae family, there are several ophiophagous species. The genus Erythrolamprus (Tribe Xenodontini) and the genera Clelia and Boiruna (Tribe Pseudoboini) contain species that are specialists in snakes. However, due to their relatively low medical relevance, they have been poorly studied and there is scarce information about their venoms and possible resistance factors or mechanisms. In the present work, the toxin composition of the venom gland transcriptome of 8 genera and 19 species of the tribe Pseudoboini and 3 genera and 8 species of the tribe Xenodontini was analyzed. In addition to being the first compositional analysis of the venom of the two tribes, a new type of enzymatic toxins exclusive to the Dipsadidae family (the svMMPs) was identified and characterized in the analyzed samples, as well as high expression levels of a type of phospholipases A2 in some species of the Pseudoboini tribe was detected. On the other hand, comparisons between liver transcriptomes and plasma proteomes of ophiophagous and non-ophiophagous species of the tribe Pseudoboini allowed identifying high expression levels of several types of toxin inhibitors. However, the occurrence of overexpression of these inhibitors in ophiophagous species was not detected. The analyzes revealed, in a broad scale, the evolutionary novelties present in the compositional profile of the venoms of the two tribes and verified the occurrence of plasma toxin inhibitors in the analyzed species.

Dentre o amplo espectro de ecologias alimentares, alguns grupos de serpentes tornaram-se ofiófagos, alimentando-se principalmente de outras serpentes. Devido à relação entre a dieta e a composição do veneno, esses grupos ofiófagos provavelmente precisaram desenvolver toxinas específicas para serpentes e algum tipo de resistência aos venenos das serpentes que predavam, associando essas características ao desenvolvimento da ofiofagia. Nos neotrópicos, mais precisamente na família Dipsadidae, ocorrem diversas espécies ofiófagas. O gênero Erythrolamprus (Tribo Xenodontini) e os gêneros Clelia e Boiruna (Tribo Pseudoboini) contêm espécies especialistas em serpentes. No entanto, devido à sua relevância médica relativamente baixa, foram pouco estudados e existem poucas informações sobre seus venenos e possíveis fatores ou mecanismos de resistência. No presente trabalho foi analisada a composição de toxinas do transcriptoma da glândula de veneno de 8 gêneros e 19 espécies da tribo Pseudoboini e de 3 gêneros e 8 espécies da tribo Xenodontini. Além de ser a primeira análise composicional do veneno das duas tribos, permitiu a identificação e caracterização de um novo tipo de toxinas enzimáticas exclusivo da família Dipsadidae (as svMMPs), assim como revelou níveis de expressão elevados de um tipo de fosfolipases A2 em algumas espécies da tribo Pseudoboini. Por outro lado, as comparações entre os transcriptomas do fígado e os proteomas do plasma de espécies ofiófagas e não ofiófagas da tribo Pseudoboini indicaram níveis de expressão elevados de vários tipos de inibidores de toxinas. Porém, não foi detectada a ocorrência de sobre expressão destes inibidores nas espécies ofiófagas. As análises revelaram, em escala abrangente, as novidades evolutivas presentes no perfil composicional dos venenos das duas tribos e verificaram a ocorrência de inibidores de toxinas plasmáticos nas espécies analisadas.

Multiomics profiling of toxins in the venom of the Amazonian spider Acanthoscurria juruenicola

Acanthoscurria juruenicola is an Amazonian spider described for the first time almost a century ago. However, little is known about their venom composition. Here, we present a multiomics characterization of A. juruenicola venom by a combination of transcriptomics, proteomics, and peptidomics approaches. Transcriptomics of female venom glands resulted in 93,979 unique assembled mRNA transcript encoding proteins. A total of 92 proteins were identified in the venom by mass spectrometry, including 14 mature cysteine-rich peptides (CRPs). Quantitative analysis showed that CRPs, cysteine-rich secretory proteins, metalloproteases, carbonic anhydrases, and hyaluronidase comprise >90% of the venom proteome. Relative quantification of venom toxins was performed by DIA and DDA, revealing converging profiles of female and male specimens by both methods. Biochemical assays confirmed the presence of active hyaluronidases, phospholipases, and proteases in the venom. Moreover, the venom promoted in vivo paralytic activities in crickets, consistent with the high concentration of CRPs. Overall, we report a comprehensive analysis of the arsenal of toxins of A. juruenicola and highlight their potential biotechnological and pharmacological applications. Mass spectrometry data were deposited to the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD013149 and via the MassIVE repository with the dataset identifier MSV000087777.

Differences in PLA2 constitution distinguish the venom of two endemic brazilian mountain lanceheads, Bothrops cotiara and Bothrops fonsecai

Interspecific differences in snake venom compositions can result from distinct regulatory mechanisms acting in each species. However, comparative analyses focusing on identifying regulatory elements and patterns that led to distinct venom composition are still scarce. Among venomous snakes, Bothrops cotiara and Bothrops fonsecai represent ideal models to complement our understanding of the regulatory mechanisms of venom production. These recently diverged species share a similar specialized diet, habitat, and natural history, but each presents a distinct venom phenotype. Here, we integrated data from the venom gland transcriptome and miRNome and the venom proteome of B. fonsecai and B. cotiara to better understand the regulatory mechanisms that may be acting to produce differing venom compositions. We detected not only the presence of similar toxin isoforms in both species but also distinct expression profiles of phospholipases A2 (PLA2) and some snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) isoforms. We found evidence of modular expression regulation of several toxin isoforms implicated in venom divergence and observed correlated expression of several transcription factors. We did not find strong evidence for miRNAs shaping interspecific divergence of the venom phenotypes, but we identified a subset of toxin isoforms whose final expression may be fine-tuned by specific miRNAs. Sequence analysis on orthologous toxins showed a high rate of substitutions between PLA2s, which indicates that these toxins may be under strong positive selection or represent paralogous toxins in these species. Our results support other recent studies in suggesting that gene regulation is a principal mode of venom evolution across recent timescales, especially among species with conserved ecotypes.

An integrative view of the toxic potential of Conophis lineatus (Dipsadidae: Xenodontinae), a medically relevant rear-fanged snake

Most traditional research on snake venoms has focused on front-fanged snake families (Viperidae, Elapidae, and Atractaspididae). However, venom is now generally accepted as being a much more broadly possessed trait within snakes, including species traditionally considered harmless. Unfortunately, due to historical inertia and methodological challenges, the toxin repertoires of non-front-fanged snake families (e.g., Colubridae, Dipsadidae, and Natricidae) have been heavily neglected despite the knowledge of numerous species capable of inflicting medically relevant envenomations. Integrating proteomic data for validation, we perform a de novo assembly and analysis of the Duvernoy's venom gland transcriptome of the Central American Road Guarder (Dipsadidae: Xenodontinae: Conophis lineatus), a species known for its potent bite. We identified 28 putative toxin transcripts from 13 toxin families in the Duvernoy's venom gland transcriptome, comprising 63.7% of total transcriptome expression. In addition to ubiquitous snake toxin families, we proteomically confirmed several atypical venom components. The most highly expressed toxins (55.6% of total toxin expression) were recently described snake venom matrix metalloproteases (svMMPs), with 48.0% of svMMP expression contributable to a novel svMMP isoform. We investigate the evolution of the new svMMP isoform in the context of rear-fanged snakes using phylogenetics. Finally, we examine the morphology of the venom apparatus using μCT and explore how the venom relates to autecology and the highly hemorrhagic effects seen in human envenomations. Importantly, we provide the most complete venom characterization of this medically relevant snake species to date, producing insights into the effects and evolution of its venom, and point to future research directions to better understand the venoms of ‘harmless’ non-front-fanged snakes.

Phylogenetically diverse diets favor more complex venoms in North American pitvipers

The role of natural selection in the evolution of trait complex-ity can be characterized by testing hypothesized links betweencomplex forms and their functions across species. Predatory ven-oms are composed of multiple proteins that collectively function toincapacitate prey. Venom complexity fluctuates over evolutionarytimescales, with apparent increases and decreases in complexity,and yet the causes of this variation are unclear. We tested alterna-tive hypotheses linking venom complexity and ecological sourcesof selection from diet in the largest clade of front-fanged ven-omous snakes in North America: the rattlesnakes, copperheads,cantils, and cottonmouths. We generated independent transcrip-tomic and proteomic measures of venom complexity and collatedseveral natural history studies to quantify dietary variation. Wethen constructed genome-scale phylogenies for these snakes forcomparative analyses. Strikingly, prey phylogenetic diversity wasmore strongly correlated to venom complexity than was overallprey species diversity, specifically implicating prey species’ diver-gence, rather than the number of lineages alone, in the evolutionof complexity. Prey phylogenetic diversity further predicted tran-scriptomic complexity of three of the four largest gene familiesin viper venom, showing that complexity evolution is a concertedresponse among many independent gene families. We suggest thatthe phylogenetic diversity of prey measures functionally relevantdivergence in the targets of venom, a claim supported by sequencediversity in the coagulation cascade targets of venom. Our resultssupport the general concept that the diversity of species in an eco-logical community is more important than their overall number indetermining evolutionary patterns in predator trait complexity.

Recombinant production and characterization of a new toxin from cryptops iheringi centipede venom revealed by proteome and transcriptome analysis

Among the Chilopoda class of centipede, the Cryptops genus is one of the most associated with envenomation in humans in the metropolitan region of the state of São Paulo. To date, there is no study in the literature about the toxins present in its venom. Thus, in this work, a transcriptomic characterization of the Cryptops iheringi venom gland, as well as a proteomic analysis of its venom, were performed to obtain a toxin profile of this species. These methods indicated that 57.9% of the sequences showed to be putative toxins unknown in public databases; among them, we pointed out a novel putative toxin named Cryptoxin-1. The recombinant form of this new toxin was able to promote edema in mice footpads with massive neutrophils infiltration, linking this toxin to envenomation symptoms observed in accidents with humans. Our findings may elucidate the role of this toxin in the venom, as well as the possibility to explore other proteins found in this work.

Latency-associated DNA methylation patterns among HIV-1 infected individuals with distinct disease progression courses or antiretroviral virologic response

DNA methylation is one of the epigenetic modifications that configures gene transcription programs. This study describes the DNA methylation profile of HIV-infected individuals with distinct characteristics related to natural and artificial viremia control. Sheared DNA from circulating mononuclear cells was subjected to target enrichment bisulfite sequencing designed to cover CpG-rich genomic regions. Gene expression was assessed through RNA-seq. Hypermethylation in virologic responders was highly distributed closer to Transcription Start Sites (p-value = 0.03). Hyper and hypomethylation levels within TSS adjacencies varied according to disease progression status (Kruskal–Wallis, p < 0.001), and specific differentially methylated regions associated genes were identified for each group. The lower the promoter methylation, the higher the gene expression in subjects undergoing virologic failure (R = − 0.82, p = 0.00068). Among the inversely correlated genes, those supporting glycolysis and its related pathways were hypomethylated and up-regulated in virologic failures. Disease progression heterogeneity was associated with distinct DNA methylation patterns in terms of rates and distribution. Methylation was associated with the expression of genes sustaining intracellular glucose metabolism in subjects undergoing antiretroviral virologic failure. Our findings highlight that DNA methylation is associated with latency, disease progression, and fundamental cellular processes.

Tracking the recruitment and evolution of snake toxins using the evolutionary context provided by the Bothrops jararaca genome

Venom is a key adaptive innovation in snakes, and how nonvenom genes were co-opted to become part of the toxin arsenal is a significant evolutionary question. While this process has been investigated through the phylogenetic reconstruction of toxin sequences, evidence provided by the genomic context of toxin genes remains less explored. To investigate the process of toxin recruitment, we sequenced the genome of Bothrops jararaca, a clinically relevant pitviper. In addition to producing a road map with canonical structures of genes encoding 12 toxin families, we inferred most of the ancestral genes for their loci. We found evidence that 1) snake venom metalloproteinases (SVMPs) and phospholipases A2 (PLA2) have expanded in genomic proximity to their nonvenomous ancestors; 2) serine proteinases arose by co-opting a local gene that also gave rise to lizard gilatoxins and then expanded; 3) the bradykinin-potentiating peptides originated from a C-type natriuretic peptide gene backbone; and 4) VEGF-F was co-opted from a PGF-like gene and not from VEGF-A. We evaluated two scenarios for the original recruitment of nontoxin genes for snake venom: 1) in locus ancestral gene duplication and 2) in locus ancestral gene direct co-option. The first explains the origins of two important toxins (SVMP and PLA2), while the second explains the emergence of a greater number of venom components. Overall, our results support the idea of a locally assembled venom arsenal in which the most clinically relevant toxin families expanded through posterior gene duplications, regardless of whether they originated by duplication or gene co-option

ToxCodAn: a new toxin annotator and guide to venom gland transcriptomics

Motivation: Next-generation sequencing has become exceedingly common and has transformed our ability to explore nonmodel systems. In particular, transcriptomics has facilitated the study of venom and evolution of toxins in venomous lineages; however, many challenges remain. Primarily, annotation of toxins in the transcriptome is a laborious and time-consuming task. Current annotation software often fails to predict the correct coding sequence and overestimates the number of toxins present in the transcriptome. Here, we present ToxCodAn, a python script designed to perform precise annotation of snake venom gland transcriptomes. We test ToxCodAn with a set of previously curated transcriptomes and compare the results to other annotators. In addition, we provide a guide for venom gland transcriptomics to facilitate future research and use Bothrops alternatus as a case study for ToxCodAn and our guide. Results: Our analysis reveals that ToxCodAn provides precise annotation of toxins present in the transcriptome of venom glands of snakes. Comparison with other annotators demonstrates that ToxCodAn has better performance with regard to run time (⁠>20x faster), coding sequence prediction (⁠>3x more accurate) and the number of toxins predicted (generating >4x less false positives). In this sense, ToxCodAn is a valuable resource for toxin annotation. The ToxCodAn framework can be expanded in the future to work with other venomous lineages and detect novel toxins.