Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, often diagnosed at advanced stages when curative options are limited and conventional therapies like sorafenib show modest efficacy and significant toxicity. This highlights the urgent need for innovative, targeted treatments with improved selectivity and safety. Snake venoms are a rich source of bioactive molecules with therapeutic potential, particularly in the field of oncology. This study explores the anticancer properties of Moroccan Naja haje venom and its purified fractions against HCC using both conventional monolayer (2D) cultures and physiologically relevant multicellular tumor spheroid (3D) models. Crude venom was fractionated by gel filtration and RP-HPLC, and the resulting components were tested for cytotoxicity on HepG2 and Huh7.5 liver cancer cell lines. Their efficacy was further evaluated in 3D spheroids co-culturing cancer cells with human fibroblasts (WI38), hepatic stellate cells (LX2), and endothelial cells (HUVECs). Normal hepatocytes (Fa2N-4) were used to assess off-target toxicity. Several fractions, particularly Fraction 7 and its subfraction 7d, significantly inhibited cancer cell proliferation and reduced spheroid size, with minimal toxicity to normal liver cells,demonstrating greater selectivity than sorafenib. Proteomic analysis identified cytotoxins 2, 5, and 10 as key components responsible for this activity. These findings underscore the potential of Naja haje venom-derived peptides as promising candidates for selective HCC therapy,warranting further investigation into their mechanisms of action and therapeutic development.

Angiogenesis, the formation of new blood vessels, is a fundamental process in tumor development, progression, and metastasis, making it a critical target in modern cancer therapy. Among natural bioactive compounds, animal venoms have emerged as a rich source of pharmacologically active peptides with potential anti-angiogenic effects. In this in silico study, we explored the therapeutic potential of bee venom peptides, focusing particularly on melittin and tertiapin, as inhibitors of tumor-induced angiogenesis. Using molecular docking techniques, these peptides were evaluated for their binding affinity toward key angiogenesis-related receptors, including platelet-derived growth factor receptor alpha (PDGFR-α), vascular endothelial growth factor receptors (VEGFRs), fibroblast growth factor receptors (FGFRs), and integrin αvβ3. Both melittin and tertiapin exhibited strong and specific binding affinities, with melittin showing notable interaction with PDGFR-α and tertiapin demonstrating a high affinity for integrin αvβ3. Molecular dynamics simulations further confirmed the stability and integrity of these peptide–receptor complexes over time, suggesting the potential for sustained biological activity. Additionally, functional enrichment analysis using the Reactome database revealed significant modulation of angiogenesis-related pathways, particularly those involved in cell migration and vascular remodeling. These findings highlight the potential of bee venom peptides as natural, targeted modulators of angiogenesis and support their further development as candidates for anti-cancer drug discovery.

Epsilon toxin (ETX) is a pore-forming toxin (PFT) capable of crossing the blood–brain barrier and binding to myelin structures. In vitro assays have demonstrated that ETX impairs oligodendrocytes and induces demyelination. Notably, ETX has been implicated in the pathogenesis of multiple sclerosis (MS), with ETX-specific antibodies detected in sera from MS patients.

Myelin and lymphocyte protein (MAL) is widely recognized as the receptor for ETX. Its presence is essential for ETX-induced pore formation in the plasma membrane of host cells, ultimately leading to cell death. Recent findings have shown that ETX also binds to and kills primary human lymphocytes, which express elevated levels of MAL. This suggests that ETX may influence immune responses associated with MS, although its precise mechanism of action remains unclear.

To counteract the damage caused by PFTs, some host cells release extracellular vesicles (EVs) to reduce pore insertion into the plasma membrane. ETX has been shown to induce EV production in HeLa cells overexpressing MAL-GFP. Similar effects have been observed in MOLT4 cells, a T-lymphocyte line that endogenously expresses MAL protein. Our observations confirm that ETX also stimulates EV formation in MOLT4 cells. Moreover, both MAL protein and ETX oligomers are present in these EVs, making them a valuable tool for investigating ETX’s mode of action and its interaction with its receptor. We speculate that these EVs may play a role in the induction of MS.

Introduction: Pore-forming toxins (PFTs) play a pivotal role in bacterial pathogenicity, yet the cellular responses they elicit remain incompletely understood.

Methods: Using in vivo Mean Shift Super-Resolution (MSSR) microscopy, we analyzed mosquito larval midgut cells exposed to Bacillus thuringiensis Cry11Aa toxin, achieving unprecedented nanoscale visualization of the intoxication process. Transmission electron microscopy was also used in this study.

Results: Our observations uncovered a sequential cascade beginning with toxin binding to brush border microvilli (BBM), followed by membrane pore formation that disrupts actin cytoskeletal integrity and stimulates massive endocytosis. An additional observed response was the release of large (5–8 µm) membrane-enclosed vesicles into the midgut lumen. These vesicles, segmented by endoplasmic reticulum (ER) membranes and actin filaments, carried damaged organelles—such as mitochondria, lysosomes, and endosomes—yet lacked nuclear material. Notably, the toxin was found in close association with ER membranes within these vesicles, suggesting this process may represent a toxin-clearing defense mechanism. Transmission electron microscopy confirmed vesicle morphology and contents. A comparison with a non-toxic Cry11E97A mutant, defective in oligomerization and membrane insertion, confirmed that pore formation is essential for triggering the observed cellular events. Vesicles of similar morphology were rarely detected in untreated controls, implying that their production may reflect an amplified version of a natural epithelial renewal process.

Conclusions: These findings shed light on a previously unrecognized aspect of midgut epithelial defense against Cry toxins, emphasizing the complexity of the insect cellular response to PFTs. Understanding this pathway offers new perspectives for enhancing the effectiveness of Bt-based pest control and managing resistance in insect populations.

The high rate of accidents involving venomous animals, particularly in developing countries, represents a serious public health problem. In Brazil, crotalic snakes are the most lethal and have the highest accident incidence rates. Serotherapy is the most appropriate method of treating crotalic envenomation. Despite the high incidence and severity of accidents, there have been very few studies on the effect of venom on the fetus when pregnant women are involved. This project aims to study and elucidate the perinatal effects of crotalic envenomation.

Pregnant Wistar rats were treated with either saline (1.0 ml/kg sc, n=8) or crude venom from Crotalus durissus terrificus (2.5 mg/kg sc, n=8) on either the 5th (V5) or 10th (V10) gestational day (GD). On the 21st GD, they underwent a laparotomy, and maternal reproductive performance; the weights of the embryos and placentas; fetal deaths; and visceral and skeletal alterations and/or malformations were analyzed.

The data obtained was analyzed using Student's t-test and Fisher’s exact test, with the results considered significant at p>0.05. In the V5 experimental group, an increase in the average weight of the male and female fetuses, as well as in the weight of the kidneys and livers of the female fetuses, was observed. Some fetuses exhibited incomplete skull ossification. In the V10 experimental group, pregnant females experienced reductions in weight and in their consumption of food and water between GD10 and GD16. The average placental weight of the male fetuses increased, and the average weight of males and females decreased. Female fetuses showed reduced weights in their kidneys, livers, and lungs.

This study demonstrated the embryotoxic potential of Crotalus durissus terrificus venom during embryogenesis and organogenesis. Future experiments will evaluate the visceral effects of the venom in order to investigate the presence of malformations and other alterations.

Supported by Fundação Butantan.

Introduction
Bothrops snakes are the main cause of snakebites in Brazil, with envenomation leading to severe effects, especially hemostasis disorders. Bothrops jararaca venom induces inflammatory, coagulant, and hemorrhagic actions through components such as metalloproteinases, serine proteases, C-type lectin-like proteins (CTLPs), and phospholipases A2. CTLPs alter platelet aggregation and coagulation. Recent studies isolated and initially characterized two CTLPs (E6 and D4) from B. jararaca venom that induce thrombocytopenia. This project aims to characterize these proteins and understand how they reduce platelet counts during envenomation.

Objectives:
The aim of our work was to further purify and characterize CTLP-E6 and CTLP-D4 and elucidate which of their mechanisms affect platelets.

Methods:
In vivo experiments were approved by CEUAIB (protocol 8227240723) using C57BL/6 mice (30-35g, n=5-8/group). Human blood samples were obtained from volunteers (aged 18-45 years) in accordance with CONEP guidelines (CAAE: 52787521.0.0000.5469). Venom was subjected to anion exchange chromatography (Q-Sepharose), followed by cation exchange (SP—Sepharose). Fractions were analyzed by SDS-PAGE (15%) and tested in vivo: mice received subcutaneous injections of CTLP fractions or saline. Blood samples were collected 3 hours later to determine the platelet count and facilitate fibrinogen measurement.

Results:
Anion exchange chromatography showed most proteins of interest eluted in the flow-through. SDS-PAGE revealed the presence of CTLP-E6 and CTLP-C4 at ~30 kDa under non-reducing conditions, splitting into 16 kDa and 13 kDa bands under reducing conditions. These fractions underwent further purification by cation exchange. SDS-PAGE and optical density confirmed purification. Injected mice exhibited thrombocytopenia without fibrinogen changes, indicating successful CTLP purification.

Conclusions:
The study effectively purified CTLPs that induce thrombocytopenia. Further research will investigate their mechanisms of action.

Acknowledgments:
Financial support: CAPES, Fundação Butantan, and CNPq (grant 309980/2021-6).

Introduction: Bothrops jararaca (Bj) snakebites cause hemostatic and inflammatory disturbances in victims. Bj venom contains various proteins that induce local and systemic bleeding, thrombocytopenia, and consumption coagulopathy. Parenteral administration of rutin, a bioflavonoid, has shown potential in mitigating venom effects by reducing bleeding time, delaying coagulopathy, and decreasing local hemorrhage (doi: 10.3389/fphar.2022.828269), and thereby may accelerate the recovery of patients treated with specific antivenom therapy (SAB).

Aims: To evaluate the potential use of orally administered nano-rutin in reducing the hemorrhagic effects and hemostatic disturbances caused by Bj envenomation. Additionally, this study aimed to explore the role of cfDNA in the pathophysiology of envenomation and assess the levels of inflammatory markers in animals treated with a combination of nano-rutin and SAB.

Methods: The use of male C57BL/6 mice was approved by IACUC (4491070319). Mice were divided into experimental groups (n=10/group) and evaluated at 6 and 12h after s.c. administration of saline or Bj venom. Nano-rutin (v.o.) and/or SAB therapy (i.v.) were administered following venom administration.

Results: Marked hypofibrinogenemia, increased IL-6 levels, and intense local hemorrhage were observed in envenomed mice. The association of nano-rutin with SAB restored fibrinogen and IL-6 levels faster than SAB or rutin alone. This association also blocked the development of local hemorrhage. Nano-rutin alone increased NO levels. The levels of cfDNA were not significantly different from those of the controls, but were higher with this association at 6h.

Conclusions: The association of the administration of nano-rutin followed by SAB enhanced the recovery of inflammatory and hemostatic markers after Bj envenomation, representing a promising strategy for snakebite treatment.
Acknowledgements: CAPES (#88887.886144/2023-00), Fundação Butantan, and (CNPq, # 309980/2021-6).

Introduction: Bothrops jararaca snake venom (BjV) contains toxins that activate and inhibit blood coagulation, causing consumption coagulopathy, thrombocytopenia, endothelial dysfunction, and secondary fibrinolysis in human and animal victims of snakebites. Objectives: The objective of this work was to study the in vitro coagulant activity of BjV in human, mouse, and rat plasma. Methods: The coagulant activity of BjV was assessed in normal and coagulation-factor-deficient plasma from humans, rats, and mice (C57BL/6, F8^-/-, F9^-/-, Vwf^-/-, and pearl mice (Ap3b1^-/-)). The roles of calcium ions, phospholipids, and blood cells in the coagulant activity of BjV were evaluated. Pharmacological inhibitors were used to analyze the generation of factor Xa and/or thrombin and their contribution to the coagulant activity of BjV. Results: Human plasma showed greater sensitivity to BjV than rat and mouse plasma. Calcium ions and phospholipids influenced its coagulant activity. Pre-incubation with rivaraxoban and/or dabigatran showed that the coagulant activity of BjV in rodent plasmas primarily depends on prothrombin activation, whereas in human plasma, it involves both thrombin-like-enzymes and prothrombin activators. Conclusions: This study highlighted important differences in the coagulant activity of BjV in human and rodent plasmas, underscoring the need to consider these disparities in comparative envenomation studies using animal models.

The genus Hypnale, endemic to the Western Ghats of India and Sri Lanka, comprises three valid species: Hypnale hypnale, Hypnale nepa, and Hypnale zara. Of these, H. nepa and H. zara are restricted to Sri Lanka, while H. hypnale is the only species present in India, where it forms a monophyletic lineage endemic to the Western Ghats and Sri Lanka. Despite its medical significance, the hump-nosed pit viper (Hypnale hypnale) remains understudied in venom research and neglected in antivenom development. Currently, India’s polyvalent antivenom targets only the "big four" Indian snakes [Russell’s viper (Daboia russelii), Echis carinatus, Naja naja, and Bungarus caeruleus), showing limited efficacy against Hypnale envenomation. Misidentification and inadequate clinical management further exacerbate the threat posed by this species, whose venom is known to induce coagulopathy, local tissue damage, acute kidney injury (AKI), and multi-organ failure. The Western Ghats is a recognized biodiversity hotspot, presenting significant potential for the discovery of cryptic or previously undocumented Hypnale species due to their complex topography and underexplored habitats. In this study, for the first time, we collected 35 venom samples of H. hypnale from diverse locations across the Western Ghats. We generated venom gland transcriptomes and undertook biochemical assays to investigate molecular diversity and assess regional venom variation. Our findings underscore the urgent need for Hypnale-specific antivenom formulation to improve treatment outcomes in affected regions.

Introduction

Ciguatoxins (CTXs), produced by certain marine microalgae, can bioaccumulate and biomagnify through the marine food web, ultimately posing a health risk to humans. Previous studies have identified a high prevalence of CTX-producing microalgae in the coastal areas of Risco Verde and Sardina del Norte (Gran Canaria, Spain). These findings prompted a focused investigation of fish species from these regions over two consecutive years, 2023 and 2024.

Methodology

Fish specimens representing various trophic levels were collected from both areas, a total of 72 fish in 2023 (35 from Risco Verde and 37 from Sardina del Norte) and 82 in 2024 (41 from each area). Liver and muscle tissues were selected for CTX extraction and analysed using a neuro-2a MTT cell-based assay.

Results and conclusion

In 2023, 35 specimens from Risco Verde comprised 10 species, with 8 testing positive for CTXs. All of them showed liver toxicity, and only 3 of them also showed muscle toxicity. From Sardina del Norte, 37 specimens representing 11 species were analysed, of which 6 species were CTX-positive. All of them showed liver toxicity, and only 3 of them also showed muscle toxicity.

In 2024, 41 specimens from Risco Verde included 18 species, with 10 testing positive. Similarly, the 41 specimens from Sardina del Norte represented 15 species, of which 9 tested positive. At both locations, the Parrotfish tested positive for CTXs. Redporgy showed toxicity to CTXs in the liver at both sites as well.

The results demonstrate the widespread presence of CTXs across various fish species and trophic levels in both locations. These findings underscore the importance of ongoing monitoring to assess the risk of ciguatera poisoning in the Canary Islands.