Abstract Background: This study examines the impact of Phα1β, a spider peptide derived from the venom of Phoneutria nigriventer, on the Kv11.1 potassium channel in HEK293 cells transfected with the human ERG potassium channel. Phα1β inhibits high-voltage calcium channels and acts as an antagonist of the TRPA1 receptor, both of which play crucial roles in pain transduction pathways. Over the past 15 years, our research has demonstrated the potential of Phα1β, in both its native and recombinant forms, as a promising analgesic drug through preclinical tests conducted on rodent pain models. Regulatory agencies require the evaluation of new drugs on human ERG channels. Methods: To assess hERG potassium channel inhibition, we utilized the FLIPR® Potassium Assay, a commercially available kit. The assay involved testing the effects of Phα1β alongside the well-established hERG potassium channel blocker dofetilide, which served as a positive control. The viability of HEK-293 cells was assessed using the colorimetric MTT reduction test (3-(4, dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), whereby viable cells reduce the MTT salt, forming a formazan complex within their mitochondria, as previously described. Results: Phα1β was tested at concentrations of 56, 225, 450, and 900 pMol, resulting in a discreet inhibition of hERG potassium channel activity at higher concentrations, approximately 13.47%, with an IC50 value exceeding 900 pMol. Dofetilide, administered at concentrations ranging from 0.0001 to 10 µM, displayed a concentration-dependent inhibition of the hERG potassium channel, with a mean IC50 value of 0.1642 µM (0.1189-0.2282 µM). To evaluate cytotoxicity, HEK293-hERG cells were exposed to Phα1β concentrations of 56/900 pMol for 24 hours, resulting in no significant alteration in cell viability. Conclusion: Our findings indicate that even at high concentrations, Phα1β does not impede the functionality of the hERG potassium channel nor affect cell viability.

Abstract Background: Non-small cell lung cancers (NSCLC) represent the primary cause of cancer-related deaths worldwide. Rhopalurus junceus venom has been shown to exert cytotoxic effects against a panel of epithelial cancer cells in vitro and suggested that NSCLC was the subtype most susceptible to the treatment. Methods: This study evaluated the effect of Rhopalurus junceus scorpion venom on cell viability, in non-cancerous (MRC-5, lung; CHO-K1, ovary) and NSCLC (A549; NCI-H460) cell lines. The effects on cell cycle, apoptosis, and cell signaling-related proteins were determined by flow cytometry and WB. Protein fractions responsible for the observed effect were identified using HPLC. Results: Scorpion venom was more effective against NSCLC than non-cancerous cells. Emax values were 20.0 ± 5.8% and 22.47 ± 6.02% in A549 and NCI-H460 cancer cells, respectively, as compared to 50 ± 8.1% in MRC-5 and 54.99 ± 7.39% in CHO-K1 cells. It arrested NSCLC cells in the G2/M phase, while non-cancerous cells were arrested in the S (MRC-5) or G0/G1 (CHO-K1) phases. No changes were observed in the Bax/Bcl-2 or the cleaved-caspase 3/Total caspase 3 ratios in cells treated with venom. Likewise, the scorpion venom treatment did not affect p-ERK, p-AKT, or p-38MAPK protein levels. In contrast, scorpion venom treatment increased the cytosolic apoptosis-inducing factor (AIF) in A549 cells, indicating caspase-independent apoptosis. Additionally, combined etoposide/venom exposure provoked G2/M arrest and apoptosis in NSCLC more strongly than either substance alone. Furthermore, upon crude venom fractioning through RP-HPLC, we found two soluble fractions with high cytotoxic effects. Conclusion: The present study concludes that a specific fraction of Rhopalurus junceus venom reduces cell viability of NSCLC cells. The AIF protein plays a key role in mediating caspase-independent apoptotic cell death. These findings suggest that Rhopalurus junceus venom enhances the anticancer effect of etoposide in vitro by causing cell cycle arrest and caspase-independent apoptosis.

Abstract Background: Fish venoms have been poorly characterized and the available information about their composition suggests they are uncomplicated secretions that, combined with epidermal mucus, could induce an inflammatory reaction, excruciating pain, and, in some cases, local tissue injuries. Methods: In this study, we characterized the 24-hour histopathological effects of lionfish venom in a mouse experimental model by testing the main fractions obtained by size exclusion-HPLC. By partial proteomics analysis, we also correlated these in vivo effects with the presence of some potentially toxic venom components. Results: We observed a strong lesion on the skin and evident necrosis in the skeletal muscle. None of the tissue-damaging effects were induced by the fraction containing cytolysins, membrane pore-forming toxins ubiquitously present in species of scorpionfish, stonefish, and lionfish, among others. On the contrary, injuries were associated with the presence of other components, which have remained practically ignored so far. This is the case of an abundant protein, present in venom, with homology to a Golgi-associated plant pathogenic protein 1-like (GAPR1), which belongs to the same protein superfamily as venom CRISPs and insect allergens. Conclusion: This GAPR1-like protein and the hyaluronidase are probably responsible for the hemostasis impairment and hemorrhagic lesions observed in mouse skin, whereas muscle injuries can be indirectly caused by a combination of inflammatory and hemorrhagic events. More information is required to establish the components accountable for the myonecrotic effect.

Abstract Background: Acid-sensing ion channel 1a (ASIC1a) plays a critical role in physiological and pathological processes. To further elucidate the biological functions of ASICs and their relationships with disease occurrence and development, it is advantageous to investigate and develop additional regulatory factors for ASICs. Methods: In this study, cation exchange chromatography was used to separate seven chromatographic components from Naja naja atra venom. Capillary electrophoresis was employed to detect that Ⅶ peak component containing a main protein Ⅶ-2, which could bind to ASIC1a. The analgesic effects of Ⅶ-2 protein were determined using hot plate methods, and ASIC1a expression in spinal cord tissue from rats with inflammatory pain was detected using western blot. Results: The purified Ⅶ-2 protein named Naja naja atra venom-Ⅶ-2 (NNAV-Ⅶ-2) was obtained by Sephadex G-50 gel filtration, which exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 6.7 kD. Remarkably, the NNAV-Ⅶ-2 protein demonstrated a significant analgesic effect and downregulated ASIC1a expression in the spinal cord tissue of rats with inflammatory pain. Conclusions: The analgesic mechanism of the NNAV-Ⅶ-2 protein may be associated with its binding to ASIC1a, consequently downregulating ASIC1a expression in neural tissues.

Abstract Background: Paracoccidioidomycosis (PCM) is a severe granulomatous disease. The hallmark of this mycosis is fibrin degradation and granuloma formation as a result of a wound-healing process in the context of excessive inflammation. Therefore, as the content of collagen can be assessed by the methodology described in this manuscript, we propose that the content of hydroxyproline (HYP) be employed as a new and efficient measurement of granulomatous lesions developed. To estimate the level of HYP the major byproduct of the degradation process, we hypothesized that this simple and efficient technique could serve as a marker of disease severity. Methods: Five B10.A female mice were infected with P. brasiliensis and, after 15 days, the omentum was removed, subjected to histopathological analysis or processed (i.e. deproteinized and derivatized), and further analyzed on a reverse phase HPLC using a C-18 column. The omentum of five uninfected controls was also collected and similarly analyzed. Results: Infected mice showed numerous, disseminated paracoccidioidomycotic lesions, as well as marked collagen deposits, as observed in histopathologic analysis, and high levels of HYP. Normal uninfected mice showed no granulomas, little or no deposits of collagen fibers, and very low levels of HYP, as evaluated by HPLC. Our results show that the disease intensity as evaluated number and the morphology of the granulomatous lesions were correlated to the HYP levels using small tissue samples from the omentum, the main target organ of P. brasiliensis. Conclusions: Here we propose an alternative methodology to follow disease evolution and, to some extent, fungal load in experimental P. brasiliensis infection and suggest its usefulness to other diseases with pronounced fibrin degradation.

Abstract Background: Sea anemones are well known to contain multiple peptide toxins. However, of more than 1100 species of sea anemones distributed worldwide, only a little over 50 have been studied for peptide toxins. Therefore, innumerable unique and novel peptide toxins remain to be discovered in unstudied sea anemones. Methods: Isolation of peptide toxins in the sea anemone Heteractis aurora was attempted by gel filtration and reverse-phase high performance liquid chromatography, using the toxicity to crabs as an index. The amino acid sequences of the isolated four toxins (Hau I-IV) and their precursors were determined using a combination of protein sequencing and cDNA cloning. Results: Hau I and IV were potently lethal to crabs, whereas Hau II and III were only paralytic. The precursor proteins of the four toxins were commonly composed of a signal peptide, a propart, and the remaining region including a mature peptide. Interestingly, four and two copies of the mature peptide were present in the precursor proteins of Hau II and III, respectively. Homology searches revealed that Hau I (30 amino acid residues) is a novel peptide toxin, although it has the same cysteine pattern CXXC-C-C as the boundless β-hairpin (BBH) family. Hau II (27 amino acid residues) and III (28 amino acid residues) were homologous with the BBH family, whereas Hau IV (49 amino acid residues) was a new member of the well-known type 1 sodium channel toxin family. Conclusion: This study showed that a novel class of toxin (Hau I), two BBH family toxins (Hau II and III), and a type 1 sodium channel toxin (Hau IV) are present in the toxin of the sea anemone H. aurora.

Abstract Background: Micrurus mipartitus is a coral snake of public health concern in Colombia. Its venom is mainly composed of three-finger toxins (3FTxs), Mipartoxin-1 being the most abundant protein partially responsible for its lethal effect. In this work, we present the production of Mipartoxin-1 in a recombinant form and evaluate its immunogenic potential. Methods: A genetic construct HisrMipartoxin-1 was cloned into the pET28a vector and heterologous expression was obtained in E. coli BL21 (DE3). The recombinant HisrMipartoxin-1 protein was extracted from inclusion bodies, refolded in vitro, and isolated by affinity and RP-HPLC chromatography. The lethal effect of HisrMipartoxin-1 was tested, and antibodies against HisrMipartoxin-1 were produced by immunization in rabbits. The antibody titers were monitored by an ELISA test. The neutralizing ability of the antibodies, against the lethal effect of native toxins and M. mipartitus venom, was also assessed. Results: HisrMipartoxin-1 was detected on SDS-PAGE, with a molecular mass of around 11 kDa. The retention time was 16.0 minutes. HisrMipartoxin-1 did not exhibit lethality in mice; however, antibodies against HisrMipartoxin-1 recognized the native toxin, the whole venom of M. mipartitus, and a 3FTx from another species within the Micrurus genus. Furthermore, antibodies against HisrMipartoxin-1 completely neutralized the lethal effect of native Mipartoxin-1 in mice but not M. mipartitus whole venom. Conclusion: These findings indicate that HisrMipartoxin-1 might be used as an immunogen to develop anticoral antivenoms or complement them. This work is the first report of the heterologous expression of 3FTx from M. mipartitus.

ABSTRACT Visceral leishmaniasis (VL) is a neglected disease that is typical of tropical and subtropical parts of the world and is caused by the trypanosomatid Leishmania donovani complex. This disease is a multifactorial condition that involves parasitic, environmental, and immunogenetic characteristics. Genetic changes in genes encoding cytokines may be associated with changes in their expression and, consequently, with the development of clinical resistance or susceptibility to the disease. This systematic review and meta-analysis aimed to assess whether single nucleotide polymorphisms (SNPs) in interleukin genes influence the clinical consequences of visceral leishmaniasis infection. To this end, we carried out a systematic review and meta-analysis with structured searches in the EMBASE, PubMed, Scopus, SciELO, and Web of Science databases without time restrictions. Two independent reviewers examined the studies, performed data extraction, and assessed quality by assigning scores. If there were any discrepancies, a third reviewer with more experience was consulted. After the screening process, 28 articles were included in the systematic review and 9 in the final analysis of the meta-analysis. Statistical analyses were carried out using various genetic models. The odds ratio (OR) and corresponding 95% confidence intervals (CIs) were calculated to estimate the associations. Overall, the main clinical outcomes were classified as not associated or associated when they presented susceptibility, resistance, risk, or protective factors for the development of the disease. Associations between IFN-γ +874T/A polymorphisms in the dominant model (OR 1.64, 95% CI 1.13-2.38, I2 = 0%, p < 0.01) and heterozygous model (OR 1.72, 95% CI 1.15-2.57, I2 = 0%, p < 0.01) and IL-18 -137G/C in the recessive model (OR 1.33, 95% CI 1.02-1.71, I2 = 9%, p = 0.03) and VL were observed. For the IL-10 gene SNPs, there was no significant association. Our findings suggest that SNPs in the IFN-γ and IL-18 genes may be associated with the risk of developing VL.

ABSTRACT Background: Pathophysiological mechanisms of rheumatoid arthritis arise because of a proinflammatory environment, generated by the interaction of autoreactive lymphocytes and proinflammatory mediators. Current strategies to mitigate the progression of the disease produce adverse effects, so there is a need for new therapeutic strategies and molecular targets to treat this disease. In this context, evidence suggests that scorpion venoms could modulate the immune response and some important cellular mechanisms of pharmacological interest. To evaluate the immunomodulatory effect of the venom of Tityus sp. (a possible new species close to Tityus metuendus) peripheral blood mononuclear cells of women diagnosed with RA were compared to cells of a control group. Methods: A case-control study was conducted with a sample of 10 women with a confirmed diagnosis of RA and controls matched by sex and age. The cytotoxicity of the venom was evaluated to find sublethal concentrations of the venom, and subsequently, their immunomodulatory capacity in terms of percentage of proliferation, cell activation, and cytokines production. Results: the venom of Tityus sp. produced a decrease in the percentage of proliferation in the CD3+, CD3+CD4+, and CD3+CD8+ cell subpopulations of RA patients and healthy controls, at concentrations of 252 and 126 µg/mL. However, the venom did not induce significant differences in the percentage of cell activation markers. The venom caused a decrease in IL-10 at a concentration of 252 µg/mL compared to untreated cells from patients and controls. The remaining cytokines did not show significant differences. Conclusion: the venom of Tityus sp. is a potential source of molecules with immunomodulatory ability in CD4 and CD8 T lymphocytes. This result directs venom characterization studies to identify pharmacological targets with immunomodulatory capacity in T lymphocytes to enhance research in the treatment of autoimmune disorders such as RA.

Abstract Background: Members of the genus Cupiennius Simon, 1891 are categorized as wandering spiders and are part of the family Trechaleidae. The genomics and proteomics of Cupiennius spiders from North America remain uncharacterized. The present study explores for the first time molecular data from the endemic species Cupiennius chiapanensis Medina, 2006, and also presents new data for Cupiennius salei (Keyserling, 1878), both collected in southern Mexico. Methods: In total, 88 Cupiennius specimens were collected from southern Mexico and morphologically identified. DNA was extracted and the mitochondrial COI fragment was amplified. COI sequences were analyzed, and a phylogenetic tree was inferred for species from the Americas. Genetic diversity was analyzed using haplotype networks and gene distances. Venom was obtained from C. chiapanensis and C. salei by electrostimulation. The venom was separated by HPLC, visualized using SDS-PAGE, and quantified for use in toxicity bioassays in mice and insects. Results: Analysis of COI sequences from C. chiapanensis showed 94% identity with C. salei, while C. salei exhibited 94-97% identity with sequences from Central and South American conspecifics. The venom from C. chiapanensis exhibited toxic activity against crickets. Venoms from C. chiapanensis and C. salei caused death in Anastrepha obliqua flies. Analysis of venom fractions from C. salei and C. chiapanensis revealed molecular masses of a similar size as some previously reported toxins and neurotoxic components. We determined the amino acid sequences of ChiaTx1 and ChiaTx2, toxins that are reported here for the first time and which showed toxicity against mice and insects. Conclusion: Our work is the first to report COI-based DNA barcoding sequences from southern Mexican Cupiennius spiders. Compounds with toxic activity were identified in venom from both species.

Abstract Background: Loxoscelism refers to a set of clinical manifestations caused by the bite of spiders from the Loxosceles genus. The classic clinical symptoms are characterized by an intense inflammatory reaction at the bite site followed by local necrosis and can be classified as cutaneous loxoscelism. This cutaneous form presents difficult healing, and the proposed treatments are not specific or effective. This study aimed to evaluate the protective effect of mesenchymal stromal cells-derived secretome on dermonecrosis induced by Loxosceles intermedia spider venom in rabbits. Methods: Sixteen rabbits were distributed into four groups (n = 4). Except for group 1 (G1), which received only PBS, the other three groups (G2, G3, and G4) were initially challenged with 10 μg of L. intermedia venom, diluted in 100 μL of NaCl 0.9%, by intradermic injection in the interscapular region. Thirty minutes after the challenge all groups were treated with secretome, except for group 2. Group 1 (G1-control group) received intradermal injection (ID) of 60 μg of secretome in 0.15 M PBS; Group 2 (G2) received 0.9% NaCl via ID; Group 3 (G3) received 60 μg of secretome, via ID and Group 4 (G4), received 60 μg of secretome by intravenous route. Rabbits were evaluated daily and after 15 days were euthanized, necropsied and skin samples around the necrotic lesions were collected for histological analysis. Results: Rabbits of G1 did not present edema, erythema, hemorrhagic halo, or necrosis. In animals from G2, G3, and G4, edema appeared after 6h. However, minor edema was observed in the animals of G2 and G3. Hemorrhagic halo was observed in animals, six hours and three days after, on G2, G3, and G4. Macroscopically, in G4, only one animal out of four had a lesion that evolved into a dermonecrotic wound. No changes were observed in the skin of the animals of G1, by microscopic evaluation. All animals challenged with L. intermedia venom showed similar alterations, such as necrosis and heterophilic infiltration. However, animals from G4 showed fibroblast activation, early development of connective tissue, neovascularization, and tissue re-epithelialization, indicating a more prominent healing process. Conclusion: These results suggest that secretome from mesenchymal stromal cells cultured in a xeno-free and human component-free culture media can be promising to treat dermonecrosis caused after Loxosceles spiders bite envenoming.

Abstract Glioblastoma (GB) is the most common type of malignant tumor of the central nervous system, responsible for significant morbidity and with a 5-year overall relative survival of only 6.8%. Without advances in treatment in the last twenty years, the standard of care continues to be maximum safe resection, Temozolomide (TMZ), and radiotherapy. Many new trials are ongoing, and despite showing increased progression-free survival, these trials did not improve overall survival. They did not consider the adverse effects of these therapies. Therefore, an increasing number of bioprospecting studies have used snake venom molecules to search for new strategies to attack GB selectively without producing side effects. The present review aims to describe GB characteristics and current and new approaches for treatment considering their side effects. Besides, we focused on the antitumoral activity of snake venom proteins from the Viperidae family against GB, exploring the potential for drug design based on in vitro and in vivo studies. This review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. In January 2024, a systematic search was performed in the PubMed, EMBASE, and Web of Science databases from January 2000 to December 2023. Search terms were selected based on the population/exposure/outcome (PEO) framework and combined using Boolean operators ("AND", "OR"). The search strategy used these terms: glioblastoma, glioma, high-grade glioma, WHO IV glioma, brain cancer, snake venom, Viperidae, and bioprospection. We identified 10 in vivo and in vitro studies with whole and isolated proteins from Viperidae venom that could have antitumor activity against glioblastoma. Studies in bioprospecting exploring the advantage of snake venom proteins against GB deserve to be investigated due to their high specificity, small size, inherent bioactivity, and few side effects to cross the blood-brain barrier (BBB) to reach the tumor microenvironment.

Abstract The effect of peptide toxins on voltage-gated ion channels can be reliably assessed using electrophysiological assays, such as the patch-clamp technique. However, much of the toxinological research done in Central and South America aims at purifying and characterizing biochemical properties of the toxins of vegetal or animal origin, lacking electrophysiological approaches. This may happen due to technical and infrastructure limitations or because researchers are unfamiliar with the techniques and cellular models that can be used to gain information about the effect of a molecule on ion channels. Given the potential interest of many research groups in the highly biodiverse region of Central and South America, we reviewed the most relevant conceptual and methodological developments required to implement the evaluation of the effect of peptide toxins on mammalian voltage-gated ion channels using patch-clamp. For that, we searched MEDLINE/PubMed and SciELO databases with different combinations of these descriptors: “electrophysiology”, “patch-clamp techniques”, “Ca2+ channels”, “K+ channels”, “cnidarian venoms”, “cone snail venoms”, “scorpion venoms”, “spider venoms”, “snake venoms”, “cardiac myocytes”, “dorsal root ganglia”, and summarized the literature as a scoping review. First, we present the basics and recent advances in mammalian voltage-gated ion channel’s structure and function and update the most important animal sources of channel-modulating toxins (e.g. cnidarian and cone snails, scorpions, spiders, and snakes), highlighting the properties of toxins electrophysiologically characterized in Central and South America. Finally, we describe the local experience in implementing the patch-clamp technique using two models of excitable cells, as well as the participation in characterizing new modulators of ion channels derived from the venom of a local spider, a toxins’ source less studied with electrophysiological techniques. Fostering the implementation of electrophysiological methods in more laboratories in the region will strengthen our capabilities in many fields, such as toxinology, toxicology, pharmacology, natural products, biophysics, biomedicine, and bioengineering.

Abstract 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 β), 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.

Abstract Background: The bioactive peptides derived from snake venoms of the Viperidae family species have been promising as therapeutic candidates for neuroprotection due to their ability to prevent neuronal cell loss, injury, and death. Therefore, this study aimed to evaluate the cytoprotective effects of a synthetic proline-rich oligopeptide 7a (PRO-7a; <EDGPIPP) from Bothrops jararaca snake, on oxidative stress-induced toxicity in neuronal PC12 cells and astrocyte-like C6 cells. Methods: Both cells were pre-treated for four hours with different concentrations of PRO-7a, submitted to H2O2-induced damage for 20 h, and then the oxidative stress markers were analyzed. Also, two independent neuroprotective mechanisms were investigated: a) L-arginine metabolite generation via argininosuccinate synthetase (AsS) activity regulation to produce agmatine or polyamines with neuroprotective properties; b) M1 mAChR receptor subtype activation pathway to reduce oxidative stress and neuron injury. Results: PRO-7a was not cytoprotective in C6 cells, but potentiated the H2O2-induced damage to cell integrity at a concentration lower than 0.38 μM. However, PRO-7a at 1.56 µM, on the other hand, modified H2O2-induced toxicity in PC12 cells by restoring cell integrity, mitochondrial metabolism, ROS generation, and arginase indirect activity. The α-Methyl-DL-aspartic acid (MDLA) and L-NΩ-Nitroarginine methyl ester (L-Name), specific inhibitors of AsS and nitric oxide synthase (NOS), which catalyzes the synthesis of polyamines and NO from L-arginine, did not suppress PRO-7a-mediated cytoprotection against oxidative stress. It suggested that its mechanism is independent of the production of L-arginine metabolites with neuroprotective properties by increased AsS activity. On the other hand, the neuroprotective effect of PRO-7a was blocked in the presence of dicyclomine hydrochloride (DCH), an M1 mAChR antagonist. Conclusions: For the first time, this work provides evidence that PRO-7a-induced neuroprotection seems to be mediated through M1 mAChR activation in PC12 cells, which reduces oxidative stress independently of AsS activity and L-arginine bioavailability.