Introduction:
Experimental evidence has shown that the rate of ATP synthesis correlates more strongly with proton movement along the surface of the inner mitochondrial membrane (IMM) than with the transmembrane proton concentration gradient in the bulk aqueous phase. This has prompted a revision of the classical chemiosmotic theory of mitochondrial ATP production. It has been proposed that lateral proton transport along the IMM surface generates an electric potential that drives ATP synthase activity. Moreover, recent studies have reported proton accumulation and storage on the membranes of the myelin sheath and endoplasmic reticulum (ER), suggesting that surface-based energy storage via absorbed protons may be a fundamental principle of cellular bioenergetics. In this study, we examined whether membranes composed of different types of phospholipids and those enriched with either acidic or basic bee venom proteins differ in their capacity to enhance proton absorption at the membrane surface.

Methods:
Five types of phospholipids commonly found in myelin sheath and ER membranes were purified using immunoaffinity chromatography. Anionic and basic protein isoforms were isolated from bee venom via CM Sephadex C-50 chromatography. Liposomal membranes were prepared through ultrasonic frequency dispersion of phospholipid suspensions. Proton absorption at the membrane surface was assessed by measuring pH differences between liposome suspensions and pure water.

Results:
Membranes of liposomes enriched with anionic proteins showed enhanced proton absorption compared to protein-free liposomal membranes. Among all tested conditions, phosphatidylethanolamine membranes enriched with basic proteins exhibited the highest proton absorption, while other basic protein-enriched membranes showed minimal absorption.

Conclusions:
These findings support a revised model of membrane-associated energy storage, which holds potential pharmacological implications. A deeper understanding of membrane-based energy accumulation mechanisms may facilitate the development of novel therapies targeting age-related and pathological impairments in cellular bioenergetics.

Introduction: CYGNOS BioTech is pioneering the development of broad-spectrum antiviral (PVX) and antitumor (MMX) biologic products for both veterinary and human applications. Our innovative technology has led to the issuance of nine patents covering composition, process, and usage. The US FDA has granted two Investigational New Animal Drug Applications for 1) a non-specific antiviral drug for companion animals and 2) an antitumor drug for stage II and III canine oral malignant melanoma.

Methods: PVX and MMX are derived from cobra venom toxin (NT3) through fractionation followed by proprietary chemical processes to achieve detoxification and specific biologic activities.

Results: Preliminary data suggest that PVX's antiviral activity targets host-cell mechanisms rather than the virus. PVX inhibits Protein Kinase activities, potentially affecting cellular pathways related to transcription and translation. Clinical studies in dogs, cats, and horses demonstrate significant antiviral and antitumor effects without adverse events. PVX appears effective for most viral infections, including enveloped RNA and DNA viruses such as canine distemper virus (paramyxovirus), Human and Feline Herpes viruses, and Feline Corona virus causing Feline Infectious Peritonitis (FIP).

Conclusions: The global community faces increasing threats from emerging zoonotic diseases and bio-terrorism activities. The potential for a new pandemic or the release of genetically engineered viruses is a real and devastating concern. CYGNOS BioTech’s technology serves as a potential platform to treat infectious diseases, applicable across veterinary, public health, and military sectors. The availability of a drug capable of addressing emerging and genetically engineered viruses as a first line of defense could have a profound impact.

Throughout history, there have been several instances of toxins being used as biological weapons, including marine toxins produced by dinoflagellates and other filter-feeding organisms, such as cyanobacteria. Furthermore, they may be associated with food seafood poisoning. Despite being potentially toxic, marine toxins present a vast chemical and biological diversity, making them an exceptional reservoir for discovering new medicines. Saxitoxin (STX) is a powerful paralyzing marine toxin from dinoflagellates and cyanobacteria. Aiming at exploring the value of marine toxins and other associated bioactive molecules as a source of potential therapeutic agents for biotechnological applications, the objective of this study is to evaluate the binding mode of STX to different targets and the in vitro consequences of their interaction. These results will help us propose paths of therapeutic activities for STX. We used the molecular docking methodology for in silico assays. The structures of SXT and neosaxitoxin, obtained from the Protein Data Bank (PDB) database, were used as ligands. To identify new therapeutic targets in humans, we used the SwissTargetPrediction server, which reported 18 possible targets. We then performed molecular docking simulations with the AutoDock Vina 1.1.2 program with default parameters. Of all the targets, the best pose was in serotonin-6 receptor reported by AutoDock Vina, which presented a score of -5.5 and -5.7, and hydrogen bond interactions with Ser111, Leu183, Val107, Asn288, Thr196, Tyr310, and Leu183 residues, to saxitoxin and neosaxitoxin. The second-best pose in the carbonic anhydrase XII receptor reported presented a score of -8.06 and -6.03 and hydrogen bond interactions with Ala129, Ser130, Thr199, and Pro201 residues to saxitoxin and neosaxitoxin. From this perspective, molecular dynamics simulations will be performed for the most promising complexes, and atomic force microscopy tests will be performed to identify the interaction of STX and neosaxitoxin with their targets.

Pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs) are natural toxins which can be found in honey and are recognized for their hepatoxic and neurotoxic properties. Given honey's widespread consumption as part of the human diet, ensuring its safety is mandatory [1]. Therefore, an accredited method [2] was employed to detect 33 PAs and 21 TAs in honey, based on ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) with a quadrupole linear ion trap (Q-TRAP) system. Honey samples were extracted using a solvent mixture of methanol and water acidified with formic acid. In a comprehensive analysis of 33 Czech and Spanish honey samples from diverse botanical origins, it was found that among the 21 Czech samples, 6 tested positive for PAs and TAs. However, only two of them exhibited quantifiable residues of echimidine (1.4 µg/kg), lycopsamine (197 µg/kg), and intermedine (140 µg/kg). Among the 12 analyzed Spanish honeys, 5 tested positive for nine PAs (intermedine, lycopsamine, heliotrine, europine, seneciphylline, senecionine, senecivernine, echimidine and lasiocarpine). Total PA concentrations in Spanish honeys ranged from 0.4 to 65.1 µg/kg. Contrastingly, none of the monitored TAs were detected in these analyzed samples. The new Commission Regulation 2023/915 does not currently include maximum residue limits for these compounds in honey. Instead, it outlines limits solely for pollen-based food supplements. In light of the demonstrable potential health hazards associated with these toxins, it is expected that accurate analytical methods and more extensive investigations for PAs and TAs in honey-based products will be developed to enhance their safety for potential consumers.

The occurrence of marine toxins such as tetrodotoxin (TTX) and saxitoxin (STX) in seafood represents a serious threat to food safety due to their high neurotoxicity, which can result in severe poisoning incidents and, in some cases, fatalities. Most conventional detection techniques are tailored to identify toxins from a single family, making them less effective when both toxins are present simultaneously. Given recent reports of co-occurrence of TTX and STX in seafood, there is a clear demand for advanced bioanalytical multiplexed platforms capable of detecting both toxins in a single assay. This study introduces a novel electrochemical immunosensor designed to detect concurrent TTX and STX. The biosensor features a four-electrode array: two electrodes dedicated to identifying the presence of the toxins in seafood samples, and two control electrodes serving as reference points aligned with established regulatory safety thresholds. By integrating the output signals from the biosensor, the system enables not only toxin identification but also rapid assessment of whether concentrations exceed acceptable safety levels. This multiplexed detection strategy provides a user-friendly, time-efficient tool for screening seafood, contributing to enhanced consumer safety. Moreover, it represents a promising step toward the development of comprehensive biosensing platforms for multi-toxin detection in marine environments.

Introduction: Phospholipase A₂ (PLA2) is one of the largest families of snake toxins. Snake venom PLA2s affect various systems in prey, including the cardiovascular one; however, data on their effect on cardiomyocytes are practically absent. In this work, we investigated the influence of heterodimeric PLA2s HDP-I and HDP-II from the venom of Vipera nikolskii on isolated rat cardiomyocytes.

Methods: Cardiomyocytes were prepared from the hearts of anesthetized animals. Only rod-shaped cardiomyocytes with clear striations were used. They were stained with a fluorescent probe, Fura-2 or Fura-4. The fluorescence in cardiomyocytes was measured using the Cell Observer fluorescent station based on an AxioVert 200M motorized inverted microscope equipped with a 10x PlanApochromat objective and Orca-Flash R2 monochrome camera.

Results: After HDP2 (10 μM) application, there was a prolonged lag phase with no changes in cell morphology or probe signal. Then, fluctuations in the cytosolic calcium level ([Ca2+]i) were observed, followed by a sharp increase in [Ca2+]i and the onset of hypercontracture, with subsequent disruption of the plasma membrane. In the experiments under conditions of electrical stimulation, after recording control values of the fluorescent response, HDP II was added and changes in the fluorescence were recorded until cell death. In all cases, a “pause” in contractions was observed, characterized by an increase in intracellular calcium and decrease in the amplitude of the fluorescent response. Then, the rhythm recovered, and later, hypercontracture was observed followed by cell destruction. The times to these events depended on the HDP II concentration and the efficiency of stimulation. No effects on cardiomyocytes were observed upon application of 1 µM HDP I.

Conclusions: In isolated rat cardiomyocytes, PLA2 HDP II from Vipera nikolskii venom induced an elevation in cytosolic calcium level followed by hypercontracture and cell destruction. This is the first indication for the direct effect of snake venom D49 PLA2 on cardiomyocytes.

Introduction

Co-expression of the Kv1.1 and Kv1.2 subunits in the cells of the central nervous system results in the predominant formation of heterotetrameric voltage-gated potassium Kv1.1/Kv1.2 channels, which regulate neuronal excitability. An inherited mutation of the Kv1.1 channel, T226R, which is associated with episodic ataxia and epilepsy, disrupts the Kv1.1 channel's activity. The influence of this mutation on the properties of the heterotetrameric Kv1.1/Kv1.2 channel needs to be investigated.

Methods

Expression plasmids encoding fluorescently labeled mKate2-Kv1.1(T226R) and tandem dimers mKate2-Kv1.1-Kv1.2 and mKate2-Kv1.1(T226R)-Kv1.2 were constructed and used to transfect Neuro-2a cells. Recombinant peptide toxins and the fluorescent ligand HgTx-GFP were produced. Confocal microscopy was used to study the ligand binding and distribution of the channels in the cells. The electrical activity of the heterotetrameric channels formed by the tandem dimers was studied using a whole-cell patch-clamp technique.

Results

The expression of mKate2-Kv1.1(T226R) and mKate2-Kv1.1(T226R)-Kv1.2 in the plasma membrane and the ability of the dimers to form heterotetrameric channels with a preserved pore structure were studied by binding the fluorescent ligand HgTx-GFP. The binding affinities of peptide blockers for the wild type and the mutant heteromers were studied in a competitive assay. The activation and deactivation constants, as well as the voltage dependence, were measured for the heteromeric channels formed by the Kv1.1(T226R)-Kv1.2 and Kv1.1-Kv1.2 dimers. The essential differences that were identified in these studies are discussed.

Conclusions

The data obtained provide insights into the molecular mechanisms of epilepsy associated with the T226R mutation in the Kv1.1 subunit of the Kv1 channels.

Acknowledgements. This work was supported by the Russian Science Foundation (project 25-24-00191).

Aspergillus flavus is a major fungal pathogen in maize, significantly impacting kernel quality and food safety through mycotoxin contamination. This study examined the physiological and biochemical effects of A. flavus inoculation on maize, assessing kernel production, fungal proliferation, mycotoxin accumulation, and key biochemical parameters. A controlled field experiment was conducted, comparing inoculated (IN) and control (CT) maize. Inoculated maize exhibited a significant reduction in kernel number per ear length (20.14 ± 0.43 vs. 21.25 ± 0.33; p < 0.05), indicating compromised reproductive success. Mold count was significantly higher in IN maize (6.32 ± 0.13 log CFU/g) compared to CT (5.55 ± 0.17 log CFU/g; p < 0.01), confirming enhanced fungal colonization. The concentration of Aflatoxin B1 (AFB1) increased drastically in IN maize (139.46 ± 38.64 µg/kg) compared to CT (0.11 ± 0.07 µg/kg; p < 0.01), posing serious food safety concerns. However, no significant differences were observed in fumonisin B1 (FB1), deoxynivalenol (DON), zearalenone (ZEA), starch, protein, or total polyphenols between treatments, suggesting that short-term fungal infection primarily affects kernel formation and mycotoxin accumulation rather than biochemical composition. These findings emphasize the critical role of A. flavus in maize contamination and highlight the necessity of mitigation strategies to reduce fungal colonization and aflatoxin accumulation, ensuring maize safety and quality.

Acknowledgement

Project No. TKP2021-NKTA-32 has been implemented with the support provided by the Ministry of Culture and Innovation of Hungary from the National Research, Development and Innovation Fund, financed under the TKP2021-NKTA funding scheme. This research is funded by the National Research, Development and Innovation Fund of Hungary project No. 2018-1.2.1-NKP-2018-00002.

Naja sumatrana venom cytotoxin (CTX) is a three-finger toxin which has been reported to exhibit caspase-dependent and mitochondrial-mediated apoptosis without transitioning into necrosis in human breast cancer MCF-7 cells. It has also been found to cause membrane permeabilization, leading to secretory phenotype changes. This study investigates the time- and dose-dependent alterations of the intracellular proteome by the cytotoxin from system toxicology and mechanistic perspectives. The cytotoxicity of CTX was determined at 8 and 24 h. MCF-7 cells were treated with IC₂₀, IC₅₀, and IC₈₀ of CTX at 8 h and 24 h. Label-free quantitative (LFQ) proteomics was performed to determine the intracellular proteome changes caused by CTX. Bioinformatic analyses were then carried out to identify the significantly distinguished proteins and pathway enrichment for protein–protein interaction networks. The LFQ proteomics revealed distinctive protein families significantly distinguished at 8 and 24 h. At 8 h, CTX targeted the cell cycle checkpoints and estrogen signalling, Notch, WNT/ β-catenin, and Rho GTPase pathways. On the other hand, at 24 h, proteins related to oxidative stress and MAPK-mediated apoptosis pathways were detected. There was also a concentration-dependent substantial elevation in different protein families associated with chromatin organization, DNA damage checkpoints, and DNA fragmentation during apoptosis. The protein–protein interaction (PPI) networks demonstrated several potential clusters related to toxin internalization, which could lead to intracellular accumulation that triggers cytotoxicity. Nevertheless, the exposure to CTX did not have notable effects on cellular metabolism. In conclusion, CTX induced significant time- and dose-dependent alterations of the intracellular proteome, revealing the involvement of apoptosis-related pathways and the suppression of proliferation in MCF-7. Integrative proteomics and system toxicology uncovered the mechanistic insights of CTX’s progressive actions in causing cell death.

Bacillus thuringiensis is a Gram-positive, spore-forming bacterium widely recognized for its potent insecticidal properties. It is recognized as a key biological control agent and a more sustainable alternative to chemical pesticides. This study focuses on the INTA Mo1-10 strain, preserved in our collection and originally isolated from grain milling residues in Argentina. The objective was to elucidate the relationship between its genetic profile and biocidal activity through comprehensive genotypic and phenotypic characterization. Genomic DNA analysis, performed via Illumina sequencing, identified nine insecticidal protein genes: cry1Aa, cry1Ab1, cry1Ca, cry1Da, cry1Ia, cry2Ab, cry9Ea, vip3Aa, and spp1Aa. Also, the amino acid sequence identities ranged from 77% to 100%. Microscopic analyses revealed that sporulated cultures had bipyramidal crystals that were fully separated from spores, as observed using phase contrast and scanning electron microscopy. The presence of a parasporal crystal protein band of approximately 130 kDa was further confirmed via SDS-PAGE analysis. Bioassays demonstrated the strain's broad-spectrum biocidal activity, achieving over 83% efficacy against Cydia pomonella (Lepidoptera: Tortricidae) and Spodoptera frugiperda (Lepidoptera: Noctuidae) when incorporated into insect diets. For Panagrellus redivivus (Tylenchida: Panagrolaimidae), the strain was grown as a lawn on TSA agar plates before nematode inoculation, which resulted in comparably high mortality. Furthermore, the strain exhibited 45% larvicidal activity against Aedes aegypti (Diptera: Culicidae) when applied to water-filled containers. All bioassays utilized spore/crystal complexes at appropriately high doses to ensure effectiveness. The findings establish a clear correlation between the strain's genetic makeup and its biocidal efficacy. Notably, the insecticidal protein gene profile of INTA Mo1-10 closely aligns with that of other B. thuringiensis serovar galleriae strains. These results underscore the considerable potential of INTA Mo1-10 as an effective and versatile biocontrol agent, contributing to sustainable pest control strategies.