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Toxins
Volume 16
Issue 8
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Toxins, Volume 16, Issue 8 (August 2024) – 47 articles

Cover Story (view full-size image): Conotoxins are powerful neuroactive components discovered in the venom of the marine gastropods of the genus Conus and present in many other conoidean taxa. From the transcriptome of the salivary glands and venom duct of Raphitoma purpurea, >100 putative venom components and 20 novel toxin families were retrieved. Among them, some salivary peptides showed high resemblance to cone snail toxins and toxin chaperones. Conversely, the peptides expressed in the venom duct had little similarity with known toxins and may represent bioactive substances new to science. These findings suggest that some of the earliest adaptations for venom production in conoideans may actually concern their salivary secretions. View this paper
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17 pages, 1616 KiB  
Systematic Review
Resistance of Transgenic Maize Cultivars to Mycotoxin Production—Systematic Review and Meta-Analysis
by Ana Silvia de Lara Pires Batista Gomes, Saulo Henrique Weber and Fernando Bittencourt Luciano
Toxins 2024, 16(8), 373; https://doi.org/10.3390/toxins16080373 - 22 Aug 2024
Viewed by 633
Abstract
Approximately 25% of cereal grains present with contamination caused by fungi and the presence of mycotoxins that may cause severe adverse effects when consumed. Maize has been genetically engineered to present different traits, such as fungal or insect resistance and herbicide tolerance. This [...] Read more.
Approximately 25% of cereal grains present with contamination caused by fungi and the presence of mycotoxins that may cause severe adverse effects when consumed. Maize has been genetically engineered to present different traits, such as fungal or insect resistance and herbicide tolerance. This systematic review compared the observable quantities, via meta-analysis, of four mycotoxins (aflatoxins—AFL, fumonisins—FUM, deoxynivalenol—DON, zearalenone—ZEA) between genetically modified (GM) and conventional maize kernels. This study was conducted following the PRISMA guidelines, with searches performed using PubMed, Web of Science, Scopus, Google Scholar, and CAPES journals databases. Analyses were conducted using RevMan v.5.4 software. Transgenic maize showed a 58% reduction in total mycotoxins (p < 0.001) compared to conventional maize. FUM were the most impacted, with a 59% reduction (p < 0.001) in GM maize. AFL and ZEA levels were also lower in GM maize by 49% (p = 0.02) and 51% (p < 0.001), respectively. On the other hand, DON levels increased by 6% (p < 0.001) in GM maize compared to conventional maize. However, results for ZEA and DON were inconclusive due to the limited research and sample sizes. We conclude that transgenic maize reduces total mycotoxins by over 50%, primarily fumonisin and aflatoxin. Most studies presented maize varieties that were resistant to insects or herbicides, not fungal pathogens, showing a positive collateral effect of these genetic alterations. Therefore, transgenic maize appears to be a safer product for animal and human consumption from a toxicological point of view. Further studies with larger sample sizes are needed to confirm our findings for ZEA and DON in transgenic maize. Full article
(This article belongs to the Special Issue Effect of Mycotoxins on Crops and Their Prevention)
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<p>Number of articles evaluating mycotoxin concentration in maize. Total number of articles per mycotoxin is presented in green. Intersections represent the number of studies that shows the concomitant evaluation of mycotoxins. AFL—aflatoxins. DON—deoxynivalenol. FUM—fumonisins. ZEA—zearalenone.</p> Full article ">Figure 2
<p>Methods of mycotoxin quantification used in the selected articles. Numbers presented in the oval/circular elements represent the number of studies that used determined method. Intersections represent the number of studies that used two or more methods to analyze mycotoxins. The figure was created using Lucidchart<sup>©</sup> software (online version <a href="http://www.lucidchart.com" target="_blank">www.lucidchart.com</a> (accessed on 14 September 2022)). DAD/UV—diode array detector/ultraviolet. ECD—electron-capture dissociation. ELISA—enzyme-linked immunosorbent assay. FLD—fluorescence detection; MS/MS—tandem mass spectrometry. ROSA—rapid one-step assay.</p> Full article ">Figure 3
<p>Flow diagram of study selection based on PRISMA [<a href="#B82-toxins-16-00373" class="html-bibr">82</a>].</p> Full article ">
14 pages, 1712 KiB  
Article
A Multi-Year Study of Mycotoxin Co-Occurrence in Wheat and Corn Grown in Ontario, Canada
by Megan J. Kelman, J. David Miller, Justin B. Renaud, Daria Baskova and Mark W. Sumarah
Toxins 2024, 16(8), 372; https://doi.org/10.3390/toxins16080372 - 22 Aug 2024
Viewed by 594
Abstract
Mycotoxin emergence and co-occurrence trends in Canadian grains are dynamic and evolving in response to changing weather patterns within each growing season. The mycotoxins deoxynivalenol and zearalenone are the dominant mycotoxins detected in grains grown in Eastern Canada. Two potential emerging mycotoxins of [...] Read more.
Mycotoxin emergence and co-occurrence trends in Canadian grains are dynamic and evolving in response to changing weather patterns within each growing season. The mycotoxins deoxynivalenol and zearalenone are the dominant mycotoxins detected in grains grown in Eastern Canada. Two potential emerging mycotoxins of concern are sterigmatocystin, produced by Aspergillus versicolor, and diacetoxyscirpenol, a type A trichothecene produced by a number of Fusarium species. In response to a call from the 83rd Joint Expert Committee on Food Additives and Contaminants, we conducted a comprehensive survey of samples from cereal production areas in Ontario, Canada. Some 159 wheat and 160 corn samples were collected from farms over a three-year period. Samples were extracted and analyzed by LC-MS/MS for 33 mycotoxins and secondary metabolites. Ergosterol was analyzed as an estimate of the overall fungal biomass in the samples. In wheat, the ratio of DON to its glucoside, deoxynivalenol-3-glucoside (DON-3G), exhibited high variability, likely attributable to differences among cultivars. In corn, the ratio was more consistent across the samples. Sterigmatocystin was detected in some wheat that had higher concentrations of ergosterol. Diacetoxyscirpenol was not detected in either corn or wheat over the three years, demonstrating a low risk to Ontario grain. Overall, there was some change to the mycotoxin profiles over the three years for wheat and corn. Ongoing surveys are required to reassess trends and ensure the safety of the food value chain, especially for emerging mycotoxins. Full article
(This article belongs to the Section Mycotoxins)
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<p>Concentration (µg/g) ratios of DON-3G:DON in wheat and corn over the 2015, 2016, and 2017 sampling years.</p> Full article ">Figure 2
<p>Number and distribution of mycotoxins (blue) and co-occurring mycotoxins (grey) in wheat samples between 2015 and 2017.</p> Full article ">Figure 3
<p>Ergosterol (µg/g) in wheat samples between 2015 and 2017. Outlier values (°) are shown as points outside of the maximum or minimum whisker of the dataset.</p> Full article ">Figure 4
<p>Number and distribution of mycotoxins (blue) and co-occurring mycotoxins (grey) in corn samples between 2015 and 2017.</p> Full article ">Figure 5
<p>Ergosterol (µg/g) in corn samples between 2015 and 2017. Outlier values (°) are shown as points outside of the maximum or minimum whisker of the dataset.</p> Full article ">
12 pages, 618 KiB  
Article
Delphi Analysis: Optimizing Anatomy Teaching and Ultrasound Training for Botulinum Neurotoxin Type A Injection in Spasticity and Dystonia
by Kimberly Heckert, Bo Biering-Sørensen, Tobias Bäumer, Omar Khan, Fernando Pagan, Mitchell Paulin, Todd Stitik, Monica Verduzco-Gutierrez and Rajiv Reebye
Toxins 2024, 16(8), 371; https://doi.org/10.3390/toxins16080371 - 21 Aug 2024
Viewed by 830
Abstract
Our objective was to provide expert consensus on best practices for anatomy teaching and training on ultrasound-guided botulinum neurotoxin type A (BoNT-A) injection for specialists involved in treating spasticity and dystonia. Nine experts (three neurologists; six physical medicine and rehabilitation physicians) participated in [...] Read more.
Our objective was to provide expert consensus on best practices for anatomy teaching and training on ultrasound-guided botulinum neurotoxin type A (BoNT-A) injection for specialists involved in treating spasticity and dystonia. Nine experts (three neurologists; six physical medicine and rehabilitation physicians) participated in a three-round modified Delphi process. Over three rounds, experts reached consensus on 15 of 16 statements describing best practices for anatomy and BoNT-A injection training. They unanimously agreed that knowledge of the target audience, including their needs and current competency, is crucial when designing training programs. Experts also agreed that alignment between instructors is essential to ensure consistency of approach over time and between regions, and that training programs should be simple, adaptable, and “hands-on” to enhance engagement and learning. Consensus was also reached for several other key areas of training program development. The best-practice principles identified by expert consensus could aid in the development of effective, standardized programs for anatomy teaching and BoNT-A injection training for the purposes of treating spasticity and dystonia. This will enhance the exchange of knowledge, skills, and educational approaches between global experts, allowing more specialists to treat important movement disorders and ultimately improving patient outcomes. Full article
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<p>Example goals for a simple curriculum design <sup>1</sup> for anatomy and BoNT-A injection training courses. <sup>1</sup> Based on the Scandinavian Diploma Education in Dystonia and Spasticity Treatment (SKANDYSPAS) training program [<a href="#B21-toxins-16-00371" class="html-bibr">21</a>].</p> Full article ">Figure 2
<p>Modified Delphi process.</p> Full article ">
19 pages, 3710 KiB  
Article
Nanofractionation Analytics for Comparing MALDI-MS and ESI-MS Data of Viperidae Snake Venom Toxins
by Haifeng Xu, Jesse Mastenbroek, Natascha T. B. Krikke, Susan El-Asal, Rama Mutlaq, Nicholas R. Casewell, Julien Slagboom and Jeroen Kool
Toxins 2024, 16(8), 370; https://doi.org/10.3390/toxins16080370 - 21 Aug 2024
Viewed by 752
Abstract
Worldwide, it is estimated that there are 1.8 to 2.7 million cases of envenoming caused by snakebites. Snake venom is a complex mixture of protein toxins, lipids, small molecules, and salts, with the proteins typically responsible for causing pathology in snakebite victims. For [...] Read more.
Worldwide, it is estimated that there are 1.8 to 2.7 million cases of envenoming caused by snakebites. Snake venom is a complex mixture of protein toxins, lipids, small molecules, and salts, with the proteins typically responsible for causing pathology in snakebite victims. For their chemical characterization and identification, analytical methods are required. Reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (RP-LC-ESI-MS) is a widely used technique due to its ease of use, sensitivity, and ability to be directly coupled after LC separation. This method allows for the efficient separation of complex mixtures and sensitive detection of analytes. On the other hand, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is also sometimes used, and though it typically requires additional sample preparation steps, it offers desirable suitability for the analysis of larger biomolecules. In this study, seven medically important viperid snake venoms were separated into their respective venom toxins and measured by ESI-MS. In parallel, using nanofractionation analytics, post-column high-resolution fractionation was used to collect the eluting toxins for further processing for MALDI-MS analysis. Our comparative results showed that the deconvoluted snake venom toxin masses were observed with good sensitivity from both ESI-MS and MALDI-MS approaches and presented overlap in the toxin masses recovered (between 25% and 57%, depending on the venom analyzed). The mass range of the toxins detected in high abundance was between 4 and 28 kDa. In total, 39 masses were found in both the ESI-MS and/or MALDI-MS analyses, with most being between 5 and 9 kDa (46%), 13 and 15 kDa (38%), and 24 and 28 kDa (13%) in size. Next to the post-column MS analyses, additional coagulation bioassaying was performed to demonstrate the parallel post-column assessment of venom activity in the workflow. Most nanofractionated venoms exhibited anticoagulant activity, with three venoms additionally exhibiting toxins with clear procoagulant activity (Bothrops asper, Crotalus atrox, and Daboia russelii) observed post-column. The results of this study highlight the complementarity of ESI-MS and MALDI-MS approaches for characterizing snake venom toxins and provide a complementary overview of defined toxin masses found in a diversity of viper snake venoms. Full article
(This article belongs to the Special Issue From Animal Venoms to Solutions: In Honor of Professor Lourival D. Possani on the Occasion of His 85th Birthday)
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<p>MALDI-MS spectrum obtained for a nanofractionated toxin fraction. (<b>A</b>) shows a typical MALDI-MS spectrum obtained for nanofractionated toxins of <span class="html-italic">D. russelii</span> venom at the retention time fraction of 20.0 min. The toxin ions with the mass of 13,644 Da presented themselves as singly (M + H)<sup>+</sup> and doubly charged (M + 2H)<sup>2+</sup> ions (which were used for toxin mass deconvolution) with <span class="html-italic">m</span>/<span class="html-italic">z</span> values of 13,644 and 6822 based on manual deconvolution. (<b>B</b>) shows zoomed-in toxin-ion peaks with a <span class="html-italic">m</span>/<span class="html-italic">z</span> of 40,859.</p> Full article ">Figure 2
<p>Integrated <span class="html-italic">Bitis arietans</span> venom results: superimposed results of LC-UV data, LC-(ESI)-MS data, and nanofractionation MALDI-MS data plotted chromatographically and nanofractionation coagulation bioassay data plotted chromatographically. (<b>A</b>) LC-UV trace of separated snake venom at 220 nm (orange) and 254 nm (blue). (<b>B</b>) Coagulation bioactivity chromatograms representing anticoagulation (upper trace) and procoagulation activity (lower trace). (<b>C</b>) Chromatographically plotted MALDI-MS data of the identified toxins in the wells with nanofractionated toxins. For each toxin identified in different wells, the measured intensity from the MALDI data was plotted on the y-axis versus the retention time of fractionation on the <span class="html-italic">x</span>-axis. As all toxins were eluted over a series of subsequent wells, so-called MALDI-MS chromatograms of each toxin were the result. (<b>D</b>) Extracted Ion Currents (EICs) from the LC-ESI-MS data.</p> Full article ">Figure 3
<p>Integrated <span class="html-italic">Bothrops jararaca</span> venom results: superimposed results of LC-UV data, LC-(ESI)-MS data, and nanofractionation MALDI-MS data plotted chromatographically and nanofractionation coagulation bioassay data plotted chromatographically. (<b>A</b>) LC-UV trace of separated snake venom at 220 nm (orange) and 254 nm (blue). (<b>B</b>) Coagulation bioactivity chromatograms representing anticoagulation (upper trace) and procoagulation activity (lower trace). (<b>C</b>) Chromatographically plotted MALDI-MS data of the identified toxins in the wells with nanofractionated toxins. For each toxin identified in different wells, the measured intensity from the MALDI data was plotted on the y-axis versus the retention time of fractionation on the <span class="html-italic">x</span>-axis. As all toxins were eluted over a series of subsequent wells, so-called MALDI-MS chromatograms of each toxin were the result. (<b>D</b>) Extracted Ion Currents (EICs) from the LC-ESI-MS data.</p> Full article ">Figure 4
<p>Integrated <span class="html-italic">Bothrops asper</span> venom results: superimposed results of LC-UV data, LC-(ESI)-MS data, and nanofractionation MALDI-MS data plotted chromatographically and nanofractionation coagulation bioassay data plotted chromatographically. (<b>A</b>) LC-UV trace of separated snake venom at 220 nm (orange) and 254 nm (blue). (<b>B</b>) Coagulation bioactivity chromatograms representing anticoagulation (upper trace) and procoagulation activity (lower trace). (<b>C</b>) Chromatographically plotted MALDI-MS data of the identified toxins in the wells with nanofractionated toxins. For each toxin identified in different wells, the measured intensity from the MALDI data was plotted on the y-axis versus the retention time of fractionation on the <span class="html-italic">x</span>-axis. As all toxins were eluted over a series of subsequent wells, so-called MALDI-MS chromatograms of each toxin were the result. (<b>D</b>) Extracted Ion Currents (EICs) from the LC-ESI-MS data.</p> Full article ">Figure 5
<p>Integrated <span class="html-italic">Crotalus atrox</span> venom results: superimposed results of LC-UV data, LC-(ESI)-MS data, and nanofractionation MALDI-MS data plotted chromatographically and nanofractionation coagulation bioassay data plotted chromatographically. (<b>A</b>) LC-UV trace of separated snake venom at 220 nm (orange) and 254 nm (blue). (<b>B</b>) Coagulation bioactivity chromatograms representing anticoagulation (upper trace) and procoagulation activity (lower trace). (<b>C</b>) Chromatographically plotted MALDI-MS data of the identified toxins in the wells with nanofractionated toxins. For each toxin identified in different wells, the measured intensity from the MALDI data was plotted on the y-axis versus the retention time of fractionation on the <span class="html-italic">x</span>-axis. As all toxins were eluted over a series of subsequent wells, so-called MALDI-MS chromatograms of each toxin were the result. (<b>D</b>) Extracted Ion Currents (EICs) from the LC-ESI-MS data.</p> Full article ">Figure 6
<p>Integrated <span class="html-italic">Daboia russelii</span> venom results: superimposed results of LC-UV data, LC-(ESI)-MS data, and nanofractionation MALDI-MS data plotted chromatographically and nanofractionation coagulation bioassay data plotted chromatographically. (<b>A</b>) LC-UV trace of separated snake venom at 220 nm (orange) and 254 nm (blue). (<b>B</b>) Coagulation bioactivity chromatograms representing anticoagulation (upper trace) and procoagulation activity (lower trace). (<b>C</b>) Chromatographically plotted MALDI-MS data of the identified toxins in the wells with nanofractionated toxins. For each toxin identified in different wells, the measured intensity from the MALDI data was plotted on the y-axis versus the retention time of fractionation on the <span class="html-italic">x</span>-axis. As all toxins were eluted over a series of subsequent wells, so-called MALDI-MS chromatograms of each toxin were the result. (<b>D</b>) Extracted Ion Currents (EICs) from the LC-ESI-MS data.</p> Full article ">Figure 7
<p>Integrated <span class="html-italic">Echis carinatus</span> venom results: superimposed results of LC-UV, LC-(ESI)-MS, and nanofractionated MALDI-MS data plotted chromatographically and nanofractionated coagulation bioassay data plotted chromatographically. (<b>A</b>) LC-UV trace of separated snake venom at 220 nm (orange) and 254 nm (blue). (<b>B</b>) Coagulation bioactivity chromatograms representing anticoagulation (upper trace) and procoagulation activity (lower trace). (<b>C</b>) Chromatographically plotted MALDI-MS data of the identified toxins in the wells with nanofractionated toxins. For each toxin identified in different wells, the measured intensity from the MALDI data was plotted on the y-axis versus the retention time of fractionation on the <span class="html-italic">x</span>-axis. As all toxins were eluted over a series of subsequent wells, so-called MALDI-MS chromatograms of each toxin were the result. (<b>D</b>) Extracted Ion Currents (EICs) from the LC-ESI-MS data.</p> Full article ">Figure 8
<p>Integrated <span class="html-italic">Echis ocellatus</span> venom results: superimposed results of LC-UV, LC-(ESI)-MS, and nanofractionated MALDI-MS data plotted chromatographically and nanofractionated coagulation bioassay data plotted chromatographically. (<b>A</b>) LC-UV trace of separated snake venom at 220 nm (orange) and 254 nm (blue). (<b>B</b>) Coagulation bioactivity chromatograms representing anticoagulation (upper trace) and procoagulation activity (lower trace). (<b>C</b>) Chromatographically plotted MALDI-MS data of the identified toxins in the wells with nanofractionated toxins. For each toxin identified in different wells, the measured intensity from the MALDI data was plotted on the y-axis versus the retention time of fractionation on the <span class="html-italic">x</span>-axis. As all toxins were eluted over a series of subsequent wells, so-called MALDI-MS chromatograms of each toxin were the result. (<b>D</b>) Extracted Ion Currents (EICs) from the LC-ESI-MS data.</p> Full article ">
12 pages, 1411 KiB  
Article
Scavenger Receptor C1 Mediates Toxicity of Binary Toxin from Lysinibacillus sphaericus to Ag55 Cells
by Qi Zhang, Gang Hua, Laramie Smith and Michael J. Adang
Toxins 2024, 16(8), 369; https://doi.org/10.3390/toxins16080369 - 21 Aug 2024
Viewed by 558
Abstract
Lysinibacillus sphaericus harboring Binary (BinA and BinB) toxins is highly toxic against Anopheles and Culex mosquito larvae. The Anopheles Ag55 cell line is a suitable model for investigating the mode of Bin toxin action. Based on the low-levels of α-glycosidase Agm3 mRNA in [...] Read more.
Lysinibacillus sphaericus harboring Binary (BinA and BinB) toxins is highly toxic against Anopheles and Culex mosquito larvae. The Anopheles Ag55 cell line is a suitable model for investigating the mode of Bin toxin action. Based on the low-levels of α-glycosidase Agm3 mRNA in Ag55 cells and the absence of detectable Agm3 proteins, we hypothesized that a scavenger receptor could be mediating Bin cytotoxicity. Preliminary RNA interference knockdown of the expressed scavenger receptors, combined with Bin cytotoxicity assays, was conducted. The scavenger Receptor C1 (SCRC1) became the focus of this study, as a putative receptor for Bin toxins in Ag55 cells, and SCRBQ2 was selected as a negative control. Open reading frames encoding SCRC1 and SCRBQ2 were cloned and expressed in vitro, and polyclonal antibodies were prepared for immunological analyses. The RNAi silencing of SCRC1 and SCRBQ2 resulted in the successful knockdown of both SCRC1 and SCRBQ2 transcripts and protein levels. The cytolytic toxicity of Bin against Ag55 cells was severely reduced after the SCRC1-RNAi treatment. The phagocytic receptor protein SCRC1 mediates endocytosis of the Bin toxin into Ag55 cells, thereby facilitating its internal cytological activity. The results support a mechanism of the Bin toxin entering Ag55 cells, possibly via SCRC1-mediated endocytosis, and encourage investigations into how Bin is transferred from its bound form on the midgut epithelial cells into the epithelial endocytic system. Full article
(This article belongs to the Special Issue Entomopathogenic Bacteria and Toxin: Utilization or Prevention?)
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<p>Knockdown of SCRC1 and SCRBQ2 expression in Ag55 cells. The dsRNAs, dseGFP, dsSCRC1 and dsSCRBQ2 were synthesized as described in <a href="#sec4-toxins-16-00369" class="html-sec">Section 4</a>. Relative amount of SCRC1 and SCRBQ2 transcripts in each treatment group was compared to that of eGFP-dsRNA after normalization to the expression of AgRPS3. Statistically different values are designated by different letters, whereas values statistically the same as eGFP controls are designated ns.</p> Full article ">Figure 2
<p>Western blot analysis of SCRC1 and SCRBQ2 produced in <span class="html-italic">E. coli</span> and in Ag55 cells after RNAi treatments. (<b>A</b>,<b>B</b>) Diagrams of SCRC1 and SCRBQ2 proteins and regions expressed for use as antigens. (<b>C</b>,<b>D</b>) Partially purified SCRC1 and SCRBQ2 proteins produced in <span class="html-italic">E. coli</span>, stained with Coomassie brilliant blue (CBB), and detected by Western blot as described in <a href="#sec4-toxins-16-00369" class="html-sec">Section 4</a>. (<b>E</b>) SCRC1 and SCRBQ2 proteins in Ag55 cells stained with CBB and detected on Western blots after dsRNA interference as described in <a href="#sec4-toxins-16-00369" class="html-sec">Section 4</a>.</p> Full article ">Figure 3
<p>Toxicity of Bin toxin on Ag55 cells treated with dsRNAs. After dsRNA treatments for three days, Ag55 cells from the control (buffer or eGFPdsRNA) and experimental groups (SCRC1-dsRNA and SCRBQ2-dsRNA) were bioassayed. (<b>A</b>) Coomassie brilliant blue (CBB) stained BinA and BinB subunits of Binary toxin. (<b>B</b>) Bioassays were performed with 25 nM of Bin toxin and cell mortality was recorded on day 2. Each data point represents the mean ± standard error of the results from bioassay. A significant difference (chi-square analysis; <span class="html-italic">p</span> &lt; 0.001) was obtained between cell mortality with ds-eGFP and ds-SCRC1 (a vs. b). The significances (<span class="html-italic">p</span> &lt; 0.001) are also presented between ds-eGFP (or ds-SCRBQ2) and ds-SCRC1 + eGFP (or ds-eGFP and ds-SCRC1 + BQ2) (a vs. c).</p> Full article ">Figure 4
<p>Bin toxicity to Ag55 cells treated with or without SCRC1-dsRNA. Ag55 cells were exposed to Bin toxin at a final concentration of 0, 6.25, 12.5, or 25 nM for 48 h and cell mortalities were measured by trypan blue exclusion. Cell mortality is expressed as the % dead cells relative to total cells without Bin treatment. Error bars show standard error of the means. Letters a–g denote significant differences between compared values (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">
8 pages, 462 KiB  
Review
Bacterial Porins and Their Procoagulant Role: Implication in the Pathophysiology of Several Thrombotic Complications during Sepsis
by Carmine Siniscalchi, Alessandro Perrella, Ugo Trama, Francesca Futura Bernardi, Egidio Imbalzano, Giuseppe Camporese, Vincenzo Russo, Olga Scudiero, Tiziana Meschi and Pierpaolo Di Micco
Toxins 2024, 16(8), 368; https://doi.org/10.3390/toxins16080368 - 20 Aug 2024
Viewed by 552
Abstract
The association between sepsis and thrombotic complications is still not well known. Different mechanisms have been shown to be involved in the sepsis-induced prothrombotic state, but clinical scenarios may differ. In this review, we have summarized the role that bacterial products such as [...] Read more.
The association between sepsis and thrombotic complications is still not well known. Different mechanisms have been shown to be involved in the sepsis-induced prothrombotic state, but clinical scenarios may differ. In this review, we have summarized the role that bacterial products such as porins and toxins can have in the induction of the prothrombotic state during sepsis and the interaction that they can have with each other. Furthermore, the above-mentioned mechanisms might be involved in the pattern of the clinical presentation of thrombotic events during bacterial sepsis, which would secondarily explain the association between sepsis and venous thromboembolism, the association between sepsis and disseminated intravascular coagulation, and the association between sepsis and microangiopathic venous thromboembolism. Full article
(This article belongs to the Topic Bacterial Porins and Their Implication in Pathophysiology of Septic Complications In Vitro and In Vivo)
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<p>Bacterial infection and hypercoagulable state.</p> Full article ">
14 pages, 1904 KiB  
Article
Aflatoxin B1 Control by Various Pseudomonas Isolates
by Dóra Anna Papp, Sándor Kocsubé, Zoltán Farkas, András Szekeres, Csaba Vágvölgyi, Zsuzsanna Hamari and Mónika Varga
Toxins 2024, 16(8), 367; https://doi.org/10.3390/toxins16080367 - 20 Aug 2024
Viewed by 664
Abstract
The climate-change-coupled fungal burden in crop management and the need to reduce chemical pesticide usage highlight the importance of finding sustainable ways to control Aspergillus flavus. This study examines the effectiveness of 50 Pseudomonas isolates obtained from corn rhizospheres against A. flavus [...] Read more.
The climate-change-coupled fungal burden in crop management and the need to reduce chemical pesticide usage highlight the importance of finding sustainable ways to control Aspergillus flavus. This study examines the effectiveness of 50 Pseudomonas isolates obtained from corn rhizospheres against A. flavus in both solid and liquid co-cultures. The presence and quantity of aflatoxin B1 (AFB1) and AFB1-related compounds were determined using high-performance liquid chromatography–high resolution mass spectrometry analysis. Various enzymatic- or non-enzymatic mechanisms are proposed to interpret the decrease in AFB1 production, accompanied by the accumulation of biosynthetic intermediates (11-hydroxy-O-methylsterigmatocystin, aspertoxin, 11-hydroxyaspertoxin) or degradation products (the compounds C16H10O6, C16H14O5, C18H16O7, and C19H16O8). Our finding implies the upregulation or enhanced activity of fungal oxidoreductases and laccases in response to bacterial bioactive compound(s). Furthermore, non-enzymatic reactions resulted in the formation of additional degradation products due to acid accumulation in the fermented broth. Three isolates completely inhibited AFB1 or any AFB1-related compounds without significantly affecting fungal growth. These bacterial isolates supposedly block the entire pathway for AFB1 production in the fungus during interaction. Apart from identifying effective Pseudomonas isolates as potential biocontrol agents, this work lays the foundation for exploring new bacterial bioactive compounds. Full article
(This article belongs to the Section Mycotoxins)
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<p>Monitoring of interaction phenotypes and production of AFB1 and AFB1-related compounds in solid and liquid co-cultures of <span class="html-italic">A. flavus</span> with 50 <span class="html-italic">Pseudomonas</span> isolates. (<b>A</b>): The morphology of interaction phenotypes in solid co-cultures. Axenic control shows the morphology of <span class="html-italic">A. flavus</span> mono-culture. (<b>B</b>): Graphical presentation of AFB1 content of agar disks cut from the center of fungal colonies of solid co-cultures in % of the AFB1 content of the axenic <span class="html-italic">A. flavus</span> control. Colors indicate the type of interaction presented in <a href="#toxins-16-00367-f001" class="html-fig">Figure 1</a>A. CT on the <span class="html-italic">X</span>-axis denotes the axenic control. The numbers on the <span class="html-italic">X</span>-axis confer to the collection names of the co-cultured <span class="html-italic">Pseudomonas</span> isolates. A dashed line marks the reference value of the axenic control (100%). Raw data points of the three biological replicates (empty circles) and mean values (full diamonds) are shown. Significant differences (Student’s <span class="html-italic">t</span>-test) are marked with asterisks (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; ns: non-significant). For raw data, see <a href="#app1-toxins-16-00367" class="html-app">Table S1</a>. (<b>C</b>): Graphical presentation of the AFB1 content of the fermented broth of liquid co-cultures in % of the AFB1 content of the axenic <span class="html-italic">A. flavus</span> control. Color and figure codes are shown in the figure legend. The colors indicate the type of liquid interactions (explained in the main text). The size of the circles indicates the dry weight of the fungal biomass derived from liquid co-cultures in % of that of the axenic control. Explanation of X-axis, dashed line and significant differences is the same as in <a href="#toxins-16-00367-f001" class="html-fig">Figure 1</a>A. The raw data points of the three biological replicates (empty circles) and mean values (full diamonds) are shown. For raw data, see <a href="#app1-toxins-16-00367" class="html-app">Table S1</a>. (<b>D</b>): Heat map presentation of the appearance of AFB1-related compounds in the solid and liquid co-cultures and in the axenic control. Color codes are denoted in the legend. The detected compounds were OH-OMeSTC (11-hydroxy-O-methylsterigmatocystin), OH-ASP (11-hydroxyaspertoxin), Me-AFB2a<sub>RT=12.5</sub>, and Me-AFB2a<sub>RT=12.3</sub> (two isomers of methoxy-aflatoxin B2a), ASP (aspertoxin), AFP1 (aflatoxin P1), and AFD1 (aflatoxin D1). For raw data, see <a href="#app1-toxins-16-00367" class="html-app">Table S2</a>.</p> Full article ">Figure 2
<p>Biosynthetic pathway for aflatoxins (according to Udwary [<a href="#B37-toxins-16-00367" class="html-bibr">37</a>], and Yabe [<a href="#B38-toxins-16-00367" class="html-bibr">38</a>]) and downstream transformation reactions. Arrows and dashed arrows denote single and multiple enzyme reaction steps, respectively. Enzymes of reaction steps are indicated. Biosynthetic, enzymatic, and non-enzymatic degradation reactions are drawn with green, pink, and gray background colors, respectively. NE denotes non-enzymatic reactions (these steps are emphasized with double-line arrows). VA, versicolorin A; OMeSTC, O-methylsterigmatocystin; OH-OMeSTC, 11-hydroxy-O-methylsterigmatocystin; ASP, aspertoxin; OH-ASP, 11-hydroxyaspertoxin; DH-OH-ASP, dihydro-hydroxyaspertoxin; AFG1, AFB1, AFM1, aflatoxin G1, B1, M1.</p> Full article ">
15 pages, 2218 KiB  
Article
Real-World Observational Analysis of Clinical Characteristics and Treatment Patterns of Patients with Chronic Sialorrhea
by Michael A. Hast, Amanda M. Kong, Jenna Abdelhadi, Rohan Shah, Andrew Szendrey and Jordan Holmes
Toxins 2024, 16(8), 366; https://doi.org/10.3390/toxins16080366 - 17 Aug 2024
Viewed by 623
Abstract
Chronic sialorrhea is a condition characterized by excessive drooling, often associated with neurological and neuromuscular disorders such as Parkinson’s disease, cerebral palsy, and stroke. Despite its prevalence, it remains underdiagnosed and poorly understood, leading to a lack of comprehensive data on patient demographics, [...] Read more.
Chronic sialorrhea is a condition characterized by excessive drooling, often associated with neurological and neuromuscular disorders such as Parkinson’s disease, cerebral palsy, and stroke. Despite its prevalence, it remains underdiagnosed and poorly understood, leading to a lack of comprehensive data on patient demographics, clinical characteristics, and treatment patterns. This study aimed to help fill these existing gaps by analyzing real-world data using Optum’s de-identified Clinformatics® Data Mart Database. Patients were required to have a diagnosis indicative of sialorrhea plus evidence of sialorrhea treatment between 1/1/2007 and 5/31/2022. Two cohorts were analyzed: patients with evidence of newly diagnosed sialorrhea and associated treatment, and sialorrhea patients initiating incobotulinumtoxinA. Clinical characteristics, comorbidities, symptoms, and treatment utilization were described before and after diagnosis and incobotulinumtoxinA initiation. No formal statistical comparisons were performed. Patients were predominantly aged 65 or older, male, and non-Hispanic white. Parkinson’s disease and cerebral palsy were the most common comorbidities among adults and children, respectively. Treatment patterns suggest that anticholinergics are more commonly used than botulinum toxin therapy. The findings offer valuable information for improving diagnosis and treatment approaches and suggest a need for further research into treatment effectiveness, safety, and disease burden. Full article
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<p>(<b>A</b>,<b>B</b>) Patient attrition of overall chronic sialorrhea cohort and incobotulinumtoxinA cohort.</p> Full article ">Figure 1 Cont.
<p>(<b>A</b>,<b>B</b>) Patient attrition of overall chronic sialorrhea cohort and incobotulinumtoxinA cohort.</p> Full article ">
14 pages, 928 KiB  
Article
Treatment of Acquired Deforming Hypertonia with Botulinum Toxin in Older Population: A Retrospective Study
by Pablo Maldonado, Hugo Bessaguet, Cédric Chol, Pascal Giraux, Ludovic Lafaie, Ahmed Adham, Romain David, Thomas Celarier and Etienne Ojardias
Toxins 2024, 16(8), 365; https://doi.org/10.3390/toxins16080365 - 16 Aug 2024
Viewed by 631
Abstract
Acquired deforming hypertonia (ADH) affects the daily care of numerous nursing home residents. The aim of this study was to analyze the practice, aims, and effectiveness of botulinum toxin injections (BTxis) in the treatment of older patients with contractures, an indication for which [...] Read more.
Acquired deforming hypertonia (ADH) affects the daily care of numerous nursing home residents. The aim of this study was to analyze the practice, aims, and effectiveness of botulinum toxin injections (BTxis) in the treatment of older patients with contractures, an indication for which BTxis are still underused. Data were extracted retrospectively from medical records regarding population, contractures, and injections. A prospective analysis was conducted to evaluate treatment goals set by goal attainment scaling (GAS) at T0 and at T1, to evaluate the therapeutic effects. We also recorded the occurrence of side effects, using a telephone questionnaire. This study included 41 patients older than 70 years who had received one or more BTxis for the first time between January 2018 and December 2021. Most of the older people we included lived in an institution (66%), manifested severe dependence, and presented significant morbi-mortality (37% of the patients died in the year after the last injection). The main objectives of these injections were purely comfort, without any functional goals. The GAS scores suggested effectiveness for comfort GAS scores. No complications were recorded. This study highlights the BTxis potential to address the needs of a larger number of older patients with ADH. Full article
(This article belongs to the Special Issue Uses of Botulinum Toxin Injection in Medicine)
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<p>Survival curve of patients receiving botulinum toxin injections (BTxis) to treat acquired deforming hypertonia (ADH); (time in months elapsed between the first injection and death). Legend F1: This survival curve represents the survival of the population during the treatment follow-up period. Time 0 on the <span class="html-italic">x</span>-axis corresponds to the first BTxi for each patient (100% of the population alive, <span class="html-italic">y</span>-axis). At the end of the study period (24 months), 50% of the population is alive.</p> Full article ">Figure 2
<p>Mean GAS score for each goal (with minimum and maximum values) at the time of individual re-evaluation (T1) (aggregate data for all individuals; symbols) and percentage frequency of each goal among all the goals chosen (histograms). Legend F2: This graph shows the frequency of goals set at the start of treatment with BTxi (right <span class="html-italic">y</span>-axis) and the mean of GAS T1 scores by goal category after treatment (left <span class="html-italic">y</span>-axis) with minimum and maximum values.</p> Full article ">
13 pages, 3988 KiB  
Article
Intramuscular Botulinum Toxin as an Adjunct to Arthrocentesis with Viscosupplementation in Temporomandibular Disorders: A Proof-of-Concept Case–Control Investigation
by Luca Guarda Nardini, Daniele Manfredini, Anna Colonna, Edoardo Ferrari Cagidiaco, Marco Ferrari and Matteo Val
Toxins 2024, 16(8), 364; https://doi.org/10.3390/toxins16080364 - 16 Aug 2024
Viewed by 557
Abstract
Background: The reduction in joint load is a potential beneficial factor in managing osteoarthritis of the temporomandibular joint (TMJ). This paper aims to compare the effectiveness of the intramuscular injection of botulinum toxin (BTX-A) as an adjunct to TMJ arthrocentesis plus viscosupplementation with [...] Read more.
Background: The reduction in joint load is a potential beneficial factor in managing osteoarthritis of the temporomandibular joint (TMJ). This paper aims to compare the effectiveness of the intramuscular injection of botulinum toxin (BTX-A) as an adjunct to TMJ arthrocentesis plus viscosupplementation with arthrocentesis plus viscosupplementation alone in the management of TMJ osteoarthritis. Methods: A pilot clinical retrospective study examined TMJ osteoarthritis treatments. Patients were divided into two groups: Group A received BTX-A injections and arthrocentesis with viscosupplementation, while Group B received only arthrocentesis with viscosupplementation. The study assessed outcomes based on mouth opening (MO), pain at rest (PR), pain at mastication (PF), and masticatory efficiency (ME) at various time points (baseline (T0), 1 week (T1), 2 weeks (T2), 3 weeks (T3), and 4 weeks (T4)) up to 2 months after treatment. Results: The study included two groups, each with five patients. Group A received five weekly sessions of arthrocentesis plus viscosupplementation and a single BTX-A injection during the first arthrocentesis appointment. Group B underwent the five-session protocol of arthrocentesis plus viscosupplementation alone. MO, PF, PR, and ME improved quickly in T2 in both groups, but the improvement was of greater importance over the following weeks and lasted longer in Group A. Conclusions: Arthrocentesis with viscosupplementation associated with BTX-A was found to be more effective than arthrocentesis alone in improving clinical outcomes. This suggests that patients with TMJ osteoarthritis and myofascial pain may benefit from reduced muscle tone and joint load. Full article
(This article belongs to the Section Bacterial Toxins)
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<p>Variations in pain at rest (score according to VAS scale) in T0 (before treatment), T1, T2, T3, T4, and T5 (2 months after last arthrocentesis in T4) in Group A.</p> Full article ">Figure 2
<p>Variations in pain at rest (score according to VAS scale) in T0 (before treatment), T1, T2, T3, T4, and T5 (2 months after last arthrocentesis in T4) in Group B.</p> Full article ">Figure 3
<p>Variations in pain during chewing (score according to VAS scale) in T0 (before treatment), T1, T2, T3, T4, and T5 (2 months after last arthrocentesis in T4) in Group A.</p> Full article ">Figure 4
<p>Variations in pain during chewing (score according to VAS scale) in T0 (before treatment), T1, T2, T3, T4, and T5 (2 months after last arthrocentesis in T4) in Group B.</p> Full article ">Figure 5
<p>Mastication efficiency variations (score according to VAS scale) in T0 (before treatment), T1, T2, T3, T4, and T5 (2 months after last arthrocentesis in T4) in Group A.</p> Full article ">Figure 6
<p>Mastication efficiency variations (score according to VAS scale) in T0 (before treatment), T1, T2, T3, T4, and T5 (2 months after last arthrocentesis in T4) in Group B.</p> Full article ">Figure 7
<p>Functional limitation variations (0, absent; 1, slight; 2, moderate; 3, intense; 4, severe) in T0 (before treatment), T1, T2, T3, T4, and T5 (2 months after last arthrocentesis in T4) in Group A.</p> Full article ">Figure 8
<p>Functional limitation variations (0, absent; 1, slight; 2, moderate; 3, intense; 4, severe) in T0 (before treatment), T1, T2, T3, T4, and T5 (2 months after last arthrocentesis in T4) in Group B.</p> Full article ">Figure 9
<p>Site of injection of BTX in masseter and temporalis muscle. The dots indicate the injection sites for BTX, while the lines outline the recommended areas for injecting the masseter muscle inferiorly and the anterior portion of the temporalis muscle superiorly.</p> Full article ">
17 pages, 1701 KiB  
Article
Effect of Bioactive Ingredients on Urinary Excretion of Aflatoxin B1 and Ochratoxin A in Rats, as Measured by Liquid Chromatography with Fluorescence Detection
by Pilar Vila-Donat, Dora Sánchez, Alessandra Cimbalo, Jordi Mañes and Lara Manyes
Toxins 2024, 16(8), 363; https://doi.org/10.3390/toxins16080363 - 16 Aug 2024
Viewed by 556
Abstract
Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are highly toxic mycotoxins present in food and feed, posing serious health risks to humans and animals. This study aimed to validate an efficient and cost-effective analytical method for quantifying AFB1 and OTA in rat urine [...] Read more.
Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are highly toxic mycotoxins present in food and feed, posing serious health risks to humans and animals. This study aimed to validate an efficient and cost-effective analytical method for quantifying AFB1 and OTA in rat urine using immunoaffinity column extraction and liquid chromatography with fluorescence detection (IAC-LC-FD). Additionally, the study evaluated the effect of incorporating fermented whey and pumpkin into the feed on the urinary excretion of these mycotoxins. The limits of detection and quantification were determined to be 0.1 µg/kg and 0.3 µg/kg, respectively, for both mycotoxins in feed, and 0.2 ng/mL and 0.6 ng/mL, respectively, in urine. The method demonstrated robust recovery rates ranging from 74% to 119% for both AFB1 and OTA in both matrices. In feed samples, the levels of AFB1 and OTA ranged from 4.3 to 5.2 µg/g and from 5.4 to 8.8 µg/g, respectively. This validated method was successfully applied to analyze 116 urine samples from rats collected during the fourth week of an in vivo trial. The results indicated that the addition of fermented whey and pumpkin to the feed influenced mycotoxin excretion in urine, with variations observed based on the sex of the rats, type of mycotoxin, and exposure dosage. Full article
(This article belongs to the Special Issue Advances in Contamination, Detection and Risk Assessment of Mycotoxins)
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<p>Effects of fermented whey (FW) and pumpkin (P) on urinary aflatoxin B1 (AFB1) levels in male and female Wistar rats. (*) indicates statistically significant differences (<span class="html-italic">p</span> ≤ 0.05) in AFB1 urinary levels between experimental groups.</p> Full article ">Figure 2
<p>Effects of fermented whey (FW) and pumpkin (P) on ochratoxin A (OTA) urinary levels in male and female rats. (**) indicate statistically significant differences (<span class="html-italic">p</span> ≤ 0.01) in OTA urinary levels between experimental groups.</p> Full article ">Figure 3
<p>In vivo study scheme using metabolic cages for 24 h once per week starting from second week of exposure.</p> Full article ">Figure 4
<p>Extraction method M2 of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in urine using AflaOchra immunoaffinity columns (IACs) and liquid chromatography with fluorescence detection (LC-FLD).</p> Full article ">
13 pages, 1759 KiB  
Article
Patient Characteristics and Real-World Use of Botulinum Toxins for the Treatment of Cervical Dystonia, Blepharospasm, and Hemifacial Spasm
by Michael A. Hast, Amanda M. Kong, Shaina Desai, Soo Back, Sahar Syed and Jordan Holmes
Toxins 2024, 16(8), 362; https://doi.org/10.3390/toxins16080362 - 16 Aug 2024
Viewed by 610
Abstract
Movement disorders such as cervical dystonia, blepharospasm, and hemifacial spasm negatively impact the quality of life of people living with these conditions. Botulinum toxin (BoNT) injections are commonly used to treat these disorders. We sought to describe patient characteristics, BoNT utilization, and potential [...] Read more.
Movement disorders such as cervical dystonia, blepharospasm, and hemifacial spasm negatively impact the quality of life of people living with these conditions. Botulinum toxin (BoNT) injections are commonly used to treat these disorders. We sought to describe patient characteristics, BoNT utilization, and potential adverse events (AEs) among patients with cervical dystonia, blepharospasm, and hemifacial spasm using Optum’s de-identified Clinformatics® Data Mart Database. Patients were required to have a diagnosis of the specific condition plus evidence of treatment with BoNT between 8/1/2010 and 5/31/2022. Cervical dystonia patients were commonly females (76%) and aged 45 and older (78%); both blepharospasm and hemifacial spasm patients were commonly females (both 69%) and aged 65 and older (61% and 56%, respectively). Anticholinergics were commonly used (65–82% across cohorts), as were peripheral muscle relaxants for cervical dystonia patients specifically (31%). The median number of injections per year was 2 with the median weeks between injections being between 13 and 15. Of the AEs evaluated, dyspnea was identified frequently across all the cohorts (14–20%). The findings were similar for different BoNT formulations. More research is needed to thoroughly describe BoNT utilization, such as the doses injected, and to optimize treatment for patients with these conditions. Full article
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<p>Patient attrition for cervical dystonia, blepharospasm, and hemifacial spasm patient populations.</p> Full article ">Figure 2
<p>(<b>A</b>,<b>B</b>) Proportions of Patients Experiencing a Potential AE within 5 Months of Botulinum Toxin Administration.</p> Full article ">
21 pages, 5340 KiB  
Article
Importance of the Cysteine-Rich Domain of Snake Venom Prothrombin Activators: Insights Gained from Synthetic Neutralizing Antibodies
by Laetitia E. Misson Mindrebo, Jeffrey T. Mindrebo, Quoc Tran, Mark C. Wilkinson, Jessica M. Smith, Megan Verma, Nicholas R. Casewell, Gabriel C. Lander and Joseph G. Jardine
Toxins 2024, 16(8), 361; https://doi.org/10.3390/toxins16080361 - 15 Aug 2024
Viewed by 696
Abstract
Snake venoms are cocktails of biologically active molecules that have evolved to immobilize prey, but can also induce a severe pathology in humans that are bitten. While animal-derived polyclonal antivenoms are the primary treatment for snakebites, they often have limitations in efficacy and [...] Read more.
Snake venoms are cocktails of biologically active molecules that have evolved to immobilize prey, but can also induce a severe pathology in humans that are bitten. While animal-derived polyclonal antivenoms are the primary treatment for snakebites, they often have limitations in efficacy and can cause severe adverse side effects. Building on recent efforts to develop improved antivenoms, notably through monoclonal antibodies, requires a comprehensive understanding of venom toxins. Among these toxins, snake venom metalloproteinases (SVMPs) play a pivotal role, particularly in viper envenomation, causing tissue damage, hemorrhage and coagulation disruption. One of the current challenges in the development of neutralizing monoclonal antibodies against SVMPs is the large size of the protein and the lack of existing knowledge of neutralizing epitopes. Here, we screened a synthetic human antibody library to isolate monoclonal antibodies against an SVMP from saw-scaled viper (genus Echis) venom. Upon characterization, several antibodies were identified that effectively blocked SVMP-mediated prothrombin activation. Cryo-electron microscopy revealed the structural basis of antibody-mediated neutralization, pinpointing the non-catalytic cysteine-rich domain of SVMPs as a crucial target. These findings emphasize the importance of understanding the molecular mechanisms of SVMPs to counter their toxic effects, thus advancing the development of more effective antivenoms. Full article
(This article belongs to the Special Issue Venom Components Acting on the Hemostatic System: Structural and Mechanistic Insights)
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<p>Isolation of rEcarin neutralizing antibodies. (<b>A</b>) Sorting strategy for the selection of high-affinity rEcarin binders and cross-rEcarin/rEoMP06 binders. rEcarin was used as bait in MACS. For FACS, iterations of positive selections with rEcarin and depletions with polyspecific reagent (PSR) were used to remove nonspecific antibodies. The final AFF sort (X-AFF) was performed with rEoMP06 to select for enriched cross-reactive clones. Created with BioRender.com. (<b>B</b>) Selection of antibodies for functional assay by ELISA. The highest tested concentration (0.1 mg/mL, 0. 67 µM) is shown for each construct. Antibodies highlighted in red color were not chosen either because they did not bind to rEcarin (circles) or bound to PSR preparations (triangles). A threshold is set at Abs405 = 0.25 AU. (<b>C</b>) Identification of three rEcarin neutralizing antibodies. Reaction conditions: PBS pH 7.4, 37 °C, rEcarin 2 nM, recombinant human PT 0.2 µM, S-2238 0.5 mM, antibody 0.1 mg/mL (0.67 µM). The Abs405 after 30 min reaction time is depicted. Positive (green dot) and negative (red dot) controls were performed in the absence of antibody and in the presence of 20 mM EDTA, respectively. Three antibodies (B3, H11 and H12, black dots) show a similar inhibition to 20 mM EDTA (dotted line).</p> Full article ">Figure 2
<p>Characterization of rEcarin and rEoMP06 neutralizing antibodies. (<b>A</b>) Relative activity of rSVMP (rEcarin, left, and rEoMP06, right) in the presence of different concentrations of B3, H11 and H12. Reaction conditions: PBS pH 7.4, 37 °C, rEcarin 50 nM (<b>left</b>) or rEoMP06 50 nM (<b>right</b>), recombinant human PT 0.2 µM, S-2238 0.5 mM, antibody 0.5–500 nM. We set 100% activity as the reference rate for each SVMP in the absence of antibody. Reactions were run in biological triplicate. The errors bars represent the standard deviations of the measurements. (<b>B</b>) CDRH3 sequence alignment of B3, H11 and H12. (<b>C</b>) Epitope binning showing competition between B3, H11 and H12 for ecarin binding. Step 1: rEcarin immobilized on the Penta-His sensor, step 2: saturating antibodies (0.1 mg/mL) bind to ecarin, and step 3: competing antibodies (0.05 mg/mL) bind to ecarin to assess competition (see <a href="#sec5dot10-toxins-16-00361" class="html-sec">Section 5.10</a> for details). Six different combinations of antibodies are depicted in distinct colors across the three steps. None of the competing antibodies (step 3) bind to ecarin, which indicates that they recognize the same or overlapping epitope as the saturating antibodies (step 2). (<b>D</b>) BLI sensorgrams depicting rEcarin binding to B3, H11 and H12. The concentrations of rEcarin are indicated on the traces (31–1000 nM). Raw and fitted data are shown in black and red, respectively.</p> Full article ">Figure 3
<p>Michaelis–Menten curve of PT activation catalyzed by rEcarin. Reaction conditions: PBS pH 7.4, 37 °C, rEcarin 50 nM, recombinant human PT 0.2–3 µM, S-2238 0.5 mM. Reactions were carried out in duplicate for each data point.</p> Full article ">Figure 4
<p>H11 inhibits nEcarins from African saw scale vipers. (<b>A</b>) H11 binds to whole venoms of <span class="html-italic">E. leucogaster</span> (Mali), <span class="html-italic">E. p. leakeyi</span> (Kenya) and <span class="html-italic">E. romani</span> (Nigeria), but not <span class="html-italic">E. carinatus</span> (India). (<b>B</b>) SDS PAGE gels in reducing conditions showing the isolation of SVMP PIIIs from <span class="html-italic">E. leucogaster</span> and <span class="html-italic">E. p. leakeyi</span> after SEC. (<b>C</b>) Inhibition of nEcarins from <span class="html-italic">E. leucogaster</span> and <span class="html-italic">E. p. leakeyi</span> venoms. Reaction conditions: PBS pH 7.4, 37 °C, isolated SVMP PIII fractions 50 nM, recombinant human PT 0.2 µM, S-2238 0.5 mM, inhibitor concentrations are indicated on the graph. We set 100% activity as the reference rate for each SVMP in the absence of inhibitor. Reactions were performed in duplicate. The error bars indicate the standard deviations of the measurements.</p> Full article ">Figure 5
<p>Cryo-EM structure of H11 Fab bound to ecarin. (<b>A</b>) Overview of the sharpened cryo-EM map of H11 Fab–ecarin complex. The map is segmented and colored according to domain architecture (M domain, salmon; D domain, pink; C domain, pale cyan; H11 Fab V<sub>H</sub>, orange; H11 Fab V<sub>L</sub>, light orange). (<b>B</b>) Overview of the atomic model generated using the cryo-EM density. The coloring scheme matches the same domain colors in panel A except that the CDRH3 loop, which contains the diversity in our antibody library, is colored coral. Perspective eyes denote the viewing angle for panels (<b>C</b>–<b>F</b>) and the black box provides the viewing perspective for panel (<b>G</b>). (<b>C</b>–<b>E</b>) Interface interactions between H11 and the C domain of ecarin. (<b>F</b>) Modeled polyalanine peptide in the M domain proteolytic active site with associated cryo-EM density at a binarization threshold of 0.2. The peptide is rendered as magenta, and the M domain is salmon. (<b>G</b>) Interactions between the C domain and M domain of ecarin corresponding to the black box in panel B. All interacting residues were selected using a distance cutoff of 4 Å between the two domains.</p> Full article ">Figure 6
<p>AlphaFold 2 complex of PT–ecarin. (<b>A</b>) Overview of the highest-scored ecarin–PT complex from AlphaFold 2. Ecarin is represented as a surface and rendered with the M domain as salmon, D domain as pink and C domain as pale cyan. The Kringle 2 domain of PT (yellow) associates with the C domain (pale cyan) of ecarin in our predicted model. The loop containing the secondary ecarin cut site bound in the M domain active site is colored magenta. (<b>B</b>) Electrostatic potential surfaces of the predicted ecarin–PT AlphaFold model. Ecarin and PT present complementary charged surfaces to facilitate complex formation. (<b>C</b>) The predicted PT–ecarin model overlaid with our H11 Fab–ecarin cryo-EM structure, demonstrating significant clashes (red X) between H11 (V<sub>H</sub> orange and V<sub>L</sub> pale orange, surface representation) and PT (yellow, cartoon representation) at the C domain interface.</p> Full article ">
17 pages, 3128 KiB  
Article
Continuous and Intermittent Exposure to the Toxigenic Cyanobacterium Microcystis aeruginosa Differentially Affects the Survival and Reproduction of Daphnia curvirostris
by Fernando Martínez-Jerónimo, Lizabeth Gonzalez-Trujillo and Miriam Hernández-Zamora
Toxins 2024, 16(8), 360; https://doi.org/10.3390/toxins16080360 - 15 Aug 2024
Viewed by 682
Abstract
Anthropic eutrophication leads to water quality degradation because it may cause the development of harmful cyanobacterial blooms, affecting aquatic biota and threatening human health. Because in the natural environment zooplankters are exposed continuously or intermittently to cyanotoxins in the water or through cyanobacterial [...] Read more.
Anthropic eutrophication leads to water quality degradation because it may cause the development of harmful cyanobacterial blooms, affecting aquatic biota and threatening human health. Because in the natural environment zooplankters are exposed continuously or intermittently to cyanotoxins in the water or through cyanobacterial consumption, this study aimed to assess the effects of the toxigenic Microcystis aeruginosa VU-5 by different ways of exposure in Daphnia curvirostris. The acute toxicity produced by the cells, the aqueous crude extract of cells (ACE), and the cell-free culture medium (CFM) were determined. The effect on the survival and reproduction of D. curvirostris under continuous and intermittent exposure was determined during 26 d. The LC50 was 407,000 cells mL−1; exposure to the ACE and CFM produced mortality lower than 20%. Daphnia survivorship and reproduction were significantly reduced. Continuous exposure to Microcystis cells caused 100% mortality on the fourth day. Exposure during 4 and 24 h in 48 h cycles produced adult mortality, and reproduction decreased as the exposure time and the Microcystis concentrations increased. The higher toxicity of cells than the ACE could mean that the toxin’s absorption is higher in the digestive tract. The temporary exposure to Microcystis cells produced irreversible damage despite the recovery periods with microalgae as food. The form and the continuity in exposure to Microcystis produced adverse effects, warning about threats to the zooplankton during HCBs. Full article
(This article belongs to the Section Marine and Freshwater Toxins)
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<p>Survival of <span class="html-italic">Daphnia curvirostris</span> in alternating 24 h exposure to <span class="html-italic">Microcystis aeruginosa</span> with 24 h recovery fed on <span class="html-italic">Pseudokirchneriella subcapitata</span>. Different letters indicate significant differences according to the Holm–Sidak test, <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 2
<p>Survival of <span class="html-italic">Daphnia curvirostris</span> in alternating 4 h exposure to <span class="html-italic">Microcystis aeruginosa</span> with 44 h recovery fed on <span class="html-italic">Pseudokirchneriella subcapitata</span>. Different letters indicate significant differences according to the Holm–Sidak test, <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 3
<p>Reproductive responses of <span class="html-italic">Daphnia curvirostris</span> in alternating 24 h exposure to <span class="html-italic">Microcystis aeruginosa</span> with 24 h recovery fed on <span class="html-italic">Pseudokirchneriella subcapitata</span>. Bars are for average values ± standard error limits. Asterisks indicate significant differences from the control (Dunnett’s test, <span class="html-italic">p</span> &lt; 0.05); different letters above the columns are for treatments differing significantly (pairwise Tukey’s test, <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 4
<p>Reproductive responses of <span class="html-italic">Daphnia curvirostris</span> in alternating 4 h exposure to <span class="html-italic">Microcystis aeruginosa</span> with 44 h recovery fed on <span class="html-italic">Pseudokirchneriella subcapitata</span>. Bars are for average values ± standard error limits. Asterisks indicate significant differences from the control (Dunnett’s test, <span class="html-italic">p</span> &lt; 0.05). Different letters above the columns are for treatments differing significantly (pairwise Tukey’s test, <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 5
<p>Total length, body length, and body width of <span class="html-italic">D. curvirostris</span> adults alternately exposed to the toxigenic cyanobacterium <span class="html-italic">M. aeruginosa</span> for 24 h and 24 h recovery, and 4 h with 44 h recovery. Bars represent mean values ± standard error. Asterisks indicate significant differences compared to the control group for each measure (Dunnett’s test, <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 6
<p>The concentration of microcystins (expressed as microcystin-LR), recorded in the different cell densities of <span class="html-italic">M. aeruginosa</span> used to determine the LC<sub>50</sub> in <span class="html-italic">D. curvirostris</span>.</p> Full article ">
14 pages, 2730 KiB  
Article
Rattlesnake Crotalphine Analgesic Active on Tetrodotoxin-Sensitive Na+ Current in Mouse Dorsal Root Ganglion Neurons
by Aurélie Antunes, Philippe Robin, Gilles Mourier, Rémy Béroud, Michel De Waard, Denis Servent and Evelyne Benoit
Toxins 2024, 16(8), 359; https://doi.org/10.3390/toxins16080359 - 15 Aug 2024
Viewed by 573
Abstract
Crotalphine is an analgesic peptide identified from the venom of the South American rattlesnake Crotalus durissus terrificus. Although its antinociceptive effect is well documented, its direct mechanisms of action are still unclear. The aim of the present work was to study the [...] Read more.
Crotalphine is an analgesic peptide identified from the venom of the South American rattlesnake Crotalus durissus terrificus. Although its antinociceptive effect is well documented, its direct mechanisms of action are still unclear. The aim of the present work was to study the action of the crotalid peptide on the NaV1.7 channel subtype, a genetically validated pain target. To this purpose, the effects of crotalphine were evaluated on the NaV1.7 component of the tetrodotoxin-sensitive Na+ current in the dorsal root ganglion neurons of adult mice, using the whole-cell patch-clamp configuration, and on cell viability, using propidium iodide fluorescence and trypan blue assays. The results show that 18.7 µM of peptide inhibited 50% of the Na+ current. The blocking effect occurred without any marked change in the current activation and inactivation kinetics, but it was more important as the membrane potential was more positive. In addition, crotalphine induced an increase in the leakage current amplitude of approximately 150% and led to a maximal 31% decrease in cell viability at a high 50 µM concentration. Taken together, these results point out, for the first time, the effectiveness of crotalphine in acting on the NaV1.7 channel subtype, which may be an additional target contributing to the peptide analgesic properties and, also, although less efficiently, on a second cell plasma membrane component, leading to cell loss. Full article
(This article belongs to the Special Issue Toxins: From the Wild to the Lab)
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<p>Chemical structure of crotalphine. C: cysteine; E: glutamic acid; F: phenylalanine; G: glycine; N: asparagine; P: proline; Q: glutamine; S: serine; and PCA: pyroglutamic acid (non-standard amino acid).</p> Full article ">Figure 2
<p>Concentration–response curve of crotalphine’s steady-state effect on the TTX-sensitive Na<sup>+</sup> current. Each value of peak Na<sup>+</sup> current, expressed as a percentage of that determined before peptide application, represents the mean ± S.E.M. of data obtained from four to six neurons (numbers in parentheses). The theoretical curve was calculated according to Equation (1), with IC<sub>50</sub> and n<sub>H</sub> values of 18.7 µM and 0.73 (r<sup>2</sup> = 0.999), respectively.</p> Full article ">Figure 3
<p>Effect of crotalphine on the activation and inactivation kinetics of the TTX-sensitive Na<sup>+</sup> current. Representative traces of TTX-sensitive Na<sup>+</sup> currents recorded before (in black) and after (in blue) the application of 50 µM of crotalphine. The leakage current was compensated for. On the right, the peptide peak current was normalized to that of the control to allow for a better comparison of the kinetics.</p> Full article ">Figure 4
<p>Effect of crotalphine on the activation and inactivation voltage dependencies of the TTX-sensitive Na<sup>+</sup> current. (<b>a</b>) Representative current–voltage relationships before (black circles) and after (blue circles) the exposure of neurons to 50 µM of crotalphine. The black and blue arrows indicate the maximal peak Na<sup>+</sup> current amplitude under the control conditions (at −20 mV) and in the presence of crotalphine (at −10 mV), respectively. (<b>b</b>) Representative steady-state inactivation–voltage (open circles) and conductance–voltage (filled circles) relationships before (black circles) and after (blue circles) the exposure of neurons to 50 µM of crotalphine. Each value is expressed as a percentage of either the maximal peak amplitude of the current at strongly negative pre-pulse voltages or the maximal conductance calculated at strongly positive test-pulse voltages. The theoretical curves correspond to data point fits according to Equations (3) and (4), with V<sub>P50%</sub>, k<sub>H</sub>, V<sub>T50%</sub>, and k<sub>G</sub> values of −81.7 mV, 10.3 mV<sup>−1</sup>, −36.4 mV, and 2.9 mV<sup>−1</sup> (r<sup>2</sup> ≥ 0.994), respectively, for the control, and −87.2 mV, 10.2 mV<sup>−1</sup>, −30.9 mV, and 5.5 mV<sup>−1</sup> (r<sup>2</sup> ≥ 0.996), respectively, in the presence of crotalphine.</p> Full article ">Figure 5
<p>Effect of crotalphine on the leakage current. Representative experiment of the time course of crotalphine’s effect on the leakage current’s amplitude, before (black circles) and after (blue circles) the addition of 50 μM of the peptide to the standard physiological medium bathing the neurons (arrow).</p> Full article ">Figure 6
<p>Effect of crotalphine (50 µM) on cell viability, using the propidium iodide fluorescence (<b>a</b>–<b>c</b>) or the trypan blue (<b>d</b>) assay. (<b>a</b>) Images of cultured DRG neurons and CHO epithelial cells acquired under the indicated conditions using an inverted epifluorescence microscope. The nuclei of dead cells are colored in red. (<b>b</b>) Histogram of cell viability (DRG neurons and CHO cells) in the presence of either crotalphine or triton X-100 (1%), as a function of time (from 5 to 75 min). Cell viability (number of non-fluorescent cells normalized to that of total cells) is expressed relative to that measured in the presence of the peptide vehicle, i.e., 1% of distilled water (dashed line). (<b>c</b>) Histogram of cell death (DRG neurons and CHO cells) induced by either crotalphine or triton X-100 (1%) and measured just after the end of image acquisition. The cell death (i.e., the fluorescence intensity) is expressed relatively to that measured in the presence of the peptide vehicle (dashed line). (<b>d</b>) Histogram of cell viability (DRG neurons) in the presence of either crotalphine or the peptide vehicle, as a function of time (from 5 to 75 min). Under each condition, the cell viability (number of cells having a clear cytoplasm normalized to the number of total cells) is expressed relative to that measured before peptide or vehicle addition to the cell suspension (dashed line). (<b>b</b>–<b>d</b>) Means ± S.E.M. of data obtained from three to five different experiments. *: <span class="html-italic">p</span> ≤ 0.0457; **: <span class="html-italic">p</span> ≤ 0.0069; and ***: <span class="html-italic">p</span> ≤ 0.0001.</p> Full article ">
17 pages, 2423 KiB  
Article
Cell Penetrating Peptide Enhances the Aphidicidal Activity of Spider Venom-Derived Neurotoxin
by Wenxian Wu, Abid Ali, Jinbo Shen, Maozhi Ren, Yi Cai and Limei He
Toxins 2024, 16(8), 358; https://doi.org/10.3390/toxins16080358 - 14 Aug 2024
Viewed by 442
Abstract
HxTx-Hv1h, a neurotoxic peptide derived from spider venom, has been developed for use in commercial biopesticide formulations. Cell Penetrating Peptides (CPPs) are short peptides that facilitate the translocation of various biomolecules across cellular membranes. Here, we evaluated the aphidicidal efficacy of a conjugated [...] Read more.
HxTx-Hv1h, a neurotoxic peptide derived from spider venom, has been developed for use in commercial biopesticide formulations. Cell Penetrating Peptides (CPPs) are short peptides that facilitate the translocation of various biomolecules across cellular membranes. Here, we evaluated the aphidicidal efficacy of a conjugated peptide, HxTx-Hv1h/CPP-1838, created by fusing HxTx-Hv1h with CPP-1838. Additionally, we aimed to establish a robust recombinant expression system for HxTx-Hv1h/CPP-1838. We successfully achieved the secretory production of HxTx-Hv1h, its fusion with Galanthus nivalis agglutinin (GNA) forming HxTx-Hv1h/GNA and HxTx-Hv1h/CPP-1838 in yeast. Purified HxTx-Hv1h exhibited contact toxicity against Megoura crassicauda, with a 48 h median lethal concentration (LC50) of 860.5 μg/mL. Fusion with GNA or CPP-1838 significantly enhanced its aphidicidal potency, reducing the LC50 to 683.5 μg/mL and 465.2 μg/mL, respectively. The aphidicidal efficacy was further improved with the addition of surfactant, decreasing the LC50 of HxTx-Hv1h/CPP-1838 to 66.7 μg/mL—over four times lower compared to HxTx-Hv1h alone. Furthermore, we engineered HxTx-Hv1h/CPP-1838 multi-copy expression vectors utilizing the BglBrick assembly method and achieved high-level recombinant production in laboratory-scale fermentation. This study is the first to document a CPP fusion strategy that enhances the transdermal aphidicidal activity of a natural toxin like HxTx-Hv1h and opens up the possibility of exploring the recombinant production of HxTx-Hv1h/CPP-1838 for potential applications. Full article
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<p>Expression and detection of recombinant proteins. (<b>A</b>) Schematic representation of constructs engineered to encode HxTx-Hv1h, HxTx-Hv1h/GNA, and HxTx-Hv1h/CPP-1838 in yeast. The α-factor leader sequence facilitates targeting of the expressed proteins to the yeast secretory pathway, thus enabling their isolation from the fermentation culture supernatant. The His-tag indicates an incorporated hexahistidine motif, which facilitates protein purification via nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and permits detection through Western blot analysis. (<b>B</b>) Resolution of purified recombinant proteins on a 15% SDS-PAGE gel, visualized post-staining with Coomassie ultrafast. ‘M’ designates the molecular weight marker; lane 1 contains 5 μg of HxTx-Hv1h; lane 2 contains 7.5 μg of HxTx-Hv1h/GNA; lane 3 features 10 μg of HxTx-Hv1h/CPP-1838. (<b>C</b>) Immunoblot analysis of the recombinant proteins utilizing an anti-His tag antibody. ‘M’ comprises molecular weight standards, with lanes 1–3 corresponding to the samples detailed in (<b>B</b>), loaded with approximately 50 ng of HxTx-Hv1h, 200 ng of HxTx-Hv1h/GNA, and 100 ng of HxTx-Hv1h/CPP-1838, respectively. (<b>D</b>) Western blot detection of the recombinant proteins using an anti-HxTx-Hv1h antibody. Lanes 1–3 follow the pattern established in (<b>B</b>), with loading quantities at approximately 50 ng for HxTx-Hv1h and HxTx-Hv1h/CPP-1838, while 100 ng of HxTx-Hv1h/GNA is introduced in lane 2.</p> Full article ">Figure 2
<p>Evaluation of the contact toxicity of recombinant proteins against aphids. (<b>A</b>–<b>C</b>) Dose–response curves depicting the contact toxicity of HxTx-Hv1h, HxTx-Hv1h/GNA, and HxTx-Hv1h/CPP-1838 on aphid populations, in the absence of the surfactant Silwet L-77. The curves illustrate the percentage of mortality across a spectrum of concentrations, highlighting the aphidicidal potency of the recombinant proteins without surfactant assistance. (<b>D</b>–<b>F</b>). Dose–response curves showcasing the augmented contact toxicity of HxTx-Hv1h, HxTx-Hv1h/GNA, and HxTx-Hv1h/CPP-1838 when used in conjunction with the surfactant Silwet L-77. These curves demonstrate the synergy between the bioactive proteins and the surfactant, culminating in enhanced lethality towards the aphids.</p> Full article ">Figure 3
<p>Immunoblot analysis of protein extracts from insects. ‘C’ denotes the lanes loaded with extracts from control aphids that did not undergo exposure to any recombinant proteins. Above each lane, the identity of the specific recombinant protein that the aphids contacted is labeled. The protein samples were derived from groups of 20 aphids harvested 8 h following contact with a 200 μM concentration of each respective recombinant protein. ‘S1’, ‘S2’, and ‘S3’ correspond to the protein standards for HxTx-Hv1h, HxTx-Hv1h/GNA, and HxTx-Hv1h/CPP-1838, each at a concentration of 100 ng.</p> Full article ">Figure 4
<p>Confirmation of plasmids harboring single and multiple HxTx-Hv1h/CPP-1838 expression cassettes and copy number assessment in transformed yeast clones. (<b>A</b>) plasmid shift analysis. ‘M’ denotes the molecular weight standards. Lanes 1C through 12C contain plasmids pGAPZαA-HxTx-Hv1h/CPP-1838 with varying copy counts, notationally indicated as 1C (one copy), 2C (two copies), 4C (four copies), 6C (six copies), 8C (eight copies), 10C (ten copies), and 12C (twelve copies), respectively. (<b>B</b>) Analysis of plasmid conformation subsequent to double restriction digestion with <span class="html-italic">Bgl</span> II and <span class="html-italic">Bam</span>H I enzymes, with each lane corresponding to the respective plasmid construct detailed in (<b>A</b>). (<b>C</b>) qPCR evaluation of yeast clones transformed with plasmids encompassing single and assorted copy numbers of the HxTx-Hv1h/CPP-1838 expression cassette. The RQ (Relative Quantification) values signify the comparative determination of copy numbers of the HxTx-Hv1h/CPP-1838 construct in relation to a reference housekeeping gene.</p> Full article ">Figure 5
<p>Evaluation of protein expression levels in <span class="html-italic">P. pastoris</span> transformants with multiple gene copies. (<b>A</b>) Depiction of cultured supernatant protein profiles from <span class="html-italic">P. pastoris</span> transformants incorporating varying copy numbers of the HxTx-Hv1h/CPP-1838 expression cassettes. ‘C’ represents the expression cassette featuring a GAP promoter, the HxTx-Hv1h/CPP-1838 gene, and an AOX1 terminator. ‘1C’ denotes the yeast transformant harboring a single expression cassette. ‘2C’, ‘4C’, ‘6C’, ‘8C’, ‘10C’, and ‘12C’ indicate transformants encompassing two, four, six, eight, ten, and twelve repeats of the expression cassette, respectively. (<b>B</b>) Chronological analysis of HxTx-Hv1h/CPP-1838 protein expression in yeast transformants across diverse fermentation time frames. ‘M’ represents the molecular weight marker.</p> Full article ">
20 pages, 4904 KiB  
Article
Community Structure and Toxicity Potential of Cyanobacteria during Summer and Winter in a Temperate-Zone Lake Susceptible to Phytoplankton Blooms
by Łukasz Wejnerowski, Tamara Dulić, Sultana Akter, Arnoldo Font-Nájera, Michał Rybak, Oskar Kamiński, Anna Czerepska, Marcin Krzysztof Dziuba, Tomasz Jurczak, Jussi Meriluoto, Joanna Mankiewicz-Boczek and Mikołaj Kokociński
Toxins 2024, 16(8), 357; https://doi.org/10.3390/toxins16080357 - 14 Aug 2024
Viewed by 615
Abstract
Cyanobacterial blooms are increasingly common during winters, especially when they are mild. The goal of this study was to determine the summer and winter phytoplankton community structure, cyanotoxin presence, and toxigenicity in a eutrophic lake susceptible to cyanobacterial blooms throughout the year, using [...] Read more.
Cyanobacterial blooms are increasingly common during winters, especially when they are mild. The goal of this study was to determine the summer and winter phytoplankton community structure, cyanotoxin presence, and toxigenicity in a eutrophic lake susceptible to cyanobacterial blooms throughout the year, using classical microscopy, an analysis of toxic cyanometabolites, and an analysis of genes involved in biosynthesis of cyanotoxins. We also assessed whether cyanobacterial diversity in the studied lake has changed compared to what was reported in previous reports conducted several years ago. Moreover, the bloom-forming cyanobacterial strains were isolated from the lake and screened for cyanotoxin presence and toxigenicity. Cyanobacteria were the main component of the phytoplankton community in both sampling times, and, in particular, Oscillatoriales were predominant in both summer (Planktothrix/Limnothrix) and winter (Limnothrix) sampling. Compared to the winter community, the summer community was denser; richer in species; and contained alien and invasive Nostocales, including Sphaerospermopsis aphanizomenoides, Raphidiopsis raciborskii, and Raphidiopsis mediterranea. In both sampling times, the blooms contained toxigenic species with genetic determinants for the production of cylindrospermopsin and microcystins. Toxicological screening revealed the presence of microcystins in the lake in summer but no cyanotoxins in the winter period of sampling. However, several cyanobacterial strains isolated from the lake during winter and summer produced anabaenopeptins and microcystins. This study indicates that summer and winter blooms of cyanobacteria in the temperate zone can differ in biomass, structure, and toxicity, and that the toxic hazards associated with cyanobacterial blooms may potentially exist during winter. 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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<p>The proportion of cyanobacteria in the phytoplankton (<b>a</b>) and quantitative structure of cyanobacterial communities (<b>b</b>) of the Lubosińskie Lake during summer (left panel) and winter (right panel). No bar means that a given taxon was absent.</p> Full article ">Figure 2
<p>Micrographs of <span class="html-italic">Limnothrix</span> and <span class="html-italic">Planktothrix</span> specimens from summer (<b>a</b>) and winter (<b>b</b>) samples, conducted using light microscope with Nomarski interference contrast.</p> Full article ">Figure 3
<p>Micrographs of cyanobacterial strains isolated from summer and winter phytoplankton of Lubosińskie Lake, conducted using light microscope with integrated modulation contrast.</p> Full article ">Figure 4
<p>Phylogenetic reconstruction based on the <span class="html-italic">rpo</span>B gene for the isolated cyanobacterial strains from the Lubosińskie Lake. The tree was constructed using the NJ topology and the numbers associated with the nodes represent the bootstrap support values of NJ and ML analyses, respectively. Only bootstrap supports ≥ 50% were reported. The bar above the tree represents nucleotide substitutions per position. Accession numbers were given for <span class="html-italic">rpo</span>B sequences obtained from GenBank. The sequences of <span class="html-italic">Microcystis aeruginosa</span> PCC7806 and <span class="html-italic">Thermus thermophilus</span> AK1 were used as outgroups to cluster the representative strains in the phylum Cyanobacteria. Red and blue colors indicate summer and winter strains, respectively.</p> Full article ">Figure 5
<p>Qualitative amplification of toxigenic genes in summer and winter samples from Lubosińskie Lake: (<b>a</b>) <span class="html-italic">mcy</span>E (405 bp)—potential for production of MCs; (<b>b</b>) <span class="html-italic">cyr</span>J (578 bp)—potential for production of CYN; and (<b>c</b>) <span class="html-italic">ana</span>F (467 bp)—potential for production of ATX-a. SS in red color—summer lake-water sample, WS in blue color—winter lake-water sample, PC—positive control (<span class="html-italic">mcy</span>E, <span class="html-italic">Microcystis aeruginosa</span> PCC7806; <span class="html-italic">cyr</span>J, <span class="html-italic">Raphidiopsis raciborskii</span> CS505 Australia; <span class="html-italic">ana</span>F, <span class="html-italic">Cuspidothrix issatschenkoi</span> NIVA-CYA 711); NC—negative control; M—DNA size marker.</p> Full article ">Figure 6
<p>Visualized HPLC-DAD chromatograms and compound spectra of dmMC-RR (1, RT = 3.776 min), MC-YR (3, RT = 4.945 min), and dmMC-LR (4, RT = 5.086 min) in summer <span class="html-italic">P. agardhii</span> strain W67 (<b>a</b>); dmMC-RR (1, RT = 3.771 min) and dmMC-LR (4, RT = 5.081 min) in winter <span class="html-italic">P. agardhii</span> strain W70 (<b>b</b>); and dmMC-RR (1, RT = 3.766 min), MC-RR (2, RT = 3.955 min), MC-YR (3, RT = 5.001 min), dmMC-LR (4, RT = 5.071 min), and MC-LR (5, RT = 5.260 min) in the standard NIES107 (<b>c</b>). Summer and winter MCs-producing strains are marked, respectively, in red and blue.</p> Full article ">Figure 7
<p>Visualized chromatograms and MS scan spectra of (<b>a</b>) dmMC-RR in the summer <span class="html-italic">P. agardhii</span> W67 (RT = 4.5 min), winter <span class="html-italic">P. agardhii</span> W70 (RT = 4.5 min), and NIES107 standard (RT = 4.5 min); and (<b>b</b>) MC-YR in <span class="html-italic">P. agardhii</span> W67 (RT = 5.6 min) and NIES107 standard (RT = 5.7 min). Summer and winter MCs-producing strains are marked, respectively, in red and blue.</p> Full article ">Figure 8
<p>Preliminary data obtained from strain culturing about chlorophyll-<span class="html-italic">a</span> (filled circles) and pheophytin (empty circles) concentration (<b>a</b>), pH (<b>b</b>), total nitrogen concentration (<b>c</b>), and total phosphorus concentration (<b>d</b>) in 40-day-old cyanobacterial cultures. Red and blue colors indicate the origin of strains (summer and winter, respectively).</p> Full article ">
14 pages, 2658 KiB  
Article
Mitigation of Deoxynivalenol (DON)- and Aflatoxin B1 (AFB1)-Induced Immune Dysfunction and Apoptosis in Mouse Spleen by Curcumin
by Azhar Muhmood, Jianxin Liu, Dandan Liu, Shuiping Liu, Mahmoud M. Azzam, Muhammad Bilawal Junaid, Lili Hou, Guannan Le and Kehe Huang
Toxins 2024, 16(8), 356; https://doi.org/10.3390/toxins16080356 - 13 Aug 2024
Viewed by 824
Abstract
In the context of the potential immunomodulatory properties of curcumin in counteracting the detrimental effects of concurrent exposure to Deoxynivalenol (DON) and Aflatoxin B1 (AFB1), a comprehensive 28-days trial was conducted utilizing 60 randomly allocated mice divided into four groups. Administration of curcumin [...] Read more.
In the context of the potential immunomodulatory properties of curcumin in counteracting the detrimental effects of concurrent exposure to Deoxynivalenol (DON) and Aflatoxin B1 (AFB1), a comprehensive 28-days trial was conducted utilizing 60 randomly allocated mice divided into four groups. Administration of curcumin at a dosage of 5 mg/kg body weight in conjunction with DON at 0.1 mg/kg and AFB1 at 0.01 mg/kg body weight was undertaken to assess its efficacy. Results indicated that curcumin intervention demonstrated mitigation of splenic structural damage, augmentation of serum immunoglobulin A (IgA) and immunoglobulin G (IgG) levels, elevation in T lymphocyte subset levels, and enhancement in the mRNA expression levels of pro-inflammatory cytokines TNF-α, IFN-γ, IL-2, and IL-6. Furthermore, curcumin exhibited a suppressive effect on apoptosis in mice, as evidenced by decreased activity of caspase-3 and caspase-9, reduced expression levels of pro-apoptotic markers Bax and Cytochrome-c (Cyt-c) at both the protein and mRNA levels, and the maintenance of a balanced expression ratio of mitochondrial apoptotic regulators Bax and Bcl-2. Collectively, these findings offer novel insights into the therapeutic promise of curcumin in mitigating immunosuppression and apoptotic events triggered by mycotoxin co-exposure. Full article
(This article belongs to the Section Mycotoxins)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Microscopic changes in mouse spleen treated with DON, AFB1, and curcumin. The image magnification is 50×; WP, white pulp; RP, red pulp; black arrows indicate the splenic demarcation between white pulp and red pulp, (<b>A</b>) represents well-organized structure and distinct nucleus, (<b>B</b>) represents unclear boundaries between white pulp and red pulp, (<b>C</b>) represents distinct and clear margins between white pulp and red pulp, and (<b>D</b>) represents increased lymphocytes and evident margins as compared with the DON + AFB1 group.</p> Full article ">Figure 2
<p>Effects of curcumin on the immune system of mice exposed to DON + AFB1. (<b>A</b>,<b>B</b>) IgA and IgG were detected by an ELISA KIT in the serum of mice. (<b>C</b>–<b>F</b>) The mRNA levels of <span class="html-italic">TNF-α</span>, <span class="html-italic">IFN-γ</span>, <span class="html-italic">IL-2</span>, and <span class="html-italic">IL-6</span> cytokines in the spleen tissue of mice were quantified by qRT-PCR. (<b>G</b>,<b>H</b>) The percentage of CD4<sup>+</sup> and CD8<sup>+</sup> T lymphocytes were assessed by flow cytometry, and the CD4<sup>+</sup>/CD8<sup>+</sup> ratio is presented (<b>I</b>). (<b>J</b>) The representative CD4<sup>+</sup> and CD8<sup>+</sup> flow cytometry profiles of splenic cells are shown. The data are represented as the means ± standard deviation (<span class="html-italic">n</span> = 15). Different lowercase letters represent <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 3
<p>Curcumin treatment restrains the DON + AFB1-induced mitochondrial-mediated pathway in the spleen tissue of mice. (<b>A</b>–<b>D</b>) The mRNA expression of <span class="html-italic">Bcl-2</span>, <span class="html-italic">Bax</span>, <span class="html-italic">Cas-3</span>, <span class="html-italic">Cyt-c</span> was quantified by qRT-PCR. (<b>E</b>–<b>J</b>) The protein expression of Bcl-2, Bax, Cas-3, Cyt-c was measured by Western blotting. The lower bands represent β-actin for all above-validated proteins. (<b>K</b>) The splenocytes’ apoptosis was analyzed by flow cytometry. (<b>L</b>) Quantitative analysis of the apoptosis rate. The results are shown as the means ± standard deviation (<span class="html-italic">n</span> = 15). Different lowercase letters represent <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">
10 pages, 418 KiB  
Review
The Potential Therapeutic Effects of Botulinum Neurotoxins on Neoplastic Cells: A Comprehensive Review of In Vitro and In Vivo Studies
by Delaram Safarpour, Fattaneh A. Tavassoli and Bahman Jabbari
Toxins 2024, 16(8), 355; https://doi.org/10.3390/toxins16080355 - 13 Aug 2024
Viewed by 628
Abstract
A systematic review of the literature found fifteen articles on the effect of a botulinum toxin on neoplastic cell lines and eight articles on in vivo neoplasms. The reported in vitro effects rely on high doses or the mechanical disruption of cell membranes [...] Read more.
A systematic review of the literature found fifteen articles on the effect of a botulinum toxin on neoplastic cell lines and eight articles on in vivo neoplasms. The reported in vitro effects rely on high doses or the mechanical disruption of cell membranes to introduce the botulinum neurotoxin into the cell cytoplasm. The potency of the botulinum neurotoxin to intoxicate non-neuronal cells (even cell lines expressing an appropriate protein receptor) is several orders of magnitude lower compared to that to intoxicate the primary neurons. The data suggest that the botulinum toxin disrupts the progression of cancer cells, with some studies reporting apoptotic effects. A majority of the data in the in vivo studies also showed similar results. No safety issues were disclosed in the in vivo studies. Limited studies have suggested similar anti-neoplastic potential for the clostridium difficile. New modes of delivery have been tested to enhance the in vivo delivery of the botulinum toxin to neoplastic cells. Careful controlled studies are necessary to demonstrate the efficacy and safety of this mode of anti-neoplastic treatment in humans. Full article
(This article belongs to the Special Issue Intracellular Transport of Toxins: Insights into Mechanisms of Cytotoxicity and Applications in Cell Biology and Medicine)
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<p>Prisma.</p> Full article ">
17 pages, 4494 KiB  
Article
Sága, a Deep Learning Spectral Analysis Tool for Fungal Detection in Grains—A Case Study to Detect Fusarium in Winter Wheat
by Xinxin Wang, Gerrit Polder, Marlous Focker and Cheng Liu
Toxins 2024, 16(8), 354; https://doi.org/10.3390/toxins16080354 - 13 Aug 2024
Viewed by 766
Abstract
Fusarium head blight (FHB) is a plant disease caused by various species of the Fusarium fungus. One of the major concerns associated with Fusarium spp. is their ability to produce mycotoxins. Mycotoxin contamination in small grain cereals is a risk to human and [...] Read more.
Fusarium head blight (FHB) is a plant disease caused by various species of the Fusarium fungus. One of the major concerns associated with Fusarium spp. is their ability to produce mycotoxins. Mycotoxin contamination in small grain cereals is a risk to human and animal health and leads to major economic losses. A reliable site-specific precise Fusarium spp. infection early warning model is, therefore, needed to ensure food and feed safety by the early detection of contamination hotspots, enabling effective and efficient fungicide applications, and providing FHB prevention management advice. Such precision farming techniques contribute to environmentally friendly production and sustainable agriculture. This study developed a predictive model, Sága, for on-site FHB detection in wheat using imaging spectroscopy and deep learning. Data were collected from an experimental field in 2021 including (1) an experimental field inoculated with Fusarium spp. (52.5 m × 3 m) and (2) a control field (52.5 m × 3 m) not inoculated with Fusarium spp. and sprayed with fungicides. Imaging spectroscopy data (hyperspectral images) were collected from both the experimental and control fields with the ground truth of Fusarium-infected ear and healthy ear, respectively. Deep learning approaches (pretrained YOLOv5 and DeepMAC on Global Wheat Head Detection (GWHD) dataset) were used to segment wheat ears and XGBoost was used to analyze the hyperspectral information related to the wheat ears and make predictions of Fusarium-infected wheat ear and healthy wheat ear. The results showed that deep learning methods can automatically detect and segment the ears of wheat by applying pretrained models. The predictive model can accurately detect infected areas in a wheat field, achieving mean accuracy and F1 scores exceeding 89%. The proposed model, Sága, could facilitate the early detection of Fusarium spp. to increase the fungicide use efficiency and limit mycotoxin contamination. Full article
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<p>Result of the wheat ear bounding box detection and wheat ear segmentation. The left image shows the detected wheat ears with bounding boxes. The right image shows the segmented wheat ears with colored masks.</p> Full article ">Figure 2
<p>The comparison of the mean wavelength reflectance between the infected ears (red) and healthy ears (blue). The shaded areas around each line represent the standard deviation, which illustrates the variability or dispersion of the reflectance values around the mean.</p> Full article ">Figure 3
<p>Confusion matrices for model evaluation. Confusion matrices illustrating the performance of the XGBoost classifier on both the training set (<b>left</b>) and the validation set (<b>right</b>). The matrices display the counts of true positives, true negatives, false positives, and false negatives. The colorbar represents the number of samples in each category. Class labels are “Healthy” (0) and “Infected” (1).</p> Full article ">Figure 4
<p>The twenty most important features (wavelengths). The left figure shows the wavelengths sorted based on their importance on the contribution of each feature (wavelength in this case) to the model’s predictive performance. The right figure shows what is the wavelength’s impact on the infected wheat ear.</p> Full article ">Figure 5
<p>The study area and spectral image acquired by the Netherlands Plant Eco-phenotyping Centre (NPEC) facility TraitSeeker. (<b>a</b>): this panel shows the study area, highlighted in yellow, with the upper 10 plots serving as the control plots and the lower 10 plots as the experimental plots. (<b>b</b>): this panel provides a zoomed-in view of the two plots for better spatial context. (<b>c</b>): This panel displays an example of imaging spectroscopy from one spot within the wheat field, collected from an experimental plot. The image depicts the reflectance at 460 nm, extracted from the full spectral image, with a spatial resolution of 0.2 cm × 0.2 cm.</p> Full article ">Figure 6
<p>Model development procedure including two modules. The first module consists of the detection and segmentation of wheat ears as well as the extraction of the spectral information from the segmented wheat ears; the second module consists of the creation of a predictive model using the spectral data extracted from the segmented wheat ears.</p> Full article ">Figure A1
<p>Field experiment plan 2021. Experimental field (yellow): The experimental field, measuring 52.5 m by 3 m, where inoculation was performed to facilitate fungal infection. This field is represented in yellow. Space between fields (green): a 6 m buffer zone separates the inoculated field from the control field to minimize cross-contamination. Control field (blue): The control field, also measuring 52.5 m by 3 m, where fungicide spray was applied and no inoculation was performed. This field is represented in blue.</p> Full article ">Figure A2
<p>Field experiment plan 2021. Green color shows the wheat fields at different period. Red dots show the observed fusarium head blight spots.</p> Full article ">Figure A3
<p>The wavelength reflectance of all the wheat ears between the healthy ear and infected ear.</p> Full article ">Figure A4
<p>TraitSeeker in the wheat field.</p> Full article ">
17 pages, 8497 KiB  
Article
Investigation of Deoxynivalenol Contamination in Local Area and Evaluation of Its Multiple Intestinal Toxicity
by Yebo Wang, Minjie Zhang, Ke Li, Chune Zhang, Honglei Tian and Ying Luo
Toxins 2024, 16(8), 353; https://doi.org/10.3390/toxins16080353 - 12 Aug 2024
Viewed by 613
Abstract
Deoxynivalenol (DON) is a mycotoxin produced by Fusarium fungi widespread in wheat, corn, barley and other grain crops, posing the potential for being toxic to human and animal health, especially in the small intestine, which is the primary target organ for defense against [...] Read more.
Deoxynivalenol (DON) is a mycotoxin produced by Fusarium fungi widespread in wheat, corn, barley and other grain crops, posing the potential for being toxic to human and animal health, especially in the small intestine, which is the primary target organ for defense against the invasion of toxins. This study firstly investigated DON contamination in a local area of a wheat production district in China. Subsequently, the mechanism of DON toxicity was analyzed through cellular molecular biology combining with intestinal flora and gene transcription analysis; the results indicated that DON exposure can decrease IPEC−J2 cell viability and antioxidant capacity, stimulate the secretion and expression of proinflammatory factors, destroy the gut microbiota and affect normal functions of the body. It is illustrated that DON could induce intestinal damage through structural damage, functional injury and even intestinal internal environment disturbance, and, also, these intestinal toxicity effects are intrinsically interrelated. This study may provide multifaceted information for the treatment of intestinal injury induced by DON. Full article
(This article belongs to the Section Mycotoxins)
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<p>Investigation on DON contamination in the main wheat production areas of Shaanxi. (<b>a</b>) The main contamination distribution of DON in Shaanxi; (<b>b</b>) detection process of DON in wheat; (<b>c</b>) DON standard curve; (<b>d</b>) DON contamination rate.</p> Full article ">Figure 2
<p>Effects of DON on IPEC−J2 cell viability, LDH release and inflammation. (<b>a</b>) DON IPEC−J2 cytotoxicity assay flow; (<b>b</b>) effect of DON on cell viability at 24, 48 and 72 h; (<b>c</b>) effect of DON on drug inhibition at 24, 48 and 72 h; (<b>d</b>) effect of DON on LDH release at 24, 48 and 72 h; (<b>e</b>) the effect of DON on NF-κβ expression; (<b>f</b>–<b>h</b>) the effect of DON on inflammation factors of IL-6, COX-2 and IL-10. Bars marked with different lowercase letters were significantly different (<span class="html-italic">p</span> &lt; 0.05). Bars marked with * and *** were significantly different with <span class="html-italic">p</span> &lt; 0.05 and <span class="html-italic">p</span> &lt; 0.001, respectively. Notes: the negative co-ordinates in Figure h represent the negative feedback relationship between IL-10 and induced inflammation.</p> Full article ">Figure 3
<p>Effects of DON on IPEC−J2 oxidative stress and cell apoptosis. (<b>a</b>,<b>b</b>) Effect of DON on level of ROS; (<b>c</b>–<b>f</b>) effect of DON on SOD, GSH, CAT and MDA content; (<b>g</b>–<b>l</b>) the cell apoptosis of IPEC−J2 after DON exposure; (<b>m</b>,<b>n</b>) comparison of living cell ratio and apoptosis ratio between control group and DON group. Bars marked with ** and *** were significantly different with <span class="html-italic">p</span> &lt; 0.01 and <span class="html-italic">p</span> &lt; 0.001, respectively. Bars marked with different lowercase letters were significantly different (<span class="html-italic">p</span> &lt; 0.05). Notes: the negative co-ordinates in Figure (<b>f</b>) represent the negative feedback relationship between MDA and antioxidant.</p> Full article ">Figure 4
<p>Effects of DON on basic indicators of Kunming mice. (<b>a</b>) DON animal toxicity experimental process; (<b>b</b>) effect of DON on initial and final body weights; (<b>c</b>) effect of DON on small intestine length; (<b>d</b>–<b>f</b>) effects of DON on the body weight ratio of kidney, liver and spleen, respectively; (<b>g</b>) effects of DON on counts of neutrophil; (<b>h</b>) effects of DON on counts of white blood cells; (<b>i</b>) effects of DON on counts of monocytes; (<b>j</b>) effects of DON on concentration of aspartate aminotransferase; (<b>k</b>) effects of DON on concentration of alanine aminotransferase. (<b>l</b>) The histopathology of liver, kidney and intestinal tissues, with magnification 10× and 20×; (<b>m</b>) effect of DON on the number of goblet cells; (<b>n</b>) effect of DON on villus height; (<b>o</b>) effect of DON on crypt depth; (<b>p</b>) effect of DON on villus height/crypt depth. Bars marked with *** and **** were significantly different with <span class="html-italic">p</span> &lt; 0.001 and <span class="html-italic">p</span> &lt; 0.0001, respectively. Bars marked with different lowercase letters were significantly different (<span class="html-italic">p</span> &lt; 0.05). Notes: the box and line segments in Figure (<b>a</b>) indicate that the area is magnified in the following figure.</p> Full article ">Figure 5
<p>Transcriptomic analysis with DON exposure. (<b>a</b>) The intra-group and extra-group correlations among samples; (<b>b</b>) volcano plot of differentially expressed genes; (<b>c</b>) KEGG pathway enrichment analysis; (<b>d</b>) GO enrichment plot analysis; (<b>e</b>) the different expressed genes related to inflammation response; (<b>f</b>) the different expressed genes related to immune response. The different expressed genes related to immune response. Notes: the red boxes indicate several related pathways involved in the article.</p> Full article ">Figure 6
<p>Gut microbiota analysis with DON exposure. (<b>a</b>) Goods coverage plot; (<b>b</b>) observed otus plot; (<b>c</b>) Chao 1 plot; (<b>d</b>) Shannon plot. (<b>e</b>) NMDS analysis; (<b>f</b>) PCOA score plot of the features; (<b>g</b>) Venn diagram; (<b>h</b>) gut microbial composition at the phylum level; (<b>i</b>) gut microbial composition at the genus level; (<b>j</b>) effect of DON on relative abundance of <span class="html-italic">firmicutes</span>; (<b>k</b>) effect of DON on relative abundance of <span class="html-italic">Bacteroidates</span>; (<b>l</b>) effect of DON on ratio of <span class="html-italic">Firmicutes</span>/<span class="html-italic">Bacteroidetes</span> (F/B); (<b>m</b>) effect of DON on relative abundance of <span class="html-italic">Ligilactobacillus</span>; (<b>n</b>) effect of DON on relative abundance of <span class="html-italic">Muribaculaceae</span>; (<b>o</b>) effect of DON on relative abundance of <span class="html-italic">Lachnospiraceae_NK4A136_group</span>; (<b>p</b>) effect of DON on relative abundance of <span class="html-italic">Helicobacter</span>. Bars marked with *, ** and *** were significantly different with <span class="html-italic">p</span> &lt; 0.05, <span class="html-italic">p</span> &lt; 0.01 and <span class="html-italic">p</span> &lt; 0.001, respectively.</p> Full article ">Figure 7
<p>Correlation analysis between gut microbiota and other indicators. (<b>a</b>) Biomarker analysis by LefSe; (<b>b</b>) functional analysis of gut microbiota prediction; (<b>c</b>) predictive analysis of gut microbiota phenotypes; (<b>d</b>) correlation analysis heatmap between gut microbiota and serum indicators; (<b>e</b>) correlation analysis network between gut microbiota and serum indicator; (<b>f</b>) correlation analysis heatmap between gut microbiota and genes; (<b>g</b>) correlation analysis network between gut microbiota and genes. Bars marked with *, ** and *** were significantly different with <span class="html-italic">p</span> &lt; 0.05, <span class="html-italic">p</span> &lt; 0.01 and <span class="html-italic">p</span> &lt; 0.001, respectively.</p> Full article ">
19 pages, 9937 KiB  
Article
Oxidative Stress, Oxidative Damage, and Cell Apoptosis: Toxicity Induced by Arecoline in Caenorhabditis elegans and Screening of Mitigating Agents
by Kaiping Xiang, Bing Wang, Lanying Wang, Yunfei Zhang, Hanzeng Li and Yanping Luo
Toxins 2024, 16(8), 352; https://doi.org/10.3390/toxins16080352 - 12 Aug 2024
Viewed by 631
Abstract
As the areca nut market is expanding, there is a growing concern regarding areca nut toxicity. Areca nut alkaloids are the major risky components in betel nuts, and their toxic effects are not fully understood. Here, we investigated the parental and transgenerational toxicity [...] Read more.
As the areca nut market is expanding, there is a growing concern regarding areca nut toxicity. Areca nut alkaloids are the major risky components in betel nuts, and their toxic effects are not fully understood. Here, we investigated the parental and transgenerational toxicity of varied doses of areca nut alkaloids in Caenorhabditis elegans. The results showed that the minimal effective concentration of arecoline is 0.2–0.4 mM. First, arecoline exhibited transgenerational toxicity on the worms’ longevity, oviposition, and reproduction. Second, the redox homeostasis of C. elegans was markedly altered under exposure to 0.2–0.4 mM arecoline. The mitochondrial membrane potential was thereafter impaired, which was also associated with the induction of apoptosis. Moreover, antioxidant treatments such as lycopene could significantly ameliorate the toxic effects caused by arecoline. In conclusion, arecoline enhances the ROS levels, inducing neurotoxicity, developmental toxicity, and reproductive toxicity in C. elegans through dysregulated oxidative stress, cell apoptosis, and DNA damage-related gene expression. Therefore, the drug-induced production of reactive oxygen species (ROS) may be crucial for its toxic effects, which could be mitigated by antioxidants. Full article
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<p>Effect of arecoline on the toxicity to <span class="html-italic">C. elegans.</span> (<b>A</b>) F0 generation lifespan; (<b>B</b>) F1 generation lifespan; (<b>C</b>) average longevity; (<b>D</b>) body length; (<b>E</b>) body width; (<b>F</b>) average egg production; (<b>G</b>) head thrashes; (<b>H</b>) body bends; (<b>I</b>) muscarinic acetylcholine receptor content. Data are presented as mean ± SEM. Values followed by the different lowercase letters within a row represent the significant difference (Tukey tests, <span class="html-italic">p</span> &lt; 0.05). One-way ANOVA with Tukey post hoc test.</p> Full article ">Figure 2
<p>Effects of arecoline on ROS, lipofuscin accumulation, and gonad cell corpses in <span class="html-italic">C. elegans</span>. (<b>A</b>) Fluorescence pictures of ROS and lipofuscin accumulation and the gonad cell corpses assay. (<b>B</b>) Quantification of ROS accumulation. (<b>C</b>) Quantification of lipofuscin accumulation. (<b>D</b>) Quantification of gonad cell corpses. (<b>E</b>,<b>F</b>) Cell corpses were counted in N2. (<b>G</b>,<b>H</b>) Cell corpses were counted in the <span class="html-italic">ced-1(e1735)</span> strains. Values followed by the different lowercase letters within a row represent the significant difference (Tukey tests, <span class="html-italic">p &lt;</span> 0.05). One-way ANOVA with Tukey post hoc test.</p> Full article ">Figure 3
<p>Effect of arecoline on the antioxidant enzyme system and non-enzymatic antioxidant system of <span class="html-italic">C. elegans</span>. (<b>A</b>) Oxidizing enzyme; (<b>B</b>) GSSG and GSH content; (<b>C</b>) MDA content, 8-OHdG content and protein carbonyl content; (<b>D</b>) UV stress; (<b>E</b>) mean survival time of UV stress. Values followed by the different lowercase letters within a row represent the significant difference (Tukey tests, <span class="html-italic">p &lt;</span> 0.05). *** Statistical significance at <span class="html-italic">p</span> &lt; 0.001. One-way ANOVA with Tukey post hoc test.</p> Full article ">Figure 4
<p>Effect of arecoline on mitochondria in <span class="html-italic">C. elegans.</span> (<b>A</b>) Electron transport chain complex I; (<b>B</b>) electron transport chain complex III. (<b>C</b>) MMP. (<b>D</b>) Quantitative analysis of green/red fluorescence intensity ratio. Values followed by the different lowercase letters within a row represent the significant difference (Tukey tests, <span class="html-italic">p &lt;</span> 0.05). One-way ANOVA with Tukey post hoc test.</p> Full article ">Figure 5
<p>Effect of arecoline on gene expression in <span class="html-italic">C. elegans</span>. (<b>A</b>) <span class="html-italic">daf-16</span> gene, <span class="html-italic">skn-1</span> gene and their downstream genes. (<b>B</b>) DNA damage genes. (<b>C</b>) Apoptosis genes.</p> Full article ">Figure 6
<p>Effect of arecoline + antioxidants on ROS levels in <span class="html-italic">C. elegans</span>. (<b>A</b>) Fluorescence pictures of ROS. (<b>B</b>) Quantification of ROS accumulation. CK: 0 mM; NC: 0.04 mM antioxidants: curcumin; phillyrin; vitamin C; melatonin; lycopene; PC: 0.4 mM arecoline. Values followed by the different lowercase letters within a row represent the significant difference (Tukey tests, <span class="html-italic">p &lt;</span> 0.05). One-way ANOVA with Tukey post hoc test.</p> Full article ">Figure 7
<p>Effect of arecoline + lycopene on the toxicity in <span class="html-italic">C. elegans</span>. (<b>A</b>) Lifespan. (<b>B</b>) Mean lifespan. (<b>C</b>) Body length. (<b>D</b>) Body width. (<b>E</b>) Head thrashes. (<b>F</b>) Body bends. (<b>G</b>) Average number of progent. CK: 0 mM; NC: 0.04 mM lycopene; PC: 0.4 mM arecoline. Values followed by the different lowercase letters within a row represent the significant difference (Tukey tests, <span class="html-italic">p &lt;</span> 0.05). One-way ANOVA with Tukey post hoc test.</p> Full article ">Figure 8
<p>Effect of arecoline + lycopene on apoptosis. (<b>A</b>) Gonad apoptosis, the arrows indicate the number of apoptotic cells. (<b>B</b>) The gonad apoptosis in the worms was quantified using ImageJ 1.51 software. (<b>C</b>,<b>D</b>) Cell corpses were counted in N2. (<b>E</b>,<b>F</b>) Cell corpses were counted in <span class="html-italic">ced-1</span>(e1735) strains in <span class="html-italic">C. elegans.</span> CK: 0 mM; NC: 0.04 mM lycopene; PC: 0.4 mM arecoline, a &lt; 0.05, b &lt; 0.01, c &lt; 0.001. One-way ANOVA with Tukey post hoc test.</p> Full article ">
18 pages, 308 KiB  
Review
Closed Loop Ultrafiltration Feedback Control in Hemodialysis: A Narrative Review
by Zijun Dong, Lemuel Rivera Fuentes, Sharon Rao and Peter Kotanko
Toxins 2024, 16(8), 351; https://doi.org/10.3390/toxins16080351 - 10 Aug 2024
Viewed by 529
Abstract
While life-sustaining, hemodialysis is a non-physiological treatment modality that exerts stress on the patient, primarily due to fluid shifts during ultrafiltration. Automated feedback control systems, integrated with sensors that continuously monitor bio-signals such as blood volume, can adjust hemodialysis treatment parameters, e.g., ultrafiltration [...] Read more.
While life-sustaining, hemodialysis is a non-physiological treatment modality that exerts stress on the patient, primarily due to fluid shifts during ultrafiltration. Automated feedback control systems, integrated with sensors that continuously monitor bio-signals such as blood volume, can adjust hemodialysis treatment parameters, e.g., ultrafiltration rate, in real-time. These systems hold promise to mitigate hemodynamic stress, prevent intradialytic hypotension, and improve the removal of water and electrolytes in chronic hemodialysis patients. However, robust evidence supporting their clinical application remains limited. Based on an extensive literature research, we assess feedback-controlled ultrafiltration systems that have emerged over the past three decades in comparison to conventional hemodialysis treatment. We identified 28 clinical studies. Closed loop ultrafiltration control demonstrated effectiveness in 23 of them. No adverse effects of closed loop ultrafiltration control were reported across all trials. Closed loop ultrafiltration control represents an important advancement towards more physiological hemodialysis. Its development is driven by innovations in real-time bio-signals monitoring, advancement in control theory, and artificial intelligence. We expect these innovations will lead to the prevalent adoption of ultrafiltration control in the future, provided its clinical value is substantiated in adequately randomized controlled trials. Full article
18 pages, 2701 KiB  
Article
Fungal Laccases and Fumonisin Decontamination in Co-Products of Bioethanol from Maize
by Marianela Bossa, Noelia Edith Monesterolo, María del Pilar Monge, Paloma Rhein, Sofía Noemí Chulze, María Silvina Alaniz-Zanon and María Laura Chiotta
Toxins 2024, 16(8), 350; https://doi.org/10.3390/toxins16080350 - 10 Aug 2024
Viewed by 788
Abstract
Maize (Zea mays L.) may be infected by Fusarium verticillioides and F. proliferatum, and consequently contaminated with fumonisins (FBs), as well as the co-products of bioethanol intended for animal feed. Laccase enzymes have a wide industrial application such as mycotoxin degradation. [...] Read more.
Maize (Zea mays L.) may be infected by Fusarium verticillioides and F. proliferatum, and consequently contaminated with fumonisins (FBs), as well as the co-products of bioethanol intended for animal feed. Laccase enzymes have a wide industrial application such as mycotoxin degradation. The aims were to isolate and identify fungal laccase-producing strains, to evaluate laccase production, to determine the enzymatic stability under fermentation conditions, and to analyse the effectiveness in vitro of enzymatic extracts (EEs) containing laccases in degrading FB1. Strains belonging to Funalia trogii, Phellinus tuberculosus, Pleurotus ostreatus, Pycnoporus sanguineus and Trametes gallica species showed laccase activity. Different isoforms of laccases were detected depending on the evaluated species. For the FB1 decontamination assays, four enzymatic activities (5, 10, 15 and 20 U/mL) were tested, in the absence and presence of vanillic acid (VA) and 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) as redox mediators (1 and 10 mM). Trametes gallica B4-IMICO-RC EE was the most effective strain in buffer, achieving a 60% of FB1 reduction. Laccases included in EEs remained stable at different alcoholic degrees in maize steep liquor (MSL), but no significant FB1 reduction was observed under the conditions evaluated using MSL. This study demonstrate that although laccases could be good candidates for the development of a strategy to reduce FB1, further studies are necessary to optimise this process in MSL. Full article
(This article belongs to the Special Issue Effect of Mycotoxins on Crops and Their Prevention)
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<p>Specific and enzymatic activities of laccase enzymes from different enzymatic extracts of the laccase producing fungal isolates and the control strain (<span class="html-italic">Funalia trogii</span> B1-IMICO-RC), inoculated in broth for the laccase production. The enzymatic substrate used for the enzymatic activity determination was 2,2′-azino-di-3 ethylbenzothiazoline (ABTS). Error bars indicate the standard deviation within each treatment. Different capital letters denote significant differences between enzymatic activities (<span class="html-italic">p</span> value &lt; 0.01), and different lowercase letters indicate significant differences between specific activities (<span class="html-italic">p</span> value &lt; 0.01).</p> Full article ">Figure 2
<p>Maximum specific activity observed during the 24-day incubation period of the laccase enzymes contained in the different enzymatic extracts of the laccase producing fungal isolates and the control strain (<span class="html-italic">Funalia trogii</span> B1-IMICO-RC), inoculated in broth for the laccase production. The enzymatic substrate used for the enzymatic activity determination was ABTS. Error bars indicate the standard deviation within each treatment. Different letters indicate significant differences between specific activities (<span class="html-italic">p</span> value &lt; 0.01).</p> Full article ">Figure 3
<p>Native-PAGE (left side of each strain) vs. zymogram (right side of each strain) of the concentrated enzymatic extracts. Images of both gels were cut and presented one next to the other to show a better comparison of the results. Numbered arrows indicate the laccase or laccase isoenzyme synthesised by each strain. MW: molecular weight marker.</p> Full article ">Figure 4
<p>Reduction percentages of FB<sub>1</sub> in the in vitro assays using buffer medium without redox mediators. A value of 100% signifies the total reduction of FB<sub>1</sub> compared to the control (0% reduction). Error bars represent the standard deviation of the replicates for each treatment. Different letters on the bars denote significant differences between treatments (<span class="html-italic">p</span> value &lt; 0.01).</p> Full article ">Figure 5
<p>Stability of laccases from <span class="html-italic">Trametes gallica</span> B4-IMICO-RC (<b>a</b>) and <span class="html-italic">T. gallica</span> B7-IMICO-RC (<b>b</b>) under different alcoholic degrees in maize steep liquor (MSL) which was obtained from a bioethanol production process. M0, M12, M24 and M36 are samples collected at different times from the fermentation stage, corresponding to 0, 12, 24 and 36 h, respectively. The bars indicate the enzymatic activity (time 0 and 1 h) and the dotts linked by a dotted line represent the alcoholic degree of the MSL samples, measured at 0 h. Error bars indicate the standard deviation within each treatment. Different letters show significant differences between treatments (<span class="html-italic">p</span> value &lt; 0.01).</p> Full article ">
11 pages, 972 KiB  
Article
Assessment of Within- and Inter-Patient Variability of Uremic Toxin Concentrations in Children with CKD
by Evelien Snauwaert, Stefanie De Buyser, An Desloovere, Wim Van Biesen, Ann Raes, Griet Glorieux, Laure Collard, Koen Van Hoeck, Maria Van Dyck, Nathalie Godefroid, Johan Vande Walle and Sunny Eloot
Toxins 2024, 16(8), 349; https://doi.org/10.3390/toxins16080349 - 9 Aug 2024
Viewed by 551
Abstract
To promote improved trial design in upcoming randomized clinical trials in childhood chronic kidney disease (CKD), insight in the within- and inter-patient variability of uremic toxins with its nutritional, treatment- and patient-related confounding factors is of utmost importance. In this study, the within- [...] Read more.
To promote improved trial design in upcoming randomized clinical trials in childhood chronic kidney disease (CKD), insight in the within- and inter-patient variability of uremic toxins with its nutritional, treatment- and patient-related confounding factors is of utmost importance. In this study, the within- and inter-patient variability of a selection of uremic toxins in a longitudinal cohort of children diagnosed with CKD was assessed, using the intraclass correlation coefficient (ICC) and the within-patient coefficient of variation (CV). Subsequently, the contribution of anthropometry, estimated glomerular filtration rate (eGFR), dietary fiber and protein, and use of (prophylactic) antibiotics to uremic toxin variability was evaluated. Based on 403 observations from 62 children (median seven visits per patient; 9.4 ± 5.3 years; 68% males; eGFR 38.5 [23.1; 64.0] mL/min/1.73 m2) collected over a maximum of 2 years, we found that the within-patient variability is high for especially protein-bound uremic toxins (PBUTs) (ICC < 0.7; within-patient CV 37–67%). Moreover, eGFR was identified as a predominant contributor to the within- and inter-patient variability for the majority of solutes, while the impact of the child’s anthropometry, fiber and protein intake, and antibiotics on the variability of uremic toxin concentrations was limited. Based on these findings, we would recommend future intervention studies that attempt to decrease uremic toxin levels to select a (non-dialysis) CKD study population with a narrow eGFR range. As the expected effect of the selected intervention should exceed the inter-patient variability of the selected uremic toxins, a narrow eGFR range might aid in improving the trial design. Full article
(This article belongs to the Section Uremic Toxins)
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<p>Plots of the observed protein-bound toxin concentrations in the function of ascending within-subject geometric mean toxin concentrations. Abbreviations: HA: hippuric acid; IAA: indole acetic acid; IxS: indoxyl sulfate; PCS: p-cresyl sulfate; CMPF: 3-carboxy-4-methyl-5-propyl-2furanpropionic acid.</p> Full article ">Figure 2
<p>Study flow chart. CKD: chronic kidney disease, TX: kidney transplant.</p> Full article ">
26 pages, 5099 KiB  
Article
Potential Ancestral Conoidean Toxins in the Venom Cocktail of the Carnivorous Snail Raphitoma purpurea (Montagu, 1803) (Neogastropoda: Raphitomidae)
by Giacomo Chiappa, Giulia Fassio, Maria Vittoria Modica and Marco Oliverio
Toxins 2024, 16(8), 348; https://doi.org/10.3390/toxins16080348 - 9 Aug 2024
Viewed by 693
Abstract
Venomous marine gastropods of the superfamily Conoidea possess a rich arsenal of toxins, including neuroactive toxins. Venom adaptations might have played a fundamental role in the radiation of conoideans; nevertheless, there is still no knowledge about the venom of the most diversified family [...] Read more.
Venomous marine gastropods of the superfamily Conoidea possess a rich arsenal of toxins, including neuroactive toxins. Venom adaptations might have played a fundamental role in the radiation of conoideans; nevertheless, there is still no knowledge about the venom of the most diversified family of the group: Raphitomidae Bellardi, 1875. In this study, transcriptomes were produced from the carcase, salivary glands, and proximal and distal venom ducts of the northeastern Atlantic species Raphitoma purpurea (Montagu, 1803). Using a gut barcoding approach, we were also able to report, for the first time, molecular evidence of a vermivorous diet for the genus. Transcriptomic analyses revealed over a hundred putative venom components (PVC), including 69 neurotoxins. Twenty novel toxin families, including some with high levels of expansion, were discovered. No significant difference was observed between the distal and proximal venom duct secretions. Peptides related to cone snail toxins (Cerm06, Pgam02, and turritoxin) and other venom-related proteins (disulfide isomerase and elevenin) were retrieved from the salivary glands. These salivary venom components may constitute ancestral adaptations for venom production in conoideans. Although often neglected, salivary gland secretions are of extreme importance for understanding the evolutionary history of conoidean venom. Full article
(This article belongs to the Special Issue Structure, Function and Evolution of Conotoxins)
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<p>Results of the differential expression analysis; (<b>a</b>) Correlation matrix among samples, yellow colour represents high correlation; (<b>b</b>) Tissue-specific gene expression plots for venom duct vs. salivary glands (above) and distal vs. proximal venom duct (below), red dots represent differentially expressed transcripts.</p> Full article ">Figure 2
<p>UpsetR diagram of candidate toxic components identified by Detox with relative frequencies of all score combinations. Constructed in RStudio [<a href="#B46-toxins-16-00348" class="html-bibr">46</a>]. Bars scaled with the “log2” parameter. Values above the bars represent the frequencies. The pie chart shows the results of the PVC functional annotation (marked with “*” over the upsetR diagram bars). Score legend: S = signal sequence and no transmembrane domain; C = cysteine framework; D = domain match with Pfam; B = similarity match with the reference database; T-x: TPM in salivary glands or venom duct equal to or greater than x.</p> Full article ">Figure 3
<p>Maximum likelihood relationships of the amino acid sequences of (<b>a</b>) disulfide isomerases and (<b>b</b>) elevenin from <span class="html-italic">Raphitoma purpurea</span> (highlighted in red) with the gastropod sequences from NCBI and UniProtKB. Black dots mark nodes with Ultrafast support &gt; 95%.</p> Full article ">Figure 4
<p>Peptide sequence alignment of (<b>a</b>) turritoxin, (<b>b</b>) conotoxin Pmag02, and (<b>c</b>) conotoxin Cerm06 from <span class="html-italic">Raphitoma purpurea</span> (highlighted in red) with gastropod sequences from NCBI and UniProtKB. The green arrow points at the predicted signal sequence cleavage site and the red arrows highlight the cysteine framework.</p> Full article ">
23 pages, 928 KiB  
Systematic Review
The Effect of Botulinum Neurotoxin-A (BoNT-A) on Muscle Strength in Adult-Onset Neurological Conditions with Focal Muscle Spasticity: A Systematic Review
by Renée Gill, Megan Banky, Zonghan Yang, Pablo Medina Mena, Chi Ching Angie Woo, Adam Bryant, John Olver, Elizabeth Moore and Gavin Williams
Toxins 2024, 16(8), 347; https://doi.org/10.3390/toxins16080347 - 8 Aug 2024
Viewed by 763
Abstract
Botulinum neurotoxin-A (BoNT-A) injections are effective for focal spasticity. However, the impact on muscle strength is not established. This study aimed to investigate the effect of BoNT-A injections on muscle strength in adult neurological conditions. Studies were included if they were Randomised Controlled [...] Read more.
Botulinum neurotoxin-A (BoNT-A) injections are effective for focal spasticity. However, the impact on muscle strength is not established. This study aimed to investigate the effect of BoNT-A injections on muscle strength in adult neurological conditions. Studies were included if they were Randomised Controlled Trials (RCTs), non-RCTs, or cohort studies (n ≥ 10) involving participants ≥18 years old receiving BoNT-A injection for spasticity in their upper and/or lower limbs. Eight databases (CINAHL, Cochrane, EMBASE, Google Scholar, Medline, PEDro, Pubmed, Web of Science) were searched in March 2024. The methodology followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and was registered in the Prospective Register of Systematic Reviews (PROSPERO: CRD42022315241). Quality was assessed using the modified Downs and Black checklist and the PEDro scale. Pre-/post-injection agonist, antagonist, and global strength outcomes at short-, medium-, and long-term time points were extracted for analysis. Following duplicate removal, 8536 studies were identified; 54 met the inclusion criteria (3176 participants) and were rated as fair-quality. Twenty studies were analysed as they reported muscle strength specific to the muscle injected. No change in agonist strength after BoNT-A injection was reported in 74% of the results. Most studies’ outcomes were within six weeks post-injection, with few long-term results (i.e., >three months). Overall, the impact of BoNT-A on muscle strength remains inconclusive. Full article
(This article belongs to the Special Issue The Botulinum Toxin and Spasticity: Exploring New Horizons)
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<p>Flow diagram of study identification to obtain articles for review inclusion.</p> Full article ">
24 pages, 4644 KiB  
Article
Feasibility of Adjunct Therapy with a Robotic Hand Orthosis after Botulinum Toxin Injections in Persons with Spasticity: A Pilot Study
by Raffaele Ranzani, Margherita Razzoli, Pierre Sanson, Jaeyong Song, Salvatore Galati, Carlo Ferrarese, Olivier Lambercy, Alain Kaelin-Lang and Roger Gassert
Toxins 2024, 16(8), 346; https://doi.org/10.3390/toxins16080346 - 8 Aug 2024
Viewed by 750
Abstract
Upper-limb spasticity, frequent after central nervous system lesions, is typically treated with botulinum neurotoxin type A (BoNT-A) injections to reduce muscle tone and increase range of motion. However, performing adjunct physical therapy post-BoNT-A can be challenging due to residual weakness or spasticity. This [...] Read more.
Upper-limb spasticity, frequent after central nervous system lesions, is typically treated with botulinum neurotoxin type A (BoNT-A) injections to reduce muscle tone and increase range of motion. However, performing adjunct physical therapy post-BoNT-A can be challenging due to residual weakness or spasticity. This study evaluates the feasibility of hand therapy using a robotic hand orthosis (RELab tenoexo) with a mobile phone application as an adjunct to BoNT-A injections. Five chronic spastic patients participated in a two-session pilot study. Functional (Box and Block Test (BBT), Action Research Arm Test (ARAT)), and muscle tone (Modified Ashworth Scale (MAS)) assessments were conducted to assess functional abilities and impairment, along with usability evaluations. In the first session, subjects received BoNT-A injections, and then they performed a simulated unsupervised therapy session with the RELab tenoexo in a second session a month later. Results showed that BoNT-A reduced muscle tone (from 12.2 to 7.4 MAS points). The addition of RELab tenoexo therapy was safe, led to functional improvements in four subjects (two-cube increase in BBT as well as 2.8 points in grasp and 1.3 points in grip on ARAT). Usability results indicate that, with minor improvements, adjunct RELab tenoexo therapy could enhance therapy doses and, potentially, long-term outcomes. Full article
(This article belongs to the Special Issue The Botulinum Toxin and Spasticity: Exploring New Horizons)
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<p>Checklist results represented as a heatmap. The results averaged over the subjects, and items are presented on the right and at the bottom of the heat map, respectively. The items “DN”, “CO”, and “DF” were evaluated during the assessments at S0 and S1, the other items only at S1. The tasks are ranked successful (1, green, i.e., no problem/issue in item completion without external intervention) or failed (0, red, i.e., failure and/or external intervention required to solve the item). Intermediate nuances between green and red are average values (Av) between 0 and 1 over subjects and items, respectively. DN: donning; CO: start-up and control; TH: all therapy exercises; ST: Stretching; PY: Pyramid; PA: Painting; *: item that could not be performed without external intervention before BoNT-A injections (S0) but could be performed after BoNT-A injections (S1).</p> Full article ">Figure 2
<p>Subject one performing the painting exercise (difficulty level 2) and the resulting sheet after 10 min of exercise.</p> Full article ">Figure 3
<p>Box and Block Test box-plot results for all five subjects (<b>A</b>) and for the four subjects with muscle hypertonia in finger flexors (i.e., MAS ≥ 1) (<b>B</b>). On the left of the black line are the results at the baseline (S0), while on the right of the line are the changes in performance when using the RELab tenoexo before BoNT-A injections (S0), four to six weeks after BoNT-A injections without RELab tenoexo (S1), and adjunct BoNT-A injections and RELab tenoexo (S1). °: individual scores; ×: mean value.</p> Full article ">Figure 4
<p>Action Research Arm Test (ARAT) results for all the subjects (<b>top</b>) and for the four subjects with muscle hypertonia in finger flexors (i.e., MAS ≥ 1) (<b>bottom</b>). On the left of the black line are the results at the baseline (S0), while on the right of the line are the changes in performance when using the RELab tenoexo (S0), BoNT-A injections (S1), and adjunct BoNT-A injections and RELab tenoexo (S1). °: individual scores; ×: mean value.</p> Full article ">Figure 5
<p>One of the study participants (subject 5) performs the hand muscle tone assessment with ReHandyBot (RHB), a haptic device including a physical (i.e., instrumented finger pads) and a graphical user interface to perform assessments and therapy exercises. During the muscle tone assessment, the subject has to relax their hand while the robot applies six 150 ms hand opening perturbations of 20 mm amplitude.</p> Full article ">Figure 6
<p>RELab tenoexo overview: the RELab tenoexo can be worn by the user or mounted on a wheelchair (<b>left</b>). The hand module (148 g) can be placed on a user’s hand using a glove with Velcro and button-clips and is actuated by motors placed in the actuation unit (720 g) together with the electronics. Different intention detection strategies (e.g., push button, mobile phone application) can be used to trigger the motion of the RHO (<b>right</b>).</p> Full article ">Figure 7
<p>Grasp functions of the RELab tenoexo (adapted from [<a href="#B31-toxins-16-00346" class="html-bibr">31</a>]): the compliant structure of the finger mechanism allows for adapting to the shape of different objects. Four grasp types (denomination according to [<a href="#B71-toxins-16-00346" class="html-bibr">71</a>]) can be executed with the RELab tenoexo by moving the thumb slider at the back of the hand module from medial to lateral.</p> Full article ">Figure 8
<p>RELab tenoexo mobile phone application. (<b>A</b>) Main screen offering five functions: (<b>i</b>) battery information, (<b>ii</b>) emergency stop, (<b>iii</b>) opening and closing of the hand, (<b>iv</b>) changing of the grip type (i.e., cylindrical grip, key grip), and (<b>v</b>) force selection. (<b>B</b>) Therapy screen: the user can navigate between different therapy exercises (represented with an icon and a short description (<b>i</b>)) using left/right arrows (<b>ii</b>) and start the exercise with the green button (<b>iii</b>). During the exercise, the application records end-point accelerations from an XSens DOT accelerometer, which can be attached to the dorsum of the RELab tenoexo, but this feature was not used for the scope of this study.</p> Full article ">Figure 9
<p>Example of therapy setup with the RELab tenoexo. (<b>A</b>) Based on a predefined therapy plan (i), the user can use the mobile phone application (ii) to perform a set of therapy exercises with the RELab tenoexo (iii). (i.e., Cleaning (iv), Pyramid (v)). Mildly impaired subjects will perform fine functional tasks (i.e., Puzzle (vi), Painting (vii)). (<b>B</b>) The therapy exercises have increasing difficulty, depending on the subject’s ability level. Severely impaired subjects will perform hand opening–closing motor training or the Stretching exercise. Moderately impaired subjects will perform gross functional tasks requiring the recruitment of hand and proximal upper limb joints. (<b>C</b>) Tabular therapy plan used in the study. Participants identified “Wednesday” and performed the three 10 min mandatory exercises: Stretching, Pyramid (difficulty level 1: glasses of 3 cm), and Painting (difficulty level 2).</p> Full article ">Figure 10
<p>The pilot study consisted of two sessions, S0 and S1. In the morning of S0, subjects performed baseline assessments while the experimenter evaluated whether the subject could autonomously don/doff and control the RELab tenoexo using the mobile phone application. In the afternoon, subjects received BoNT-A injections. After four to six weeks (session S1), when the BoNT-A effect peaks, the subjects repeated the assessments. In the afternoon, they performed a simulated unsupervised therapy session with the RELab tenoexo consisting of three 10 min exercises (Stretching, Pyramid, and Painting) performed with the mobile phone application while the experimenter recorded which tasks the subject could not perform autonomously. At the end of the study, the subjects answered usability questionnaires. * = assessment performed with and without the RELab tenoexo. Abbreviations: MAS = Modified Ashworth Scale, BBT = Box and Block Test, SUS = System Usability Scale, rawTLX = raw Task Load Index, MAUQ = mHealth App Usability Questionnaire, QUEST 2.0 = Quebec User Evaluation of Satisfaction with Assistive Technology 2.0.</p> Full article ">
19 pages, 2280 KiB  
Systematic Review
A Systematic Review of Uremic Toxin Concentrations and Cardiovascular Risk Markers in Pediatric Chronic Kidney Disease
by Heshini Dalpathadu, Aly Muhammad Salim, Andrew Wade and Steven C. Greenway
Toxins 2024, 16(8), 345; https://doi.org/10.3390/toxins16080345 - 8 Aug 2024
Viewed by 762
Abstract
Chronic kidney disease (CKD) can lead to cardiac dysfunction in a condition known as cardiorenal syndrome (CRS). It is postulated that the accumulation of uremic toxins in the bloodstream, as a consequence of declining kidney function, may contribute to these adverse cardiac effects. [...] Read more.
Chronic kidney disease (CKD) can lead to cardiac dysfunction in a condition known as cardiorenal syndrome (CRS). It is postulated that the accumulation of uremic toxins in the bloodstream, as a consequence of declining kidney function, may contribute to these adverse cardiac effects. While CRS in adults has been extensively studied, there is a significant knowledge gap with pediatric patients. Uremic toxin levels in children remain inadequately characterized and quantified compared to adults. This review aims to systematically evaluate the association between uremic toxin concentrations and cardiac changes in pediatric CRS and to examine the impact of different dialysis modalities, specifically hemodialysis and peritoneal dialysis, on uremic toxin clearance and cardiovascular parameters. To address this, we conducted a systematic literature search of PubMed, following PRISMA guidelines. We used the terms “uremic toxins” and “cardiorenal syndrome” with variations in syntax to search for studies discussing the relationship between uremic toxin levels in CKD, the subsequent impact on cardiac parameters, and the emergence of cardiac dysfunction. Full-text articles written in English, conducted on humans aged from birth to 18 years, and published until December 2021 were included. A comprehensive literature search yielded six studies, and their risk of bias was assessed using JBI Critical Appraisal Checklists. Our systematic review is registered on PROSPERO, number CRD42023460072. This synthesis intends to provide an understanding of the role of uremic toxins in pediatric CRS. The findings reveal that pediatric patients with end-stage CKD on dialysis exhibit elevated uremic toxin levels, which are significantly associated with cardiovascular disease parameters. Additionally, the severity of CKD correlated with higher uremic toxin levels. No conclusive evidence was found to support the superiority of either hemodialysis or peritoneal dialysis in terms of uremic toxin clearance or cardiovascular outcomes. More pediatric-specific standardized and longitudinal studies are needed to develop targeted treatments and improve clinical outcomes and the quality of life for affected children. Full article
(This article belongs to the Section Uremic Toxins)
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<p>The journey of uremic toxins from their generation to elimination. Uremic toxins are metabolic by-products or waste substances originating from dietary intake, gastrointestinal tract, and liver. These toxins enter the bloodstream and are subsequently transported to the kidneys for filtration and removal from the body in the urine. In chronic kidney disease, the effective elimination of these toxins is compromised. The accumulated uremic toxins can cause cardiovascular complications through myocardial toxicity and systemic inflammatory responses. This cardiac dysfunction can reduce renal perfusion and enhance fluid retention due to decreased cardiac output, further exacerbating cardiac load. The resulting renal dysfunction, characterized by inadequate filtration and fluid imbalance, perpetuates a feedback loop, worsening the progression of cardiorenal syndrome. Created with BioRender.</p> Full article ">Figure 2
<p>PRISMA flow diagram of the study selection process.</p> Full article ">Figure 3
<p>Risk of bias. Joanna Briggs Institute Critical Appraisal Checklist. Created with BioRender [<a href="#B19-toxins-16-00345" class="html-bibr">19</a>,<a href="#B20-toxins-16-00345" class="html-bibr">20</a>,<a href="#B21-toxins-16-00345" class="html-bibr">21</a>,<a href="#B22-toxins-16-00345" class="html-bibr">22</a>,<a href="#B23-toxins-16-00345" class="html-bibr">23</a>,<a href="#B24-toxins-16-00345" class="html-bibr">24</a>].</p> Full article ">Figure 4
<p>Venn diagram depicting the categorization of studies based on the classes of uremic toxins examined and their association with cardiac dysfunction. The diagram illustrates the overlap between studies investigating small water-soluble uremic toxins, middle molecule uremic toxins, and protein-bound uremic toxins. The adjacent table provides a summary of each study’s focus, indicating the specific uremic toxin classes examined and whether cardiac dysfunction was assessed. Created with BioRender [<a href="#B19-toxins-16-00345" class="html-bibr">19</a>,<a href="#B20-toxins-16-00345" class="html-bibr">20</a>,<a href="#B21-toxins-16-00345" class="html-bibr">21</a>,<a href="#B22-toxins-16-00345" class="html-bibr">22</a>,<a href="#B23-toxins-16-00345" class="html-bibr">23</a>,<a href="#B24-toxins-16-00345" class="html-bibr">24</a>].</p> Full article ">
17 pages, 2302 KiB  
Article
Modulation of Growth and Mycotoxigenic Potential of Pineapple Fruitlet Core Rot Pathogens during In Vitro Interactions
by Manon Vignassa, Christian Soria, Noël Durand, Charlie Poss, Jean-Christophe Meile, Marc Chillet and Sabine Schorr-Galindo
Toxins 2024, 16(8), 344; https://doi.org/10.3390/toxins16080344 - 7 Aug 2024
Viewed by 562
Abstract
Pineapple Fruitlet Core Rot (FCR) is a fungal disease characterized by a multi-pathogen pathosystem. Recently, Fusarium proliferatum, Fusarium oxysporum, and Talaromyces stollii joined the set of FCR pathogens until then exclusively attributed to Fusarium ananatum. The particularity of FCR relies on the presence of [...] Read more.
Pineapple Fruitlet Core Rot (FCR) is a fungal disease characterized by a multi-pathogen pathosystem. Recently, Fusarium proliferatum, Fusarium oxysporum, and Talaromyces stollii joined the set of FCR pathogens until then exclusively attributed to Fusarium ananatum. The particularity of FCR relies on the presence of healthy and diseased fruitlets within the same infructescence. The mycobiomes associated with these two types of tissues suggested that disease occurrence might be triggered by or linked to an ecological chemical communication-promoting pathogen(s) development within the fungal community. Interactions between the four recently identified pathogens were deciphered by in vitro pairwise co-culture bioassays. Both fungal growth and mycotoxin production patterns were monitored for 10 days. Results evidenced that Talaromyces stollii was the main fungal antagonist of Fusarium species, reducing by 22% the growth of Fusarium proliferatum. A collapse of beauvericin content was observed when FCR pathogens were cross-challenged while fumonisin concentrations were increased by up to 7-fold. Antagonism between Fusarium species and Talaromyces stollii was supported by the diffusion of a red pigmentation and droplets of red exudate at the mycelium surface. This study revealed that secondary metabolites could shape the fungal pathogenic community of a pineapple fruitlet and contribute to virulence promoting FCR establishment. Full article
(This article belongs to the Special Issue Toxins: 15th Anniversary)
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<p>FCR pathogen colony aspects on PDA after 10 days of incubation in single culture of (<b>A</b>) <span class="html-italic">Fusarium ananatum</span> (<span class="html-italic">Fa</span>), (<b>B</b>) <span class="html-italic">Fusarium oxysporum</span> (<span class="html-italic">Fo</span>), (<b>C</b>) <span class="html-italic">Fusarium proliferatum</span> (<span class="html-italic">Fp</span>), and (<b>D</b>) <span class="html-italic">Talaromyces stollii</span> (<span class="html-italic">Ts</span>) or in condition of co-culture corresponding to (<b>E</b>) <span class="html-italic">F. ananatum</span> versus <span class="html-italic">F. proliferatum</span> (<span class="html-italic">Fa</span> vs. <span class="html-italic">Fp</span>), (<b>F</b>) <span class="html-italic">F. oxysporum</span> versus <span class="html-italic">F. proliferatum</span> (<span class="html-italic">Fo</span> vs. <span class="html-italic">Fp</span>), (<b>G</b>) <span class="html-italic">F. ananatum</span> versus <span class="html-italic">F. oxysporum</span> (<span class="html-italic">Fa</span> vs. <span class="html-italic">Fo</span>), (<b>H</b>) <span class="html-italic">F. ananatum</span> versus <span class="html-italic">T. stollii</span> (<span class="html-italic">Fa</span> vs. <span class="html-italic">Ts</span>), (<b>I</b>) <span class="html-italic">F. oxysporum</span> versus <span class="html-italic">T. stollii</span> (<span class="html-italic">Fo</span> vs. <span class="html-italic">Ts</span>), and (<b>J</b>) <span class="html-italic">F. proliferatum</span> versus <span class="html-italic">T. stollii</span> (<span class="html-italic">Fp</span> vs. <span class="html-italic">Ts</span>), well diameter = 2 cm.</p> Full article ">Figure 2
<p>Influence of FCR pathogen interactions on colony growth parameters. (<b>A</b>–<b>D</b>) panels show fungal growth evolution over 10 days for co-cultures and corresponding single cultures. Gray dashed lines represent 50% of well area (1.57 cm<sup>2</sup>). (<b>E</b>–<b>H</b>) panels indicate associated growth inhibition ratio calculated from data at day 10 for each co-culture condition. (<b>A</b>,<b>E</b>); (<b>B</b>,<b>F</b>); (<b>C</b>,<b>G</b>) and (<b>D</b>,<b>H</b>) show growth profiles and inhibition potential on <span class="html-italic">F. ananatum</span>, <span class="html-italic">F. oxysporum</span>, <span class="html-italic">F. proliferatum</span>, and <span class="html-italic">T. stollii</span>, respectively. Vertical bars represent standard error of means (<span class="html-italic">n</span> = 9). Letters show significantly different inhibition ratios for each species following Tukey’s multiple comparison test at <span class="html-italic">p</span> &lt; 0.05. <span class="html-italic">Fa</span>: <span class="html-italic">Fusarium ananatum</span>, <span class="html-italic">Fo</span>: <span class="html-italic">Fusarium oxysporum</span>, <span class="html-italic">Fp</span>: <span class="html-italic">Fusarium proliferatum</span>, and <span class="html-italic">Ts</span>: <span class="html-italic">Talaromyces stollii</span>.</p> Full article ">Figure 3
<p>Dynamics of fumonisin B<sub>1</sub> (FB<sub>1</sub>), fumonisin B<sub>2</sub> (FB<sub>2</sub>), and beauvericin (BEA) during co-culture bioassays between <span class="html-italic">Fusarium</span> spp. and <span class="html-italic">Talaromyces stollii</span>. (<b>A</b>) <span class="html-italic">F. ananatum</span> versus <span class="html-italic">T. stollii</span> (<span class="html-italic">Fa</span> vs. <span class="html-italic">Ts</span>), (<b>B</b>) <span class="html-italic">F. oxysporum</span> vs. <span class="html-italic">T. stollii</span> (<span class="html-italic">Fo</span> vs. <span class="html-italic">Ts</span>), and (<b>C</b>) <span class="html-italic">F. proliferatum</span> versus <span class="html-italic">T. stollii</span> (<span class="html-italic">Fp</span> vs. <span class="html-italic">Ts</span>). Vertical bars represent standard deviation (<span class="html-italic">n</span> = 3). Differences between single and co-cultures were either significant at <span class="html-italic">p</span> &lt; 0.05 (*), <span class="html-italic">p</span> &lt; 0.01 (**), and <span class="html-italic">p</span> &lt; 0.001 (***) or non-significant (ns) for each toxin and each time point. Dot colors indicate significant differences according to Tukey’s multiple comparison test at <span class="html-italic">p</span> &lt; 0.05 and correspond to ‘a’ (yellow dots), ‘b’ (red dots), ‘ab’ (blue dots), and ‘c’ (green dots).</p> Full article ">Figure 4
<p>Dynamics of fumonisin B<sub>1</sub> (FB<sub>1</sub>), fumonisin B<sub>2</sub> (FB<sub>2</sub>), and beauvericin (BEA) during co-culture bioassays between <span class="html-italic">Fusarium</span> spp. reported as FCR pathogens. (<b>A</b>) <span class="html-italic">F. oxysporum</span> versus <span class="html-italic">F. ananatum</span> (<span class="html-italic">Fo</span> vs. <span class="html-italic">Fa</span>), (<b>B</b>) <span class="html-italic">F. ananatum</span> vs. <span class="html-italic">F. proliferatum</span> (<span class="html-italic">Fa</span> vs. <span class="html-italic">Fp</span>), and (<b>C</b>) <span class="html-italic">F. oxysporum</span> versus <span class="html-italic">F. proliferatum</span> (<span class="html-italic">Fo</span> vs. <span class="html-italic">Fp</span>). Vertical bars represent standard deviation (<span class="html-italic">n</span> = 3). Differences between single and co-cultures were either significant at <span class="html-italic">p</span> &lt; 0.05 (*), <span class="html-italic">p</span> &lt; 0.01 (**), and <span class="html-italic">p</span> &lt; 0.001 (***) or non-significant (ns) for each toxin and each time point. Dot colors indicate significant differences according to Tukey’s multiple comparison test at <span class="html-italic">p</span> &lt; 0.05 and correspond to ‘a’ (yellow dots), ‘b’ (red dots), and ‘ab’ (blue dots).</p> Full article ">
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