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.

Evaluation of the Inhibitory Potential of Synthetic Peptides Homologous to CDR3 Regions of a Monoclonal Antibody against Bothropic Venom Serine Proteases.

Snakebite accidents, neglected tropical diseases per the WHO, pose a significant public health threat due to their severity and frequency. Envenomation by Bothrops genus snakes leads to severe manifestations due to proteolytic enzymes. While the antibothropic serum produced by the Butantan Institute saves lives, its efficacy is limited as it fails to neutralize certain serine proteases. Hence, developing new-generation antivenoms, like monoclonal antibodies, is crucial. This study aimed to explore the inhibitory potential of synthetic peptides homologous to the CDR3 regions of a monoclonal antibody targeting a snake venom thrombin-like enzyme (SVTLE) from B. atrox venom. Five synthetic peptides were studied, all stable against hydrolysis by venoms and serine proteases. Impressively, four peptides demonstrated uncompetitive SVTLE inhibition, with Ki values ranging from 10-6 to 10-7 M. These findings underscore the potential of short peptides homologous to CDR3 regions in blocking snake venom toxins, suggesting their promise as the basis for new-generation antivenoms. Thus, this study offers potential advancements in combatting snakebites, addressing a critical public health challenge in tropical and subtropical regions.

TnP and AHR-CYP1A1 Signaling Crosstalk in an Injury-Induced Zebrafish Inflammation Model.

Aryl Hydrocarbon Receptor (AHR) signaling is crucial for regulating the biotransformation of xenobiotics and physiological processes like inflammation and immunity. Meanwhile, Thalassophryne nattereri Peptide (TnP), a promising anti-inflammatory candidate from toadfish venom, demonstrates therapeutic effects through immunomodulation. However, its influence on AHR signaling remains unexplored. This study aimed to elucidate TnP's molecular mechanisms on the AHR-cytochrome P450, family 1 (CYP1) pathway upon injury-induced inflammation in wild-type (WT) and Ahr2-knockdown (KD) zebrafish larvae through transcriptomic analysis and Cyp1a reporters. TnP, while unable to directly activate AHR, potentiated AHR activation by the high-affinity ligand 6-Formylindolo [3,2-b]carbazole (FICZ), implying a role as a CYP1A inhibitor, confirmed by in vitro studies. This interplay suggests TnP's ability to modulate the AHR-CYP1 complex, prompting investigations into its influence on biotransformation pathways and injury-induced inflammation. Here, the inflammation model alone resulted in a significant response on the transcriptome, with most differentially expressed genes (DEGs) being upregulated across the groups. Ahr2-KD resulted in an overall greater number of DEGs, as did treatment with the higher dose of TnP in both WT and KD embryos. Genes related to oxidative stress and inflammatory response were the most apparent under inflamed conditions for both WT and KD groups, e.g., Tnfrsf1a, Irf1b, and Mmp9. TnP, specifically, induces the expression of Hspa5, Hsp90aa1.2, Cxcr3.3, and Mpeg1.2. Overall, this study suggests an interplay between TnP and the AHR-CYP1 pathway, stressing the inflammatory modulation through AHR-dependent mechanisms. Altogether, these results may offer new avenues in novel therapeutic strategies, such as based on natural bioactive molecules, harnessing AHR modulation for targeted and sustained drug effects in inflammatory conditions.

Transcriptome profiling and Calreticulin expression in Zika virus -infected Aedes aegypti.

Zika virus (ZIKV) may cause febrile illness and neurological damage, such as microcephaly in fetuses. ZIKV is transmitted to humans by Aedes aegypti, a nearly cosmopolitan mosquito. Understanding the virus-vector molecular interactions has been promising to enhance the knowledge towards disease mitigation. Since ZIKV infection alters gene physiology of mosquitoes, we examined the expression profile of ZIKV-infected Ae. aegypti by several approaches to identify genes altered by viral infection. Transcriptomics were performed by comparing between ZIKV-infected and uninfected Ae. aegypti females, which revealed some differentially expressed genes. Most of these genes appear to be involved with immune response as evidenced by an interactome analysis, and a prominent finding was a calreticulin-like (CRT) gene, which was upregulated during the infection. Expression of CRT was also experimentally quantified by qPCR, however, it revealed no significant differences between infected and uninfected females. Instead, expression levels were highly variable among individuals and negatively correlated to viral load. We also tested the possibility of this gene to be silenced, but the double-stranded RNA did not reduce CRT expression, and actually increased the inter-individuals' expressional variability. Present results differed from our original hypothesis of upregulation by infection. They also diverged between them (comparing qPCR to Transcriptomics) and from the literature which reported augmented CRT levels in Aedes species during viral infection. Present case probably underlies a more complex virus-host interaction system than we expected. Regulation of this gene seems not to be a linear correlation between expression and viremy. As infection takes place, a complex homeostatic mechanism may act to prevent expression and other cellular tasks from drifting. It is also possible that CRT expression is simply randomly disturbed by viral infection. Taken together, results show that CRT expression profile during ZIKV infection is complex and requires different investigative approaches to be understood. Studies focused on the biochemical function of CRT protein and on its role in the native mosquito metabolic network could unravel how it is actually influenced by ZIKV. Current work contributes more by getting incidental findings and by posing new hypotheses than by answering the original questions.

ECM proteins involved in cell migration and vessel formation compromise bovine cloned placentation.

Advances in Artificial Reproductive Technologies (ARTs) in bovine embryos to produce cloned pregnancies have been developed in the last years, however high pregnancy losses rates still present. Those rates are associated to placental morphology alterations that are majorly focused on extracellular matrix (ECM) alterations and consequently placentome hyperplasia, increased trophoblast cell migration and vascular defects. Herein, we aimed to search, at protein level, pathways altered by ART that can modify the placental development harmony. For this, we used 4-month-old control (n = 3), SDS-decellularized (n = 3) and cloned (n = 3) cotyledons for proteomic analysis. Samples were grouped by condition and were washed, lysed, urea-reduced, acetone-precipitated, DTT-educed, iodoacetamide-alkylated, trypsin digested, and C-18 column purified. At the end, 3 µg protein were loaded in Orbitrap Fusion Lumos spectrometer (ThermoScientific). Generated spectra were exported to MaxQuant software (v1.6.10.43) to produce the protein list of each sample, and the LFQ intensity were statistically analyzed by Inferno software (v.1.1.6970). After this, proteins related to ECM and cellular junction ontologies were filtered and manually annotated using DAVID Bioinformatics Resources 6.8. From 2577 identified protein sequences by MaxQuant software, 165 (7.1%) were filtered by selected ontologies. We found 10 proteins (B2M, COL6A6, FERMT3, LGALS3BP, NIBAN2, PDLIM5, PON1, PRP9, RASIP1 and SPARC) upregulated in clone, when compared to control condition. The ten pathways that enriched more proteins were: focal adhesion, ECM-receptor interaction, PI3K-Akt signaling pathway, protein digestion and absorption, amoebiasis, pathways in cancer, small cell lung cancer, platelet activation, regulation of actin cytoskeleton, and proteoglycans in cancer. Functionally, detected proteins, signaling pathways and ontologies are orchestrated to permit the binucleated trophoblastic cells migration and blood vessels modelling. In conclusion, the cloned condition presents the same mechanisms as control one, however overexpression of some specific ECM proteins could be responsible to exacerbate those mechanisms and can explain all morphophysiological alterations presented in cloned pregnancies associated to high pregnancies losses rates in this condition.

Proteomic analysis reveals rattlesnake venom modulation of proteins associated with cardiac tissue damage in mouse hearts.

Snake envenomation is a common but neglected disease that affects millions of people around the world annually. Among venomous snake species in Brazil, the tropical rattlesnake (Crotalus durissus terrificus) accounts for the highest number of fatal envenomations and is responsible for the second highest number of bites. Snake venoms are complex secretions which, upon injection, trigger diverse physiological effects that can cause significant injury or death. The components of C. d. terrificus venom exhibit neurotoxic, myotoxic, hemotoxic, nephrotoxic, and cardiotoxic properties which present clinically as alteration of central nervous system function, motor paralysis, seizures, eyelid ptosis, ophthalmoplesia, blurred vision, coagulation disorders, rhabdomyolysis, myoglobinuria, and cardiorespiratory arrest. In this study, we focused on proteomic characterization of the cardiotoxic effects of C. d. terrificus venom in mouse models. We injected venom at half the lethal dose (LD50) into the gastrocnemius muscle. Mouse hearts were removed at set time points after venom injection (1 h, 6 h, 12 h, or 24 h) and subjected to trypsin digestion prior to high-resolution mass spectrometry. We analyzed the proteomic profiles of >1300 proteins and observed that several proteins showed noteworthy changes in their quantitative profiles, likely reflecting the toxic activity of venom components. Among the affected proteins were several associated with cellular deregulation and tissue damage. Changes in heart protein abundance offer insights into how they may work synergistically upon envenomation. SIGNIFICANCE: Venom of the tropical rattlesnake (Crotalus durissus terririficus) is known to be neurotoxic, myotoxic, nephrotoxic and cardiotoxic. Although there are several studies describing the biochemical effects of this venom, no work has yet described its proteomic effects in the cardiac tissue of mice. In this work, we describe the changes in several mouse cardiac proteins upon venom treatment. Our data shed new light on the clinical outcome of the envenomation by C. d. terrificus, as well as candidate proteins that could be investigated in efforts to improve current treatment approaches or in the development of novel therapeutic interventions in order to reduce mortality and morbidity resulting from envenomation.

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.

Bothrops Jararaca Snake Venom Modulates Key Cancer-Related Proteins in Breast Tumor Cell Lines.

Cancer is characterized by the development of abnormal cells that divide in an uncontrolled way and may spread into other tissues where they may infiltrate and destroy normal body tissue. Several previous reports have described biochemical anti-tumorigenic properties of crude snake venom or its components, including their capability of inhibiting cell proliferation and promoting cell death. However, to the best of our knowledge, there is no work describing cancer cell proteomic changes following treatment with snake venoms. In this work we describe the quantitative changes in proteomics of MCF7 and MDA-MB-231 breast tumor cell lines following treatment with Bothrops jararaca snake venom, as well as the functional implications of the proteomic changes. Cell lines were treated with sub-toxic doses at either 0.63 µg/mL (low) or 2.5 µg/mL (high) of B. jararaca venom for 24 h, conditions that cause no cell death per se. Proteomics analysis was conducted on a nano-scale liquid chromatography coupled on-line with mass spectrometry (nLC-MS/MS). More than 1000 proteins were identified and evaluated from each cell line treated with either the low or high dose of the snake venom. Protein profiling upon venom treatment showed differential expression of several proteins related to cancer cell metabolism, immune response, and inflammation. Among the identified proteins we highlight histone H3, SNX3, HEL-S-156an, MTCH2, RPS, MCC2, IGF2BP1, and GSTM3. These data suggest that sub-toxic doses of B. jararaca venom have potential to modulate cancer-development related protein targets in cancer cells. This work illustrates a novel biochemical strategy to identify therapeutic targets against cancer cell growth and survival.

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.

Rhythms of Transcription in Field-Grown Sugarcane Are Highly Organ Specific.

Circadian clocks improve plant fitness in a rhythmic environment. As each cell has its own circadian clock, we hypothesized that sets of cells with different functions would have distinct rhythmic behaviour. To test this, we investigated whether different organs in field-grown sugarcane follow the same rhythms in transcription. We assayed the transcriptomes of three organs during a day: leaf, a source organ; internodes 1 and 2, sink organs focused on cell division and elongation; and internode 5, a sink organ focused on sucrose storage. The leaf had twice as many rhythmic transcripts (>68%) as internodes, and the rhythmic transcriptomes of the internodes were more like each other than to those of the leaves. Among the transcripts expressed in all organs, only 7.4% showed the same rhythmic pattern. Surprisingly, the central oscillators of these organs - the networks that generate circadian rhythms - had similar dynamics, albeit with different amplitudes. The differences in rhythmic transcriptomes probably arise from amplitude differences in tissue-specific circadian clocks and different sensitivities to environmental cues, highlighted by the sampling under field conditions. The vast differences suggest that we must study tissue-specific circadian clocks in order to understand how the circadian clock increases the fitness of the whole plant.

The impact of rattlesnake venom on mice cerebellum proteomics points to synaptic inhibition and tissue damage.

Snake envenomation is responsible for more than 130,000 deaths worldwide. In Brazil, the Crotalus rattlesnake is responsible for the second largest number of accidental snake bites in the country. Although there are many descriptions of the clinical and biochemical effects of Crotalus envenoming, there are few works describing the molecular events in the central nervous system of an organism due to envenomation. In this study, we analyzed the proteomic effect of Crotalus durissus terrificus snake venom on mice cerebellums. To monitor the envenomation over time, changes in the protein abundance were evaluated at 1 h, 6 h, 12 h and 24 h after venom injection by mass spectrometry. The analysis of the variation of over 4600 identified proteins over time showed a reduction in components of inhibitory synapse signaling, oxidative stress, and maintenance of neuronal cells, which paralleled increasing tissue damage and apoptosis factors. These analyses revealed the potential protein targets of the C. d. terrificus venom on the murine cerebellum, showing new aspects of the snake envenomation effect. These data may contribute to new therapeutic approaches (i.e., approaches directed at protein targets affected by the envenomation) on the treatment of envenomation by the neurotoxic C. d. terrificus snake venom. SIGNIFICANCE: Snakebites are a neglected global health problem that affects mostly rural and tropical areas of developing countries. It is estimated that over 5.4 million people are bitten by snakes each year, from which 2.7 million people are bitten by venomous snakes, resulting in disabilities such as amputations and in some cases leading to death. The C. d. terrificus snake is the most lethal snake in Brazil. Studying the molecular changes upon envenomation in a specific tissue may lead to a better understanding of the envenomation process by C. d. terrificus snakebites.