ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming.

Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom-polyclonal antibodies isolated from the plasma of hyperimmunised animals-which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability.

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.

Insights into structure and function of CdcVEGFs, the vascular endothelial growth factor from Crotalus durissus collilineatus snake venom.

Vascular endothelial growth factors (VEGFs) are crucial molecules involved in the modulation of angiogenesis. Snake venom-derived VEGFs (svVEGFs) are known to contribute significantly to the envenoming due to their capacity of increasing vascular permeability. In our work, we isolated and analyzed the biochemical and functional properties of the VEGF from Crotalus durissus collilineatus venom (CdcVEGF). The venom was fractionated by reversed phase chromatography on FPLC system (Fast Protein Liquid Chromatography) and the eluted fractions were submitted to an ELISA assay using an anti-VEGF-F antibody, for identification of svVEGF. Positive fractions for svVEGF were submitted to SDS-PAGE and to an anion exchange chromatography to isolate the molecule. The subfractions were analyzed by ELISA and SDS-PAGE and six of them presented svVEGFs, named CdcVEGF1 (Q23-3), CdcVEGF2 (Q24-3), CdcVEGF3 (Q24-4), CdcVEGF4 (Q25-3), CdcVEGF5 (Q25-4), and CdcVEGF6 (Q25-5). Their structural characterization was accomplished by mass spectrometry analysis using MALDI-TOF to determine their molecular masses and UPLC-ESI-QTOF to determine their amino acid sequence. Interestingly, all isolated CdcVEGFs induced angiogenesis on HUVEC cells through tube formation on Matrigel when compared to culture medium (negative control). Moreover, CdcVEGF2 and CdcVEGF3 also induced a significant increase in tube formation when compared to the positive control (basic fibroblast growth factor - bFGF). Additionally, crotalid antivenom produced by the Instituto Butantan was able to recognize CdcVEGFs, demonstrating to be immunogenic. This study demonstrates that snake venom cocktail can reveal novel and important molecules, which are potential molecular tools to study diverse biological processes, such as angiogenesis.

Bothrops leucurus snake venom protein profile, isolation and biological characterization of its major toxin PLA2s-like.

Bothrops leucurus is considered as a snake of medical interest in the State of Bahia, Brazil. However, so far, there are no studies that provide a refined mapping of the composition of this venom. The aim of this work was to better understand the protein composition of B. leucurus snake venom and to isolate and biologically characterize the most abundant toxin, a basic PLA2-like. Shotgun proteomics approach identified 137 protein hits in B. leucurus venom subdivided into 19 protein families. The new basic PLA2-like toxin identified was denominated Bleu-PLA2-like, it and other proteoforms represents about 25% of the total proteins in the venom of B. leucurus and induces myotoxicity, inflammation and muscle damage. Immunoreactivity assays demonstrated that B. leucurus venom is moderately recognized by bothropic and crotalic antivenoms, and on the other hand, Bleu-PLA2-like and its proteoforms are poorly recognized. Our findings open doors for future studies in order to assess the systemic effects caused by this snake venom in order to better understand the toxinological implications of this envenomation and, consequently, to assist in the clinical treatment of victims.

Towards toxin PEGylation: The example of rCollinein-1, a snake venom thrombin-like enzyme, as a PEGylated biopharmaceutical prototype.

PEGylation was firstly described around 50 years ago and has been used for more than 30 years as a strategy to improve the drugability of biopharmaceuticals. However, it remains poorly employed in toxinology, even though it may be a promising strategy to empower these compounds in therapeutics. This work reports the PEGylation of rCollinein-1, a recombinant snake venom serine protease (SVSP), able to degrade fibrinogen and inhibit the hEAG1 potassium channel. We compared the functional, structural, and immunogenic properties of the non-PEGylated (rCollinein-1) and PEGylated (PEG-rCollinein-1) forms. PEG-rCollinein-1 shares similar kinetic parameters with rCollinein-1, maintaining its capability of degrading fibrinogen, but with reduced activity on hEAG1 channel. CD analysis revealed the maintenance of protein conformation after PEGylation, and thermal shift assays demonstrated similar thermostability. Both forms of the enzyme showed to be non-toxic to peripheral blood mononuclear cells (PBMC). In silico epitope prediction indicated three putative immunogenic peptides. However, immune response on mice showed PEG-rCollinein-1 was devoid of immunogenicity. PEGylation directed rCollinein-1 activity towards hemostasis control, broadening its possibilities to be employed as a defibrinogenant agent.

Unraveling the structure and function of CdcPDE: A novel phosphodiesterase from Crotalus durissus collilineatus snake venom.

This study reports the isolation, structural, biochemical, and functional characterization of a novel phosphodiesterase from Crotalus durissus collilineatus venom (CdcPDE). CdcPDE was successfully isolated from whole venom using three chromatographic steps and represented 0.7% of total protein content. CdcPDE was inhibited by EDTA and reducing agents, demonstrating that metal ions and disulfide bonds are necessary for its enzymatic activity. The highest enzymatic activity was observed at pH 8-8.5 and 37 °C. Kinetic parameters indicated a higher affinity for the substrate bis(p-nitrophenyl) phosphate compared to others snake venom PDEs. Its structural characterization was done by the determination of the protein primary sequence by Edman degradation and mass spectrometry, and completed by the building of molecular and docking-based models. Functional in vitro assays showed that CdcPDE is capable of inhibiting platelet aggregation induced by adenosine diphosphate in a dose-dependent manner and demonstrated that CdcPDE is cytotoxic to human keratinocytes. CdcPDE was recognized by the crotalid antivenom produced by the Instituto Butantan. These findings demonstrate that the study of snake venom toxins can reveal new molecules that may be relevant in cases of snakebite envenoming, and that can be used as molecular tools to study pathophysiological processes due to their specific biological activities.

Characterization of class II fumarase from Schistosoma mansoni provides the molecular basis for selective inhibition.

Schistosomiasis is a neglected tropical disease that affects more than 250 million people worldwide. The only drug available for its treatment undergoes first-pass hepatic metabolism and is not capable of preventing reinfection, which makes the search of new therapies urgently needed. Due to the essential role of fumarases in metabolism, these enzymes represent potential targets for developing novel schistosomiasis treatments. Here, we evaluate the expression profiles for class I and class II fumarases from Schistosoma mansoni (SmFHI and SmFHII, respectively), and report the complete characterization of SmFHII. The first SmFHII structure in complex with L-malate was determined at 1.85 Å resolution. The significant thermoshift observed for SmFHII in the presence of identified ligands makes the differential scanning fluorimetry an adequate technique for ligand screening. A complete kinetic characterization of SmFHII was performed, and comparison with the human fumarase (HsFH) revealed differences regarding the turnover number (kcat). Structural characterization allowed us to identify differences between SmFHII and HsFH that could be explored to design new selective inhibitors. This work represents the very first step towards validate the fumarases as drug targets to treat schistosomiasis. Our results provide the structural basis to rational search for selective ligands.

From Animal Poisons and Venoms to Medicines: Achievements, Challenges and Perspectives in Drug Discovery.

Animal poisons and venoms are comprised of different classes of molecules displaying wide-ranging pharmacological activities. This review aims to provide an in-depth view of toxin-based compounds from terrestrial and marine organisms used as diagnostic tools, experimental molecules to validate postulated therapeutic targets, drug libraries, prototypes for the design of drugs, cosmeceuticals, and therapeutic agents. However, making these molecules applicable requires extensive preclinical trials, with some applications also demanding clinical trials, in order to validate their molecular target, mechanism of action, effective dose, potential adverse effects, as well as other fundamental parameters. Here we go through the pitfalls for a toxin-based potential therapeutic drug to become eligible for clinical trials and marketing. The manuscript also presents an overview of the current picture for several molecules from different animal venoms and poisons (such as those from amphibians, cone snails, hymenopterans, scorpions, sea anemones, snakes, spiders, tetraodontiformes, bats, and shrews) that have been used in clinical trials. Advances and perspectives on the therapeutic potential of molecules from other underexploited animals, such as caterpillars and ticks, are also reported. The challenges faced during the lengthy and costly preclinical and clinical studies and how to overcome these hindrances are also discussed for that drug candidates going to the bedside. It covers most of the drugs developed using toxins, the molecules that have failed and those that are currently in clinical trials. The article presents a detailed overview of toxins that have been used as therapeutic agents, including their discovery, formulation, dosage, indications, main adverse effects, and pregnancy and breastfeeding prescription warnings. Toxins in diagnosis, as well as cosmeceuticals and atypical therapies (bee venom and leech therapies) are also reported. The level of cumulative and detailed information provided in this review may help pharmacists, physicians, biotechnologists, pharmacologists, and scientists interested in toxinology, drug discovery, and development of toxin-based products.

Global proteomic and functional analysis of Crotalus durissus collilineatus individual venom variation and its impact on envenoming.

Individual variations studies are important to understand the snakebite envenoming and to improve the antivenom production and its effectiveness. In this way, the objective of this study was a comparative analysis of intraspecific variation in the venom composition of 22 Crotalus durissus collilineatus specimens through proteomic techniques. Venoms were fractionated by RP-FPLC, and analyzed by SDS-PAGE and mass spectrometry. Although similar, chromatographic and electrophoretic profiles showed significant qualitative and quantitative differences. Some venom components were identified for the very first time in C. d. collilineatus, such as glutathione peroxidase, nerve growth factor, 5'-nucleotidase, angiotensin-converting enzyme, carboxypeptidase, phosphodiesterase, glutaminyl cyclase and phospholipase B. Regarding hyaluronidase activity, 2 venoms did not present detectable enzyme activity in the tested amounts. Additionally, in vivo crotalic envenoming in mice showed that venoms from different specimens resulted in diversified changes of biochemical and immunological parameters, such as creatine kinase and interleukin 6. This study demonstrated significant intraspecific variations in the venom of C. d. collilineatus, which may impact the production and effectiveness of the antivenom therapy. BIOLOGICAL SIGNIFICANCE: This study performed the proteomic and functional analyzes of 22 C. d. collilineatus individual venoms and verified the occurrence of quali and quantitative variations among them. The venoms evaluated caused envenomings with different changes in biochemical and immunological parameters. These results confirm the need to use a pool of venoms with the greatest possible variability in the preparation of antivenoms, in order to improve their effectiveness. In addition, this study was able to identify for the first time 8 different proteins in this subspecies venom, increasing knowledge about its composition and showing that it is a source of these proteins with possible biotechnological applications.

Cell migration inhibition activity of a non-RGD disintegrin from Crotalus durissus collilineatus venom.

BACKGROUND: In recent decades, snake venom disintegrins have received special attention due to their potential use in anticancer therapy. Disintegrins are small and cysteine-rich proteins present in snake venoms and can interact with specific integrins to inhibit their activities in cell-cell and cell-ECM interactions. These molecules, known to inhibit platelet aggregation, are also capable of interacting with certain cancer-related integrins, and may interfere in important processes involved in carcinogenesis. Therefore, disintegrin from Crotalus durissus collilineatus venom was isolated, structurally characterized and evaluated for its toxicity and ability to interfere with cell proliferation and migration in MDA-MB-231, a human breast cancer cell line. METHODS: Based on previous studies, disintegrin was isolated by FPLC, through two chromatographic steps, both on reversed phase C-18 columns. The isolated disintegrin was structurally characterized by Tris-Tricine-SDS-PAGE, mass spectrometry and N-terminal sequencing. For the functional assays, MTT and wound-healing assays were performed in order to investigate cytotoxicity and effect on cell migration in vitro, respectively. RESULTS: Disintegrin presented a molecular mass of 7287.4 Da and its amino acid sequence shared similarity with the disintegrin domain of P-II metalloproteases. Using functional assays, the disintegrin showed low cytotoxicity (15% and 17%, at 3 and 6 µg/mL, respectively) after 24 h of incubation and in the wound-healing assay, the disintegrin (3 µg/mL) was able to significantly inhibit cell migration (24%, p < 0.05), compared to negative control. CONCLUSION: Thus, our results demonstrate that non-RGD disintegrin from C. d. collilineatus induces low cytotoxicity and inhibits migration of human breast cancer cells. Therefore, it may be a very useful molecular tool for understanding ECM-cell interaction cancer-related mechanisms involved in an important integrin family that highlights molecular aspects of tumorigenesis. Also, non-RGD disintegrin has potential to serve as an agent in anticancer therapy or adjuvant component combined with other anticancer drugs.

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.

Cell migration inhibition activity of a non-RGD disintegrin from Crotalus durissus collilineatus venom

In recent decades, snake venom disintegrins have received special attention due to their potential use in anticancer therapy. Disintegrins are small and cysteine-rich proteins present in snake venoms and can interact with specific integrins to inhibit their activities in cell-cell and cell-ECM interactions. These molecules, known to inhibit platelet aggregation, are also capable of interacting with certain cancer-related integrins, and may interfere in important processes involved in carcinogenesis. Therefore, disintegrin from Crotalus durissus collilineatus venom was isolated, structurally characterized and evaluated for its toxicity and ability to interfere with cell proliferation and migration in MDA-MB-231, a human breast cancer cell line. Methods: Based on previous studies, disintegrin was isolated by FPLC, through two chromatographic steps, both on reversed phase C-18 columns. The isolated disintegrin was structurally characterized by Tris-TricineSDS-PAGE, mass spectrometry and N-terminal sequencing. For the functional assays, MTT and wound-healing assays were performed in order to investigate cytotoxicity and effect on cell migration in vitro, respectively. Results: Disintegrin presented a molecular mass of 7287.4 Da and its amino acid sequence shared similarity with the disintegrin domain of P-II metalloproteases. Using functional assays, the disintegrin showed low cytotoxicity (15% and 17%, at 3 and 6 µg/mL, respectively) after 24 h of incubation and in the wound-healing assay, the disintegrin (3 µg/mL) was able to significantly inhibit cell migration (24%, p < 0.05), compared to negative control. Conclusion: Thus, our results demonstrate that non-RGD disintegrin from C. d. collilineatus induces low cytotoxicity and inhibits migration of human breast cancer cells. Therefore, it may be a very useful molecular tool for understanding ECM-cell interaction cancer-related mechanisms involved in an important integrin family that highlights molecular aspects of tumorigenesis. Also, non-RGD disintegrin has potential to serve as an agent in anticancer therapy or adjuvant component combined with other anticancer drugs.(AU)

Minor snake venom proteins: Structure, function and potential applications.

Snake venoms present a great diversity of pharmacologically active compounds that may be applied as research and biotechnological tools, as well as in drug development and diagnostic tests for certain diseases. The most abundant toxins have been extensively studied in the last decades and some of them have already been used for different purposes. Nevertheless, most of the minor snake venom protein classes remain poorly explored, even presenting potential application in diverse areas. The main difficulty in studying these proteins lies on the impossibility of obtaining sufficient amounts of them for a comprehensive investigation. The advent of more sensitive techniques in the last few years allowed the discovery of new venom components and the in-depth study of some already known minor proteins. This review summarizes information regarding some structural and functional aspects of low abundant snake venom proteins classes, such as growth factors, hyaluronidases, cysteine-rich secretory proteins, nucleases and nucleotidases, cobra venom factors, vespryns, protease inhibitors, antimicrobial peptides, among others. Some potential applications of these molecules are discussed herein in order to encourage researchers to explore the full venom repertoire and to discover new molecules or applications for the already known venom components.

Tityus serrulatus envenoming in non-obese diabetic mice: a risk factor for severity.

BACKGROUND: In Brazil, accidents with venomous animals are considered a public health problem. Tityus serrulatus (Ts), popularly known as the yellow scorpion, is most frequently responsible for the severe accidents in the country. Ts envenoming can cause several signs and symptoms classified according to their clinical manifestations as mild, moderate or severe. Furthermore, the victims usually present biochemical alterations, including hyperglycemia. Nevertheless, Ts envenoming and its induced hyperglycemia were never studied or documented in a patient with diabetes mellitus (DM). Therefore, this is the first study to evaluate the glycemia during Ts envenoming using a diabetic animal model (NOD, non-obese diabetic). METHODS: Female mice (BALB/c or NOD) were challenged with a non-lethal dose of Ts venom. Blood glucose level was measured (tail blood using a glucose meter) over a 24-h period. The total glycosylated hemoglobin (HbA1c) levels were measured 30 days after Ts venom injection. Moreover, the insulin levels were analyzed at the glycemia peak. RESULTS: The results demonstrated that the envenomed NOD animals presented a significant increase of glycemia, glycosylated hemoglobin (HbA1c) and insulin levels compared to the envenomed BALB/c control group, corroborating that DM victims present great risk of developing severe envenoming. Moreover, the envenomed NOD animals presented highest risk of death and sequelae. CONCLUSIONS: This study demonstrated that the diabetic victims stung by Ts scorpion should be always considered a risk group for scorpion envenoming severity.

Experimental Lachesis muta rhombeata envenomation and effects of soursop (Annona muricata) as natural antivenom.

BACKGROUND: In the Atlantic forest of the North and Northeast regions of Brazil, local population often uses the fruit juice and the aqueous extract of leaves of soursop (Annona muricata L.) to treat Lachesis muta rhombeata envenomation. Envenomation is a relevant health issue in these areas, especially due to its severity and because the production and distribution of antivenom is limited in these regions. The aim of the present study was to evaluate the relevance of the use of soursop leaf extract and its juice against envenomation by Lachesis muta rhombeata. METHODS: We evaluated the biochemical, hematological and hemostatic parameters, the blood pressure, the inflammation process and the lethality induced by Lachesis muta rhombeata snake venom. We also assessed the action of the aqueous extract of leaves (AmL) and juice (AmJ) from A. muricata on the animal organism injected with L. m. rhombeata venom (LmrV) in the laboratory environment. RESULTS: LmrV induced a decrease of total protein, albumin and glucose; and increase of creatine kinase, aspartate aminotransferase, and urea concentrations. It provoked hemoconcentration followed by reduction of hematocrit, an increase in prothrombin time and partial thromboplastin time and a decrease of the blood pressure. LmrV induced the release of interleukin-6, an increase in neutrophils and changes in the serum protein profile, characteristic of the acute inflammatory process. LD50 values were similar for the groups injected with LmrV and treated or untreated with AmJ and AmL. Both treatments play a role on the maintenance of blood glucose, urea and coagulation parameters and exert a protective action against the myotoxicity. However, they seem to worsen the hypotension caused by LmrV. CONCLUSION: The treatments with AmJ and AmL present some beneficial actions, but they might intensify some effects of the venom. Therefore, additional studies on A. muricata are necessary to enable its use as natural antivenom for bushmaster snakebite.

Tityus serrulatus envenoming in non-obese diabetic mice: a risk factor for severity

Background In Brazil, accidents with venomous animals are considered a public health problem. Tityus serrulatus (Ts), popularly known as the yellow scorpion, is most frequently responsible for the severe accidents in the country. Ts envenoming can cause several signs and symptoms classified according to their clinical manifestations as mild, moderate or severe. Furthermore, the victims usually present biochemical alterations, including hyperglycemia. Nevertheless, Ts envenoming and its induced hyperglycemia were never studied or documented in a patient with diabetes mellitus (DM). Therefore, this is the first study to evaluate the glycemia during Ts envenoming using a diabetic animal model (NOD, non-obese diabetic). Methods Female mice (BALB/c or NOD) were challenged with a non-lethal dose of Ts venom. Blood glucose level was measured (tail blood using a glucose meter) over a 24-h period. The total glycosylated hemoglobin (HbA1c) levels were measured 30 days after Ts venom injection. Moreover, the insulin levels were analyzed at the glycemia peak. Results The results demonstrated that the envenomed NOD animals presented a significant increase of glycemia, glycosylated hemoglobin (HbA1c) and insulin levels compared to the envenomed BALB/c control group, corroborating that DM victims present great risk of developing severe envenoming. Moreover, the envenomed NOD animals presented highest risk of death and sequelae. Conclusions This study demonstrated that the diabetic victims stung by Ts scorpion should be always considered a risk group for scorpion envenoming severity.(AU)

Tityus serrulatus envenoming in non-obese diabetic mice: a risk factor for severity

In Brazil, accidents with venomous animals are considered a public health problem. Tityus serrulatus (Ts), popularly known as the yellow scorpion, is most frequently responsible for the severe accidents in the country. Ts envenoming can cause several signs and symptoms classified according to their clinical manifestations as mild, moderate or severe. Furthermore, the victims usually present biochemical alterations, including hyperglycemia. Nevertheless, Ts envenoming and its induced hyperglycemia were never studied or documented in a patient with diabetes mellitus (DM). Therefore, this is the first study to evaluate the glycemia during Ts envenoming using a diabetic animal model (NOD, non-obese diabetic). Methods Female mice (BALB/c or NOD) were challenged with a non-lethal dose of Ts venom. Blood glucose level was measured (tail blood using a glucose meter) over a 24-h period. The total glycosylated hemoglobin (HbA1c) levels were measured 30 days after Ts venom injection. Moreover, the insulin levels were analyzed at the glycemia peak. Results The results demonstrated that the envenomed NOD animals presented a significant increase of glycemia, glycosylated hemoglobin (HbA1c) and insulin levels compared to the envenomed BALB/c control group, corroborating that DM victims present great risk of developing severe envenoming. Moreover, the envenomed NOD animals presented highest risk of death and sequelae. Conclusions This study demonstrated that the diabetic victims stung by Ts scorpion should be always considered a risk group for scorpion envenoming severity.

Influence of post-starvation extraction time and prey-specific diet in Tityus serrulatus scorpion venom composition and hyaluronidase activity.

The role of diet in venom composition has been a topic of intense research interest. This work presents evidence that the variation in the venom composition from the scorpion Tityus serrulatus (Ts) is closely associated with post-starvation extraction time and prey-specific diet. The scorpions were fed with cockroach, cricket, peanut beetle or giant Tenebrio. The venoms demonstrated a pronounced difference in the total protein and toxins composition, which was evaluated by electrophoresis, reversed-phase chromatography, densitometry, hyaluronidase activity and N-terminal sequencing. Indeed, many toxins and peptides, such as Ts1, Ts2, Ts4, Ts5, Ts6, Ts15, Ts19 frag. II, hypotensins 1 and 3, PAPE peptide and peptide 9797 (first described in Ts venom), were all identified in different proportions in the analyzed Ts venoms. This study is pioneer on assessing the influence of the starvation time and the prey diet on hyaluronidase activity as well as to describe a modification of Tricine-gel-electrophoresis to evaluate this enzyme activity. Altogether, this study reveal a large contribution of the extraction time and diet on Ts venom variability as well as present a background to recommend the cockroach diet to obtain higher protein content and the cricket diet to obtain higher hyaluronidase specific activity.