Lipidomic profiling of the Brazilian yellow scorpion venom: new insights into inflammatory responses following Tityus serrulatus envenomation.

Due to the high prevalence and clinical relevance, scorpionism is a critical public health issue in several Brazilian regions. Tityus serrulatus, commonly known as the Brazilian yellow scorpion, is the most venomous genus found in Brazilian fauna and associated with severe clinical manifestations such as localized pain, hypertension, sweating, tachycardia and complex hyperinflammatory responses. In general, T. serrulatus venom contains a complex mixture of active compounds, including proteins, peptides, and amino acids. Although knowledge of the protein fractions of scorpion venom is available, venom lipid components are not yet comprehensively known. The aim of the present study was to determine and characterize the lipid constituents/profile of the T. serratus venom utilizing liquid chromatography coupled with high-resolution mass spectrometry. Lipid species (164 in total) belonging to 3 different lipid categories, glycerophospholipids, sphingolipids, and glycerolipids, were identified. A further search on MetaCore/MetaDrug platform, which is based upon a manually curated database of molecular interactions, molecular pathways, gene-disease associations, chemical metabolism, and toxicity information, exhibited several metabolic pathways for 24 of previously identified lipid species, including activation of nuclear factor kappa B and oxidative stress pathways. Further several bioactive compounds, such as plasmalogens, lyso-platelet-activating factors, and sphingomyelins, associated with systemic responses triggered by T. serrulatus envenomation were detected. Finally, lipidomic data presented provide advanced and valuable information to better comprehend the mechanisms underlying the complex pathophysiology induced by T. serrulatus envenomation.

Tityus serrulatus scorpion venom as a potential drug source for Chagas' disease: Trypanocidal and immunomodulatory activity.

Current chemical therapies for Chagas Disease (CD) lack ability to clear Trypanosoma cruzi (Tc) parasites and cause severe side effects, making search for new strategies extremely necessary. We evaluated the action of Tityus serrulatus venom (TsV) components during Tc infection. TsV treatment increased nitric oxide and pro-inflammatory cytokine production by Tc-infected macrophages (MØ), decreased intracellular parasite replication and trypomastigotes release, also triggering ERK1/2, JNK1/2 and p38 activation. Ts7 demonstrated the highest anti-Tc activity, inducing high levels of TNF and IL-6 in infected MØ. TsV/Ts7 presented synergistic effect on p38 activation when incubated with Tc antigen. KPP-treatment of MØ also decreased trypomastigotes releasing, partially due to p38 activation. TsV/Ts7-pre-incubation of Tc demonstrated a direct effect on parasite decreasing MØ-trypomastigotes releasing. In vivo KPP-treatment of Tc-infected mice resulted in decreased parasitemia. Summarizing, this study opens perspectives for new bioactive molecules as CD-therapeutic treatment, demonstrating the TsV/Ts7/KPP-trypanocidal and immunomodulatory activity during Tc infection.

Moving Pieces in a Cellular Puzzle: A Cryptic Peptide from the Scorpion Toxin Ts14 Activates AKT and ERK Signaling and Decreases Cardiac Myocyte Contractility via Dephosphorylation of Phospholamban.

Cryptic peptides (cryptides) are biologically active peptides formed after proteolysis of native precursors present in animal venoms, for example. Proteolysis is an overlooked post-translational modification that increases venom complexity. The tripeptide KPP (Lys-Pro-Pro) is a peptide encrypted in the C-terminus of Ts14-a 25-mer peptide from the venom of the Tityus serrulatus scorpion that has a positive impact on the cardiovascular system, inducing vasodilation and reducing arterial blood pressure of hypertensive rats among other beneficial effects. A previous study reported that KPP and its native peptide Ts14 act via activation of the bradykinin receptor B2 (B2R). However, the cellular events underlying the activation of B2R by KPP are unknown. To study the cell signaling triggered by the Ts14 cryptide KPP, we incubated cardiac myocytes isolated from C57BL/6 mice with KPP (10-7 mol·L-1) for 0, 5, or 30 min and explored the proteome and phosphoproteome. Our results showed that KPP regulated cardiomyocyte proteins associated with, but not limited to, apoptosis, muscle contraction, protein turnover, and the respiratory chain. We also reported that KPP led to AKT phosphorylation, activating AKT and its downstream target nitric oxide synthase. We also observed that KPP led to dephosphorylation of phospholamban (PLN) at its activation sites (S16 and T17), leading to reduced contractility of treated cardiomyocytes. Some cellular targets reported here for KPP (e.g., AKT, PLN, and ERK) have already been reported to protect the cardiac tissue from hypoxia-induced injury. Hence, this study suggests potential beneficial effects of this scorpion cryptide that needs to be further investigated, for example, as a drug lead for cardiac infarction.

Heterologous expression of rTsHyal-1: the first recombinant hyaluronidase of scorpion venom produced in Pichia pastoris system.

In general, hyaluronidases have a broad potential application on medicine and esthetics fields. Hyaluronidases from animal venoms cleave hyaluronan present in the extracellular matrix, acting as spreading factors of toxins into the tissues of the victim. However, the in-depth characterization of hyaluronidase from animal venoms has been neglected due to its instability and low concentration in the venom, which hamper its isolation. Thus, heterologous expression of hyaluronidase acts as a biotechnological tool in the obtainment of enough amounts of the enzyme for structural and functional studies. Therefore, this study produced a recombinant hyaluronidase from Tityus serrulatus scorpion venom, designated as rTsHyal-1, in the Pichia pastoris system. Thus, a gene for TsHyal-1 (gb|KF623285.1) was synthesized and cloned into the pPICZαA vector (GenScript Corporation) for heterologous expression in P. pastoris. rTsHyal-1 was expressed in laboratorial scale in a buffered minimal medium containing methanol (BMM) for 96 h with daily addition of methanol. Expression of rTsHyal-1 resulted in a total protein yield of 0.266 mg/mL. rTsHyal-1 partially purified through cation exchange chromatography presented a specific activity of 1097 TRU/mg, against 838 TRU/mg for the final expressed material, representing a 1.31-fold purification. rTsHyal-1 has molecular mass of 49.5 kDa, and treatment with PNGase F and analysis by mass spectrometry (MALDI-TOF) indicated a potential N-glycosylation of 4.5 kDa. Additionally, de novo sequencing of rTsHyal-1, performed in MALDI-TOF and Q Exactive Orbitrap MS, resulted in 46.8% of protein sequence coverage. rTsHyal-1 presents the highest substrate specificity to hyaluronan followed by chondroitin-6-sulfate, chondroitin-4-sulfate, and dermatan sulfate and showed an optimum activity at pH 6.0 and 40 °C. These results validate the biotechnological process for the heterologous expression of rTsHyal-1. This is the first recombinant hyaluronidase from scorpion venoms expressed in the P. pastoris system with preserved enzyme activity.

Aspidosperma pyrifolium Has Anti-Inflammatory Properties: An Experimental Study in Mice with Peritonitis Induced by Tityus serrulatus Venom or Carrageenan.

Scorpions of the genus Tityus are responsible for the majority of envenomation in Brazil, the Tityus serrulatus species being the most common and dangerous in South America. In this approach, we have investigated the ability of the aqueous extract from the leaves of Aspidosperma pyrifolium in reducing carrageenan-induced inflammation and the inflammation induced by T. serrulatus envenomation in mice. We also evaluated the cytotoxic effects of this extract, using the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-2H-tetrazolium (MTT) assay and the results revealed that the extract is safe. Analysis by High Performance Liquid Chromatography coupled with Diode Array Detector (HPLC-DAD) and Liquid Chromatography Coupled with Mass Spectrometry with Diode Array Detection (LC-DAD-MS) showed one major chemical component, the flavonoid rutin and phenolics compounds. For in vivo studies in carrageenan-induced peritonitis model, mice received extracts, dexamethasone, rutin or saline, before administration of carrageenan. For venom-induced inflammation model, animals received T. serrulatus venom and were, simultaneously, treated with extracts, antivenom, rutin or saline. The extract and rutin showed a reduction in the cell migration into the peritoneal cavity, and in the same way the envenomated animals also showed reduction of edema, inflammatory cell infiltration and vasodilation in lungs. This is an original study revealing the potential action of A. pyrifolium against inflammation caused by Tityus serrulatus venom and carrageenan, revealing that this extract and its bioactive molecules, specifically rutin, may present potential anti-inflammatory application.

Immunosuppressive evidence of Tityus serrulatus toxins Ts6 and Ts15: insights of a novel K(+) channel pattern in T cells.

The voltage-gated potassium channel Kv1.3 is a novel target for immunomodulation of autoreactive effector memory T cells, which play a major role in the pathogenesis of autoimmune diseases. In this study, the Ts6 and Ts15 toxins isolated from Tityus serrulatus (Ts) were investigated for their immunosuppressant roles on CD4(+) cell subsets: naive, effector (TEF ), central memory (TCM) and effector memory (TEM). The electrophysiological assays confirmed that both toxins were able to block Kv1.3 channels. Interestingly, an extended Kv channel screening shows that Ts15 blocks Kv2.1 channels. Ts6 and Ts15 significantly inhibit the proliferation of TEM cells and interferon-γ production; however, Ts15 also inhibits other CD4(+) cell subsets (naive, TEF and TCM). Based on the Ts15 inhibitory effect of proliferation of all CD4(+) cell subsets, and based on its blocking effect on Kv2.1, we investigated the Kv2.1 expression in T cells. The assays showed that CD4(+) and CD8(+) cells express the Kv2.1 channels mainly extracellularly with TCM cells expressing the highest number of Kv2.1 channels. We also provide in vivo experimental evidence to the protective effect of Ts6 and Ts15 on delayed-type hypersensitivity reaction. Altogether, this study presents the immunosuppressive behaviour of Ts6 and Ts15 toxins, indicating that these toxins could be promising candidates for autoimmune disease therapy. Moreover, this is the first report illustrating the involvement of a novel K(+) channel subtype, Kv2.1, and its distribution in T-cell subsets.

Understanding the complexity of Tityus serrulatus venom: A focus on high molecular weight components.

Tityus serrulatus scorpion is responsible for a significant number of envenomings in Brazil, ranging from mild to severe, and in some cases, leading to fatalities. While supportive care is the primary treatment modality, moderate and severe cases require antivenom administration despite potential limitations and adverse effects. The remarkable proliferation of T. serrulatus scorpions, attributed to their biology and asexual reproduction, contributes to a high incidence of envenomation. T. serrulatus scorpion venom predominantly consists of short proteins acting as neurotoxins (α and ß), that primarily target ion channels. Nevertheless, high molecular weight compounds, including metalloproteases, serine proteases, phospholipases, and hyaluronidases, are also present in the venom. These compounds play a crucial role in envenomation, influencing the severity of symptoms and the spread of venom. This review endeavors to comprehensively understand the T. serrulatus scorpion venom by elucidating the primary high molecular weight compounds and exploring their potential contributions to envenomation. Understanding these compounds' mechanisms of action can aid in developing more effective treatments and prevention strategies, ultimately mitigating the impact of scorpion envenomation on public health in Brazil.

Use of point-of-care ultrasound to assess the severity of scorpion stings in hospitalized patients.

INTRODUCTION: Scorpionism is a public health problem, especially in tropical regions. In Brazil, the prevalence of envenomation by scorpions is high, and the average national lethality is around 0.16 percent. The Tityus serrulatus scorpion is the primary species of medical importance. However, objective tools to predict and define the severity of these envenomations are lacking. MATERIALS AND METHODS: This was an observational study conducted among patients aged 0-19 years with scorpionism. Patients were admitted to a reference hospital between December 2020 and May 2022. Point-of-care ultrasound was performed within 24 hours of the scorpion sting. RESULTS: Forty-nine patients were included, with a median age of 3.6 (interquartile range 2.3-5.3) years and a predominance of females (51 percent). Fifteen patients (30.6 percent) presented major life-threatening signs, 32 (65.3 percent) minor systemic manifestations, and two (4.1 percent) only local manifestations. Left ventricular dysfunction was identified in 13 patients (26.5 percent). Ten patients (20.4 percent) presented pattern B (visualization of three or more B lines in the evaluated quadrant) in at least one lung window. The sensitivity and specificity of cardiac and pulmonary ultrasound to identify the most severely ill patients were 86 percent and 94 percent, respectively. DISCUSSION: The changes found on point-of-care ultrasound were associated with life-threatening signs. All patients with class III envenomation were referred to the intensive care unit, showing the importance of early identification of this subgroup. The main limitations were the small sample size and the fact that admission to intensive care was not based on systematic criteria. CONCLUSIONS: Point-of-care ultrasound is able to identify early signs of pulmonary congestion and heart failure in scorpionism. It can be useful for the objective selection of patients who are at a higher risk of complications and death and who require intensive support; it may also be valuable for periodic reassessments. Point-of-care ultrasound is a valuable tool for identifying and monitoring severe cases of scorpionism.

Analysis of antibodies avidity for Tityus serrulatus scorpion venom in antivenom production and its potential for application as a potency test.

Antivenoms are the only specific medication for neutralizing toxins present in venom of animals such scorpions and snakes through antigen-antibody binding. Several analyses are carried out throughout its production in order to ensure the quality and effectiveness of the antivenom that will be administered to the patient. One of these is the potency assay, which is performed to assess the ability of antivenoms to neutralize the toxic effects of the venom injected in mice. The substitution of in vivo for in vitro assays such as ELISA has been presented by other authors, bringing several advantages such as the reduction in the use of animals, in costs and in the duration of the assays. However, the avidity index of antivenom antibodies determined by ELISA has not yet been applied for this purpose. Therefore, the objective of this study was to evaluate the avidity of sera from hyperimmunized horses with crude Tityus serrulatus venom, a scorpion species associated with the most serious accidents in Brazil, and its potential for application as a potency test replacing the in vivo assay. The avidity ELISA proved to be interesting for monitoring the binding strength of antibodies produced by horses in hyperimmune plasma production programs. It was possible to verify oscillations in antibody avidity that occurred along the immunization cycles, differences between novice and veteran horses, maturation of antibody avidity, and correlation between avidity index and antibody titre. Similar results were obtained for crude venom and purified Ts1 toxin. In addition, the avidity ELISA apparently demonstrated potential for application as a potency test in the initial stage of antivenom production. However, more studies are necessary.

Mitochondrial swelling in cardiomyocytes: Insights from a murine model of Tityus serrulatus scorpion envenomation.

Immune system hyperactivation is involved with clinical severity and pathological alterations during scorpion envenomation. In a murine model, mice inoculated with a lethal dose of Tityus serrulatus scorpion venom presented mitochondrial swelling in cardiomyocytes, with other structures such as sarcomeres and intercalated disks preserved. Treatment with dexamethasone or knockout animals to the interleukin-1ß receptor do not undergo mitochondrial changes in cardiomyocytes during envenomation.

Ts17, a Tityus serrulatus ß-toxin structurally related to α-scorpion toxins.

Ts17 was purified from the venom of the scorpion Tityus serrulatus, the most dangerous scorpion species in Brazil. The activity on Nav1.1-Nav1.7 channels was electrophysiologically characterized by patch-clamp technique. Ts17 amino acid sequence indicated high similarity to alpha-scorpion toxins; however, it presented beta-toxin activity, altering the kinetics of the Na+-channels. The most affected subtypes during activation (with and without prepulse) and inactivation phases were Nav1.2 and Nav1.5, respectively. For recovery from inactivation, the most affected voltage-gated sodium channel was Nav1.5. Circular dichroism spectra showed that Ts17 presents mainly ß-sheet and unordered structures at all analyzed pHs, and the maximum value of α-helix was found at pH 4.0 (13.3 %). Based on the results, Ts17 might be used as a template to develop a new cardiac drug. Key contribution Purification of Ts17 from Tityus serrulatus, electrophysiological characterization of Ts17 on voltage-gated sodium channel subtypes, ß-toxin classification.

Back to Tityus serrulatus Lutz & Mello, 1922 (Scorpiones: Buthidae): new comments about an old species.

A synopsis on the historical, geographical and ecological aspects related to the most conspicuous scorpion species of the genus Tityus known from Brazil is proposed. Tityus serrulatus Lutz & Mello, 1922 was described precisely one century ago, nevertheless many questions related to its ecological adaptations and geographical expansion remain without a precise response. This species, well known for its infamous reputation of noxious species, is also known for its capacity to reproduce asexually, by parthenogenesis. Although the individuals of a given population are considered clones, a new hypothesis could suggest the occurrence of mutations within isolated individuals, leading to distinct subpopulations that could present better phenotypic performances in ecological habitats distinct from those of the original area of distribution of the species.

Detection of areas vulnerable to scorpionism and its association with environmental factors in São Paulo, Brazil.

Accidents caused by scorpions are considered a neglected condition and represent a major health problem in most tropical countries, especially for children and elderly people. In Brazil, scorpionism is recurrent in the southeast region, mainly in the state of São Paulo, due to the progressive increase in scorpions found in urban habitats. Thus, our study aimed to provide better insights into the geographic and epidemiological characteristics of scorpion envenomation in São Paulo state and identify the environmental factors that are associated with these accidents. This is an ecological and retrospective study with secondary data on scorpion accidents in the state of São Paulo from 2008 to 2018 obtained from the Notifiable Disease Information System. The SatScan software was used to identify the higher- and lower-risk spatiotemporal clusters. A total of 145,464 scorpion sting cases were recorded in the state of São Paulo, between 2008 and 2018; there was a four-fold increase in the incidence rate. Accidents occurred more frequently in the spring season, wherein higher-risk clusters were in the north and northwest regions of the state. High temperatures, low precipitation, and poor natural vegetation are associated with higher risk areas. Our study mapped vulnerable areas for scorpion accidents that can aid in the design of efficient public health policies, which should be intensified during the spring season.

Heterologous expression of Ts8, a neurotoxin from Tityus serrulatus venom, evidences its antifungal activity.

In this study we expressed the Ts8, a neurotoxin from Tityus serrulatus scorpion venom, in Pichia pastoris yeast. We evaluated the peptide expression in different conditions, such as pH, temperature, and addition of casamino acids supplement. Analyses of expressed products by mass spectrometry and Edman degradation showed that rTs8 has sites that allow its cleavage by yeast proteases released into the culture medium. The casamino acids addition was favourable for toxin expression, however, was not sufficient to minimize proteolytic degradation. Functional assays with recombinant toxin fragments and native toxins have demonstrated the release of cytokines such as TNF-α and IL-1ß in some peptides tested. In addition, the toxins were shown to inhibit the Pichia pastoris growth in antifungal test and were not toxic to alveolar macrophages cells at the concentrations analyzed The electrophysiological screening, by voltage clamp technique, showed that the rTs8 fragment with the highest molecular weight inhibited the Kv1.3 channel, whereas the N-terminal fragment had no activity on the ion channels tested.

New Insights into the Hypotensins from Tityus serrulatus Venom: Pro-Inflammatory and Vasopeptidases Modulation Activities.

The Tityus serrulatus scorpion is considered the most dangerous of the Brazilian fauna due to the severe clinical manifestations in injured victims. Despite being abundant components of the venom, few linear peptides have been characterized so far, such as hypotensins. In vivo studies have demonstrated that hypotensin I (TsHpt-I) exerts hypotensive activity, with an angiotensin-converting enzyme (ACE)-independent mechanism of action. Since experiments have not yet been carried out to analyze the direct interaction of hypotensins with ACE, and to deepen the knowledge about these peptides, hypotensins I and II (TsHpt-II) were studied regarding their modulatory action over the activities of ACE and neprilysin (NEP), which are the peptidases involved in blood pressure control. Aiming to search for indications of possible pro-inflammatory action, hypotensins were also analyzed for their role in murine macrophage viability, the release of interleukins and phagocytic activity. TsHpt-I and -II were used in kinetic studies with the metallopeptidases ACE and NEP, and both hypotensins were able to increase the activity of ACE. TsHpt-I presented itself as an inhibitor of NEP, whereas TsHpt-II showed weak inhibition of the enzyme. The mechanism of inhibition of TsHpt-I in relation to NEP was defined as non-competitive, with an inhibition constant (Ki) of 4.35 µM. Concerning the analysis of cell viability and modulation of interleukin levels and phagocytic activity, BALB/c mice's naïve macrophages were used, and an increase in TNF production in the presence of TsHpt-I and -II was observed, as well as an increase in IL-6 production in the presence of TsHpt-II only. Both hypotensins were able to increase the phagocytic activity of murine macrophages in vitro. The difference between TsHpt-I and -II is the residue at position 15, with a glutamine in TsHpt-I and a glutamic acid in TsHpt-II. Despite this, kinetic analyzes and cell assays indicated different actions of TsHpt-I and -II. Taken together, these results suggest a new mechanism for the hypotensive effects of TsHpt-I and -II. Furthermore, the release of some interleukins also suggests a role for these peptides in the venom inflammatory response. Even though these molecules have been well studied, the present results suggest a new mechanism for the hypotensive effects of TsHpt-I.

Tityus serrulatus (Scorpion): From the Crude Venom to the Construction of Synthetic Peptides and Their Possible Therapeutic Application Against Toxoplasma gondii Infection.

Toxoplasmosis, caused by Toxoplasma gondii, is a major public concern owing to its neurotropic nature and high morbidity and mortality rates in immunocompromised patients and newborns. Current treatment for this disease is inefficient and produces side effects. Inflammatory mediators produced during T. gondii infection (e.g., cytokines and nitric oxide) are crucial in controlling parasite replication. In this context, Tityus serrulatus venom (TsV) induces the production of inflammatory mediators by immune cells. Thus, this study aimed to isolate and identify the components of TsV with potential anti-T. gondii activity. TsV was extracted from scorpions and lyophilized or loaded onto a column to obtain its fractions. TsV subfractions were obtained using chromatography, and its amino acid sequence was identified and applied to peptide design using bioinformatics tools. The C57BL/6 mice and their harvested macrophages were used to test the anti-Toxoplasma activity of TsV components and peptides. TsV and its fraction F6 attenuated the replication of tachyzoites in macrophages and induced nitric oxide and cytokine (IL-12, TNF, and IL-6) production by infected cells, without host cell toxicity. Moreover, Su6-B toxin, a subfraction of F6, demonstrated anti-T. gondii activity. The partially elucidated and characterized amino acid sequence of Sub6-B demonstrated 93% similarity with T. serrulatus 2 toxin (Ts2). Ts2 mimetic peptides ("Pep1," "Pep2a," and "Pep2b") were designed and synthesized. Pep1 and Pep2a, but not Pep2b, reduced the replication of tachyzoites in macrophages. In vivo, treatment of T. gondii-infected mice with Pep1, Pep2a, or Pep2b decreased the number of cerebral cysts and did not induce hepatotoxicity in the animals. Taken together, our data show promising immunomodulatory and antiparasitic activity of TsV that could be explored and applied in future therapies for treating infectious parasitic diseases such as toxoplasmosis.

Toads prey upon scorpions and are resistant to their venom: A biological and ecological approach to scorpionism.

In recent years, SE Brazil, the most populous region in the country with an estimated population of 88 million, has been experiencing an alarming increase in scorpions accidents (scorpionism), mainly caused by the yellow scorpion (Tityus serrulatus), or "escorpião amarelo" in Portuguese. This species is considered particularly dangerous to humans and can reproduce by parthenogenesis favouring rapid dispersal and colonization of new environments. Since the 1940s, owing to the growing danger represented by scorpionism, public control policies have been developed, including active search for scorpions, together with the use of toxic substances applied in places most likely to serve as their refuges. Even so, the number of accidents is increasing year by year, presently at an alarming rate. It seems evident that the increase in accidents is directly (or primarily) related to the lack of predators that in healthy environmental conditions would naturally control scorpion populations. However, due to environmental changes, leading to a lack of predators, scorpions have been gradually invading the urban environment. Arachnids and insects in general, as well as some other invertebrates, are preyed upon by anuran amphibians (toads, frogs and tree frogs). Toads (family Bufonidae) are nocturnal, large, and highly voracious animals, capable of actively exploring extensive areas and consuming large numbers of insects and arachnids daily. One of the most common toad species in southeastern Brazil is Rhinella icterica. Both R. icterica and T. serrulatus inhabit the same nocturnal environment. The predatory action of toads, specifically on scorpions, is practically unknown from behavioural and toxinological points of view. Thus, we studied the predatory behaviour of this toad against the yellow scorpion and evaluated the resistance of the amphibian to scorpion venom. Our results show that R. icterica is a voracious predator of T. serrulatus and is extremely resistant to its venom. Human/toad relationship throughout western history has always been very conflicted and possibly one of the factors that most has contributed to human ignorance of the role of these amphibians in maintaining ecological balance. Presently, the control of scorpionism is being performed through active search and/or the use of chemical agents, although showing little efficacy in reducing human accidents. In the medium or long term, more effective actions taking into account the biology of scorpions and their predators have never been taken to reduce these accidents.

Self-Assembled Cationic-Covered Nanoemulsion as A Novel Biocompatible Immunoadjuvant for Antiserum Production Against Tityus serrulatus Scorpion Venom.

This study assesses the efficacy of different nanoemulsion formulations as new and innovative adjuvants for improving the in vivo immunization against the Tityus serrulatus scorpion venom. Nanoemulsions were designed testing key-variables such as surfactants, co-solvents, and the influence of the temperature, which would be able to induce the phase transition from a liquid crystal to a stable nanoemulsion, assessed for four months. Additionally, cationic-covered nanoemulsion with hyper-branched poly(ethyleneimine) was prepared and its performance was compared to the non-cationic ones. The physicochemical properties of the selected nanoemulsions and the interactions among their involved formulation compounds were carefully monitored. The cytotoxicity studies in murine macrophages (RAW 264.7) and red blood cells were used to compare different formulations. Moreover, the performance of the nanoemulsion systems as biocompatible adjuvants was evaluated using mice immunization protocol. The FTIR shifts and the zeta potential changes (from -18.3 ± 1.0 to + 8.4 ± 1.4) corroborated with the expected supramolecular anchoring of venom proteins on the surface of the nanoemulsion droplets. Cell culture assays demonstrated the non-toxicity of the formulations at concentrations less than 1.0 mg/mL, which were able to inhibit the hemolytic effect of the scorpion venom. The cationic-covered nanoemulsion has shown superior adjuvant activity, revealing the highest IgG titer in the immunized animals compared to both the non-cationic counterpart and the traditional aluminum adjuvant. In this approach, we demonstrate the incredible potential application of nanoemulsions as adjuvants, using a nanotechnology platform for antigen delivery system on immune cells. Additionally, the functionalization with hyper-branched poly(ethyleneimine) enhances this recognition and improves its action in immunization.

Effect of Tityus serrulatus scorpion venom on isolated jejunum: A very useful tool to study the interaction between neurons in the enteric nervous system.

Scorpion envenomation is a public health problem in tropical and subtropical areas. In Brazil, Tityus serrulatus is the biggest cause of accidents with venomous animals. Tityus serrulatus venom causes symptoms related to a great activation of the autonomic system attributed to a massive release of sympathetic and parasympathetic mediators. This effect is attributed to the presence of toxins acting in Na+ and K+ ion channels, leading to an increase in cell excitability. Although gastrointestinal symptoms, like diarrhoea and sialorrhea, is observed in moderate to severe cases, little attention is given in clinical reports. Gastrointestinal motility is controlled by the enteric nervous system which is composed of a wide variety of interconnected neurons that are influenced by the sympathetic and parasympathetic nervous systems. Thus, this work aimed to characterize the effects of Tityus serrulatus venom on sympathetic and parasympathetic neurotransmission of rat jejunum, as well as to investigate possibles effects on other neurons of the enteric nervous system. To this, we verify the effects of Tityus serrulatus venom on the contractility of isolated rat jejunum through organ-bath experiments. We observed that venom can induce both contraction and relaxation. The contraction was partially inhibited by atropine (1 µM) and by suramin (0.1 mM) through tetrodotoxin-resistant and sensitive mechanisms. The relaxation was completely inhibited by 3 µM propranolol and partially inhibited by 1 µM phentolamine. Suramin induced a slowing of relaxation curve. Tetrodotoxin completely inhibits the relaxation induced by Tityus serrulatus venom, but the contraction curves were only partially reduced in their initial portion. The final part of the curve was largely enhanced by Tetrodotoxin. Atropine blocks almost completely the contraction curve in the presence of Tetrodotoxin. These results indicate that Tityus serrulatus venom induces the release of both excitatory (predominantly acetylcholine) and inhibitory (mainly noradrenaline) neurotransmitters. The effects of Tityus serrulatus venom on organ contractility was quite complex and seem to derive from a diffuse and nonspecific release of mediators from autonomic and enteric nervous systems. Further investigation of venom action and its isolated toxins can reveal important aspects to deepen our knowledge about the enteric nervous system transmission and the interaction between excitatory and inhibitory mediators as well as the physiological role of Na+ and K+ ion channels in gut motility.

Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the Tityus serrulatus Venom.

Although omics studies have indicated presence of proteases on the Tityus serrulatus venom (TsV), little is known about the function of these molecules. The TsV contains metalloproteases that cleave a series of human neuropeptides, including the dynorphin A (1-13) and the members of neuropeptide Y family. Aiming to isolate the proteases responsible for this activity, the metalloserrulase 3 and 4 (TsMS 3 and TsMS 4) were purified after two chromatographic steps and identified by mass spectrometry analysis. The biochemical parameters (pH, temperature and cation effects) were determined for both proteases, and the catalytic parameters (Km, kcat, cleavage sites) of TsMS 4 over fluorescent substrate were obtained. The metalloserrulases have a high preference for cleaving neuropeptides but presented different primary specificities. For example, the Leu-enkephalin released from dynorphin A (1-13) hydrolysis was exclusively performed by TsMS 3. Neutralization assays using Butantan Institute antivenoms show that both metalloserrulases were well blocked. Although TsMS 3 and TsMS 4 were previously described through cDNA library studies using the venom gland, this is the first time that both these toxins were purified. Thus, this study represents a step further in understanding the mechanism of scorpion venom metalloproteases, which may act as possible neuropeptidases in the envenomation process.