Non-mammalian Organisms Research Articles

Seminar: Functional Exposomics and Mechanisms of Toxicity-Insights from Model Systems and NAMs.

Significant progress has been made over the past decade in measuring the chemical components of the exposome, providing transformative population-scale frameworks in probing the etiologic link between environmental factors and disease phenotypes. While the analytical technologies continue to evolve with reams of data being generated, there is an opportunity to complement exposome-wide association studies (ExWAS) with functional analyses to advance etiologic search at organismal, cellular, and molecular levels. Exposomics is a transdisciplinary field aimed at enabling discovery-based analysis of the nongenetic factors that contribute to disease, including numerous environmental chemical stressors. While advances in exposure assessment are enhancing population-based discovery of exposome-wide effects and chemical exposure agents, functional screening and elucidation of biological effects of exposures represent the next logical step toward precision environmental health and medicine. In this work, we focus on the use, strategies, and prospects of alternative approaches and model systems to enhance the current human exposomics framework in biomarker search and causal understanding, spanning from bench-based nonmammalian organisms and cell culture to computational new approach methods (NAMs). We visit the definition of the functional exposome and exposomics and discuss a need to leverage alternative models as opposed to mammalian animals for delineating exposome-wide health effects. Under the "three Rs" principle of reduction, replacement, and refinement, model systems such as roundworms, fruit flies, zebrafish, and induced pluripotent stem cells (iPSCs) are advantageous over mammals (e.g., rodents or higher vertebrates). These models are cost-effective, and cell-specific genetic manipulations in these models are easier and faster, compared to mammalian models. Meanwhile, in silico NAMs enhance hazard identification and risk assessment in humans by bridging the translational gaps between toxicology data and etiologic inference, as represented by in vitro to in vivo extrapolation (IVIVE) and integrated approaches to testing and assessment (IATA) under the adverse outcome pathway (AOP) framework. Together, these alternatives offer a strong toolbox to support functional exposomics to study toxicity and causal mediators underpinning exposure-disease links. https://doi.org/10.1289/EHP13120.

Cross-Species Applications of Peptide Substrate Reporters to Quantitative Measurements of Kinase Activity.

Peptide substrate reporters are short chains of amino acids designed to act as substrates for enzymes of interest. Combined with capillary electrophoresis and laser-induced fluorescence detection (CE-LIF), they are powerful molecular tools for quantitative measurements of enzyme activity even at the level of single cells. Although most peptide substrate reporters have been optimized for human or murine cells in health-related applications, their performance in nonmammalian organisms remains largely unexplored. In this study, we evaluated three peptide substrate reporters for protein kinase B (PKB) in two eukaryotic microbes, Dictyostelium discoideum and Tetrahymena thermophila, which are evolutionarily distant from mammals and from each other yet express PKB homologues. All three peptide substrate reporters were phosphorylated in lysates from both organisms but with varying phosphorylation kinetics and stability. To demonstrate reporter utility, we used one to screen for and identify the previously unknown stimulus needed to activate PHK5, the PKB homologue in T. thermophila. In D. discoideum, we employed the highly quantitative nature of these assays using CE-LIF to make precise measurements of PKB activity in response to transient stimulation, drug treatment, and genetic mutation. These results underscore the broad applicability of peptide substrate reporters across diverse species while highlighting the need for further research to determine effective peptide stabilization strategies across different biological contexts.

Exploiting fly models to investigate rare human neurological disorders.

Rare neurological diseases, while individually are rare, collectively impact millions globally, leading to diverse and often severe neurological symptoms. Often attributed to genetic mutations that disrupt protein function or structure, understanding their genetic basis is crucial for accurate diagnosis and targeted therapies. To investigate the underlying pathogenesis of these conditions, researchers often use non-mammalian model organisms, such as Drosophila (fruit flies), which is valued for their genetic manipulability, cost-efficiency, and preservation of genes and biological functions across evolutionary time. Genetic tools available in Drosophila, including CRISPR-Cas9, offer a means to manipulate gene expression, allowing for a deep exploration of the genetic underpinnings of rare neurological diseases. Drosophila boasts a versatile genetic toolkit, rapid generation turnover, and ease of large-scale experimentation, making it an invaluable resource for identifying potential drug candidates. Researchers can expose flies carrying disease-associated mutations to various compounds, rapidly pinpointing promising therapeutic agents for further investigation in mammalian models and, ultimately, clinical trials. In this comprehensive review, we explore rare neurological diseases where fly research has significantly contributed to our understanding of their genetic basis, pathophysiology, and potential therapeutic implications. We discuss rare diseases associated with both neuron-expressed and glial-expressed genes. Specific cases include mutations in CDK19 resulting in epilepsy and developmental delay, mutations in TIAM1 leading to a neurodevelopmental disorder with seizures and language delay, and mutations in IRF2BPL causing seizures, a neurodevelopmental disorder with regression, loss of speech, and abnormal movements. And we explore mutations in EMC1 related to cerebellar atrophy, visual impairment, psychomotor retardation, and gain-of-function mutations in ACOX1 causing Mitchell syndrome. Loss-of-function mutations in ACOX1 result in ACOX1 deficiency, characterized by very-long-chain fatty acid accumulation and glial degeneration. Notably, this review highlights how modeling these diseases in Drosophila has provided valuable insights into their pathophysiology, offering a platform for the rapid identification of potential therapeutic interventions. Rare neurological diseases involve a wide range of expression systems, and sometimes common phenotypes can be found among different genes that cause abnormalities in neurons or glia. Furthermore, mutations within the same gene may result in varying functional outcomes, such as complete loss of function, partial loss of function, or gain-of-function mutations. The phenotypes observed in patients can differ significantly, underscoring the complexity of these conditions. In conclusion, Drosophila represents an indispensable and cost-effective tool for investigating rare neurological diseases. By facilitating the modeling of these conditions, Drosophila contributes to a deeper understanding of their genetic basis, pathophysiology, and potential therapies. This approach accelerates the discovery of promising drug candidates, ultimately benefiting patients affected by these complex and understudied diseases.

Importance of Zebrafish as a Non-Mammalian Vertebrate Model for Studying Neurodegenerative Disorders Using Ayurvedic Medicinal Plants: A Review

Alzheimer's disease and Parkinson's disease are debilitating neurodegenerative disorders that impose a significant burden on the affected individuals and the society as a whole. Despite extensive research efforts, effective treatments for these diseases remain elusive till date. Ayurveda, a traditional system of medicinal practice which originated in ancient India, has long touted the potential of various plants possessing neuroprotective properties. The effects of a few relevant Ayurvedic plants viz. Guduchi, Kapikachhu, Shankhapushpi, Turmeric, Mandukparni, Ashwagadha, Brahmi, Jatamansi etc. on several non-human primates and rodents have been reported. The therapeutic potential of those plants to mitigate the pathological hallmarks associated with Alzheimer's disease and Parkinson's disease was assessed. We have outlined the most recent research on Ayurveda's effects on neurodegenerative brain illnesses in this review article, with an emphasis on Alzheimer's disease and Parkinson's disease. As the zebrafish (Danio rerio Hamilton) has emerged as a useful, non-mammalian vertebrate model organism for studying a wide range of diseases, the effect of few other medicinal plants on zebrafish was also mentioned. Powerful live imaging and genetic tools which is currently available for zebrafish has transformed this fish to a top model in biomedical research and toxicity testing. However, further research is needed to elucidate the precise mechanisms of action and to translate these promising results into clinical applications. The exploration of traditional medicinal systems, in conjunction with modern scientific methodologies using novel model organisms, such as zebrafish offers a valuable avenue for the development of innovative treatments for neurodegenerative disorders.

Uncovering the essential roles of glutamate carboxypeptidase 2 orthologs in Caenorhabditis elegans.

Human glutamate carboxypeptidase 2 (GCP2) from the M28B metalloprotease group is an important target for therapy in neurological disorders and an established tumor marker. However, its physiological functions remain unclear. To better understand general roles, we used the model organism Caenorhabditis elegans to genetically manipulate its three existing orthologous genes and evaluate the impact on worm physiology. The results of gene knockout studies showed that C. elegans GCP2 orthologs affect the pharyngeal physiology, reproduction, and structural integrity of the organism. Promoter-driven GFP expression revealed distinct localization for each of the three gene paralogs, with gcp-2.1 being most abundant in muscles, intestine, and pharyngeal interneurons, gcp-2.2 restricted to the phasmid neurons, and gcp-2.3 located in the excretory cell. The present study provides new insight into the unique phenotypic effects of GCP2 gene knockouts in C. elegans, and the specific tissue localizations. We believe that elucidation of particular roles in a non-mammalian organism can help to explain important questions linked to physiology of this protease group and in extension to human GCP2 involvement in pathophysiological processes.

Animal models of inflammatory bowel disease: category and evaluation indexes.

Inflammatory bowel disease (IBD) research often relies on animal models to study the etiology, pathophysiology, and management of IBD. Among these models, rats and mice are frequently employed due to their practicality and genetic manipulability. However, for studies aiming to closely mimic human pathology, non-human primates such as monkeys and dogs offer valuable physiological parallels. Guinea pigs, while less commonly used, present unique advantages for investigating the intricate interplay between neurological and immunological factors in IBD. Additionally, New Zealand rabbits excel in endoscopic biopsy techniques, providing insights into mucosal inflammation and healing processes. Pigs, with their physiological similarities to humans, serve as ideal models for exploring the complex relationships between nutrition, metabolism, and immunity in IBD. Beyond mammals, non-mammalian organisms including zebrafish, Drosophila melanogaster, and nematodes offer specialized insights into specific aspects of IBD pathology, highlighting the diverse array of model systems available for advancing our understanding of this multifaceted disease. In this review, we conduct a thorough analysis of various animal models employed in IBD research, detailing their applications and essential experimental parameters. These include clinical observation, Disease Activity Index score, pathological assessment, intestinal barrier integrity, fibrosis, inflammatory markers, intestinal microbiome, and other critical parameters that are crucial for evaluating modeling success and drug efficacy in experimental mammalian studies. Overall, this review will serve as a valuable resource for researchers in the field of IBD, offering insights into the diverse array of animal models available and their respective applications in studying IBD.

Pesticide residues in adults living near a bioenergy plant with 85,000 tons of contaminated wetcake

Neonicotinoid insecticide use is on the rise worldwide due to its broad-spectrum insecticidal action and exclusive approach of neurotoxic action. Besides application during the cultivation of several crops, all seed companies coat their seeds with neonicotinoids to have increased protection against insects during germination. Despite reduced mammalian toxicity, neonicotinoids have harmful effects on non-target non-mammalian organisms such as bees, an essential part of maintaining the ecosystem. In addition, epidemiologic studies have linked human exposure to neonicotinoids with poor developmental and neurological outcomes. Starting in 2015, the AltEn bioenergy plant near Mead, Nebraska, USA, used coated seeds for their ethanol production and failed to properly dispose of byproducts, causing environmental contamination that still exists. This pilot study reports the human urinary levels of neonicotinoids in samples collected during 2022–2023 in the population living in areas close to this now-closed bioenergy plant. Our results show that approximately 30% of the urine samples are contaminated with at least one of the targeted neonicotinoids or their transformed products. The most frequently detected parent neonicotinoid was clothianidin, which accounts for 13% of the samples. However, 5-hydroxy-imidacloprid, the transformed imidacloprid product, is detected in 27% of the samples, ranging from 1.2 to 42 ng/mL. In conclusion, the environmental contamination near Mead, Nebraska, due to improper storage and disposal of highly contaminated byproducts, puts the nearby population at risk from continuous exposure to neonicotinoids through air and dust particles and possible water contamination.

The wide world of non-mammalian phospholipase D enzymes

Phospholipase D (PLD) hydrolyses phosphatidylcholine (PtdCho) to produce free choline and the critically important lipid signaling molecule phosphatidic acid (PtdOH). Since the initial discovery of PLD activities in plants and bacteria, PLDs have been identified in a diverse range of organisms spanning the taxa. While widespread interest in these proteins grew following the discovery of mammalian isoforms, research into the PLDs of non-mammalian organisms has revealed a fascinating array of functions ranging from roles in microbial pathogenesis, to the stress responses of plants and the developmental patterning of flies. Furthermore, studies in non-mammalian model systems have aided our understanding of the entire PLD superfamily, with translational relevance to human biology and health. Increasingly, the promise for utilization of non-mammalian PLDs in biotechnology is also being recognized, with widespread potential applications ranging from roles in lipid synthesis, to their exploitation for agricultural and pharmaceutical applications.

Integrating non-mammalian model organisms in the diagnosis of rare genetic diseases in humans.

Next-generation sequencing technology has rapidly accelerated the discovery of genetic variants of interest in individuals with rare diseases. However, showing that these variants are causative of the disease in question is complex and may require functional studies. Use of non-mammalian model organisms - mainly fruitflies (Drosophila melanogaster), nematode worms (Caenorhabditis elegans) and zebrafish (Danio rerio) - enables the rapid and cost-effective assessment of the effects of gene variants, which can then be validated in mammalian model organisms such as mice and in human cells. By probing mechanisms of gene action and identifying interacting genes and proteins in vivo, recent studies in these non-mammalian model organisms have facilitated the diagnosis of numerous genetic diseases and have enabled the screening and identification of therapeutic options for patients. Studies in non-mammalian model organisms have also shown that the biological processes underlying rare diseases can provide insight into more common mechanisms of disease and the biological functions of genes. Here, we discuss the opportunities afforded by non-mammalian model organisms, focusing on flies, worms and fish, and provide examples of their use in the diagnosis of rare genetic diseases.

Human Mutated MYOT and CRYAB Genes Cause a Myopathic Phenotype in Zebrafish.

Myofibrillar myopathies (MFMs) are a group of hereditary neuromuscular disorders sharing common histological features, such as myofibrillar derangement, Z-disk disintegration, and the accumulation of degradation products into protein aggregates. They are caused by mutations in several genes that encode either structural proteins or molecular chaperones. Nevertheless, the mechanisms by which mutated genes result in protein aggregation are still unknown. To unveil the role of myotilin and αB-crystallin in the pathogenesis of MFM, we injected zebrafish fertilized eggs at the one-cell stage with expression plasmids harboring cDNA sequences of human wildtype or mutated MYOT (p.Ser95Ile) and human wildtype or mutated CRYAB (p.Gly154Ser). We evaluated the effects on fish survival, motor behavior, muscle structure and development. We found that transgenic zebrafish showed morphological defects that were more severe in those overexpressing mutant genes. which developed a myopathic phenotype consistent with that of human myofibrillar myopathy, including the formation of protein aggregates. Results indicate that pathogenic mutations in myotilin and αB-crystallin genes associated with MFM cause a structural and functional impairment of the skeletal muscle in zebrafish, thereby making this non-mammalian organism a powerful model to dissect disease pathogenesis and find possible druggable targets.

A ligand-receptor interactome atlas of the zebrafish

Studies in zebrafish can unravel the functions of cellular communication and thus identify novel bench-to-bedside drugs targeting cellular communication signaling molecules. Due to the incomplete annotation of zebrafish proteome, the knowledge of zebrafish receptors, ligands, and tools to explore their interactome is limited. To address this gap, we de novo predicted the cellular localization of zebrafish reference proteome using deep learning algorithm. We combined the predicted and existing annotations on cellular localization of zebrafish proteins and created repositories of zebrafish ligands, membrane receptome, and interactome as well as associated diseases and targeting drugs. Unlike other tools, our interactome atlas is based on both the physical interaction data of zebrafish proteome and existing human ligand-receptor pair databases. The resources are available as R and Python scripts. DanioTalk provides a novel resource for researchers interested in targeting cellular communication in zebrafish, as we demonstrate in applications studying synapse and axo-glial interactome. DanioTalk methodology can be applied to build andexplore the ligand-receptor atlas of other non-mammalian model organisms.

Investigating the functional roles of TNFSF13 family members in Cartilaginous Fishes

Abstract The TNFSF13 cytokines BAFF and APRIL along with their cognate receptors BAFFR, TACI, and BCMA play critical parts in the development, function, and maintenance of B cells in mammals. Despite the well-studied roles that BAFF and APRIL play in the immune systems of higher vertebrates, their evolutionary history and functions in non-mammalian organisms are not well understood. Recently, our lab has demonstrated that collectively the TNFSF is evolutionarily ancient, with most of its members having evolved over 450 million years ago in the jawed vertebrate ancestor. Interestingly, we found that despite the evolutionary ancient position of cartilaginous fishes, their repertoire of TNFSF is just as complex as higher vertebrates with evidence of lineage specific TNFSF members that are absent in more evolutionary recent taxa. Specifically, cartilaginous fishes possess two additional TNFSF13 family members, namely BALM (TNFSF13C) and a retrotransposed duplicate of BAFF (iBAFF). Thus, cartilaginous fishes possess four TNFSF13 members whose functional roles remain unexplored. Here we demonstrate the tissue specific expression of BAFF, iBAFF, BALM, and APRIL, as well as their anticipated receptors BAFFR, TACI, and BCMA in the nurse shark. Additionally, we explore receptor-ligand interactions using a yeast surface display assay aiming to understand the role(s) each plays in the development and maintenance of shark B cells. Such information is key to understanding the evolutionary history of this key cytokine family.

Transgenerational epigenetic inheritance of axonal regeneration after spinal cord injury.

Human epidemiological studies reveal that dietary and environmental alterations influence the health of the offspring and that the effect is not limited to the F1 or F2 generations. Non-Mendelian transgenerational inheritance of traits in response to environmental stimuli has been confirmed in non-mammalian organisms including plants and worms and are shown to be epigenetically mediated. However, transgenerational inheritance beyond the F2 generation remains controversial in mammals. Our lab previously discovered that the treatment of rodents (rats and mice) with folic acid significantly enhances the regeneration of injured axons following spinal cord injury in vivo and in vitro, and the effect is mediated by DNA methylation. The potential heritability of DNA methylation prompted us to investigate the following question: Is the enhanced axonal regeneration phenotype inherited transgenerationally without exposure to folic acid supplementation in the intervening generations? In the present review, we condense our findings showing that a beneficial trait (i.e., enhanced axonal regeneration after spinal cord injury) and accompanying molecular alterations (i.e., DNA methylation), triggered by an environmental exposure (i.e., folic acid supplementation) to F0 animals only, are inherited transgenerationally and beyond the F3 generation.

Regenerative Strategies for Retinal Neurons: Novel Insights in Non-Mammalian Model Organisms.

A detailed knowledge of the status of the retina in neurodegenerative conditions is a crucial point for the development of therapeutics in retinal pathologies and to translate eye research to CNS disease. In this context, manipulating signaling pathways that lead to neuronal regeneration offers an excellent opportunity to substitute damaged cells and, thus, restore the tissue functionality. Alternative systems and methods are increasingly being considered to replace/reduce in vivo approaches in the study of retina pathophysiology. Herein, we present recent data obtained from the zebrafish (Danio rerio) and the fruit fly Drosophila melanogaster that bring promising advantages into studying and modeling, at a preclinical level, neurodegeneration and regenerative approaches in retinal diseases. Indeed, the regenerative ability of vertebrate model zebrafish is particularly appealing. In addition, the fruit fly is ideal for regenerative studies due to its high degree of conservation with vertebrates and the broad spectrum of genetic variants achievable. Furthermore, a large part of the drosophila brain is dedicated to sight, thus offering the possibility of studying common mechanisms of the visual system and the brain at once. The knowledge acquired from these alternative models may help to investigate specific well-conserved factors of interest in human neuroregeneration after injuries or during pathologies.

Expression and Development of TARP γ-4 in Embryonic Zebrafish

Fast excitatory synaptic transmission in the CNS is mediated by the neurotransmitter glutamate, binding to and activating AMPA receptors (AMPARs). AMPARs are known to interact with auxiliary proteins that modulate their behavior. One such family of proteins is the transmembrane AMPAR-related proteins, known as TARPs. Little is known about the role of TARPs during development or about their function in nonmammalian organisms. Here, we report on the presence of TARP γ-4 in developing zebrafish. We find that zebrafish express 2 forms of TARP γ-4: γ-4a and γ-4b as early as 12 h post-fertilization. Sequence analysis shows that both γ-4a and γ-4b shows great level of variation particularly in the intracellular C-terminal domain compared to rat, mouse, and human γ-4. RT-qPCR showed a gradual increase in the expression of γ-4a throughout the first 5 days of development, whereas γ-4b levels were constant until day 5 when levels increased significantly. Knockdown of TARP γ-4a and γ-4b via either splice-blocking morpholinos or translation-blocking morpholinos resulted in embryos that exhibited deficits in C-start escape responses, showing reduced C-bend angles. Morphant larvae displayed reduced bouts of swimming. Whole-cell patch-clamp recordings of AMPAR-mediated currents from Mauthner cells showed a reduction in the frequency of mEPCs but no change in amplitude or kinetics. Together, these results suggest that γ-4a and γ-4b are required for proper neuronal development.

Role of foxn1 in Xenopus laevis thymopoiesis.

Abstract The transcription factor forkhead box N1 (FOXN1) plays a pivotal role in differentiation of thymic epithelial cells (TECs) essential for intrathymic T cell education and keratinocyte development. Mutations in murine foxn1 are associated with T− B+ NK+ nude/severe combined immunodeficiency (SCID) phenotype. In humans FOXN1 mutations also lead to thymic aplasia or hypoplasia, hair loss and dysmorphic nails. In the amphibian Xenopus tropicalis, foxn1−/− line exhibited loss of thymi formation and absence of mature T-cells, similar with Foxn1−/− murine line. Due to expanded resources in immunobiology of X. laevis (a close relative of X. tropicalis), we generated a novel foxn1-deficient X. laevis model by using the CRISPR/Cas9. foxn1 crispants shows a high mutational efficiency (30–90%) and about 25% of tadpoles (stage 55–56), exhibit hypoplastic thymi. foxn1 disruption resulted in significant reduction of T-cell markers transcript levels, detected by RT-PCR. Furthermore, spleen of foxn1 crispants had markedly reduced CD3e staining compared to controls. Interestingly, Mycobacterium marinum (M. marinum) infection of tadpoles results in noninflammatory CD8 negative- and iT cell–mediated response, while foxn1 mutations in X. laevis appeared to affect tolerigenic innate immune response of infected tadpoles. This foxn1-deficient transgenic line will empower X. laevis as nonmammalian alternative experimental organism for studying host interaction with to non-tuberculosis mycobacteria. Currently, we are investigating mechanisms by which foxn1 mutation alter innate immunity of M. marinum inoculated tadpoles and evaluating tolerance/resistance by analysing the inflammatory/anti-inflammatory cytokine profile.

A Model of Discovery: The Role of Imaging Established and Emerging Non-mammalian Models in Neuroscience.

Rodents have been the dominant animal models in neurobiology and neurological disease research over the past 60 years. The prevalent use of rats and mice in neuroscience research has been driven by several key attributes including their organ physiology being more similar to humans, the availability of a broad variety of behavioral tests and genetic tools, and widely accessible reagents. However, despite the many advances in understanding neurobiology that have been achieved using rodent models, there remain key limitations in the questions that can be addressed in these and other mammalian models. In particular, in vivo imaging in mammals at the cell-resolution level remains technically difficult and demands large investments in time and cost. The simpler nervous systems of many non-mammalian models allow for precise mapping of circuits and even the whole brain with impressive subcellular resolution. The types of non-mammalian neuroscience models available spans vertebrates and non-vertebrates, so that an appropriate model for most cell biological questions in neurodegenerative disease likely exists. A push to diversify the models used in neuroscience research could help address current gaps in knowledge, complement existing rodent-based bodies of work, and bring new insight into our understanding of human disease. Moreover, there are inherent aspects of many non-mammalian models such as lifespan and tissue transparency that can make them specifically advantageous for neuroscience studies. Crispr/Cas9 gene editing and decreased cost of genome sequencing combined with advances in optical microscopy enhances the utility of new animal models to address specific questions. This review seeks to synthesize current knowledge of established and emerging non-mammalian model organisms with advances in cellular-resolution in vivo imaging techniques to suggest new approaches to understand neurodegeneration and neurobiological processes. We will summarize current tools and in vivo imaging approaches at the single cell scale that could help lead to increased consideration of non-mammalian models in neuroscience research.

Meta-analysis of Bioaccumulation Data for Nondissolvable Engineered Nanomaterials in Freshwater Aquatic Organisms.

Understanding the bioaccumulation of engineered nanomaterials (ENMs) is essential for making regulatory decisions on potential environmental risks. Research in the field of ENM bioaccumulation has increased in recent years, but the compilation and statistical analysis of the available experimental data have not been updated. We therefore performed a meta‐analysis of the existing literature on the bioaccumulation of eight types of nondissolvable ENMs (titanium dioxide [TiO2], aluminum oxide [Al2O3], gold [Au], fullerene [C60], carbon nanotubes, iron oxide [FeO x ], graphene, and polystyrene) in nonmammalian freshwater aquatic organisms across three trophic levels including phytoplankton, zooplankton, and fish. Three typical endpoints were used to assess the bioaccumulation potential: the bioconcentration factor (BCF), the bioaccumulation factor (BAF), and the biomagnification factor (BMF). Our results suggest that zooplankton has greater mean logarithmic BCF and BAF values than phytoplankton (3.31 vs. 1.42) and fish (2.04). The ENMs are biomagnified in zooplankton, with a mean BMF of 17.4, whereas trophic transfer from primary consumers (zooplankton) to secondary consumers (fish) was not observed (mean BMF of 0.13). No clear dependency was identified between the physicochemical characteristics of ENMs (e.g., primary particle size, zeta potential, or shape) and bioaccumulation, except for coated versus uncoated particles accumulated in phytoplankton. Carbonaceous ENMs were found to be more bioaccumulated than the other ENMs we considered, except for TiO2. A meta‐analysis of bioaccumulation data can (1) deepen the understanding of bioconcentration, bioaccumulation, and biomagnification of ENMs, (2) be used to support grouping strategies as a basis for a safer‐by‐design approach for ENMs, (3) be integrated into comprehensive hazard and risk assessments, (4) promote the standardization of testing guidelines, and (5) enhance future kinetic bioaccumulation modeling. Environ Toxicol Chem 2022;41:1202–1214. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies.

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical functions. However, many difficulties remain in mammalian research, which necessitates the development of non-mammalian model organisms for cannabinoid research. The authors suggest the tobacco hornworm Manduca sexta as a novel insect model system. This protocol provides information on preparing the artificial diet with varying amounts of cannabidiol (CBD), setting up a cultivation environment, and monitoring their physiological and behavioral changes in response to CBD treatment. Briefly, upon receiving hornworm eggs, the eggs were allowed 1-3 days at 25 °C on a 12:12 light-dark cycle to hatch before being randomly distributed into control (wheat germ-based artificial diet; AD), vehicle (AD + 0.1% medium-chain triglyceride oil; MCT oil) and treatment groups (AD + 0.1% MCT + 1 mM or 2 mM of CBD). Once the media was prepared, 1st instar larvae were individually placed in a 50 mL test tube with a wooden skewer stick, and then the test tube was covered with a cheesecloth. Measurements were taken in 2-day intervals for physiological and behavioral responses to the CBD administration. This simple cultivation procedure allows researchers to test large specimens in a given experiment. Additionally, the relatively short life cycles enable researchers to study the impact of cannabinoid treatments over multiple generations of a homogenous population, allowing for data to support an experimental design in higher mammalian model organisms.

Spinal Cord Transection In Xenopus laevis Tadpoles.

Spinal cord injury (SCI) is a permanent affliction, which affects the central nervous system (CNS) motor and sensory nerves, resulting in paralysis beneath the injury site. To date, there is no functional recovery therapy for SCI, and there is a lack of clarity regarding the many complexes and dynamic events occurring after SCI. Many non-mammalian organisms can regenerate after severe SCI, such as teleost fishes, urodele amphibians, and larval stages of anuran amphibians, including Xenopus laevis tadpoles. These are bona fide model organisms to study and understand the response to SCI and the mechanisms underlying successful regenerative processes. This type of research can lead to the identification of potential targets for SCI therapeutic intervention. This article describes how to perform Xenopus laevis tadpole spinal cord transection, including husbandry, surgery, postsurgery care, and functional test evaluation. This injury method can be applied for elucidating the different steps of spinal cord regeneration by studying the cellular, molecular, and genetic mechanisms, as well as histological and functional evolution after SCI and during spinal cord regeneration.