Staphylococcal Enterotoxin B (SEB) and Shiga Toxins (STX1 and STX2) pose significant threats to food and water safety and are associated with bacterial strains that are considered to be potential bioterrorism agents (BA). Given their public health relevance and misuse potential, the rapid and accurate detection of toxin genes is essential for effective surveillance and response. The Brazilian Army plays a crucial role in biodefense efforts, including BA identification methods. This study focused on developing a multiplex qPCR assay for seb, stx1, stx2, and 16S rRNA gene targets. The design of the Stx2 primer was performed using PrimerQuest Tool; its specificity was evaluated in Primer-BLAST, and its secondary structure was analyzed using the OligoAnalyzer Tool. Staphylococcus aureus ATCC 25923, S. aureus NCTC 12493, Escherichia coli O157:H7, and E. coli ATCC 25922 strains were used for validation tests. Bacteria were grown in BHI media and incubated at 37 °C for 24 h. DNA extraction was performed using the MPTA016 pathogen kit in the Extracta32 system, and amplification was carried out on the Amplio96™ equipment. The probe concentration and DNA mass were 250 nM and 10 ng, respectively. The optimal temperatures revealed were 58.4 °C, 60.2 °C, and 58.4 °C for stx1 (Cq=22.49), seb (Cq=26.10), and 16S rRNA (Cq=14.91), respectively, supporting their inclusion in a multiplex setup. Therefore, 58.4 °C was selected for the assay. The 16S rRNA primer concentration showed no significant difference between 250 nM and 350 nM, allowing for the selection of 250 nM. For stx1, 350 nM provided a superior performance. No amplification was observed in non-toxin-producing strains, confirming the stx1 and seb primers' specificity. The stx2 primer and probe exhibited melting temperatures of 62 °C and 67 °C, respectively. Only heterodimer formation was observed (–13.29 kcal/mol). Our results showed that the primers were specific, allowing for their application to identifying the target genes proposed. This protocol was integrated into the Brazilian Army's biodefense system workflows to enhance its national and military biosurveillance.

Introduction: Cereulide toxin, a cyclic dodecadepsipeptide, is produced by Bacillus cereus and is a causative agent in food-borne outbreaks (FBOs). More than 470 FBOs related to B.c. toxins have been reported in 2023 (European Union One Health Zoonoses Report, EFSA and ECDC). Insufficient hygiene practices are the main reasons for intoxications. Cereulide affects several foods, including starch-rich foods such as pasta and rice. Along with validated methods and proficiency testing schemes, certified reference materials (CRMs) are key to ensure reliable measurements. The JRC aimed to produce a rice CRM containing cereulide at a low ng/g concentration.

Methods: Highly pure cereulide (Chiralix; Nijmegen, NL) was used to spike cooked rice. Thereafter, a rice powder candidate CRM was processed, which exhibits appropriate homogeneity and short-term stability. A characterisation exercise with ten expert laboratories using LC-MS/MS methods mostly based on ISO 18465 (Microbiology of the food chain – quantitative determination of emetic toxin (cereulide) using LC-MS/MS) was carried out. Each laboratory was requested to analyse nine independent sample replicates together with two QC samples (blank rice powder and rice powder contaminated at a very low cereulide concentration) under intermediate precision conditions.

Results: First, the measurement results were technically scrutinised. Some data sets had to be excluded from further evaluation (e.g. working range of method inadequate, QC results incorrect, and non-adherence to the provided instructions). After statistical evaluation, it could be concluded that the laboratory mean results were about normally distributed. No mean and variance outliers were detected. The mean of laboratory means amounted to 11.25 μg cereulide per kg rice, with an uncertainty related to characterisation of 0.23 μg/kg (2.04 %).

Conclusion: Once long-term stability data is available, a full uncertainty budget will be drawn up. The CRM will be used for method validation and quality control; therefore, it will enable laboratories to set up and safeguard reliable cereulide measurements in food and facilitate effective consumer protection.

The human neuromuscular junction (NMJ) is a specialized synapse responsible for modulating neurological influences on muscle contraction and enabling precise motor neuron-to-muscle cell communication. Compared to rodent NMJs, human neurophysiology remains poorly characterized, highlighting the need for more translatable disease models.

To study the NMJ's development and organization in humans, we employed a previously established system of neuromuscular organoids derived from induced pluripotent stem cells. This 3D co-culture system supports the concurrent development of motor neurons and skeletal muscle, exhibiting key molecular and structural hallmarks of NMJ formation, including characteristic synaptic architectures, acetylcholine receptor clustering, and spontaneous contractile activity.

To validate the physiological relevance of this model, we evaluated its response to known neurotoxins that disrupt neuromuscular transmission: botulinum neurotoxin A (BoNT/A), tetrodotoxin (TTX), and curare. Exposure to these agents resulted in reproducible impairments in synaptic communication and contractility, consistent with their known mechanisms of action. These findings demonstrate the model’s sensitivity to both presynaptic and postsynaptic disruption and support its utility for studying NMJ pathology under controlled conditions.

This system enables detailed investigations into the mechanisms underlying NMJ dysfunction and offers a manipulatable platform for screening therapeutic interventions. It advances our ability to replicate human NMJ biology and disease, including disruptions to neurotransmitter release and synaptic signaling, and provides a promising tool for identifying molecular targets in neuromuscular and motor neuron disorders.

Physalia physalis, or Portuguese Man O’ War, is one of the most venomous cnidarians. Despite its known toxicity, detailed studies on its venom components remain limited, specifically concerning their neuroactive effects. This study seeks to explore the effects of total/crude venom (CV), as well as high-molecular-weight (HMW) and low-molecular-weight (LMW) venom fractions, using Drosophila melanogaster as an in vivo model. Oral administration enabled the assessment of fly mortality and locomotor behavior. CV and LMW venom fractions induced similar levels of mortality, while HMW and control groups showed comparably lower rates. The CV-treated group displayed acute locomotor impairment, with an acute toxicity pattern in climbing and moderate symptom variability. In contrast, the HMW-treated group showed minimal impairment, characterized by a predominantly straight trailing climbing pattern and reduced symptom variability. The LMW-treated group exhibited pronounced locomotor dysfunction, displaying a variability of abnormal climbing behaviors and symptoms. Real-time monitoring revealed an initial phase of hyperactivity followed by prolonged paralysis, resulting in reduced locomotor activity and altered circadian rhythm. The results indicate that neurotoxicity resides primarily in LMW components, demonstrating the utility of Drosophila for venom screening and neurodegenerative-related research. This work supports future proteomic and transcriptomic studies identifying novel bioactive compounds with potential therapeutic applications.

Aflatoxin B1 (AFB1) is a major global health concern due to its carcinogenic effects in humans and animals. Because AFB1 is highly stable once produced, the most effective strategy is to prevent its synthesis by toxigenic fungi. Currently, plant extracts have been increasingly studied for this purpose. These extracts often contain polyphenols as active compounds against AFB1, which are effective but unstable over time. Therefore, it is necessary to develop new formulations that enhance their preservation, storage, and applicability.

Microencapsulation through spray drying is a promising approach. This technique converts a liquid into a dry powder by spraying it into a hot air chamber, embedding the bioactive compounds in a protective matrix. Spray drying offers several advantages, including high encapsulation efficiency, stability, and low production costs, making it a practical solution for preserving plant-derived antifungal agents targeting AFB1.

This study aimed to evaluate the potential of spray drying encapsulation to preserve the anti-aflatoxin activity of different plant aqueous extracts able to inhibit AFB1 synthesis. A total of 11 coating materials were screened based on their impact on Aspergillus flavus growth and AFB1 inhibition. Pectin, maize starch, and maltodextrin were identified as the most suitable, showing no effect on fungal development and AFB1 synthesis. Maltodextrin was selected to encapsulate aqueous extracts of Mimosa tenuiflora and Aloysia citrodora. These extracts inhibited AFB1 by 66% and 76 % at the concentration of 0.3 mg of dry matter /mL and displayed IC50_AFB1 of 0.15 and 0.11 mg/mL, respectively. After encapsulation, the extracts inhibited AFB1 by 60% (IC50 _AFB1= 0.29 mg/mL) and 65% (IC50 _AFB1= 0.26 mg/mL) in a dose-dependent manner.

Different core-to-wall ratios were tested, with 1:2 being the most effective, preserving 92% of anti-AFB1 activity. After 1 year of storage, the encapsulated extract preserved 90% of its activity, which is better than freezing (61%). Spray drying proved to be an efficient, low-cost method for preserving anti-aflatoxin properties of plant-based extracts.

Ciguatera poisoning (CP) is a foodborne illness of significant public health concern, which is caused by the consumption of fish contaminated with ciguatoxins (CTXs), lipophilic neurotoxins that bioaccumulate throughout the marine food web. Despite its global relevance, the specific morphological and ultrastructural effects of CTX exposure on fish tissues remain poorly understood. This study aimed to characterize histological and ultrastructural alterations induced by dietary CTX exposure and how toxins accumulate in tissues of the dusky grouper (Epinephelus marginatus).

Captive-born dusky groupers were fed an experimental diet composed of CTX-contaminated amberjack and dusky grouper tissue, with an average concentration of (0.109 ± 0.003 ng Eq. CTX1B/g of flesh). Tissue samples were collected at 24, 60, and 108 days. CTX estimation was performed using a cell-based assay in the liver, muscle and gonads, and histological examinations of liver, kidney, gonads, muscle, and intestinal tissues were conducted. Additionally, hepatic samples were analyzed by light and transmission electron microscopy.

Histopathological findings included cytoplasmic vacuolization, localized inflammation, and increased connective tissue development. Ultrastructural analysis showed signs of hepatocyte necrosis, nuclear condensation, and displacement of cellular organelles in CTX-exposed individuals. Despite toxin exposure, tissue alterations were mild and predominantly localized to the liver, with no significant histological changes detected in the intestine, gonads, or muscle. CTX accumulation was first detected in the liver and gonads, with hepatic toxin levels peaking at 60 days (0.868 ± 0.121 ng Eq. CTX1B/g) and subsequently declining, while gonads exhibited a delayed increase in toxin concentration.

These results demonstrate that dietary exposure to CTXs induces significant hepatic structural damage in E. marginatus and that toxin distribution across tissues evolves over time. This study provides experimental evidence supporting the role of sublethal CTX exposure in the onset of tissue-specific damage in adult reef fish under toxin concentrations relevant to natural environmental conditions.

The toxicity of snake venoms is mostly determined in mice, the most frequently used mammalian model. These assays are expensive and subject to strict ethical regulations. To overcome these drawbacks, we evaluated the potential use of alternative models for snake venom testing. Caenorhabditis elegans is a free-living nematode widely used in biomedical research.

The wild-type N2 strain was used in all experiments. Ten L4-stage worms were pipetted into each well of a 96-well microplate containing M9 buffer. Worms were then exposed to six different concentrations of snake venom (0, 200, 400, 600, 800, and 1000 µg/ml) in a M9 buffer. The venom used was from Naja kaouthia (monocled cobra). Plates were incubated at 20 °C for 24 hours, after which motility and viability were assessed. Worms exhibiting movement were considered alive, while those that became straight and rigid were considered dead. Motility was quantified using the Wmicrotracker device (Phylumtech).

The results show lethality of N. kaouthia venom in a dose-dependent manner, and the LD50 was estimated to be 520 μg/mL. Increased mortality was preceded by a decrease in motility, and this could be attributed to the neurotoxic activity of the venom.

High doses of N. kaouthia exhibited significant effects on the viability and motility of the free-living nematode C. elegans, suggesting a potential utility for studies on snake venom toxicity and their neutralization. Nevertheless, the mass of venom required for the LD50 determination of L4 mortality was high. Despite this drawback, considering the cost and difficulty of venom obtaining, a study on other early larval stages must be tested in order to determine if the doses of venom required could be lower. Nevertheless, the usefulness of testing toxic mechanisms in wild-type, transgenic, or mutant strains makes C. elegans a suitable model for snake venom toxicity testing.

Venoms have evolved at least 100 times in the animal kingdom, with their main biological roles being prey aquisition and self-defence. Snake venoms show extreme variability in composition at all taxonomic levels, both within and between species. The selective pressures underlying this variation have long been debated. Since snake venoms are used for both foraging and defence, we hypothesise that venom composition may represent a trade-off between lethal/immobilising predatory toxins and pain-inducing defensive toxins. Here, we use the Eurasian viper genus Vipera to test the hypothesis that snakes feeding on weakly defended prey, such as insects, should have venoms more adapted for defence than snakes feeding on well-defended prey such as rodents. We tested the nociceptor-activating (pain-inducing) capacity of the venoms of populations of the largely insect-eating Vipera ursinii complex and of the primarily mammal-eating V. ammodytes, V. aspis and V. berus. We used high-throughput Ca²⁺ imaging and deconvolution microscopy on F11 cells and native dorsal root ganglia cells, respectively, to test the algesic activity of the venoms of European vipers. Contrary to our expectations, none of the venoms resulted in nociceptor activation, irrespective of diet. We conclude that selection for defence is unlikely to have played a significant role in driving the evolution of venom composition in this genus.

Food contamination risks from mycotoxins pose a serious threat to public health. These toxic compounds are produced by filamentous fungi such as Aspergillus, Penicillium, Fusarium and Alternaria under specific temperature and humidity conditions. They can affect a wide range of agricultural products (nuts, cereals and their derivatives) during both the pre- and post-harvest stages. Among these, aflatoxin B1 is especially dangerous and has been classified as carcinogenic to humans. As part of the project PRIN 2022 PNRR “P20223P48S” entitled “Implementing advanced elemental and chemical analysis for quality, safety and traceability assessment of PGI and PDO agri-food products” funded by the European Union NextGenerationEU, we evaluated the feasibility of detecting aflatoxins in pistachio seeds using Fourier-Transform Infrared (FT-IR) spectroscopy. As a rapid and non-destructive technique, FT-IR holds significant potential for identifying contaminants in food, including mycotoxins. In the framework of the above project, FT-IR spectroscopy was initially applied to investigating the responses of pistachio samples from different locations in Sicily. The results demonstrate the ability of the FT-IR technique to discriminate between pistachios of different geographical origins, highlighting the connection between terroir and the chemical composition of the crops. Further studies are underway to evaluate the method’s potential to distinguish between contaminated and uncontaminated seeds by detecting the presence of aflatoxins B1, B2, G1, and G2. FT-IR analysis is being used to compare the chemical profiles of contaminated samples with those of mycotoxin-free ones.

Introduction: Snake venom represents a relevant source of bioactive molecules with therapeutical potential, particularly for modulating complex pathways such as the coagulation cascade. Snake venom metalloproteinases (SVMPs) are enzymatic proteins classified according to their structural domains: (1) class P-I SVMPs, which exclusively contain the metalloproteinase catalytic domain; (2) class P-II SVMPs, which possess, in addition to the catalytic domain, a disintegrin domain; and (3) class P-III SVMPs, which contain metalloproteinase, disintegrin-like, and cysteine-rich domains. Class P-III SVMPs may also associate with C-type lectin-like domains, which are then classified as P-IIId. Snake venom C-type lectin-like proteins (snaclecs) are non-enzymatic proteins that are structurally organized as heterodimers (α and β subunits). They play a fundamental role in hemostasis through receptor-mediated interactions despite lacking carbohydrate-binding properties. In previous work from our group, a proteolytically active fraction from Bothrops atrox venom showed reactivity with anti-C-type lectin (CTL) antibodies. The present study aimed to characterize the biochemical and functional properties.

Methods: The identified fraction from B. atrox venom was evaluated using SDS-PAGE, plasminogen activation assays, hemagglutinating-activity assays, platelet aggregation assays using washed platelets and platelet-rich plasma (PRP), Western blot analysis for reactivity with anti-SVMP-P-I and -P-III antibodies, and proteolytic-activity assays on extracellular matrix (ECM) components.

Results: The selected fraction induced aggregation in PRP, but only weakly in washed platelets. However, it strongly promoted the aggregation of washed platelets in the presence of human fibrinogen. Western blot analysis revealed a ~26 kDa band recognized by anti-CTL and anti-SVMP-P-I antibodies, while the anti-SVMP-P-III antibody identified bands of ~26 kDa and between 43 and 72 kDa. The fraction showed no hemagglutinating activity, did not activate plasminogen, but effectively degraded ECM components.

Conclusion: These results suggest the presence of a P-IIId in B. atrox venom. Further studies are necessary to fully elucidate its structure and biological activities.