Animal venoms are increasingly recognized as valuable molecular toolboxes for the discovery of novel compounds with therapeutic potential. Among the most promising therapeutic targets for venom-derived molecules are voltage-gated ion channels, which are involved in multiple physiological processes and implicated in various diseases. Here, we report the discovery of novel peptides from the venom of Physalia physalis, predicted to target vertebrate Kv channels.

A proteomic analysis of P. physalis tentacles led to the identification of 30 ShK-like peptides belonging to the ShK family, based on their conserved primary structure and cysteine-rich scaffold. Some of them showed potential as therapeutic leads due to their ability to selectively target specific Kv channel subtypes. Their 3D structures were predicted using the deep-learning-based tool AlphaFold and subjected to molecular docking against a panel of human potassium channels. Several candidates exhibited low binding energies and high predicted specificity for the Kv1.3 channel. A high-affinity peptide was produced in recombinant form and functionally validated through whole-cell patch-clamp electrophysiology, confirming its inhibitory activity against Kv1.3 at nanomolar concentrations. Additionally, a radioligand competition assay with α-dendrotoxin in human brain tissue revealed low competition for Kv1.1, Kv1.2, and Kv1.6, supporting the peptide’s selectivity.

These findings underscore the potential of P. physalis venom components as a source of new and selective ion channel modulators. Given the role of the Kv1.3 channel in a range of human diseases—including pain, autoimmune disorders, and neurological conditions—these molecules hold great promise for the development of new therapeutic drugs.

Introduction: One of the main causes of Alzheimer’s disease (AD) is the accumulation of β-amyloid peptide in brain, leading to several consequences for neurons, including oxidative stress. To counteract oxidative stress, new antioxidants have been studied; however, few are able to cross the blood–brain barrier or demonstrate effectiveness. This study aimed to obtain new antioxidants from the sea anemone Anthopleura cascaia, whose venom has the potential to inhibit neuronal death caused by Aβ42. Methods: A. cascaia was collected in São Sebastião/SP/Brazil, and its venom was extracted through chemical stimulation. The venom was subsequently fractionated based on its antioxidant activity and assessed by DPPH and PFRAP tests. The first fractionation step was a solid-phase extraction (SPE, C18 cartridge), with elution at 0,25, 50, 75 and 100% acetonitrile/0.1% trifluoroacetic acid (TFA). A second fractionation was conducted using HPLC with a C18 column and a 0-100% acetonitrile/0.1% TFA gradient, followed by a third fractionation using an HILIC column. Thepurified active molecule was analyzed by mass spectrometry and incubated with differentiated-SH-SY5Y neurons, pre-exposed to Aβ42 for 48 h. Cell viability was assessed using the LDH test. Results: The 0% SPE fraction and the first peak of C18-HPLC exhibited antioxidant activity in both tests. Due to peak impurities, a third fractionation step was performed, yielding six fractions, and one of them demonstrated 40% and 50% antioxidant activity in the DPPH and PFRAP tests, respectively. Mass spectrometry analysis confirmed high purity and an ion at 232.943 m/z, with no matches in natural products databases. Furthermore, this molecule successfully inhibited neuronal death induced by Aβ42, with LDH values comparable to the control. Conclusion: A novel antioxidant molecule with neuroprotective potential was successfully isolated from a sea anemone. This discovery could contribute to the development of a prototype for a new AD treatment by targeting a key mechanism associated with the disease.

Mycotoxins are secondary metabolites from filamentous fungi contaminating food and feed. One major mycotoxin produced by some Fusarium species is zearalenone (ZEN). Due to its estrogen-like structure, ZEN shows strong estrogenic and anabolic effects in addition to immunotoxic and hepatotoxic effects in humans and animals. One pillar of mycotoxin risk management includes effective strategies to eliminate ZEN. The zearalenone hydrolase ZenA (ZENzyme®) can be used for the enzymatic detoxification of ZEN in animal feed to mitigate the negative effects of ZEN on farm animals. ZENzyme® cleaves the lactone ring of ZEN to form the non-estrogenic metabolite hydrolyzed zearalenone (HZEN).

For product registration, the efficacy of ZENzyme® has to be proven to authorities in the form of the reduced systemic absorption of ZEN in the presence of ZENzyme®. Therefore, in a feeding trial, 72 weaned piglets were exposed to feed contaminated with 200 µg/kg ZEN for 42 days. In one group, the feed was additionally supplemented with 10 Units/kg ZENzyme®. For assessment of ZENzyme®’s efficacy, exposure-based biomarkers were measured in plasma, urine, and feces at different time points of the feeding trial by LC-MS/MS. Applied methods did not only cover ZEN, but also the metabolites α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL), as well as the enzymatic degradation products HZEN and decarboxylated hydrolyzed zearalenone (DHZEN). A significant reduction of ZEN in the presence of ZENzyme® was observed in all tested matrices. HZEN levels in feces increased significantly, if ZENzyme® was present in animal feed. DHZEN concentrations above the limit of quantification were scarcely found and most prevalent in urine samples.

These data conclusively showed both the reduction of systemic absorption of ZEN from feed in the gastro-intestinal tract of piglets and the biotransformation of ZEN to HZEN.

Introduction

Ciguatoxins (CTXs) are marine toxins produced by microalgae of the genera Gambierdiscus and Fukuyoa that cause harmful algal blooms. These blooms are a threat to food safety due to the consumption of fish or shellfish containing CTXs. Liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) allows the identification of CTXs congeners. Instrument sensitivity can be affected by sample matrix effects; however, sample purification could prevent erroneous results.

Methods

Different samples of Gambierdiscus have been analyzed by LC-MS/MS after suspension and column-cleaning protocols were tested with seven resins (Florisil, C18, Diaion® γ-CD-HDI, γ-CD-EPI, β-CD-HDI and β-CD-EPI). Firstly, solid-phase extraction (SPE) was performed on suspension and columns using established standards to test the efficacy of each resin. Secondly, SPE was performed on the column using the four most effective resins from the first phase for one Gambierdiscus sample. Thirdly, all Gambierdiscus samples were subjected to the four resins used in the second phase.

Results and conclusion

The performances of all resins and two SPE formats were evaluated using gambierone and 44-methylgambierone standards at different concentrations (2.5 ng/mL, 12.5 ng/mL and 200 ng/mL). The recovery rates obtained with 12.5 ng/mL of C18, Diaion, and γ-CD-EPI in the column were very good. Florisil was associated with the lowest number of recoveries, while β-CD-HDI and β-CD-EPI did not perform well. The clean-up in suspension worked well, but the recovery rates were lower.

The performance of C18, Diaion, γ-CD-EPI and γ-CD-HDI was evaluated by SPE in column with the addition of gambierone, 44-methylgambierone and CTX1B to a G. excentricus extract. Recoveries were similar for C18, Diaion and γ-CD-EPI. Gambierone had a greater recovery with C18, while 44-methylgambierone and CTX1B achieved greater recovery with diaion. Finally, all Gambierdiscus samples were passed through the four resins and analyzed. To compare the matrix effect and establish a toxin profile, these samples were also analyzed without SPE.

Physalia physalis, commonly known as the Portuguese man o’ war, is a venomous cnidarian frequently observed in the Atlantic Ocean. Contact with its tentacles may result in local and systemic symptoms, including interference with the human hemostatic system. Despite increasing clinical reports, the molecular basis of these effects remains poorly understood. In this study, we characterized the enzymatic properties of P. physalis venom obtained through electrostimulation-induced cnidocyte discharge, a method that enables the selective release of venom with minimal structural contamination.

The venom demonstrated a dual modulatory effect on hemostasis. One fraction exhibited potent fibrinolytic and fibrinogenolytic activity, capable of degrading fibrin clots and completely hydrolyzing the α, β, and γ chains of human fibrinogen. The second fraction promoted rapid clot formation through the generation of ~23 kDa fibrin fragments, a hallmark of thrombin-like activity. Enzymatic assays and inhibitor profiling revealed that the fibrinolytic effect was primarily associated with a metalloprotease, whereas the thrombin-like activity was attributed to a serine protease. These enzymes act on different components of the coagulation cascade, suggesting distinct biochemical mechanisms within the same venom source.

The discovery of both fibrinolytic and thrombin-like functions within P. physalis venom represents a novel insight into cnidarian envenomation. It also emphasizes the biochemical diversity of marine venoms and their potential application in therapeutic development. These results not only improve our understanding of venom-induced coagulopathies but also support the exploration of marine-derived enzymes as lead candidates for new anticoagulant or procoagulant drugs.

Coral snakes are the American representatives of the Elapidae family. Micrurus surinamensis stands out as the largest coral snake species found in Brazil, notable for its semiaquatic habits and a diet consisting exclusively of fish. Its venom is primarily made up of three-finger toxins (3FTx), while the presence of phospholipases A₂ (PLA₂) appears to be inconsistent across different studies.

Methods:
This study investigated the neuromuscular effects of M. surinamensis venom using isolated neuromuscular models: the mouse phrenic nerve–diaphragm (PND) and chick biventer cervicis (BC) models. Mouse sciatic nerve preparations were also used.

Results:
The venom-induced neuromuscular blockade occurred in both the PND and BC preparations in a concentration- and time-dependent fashion, with the BC model demonstrating greater sensitivity. In the PND model, the findings included the following: the concentration- and time-dependent inhibition of tetanic contractions, without evidence of a tetanic fade; the full recovery of neuromuscular function following washing; reduced muscle depolarization induced by carbachol; and the suppression of miniature endplate potentials. In the BC preparation, the venom abolished contractile responses to both acetylcholine and carbachol, and washing led to partial recovery from the neuromuscular blockade. In both models, the partial and temporary restoration of neuromuscular function was achieved using neostigmine or 3,4-diaminopyridine. The compound action potentials in the mouse sciatic nerve remained unaffected. Additionally, no signs of myotoxicity were detected in either the PND or BC preparations, as evidenced bya stable muscle tension baseline; a preserved response to direct muscle stimulation; an unchanged muscle resting membrane potential in the PND model; an unaltered contractile response to exogenous potassium in the BC model; and the absence of histological damage in the muscle tissue. The venom showed no PLA₂ enzymatic activity.

Conclusion:
These findings support the conclusion that the neuromuscular blockade caused by M. surinamensis venom is predominantly due to α-neurotoxins, probably of the 3FTx class. There was no contribution from β-neurotoxins (such as PLA₂) or significant muscle tissue injury .

Introduction

Approximately 80% of the 8,000 annual envenomation cases reported in Argentina are attributed to scorpion stings, with Tityus carrilloi being the most medically significant species. Antivenom is the only specific treatment for severe cases. It is produced from the plasma of horses hyperimmunized with T. carrilloi venom. However, the venom supply represents a bottleneck in antivenom production. In Tityus serrulatus, a related species, recombinant toxins have been investigated as potential replacements or complements for native venom. A similar approach could be applied to T. carrilloi by identifying key toxin candidates and optimizing expression systems to improve antigenicity. Sodium channel-targeting toxins, which drive the most severe symptoms, have complex structures stabilized by four disulfide bonds. This study evaluated how antigenicity is influenced by refolding conditions that promote native-like conformations.

Methods

Fusion proteins 6xHis_MBP_TsNTxP and 6xHis_MBP_Tt1G were expressed in E. coli Shuffle® cells and purified using immobilized metal affinity chromatography under denaturing conditions. Protein expression, molecular weight, and purity were confirmed via SDS–PAGE and Western blotting. Antigenic recognition was assessed via ELISA using six independent antivenom batches against reduced/alkylated, refolded, and non-refolded protein versions.

Results

Soluble recombinant TsNTxP and Tt1G fused to MBP were successfully expressed in E. coli. All six antivenom batches showed stronger recognition of the refolded proteins, confirming the relevance of conformational epitopes. Moreover, TsNTxP exhibited stronger reactivity than Tt1G, supporting its potential as a complementary immunogen to T. carrilloi venom in antivenom production.

Conclusions

Our findings demonstrate that refolding significantly increased recognition by T. carrilloi antivenom for both TsNTxP (from T. serrulatus) and Tt1G (from T. carrilloi), highlighting the role of conformational epitopes in immune recognition.

Nanocrystals are unique crystalline particles with a highly ordered atomic structure and distinctive physicochemical properties which arise from their high surface-area-to-volume ratios. These characteristics have made nanocrystalline systems increasingly attractive for biomedical applications, particularly in biomanufacturing, drug delivery and therapeutic protein stabilisation. A biologically derived example of such nanocrystals is found in the parasporal inclusions of Bacillus thuringiensis (Bt), composed of Cry proteins. These crystalline protein structures have traditionally been employed as bioinsecticides due to their specificity and toxicity toward target insect species. However, studies have demonstrated their non-toxicity in humans and non-target insects, thereby highlighting their potential for safe biotechnological applications. The inherent crystallinity and high stability of Cry proteins have prompted exploration into their use as platforms for recombinant protein encapsulation.

This study investigates the feasibility of utilising Cry protein nanocrystals as stable scaffolds for the display of functional peptide tags. Specifically, the SpyTag peptide sequence was genetically fused to Cry proteins, and the resulting recombinant nanocrystals were produced in E. coli, purified, and characterised. The structural analysis via electron microscopy revealed irregularly shaped particles ranging from 700 to 800 nm in size. Functional validation was performed by incubating the SpyTag-functionalised crystals with SpyCatcher, confirming covalent complex formation through SDS-PAGE. These findings demonstrate that Cry-based nanocrystals can preserve the functionality of fused peptide tags, supporting their use as versatile platforms for recombinant protein stabilisation. This approach offers promising implications for the easy production and stable formulation of therapeutically relevant proteins with poor intrinsic stability.

This study investigated the effect of vomitoxin (DON) on intestinal motility in 6-week-old male C57BL/6J mice. Sixty mice were randomized into control and DON-treated groups, with six replicates of five mice each. The control group was gavaged with saline and the DON-treated group was gavaged with 3 mg/kg body weight of DON for 14 days. The results showed that DON significantly decreased the feed intake and weight gain (P＜0.001) and inhibited the growth of mice; shortened the intestinal propulsive distance, decreased the villus height, villus height/crypt depth, and intestinal wall thickness (P＜0.001), and disrupted the intestinal morphology and structure; down-regulated the expression of intestinal motility-related genes and proteins (P＜0.001); increased the number of M1-type inflammatory macrophages and activated the TLR-MAPK signaling pathway, up-regulated pro-inflammatory cytokine expression, and down-regulated anti-inflammatory cytokine expression (P＜0.001); activated the TNFR1-Casp3 signaling pathway and increased the secretion of apoptotic marker proteins (P＜0.001); and decreased the number of ICC cells (P＜0.001). This study demonstrated that DON causes intestinal motility disorders and morphological and structural damage and is associated with changes in the expression of related gene proteins, activation of inflammatory signaling pathways, activation of pro-apoptotic signaling pathways, and a decrease in the number of ICC cells. This study provides a theoretical basis for understanding the mechanism of DON toxicity and developing prevention and control strategies.