RESUMO

Skeletal muscle regeneration after injury is a complex process involving inflammatory signaling and myoblast activation. Pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) are key mediators, but their effects on gene expression in proliferating myoblasts are unclear. We performed the RNA sequencing of TNF-α treated C2C12 myoblasts to elucidate the signaling pathways and gene networks regulated by TNF-α during myoblast proliferation. The TNF-α (10 ng/mL) treatment of C2C12 cells led to 958 differentially expressed genes compared to the controls. Pathway analysis revealed significant regulation of TNF-α signaling, along with the chemokine and IL-17 pathways. Key upregulated genes included cytokines (e.g., IL-6), chemokines (e.g., CCL7), and matrix metalloproteinases (MMPs). TNF-α increased myogenic factor 5 (Myf5) but decreased MyoD protein levels and stimulated the release of MMP-9, MMP-10, and MMP-13. TNF-α also upregulates versican and myostatin mRNA. Overall, our study demonstrates the TNF-α modulation of distinct gene expression patterns and signaling pathways that likely contribute to enhanced myoblast proliferation while suppressing premature differentiation after muscle injury. Elucidating the mechanisms involved in skeletal muscle regeneration can aid in the development of regeneration-enhancing therapeutics.

Assuntos

RESUMO

Although non-front fanged snakes account for almost two-thirds of snake diversity, most studies on venom composition and evolution focus exclusively on front-fanged species, which comprise most of the clinically relevant accidents. Comprehensive reports on venom composition of non-front fanged snakes are still scarce for several groups. In this study, we address such shortage of knowledge by providing new insights about the venom composition among species of Phalotris, a poorly studied Neotropical dipsadid genus. Phalotris are known for their specialized venom delivery system and toxic venoms, which can cause life-threatening accidents in humans. We evaluate the venom-gland transcriptome of Phalotris, comparing the following three South American species: P. reticulatus for the Araucaria Pine forests, P. lemniscatus for the Pampa grasslands, and P. mertensi for the Brazilian Cerrado. Our results indicate similar venom profiles, in which they share a high expression level of Kunitz-type inhibitors (KUNZ). On the other hand, comparative analyses revealed substantial differences in the expression levels of C-type lectins (CTL) and snake venom metalloproteinases (SVMP). The diverse set of SVMP and CTL isoforms shows signals of positive selection, and we also identified truncated forms of type III SVMPs, which resemble type II and type I SVMPs of viperids. Additionally, we identified a CNP precursor hosting a proline-rich region containing a BPP motif resembling those commonly detected in viperid venoms with hypotensive activity. Altogether, our results suggest an evolutionary history favoring high expression levels of few KUNZ isoforms in Phalotris venoms, contrasting with a highly diverse set of SVMP and CTL isoforms. Such diversity can be comparable with the venom variability observed in some viperids. Our findings highlight the extreme phenotypic diversity of non-front fanged snakes and the importance to allocate greater effort to study neglected groups of Colubroidea.

Assuntos

RESUMO

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.

Assuntos

RESUMO

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/ToxCodAn-Genome.

Assuntos

RESUMO

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.

Assuntos

RESUMO

Although non-front fanged snakes account for almost two-thirds of snake diversity, most studies on venom composition and evolution focus exclusively on front-fanged species, which comprise most of the clinically relevant accidents. Comprehensive reports on venom composition of non-front fanged snakes are still scarce for several groups. In this study, we address such shortage of knowledge by providing new insights about the venom composition among species of Phalotris, a poorly studied Neotropical dipsadid genus. Phalotris are known for their specialized venom delivery system and toxic venoms, which can cause life-threatening accidents in humans. We evaluate the venom-gland transcriptome of Phalotris, comparing the following three South American species: P. reticulatus for the Araucaria Pine forests, P. lemniscatus for the Pampa grasslands, and P. mertensi for the Brazilian Cerrado. Our results indicate similar venom profiles, in which they share a high expression level of Kunitz-type inhibitors (KUNZ). On the other hand, comparative analyses revealed substantial differences in the expression levels of C-type lectins (CTL) and snake venom metalloproteinases (SVMP). The diverse set of SVMP and CTL isoforms shows signals of positive selection, and we also identified truncated forms of type III SVMPs, which resemble type II and type I SVMPs of viperids. Additionally, we identified a CNP precursor hosting a proline-rich region containing a BPP motif resembling those commonly detected in viperid venoms with hypotensive activity. Altogether, our results suggest an evolutionary history favoring high expression levels of few KUNZ isoforms in Phalotris venoms, contrasting with a highly diverse set of SVMP and CTL isoforms. Such diversity can be comparable with the venom variability observed in some viperids. Our findings highlight the extreme phenotypic diversity of non-front fanged snakes and the importance to allocate greater effort to study neglected groups of Colubroidea.

RESUMO

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.

RESUMO

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.

RESUMO

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.

RESUMO

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.