Cyanobacteria are infamous producers of toxins. While the toxic potential of planktonic cyanobacterial blooms is well documented, the ecosystem level effects of toxigenic benthic and epiphytic cyanobacteria are an understudied threat. The freshwater epiphytic cyanobacterium Aetokthonos hydrillicola has recently been shown to produce the “eagle killer” neurotoxin aetokthonotoxin (AETX) causing the fatal neurological disease vacuolar myelinopathy. The disease affects a wide array of wildlife in the southeastern United States, most notably waterfowl and birds of prey, including the bald eagle. In an assay for cytotoxicity, we found the crude extract of the cyanobacterium to be much more potent than pure AETX, prompting further investigation. Here, we describe the isolation and structure elucidation of the aetokthonostatins (AESTs), linear peptides belonging to the dolastatin compound family, featuring a unique modification of the C-terminal phenylalanine-derived moiety. Using immunofluorescence microscopy and molecular modeling, we confirmed that AEST potently impacts microtubule dynamics and can bind to tubulin in a similar matter as dolastatin 10. We also show that AEST inhibits reproduction of the nematode Caenorhabditis elegans . Bioinformatic analysis revealed the AEST biosynthetic gene cluster encoding a nonribosomal peptide synthetase/polyketide synthase accompanied by a unique tailoring machinery. The biosynthetic activity of a specific N-terminal methyltransferase was confirmed by in vitro biochemical studies, establishing a mechanistic link between the gene cluster and its product.

Aims: Taste modifies eating behaviour, impacting body weight and potentially obesity development. The Obese Taste Bud (OTB) Study is a prospective cohort study launched in 2020 at the University of Leipzig Obesity Centre in cooperation with the HI‐MAG Institute. OTB will test the hypothesis that taste cell homeostasis and taste perception are linked to obesity. Here, we provide the study design, data collection process and baseline characteristics. Materials and Methods: Participants presenting overweight, obesity or normal weight undergo taste and smell tests, anthropometric, and taste bud density (TBD) assessment on Day 1. Information on physical and mental health, eating behaviour, physical activity, and dental hygiene are obtained, while biomaterial (saliva, tongue swap, blood) is collected in the fasted state. Further blood samples are taken during a glucose tolerance test. A stool sample is collected at home prior to Day 2, on which a taste bud biopsy follows dental examination. A subsample undergoes functional magnetic resonance imaging while exposed to eating‐related cognitive tasks. Follow‐up investigations after conventional weight loss interventions and bariatric surgery will be included. Results: Initial results show that glycated haemoglobin levels and age are negatively associated with TBD, while an unfavourable metabolic profile, current dieting, and vegan diet are related to taste perception. Olfactory function negatively correlates with age and high‐density lipoprotein cholesterol. Conclusion: Initial findings suggest that metabolic alterations are relevant for taste and smell function and TBD. By combining omics data from collected biomaterial with physiological, metabolic and psychological data related to taste perception and eating behaviour, the OTB study aims to strengthen our understanding of taste perception in obesity.

Background and aims: Saliva is essential for the proper dilution and distribution of taste molecules on the tongue. It harbors extracellular vesicles (EVs), which mediate cell–cell communication. Changes in the composition of salivary EVs may arise under obese conditions and may potentially be involved in taste sensation and dysregulated eating behavior. Therefore, this study addresses the relationship between the size and concentration of salivary EVs and metabolic shifts in obesity or factors of taste sensation. Materials and methods: A total of 119 participants in the Obese Taste Bud (OTB) Study were included, who performed a standardized taste test, underwent taste bud density assessment, and were phenotypically characterized for anthropometrics, blood- and saliva adipokine levels, and various metabolic factors. Utilizing size exclusion chromatography followed by ultrafiltration, EVs were extracted from 2 mL of actively secreted saliva. EVs were characterized using nanoparticle tracking analyses, Western blot, and scanning transmission electron microscopy. Finally, group comparisons and bivariate correlation analyses were conducted. Results: Among the total cohort, the median size of salivary EVs was 190.05 nm, and the overall concentration ranged from 1.4 × 10⁷ to 1.76 × 10⁹ per mL of saliva. The size range and concentration of EVs per mL are negatively correlated (p = 0.0002, r = −0.264). Comparing lean participants (mean rank of 45.98) with those presenting obesity (mean rank of 34.46), a significant difference in the salivary EV content was observed (p = 0.029). Body weight, BMI, arm and calf circumferences, as well as the percentage of body fat were all negatively related to the concentration of EVs in all study participants (all p < 0.05, r > −0.2). No associations were found between the EV parameters and taste perception but serum alkaline phosphatase levels were negatively correlated (p = 0.007, r = −0.284) and adiponectin serum levels were positively correlated to the EV concentration (p = 0.036, r = 0.208). Conclusion: The current study provides evidence for the relation between salivary EVs and anthropometric as well as metabolic parameters of obesity. This can provide the basis for further research on the cargo of salivary EVs and how they may influence taste sensation, and may elucidate their potential connection to altered eating habits in obesity.

Cyanobacteria are infamous producers of toxins. While the toxic potential of planktic cyanobacterial blooms is well documented, the ecosystem level effects of toxigenic benthic and epiphytic cyanobacteria are an understudied threat. The freshwater epiphytic cyanobacterium Aetokthonos hydrillicola has recently been shown to produce the “eagle killer” neurotoxin aetokthonotoxin causing the fatal neurological disease Vacuolar Myelinopathy. The disease affects a wide array of wildlife in the southeastern United States, most notably waterfowl and birds of prey, including the bald eagle. In an assay for cytotoxicity, we found the crude extract of the cyanobacterium to be much more potent than pure aetokthonotoxin, prompting further investigation. Here, we describe the isolation and structure elucidation of the aetokthonostatins, linear peptides belonging to the dolastatin compound family, featuring a unique modification of the C-terminal phenylalanine derived moiety. Using immunofluorescence microscopy and molecular modeling, we confirmed that aetokthonostatin acts as a potent tubulin binder. We also show that aetokthonostatin inhibits reproduction of the nematode C. elegans , resulting in increased population lethality of the combined action of the two toxins produced by A. hydrillicola . Bioinformatic analysis revealed the aetokthonostatin biosynthetic gene cluster encoding a non-ribosomal peptide synthe-tase/polyketide synthase accompanied by a unique tailoring machinery. The biosynthetic activity of a specific N-terminal methyltransferase was confirmed by in vitro biochemical studies, establishing a mechanistic link between the gene cluster and its product. Significance Statement: Cyanotoxins have adverse effects on ecosystems. Our understanding of their potential risk has recently been expanded by the discovery of aetokthonotoxin, produced by the cyanobacterium Aetokthonos hydrillicola growing on invasive plants. Via trophic transfer, it acts as a neurotoxin causing mortality in animals including top predators like Bald Eagles. Closer examination of A. hydrillicola revealed that it also produces highly toxic dolastatin derivatives. A. hydrillicola is the first cultured cyanobacterium producing dolastatin derivatives, allowing us to uncover biosynthetic gene clusters of this compound family. In contrast to all other known dolastatin-producers, which are marine cyanobacteria, A. hydrillicola thrives in freshwater reservoirs, making it a potential threat also for human health. Monitoring of the cyanobacterium and its toxins is strongly recommended.

Objective Obesity might lead to alterations in salivary extracellular vesicle (EV) composition, with consequences on taste-cell transcriptomics. This project aims to characterize alterations in salivary EV number, size distribution and microRNA content from subjects with obesity and normal weight and correlate them with parameters of the metabolic syndrome. With this, we want to identify and validate EV microRNA-target gene interactions in human taste-cells. Methods Three methods of EV isolation were tested and validated - first, ultracentrifugation, precipitation using ExoQuick chemistry and size exclusion chromatography (SEC). Quantification and measurement of particle size distribution was done using nanosight technology and western blot analyses. Extraction of total RNA was carried out by miRNeasy Micro Kit. Quantification and quality control were done with Bioanalyzer Small RNA and Qubit™ microRNA Assays. RNA with good integrity was taken forward for small RNA sequencing using NovaSeq6000 platform. Results The best method to isolate EVs from saliva samples is SEC. This enabled the isolation of the highest concentration and purity of the EVs as well as the best yield of RNA including the microRNA fraction. First small RNA sequencing analyses also confirmed the presence of microRNAs within the isolated EV fraction [1] [2]. Conclusion Our established protocol allows us to isolate and characterize the EV fraction of a small volume of human saliva with consequent RNA sequencing methods. With this we are able to identify differentially expressed microRNAs in EVs from the study subjects which may offer diagnostic and therapeutic potential. Article published online: 02 May 2023 © 2023. Thieme. All rights reserved. Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany

Research Question: Can the intricate relationship between metabolic changes in obesity, alterations in taste perception, and the distribution and composition of extracellular vesicles (EVs) be unraveled? This study seeks to explore this uncharted territory by enrolling 111 subjects from the Obese Taste Bud (OTB) Study, subjecting them to both quantitative and qualitative analyses of salivary EVs. As we delve into the correlation between metabolic changes in obesity and shifts in taste perception, the focus on EVs aims to uncover their potential role as carriers of molecules, particularly microRNAs, and their influence on eating behavior. Through this exploration, we aim to contribute valuable insights into the medical prospects of EVs for targeted therapeutic and diagnostic purposes on a cellular level within the context of obesity research. Methods: Using ÄKTA, a size- and molecular weight-based separation system, EVs were isolated from 2 ml saliva per subject. Nanosight technology and western blot analyses were performed for quantification and characterization of EVs. A standardized taste test was used to assess taste perception of study participants, while data on eating behaviour were obtained using standardized questionnaires. The study participants were extensively phenotyped for parameters such as body mass index, waist-to-hip circumference and further anthropometrics including data from bioimpedance analyses and clinically relevant parameters of obesity (e.g. blood pressure, lipid status, parameters of glucose and insulin metabolism, adipokine level in serum). Results: The findings indicate a relationship between the EV size and markers of nutritional status (all P<0.05). Increased EV concentration is associated with lower adiponectin serum level (P=0.017). Furthermore, EV concentration correlates negatively with reduced arm (P=0.025) and calf circumferences (P=0.035), suggesting that body composition, rather than obesity per se, is related to EVs in saliva. The study successfully demonstrated the isolation of clean vesicles from saliva, confirmed by the presence of characteristic EV markers and the intriguing presence of PPARg, hinting at a possible origin EVs from fat cells. Conclusion: These findings provide valuable insights into the intricate interplay between salivary EVs and obesity. Ongoing analyses on microRNA composition of salivary EVs might enlighten our understanding of how these particles may affect taste buds and eating behavior in obesity. These results may contribute to the development of effective interventions for obesity as burgeoning health concern. Article published online: 18 April 2024 © 2024. Thieme. All rights reserved. Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany