BACKGROUND: Thrombin is produced by the prothrombinase complex, composed of factor (f) Xa and fVa on a phospholipid (PL) membrane surface. Snakes of the Elapidae family have venom versions of these factors that cause coagulopathy in prey. Group C venoms contain both fXa and fVa orthologues. Group D venoms only contain a fXa orthologue and hijack fV(a) of the prey. Hopsarin D (HopD) is the venom fXa of the Stephen\'s Banded snake (Hoplocephalus stephensii).
OBJECTIVE: We set out to address the following: Does HopD bind to human fVa with high affinity in the absence of PL? Does it process prothrombin through the meizothrombin pathway? Is the order of cleavage PL-dependent? Can HopD activate fV?
METHODS: We produced and characterized full-length and truncated HopD.
RESULTS: HopD is only able to clot plasma that contains fV, and competes with human fXa for fVa binding. HopD binds to both human fVa and fV with high affinity (Kd∼10nM), in contrast to fXa. HopD processes prothrombin down the meizothrombin route in the absence and presence of PL. Although HopD can bind to fV, conversion to fVa is necessary for prothrombin processing. HopD initiates clotting in the blood of prey by activating fV.
CONCLUSIONS: HopD binds to fVa with high affinity and rapidly activates prothrombin in the absence of PL, exclusively through the meizothrombin intermediate. HopD binds with high affinity to both fVa and fV, suggesting that the B-domain does not sterically block fXa binding, but inhibits productive interaction in another way, and additionally prevents prothrombin binding.

BACKGROUND: Venoms have repeatedly evolved over 100 occasions throughout the animal tree of life, making them excellent systems for exploring convergent evolutionary novelty. Growing evidence supports that venom evolution is predominantly driven by prey or host-related selection pressures, and the expression patterns of venom glands reflect adaptive evolution. However, it remains elusive whether the evolution of expression patterns in venom glands is likewise a convergent evolution driven by their prey/host species.
RESULTS: We utilized parasitoid wasps that had independently adapted to Drosophila hosts as models to investigate the convergent evolution of venom gland transcriptomes in 19 hymenopteran species spanning ~ 200 million years of evolution. Comparative transcriptome analysis reveals that the global expression patterns among the venom glands of Drosophila parasitoid wasps do not achieve higher similarity compared to non-Drosophila parasitoid wasps. Further evolutionary analyses of expression patterns at the single gene, orthogroup, and Gene Ontology (GO) term levels indicate that some orthogroups/GO terms show correlation with the Drosophila parasitoid wasps. However, these groups rarely include genes highly expressed in venom glands or putative venom genes in the Drosophila parasitoid wasps.
CONCLUSIONS: Our study suggests that convergent evolution may not play a predominant force shaping gene expression levels in the venom gland of the Drosophila parasitoid wasps, offering novel insights into the co-evolution between venom and prey/host.

Three-finger proteins are the most abundant toxins in the venom of Naja ashei, a snake species from the Elapidae family. This research aimed to describe the effects of varying charges of these proteins, isolated from Naja ashei venom using SEC and IEX chromatography. The study examined how differently charged three-finger toxin fractions interact with and affect neuroblastoma (SK-N-SH) and promyeloblast (HL-60) cells, as well as model Langmuir membranes and liposomes designed to mimic cellular lipid composition. Findings revealed that protein surface charges significantly impact cell survival (MTT assay), membrane damage (lactate dehydrogenase release, malondialdehyde formation), and the structural and electrochemical properties of model membranes (Langmuir membranes and zeta potential for liposomes and cancer cell lines). Results indicated that SK-N-SH cells, characterized by a higher negative charge on their cell membranes, interacted more effectively with positively charged toxins than HL-60 cells. However, the mechanism of these electrostatic interactions is complex. The research demonstrated that electrostatic and mechanical membrane modifications induced by venom proteins can significantly affect cell metabolism. Additionally, the total charge of the membrane, influenced by polar lipid components and phospholipid saturation, plays a decisive role in toxin interaction.

Envenomation by reptile venom, particularly from lizards, poses significant health risks and can lead to physiological and cardiovascular changes. The venom of Heloderma horridum horridum, endemic to Colima, Mexico, was tested on Wistar rats. Electrocardiographic (ECG) data were collected pre-treatment and at 5-min intervals for 1 h post-envenomation. A specially designed computational linear regression algorithm (LRA) was used for the segmentation analysis of the ECG data to improve the detection of fiducial points (P, Q, R, S, and T) in ECG waves. Additionally, heart tissue was analyzed for macroscopic and microscopic changes. The results revealed significant electrocardiographic alterations, including pacemaker migration, junctional extrasystoles, and intraventricular conduction aberrations. By applying a linear regression algorithm, the study compensated for noise and anomalies in the isoelectric line in an ECG signal, improving the detection of P and T waves and the QRS complex with an efficiency of 97.5%. Cardiac enzyme evaluation indicated no statistically significant differences between the control and experimental groups. Macroscopic and microscopic examination revealed no apparent signs of damage or inflammatory responses in heart tissues. This study enhances our understanding of the cardiovascular impact of Heloderma venom, suggesting a greater influence on changes in conduction and arrhythmias than on direct cardiac damage to the myocardium.

Loxoscelism is the pathological condition triggered by a brown spider bite. The venom of these spiders is rich in phospholipases D (PLDs), which can induce virtually all local and systemic manifestations. Recombinant mutated PLDs from clinically relevant Loxosceles species in South America have been investigated as potential antigens to develop novel therapeutic strategies for loxoscelism. However, certain gaps need to be addressed before a clinical approach can be implemented. In this study, we examined the potential of these recombinant mutated PLDs as antigens by testing some variations in the immunization scheme. Furthermore, we evaluated the efficacy of the produced antibodies in neutralizing the nephrotoxicity and sphingomyelinase activity of brown spider venoms. Our findings indicate that the number of immunizations has a greater impact on the effectiveness of neutralization compared to the amount of antigen. Specifically, two or three doses were equally effective in reducing dermonecrosis and edema. Additionally, three immunizations proved to be more effective in neutralizing mice lethality than one or two. Moreover, immunizations mitigated the signs of kidney injury, a crucial aspect given that acute renal failure is a serious systemic complication. In vitro inhibition of the sphingomyelinase activity of Loxosceles venoms, a key factor in vivo toxicity, was nearly complete after incubation with antibodies raised against these antigens. These findings underscore the importance of implementing an effective immunization scheme with multiple immunizations, without the need for high antigen doses, and enhances the spectrum of neutralization exhibited by antibodies generated with these antigens. In summary, these results highlight the strong potential of these antigens for the development of new therapeutic strategies against cutaneous and systemic manifestations of loxoscelism.

BACKGROUND: India experiences the highest snakebite burden globally, with 58 000 predicted deaths annually. The central Indian state of Madhya Pradesh is thought to have a substantial snakebite burden and provides compensation to families who can demonstrate by postmortem and hospital treatment reports that their relatives have died due to snakebite. This study represents the first report on the frequency of distribution of compensation for snakebite deaths in Madhya Pradesh.
METHODS: Statewide snakebite death compensation data from 2020-2021 and 2021-2022, provided by the Madhya Pradesh health authorities, were analysed alongside interviews with 15 families that described the events that ultimately led to their compensation claims.
RESULTS: Compensation was paid to a total of 5728 families, with a total value equating to 22 912 Lakhs (approximately US${\\$}$27.94 million). Families described commonly recognised snakebite risk factors and behaviours in the events that resulted in their relatives\' deaths.
CONCLUSIONS: The snakebite burden in Madhya Pradesh is significant, both in terms of mortality and economic expenditure of the state. Sustained investment in preventative interventions, as well as monitoring of the rate of compensation payouts due to snakebite death as a measure of intervention effectiveness, should be considered to substantially reduce snakebite incidence and mortality.

Scorpions are predatory arachnids whose venomous sting primarily affects people in tropical and subtropical regions. Most scorpion stings can only cause localized pain without severe envenomation. Less than one-third of the stings cause systemic envenoming and possibly lead to death. About 350,000 scorpion stings in Northern Africa are recorded yearly, resulting in about 810 deaths. In Eastern/Southern Africa, there are about 79,000 stings recorded yearly, resulting in 245 deaths. Farmers and those living in poverty-stricken areas are among the most vulnerable to getting stung by scorpions. However, compared to adults, children are at greater risk of severe envenomation. Scorpion venom is made up of complex mixtures dominated by peptides and proteins that confer its potency and toxicity. These venom toxins have intra- and interspecies variations associated with the scorpion\'s habitat, sex, diet, and age. These variations alter the activity of antivenoms used to treat scorpion sting envenomation. Thus, the study of the proteome composition of medically important scorpion venoms needs to be scaled up along their geographical distribution and contributions to envenomation in Southern and Northern Africa. This will help the production of safer, more effective, and broad-spectrum antivenoms within these regions. Here, we review the clinical implications of scorpion sting envenomation in Southern and Northern Africa. We further highlight the compositions of scorpion venoms and tools used in scorpion venomics. We discuss current antivenoms used against scorpion sting envenomation and suggestions for future production of better antivenoms or alternatives. Finally, we discuss the therapeutic properties of scorpion venom.

Understanding how environmental factors affect the performance of predators can provide profound insights into predator-prey interactions from evolutionary and ecological perspectives and the global distributional patterns of each taxon. Almost all venomous predators are ectotherms, with muscle contraction properties depending on temperature. For predators having venom transportation systems driven by muscle contraction, temperature may have quite large effects on envenomation performance for prey subjugation. Here, we used videography and enzyme-linked immunosorbent assay to examine thermal effects on envenomation kinematics and venom expenditure in predatory strikes of a venomous snake, the Mamushi Gloydius blomhoffii, to its main rodent prey at various body temperatures under both field and laboratory experimental conditions. Unexpectedly, we found that the thermal effects on envenomation performance are limited over nearly the entire ecologically relevant range of temperature (from 13.2°C to 26.2°C). Although temperature statistically significantly affected the mass of venom injected under field conditions, temperature explained only a minor proportion of the variation in venom expenditure. These findings suggest that the Mamushi is able to maintain prey subjugation performance across a wide range of temperatures, which is highly advantageous for ectothermic predators. Further studies should examine the underlying mechanisms of the limited thermal effects and their ubiquity across venomous predators.

This study focused on developing an optimal formulation of liposomes loaded with bee venom (BV) and coated with PEG (BV-Lipo-PEG). The liposomes were characterized using dynamic light scattering, transmission electron microscopy, and Fourier transform infrared spectroscopy. Among the liposomal formulations, F3 exhibited the narrowest size distribution with a low PDI value of 193.72 ± 7.35, indicating minimal agglomeration-related issues and a more uniform size distribution. BV-Lipo-PEG demonstrated remarkable stability over 3 months when stored at 4 °C. Furthermore, the release of the drug from the liposomal formulations was found to be pH-dependent. Moreover, BV-Lipo-PEG exhibited favorable entrapment efficiencies, with values reaching 96.74 ± 1.49. The anticancer potential of the liposomal nanocarriers was evaluated through MTT assay, flow cytometry, cell cycle analysis, and real-time experiments. The functionalization of the liposomal system enhanced endocytosis. The IC50 value of BV-Lipo-PEG showed a notable decrease compared to both the free drug and BV-Lipo alone, signifying that BV-Lipo-PEG is more effective in inducing cell death in A549 cell lines. BV-Lipo-PEG exhibited a higher apoptotic rate in A549 cell lines compared to other samples. In A549 cell lines treated with BV-Lipo-PEG, the expression levels of MMP-2, MMP-9, and Cyclin E genes decreased, whereas the expression levels of Caspase3 and Caspase9 increased. These findings suggest that delivering BV via PEGylated liposomes holds significant promise for the treatment of lung cancer.

This study investigated the intraspecific and interspecific variability in the venom effects of Agkistrodon viperid snake species and subspecies (eleven venoms total) on plasma clotting times, fibrinogen levels, and fibrin clot strength. Significant delays in plasma clotting time were observed for A. conanti, A. contortrix mokasen, A. contortrix phaeogaster, A. howardgloydi, A. piscivorus leucostoma, and A. piscivorus piscivorus. Notably, the phylogenetically disjunct lineages A. conanti, A. contortrix mokasen, and A. howardgloydi exhibited the most potent anticoagulant effects, indicating the independent amplification of a basal trait. Inhibition assays with the activated clotting enzymes Factors XIa, IXa, Xa, and IIa (thrombin) revealed that FXa inhibition is another basal trait amplified independently on multiple occasions within the genus, but with A. howardgloydi, notably more potent than all others. Phospholipid degradation and zymogen destruction were identified as mechanisms underlying the variability in venom effects observed experimentally and in previous clinical reports. Thromboelastography demonstrated that the venoms did not clot fibrinogen directly but affected fibrin clot strength by damaging fibrinogen and that thrombin was subsequently only able to cleave into weak, unstable clots. The ability to activate Protein C, an endogenous anticoagulant enzyme, varied across species, with some venoms exceeding that of A. contortrix contortrix, which previously yielded the protein diagnostic agent Protac®. Phylogenetic analysis suggested that both fibrinogen degradation and Protein C activation were each amplified multiple times within the genus, albeit with negative correlation between these two modes of action. This study highlights the evolutionary, clinical, and biodiscovery implications of venom variability in the Agkistrodon species, underscoring their dynamic evolution, emphasising the need for tailored clinical approaches, and highlighting the potential for novel diagnostic and therapeutic developments inspired by the unique properties of snake venoms.