Salmonella is a major cause of foodborne illness, hospitalization, and death worldwide. This high-priority pathogen produces multiple virulence factors that enable it to evade host immune defenses. Among these are AB₅ toxins, a family of multi-subunit secreted proteins known to be virulence factors in other Gram-negative bacteria. AB₅ toxins have been identified in both typhoidal and non-typhoidal Salmonella (NTS) serovars; however, their molecular role in pathogenesis in NTS remains poorly understood. We have previously purified an AB₅-like toxin, ArtAB, and its B₅ subunit (ArtB) from Salmonella Typhimurium. In this study, we treated Chinese hamster ovary (CHO) cells with ArtAB, ArtB, and other AB₅ toxins to examine cellular morphology, such as cell elongation or clustering. We also performed a cytotoxicity assay with 2-fold dilutions of ArtAB and ArtB on CHO cells. Based on these results, we treated CHO cells with 10 µg/mL of ArtAB and 7.25 µg/mL of ArtB (equimolar concentrations) for 4 hours, followed by mRNA sequencing and whole-cell proteomics to investigate differential gene and protein expression. Preliminary mRNA-seq results indicate that ArtAB significantly alters the expression of genes involved with transcription factors, cell signaling, intracellular transport of biomolecules, metabolism, and extracellular matrix remodeling. In contrast, ArtB behaves similarly to the buffer control (1X PBS + 5% glycerol) and does not significantly modulate gene expression. Proteomics analysis thus far supports these findings. Uniquely, enterotoxin B₅ subunits, such as cholera toxin B (CTB), are known to serve as both vaccine antigens and protein adjuvants. Further defining the cellular signaling pathways induced by these toxins is essential to understanding how they contribute to Salmonella pathogenesis and may lead to the development of more effective therapeutics and vaccines.

Introduction

Systemic loxoscelism, caused by the venom of the Loxosceles laeta spider, can be life-threatening. Current specific treatment relies on the administration of antivenom produced from the plasma of horses that have been hyperimmunized with spider venom. However, limited venom availability is the major production bottleneck. This study evaluates whether a genetically detoxified version of Sphingomyelinase D (SphD), the main toxin responsible for severe loxoscelism, could improve antivenom production.

Methods

Two versions of SphD were engineered using the baculovirus-insect larvae (BIL) platform: r(wtSphD), retaining its toxic functions, and r(dSphD), a detoxified variant (D259G mutation). Two horses were immunized over three cycles with r(dSphD), and their specific serum antibody responses were evaluated using an indirect ELISA. After each cycle, plasma was collected to produce pilot-scale Active Pharmaceutical Ingredients (APIs) of antivenom. Neutralizing activities of sera and APIs were tested against r(wtSphD) and native L. laeta venom using rabbit dermonecrotic assays and direct human red blood cell hemolysis test.

Results

A specific immune humoral response was developed after each immunization cycle. Higher levels of antibodies were detected in Horse 1. Dermonecrotic injuries of native L. laeta venom were partially neutralized by the sera and APIs from both animals. Moreover, sera and APIs from Horse 1 at cycles 2 and 3 effectively neutralized the r(wtSphD)´s dermonecrotic and hemolytic activities, while Horse 2 only showed similar neutralization capacity at cycle 3.

Conclusions

These promising results demonstrate that theBIL platform can be used to produce a genetically detoxified version of SphD, capable of inducing an immune-effective neutralization response of its homologous toxic form. Further ongoing research is being conducted to evaluate other detoxified versions of SphD isoforms and achieve complete protection against whole spider venom. This strategy offers a scalable, affordable, and safer alternative for venom complement or replacement in antivenom production.

Botulinum neurotoxins (BoNTs) are among the most potent protein toxins known to humans, yet the intracellular trafficking of BoNT-Light Chain A1 (LC/A1) to its substrate, Synaptosome Associated Protein of 25-kDa (SNAP-25), remains poorly understood. A mouse neuroblastoma-2A (N2A) cell-based assay was used to track the intracellular trafficking of cytosolic EGFP-tagged LC/A1. The results revealed that LC/A1 associates with intracellular vesicles as a function of the LC/A1-N terminus, while plasma membrane binding is facilitated by an internal region of LC/A1 that can target LC/A1 to the plasma membrane from the cytosol. An internal LC/A domain, termed the Membrane Localization Domain (MLD), is responsible for the movement of the LC/A1 to the plasma membrane, where the association may be stable or reversible depending on the LC/A subtype. Recent studies have detected the physical trafficking of EGFP-LC/A1 from the cytosol to the intracellular plasma membrane. Thus, in addition to known steps such as host receptor binding and catalysis, the intracellular trafficking of LC to the target substrate may be a critical determinant of BoNT potency. Mechanistic insights into BoNT intracellular trafficking will clarify fundamental aspects of toxin action towards the development of novel therapeutic strategies to mitigate BoNT toxicity.

Introduction: Snake venom lectins are categorized into classical sugar-binding C-type lectins and non-sugar binding C-type-like lectins. C-type lectins are typically homodimeric proteins with affinity for beta-galactoside residues and are known to exhibit diverse biological activities, including the inhibition of platelet aggregation. Platelets play a crucial role in several physiological and pathological processes, such as thrombosis and metastasis. This study aimed to elucidate the biochemical and functional properties of leuccetin, a C-type lectin isolated from Bothrops leucurus venom, with a particular focus on its effects on platelet function.

Methods: Leuccetin was purified via affinity chromatography using D-galactose gel resin. Its effects on platelet function were assessed using washed platelets and platelet-rich plasma. The potential anti-metastatic activity of leuccetin was evaluated using MDA-MB-231 breast cancer cells. Platelet–tumor cell interactions were quantified using a plate reader-based assay.

Results: Leuccetin displayed a molecular mass of approximately 15 kDa under reducing conditions and 30 kDa under non-reducing conditions. Functionally, leucceytin inhibits platelet aggregation induced by convulxin (CVX) and the von Willebrand factor (vWF). Western Blot analysis demonstrated that leuccetin binds to key platelet receptors—GPVI, GPIb, αV integrin, and CLEC-2—correlating with its inhibitory effects on platelet aggregation. Notably, leuccetin also inhibited platelet aggregation induced by MDA-MB-231 and reduced the adhesion of these cells at non-cytotoxic concentrations. Furthermore, leuccetin disrupted platelet–tumor cell interaction.

Conclusion: These findings suggest that leuccetin has biotechnological potential as a novel antiplatelet and/or anti-metastatic agent, due to its ability to inhibit platelet aggregation and platelet–tumor cell interactions.

Introduction

Nanomedicine has helped in the development of novel therapeutics. For example, the encapsulation of bioactive components such as peptides/proteins into polymeric nanoparticles can lead to increased bioavailability, passive targeting and sustained release. Snake venoms have proven to be rather interesting for the R&D of new medicines, as they can present antifungal, antiparasitic, antibacterial or antiproliferative activity. In the present study, we encapsulated venom from C. molossus in PLGA NPs and evaluated them for hemolytic activity, kinetic release profile and cytotoxic activity against T47D breast carcinoma cells.

Methods

PLGA NPs were produced by a double emulsion-solvent evaporation method. Briefly, PLGA was dissolved in dichloromethane, into which a small volume of snake venom resuspended in water was added. This solution was then emulsified by ultrasonication, forming the organic phase. Later, the organic phase was added to a stirring aqueous phase containing 2.5% PVA, and then emulsified again by ultrasonication. NPs were washed three times by ultracentrifugation, freeze-dried and stored at -20 ºC until use.

Conclusions

PLGA NPs serve as an efective encapsulation system for bioactive components such as snake venom toxins. We obtained NPs with sizes of ~250 nm, PDI of 0.10, a zeta-potential of -26 mV and an EE% of ~85%. PLGA-Venom NPs were not hemolytic unlike C. molossus venom and presented a sustained release profile. Finally, they were cytotoxic against T47D cells, providing helpful insights into the development of these novel drug delivery systems. More studies are requiered to evaluate the feasilibity of using these agents alongside nanoparticle systems.

Introduction

Mexico is the country with the highest diversity of rattlesnakes worldwide, and their venom represents a valuable source of molecules with biomedical potential. Documented bioactivities include antiproliferative, analgesic and microbicide properties. Following that, the World Health Organization considers antibiotic resistance as a global threat. Therefore, studying alternatives to these drugs is imperative. The Veracruz neotropical rattlesnake (Crotalus mictlantecuhtli) is an endemic species from Central Mexico whose venom has been characterized before, but with no reports of its antibacterial properties, to the best of our knowledge.

Methods

Four protein fractions from C. mictlantecuhtli venom were obtained by size exclusion chromatography (SE-LC). The fractions were lyophilized and stored at -20°C until analysis. Protein quantification was performed using a Bicinchoninic Acid (BCA) Protein Assay, with bovine serum albumin (BSA) as a standard. All fractions were adjusted by protein content, diluted with saline solution (NaCl 0.9%) and added to 96-well microplates with Müller–Hinton (MH) in concentrations ranging from 10 to 0.001 µg/µL. To evaluate the antimicrobial activity, the Minimal Inhibitory Concentration (MIC) was determined via the microdilution method, which was performed by inoculating each well with ATCC strains E. coli (25922) or S. aureus (25923) standardized in brain–heart infusion to 0.5 McFarland. The results were corroborated using the agar well diffusion method in MH. Additionally, blood samples were drawn and anticoagulated with calcium-balanced lithium heparin to evaluate indirect hemolysis in 5% blood agar. The data was analyzed by two-way ANOVA and direct observation.

Results

Venom fractions 1, 3 and 4 did not differ significantly from the gentamicin control below 1 µg/µL for E. coli and S. aureus (P > 0.05). Fraction 1 exhibited hemolytic halos with diameters of 0.7 mm and 14 mm, respectively.

Conclusions

C. mictlantecuhtli venom contains proteins of medical interest, showing antimicrobial potential; hence, it is imperative to continue studying it.

Lectins are non-enzymatic proteins that bind reversibly and specifically to carbohydrates. In snake venoms, lectins are grouped into classical lectins, which bind Ca2+ and sugars (CTLs), and non-sugar-binding snake venom C-type lectin-related proteins (CLRPs). These proteins target distinct molecules, such as coagulation factors and receptors, on platelet surfaces and endothelial or immune cells, which are involved in hemostasis, inflammation and metastasis. Because of their specificity and versatility, C-type lectins are used as models in biotechnological studies and applications. Toward this goal, the current study aims to determine the molecular and biochemical properties of the C-type lectin isolated from Bothrops neuwiedi venom (CTL-Bn). The protein was isolated by D-galactose column affinity chromatography and analyzed by SDS-PAGE and mass spectrometry. We demonstrate that CTL-Bn forms oligomers whose monomers have a molecular mass of approximately 15 kDa. CTL-Bn induced hemagglutination at a minimum dose of 2.5 μg, which was inhibited by galactose, lactose and EDTA. The protein is stable at a wide pH range and shows optimal activity at 20 °C. Furthemore, CTL-Bn inhibits platelet aggregation induced by convulxin and binds to fibrinogen and glycoprotein GPVI (the main collagen receptor), which probably explains its inhibitory effect on convulxin-induced platelet aggregation. Based on docking molecular studies, we proposed that CTL-Bn binds to GPVI in a region formed by hydrophobic residues with polar and basic residues around the periphery. To evaluate the role of CTL-Bn in the metastatic process, we tested CTL-Bn on platelet aggregation induced by MDA-MB-231 tumor cells. CTL-Bn inhibited this aggregation; however, in the MTT assay, the protein did not affect the viability of the tumor cells. Altogether, our results provided a comprehensive biochemical, biological and structural characterization of CTL-Bn.

Snake venom disintegrins are a group of small, non-enzymatic proteins that act by binding to integrins on platelet surfaces and other cells, preventing the interactions required for cell adhesion and platelet aggregation. We expressed and purified three disintegrins from Echis coloratus, containing different integrin-binding motifs: Dis_VGD (VGD motif), Dis_KGD (KGD motif), and Dis_RGD (RGD motif). The expression was carried out using the E. coli expression system, and the purification steps included immobilised metal affinity, anion exchange, and size exclusion chromatography. The ability to inhibit platelet aggregation was evaluated by plate-based aggregometry, using 2-MeS-ADP and CRP-XL as agonists. Dis_VGD presented no effect on platelet aggregation under the tested conditions, while Dis_RGD and Dis_KGD were capable of inhibiting platelet aggregation in a non-competitive and dose-dependent manner. When the platelet aggregation was induced by 2-MeS-ADP, Dis_RGD presented almost complete inhibition at 250 nM, while Dis_KGD presented only 20% of inhibition at the same concentration. The effect on platelet aggregation induced by CRP-XL was less pronounced, with Dis-RGD and Dis-KGD reaching 70% and 10% of inhibition at 500 nM, respectively. The VGD motif binds to integrin α5β1, a fibronectin receptor involved in platelet adhesion and spreading, but not in aggregation. In contrast, the RDG and KGD motifs interact with the αIIbβ3 integrin, a key mediator of platelet aggregation, and are therefore expected to inhibit this process. Our study confirmed that the expressed E. coloratus disintegrins are functional and present the expected effect on platelet aggregation. Further analyses are underway to evaluate their impact on cell adhesion and to gain a more comprehensive understanding of their broader activity.

Snakebite envenomation (SBE)—the injection of venom through the bite of a venomous snake—in domestic animals is a neglected and under-reported problem. Whilst SBE is a major public health issue in humans, its impact on livestock remains poorly understood. This pilot study explored the clinical features and socioeconomic impacts of SBE in livestock in Tamil Nadu, India—a country with the world’s highest snakebite incidence. Between September and October 2024, 23 farmers from Coimbatore and Pollachi districts were interviewed using a structured questionnaire, reporting 21 cattle and 4 goat SBE cases.

Reported mortality rates were 52% in cattle and 100% in goats. Common clinical signs included local swelling, hypersalivation, and bleeding, with bites occurring mainly on the limbs or face. Bites predominantly occurred during the monsoon season, and in fields or sheds. Surviving cattle recovered in an average of four days, and farmers reported impacts on milk yield, fertility, and carcass weight.

Treatment was provided to 81% of cattle and 50% of goats. Financial losses were reported by 72% of farmers, averaging INR 42,000 per cow and INR 10,750 per goat, and were particularly impactful given the small herd sizes. These findings highlight the need for further research into the incidence and impact of livestock SBE, alongside the development of prevention strategies and improved access to treatment. Preventing livestock SBE is especially critical given limited antivenom availability and supports a One Health approach to snakebite control.

Toxic organisms are capable of producing harmful substances that can adversely affect other living organisms, including humans. Some mushrooms produce powerful toxins that can cause serious poisoning. Boletus satanas, with its impressive size and colour, is also renowned for its toxicity. It causes severe gastrointestinal disorders, with violent symptoms. It deserves particular attention due to its resemblance to certain edible boletus. This survey was carried out near the coastal region of Ghazaouet, in the Traras mountains, Tlemcen wilaya. The aim of the present study is to characterize the morphological features of this species and subsequently investigate the myco-chemical screening of the hydro-methanolic extract for the purpose of determining the predominant chemical families of its secondary metabolites using simple methods and techniques that can be rapidly applied. The results of mycochemical screening performed on the extract revealed the presence of substances belonging to classes of active compounds, including alkaloids, coumarins, free quinones, flavonoids, reducing compounds, terpenoids, and saponins. Tannins, anthraquinones and anthocyanins were absent. The morphological determination of this mushroom is based on a series of macroscopic and microscopic criteria. This mushroom is an ectomycorrhizal basidiomycete that grows mainly on calcareous soils, in symbiosis with some broadleaved trees. Its distinctive features include a massive morphology and contrasting colors. It can be recognized by its large pale cap, its red pores, its pot-bellied yellow stem with a red network, and its flesh, which turns blue when cut. Under the effect of Melzer's solution, the stipe undergoes an inamyloid reaction. The spore is olive brown to brown in colour. The spores measure 10-15 x 5.5-6.5 µm and are elliptical to sub-fusiform. These preliminary results encourage further research to assess and evaluate their biological activities.