Toxins doi: 10.3390/toxins18060250

Authors: Swapnil Kiran Siripuram Srinivas Karthikeyan Vasudevan

Snakebite envenoming is a major public health concern in India that causes economic hardship for the rural populations. We estimated the per capita economic burden of snakebites in a rural population by quantifying mortality and morbidity rates. We interviewed for outcomes of snake envenomation of 541 participants from 205 villages in Jagtial, Telangana, from 2010 to 2020 using a community-based snowball sampling approach. Snakebites caused 24.21% morbidity and 12.75% mortality. The age-adjusted mortality rate and age-adjusted morbidity rate were 11.72 and 22.8 per 100,000 people, respectively. The overall annual burden of snakebites was 31.96 Disability-Adjusted Life Years (DALYs) per 100,000 people. The mean annual earning opportunity cost and the mean annual mortality cost were USD 321.27 and USD 24,016.54 per person, respectively. We highlight the need for targeted public health interventions such as monetary compensation and community support schemes to reduce the morbidity and mortality rates in rural areas.

Toxins doi: 10.3390/toxins18060249

Authors: Rongyan He Shuting Xiao Xiaoying Liang Qiuyu Cao Shaoxian Wu Sulan Luo

Chronic neuropathic pain, particularly diabetic neuropathic pain and postherpetic neuralgia, severely impairs patients’ quality of life due to their complex mechanisms and recurrent, long-term nature, making treatment challenging. This study aimed to evaluate the analgesic efficacy of α-conotoxin GeXIVA[1,2], a selective antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR), in rat models of diabetic neuropathic pain and postherpetic neuralgia and investigate its associated physiological and pathological effects. GeXIVA[1,2] was administered continuously for three weeks, with mechanical hypersensitivity assessed through pain sensitivity tests, and behavioral assessments conducted to examine motor coordination and gait. Additionally, neural tissue structure and inflammation were analyzed. The results demonstrated that GeXIVA[1,2] significantly alleviated mechanical hypersensitivity in both diabetic neuropathic pain and postherpetic neuralgia models, with greater efficacy than gabapentin and no signs of tolerance. Behavioral tests indicated no significant effects on motor coordination or gait. Further analysis revealed that GeXIVA[1,2] reduced pro-inflammatory cytokine levels, decreased immune cell infiltration, and promoted repair of damaged nerve fibers. Overall, these findings suggest that GeXIVA[1,2] exerts analgesic effects through anti-inflammatory and neuroprotective mechanisms, providing a potential new therapeutic strategy for diabetic neuropathic pain and postherpetic neuralgia.

Toxins doi: 10.3390/toxins18060248

Authors: Andreas A. Argyriou Emmanouil V. Dermitzakis Dimitrios Rikos Georgia Xiromerisiou Panagiotis Soldatos Maria Chondrogianni Eleni Mavraki Michail Vikelis

Trigeminal neuralgia (TN) ranks among the most excruciating neuropathic pain syndromes, characterized clinically by multiple daily episodes of unilateral, paroxysmal, electric shock-like facial pain. The daily activities and quality of life of affected patients are profoundly diminished. First-line pharmacological agents, such as carbamazepine and oxcarbazepine, provide initial relief for many patients. However, a significant proportion eventually develops refractory symptoms or experience intolerable adverse effects, leading to the discontinuation of traditional oral medications. For these patients with complex clinical phenotypes who fail to respond or are intolerant to these therapies, alternative pharmacological strategies are required before considering invasive surgical procedures. Over the past two decades, botulinumtoxin type-A (BoNTA) has become an effective and safe, minimally invasive therapeutic option for refractory TN. This review provides a practical framework for BoNTA use in the clinical setting of refractory TN. To connect the pathophysiological background with clinical patient care, we summarize the current understanding of TN pathophysiology, the proposed mechanisms by which BoNTA exerts its antinociceptive effects and the evolving clinical evidence supporting its efficacy and safety. We also critically examine dosing protocols, injection techniques, long-term outcomes and the integration of BoNTA into the management algorithm of refractory TN.

Toxins doi: 10.3390/toxins18060247

Authors: Yaning Huang Qiao Li Jiaqi Wang Yulei Wang Daolong Liao Xiaodong Sun Haitao Li

To elucidate the mode of action of Mpp5Ab1 against Colaphellus bowringi Baly larvae, this study aimed to identify midgut proteins interacting with the toxin. A validated bait plasmid, pBT3-SUC-mpp5Ab1, was used to screen a larval midgut cDNA library via the split-ubiquitin yeast two-hybrid system. A total of 33 positive clones representing five distinct proteins were obtained, among which bioinformatic analyses prioritized three candidates: Cb-RP-L23e, Cb-CTSL, and Cb-TsetseEP. Subsequent bimolecular fluorescence complementation (BiFC) assays in Sf9 cells specifically confirmed interactions between Mpp5Ab1 and both Cb-CTSL and Cb-TsetseEP, whereas no fluorescence signal was observed for Cb-RP-L23e. Molecular docking further supported stable interactions between Mpp5Ab1 and the validated candidate proteins through hydrogen bonds, salt bridges, and hydrophobic interactions. These findings suggest that Cb-CTSL and Cb-TsetseEP may function as candidate interacting proteins associated with the activity of Mpp5Ab1 in the larval midgut of C. bowringi. Overall, this study provides new insight into the molecular interactions of Mpp5Ab1 and establishes a foundation for future functional studies on its insecticidal mechanism and receptor validation.

Toxins doi: 10.3390/toxins18060246

Authors: Ho-Hsiang Chang Yu-Li Lin Chi-Chong Tang Chiu-Huang Kuo Chih-Hsien Wang Bang-Gee Hsu

Background: Arterial stiffness is a major predictor of cardiovascular-related mortality in chronic kidney disease (CKD). While acrolein—an endogenous reactive aldehyde that accumulates with declining renal function—may be linked to vascular injury, its association with high peripheral arterial stiffness (HPAS) remains unclear. Methods: This cross-sectional study used the cardio-ankle vascular index (CAVI) to examine the association between serum acrolein levels and HPAS (CAVI ≥ 9.0) in 204 adults with non-dialysis CKD stages 3–5. Results: HPAS was identified in 90 patients (44.1%) and was associated with a higher prevalence of diabetes (p = 0.047), older age (p = 0.023), higher spot urine protein–creatinine ratio (UPCR, p = 0.046), higher serum fasting glucose (p = 0.041), higher interleukin-6 (p = 0.025), and higher acrolein (p = 0.008). In multivariable logistic regression analysis, serum acrolein (odds ratio, 1.015; 95% confidence interval, 1.002–1.028; p = 0.025), age (p = 0.010), and UPCR (p = 0.046) remained independently associated with HPAS. Log-transformed acrolein was positively correlated with bilateral CAVI (all p < 0.001) and log-transformed UPCR (p < 0.001) but negatively correlated with the estimated glomerular filtration rate (p = 0.001). Conclusions: Elevated serum acrolein is independently associated with HPAS in non-dialysis CKD stages 3–5.

Toxins doi: 10.3390/toxins18060245

Authors: Stefanie Honndorf Jessica Moser Klaus Fink

The neurotoxin botulinum toxin type A (BoNT/A), produced by the bacteria Clostridium botulinum, is commonly injected intramuscularly (IM) for the management of chronic muscle hyperactivity, such as post-stroke spasticity. New indications such as peripheral neuropathic pain require alternative routes of BoNT/A administration, such as subcutaneous (SC) or intradermal (ID). While IM BoNT/A injections may elicit anti-drug-antibodies (ADAs), their occurrence following SC or ID administration is unknown. Therefore, we investigated whether repeated SC or ID injections of 150 kDa BoNT/A can elicit ADAs in a dose-dependent manner, and whether these differ depending upon the route of administration. Mice were injected 5 times ID or SC with 150 kDa BoNT/A or, for higher doses, inactive mutant BoNT/A (DRBoNT/A) or inactivated toxoid (IA-BoNT/A). Total ADAs were analyzed by an immunoassay and the subgroup of neutralizing ADAs by an in vivo digit abduction score (DAS) assay following a challenge of 0.6 U BoNT/A IM. DRBoNT/A and IA-BoNT/A injections elicit ADAs (22.7 U/mL vs. 460.5 U/mL for ID; 4.7 U/mL vs. 339.4 U/mL for SC), while therapeutic doses of 150 kDa BoNT/A do not. Whereas mice with repeated 150 kDa BoNT/A injections at therapeutic dose show an unrestricted DAS of 3.7 (ID) or 3.4 (SC), mice injected repeatedly with 1.8 µg/kg DRBoNT/A or 500 µg/kg IA-BoNT/A show only a minimal DAS of ≤0.7, indicating a high titer of neutralizing ADAs. No differences were observed between administration routes. Accordingly, repeated ID or SC injections of pure 150 kDa BoNT/A at therapeutic doses fail to induce ADA formation in mice. On the other hand, DRBoNT/A ID injections induce higher ADA concentrations than SC, but generate similar amounts of neutralizing ADAs. IA-BoNT/A injections induce ADAs and neutralizing ADAs similarly after ID and SC injections. ADA development at intermediate BoNT/A doses can be higher after ID injection, but does not lead to differences in neutralizing ADAs. Our data demonstrate that the antibody response to botulinum toxin depends predominantly on the protein load, and less on the administration route.

Toxins doi: 10.3390/toxins18060244

Authors: Yilin Tang Lu Ding Shujuan Sun Mengmeng Mi Minqi Shao Yan Zhao Mingxia Zhu Yun Wang Muhammad Zahoor Khan Changfa Wang Mengmeng Li

Aflatoxins (AFs), toxic secondary metabolites produced by Aspergillus species, represent a major threat to food safety and public health due to their pronounced hepatotoxic, carcinogenic, and mutagenic effects. With increasing global contamination risks driven by climate change and agricultural practices, the development of effective detoxification strategies has become a critical priority. This review provides a comprehensive and mechanistic overview of current aflatoxin (AF) decontamination approaches, focusing on two principal pathways: adsorption and inhibition strategies. Adsorption mechanisms involve the physicochemical sequestration of aflatoxins by inorganic materials, biological adsorbents, and engineered nanocomposites, thereby reducing toxin bioavailability. In contrast, inhibition strategies target fungal growth, toxin biosynthesis pathways, or promote enzymatic and microbial degradation of aflatoxins, offering more specific and potentially sustainable control. We critically analyze the underlying mechanisms, advantages, and limitations of each approach, including issues related to specificity, environmental stability, safety, and interactions with food matrices. Particular emphasis is placed on the toxicological implications of detoxification processes, including the reduction in aflatoxin-induced health risks and the safety of degradation products. Finally, this review highlights the importance of integrating adsorption and inhibition strategies to achieve synergistic decontamination and detoxification effects. Future perspectives on multifunctional materials, biological control systems, and intelligent monitoring technologies are discussed to advance the development of efficient, safe, and sustainable aflatoxin mitigation strategies.

Toxins doi: 10.3390/toxins18060243

Authors: Simone Aloisio Massimiliano Passaretti Luca Angelini Martina De Riggi Francesca Santachiara Anna Sofia Grandolfo Daniele Birreci Matteo Bologna

Cervical dystonia (CD) is increasingly recognized as a disorder with a dynamic clinical course rather than a fixed phenomenological profile. This report describes the long-term observation of a man with isolated CD followed for nearly 12 years from symptom onset. The course was characterized by multiple pattern changes, including a clear shift from left- to right-sided torticollis in 2019, followed by alternating and mixed dystonic patterns. Three remission phases were documented during follow-up: two prolonged periods in 2017–2018 and 2020–2021, and a further phase from March to November 2022. Over time, treatment requirements decreased from higher initial doses (500–700 U abobotulinumtoxinA and up to 130 U onabotulinumtoxinA) to lower maintenance doses of 30–50 U incobotulinumtoxinA/onabotulinumtoxinA, with longer injection intervals and no obvious documented need for dose adjustment to maintain clinical responsiveness. This case highlights the coexistence of pattern changes and recurrent remissions within a single continuously followed patient over an unusually long period. Although a treatment-related mechanism remains speculative and alternative explanations cannot be excluded, the observation underscores the value of prolonged follow-up and individualized treatment strategies in CD.

Toxins doi: 10.3390/toxins18060242

Authors: Oriana Nobus Aurélie Carlier Silvia M. Mihăilă Vanessa Dubois

Chronic kidney disease (CKD) is characterized by the progressive accumulation of uremic toxins (UTs), which contribute to systemic complications, increased cardiovascular risk, and disease progression. Epidemiological and experimental evidence demonstrate pronounced sex differences in CKD progression and outcomes, yet the mechanisms underlying sex-specific uremic toxicity remain unclear. This review synthesizes current knowledge on sex differences in the origin, metabolism, transport, and biological effects of UTs, with a focus on sex-dependent regulatory mechanisms along the gut–liver–kidney axis. Sex hormones influence key determinants of toxin handling, including gut microbiota composition, hepatic enzyme activity, plasma protein binding, membrane transporter expression, and intracellular signaling pathways. Together, these factors regulate systemic toxin exposure and tissue susceptibility to injury. CKD also disrupts endocrine homeostasis, creating bidirectional interactions between hormonal regulation and toxin accumulation. Experimental and limited clinical evidence suggest that sex may influence circulating toxin profiles and susceptibility to toxin-associated complications. Collectively, sex is an important modulator of uremic toxicity, with sex hormones mediating at least part of the sex differences. A sex-informed framework may improve fundamental understanding through mechanistic studies and future clinical research may help clarify its relevance for biomarker development and support the development of personalized therapeutic strategies for CKD.

Toxins doi: 10.3390/toxins18060241

Authors: Eva María Mateo Fernando Mateo Andrea Tarazona María Ángeles García-Esparza José Miguel Soria Misericordia Jiménez

Mycotoxins are compounds produced by the secondary metabolism of certain fungi. These compounds contaminate foods worldwide and pose a severe threat to the health of humans and animals. They also cause huge economic losses. A plethora of methodologies, encompassing agricultural, biological, chemical, and physical approaches, have been devised to curtail the presence of mycotoxins in food commodities. Among the physical processes, cold plasma (CP) has emerged as a useful technique for controlling the presence of toxigenic fungi in foods and for degrading the mycotoxins occurring in them without significantly affecting the quality and organoleptic properties of the treated commodities. The present review endeavors to demonstrate the efficacy of CP as a method of eradicating or reducing both the toxigenic mycobiota and the mycotoxins present in the most contaminated foods, including nuts, dried fruits, and cereal grains. The mechanisms of toxin degradation proposed by the different researchers are also examined and compared. Furthermore, the impact of the CP effect on the quality, sensorial characteristics, and toxicological properties of the treated food is thoroughly examined.

Toxins doi: 10.3390/toxins18060240

Authors: Vitelbina Núñez Rangel Paola Rey-Suárez Daniel Buitrago-Chinchilla Laura Reyes-Méndez Leidy Gómez-Sampedro Alejandro Carmona-Jiménez Mateo Rivillas-Ochoa Adriana Muñoz-Bravo

Phytopathogenic bacteria and fungi significantly reduce fruit and vegetable yields, resulting in substantial economic losses. Conventional management relies on synthetic agrochemicals; however, their intensive use poses risks to human health, environmental integrity, and biodiversity. Snake venoms have evolved under selective pressure, developing specialized components with potent antimicrobial properties as part of a defense mechanism against prey-borne microorganisms. This study evaluated the inhibitory potential of Micrurus venoms against pathogens of agricultural interest and developed bioactive gelatin-based films incorporated with purified L-amino acid oxidases (LAAOs) as a novel biocontrol strategy. Venoms from M. ancoralis, M. mipartitus, and M. dumerilii exhibited significant growth inhibition against Xanthomonas and Fusarium strains. The primary active component was identified as LAAO through biological activity and mass spectrometry. Biofilms were formulated by incorporating M. ancoralis venom and its purified LAAO into a gelatin matrix. Physicochemical and microbiological characterization, alongside in situ assays on strawberries, demonstrated that the functionalized biofilms retained potent antimicrobial activity. Furthermore, LAAO incorporation did not significantly alter the physicochemical properties of the fruit but effectively extended shelf life by reducing weight loss and maintaining sensory appearance. These findings highlight the biotechnological potential of elapid venom components in the development of alternatives for phytopathogen control and active food packaging.

Toxins doi: 10.3390/toxins18060239

Authors: Kristi L. Hill Andrea C. Jaegge Devin M. Moore Thomas D. Byl

Cyanobacterial toxins (cyanotoxins) threaten aquatic ecosystems and human health, yet the factors influencing their production and distribution in freshwater remain unclear. In north-central Tennessee, nutrient-rich runoff from agricultural and urban areas, combined with a karst landscape that supports drinking and recreational water use, heightens the need to understand cyanotoxin behavior. To examine cyanotoxin patterns, the U.S. Geological Survey and the Tennessee Department of Environment and Conservation monitored 18 sites, including two wells under the influence of surface water, every two weeks from September 2022 to November 2024. At least one cyanotoxin was detected at all sites, with the highest concentrations in deep reservoirs and lower levels in shallow systems. Most detections occurred during summer and fall, aligning with high temperatures and rapid-onset drought. Statistical analysis indicated that increased specific conductivity and pH raised the likelihood of detecting total microcystin, likely resulting from drought conditions and nutrient-laden runoff. Additionally, dissolved microcystin showed an inverse relationship with Cumberland River water levels, and principal component analysis showed that Secchi depth, chlorophyll a, pH, temperature, and conductivity explained most water quality variability. These results help increase understanding of cyanotoxin distribution and associated water quality conditions during detections to guide future freshwater cyanotoxin monitoring studies.

Toxins doi: 10.3390/toxins18060238

Authors: Domenico Antonio Restivo Mario Stampanoni Bassi Rosario Marchese-Ragona Giovanni Castelnovo Angelo Alito Demetrio Milardi Stefano Masiero Daniele Coraci

The efficacy of botulinum neurotoxin (BoNT) is strongly dependent on its accurate delivery to hyperactive muscles and, ideally, to motor endplate regions. Although guidance techniques such as electromyography (EMG) and ultrasound (US) improve injection precision, each technique provides only partial information—either functional or anatomical. Integrating these techniques could enhance targeting accuracy, optimize dose distribution, and reduce off-target effects. A structured PubMed search was performed using terms related to BoNT, spasticity/dystonia, EMG, and US. Filters included clinical trials, randomized controlled trials, meta-analyses and reviews published within the last decade. Fifty-nine studies met the inclusion criteria. The publications were predominantly in neuroscience and rehabilitation journals. Only 17 studies reported combined EMG–US guidance. These focused mainly on stroke and cervical dystonia. While EMG-US integration is a promising strategy, we emphasize the added value of EMG guidance for US approaches, which is particularly important when treating complex neurological conditions involving complex, overlapping muscle activation patterns, or when targeting structures that are inaccessible to conventional imaging techniques. The EMG-US integrated approach is a promising strategy for optimizing BoNT therapy by combining structural visualization with real-time functional assessment. Despite its promising advantages in terms of accuracy and dose optimization, its clinical adoption is limited by a lack of high-quality evidence.

Toxins doi: 10.3390/toxins18050237

Authors: Paweł Kwiatkowski Helena Masiuk Agata Pruss Stefania Giedrys-Kalemba Piotr Baszuk Iwona Wojciechowska-Koszko Monika Sienkiewicz

Staphylococcus aureus nasal carriage contributes to asymptomatic transmission in both community and healthcare settings. This study aimed to characterize S. aureus strains isolated from students of the Pomeranian Medical University in Szczecin, Poland, using phenotypic and genotypic methods. A total of 175 S. aureus strains were isolated from the nasal vestibules of 800 students between 2014 and 2015. Species identification and antimicrobial susceptibility testing were performed using standard microbiological methods, while virulence-associated genes and agr groups were analyzed using Single-PCR and Multiplex-PCR assays. Genotypic diversity was assessed by pulsed-field gel electrophoresis (PFGE). The prevalence of S. aureus nasal carriage among students was 21.9% and did not differ according to faculty or year of study. Most isolates (84.0%) were susceptible to all tested antibiotics, and no methicillin-resistant S. aureus (MRSA) strains were detected. All strains carried the hla gene, whereas hld and hlg were identified in 93.7% and 93.1% of isolates, respectively. In addition, the tst gene was detected in 22.3% of strains, while the lukS-PV/lukF-PV genes were identified in only one isolate (0.6%). The most prevalent enterotoxin genes were sep (17.1%) and sea (13.7%), whereas genes of the egc cluster, including seg, sei, and seo, were detected in 53.7% of isolates. Significant associations were observed between specific egc gene combinations and superantigen gene profiles, including increased frequencies of sec, sel, and tst genes (p < 0.001). The predominant agr type was agr-1 (49.7%), followed by agr-3 (28.6%) and agr-2 (20.0%). Strains carrying agr-1 more frequently harbored the g i m n o cluster as well as the sec, sel, and sep genes, whereas agr-3-positive isolates were significantly associated with the g i m o u and g i o u clusters and with the presence of tst, sea, and seh genes (p < 0.05). PFGE analysis demonstrated substantial genetic heterogeneity among the isolates, with no evidence of a predominant clonal lineage. These findings indicate a heterogeneous, non-epidemic population structure of S. aureus strains circulating among university students and highlight the considerable diversity and interrelationships of virulence-associated genetic profiles within this population.

Toxins doi: 10.3390/toxins18050236

Authors: Nazia Tabassum Minji Kim Tae-Hee Kim Du-Min Jo Won-Kyo Jung Young-Mog Kim Fazlurrahman Khan

Mycotoxins are one of the biggest threats to global food safety, public health, and economic stability. More than 400 mycotoxins have been found to be secondary metabolites of toxigenic fungi, mostly from the genera Aspergillus, Fusarium, Penicillium, and Alternaria. Aflatoxins (AFs), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEA), fumonisins (FBs), patulin (PAT), and T-2/HT-2 toxins are the most dangerous to the health of people and animals. Conventional physical and chemical decontamination methods are only partially effective and can reduce food quality, leave toxic residues, or be too expensive for smallholder food systems. Recent studies have shown that the application of lactic acid bacteria (LAB) as a biological detoxification method is a safe, cost-effective, and environmentally friendly option, and has a long history of safe use in fermented foods. Selected strains or taxonomic units have been granted GRAS status by the FDA or QPS (Qualified Presumption of Safety) status by EFSA. However, their use for mycotoxin detoxification still requires strain-level safety assessment and efficacy validation in the intended food matrix. There are several mechanisms by which LAB employ to reduce the bioavailability of mycotoxins in food systems: (i) physical adsorption via cell wall components such as peptidoglycan, teichoic acids, and exopolysaccharides; (ii) enzymatic biotransformation that may produce non-toxic or less-toxic metabolites, though the safety of degradation products requires case-by-case toxicological assessment; (iii) antifungal metabolite production that inhibits fungal growth and mycotoxin biosynthesis; and (iv) competitive exclusion of toxigenic fungi during fermentation. This comprehensive review examines the existing evidence on the detoxification of major food mycotoxins by LAB, with an emphasis on mechanisms, strain-specific efficacy, food-matrix applications, and factors that affect detoxification efficacy. Discussion has also been made of translating in vitro findings to in vivo settings and food-scale applications, alongside regulatory frameworks, current challenges, and future research directions. The review also suggests ways to combine LAB with new technologies, such as encapsulation, genetic engineering, and fermentation optimization, to make food systems safer by synergistically controlling mycotoxins.

Toxins doi: 10.3390/toxins18050235

Authors: Jack W. Creagh Lily L. Givens David C. Reetz Sarah A. Coss Rodolfo Bizarria Siti Aisyah Alias Mohammed Rizman-Idid Jagdish S. Patel Andre Rodrigues F. Marty Ytreberg Paul A. Rowley

Antifungal killer toxins are cytotoxic proteins that have the potential to combat the growing threat of fungi to human health and agriculture. A lack of empirical tertiary structures has limited understanding of their mechanisms of action and their ability to target pathogens. In this study, AlphaFold and molecular dynamics simulations were used to generate tertiary structure models of all canonical Saccharomyces killer toxins and to place them in the context of historical empirical data. These models enabled the prediction of functional domains and posttranslational modifications, including proteolytic cleavage sites and disulfide bonds. They also revealed unexpected homology between Saccharomyces killer toxins, suggesting that all but K28 are likely ionophores. Structural homology to the well-studied killer toxins K1 and K2 enabled the prediction of the antifungal and immunity mechanisms of K1L, K21, K45, K74, and KHS. The understudied killer toxins Klus, KHR, and K62 were found to have homology to bacterial and plant toxins, including members of the aerolysin family and antifungal lectins. These structural similarities provide clues for the mechanisms of killer toxin carbohydrate binding, oligomerization, and membrane attack. This modeling approach will help guide the continued use of the model yeast S. cerevisiae to study killer toxins in the context of the wealth of functional data gathered in the decades since their first discovery.

Toxins doi: 10.3390/toxins18050234

Authors: Tatiana V. Antipova Vsevolod R. Dubovik Anton N. Yurchenko Olesya I. Zhuravleva Valentina P. Zhelifonova Elizaveta G. Lukina Boris P. Baskunov Oussama Abdelhamid Mammeri Sergey N. Smirnov Natalya E. Ivanushkina Kirill V. Zaitsev Qunfang Weng Mikhail B. Vainshtein Alexander O. Berestetskiy

Aspergillus fungi are a source of low-molecular compounds of various structures possessing biological activities. We investigated the secondary metabolite profile of the soil fungus A. calidoustus VKM F-4916. The strain was found to synthesize new metabolites attributed to furoisocoumarins, which we named asperisocoumarin J and K, and a known siderophore desferritriacetylfusigen. The structure of asperisocoumarin J and K were determined by mass spectrometry and NMR spectroscopy. Asperisocoumarins J, K and desferritriacetylfusigen possessed a phytotoxicity, inhibiting the lettuce root growth. Sow thistle leaf and wheat leaf cuttings were sensitive to the action of asperisocoumarin J and K at a concentration of 5 mg/mL. Analysis of the structures of furoisocoumarins (asperisocoumarins J and K) using the online resource Pesti-DGI-Net showed that compounds had the physico-chemical properties favorable for pesticide development, in particular, fungicides and herbicides. An in-depth study of the phytotoxic properties of furoisocoumarins and their natural analogs is of interest in the context of the search for new herbicide compounds.

Toxins doi: 10.3390/toxins18050233

Authors: Woo-Hyeon Jeong

Ricin and abrin are highly lethal Type II ribosome-inactivating proteins. They depurinate the same site of the 28S rRNA to inhibit protein synthesis. Consequently, standard molecular-level activity assays used to detect the toxic activity of ricin or abrin do not distinguish between the two in mixed samples without prior physical separation or specially designed substrates. This study proposes a novel, cost-effective method to separately and simultaneously quantify the activities of ricin and abrin in mixtures by exploiting their distinct thermal stabilities. Thermal inactivation was used to demonstrate that heating samples at 80 °C for 5 min maximized the difference in their activities; while ricin retained most of its activity, abrin activity dropped to 20% after thermal treatment. This thermal treatment yielded 4 standard curves—ricin or abrin, thermally treated or not treated—in the 0.3 to 50 µg/mL range. By applying Cramer’s rule, the individual concentrations of active ricin and abrin in mixed samples were successfully calculated. However, this method should be used with a method detecting presence of ricin/abrin, to avoid unexpected reactivity due to contaminating RIPs.

Toxins doi: 10.3390/toxins18050232

Authors: Andrea Felice Armenti

Botulinum toxin A (BoNT-A) is well established for the esthetic treatment of upper-face dynamic lines, and long-term adult studies suggest that repeated treatment may remain effective and acceptable over time in many patients. However, wrinkle efficacy and patient satisfaction do not by themselves determine whether repeated treatment remains acceptable in broader morphologic, dynamic–expressive, and longitudinal terms. At the same time, the toxin’s pharmacology entails finite presynaptic blockade followed by active synaptic and architectural recovery, and imaging, histological, and neurophysiological studies indicate that repeated chemodenervation cannot be assumed to be biologically neutral at the muscle level. The resulting problem is not whether BoNT-A works, but whether current outcome frameworks are sufficient to judge repeated-treatment compatibility in full. Here, structural tolerance is proposed as a provisional clinical lens for an underdescribed boundary: whether repeated-treatment compatibility is fully captured by wrinkle reduction and patient acceptability alone. The paper organizes this problem across morphologic, dynamic–expressive, and longitudinal domains, outlines candidate warning signs, and develops a pragmatic tiered approach to assessment spanning routine care, structured clinical follow-up, and research-oriented evaluation. The aim is to support more complete longitudinal thinking in upper-face aesthetic BoNT-A, to clarify what current outcome frameworks remain unaddressed, and to identify priorities for future empirical study.

Toxins doi: 10.3390/toxins18050231

Authors: Jeong-Sun Nam Daewon Yoon Yujin Kim Su-Young Kim Jae-Young Kim Yoonkyoung Cha Joon Seok Beom Joon Kim

Botulinum toxin suppresses neurotransmitter release, thereby inhibiting muscle contraction and inducing flaccid paralysis. Botulinum toxin type A (BoNT/A) is widely used for neuromuscular blockade but, upon repeated administration, may cause long-lasting muscle atrophy, fibrosis, and inflammation. It is produced as a single peptide chain that becomes activated through cleavage into a heavy and light chain. BoNT/E, like BoNT/A, is produced as a single-chain polypeptide and requires cleavage to generate the active dichain form. Although BoNT/E is known to have a faster onset and shorter duration of action compared with BoNT/A, its efficacy and safety have not been thoroughly investigated. We compared BoNT/E and BoNT/A in SKH-1 hairless mice. Neuromuscular blockade, recovery pattern, and changes in muscle weight, volume, fiber size, fibrosis, mast cell infiltration, and diffusion to adjacent muscles were evaluated over time. BoNT/E induced maximal neuromuscular blockade on day 3 and fully recovered by day 35, whereas BoNT/A reached maximal effect on day 7 and showed only 20% recovery of the vehicle group by day 35. BoNT/E caused transient, dose-dependent reductions in muscle weight, volume, fiber size, and fibrosis, which largely normalized by day 35. In contrast, BoNT/A, administered at a dose of 0.5 U per injection site, induced persistent muscle atrophy, fibrosis, and significantly increased mast cell infiltration under the experimental conditions used in this study. Neither BoNT/E nor BoNT/A showed diffusion to adjacent muscles or changes in body weight. These findings suggest that BoNT/E provides rapid onset, short duration, and favorable safety, supporting its potential as an alternative therapeutic option for indications requiring temporary muscle relaxation with minimized long-term adverse effects.

Toxins doi: 10.3390/toxins18050230

Authors: Khouloud Ben Hassouna Jalila Ben Salah-Abbès Samir Abbès Ferjeni Zouidi Mourad Jridi Albert Sebastià Noelia Pallarés Houda Berrada

The contamination of cereals by mycotoxins represents a major concern due to their harmful effects on human health and food quality. The current study investigated the occurrence of major mycotoxins (AFB1, AFB2, AFG1, AFG2, OTA, ENA, ENA1, ENB, and ENB1) in 158 raw cereal samples (durum wheat, barley, and maize) collected from two Tunisian regions: Beja (continental region) and Mahdia (coastal region). Mycotoxins were extracted using the QuEChERS method and quantified by UHPLC–MS/MS. Several mycotoxins were detected at high levels across all the cereals. In the Beja region, durum wheat was contaminated with AFB1, AFG1, ENA, ENA1, ENB, and ENB1, with ENB being the most frequent (70%). Mahdia durum wheat was contaminated only with ENA, ENA1, ENB, and ENB1, with ENB1 being the most prevalent (22.6%). Barley from both regions was contaminated only with ENs. The ENB was the most frequent (Beja 66%, Mahdia 28.6%). Maize from Beja was contaminated by OTA and ENs, with ENA1 being the most frequent (22.5%), while maize from Mahdia was contaminated by AFB1, AFB2, AFG2, and ENs (AFB1 was the most frequent, 35%). All wheat samples contaminated with AFG1 (6.55%) exceeded the European Union maximum limit for AFs in cereals (4 µg/kg). Similarly, maize samples contaminated with AFB1 (17.5%), AFB2 (10%), and AFG2 (2.5%) exceeded the EU maximum limit for aflatoxins in maize (10 µg/kg). Additionally, maize samples contaminated with OTA (5%) exceeded the EU maximum limit for OTA in unprocessed cereals (5 µg/kg). The co-occurrence of multiple mycotoxins was observed in all cereal types, with up to six different mycotoxins detected in a single sample. Dietary risk assessment revealed high EDIs of AFB1, AFG1, and ENs through the consumption of wheat and barley by Tunisian adults. The calculated MOE values for AFB1 and AFG1 in wheat were below 10,000 (MOE = 1190 for AFB1 and 2.5 for AFG1), suggesting a potential health concern associated with dietary exposure. Despite this potential risk, AFB1 and AFG1 were detected in only 3% and 7% of the analyzed samples, respectively. These results highlight the need for regular monitoring and the establishment of regulations to control mycotoxins in Tunisian cereals.

Toxins doi: 10.3390/toxins18050229

Authors: Ping Ding Wenchao Liao Chenyu Chen Xincheng Chen Chengfei Wang Xiaoyang Li

Zearalenone (ZEN) is a pervasive mycotoxin contaminating global food and feed. While enzymatic degradation offers a promising, specific, and eco-friendly strategy for mycotoxin mitigation, the biotransformation of ZEN within acidic food matrices remains challenging due to the intrinsically low activity of zearalenone lactonase (ZENG). In this work, we synthesized a ZENG–hydroxyapatite (Ca10(PO4)6(OH)2) hybrid nanoflower (CaNF) via biomineralization under alkaline conditions. Compared to free ZENG, the as-prepared biohybrid nanoflower exhibited markedly enhanced acid tolerance and catalytic activity, achieving a 12-fold increase in ZEN degradation efficiency at pH 5.0. Furthermore, the biohybrid nanoflower demonstrated robust performance in various acidic food matrices, including corn juice, wort, beer, and corn steep liquor. This study presents a powerful enzymatic tool for the efficient biotransformation of ZEN in acidic food-related systems.

Toxins doi: 10.3390/toxins18050228

Authors: Farris Kassam Fraser MacRae Linden Lechner Heather Dow Ève Boissonnault Fiona Huang Paul Winston

Objectives: To investigate current Canadian physicians’ practice patterns of treating upper limb post-stroke spasticity (PSS) and hemiplegic shoulder pain (HSP) acutely after a stroke. In addition, by examining Canadian physicians’ diagnostic capabilities, time till treatment, minimum criteria to begin treatment, mechanisms of treatment, targeting of muscles, and benefits and adverse effects of treatment, we aim to learn about areas of improvement to optimize PSS management for Canadians. Design: The present study was a cross-sectional survey, polling practicing Canadian physicians. Results: A total of 17 physicians completed the survey, all PM&R specialists, save one neurologist. Four provinces were represented in the responses. Participants had, on average, over ten years of experience managing post-stroke spasticity in outpatient and inpatient clinics. All 17 perform botulinum neurotoxin A (BoNT-A) injections for HSP associated with PSS. Most participants reported that they will begin BoNT-A treatments 2–3 weeks post-stroke, most commonly targeting the pectoralis major, subscapularis, and latissimus dorsi. Participants reported the mean median dosage they use as onabotulinum toxin A (169.12 units, SD = 73.70), incobotulinum toxin A (178.13 units, SD = 65.75), and abobotulinum toxin A (470.83 units, SD = 171.17). For injection guidance, participants responded that they use ultrasound for the largest percentage of their caseload, followed by electromyography, then electrical stimulation, then palpation. Very seldom did participants use palpation alone. Conclusions: From the limited sample included in analyses, the Canadian physicians respondents seem to be treating HSP and associated PSS with variable strategies. Further research is required to align dosages, targets, and guidance strategies as they vary considerably.

Toxins doi: 10.3390/toxins18050227

Authors: Chao Chen Yifan Qin Zijun Luo Peiqiang Mu Jikai Wen Yiqun Deng

Deoxynivalenol (DON) is a globally prevalent mycotoxin that threatens food and feed safety via severe multi-organ toxicity. Previous studies indicate that DON induces cellular energy metabolism dysregulation by triggering oxidative stress and impairing mitochondrial function. During this process, nicotinamide adenine dinucleotide (NAD+), a central coenzyme in cellular energy metabolism, frequently exhibits significantly decreased intracellular levels or even complete depletion. However, the molecular mechanisms underlying the disruption of NAD+ homeostasis by DON exposure, as well as the development of targeted countermeasures, remain elusive. Using human embryonic kidney 293T (HEK293T) cells as an in vitro renal toxicity model, we dissected DON-induced NAD+ dysregulation and evaluated the protective potential of nicotinamide (NAM). DON caused significant NAD+ depletion in porcine serum (in vivo) and HEK293T cells (in vitro), which was confirmed as a key driver of cytotoxicity. Mechanistically, although DON binds and inhibits nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway, neither NAMPT knockdown and overexpression nor nicotinamide mononucleotide (NMN) supplementation rescued DON-induced toxicity. Instead, DON dose-dependently activated poly(ADP-ribose) polymerase 1 (PARP1), the primary intracellular NAD+-consuming enzyme, to accelerate NAD+ depletion. PARP1 knockdown markedly attenuated DON-induced cytotoxicity, identifying PARP1 hyperactivation as the core toxic mechanism. NAM dose-dependently suppressed PARP1 activity, replenished NAD+ pools, and reversed cell injury. These findings establish PARP1-driven NAD+ depletion as an important mechanism of DON-induced renal toxicity, providing a promising intervention candidate for mitigating DON toxicity in food safety.

Toxins doi: 10.3390/toxins18050226

Authors: Xinyi Wang Kehan Yi Yongjun Jiang Mengmeng Tong

Chattonella marina is an ichthyotoxic, bloom-forming raphidophyte known for its hemolytic activity. However, the mechanisms by which nitrogen (N) and phosphorus (P) limitation influence this hemolytic toxicity remain poorly understood. In this study, both N and P limitation reduced growth, photosynthetic efficiency (Fv/Fm, YII, rETRmax), and the expression of nutrient-uptake, tetrapyrrole/chlorophyll biosynthesis genes. Nevertheless, the two nutrients produced opposite effects on toxicity: N limitation lowered hemolytic activity and ROS levels to near zero, whereas P limitation kept both relatively high, similar to nutrient-replete controls. The addition of the antioxidant NAC (N-Acetyl-L-cysteine) reduced hemolytic activity, confirming that ROS contributes to toxicity. Transcriptome data showed that under N limitation, genes for nitrogen uptake and initial reduction (NRT, NR, glnA) were upregulated, while downstream assimilation genes (nirA, GLT1) were downregulated. In contrast, under P limitation, all the nitrogen-metabolism-related genes (NRT, NR, glnA, nirA, GLT1) were downregulated. In the tetrapyrrole pathway, most genes were downregulated under both nutrient-limited conditions, except for HemD, suggesting a bottleneck that may result in the accumulation of porphyrin intermediates within the tetrapyrrole/chlorophyll biosynthesis pathway. Together, the secondary products derived primarily from the reaction of ROS with tetrapyrrole-based compounds appear to be the main contributors to hemolytic toxicity. Consequently, high levels of both ROS and porphyrin intermediates under P-limited conditions, as well as high ROS levels but low porphyrin intermediates under nutrient-sufficient conditions, may both contribute to the high hemolytic toxicity of C. marina. In contrast, under N limitation, despite the accumulation of porphyrin intermediates, the strong suppression of photosynthetic electron transport limits both ROS production and the synthesis of nitrogen-containing toxins, resulting in low hemolytic activity. These findings demonstrate that nutrient conditions regulate hemolytic activity in C. marina in a nutrient-specific manner.

Toxins doi: 10.3390/toxins18050225

Authors: Søren H. Elsborg Jasmine C. L. Atay Johan Palmfeldt Christian Daugaard Peters Krista Dybtved Kjærgaard Henricus A. M. Mutsaers Rikke Nørregaard

Chronic kidney disease (CKD) affects nearly 10% of the global population, yet diagnosis and disease monitoring still rely primarily on plasma creatinine. Because creatinine levels are strongly influenced by non–renal factors, such as age, sex, muscle mass, and diet, its accuracy as a kidney function marker is limited. To identify plasma biomarkers that reflect kidney injury, we applied untargeted and targeted metabolomics in the adenine-induced CKD mouse model, a well-known tubular damage model, and validated the findings in plasma from patients with advanced CKD and healthy controls. We identified five metabolites that showed altered plasma levels in both experimental and human CKD, of which galactonic acid, pipecolic acid, and N-acetylneuraminic acid were significantly associated with measured glomerular filtration rate (GFR). As a proof-of-concept, we demonstrated that integrating these metabolites into a biomarker panel alongside creatinine could improve GFR estimation compared with creatinine alone. Our study introduces a promising metabolite-based biomarker panel that might enhance the accuracy of kidney function assessment and could potentially support diagnosis, risk stratification, and monitoring of disease progression; however, validation in a broader CKD cohort is needed.

Toxins doi: 10.3390/toxins18050224

Authors: Magdalena Gajęcka Łukasz Zielonka Maciej T. Gajęcki

The aim of this study was to determine whether exposure to low doses of zearalenone (ZEN) over a period of six weeks affects the values of the carry-over factor (COF) of ZEN in the roof of the third cerebral ventricle (RTCV) and in selected skeletal muscles (longissimus and quadriceps) in prepubertal gilts. The study was conducted on 60 clinically healthy prepubertal gilts with an initial body weight (BW) of 14.5 ± 2 kg. Gilts were randomly assigned to a control group (group C; n = 15) and three experimental groups (ZEN5, ZEN10, and ZEN15; n = 15 each). Groups ZEN5, ZEN10, and ZEN15 were administered ZEN per os at doses of 5 µg/kg BW, 10 µg/kg BW, and 15 µg/kg BW, respectively. Group C animals were orally administered a placebo. Tissue samples (brain and skeletal muscles) were collected post-mortem for toxicological analyses on exposure days 7 (D1), 21 (D2), and 42 (D3). The concentrations of ZEN and its metabolites, α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL), were determined in the collected samples. All examined tissues contained the parent compound, but ZEN metabolites were not detected in any of the samples. The absence of ZEN metabolites may have resulted from a physiological deficit of estradiol (E2) and, consequently, testosterone (T) and progesterone (P4) in prepubertal gilts. Low-dose ZEN mycotoxicosis led to a persistent presence of ZEN in the RTCV (COF from 1 × 10−6 on D1 to 7 × 10−7 on D3) and somewhat lower ZEN levels in skeletal muscles (COF from 8 × 10−6 on D1 to 6 × 10−7 on D3). The presence of ZEN in the RTCV confirms that it crosses the blood–brain barrier and may therefore participate in the hormonal homeostasis of the brain.

Toxins doi: 10.3390/toxins18050223

Authors: Fanny Jouve Christophe O. Soulage Laetitia Koppe

Chronic kidney disease (CKD) is characterized by persistent exposure to uremic toxins (UTs), many of which originate from gut microbial metabolism and contribute to renal, cardiovascular, and metabolic complications. Current evidence indicates that CKD is associated with dysbiosis and the enrichment of microbial taxa carrying genes involved in UT precursor production. Diet is a major modulator of the gut microbiota and therefore represents a promising lever to reduce UT generation in synergy with current nephroprotective therapies. Beyond simple protein restriction, more specific dietary approaches, particularly plant-based low-protein diets, appear especially relevant. Additional factors, including amino acid composition, lipid quality, food processing, constipation, transit time, meal timing, and circadian rhythms, may also influence microbial metabolism and UT production. This review examines the role of nutrition in shaping the gut microbiota–UT–kidney axis and discusses how dietary modulation may support precision nutrition in the context of CKD. It also highlights future directions based on multidimensional phenotyping and robust biomarkers to capture interindividual variability, guide personalized interventions, and ultimately improve renal and cardiovascular outcomes in CKD.

Toxins doi: 10.3390/toxins18050222

Authors: Zixiao Zhou Ziyi Wang Chundi Mu Yan Qi Jing Zhang Xia Cui Sai Fan Jing Xiao Rong Zhao

Cereulide is a heat-stable cyclic depsipeptide toxin produced by Bacillus cereus and is responsible for foodborne emetic syndrome. Recent reports of Bacillus cereus contamination and cereulide occurrence in infant formula have raised increasing food safety concerns. Due to the immature immune and metabolic systems of infants, exposure to cereulide through contaminated formula may lead to potential health risks. However, direct application of existing cereulide analytical methods to infant formula remains challenging because of the unique processing technologies, encapsulated nutrients, and variable matrix composition of this product category, which may hinder toxin release and cause significant matrix interference. In practical analysis, inter-laboratory comparisons revealed that existing methods exhibited relatively large deviations and insufficient sensitivity, making them not specifically optimized for infant formula matrices. The present study was motivated by the need for a matrix-specific, sensitive, and reliable analytical method for cereulide determination in infant formula. In this study, a method based on solid-phase extraction coupled with ultra-performance liquid chromatography–tandem mass spectrometry (SPE–UPLC–MS/MS) was developed and validated. To improve the applicability of cereulide analysis to infant formula, this method incorporates a hydration-assisted extraction step tailored to infant formula, which increased the detected cereulide response by approximately fourfold, together with optimized SPE clean-up and improved chromatographic conditions to reduce matrix effects and enhance quantitative reproducibility. The method showed good linearity (0.1–10 μg·L−1, R2 > 0.999), low values for limit of detection (LOD) (0.03 μg·kg−1) and limit of quantification (LOQ) (0.1 μg·kg−1), and acceptable recoveries (94.4–110.3%) with RSDs below 3.7%. The developed method was successfully applied to commercial infant formula samples, and cereulide-positive samples were identified. This method provides a reliable analytical tool for the monitoring of cereulide in infant formula and contributes to improved food safety surveillance and exposure risk assessment.

Toxins doi: 10.3390/toxins18050221

Authors: Akindele Oluwatosin Adeyi Oluwatimilehin Stephen Emmanuel Samuel Itang Itang Babafemi Siji Ajisebiola Mihir Kumar Gotravalli V. Rudresha Prasad Gopalkrishna Gond Thomas Crasset Damien Redureau Fernanda Gobbi Amorim Kartik Sunagar Loïc Quinton

Snakebite envenoming is a major yet neglected tropical disease in sub-Saharan Africa, where antivenom efficacy is critically limited by intraspecific venom variation shaped by local ecological pressures. Nigeria’s sharply contrasting Sudan Savanna (North) and Lowland Rainforest (South) provide an ideal natural system to investigate this variation, yet a comparative analysis of its medically important snakes has been lacking. We conducted an integrated proteomic and functional characterization of venoms from the puff adder (Bitis arietans) and black-necked spitting cobra (Naja nigricollis) collected in Kaduna (North) and Ibadan (South). Using high-resolution LC-MS/MS, SDS-PAGE, and biochemical assays (phospholipase A2, protease, fibrinogenolytic, hemolytic, and coagulation activities), we mapped region-specific venom compositions and characterized their functional activities. Bitis arietans displayed region-associated divergence: southern venom was enriched in serine proteases, whereas northern venom was dominated by lectins and distinct snake venom metalloproteinase isoforms. Naja nigricollis showed a conserved phospholipase A2/three-finger toxins backbone, yet southern venoms exhibited elevated snake venom metalloproteinase III and L-amino acid oxidase. These molecular differences manifested functionally, with southern B. arietans venom showing higher protease activity than northern B. arietans, whereas southern and northern N. nigricollis venom exhibited similar protease activity but enhanced phospholipase A2 activity in southern N. nigricollis. This work provides the first integrated proteomic and functional comparison of venoms from northern and southern Nigerian venom sample of B. arietans and N. nigricollis. While based on a limited number of individuals, the observed differences should be considered preliminary and indicative of potential regional trends rather than population-level characteristics.

Toxins doi: 10.3390/toxins18050220

Authors: David Faustino Ângelo Henrique José Cardoso Marcella Sarkis Kelly Santos Francesco Maffia David Sanz Francisco Salvado

Background: Myogenous temporomandibular disorders (TMDs) are a common subtype of orofacial pain. Evidence regarding treatment with botulinum toxin type A (BoNT-A) remains heterogeneous, and its use is generally limited to refractory cases. This study evaluated 12-month clinical outcomes following an incobotulinumtoxinA protocol (the Ângelo Botulinum Toxin Protocol®) in adults with DC/TMD-confirmed myogenous TMD unresponsive to conservative therapy. Methods: This retrospective observational study reviewed records from 98 adults treated with incobotulinumtoxinA following the predefined injection protocol. All patients had failed ≥ 3 months of conservative management and completed ≥ 12 months of follow-up. Outcomes included myalgia severity (0–3), patient-reported orofacial pain intensity (VAS 0–10), and maximum mouth opening (MMO). Favorable outcome criteria required myalgia 0–1 or VAS ≤ 2 and MMO ≥ 35 mm. Results: Myalgia significantly decreased at 12 months (2.69 ± 0.64 to 0.43 ± 0.85; p < 0.001). Patient-reported orofacial pain intensity also improved (2.44 ± 2.54 to 0.37 ± 1.33; p < 0.001). MMO remained stable, indicating preserved mandibular mobility. Overall, 79.6% of patients met the predefined favorable outcome criteria. Reintervention was required in 12 patients; 7 received additional incobotulinumtoxinA injections, and 5 underwent TMJ arthrocentesis. No complications were observed. Conclusion: This protocol was associated with improvements in muscular pain and orofacial discomfort while preserving mandibular mobility. However, given the retrospective design and absence of a control group, these findings should be interpreted as hypothesis generating.

Toxins doi: 10.3390/toxins18050219

Authors: Bin Gao Jialin Sun Zechao Xu Xiaohui Li Jianxin Ma Xiaomin Han Shuo Wang

Mycotoxin contamination in wheat products has consistently been a key issue of concern in food safety, and urinary biomonitoring provides an effective approach for assessing internal human exposure. In this study, a sensitive ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of 28 mycotoxins in wheat products and human urine. For the two matrices, the extraction solvent, acid concentration, solid-phase extraction cartridge type, and enzymatic hydrolysis parameters were optimized. Under the optimized conditions, all target compounds showed excellent linear relationships within the tested concentration ranges (R2 > 0.99). In wheat products, the spiked recoveries ranged from 70.2% to 120%, the repeatabilities ranged from 1.6% to 9.1%, and the limits of detection and limits of quantification were 0.001~8.3 μg/kg and 0.002~25.0 μg/kg, respectively. In urine, the spiked recoveries ranged from 79.3% to 120%, the repeatabilities ranged from 0.7% to 9.4%, and the limits of detection and limits of quantification were 0.0001~1.0 μg/L and 0.0002~3.0 μg/L, respectively. Analysis of real samples showed that at least seven mycotoxins were detected in wheat product samples, and at least five were detected in urine samples. In wheat products, the detection rates of deoxynivalenol, enniatin B, enniatin A1, enniatin B1, tenuazonic acid, and tentoxin were all 100%, whereas in urine, the detection rate of fumonisin B1 reached 100%, and tenuazonic acid showed the highest mean concentration in both matrices. In conclusion, the developed ultra-performance liquid chromatography–tandem mass spectrometry method is suitable for the simultaneous quantification of 28 mycotoxins in wheat products and human urine, and its preliminary application demonstrates good practical applicability.

Toxins doi: 10.3390/toxins18050218

Authors: Terenzio Bertuzzi Lorena Schiavi Federico Siboni Roberta Battaglia Paola Giorni Domenica Iraci Capuccinello Massimo Montanari

Hemp seeds and derived products were recently re-evaluated in the food sector, thanks to their high nutritional value and absence of gluten. This increasing diffusion required to investigate the occurrence of mycotoxins, in particular of Alternaria toxins (ALTs), has been uncovered at high levels in previous work. An integrated approach was involved in this study. First, Alternaria spp. incidence and ALTs were determined in hemp seeds harvested in different fields during 2024 and 2025 and the influence of meteorological conditions and of varieties was evaluated. Then, their distribution in hemp oil and defatted flour was studied after a cold pressing process of naturally contaminated hemp seeds, finding a high percentage, over 85%, in hemp cake. Finally, a small survey was conducted on different hemp products intended for direct human consumption, confirming the risk of contamination in seeds, flour and derived bakery products.

Toxins doi: 10.3390/toxins18050217

Authors: Stefano Carda Elisa Grana

Background: Post-stroke neuropathic pain, particularly central post-stroke pain and facial pain syndromes, continues to be challenging to manage with conventional pharmacological approaches. While botulinum toxin A (BoNT-A) is well established for treating spasticity after stroke, its use in the management of central neuropathic pain remains less well established. Methods: This report presents two cases of patients with refractory neuropathic pain following ischemic cerebrovascular accidents who achieved significant pain relief through subcutaneous botulinum toxin administration, after failure of multiple conventional and intramuscular BoNT-A approaches. Results: Case 1 involves a 66-year-old patient with 18 years of post-stroke hemicorporeal pain who responded dramatically to subcutaneous BoNT-A injections after extensive prior treatment failures. Case 2 describes a 54-year-old with trigeminal-region and mandibular pain following ICA dissection who achieved complete pain resolution at facial sites with subcutaneous administration of BoNT-A. Conclusions: These cases demonstrate the potential efficacy of subcutaneous botulinum toxin for managing post-stroke neuropathic pain in selected patients and suggest a mechanism of action related to peripheral pain sensitization rather than motor denervation. Our findings support further investigation of subcutaneous administration techniques for pain management in specialized centers.

Toxins doi: 10.3390/toxins18050216

Authors: Tsonka Dimitrova Djeni Cherneva Kaloyan Mihalev Ivelin Iliev Galina Yaneva Svetlana Georgieva

Wild mushroom poisoning remains a major medical and toxicological challenge worldwide because of the diversity of toxic compounds, the broad spectrum of clinical manifestations, and the risk of severe hepatic or renal injury. Early differentiation between self-limiting gastrointestinal syndromes and potentially fatal intoxications with progressive organ failure remain a central clinical challenge. This review examines recent advances in the diagnosis, risk stratification, and therapeutic management of wild mushroom poisoning, with amatoxin intoxication serving as the principal clinical focus. Selected evidence from other mushroom toxic syndromes is also included to support differential diagnosis, highlight syndrome-specific variability, and provide comparative clinical and methodological context. The recent literature indicates a shift from predominantly symptom-based diagnosis toward integrated models combining clinical evaluation, laboratory biomarkers, toxicological testing, and analytical and molecular methods. Liquid chromatography, mass spectrometry, immunoassays, and the molecular identification of fungal species have improved diagnostic precision, particularly in cases with uncertain exposure history or delayed presentation. Current management relies on early multimodal strategies including intensive supportive care, targeted pharmacological interventions, extracorporeal detoxification, and, in selected severe cases, liver transplantation. Overall, clinical outcome depends not only on toxin profile, but also on timely diagnosis, accurate early risk stratification, and prompt coordinated treatment. Future research should prioritize standardized diagnostic pathways, validated prognostic models, and clinically applicable treatment algorithms that support earlier escalation of care in severe mushroom intoxication.

Toxins doi: 10.3390/toxins18050215

Authors: Warissara Kasikonsunthonchai Saowalak Adunphatcharaphon Chris Elliott Doris Marko Dino Grgic Florian Call Awanwee Petchkongkaew

Multiple mycotoxins in feed threaten animal health and food safety, demanding sustainable mitigation strategies. This study evaluated acid-modified mangosteen peel (AMP), an agricultural by-product, as a potential multi-mycotoxin adsorbent. Physicochemical characterization using scanning electron microscopy (SEM), Brunauer–Emmett–Teller (BET) surface area analysis, and Fourier transform infrared spectroscopy (FTIR) analyses demonstrated that acid modification increased surface area (1.9 to 9.03 m2/g), pore volume (0.005 to 0.027 cm3/g), and surface negativity, indicating enhanced adsorption properties. In vitro binding experiments assessed adsorption of aflatoxin B1 (AFB1), zearalenone (ZEA), ochratoxin A (OTA), T-2 toxin, deoxynivalenol (DON) and fumonisin B1 (FB1) under different pH conditions. AMP exhibited high adsorption efficiencies for AFB1, ZEA, OTA, and T-2 toxin, particularly at pH 3, whereas DON and FB1 showed limited binding. Adsorption behavior was dose-dependent and best described by Langmuir and Freundlich isotherm models. Simulated gastrointestinal digestion indicated stable binding of AFB1 and ZEA under gastric conditions, with partial release of some toxins at neutral pH. Cytotoxicity assessment in porcine intestinal epithelial cells (IPEC J2) showed no apparent cytotoxic effects at 0.25–1 mg/mL. Therefore, AMP demonstrated improved multi-mycotoxin adsorption compared to the untreated material and showed no apparent cytotoxic effects in vitro within the tested concentration range, indicating its potential as a promising feed additive candidate.

Toxins doi: 10.3390/toxins18050214

Authors: Krzysztof Waśkiewicz Michał Dąbrowski Michał Łuczyński Marcin Wróbel Łukasz Zielonka

Zearalenone (ZEN) is an estrogenic mycotoxin produced by Fusarium spp. and commonly found in cereals and feed materials. This study evaluated the ability of Metschnikowia pulcherrima KKP 1368 biomass to reduce extractable ZEN under controlled buffered pH conditions (pH 3.50 and 7.00) selected as simplified conditions relevant to the porcine gastrointestinal environment. ZEN was quantified by LC-MS/MS, whereas LC-MS-QTOF was used as a qualitative/semi-quantitative screening approach for tentatively assigned transformation-related features. In the presence of yeast biomass, extractable ZEN was already lower than in the corresponding controls at the first sampling point, indicating a rapid biomass-associated effect. After 12 h, reductions relative to the corresponding controls reached 63.0% at pH 3.50 (p < 0.0001) and 51.6% at pH 7.00 (p = 0.0001). ZEN remained stable in control samples, and the strain remained viable under both pH conditions throughout incubation. LC-MS-QTOF detected several tentatively assigned features consistent with zearalanone, zearalenone-14-glucuronide, and zearalenol O-glucoside; these assignments require confirmation with authentic standards. Overall, M. pulcherrima KKP 1368 reduced extractable ZEN in a simplified buffered in vitro system, probably through rapid adsorption/reduced extractability and possible biotransformation. Further studies using biomass fractions or inactivated biomass, mass-balance experiments, authentic standards, and toxicological assays are needed to clarify the relative contribution of adsorption and transformation and to assess the practical relevance of this approach.

Toxins doi: 10.3390/toxins18050213

Authors: Paulo Lee Ho

Transcriptomic and proteomic studies concerning venom and venom glands have provided major breakthroughs in the characterization and knowledge of global crude venom compositions and have also allowed for the identification of new toxins and activities [...]

Toxins doi: 10.3390/toxins18050212

Authors: Evoli N. Lopez Guadalupe Delgado-López Paola Maycotte Pablo Hernández-Jáuregui Irma Herrera-Camacho Nora Hilda Rosas-Murrieta Eunice López-Muñoz Claudia Teresita Gutiérrez-Quiroz Uriel Ramírez-Carrera Cindy Bandala Lourdes Millán-Pérez-Peña Maricruz Anaya-Ruiz

Triple-negative breast cancer (TNBC) continues to be a medical challenge requiring multiple treatment options. SV2A, a protein involved in vesicular release, has emerged as a promising biomarker for various cancers. Research shows that botulinum neurotoxin type A (BoNT/A), which binds to SV2A, the BoNT/A receptor, can inhibit the growth of prostate and breast cancer cells, suggesting its potential as an alternative treatment for breast cancer. The purpose of this study was to determine the potential of BoNT/A to inhibit tumor growth in a mouse preclinical model. BoNT/A was evaluated for its effects in an in vitro model employing 4T1 cells and in an in vivo model of orthotopically inoculated 4T1 cells in BALB/c mice. BoNT/A inhibited the proliferation of 4T1 cells, which express the SV2A protein; decreased tumor growth in the preclinical model; and decreased inflammation, associated with fewer blood neutrophils and monocytes, suggesting an immunomodulatory and anti-inflammatory effect. The effect of BoNT/A on the TNBC model supports its use as a repurposed drug for this type of aggressive cancer. Our results emphasize the significance of the SV2A receptor and its interaction with BoNT/A as promising therapeutic targets, particularly for TNBC.

Toxins doi: 10.3390/toxins18050211

Authors: Mahtab Khatibi José R. Almeida Ashifa Al Juwaiser Soheil Gilabadi Ketan Patel Sakthivel Vaiyapuri

The Cape cobra (Naja nivea), one of Africa’s most lethal snakes, can cause rapid, life-threatening paralysis. However, the impact of this venom on platelet function and blood coagulation remains poorly understood. To address this gap, we investigated the enzymatic profiles and the impacts of N. nivea venom on multiple aspects of haemostasis using human whole blood. Our results illustrate that Cape cobra venom significantly increases clotting time in rotational thromboelastometry without affecting other coagulation parameters. This venom significantly inhibits platelet aggregation and activation yet does not exert cytotoxic effects on platelets. The venom was subsequently fractionated using reverse-phase high-performance liquid chromatography, and the most potent purified fraction was identified as a cytotoxin (three-finger toxin) through mass spectrometry. This purified fraction showed an inhibitory effect on platelet activity. These findings highlight that N. nivea venom can induce haemotoxicity in addition to neurotoxicity. Moreover, three-finger toxins may be promising candidates for bioprospecting to develop novel antithrombotic agents.

Toxins doi: 10.3390/toxins18050210

Authors: Yamil Liscano Juan M. Álvarez-Caballero Alberto Aragón-Muriel

The antimicrobial resistance of Acinetobacter baumannii necessitates the development of novel therapeutic strategies targeting essential enzymes such as Undecaprenyl Pyrophosphate Phosphatase (UppP). This study explored spider venom peptides in silico as potential allosteric inhibitors of A. baumannii UppP. A systematic literature review was conducted to select eight α-helical peptides with reported anti-A. baumannii activity, followed by their computational physicochemical characterization. Three-dimensional models of A. baumannii UppP and the candidate peptides were generated, and a putative allosteric binding site was validated through molecular docking of a known inhibitor of the BacA homolog. The eight peptides were subsequently docked to this validated site using HADDOCK. Results revealed variable binding affinities; peptides LC-AMP-I1, Lycosin-II, and GK37 exhibited the most favorable HADDOCK scores and extensive interaction networks, consistent with their reported high antimicrobial potency. Other candidates, notably Lt-MAP2, showed low binding affinity but high predicted synergistic potential. These findings identify promising spider venom peptide candidates, suggesting dual (membrane disruption/UppP inhibition) or synergistic mechanisms of action, and validate UppP as a viable pharmacological target for peptide-based inhibitors.

Toxins doi: 10.3390/toxins18050209

Authors: Francesca De Battistis Chiara Civitelli Valentina Prota Francesca Caloni Alberto Mantovani Olimpia Vincentini

Emerging mycotoxins are unregulated natural toxins, often detected in small-grain cereal crops. They are produced by various Fusarium molds and have been reported in surveys conducted across Europe. Many Fusarium species that produce mycotoxins thrive and exhibit greater pathogenicity under relatively warm and humid conditions. Environmental conditions that promote fungal growth often also enhance mycotoxin accumulation. Various abiotic factors influence both Fusarium growth and mycotoxin biosynthesis, and several studies have associated these environmental conditions with the occurrence of enniatins (ENNs) and beauvericin (BEA) in cereal crops. Ongoing climate change in Europe may further support the spread and development of Fusarium species, potentially increasing the production of emerging mycotoxins. Following recent updates on the occurrence of these mycotoxins, this review evaluates the scientific literature concerning Fusarium species responsible for ENNs and BEA production.

Toxins doi: 10.3390/toxins18050208

Authors: Stefanie Lietz Holger Barth

The life-threatening disease pertussis, also known as whooping cough, is caused by a complex interplay of several virulence factors produced by the bacterium Bordetella (B.) pertussis. These include the AB-type protein toxin pertussis toxin (PT), the main causative agent of pertussis. After infection with B. pertussis, PT is released and binds to its human target cells, which internalize PT. The enzyme subunit of PT is then taken up into the cytosol, where it catalyzes the ADP-ribosylation of the α-subunit of inhibitory GTP-binding proteins from the Gαi type. This ultimately leads to the development of the characteristic clinical symptoms associated with pertussis. Pertussis is a vaccine-preventable but highly infectious respiratory disease, and especially younger children are prone to develop severe pertussis. Despite the vaccination, over the past few years, increasing case numbers have been reported globally. Moreover, treatment options are strongly limited to antibiotics and symptomatic treatment. Therefore, novel therapies against toxin-mediated diseases are urgently required, while AB-type toxins such as PT are promising pharmacological targets to combat these associated diseases. To identify novel pharmacological inhibitors for AB-type toxins, huge potential lies within the human proteome/peptidome. Endogenous protein or peptide inhibitors for bacterial toxins might have evolved as part of the innate immunity and are awaited to be discovered. The scientific community is committed to identify potential candidates through targeted screening or explorative hypothesis-driven approaches. This review summarizes the recent efforts in the identification and characterization of the human body’s own proteins and peptides that inhibit PT. PT-inhibiting peptides were found by unbiased screening of peptide libraries from human hemofiltrate or hypothesis-driven evaluation, and PT-neutralizing mechanisms were discovered in cell-based approaches. The identification of endogenous peptides and proteins, e.g., defensins and α1-antitrypsin, as potent inhibitors of PT paves the way towards the development of novel therapeutic options against pertussis.

Toxins doi: 10.3390/toxins18050207

Authors: Nela Drača Sunčana Včelik Rudolf Krska Drago Šubarić Tihomir Kovač

For centuries, bee pollen has been known for its medicinal value and regarded as a rich source of bioactive compounds, including essential nutrients and phytochemicals. Its putative therapeutic and health-promoting benefits include antioxidant, antibacterial, anticarcinogenic, anti-inflammatory, hypoglycaemic, and numerous additional properties. However, the beneficial qualities can only be guaranteed if potential contaminants do not detract from its superfood image. Recent research has indicated frequent occurrence of mycotoxins in bee pollen, occasionally at concentration levels exceeding safe intakes. There are very few published publications in the literature related to the research of mycotoxin concentrations in bee pollen. Based on that, the aim of this review is to provide an overview update of existing scientific research on the presence, prevalence and types of mycotoxins in bee pollen, with particular emphasis on toxins produced by fungi. Furthermore, the aim is to compile the available data on mycotoxin contamination of pollen in order to identify factors relevant to the safety and quality of bee pollen as a food product and dietary supplement.

Toxins doi: 10.3390/toxins18050206

Authors: Rachel Whiting Isobel Picken Grace Howells A. Christopher Green Chris Elliott Graeme C. Clark

Saxitoxin (STX) is one of the most potent natural neurotoxins known and is the only marine toxin to be declared a chemical weapon. In both marine and freshwater systems filter feeding organisms can accumulate saxitoxin and human consumption of toxin-contaminated food can result in paralytic shellfish poisoning. Here we highlight for the first time a human cell-based assay for the detection and neutralisation of STX activity on an automated patch clamp (APC) system. We demonstrate that a human embryonic kidney (HEK) cell line expressing human Nav1.6 can rapidly and sensitively detect the presence of a range of sodium ion channel blockers including STX. The use of neutralising monoclonal antibody GT13-A and/or saxiphilin was found to confer specificity to the assay by being able to dissociate between STX (along with closely related analogues) and tetrodotoxin. Finally, the application of the functional assay for the detection of STX in complex samples was evaluated during an international exercise led by the Organisation for the Prohibition of Chemical Weapons (OPCW). The neutralisation of STX activity in blinded samples enabled the indirect detection of the toxin in the relevant samples and provided an alternative orthogonal technique to corroborate the findings of liquid chromatography–mass spectrometry (LC-MS). Collectively this work demonstrates the significant potential for functional assays in the analysis of samples suspected of being contaminated with STX and related sodium ion channel targeting toxins; complementing traditional direct identification methods such as high-performance liquid chromatography with fluorescence detection (HPLC-FLD), LC-MS or enzyme-linked immunosorbent assay (ELISA).

Toxins doi: 10.3390/toxins18050205

Authors: Sihan Pan Yuanzhi Ye Yang Li Hongxin Fu Jufang Wang

Botulinum neurotoxins (BoNTs) act on peripheral cholinergic nerve terminals, inducing reversible muscle paralysis and profound therapeutic effects. However, their limited cell-type specificity and narrow therapeutic window have motivated the development of engineered variants. Here, a modular strategy was employed to construct full-length chimeric BoNTs, grafting receptor-binding segments from BoNT/B or BoNT/F onto the BoNT/A framework. The novel chimeras AAAF and AAFF efficiently cleaved rSNAP-25 in cell-free assays. Firstly, both toxins showed effective cellular uptake and cleaved endogenous SNAP-25 in Neuro-2a cells, with cleavage efficiencies of approximately 46% for AAAF and 73% for AAFF, highlighting the enhanced activity of AAFF. Secondly, AAAF induced faster recovery from reversible muscle paralysis compared to rBoNT/A-WT, whereas AAFF produced more sustained paralysis, with both exhibiting reduced systemic toxicity. Despite these altered pharmacological profiles, the chimeras required higher doses than rBoNT/A-WT to induce neuromuscular effects. Collectively, this study presents the design of novel chimeric BoNT/A-F proteins, characterizes their functional activities, and provides a preliminary exploration of how domain grafting affects cellular uptake, enzymatic activity, and neuromuscular pharmacodynamics.

Toxins doi: 10.3390/toxins18050204

Authors: Svetlana V. Malysheva

Natural toxins constitute a large group of toxic compounds produced by plants, fungi, bacteria and other organisms [...]

Toxins doi: 10.3390/toxins18050203

Authors: Jiangbin Wang Min Jiang Shuhua Wang Zixin Wang Yikun Cui Ying Feng Shanshan Zhang Mingjiang Cai Yanping Zhong

Algal proliferation in subtropical drinking water reservoirs has become increasingly severe, and developing a reliable prediction for algal abundance through high-frequency in situ data is essential for early risk warning and effective management. This study analyzed the interannual variations in algal abundance in the Shanmei (SM) Reservoir, located in Quanzhou City, Fujian Province, China, based on the high-frequency data between 2020 and 2025, and forecasted algal abundance 24 h ahead via the optimized Transformer model. Results revealed that the SM reservoir exhibited seasonal variability in environmental factors, with persistently elevated pH during spring and summer, ranging from 7.12 to 9.66, and relatively high total nitrogen concentrations, ranging from 1.17 to 2.28 mg/L. Overall, algal abundance increased throughout the study period, and the annual average algal abundance in 2025 was 8.18 × 106 cells/L, which was twice that in 2021. Model comparisons revealed that the optimized Transformer model exhibited the highest performance in terms of R2 = 0.88 when predicting the next hour using 12 days of data. Feature importance analysis based on SHapley Additive exPlanations (SHAPs) revealed that the predictions of algal dynamics were primarily influenced by previous-hours algal abundance, permanganate index, dissolved oxygen, air temperature, wind speed, and pH. This study revealed that the optimized independent learning model with integrated multi-scale features can significantly enhance the predictive performance of algal dynamics, offering a technical basis for early warning of algal blooms and refined reservoir management.

Toxins doi: 10.3390/toxins18050202

Authors: Frances Marie Tamayo Raymond Rosales Jörg Wissel Bo Biering-Sørensen Joshua Nathaniel Ellano David Simpson

In the original publication of this article [...]

Toxins doi: 10.3390/toxins18050201

Authors: Zichen Qiao Yong Tang Qianshun Zhou Bryan G. Fry

Azemiops feae (Fea’s viper) is a phylogenetically distinctive Asian viper with poorly defined medical significance, and human envenomations remain rarely reported in the English-language literature. We describe a new case of A. feae envenoming from Chongqing, China, and present a scoping review of published clinical case reports and case series to better characterize its epidemiology, clinical manifestations, and management. A 53-year-old male developed marked local pain and swelling following a bite to the hand, accompanied by transient neurotoxic symptoms, as well as mild hypofibrinogenemia. Treatment with a single vial of Gloydius brevicaudus monovalent antivenom was followed by clinical improvement and full recovery. Review of the literature identified nine previously published studies from China and one captive case from Europe. Envenoming typically occurred during agricultural activities, most commonly affected the lower extremities, and was characterized by prominent local effects with occasional mild neurotoxic features and inconsistent, generally mild coagulation abnormalities. Antivenom use was highly variable, involving multiple heterologous monovalent antivenoms, and outcomes were uniformly favourable regardless of antivenom administration. Collectively, available evidence indicates that A. feae envenoming is usually self-limited, with predominantly local effects and infrequent, mild systemic involvement. However, the absence of species-specific antivenom and the heterogeneity of current treatment practices highlight the need for systematic venom characterization and functional antivenom efficacy studies to inform evidence-based clinical management.

Toxins doi: 10.3390/toxins18050200

Authors: Ju-Eun Hong Soonjae Hwang

Metabolic dysfunction-associated steatohepatitis (MASH) is a complex, multifactorial disease heavily influenced by the gut–liver axis. While enterotoxigenic Bacteroides fragilis (ETBF) and its principal virulence factor, B. fragilis toxin (BFT)—a zinc-dependent metalloprotease—are well-known for disrupting intestinal barriers, their potential systemic impact on distant organs remains an emerging area of interest. Although various gut-derived factors contribute to hepatic inflammation, the precise molecular triggers that exacerbate the transition from simple steatosis to progressive fibrosis remain incompletely understood. This review proposes the “Direct Structural Disruption” hypothesis, examining the biological activity of BFT and its proposed role in MASH pathogenesis. We postulate that under permissive conditions, systemic BFT may target hepatic structural proteins (e.g., cadherins). This hypothesized architectural impairment amplifies canonical fibrogenic signaling and hepatic stellate cell (HSC) activation. In addition, we discuss current challenges in the detection and characterization of systemic BFT, particularly the technical limitations in clinical diagnostics stemming from its profound structural homology with host metalloproteinases. Future research integrating advanced diagnostic methodologies and liver-specific in vivo models is essential to elucidate these pathophysiological mechanisms and evaluate the ETBF-BFT axis as a complementary target in progressive MASH.

Toxins doi: 10.3390/toxins18050199

Authors: Andrew Davis Kamaljit Bajwa Zachary Martinez Ryan R. Davis April Green Fletcher Suber Shauna Farr-Jones Milan T. Tomic

A highly potent antitoxin for botulinum neurotoxin (BoNT) serotypes A and B has been developed that comprises three monoclonal antibodies (mAbs) targeting BoNT/A and three targeting BoNT/B. These oligoclonal antibody combinations neutralize toxin by simultaneously binding non-overlapping epitopes, thereby promoting rapid toxin clearance. All six mAbs use the same human Fc and framework and have been individually manufactured using the same expression platform and purification process. To minimize the time and labor required to produce the divalent antitoxin, we tested a co-expression and co-purification strategy for the three mAbs per serotype. The mAbs were expressed in CHO-K1 cells, and the media were optimized for co-expression in 10 L bioreactors. Chromatographic co-purification consisted of Protein A capture, followed by strong anion exchange chromatography in flow-through mode and cation-exchange chromatography in bind-elute mode. Co-expression experiments demonstrated that expression of the three anti-BoNT/A antibodies remained within approximately ±30% of the optimal equimolar ratio, whereas the anti-BoNT/B antibodies showed greater variability. Downstream purification steps achieved recoveries greater than 95% per chromatographic step, resulting in overall process yields of approximately 63–75%. This strategy provided sufficient purity of all six mAbs while largely preserving their relative ratios. These results demonstrate the feasibility of producing oligoclonal antitoxin antibodies using co-expression and shared purification strategies. Such approaches may simplify the manufacturing of antibody cocktails while maintaining product quality and biological activity.

Toxins doi: 10.3390/toxins18050198

Authors: Jisu Jin Guangyuan Jiao Xiaolei Huang Yingying Sun Chao Chen Hong Zhang

Venom-mediated systemic toxicity is not fully understood. This study explored the dose-dependent effects of Vespa mandarinia venom (VMV) on mice via integrated transcriptomic and metabolomic analyses. Subcutaneous VMV injection induced dose-dependent hypothermia: 80 μg caused severe transient hypothermia and partial mortality, while 40/60 μg led to reversible hypothermia within 24 h. Whole-blood sequencing identified 2400–3281 differentially expressed genes (DEGs) per group, including 1764 shared DEGs. Immune-related pathways were significantly activated, with hub genes validated by qRT-PCR. Serum metabolomics revealed alterations in organic acids, alkaloids, and other metabolites. Integrative transcriptome–metabolome analysis predicted the potential involvement of various pathways in VMV-induced toxicity, including ferroptosis (shared in low-dose VMV groups) and apoptosis. Cumulatively, this study confirms that VMV induces immunometabolic reprogramming, providing a molecular framework for understanding venom-induced systemic toxicity.

Toxins doi: 10.3390/toxins18050197

Authors: Yaqian Liu Shimin Lei Yixuan Peng Yuan Li Xingxiang Chen Xinyi Xu Sichao Mao

Aflatoxin B1 (AFB1) and patulin (PAT) are prevalent foodborne mycotoxins with hepatotoxic potential, but the hepatic effects of combined exposure remain largely unclear. This study investigated the hepatotoxic consequences of co-exposure to AFB1 and PAT using no-observed adverse effect levels (NOAELs) in C57BL/6 mice and low-cytotoxic concentrations in HepG2 cells selected by viability screening. Mice and cells were assigned to four groups: control, AFB1, PAT and AFB1 + PAT. Exposure to either toxin individually did not cause evident liver injury, whereas co-exposure significantly elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, reduced liver index, and induced clear histopathological alterations. Co-exposure markedly aggravated oxidative stress, characterized by increased reactive oxygen species (ROS) and malondialdehyde (MDA) and decreased superoxide dismutase (SOD). In parallel, the levels of interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) were elevated, together with the early fibrosis-related markers alpha-smooth muscle actin (α-SMA) and vimentin. The apoptotic response was characterized by increased Bcl-2-associated X protein (Bax) and reduced B-cell lymphoma-2 (Bcl-2), together with cysteine-dependent aspartate-specific protease-3 (caspase-3) activation. These findings indicate that co-exposure to AFB1 and PAT elicits hepatotoxicity through amplified oxidative stress, inflammation, and caspase-dependent apoptosis, supporting the need to further consider mycotoxin co-exposure in toxicological evaluation.

Toxins doi: 10.3390/toxins18050196

Authors: Katia Comte

Aquatic environments, whether freshwater, brackish, or marine, are increasingly disrupted, in terms of frequency, extent, geographic distribution, and duration, by the massive, worldwide proliferation of harmful and/or nuisance algae, the so-called Harmful algal blooms (HABs), which are a global phenomenon that poses a major threat to human and animal health and ecosystems [...]

Toxins doi: 10.3390/toxins18050195

Authors: Zixue Yuan Min Zhou Yue Luan Lei Kong Weiren Yang Shuzhen Jiang

Zearalenone (ZEA) is a potent estrogenic mycotoxin known to disrupt reproductive functions, but its precise central neuroendocrine mechanisms remain unclear. This study investigated the effects of ZEA on the hypothalamic-pituitary Kiss1/GPR54 signaling pathway in weaned gilts. A total of 32 gilts were randomly assigned to four dietary treatments contained with 0, 0.15, 1.5, or 3.0 mg/kg ZEA for a 32-day feeding trial. Histopathology, immunohistochemistry, and mRNA/protein expression analyses of GPR30, Kiss1, GPR54, GnRH, and GnRHR in the hypothalamus and pituitary gland were conducted. ZEA exposure induced significant histological damage in both tissues. In the hypothalamus, Kiss1, GPR54, GnRH, and GnRHR exhibited a non-linear response, increasing at moderate doses and decreasing at 3.0 mg/kg ZEA, whereas GPR30 expression was continuously upregulated. In the pituitary gland, GnRHR showed a similar non-linear pattern. Furthermore, high-dose ZEA down-regulated pituitary Kiss1 and GPR54 while up-regulating GnRH and GPR30 expressions. In conclusion, ZEA induces reproductive neuroendocrine toxicity through a complex, dose-dependent modulation of the Kiss1/GPR54 signaling axis. The persistent upregulation of GPR30 suggests it acts as a crucial mediator in disrupting this endocrine feedback loop within the hypothalamus and pituitary gland.

Toxins doi: 10.3390/toxins18050194

Authors: Cuiping Shao Yalin Li Ying Wu Lina Zhao Pingping Tian Shaobin Gu

This study investigates the degradation characteristics, pathways, and mechanisms of ochratoxin A (OTA) by Weizmannia coagulans CGMCC 9951 (W. coagulans CGMCC 9951), as well as its detoxification effect on Cornus officinalis pulp through fermentation. The strain efficiently degraded 300 ng/mL of OTA within 72 h (98% degradation) under optimal conditions of 37 °C, pH 5.0, and 180 rpm. Active degradation substances were primarily localized in the cell-free supernatant (CF). The degradation activity was significantly inhibited by heat treatment, proteinase K, EDTA, Cu2+, and organic reagents, suggesting an enzymatic mechanism. UHPLC-MS and MS/MS analysis indicated that OTA appears to be degraded to a product consistent with ochratoxin α (OTα). Based on homology to known OTA-degrading carboxypeptidases, the gene encoding WGU28473.1 was selected, expressed in E. coli, and confirmed to possess OTA-degrading activity. Molecular docking suggested potential interactions between the enzyme and OTA. Under optimal conditions, co-fermentation with Cornus officinalis pulp contaminated with 300 ng/mL OTA for 96 h resulted in a 74% degradation of OTA. The fermentation process increased the pulp’s sugar content and ABTS+ free radical scavenging capacity, reduced acidity, and improved the safety of the pulp. These findings demonstrate that W. coagulans CGMCC 9951 efficiently degrades OTA and improves pulp quality, highlighting its potential as a starter culture for detoxifying OTA-contaminated food.

Toxins doi: 10.3390/toxins18050193

Authors: Xinyue Zhang Yufan Yang Yongping Jiang Lingfang Shi Haolan Du Antonio Francesco Logrieco Antonio Moretti Susu Han Fuguo Xing

Aflatoxin B1 (AFB1) is a highly stable mycotoxin that can persist during conventional food processing and therefore poses a serious risk to food and feed safety. In this study, two enzymes (CotA and Prx) were heterologously expressed in Bacillus subtilis, purified by Ni–NTA affinity chromatography, and evaluated for their ability to degrade AFB1. Both enzymes exhibited remarkable thermostability and distinct catalytic optima. CotA exhibited its highest activity at 80 °C with an AFB1 removal of 38.4%, whereas Prx showed its highest activity at 90 °C with a removal of 82.6%. The optimal pH values were near neutral, with CotA performing best at pH 7.0 and Prx at pH 7.5, and both reactions approached maximal conversion within approximately 10 h. When the two enzymes were combined, a clear cooperative effect was observed. The mixed system achieved 91.0% AFB1 removal at 80 °C after 10 h, with the best degradation activity occurring at a CotA to Prx ratio of 1:3. At 50 °C, neither enzyme alone caused appreciable AFB1 degradation, but the addition of hydrogen peroxide markedly enhanced catalytic activity. Both enzymes also retained substantial activity after boiling and autoclaving. In a maize flour model, the mixed-enzyme system showed strong AFB1 degradation capacity, and peroxide-assisted treatment further improved activity. These results establish a thermostable and peroxide-responsive enzymatic platform for AFB1 degradation and support future development of enzyme-based detoxification strategies for food and feed applications. Product identification and toxicological validation will be needed to confirm the safety of the treated products.

Toxins doi: 10.3390/toxins18040192

Authors: Małgorzata Cisowska-Adamiak Magdalena Mackiewicz-Milewska Elżbieta Dorota Miller

Background: Botulinum neurotoxin type A is widely used in the management of post-stroke upper-limb spasticity; however, many studies report total injected doses rather than muscle-specific dosing, limiting clinical applicability. This study aimed to evaluate how frequently muscle-level dosing protocols of onabotulinumtoxinA are reported and to assess consistency of dosing patterns across published studies. Methods: A literature search was conducted in PubMed, Wiley/Cochrane Library, and EBSCO/CINAHL using a structured search strategy informed by PRISMA guidelines. Studies published within the last 10 years reporting on onabotulinumtoxinA treatment in post-stroke upper-limb spasticity with muscle-specific dosing data were included. Studies not providing muscle-level dosing or not allowing extraction of post-stroke upper-limb data were excluded. Data were summarized descriptively and compared across studies. Results: Twenty-seven full-text articles were assessed, and five studies met the inclusion criteria. Muscle-specific dosing was consistently reported for commonly treated muscles such as biceps brachii and wrist and finger flexors, whereas other muscles were less frequently targeted. Variability in dosing between studies was observed, particularly in multicenter real-world datasets. Standardized high-dose protocols contrasted with individualized dosing strategies, which generally showed more moderate dose ranges. Expert recommendations often suggest higher doses than those observed in routine clinical practice. Conclusions: Muscle-specific dosing of onabotulinumtoxinA in post-stroke upper-limb spasticity is reported infrequently, and substantial variability exists between studies and clinical practice. Standardized reporting of muscle-level dosing and its relationship to baseline spasticity severity is needed to improve clinical applicability and reproducibility.

Toxins doi: 10.3390/toxins18040191

Authors: Leqi Wang Zhenyi Liu Ivan M. Dubovskiy Changlong Shu Jie Zhang Junjie Zhang Wenmei Du Qi Peng

Phyllotreta striolata is a global pest of cruciferous vegetables, and controlling its soil-dwelling larvae is challenging. The lack of standardized larval bioassay methods hinders the screening of effective biocontrol agents. In this study, we established a stable and standardized laboratory-efficacy trial system for P. striolata larvae. Indoor rearing techniques were optimized for Brassica juncea var. foliosa and Brassica juncea var. megarrhiza were identified as the optimal host plants, with ideal oviposition conditions at 26–28 °C using black flannel substrate, and soil-cultured Brassica rapa var. pekinensis as the host plant. Based on these findings, a larval bioactivity assay was established using B. juncea var. megarrhiza slices on water-agar. This system maintained a natural larval mortality rate below 5% within 48 h, meeting the bioassay requirements. The reliability of the system was validated by evaluating the activity of the engineered Bacillus thuringiensis (Bt) strain G033A against larvae, where the LC50 value decreased from 23.013 mg/mL to 7.295 mg/mL with an extended treatment time (12–48 h). Using this standardized method, novel Cry proteins with high activity against P. striolata larvae were screened. Cry8Ca and Cry8Ga proteins exhibited LC50 values of 2.243 mg/mL and 1.649 mg/mL, respectively.

Toxins doi: 10.3390/toxins18040190

Authors: María Morales Alicia Jordá Marín Bárbara Cases Louise Wallace Dolores Hernández Fernández De Rojas

A homologous classification for vespid venoms is missing. This study compared Polistes dominula and Vespula spp. venoms to evaluate their homology level. P. dominula and Vespula spp. extracts, including V. germanica, V. maculifrons, V. pensylvanica, V. alascensis, and V. squamosa in equal proportions, were generated from venom sacs and were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot using Vespula-positive sera. Bands described as allergenic were excised and sequenced through Liquid Chromatography–Mass Spectrometry tandem analysis (LC-MS/MS) to confirm their identity. Phospholipase (group 1) and hyaluronidase (group 2) enzymatic activities were measured. Group 1 and 5 3-D structures and sequence identity were analyzed in silico. The results showed that the P. dominula and Vespula spp. venom extracts exhibit similar protein profiles and comparable allergen composition, with phospholipase and hyaluronidase activities. The structures of Pol d 1 and Ves v 1 and Pol d 5 and Ves v 5 were highly similar, and the identity levels were high across and within the Polistes and Vespula genera (≥50%). These results suggest the inclusion of venoms from Polistes and Vespula genera as candidates to create a new homologous group for wasp venoms and indicate that the currently described homologous groups require revision.

Toxins doi: 10.3390/toxins18040189

Authors: Woo-Seung Kim Soohyun Lee Ki-Ju Kwon So-Min Kim Ki-Jong Rhee

Bacteroides fragilis is a major commensal bacterium of the human colon. However, enterotoxigenic B. fragilis (ETBF) secretes B. fragilis toxin (BFT), a zinc-dependent metalloprotease that cleaves E-cadherin and promotes chronic inflammation and colorectal tumorigenesis. Despite extensive research, the cellular receptor for BFT remains unidentified. In this study, we developed His-tagged recombinant BFT variants including both catalytically active and inactive forms to facilitate biochemical and functional analyses. Functional assays confirmed that the active variant retained proteolytic activity and induced characteristic cellular responses, while the inactive variant served as an effective negative control. These results establish a robust experimental platform for BFT receptor identification and mechanistic studies of BFT-host interactions. The active and inactive BFT variants provide essential molecular tools for investigating ETBF pathogenicity and developing therapeutic interventions.

Toxins doi: 10.3390/toxins18040188

Authors: Ibrahim Güler Armin Kraus Gerrit Grieb Henrik Stelling

Botulinum neurotoxin (BoNT) treatment outcomes are commonly assessed through visual evaluation of facial wrinkle patterns, a process that remains inherently subjective despite structured grading systems. This study evaluated whether contemporary multimodal artificial intelligence (AI) systems can identify facial changes associated with BoNT treatment, using region-specific wrinkle patterns as surrogate markers of underlying muscle activity. A dataset of 46 facial images (23 pre-treatment, 23 post-treatment) was analyzed using four multimodal models, each assessed across five independent runs. Models were tasked with classifying treatment state from single images, detecting wrinkle presence in the forehead, glabella, and periorbital regions, and generating exploratory severity scores and age estimates. Two models achieved 100% accuracy in distinguishing pre- from post-treatment images in this dataset, while region-specific wrinkle detection was variable and frequently did not exceed majority-class baselines. Inter-run reliability varied substantially across models. Exploratory wrinkle severity scores showed directional differences between treatment states, whereas apparent age estimates demonstrated minimal systematic variation. These findings suggest that global facial changes associated with BoNT treatment appear to be detectable in model outputs, but region-specific assessment remains limited, underscoring the need for cautious interpretation and further validation.

Toxins doi: 10.3390/toxins18040187

Authors: Claudia Panzera Sebastiano Gangemi Luisa Ricciardi

Hymenoptera venom allergy is a cause of anaphylaxis, which significantly affects patients’ daily lives due to the constant fear of accidental stings. Venom immunotherapy (VIT) is the only treatment capable of preventing severe systemic reactions (SSRs). Limited long-term real-life data are available, integrating both clinical and immunological outcomes. A five-year prospective observational study was conducted on 35 patients with a history of SSR who underwent VIT at a tertiary allergy center in Southern Italy; two of them had a diagnosis of systemic mastocytosis. Most patients were sensitized to Vespula, but others to Apis, Polistes dominula and Vespa crabro, reflecting the exposure pattern characteristic of Mediterranean regions. Clinical outcomes following accidental re-stings and serological trends, including total IgE, venom-specific IgE, and baseline serum tryptase, were assessed at treatment initiation and after five years of maintenance therapy. During the entire follow-up, all patients tolerated VIT. No SSRs occurred after accidental stings in 17/35 patients, confirming clinical protection achieved with VIT. Vespula serum-specific IgE presented a highly significant decrease; total IgE, tryptase and specific IgE for Apis, Polistes dominula and Vespa crabro showed a statistically significant decrease. Our findings reinforce the role of VIT as a well-tolerated, effective and disease-modifying treatment in a real-world setting.

Toxins doi: 10.3390/toxins18040186

Authors: Anna Langejürgen Gudula Schmidt Leon Unsöld Helma Tatge Ethel Oyson Ralf Gerhard

Toxin A and B from Clostridioides difficile are the main pathogenicity factors for clinical symptoms of C. difficile infections. Receptor-mediated endocytosis and endosomal escape are required for targeting substrate proteins of the Rho-GTPase family. We previously reported that Toxin B (TcdB) affects endo-lysosomal transport and autophagic flux of target cells. These effects are independent from pathogenic Rho inhibition. Here, we aimed at further characterization of this event by immunofluorescent characterization of the vesicular structures that are affected. We found large aggregates of damaged endolysosomal structures positive for EEA1, LAMP1, CHMP4B and TcdB, as well as an increase in perinuclear concentration of non-mature autophagosomes (amphisomes) positive for SQSTM, Rab7, and LC3B. We investigated whether Rab7, a regulator of late endosome transport, is causative for decreased lysosome function. Although TcdB induced an increase in active Rab7, as tested by an RILP pull-down assay, inhibition of Rab7 did not prevent TcdB-induced decrease in cathepsin D as a surrogate for lysosome dysfunction. It also indicates that the observed increase in Rab7 positive amphisomes is secondary to lysosomal dysfunction. By applying an autoproteolytic deficient mutant of TcdB we proved that the release of the glucosyltransferase domain is mandatory for triggering all of these effects. This suggests that after membrane perforation the toxin remnants leave an open leak in endolysosomes affecting ion homeostasis. Investigation of all large clostridial glucosyltransferases and other toxins revealed lysosomal dysfunction as a general effect of many but not of all toxins that integrate into the endosome membrane.

Toxins doi: 10.3390/toxins18040185

Authors: Manisha Mishra Leonor Georgette Farias Steven Song Steven Nguyen Purav Shah Adler R. Dillman

The use of Steinernema carpocapsae infective juveniles as biological control agents is a long-standing practice, yet the oral impact of their secreted venom proteins on crop pests remains largely unknown. We evaluated the oral toxicity of S. carpocapsae venom proteins against Spodoptera frugiperda using artificial diet assays. Ingestion caused significant dose-dependent toxicity in early-instar larvae, resulting in mortality and a prolonged developmental duration. Carry-over effects were profound; treated pupae were smaller and malformed, with only 19% of larvae fed on 1000 ng g−1 venom protein-supplemented diet reaching adulthood compared to 92% in controls. Surviving adults lived 30% fewer days and laid over 90% fewer morphologically normal eggs. These physiological disruptions coincided with elevated oxidative stress and detoxification enzyme activity, suggesting the venom induces oxidative and detoxification responses, which may be associated with the observed phenotypic alterations. This study provides the first demonstration of the oral toxicity of entomopathogenic nematode venom proteins, positioning them as a promising resource for the discovery of novel insecticidal proteins for sustainable pest management.

Toxins doi: 10.3390/toxins18040184

Authors: Harald Hefter Sara Samadzadeh

Patients with cervical dystonia (CD) often believe that disease severity would have progressed beyond the pre-treatment level if botulinum neurotoxin (BoNT) therapy had not been initiated. The aim of the present study was to assess the perceptions of long-term BoNT-treated patients with CD regarding the expected course of disease severity over the next 10 years under the hypothetical assumption that BoNT therapy is discontinued. Fifty patients with idiopathic CD receiving long-term BoNT therapy were screened, and 43 patients were included. Disease severity at the day of recruitment was assessed as a percentage of CD severity at the onset of BoNT therapy (PAS-%). Patients also generated, in a standardized manner, a graph illustrating the development of CD severity since initiation of BoNT therapy. Subsequently, patients estimated the expected severity of CD after 10 years, expressed as a percentage of severity at BoNT therapy onset (PAS-STD), under the hypothetical assumption that BoNT therapy was discontinued at the time of recruitment. They additionally drew a graph depicting the anticipated progression of CD severity over the subsequent 10 years under this assumption. Furthermore, 33 of these 43 patients had previously assessed the expected development of CD severity under the assumption that no BoNT therapy had ever been performed (PAS-NO%) in an earlier study. Mean PAS-STD was significantly higher than mean PAS-% (p < 0.001). Comparison of mean PAS-STD with mean PAS-NO% in the 33 patients who participated in both studies demonstrated that mean PAS-STD was significantly lower than mean PAS-NO% (p < 0.001). Long-term BoNT-treated patients with CD believe that disease severity would worsen again if BoNT therapy were discontinued. However, they do not expect CD severity to deteriorate to the level that they believe would have been reached if no BoNT therapy had been administered. We interpret this finding as suggesting that patients perceive a preventive effect of long-term BoNT therapy.

Toxins doi: 10.3390/toxins18040183

Authors: Stephen J. Knabel Aubrey Mendonca

A recent publication proposed that the main biological function of chromosomal toxin–antitoxin systems (TASs) in free-living bacteria is to optimize fitness by mediating K Sensing and Control via a Nutrient-Responsive Cybernetic System. Viable cell density data were consistent with analog (continuous) regulation of population dynamics and cellular physiology throughout the life cycle; however, exactly how bacteria utilize TASs to regulate this was not explained in that publication. Two different concepts of injury have been proposed in the field of microbiology: (1) injury due to external physical and chemical stresses, which lead to sublethal (reversible) or lethal (irreversible) injury depending on the degree of injury, and (2) injury due to internal, self-inflicted stresses mediated by TA toxins. While self-inflicted injury due to TA toxins has been recognized as playing a role in growth arrest and dormancy, which can be reversed by repair, there is little support for TA toxins causing irreversible programmed cell death under normal physiological conditions. The purpose of the present paper was to explain how merging the above two concepts of injury might reveal how TASs optimize the fitness of free-living bacteria under normal physiological conditions by continuously regulating the ratio of injury: repair throughout the life cycle.

Toxins doi: 10.3390/toxins18040182

Authors: Michel Kawayidiko Kasongo Arthur Mpanzu Duki Christophe Tsobo Masiala Sarah De Saeger José Diana Di Mavungu

Mycotoxin contamination remains a persistent threat to food safety in the Democratic Republic of the Congo (DRC) and neighboring countries, driven by conducive tropical agroecological conditions, inadequate post-harvest practices, and limited regulatory governance. This critical narrative review (2009–2024) synthesizes the occurrence data for major staple foods (maize, peanuts, cassava, sorghum, millet, and beans) and dairy products compiled from Google Scholar, ScienceDirect, MDPI and institutional sources. It examines the co-occurrence patterns, exposure pathways, and analytical and regulatory gaps. Warm, humid lowland environments favor Aspergillus and aflatoxins, whereas cooler, humid highland zones promote Fusarium, fumonisins, and deoxynivalenol. Across commodities, contamination intensifies along food value chains through inadequate drying, non-hermetic storage, insect damage, and prolonged handling, with processed products generally exhibiting the highest levels of mycotoxins. Regulated mycotoxins, including aflatoxins, fumonisins, trichothecenes, ochratoxins, and zearalenone, frequently exceed European Union (EU), East African Community (EAC), and Codex Alimentarius Commission (CAC) limits in staple foods. Their co-occurrence is widespread, including emerging mycotoxins such as beauvericin and enniatins, particularly in maize- and peanut-based products, raising concerns about potential additive or synergistic effects. Aflatoxin M1 in milk highlights plant–feed–animal–human transfer within a One Health framework. Despite increasing evidence, the available data remain fragmented and heterogeneous; rapid tests dominate, while few studies employ multi-mycotoxin LC-MS/MS methods. Cross-border trade between countries, such as Uganda, Tanzania, Zambia and Angola, facilitates the circulation of contaminated commodities in the absence of harmonized standards and risk-based controls. Priorities include harmonized regional surveillance, biomarker-based co-exposure assessment, cost-effectiveness evaluation of mitigation strategies, and regulatory alignment at borders. Coordinated, multisectoral action is essential to reduce chronic dietary exposure and improve food safety across the region.

Toxins doi: 10.3390/toxins18040181

Authors: Mikela Vlachou Nikolaos Solomakos Alexander Govaris Vassilis Athanasiadis Stavros I. Lalas Andreana Pexara

This study aimed to assess the occurrence and contamination levels of OTA in edible tissues of slaughtered pigs in Greece using high-performance liquid chromatography with fluorescence detection (HPLC-FD). Kidney, liver, muscle, and fat samples were collected from 1695 healthy slaughtered pigs originating from 113 swine farms across eight geographical regions of Greece and analyzed for OTA. OTA was not detected in muscle or fat samples. In contrast, OTA was detected in 99 of 1695 kidney samples (5.8%), with concentrations ranging from 0.36 to 1.36 μg/kg (mean 0.73 μg/kg; median 0.70 μg/kg). OTA-positive kidney samples were identified in four regions, with the highest prevalence recorded in the regional unit of Karditsa, within the region of Thessaly (75/105 samples; 71.4%), where the maximum OTA concentration in kidneys was observed (1.36 μg/kg). Karditsa was also the only regional unit where OTA was detected in liver samples (40/1695; 2.4%), with concentrations ranging from 0.42 to 1.08 μg/kg (mean 0.61 μg/kg; median 0.53 μg/kg). The lack of detectable OTA levels in muscle and fat indicates minimal consumer exposure through pork; nevertheless, the presence of low-level residues in kidneys and liver emphasizes the necessity for ongoing monitoring using sensitive analytical methods. Overall, OTA contamination in edible tissues was low and unevenly distributed, reflecting localized exposure likely associated with region- or farm-specific feed contamination.

Toxins doi: 10.3390/toxins18040180

Authors: Kendra L. Lawrence Jeffery L. Owen Lindsey S. Garver Brandi A. Ritter Christopher M. Wilson Ginger R. Boatright F. Y. Bowling Timothy F. Platts-Mills Andrea K. Renner Rebecca W. Carter

Snakebite envenoming causes an estimated 138,000 deaths annually worldwide, with approximately 75% of fatalities occurring prior to arrival at definitive medical care. Even in regions where antivenom is available in hospitals, the absence of treatment options before a victim can reach definitive care results in delays of many hours before therapy is initiated. Manufacturing complexity, region-specific products, and the risk of anaphylaxis further limit the availability and use of antivenom in many regions. Reducing the persistently high mortality of snakebite envenoming requires both novel scientific approaches and partnerships that extend beyond traditional disciplinary and funding silos. This article describes the collaboration between Ophirex, a Public Benefit Corporation developing the oral secretory phospholipase A2 (sPLA2) inhibitor varespladib, and the United States military, which has identified a capability gap in snakebite treatment for forward-deployed personnel. The partnership was driven by a shared requirement for a shelf-stable, easy-to-administer, snake-species-agnostic therapy suitable for use prior to definitive medical care. A central insight of the program was that military operational requirements and global public health needs converged around the same product characteristics, enabling a strategically aligned development effort. From early proof-of-concept studies through regulatory pathway definition and advanced development, the Military–Ophirex partnership integrated operational requirements, regulatory planning, and iterative risk mitigation to advance manufacturing, nonclinical, and clinical development. This work provides both practical insights into complex drug development and a case study in how structured partnerships can carry innovation through translation in underfunded and operationally challenging conditions.

Toxins doi: 10.3390/toxins18040179

Authors: John T. Daugirdas

Hyperphosphatemia is a major complication in patients with kidney failure undergoing dialysis and is strongly associated with cardiovascular disease, vascular calcification, and increased mortality. Conventional management relies on dietary phosphate restriction, oral phosphate binders, and dialysis, yet persistent hyperphosphatemia affects a substantial proportion of patients. High-volume hemodiafiltration, combining diffusive and convective clearances, achieves greater phosphate removal than standard hemodialysis, with kinetic modeling predicting ~15–20% higher dialytic phosphate clearance (and ~0.5 mg/dL lower predialysis serum phosphate when nondialytic factors are constant). In this narrative review, we quantify the magnitude of improvement in dialytic clearance of phosphate with hemodiafiltration relative to hemodialysis and evaluate its effects on phosphate control measures. We also analyze phosphate balance in selected hemodiafiltration vs. hemodialysis comparisons and demonstrate why predialysis serum phosphate levels are sometimes only modestly lower or similar when hemodiafiltration is compared with hemodialysis. These findings are largely attributable to nondialytic factors—minor differences in phosphate binder equivalent dose, dietary phosphate ingestion, or residual kidney function—as predicted by phosphate kinetic modeling and supported by clinical trial data. Recognizing these confounders is essential for interpreting hemodiafiltration’s phosphate-lowering potential in real-world practice.

Toxins doi: 10.3390/toxins18040178

Authors: Mihaela Čikeš Šimunković Adrijana Leonardi Igor Križaj Svjetlana Karabuva

In Croatia, the European Viper Venom Antiserum®, produced by the Institute of Immunology Zagreb, was the only antiserum used to treat Vipera ammodytes envenomation. When production of the Zagreb antivenom ceased, three other antivenoms, Viperfav®, BulBio®, and Viekvin®, replaced it in clinical practice at the Department of Infectious Diseases, University Hospital Split. This study includes 34 patients envenomed by Vipera ammodytes during the period between 2020 and 2025: 24 (71%) suffered grade 2a envenomation, nine (26%) grade 2b, and one grade 3 (severe envenomation). None were admitted to the Intensive Care Unit. All patients received antivenom: 16 received Viperfav®, 17 BulBio®, and one Viekvin®. All grade 2a patients were treated with a single dose of antivenom. Among grade 2b patients, four received one dose and two received two doses of Viperfav®, while one received one dose and two received two doses of BulBio®. The grade 3 patient received two doses of BulBio®. In all cases, treatment was successful and patients were discharged from hospital after an average of 3.97 days. Patients with pronounced neurotoxic signs did not require treatment with multiple doses of antivenom. All antivenoms proved effective. No adverse reactions or fatalities were observed.

Toxins doi: 10.3390/toxins18040177

Authors: Phantakan Tansuwannarat Satariya Trakulsrichai Juthathip Pathumarak Achara Tongpoo

Asian coral snakes are distributed throughout Southeast Asia, including Thailand, but clinical data on their envenomation remain limited. Using a 10-year retrospective dataset from the Ramathibodi Poison Center, we investigated the epidemiology, clinical characteristics, management, and outcomes of Asian coral snake envenomation in Thailand. Patient demographics, clinical and laboratory data, treatments, and outcomes were analyzed descriptively. Fifty-two patients were included. Sinomicrurus macclellandi was the most frequently reported species. Most bites occurred during the rainy season and involved the lower extremities. Clinical manifestations were predominantly mild and localized. No cases of systemic neurotoxicity, bulbar weakness, respiratory compromise, or death were observed. Laboratory results were generally within normal limits. Two patients developed anaphylaxis, which resolved with standard emergency treatment, while two experienced severe pain. Calliophis intestinalis lineata was associated with a higher proportion of tachycardia at presentation and longer hospitalization. No patients required mechanical ventilation or antivenom therapy. Supportive care and short-term hospital observation are generally sufficient in confirmed cases. The median duration of hospitalization was 1–3 day. Local manifestations were the predominant clinical findings following Asian coral snake envenomation in Thailand, and systemic neurotoxicity was not observed. These findings differ from reports of Micrurus envenomation, which primarily involve New World coral snakes, whereas the species implicated in Thailand belong to Old World genera.

Toxins doi: 10.3390/toxins18040175

Authors: Caiyao Han Hong Yuan Sixian Chen Zhuohua Huang He Gong Lin Lv Xinpeng Zhou Jiang Ma Xin He

Tripterygium glycoside tablets (TGT), a representative formulation derived from Tripterygium wilfordii Hook F, have limited clinical application due to adverse reproductive toxicity. In previous studies investigating the effects of TGT on chronic kidney disease (CKD), it was found that both TGT and its alkaloid-enriched fraction (AEF) induced testicular atrophy, suggesting that AEF may be the material basis for the reproductive toxicity of TGT. Therefore, the reproductive toxicity of AEF was investigated in depth. This study established a CKD rat model to investigate the toxic effects of TGT, AEF, and the non-alkaloid-enriched fraction (NAEF) on the reproductive system during CKD treatment. Network toxicology and metabolomics were combined to elucidate the underlying mechanisms of AEF-induced reproductive toxicity. The results showed that both TGT and AEF significantly reduced testicular index and sperm concentration, causing seminiferous tubule atrophy and disrupting the levels of testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). Furthermore, TGT, AEF, and NAEF all significantly inhibited the proliferation of GC-1 cells. Network toxicology indicated that AEF modulates targets such as SRC, AKT, and HSP90AA1, thereby influencing pathways including the PI3K-AKT signaling pathway and pathways in cancer. Metabolomics obtained 89 differential metabolites of AEF, which were enriched in glycerophospholipid, linoleic acid, and arachidonic acid metabolism, a finding consistent with the constructed “metabolite–enzyme–reaction–gene” network. In summary, AEF exerts reproductive toxicity primarily by disrupting hypothalamic–pituitary–testicular axis homeostasis and perturbing glycerophospholipid, linoleic acid, and arachidonic acid metabolism.

Toxins doi: 10.3390/toxins18040176

Authors: Mara Lauriola Sophie Valkenburg Sander Dejongh Ward Zadora Hubert Krukowski Pieter Evenepoel Jeroen Raes Ricard Farré Griet Glorieux Björn Meijers

Growing evidence suggests that chronic kidney disease (CKD) profoundly disrupts gut microbiome and its activity. This study explores how CKD affects colon microbial metabolism, focusing on (1) the representativeness of fecal metabolomics, (2) saccharolytic and proteolytic fermentation metabolites, and (3) the gut microbiome’s role in the partitioning of tryptophan in its metabolic pathways. Tryptophan’s main metabolic pathways include the indolic and the kynurenine pathways, which lead, respectively, to the formation of indoxyl sulfate and kynurenine, both contributing to uremic toxicity. Using a rat model of CKD, we evaluated whether fecal concentrations of microbial compounds, on which most studies are based, reflect the colonic concentrations in contact with the gut mucosa. Thus, we quantified the concentration and content of amino acids, indole, p-cresol, and also short-chain fatty acids, in different colon sections. We demonstrated that CKD promotes increased proteolytic fermentation and an augmented tryptophan partitioning into both the indolic and kynurenine pathways. Depletion of the indolic pathway obtained upon antibiotic treatment leads to a further enhancement of the kynurenine pathway.

Toxins doi: 10.3390/toxins18040174

Authors: Eve Corrie Rebecca Bresnahan Ciara Doran Charlotte Leese Matthew R. Balmforth Anna Andreou Aisha Zhantleuova Elizabeth P. Seward Michael E. Webb W. Bruce Turnbull Bazbek Davletov

Botulinum neurotoxin injections are used off-label to treat chronic pain, but their efficacy is limited and paralytic effects restrict clinical utility in these applications. Here, we investigated whether combining the light chain and translocation domains of botulinum neurotoxin A (BoNT/A) with the GM1-binding B subunit of cholera toxin would be beneficial in silencing pain-associated sensory neurons. Chimeric ChoBot was assembled via a coiled-coil linking technology and was shown to retain the enzymatic activity of BoNT/A in vitro and in vivo. In cultured dorsal root ganglion neurons, ChoBot cleaved SNAP25 in a calcitonin gene-related peptide (CGRP)-rich subpopulation of sensory neurons, resulting in marked inhibition of CGRP release. ChoBot had a lesser effect on the compound muscle action potentials of the rat gastrocnemius muscle than BoNT/A following subcutaneous injections. In rat models of pain, including chemotherapy-induced peripheral neuropathy, intraplantar administration of ChoBot significantly attenuated mechanical allodynia. Immunohistochemical analysis confirmed SNAP25 cleavage in NF200- and CGRP-expressing sensory fibres in the epidermis following a single injection. ChoBot also mediated SNAP25 cleavage in human neuroblastoma cells in culture. Together, these findings indicate that ChoBot enables a silencing of pain-associated sensory pathways, providing a new strategy for the development of new long-lasting analgesics for chronic pain.

Toxins doi: 10.3390/toxins18040173

Authors: Prosper Amuzu Gan Gu Xuwen Hou Jiahang Sun Muhammad Abubakar Jakada Eromosele Odigie Daowan Lai Ligang Zhou

Fusarium oxysporum f. sp. radicis-lycopersici (Forl) is the etiological agent of tomato Fusarium crown and root rot (FCRR), a devastating soil-borne disease that severely compromises global tomato production. The pathogenicity of Forl has been increasingly linked to its capacity to produce phytotoxic isocassadiene-type diterpenoids. In this study, Forl was cultured in rice medium to obtain Forl cultures, which were used for the separation and identification of secondary metabolites. After removing the known metabolites, two new isocassadiene-type diterpenoid compounds, namely fusariumic acids I (1) and J (2), were isolated from the ethyl acetate extract. Their structures were identified using spectroscopic data analyses and quantum chemical calculations. This is the first report of the fusariumic acid analogs containing a hydroxyl group at position C–1 in the molecule. Fusariumic acids I (1) and J (2) exhibited significantly inhibitory activities on the hypocotyl elongation of tomato (Solanum lycopersicum) and sesame (Sesamum indicum) seedlings, as well as on the coleoptile elongation of rice (Oryza sativa var. japonica) seedlings at concentrations from 10 to 100 µg/mL. The discovery of two new phytotoxic isocassadiene-type diterpenoids expanded the diversity of secondary metabolites of Forl. Meanwhile, it provided critical insights into Forl-tomato interactions and the candidate lead compounds for the development of new herbicides as well.

Toxins doi: 10.3390/toxins18040172

Authors: Shusen Liu Xiaojuan Zheng Shuo Zhang Ning Guo Haijian Zhang Jie Shi

Highly sensitive methods for trace-level aflatoxin determination are indispensable for cereal food safety and public health protection. This study developed a ZrO2-assisted QuEChERS-UHPLC-MS/MS method for the simultaneous determination of AFB1, AFB2, AFG1, and AFG2 in maize, wheat, rice, and soybean. Systematic optimization identified acetonitrile as the optimal extraction solvent and 10 mg ZrO2 in combination with PSA, C18, and GCB as the optimal cleanup formulation, providing recoveries of 107.33–111.60%. Chromatographic baseline separation was achieved within 8.0 min using a moderate gradient program. The method exhibited excellent linearity (R2 > 0.999) with LODs of 0.15–0.25 µg/kg and LOQs of 0.50–0.75 µg/kg. Negligible matrix effects (0.85–1.02) validated the efficacy of ZrO2-assisted cleanup in eliminating co-extractive interferences in maize. Satisfactory accuracy (recoveries of 86.66–111.04%) and precision (RSDs < 14%) were obtained across all matrices. The method demonstrated consistent performance across diverse cereal and soybean matrices, fulfilling international regulatory requirements for routine aflatoxin monitoring in agricultural commodities.

Toxins doi: 10.3390/toxins18040171

Authors: Sylwia Barbara Okorska Magdalena Serafin-Andrzejewska Agnieszka Pszczółkowska Agnieszka Falkiewicz Marcin Włodarczyk Mengcen Wang Adam Okorski Marcin Kozak

Winter barley (Hordeum vulgare L.) is an important crop used for feed, food, malting, and bioethanol production. Recent research indicates that the seed mycobiome significantly influences seed health and usability, affecting its potential applications. This study examined the fungal species present in seven barley cultivars grown under two agrotechnical regimes. Fungal species were classified according to their effects on seeds and plants, and biodiversity indices were calculated for each group. Enhanced agrotechnical practices increased yields and improved grain quality. Higher DON concentrations were observed in low-yield treatments. Mycological analysis revealed that field fungi, particularly Fusarium, dominated the grain mycobiome and were associated with infection and reduced seed quality. High Dominance (Y), Margalef, and Shannon–Wiener indices for quality-deteriorating fungi correlated with lower yields, while the Dominance index (λ) for these fungi was negatively correlated with grain protein content. The prevalence of specific fungi on seeds depends on storage conditions and fungal adaptations, which may result in complementary consortia. Understanding these interactions can support the development of effective seed storage strategies and inform material classification and future use decisions.

Toxins doi: 10.3390/toxins18040170

Authors: Francisco Cruz Martin C. Michel Yasuhiko Igawa

Botulinum toxin type A (BoNT/A) injection into the bladder wall is a milestone in the treatment of urinary incontinence in patients with neurogenic detrusor overactivity (NDOi) or overactive bladder syndrome (OABi) who are refractory to or unable to tolerate oral or transdermal therapies. However, the efficacy of BoNT/A is hampered by the low long-term adherence of patients to a treatment that requires repeated bladder injections under cystoscopy control. The discontinuation is particularly evident among incontinent patients with spontaneous voluntary voiding, regardless of whether the cause is NDOi or OABi, although clearly more marked among the latter group. In addition to the bother and pain associated with repeated cystoscopies, these patients show low tolerance to the high incidence of urinary tract infections (UTIs) and transient urinary retention, the two most common adverse events. Fewer injection points may render treatments less painful, apparently without reducing efficacy, but will not avoid the need for repeated cystoscopies, and no studies have demonstrated that such modification increases adherence. Eventually, accessing the bladder wall for BoNT/A administration via a transabdominal approach, under real-time ultrasound guidance, may overcome trans-urethral limitations, but the technique’s reproducibility remains unknown. An intensive investigation is ongoing to identify aids that facilitate the passage of the large, fragile BoNT/A molecule across the urothelium to reach the bladder nerves without injections. Electromotive Drug Administration (EMDA) of BoNT/A demonstrated efficacy and safety over a 6-year follow-up in NDOi patients at a single center, but the results were not reproduced at other institutions. The application of shock waves to the bladder using shock waves generated by Extracorporeal Shock Wave Lithotripsy (ESWL) machines to tear the urothelium and facilitate the passage of BoNT/A instilled in the bladder is ingenious, but the experience is very limited. Dimethyl sulfoxide, liposomes, and thermal-reversal hydrogel to deliver the toxin failed in pilot trials. BoNT/A in nano-formulations has high heat stability, resistance to pH changes, and to enzymatic degradation. Extended efficacy in dermal and intramuscular pilot applications is promising but needs to be replicated in the bladder.

Toxins doi: 10.3390/toxins18040169

Authors: Hanish Polavarapu Walter A. Hall

Malignant gliomas remain highly treatment-resistant brain tumors despite surgery and adjuvant therapies. Targeted toxin therapies represent a unique strategy that exploits receptor-mediated cellular internalization to deliver cytotoxic components that result in the irreversible inhibition of protein synthesis independent of DNA damage or cell-cycle status. Advances in molecular profiling, toxin engineering, and delivery development have refined components targeting IL4Rα, IL13Rα2, EGFR/EGFRvIII, uPAR, and the transferrin receptor. Early clinical studies demonstrated biological activity, acceptable safety, and durable responses in subsets of patients, validating the fundamental mechanism of this approach. However, late-phase trials failed to demonstrate a population-level survival benefit, largely due to variability in delivery, receptor heterogeneity, and limitations in trial design rather than insufficient cytotoxic potency. Recent progress has focused on multiple receptor-targeting and delivery systems capable of achieving reliable intratumoral distribution. MRI-guided convection-enhanced delivery, vector-mediated toxin expression, and blood–brain barrier penetrant nanocarriers now enable more precise tumor targeting. Emerging evidence also reveals that toxin-mediated cytotoxicity can enhance antitumor immune responses, supporting their integration with immunotherapy. These advances position targeted toxins as precision cytotoxic compounds whose success depends on coordinated molecular targeting, delivery optimization, and biologically stratified patient selection, establishing a translational pathway for future glioma therapy.

Toxins doi: 10.3390/toxins18040168

Authors: Yixuan Fan Ziteng Liang Lingli Zou Luyao Wang Lei Ge Kai Zhao Yu Sun Peng Li

With the large-scale cultivation of transgenic Bacillus thuringiensis (Bt) crops, the Bt toxin released from Bt crops is continuously introduced into the soil. Its environmental fate represents a key indicator for assessing the ecological safety of transgenic crops. However, the persistence of Bt toxin in soil is influenced by both biotic and abiotic processes, and their respective contributions under natural conditions remain unclear. This study measured water-dissolved Bt toxin concentrations in paddy soil (PS) and red soil (RS) to compare the influence of biotic and abiotic factors on the dynamic retention of exogenous Bt toxin under different sterilization methods: no sterilization, heat sterilization (HT), and irradiation sterilization (IS). The water-dissolved Bt toxin exhibited a dynamic decrease–increase–decrease trend across all three treatments in both soil types during the 30 day experimental period. Bt toxin displayed rapid adsorption during the initial 2 h stage in RS, but subsequently showed a high desorption, whereas PS probably achieved more stable bonding through soil organic matter (SOM). Different sterilization methods significantly influenced the results by altering abiotic factors: Compared to CK, HT affected soil physicochemical properties and enhanced adsorption resilience, whereas IS caused minimal impact on the soil physicochemical properties, thereby providing a more accurate reflection of abiotic processes. And microbial, as biotic facters, also influence the reduction process of Bt toxin by participating in the adsorption–desorption–degradation equilibrium process. Therefore, we infer that over time, the concentration of water-soluble Bt proteins in the soil will tend toward zero. Additionally, the initial Bt toxin concentration influenced dynamic balance by adjusting adsorption site saturability, with more pronounced desorption reversibility at 500 ng/g concentrations. Overall, this study systematically reveals the effects of soil properties, microorganisms, and sterilization methods on Bt toxin persistence. The findings underscore the importance of selecting and justifying sterilization methods in related environmental behavior studies, while providing essential guidance for the scientific assessment of environmental risks posed by transgenic crops.

Toxins doi: 10.3390/toxins18040167

Authors: Jiahao Chen Qin Liu Songwen Tan Peng Guo Lianming Du

Rhabdophis lateralis is a snake species within the family Natricidae, which is widely distributed across mainland China, Russia, and Korea. Although this species was once thought to be non-venomous, there are quite a few cases demonstrating its bite could be fatal. In this study, we performed de novo assembly and analysis of the transcriptome data from the Duvernoy’s gland of R. lateralis, aiming to characterize its venom transcriptome and reveal the molecular basis of its toxicity. Among 6196 annotated transcripts, 77 were identified as potential toxin transcripts belonging to 26 toxin families. The most highly expressed toxin family was the SVMP family, accounting for 51.10% of the total toxin expression. The other notable toxins included cysteine-rich secretory proteins (CRISPs, 22.36%), c-type lectins (CTLs and snaclecs, 12.13%), and three-finger toxins (3Ftxs, 6.36%). Phylogenetic analyses indicated that SVMPs, CRISPs, and three-finger toxins (3FTxs) are evolutionarily conserved within Colubridae, whereas CTLs likely arose through convergent evolution. All identified SVMPs were classified as P-III type, with one sequence displaying a unique deletion distinct from conventional truncation patterns. The predominantly expressed CTLs are more likely to combine into dimers, exerting coagulation activity. This study provides an insight into the toxin gene expression in the Duvernoy’s gland of R. lateralis, which will benefit future research into the ecological and pharmacological significance of toxins in the genus Rhabdophis.

Toxins doi: 10.3390/toxins18040166

Authors: Kun-Hwa Kang Jae-Kwang Jung Jin-Seok Byun Ji Rak Kim

We thank the authors for their thoughtful comments [...]

Toxins doi: 10.3390/toxins18040165

Authors: Elif Tarihci Cakmak Fatma Merih Akpinar Ekin Ilke Sen Nalan Capan

We read with great interest and some concerns the article by Kang and colleagues [...]

Toxins doi: 10.3390/toxins18040164

Authors: Agnieszka Pszczółkowska Elżbieta Suchowilska Michael Sulyok Wolfgang Kandler Adam Okorski Rudolf Krska Marian Wiwart

A three-year experiment was conducted over the years 2020–2022 to determine the spectrum of microscopic fungi colonizing the grain of two fungicide-treated cultivars of spring barley and the profiles of mycotoxins identified in grain. In comparison with the unprotected control, fungicide treatment significantly increased grain yield by an average of approximately 10% in cv. Atrika and approximately 20% in cv. Vermont. The most abundantly isolated species were Alternaria alternata and Bipolaris sorokiniana. Fungi of the genus Fusarium were also widely represented, accounting for 7% to 27% of all isolates, depending on the year. Each year, 45 secondary fungal metabolites produced mainly by Fusarium and Alternaria species were identified. Fungicide protection did not reduce the overall concentration of Fusarium toxins and even caused a slight increase, while contributing to a decrease in the levels of nivalenol-3-glucoside, nivalenol, and deoxynivalenol. Concurrently, the concentrations of group A trichothecenes and moniliformin increased. The grain of spring barley cv. Vermont contained higher levels of the major Fusarium toxins than the grain of cv. Atrika. Non-parametric Friedman ANOVA revealed significant differences between years for eight mycotoxin concentrations. These results confirm the complex effects of chemical protection on the composition of grain microflora and mycotoxin profiles, indicating the need for further research into interactions between cultivars, environmental conditions, and integrated plant protection strategies in the production of food and feed cereals to improve food safety.

Toxins doi: 10.3390/toxins18040163

Authors: Yang Li Muling Shi Wenyue Li Yiqing Yang Xiang Li Chen Shen Xiong Wang Shuanglin Zhang Jie Du

Deaths from the accidental ingestion of poisonous Amanita mushrooms occur every year due to the lack of a specific antidote against α-amanitin poisoning. Intervention and treatment can be promptly carried out to avoid serious consequences when the toxin can be effectively detected in whole blood before liver toxicity develops. Aptamers are molecular recognition units similar to antibodies, capable of specifically recognizing and detecting small molecules such as α-amanitin for which monoclonal antibodies are difficult to prepare. However, α-amanitin has a small molecular size and limited binding sites, which bring difficulties to aptamer selection. Moreover, achieving highly specific detection of α-amanitin in whole blood remains challenging due to the presence of potentially interfering components, such as human serum albumin (HSA). For these problems, we propose an aptamer selection method for small-molecule target α-amanitin, combining target-immobilized and library-immobilized SELEX to select high-affinity aptamers. To exclude HSA interference, counter-selection was introduced to remove HSA-bound sequences. Through these strategies, we successfully selected a highly specific α-amanitin aptamer with nanomolar affinity.

Toxins doi: 10.3390/toxins18040162

Authors: Agnieszka Bańka-Wrona Witold Wrona Wioletta Barańska-Rybak

Rosacea-associated erythema and flushing often remain inadequately controlled by standard therapies. Intradermal botulinum toxin A has emerged as a potential treatment targeting the neurovascular component of rosacea. This pilot study aimed to evaluate the safety and preliminary efficacy of intradermal letibotulinumtoxinA for persistent erythema and flushing in rosacea. Eleven patients with refractory erythematotelangiectatic rosacea received a single session of intradermal letibotulinumtoxinA (20 U total dose). Outcomes at 2 weeks included clinician- and patient-rated erythema severity, patient-reported flushing, skin hydration, sebum, elasticity, and Dermatology Life Quality Index (DLQI). Safety assessments included adverse events and pain. Two weeks post-treatment, 73% of patients showed improvement in Clinician’s Erythema Assessment score and 100% reported reduced flushing. Median hydration and elasticity increased, while the level of sebum decreased. Median DLQI improved from 9 to 2. No serious adverse effects occurred; mild, transient cheek heaviness and dryness were noted in three cases. Intradermal letibotulinumtoxinA was well tolerated, with few reported side effects/complications. The treatment demonstrated preliminary efficacy in reducing rosacea erythema and flushing, and improving skin physiology and quality of life; however, these require confirmation in a larger, controlled study.

Toxins doi: 10.3390/toxins18040161

Authors: Jiaqi Shi Kejie Gao Wenjing Wang Shengjie Shi Shuzhen Jiang Lijie Yang

Zearalenone (ZEN) is a widely distributed estrogenic mycotoxin that compromises intestinal health in pigs, but its spatial difference ZEN and niche-specific regulatory effects on the intestinal microbiota remain largely unelucidated. In this study, 12 healthy three-way crossbred weaned piglets (Duroc × Landrace × Yorkshire) were randomly divided into two treatments. The control group (CON) was fed with the basal diet, and the treatment group (ZEN) was supplemented with 1.5 mg ZEA/kg of the basal diet for 28 days. Chyme and mucosal microorganisms in the duodenum, jejunum, ileum, colon and cecum were profiled by using 16S rDNA sequencing. The results indicated that ZEN significantly reduced the α-diversity of ileal chyme, while the abnormal increase in α-diversity of ileal and cecal mucosa represented a pathological signature of intestinal mucosal barrier damage induced by ZEN, which was detrimental to intestinal health. β-Diversity analysis revealed ZEN altered the microbial community composition of the cecal chyme. LEfSe analysis revealed gut segment-specific and niche-specific biomarker taxa among the groups, and functional prediction further indicated that ZEN exposure significantly perturbed key metabolic pathways: it downregulated nicotinate and nicotinamide metabolism as well as the citrate cycle in ileal chyme and upregulated the pentose and glucuronate interconversions pathway in cecal chyme. Collectively, this study demonstrated the effects of ZEN on the intestinal microbiota across spatial difference and ecological niches in weaned piglets, providing a basis for elucidating the microecological mechanisms underlying ZEN-induced intestinal injury in pigs.

Toxins doi: 10.3390/toxins18040159

Authors: María Agustina Pavicich Claudia Giménez-Campillo José Diana Di Mavungu Sarah De Saeger Andrea Patriarca

Apples are highly susceptible to fungal infections, particularly by Alternaria species, which can lead to fruit deterioration and mycotoxin contamination during storage. This study aimed to evaluate the potential of untargeted liquid chromatography–high-resolution mass spectrometry (LC-HRMS) as a control-oriented strategy to detect Alternaria-infected apples under retail and long-term storage conditions. Healthy Red Delicious apples were artificially inoculated with three Alternaria tenuissima strains on the fruit surface or core and incubated at 25 °C or 4 °C. Extracts were analysed by UPLC-HRMS in both positive and negative electrospray ionisation modes, followed by multivariate chemometric analysis. Principal component analysis and partial least squares discriminant analysis consistently discriminated infected from non-infected apples, independent of strain, infection site, or incubation temperature. Feature selection based on variable importance values significantly improved model robustness and predictive performance. The metabolomic profiles also enabled discrimination according to Alternaria strain, infection site, storage temperature, and selected combinations of these factors. The results demonstrate that LC-HRMS-based untargeted metabolomics could provide a statistically robust framework for detecting Alternaria tenuissima infection in apples under the studied conditions.

Toxins doi: 10.3390/toxins18040160

Authors: Areerat Suputtitada

Complex regional pain syndrome (CRPS) is a heterogeneous and disabling chronic pain condition characterized by maladaptive neuroplasticity involving persistent peripheral nociceptive input, autonomic dysregulation, and central sensitization. Despite increasing clinical use, the role of botulinum toxin in CRPS remains controversial, with inconsistent outcomes reported across studies. This review synthesizes mechanistic, translational, and clinical evidence suggesting that these apparent inconsistencies may be partly explained by heterogeneity in anatomical targeting and route of administration rather than absence of biological efficacy. Available evidence suggests that botulinum toxin may exhibit its most consistent therapeutic signal when delivered to neural structures directly implicated in dominant CRPS pathophysiology, particularly the sympathetic nervous system and proximal somatic afferents, whereas superficial or non-specific delivery strategies appear to yield more variable responses. Importantly, differences across anatomical targets should not be interpreted as evidence of comparative effectiveness, as observed variation may reflect phenotype selection, procedural heterogeneity, confounding, and differences in outcome reporting. By integrating experimental data, randomized trials, and case-based clinical evidence, an anatomy-informed, route-specific neuromodulation framework is proposed to reconcile existing findings and inform future research. This mechanism-informed perspective is intended to guide rational trial design and phenotype-aligned clinical application of botulinum toxin in CRPS, rather than to provide a definitive evidence-closing synthesis.

Toxins doi: 10.3390/toxins18040158

Authors: Jiali Liu Huibing Chi Xiaoyu Zhu Qingwei Jiang Zhaoxin Lu Ping Zhu Fengxia Lu

Trichothecene mycotoxins, especially T-2 toxin, represent a significant threat to food safety and public health. Although the enzymatic degradation of deoxynivalenol has been extensively investigated, there are few reports of enzymes capable of efficiently degrading T-2 toxin. This study identified that the aldo-keto reductase AKR13B3 from Devosia A6-243 exhibits 3-keto-DON-degrading and a little T-2 toxin-degrading activity. To address this limitation, a rational design strategy targeting the substrate-binding pocket was employed to enhance its activity. Utilizing site-directed and combinatorial mutagenesis, a double mutant R134F/D217A was successfully screened. R134F/D217A retains catalytic activity towards 3-keto-DON while significantly enhancing its catalytic capacity for T-2. Specifically, the R134F/D217A variant exhibited a 2.88-fold increase in catalytic activity and a 3.15-fold enhancement in catalytic efficiency (kcat/Km) relative to the wild type enzyme. Notably, a substantial improvement in thermal stability was also observed. After incubation at 55 °C, the residual activity of the R134F/D217A mutant was 2.63 times that of the wild type. Molecular dynamics (MD) simulations and three-dimensional structural modeling suggested the mechanistic basis for the enhanced performance of the R134F/D217A double mutant. Catalytic enhancement stems from a shortened nucleophilic attack distance, a positively biased electrostatic environment, combined with an enlarged pocket and reduced binding free energy. Concurrently, the increased thermal stability results from decreased flexibility and a more rigid structural architecture. This work presents the first report of AKR13B3 as an effective enzyme for T-2 toxin transformation, and its catalytic activity was significantly enhanced through rational design. Thus, a novel enzymatic strategy was proposed, and could inform future approaches to study issues related to T-2 toxin contamination.

Toxins doi: 10.3390/toxins18040157

Authors: Malgorzata Slocinska Justyna Mirek Zbigniew Adamski Jan Lubawy

Steroidal glycoalkaloids (GAs) are key plant defense compounds, yet their effects on insect gut physiology are not fully understood. We investigated how purified α-chaconine and Solanum tuberosum leaf extract influence the gut function and growth of the mealworm Tenebrio molitor. Larvae were exposed to sublethal doses of GAs, and gut contractility, midgut digestive enzyme activity and body weight were analysed over time. Both α-chaconine and potato extract caused a rapid decrease in digestive enzyme activity 2 h after exposure, followed by a clear increase above control levels after 24 h, indicating a time-dependent compensatory response of the digestive system. Gut contractility was significantly enhanced in treated larvae, and larvae exposed to both treatments exhibited a body weight loss over 72 h. These results show that potato glycoalkaloids strongly modulate the gut physiology of T. molitor while allowing continued growth, highlighting both the plasticity of insect digestive responses and the need to consider sublethal, gut-centered effects when evaluating glycoalkaloids as candidates for bioinsecticidal agents.

Toxins doi: 10.3390/toxins18040156

Authors: Mariana Murea Alaa S. Awad Vandana D. Niyyar Tibor Fülöp Akihiro C. Yamashita Tadashi Tomo Masanori Abe

Extracorporeal dialysis for uremic toxin removal and fluid regulation relies on specialized dialyzers whose membranes differ markedly in polymer chemistry, pore architecture, adsorption capacity, surface bioactivity, and convective performance. These structural and material distinctions result in wide variation in the clearance of chemically diverse uremic solutes. Despite the expanding range of dialyzer options, membrane selection in clinical practice remains largely non-individualized. In this review, we propose a phenotype-based model for dialyzer membrane selection. We outline how distinct membrane families achieve differential solute clearance and integrate these functional characteristics into a framework that considers residual kidney function, nutritional and inflammatory status, cardiovascular physiology, protein-bound toxin burden, and hemodynamic vulnerability. Because access to advanced membranes varies across regions and dialysis providers, implementation will require adaptation to local formulary constraints. Nevertheless, aligning membrane properties with patient-specific toxin profiles offers a promising strategy to optimize extracorporeal therapy and improve outcomes in chronic dialysis.

Toxins doi: 10.3390/toxins18040155

Authors: Jiahao Liu Zejing Wen Sunkun Tang Jiajia Wu Xiaowen Bi Yang Yang Chunhong Huang

Snakebite envenoming remains a major global health challenge. Naja atra (N. atra) envenomation induces severe acute kidney injury (AKI), largely driven by snake venom phospholipase A2 (SVPLA2). Increasing evidence suggests that immune dysregulation, in addition to direct cytotoxicity, contributes to delayed renal injury. Here, we investigated whether N. atra SVPLA2 exposure is associated with macrophage immunometabolic remodeling and functional changes relevant to AKI progression. In vivo, AKI was induced in C57BL/6J mice by intraperitoneal administration of N. atra venom, followed by treatment with the SVPLA2 inhibitor varespladib. In vitro, bone marrow–derived macrophages were exposed to venom with or without varespladib. N. atra venom exposure was associated with extensive tubular apoptosis, increased renal macrophage abundance, and elevated kidney injury biomarkers. Macrophages exhibited a shift toward a pro-inflammatory polarization signature accompanied by reduced efferocytic capacity. Targeted metabolomics revealed coordinated increases in glycolytic intermediates together with upregulation of key glycolytic enzymes. Pharmacological inhibition of SVPLA2 partially restored macrophage metabolic features and efferocytic capacity and was accompanied by attenuation of renal injury. Together, these findings support a model in which SVPLA2 exposure is associated with macrophage immunometabolic remodeling and impaired apoptotic cell clearance during venom-induced AKI.

Toxins doi: 10.3390/toxins18040154

Authors: Maryam Tavakol Noorabadi Ishara S. Manawasinghe Jiayu Xu Caiqing Zhao Naghmeh Afshari Wei Dong Antonio Francesco Logrieco Kevin D. Hyde

This study provides a combined profile of fungal isolates from fresh and dried chili peppers in markets in Guangzhou. Multilocus sequence analysis revealed a wide variety of species, seven of which were reported for the first time from chili pepper (F. annulatum, F. compactum, F. pernambucanum, F. ramsdenii, and F. tardichlamydosporum, P. citrinum and P. steckii). In this research work, quantitative determination using targeted LC–MS/MS of dried chili peppers showed a significantly higher frequency of contamination and higher toxin concentrations than fresh samples. The predominant mycotoxins in dried peppers were DON and FB1, which were present in all the samples at mean levels of 0.56 µg/g and 0.067 µg/g, respectively. AFB1 and OTA were present in all dried samples but were detected only occasionally in fresh peppers. ZEN and CIT were detected at lower concentrations, but more prevalent among dried products (63.6% and 81.8% of all samples, respectively). The aflatoxin B1 (AFB1) level of 180 µg/kg in dried chili samples was 36 times above the EU maximum limit (5 µg/kg), and the OTA level reached 54 µg/kg, exceeding the EU limit by a factor of 2.7 (20 µg/kg). Statistical analysis also showed that all six mycotoxins were statistically higher in dried pepper than in fresh pepper. In vitro evaluation demonstrated that certain Fusarium isolates synthesized FB1. At the same time, Penicillium species, including P. citrinum and P. steckii, consistently produced citrinin, confirming the strong influence of growth substrate on toxin biosynthesis. The frequent occurrence and elevated levels of regulated mycotoxins highlight significant public health concerns and underscore the need for improved postharvest handling and drying practices. These findings provide critical baseline data linking fungal diversity with toxin production dynamics, developing essential guidance for targeted mitigation strategies.

Toxins doi: 10.3390/toxins18040153

Authors: Mauro Silvestrini Giovanna Viticchi Sergio Salvemini Gioacchino De Vanna Marco Bartolini Simona Luzzi

Chronic migraine (CM) is a debilitating neurological disorder characterized not only by persistent and severe pain, but also by substantial cognitive dysfunction that affects attention, working memory, processing speed, and executive functions. These neuropsychological disturbances are likely influenced by overall disease burden and are further modulated by affective comorbidities, sleep disturbances, and medication overuse. OnabotulinumtoxinA (BoNT-A) is an established preventive therapy for CM, supported by strong evidence of both efficacy and safety. This narrative review synthesizes findings from studies examining the relationship between BoNT-A treatment and domain-specific cognitive improvements in CM. It also outlines the potential pathophysiological mechanisms underlying these effects, summarizes the limitations of the existing literature, and highlights priorities for future research. Current evidence suggests that BoNT-A may confer neurocognitive benefits, particularly in working memory and processing speed, and that these improvements may occur partly independently of reductions in headache frequency. These favorable cognitive effects appear to be plausibly linked to decreased nociceptive “noise” and improved cortical inhibition, potentially mediated through modulation of central sensitization, nociceptive signaling, and affective states.

Toxins doi: 10.3390/toxins18030152

Authors: Timur Yu. Magarlamov Grigorii V. Malykin

Tetrodotoxin (TTX), widely known as pufferfish venom, is a low-molecular-weight guanidinium neurotoxin. It can accumulate to extremely high concentrations in certain animals, including pufferfish, blue-ringed octopuses, flatworms, and nemerteans. However, the origin of TTX and the mechanisms that enable such extreme accumulation in these animals remain poorly understood. In this study, using confocal laser scanning microscopy combined with electron immunocytochemistry and ultrastructural analysis, we demonstrate the presence of TTX-positive bacteria associated with specialized cellular structures—type II phagosomes of gut enterocytes—in the highly toxic nemertean Cephalothrix cf. simula. We hypothesize that TTX production in C. cf. simula results from interactions between the nemertean host and its endosymbionts. These findings clarify the origin and accumulation of the toxin in nemerteans and have broader implications for other TTX-bearing species.

Toxins doi: 10.3390/toxins18030151

Authors: Akihiko Yamamoto Takashi Ito Toru Hifumi

Yamakagashi (Rhabdophis tigrinus) is a widely distributed snake species in Japan. Yamakagashi causes venom-induced consumption coagulopathy (VICC) when the amount of infused venom is high, and bites can be fatal if antivenom treatment is delayed. However, yamakagashi antivenom is an unapproved treatment, and its storage capacity is limited, preventing its prompt administration. Therefore, we investigated the application of commercially available drugs, namely tranexamic acid and antithrombin III, in the treatment of VICC caused by yamakagashi venom in a rat model. Furthermore, we investigated the combination of each drug with recombinant thrombomodulin α. Administration of tranexamic acid or antithrombin III alone failed to extend rat survival or correct changes in blood coagulation markers, such as prothrombin time, fibrinogen concentrations, and D-dimer levels, in yamakagashi venom-treated rats. However, combined administration of recombinant thrombomodulin α and tranexamic acid extended rat survival and partially restored blood coagulation markers. Therefore, the combination of recombinant thrombomodulin α and tranexamic acid might represent a useful therapeutic regimen for yamakagashi venom exposure.