Marine biotoxins are natural chemical contaminants produced by a wide variety of microorganisms, such as bacteria and algae, among which ciguatoxins (CTXs) stand out.

CTXs enter the human food chain through the consumption of contaminated fish and other marine organisms, resulting in Ciguatera poisoning (CP), which is one of the most prevalent foodborne intoxications worldwide.

Although CP was historically confined to tropical and subtropical regions, the ongoing process of global warming is contributing to its geographical expansion. This occurs not only through the enhancement of environmental conditions favorable to CP-associated dinoflagellates but also via the introduction of non-native species. These invasive organisms may disrupt local ecosystems, establish new toxin reservoirs, and facilitate the persistence and spread of CTXs in previously unaffected marine environments.

This study focuses on the Canary Islands, a recognized hotspot for CTXs and one of the few regions with an established official CTX monitoring program. Seven specimens from six different species [(Caranx crysos, n=1), (Acanthurus monroviae, n=2), (Synaphobranchus sp., n=1), (Elagatis bipinnulata, n=1), (Cephalopholis taeniops, n=1), and (Lutjanus endecacanthus, n=1)] were analyzed. CTX-like activity was assessed in muscle, liver, and gonadal tissues (when available) using the Neuro-2a MTT cell-based assay.

CTX-like toxicity was detected in six of the seven fish analyzed. Liver and gonad samples showed higher levels of toxicity compared to muscle tissue. No significant differences were observed between cranial and caudal samples in terms of toxic potential.

In conclusion, the detection of CTX-like toxicity in the majority of the specimens analyzed underscores the importance of continued surveillance of and research on non-native fish species that might act as new vectors and reservoirs for the toxin. The early detection of CTXs in exotic marine organisms is essential, as it may pose an emerging risk to food safety, public health, and the environment.

Introduction: Edible mushrooms have many nutritional goods; they are rich in carbohydrates, proteins, minerals, and flavor compounds and are low in fat and calories. Moreover, mushrooms are rich in beneficial bioactive components with pharmacological properties, but they are also known to contain toxic substances. Recently, a new protein synthesis inhibitor enzyme from Boletus edulis, named edulitin 2, has been identified and characterized as a ribotoxin-like protein. The aim of this paper is to evaluate the cytotoxic profile of edulitin 2 on two colon adenocarcinoma cell lines, Caco-2 and HT29.

Methods: Edulitin 2 cytotoxicity was evaluated on Caco-2 and HT29 cells measuring cell viability and apoptotic/necrotic cell death in dose–response and time–response experiments. Furthermore, the effect of edulitin 2 on the integrity of the cultured cell monolayer was assessed using Transepithelial Electrical Resistance (TEER) measurements. TEER was evaluated in both Caco-2 monocultures and Caco-2/HT29 co-cultures, where the latter more closely resembled the intestinal epithelium.

Results: Edulitin 2 seems to require a quite long delay to exert its toxic effects. In fact, in our models, viability was slightly reduced after 24h of incubation with edulitin 2, but it was strongly affected after 48 and 72h. The half-maximal effective concentration (EC₅₀) values, calculated from dose–response curves at 72h, were in the 0.1 µM range. Flow cytometric analysis showed apoptotic cell death (early and/or late), but not necrosis. After 72 h, 1 µM of edulitin 2 strongly reduced TEER values in both models, thus suggesting the damage of cell monolayer integrity.

Conclusions: Our data demonstrate that edulitin 2 exerts a cytotoxic effect on intestinal cells after long time incubations (48-72 h). No necrotic lesions were observed in intoxicated cells at any tested concentration. The discrimination between apoptosis and necrosis is important as apoptosis does not cause inflammation.

Introduction: Ricin, a type 2 ribosome-inactivating protein, is a potent lethal toxin purified from castor bean seeds and has been classed as a chemical and biological weapon according to Schedule 1 by the OPCW. Ricin's cytotoxicity results from its lectin chain, which enables cell entry, and its active chain, which can depurinate different substrates, leading to multiple cell death pathways. Although the systemic toxicity of ricin has been extensively studied, its localized effects on the gastrointestinal tract remain a critical concern, particularly in cases of oral ingestion. The aim of this study is to elucidate ricin intoxication in human intestinal cell lines and determine its impact on the integrity of the epithelial barrier.

Methods: Ricin cytotoxicity in intestinal-derived HT29 and Caco-2 cells was evaluated in dose– and time–response experiments, with cell viability determined via an MTS-reduction assay. The effect of ricin on Caco-2 cell layer integrity was monitored by Trans-Epithelial-Electrical Resistance (TEER) measurements. The involvement of oxidative stress in ricin-treated cells was indirectly investigated by pretreating cells with reactive oxygen species scavengers. Cell death was determined through cytofluorimetric analysis of AnnexinV/PI positivity.

Results: Ricin showed high cytotoxicity toward HT29 and Caco-2 cells, with EC₅₀s ranging from 10 to 0.1 nM after 24-72 h of intoxication. Ricin strongly reduced TEER values in Caco-2 cells at 1-0.1 nM after 24 h of treatment. Ricin cytotoxicity (at 1 nM concentration) can be completely prevented by pre-incubating cells with antioxidant scavengers, demonstrating the involvement of oxidative stress in ricin cell death mechanisms. Moreover, ricin triggered only apoptosis without necrosis involvement.

Conclusions: Ricin has a potent cytotoxic effect on human intestinal cell lines that can be prevented by antioxidant scavengers. This study could pave the way for new pharmacological strategies to tackle ricin intoxication.

Saxitoxin (STX) is a potent neurotoxin produced by certain species of seaweed (dinoflagellates) and cyanobacteria. Saxitoxin has more than 50 analogs, but in this work, we studied only saxitoxin and its analogs GTX5, GTX2/3, and C1/2, which are mollusk paralyzing toxins (PSTs). Because it is highly lethal and its mechanism of action has not yet been fully elucidated, it can be used as a biological weapon. This project addresses an in silico study on the interactions of STX and its analogs selected with potential targets in order to understand its mechanism of action. Initially, the 3D structure selected in the PDB database was the Saxiphilin protein, identified by the PDB code ID 8D6M. The results obtained in this project through molecular docking performed by AMDockVina show us that some residues are present in some interactions of Saxifilin with STX and its analogs, and the residues that appear repeatedly are aspartic acid, glutamic acid, and glycine. Furthermore, we performed a search against the BLAST database for human sequences homologous to Saxiphilin. The sequence that presented the greatest identity of 41.22% and coverage of 84% was the Lactoferrin sequence. The next will step will be to perform new docking simulations with STX and its analogs using the 3D structre of Lactoferrin.

Bacteroidales are abundant Gram-negative bacteria that are present in the gut microbiota of most animals, including humans, where they carry out functions that are vital to the health of their host. To thrive in this competitive environment, Bacteroidales use sophisticated weapons to outmatch their competitors. Among these, BSAPs (Bacteroidales Secreted Antimicrobial Proteins) represent a novel class of bactericidal pore-forming toxins that are highly specific to their receptor, typically targeting only a single membrane protein or lipopolysaccharide. The molecular determinants conferring this high selectivity remain unknown. In this study, we therefore investigated the model protein BSAP-1 and determined which of its domains is involved in providing receptor specificity. We demonstrate that receptor recognition is entirely driven by the C-terminal domain (CTD) of BSAP-1 using a combination of in vivo competition assays, in vitro protein-binding studies, and structural analysis. Specifically, we show that deletion of the CTD abrogates BSAP-1 bactericidal activity by preventing receptor binding, while grafting the CTD to unrelated carrier proteins enables CTD-driven interaction with the BSAP-1 receptor. Building upon this discovery, we show that BSAPs can be categorized according to the structure of their CTD and that BSAPs within the same cluster are likely to target the same type of receptor. Additionally, we show that the CTD of BSAP-1 can be repurposed to generate probes for fluorescent labelling of membrane proteins in live cells. In summary, our research demonstrates that BSAP receptor recognition is driven by their CTD and that these can be engineered to develop novel tools for the investigation of Bacteroidales biology.

This study aimed to assess the occurrence of aflatoxin B₁ (AFB₁) and the total amount of aflatoxins (AFs) in commercial sweet potato and ready-to-eat sweet potato samples, collected from Punjab, Pakistan. A total of 480 commercial sweet potato and 365 ready-to-eat sweet potato samples were analyzed using HPLC. The results showed that 28.5% and 19.7% of commercial sweet potato and ready-to-eat sweet potato samples were found to be contaminated with AFs. The highest mean level of total AFs was 28.5 ± 4.2 µg/kg in commercial sweet potato samples from Daska (city), and the highest mean level in ready-to-eat samples of sweet potato was 14.50 ± 2.50 µg/kg, from the same city. The results showed that 40% of samples have total AF levels in the range of 1-10 µg/kg in commercial sweet potato samples (Jamki), and 13.3% of samples have total AF levels above 20 µg/kg from the city (Kila Soba Singh). The highest mean dietary intake levels of 3.16 µg/kg body weight/day were found in female volunteers in the age group 21-30 years. The results emphasized the need to implement effective storage practices that could be beneficial in minimizing the exposure of AFs to crops. Furthermore, the results would help implement strict laws for aflatoxins in food and food products.

Globally, mycotoxin contamination of food poses a severe hazard to public health and food safety. One of the most serious hazards to human health is the contamination of agricultural products with mycotoxins, which are toxic secondary metabolites generated by fungi. Chromatographic separation is a popular approach to detecting mycotoxins, often used in conjunction with mass spectrometry, which is an accurate method but requires specialized staff and a lengthy sample preparation process. Artificial intelligence (AI) is a highly precise and reliable technology for identifying mycotoxins in food. This unique method shows how multiple AI systems can be merged. Neural networks, machine learning approaches, and deep learning models were utilized to analyze complex datasets from various analytical platforms. Furthermore, we have emphasized the need for AI in conjunction with smart sensing technologies or other unconventional methods, such as spectroscopy, biosensors, and imaging methodologies, to identify mycotoxins more quickly and safely. Among other vital challenges in this area, we question the importance of employing large and diverse datasets to train AI models, debate the need to standardize the analytical approaches, and explore strategies for obtaining regulatory approval for AI-based procedures. Furthermore, this study provides some intriguing use cases and real-world business applications in which AI outperformed the more traditional methodologies in terms of its sensitivity and specificity and the time required by incorporating the most recent research findings and emphasizing the value of interdisciplinary collaboration among food scientists, engineers, and computer scientists for future paths in AI-enabled mycotoxin detection. Ultimately, AI has the potential to transform mycotoxin monitoring systems, enhancing food safety and public health globally.

The Pseudechis genus (black snakes) occurs throughout mainland Australia and Papua New Guinea, with the most widespread species being P. australis (Mulga snake). Black snakes in Australia are known to have a significant number of phospholipase A2 (PLA2) toxins, with ten previously being identified. P. colletti (Collett’s snake) and P. papuanus (Papuan black snake) both have PLA2-dominant venoms, with more than 90% of the whole venom being PLA2. This is consistent with in vitro studies demonstrating high PLA2 activity in the genus. We aimed to characterise the venom proteome of P. australis with a two-dimensional fractionation method involving reverse-phase high-performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis alongside bottom-up proteomics. After matching the venom peptides to the transcriptome, we characterised 98% of the venom and found that 89.8% was PLA2, and the remainder of the venom consisted of only three other protein families, snake venom metalloprotease (SVMP), l-amino acid oxidase (LAAO) and triosephosphate isomerase, which made up 6.0%, 2.5% and 0.03% of the whole venom, respectively. LAAO usually occurs in abundances less than 1.6% in snake venoms worldwide, except in Hoplocephalus stephensii, another Australian elapid, making it the second highest abundance in Australian snake venoms. In addition, the presence of SVMP was unusual for an elapid, again consistent with other Australian elapids. We identified eleven different PLA2 toxins in the venom of P australis, but the other three protein families all had one toxin. The study again demonstrates the unusual characteristics of the venoms of Australian snake venoms, and in particular the PLA2-dominant venom of the genus Pseudechis, consistent with its clinical effects of myotoxicity and anticoagulant activity.

Microcystis aeruginosa is a freshwater cyanobacterium capable of forming massive blooms and producing microcystins, hepatotoxins with adverse effects on aquatic organisms and human health. Among the emerging contaminants in aquatic environments, titanium dioxide (TiO₂) in nanoparticle (NP) form has garnered increasing attention due to its widespread industrial application and growing presence in natural water bodies. Its impact on the physiology and secondary metabolism of photosynthetic microorganisms remains insufficiently understood. In this study, M. aeruginosa (LEGE-91096) was cultured in the presence of TiO₂ NPs (commercial P25) at concentrations of 0.1 and 1 mg/L. Control treatments included humic acid as a surface coating (1:1 ratio). The stability of the nanoparticles in the culture medium was characterized by DLS, and their growth rate was assessed using the OECD 201 protocol. Metabolite profiling was performed via UHPLC-MS/MS, and data analysis was conducted using Compound Discoverer software to identify variations in bioactive compound production. According to OECD 201 results, TiO₂ nanoparticles at 0.1 and 1 mg/L did not significantly affect Microcystis aeruginosa growth. Ongoing analysis will further allow us to explain the molecular mechanisms underlying the interaction between TiO₂ nanoparticles and cyanobacterial metabolic pathways, particularly those involved in microcystin and other bioactive compound biosynthesis.

Acknowledgement: ACL wishes to thank AUIP for the awarding of the mobility scholarship within the Magallanes Program (2025). This work was also supported by the Portuguese Foundation for the Science and Technology (FCT) through the project NanoPlanet 2022.02340.PTDC and UIDB/04423/2020 and UIDP/04423/2020 contracts. IBO and MJA also acknowledge FCT funding for the Scientific Employment Stimulus Program (10.13039/501100001871.CEECIND/01368/2018 and 2023.06491.CEECIND).

Introduction: Recently, hemp products—seeds, oil, and tea—have been gaining popularity as food due to their high content of essential micro- and macro-nutrients. While hemp products offer significant nutritional benefits, they might pose a risk to consumers due to natural toxins such as cannabinoids. Cannabinoids include both psychoactive substances, such as Δ9-tetrahydrocannabinol (Δ9-THC), and non-psychoactive ones like cannabidiol (CBD). Even though hemp typically contains low levels of Δ9-THC, even trace amounts in food products can be concerning. Because of this, Δ9-THC content in products for human consumption has been regulated, with the maximum limits introduced under Commission Regulation (EU) 2023/915.

Methods: Nine cannabinoids—Δ9-tetrahydrocannabinol, Δ8-tetrahydrocannabinol, cannabinol, cannabidiol, Δ9-tetrahydrocannabivarin, cannabigerol, Δ9-tetrahydrocannabinolic acid (Δ9-THCA), cannabidiolic acid (CBDA), and cannabigerolic acid—were determined in 25 samples, including hemp seeds, oil, and teas. Samples were extracted with acetonitrile and diluted before analysis. Measurements were conducted using LC-MS/MS, operating in both positive and negative electrospray ionization (ESI+/-).

Results: Cannabinoid content varied with the tested materials. The highest concentrations of Δ9-THC and Δ9-THCA were found in tea samples (37.6–168.0 mg/kg and 28.2–69.7 mg/kg). Three of five oils contained significantly more (up to 398.3 mg/kg) Δ9-THC and Δ9-THCA than allowed. Only one hemp seed sample had Δ9-THC equivalents exceeding the maximum limit. Among all compounds, CBD and CBDA were found in the highest concentrations across all samples.

Conclusions: While hemp products offer health benefits, the risks from cannabinoids must be managed. Regulatory oversight, standardized testing, and public awareness are crucial for ensuring the safety of hemp-based foods. Although hemp contains low Δ9-THC levels, some tested foods exceeded the allowed concentrations, posing a risk to consumers.