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[Display omitted] • CQDs were non-toxic at low doses but harmful at higher concentrations to zebrafish. • The locomotor activity of zebrafish exposed to CQDs varies dose-dependently. • At high concentrations, CQDs cause morphological defects in zebrafish embryos. • In larvae, high CQD dosages result in dopamine decrease and organ damage. Fluorescent carbon dots have emerged as promising nanomaterials for various applications, including bioimaging, food safety detection and drug delivery. However, their potential impact on neurological systems, especially in-vivo models, remains a critical area of investigation. This review focuses on the neurological effects of carbon dots and carbon quantum dots on zebrafish, an established vertebrate model with a conserved central nervous system. Recent studies have demonstrated the efficient uptake and distribution of carbon dots in zebrafish tissues, with a particular affinity for neural tissues. The intricate neural architecture of zebrafish allows for the precise examination of behavioral changes and neurodevelopmental alterations induced by fluorescent carbon dots. Neurotoxicity assessments reveal both short-term and long-term effects, ranging from immediate behavioral alterations to subtle changes in neuronal morphology. The review discusses potential mechanisms underlying these effects highlights the need for standardized methodologies in assessing neurological outcomes and emphasizes the importance of ethical considerations in nanomaterial research. As the field of nanotechnology continues to advance, a comprehensive understanding of the impact of fluorescent carbon dots on neurological function in zebrafish is crucial for informing safe and sustainable applications in medicine and beyond. [ABSTRACT FROM AUTHOR]

Background: Natural herbs have been widely considered a reservoir for skin‐lightening ingredients, but discovery of the effective ingredients from herbs remains a large challenge. Aim: This research aimed to rapidly identify compounds with skin‐lightening activity in Chinese herbs. Methods: The structure information of herbal compounds was collected and selected from the open‐source data. High throughput virtual screening (HTVS) and Extra precision (XP) docking modes were used to screen for compounds that could bind to the mushroom tyrosinase involved in melanin synthesis. Furthermore, molecular dynamics (MD) simulations were introduced to assess the binding stability of those compounds with the key target protein. The candidate compounds found by this kind of multidimensional molecular screening were finally tested for their ability to inhibit pigmentation and potential toxicity using an in vivo zebrafish animal model. Results: A Natural Compounds Database was established with 5616 natural compounds. Fourteen compounds with favorable binding capability were screened by the XP docking mode with mushroom tyrosinase and five compounds among them were found to have superior dynamic binding performance through MD simulations. Then the Zebrafish animal experiments revealed that two components, sennoside B (SB) and sennoside C (SC), could significantly inhibit melanogenesis rather than the other three compounds. Meanwhile, there were no obvious side effects observed in SB and SC about the morphology, heart rate, or body length of zebrafish. Conclusion: A strategy for rapid screening of compounds with whitening activity has been established, and two potent skin‐lightening compounds, SB and SC, have been identified from a vast library of herbal compounds. This study revealed that SB and SC have potential for topical use in skin lightening for the first time. The findings of this study would provide an important theoretical basis for the application of these two compounds in the cosmetic field in the future. [ABSTRACT FROM AUTHOR]

Objectives: Despite some existing studies on the safety of high static magnetic fields (SMFs), the effects of ultra-high SMFs above 20.0 T for embryonic development in early pregnancy are absent. The objective of this study is to evaluate the influence of 16.8–22.0 T SMF on the development of zebrafish embryos, which will provide important information for the future application of ultra-high field magnetic resonance imaging (MRI). Methods: Two-hour exposure to homogenous (0 T/m) 22.0 T SMF, or 16.8 T SMFs with 123.25 T/m spatial gradient of opposite magnetic force directions was examined in the embryonic development of 200 zebrafish. Their body length, heart rate, spontaneous tail-wagging movement, hatching and survival rate, photomotor response, and visual motor response (VMR) were analyzed. Results: Our results show that these ultra-high SMFs did not significantly affect the general development of zebrafish embryos, such as the body length or spontaneous tail-wagging movement. However, the hatching rate was reduced by the gradient SMFs (p < 0.05), but not the homogenous 22.0 T SMF. Moreover, although the zebrafish larva activities were differentially affected by these ultra-high SMFs (p < 0.05), the expression of several visual and neurodevelopmental genes (p < 0.05) was generally downregulated in the eyeball. Conclusions: Our findings suggest that exposure to ultra-high SMFs, especially the gradient SMFs, may have adverse effects on embryonic development, which should cause some attention to the future application of ultra-high field MRIs. Clinical relevance statement: As technology advances, it is conceivable that very strong magnetic fields may be adapted for use in medical imaging. Possible dangers associated with these higher Tesla fields need to be considered and evaluated prior to human use. Key Points: Ultra-High static magnetic field may affect early embryonic development. High strength gradient static magnetic field exposure impacted zebrafish embryonic development. The application of very strong magnetic fields for MR technologies needs to be carefully evaluated. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds frequently detected in the environment with widely varying toxicities. Many PAHs activate the aryl hydrocarbon receptor (AHR), inducing the expression of a battery of genes, including xenobiotic metabolizing enzymes like cytochrome P450s (CYPs); however, not all PAHs act via this mechanism. We screened several parent and substituted PAHs in in vitro AHR activation assays to classify their unique activity. Retene (1-methyl-7-isopropylphenanthrene) displays Ahr2-dependent teratogenicity in zebrafish, but did not activate human AHR or zebrafish Ahr2, suggesting a retene metabolite activates Ahr2 in zebrafish to induce developmental toxicity. To investigate the role of metabolism in retene toxicity, studies were performed to determine the functional role of cyp1a , cyp1b1 , and the microbiome in retene toxicity, identify the zebrafish window of susceptibility, and measure retene uptake, loss, and metabolite formation in vivo. Cyp1a-null fish were generated using CRISPR-Cas9. Cyp1a-null fish showed increased sensitivity to retene toxicity, whereas Cyp1b1-null fish were less susceptible, and microbiome elimination had no significant effect. Zebrafish required exposure to retene between 24 and 48 hours post fertilization (hpf) to exhibit toxicity. After static exposure, retene concentrations in zebrafish embryos increased until 24 hpf, peaked between 24 and 36 hpf, and decreased rapidly thereafter. We detected retene metabolites at 36 and 48 hpf, indicating metabolic onset preceding toxicity. This study highlights the value of combining molecular and systems biology approaches with mechanistic and predictive toxicology to interrogate the role of biotransformation in AHR-dependent toxicity. [ABSTRACT FROM AUTHOR]

[Display omitted] • Temperature jump microscopy of live zebrafish larvae can study stress response. • Clean imaging is obtained by expressing fluorescent proteins in just a few cells. • Automated feedback and focusing mitigates temperature drift during experiments. • Hsp70 binds a client protein in vivo , while an inactive mutant does not. Biomolecular processes such as protein–protein interactions can depend strongly on cell type and even vary within a single cell type. Here we develop a microscope with a Peltier-controlled temperature stage, a laser temperature jump to induce heat stress, and an autofocusing feature to mitigate temperature drift during experiments, to study a protein–protein interaction in a selected cell type within a live organism, the zebrafish larva. As an application of the instrument, we show that there is considerable cell-to-cell variation of the heat shock protein Hsp70 binding to one of its clients, phosphoglycerate kinase in vivo. We adapt a key feature from our previous folding study, rare transformation of cells within the larva, so that individual cells can be imaged and differentiated for cell-to-cell response. Our approach can be extended to other organisms and cell types than the ones demonstrated in this work. [ABSTRACT FROM AUTHOR]

A supplemented collagen peptide composition (SCPC) containing microbe-fermented collagen peptide, collagen tripeptide, elastin peptide, and sodium hyaluronate was investigated to determine its effects on skin aging, skin elasticity, and tissue regeneration in zebrafish. β-Galactosidase activity was inhibited, while telomerase activity was elevated, by exposure to 0.977 μL/mL of SCPC in an H 2 O 2 -induced aging zebrafish model, indicating that SCPC can delay cell senescence. Expression of the collagen I gene col1a1a was up-regulated by exposure to 62.5 μL/mL of SCPC, indicating that SCPC can improve skin elasticity. Regrowth of the injured caudal fin was enhanced by exposure to only 0.122 μL/mL of SCPC. These improvements to skin aging, elasticity, and tissue regeneration in zebrafish suggest that this SCPC has potential as a functional food with beneficial effects on skin health and aging resistance in humans. [Display omitted] • Zebrafish were used for building animal models. • Effects of the supplemented collagen peptide composition on the skin. • Inhibiting β-galactosidase activity and elevated telomerase activity. • Up-regulated the expression of collagen I gene col1a1a. • Enhanced the regrowth of injured caudal fin. [ABSTRACT FROM AUTHOR]

In 2015, we reported primed conversion, a novel way to convert green‐to‐red photoconvertible fluorescent proteins, which emerges as a powerful tool for precision optical imaging. Primed conversion uses the intercept of blue and red‐to‐far‐red light instead of traditional violet or near‐UV light illumination which offers a series of advantages. Here, we review the fundamental principles and applications of primed conversion with a focus on its use in single‐cell labelling and lineage tracing. We provide a historical perspective of lineage tracing techniques, thereby covering basic principles of fluorescence, photoconvertible fluorescent proteins, and eventually primed conversion. We then present the molecular requirements for primed conversion to take place and showcase how it can be used for dual‐colour high‐fidelity lineage tracing. Further, we discuss potential future developments of the primed conversion imaging toolkit that can benefit the study of both development and disease progression. [ABSTRACT FROM AUTHOR]

Regenerating tissues must remember or interpret their spatial position, using this information to restore original size and patterning. The external skeleton of the zebrafish caudal fin is composed of 18 rays; after any portion of the fin is amputated, position-dependent regenerative growth restores each ray to its original length. We tested for transcriptional differences during regeneration of proximal versus distal tissues and identified 489 genes that differed in proximodistal expression. Thyroid hormone directs multiple aspects of ray patterning along the proximodistal axis, and we identified 364 transcripts showing a proximodistal expression pattern that was dependent on thyroid hormone context. To test what aspects of ray positional identity are directed by extrinsic environental cues versus remembered identity autonomous to the tissue, we transplanted distal portions of rays to proximal environments and evaluated regeneration within the new location. Native regenerating proximal tissue showed robust expression of scpp7 , a transcript with thyroid-regulated proximal enrichment; in contrast, regenerating rays originating from transplanted distal tissue showed reduced (distal-like) expression during outgrowth. These distal-to-proximal transplants regenerated far beyond the length of the graft itself, indicating that cues from the proximal environment promoted additional growth. Nonetheless, these transplants initiated regeneration at a much slower rate compared to controls, suggesting memory of distal identity was retained by the transplanted tissue. This early growth retardation caused rays that originated from transplants to grow noticeably shorter than neighboring native rays. While several aspects of fin ray morphology (bifurcation, segment length) were found to be determined by the environment, we found that both regeneration speed and ray length are remembered autonomously by tissues, and that persist through multiple rounds of amputation and regeneration. [Display omitted] • Gene expression patterns during fin regeneration correspond to proximodistal location. • Distal portions of rays transplanted to proximal regions retain positional identity that influences growth rate and length during regeneration. • Ray patterning along the proximodistal axis is determined by the regenerative environment, not remembered identity. [ABSTRACT FROM AUTHOR]

The somatosensory system detects peripheral stimuli that are translated into behaviors necessary for survival. Fishes and amphibians possess two somatosensory systems in the trunk: the primary somatosensory system, formed by the Rohon-Beard neurons, and the secondary somatosensory system, formed by the neural crest cell-derived neurons of the Dorsal Root Ganglia. Rohon-Beard neurons have been characterized as a transient population that mostly disappears during the first days of life and is functionally replaced by the Dorsal Root Ganglia. Here, I follow Rohon-Beard neurons in vivo and show that the entire repertoire remains present in zebrafish from 1-day post-fertilization until the juvenile stage, 15-days post-fertilization. These data indicate that zebrafish retain two complete somatosensory systems until at least a developmental stage when the animals display complex behavioral repertoires. [Display omitted] • Rohon-Beard neurons are primary somatosensory neurons in the dorsal spinal cord of fishes and amphibians. • The entire population of Rohon-Beard neurons survives until free-swimming and juvenile stages in zebrafish. • Concomitant processes may have produced the illusion of disappearance over time. • Canonical cell death markers are absent in Rohon-Beard neurons. • Rohon-Beard neurons co-exist with neurons of the Dorsal Root Ganglia, which form later and are part of the secondary somatosensory system [ABSTRACT FROM AUTHOR]

During neural development, sculpting of early formed circuits by cell death and synaptic pruning is necessary to generate a functional and efficient nervous system. This allows for the establishment of rudimentary circuits which necessitate early organism survival to later undergo subsequent refinement. These changes facilitate additional specificity to stimuli which can lead to increased behavioral complexity. In multiple species, Rohon-Beard neurons (RBs) are the earliest mechanosensory neurons specified and are critical in establishing a rudimentary motor response circuit. Sensory input from RBs gradually becomes redundant as dorsal root ganglion (DRG) neurons develop and integrate into motor circuits. Previous studies demonstrate that RBs undergo a dramatic wave of cell death concurrent with development of the DRG. However, contrary to these studies, we show that neurogenin1 + (ngn1) RBs do not undergo a widespread wave of programmed cell death during early zebrafish development and instead persist until at least 15 days post fertilization (dpf). Starting at 2 dpf, we also observed a dramatic medialization and shrinkage of ngn1 + RB somas along with a gradual downregulation of ngn1 in RBs. This alters a fundamental premise of early zebrafish neural development and opens new avenues to explore mechanisms of RB function, persistence, and circuit refinement. [Display omitted] • Rohon-Beard neurons do not succumb to cell death during early zebrafish development. • The vast majority of Rohon-Beard neurons do not express markers of cell death. • Rohon-Beard neurons persist until 15 days post fertilization. [ABSTRACT FROM AUTHOR]

Aminoglycoside antibiotics, including gentamicin (GM), induce delayed ototoxic effects such as hearing loss after prolonged use, which results from the death of hair cells. However, the mechanisms underlying the ototoxicity of aminoglycosides warrant further investigation, and there are currently no effective drugs in the clinical setting. Herein, the therapeutic effect of the flavonoid compound rutin against the ototoxic effects of GM in zebrafish hair cells was investigated. Animals incubated with rutin (100–400 µmol/L) were protected against the pernicious effects of GM (200 µmol/L). We found that rutin improves hearing behavior in zebrafish, and rutin was effective in reducing the number of Tunel-positive cells in the neuromasts of the zebrafish lateral line and promoting cell proliferation after exposure to GM. Subsequently, rutin exerted a protective effect against GM-induced cell death in HEI-OC1 cells and could limit the production of cytosolic reactive oxygen species (ROS) and diminish the percentage of apoptotic cells. Additionally, the results of the proteomic analysis revealed that rutin could effectively inhibit the expression of necroptosis and apoptosis related genes. Meanwhile, molecular docking analysis revealed a high linking activity between the molecular docking of rutin and STAT1 proteins. The protection of zebrafish hair cells or HEI-OC1 cells from GM-induced ototoxicity by rutin was attenuated by the introduction of STAT1 activator. Finally, we demonstrated that rutin significantly improves the bacteriostatic effect of GM by in vitro experiments, emphasising its clinical application value. In summary, these results collectively unravel a novel therapeutic role for rutin as an otoprotective drug against the adverse effects of GM. [ABSTRACT FROM AUTHOR]

The development of techniques to reduce stress in the aquaculture industry is essential for the good growth, low mortality, and high flesh quality of farmed fish. Shochu is a traditional Japanese alcoholic beverage, and the disposal of large amounts of Shochu lees waste has been a problem. As Shochu lees contain low-molecular-weight compounds, which are thought to have health benefits, the lactic acid-fermented by-product of Shochu distillery (FBPSD) was tested to estimate its anti-stress effects in neuropeptide Y-knockout zebrafish (NPY-KO), which exhibit high anxiety and low sociability, as a farmed fish model. In the Black-White preference test, NPY-KO preferred the black area over the white area due to fear, whereas NPY-KO fed the FBPSD showed a higher preference for the white area with more swimming distance than the control group. In the 3-chambers test, NPY-KO showed low sociability with the unfamiliar zebrafish group, whereas FBPSD-fed NPY-KO showed high sociability with the group. The mRNA level of isotocin, the homologue of human oxytocin, was significantly increased in the brains of FBPSD-fed zebrafish. The oxytocin receptor antagonists L-368,899 attenuated the effects of FBPSD in the 3-chambers test. These results suggest that FBPSD attenuates anxiety in NPY-KO zebrafish by activating isotocin neurons. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases are a rampant public health threat. Environmental contaminants, such as Cadmium (Cd), a toxic metal, are risk factors for cardiovascular diseases. Given that human exposure to Cd is increasing, there is a need for therapies to ameliorate Cd toxicity. Selenium (Se), an essential trace element, has been proposed to rescue the effects of Cd toxicity, with mixed effects. Se's narrow therapeutic window necessitates precise dosing to avoid toxicity. Here, we assessed the effects of various waterborne Cd and Se concentrations and sequences on cardiac function using zebrafish (Danio rerio). We showed that Cd induced pericardial edemas and modified heart rates in zebrafish larvae in a concentration-dependent manner. To identify the therapeutic range of Se for Cd-induced cardiotoxicity, zebrafish embryos were treated with 0, 10, 50, 100, 150, or 200 μg/L Se for 1–4 days prior to exposure to 2.5 and 5 μg/L Cd. We found that a 50 µg/L Se pre-treatment before 2.5 μg/L Cd, but not 5 μg/L Cd, reduced the prevalence of pericardial edemas and ameliorated Cd-induced bradycardia in zebrafish. Zebrafish exposed to 10 and 50 μg/L of Se for up to 4 days showed typical heart morphology, whereas other Se-exposed and control fish presented pericardial edemas. Longer Se pre-treatment durations led to fewer incidences of pericardial edemas. Overall, this study highlights the importance of optimizing Se concentrations and pre-treatment periods to harness its protective effects against Cd-induced cardiotoxicity. These findings provide insights into potential therapeutic strategies for reducing Cd-related cardiovascular damage in humans. [ABSTRACT FROM AUTHOR]

Introduction: Hair cells (HCs) are the sensory receptors of the auditory and vestibular systems in the inner ears of vertebrates that selectively transduce mechanical stimuli into electrical activity. Although all HCs have the hallmark stereocilia bundle for mechanotransduction, HCs in non-mammals and mammals differ in their molecular specialization in the apical, basolateral, and synaptic membranes. HCs of non-mammals, such as zebrafish (zHCs), are electrically tuned to specific frequencies and possess an active process in the stereocilia bundle to amplify sound signals. Mammalian HCs, in contrast, are not electrically tuned and achieve amplification by somatic motility of outer HCs (OHCs). Methods: To understand the genetic mechanisms underlying differences between adult zebrafish and mammalian HCs, we compared their RNA-seq- characterized transcriptomes, focusing on protein-coding orthologous genes related to HC specialization. Results: There was considerable shared expression of gene orthologs among the HCs, including those genes associated with mechanotransduction, ion transport/channels, and synaptic signaling. However, there were some notable differences in expression among zHCs, OHCs, and inner HCs (IHCs), which likely underlie the distinctive physiological properties of each cell type. For example, OHCs highly express Slc26a5 which encodes the motor protein prestin that contributes toOHC electromotility. However, zHCs have only weak expression of slc26a5, and subsequently showed no voltage-dependent electromotility when measured. Notably, the zHCs expressed more paralogous genes including those associated with HC-specific functions and transcriptional activity, though it is unknown whether they have functions similar to their mammalian counterparts. There was overlap in the expressed genes associated with a known hearing phenotype. Discussion: Our analyses unveil substantial differences in gene expression patterns thatmay explain phenotypic specialization of zebrafish andmouse HCs. This dataset also includes several protein-coding genes to further the functional characterization of HCs and study of HC evolution from non-mammals to mammals. [ABSTRACT FROM AUTHOR]

Receptors for activated C kinases (RACKs) have been shown to coordinate PKC-mediated hypertrophic signalling in mice. However, little information is available on its participation in embryonic gene expression. This study investigated the involvement of RACK1 in the expression of embryonic genes in a zebrafish (ZF) ex vivo heart culture model by using phenylephrine (PE) or a growth factors cocktail (GFs) as a prohypertrophic/regeneration stimulus. Blebbistatin (BL) inhibition has also been studied for its ability to block the signal transduction actions of some PEs. qRT‒PCR and immunoblot analyses confirmed the upregulation of RACK1 in the PE- and GFs-treated groups. BL administration counteracted PE-induced hypertrophy and downregulated RACK1 expression. Immunohistochemical analyses of the heart revealed the colocalization of RACK1 and embryonic genes, namely, Gata4, Wt1, and Nfat2, under stimulation, whereas these genes were expressed at lower levels in the BL treatment group. Culturing ZF heart cells activated via GFs treatment increased the expression of RACK1. The overexpression of RACK1 induced by the transfection of recombinant RACK1 cDNA in ZF heart cells increased the expression of embryonic genes, especially after one week of GFs treatment. In summary, these results support the involvement of RACK1 in the induction of embryonic genes during cardiac hypertrophy/GFs stimulation in a fish heart model, which can be used as an alternative study model for mammals. [ABSTRACT FROM AUTHOR]

AbstractZinc oxide quantum dots, also known as ZnO QDs, are highly desirable due to their numerous favorable characteristics, such as their beneficial photoluminescence, solubility in water, along with sunlight absorption. They are well-suited for use in biomedical applications, drugs, and bioimaging. However, study on the in-vivo toxicology of these QDs is needed before they can be used in humans. Zebrafish (Danio rerio) are cheap, fast-growing, and similar to humans, which makes them ideal as in vivo model for studying the toxicity of nanomaterials. The toxicity investigations involving zinc oxide QDs (ZnO QDs) and zinc oxide bionanocomposite (ZnO BC) in zebrafish that were concentration-dependent are evaluated, and the Box-Behnken design (BBD) was utilized to optimize the results. To determine the proper dosage, a study on cell line as well as hemocompatibility was carried out prior to testing the toxic effects of ZnO QDs along with ZnO BC upon zebrafish. When administered at 2.5 μg/l of ZnO BC and 2 μg/l of ZnO QDs, neither ZnO BC nor ZnO QDs appeared to be toxic to embryos during hatching and development. The testing of larval behavior in visible light revealed a dose-dependent decrease in both the total diving distance as well as speed. Nevertheless, at ZnO BC and ZnO QDs levels >250 μg/l and >200 μg/l, respectively, notable effects were seen in zebrafish embryos. Hence, ZnO QDs and BC at low concentrations were notably nontoxic. In order to guarantee the safety of nanomaterials in bio applications, this research supports upcoming in-vivo imaging investigations on their harmful effects. [ABSTRACT FROM AUTHOR]

Introduction: Congenital hypopituitarism (CH) is characterized by the deficiency of pituitary hormones. Among CH patients, 85% lack a molecular diagnosis. Whole Exome Sequencing (WES) identified a homozygous variant (c.865G>A, p.Val289Ile) in the CDH2 gene, responsible for N-Cadherin production, crucial for cell-cell adhesion. Predicted to be likely pathogenic, the variant was found in a patient deficient in GH, TSH, ACTH, and LH/FSH. Its impact on cell adhesion was confirmed in L1 fibroblast cell lines. Objective: Create a cdh2 knockdown in zebrafish for investigating its role in pituitary development through growth hormone and transcription factors expression. Methods: Utilized pET28B-RfxCas13d-His plasmid for Cas13 mRNA production via in vitro transcription, guiding Cas13 to cdh2 with three RNAs. Injected the complex into single-cell embryos for analysis up to 96 hpf. Assessed gene expression of cdh2, prop1, pit1, and gh1 using RT-qPCR. Evaluated cdh2 protein expression through the western blot technique. Results: Knockdown animals displayed developmental delay. The cdh2 expression decreased by 75% within 24 hours, rebounded by 48 hours, and reached wild-type levels by 96 hpf. gh1 expression decreased at 48h but increased by 96 hpf, aligning with WT. No significant differences in prop1 and pit1 expression were observed. Conclusion: Our findings underscore cdh2's role in pituitary development and hormonal regulation, offering insights for developmental biology research. [ABSTRACT FROM AUTHOR]

Inherited and acquired muscle diseases are an important cause of morbidity and mortality in human medical and veterinary patients. Researchers use models to study skeletal muscle development and pathology, improve our understanding of disease pathogenesis and explore new treatment options. Experiments on laboratory animals, including murine and canine models, have led to huge advances in congenital myopathy and muscular dystrophy research that have translated into clinical treatment trials in human patients with these debilitating and often fatal conditions. Whilst animal experimentation has enabled many significant and impactful discoveries that otherwise may not have been possible, we have an ethical and moral, and in many countries also a legal, obligation to consider alternatives. This review discusses the models available as alternatives to mammals for muscle development, biology and disease research with a focus on inherited myopathies. Cell culture models can be used to replace animals for some applications: traditional monolayer cultures (for example, using the immortalised C2C12 cell line) are accessible, tractable and inexpensive but developmentally limited to immature myotube stages; more recently, developments in tissue engineering have led to three-dimensional cultures with improved differentiation capabilities. Advances in computer modelling and an improved understanding of pathogenetic mechanisms are likely to herald new models and opportunities for replacement. Where this is not possible, a 3Rs approach advocates partial replacement with the use of less sentient animals (including invertebrates (such as worms Caenorhabditis elegans and fruit flies Drosophila melanogaster) and embryonic stages of small vertebrates such as the zebrafish Danio rerio) alongside refinement of experimental design and improved research practices to reduce the numbers of animals used and the severity of their experience. An understanding of the advantages and disadvantages of potential models is essential for researchers to determine which can best facilitate answering a specific scientific question. Applying 3Rs principles to research not only improves animal welfare but generates high-quality, reproducible and reliable data with translational relevance to human and animal patients. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA) is an environmental estrogen which perturbs hormone signaling pathways adversely affecting aquatic organisms. To evaluate the impact of developmental exposure to long term yet environmentally relevant low doses of BPA, wild-type juvenile zebrafish of 35 days post fertilization were treated with BPA (1 and 10 µg/L), treatment control (0.5% v/v methanol) and control for 60 days. Both BPA treatments led to significantly increased morality overtime. Length increment and specific growth rates became significantly high in BPA exposed zebrafish overtime. Obesogenic property of BPA was not evident with longexposure to low BPA doses. A significantly high and BPA dose-dependent female-biased sex ratios were observed following the juvenile exposure. Significantly low swimming speed was recorded in the fish of both BPA-treated tanks than that of control. Condition factor was significantly low in BPA exposed fish indicating the poor-wellness. There were numerous histopathological alterations of gonads, liver and kidney indicating impacts of juvenile exposure in zebrafish. Altered growth, swimming, mortality, feminization and histopathological changes in zebrafish induced by BPA indicate the risks associated with developmental exposures. The findings call for more comprehensive studies to comprehend the ecological risks imposed by low concentrations of environmental estrogens in urban aquatic ecosystems. [ABSTRACT FROM AUTHOR]

Paqr5b is a gene encoding membrane progesterone receptor γ (mPRγ), which is one of five mPR subtypes. Paqr5b belongs to the progestin and adipoQ receptor (PAQR) family, which consists of 11 genes. To elucidate the physiological functions of the mPR subtypes, we established gene knockout (KO) zebrafish strains by genetically editing seven paqr genes and analyzed their phenotypes. The null-mutant strain of paqr5b (paqr5b−/−) that we established in this study showed low fecundity, reduced chorion elevation and a high percentage of abnormal embryos. Embryos showed curvature of the spine and an abnormal head morphology. Individuals with abnormal head morphology continued to develop a phenotype of markedly abnormal palatine bone. The length of the brain of paqr5b−/− zebrafish was short, and the position of the cerebellum moved to the front and overlapped with that of the midbrain. Micro-CT scans revealed that the olfactory rosettes (ORs) were so shrunken that they were difficult to identify and connected with the olfactory bulbs (OBs) by thread-like structures. Immunohistochemical staining of OR with an anti-Paqr5b antibody revealed that Paqr5b was extensively expressed in neurons in the OR in wild-type zebrafish, whereas signals were not detected in paqr5b−/− zebrafish. In histological sections, the neurons disappeared, and the lamellar layer of the OR became thinner. These results indicate that Paqr5b is required for the formation of neurons in the OR. This is the first report demonstrating a distinct role for the mPR gene. [ABSTRACT FROM AUTHOR]

Inherited retinal diseases (IRDs) are a rare group of eye disorders characterized by progressive dysfunction and degeneration of retinal cells. In this study, we characterized the raifteirí (raf) zebrafish, a novel model of inherited blindness, identified through an unbiased ENU mutagenesis screen. A mutation in the largest subunit of the endoplasmic reticulum membrane protein complex, emc1 was subsequently identified as the causative raf mutation. We sought to elucidate the cellular and molecular phenotypes in the emc1−/− knockout model and explore the association of emc1 with retinal degeneration. Visual behavior and retinal electrophysiology assays demonstrated that emc1−/− mutants had severe visual impairments. Retinal histology and morphometric analysis revealed extensive abnormalities, including thinning of the photoreceptor layer, in addition to large gaps surrounding the lens. Notably, photoreceptor outer segments were drastically smaller, outer segment protein expression was altered and hyaloid vasculature development was disrupted. Transcriptomic profiling identified cone and rod‐specific phototransduction genes significantly downregulated by loss of emc1. These data shed light on why emc1 is a causative gene in inherited retinal disease and how outer segment morphogenesis is regulated. [ABSTRACT FROM AUTHOR]

Currently, clinically available cancer chemopreventive drug options are limited to mostly tamoxifen and its derivatives, such as raloxifene, and approved specifically for breast cancer. Thus, the availability of chemopreventive drug molecules for other types of malignant cancers would be desirable. In previous reports, the arils of Myristica fragrans (mace) have been found to exhibit cancer chemopreventive activity. Therefore, the purpose of the present study was to identify a natural product from this species with potential chemopreventive activity guided by chemoinformatic sample analysis via Global Natural Products Social (GNPS) molecular networking and molecular docking. The neolignan licarin A (1) was identified as a potential chemopreventive constituent, and subsequently submitted to several in vitro bioassays and a zebrafish toxicity evaluation. In this work, 1 afforded superior phosphoNF-κBp65 phosphorylation activity in DU-145 prostate cancer cells compared to isoliquiritigenin (2), which was used as a natural product chemopreventive control. Both 1 and 2 showed a longer-lasting reduction in cellular stress in a cell oxidative stress real-time dose–response assay than the positive control using Hepa1c1c7 mouse hepatoma cells. In addition, 1 displayed similar activities to 2, while also being less toxic to zebrafish (Danio rerio) than both this chalcone and the clinically used chemopreventive drug tamoxifen. [ABSTRACT FROM AUTHOR]

Diisononyl phthalate, classified as endocrine disruptor, has been investigate to trigger lipid biosynthesis in both mammalian and teleostean animal models. Despite this, little is known about the effects of DiNP exposure at tolerable daily intake level and the possible mechanisms of its toxicity. Probiotics, on the other hand, were demonstrated to have beneficial effects on the organism's metabolism and recently emerged as a possible tool to mitigate the EDC toxicity. In the present study, using a metabolomic approach, the potential hepatic sex-related toxicity of DiNP was investigated in adult zebrafish together with the mitigating action of the probiotic formulation SLAB51, which has already demonstrated its ability to ameliorate gastrointestinal pathologies in animals including humans. Zebrafish were exposed for 28 days to 50 µg/kg body weight (bw)/day of DiNP (DiNP) through their diet and treated with 109 CFU/g bw of SLAB51 (P) and the combination of DiNP and SLAB51 (DiNP + P), and the results were compared to those of an untreated control group (C). DiNP reduced AMP, IMP, and GMP in the purine metabolism, while such alterations were not observed in the DiNP + P group, for which the phenotype overlapped that of C fish. In addition, in male, DiNP reduced UMP and CMP levels in the pyrimidine metabolism, while the co-administration of probiotic shifted the DiNP + P metabolic phenotype toward that of P male and closed to C male, suggesting the beneficial effects of probiotics also in male fish. Overall, these results provide the first evidence of the disruptive actions of DiNP on hepatic nucleotide metabolism and mitigating action of the probiotic to reduce a DiNP-induced response in a sex-related manner. [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI) is a debilitating central nervous system (CNS) disorder that leads to significant motor and sensory impairments. Given the limited regenerative capacity of adult mammalian neurons, this study presents an innovative strategy to enhance axonal regeneration and functional recovery by identifying a novel factor that markedly promotes axonal regeneration. Employing a zebrafish model with targeted single axon injury in Mauthner cells (M-cells) and utilizing the Tg (Tol056: EGFP) transgenic line for in vivo monitoring, we investigate the intrinsic mechanisms underlying axonal regeneration. This research specifically examines the role of amino acid transport, emphasizing the role of the solute carrier 1A4 amino acid transporter in axonal regeneration. Our findings demonstrate that Slc1a4 overexpression significantly enhances axonal regeneration in M-cells, whereas Slc1a4 deficiency impedes this process, which is concomitant with the downregulation of the P53/Gap43 signaling pathway. By elucidating the fundamental role of Slc1a4 in axonal regeneration and uncovering its underlying mechanisms, this study thus provides novel insights into therapeutic strategies for SCI. [ABSTRACT FROM AUTHOR]

Individuals of many species prefer to associate with familiar conspecifics from their established social group over unfamiliar conspecifics. Such familiarity preferences are thought to be adaptive and have been documented widely in many social fishes. Zebrafish (Danio rerio) are extensively studied, highly social fish that form stable shoals in the wild, however there is only mixed evidence for familiarity preferences in this species. Here, we test how a small variation in experimental design can influence preferences for familiar conspecifics in D. rerio by varying the distance between two stimulus shoals of fish in a shoaling choice paradigm. Individual subjects tested for their preference to shoal with familiar or unfamiliar groups of conspecifics showed a preference for familiar fish when the two shoals were 30 cm apart, but not when they were 45 or 60 cm apart. Thus, choice tests can be used to detect familiarity preferences in D. rerio, but only when alternate shoals are close together, as increased intershoal distances seemingly prevent subjects from displaying their preference. Longer distances may inhibit preference behavior due to the higher risk of crossing between shoals, alternatively subjects may be unable to reliably discern distinguishing cues of familiarity when the shoals are further apart. Our results demonstrate that while familiarity preferences exist in D. rerio, experimental test design is critical for detecting and measuring these successfully. [ABSTRACT FROM AUTHOR]

Chitin is converted into chitosan by the gladius of the Uroteuthis sibogae (U. sibogae). Chitosan (CH) is then converted into carboxymethyl chitosan (CMC). The structural and functional groups of CH and CMC, respectively, are ascertained using FT-IR and NMR spectroscopy. The molecular weight of the isolated CMC is 731.87 Da. The degree of deacetylation (DDA) is confirmed at about 83%, and the CMC degree of substitution (DS) of 0.8 is determined. Moreover, using the crosslinker sodium tripolyphosphate (TPP), nano-CMC (N-CMC) is synthesized from the obtained CMC. The N-CMC particle size is 146 nm, and 68% of N-CMC is soluble in distilled water. N-CMC is thermostable up to 194.25°C as determined by thermogravimetric analysis (TGA). Zebrafish embryos (ZFE) exposed to N-CMC at a concentration of 4 μg/mL revealed teratogenic effects. The locomotor activity of the N-CMC (1 to 4 mg/L) treated zebrafish was significantly (P<0.05) decreased during the third week of the experiment by using ToxTrac software. [ABSTRACT FROM AUTHOR]

The widely used non-steroidal anti-inflammatory drug, diclofenac, detected in increasing concentrations in freshwater ecosystems, is among the most pressing environmental problems today. In this study, the bacterial isolate Stenotrophomonas humi strain DIC_5 was capable of degrading diclofenac. It eliminated 75.1% of diclofenac at an initial concentration of 1.5 mg/L after 8 days in the presence of glucose (3.0 g/L). During the process, nitro-diclofenac was identified as a resulting metabolite, whose concentration increased significantly in the bacterial medium from the 7th day of the experiment, while the concentration of diclofenac decreased correspondingly. The ecotoxicological tests on Aliivibrio fischeri and zebrafish embryos showed that the bacterial metabolites without diclofenac have a higher toxicity (up to 35.5% bacterial bioluminescence inhibition and 36.7% embryo mortality) than the diclofenac degradation residues (28% and 26.7%, respectively). Based on these results, neither diclofenac nor its degradation products exhibit toxic effects on the test organisms. Conversely, the toxic effect caused by the bacteria was reduced in the presence of diclofenac. Our work highlights the importance of using biotic controls in biotransformation trials, especially when the foreign material is applied in intermediate or environmentally relevant concentration ranges. Key points: • Biotransformation of diclofenac by bacteria isolated from a bacterial biofilm. • Biotransformation of diclofenac led to the formation of nitro-diclofenac. • Microorganisms are alternatives for reducing the concentration of diclofenac in water. [ABSTRACT FROM AUTHOR]

Dopaminergic (DAnergic) dysfunction and imbalanced dopamine (DA) levels are known contributors to the pathogenesis of numerous psychiatric and neurodegenerative disorders. Of the many identified risk factors for DA‐associated disorders, nuclear receptor subfamily 4 group A2 (NR4A2; or nuclear receptor related‐1 protein (NURR1)), a transcription factor involved in DAnergic differentiation, has been associated with Parkinson's disease and attention deficit hyperactive disorder (ADHD). In zebrafish, transient loss of nr4a2 was previously shown to decrease tyrosine hydroxylase (TH) expression and impair locomotion. To further characterize the roles of the two zebrafish nr4a2 paralogs, nr4a2a, and nr4a2b, we produced targeted loss‐of‐function mutants and examined DAnergic neuron regeneration, oxidative respiration, and behavioral traits. The loss of nr4a2a function more closely recapitulated Parkinsonian phenotypes and affected neurotrophic factor gene expression. Conversely, nr4a2b mutants displayed behavioral symptoms reminiscent of mice deficient in Nr4a2 with increased neurotrophic output. In contrast, nr4a2b mutants also displayed increased metabolic input from non‐mitochondrial sources indicative of high cytosolic reactive oxygen species and perturbed mitochondrial function. The nr4a2a mutants also showed increased maximal respiration, which may suggest a compensatory mechanism to meet the metabolic requirements of DAnergic neuron health. Overall, the zebrafish mutants generated in this study helped uncover molecular mechanisms involved in DA‐related disease pathologies, and in the regeneration of DAnergic neurons. [ABSTRACT FROM AUTHOR]

Key points Sensory hair cells of the inner ear utilize specialized ribbon synapses to transmit sensory stimuli to the central nervous system. This transmission necessitates rapid and sustained neurotransmitter release, which depends on a large pool of synaptic vesicles at the hair‐cell presynapse. While previous work in neurons has shown that kinesin motor proteins traffic synaptic material along microtubules to the presynapse, the mechanisms of this process in hair cells remain unclear. Our study demonstrates that the kinesin motor protein Kif1a, along with an intact microtubule network, is essential for enriching synaptic vesicles at the presynapse in hair cells. Through genetic and pharmacological approaches, we disrupt Kif1a function and impair microtubule networks in hair cells of the zebrafish lateral‐line system. These manipulations led to a significant reduction in synaptic‐vesicle populations at the presynapse in hair cells. Using electron microscopy, in vivo calcium imaging, and electrophysiology, we show that a diminished supply of synaptic vesicles adversely affects ribbon‐synapse function. Kif1aa mutants exhibit dramatic reductions in spontaneous vesicle release and evoked postsynaptic calcium responses. Furthermore, kif1aa mutants exhibit impaired rheotaxis, a behaviour reliant on the ability of hair cells in the lateral line to respond to sustained flow stimuli. Overall, our results demonstrate that Kif1a‐mediated microtubule transport is critical to enrich synaptic vesicles at the active zone, a process that is vital for proper ribbon‐synapse function in hair cells. Kif1a mRNAs are present in zebrafish hair cells. Loss of Kif1a disrupts the enrichment of synaptic vesicles at ribbon synapses. Disruption of microtubules depletes synaptic vesicles at ribbon synapses. Kif1aa  mutants have impaired ribbon‐synapse and sensory‐system function. [ABSTRACT FROM AUTHOR]

Saccades are rapid eye movements that are used by all species with good vision. In this study, we set out to characterize the complete repertoire of larval zebrafish horizontal saccades to gain insight into their contributions to visually guided behavior and underlying neural control. We identified five saccade types, defined by systematic differences in kinematics and binocular coordination, which were differentially expressed across a variety of behavioral contexts. Conjugate saccades formed a large group that serves at least four functions. These include fast phases of the optokinetic nystagmus, visual scanning in stationary animals, and shifting gaze in coordination with body turns. In addition, we discovered a previously undescribed pattern of eye-body coordination in which small conjugate saccades partially oppose head rotation to maintain gaze during forward locomotion. Convergent saccades were coordinated with body movements to foveate prey targets during hunting. Detailed kinematic analysis showed that conjugate and convergent saccades differed in the millisecond coordination of the eyes and body and followed distinct velocity main sequence relationships. This challenges the prevailing view that all horizontal saccades are controlled by a common brainstem circuit and instead indicates saccade-type-specific neural control. • Kinematic analysis of thousands of rapid eye movements reveals five saccade types • Conjugate saccades have at least four identifiable visual functions • Convergent saccades are coordinated with body movements to foveate prey • Distinct main sequence relationships imply saccade-type-specific neural control Dowell et al. characterize the repertoire of saccadic eye movements of larval zebrafish and examine their coordination with swims during visuomotor behaviors. Differences in timing, kinematics, and velocity main sequences indicate that different saccade types are controlled by distinct patterns of brainstem activity. [ABSTRACT FROM AUTHOR]

Enhancers are non‐coding cis‐regulatory elements crucial for transcriptional regulation. Mutations in enhancers can disrupt gene regulation, leading to disease phenotypes. Identifying enhancers and their tissue‐specific activity is challenging due to their lack of stereotyped sequences. This study presents a sequence‐based computational model that uses combinatorial transcription factor (TF) genomic occupancy to predict tissue‐specific enhancers. Trained on diverse datasets, including ENCODE and Vista enhancer browser data, the model predicted 25 000 forebrain‐specific cis‐regulatory modules (CRMs) in the human genome. Validation using biochemical features, disease‐associated SNPs, and in vivo zebrafish analysis confirmed its effectiveness. This model aids in predicting enhancers lacking well‐characterized chromatin features, complementing experimental approaches in tissue‐specific enhancer discovery. [ABSTRACT FROM AUTHOR]

Background: One of the etiologies of stroke is intracerebral hemorrhage and the demand for potential therapeutic agents to alleviate symptoms and improve quality of life in patients with ICH is growing. Several phytocompounds capable of such potency with minimal adverse effects can replace drugs in market. This requires high throughput screening and in vivo experimental demonstration of the potency in ICH models that simulate mammalian pathophysiology. We aimed to study the neuroprotective activity of Asiaticoside using an adult zebrafish ICH model. Materials and Methods: Atorvastatin-induced ICH models were treated with non-toxic concentrations of Asiaticoside. Behavioural, biochemical, and cellular assays were performed to validate the developed model. Antioxidant potency was identified by testing the levels of lipid peroxidation, superoxide dismutase and xanthine oxidase in the brain homogenate. Iron chelating potency was assessed in Perl's-stained brain tissue sections. The anti-inflammatory potency was observed by measuring fold change in the expression of inflammatory gene IL-1β in brain homogenates by RT-PCR method and by immunohistochemistry techniques. The neuroprotective role of the compound was studied by fluorescent staining of the brain whole cell suspension to check apoptotic and necrotic population post-treatment. Results and Discussion: The iron accumulation in tissue sections and brain homogenates reduced 3-fold post treatment. With 600 μg/mL concentration of Asiaticoside, the malondialdehyde and xanthine levels reduced 3-fold, and scavenging activity was found to increase after 24 hr, indicating antioxidant potency. This concentration also resulted in one third reduction in the expression of pro-inflammatory cytokine IL-1β and reduced inflammatory cell infiltration showing anti-inflammatory properties. The compound was found to be neuroprotective by reducing the apoptotic cell population from 84% to 20% in comparison to untreated control. In addition, post-treatment the nitric oxide levels were restored better than the untreated control indicating modulation in cellular signalling mechanisms. Conclusion: The adult Zebrafish ICH model was developed to present the multifaceted mammalian pathophysiology of ICH in one system. The phytocompound Asiaticoside is shown to modulate neuroinflammation through a cycle of biological properties thus being neuroprotective, opening avenues for further pre-clinical testing. [ABSTRACT FROM AUTHOR]

Context: Spinal cord injury (SCI) is a devastating condition that results in severe disability and significant comorbidities. The complex pathophysiology of SCI repair and difficulties understanding neural regeneration are treatment challenges. Objectives: The aim of this study is to systematically review the diverse applications of various fish species as models for SCI research. Evidence Acquisition: PRISMA guidelines were used to review observational and interventional studies that utilized fish species as SCI models, published from inception to July 2023. Two independent reviewers screened and performed the data extraction. One independent reviewer assessed the risk of bias for the included studies. Results: Five thousand six hundred and thirty-three records were reviewed, and 144 met the inclusion criteria and were categorized by fish species. The majority of studies employed complete transection injuries, with the remainder being crush injuries, laser injuries, electro-ablations, and demyelination with substances. Zebrafish (Danio rerio) were most commonly utilized 102/144 (71%), primarily with larval models. Other models included Lamprey (Petromyzon marinus and Lethenteron reissued); Goldfish (Carassius auratus); European eel (Anguilla Anguilla); Knifefish (Apteronotus leptorhynchus and Apteronotus albifrons); Sailfin Molly (Poecilia latipinna); and African turquoise killifish (Nothobranchiusfurzeri). Conclusions: This systematic review highlights that fish models, particularly zebrafish, goldfish, and European eels, are important models for further defining SCI pathophysiology and regenerative processes. These models provide a less complex model to gain insights into apoptosis and glial networks. [ABSTRACT FROM AUTHOR]

Among the widespread trichothecene mycotoxins, T-2 toxin is considered the most toxic congener. In the present study, we utilized high-resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR), coupled to the zebrafish (Danio rerio) embryo model, as a toxicometabolomics approach to elucidate the cellular, molecular and biochemical pathways associated with T-2 toxicity. Aligned with previous studies in the zebrafish embryo model, exposure to T-2 toxin was lethal in the high parts-per-billion (ppb) range, with a median lethal concentration (LC50) of 105 ppb. Exposure to the toxins was, furthermore, associated with system-specific alterations in the production of reactive oxygen species (ROS), including decreased ROS production in the liver and increased ROS in the brain region, in the exposed embryos. Moreover, metabolic profiling based on HRMAS NMR revealed the modulation of numerous, interrelated metabolites, specifically including those associated with (1) phase I and II detoxification, and antioxidant pathways; (2) disruption of the phosphocholine lipids of cell membranes; (3) mitochondrial energy metabolism, including apparent disruption of the tricarboxylic acid (TCA) cycle, and the electron transport chain of oxidative phosphorylation, as well as "upstream" effects on carbohydrate, i.e., glucose metabolism; and (4) several compensatory catabolic pathways. Taken together, these observations enabled development of an integrated, system-level model of T-2 toxicity in relation to human and animal health. [ABSTRACT FROM AUTHOR]

Background/Objectives: Brazilian red propolis has attracted attention for its pharmacological properties. However, signs of toxicity were recently observed in long-term studies using the hydroalcoholic extract of red propolis (RPHE), likely due to polyprenylated benzophenones. This study aimed to develop a benzophenone-free red propolis extract (BFRP) and validate an HPLC-PDA method to quantify its main constituents: isoliquiritigenin, vestitol, neovestitol, medicarpine, and 7-O-methylvestitol. Methods: BFRP's toxicity was assessed in zebrafish larvae through a vibrational startle response assay (VSRA) and morphological analysis. Genotoxicity was evaluated using the micronucleus test in rodents, and the extract's effects on chemically induced preneoplastic lesions in rat colon were studied. An HPLC-PDA method was used to quantify BFRP's main compounds. Results: BFRP primarily contained vestitol (128.24 ± 1.01 μg/mL) along with isoliquiritigenin, medicarpin, neovestitol, and 7-O-methylvestitol. Zebrafish larvae exposed to 40 µg/mL of BFRP exhibited toxicity, higher than the 10 µg/mL for RPHE, though no morphological differences were found. Fluorescent staining in the notochord, branchial arches, and mouth was observed in larvae treated with both BFRP and RPHE. No genotoxic or cytotoxic effects were observed up to 2000 mg/kg in rodents, with no impact on hepatotoxicity or nephrotoxicity markers. Chemoprevention studies showed a 41.6% reduction in preneoplastic lesions in rats treated with 6 mg/kg of BFRP. Conclusions: These findings indicate that BFRP is a safe, effective propolis-based extract with potential applications for human health, demonstrating reduced toxicity and chemopreventive properties. [ABSTRACT FROM AUTHOR]

The first studies on ion regulation in fish exposed to low pH, which were inspired by the Acid Rain environmental crisis, seemed to indicate that ion transport at the gills was completely and irreversibly inhibited at pH 4.0–4.5 and below. However, work on characid fish native to the Rio Negro, a naturally acidic, blackwater tributary of the Amazon River, found that they possess ion transport mechanisms that are completely insensitive to pHs as low as 3.25. As more species were examined it appeared that pH-insensitive transport was a trait shared by many, if not most, species in the Order Characiformes. Subsequently, a few other species of fish have been shown to be able to transport ions at low pH, in particular zebrafish (Danio rerio), which show rapid recovery of Na+ uptake at pH 4.0 after initial inhibition. Measurements of rates of Na+ transport during exposure to pharmacological agents that inhibit various transport proteins suggested that characiform fish do not utilize the generally accepted mechanisms for Na+ transport that rely on some form of H+ extrusion. Examination of zebrafish transport at low pH suggest the rapid recovery may be due to a novel Na+/K+ exchanger, but after longer term exposure they may rely on a coupling of Na+/H+ exchangers and NH3 excretion. Further work is needed to clarify these mechanisms of transport and to find other acid-tolerant species to fully gain an appreciation of the diversity of physiological mechansisms involved. [ABSTRACT FROM AUTHOR]

Seminal studies from the early 20th century defined the structural changes associated with development and regeneration of the gills in goldfish at the gross morphological and cellular levels using standard techniques of light and electron microscopy. More recently, investigations using cell lineage tracing, molecular biology, immunohistochemistry and single-cell RNA-sequencing have pushed the field forward and have begun to reveal the cellular and molecular processes that orchestrate cell proliferation and regeneration in the gills. The gill is a multifunctional organ that mediates an array of important physiological functions, including respiration, ion regulation and excretion of waste products. It is comprised of unique cell types, such as pavement cells, ionocytes, chemoreceptors and undifferentiated stem or progenitor cells that regulate growth and replenish cell populations. The gills develop from the embryonic endoderm and are rich in cell types derived from the neural crest. The gills have the capacity to remodel themselves in response to environmental change, such as in the case of ionocytes, chemoreceptors and the interlamellar cell mass, and can completely regenerate gill filaments and lamellae. Both processes of remodeling and regeneration invariably involve cell proliferation. Although gill regeneration has been reported in only a limited number of fish species, the process appears to have many similarities to regeneration of other organs in fish and amphibians. The present article reviews the studies that have described gill development and growth, and that demonstrate a suite of genes, transcription factors and other proteins involved in cell proliferation and regeneration in the gills. [ABSTRACT FROM AUTHOR]

Hair cells of the inner ear and lateral-line system rely on specialized ribbon synapses to transmit sensory information to the central nervous system. The molecules required to assemble these synapses are not fully understood. We show that Nrxn3, a presynaptic adhesion molecule, is crucial for ribbon-synapse maturation in hair cells. In both mouse and zebrafish models, the loss of Nrxn3 results in significantly fewer intact ribbon synapses. We show in zebrafish that, initially, Nrxn3 loss does not alter pre- and postsynapse numbers but, later, synapses fail to pair, leading to postsynapse loss. We also demonstrate thatNrxn3 subtly influences synapse selectivity in zebrafish lateral-line hair cells that detect anterior flow. Loss of Nrxn3 leads to a 60%loss of synapses in zebrafish, which dramatically reduces pre- and postsynaptic responses. Despite fewer synapses, auditory responses in zebrafish andmice are unaffected. This work demonstrates that Nrxn3 is a crucial and conservedmolecule required for thematuration of ribbon synapses. Understanding how ribbon synapses mature is essential to generating new therapies to treat synaptopathies linked to auditory or vestibular dysfunction. [ABSTRACT FROM AUTHOR]

A nanoemulsion containing CdTe quantum dots (NE-CdTe-QD) was developed to shield cells from cadmium toxicity and shown to be a promising candidate for brain tumor diagnosis. CdTe-QD was characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy. CdTe-QD exhibited high luminescence emission at 700 nm, and their stability was maintained when encapsulated in lipidic/polymeric nanoemulsions (198 ± 2.0 nm; PDI = 0.174; − 49.0 mV). The biological effects of free and nanoemulsified CdTe-QD were tested in normal cells (NHF) and glioblastoma cell lines (U87-MG and T98G). Membrane colocalization of NE-CdTe-QD by T98G cells was observed. Instead, intracellular endoplasmic reticulum localization of NE-CdTe-QD was verified in U87-MG cells. Cell viability was reduced only when NE-CdTe-QD permeated the membrane of GBM cells, as observed in U87-MG cells, whereas no cytotoxic effects were observed in normal fibroblasts. Incorporating quantum dots directly into the brain cells is difficult. However, the nanoemulsions reduced the toxicity of CdTe-QD in zebrafish larvae and increased their circulation time, and direct injection into the zebrafish brain did not affect neural cell viability. This validates the potential application of these nanomaterials as diagnostic agents and satisfies the necessary criteria for their use as photosensitizers in photodynamic therapy. [ABSTRACT FROM AUTHOR]

Zebrafish (Danio rerio) is an important animal model for a wide range of neurodegenerative diseases. However, obtaining the cellular resolution that is essential for studying the zebrafish brain remains challenging as it requires high spatial resolution and signal-to-noise ratios (SNR). In the current study, we present the first MRI results of the zebrafish brain at the state-of-the-art magnetic field strength of 28.2 T. The performance of MRI at 28.2 T was compared to 17.6 T. A 20% improvement in SNR was observed at 28.2 T as compared to 17.6 T. Excellent contrast, resolution, and SNR allowed the identification of several brain structures. The normative T1 and T2 relaxation values were established over different zebrafish brain structures at 28.2 T. To zoom into the white matter structures, we applied diffusion tensor imaging (DTI) and obtained axial, radial, and mean diffusivity, as well as fractional anisotropy, at a very high spatial resolution. Visualisation of white matter structures was achieved by short-track track-density imaging by applying the constrained spherical deconvolution method (stTDI CSD). For the first time, an algorithm for stTDI with multi-shell multi-tissue (msmt) CSD was tested on zebrafish brain data. A significant reduction in false-positive tracks from grey matter signals was observed compared to stTDI with single-shell single-tissue (ssst) CSD. This allowed the non-invasive identification of white matter structures at high resolution and contrast. Our results show that ultra-high field DTI and tractography provide reproducible and quantitative maps of fibre organisation from tiny zebrafish brains, which can be implemented in the future for a mechanistic understanding of disease-related microstructural changes in zebrafish models of various brain diseases. [ABSTRACT FROM AUTHOR]

Pharmaceutical drugs and other chemicals can impact organogenesis, either during pregnancy or by postnatal exposure of very preterm infants. Corticosteroids are administered to pregnant women at risk of preterm delivery in order to reduce neonatal morbidity and mortality. In addition, high-dose corticosteroid exposure of very preterm infants regularly serves to maintain blood pressure and to prevent and treat bronchopulmonary dysplasia, a form of chronic lung disease in prematurely born infants. Despite clinical benefits, there is increasing evidence of corticosteroid-mediated short- and long-term detrimental developmental effects, especially in the kidney. Here, we performed a detailed morphological and functional analysis of corticosteroid-mediated effects on pronephros development in larval zebrafish. About 24-h postfertilization (hpf) transgenic Tg(wt1b: EGFP) zebrafish larvae were exposed to a set of natural and synthetic corticosteroids (hydrocortisone, dexamethasone, 6α-methylprednisolone, betamethasone, prednisolone, fludrocortisone, 11-deoxycorticosterone) with varying glucocorticoid and mineralocorticoid potency for 24 h at different concentrations. A semiautomated, multiparametric in vivo workflow enabled simultaneous assessment of kidney morphology, renal FITC-inulin clearance, and heart rate within the same larva. All corticosteroids exerted significant morphological and functional effects on pronephros development, including a significant hypertrophy of the pronephric glomeruli as well as dose-dependent increases in FITC-inulin clearance as a marker of glomerular filtration rate. In conclusion, the present study demonstrates a significant impact of corticosteroid exposure on kidney development and function in larval zebrafish. Hence, these studies underline that corticosteroid exposure of the fetus and the preterm neonate should be carefully considered due to potential short- and long-term harm to the kidney. [ABSTRACT FROM AUTHOR]

Simple Summary: Lead (Pb) and mercury (Hg) are two of the major heavy metals of antiquity and have gained considerable importance as potent pollutants in aquatic environments. It is known that fish embryos or larvae are more sensitive to the monitoring of heavy metal contamination. Thus, RNA sequencing (RNA-seq) analysis based on physiological changes in larval zebrafish was conducted to investigate the toxic mechanisms of lead or mercury in fish during early life stages. Our results showed that acute lead exposure significantly decreased survival but increased the malformation rates of developing zebrafish from 48 hpf to 120 hpf. Transcriptomic analysis revealed that lead-triggered biological processes included cellular process, metabolic process, biological regulation, and response to stimulus. The most enriched lead-regulated pathways included cytochrome P450, glutathione metabolism, and lipid metabolism. Moreover, a series of differentially expressed genes (DEGs) were identified by both mercury and lead treatment, which could be useful for searching potential molecular markers against the evaluation of heavy metals contamination. This study was first conducted to investigate the effects of acute lead exposure on developing zebrafish embryos or larvae from 24 to 120 h post-fertilization (hpf). Our data showed that treatment with 50–200 μM lead significantly affected larval survivability and morphology compared to the respective control. Second, we chose 120 hpf larvae treated with 12.5 μM lead for RNA sequencing due to its exposure level being sufficient to produce toxic effects with minimum death and lead bioaccumulation in developing zebrafish. A total of 137.45 million raw reads were obtained, and more than 86% of clean data were mapped to the zebrafish reference genome. Differential expression profiles generated 116 up- and 34 down-regulated genes upon lead exposure. The most enriched GO terms for representative DEGs were ion transport and lipid metabolism. Third, a comparison with the dataset of mercury-regulated gene expression identified 94 genes (64 up-regulated and 30 down-regulated) for exposure specific to lead, as well as 422 genes (338 up-regulated and 84 down-regulated) for exposure specific to mercury. In addition, 56 genes were co-regulated by micromolar mercury and lead treatment, and the expression of thirteen genes, including mt2, ctssb.1, prdx1, txn, sqrdl, tmprss13a, socs3a, trpv6, abcb6a, gsr, hbz, fads2, and zgc:92590 were validated by qRT-PCR. These genes were mainly associated with metal ion binding, proteolysis, antioxidant activity, signal transduction, calcium ion or oxygen transport, the fatty acid biosynthetic process, and protein metabolism. Taken together, these findings help better understand the genome-wide responses of developing zebrafish to lead or mercury and provide potential biomarkers for acute exposure to toxic metals. [ABSTRACT FROM AUTHOR]

Erythropoietic protoporphyria (EPP1) results in painful photosensitivity and severe liver damage in humans due to the accumulation of fluorescent protoporphyrin IX (PPIX). While zebrafish (Danio rerio) models for porphyria exist, the utility of ferrochelatase (fech) knockout zebrafish, which exhibit EPP, for therapeutic screening and biological studies remains unexplored. This study investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated fech-knockout zebrafish larvae as a model of EPP1 for drug screening. CRISPR/Cas9 was employed to generate fech-knockout zebrafish larvae exhibiting morphological defects without lethality prior to 9 days post-fertilization (dpf). To assess the suitability of this model for drug screening, ursodeoxycholic acid (UDCA), a common treatment for cholestatic liver disease, was employed. This treatment significantly reduced PPIX fluorescence and enhanced bile-secretion-related gene expression (abcb11a and abcc2), indicating the release of PPIX. Acridine orange staining and quantitative reverse transcription polymerase chain reaction analysis of the bax/bcl2 ratio revealed apoptosis in fech−/− larvae, and this was reduced by UDCA treatment, indicating suppression of the intrinsic apoptosis pathway. Neutral red and Sudan black staining revealed increased macrophage and neutrophil production, potentially in response to PPIX-induced cell damage. UDCA treatment effectively reduced macrophage and neutrophil production, suggesting its potential to alleviate cell damage and liver injury in EPP1. In conclusion, CRISPR/Cas9-mediated fech−/− zebrafish larvae represent a promising model for screening drugs against EPP1. [ABSTRACT FROM AUTHOR]

In recent years, the health benefits of lactic acid bacteria have garnered attention, but their antioxidant activity remains relatively underexplored. We have been analyzing the antioxidant activities of various dietary phytochemicals by assessing their ability to mitigate oxidative stressor-induced toxicity in zebrafish larvae through pretreatment. In this study, the antioxidant activities of 24 strains of heat-killed lactic acid bacteria from various origins were examined using this zebrafish assay system. The results revealed that all 24 strains possessed antioxidant activity that reduces hydrogen peroxide toxicity. Further detailed analysis using the H61 strain, which exhibited the strongest activity, showed that no direct antioxidant activity was observed in the assay system, suggesting that the detected antioxidant activity was entirely indirect. Moreover, it was found that pretreatment of zebrafish larvae with the H61 strain for more than 6 h was required to exert its antioxidant activity. This duration was similar to that required by dietary antioxidants that activate the Keap1-Nrf2 pathway, suggesting potential involvement of this pathway. However, analysis using Nrf2-knockout zebrafish revealed that the antioxidant activity of strain H61 is independent of Nrf2, indicating that it represents a novel indirect antioxidant activity that does not involve the Keap1-Nrf2 pathway. To further characterize this activity, the ability to mitigate the toxicity of oxidative stressors other than hydrogen peroxide was examined. The results indicated that while the toxicity of tert-butyl hydroperoxide was reduced, unlike with the Keap1-Nrf2 pathway, it was not effective in counteracting the toxicity of paraquat or arsenite, which generate superoxide radicals. In conclusion, we have identified a novel indirect antioxidant activity in lactic acid bacteria. [ABSTRACT FROM AUTHOR]

Traumatic injury to the spinal cord can lead to significant, permanent disability. Mammalian spinal cords are not capable of regeneration; in contrast, adult zebrafish are capable of such regeneration, fully recovering motor function. Understanding the mechanisms underlying zebrafish neuroregeneration may provide useful information regarding endogenous regenerative potential and aid in the development of therapeutic strategies in humans. DELTEX proteins (DTXs) regulate a variety of cellular processes. However, their role in neural regeneration has not been described. We found that zebrafish dtx2, encoding Deltex E3 ubiquitin ligase 2, is expressed in ependymo-radial glial cells in the adult spinal cord. After spinal cord injury, the heterozygous dtx2 mutant fish motor function recovered quicker than that of the wild-type controls. The mutant fish displayed increased ependymo-radial glial cell proliferation and augmented motor neuron formation. Moreover, her gene expression, downstream of Notch signaling, increased in Dtx2 mutants. Notch signaling inactivation by dominant-negative Rbpj abolished the increased ependymo-radial glia proliferation caused by Dtx2 deficiency. These results indicate that ependymo-radial glial proliferation is induced by Dtx2 deficiency by activating Notch-Rbpj signaling to improve spinal cord regeneration and motor function recovery. [ABSTRACT FROM AUTHOR]

This paper introduces the Fish 3D Locomotion app (F3LA), a Python‐based, Graphical User Interface (GUI)‐equipped tool designed to automate behavioral endpoint extraction in zebrafish locomotion assays. Building on our previous work, which utilized a specialized aquatic tank with a mirror and a single camera for fish movement tracking in three dimensions, F3LA significantly enhances data processing efficiency. Its accuracy was tested by reanalyzing and comprehensively comparing the calculated data with the previously published data from prior publications. From the comparison results, 90% of endpoints showed a similar statistical difference result. These minor differences were due to the different starting points for the dataset and updated calculation formulas that are implemented in F3LA. In addition, shoaling area or shoaling volume calculations are also included in F3LA as a new feature that can serve as sensitive indicators of social cohesion, group dynamics, or stress responses, offering insights into neuropsychological conditions or the effects of pharmacological interventions. Furthermore, F3LA offers a marked improvement over manual operations, being at least five times faster, while maintaining consistent accuracy as it reduces human‐induced errors, ensuring a higher degree of reliability in the results. Finally, the potency of F3LA was tested to evaluate the toxicities of 14 rare earth elements (REEs) to the adult zebrafish behaviors. Based on the results, our findings suggested that each tested REE altered fish behaviors in different patterns and magnitudes to each other. However, among the tested light rare earth elements (LREEs), neodymium was demonstrated to cause more relatively severe behavior alterations than other LREEs, indicated by the statistically higher value of entropy (0.2695 ± 0.04977 (mean with a standard deviation)) than the control group (0.2352 ± 0.05896). Meanwhile, in terms of heavy rare earth elements (HREEs), erbium seemed to lead to more distinct behavior toxicities than other HREEs, which was shown by the statistically lower level of fractal dimension (2.022 ± 0.3412) than the untreated group (2.255 ± 0.1661). Taken together, F3LA's development marks a significant advance in high‐throughput toxicological and pharmacological assessments in zebrafish, leveraging three‐dimensional locomotion data for a more comprehensive analysis of fish behavior performance, providing a significant contribution to research in various fields. [ABSTRACT FROM AUTHOR]

Developmental biology has greatly profited from genetic and reverse genetic approaches to indirectly studying protein function. More recently, nanobodies and other protein binders derived from different synthetic scaffolds have been used to directly dissect protein function. Protein binders have been fused to functional domains, such as to lead to protein degradation, relocalization, visualization, or posttranslational modification of the target protein upon binding. The use of such functionalized protein binders has allowed the study of the proteome during development in an unprecedented manner. In the coming years, the advent of the computational design of protein binders, together with further advances in scaffold engineering and synthetic biology, will fuel the development of novel protein binder–based technologies. Studying the proteome with increased precision will contribute to a better understanding of the immense molecular complexities hidden in each step along the way to generate form and function during development. [ABSTRACT FROM AUTHOR]

Red pitaya fruit has become a source of natural colorant, because it is rich in betalains, a pigment that imparts a red‐purple color that interests the food and cosmetics industries. This fruit also possesses high nutritional value, with a range of bioactive compounds known to confer potential health benefits and prevent chronic diseases, such as diabetes, which makes it useful for use as pharmaceutical agents and dietary supplements. In order to improve its technological and biological effects, a concentration will be required. Thus, the microfiltration, followed by vacuum concentration, can be an interesting strategy for this purpose. This study aimed to explore tangential microfiltration to produce microfiltered material, which is an important step to obtain the microfiltered red‐purple pitaya concentrate. Therefore, physicochemical and chemical characterization (including 1H NMR analysis) and biological properties (toxicity and diabetes) of this concentrate were assessed, using adult zebrafish as a model. The results show that microfiltration was carried out efficiently, with an average consumption of 95.75 ± 3.13 and 74.12 ± 3.58 kW h m−3, varying according to the material used ("unpeeled pitaya pulp" or "pitaya pulp with peel," respectively). The in vivo tests indicated non‐toxicity and hypoglycemic effect of the concentrate, since the blood glucose levels were significantly lower in the zebrafish groups treated with this concentrate in comparison with that of control group. Thus, this study suggests the potential of microfiltered red‐purple pitaya concentrate as a promising multifunctional food‐derived colorant, exhibiting beneficial biological effects far beyond its attractive color. Practical Application: Hylocereus polyrhizus (F.A.C. Weber) Britton & Rose has attracted attention as a potential source of natural colorants because of its red‐purple skin and flesh color. In addition, this fruit has a range of bioactive compounds, which make it a valuable resource for providing potential health benefits and preventing chronic diseases such as diabetes. In this paper, the microfiltered red‐purple pitaya concentrate showed beneficial biological effects far beyond its attractive color. Thus, this product can be considered a promising multifunctional food‐derived colorant to use in the food, pharmaceutical, or cosmetics industries. [ABSTRACT FROM AUTHOR]

Regeneration, the complex process of restoring damaged or absent cells, tissues, and organs, varies considerably between species. The zebrafish is a remarkable model organism for its impressive regenerative abilities, particularly in organs such as the heart, fin, retina, spinal cord, and brain. Unlike mammals, zebrafish can regenerate with limited or absent scarring, a phenomenon closely linked to the activation of stem cells and immune cells. This review examines the unique roles played by the immune response and inflammation in zebrafish and mouse during regeneration, highlighting the cellular and molecular mechanisms behind their divergent regenerative capacities. By focusing on zebrafish telencephalic regeneration and comparing it to that of the rodents, this review highlights the importance of a well‐controlled, acute, and non‐persistent immune response in zebrafish, which promotes an environment conducive to regeneration. The knowledge gained from understanding the mechanisms of zebrafish regeneration holds great promises for the treatment of human neurodegenerative diseases and brain damage (stroke and traumatic brain injuries), as well as for the advancement of regenerative medicine approaches. [ABSTRACT FROM AUTHOR]

Isl1 has been described as an embryonic master control gene expressed in the pericloacal mesenchyme. Deletion of Isl1 from the genital mesenchyme in mice leads to an ectopic urethral opening and epispadias-like phenotype. Using genome wide association methods, we identified ISL1 as the key susceptibility gene for classic bladder exstrophy (CBE), comprising epispadias and exstrophy of the urinary bladder. The most significant marker (rs6874700) identified in our recent GWAS meta-analysis achieved a p value of 1.48 × 10− 24 within the ISL1 region. In silico analysis of rs6874700 and all other genome-wide significant markers in Linkage Disequilibrium (LD) with rs6874700 (D' = 1.0; R2 > 0.90) revealed marker rs2303751 (p value 8.12 × 10− 20) as the marker with the highest regulatory effect predicted. Here, we describe a novel 1.2 kb intragenic promoter residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site, which is located in the reverse DNA strand and harbors a binding site for EZH2 at the exact region of marker rs2303751. We show, that EZH2 silencing in HEK cells reduces ISL1 expression. We show that ezh2−/− knockout (KO) zebrafish larvae display tissues specificity of ISL1 regulation with reduced expression of Isl1 in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2−/− ko zebrafish Tg(wt1ß:eGFP) reporter lines. Our study shows, that Ezh2 is a key regulator of Isl1 during urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation. [ABSTRACT FROM AUTHOR]