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Gas permeable and biocompatible soft polymers are convenient for biological applications. Using the soft polymer poly(dimethylsiloxane) (PDMS), we established a straightforward technique for in-house production of self-adhesive and optical grade microculture devices. A gas permeable PDMS layer effectively protects against medium evaporation, changes in osmolarity, contamination and drug diffusion. These chip-based devices can be used effectively for long term mammalian cell culture and support a range of bioassays used in pharmacological profiling of anti-cancer drugs. Results obtained on a panel of hematopoietic and solid tumor cell lines during screening of investigative anti-cancer agents corresponded well to those obtained in a conventional cell culture on polystyrene plates. The cumulative correlation analysis of multiple cell lines and anti-cancer drugs showed no adverse effects on cell viability or cell growth retardation during microscale static cell culture. PDMS devices also can be custom modified for many bio-analytical purposes and are interfaced easily with both inverted and upright cell imaging platforms. Moreover, PDMS microculture devices are suitable for extended real time cell imaging. Data from the multicolor, real time analysis of apoptosis on human breast cancer MCF-7 cells provided further evidence that elimination of redundant centrifugation/washing achieved during microscale real time analysis facilitates preservation of fragile apoptotic cells and provides dynamic cellular information at high resolution. Because only small reaction volumes are required, such devices offer reduced use of consumables as well as simplified manipulations during all stages of live cell imaging. [ABSTRACT FROM AUTHOR]

This study aimed to assess the behavioral responses (immobilization, horizontal and vertical motility, and response to light) of Chironomus aprilinus larvae exposed to individual cyanobacterial metabolites aeruginosin 98B (AER-B), anabaenopeptin-B (ANA-B), and cylindrospermopsin (CYL), and their binary and ternary mixtures. The investigation revealed that single metabolites ANA-B and CYL exhibited the highest potency in immobilizing the larvae. Notably, the binary mixture AER-B+CYL induced a remarkably strong synergistic interaction, while other tested binary and ternary mixtures demonstrated antagonistic effects. Both individual metabolites and their mixtures led to a decrease in larval movement speed, with the AER-B+CYL combination showing a very synergistic effect, and strong antagonistic interactions between the oligopeptides in the ternary mixture. Conversely, while AER-B and the binary mixture ANA-B+CYL stimulated vertical movement, other single metabolites and binary and ternary mixtures decreased this parameter. Antagonistic interactions were observed in all mixtures. ANA-B emerged as the most potent inhibitor, yet all tested metabolites and their mixtures decreased larval response to light, displaying synergistic interactions, except for the AER-B+ANA-B mixture at 250 μg L -1 + 250 μg L -1 . These findings underscore the sensitivity of Chironomus larvae behavioral parameters as indicators of environmental stressors and mixtures. Consequently, they are recommended for assessing toxic effects induced by cyanobacterial products and other bioactive chemicals.

Behavioural analysis has been attracting significant attention as a broad indicator of sub-lethal toxicity and has secured a place as an important subdiscipline in ecotoxicology. Among the most notable characteristics of behavioural research, compared to other established approaches in sub-lethal ecotoxicology (e.g. reproductive and developmental bioassays), are the wide range of study designs being used and the diversity of endpoints considered. At the same time, environmental hazard and risk assessment, which underpins regulatory decisions to protect the environment from potentially harmful chemicals, often recommends that ecotoxicological data be produced following accepted and validated test guidelines. These guidelines typically do not address behavioural changes, meaning that these, often sensitive, effects are not represented in hazard and risk assessments. Here, we propose a new tool, the EthoCRED evaluation method, for assessing the relevance and reliability of behavioural ecotoxicity data, which considers the unique requirements and challenges encountered in this field. This method and accompanying reporting recommendations are designed to serve as an extension of the "Criteria for Reporting and Evaluating Ecotoxicity Data (CRED)" project. As such, EthoCRED can both accommodate the wide array of experimental design approaches seen in behavioural ecotoxicology, and could be readily implemented into regulatory frameworks as deemed appropriate by policy makers of different jurisdictions to allow better integration of knowledge gained from behavioural testing into environmental protection. Furthermore, through our reporting recommendations, we aim to improve the reporting of behavioural studies in the peer-reviewed literature, and thereby increase their usefulness to inform chemical regulation., (© 2024 The Author(s). Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Chemobehavioural phenotyping presents unique opportunities for analyzing neurotoxicants and discovering behavior-modifying neuroceuticals in small aquatic model organisms such as zebrafish (Danio rerio). A recently popularized approach in this field involves the utilization of zebrafish embryos for a photo-motor response (PMR) bioassay. The PMR bioassay entails stimulating zebrafish embryos between 24 and 36 h post fertilization (hpf) with a high-intensity light stimulus, inducing a transient increase in the frequency of photo-induced embryo body flexions. These flexions can be computationally analyzed to derive behavioral signatures, enabling the categorization of neuromodulating chemicals. Despite the significant advantages of the PMR bioassay, its widespread implementation is hindered by lack of well described and straightforward high-throughput bioinformatic analysis of behavioral data. In this methods article, we present an easily implementable bioinformatics protocol specifically designed for rapid behavioral analysis of large cohorts of zebrafish specimens in PMR bioassays. We also address common pitfalls encountered during PMR analysis, discuss its limitations, and propose future directions for developing next-generation biometric analysis techniques in chemobehavioural assays utilizing zebrafish embryos., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We aimed to determine the effects of single cyanobacterial metabolites aeruginosin-B (AER-B), anabaenopeptin-B (ANA-B), cylindrospermopsin (CYL), their binary and ternary mixtures on biomarkers of Chironomus aprilinus larvae: oxygen consumption, fat body structure and two novel fluorescent indicators: imaging of nuclei in cells of body integument, and the catecholamine level. The obtained results showed that oxygen consumption was inhibited by single tested cyanobacterial metabolites except for ANA-B at the lowest concentration (250 μg/L). Although the mixtures of the metabolites inhibited oxygen consumption with antagonistic interactions between the components stimulation was noted in the group exposed to the lowest concentrations of AER-B + CYL (125 μg/L + 125 μg/L, respectively) and the ternary mixture of AER-B + ANA-B + CYL (83.3 μg/L + 83.3 μg/L + 83.3 μg/L, respectively). In vivo fluorescent staining with Hoechst 34580 showed that single AER-B had lower cytotoxic potential on body integument cells than ANA-B and CYL and most binary mixtures except for AER-B + CYL induced synergistic toxicity. Catecholamine level was decreased in animals exposed to single metabolites, their binary and ternary mixtures; however, the interactions between the components in the ternary mixture were antagonistic. Fat body was found to be disrupted in the larvae exposed to single metabolites and their combinations. Antagonistic toxic interactions between the oligopeptide components were found in most binary and the ternary mixtures; however, synergistic effect was noted in the binary mixture of AER-B + CYL. The results suggest that in natural conditions Chironomus larvae and possibly other benthic invertebrates may be affected by cyanobacterial metabolites, however various components and in mixtures and their concentrations may determine varied physiological effects and diverse interactions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Assessment of animals' sensory-motor functions requires precise and electronically controlled stimuli to induce and quantify specific behavioral phenotypes. However, accessible and inexpensive tools for conducting diverse sensory-motor biotests with fish are lacking. In this work, we present an open-source software and hardware interface that enables automated delivery of three independent and fully programmable stimuli for behavioral bioassays. We demonstrate the proof-of-concept application of this low-cost technology in establishing reproducible fear responses using a mechanical tap-startle stimulus in larval zebrafish. This response is characterized by a sudden burst of motion in response to a nondirectional mechanical stimulus delivered to the fish chamber. We propose that the simplicity and flexibility of this interface offer innovative opportunities for studying sensory-motor functions in various fields, including neurobiology, neuropharmacology, neurotoxicology, and aquatic ecotoxicology.

Aquatic eco-neurotoxicology is an emerging field that requires new analytical systems to study the effects of pollutants on animal behaviors. This is especially true if we are to gain insights into one of the least studied aspects: the potential perturbations that neurotoxicants can have on cognitive behaviors. The paucity of experimental data is partly caused by a lack of low-cost technologies for the analysis of higher-level neurological functions (e.g., associative learning) in small aquatic organisms. Here, we present a proof-of-concept prototype that utilizes a new real-time animal tracking software for on-the-fly video analysis and closed-loop, external hardware communications to deliver stimuli based on specific behaviors in aquatic organisms, spanning three animal phyla: chordates (fish, frog), platyhelminthes (flatworm), and arthropods (crustacean). The system's open-source software features an intuitive graphical user interface and advanced adaptive threshold-based image segmentation for precise animal detection. We demonstrate the precision of animal tracking across multiple aquatic species with varying modes of locomotion. The presented technology interfaces easily with low-cost and open-source hardware such as the Arduino microcontroller family for closed-loop stimuli control. The new system has potential future applications in eco-neurotoxicology, where it could enable new opportunities for cognitive research in diverse small aquatic model organisms.

Recent research has found that individuals often vary in how consistently they express their behavior over time (i.e., behavioral predictability) and suggested that these individual differences may be heritable. However, little is known about the intrinsic factors that drive variation in the predictability of behavior. Indeed, whether variation in behavioral predictability is sex-specific is not clear. This is important, as behavioral predictability has been associated with vulnerability to predation, suggesting that the predictability of behavioral traits may have key fitness implications. We investigated whether male and female eastern mosquitofish ( Gambusia holbrooki ) differed in the predictability of their risk-taking behavior. Specifically, over a total of 954 behavioral trials, we repeatedly measured risk-taking behavior with three commonly used assays-refuge-use, thigmotaxis, and foraging latency. We predicted that there would be consistent sex differences in both mean-level risk-taking behavior and behavioral predictability across the assays. We found that risk-taking behavior was repeatable within each assay, and that some individuals were consistently bolder than others across all three assays. There were also consistent sex differences in mean-level risk-taking behavior, with males being bolder across all three assays compared to females. In contrast, both the magnitude and direction of sex differences in behavioral predictability were assay-specific. Taken together, these results highlight that behavioral predictability may be independent from underlying mean-level behavioral traits and suggest that males and females may differentially adjust the consistency of their risk-taking behavior in response to subtle changes in environmental conditions., (© The Author(s) 2022. Published by Oxford University Press on behalf of the International Society for Behavioral Ecology.)

Due to technical limitations, there have been minimal studies performed on thermal preferences and thermotactic behaviors of aquatic ectotherm species commonly used in ecotoxicity testing. In this work, we demonstrate an innovative, purpose-built and miniaturized electrothermal array for rapid thermal preference behavioral tests. We applied the novel platform to define thermal preferences in multiple invertebrate and vertebrate species. Specifically, Dugesia notogaea (freshwater planarians), Chironomus tepperi (nonbiting midge larvae), Ostracoda (seed shrimp), Artemia franciscana (brine shrimp), Daphnia carinata (water flea), Austrochiltonia subtenuis (freshwater amphipod), Physa acuta (freshwater snail), Potamopyrgus antipodarum (New Zealand mud snail) and larval stage of Danio rerio (zebrafish) were tested. The Australian freshwater water fleas, amphipods, snail Physa acuta as well as zebrafish exhibited the most consistent preference to cool zones and clear avoidance of zones >27 °C out of nine species tested. Our results indicate the larval stage of zebrafish as the most responsive species highly suitable for prospective development of multidimensional behavioral test batteries. We also showcase preliminary data that environmentally relevant concentrations of pharmaceutical pollutants such as non-steroidal anti-inflammatory drug (NSAID) ibuprofen (9800 ng/L) and insecticide imidacloprid (4600 ng/L) but not anti-depressant venlafaxine (2200 ng/L) and (iv) anticonvulsant medications gabapentin (400 ng/L) can perturb thermal preference behavior of larval zebrafish. Collectively our results demonstrate the utility of simple and inexpensive thermoelectric technology in rapid exploration of thermal preference in diverse species of aquatic animals. We postulate that more broadly such technologies can also have added value in ecotoxicity testing of emerging contaminants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The rapidly increasing number of new production chemicals coupled with stringent implementation of global chemical management programs necessities a paradigm shift towards boarder uses of low-cost and high-throughput ecotoxicity testing strategies as well as deeper understanding of cellular and sub-cellular mechanisms of ecotoxicity that can be used in effective risk assessment. The latter will require automated acquisition of biological data, new capabilities for big data analysis as well as computational simulations capable of translating new data into in vivo relevance. However, very few efforts have been so far devoted into the development of automated bioanalytical systems in ecotoxicology. This is in stark contrast to standardized and high-throughput chemical screening and prioritization routines found in modern drug discovery pipelines. As a result, the high-throughput and high-content data acquisition in ecotoxicology is still in its infancy with limited examples focused on cell-free and cell-based assays. In this work we outline recent developments and emerging prospects of high-throughput bioanalytical approaches in ecotoxicology that reach beyond in vitro biotests. We discuss future importance of automated quantitative data acquisition for cell-free, cell-based as well as developments in phytotoxicity and in vivo biotests utilizing small aquatic model organisms. We also discuss recent innovations such as organs-on-a-chip technologies and existing challenges for emerging high-throughput ecotoxicity testing strategies. Lastly, we provide seminal examples of the small number of successful high-throughput implementations that have been employed in prioritization of chemicals and accelerated environmental risk assessment., Competing Interests: Declaration of competing interest Marcus Jansen is a Chief Scientist - Biology at the LemnaTec GmbH. No funding or other financial support was received for this work., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Pollutants, such as endocrine disrupting chemicals (EDCs), are increasingly being detected in organisms and ecosystems globally. Agricultural activities, including the use of hormonal growth promotants (HGPs), are a major source of EDC contamination. One potent EDC that enters into the environment through the use of HGPs is 17β-trenbolone. Despite EDCs being repeatedly shown to affect reproduction and development, comparatively little is known regarding their effects on behaviour. Amphibians, one of the most imperilled vertebrate taxa globally, are at particular risk of exposure to such pollutants as they often live and breed near agricultural operations. Yet, no previous research on amphibians has explored the effects of 17β-trenbolone exposure on foraging or antipredator behaviour, both of which are key fitness-related behavioural traits. Accordingly, we investigated the impacts of 28-day exposure to two environmentally realistic concentrations of 17β-trenbolone (average measured concentrations: 10 and 66 ng/L) on the behaviour and growth of spotted marsh frog tadpoles (Limnodynastes tasmaniensis). Contrary to our predictions, there was no significant effect of 17β-trenbolone exposure on tadpole growth, antipredator response, anxiety-like behaviour, or foraging. We hypothesise that the differences in effects found between this study and those conducted on fish may be due to taxonomic differences and/or the life stage of the animals used, and suggest further research is needed to investigate the potential for delayed manifestation of the effects of 17β-trenbolone exposure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Behavioral phenotypic analysis is an emerging and increasingly important toolbox in aquatic ecotoxicology. In this regard digital video recording has recently become a standard in obtaining behavioral data. Subsequent analysis requires applications of specialized software for detecting and reconstructing animal locomotory trajectories as well as extracting quantitative biometric endpoints associated with specific behavioral traits. Despite some profound advantages for behavioral ecotoxicology, there is a notable lack of standardization of procedures and guidelines that would aid in consistently acquiring high-quality digital videos. The latter are fundamental for using animal tracking software successfully and to avoid issues such as identification switching, incorrect interpolation, and low tracking visibility. Achieving an optimized tracking not only saves user time and effort to analyze the results but also provides high-fidelity data with minimal artifacts. In the present study we, for the first time, provide an easily accessible guide on how to set up and optimize digital video acquisition while minimizing pitfalls in obtaining the highest-quality data for subsequent animal tracking. We also discuss straightforward digital video postprocessing techniques that can be employed to further enhance tracking consistency or improve the videos that were acquired in otherwise suboptimal settings. The present study provides an essential guidebook for any aquatic ecotoxicology studies that utilize digital video acquisition systems for evaluation of behavioral endpoints. Environ Toxicol Chem 2022;41:2342-2352. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Many targeted natural product isolation approaches rely on the use of pre-existing bioactivity information to inform the strategy used for the isolation of new bioactive compounds. Bioactivity information can be available either in the form of prior assay data or via Structure Activity Relationship (SAR) information which can indicate a potential chemotype that exhibits a desired bioactivity. The work described herein utilizes a unique method of targeted isolation using structure-based virtual screening to identify potential antibacterial compounds active against MRSA within the marine sponge order Verongiida. This is coupled with molecular networking-guided, targeted isolation to provide a novel drug discovery procedure. A total of 12 previously reported bromotyrosine-derived alkaloids were isolated from the marine sponge species Pseudoceratina durissima, and the compound, (+)-aeroplysinin-1 (1) displayed activity against the MRSA pathogen (MIC: <32 µg/mL). The compounds (1−3, 6 and 9) were assessed for their central nervous system (CNS) interaction and behavioral toxicity to zebrafish (Danio rerio) larvae, whereby several of the compounds were shown to induce significant hyperactivity. Anthelmintic activity against the parasitic nematode Haemonchus contorutus was also evaluated (2−4, 6−8)., Competing Interests: The authors declare no conflict of interest.

Analysis of sensorimotor behavioral responses to stimuli such as light can provide an enhanced relevance during rapid prioritisation of chemical risk. Due to technical limitations, there have been, however, only minimal studies on using invertebrate phototactic behaviors in aquatic ecotoxicity testing. In this work, we demonstrate an innovative, purpose-built analytical system for a high-throughput phototactic biotest with nauplii of euryhaline brine shrimp Artemia franciscana . We also, for the first time, present a novel and dedicated bioinformatic approach that facilitates high-throughput analysis of phototactic behaviors at scale with great fidelity. The nauplii exhibited consistent light-seeking behaviors upon extinguishing a brief programmable light stimulus (5500K, 400 lux) without habituation. A proof-of-concept validation involving the short-term exposure of eggs (24 h) and instar I larval stages (6 h) to sub-lethal concentrations of insecticides organophosphate chlorpyrifos (10 µg/L) and neonicotinoid imidacloprid (50 µg/L) showed perturbation in light seeking behaviors in the absence of or minimal alteration in general mobility. Our preliminary data further support the notion that phototactic bioassays can represent an attractive new avenue in behavioral ecotoxicology because of their potential sensitivity, responsiveness, and low cost.

Due to increasing numbers of anthropogenic chemicals with unknown neurotoxic properties, there is an increasing need for a paradigm shift toward rapid and higher throughput behavioral bioassays. In this work, we demonstrate application of a purpose-built high throughput multidimensional behavioral test battery on larval stages of Danio rerio (zebrafish) at 5 days post fertilization (dpf). The automated battery comprised of the established spontaneous swimming (SS), simulated predator response (SPR), larval photomotor response (LPR) assays as well as a new thermotaxis (TX) assay. We applied the novel system to characterize environmentally relevant concentrations of emerging pharmaceutical micropollutants including anticonvulsants (gabapentin: 400 ng/L; carbamazepine: 3000 ng/L), inflammatory drugs (ibuprofen: 9800 ng/L), and antidepressants (fluoxetine: 300 ng/L; venlafaxine: 2200 ng/L). The successful integration of the thermal preference assay into a multidimensional behavioral test battery provided means to reveal ibuprofen-induced perturbations of thermal preference behaviors upon exposure during embryogenesis. Moreover, we discovered that photomotor responses in larval stages of fish are also altered by the as yet understudied anticonvulsant gabapentin. Collectively our results demonstrate the utility of high-throughput multidimensional behavioral ecotoxicity test batteries in prioritizing emerging risks associated with neuroactive drugs that can perturb neurodevelopment. Moreover, we showcase the added value of thermotaxis bioassays for preliminary screening of emerging contaminants.

During the last decade, there has been an increase in awareness of how anthropogenic pollution can alter behavioural traits of diverse aquatic organisms. Apart from understanding profound ecological implications, alterations in neuro-behavioural indices have emerged as sensitive and physiologically integrative endpoints in chemical risk assessment. Accordingly, behavioural ecotoxicology and broader eco-neurotoxicology are becoming increasingly popular fields of research that span a plethora of fundamental laboratory experimentations as well as applied field-based studies. Despite mounting interest in aquatic behavioural ecotoxicology studies, there is, however, a considerable paucity in deciphering the mechanistic foundations underlying behavioural alterations upon exposure to pollutants. The behavioural phenotype is indeed the highest-level integrative neurobiological phenomenon, but at its core lie myriads of intertwined biochemical, cellular, and physiological processes. Therefore, the mechanisms that underlie changes in behavioural phenotypes can stem among others from dysregulation of neurotransmitter pathways, electrical signalling, and cell death of discrete cell populations in the central and peripheral nervous systems. They can, however, also be a result of toxicity to sensory organs and even metabolic dysfunctions. In this critical review, we outline why behavioural phenotyping should be the starting point that leads to actual discovery of fundamental mechanisms underlying actions of neurotoxic and neuromodulating contaminants. We highlight potential applications of the currently existing and emerging neurobiology and neurophysiology analytical strategies that should be embraced and more broadly adopted in behavioural ecotoxicology. Such strategies can provide new mechanistic discoveries instead of only observing the end sum phenotypic effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The significance of neurotoxicological risks associated with anthropogenic pollution is gaining increasing recognition worldwide. In this regard, perturbations in behavioral traits upon exposure to environmentally relevant concentrations of neurotoxic and neuro-modulating contaminants have been linked to diminished ecological fitness of many aquatic species. Despite an increasing interest in behavioral testing in aquatic ecotoxicology there is, however, a notable gap in understanding of the neurophysiological foundations responsible for the altered behavioral phenotypes. One of the canonical approaches to explain the mechanisms of neuro-behavioral changes is functional analysis of neuronal transmission. In aquatic animals it requires, however, invasive, complex, and time-consuming electrophysiology techniques. In this perspective, we highlight emerging prospects of noninvasive, in situ electrophysiology based on multielectrode arrays (MEAs). This technology has only recently been pioneered for the detection and analysis of transient electrical signals in the central nervous system of small model organisms such as zebrafish. The analysis resembles electroencephalography (EEG) applications and provides an appealing strategy for mechanistic explorative studies as well as routine neurotoxicity risk assessment. We outline the prospective future applications and existing challenges of this emerging analytical strategy that is poised to bring new vistas for aquatic ecotoxicology such as greater mechanistic understanding of eco-neurotoxicity and thus more robust risk assessment protocols.

Contamination of the environment by pharmaceutical pollutants poses an increasingly critical threat to aquatic ecosystems around the world. This is particularly true of psychoactive compounds, such as antidepressant drugs, which have become ubiquitous contaminants and have been demonstrated to modify aquatic animal behaviours at very low concentrations (i.e. ng/L). Despite raising risks to the hydrosphere, there is a notable paucity of data on the long term, multigenerational effects of antidepressants at environmentally realistic concentrations. Moreover, current research has predominantly focused on mean-level effects, with little research on variation among and within individuals when considering key behavioural traits. In this work, we used a multigenerational exposure of a freshwater snail (Physa acuta) to an environmentally relevant concentration of the antidepressant fluoxetine (mean measured concentration: 32.7 ng/L, SE: 2.3). The snails were allowed to breed freely in large mesocosm populations over 3 years. Upon completion of the exposure, we repeatedly measured the locomotory activity (624 measures total), reproductive output (234 measures total) as well as morphometric endpoints (78 measures total). While we found no mean-level differences between treatments in locomotory activities, we did find that fluoxetine exposed snails (n = 46) had significantly reduced behavioural plasticity (i.e. V W ; within-individual variation) in activity levels compared to unexposed snails (n = 32). As a result, fluoxetine exposed snails demonstrated significant behavioural repeatability, which was not the case for unexposed snails. Further, we report a reduction in egg mass production in fluoxetine exposed snails, and a marginally non-significant difference in morphology between treatment groups. These results highlight the potential detrimental effects of long-term fluoxetine exposure on non-target organisms at environmentally realistic dosages. Additionally, our findings demonstrate the underappreciated potential for psychoactive contaminants to have impacts beyond mean-level effects, with consequences for population resilience to current and future environmental challenges., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cell adhesion molecules are membrane-bound proteins predominantly expressed in the central nervous system along principal axonal pathways with key roles in nervous system development, neural cell differentiation and migration, axonal growth and guidance, myelination, and synapse formation. Here, we describe ten affected individuals with bi-allelic variants in the neuronal cell adhesion molecule NRCAM that lead to a neurodevelopmental syndrome of varying severity; the individuals are from eight families. This syndrome is characterized by developmental delay/intellectual disability, hypotonia, peripheral neuropathy, and/or spasticity. Computational analyses of NRCAM variants, many of which cluster in the third fibronectin type III (Fn-III) domain, strongly suggest a deleterious effect on NRCAM structure and function, including possible disruption of its interactions with other proteins. These findings are corroborated by previous in vitro studies of murine Nrcam-deficient cells, revealing abnormal neurite outgrowth, synaptogenesis, and formation of nodes of Ranvier on myelinated axons. Our studies on zebrafish nrcama Δ mutants lacking the third Fn-III domain revealed that mutant larvae displayed significantly altered swimming behavior compared to wild-type larvae (p < 0.03). Moreover, nrcama Δ mutants displayed a trend toward increased amounts of α-tubulin fibers in the dorsal telencephalon, demonstrating an alteration in white matter tracts and projections. Taken together, our study provides evidence that NRCAM disruption causes a variable form of a neurodevelopmental disorder and broadens the knowledge on the growing role of the cell adhesion molecule family in the nervous system., Competing Interests: Declaration of interests C.G.-J. is a full-time employee of the Regeneron Genetics Center and receives stock options as part of compensation. All other authors have no conflicts to declare., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Environmental toxicology focuses on identifying and predicting impact of potentially toxic anthropogenic chemicals on biosphere at various levels of biological organization. Presently there is a significant drive to gain deeper understanding of cellular and sub-cellular mechanisms of ecotoxicity. Most notable is increased focus on elucidation of cellular-response networks, interactomes, and greater implementation of cell-based biotests using high-throughput procedures, while at the same time decreasing the reliance on standard animal models used in ecotoxicity testing. This is aimed at discovery and interpretation of molecular pathways of ecotoxicity at large scale. In this regard, the applications of cytometry are perhaps one of the most fundamental prospective analytical tools for the next generation and high-throughput ecotoxicology research. The diversity of this modern technology spans flow, laser-scanning, imaging, and more recently, Raman as well as mass cytometry. The cornerstone advantages of cytometry include the possibility of multi-parameter measurements, gating and rapid analysis. Cytometry overcomes, thus, limitations of traditional bulk techniques such as spectrophotometry or gel-based techniques that average the results from pooled cell populations or small model organisms. Novel technologies such as cell imaging in flow, laser scanning cytometry, as well as mass cytometry provide innovative and tremendously powerful capabilities to analyze cells, tissues as well as to perform in situ analysis of small model organisms. In this review, we outline cytometry as a tremendously diverse field that is still vastly underutilized and often largely unknown in environmental sciences. The main motivation of this work is to highlight the potential and wide-reaching applications of cytometry in ecotoxicology, guide environmental scientists in the technological aspects as well as popularize its broader adoption in environmental risk assessment., (© 2021 International Society for Advancement of Cytometry.)

The use of small aquatic model organisms to investigate the behavioral effects of chemical exposure is becoming an integral component of aquatic ecotoxicology research and neuroactive drug discovery. Despite the increasing use of invertebrates for behavioral phenotyping in toxicological studies and chemical risk assessments, little is known regarding the potential for environmental factors-such as geometry, size, opacity and depth of test chambers-to modulate common behavioral responses. In this work, we demonstrate that test chamber geometry, size, opacity and depth can affect spontaneous, unstimulated behavioral responses of euryhaline crustacean Artemia franciscana first instar larval stages. We found that in the absence of any obvious directional cues, A. franciscana exhibited a strong innate wall preference behavior. Using different test chamber sizes and geometries, we found both increased wall preference and lowered overall distance traveled by the test shrimp in a smaller chamber with sharper-angled vertices. It was also determined through quantifiable changes in the chambers' color that the A. franciscana early larval stages can perceive, differentiate and react to differences in color or perhaps rather to light transmittance of the test chambers. The interaction between innate edge preference and positive phototaxis could be consistently altered with a novel photic stimulus system. We also observed a strong initial preference for depth in A. franciscana first instar larval stages, which diminished through the acclimatization. We postulate that the impact of test chamber designs on neurobehavioral baseline responses warrants further investigation, in particular considering the increased interest in behavioral eco-neurotoxicology applications., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Large-scale chemobehavioral phenotyping with zebrafish embryos is a promising avenue for accelerated neurotoxicity testing and discovery of behavior-modifying neuroceuticals. These strategies are hampered by lack of effective embryo in-test positioning, wide-field imaging, and high-throughput bioinformatic analytics. In this study, we demonstrate advantages of using custom large-density embryo arrays in conjunction with an open-source ultra-high-definition video imaging system. Moreover, we present a high-throughput bioinformatics workflow for rapid behavioral analysis of large cohorts of specimens in photomotor response bioassays. The system validation was showcased in a proof-of-concept neurotoxicity analysis.

Continuous monitoring and early warning of potential water contamination with toxic chemicals is of paramount importance for human health and sustainable food production. During the last few decades there have been noteworthy advances in technologies for the automated sensing of physicochemical parameters of water. These do not translate well into online monitoring of chemical pollutants since most of them are either incapable of real-time detection or unable to detect impacts on biological organisms. As a result, biological early warning systems have been proposed to supplement conventional water quality test strategies. Such systems can continuously evaluate physiological parameters of suitable aquatic species and alert the user to the presence of toxicants. In this regard, single cellular organisms, such as bacteria, cyanobacteria, micro-algae and vertebrate cell lines, offer promising avenues for development of water biosensors. Historically, only a handful of systems utilising single-cell organisms have been deployed as established online water biomonitoring tools. Recent advances in recombinant microorganisms, cell immobilisation techniques, live-cell microarrays and microfluidic Lab-on-a-Chip technologies open new avenues to develop miniaturised systems capable of detecting a broad range of water contaminants. In experimental settings, they have been shown as sensitive and rapid biosensors with capabilities to detect traces of contaminants. In this work, we critically review the recent advances and practical prospects of biological early warning systems based on live-cell biosensors. We demonstrate historical deployment successes, technological innovations, as well as current challenges for the broader deployment of live-cell biosensors in the monitoring of water quality.

Monitoring of freshwater quality and its potential sudden contamination is integral to human health, sustainable economic development and prediction of pollutant impact on aquatic ecosystems. Although there have been significant advances in technologies for automated sampling and continuous analysis of water physicochemical parameters, the current capabilities for real-time warning against rapidly developing unknown mixtures of chemical hazards are still limited. Conventional chemical analysis systems are not suitable for assessing unknown mixtures of chemicals as well as additive and/or synergetic effects on biological systems. From the perspective of neurotoxicology the acute exposures to chemical agents that affect nervous system and can enter the freshwater supplies accidentally or as a result of deliberate action, can only be reliably assessed using appropriate functional biological models. In this regard real-time biological early warning systems (BEWS), that can continuously monitor behavioural and/or physiological parameters of suitable aquatic bioindicator species, have been historically proposed to fill the gap and supplement conventional water quality test strategies. Alterations in sub-lethal neuro-behavioural traits have been proven as very sensitive and physiologically relevant endpoints that can provide highly integrative water quality sensing capabilities. Although BEWS are commonly regarded as non-specific and lacking both quantitative and qualitative detection capabilities, their advantages, if properly designed and implemented, lie in continuous sensing and early-warning information about sudden alteration in water quality parameters. In this work we review the future prospects of real-time biological early warning systems as well as recent developments that are anchored in historical successes and practical deployment examples. We concentrate on technologies utilizing analysis of behavioural and physiological endpoints of animal bioindicators and highlight the existing challenges, barriers to future development and demonstrate how recent advances in inexpensive electronics and multidisciplinary bioengineering can help revitalize the BEWS field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Well-quantified laboratory studies can provide a fundamental understanding of animal behavior in ecology, ethology and ecotoxicology research. These types of studies require observation and tracking of each animal in well-controlled and defined arenas, often for long timescales. Thus, these experiments produce long time series and a vast amount of data that require the use of software applications to automate the analysis and reduce manual annotation. In this review, we examine 28 free software applications for animal tracking to guide researchers in selecting the software that might best suit a particular experiment. We also review the algorithms in the tracking pipeline of the applications, explain how specific techniques can fit different experiments, and finally, expose each approach's weaknesses and strengths. Our in-depth review includes last update, type of platform, user-friendliness, off- or online video acquisition, calibration method, background subtraction and segmentation method, species, multiple arenas, multiple animals, identity preservation, manual identity correction, data analysis and extra features. We found, for example, that out of 28 programs, only 3 include a calibration algorithm to reduce image distortion and perspective problems that affect accuracy and can result in substantial errors when analyzing trajectories and extracting mobility or explored distance. In addition, only 4 programs can directly export in-depth tracking and analysis metrics, only 5 are suited for tracking multiple unmarked animals for more than a few seconds and only 11 have been updated in the period 2019-2021., (© 2021. Springer Nature America, Inc.)

Despite mounting evidence of pleiotropic ecological risks, the understanding of the eco-neurotoxic impact of most industrially relevant chemicals is still very limited. In particularly the acute and chronic exposures to industrial pollutants on nervous systems and thus potential alterations in ecological fitness remain profoundly understudied. Since the behavioral phenotype is the highest-level and functional manifestation of integrated neurological functions, the alterations in neuro-behavioral traits have been postulated as very sensitive and physiologically integrative endpoints to assess eco-neurotoxicological risks associated with industrial pollutants. Due to a considerable backlog of risk assessments of existing and new production chemicals there is a need for a paradigm shift from high cost, low throughput ecotoxicity test models to next generation systems amenable to higher throughput. In this review we concentrate on emerging aspects of laboratory-based neuro-behavioral phenotyping approaches that can be amenable for rapid prioritizing pipelines. We outline the importance of development and applications of innovative neuro-behavioral assays utilizing small aquatic biological indicators and demonstrate emerging concepts of high-throughput chemo-behavioral phenotyping. We also discuss new analytical approaches to effectively and rapidly evaluate the impact of pollutants on higher behavioral functions such as sensory-motor assays, decision-making and cognitive behaviors using innovative model organisms. Finally, we provide a snapshot of most recent analytical approaches that can be applied to elucidate mechanistic rationale that underlie the observed neuro-behavioral alterations upon exposure to pollutants. This review is intended to outline the emerging opportunities for innovative multidisciplinary research and highlight the existing challenges as well barriers to future development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N -sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgsh Δex5-6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgsh Δex5-6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgsh Δex5-6 zebrafish is largely dependent on interleukin-1β and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgsh Δex5-6 mutant larvae in a context-dependent manner. We expect the sgsh Δex5-6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.

Addressing the shift from classical animal testing to high-throughput in vitro and/or simplified in vivo proxy models has been defined as one of the upcoming challenges in aquatic toxicology. In this regard, the fish embryo toxicity test (FET) has gained significant popularity and wide standardization as one of the sensitive alternative approaches to acute fish toxicity tests in chemical risk assessment and water quality evaluation. Nevertheless, despite the growing regulatory acceptance, the actual manipulation, dispensing, and analysis of living fish embryos remains very labor intensive. Moreover, the FET is commonly performed in plastic multiwell plates under static or semistatic conditions, potentially inadequate for toxicity assessment of some organic, easily degradable or highly adsorptive toxicants. Recent technological advances in the field of mechatronics, fluidics and digital vision systems demonstrate promising future opportunities for automation of many analytical stages in embryo toxicity testing. In this review, we highlight emerging advances in fluidic and laboratory automation systems that can prospectively enable high-throughput FET testing (HT-FET) akin to pipelines commonly found in in vitro drug discovery pipelines. We also outline the existing challenges, barriers to future development and provide an outlook of ground-breaking fluidic technologies in embryo toxicity testing.

Neurotoxicity effects of industrial contaminants are currently significantly under investigated and require innovative analytical approaches to assess health and environmental risks at individual, population and ecosystem levels. Behavioral changes assessed using small aquatic invertebrates as standard biological indicators of the aggregate toxic effects, have been broadly postulated as highly integrative indicators of neurotoxicity with physiological and ecological relevance. Despite recent increase in understanding of the emerging value of behavioral biotests, their wider implementation especially in high-throughput environmental risk assessment assays, is largely limited by the lack of advances in analytical technologies. To date, most of the behavioral biotests have only been performed with larger-volumes and lacked dynamic flow-through conditions. They also lack features necessary for development of higher throughput neuro-behavioral ecotoxicity assays such as miniaturization and integration of automated components. We postulate that some contemporary analytical limitations can be effectively addressed by innovative Lab-on-a-Chip (LOC) technologies, an emerging and multidisciplinary field poised to bring significant miniaturization to aquatic ecotoxicity testing. Recent developments in this emerging field demonstrate particular opportunities to study a plethora of behavioral responses of small model organisms in a high-throughput fashion. In this review, we highlight recent advances in this budding new interdisciplinary field of research. We also outline the existing challenges, barriers to development and provide a future outlook in the evolving field of neurobehavioral ecotoxicology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The global outbreak of a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlighted a requirement for two pronged clinical interventions such as development of effective vaccines and acute therapeutic options for medium-to-severe stages of "coronavirus disease 2019" (COVID-19). Effective vaccines, if successfully developed, have been emphasized to become the most effective strategy in the global fight against the COVID-19 pandemic. Basic research advances in biotechnology and genetic engineering have already provided excellent progress and groundbreaking new discoveries in the field of the coronavirus biology and its epidemiology. In particular, for the vaccine development the advances in characterization of a capsid structure and identification of its antigens that can become targets for new vaccines. The development of the experimental vaccines requires a plethora of molecular techniques as well as strict compliance with safety procedures. The research and clinical data integrity, cross-validation of the results, and appropriated studies from the perspective of efficacy and potently side effects have recently become a hotly discussed topic. In this review, we present an update on latest advances and progress in an ongoing race to develop 52 different vaccines against SARS-CoV-2. Our analysis is focused on registered clinical trials (current as of November 04, 2020) that fulfill the international safety and efficacy criteria in the vaccine development. The requirements as well as benefits and risks of diverse types of SARS-CoV-2 vaccines are discussed including those containing whole-virus and live-attenuated vaccines, subunit vaccines, mRNA vaccines, DNA vaccines, live vector vaccines, and also plant-based vaccine formulation containing coronavirus-like particle (VLP). The challenges associated with the vaccine development as well as its distribution, safety and long-term effectiveness have also been highlighted and discussed., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Behavioral phenotyping is an essential part of neuro-active drug discovery and predictive neurotoxicology. Due to limitations of conventional rodent in vivo models, chemobehavioral phenotypic analysis utilizing innovative small model organisms; such as nematodes, planarians and zebrafish are emerging as distinctively advantageous for high-throughput phenotypic discovery of neuroceuticals and evaluating deleterious effects of industrial pollutants on the central nervous system. Digital film recording with subsequent analysis of video sequences using specialised animal tracking software has become a standard in obtaining behavioral biometric data. At present animal tracking algorithms are largely capable of detecting and tracking small number of animals and extracting quantitative endpoints associated with specific behavioral traits based on reconstruction of movement trajectories and occupancy heatmaps. However, despite recent and significant progress in development of diverse proxy biological models, the software algorithms still lack the ability to track multiple organisms on a large scale, automatically generate behavioral fingerprints and utilize intensive computational approaches to mine rich biometric data. This creates a significant bottleneck for effective high-throughput chemobehavioral screening in drug discovery and neurotoxicology. This review outlines recent advances as well as limitations of high-throughput animal tracking and provides an outlook on future developments in rapidly evolving field of neurobehavioral phenomics., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Phenol-formaldehyde plastics are used globally as floral foam and generate microplastics that can enter the environment. This study is the first to describe how aquatic animals interact with this type of microplastic, and the resultant physiological responses. We analysed "regular foam" microplastics generated from petroleum-derived phenol-formaldehyde plastic, and "biofoam" microplastics generated from plant-derived phenol-formaldehyde plastic. Regular foam and biofoam microplastics showed similar FTIR spectra. Both types of microplastics were consumed by all six invertebrate species tested: the freshwater gastropod Physa acuta, the marine gastropod Bembicium nanum, the marine bivalve Mytilus galloprovincialis, adults and neonates of the freshwater crustacean Daphnia magna, the marine amphipod Allorchestes compressa, and nauplii of the marine crustacean Artemia sp. For all species, the occurrence of ingestion was similar for regular foam and biofoam microplastics. Biofoam microplastics leached more than twice as much phenolic compounds than regular foam microplastics. The leachates from regular foam and biofoam microplastics showed the same acute toxicity to Artemia nauplii (24-h LC 50  = 27.4 mg mL - 1 , respectively) and D. magna (48-h LC - 1 , respectively) and D. magna (48-h LC 50  = 17.8 mg mL - 1 and 15.3 mg mL - 1 , respectively). However, biofoam microplastic leachate was twice as toxic to embryos of the zebrafish, Danio rerio, compared with leachate from regular foam microplastic (96-h LC 50  = 43.8 mg mL - 1 vs 27.1 mg mL - 1 ). Using M. galloprovincialis, we show that regular foam microplastic leachate and the physical presence of the microplastics exerted separate and cumulative effects on catalase (CAT) activity, glutathione-s-transferase (GST) activity and lipid peroxidation. Microplastic ingestion did not affect the activity of acetylcholinesterase (AChE). Taken together, these results show that phenol-formaldehyde microplastics can interact with a range of aquatic animals, and affect sublethal endpoints by leaching toxic compounds, and through the physical presence of the microplastics themselves., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Chemosensory avoidance behaviors of aquatic invertebrates provide a functional link between early responses to pollutants at the infraorganismal and ecologically relevant supraorganismal levels. Despite significant importance, there is, however, a notable lack of user-friendly laboratory techniques. Here, we demonstrate a scalable millifluidic platform for higher throughput quantitative chemobehavioral studies. With a proof-of-concept application of this technology, we discovered that native Australian marine amphipods Allorchestes compressa exhibit rapid avoidance behaviors against a panel of environmental stressors. This work provides a novel avenue for the development of quantitative neurobehavioral systems applicable in diverse environmental risk assessment studies., (Copyright © 2020 Author(s).)

A series of new pyrazolo-benzothiazole hybrids (7-26) were synthesised and screened for their cytotoxic activity towards several cancer cell lines [colon (HT-29), prostate (PC-3), lung (A549), glioblastoma (U87MG)] and normal human embryonic kidney cell line (Hek-293T). Compounds 8, 9, 13, 14, 18, 19, 23, and 24 displayed significant activity, with compound 14 being particularly potent towards all the tested cancer cell lines with IC 50 values in the range 3.17-6.77 μM, even better than reference drug axitinib (4.88-21.7 μM). Compound 14 also showed the strongest growth inhibition in 3D multicellular spheroids of PC-3 and U87MG cells. The mechanism of cellular toxicity in PC-3 cells was found to be cell cycle arrest and apoptosis induction through depolarisation of mitochondrial membrane potential, increased ROS production and subsequent DNA damage. Further, compound 14 displayed significant in vitro (VEGFR-2 inhibition) and in vivo [transgenic zebrafish Tg(flila:EGFP) model] antiangiogenic properties. Overall, these results provide strong evidence that compound 14 could be considered for a lead candidate in anticancer and antiangiogenic drug discovery., (Copyright © 2019. Published by Elsevier Masson SAS.)

Chemobehavioural phenotypic analysis using small aquatic model organisms is becoming an important toolbox in aquatic ecotoxicology and neuroactive drug discovery. The analysis of the organisms' behavior is usually performed by combining digital video recording with animal tracking software. This software detects the organisms in the video frames, and reconstructs their movement trajectory using image processing algorithms. In this work we investigated the impact of video file characteristics, video optimization techniques and differences in animal tracking algorithms on the accuracy of quantitative neurobehavioural endpoints. We employed larval stages of a free-swimming euryhaline crustacean Artemia franciscana ,commonly used for marine ecotoxicity testing, as a proxy modelto assess the effects of video analytics on quantitative behavioural parameters. We evaluated parameters such as data processing speed, tracking precision, capability to perform high-throughput batch processing of video files. Using a model toxicant the software algorithms were also finally benchmarked against one another. Our data indicates that variability in video file parameters; such as resolution, frame rate, file containers types, codecs and compression levels, can be a source of experimental biases in behavioural analysis. Similarly, the variability in data outputs between different tracking algorithms should be taken into account when designing standardized behavioral experiments and conducting chemobehavioural phenotyping., Competing Interests: The authors declare there are no competing interests.

The environmental impact of exposure to 3D-printed plastics as well as potential migration of toxic chemicals from 3D-printed plastics remains largely unexplored. In this work we applied leachates from plastics fabricated using a stereolithography (SLA) process to early developmental stages of zebrafish (Danio rerio) to investigate developmental toxicity and neurotoxicity. Migration of unpolymerized photoinitiator, 1-hydroxycyclohexyl phenyl ketone (1-HCHPK) from a plastic solid phase to aqueous media at up to 200 mg/L in the first 24 h was detected using gas chromatography-mass spectrometry. Both plastic extracts (LC 50 22.25% v/v) and 1-HCHPK (LC 50 60 mg/L) induced mortality and teratogenicity within 48 h of exposure. Developmental toxicity correlated with in situ generation of reactive oxygen species (ROS), an increase in lipid peroxidation and protein carbonylation markers and enhanced activity of superoxide dismutase (SOD) and glutathione-S-transferase (GST) in embryos exposed to concentrations as low as 20% v/v for plastic extracts and 16 mg/L for 1-HCHPK. ROS-induced cellular damage led to induction of caspase-dependent apoptosis which could be pharmacologically inhibited with both antioxidant ascorbic acid and a pan-caspase inhibitor. Neuro-behavioral analysis showed that exposure to plastic leachates reduced spontaneous embryonic movement in 24-36 hpf embryos. Plastic extracts in concentrations above 20% v/v induced rapid retardation of locomotion, changes in photomotor response and habituation to photic stimuli with progressive paralysis in 120 hpf larvae. Significantly decreased acetylcholinesterase (AChE) activity with lack of any CNS-specific apoptotic phenotypes as well as lack of changes in motor neuron density, axonal growth, muscle segment integrity or presence of myoseptal defects were detected upon exposure to plastic extracts during embryogenesis. Considering implications of the results for environmental risk assessment and the growing usage of 3D-printing technologies, we speculate that some 3D-printed plastic waste may represent a significant and yet very poorly uncharacterized environmental hazard that merits further investigation on a range of aquatic and terrestrial species., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Identifying novel marine-derived neuroactive chemicals with therapeutic potential is difficult due to inherent complexities of the central nervous system (CNS), our limited understanding of the molecular foundations of neuro-psychiatric conditions, as well as the limited applications of effective high-throughput screening models that recapitulate functionalities of the intact CNS. Furthermore, nearly all neuro-modulating chemicals exhibit poorly characterized pleiotropic activities often referred to as polypharmacology. The latter renders conventional target-based in vitro screening approaches very difficult to accomplish. In this context, chemobehavioural phenotyping using innovative small organism models such as planarians and zebrafish represent powerful and highly integrative approaches to study the impact of new chemicals on central and peripheral nervous systems. In contrast to in vitro bioassays aimed predominantly at identification of chemicals acting on single targets, phenotypic chemobehavioural analysis allows for complex multi-target interactions to occur in combination with studies of polypharmacological effects of chemicals in a context of functional and intact milieu of the whole organism. In this review, we will outline recent advances in high-throughput chemobehavioural phenotyping and provide a future outlook on how those innovative methods can be utilized for rapidly screening and characterizing marine-derived compounds with prospective applications in neuropharmacology and psychosomatic medicine.

A series of alkynylgold(I) phosphine complexes containing methoxy-substituted cinnamide moieties (3a-3c and 4a-4c) have been synthesized and characterized. All of the synthesized complexes were evaluated for their cytotoxicity against three human cancer cell lines A549 (lung), D24 (melanoma), and HT1080 (fibrosarcoma) and the human embryonic kidney 293 cell line (Hek293T) as a proxy model for noncancer cells. Most of the synthesized compounds showed antiproliferative activity against cancer cell lines at low micromolar concentrations. Among these, complex 3c showed a broad spectrum of anticancer activity with IC 50 values in the range of 1.53-6.05 μM against all tested cancer lines. Complex 3c possessed 20 times higher cytotoxicity than the reference drug cisplatin against D24 melanoma cells and showed significant anticancer activity in 3D spheroidal models of melanoma cells. Mechanistic investigations of 3c activity indicate thioredoxin reductase inhibition through steric and hydrogen-bonding interactions, followed by the induction of oxidative stress and a mitochondrial pathway of cell death. Compound 3c also showed significant antiangiogenic properties in a transgenic zebrafish Tg(fli1a:EGFP) in vivo model.

Zebrafish larvae are suitable in vivo models for toxicological and pharmacological screens due to their transparency, small size, ex utero development, and genetic and physiological similarity to humans. Using modern imaging techniques, cells and tissues can be dynamically visualized over several days in multiple zebrafish larvae. However, precise specimen immobilization and maintenance of homeostatic conditions remain a challenge for longitudinal studies. A highly customizable mounting configuration with inbuilt means of controlling temperature and media flow would therefore be a valuable tool to facilitate long-term imaging of a large number of specimens. Using three-dimensional printing, we have developed a millifluidic, modular homeostatic imaging plate (HIP), which consists of a customizable sample insert and a temperature-controlled incubation chamber that is continuously perfused, providing an ideal environment for long-term experiments where homeostatic conditions are desired. The HIP is cheap to produce, has a standard microtiter well plate format, and can be fitted to most microscopes. We used the device to image dynamic regeneration of spinal cord neurons. The flexibility and adaptability of the HIP facilitate long-term in vivo imaging of many samples, and can be easily adapted to suit a broad range of specimens.

Quantification of neuro-behavioural responses of intact small model organisms has been proposed as a sensitive, sub-lethal alternative to conventional toxicity testing. Such bioassays are characterized by a high physiological and ecological relevance, short response times, increased sensitivity, and non-invasive nature. Despite a significant potential for predictive aquatic toxicology analysis of behavioural traits of micro-invertebrates in microfluidic environment has received little attention. In this work, we demonstrate a new Lab-on-a-Chip technology capable of effectively caging freshwater rotifers Brachionus calyciflorus for real-time video-microscopy analysis. We demonstrate that behavioural bioassays performed under microfluidic perfusion can significantly enhance the sensitivity of conventional ecotoxicology test protocols.