Bi-allelic variants in neuronal cell adhesion molecule cause a neurodevelopmental disorder characterized by developmental delay, hypotonia, neuropathy/spasticity.

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 nrcamaΔ 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, nrcamaΔ 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.

Toward Real-Time Animal Tracking with Integrated Stimulus Control for Automated Conditioning in Aquatic Eco-Neurotoxicology.

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 progress in cytometric technologies and their applications in ecotoxicology and environmental risk assessment.

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.

Noninvasive Electrophysiology: Emerging Prospects in Aquatic Neurotoxicity Testing.

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.

Targeted Isolation of Antibiotic Brominated Alkaloids from the Marine Sponge Pseudoceratina durissima Using Virtual Screening and Molecular Networking.

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).

Sensory-Motor Perturbations in Larval Zebrafish (Danio rerio) Induced by Exposure to Low Levels of Neuroactive Micropollutants during Development.

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.

Toward High-Throughput Fish Embryo Toxicity Tests in Aquatic Toxicology.

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.

Live-Cell Systems in Real-Time Biomonitoring of Water Pollution: Practical Considerations and Future Perspectives.

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.

An Engineered sgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA.

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 sgshΔex5-6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgshΔex5-6 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 sgshΔex5-6 zebrafish is largely dependent on interleukin-1ß and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgshΔex5-6 mutant larvae in a context-dependent manner. We expect the sgshΔex5-6 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.

Synthesis of Gold(I) Complexes Containing Cinnamide: In Vitro Evaluation of Anticancer Activity in 2D and 3D Spheroidal Models of Melanoma and In Vivo Angiogenesis.

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 IC50 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.

Towards High-Throughput Chemobehavioural Phenomics in Neuropsychiatric Drug Discovery.

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.

Rapid Fabrication of Chip-Based Physiometers for Neurobehavioral Toxicity Assays Using Rotifers Brachionus calyciflorus.

An increased interest in implementations of Lab-on-a-Chip (LOC) technologies for in-situ analysis of multicellular metazoan model organisms and their embryonic stages demands development of new prototyping techniques. Due to size of multicellular organisms the fabrication of soft-lithography molds requires features with high aspect ratios as well as deposition of layers with significant thicknesses. This makes them time consuming and difficult to fabricate using conventional photolithography techniques. In this work we describe development of a rapid technique capable of generating thick films achieved with high viscosity SU-8 and used in fabricating master templates for high aspect ratio micro- and mesofluidic devices. The cost effective and rapid method eliminated the need for multiple spin coating cycles as well as edge bead artifacts while preserving low surface roughness and superior surface uniformity. Due to elimination of spin coating steps, typically constrained to clean room facilities, the new method allows to significantly reduce microfabrication costs. We have utilized the prototyping technique to develop proof-of-concept chip-based devices capable of effectively caging freshwater rotifers Brachionus calyciflorus for high-definition video-microscopy analysis. The combination of time-resolved video-microscopy and chip-based physiometers enabled us to demonstrate new applications for neurobehavioral assays utilizing non-invasive sub-lethal end-points.

Ecotoxicology Goes on a Chip: Embracing Miniaturized Bioanalysis in Aquatic Risk Assessment.

Biological and environmental sciences are, more than ever, becoming highly dependent on technological and multidisciplinary approaches that warrant advanced analytical capabilities. Microfluidic lab-on-a-chip technologies are perhaps one the most groundbreaking offshoots of bioengineering, enabling design of an entirely new generation of bioanalytical instrumentation. They represent a unique approach to combine microscale engineering and physics with specific biological questions, providing technological advances that allow for fundamentally new capabilities in the spatiotemporal analysis of molecules, cells, tissues, and even small metazoan organisms. While these miniaturized analytical technologies experience an explosive growth worldwide, with a substantial promise of a direct impact on biosciences, it seems that lab-on-a-chip systems have so far escaped the attention of aquatic ecotoxicologists. In this Critical Review, potential applications of the currently existing and emerging chip-based technologies for aquatic ecotoxicology and water quality monitoring are highlighted. We also offer suggestions on how aquatic ecotoxicology can benefit from adoption of microfluidic lab-on-a-chip devices for accelerated bioanalysis.

Bioelectric signalling via potassium channels: a mechanism for craniofacial dysmorphogenesis in KCNJ2-associated Andersen-Tawil Syndrome.

KEY POINTS: Xenopus laevis craniofacial development is a good system for the study of Andersen-Tawil Syndrome (ATS)-associated craniofacial anomalies (CFAs) because (1) Kcnj2 is expressed in the nascent face; (2) molecular-genetic and biophysical techniques are available for the study of ion-dependent signalling during craniofacial morphogenesis; (3) as in humans, expression of variant Kcnj2 forms in embryos causes a muscle phenotype; and (4) variant forms of Kcnj2 found in human patients, when injected into frog embryos, cause CFAs in the same cell lineages. Forced expression of WT or variant Kcnj2 changes the normal pattern of Vmem (resting potential) regionalization found in the ectoderm of neurulating embryos, and changes the normal pattern of expression of ten different genetic regulators of craniofacial development, including markers of cranial neural crest and of placodes. Expression of other potassium channels and two different light-activated channels, all of which have an effect on Vmem , causes CFAs like those induced by injection of Kcnj2 variants. In contrast, expression of Slc9A (NHE3), an electroneutral ion channel, and of GlyR, an inactive Cl(-) channel, do not cause CFAs, demonstrating that correct craniofacial development depends on a pattern of bioelectric states, not on ion- or channel-specific signalling. Using optogenetics to control both the location and the timing of ion flux in developing embryos, we show that affecting Vmem of the ectoderm and no other cell layers is sufficient to cause CFAs, but only during early neurula stages. Changes in Vmem induced late in neurulation do not affect craniofacial development. We interpret these data as strong evidence, consistent with our hypothesis, that ATS-associated CFAs are caused by the effect of variant Kcnj2 on the Vmem of ectodermal cells of the developing face. We predict that the critical time is early during neurulation, and the critical cells are the ectodermal cranial neural crest and placode lineages. This points to the potential utility of extant, ion flux-modifying drugs as treatments to prevent CFAs associated with channelopathies such as ATS. ABSTRACT: Variants in potassium channel KCNJ2 cause Andersen-Tawil Syndrome (ATS); the induced craniofacial anomalies (CFAs) are entirely unexplained. We show that KCNJ2 is expressed in Xenopus and mouse during the earliest stages of craniofacial development. Misexpression in Xenopus of KCNJ2 carrying ATS-associated mutations causes CFAs in the same structures affected in humans, changes the normal pattern of membrane voltage potential regionalization in the developing face and disrupts expression of important craniofacial patterning genes, revealing the endogenous control of craniofacial patterning by bioelectric cell states. By altering cells' resting potentials using other ion translocators, we show that a change in ectodermal voltage, not tied to a specific protein or ion, is sufficient to cause CFAs. By adapting optogenetics for use in non-neural cells in embryos, we show that developmentally patterned K(+) flux is required for correct regionalization of the resting potentials and for establishment of endogenous early gene expression domains in the anterior ectoderm, and that variants in KCNJ2 disrupt this regionalization, leading to the CFAs seen in ATS patients.

Real-time 2D visualization of metabolic activities in zebrafish embryos using a microfluidic technology.

Non-invasive and real-time visualization of metabolic activities in living small model organisms such as embryos and larvae of zebrafish has not yet been attempted largely due to profound analytical limitations of existing technologies. Historically, our capacity to examine oxygen gradients surrounding eggs and embryos has been severely limited, so much so that to date, most of the articles characterizing in situ oxygen gradients have described predominantly mathematical simulations. These drawbacks can, however, be experimentally addressed by an emerging field of microfluidic Lab-on-a-Chip (LOC) technologies combined with sophisticated optoelectronic sensors. In this work, we outline a proof-of-concept approach utilizing microfluidic living embryo array system to enable in situ Fluorescence Ratiometric Imaging (FRIM) on developing zebrafish embryos. The FRIM is an innovative method for kinetic quantification of the temporal patterns of aqueous oxygen gradients at a very fine scale based on signals coming from an optical sensor referred to as a sensor foil. We envisage that future integration of microfluidic chip-based technologies with FRIM represents a noteworthy direction to miniaturize and revolutionize research on metabolism and physiology in vivo.

Microfluidic device for a rapid immobilization of zebrafish larvae in environmental scanning electron microscopy.

Small vertebrate model organisms have recently gained popularity as attractive experimental models that enhance our understanding of human tissue and organ development. Despite a large body of evidence using optical spectroscopy for the characterization of small model organism on chip-based devices, no attempts have been so far made to interface microfabricated technologies with environmental scanning electron microscopy (ESEM). Conventional scanning electron microscopy requires high vacuum environments and biological samples must be, therefore, submitted to many preparative procedures to dehydrate, fix, and subsequently stain the sample with gold-palladium deposition. This process is inherently low-throughput and can introduce many analytical artifacts. This work describes a proof-of-concept microfluidic chip-based system for immobilizing zebrafish larvae for ESEM imaging that is performed in a gaseous atmosphere, under low vacuum mode and without any need for sample staining protocols. The microfabricated technology provides a user-friendly and simple interface to perform ESEM imaging on zebrafish larvae. Presented lab-on-a-chip device was fabricated using a high-speed infrared laser micromachining in a biocompatible poly(methyl methacrylate) thermoplastic. It consisted of a reservoir with multiple semispherical microwells designed to hold the yolk of dechorionated zebrafish larvae. Immobilization of the larvae was achieved by a gentle suction generated during blotting of the medium. Trapping region allowed for multiple specimens to be conveniently positioned on the chip-based device within few minutes for ESEM imaging.

Automated Lab-on-a-Chip Technology for Fish Embryo Toxicity Tests Performed under Continuous Microperfusion (µFET).

The fish embryo toxicity (FET) biotest has gained popularity as one of the alternative approaches to acute fish toxicity tests in chemical hazard and risk assessment. Despite the importance and common acceptance of FET, it is still performed in multiwell plates and requires laborious and time-consuming manual manipulation of specimens and solutions. This work describes the design and validation of a microfluidic Lab-on-a-Chip technology for automation of the zebrafish embryo toxicity test common in aquatic ecotoxicology. The innovative device supports rapid loading and immobilization of large numbers of zebrafish embryos suspended in a continuous microfluidic perfusion as a means of toxicant delivery. Furthermore, we also present development of a customized mechatronic automation interface that includes a high-resolution USB microscope, LED cold light illumination, and miniaturized 3D printed pumping manifolds that were integrated to enable time-resolved in situ analysis of developing fish embryos. To investigate the applicability of the microfluidic FET (µFET) in toxicity testing, copper sulfate, phenol, ethanol, caffeine, nicotine, and dimethyl sulfoxide were tested as model chemical stressors. Results obtained on a chip-based system were compared with static protocols performed in microtiter plates. This work provides evidence that FET analysis performed under microperfusion opens a brand new alternative for inexpensive automation in aquatic ecotoxicology.

Integrated chip-based physiometer for automated fish embryo toxicity biotests in pharmaceutical screening and ecotoxicology.

Transgenic zebrafish (Danio rerio) models of human diseases have recently emerged as innovative experimental systems in drug discovery and molecular pathology. None of the currently available technologies, however, allow for automated immobilization and treatment of large numbers of spatially encoded transgenic embryos during real-time developmental analysis. This work describes the proof-of-concept design and validation of an integrated 3D microfluidic chip-based system fabricated directly in the poly(methyl methacrylate) transparent thermoplastic using infrared laser micromachining. At its core, the device utilizes an array of 3D micromechanical traps to actively capture and immobilize single embryos using a low-pressure suction. It also features built-in piezoelectric microdiaphragm pumps, embryo-trapping suction manifold, drug delivery manifold, and optically transparent indium tin oxide heating element to provide optimal temperature during embryo development. Furthermore, we present design of the proof-of-concept off-chip electronic interface equipped with robotic servo actuator driven stage, innovative servomotor-actuated pinch valves, and embedded miniaturized fluorescent USB microscope. Our results showed that the innovative device has 100% embryo-trapping efficiency while supporting normal embryo development for up to 72 hr in a confined microfluidic environment. We also showed data that this microfluidic system can be readily applied to kinetic analysis of a panel of investigational antiangiogenic agents in transgenic zebrafish lines. The optical transparency and embryo immobilization allow for convenient visualization of developing vasculature patterns in response to drug treatment without the need for specimen re-positioning. The integrated electronic interfaces bring the lab-on-a-chip systems a step closer to realization of complete analytical automation.

Fishing on chips: up-and-coming technological advances in analysis of zebrafish and Xenopus embryos.

Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole-organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high-throughput cell-based in vitro assays and low-throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor-intensive. Recently, a new generation of miniaturized chip-based devices have been developed for zebrafish and Xenopus embryo on-chip culture and experimentation. In this work, we review the critical developments in the field of Lab-on-a-Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae. © 2014 International Society for Advancement of Cytometry.

Interactive effects of cyanobacterial metabolites aeruginosin-98B, anabaenopeptin-B and cylindrospermopsin on physiological parameters and novel in vivo fluorescent indicators in Chironomus aprilinus larvae.

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.