Toxins

7 pages, 347 KiB

Open AccessArticle

Food Confusion Between Edible and Poisonous Plants: A 22-Year Retrospective of the Southeastern France Poison Control Center

by Romain Torrents, Julien Reynoard, Mathieu Glaizal, Corinne Schmitt, Katharina Von Fabeck, Audrey Boulamery, Luc De Haro and Nicolas Simon

Toxins 2024, 16(12), 552; https://doi.org/10.3390/toxins16120552 (registering DOI) - 21 Dec 2024

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Abstract

Objective: In some regions of the globe, accidental food confusion regarding plants can cause severe poisoning events and deaths. The aim of this study was to report on those confusions from the Marseille Poison Control Centre’s (PCC) experience from 2002 to 2023. Results: [...] Read more.

Objective: In some regions of the globe, accidental food confusion regarding plants can cause severe poisoning events and deaths. The aim of this study was to report on those confusions from the Marseille Poison Control Centre’s (PCC) experience from 2002 to 2023. Results: Over 22 years, 2197 food confusion events were managed with 321 different species. The most frequently involved plant was Nerium oleander (289 cases, 13.1%), then Cucurbitaceae genus (3.3%), Colchicum autumnale (3.3%), Prunus amygdalus (3%), Mahinot esculenta (3%), Cytisus laburnum (2.6%), Aesculus hippocastanum (2.5%) and Narcissus Jonquilla (2%). Many botanical confusion events were also reported (n = 1386, 63%), but with fewer than five identical species. Only one death was reported for this review, in an event involving Aconitum napellus. Two antidotes were used for all the series: Datura genus and Prunus dulcis. Discussion: The most implicated plant was Nerium oleander. This is explained by its distribution. This rate is very low compared to that of suicide attempts with this plant. Many cases were symptomatic (53.6%), but very few of them described severe symptoms (only 0.5% severe poisonings). Few patients needed to be admitted to intensive care (0.4%), and even fewer needed an antidote (two cases). Only one death occurred, involving Aconitum napellus. Food confusions were more common than suicide attempts with plants, but seem to be less severe. However, death and serious complications can occur, so it is important to identify and manage the plants concerned. Material and Methods: For each food confusion event managed between 2002 and 2023 at the Southeastern France PCC based in Marseille, we performed a retrospective review. This PCC is responsible for the Provence–Alpes–Côte d’Azur Region, Corsica Island and Indian Ocean French overseas territories. For each case, severity was calculated with the Poison Severity Score (PSS). Full article

(This article belongs to the Section Plant Toxins)

19 pages, 2438 KiB

Open AccessReview

Lesser-Known Cyanotoxins: A Comprehensive Review of Their Health and Environmental Impacts

by Molham Al Haffar, Ziad Fajloun, Sami Azar, Jean-Marc Sabatier and Ziad Abi Khattar

Toxins 2024, 16(12), 551; https://doi.org/10.3390/toxins16120551 - 19 Dec 2024

Viewed by 532

Abstract

Cyanobacteria, also known as blue-green algae, are a diverse phylum of photosynthetic, Gram-negative bacteria and one of the largest microbial taxa. These organisms produce cyanotoxins, which are secondary metabolites that can have significant impacts on both human health and the environment. While toxins [...] Read more.

Cyanobacteria, also known as blue-green algae, are a diverse phylum of photosynthetic, Gram-negative bacteria and one of the largest microbial taxa. These organisms produce cyanotoxins, which are secondary metabolites that can have significant impacts on both human health and the environment. While toxins like Microcystins and Cylindrospermopsins are well-documented and have been extensively studied, other cyanotoxins, including those produced by Lyngbya and Nostoc, remain underexplored. These lesser-known toxins can cause various health issues in humans, including neurotoxicity, hepatotoxicity, and dermatotoxicity, each through distinct mechanisms. Moreover, recent studies have shown that cyanobacteria can be aerosolized and transmitted through the air over long distances, providing an additional route for human exposure to their harmful effects. However, it remains an area that requires much more investigation to accurately assess the health risks and develop appropriate public health guidelines. In addition to direct exposure to toxins, cyanobacteria can lead to harmful algal blooms, which pose further risks to human and wildlife health, and are a global concern. There is limited knowledge about these lesser-known cyanotoxins, highlighting the need for further research to understand their clinical manifestations and improve society’s preparedness for the associated health risks. This work aims to review the existing literature on these underexplored cyanotoxins, which are associated with human intoxication, elucidate their clinical relevance, address significant challenges in cyanobacterial research, and provide guidance on mitigating their adverse effects. Full article

(This article belongs to the Special Issue Advances in Cyanotoxins: Latest Developments in Risk Assessment)

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14 pages, 1375 KiB

Open AccessArticle

The Effect of Purified Opharin Isolated from the Venom of King Cobra (Ophiophagus hannah) in Modulating Macrophage Inflammatory Responses and Vascular Integrity

by Tuchakorn Lertwanakarn, Armando Reyes, Emelyn Salazar, Martha Barrientos, Elda E. Sanchez and Montamas Suntravat

Toxins 2024, 16(12), 550; https://doi.org/10.3390/toxins16120550 - 19 Dec 2024

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Abstract

King cobra (Ophiophagus hannah) venom comprises a diverse array of proteins and peptides. However, the roles and properties of these individual components are still not fully understood. Among these, Cysteine-rich secretory proteins (CRiSPs) are recognized but not fully characterized. This study [...] Read more.

King cobra (Ophiophagus hannah) venom comprises a diverse array of proteins and peptides. However, the roles and properties of these individual components are still not fully understood. Among these, Cysteine-rich secretory proteins (CRiSPs) are recognized but not fully characterized. This study investigates the biological effects of Opharin, the CRiSP from king cobra venom (KCV). The effects of Opharin on cytokine production, specifically on IL-1β, IL-6, IL-8, TNF-α, and IL-10 release, were evaluated over 24 h in monocyte-derived macrophage (MDM) cells. Notably, the levels of these inflammatory cytokines were significantly increased over 24 h, with values higher than those observed in cells treated with crude KCV at most time points. Additionally, the in vivo Miles assay in mice revealed that Opharin increased vascular permeability by 26% compared to the negative control group. These findings highlight the Opharin’s role in severe inflammatory and vascular responses observed in king cobra envenomation. Still, further research is essential to elucidate the pharmacological and toxicological effects of venom components, ultimately enhancing the clinical management of envenomation. Full article

(This article belongs to the Special Issue Advances in Venom Research: Biological Activities, Therapeutic Potential, and Molecular Mechanism)

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Two-step purification of Opharin from crude KCV. (A) An RP-HPLC chromatogram revealed the fractionation of crude venom into 48 distinct fractions. Opharin was specifically detected in the 34th fraction (shaded), with an estimated molecular mass of 28 kDa, as verified by SDS-PAGE (upper panel). The green line demonstrates the gradient of the elution buffer. (B) Subsequent fractionation of the pooled 34th fraction (PF) using aIEX chromatography produced four subfractions. Opharin was identified within the first subfraction (shaded) and further confirmed through SDS-PAGE analysis (inset). The gray line represents the gradient of the elution buffer. M = protein marker. Full article ">Figure 2
Evaluation of inflammatory cytokine production in MDM cells treated with saline (negative control), lipopolysaccharide (0.25 μg/mL; LPS, positive control), crude KCV (287 μg/mL), and Opharin (25 µg/mL). The release of inflammatory mediators, including (A) IL-1β, (B) IL-6, (C) IL-8, (D) TNF-α, and (E) IL-10, was quantified at various time points using ELISA. Results are expressed as mean ± SD, n = 4–6 per treatment (black dots). * p < 0.05 compared to the negative control, Δ p < 0.05 compared to LPS, and ξ p < 0.05 compared to KCV. Full article ">Figure 3
Assessment of in vivo vascular permeability in mice using the Miles assay. Mice received an intravenous injection of 100 μL of Evans blue dye, followed promptly by a subcutaneous injection of normal saline solution (NSS), VEGF-A (143 ng/mouse), or Opharin (163 ng/mouse). Results were assessed from five mice and are expressed as mean ± S.D. Full article ">

14 pages, 4733 KiB

Open AccessArticle

Rice Straw-Derived Biochar Mitigates Microcystin-LR-Induced Hepatic Histopathological Injury and Oxidative Damage in Male Zebrafish via the Nrf2 Signaling Pathway

by Wang Lin, Fen Hu, Wansheng Zou, Suqin Wang, Pengling Shi, Li Li, Jifeng Yang and Pinhong Yang

Toxins 2024, 16(12), 549; https://doi.org/10.3390/toxins16120549 - 18 Dec 2024

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Abstract

Microcystin-leucine arginine (MC-LR) poses a serious threat to aquatic animals during cyanobacterial blooms. Recently, biochar (BC), derived from rice straw, has emerged as a potent adsorbent for eliminating hazardous contaminants from water. To assess the joint hepatotoxic effects of environmentally relevant concentrations of [...] Read more.

Microcystin-leucine arginine (MC-LR) poses a serious threat to aquatic animals during cyanobacterial blooms. Recently, biochar (BC), derived from rice straw, has emerged as a potent adsorbent for eliminating hazardous contaminants from water. To assess the joint hepatotoxic effects of environmentally relevant concentrations of MC-LR and BC on fish, male adult zebrafish (Danio rerio) were sub-chronically co-exposed to varying concentrations of MC-LR (0, 1, 5, and 25 μg/L) and BC (0 and 100 μg/L) in a fully factorial experiment. After 30 days exposure, our findings suggested that the existence of BC significantly decreased MC-LR bioavailability in liver. Furthermore, histopathological analysis revealed that BC mitigated MC-LR-induced hepatic lesions, which were characterized by mild damage, such as vacuolization, pyknotic nuclei, and swollen mitochondria. Compared to the groups exposed solely to MC-LR, decreased malondialdehyde (MDA) and increased catalase (CAT) and superoxide dismutase (SOD) were noticed in the mixture groups. Concurrently, significant changes in the mRNA expression levels of Nrf2 pathway genes (cat, sod1, gstr, keap1a, nrf2a, and gclc) further proved that BC reduces the oxidative damage induced by MC-LR. These findings demonstrate that BC decreases MC-LR bioavailability in the liver, thereby alleviating MC-LR-induced hepatotoxicity through the Nrf2 signaling pathway in zebrafish. Our results also imply that BC could serve as a potentially environmentally friendly material for mitigating the detrimental effects of MC-LR on fish. Full article

(This article belongs to the Special Issue Toxic Cyanobacterial Bloom Detection and Removal: What's New?)

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Full article ">Figure 1
SEM images (A,B), size distribution (C), and zeta potential (D) of BC. Full article ">Figure 2
Hepatic H&E-stained sections of zebrafish. Control (A); 1 μg/L MC-LR (B); 5 μg/L MC-LR (C); 25 μg/L MC-LR (D); 100 μg/L BC (E); 1 μg/L MC-LR+ BC (F); 5 μg/L MC-LR + BC (G); and 25 μg/L MC-LR + BC (H). Pyknotic nuclei (black arrow), swollen hepatocytes (white arrowhead), and vacuolization (black arrowhead). Bar = 50 um. Full article ">Figure 3
Hepatic ultrastructural alterations in zebrafish. Control (A); 1 μg/L MC-LR (B); 5 μg/L MC-LR (C); 25 μg/L MC-LR (D); 100 μg/L BC (E); 1 μg/L MC-LR + BC (F); 5 μg/L MC-LR+ BC (G); and 25 μg/L MC-LR + BC (H). Swollen mitochondria (black arrowhead); deformed nuclei (white arrowhead); vacuolization (white arrow); and dilated endoplasmic reticulum (black box). Mt, mitochondria; Nu, nuclei; ER, endoplasmic reticulum. Bar = 1 μm. Full article ">Figure 4
Changes in MDA content (A), SOD activity (B), CAT activity (C), GPx activity (D), GST activity (E), and GSH content (F) in the liver of zebrafish. Asterisks (* p < 0.05; ** p < 0.01) indicate significant differences between the treatments and control. Hashes (# p < 0.05) indicate significant differences between the MC-LR + BC groups and the MC-LR-only groups, respectively. The values are presented as mean ± SD (n = 3). Full article ">Figure 5
Heatmap of Nrf2 signaling pathway gene expression. Asterisks (* p < 0.05; ** p < 0.01) indicate significant differences between the treatments and control. The dotted box with hashes (# p < 0.05; ## p < 0.01) indicate significant differences between the MC-LR + BC groups and the MC-LR-only groups, respectively. The values are presented as mean ± SD (n = 6). Full article ">

12 pages, 4369 KiB

Open AccessCommunication

Brefeldin A—A Major Pathogenic Factor of Peanut Pod Rot from Fusarium neocosmosporiellum

by Huiling Wang, Xiaohan Wang, Huiling Han, Quanlin Yu, Xinmiao Tan, Junlong Liu, Yiting Zhao and Weiming Sun

Toxins 2024, 16(12), 548; https://doi.org/10.3390/toxins16120548 - 18 Dec 2024

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Abstract

Fusarium neocosmosporiellum is the main pathogen of peanut pod rot in China. To investigate the type of F. neocosmosporiellum toxin and its pathogenic mechanism, a macrolide, brefeldin A, was isolated. The structure of the compound was identified by 1D and 2D nuclear magnetic [...] Read more.

Fusarium neocosmosporiellum is the main pathogen of peanut pod rot in China. To investigate the type of F. neocosmosporiellum toxin and its pathogenic mechanism, a macrolide, brefeldin A, was isolated. The structure of the compound was identified by 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization–mass spectrometry (HR-ESI-MS). At the same time, the content of the compound in healthy and diseased peanut capsules was detected, and its plant toxicity to radish, mung bean, rice, and peanut seed radicle elongation and pathogenicity to peanut pod rot were evaluated. The results showed that brefeldin A at 50 μg/mL could significantly inhibit the radicle elongation of rice seeds. Brefeldin A was detected only in pods with peanut rot. Injecting 2 mg/mL brefeldin A solution into peanut pods caused the severe decay of peanut pods at the R3R4 stage, which is consistent with the symptoms of peanut rot. Full article

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The 1H NMR of brefeldin A. Full article ">Figure 2
The 13C NMR of brefeldin A. Full article ">Figure 3
Structure of brefeldin A (1) and its correlation with 1H-1H COSY and HMBC. Full article ">Figure 4
Inhibitory activity of brefeldin A (1) on the radicle elongation of the mung bean seed. The mung bean was treated at concentrations of 1000, 500, 200, 100, 50, 25, 12.5, and 0 μg/mL, from left to right. Full article ">Figure 5
Inhibitory activity of brefeldin A (1) on the radicle elongation of the rice bean seed. The rice bean was treated at concentrations of 200, 100, 50, 12.5, 6.25, and 0 μg/mL, from left to right. Full article ">Figure 6
Toxicity of brefeldin A against different periods for peanut pods. (A1) is the photo of Yuhanghua 7 in R3R4 periods for peanut pods. (A2) is the photo of Yuhanghua 7 in R7R8 periods for peanut pods. (B1) is the photo of Zhonghua 12 in R3R4 periods for peanut pods. (B2) is the photo of Zhonghua 12 in R7R8 periods for peanut pods. All the peanut pods were treated with brefeldin A at concentrations of 0, 0.5, 1, and 2 mg/mL, from left to right. Full article ">Figure 7
HPLC profile of brefeldin A (1000 μg/mL), HPLC profile of rotten pods, and HPLC profile of healthy pods. Full article ">

10 pages, 290 KiB

Open AccessArticle

Ochratoxin A and AFM1 in Cheese and Cheese Substitutes: LC-MS/MS Method Validation, Natural Occurrence, and Risk Assessment

by María Agustina Pavicich, Stefano Compagnoni, Celine Meerpoel, Katleen Raes and Sarah De Saeger

Toxins 2024, 16(12), 547; https://doi.org/10.3390/toxins16120547 - 18 Dec 2024

Viewed by 357

Abstract

Cheese is vulnerable to contamination with mycotoxins, particularly ochratoxin A (OTA) and aflatoxin M1 (AFM1). This study aims to develop and validate an analytical method for the detection and quantification of OTA and AFM1 in cheese and to assess their prevalence and associated [...] Read more.

Cheese is vulnerable to contamination with mycotoxins, particularly ochratoxin A (OTA) and aflatoxin M1 (AFM1). This study aims to develop and validate an analytical method for the detection and quantification of OTA and AFM1 in cheese and to assess their prevalence and associated risks. A liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was validated for detecting these mycotoxins in 41 cheese samples, including firm-ripened, spreadable, and plant-based alternatives. The results showed that OTA was detected exclusively in grated Parmigiano Reggiano cheese, while AFM1 was found in both Parmigiano Reggiano and Pecorino cheeses. This study goes beyond analytical method development by providing a preliminary exposure assessment and risk characterization for OTA and AFM1 in cheese, bridging the gap between analytical chemistry and public health implications. This study identified potential health risks associated with OTA, particularly for children and adolescents categorized as high consumers of Parmigiano Reggiano cheese. The findings underscore the need for monitoring of OTA and AFM1 in cheese and further research to establish regulatory limits for these contaminants. Full article

(This article belongs to the Special Issue Advances in Contamination, Detection and Risk Assessment of Mycotoxins)

14 pages, 2413 KiB

Open AccessSystematic Review

Treatment with OnabotulinumtoxinA for Oromandibular Dystonia: A Systematic Review and Meta-Analysis

by Kazuya Yoshida and Ryuji Kaji

Toxins 2024, 16(12), 546; https://doi.org/10.3390/toxins16120546 - 16 Dec 2024

Viewed by 395

Abstract

Oromandibular dystonia (OMD) is a focal dystonia characterized by contractions of the masticatory, lingual, and other muscles of the stomatognathic system. We conducted a systematic review and meta-analysis to elucidate the impact and safety of botulinum toxin in OMD. The eligibility criteria were [...] Read more.

Oromandibular dystonia (OMD) is a focal dystonia characterized by contractions of the masticatory, lingual, and other muscles of the stomatognathic system. We conducted a systematic review and meta-analysis to elucidate the impact and safety of botulinum toxin in OMD. The eligibility criteria were full-length original articles that provided data evaluating the efficacy and adverse effects of onabotulinumtoxinA injections in patients with OMD. PubMed and Embase were searched for articles published before 31 May 2023. We analyzed cases that showed a favorable response (>0% improvement), moderate or greater response (>50% improvement), and adverse effects. A fixed-model meta-analysis of 26 studies involving 1103 patients revealed that an overall favorable effect of onabotulinumtoxinA injection was observed in 96.2% (95% confidence interval [CI], 95–97.5%, p < 0.00001) of patients, with significant heterogeneity (p < 0.00001, I² = 85%). A moderate response (>50% improvement) was observed in 88.9% of patients (95% CI, 87–90.8%, p < 0.00001) with significant heterogeneity (p < 0.00001, I² = 85%). Adverse effects were detected in 17.8% of patients, and the most common event was dysphagia (10.1%). Our systematic review found that onabotulinumtoxinA injection was effective, with a low rate of side effects. Further randomized controlled trials are required to clarify the evidence-based efficacy and adverse effects. Full article

(This article belongs to the Special Issue Botulinum Toxin Therapy for Diseases in the Oral and Maxillofacial Regions)

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10 pages, 1876 KiB

Open AccessArticle

The Choice of Anti-Inflammatory Influences the Elimination of Protein-Bound Uremic Toxins

by Víctor Joaquín Escudero-Saiz, Elena Cuadrado-Payán, María Rodriguez-Garcia, Gregori Casals, Lida María Rodas, Néstor Fontseré, María del Carmen Salgado, Carla Bastida, Nayra Rico, José Jesús Broseta and Francisco Maduell

Toxins 2024, 16(12), 545; https://doi.org/10.3390/toxins16120545 - 16 Dec 2024

Viewed by 355

Abstract

Pain is a frequent and disturbing symptom among hemodialysis patients. Protein-bound uremic toxins (PBUTs) are related to cardiovascular and overall mortality, and they are difficult to remove with current hemodialysis treatments. The PBUT displacers, such as furosemide, tryptophan, or ibuprofen, may be promising [...] Read more.

Pain is a frequent and disturbing symptom among hemodialysis patients. Protein-bound uremic toxins (PBUTs) are related to cardiovascular and overall mortality, and they are difficult to remove with current hemodialysis treatments. The PBUT displacers, such as furosemide, tryptophan, or ibuprofen, may be promising new strategies for improving their clearance. This study aims to compare ibuprofen versus other analgesic drugs in PBUT removal. A prospective study was carried out in 23 patients. Patients underwent four dialysis sessions with routine dialysis parameters, except for analgesic drugs administered (lysine acetylsalicylic acid, acetaminophen, dexketoprofen, and ibuprofen). The reduction ratios (RRs) of a wide range of molecular weight molecules were assessed, including total p-cresyl sulfate and total indoxyl-sulfate. There were no complications related to the administered drug, and pain was controlled independently of the drug. There were no differences in the RR of small-size and medium-sized molecules between all four study treatments. However, indoxyl sulfate and p-cresyl sulfate RRs when ibuprofen was administered were significantly higher than lysine acetylsalicylic acid, acetaminophen, and dexketoprofen treatments. In conclusion, patients with pain may benefit from treatment with ibuprofen instead of lysine acetylsalicylic acid, paracetamol, or dexketoprofen, since in addition to improving pain, it increases the removal of PBUTs. Full article

(This article belongs to the Section Uremic Toxins)

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18 pages, 4767 KiB

Open AccessArticle

The Ability of AST-120 to Lower the Serum Indoxyl Sulfate Level Improves Renal Outcomes and the Lipid Profile in Diabetic and Nondiabetic Animal Models of Chronic Kidney Disease: A Meta-Analysis

by Hande O. Altunkaynak, Eda Karaismailoglu and Ziad A. Massy

Toxins 2024, 16(12), 544; https://doi.org/10.3390/toxins16120544 - 16 Dec 2024

Viewed by 449

Abstract

The therapeutic benefit of the oral adsorbent drug AST-120 in chronic kidney disease (CKD) is related to an indoxyl sulfate (IS)-lowering action. Diabetes and dyslipidemia might worsen kidney damage in CKD. However, it is not known whether AST-120 influences lipid abnormalities as well [...] Read more.

The therapeutic benefit of the oral adsorbent drug AST-120 in chronic kidney disease (CKD) is related to an indoxyl sulfate (IS)-lowering action. Diabetes and dyslipidemia might worsen kidney damage in CKD. However, it is not known whether AST-120 influences lipid abnormalities as well as renal function in patients with CKD and diabetes. The objective of the present meta-analysis was to evaluate the efficacy of AST-120 treatment in CKD using data from preclinical studies. Mixed-effect or random-effect models were used to estimate the standardized mean difference (SMD) and the 95% confidence interval (CI). Publication bias was assessed with a funnel plot and Egger’s test. The potential influence of some variables (the dose and duration of AST-120 treatment, the animal species, and the CKD model’s diabetic status) was evaluated in subgroup analyses. Treatment with AST-120 was associated with a significantly lower IS level in animals with CKD (SMD = −1.75; 95% CI = −2.00, −1.49; p < 0.001). Significant improvements in markers of renal function and the lipid profile were also observed. In subgroup analyses of the cholesterol level, the diabetic status, the AST-120 dose, and the animal species were found to be influential factors. AST-120 lowered serum IS and triglyceride levels and improved renal function in animal models of CKD independent of diabetes status. However, AST-120’s ability to lower the total cholesterol level was more prominent in animals with diabetic CKD. Full article

(This article belongs to the Special Issue The Role of Uremic Toxins in Comorbidities of Chronic Kidney Disease)

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15 pages, 23082 KiB

Open AccessArticle

Reduction in Renal Heme Oxygenase-1 Is Associated with an Aggravation of Kidney Injury in Shiga Toxin-Induced Murine Hemolytic-Uremic Syndrome

by Antonio N. Mestekemper, Wiebke Pirschel, Nadine Krieg, Maria K. Paulmann, Christoph Daniel, Kerstin Amann and Sina M. Coldewey

Toxins 2024, 16(12), 543; https://doi.org/10.3390/toxins16120543 - 14 Dec 2024

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Abstract

Hemolytic-uremic syndrome (HUS) is a systemic complication of an infection with Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli, primarily leading to acute kidney injury (AKI) and microangiopathic hemolytic anemia. Although free heme has been found to aggravate renal damage in hemolytic diseases, the [...] Read more.

Hemolytic-uremic syndrome (HUS) is a systemic complication of an infection with Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli, primarily leading to acute kidney injury (AKI) and microangiopathic hemolytic anemia. Although free heme has been found to aggravate renal damage in hemolytic diseases, the relevance of the heme-degrading enzyme heme oxygenase-1 (HO-1, encoded by Hmox1) in HUS has not yet been investigated. We hypothesized that HO-1, also important in acute phase responses in damage and inflammation, contributes to renal pathogenesis in HUS. The effect of tamoxifen-induced Hmox1 gene deletion on renal HO-1 expression, disease progression and AKI was investigated in mice 7 days after HUS induction. Renal HO-1 levels were increased in Stx-challenged mice with tamoxifen-induced Hmox1 gene deletion (Hmox1^R26Δ/Δ) and control mice (Hmox1^lox/lox). This HO-1 induction was significantly lower (−43%) in Hmox1^R26Δ/Δ mice compared to Hmox1^lox/lox mice with HUS. Notably, the reduced renal HO-1 expression was associated with an exacerbation of kidney injury in mice with HUS as indicated by a 1.7-fold increase (p = 0.02) in plasma neutrophil gelatinase-associated lipocalin (NGAL) and a 1.3-fold increase (p = 0.06) in plasma urea, while other surrogate parameters for AKI (e.g., periodic acid Schiff staining, kidney injury molecule-1, fibrin deposition) and general disease progression (HUS score, weight loss) remained unchanged. These results indicate a potentially protective role of HO-1 in the pathogenesis of Stx-mediated AKI in HUS. Full article

(This article belongs to the Section Bacterial Toxins)

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12 pages, 543 KiB

Open AccessArticle

Microcystins Exposure and the Risk of Metabolic Syndrome: A Cross-Sectional Study in Central China

by Shuidong Feng, Yuke Zeng, Fengmei Song, Minxue Shen and Fei Yang

Toxins 2024, 16(12), 542; https://doi.org/10.3390/toxins16120542 - 14 Dec 2024

Viewed by 400

Abstract

A growing body of evidence indicates that microcystins (MCs) exposure may cause metabolic diseases. However, studies exploring the effects of MCs exposure on the risk of metabolic syndrome (MetS) in humans are currently lacking, and the underlying mechanisms remain unclear. Here, we conducted [...] Read more.

A growing body of evidence indicates that microcystins (MCs) exposure may cause metabolic diseases. However, studies exploring the effects of MCs exposure on the risk of metabolic syndrome (MetS) in humans are currently lacking, and the underlying mechanisms remain unclear. Here, we conducted a cross-sectional study in central China to explore the effect of serum MCs on MetS, and assessed the mediation effects of the inflammation biomarker, white blood cell (WBC) level, in this relationship. The relationships among MCs and WBC level and risk of MetS were assessed using binary logistic and linear regression. Mediation analysis was used to explore possible mechanisms underlying those associations by employing R software (version 4.3.1). Compared to the lowest quartile of MCs, the highest quartile had an increased risk of MetS (odds ratio [OR] = 2.10, 95% confidence interval [CI]: 1.19, 3.70), with a dose–response relationship (p for trend < 0.05). WBCs mediated 11.14% of the association between serum MCs and triglyceride (TG) levels, but did not mediate the association of MCs exposure with MetS. This study firstly reveals that MCs exposure is an independent risk factor for MetS in a dose–response manner, and suggests that WBC level could partially mediate the association of MCs exposure with TG levels. Full article

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17 pages, 1482 KiB

Open AccessArticle

Assessment of the Effects of Anatoxin-a In Vitro: Cytotoxicity and Uptake

by Cristina Plata-Calzado, Ana I. Prieto, Ana M. Cameán and Angeles Jos

Toxins 2024, 16(12), 541; https://doi.org/10.3390/toxins16120541 - 13 Dec 2024

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Abstract

Anatoxin-a (ATX-a) is a cyanotoxin whose toxicological profile has been underinvestigated in comparison to other cyanotoxins such as microcystins (MCs) or cylindrospermopsin (CYN). However, its wide distribution, occurrence, and toxic episodes justify more attention. It is classified as a neurotoxin, but it has [...] Read more.

Anatoxin-a (ATX-a) is a cyanotoxin whose toxicological profile has been underinvestigated in comparison to other cyanotoxins such as microcystins (MCs) or cylindrospermopsin (CYN). However, its wide distribution, occurrence, and toxic episodes justify more attention. It is classified as a neurotoxin, but it has also been reported to affect other organs and systems. Thus, the aim of this study was to establish, as a first tier in its toxicological evaluation, its cytotoxicity in a wide range of cell lines representative of potential target organs (N2a, SH-SY5Y, HepG2, Caco2, L5178Y Tk^+/−, THP-1 and Jurkat). As limited effects were observed after exposure to up to 200 µg/mL of ATX-a for 24 h (only Jurkat and THP-1 cells showed reduced cell viability), cell uptake experiments were performed by ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). The results showed that the immune system cells had the highest percentage of ATX-a in the intracellular fraction, followed by neuronal cells and finally Caco-2 and HepG2 cells. Moreover, the expression of genes related to cell death mechanisms in THP-1 cells was also analyzed by polymerase chain reaction (PCR) and showed no changes under the conditions tested. Further research is required on ATX-a’s toxic effects and toxicokinetics to contribute to its risk assessment. Full article

(This article belongs to the Special Issue Advances in Cyanotoxins: Latest Developments in Risk Assessment)

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Reduction in tetrazolium salt MTS in (a) neuronal cells (SH-SY5Y and N2a), (b) Caco-2 and HepG2 cells and (c) immune system cells (THP-1, Jurkat and L5178Y Tk+/−) after 24 h of exposure to 0–200 µg/mL ATX-a fumarate. In addition, 0.3% v/v Triton X-100 was used as the positive control. All experiments were performed at least in triplicate per concentration. Data are expressed as mean ± SD compared to negative control group. Note: * p < 0.05 and *** p < 0.001 indicated significant difference from negative control. Full article ">Figure 2
Reduction in tetrazolium salt MTS in THP-1 cells exposed to 0–3 µg/mL CYN for 24 h. In addition, 0.3% v/v Triton X-100 was used as the positive control. Experiments were performed in triplicate per concentration. Data are expressed as mean ± SD compared to negative control group. Note: * p < 0.05 and *** p < 0.001 indicate significant difference from the negative control. Full article ">Figure 3
Chromatograms obtained by UHPLC-MS/MS of ATX-a in THP-1 cells exposed to 50 µg/mL ATX-a fumarate for 24 h. (a) Negative control, (b) intracellular fraction and (c) extracellular fraction. Full article ">Figure 4
Effects of ATX-a on the expression of mRNA of genes involved in apoptosis/necrosis in THP-1 cells. Cells were exposed to 5 or 50 µg/mL ATX-a fumarate for 24 h. CPT (0.5 µM) was used as positive control and chloroform/methanol (4:1) as solvent control. Results are expressed as relative mRNA expression normalized to the negative control group. * p < 0.05 and **** p < 0.0001 indicate significantly difference from negative controls. Full article ">

17 pages, 19758 KiB

Open AccessArticle

The Protein Engineering of Zearalenone Hydrolase Results in a Shift in the pH Optimum of the Relative Activity of the Enzyme

by Anna Dotsenko, Igor Sinelnikov, Ivan Zorov, Yury Denisenko, Aleksandra Rozhkova and Larisa Shcherbakova

Toxins 2024, 16(12), 540; https://doi.org/10.3390/toxins16120540 - 13 Dec 2024

Viewed by 495

Abstract

An acidic shift in the pH profile of Clonostachys rosea zearalenone hydrolase (ZHD), the most effective and well-studied zearalenone-specific lactone hydrolase, is required to extend the range of applications for the enzyme as a decontamination agent in the feed and food production industries. [...] Read more.

An acidic shift in the pH profile of Clonostachys rosea zearalenone hydrolase (ZHD), the most effective and well-studied zearalenone-specific lactone hydrolase, is required to extend the range of applications for the enzyme as a decontamination agent in the feed and food production industries. Amino acid substitutions were engineered in the active center of the enzyme to decrease the pKa values of the catalytic residues E126 and H242. The T216K substitution provided a shift in the pH optimum by one unit to the acidic region, accompanied by a notable expansion in the pH profile under acidic conditions. The engineered enzyme demonstrated enhanced activity within the pH range of 3–5 and improved the activity within the pH ranging from 6 to 10. The D31N and D31A substitutions also resulted in a two-unit shift in the pH optimum towards acidic conditions, although this was accompanied by a significant reduction in the enzyme activity. The D31S substitution resulted in a shift in the pH profile towards the alkaline region. The alterations in the enzyme properties observed following the T216K substitution were consistent with the conditions required for the ZHD application as decontamination enzymes at acidic pH values (from 3.0 to 6.0). Full article

(This article belongs to the Special Issue Occurrence, Toxicity, Metabolism, Analysis and Control of Mycotoxins)

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Figure 1
The tertiary structure of C. rosea ZHD. The S102-H242-E126 catalytic triad in the active center of the enzyme; the residues S102, H242, and E126 are shown in green (a); (b) the pocket of the active center. The molecule of the substrate (zearalenone) is shown in gray; atoms of nitrogen, oxygen, and sulfur in the structure of the enzyme are shown in blue, red, and yellow, respectively. Full article ">Figure 2
Location of the residues G213, T216, and F221 near the entrance to the pocket and the residue D31 at the bottom of the active center pocket of C. rosea ZHD. The substrate molecule (zearalenone) is shown in gray; the surface of the pocket of the active center is shown in transparent gray; the residues D31, G213, T216, and F221 are shown in green spheres, with nitrogen and oxygen atoms shown in blue and red, respectively. Full article ">Figure 3
The tertiary structure of C. rosea ZHD with the T216K and T216R amino acid substitutions. The S102-H242-E126 catalytic triad in the active center of the enzyme is shown in green, the substrate molecule (zearalenone) is shown in gray, and the T216K and T216R substitutions are shown in dark gray. Nitrogen and oxygen atoms are shown in blue and red, respectively. Full article ">Figure 4
The tertiary structure of C. rosea ZHD with the T216K amino acid substitution (a) and the wild-type enzyme (b). The surface of the active center pocket is shown in transparent gray; the molecule of the substrate zearalenone is shown in gray and magenta for the modes at the bottom of the pocket and closer to the entrance to the pocket, respectively; the residues K216 and T216 are shown in green spheres, with nitrogen and oxygen atoms shown in blue and red, respectively. Full article ">Figure 5
The tertiary structure of C. rosea ZHD with the residue D31 at the bottom of the active center pocket. The S102-H242-E126 catalytic triad in the active center of the enzyme is shown in green, and the molecule of the substrate zearalenone is shown in gray. Nitrogen and oxygen atoms are shown in blue and red, respectively. Full article ">Figure 6
SDS-PAGE electropherogram of the crude lysates of E. coli cells (C—non-induced control; 1—native ZHD; 2—ZHD(T216K); 3—ZHD(D31A); 4—ZHD(D31S); 5—ZHD(D31N) and M—molecular weight marker). Full article ">Figure 7
The influence of pH on the zearalenone conversion by mutant and wild-type forms of C. rosea ZHD. Y-bars on curves show standard deviation (p < 0.05). Full article ">

20 pages, 3277 KiB

Open AccessArticle

Variations in Kojic Acid Production and Corn Infection Among Aspergillus flavus Isolates Suggest a Potential Role as a Virulence Factor

by Rebecca R. Sweany, Matthew K. Gilbert, Carol H. Carter-Wientjes, Geromy G. Moore and Matthew D. Lebar

Toxins 2024, 16(12), 539; https://doi.org/10.3390/toxins16120539 - 13 Dec 2024

Viewed by 661

Abstract

Kojic acid is a secondary metabolite with strong chelating and antioxidant properties produced by Aspergillus flavus and A. oryzae. Although antioxidants and chelators are important virulence factors for plant pathogens, the ecological role of kojic acid remains unclear. We previously observed a [...] Read more.

Kojic acid is a secondary metabolite with strong chelating and antioxidant properties produced by Aspergillus flavus and A. oryzae. Although antioxidants and chelators are important virulence factors for plant pathogens, the ecological role of kojic acid remains unclear. We previously observed a greater gene expression of antioxidants, especially kojic acid, by non-aflatoxigenic A. flavus when co-cultured with aflatoxigenic A. flavus. Aflatoxin production was also reduced. In this study, we investigated kojic acid production in 22 A. flavus isolates from Louisiana and compared them to four common A. flavus strains in liquid medium and on corn kernels. Corn kernel infection was assessed by quantifying the maize beta tubulin DNA content of the kernels using drop digital PCR (ddPCR). Maize beta tubulin DNA content decreased with increased corn kernel infection. Greater kojic acid production by A. flavus isolates coincided with greater levels of corn kernel infection. All isolates produced 60 and 700 times more kojic acid than aflatoxin and cyclopiazonic acid (a known virulence factor), respectively, which varied among sclerotial size categories. A. flavus strains with small sclerotia, which were rarely isolated from corn, produced the least kojic acid and infected corn kernels the least, while medium and large sclerotia strains—mainly isolated from corn—produced the most kojic acid and were more infectious. Non-aflatoxigenic isolates from Louisiana produced the most kojic acid. These results suggest that kojic acid is a potential virulence factor and may increase the pathogenic success of medium and large sclerotia-producing A. flavus, which could ultimately lead to more effective A. flavus biocontrol strains. Further studies are required to determine the effects that kojic acid has on the redox environment during corn infection and how the altered redox environment decreases aflatoxin production. Full article

(This article belongs to the Special Issue Toxicity, Mitigation and Chemical Analysis of Aflatoxins and Other Toxic Metabolites Produced by Aspergillus)

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Figure 1
Kojic acid production by Aspergillus flavus isolates in (a) standard medium and (b) corn kernels. Kojic acid was measured for isolates belonging to different vegetative compatibility groups (VCGs) and with different sclerotia sizes (small (S), mixed (M), and large (L)) grown in (a) standard medium and (b) corn kernels for five and six days, respectively. The VCG determined in Sweany et al. 2011 [<a href="#B23-toxins-16-00539" class="html-bibr">23</a>] is denoted as the first number in the isolate name followed by a dash and the SRRC collection number. Isolates are ordered based on their phylogenetic similarities reported in Sweany et al. 2024 [<a href="#B28-toxins-16-00539" class="html-bibr">28</a>]. Within (a,b), means and standard error followed by the same letters are statistically similar based on preplanned comparisons of LS-means (α < 0.05) implemented in linear models. N.D. indicates that data are missing for an isolate. Full article ">Figure 2
Aflatoxin B1 production by Aspergillus flavus isolates in (a) standard medium and (b) corn kernels. Aflatoxin B1 was measured for isolates belonging to different vegetative compatibility groups (VCGs) and with different sclerotia sizes (small (S), mixed (M), and large (L)) grown in (a) standard medium and (b) corn kernels for five and six days, respectively. The VCG determined by Sweany et al. 2011 [<a href="#B23-toxins-16-00539" class="html-bibr">23</a>] is denoted as the first number in the isolate name followed by a dash and the SRRC collection number. Isolates are ordered based on their phylogenetic similarities reported by Sweany et al. 2024 [<a href="#B28-toxins-16-00539" class="html-bibr">28</a>]. Means and standard error bars followed by the same letters are statistically similar based on preplanned comparisons of LS-means (α < 0.05) implemented in a single linear model, which included both standard medium and corn kernel aflatoxin values. N.D. indicates that data are missing for an isolate. (c) Kojic acid production values of each isolate are plotted against aflatoxin production values in both substrates, which did not have a linear relationship in either standard medium (F1,33.8 = 1.55, p = 0.221) or corn kernels (F1,41.3 = 0.00, p = 0.960). Full article ">Figure 3
Cyclopiazonic acid production (CPA) by Aspergillus flavus in corn kernels. (a) CPA was measured for isolates belonging to different vegetative compatibility groups (VCGs) and with different sclerotia sizes (small (S), mixed (M), and large (L)) grown in corn kernels for six days. The VCG determined by Sweany et al. 2011 [<a href="#B23-toxins-16-00539" class="html-bibr">23</a>] is denoted as the first number in the isolate name followed by a dash and the SRRC collection number. Isolates are ordered based on their phylogenetic similarities reported by Sweany et al. 2024 [<a href="#B28-toxins-16-00539" class="html-bibr">28</a>]. Means and standard error followed by the same letters are statistically similar based on comparisons of LS-means (α < 0.05) implemented in linear models. N.D. indicates that data are missing for an isolate. (b) In the top panel, CPA values are plotted against aflatoxin for each isolate. Estimated linear relationships between CPA and aflatoxin are depicted by lines. In the bottom panel, CPA is plotted against kojic acid for individual isolates, and since there is no significant linear relationship between CPA and kojic acid, no lines are depicted. Full article ">Figure 4
Conidiospore production of Aspergillus flavus isolates on corn kernels. Green conidia and black sclerotia on corn inoculated with isolates belonging to different vegetative compatibility groups (VCGs) and with different sclerotia sizes (small (S), mixed (M), and large (L)). The VCG determined by Sweany et al. 2011 [<a href="#B23-toxins-16-00539" class="html-bibr">23</a>] is denoted as the first number in the isolate name followed by a dash and the SRRC collection number. The same letter behind the isolate name refers to the mean conidial production that is statistically similar based on preplanned comparisons of LS-means (α < 0.05) implemented in linear models. Isolates are ordered in columns from top to bottom followed by rows from left to right based on their phylogenetic similarities reported by Sweany et al. 2024 [<a href="#B28-toxins-16-00539" class="html-bibr">28</a>]. Full article ">Figure 5
Relative levels of genomic DNA in corn kernels inoculated with A. flavus over an 11-day time course. Droplet digital PCR was used to amplify the target genomic sequence (beta tubulin), and the ratio (i.e., proportion (p)) of positive droplets that were amplified to total droplets formed was determined. The same letters reported above average ratios (p) with standard error bars are statistically similar based on comparisons of LS-means (α < 0.05) implemented in generalized linear models. Full article ">Figure 6
Ratio of maize gene content (p) is reduced during infection with isolates that produced more kojic acid, CPA, aflatoxin, and conidia. (a) The ratio of maize beta tubulin gene droplets to total droplets (p) was measured with droplet digital PCR for total DNA extracted from kernels inoculated with large (L), mixed (M), and small (S) sclerotia isolates. Smaller ratios (p) indicate less corn DNA in a sample and a greater level of corn infection. (b) The ratio of beta tubulin (p) was plotted against kojic acid, CPA, conidiospore, and aflatoxin production for each isolate. Estimated lines were also plotted if there was a statistically significant linear relationship between kojic acid, CPA, conidiospore, aflatoxin production, and maize beta tubulin within sclerotial size groups. Full article ">

16 pages, 14718 KiB

Open AccessArticle

Repair of Retrorsine-Induced DNA Damage in Rat Livers: Insights Gained from Transcriptomic and Proteomic Studies

by Yun Long, Yiwei Wang, Zijing Song, Xin He, Yisheng He and Ge Lin

Toxins 2024, 16(12), 538; https://doi.org/10.3390/toxins16120538 - 13 Dec 2024

Viewed by 436

Abstract

Pyrrolizidine alkaloids (PAs) are common phytotoxins that are found worldwide. Upon hepatic metabolic activation, the reactive PA metabolites covalently bind to DNAs and form DNA adducts, causing mutagenicity and tumorigenicity in the liver. However, the molecular basis of the formation and removal of [...] Read more.

Pyrrolizidine alkaloids (PAs) are common phytotoxins that are found worldwide. Upon hepatic metabolic activation, the reactive PA metabolites covalently bind to DNAs and form DNA adducts, causing mutagenicity and tumorigenicity in the liver. However, the molecular basis of the formation and removal of PA-derived DNA adducts remains largely unexplored. In the present study, Sprague Dawley (SD) rats were exposed to retrorsine (RTS), a representative PA, at a human-relevant dose of 3.3 mg/kg/day for 28 days. The rats were divided into three groups: control, RTS-28 (sacrificed after continuous RTS exposure), and RTS-161 (sacrificed at 133 days post-RTS-exposure). The multi-omics analyses demonstrated the involvement of homologous recombination (HR) and non-homologous end joining (NHEJ) repair pathways as a response to PA-induced DNA damage. Additionally, the characteristic guanine adducts induced by RTS exposure were in accordance with the higher expression of XPA and XPC, indicating that nucleotide excision repair (NER) and base excision repair (BER) also contributed to repairing RTS-induced DNA damage. Furthermore, we also showed that DNA damage persisted after PA exposure, and mutagenically related repair errors might occur due to the prolonged genotoxic effects. The present study lays the foundation for bridging PA-derived DNA adducts, DNA damage, DNA repair, and the follow-up mutagenesis and carcinogenesis associated with PA exposure. Full article

(This article belongs to the Special Issue Plant Toxin Emergency)

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19 pages, 5894 KiB

Open AccessArticle

Application of Pulsed Electric Field During Malting: Impact on Fusarium Species Growth and Mycotoxin Production

by Nela Prusova, Marcel Karabin, Lukas Jelinek, Jana Chrpova, Jaroslava Ovesna, Pavel Svoboda, Tereza Dolezalova, Adam Behner, Jana Hajslova and Milena Stranska

Toxins 2024, 16(12), 537; https://doi.org/10.3390/toxins16120537 - 12 Dec 2024

Viewed by 477

Abstract

The increasing contamination of cereals by micromycetes and mycotoxins during malting still poses an unresolved food safety problem. This study characterises the potential of the novel, rapidly developing food production technology of Pulsed Electric Field (PEF) to reduce the viability of Fusarium fungi [...] Read more.

The increasing contamination of cereals by micromycetes and mycotoxins during malting still poses an unresolved food safety problem. This study characterises the potential of the novel, rapidly developing food production technology of Pulsed Electric Field (PEF) to reduce the viability of Fusarium fungi and the production of mycotoxins during malting. Barley, artificially inoculated with four Fusarium species, was treated by PEF with two different intensities and then malted using a standard Pilsner-type technology. Concentrations of fungi were quantified by RT-PCR, expression of fungal growth-related genes was assessed using mRNA sequencing, and mycotoxin levels were analysed by U-HPLC-HRMS/MS. Despite the different trends for micromycetes and mycotoxins after application of variously intense PEF conditions, significant reductions were generally observed. The greatest decrease was for F. sporotrichioides and F. poae, where up to six fold lower levels were achieved for malts produced from the PEF-treated barley when compared to the control. For F. culmorum and F. graminearum, up to a two-fold reduction in the PEF-generated malts was observed. These reductions mostly correlated with a decrease in relevant mycotoxins, specifically type A trichothecenes. Full article

(This article belongs to the Section Mycotoxins)

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17 pages, 1879 KiB

Open AccessArticle

Degradation of Cylindrospermopsin Spiked in Natural Water (Paranoá Lake, Brasília, Brazil) by Fenton Process: A Bench–Scale Study

by Matheus Almeida Ferreira, Cristina Celia Silveira Brandão and Yovanka Pérez Ginoris

Toxins 2024, 16(12), 536; https://doi.org/10.3390/toxins16120536 - 12 Dec 2024

Viewed by 429

Abstract

The frequency and intensity of harmful cyanobacterial blooms have increased in the last decades, posing a risk to public health since conventional water treatments do not effectively remove extracellular cyanotoxins. Consequently, advanced technologies such as the Fenton process are required to ensure water [...] Read more.

The frequency and intensity of harmful cyanobacterial blooms have increased in the last decades, posing a risk to public health since conventional water treatments do not effectively remove extracellular cyanotoxins. Consequently, advanced technologies such as the Fenton process are required to ensure water safety. The cyanotoxin cylindrospermopsin (CYN) demands special attention, as it is abundant in the extracellular fraction and has a high toxicological potential. Hence, this study aimed to assess the application of the Fenton process for the oxidation of CYN spiked in natural water from Paranoá Lake (Brasília, Brazil). The H₂O₂/Fe(II) molar ratio was evaluated from 0.2 to 3.4, with an optimum molar ratio of 0.4, achieving a CYN degradation efficiency of 97.8% when using 100 µM of H₂O₂ and 250 µM of Fe(II). The CYN degradation efficiency, using 75 µM of H₂O₂ and 187.5 µM of Fe(II), decreased by increasing the initial pH (from 96.2% at pH 2 to 23.0% at pH 9) and the initial CYN concentration (from 93.7% at 0.05 µM of CYN to 85.0% at 0.2 µM of CYN). At the optimum H₂O₂/Fe(II) molar ratio of 0.4, the hydroxy radical scavengers tested (124.3 µM C of algogenic organic matter, 5 mg L⁻¹ of humic acid, and 513.3 µM of methanol) did not considerably affect the CYN degradation, reaching a maximum CYN degradation reduction from 98.3% to 82.2%. Full article

(This article belongs to the Special Issue Toxic Cyanobacterial Bloom Detection and Removal: What's New?)

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Figure 1
Residual concentration and/or removal of (a) CYN, (b) H2O2, (c) dissolved and total fractions of Fe(II), (d) dissolved and total fractions of Fe(III), (e) dissolved and total fractions of total iron, and (f) pH–time profile during Fenton oxidation for various H2O2/Fe(II) molar ratios. H2O2 dosage fixed at 75 µM and Fe(II) from 22.1 to 375.0 µM. Error bars represent the standard deviation of the mean based on three replicates. Full article ">Figure 2
Residual concentration and removal of CYN by the Fenton process for different H2O2 and Fe(II) dosages at the H2O2/Fe(II) molar ratio of 0.4. Initial pH of about 5.0, and 30 min reaction. Error bars represent the standard deviation of the mean based on three replicates. Full article ">Figure 3
Removal of CYN for various initial CYN concentrations. Initial pH about 5.0, 75 µM of H2O2, 187.5 µM of Fe(II), and 30 min reaction. Error bars represent the standard deviation of the mean based on three replicates. Full article ">Figure 4
Residual concentration and/or removal of (a) CYN, (b) H2O2, (c) dissolved and total fractions of Fe(II), (d) dissolved and total fractions of Fe(III), (e) dissolved and total fractions of total iron, and (f) pH–time profile during Fenton oxidation for various initial pH values. H2O2 dosage of 75 µM and Fe(II) of 187.5 µM. Error bars represent the standard deviation of the mean based on three replicates. Full article ">Figure 5
Removal and residual concentrations of CYN for different H2O2 and Fe(II) dosages at H2O2 Fe(II) of 0.4 in (a) ultrapure water, (b) ultrapure water containing 5.0 mg/L of HA, and (c) ultrapure water containing AOM (NPOC of 125 µM C). Initial pH around 5.0 and 30 min reaction. Error bars represent the standard deviation of the mean based on three replicates. Full article ">Figure 6
Residual concentration and removal of CYN by the Fenton process for various H2O2/Fe(II) molar ratios in (a) methanol–free ultrapure water and (b) ultrapure water containing 513.3 µM of methanol. Initial pH about 5.0, 25 µM of H2O2, 7.4 to 125 µM Fe(II), and 30 min reaction. Error bars represent the standard deviation of the mean based on two replicates. Full article ">Figure 7
Residual concentration and/or removal of (a) CYN, (c) H2O2, (e) Fe(II), Fe(III), and total iron in methanol–free ultrapure water, and (b) CYN, (d) H2O2, (f) Fe(II), Fe(III), and total iron in ultrapure water containing 513.3 µM of methanol. Initial pH about 5.0, H2O2/Fe(II) molar ratio of 0.4, and 30 min reaction. Error bars represent the standard deviation of the mean based on three replicates. Full article ">

15 pages, 2588 KiB

Open AccessCommunication

Quantification of Staphylococcal Enterotoxin A Variants at Low Level in Dairy Products by High-Resolution Top-Down Mass Spectrometry

by Nina Aveilla, Cécile Feraudet-Tarisse, Dominique Marcé, Abdelhak Fatihi, François Fenaille, Jacques-Antoine Hennekinne, Stéphanie Simon, Yacine Nia and François Becher

Toxins 2024, 16(12), 535; https://doi.org/10.3390/toxins16120535 - 11 Dec 2024

Viewed by 486

Abstract

Food poisoning outbreaks frequently involve staphylococcal enterotoxins (SEs). SEs include 33 distinct types and multiple sequence variants per SE type. Various mass spectrometry methods have been reported for the detection of SEs using a conventional bottom-up approach. However, the bottom-up approach cannot differentiate [...] Read more.

Food poisoning outbreaks frequently involve staphylococcal enterotoxins (SEs). SEs include 33 distinct types and multiple sequence variants per SE type. Various mass spectrometry methods have been reported for the detection of SEs using a conventional bottom-up approach. However, the bottom-up approach cannot differentiate between all sequence variants due to partial sequence coverage, and it requires a long trypsin digestion time. While the alternative top-down approach can theoretically identify any sequence modifications, it generally provides lower sensitivity. In this study, we optimized top-down mass spectrometry conditions and incorporated a fully ¹⁵N-labeled SEA spiked early in the protocol to achieve sensitivity and repeatability comparable to bottom-up approaches. After robust immunoaffinity purification of the SEA, mass spectrometry signals were acquired on a Q-Orbitrap instrument operated in full-scan mode and targeted acquisition by parallel reaction monitoring (PRM), enabling the identification of sequence variants and precise quantification of SEA. The protocol was evaluated in liquid and solid dairy products and demonstrated detection limits of 0.5 ng/mL or ng/g in PRM and 1 ng/mL or ng/g in full-scan mode for milk and Roquefort cheese. The top-down method was successfully applied to various dairy products, allowing discrimination of contaminated versus non-contaminated food, quantification of SEA level and identification of the variant involved. Full article

(This article belongs to the Section Bacterial Toxins)

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Intact purified 15N SEA3 (A) and SEA3 (B) at 10 µg/mL analyzed by HRMS/MS. Intact 15N SEA3 (A1) and SEA3 (B1) analyzed by full-scan HRMS and corresponding deconvoluted spectra (A2,B2). Spectra were deconvoluted with the software «Protein Deconvolution» version 4.0 from Thermo Scientific. The observed mass matched with a theoretical mass of 28,494.70 Da corresponding to 341 labeled nitrogens, a loss of one histidine in the His-Tag, and the expected disulfide bridge between the two cysteines. Blue star: additional peaks attributed to a dimeric form. HRMS/MS spectra of intact 15N SEA3 (C2) and SEA3 (D2) using HCD and observed retention times (C1,D1). Full article ">Figure 2
Measurement of the SEA3 to 15N SEA3 ratio using different filtration conditions, with and without the 10 kDa ultrafiltration device (UD10kDa). SEA3 was spiked at 10 ng/mL in milk, n = 3. Full article ">Figure 3
Sample preparation protocol for SEA followed by top-down analysis. Created in BioRender. <a href="https://BioRender.com/q37i192" target="_blank">https://BioRender.com/q37i192</a> accessed on 29 October 2024. Full article ">Figure 4
Isolation of multiple charge states for PRM acquisition. (A) MS spectra of native SEA3. (B) Peak area of SEA3 with 3 to 9 selected charge states using MSX. MSX was limited to 10 charge states with the Orbitrap instrument. (C) Peak area of SEA3 with 3 to 17 selected charge states using WIW isolation. In <a href="#app1-toxins-16-00535" class="html-app">Supplementary Data Table S3</a>, the different charge states isolated in MSX and WIW are reported. ** MSX3 isolation was previously used [<a href="#B17-toxins-16-00535" class="html-bibr">17</a>] and corresponded to the initial conditions in this work. Full article ">Figure 5
Linearity of the top-down assay for SEA3 spiked in milk and analyzed in PRM mode using the WIW9 isolation (A) or full-scan mode (B) (n = 4). Full article ">Figure 6
Linearity of SEA3 in Roquefort analyzed in PRM mode using the WIW9 isolation (A) or full-scan mode (B) (n = 2). Full article ">

21 pages, 4145 KiB

Open AccessArticle

Assessing the Potential Synergistic/Antagonistic Effects of Citrinin and Cannabidiol on SH-SY5Y, HepG2, HEK293 Cell Lines, and Human Lymphocytes

by Dubravka Rašić, Antonio Zandona, Maja Katalinić, Martin Češi and Nevenka Kopjar

Toxins 2024, 16(12), 534; https://doi.org/10.3390/toxins16120534 - 11 Dec 2024

Viewed by 462

Abstract

The increasing use of Cannabis sativa products for medicinal, dietary, and recreational purposes has raised concerns about mycotoxin contamination in cannabis and hemp. Mycotoxins persist in these products’ post-processing, posing health risks via multiple exposure routes. This study investigated cytotoxic and genotoxic interactions [...] Read more.

The increasing use of Cannabis sativa products for medicinal, dietary, and recreational purposes has raised concerns about mycotoxin contamination in cannabis and hemp. Mycotoxins persist in these products’ post-processing, posing health risks via multiple exposure routes. This study investigated cytotoxic and genotoxic interactions between cannabidiol (CBD) and the mycotoxin citrinin (CIT) using human cell models: SH-SY5Y, HepG2, HEK293, and peripheral blood lymphocytes. IC₅₀ values and membrane disruption were initially assessed, followed by an evaluation of genotoxicity in lymphocytes using the Comet Assay and Cytokinesis Blocked Micronucleus Cytome Assay. Obtained findings demonstrate that cell-type sensitivity varied across treatments, with combined CBD and CIT exposure exhibiting distinct interactions. Lactate dehydrogenase (LDH) release remained minimal, suggesting cytotoxicity did not stem from membrane disruption but likely involved intracellular pathways. In lymphocytes, CBD alone produced negligible cyto/genotoxic effects and weak antiproliferative responses, whereas CIT displayed clear toxic impacts. DNA damage indicates that CIT may induce genome instability through indirect mechanisms rather than direct DNA interaction, with evidence of potential aneuploidic effects from the CBMN Cyt Assay. Combined exposure led to a reduction in CIT-induced DNA and cytogenetic damage, suggesting CIT’s potential interference with the beneficial properties of CBD. These results provide a foundation for further toxicological assessments and highlight the necessity of standardized mycotoxin monitoring in cannabis-derived products. Full article

(This article belongs to the Special Issue Occurrence, Toxicity, Metabolism, Analysis and Control of Mycotoxins)

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Dose-dependent cytotoxicity and IC50 values of cannabidiol (CBD) and citrinin (CIT) on SH-SY5Y, HepG2 and HEK293 cells after 24 h exposure. Experimental data are presented as a mean (±SE) of at least three experiments. Full article ">Figure 2
Viability of SH-SY5Y, HepG2 and HEK293 cells after 24 h exposure to cannabidiol (CBD), citrinin (CIT) and their combinations at lowest-observed-adverse-effect level (LOAEL) concentrations (1 and 30 μM for SH-SY5Y and HepG2 and 2 and 0.05 μM for HEK293, respectively). Experimental data are presented as a mean (±SE) of at least two experiments. The results are expressed as percentages of corresponding control, untreated cells, and given as means ± SE. & p < 0.05; * p < 0.0001 vs. untreated control. Full article ">Figure 3
Levels of lactate dehydrogenase (LDH) release after 24 h exposure of SH-SY5Y, HepG2 and HEK293 cells after 24 h exposure to cannabidiol (CBD), citrinin (CIT) and their combinations at LOAEL concentrations (1 and 30 μM for SH-SY5Y and HepG2, and (2 and 0.05 μM for HEK293, respectively). Triton (0.08%) was used as the positive control. Experimental data are presented as a mean of percentage of LDH release (±SE) of at least two experiments. * p < 0.0001 vs. untreated control. Full article ">Figure 4
DNA damage in lymphocytes estimated by the alkaline comet assay. Lymphocyte cultures were treated for 24 h with citrinin (CIT) at 30 µM (7.50 µg/mL), cannabidiol (CBD) at 1 µM (3.15 µg/mL) and their combination (CIT + CBD, at the same concentrations). Negative controls (NCs) were non-treated lymphocytes. Solvent controls (SCs) were lymphocytes treated with ethanol (final concentration in the culture corresponded to 0.03%). Positive controls were lymphocytes treated for 24 h with bleomycin at 1.25 µg/mL. Six hundred independent comet measurements were carried out per experimental point. Results are expressed as mean/median, interquartile range, and range of measured values. Inter-group comparisons were performed using ANOVA with Tukey’s HSD post hoc test. Differences significant at p < 0.05 are marked with: *—vs. all other experimental groups; a—vs. negative control; b—vs. solvent control; c—vs. CBD; d—vs. CIT + CBD. Full article ">Figure 5
Typical lymphocyte nuclei observed under an epifluorescence microscope on microgels prepared for the alkaline comet assay. Non-damaged DNA in the negative control (a), and in the solvent control (b). Citrinin-treated lymphocyte (CIT) with damaged DNA (c). Low DNA damage in a cannabidiol-treated lymphocyte (CBD) (d), and in lymphocytes treated with combination of CIT and CBD (e). Highly fragmented DNA in a lymphocyte treated with bleomycin, positive control (f). Stained with ethidium bromide. Photomicrographs were taken under magnification ×200 using a black and white camera coupled with a computer-based image analysis system (Comet Assay IV, Instem-Perceptive Instruments Ltd., Suffolk, UK). Full article ">Figure 6
Photomicrographs of typical features observed on microscope slides prepared using Cytokinesis-Block Micronucleus (CBMN) Cytome Assay on human peripheral blood lymphocytes. Arrow indicates binucleated lymphocytes with (a) nuclear bud (NB) in the citrinin-treated sample (CIT); (b) micronucleus (MN) in the negative control sample; (c) two MNi in the citrinin + cannabidiol-treated sample (CIT + CBD); (d) nucleoplasmic bridge (NPB) in CIT-treated sample. Morphological features of dead cells in the positive control sample: (e) apoptotic cell with nuclear fragmentation; (f) a shift from apoptosis to necrosis; (g) necrotic cell. Typical features of cells scored to determine Cytokinesis-Block Proliferation Index: (h) mononucleated cell, M1 in negative control sample; (i) cell with two nuclei, M2 in negative control sample; (j) cell with three nuclei, M3 negative control sample; (k) cell with four nuclei, M4 in CIT-treated sample. Stained with Giemsa. Photographed at magnification ×1000 with Axiocam 208 color camera on Axiolab 5 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Full article ">Figure 7
Results of analysis of lymphocyte proliferation in cell cultures treated in vitro for 24 h with citrinin (CIT) at 7.50 µg/mL (30 µM), cannabidiol (CBD) at 3.15 µg/mL (1 µM) and their combination (CIT + CBD, at the same concentrations). Negative controls were non-treated lymphocytes. Solvent controls were lymphocytes treated with ethanol (final concentration in the culture corresponded to 0.03%). Positive controls were lymphocytes treated for 24 h with bleomycin at 1.25 µg/mL. Full article ">

20 pages, 3411 KiB

Open AccessArticle

Veratridine Induces Vasorelaxation in Mouse Cecocolic Mesenteric Arteries

by Joohee Park, Christina Sahyoun, Jacinthe Frangieh, Léa Réthoré, Coralyne Proux, Linda Grimaud, Emilie Vessières, Jennifer Bourreau, César Mattei, Daniel Henrion, Céline Marionneau, Ziad Fajloun, Claire Legendre and Christian Legros

Toxins 2024, 16(12), 533; https://doi.org/10.3390/toxins16120533 - 10 Dec 2024

Viewed by 806

Abstract

The vegetal alkaloid toxin veratridine (VTD) is a selective voltage-gated Na⁺ (Na_V) channel activator, widely used as a pharmacological tool in vascular physiology. We have previously shown that Na_V channels, expressed in arteries, contribute to vascular tone in mouse [...] Read more.

The vegetal alkaloid toxin veratridine (VTD) is a selective voltage-gated Na⁺ (Na_V) channel activator, widely used as a pharmacological tool in vascular physiology. We have previously shown that Na_V channels, expressed in arteries, contribute to vascular tone in mouse mesenteric arteries (MAs). Here, we aimed to better characterize the mechanisms of action of VTD using mouse cecocolic arteries (CAs), a model of resistance artery. Using wire myography, we found that VTD induced vasorelaxation in mouse CAs. This VTD-induced relaxation was insensitive to prazosin, an α1-adrenergic receptor antagonist, but abolished by atropine, a muscarinic receptor antagonist. Indeed, VTD–vasorelaxant effect was totally inhibited by the Na_V channel blocker tetrodotoxin (0.3 µM), the NO synthase inhibitor L-NNA (20 µM), and low extracellular Na⁺ concentration (14.9 mM) and was partially blocked by the NCX1 antagonist SEA0400 (45.4% at 1 µM). Thus, we assumed that the VTD-induced vasorelaxation in CAs was due to acetylcholine release by parasympathetic neurons, which induced NO synthase activation mediated by the NCX1-Ca²⁺ entry mode in endothelial cells (ECs). We demonstrated NCX1 expression in ECs by RT-qPCR and immunohisto- and western immunolabelling. VTD did not induce an increase in intracellular Ca²⁺ ([Ca²⁺]i), while SEA0400 partially blocked acetylcholine-triggered [Ca²⁺]i elevations in Mile Sven 1 ECs. Altogether, these results illustrate that VTD activates Na_V channels in parasympathetic neurons and then vasorelaxation in resistance arteries, which could explain arterial hypotension after VTD intoxication. Full article

(This article belongs to the Special Issue Toxins: From the Wild to the Lab)

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14 pages, 3562 KiB

Open AccessArticle

Venom from Loxosceles Spiders Collected in Southeastern and Northeastern Brazilian Regions Cause Hemotoxic Effects on Human Blood Components

by Rafaela Silva-Magalhães, Ayla Mel Gomes dos Santos, Ana Luiza Silva-Araújo, Pamella Luize Peres-Damásio, Valéria Gonçalves de Alvarenga, Luciana Souza de Oliveira, Eladio Flores Sanchez, Carlos Chávez-Olórtegui, Luana Silveira da Rocha Nowicki Varela, Ana Luiza Bittencourt Paiva and Clara Guerra-Duarte

Toxins 2024, 16(12), 532; https://doi.org/10.3390/toxins16120532 - 10 Dec 2024

Viewed by 466

Abstract

Spiders of the genus Loxosceles represent a public health problem in Brazil due to the severity of the cutaneous and systemic effects that may result from their bite. In the systemic form of loxoscelism, hemolytic anemia, thrombocytopenia, and disseminated intravascular coagulation can occur. [...] Read more.

Spiders of the genus Loxosceles represent a public health problem in Brazil due to the severity of the cutaneous and systemic effects that may result from their bite. In the systemic form of loxoscelism, hemolytic anemia, thrombocytopenia, and disseminated intravascular coagulation can occur. Despite the seriousness of Loxosceles accidents, the venom of some species has not yet been properly characterized considering these hemotoxic effects, such as that of Loxosceles amazonica, Loxosceles aff. Variegata, and Loxosceles similis. To better understand their toxic potential, this study aimed to characterize the hematotoxic properties of these Loxosceles venoms. The crude venom was obtained from specimens of L. amazonica, L. aff. Variegata, and L. similis available from Funed’s arachnidary. In washed platelets, L. aff. variegata inhibited platelet aggregation induced by collagen and convulxin, whereas L. amazonica and L. similis venoms were able to induce platelet aggregation. In the in vitro hemolysis assays, all venoms experimentally induced direct hemolysis of human erythrocytes in a concentration-dependent manner, with different intensities. Furthermore, evidence suggest that the ABO and Rh systems may influence hemolytic activity. Finally, the studied Loxosceles venoms degraded fibrinogen, suggesting possible alterations in the coagulation cascade. Based in the here-presented preliminary study, in vivo assays in model animals are needed to verify the real toxic potential of these species’ venom, building up knowledge to elucidate the action of Loxosceles venoms in blood. Full article

(This article belongs to the Special Issue Brown Spider Venom Toxins: Biological Activities, Molecular Mechanisms, Cell Biology, Biotechnological Applications, and Targets for the Therapeutic Management of Loxoscelism)

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Figure 1
Reported accidents involving Loxosceles spiders in Brazil (2007–2022) and locations of spider specimen collections. (A) Map of Brazil depicting the occurrence of Loxosceles-related accidents by state in 2022, along with the regions where the species used in this study were collected. (B) The number of reported accidents caused by Loxosceles spiders from 2007 to 2023, categorized by region within Brazil. (C) The change in the incidence rate of Loxosceles accidents over a decade (2013–2023) per region, with the national average represented by a dotted line. Color intensity reflects incidence rates as detailed in the legend. The map was created using QGIS 3.32.3 software. Incident data was retrieved from the Sistema de Informação de Agravos de Notificação—SINAN. * Data is subject to revision by SINAN for the period 2020–2023. Data from the Southeast region does not include Espirito Santo state, which stopped providing data to SINAN since 2020. Full article ">Figure 2
The crude venom of Loxosceles amazonica and Loxosceles similis induce platelet aggregation. Washed platelets were incubated with 100 μg/mL and 200 μg/mL of Loxosceles crude venoms. Aggregation was monitored by measuring light transmittance for 10 min by an aggregometer. The percentage of aggregation was automatically calculated by comparing the initial optical density with the optical density after the addition of the aggregating agent, using the HemoRam 1.1 software. The mean ± standard deviation is shown. The results are representative of two or three experiments with different individual donors (points of graph). (A) Platelet aggregation with Loxosceles amazonica; (B) Loxosceles aff. Variegata, and (C) Loxosceles similis. Collagen or convulxin were used as platelet-aggregation agonists (C+). Statistical analysis was performed using one-way ANOVA (Kruskal–Wallis test) with Dunn post-test for multiple comparison. (*) = p ≤ 0.05. Full article ">Figure 3
The crude venom of Loxosceles aff. variegata inhibits platelet aggregation induced by collagen and convulxin. Washed human platelets were pre-incubated with different concentrations of Loxosceles aff. variegata venom (100 and 200 μg/mL) under agitation at 600 rpm at 37 °C. After 3 min, platelet aggregation was induced by 10 μg/mL collagen or 0.3 mg/mL convulxin and monitored by aggregometer by measuring light transmittance for 7 min. The mean ± standard deviation is shown. The results are representative of three experiments with different individual donors (points of graph). (A) The crude L. amazonica venom does not have the ability to inhibit platelet aggregation induced by agonist collagen. (B) Platelet aggregation assay to assess the ability of crude Loxosceles aff. variegata venom to inhibit collagen-induced and convulxin-induced aggregation. (C) The crude L. similis venom does not have the ability to inhibit platelet aggregation induced by agonist Convulxin. Statistical analysis was performed using two-way ANOVA with Tukey post-test for multiple comparison. (*) = p ≤ 0.05 and (**) = p ≤ 0.01. Full article ">Figure 4
In vitro hemolytic assay of Loxosceles venoms. Human erythrocytes were exposed to various concentrations (7.8; 15.6; 31.25; and 62.5 μg/mL) of different Loxosceles spider venoms for 20 h at 37.0 °C and were evaluated for hemolysis. As a negative control, the erythrocytes were incubated only in Ringer’s Lactate. The positive control was incubated with a 0.1% (v/v) Triton X-100 solution. (A) The tubes containing the Ringer’s Lactate solution, the venoms, and the controls after incubation and centrifugation. (B) Percentage of hemolysis considering Triton X-100 (positive control) as 100%. Statistical analysis was performed using two-way ANOVA with Tukey post-test for multiple comparison. (*) = p ≤ 0.05, (**) = p ≤ 0.01 and (****) = p ≤ 0.0001. Full article ">Figure 5
Influence of the Rh system on the direct hemolytic activity of Loxosceles venoms. The blood types were incubated with 31.25 μg/mL of different Loxosceles venoms for 20 h. LaV—Loxosceles amazonica venom. LgV—Loxosceles gaucho venom. LvV—Loxosceles aff. variegata venom. LsV—Loxosceles similis venom. Statistical analysis was performed using two-way ANOVA, with post-test of Bonferroni. (*) = p < 0.05. Full article ">Figure 6
Zymogram of the fibrinogenolytic activity of Loxosceles venoms and the inhibition of this activity. (A) Zymography of Loxosceles venoms using 12% SDS–PAGE, containing 5 mg/mL of bovine fibrinogen integrated into the gel. (B) Gel incubated overnight in a 1 M phosphate buffer at pH 8.0 with 2 mM EDTA. (C) Gel incubated overnight in a 1 M phosphate buffer at pH 8.0 with 3 mM Phenanthroline. (D) Gel incubated overnight in a 1 M phosphate buffer at pH 8.0 with 5 mM Phenylmethylsulfonyl Fluoride (PMSF). Arrows in yellow point to regions where enzymatic degradation by the venoms is present. LaV—Loxosceles amazonica venom. LgV—Loxosceles gaucho venom. LvV—Loxosceles aff. variegata venom. LsV—Loxosceles similis venom. Full article ">Figure 7
Fibrinogenolytic activity of Brazilian Loxosceles. Proteolytic activity was determined by a fibrinogen digestion (Fg) assay described by Medina-Santos et al., 2019. Fibrinogenolytic activity was performed using 3 µg of Loxosceles venoms, incubated with bovine or human fibrinogen for 16 h at 37 °C. The fibrinogen samples, either pure or pre- incubated with the venoms, were analyzed by 12% SDS–PAGE. The graphs were plotted with the mean and standard deviation of the percentage density of the bands compared to the fibrinogen control, considered as 100%, analyzed in ImageJ, considering two independent assays. (A) On the left, the graph shows the percentage of degradation of fibrinogen chains. On the right, 12% SDS–PAGE displays the fibrinogenolytic activity of male and female L. amazonica venom. (B) On the left, the graph shows the percentage of degradation of fibrinogen chains. On the right, 12% SDS–PAGE displays the fibrinogenolytic activity of male and female L. aff. variegata venom. (C) On the left, the graph shows the percentage degradation of fibrinogen chains. On the right, 12% SDS–PAGE displays the fibrinogenolytic activity of L. similis venom. (D) On the left, the graph shows the percentage degradation of fibrinogen chains. On the right, 12% SDS–PAGE displays the fibrinogenolytic activity of L. gaucho venom. LaV—Loxosceles amazonica venom. LgV—Loxosceles gaucho venom. LvV—Loxosceles aff. variegata venom. LsV—Loxosceles similis venom. Fg—Fibrinogen. Full article ">

26 pages, 8285 KiB

Open AccessArticle

Image Analysis and Untargeted Metabolomics Reveal Potential Phytotoxins from Fusarium venenatum Against Major Parasitic Weed Phelipanche ramosa (L.) Pomel

by Ana Bendejacq-Seychelles, Lisa Martinez, Anaïs Corréard, Jean Chrisologue Totozafy, Christian Steinberg, Jean-Bernard Pouvreau, Carole Reibel, Grégory Mouille, Samuel Mondy, Lucie Poulin and Stéphanie Gibot-Leclerc

Toxins 2024, 16(12), 531; https://doi.org/10.3390/toxins16120531 - 10 Dec 2024

Viewed by 584

Abstract

Branched broomrape (Phelipanche ramosa (L.) Pomel), an obligate parasitic weed with a wide host range, is known for its devasting effects on many crops worldwide. Soil fungi, notably Fusarium sp., are described as pathogenic to broomrape, while the hypothesis of the phytotoxicity [...] Read more.

Branched broomrape (Phelipanche ramosa (L.) Pomel), an obligate parasitic weed with a wide host range, is known for its devasting effects on many crops worldwide. Soil fungi, notably Fusarium sp., are described as pathogenic to broomrape, while the hypothesis of the phytotoxicity of fusaric acid produced by F. verticillioides for parasitic weeds of the genus Orobanche has been proposed. Using image analysis and untargeted metabolomics, this study investigated fungal metabolites phytotoxic for P. ramosa and produced by the F. venenatum MIAE02836 strain, isolated from symptomatic broomrapes and identified as a promising candidate for broomrape biocontrol. Phytotoxicity tests of crude extracts from the fungus alone or in interaction with broomrape on P. ramosa microcalli and quantification of necrosis by image analysis confirmed the phytotoxic potential of F. venenatum MIAE02836 metabolites towards the early developmental stages of P. ramosa. Data analysis of a non-targeted metabolomics approach revealed numerous metabolites produced by F. venenatum MIAE02836. Four of them, accumulated during interaction with the parasitic plant, are known for their phytotoxic potential: maculosin, cyclo(Leu-Phe), phenylalanyl-D-histidine and anguidine. These results suggest that combining image acquisition of the microcalli screening test and untargeted metabolomic approach is an interesting and relevant method to characterize phytotoxic fungal metabolites. Full article

(This article belongs to the Section Mycotoxins)

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Figure 1
Example of the application of the necrosis analysis model on microcalli D6 after treatment with water or with fungal filtrates of modality A. (a,b) Images of microcalli treated with water (negative control) and filtrates of modality A (fungus–broomrape interaction) respectively after standardization step; (c,d) Images of the same wells after application of the classification model. Necrotic microcalli are shown in green, healthy ones are in red, seeds are in purple, and the background is in yellow. Full article ">Figure 2
Necrosis development on broomrape microcalli depending on fungal filtrates over treatment time. The percentage of necrosis is the corrected relative ratio after subtraction of the average percentage of necrosis observed for the control at D0, D1, D5, and D6. Boxplots are shown with means (bold stars) and standard errors. Asterisks indicate significance of adjusted p-values (ns: not significant; *: 0.05; **: 0.01; ****: <0.001) after ANOVA on quasi-logistic regression post hoc pairwise comparison test between filtrates (Control: distilled water; modality A: filtrates from fungus–broomrape interaction; modality B: filtrates from fungus alone) for each day (Emmeans test with Holm–Bonferroni correction). Full article ">Figure 3
Results of metabolomic data analysis (ESI+ mode) for AMs and NAMs. (a) Proportion of metabolites with relative abundance significantly and non-significantly different based on ANOVA by permutation; (b,c) metabolites with relative abundance significantly different distributed according to their origin of production (F. venenatum MIAE02836 or P. ramosa) for NAMs and AMS respectively. The origin of production was supposed after a simple comparison between the normalized means of the relative abundance of the metabolites produced by the fungus alone (modality B) and by the broomrape alone (modality C); (d) distribution by chemical class of annotated fungal metabolites with relative abundance significantly different. Full article ">Figure 4
Accumulation of AMs with significantly different relative abundance by modality (broomrape alone, fungus alone, and interaction) (ESI+ mode). Part of the hierarchical tree showing metabolites with relative abundance significantly different between modalities A (broomrape–fungus interaction), B (fungus alone), and C (broomrape alone) according to the one-factor ANOVA by permutation (1000 permutations, p-value < 0.01) using MeV 4.9.0 software. The basal normalized relative abundance is shown in black. When the gradient tends towards yellow, the metabolite is accumulated to a greater extent. When the gradient tends towards blue, the quantity is lower. Metabolites framed in red correspond to fungal metabolites accumulated during interaction (see <a href="#toxins-16-00531-t002" class="html-table">Table 2</a>). Full article ">Figure 5
Mean relative abundance of fungal metabolites accumulated during the interaction with broomrape. Boxplots are shown with means (bold stars) and standard errors of the relative abundance of the fungal peptides and sesquiterpenoids accumulated during the interaction. Anguidin has not been represented, as it is heavily produced (minimum relative abundance of 329,458 metabolites and 2,442,767 metabolites for the fungus alone and for the interaction, respectively). Modality_A: filtrates from fungus–broomrape interaction; Modality_B: filtrates from fungus alone; Modality_C: filtrates from broomrape alone. Full article ">Figure 6
Workflow Overview for BRoomrape’s microcalli Automated Image-based Necrosis-detection (BRAIN). This workflow supports batch processing of images following a standardization step (Step 1) where users can choose to (i) run our pre-trained model on broomrape microcalli images (Step 2; requires same image acquisition parameters), (ii) annotate their own images (Step 3) and train a new model (Step 4), or (iii) annotate their own images and further train the pre-trained model (Alternative Step 3 and Step 4). Full article ">Figure 7
Sampling plan for phytotoxicity test. Columns 1–4 correspond to the negative control (microcalli with distilled water); columns 5–8 correspond to the filtrates of modality B (fungus alone); columns 9–12 correspond to the filtrates of modality A (fungus–broomrape interaction). Full article ">

6 pages, 189 KiB

Open AccessEditorial

Botulinum Toxin and Migraine: Goals and Perspectives

by Maria Pia Prudenzano

Toxins 2024, 16(12), 530; https://doi.org/10.3390/toxins16120530 - 10 Dec 2024

Viewed by 469

Abstract

This Special Issue aims to provide an updated overview of the current state and future perspectives of botulinum toxin treatment for migraine [...] Full article

(This article belongs to the Special Issue Botulinum Toxin and Migraine: Goals and Perspectives)

14 pages, 1043 KiB

Open AccessArticle

Integrating Telemedicine in Botulinum Toxin Type-A Treatment for Spasticity Management: Perspectives and Challenges from Italian Healthcare Professionals

by Stefania Spina, Salvatore Facciorusso, Nicoletta Cinone, Luigi Santoro, Anna Castagna, Marina Ramella, Franco Molteni, Andrea Santamato and The Collaborative Working Group

Toxins 2024, 16(12), 529; https://doi.org/10.3390/toxins16120529 - 7 Dec 2024

Viewed by 681

Abstract

(1) Background: Telemedicine is a vital tool for enhancing healthcare accessibility and outcomes at reduced costs. This study aimed to assess the usability of the Maia Connected Care telemedicine platform for managing spasticity in patients receiving botulinum toxin type-A, focusing on the perspectives [...] Read more.

(1) Background: Telemedicine is a vital tool for enhancing healthcare accessibility and outcomes at reduced costs. This study aimed to assess the usability of the Maia Connected Care telemedicine platform for managing spasticity in patients receiving botulinum toxin type-A, focusing on the perspectives of Italian physiatrists with expertise in this treatment. (2) Methods: Conducted from March 2023 to June 2023, this multicenter survey involved 15 Italian physicians who used the platform for teleconsultations. Data collected included demographic details, responses to the Telemedicine Usability Questionnaire, and physician insights on patient satisfaction, treatment effectiveness, and implementation challenges in telehealth. (3) Results: The platform demonstrated high usability, with strong physician satisfaction due to its user-friendly interface and quality of interactions. A majority expressed willingness to continue telehealth for spasticity management, noting its effectiveness in improving patient satisfaction and outcomes. Challenges included replicating the depth of in-person consultations and addressing issues like reimbursement and telehealth standardization. (4) Conclusions: This study highlights telemedicine’s potential for spasticity management and clinician satisfaction, while underscoring the need for improvements in simulating in-person experiences and addressing systemic issues. The absence of patient perspectives represents a limitation, advocating for future research to optimize telemedicine practices and evaluate long-term clinical impacts. Full article

(This article belongs to the Section Bacterial Toxins)

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39 pages, 6873 KiB

Open AccessReview

Exploring Mycolactone—The Unique Causative Toxin of Buruli Ulcer: Biosynthetic, Synthetic Pathways, Biomarker for Diagnosis, and Therapeutic Potential

by Gideon Atinga Akolgo, Kingsley Bampoe Asiedu and Richard Kwamla Amewu

Toxins 2024, 16(12), 528; https://doi.org/10.3390/toxins16120528 - 6 Dec 2024

Viewed by 971

Abstract

Mycolactone is a complex macrolide toxin produced by Mycobacterium ulcerans, the causative agent of Buruli ulcer. The aim of this paper is to review the chemistry, biosynthetic, and synthetic pathways of mycolactone A/B to help develop an understanding of the mode of [...] Read more.

Mycolactone is a complex macrolide toxin produced by Mycobacterium ulcerans, the causative agent of Buruli ulcer. The aim of this paper is to review the chemistry, biosynthetic, and synthetic pathways of mycolactone A/B to help develop an understanding of the mode of action of these polyketides as well as their therapeutic potential. The synthetic work has largely been driven by the desire to afford researchers enough (≥100 mg) of the pure toxins for systematic biological studies toward understanding their very high biological activities. The review focuses on pioneering studies of Kishi which elaborate first-, second-, and third-generation approaches to the synthesis of mycolactones A/B. The three generations focused on the construction of the key intermediates required for the mycolactone synthesis. Synthesis of the first generation involves assignment of the relative and absolute stereochemistry of the mycolactones A and B. This was accomplished by employing a linear series of 17 chemical steps (1.3% overall yield) using the mycolactone core. The second generation significantly improved the first generation in three ways: (1) by optimizing the selection of protecting groups; (2) by removing needless protecting group adjustments; and (3) by enhancing the stereoselectivity and overall synthetic efficiency. Though the synthetic route to the mycolactone core was longer than the first generation, the overall yield was significantly higher (8.8%). The third-generation total synthesis was specifically aimed at an efficient, scalable, stereoselective, and shorter synthesis of mycolactone. The synthesis of the mycolactone core was achieved in 14 linear chemical steps with 19% overall yield. Furthermore, a modular synthetic approach where diverse analogues of mycolactone A/B were synthesized via a cascade of catalytic and/or asymmetric reactions as well as several Pd-catalyzed key steps coupled with hydroboration reactions were reviewed. In addition, the review discusses how mycolactone is employed in the diagnosis of Buruli ulcer with emphasis on detection methods of mass spectrometry, immunological assays, RNA aptamer techniques, and fluorescent-thin layer chromatography (f-TLC) methods as diagnostic tools. We examined studies of the structure–activity relationship (SAR) of various analogues of mycolactone. The paper highlights the multiple biological consequences associated with mycolactone such as skin ulceration, host immunomodulation, and analgesia. These effects are attributed to various proposed mechanisms of actions including Wiskott–Aldrich Syndrome protein (WASP)/neural Wiskott–Aldrich Syndrome protein (N-WASP) inhibition, Sec61 translocon inhibition, angiotensin II type 2 receptor (AT2R) inhibition, and inhibition of mTOR. The possible application of novel mycolactone analogues produced based on SAR investigations as therapeutic agents for the treatment of inflammatory disorders and inflammatory pain are discussed. Additionally, their therapeutic potential as anti-viral and anti-cancer agents have also been addressed. Full article

(This article belongs to the Section Mycotoxins)

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20 pages, 2006 KiB

Open AccessArticle

Biological and Chemical Management of Aspergillus carbonarius and Ochratoxin A in Vineyards

by Maria K. Iliadi, Maria Varveri and Dimitrios I. Tsitsigiannis

Toxins 2024, 16(12), 527; https://doi.org/10.3390/toxins16120527 - 6 Dec 2024

Viewed by 610

Abstract

Ochratoxin A (OTA) is a widely distributed mycotoxin and potent carcinogen produced by several fungal genera, but mainly by Aspergillus carbonarius. Grape contamination occurs in vineyards during the period between veraison and pre-harvest, and it is the main cause of OTA’s presence [...] Read more.

Ochratoxin A (OTA) is a widely distributed mycotoxin and potent carcinogen produced by several fungal genera, but mainly by Aspergillus carbonarius. Grape contamination occurs in vineyards during the period between veraison and pre-harvest, and it is the main cause of OTA’s presence in wine. The aim of the current study was the evaluation of 6 chemical and 11 biological plant protection products (PPPs) and biocontrol agents in commercial vineyards of the two important Greek white wine varieties cv. Malagousia and cv. Savatiano. The PPPs were applied in a 4-year vineyard study as single treatments or/and in combinations as part of IPM systems. Subsequently, nine strains of Aspergillus carbonarius were investigated for their sensitivity against seven active compounds of synthetic fungicides. During the multi-year field trials, various novel management systems, including consortia of biocontrol agents, were revealed to be effective against Aspergillus sour rot and OTA production. However, expected variability was observed in the experimental results, indicating the dynamic character of biological systems and highlighting the possible inconsistency of PPPs’ efficacy in a changing environment. Furthermore, the IPM systems developed effectuated an optimized control of A. carbonarius, leading to 100% inhibition of OTA contamination, showing the importance of using both chemical and biological PPPs for disease management and prevention of fungal fungicide resistance. Finally, the majority of A. carbonarius tested strains were found to be sensitive against the pure active compounds used (fludioxonil, azoxystrobin, chlorothalonil, tebuconazole, cyprodinil, pyrimethanil and boscalid), with only a few exceptions of developed resistance towards boscalid. Full article

(This article belongs to the Special Issue Toxins: 15th Anniversary)

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Figure 1
Disease severity of Aspergillus sour rot in grape berries of cv. Malagousia variety after PPP treatment of vines in the years 2016–2019. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison post hoc test (p < 0.05). Letters above the graphs indicate differences between treatments. Full article ">Figure 2
Disease severity of Aspergillus sour rot in grape berries of cv. Savatiano variety after PPP treatment of vines in the years 2016–2019. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison post hoc test (p < 0.05). Letters above the graphs indicate differences between treatments. Full article ">Figure 3
Ochratoxin A levels (μg kg−1) produced by Aspergillus carbonarius in cv. Malagousia after PPP treatment of vines in the years 2016–2019. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison post hoc test (p < 0.05). Letters above the graphs indicate differences between treatments. Full article ">Figure 4
Ochratoxin A levels (μg kg−1) produced by Aspergillus carbonarius in cv. Savatiano after PPP treatment of vines in the years 2016–2019. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison post hoc test (p < 0.05). Letters above the graphs indicate differences between treatments. Full article ">

19 pages, 6558 KiB

Open AccessArticle

Real-Time Observation of Clickable Cyanotoxin Synthesis in Bloom-Forming Cyanobacteria Microcystis aeruginosa and Planktothrix agardhii

by Rainer Kurmayer and Rubén Morón Asensio

Toxins 2024, 16(12), 526; https://doi.org/10.3390/toxins16120526 - 5 Dec 2024

Viewed by 575

Abstract

Recently, the use of click chemistry for localization of chemically modified cyanopeptides has been introduced, i.e., taking advantage of promiscuous adenylation (A) domains in non-ribosomal peptide synthesis (NRPS), allowing for the incorporation of clickable non-natural amino acids (non-AAs) into their peptide products. In [...] Read more.

Recently, the use of click chemistry for localization of chemically modified cyanopeptides has been introduced, i.e., taking advantage of promiscuous adenylation (A) domains in non-ribosomal peptide synthesis (NRPS), allowing for the incorporation of clickable non-natural amino acids (non-AAs) into their peptide products. In this study, time-lapse experiments have been performed using pulsed feeding of three different non-AAs in order to observe the synthesis or decline of azide- or alkyne-modified microcystins (MCs) or anabaenopeptins (APs). The cyanobacteria Microcystis aeruginosa and Planktothrix agardhii were grown under maximum growth rate conditions (r = 0.35–0.6 and 0.2–0.4 (day⁻¹), respectively) in the presence of non-AAs for 12–168 h. The decline of the azide- or alkyne-modified MC or AP was observed via pulse-feeding. In general, the increase in clickable MC/AP in peptide content reached a plateau after 24–48 h and was related to growth rate, i.e., faster-growing cells also produced more clickable MC/AP. Overall, the proportion of clickable MC/AP in the intracellular fraction correlated with the proportion observed in the dissolved fraction. Conversely, the overall linear decrease in clickable MC/AP points to a rather constant decline via dilution by growth instead of a regulated or induced release in the course of the synthesis process. Full article

(This article belongs to the Special Issue Harmful Algae in a Changing World: Where Did We Come from and Where Are We Going?)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Mean (±SE) proportion of natural and clickable MC in total MC (cellular fraction, composed of four MC structural variants: DAsp-MC-YR, MC-YR, DAsp-MC-LR, MC-LR) during time-lapse experiments using pulsed feeding of non-natural amino acids (non-AAs) in order to observe the build up (a,b) or decline (c,d) of azide- or alkyne-modified MC in M. aeruginosa strain Hofbauer. Control cells were grown and processed under identical conditions but without non-AA substrate and could not show any clickable MC synthesis. Full article ">Figure 2
Mean (±SE) proportion of natural and clickable AP in total AP (cellular fraction, composed of four AP structural variants: unknown AP, AP-C, AP-B, AP-A) during time-lapse experiments using pulsed feeding of non-natural amino acids (non-AAs) in order to observe the build up (a,b) or decline (c,d) of azide- or alkyne modified AP in P. agardhii strain no371/1. Control cells were grown and processed under identical conditions but without non-AA substrate and could not show any clickable MC synthesis. Full article ">Figure 3
Relationship between growth rate (day−1) and (a–c) the clickable MC net production rate (day−1) in M. aeruginosa strain Hofbauer (calculated from ln(x + 1) MC-LR equivalents in ng/mL) and (d–f) the clickable AP net production rate (day−1) in P. agardhii strain no371/1 (calculated from ln(x + 1) AP-B equivalents in ng/mL) during time-lapse experiments using feeding of non-natural amino acids (non-AAs) in order to observe the build up of azide- or alkyne-modified MC/AP. Details of linear regression curves are as follows: (a) MC-Phe-AzR (y = −0.08 + 0.73x, R2 = 0.95, p < 0.0001), (b) MC-Prop-LysR (y = −0.47 + 1.58x, R2 = 0.95, p < 0.0001), (c) MC-Prop-TyrR (y = −0.13 + 0.86x, R2 = 0.99, p < 0.0001), (d) AP-Phe-Az (not significant, p = 0.33), (e) AP-Prop-Lys (y = 0.29 + 0.39x, R2 = 0.21, p = 0.099), (f) AP-Prop-Tyr (y = −0.28 + 1.32x, R2 = 0.65, p = 0.0005), where y is MC/AP production rate (day−1) and x is growth rate (day−1). Full article ">Figure 4
Proportion of individual clickable MC in M. aeruginosa (a–c) or clickable AP in P. agardhii (d–e) in total MC/AP (cellular fraction) during time-lapse experiments using pulsed feeding of non-natural amino acids (non-AAs) in order to observe the decline of (a) MC-Phe-AzR, (b) MC-Prop-LysR, (c) MC-Prop-TyrR in M. aeruginosa, or (d) AP-Phe-Az, (e) AP-Prop-Lys, (f) AP-Prop-Tyr in P. agardhii strain no371/1. Using growth rates, the theoretical decline of clickable MC/AP was calculated (black symbols, straight line) and compared to the observed decline (colored symbols, dotted line). Note that the scale at the y-axis is different, as production efficiency differs between non-AAs (<a href="#toxins-16-00526-f001" class="html-fig">Figure 1</a> and <a href="#toxins-16-00526-f002" class="html-fig">Figure 2</a>). Full article ">Figure 5
Workflow of time-lapse experiments using pulsed feeding of non-AAs for real-time observation of clickable MC/AP synthesis in bloom-forming cyanobacteria (the workflow was the same for both M. aeruginosa and P. agardhii): (a) time-lapse build up experiments; (b) time-lapse decline experiments. Created with BioRender.com. Note that the labeling of clickable peptides via chemo-selective reaction with fluorophore and high-resolution microscopy and flow-cytometry analysis using Alexa Fluor488 will be reported in a follow-up article. Full article ">Figure 6
Chemical structures of non-AA molecules used for clickable microcystin (MC) synthesis in M. aeruginosa and for clickable anabaenopeptin (AP) synthesis in P. agardhii: (a) 4-Azido-L-phenylalanine (Phe-Az, MW 206.20 g/mol), (b) N-Propargyl-L-Lysine (Prop-Lys, MW 228.25 g/mol), (c) O-Propargyl-L-tyrosine (Prop-Tyr, MW 219.24 g/mol). Full article ">

13 pages, 2371 KiB

Open AccessArticle

Epsilon Toxin from Clostridium perfringens Induces the Generation of Extracellular Vesicles in HeLa Cells Overexpressing Myelin and Lymphocyte Protein

by Jonatan Dorca-Arévalo, Antonio Santana-Ruiz, Benjamín Torrejón-Escribano, Mireia Martín-Satué and Juan Blasi

Toxins 2024, 16(12), 525; https://doi.org/10.3390/toxins16120525 - 4 Dec 2024

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Abstract

Epsilon toxin (ETX) from Clostridium perfringens is a pore-forming toxin (PFT) that crosses the blood–brain barrier and binds to myelin structures. In in vitro assays, ETX causes oligodendrocyte impairment, subsequently leading to demyelination. In fact, ETX has been associated with triggering multiple sclerosis. [...] Read more.

Epsilon toxin (ETX) from Clostridium perfringens is a pore-forming toxin (PFT) that crosses the blood–brain barrier and binds to myelin structures. In in vitro assays, ETX causes oligodendrocyte impairment, subsequently leading to demyelination. In fact, ETX has been associated with triggering multiple sclerosis. Myelin and lymphocyte protein (MAL) is widely considered to be the receptor for ETX as its presence is crucial for the effects of ETX on the plasma membrane of host cells that involve pore formation, resulting in cell death. To overcome the pores formed by PFTs, some host cells produce extracellular vesicles (EVs) to reduce the amount of pores inserted into the plasma membrane. The formation of EVs has not been studied for ETX in host cells. Here, we generated a highly sensitive clone from HeLa cells overexpressing the MAL-GFP protein in the plasma membrane. We observed that ETX induces the formation of EVs. Moreover, the MAL protein and ETX oligomers are found in these EVs, which are a very useful tool to decipher and study the mode of action of ETX and characterize the mechanisms involved in the binding of ETX to its receptor. Full article

(This article belongs to the Special Issue Toxin-Host Interaction of Clostridium Toxins)

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13 pages, 6199 KiB

Open AccessArticle

Bicistronic Vector Expression of Recombinant Jararhagin-C and Its Effects on Endothelial Cells

by Karla Fernanda Ferraz, Lhiri Hanna De Lucca Caetano, Daniele Pereira Orefice, Paula Andreia Lucas Calabria, Maisa Splendore Della-Casa, Luciana Aparecida Freitas-de-Sousa, Emidio Beraldo-Neto, Sabri Saeed Sanabani, Geraldo Santana Magalhães and Patricia Bianca Clissa

Toxins 2024, 16(12), 524; https://doi.org/10.3390/toxins16120524 - 3 Dec 2024

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Abstract

Jararhagin-C (JarC) is a protein from the venom of Bothrops jararaca consisting of disintegrin-like and cysteine-rich domains. JarC shows a modulating effect on angiogenesis and remodeling of extracellular matrix constituents, improving wound healing in a mouse experimental model. JarC is purified from crude [...] Read more.

Jararhagin-C (JarC) is a protein from the venom of Bothrops jararaca consisting of disintegrin-like and cysteine-rich domains. JarC shows a modulating effect on angiogenesis and remodeling of extracellular matrix constituents, improving wound healing in a mouse experimental model. JarC is purified from crude venom, and the yield is less than 1%. The aim of this work was to obtain the recombinant form of JarC and to test its biological activity. For this purpose, the bicistronic vector pSUMOUlp1 was used. This vector allowed the expression of the recombinant toxin JarC (rJarC) in fusion with the small ubiquitin-related modifier (SUMO) as well as the SUMO protease Ulp1. After expression, this protease was able to efficiently remove SUMO from rJarC inside the bacteria. rJarC free from SUMO was purified at the expected molecular mass and recognized by polyclonal anti-jararhagin antibodies. In terms of biological activity, both the native and recombinant forms showed no toxicity to the HUVEC cell line CRL1730 and were effective in modulating cell migration activity in the experimental in vitro model. These results demonstrate the successful production of rJarC and the preservation of its biological activity, which may facilitate further investigations into the therapeutic potential of this snake venom-derived protein. Full article

(This article belongs to the Special Issue Animals Venom in Drug Discovery: A Valuable Therapeutic Tool)

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Figure 1

Figure 1
(A) 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel showing expression and purification of the recombinant JarC overexpressed in E. coli BL21 Star™ (DE3) at 37 °C. Protein was visualized on a 12.5% SDS/polyacrylamide gel under reducing conditions and stained with Coomassie blue R (Sigma-Aldrich, St. Louis, MO, USA). (B) Chromatogram of rJarC purification by Immobilized Metal Chelate Affinity Chromatography (IMAC). (C) For the analysis of rJarC expression by Western blot, proteins were separated by 12% SDS-PAGE, transferred onto nitrocellulose membrane, and stained with Ponceau red dye. (D) The nitrocellulose membrane was treated with an anti-His antibody for detection. For figures (A,C,D): The numbers on the left indicate the molecular mass markers (M). Lanes 1 and 2 show the extracts from BL21 Star™ (DE3) cells before and after induction with 1 mM isopropyl-β-D-thiogalactoside (IPTG), respectively; lane 3 shows the recombinant JarC purified by IMAC. Full article ">Figure 2
Alignment of the recombinant JarC peptides with the native JarC sequence. The predicted amino acid sequence of recombinant JarC was compared with the native JarC sequence (AAB30855.1) from the venom of Bothrops jararaca. The sequences were aligned with ClustalW (<a href="http://web.expasy.org" target="_blank">web.expasy.org</a>), with identical residues to the first sequence highlighted in red. Full article ">Figure 3
Dot blotting of native (JarC and Jararhagin) and recombinant proteins (rJarC) recognized by a polyclonal anti-jararhagin or anti-histidine antibody under reduced (10 µM Dithiothreitol—DTT) and non-reduced conditions. Full article ">Figure 4
Viability of HUVECs treated with native JarC and rJarC. Cell viability was determined using the MTT assay. HUVECs were not treated (RPMI) or treated with native JarC; rJarC; or rGFP at concentrations of 1 µM or Triton X 100 1% for 24 h. Full article ">Figure 5
Cell migration induced by JarC and rJarC in vitro (wound healing assay): HUVEC cells CRL1730 were cultured in a 24-well plate coated with collagen or without collagen (no collagen) until they reached approximately 100% confluence, and then a line was drawn down the center of the wells with a 200 µL tip. The cells in culture were treated with native JarC, rJarC, or rGFP at concentrations of 0.01 µM, 0.1 µM, and 1 µM. After 24 h, the culture medium plus the proteins were removed and the cells were fixed and stained with the Protocol Hema 3 staining kit. The plates were examined under a microscope and images were captured using a CCD camera (Leica Microsystems, DFC 310 FX, Mannheim, Germany) coupled to an inverted microscope (Leica Microsystems, DMIL LED, Mannheim, Germany). Full article ">

11 pages, 506 KiB

Open AccessArticle

The Association Between Serum Trimethylamine N-Oxide and Arterial Stiffness in Chronic Peritoneal Dialysis Patients: A Cross-Sectional Study

by Po-Yu Huang, Yu-Li Lin, Yi-Hsin Chen, Szu-Chun Hung, Hung-Hsiang Liou, Jen-Pi Tsai and Bang-Gee Hsu

Toxins 2024, 16(12), 523; https://doi.org/10.3390/toxins16120523 - 3 Dec 2024

Viewed by 546

Abstract

Trimethylamine N-oxide (TMAO), a gut microbiome-derived metabolite, participates in the atherogenesis and vascular stiffening that is closely linked with cardiovascular (CV) complications and related deaths in individuals with kidney failure undergoing peritoneal dialysis (PD) therapy. In these patients, arterial stiffness (AS) is [...] Read more.

Trimethylamine N-oxide (TMAO), a gut microbiome-derived metabolite, participates in the atherogenesis and vascular stiffening that is closely linked with cardiovascular (CV) complications and related deaths in individuals with kidney failure undergoing peritoneal dialysis (PD) therapy. In these patients, arterial stiffness (AS) is also an indicator of adverse CV outcomes. This study assessed the correlation between serum TMAO concentration quantified with high-performance liquid chromatography and mass spectrometry and central AS measured by carotid–femoral pulse wave velocity (cfPWV) in patients with chronic PD. Of the 160 participants included, 23.8% had a cfPWV of ≥10 m/s, which fulfilled the AS criteria. Multivariable logistic regression analysis revealed that TMAO, age, and waist circumference were positively associated with AS. Multivariable stepwise linear regression showed that underlying diabetes, advanced age, waist circumference, systolic blood pressure, and logarithmic-transformed TMAO were independently correlated with cfPWV. The area under the receiver operating characteristic curve for TMAO in differentiating AS from non-AS was 0.737. In conclusion, serum TMAO level was significantly independently correlated with central AS among participants undergoing PD for end-stage kidney failure. Full article

(This article belongs to the Section Uremic Toxins)

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