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Innovative for the scientific community and attracting attention in the extensive biomedical field are novel compact organic chemosensing systems built upon unique core molecular frameworks. These systems may demonstrate customized responses and may be adaptable to analytes, showing promise for potential in vivo applications. Our recent investigation focuses on a precursor of Mycophenolic acid, resulting in the development of LBM (LOD = 13 nM) – a specialized probe selective for H2O2. This paper details the synthesis, characterization, and thorough biological assessments of LBM. Notably, we conducted experiments involving living cells, daphnia, and zebrafish models, utilizing microscopy techniques to determine probe nontoxicity and discern distinct patterns of probe localization. Localization involved the distribution of the probe in the Zebrafish model within the gut, esophagus, and muscles of the antennae. [ABSTRACT FROM AUTHOR]

Background and Purpose Experimental Approach Key Results Conclusion and Implications The oxidant sensor transient receptor potential ankyrin 1 (TRPA1) channel expressed by Schwann cells (SCs) has recently been implicated in several models of neuropathic pain in rodents. Here we investigate whether the pro‐algesic function of Schwann cell TRPA1 is not limited to mammals by exploring the role of TRPA1 in a model of chemotherapy‐induced peripheral neuropathy (CIPN) in zebrafish larvae.We used zebrafish larvae and a mouse model to test oxaliplatin‐evoked nociceptive behaviours. We also performed a TRPA1 selective silencing in Schwann cells both in zebrafish larvae and mice to study their contribution in oxaliplatin‐induced CIPN model.We found that zebrafish larvae and zebrafish TRPA1 (zTRPA1)‐transfected HEK293T cells respond to reactive oxygen species (ROS) with nociceptive behaviours and intracellular calcium increases, respectively. TRPA1 was found to be co‐expressed with the Schwann cell marker, SOX10, in zebrafish larvae. Oxaliplatin caused nociceptive behaviours in zebrafish larvae that were attenuated by a TRPA1 antagonist and a ROS scavenger. Oxaliplatin failed to produce mechanical allodynia in mice with Schwann cell TRPA1 selective silencing (Plp1+‐Trpa1 mice). Comparable results were observed in zebrafish larvae where TRPA1 selective silencing in Schwann cells, using the specific Schwann cell promoter myelin basic protein (MBP), attenuated oxaliplatin‐evoked nociceptive behaviours.These results indicate that the contribution of the oxidative stress/Schwann cell/TRPA1 pro‐allodynic pathway to neuropathic pain models seems to be conserved across the animal kingdom. [ABSTRACT FROM AUTHOR]

Brain nuclei are traditionally defined by their anatomy, activity, and expression of specific markers. The hypothalamus contains discrete neuronal populations that coordinate fundamental behavioral functions, including sleep and wakefulness, in all vertebrates. Particularly, the diverse roles of hypocretin/orexin (Hcrt)-releasing neurons suggest functional heterogeneity among Hcrt neurons. Using single-cell RNA sequencing (scRNA-seq) and high-resolution imaging of the adult male and female zebrafish hypothalamic periventricular zone, we identified 21 glutamatergic and 28 GABAergic cell types. Integration of zebrafish and mouse scRNA-seq revealed evolutionary conserved and divergent hypothalamic cell types. The expression of specific genes, including npvf, which encodes a sleep-regulating neuropeptide, was enriched in subsets of glutamatergic Hcrt neurons in both larval and adult zebrafish. The genetic profile, activity, and neurite processing of the neuronal subpopulation that coexpresses both Hcrt and Npvf (Hcrt+Npvf+) differ from other Hcrt neurons. These interspecies findings provide a unified annotation of hypothalamic cell types and suggest that the heterogeneity of Hcrt neurons enables multifunctionality, such as consolidation of both wake and sleep by the Hcrt- and Npvf-releasing neuronal subpopulation. [ABSTRACT FROM AUTHOR]

Rampant industrialization has led to widespread reliance on hydrocarbon polymers for various commercial applications. While these synthetic polymers, commonly known as plastics, degrade in slowly in the environments, the toxic effects of their micro-sized particles remain underexplored. In this study, we synthesized polyisobutylene (PIB) microparticles in the lab and evaluated their toxicity and accumulation in a zebrafish model. Pristine and fluorescent PIB-microplastics (MPs), with particle sizes ranging from 2 to 10 μm, were synthesized using the solvent evaporation method. Fourier-transform infrared spectroscopy (FTIR) confirmed the stability of the suspensions. Zebrafish larvae exposed to various concentrations of PIB-MPs exhibited numerous morphological and molecular changes, including delayed hatching, impaired swimming behavior, increased reactive oxygen species levels, altered mRNA levels of genes encoding antioxidant proteins, and reduced survival rates. Dissections revealed PIB-MP accumulation in the guts of larvae and adult fish within 7–21 days, causing damage to the intestinal mucosa. These findings provide insights into how contaminants like PIB can induce pathophysiological defects in aquatic fauna and pose potential health hazards to humans. [ABSTRACT FROM AUTHOR]

Microglia are resident immune cells in the central nervous system, including the retina that surveil the environment for damage and infection. Following retinal damage, microglia undergo morphological changes, migrate to the site of damage, and express and secrete pro-inflammatory signals. In the zebrafish retina, inflammation induces the reprogramming and proliferation of Müller glia and the regeneration of neurons following damage or injury. Immunosuppression or pharmacological ablation of microglia reduce or abolish Müller glia proliferation. We evaluated the retinal architecture and retinal regeneration in adult zebrafish irf8 mutants, which have significantly depleted numbers of microglia. We show that irf8 mutants have normal retinal structure at 3 months post fertilization (mpf) and 6 mpf but fewer cone photoreceptors by 10 mpf. Surprisingly, light-induced photoreceptor ablation induced Müller glia proliferation in irf8 mutants and cone and rod photoreceptor regeneration. Light-damaged retinas from both wild-type and irf8 mutants show upregulated expression of mmp-9, il8, and tnfβ pro-inflammatory cytokines. Our data demonstrate that adult zebrafish irf8 mutants can regenerate normally following acute retinal injury. These findings suggest that microglia may not be essential for retinal regeneration in zebrafish and that other mechanisms can compensate for the reduction in microglia numbers. [ABSTRACT FROM AUTHOR]

AbstractEmerging endocrine-disrupting chemicals (EDCs) are a diverse group of toxic substances that disrupt the endocrine system. These substances can only be approved after obtaining concrete evidence of their toxicity. New approach methodologies (NAMs) offer information tailored to the intended application while reducing the use of animals. NAMs aim to develop quantitative structure-activity relationship (QSAR) and quantitive-Read-Across structure-activity relationship (q-RASAR) models to predict and categorize the acute toxicity of known and unknown EDCs. The q-RASAR model was constructed and verified using validation metrics (R2 = 0.886 and Q2 = 0.814). The substructure fingerprint was well-fitted for the classification model and the model was validated using 10-fold average accuracy (Q = 86.88%), specificity (Sp = 88.89%), Matthew’s correlation curve (MCC = 0.621) and receiver operating characteristics (ROC = 0.828). The dataset of unknown substances revealed that phenolphthalein (Php) exhibited a significant level of toxicity. The docking and simulation study results indicated that the computationally derived important features successfully bound to the target zebrafish sex hormone binding globulin (zfSHBG). The experimental LC50 value of 0.790 mg L−1 was very close to the predicted value of 0.763 mg L−1, which provides high confidence to the developed model. [ABSTRACT FROM AUTHOR]

DNA-protein crosslinks pose a significant challenge to genome stability and cell viability. Efficient repair of DPCs is crucial for preserving genomic integrity and preventing the accumulation of DNA damage. Despite recent advances in our understanding of DPC repair, many aspects of this process, especially at the organismal level, remain elusive. In this study, we used zebrafish as a model organism to investigate the role of TDP2 (Tyrosyl-DNA phosphodiesterase 2) in DPC repair. We characterized the two tdp2 orthologs in zebrafish using phylogenetic, syntenic and expression analysis and investigated the phenotypic consequences of tdp2 silencing in zebrafish embryos. We then quantified the effects of tdp2a and tdp2b silencing on cellular DPC levels and DSB accumulation in zebrafish embryos. Our findings revealed that tdp2b is the main ortholog during embryonic development, while both orthologs are ubiquitously present in adult tissues. Notably, the tdp2b ortholog is phylogenetically closer to human TDP2. Silencing of tdp2b, but not tdp2a, resulted in the loss of Tdp2 activity in zebrafish embryos, accompanied by the accumulation of DPCs and DSBs. Our findings contribute to a more comprehensive understanding of DPC repair at the organismal level and underscore the significance of TDP2 in maintaining genome stability. [ABSTRACT FROM AUTHOR]

Zebrafish (Danio rerio) is ideal for studying the effects of toxins like lead or plumbum (Pb) which persist in the environment and harm body systems when absorbed. Increasing Pb concentration could result in a higher mortality rate and alteration of behavior and metabolism. The present study evaluates the acute toxicity effect of Pb on metabolome and behavior in adult zebrafish. The zebrafish were exposed to various Pb concentrations ranging from 0 to 30 mg/L for different periods (24, 48, and 72 h) before the fish samples were subjected to Nuclear Magnetic Resonance (NMR)-multivariate data analysis (MVDA) with additional support from behavioral assessment. The behavior of zebrafish was significantly altered after Pb inducement and the differential metabolites increased in low (5 mg/L) while decreased in high (10 mg/L) Pb concentrations. An ideal Pb induction could be achieved by 5 mg/L concentration in 24 h, which induced significant metabolite changes without irreversible damage. Continuing research on the effects of lead toxicity is crucial to develop effective prevention and treatment strategies. [ABSTRACT FROM AUTHOR]

• TMA+ exposure significantly inhibited the swimming activities of larval zebrafish. • Swimming path angle is the most sensitive endpoint in response to TMA+ exposure. • Neurobehavior endpoints of larvae were significantly related with oxidative stress. • The calculated 96 h-LC 50 of TMA+ was lower for zebrafish juveniles than larvae. • TMA+ evoked greater inhibitions on multi-biomarkers of juveniles than larvae. Tetramethylammonium hydroxide (TMAH) is an important compound that utilized and released by the rapidly expanding semiconductor industry, which could hardly be removed by the conventional wastewater treatment techniques. As a cholinergic agonist, the tetramethylammonium ion (TMA+) has been reported to induce toxicity to muscular and respiratory systems of mammals and human, however the toxicity on aquatic biota remains poorly known. We investigated the neurotoxic effects of TMA+ exposure on zebrafish, based on neurobehavior tests and a series of biomarkers. Significant inhibitions on the swimming distance of zebrafish larvae were observed when the exposure level exceeded 50 mg/L, and significant alterations on swimming path angles (straight and deflective movements) occurred even at 10 mg/L. The tested neurobehavioral endpoints of zebrafish larvae were significantly positively correlated with reactive oxygen species (ROS) and malondialdehyde (MDA), significantly negatively related with the activities of antioxidant enzymes, but not significantly correlated with the level of acetylcholinesterase (AChE). Such relationship indicates that the observed neurotoxic effects on swimming behavior of zebrafish larvae is mainly driven by oxidative stress, rather than the alterations of neurotransmitter. At the highest exposure concentration (200 mg/L), TMA+ evoked more severe toxicity on zebrafish juveniles, showing significantly stronger elevation on the MDA activity, and greater inhibitions on the activities of antioxidant enzymes and AChE, suggesting juveniles were more susceptible to TMA+ exposure than larval zebrafish. [Display omitted] [ABSTRACT FROM AUTHOR]

Antibiotic success rates are decreasing as drug-resistant bacteria become more prevalent, prompting the development of new therapeutic drugs. Herein, we demonstrated the antimicrobial activity of sarsaparilla root extract fabricated silver nanoparticles (sAgNPs). The UV–Visible spectra revealed that the surface Plasmon resonance maxima of sAgNPs were at 415 nm. Transmission electron microscopy confirms that the particles are spherical with size of 12–35 nm. The minimum inhibitory concentration (MIC) of sAgNPs against Escherichia coli, uropathogenic Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus was 62.5, 62.5, 62.5, 62.5, 125 and 125 µM, respectively. At 1X MIC, sAgNPs induces excess reactive oxygen species (ROS) production and disturbs the bacteria membrane intergity, causing cytoplamic membrane depolarization. Interestingly, antibacterial activity of sAgNPs was considerably reduced in the presence of an antioxidant, N-acetyl cysteine, suggesting that ROS-induced membrane damage is a plausible cause of cell death. In contrast to many studies that only report the in vitro activity of NPs, we determined the in vivo antibacterial efficacy using the zebrafish model. It was found that sAgNPs protect fish from infection by inhibiting bacterial growth and eliminating them from the fish. In addition, the catalytic potential of sAgNPs for wastewater decontamination was demonstrated by degrading organic pollutants such as methyl orange, congo red, reactive black, and acid blue. The pollutants degraded in less than 10 min, and the reaction follows pseudo-first-order kinetics. As a proof of concept, the catalytic potential of sAgNPs in degrading mixed dyes to satisfy industrial wastewater treatment needs was established. In summary, sAgNPs have the potential to act as nanocatalysts and nano-drugs, addressing key challenges in medical and environmental research. [ABSTRACT FROM AUTHOR]

In-vivo flow cytometry is a noninvasive real-time diagnostic technique that facilitates continuous monitoring of cells without perturbing their natural biological environment, which renders it a valuable tool for both scientific research and clinical applications. However, the conventional approach for improving classification accuracy often involves labeling cells with fluorescence, which can lead to potential phototoxicity. This study proposes a label-free in-vivo flow cytometry technique, called dynamic YOLOv4 (D-YOLOv4), which improves classification accuracy by integrating absorption intensity fluctuation modulation (AIFM) into YOLOv4 to demodulate the temporal features of moving red blood cells (RBCs) and platelets. Using zebrafish as an experimental model, the D-YOLOv4 method achieved average precisions (APs) of 0.90 for RBCs and 0.64 for thrombocytes (similar to platelets in mammals), resulting in an overall AP of 0.77. These scores notably surpass those attained by alternative network models, thereby demonstrating that the combination of physical models with neural networks provides an innovative approach toward developing label-free in-vivo flow cytometry, which holds promise for diverse in-vivo cell classification applications. [ABSTRACT FROM AUTHOR]

Shigella is an important human-adapted pathogen for which there is an increasing incidence of antimicrobial resistance and no effective vaccine. Modelling Shigella infection of humans has historically been difficult, but new technologies and animal models have emerged to recapitulate key hallmarks of shigellosis. The use of organ-on-chip technology, zebrafish infection models, transgenic mouse models, and human challenge studies all uniquely contribute to our understanding of Shigella infection biology. Recent advances have illuminated our understanding of Shigella pathogenesis, guiding vaccine strategies and moving us closer to human health impact. Shigella is an important human-adapted pathogen which contributes to a large global burden of diarrhoeal disease. Together with the increasing threat of antimicrobial resistance and lack of an effective vaccine, there is great urgency to identify novel therapeutics and preventatives to combat Shigella infection. In this review, we discuss the development of innovative technologies and animal models to study mechanisms underlying Shigella infection of humans. We examine recent literature introducing (i) the organ-on-chip model, and its substantial contribution towards understanding the biomechanics of Shigella infection, (ii) the zebrafish infection model, which has delivered transformative insights into the epidemiological success of clinical isolates and the innate immune response to Shigella , (iii) a pioneering oral mouse model of shigellosis, which has helped to discover new inflammasome biology and protective mechanisms against shigellosis , and (iv) the controlled human infection model, which has been effective in translating basic research into human health impact and assessing suitability of novel vaccine candidates. We consider the recent contributions of each model and discuss where the future of modelling Shigella infection lies. [ABSTRACT FROM AUTHOR]

The increasing use of UV filters, such as benzophenone-3 (BP-3) and titanium dioxide nanoparticles (TiO2 NPs), has raised concerns regarding their ecotoxicological effects on the aquatic environment. The aim of the present study was to examine the embryo-larval toxicity attributed to BP-3 or TiO2 NPs, either alone or in a mixture, utilizing zebrafish (Danio rerio) as a model after exposure to environmentally relevant concentrations of these compounds. Zebrafish embryos were exposed to BP-3 (10, 100, or 1000 ng/L) or TiO2 NPs (1000 ng/L) alone or in a mixture (BP-3 10, 100, or 1000 ng/L plus 1000 ng/L of TiO2 NPs) under static conditions for 144 hr. After exposure, BP-3 levels were determined by high-performance liquid chromatography (HPLC). BP-3 levels increased in the presence of TiO2 NPs, indicating that the BP-3 degradation decreased in the presence of the NPs. In addition, in the presence of zebrafish, BP-3 levels in water decreased, indicating that zebrafish embryos and larvae might absorb BP-3. Data demonstrated that, in general, environmentally relevant concentrations of BP-3 and TiO2 NPs, either alone or in a mixture, did not significantly induce changes in heart and spontaneous contractions frequencies, levels of reactive oxygen species (ROS), morphological and morphometric parameters as well as mortality rates during 144 hr exposure. However, the groups exposed to TiO2 NPs alone and in a mixture with BP-3 at 10 ng/L exhibited an earlier significant hatching rate than the controls. Altogether, the data indicates that a potential ecotoxicological impact on the aquatic environment exists. [ABSTRACT FROM AUTHOR]

The Salacia reticulata, a medicinal woody climbing shrub, was utilized for our study, the green synthesis of CuO nanoparticles, which were analyzed through SEM, EDX, FTIR, XRD, and UV‒Vis spectroscopy. This study assessed the toxicity to zebrafish embryos and explored the antibacterial, cytotoxic, antidiabetic, and anti-inflammatory properties of the synthesized nanoparticles. In results, the UV absorption of the CuO NPs showed that the intensity of nanoparticle green colloidal suspension changed from blue to green, which also confirmed that the spectrum of the green CuO NPs changed from colorless to black. in FT-IR and XRD spectral analysis to identify functional groups and determine the particle size of CuO NPs prepared by green and chemical methods. Its showed that CuO NPs (green) had a size of approximately 42.2 nm, while CuO NPs (chemical) had a size of approximately 84 nm. The morphology of these NPs was analyzed using SEM–EDX. Compared with their chemically prepared counterparts, the green-synthesized CuO nanoparticles demonstrated superior dispersion. Additionally, both green and chemical CuO nanoparticles at a concentration of 200 µL/mL caused developmental anomalies and increased mortality in zebrafish embryos and larvae. The green and chemical CuO NPs inhibited α-glucosidase enzyme activity at concentrations between 10 and 50 µL/mL, with IC50 values of 22 µL/mL and 26 µL/mL, respectively. The extract exhibited anti-inflammatory activity, with IC50 values of 274 and 109 µL/mL. The authors concluded that this green nanoparticle method has potential as a more eco-friendly and cost-effective alternative to traditional synthetic methods. NPs are widely used in human contact fields (medicine and agriculture), hence synthesis methods that do not involve toxic substances are becoming increasingly important. [ABSTRACT FROM AUTHOR]

Microglia, the crucial immune cells inhabiting the central nervous system (CNS), perform a range of vital functions, encompassing immune defense and neuronal regulation. Microglia subsets with diverse functions and distinct developmental regulations have been identified recently. It is generally accepted that all microglia originate from hematopoiesis and depend on the myeloid transcription factor PU.1. However, a recent study reported the existence of mrc1+ microglia in zebrafish embryos, which are seemingly independent of Pu.1 and reliant on lymphatic vessels, sparking great interest in the possibility of lymphatic-originated microglia. To address this, we took advantage of a pu.1 knock-in zebrafish allele for a detailed investigation. Our results conclusively showed that almost all zebrafish embryonic microglia (~95% on average) express pu.1. Further, lineage tracing and mutant analysis revealed that these microglia neither emerged from nor depended on lymphatic vessels. In essence, our study refutes the presence of pu.1-independent but lymphatic-dependent microglia. [ABSTRACT FROM AUTHOR]

Objective Methods Results Interpretation Hexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A large body of evidence implicates dipeptide repeats (DPRs) proteins as one of the main drivers of neuronal injury in cell and animal models.A pure repeat‐associated non‐AUG (RAN) translation zebrafish model of C9orf72‐ALS/FTD was generated. Embryonic and adult transgenic zebrafish lysates were investigated for the presence of RAN‐translated DPR species and adult‐onset motor deficits. Using C9orf72 cell models as well as embryonic C9orf72‐ALS/FTD zebrafish, hypothermic‐therapeutic temperature management (TTM) was explored as a potential therapeutic option for C9orf72‐ALS/FTD.Here, we describe a pure RAN translation zebrafish model of C9orf72‐ALS/FTD that exhibits significant RAN‐translated DPR pathology and progressive motor decline. We further demonstrate that hypothermic‐TTM results in a profound reduction in DPR species in C9orf72‐ALS/FTD cell models as well as embryonic C9orf72‐ALS/FTD zebrafish.The transgenic model detailed in this paper provides a medium throughput in vivo research tool to further investigate the role of RAN‐translation in C9orf72‐ALS/FTD and further understand the mechanisms that underpin neuroprotective strategies. Hypothermic‐TTM presents a viable therapeutic avenue to explore in the context of C9orf72‐ALS/FTD. ANN NEUROL 2024 [ABSTRACT FROM AUTHOR]

Understanding how sublethal impacts of toxicants affect population‐relevant outcomes for organisms is challenging. We tested the hypotheses that the well‐known sublethal impacts of methylmercury (MeHg) and a polychlorinated biphenyl (PCB126) would have meaningful impacts on cohort growth and survival in yellow perch (Perca flavescens) and Atlantic killifish (Fundulus heteroclitus) populations, that inclusion of model uncertainty is important for understanding the sublethal impacts of toxicants, and that a model organism (zebrafish Danio rerio) is an appropriate substitute for ecologically relevant species (yellow perch, killifish). Our simulations showed that MeHg did not have meaningful impacts on growth or survival in a simulated environment except to increase survival and growth in low mercury exposures in yellow perch and killifish. For PCB126, the high level of exposure resulted in lower survival for killifish only. Uncertainty analyses increased the variability and lowered average survival estimates across all species and toxicants, providing a more conservative estimate of risk. We demonstrate that using a model organism instead of the species of interest does not necessarily give the same results, suggesting that using zebrafish as a surrogate for yellow perch and killifish may not be appropriate for predicting contaminant impacts on larval cohort growth and survival in ecologically relevant species. Our analysis also reinforces the notion that uncertainty analyses are necessary in any modeling assessment of the impacts of toxicants on a population because it provides a more conservative, and arguably realistic, estimate of impact. Environ Toxicol Chem 2024;00:1–12. © 2024 SETAC [ABSTRACT FROM AUTHOR]

Objective: Preclinical models of seizures and epilepsy in rodents contributed substantially to the discovery of currently available antiseizure medications. These were also broadly used for investigation of processes of epileptogenesis. Nevertheless, rodent models pose some limitations, thus, new models using alternative species are in high demand. The aim of this study was to describe a new model of seizures/epilepsy induced by the cholinomimetic agent, pilocarpine (PILO), in larval zebrafish. Methods: Local field potential (LFP) recordings were conducted to analyze electroencephalographic discharges and correlate it with larval behavior. Hematoxylin and eosin (H&E) staining, as well as TUNEL staining were performed to analyze morphology and apoptosis, respectively. Real-time quantitative polymerase chain reaction (qRT-PCR) was undertaken for gene expression analysis. Results: Acute exposure to PILO, in a concentration-dependent manner, induces electroencephalographic discharges in larval zebrafish, which behaviorally manifest as decreased locomotion and moving time, but enhanced movement velocity. The PILO-induced seizure-like activity is behaviorally distinct from this induced by the application of chemoconvulsant pentylenetetrazole (PTZ). Zebrafish larvae previously exposed to PILO (2 h), after a washing out period, exhibit spontaneous, unprovoked discharges and apoptotic changes in their brains. Significance: Here, we comprehensively investigated a new model of PILOinduced seizures/epilepsy in larval zebrafish. We propose that this model may be used to study epileptogenesis and for antiseizure drug screening purposes. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we compared the effectiveness of two anesthetics, eugenol and tricaine methanesulfonate (MS-222), in consecutive administrations on zebrafish. We evaluated zebrafish behaviour after repeated anesthesia. Eugenol induced anesthesia more quickly than MS-222 but had longer recovery times. Additionally, swimming frequency decreased after eugenol anesthesia. The buffered version of MS-222 was more effective than the non-buffered one. We recommend using buffered MS-222 for studies requiring repeated, brief-duration anesthesia. The increasing use of the zebrafish (Danio rerio) in scientific experiments has made it necessary to implement anesthesia protocols guaranteeing minimum pain and suffering for these animals and ensuring the reliability of the results obtained from their research. Therefore, we aimed to compare the effectiveness of two anesthetics, eugenol and MS-222, in consecutive administrations and evaluate the zebrafish behaviour after repeated anesthesia. Thus, several zebrafish were anaesthetized with eugenol, MS-222, and buffered MS-222 three times repeatedly with a 24-h interval between each exposure. The induction and recovery periods were also timed. Their swimming frequency was determined after each exposure to assess their behaviour after the anesthesia. Anesthesia induction was quicker with eugenol compared to MS-222. However, eugenol presented longer recovery times, which were prolonged after each exposure. Also, the swimming frequency was reduced after each anesthesia with eugenol. The buffered version of MS-222 was more efficacious than the non-buffered one. Both versions of MS-222 did not affect the swimming frequency. Based on these findings, we recommend the utilization of MS-222 buffered rather than eugenol when repeated, brief-duration anesthesia is necessitated for a study. [ABSTRACT FROM AUTHOR]

Background and Purpose: Parkinson's disease (PD) is a prevalent neurodegenerative movement disorder characterized by motor dysfunction. Environmental factors, especially manganese (Mn), contribute significantly to PD. Existing therapies are focused on motor coordination, whereas nonmotor features such as neuropsychiatric symptoms are often neglected. Daidzein (DZ), a phytoestrogen, has piqued interest due to its antioxidant, anti‐inflammatory, and anxiolytic properties. Therefore, we anticipate that DZ might be an effective drug to alleviate the nonmotor symptoms of Mn‐induced Parkinsonism. Experimental Approach: Naïve zebrafish were exposed to 2 mM of Mn for 21 days and intervened with DZ. Nonmotor symptoms such as anxiety, social behaviour, and olfactory function were assessed. Acetylcholinesterase (AChE) activity and antioxidant enzyme status were measured from brain tissue through biochemical assays. Dopamine levels and histology were performed to elucidate neuroprotective mechanism of DZ. Key Results: DZ exhibited anxiolytic effects in a novel environment and also improved intra and inter fish social behaviour. DZ improved the olfactory function and response to amino acid stimuli in Mn‐induced Parkinsonism. DZ reduced brain oxidative stress and AChE activity and prevented neuronal damage. DZ increased DA level in the brain, collectively contributing to neuroprotection. Conclusion and Implications: DZ demonstrated a promising effect on alleviating nonmotor symptoms such as anxiety and olfactory dysfunction, through the mitigation of cellular damage. These findings underscore the therapeutic potential of DZ in addressing nonmotor neurotoxicity induced by heavy metals, particularly in the context of Mn‐induced Parkinsonism. [ABSTRACT FROM AUTHOR]

In a developing nervous system, axonal arbors often undergo complex rearrangements before neural circuits attain their final innervation topology. In the lateral line sensory system of the zebrafish, developing sensory axons reorganize their terminal arborization patterns to establish precise neural microcircuits around the mechanosensory hair cells. However, a quantitative understanding of the changes in the sensory arbor morphology and the regulators behind the microcircuit assembly remain enigmatic. Here, we report that Semaphorin7A (Sema7A) acts as an important mediator of these processes. Utilizing a semi-automated three-dimensional neurite tracing methodology and computational techniques, we have identified and quantitatively analyzed distinct topological features that shape the network in wild-type and Sema7A loss-of-function mutants. In contrast to those of wild-type animals, the sensory axons in Sema7A mutants display aberrant arborizations with disorganized network topology and diminished contacts to hair cells. Moreover, ectopic expression of a secreted form of Sema7A by non-hair cells induces chemotropic guidance of sensory axons. Our findings propose that Sema7A likely functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development. [ABSTRACT FROM AUTHOR]

Overexpression of the agouti-signaling protein (asip1), an endogenous melanocortin antagonist, under the control of a constitutive promoter in zebrafish [Tg(Xla.Eef1a1:Cau.Asip1]iim4] (asip1-Tg) increases food intake by reducing sensitivity of the central satiety systems and abolish circadian activity rhythms. The phenotype also shows increased linear growth and body weight, yet no enhanced aggressiveness in dyadic fights is observed. In fact, asip1-Tg animals choose to flee to safer areas rather than face a potential threat, thus suggesting a potential anxiety-like behavior (ALB). Standard behavioral tests, i.e., the open field test (OFT), the novel object test (NOT), and the novel tank dive test (NTDT), were used to investigate thigmotaxis and ALB in male and female zebrafish. Results showed that the asip1-Tg strain exhibited severe ALB in every test, mainly characterized by pronounced freezing behavior and increased linear and angular swimming velocities. asip1-Tg animals exhibited low central serotonin (5-HT) and dopamine (DA) levels and high turnover rates, thus suggesting that central monoaminergic pathways might mediate melanocortin antagonist-induced ALB. Accordingly, the treatment of asip1-Tg animals with fluoxetine, a selective serotonin reuptake inhibitor (SSRI), reversed the ALB phenotype in NTDT as well as 5-HT turnover. Genomic and anatomical data further supported neuronal interaction between melanocortinergic and serotonergicsystems. These results suggest that inhibition of the melanocortin system by ubiquitous overexpression of endogenous antagonist has an anxiogenic effect mediated by serotonergic transmission. [ABSTRACT FROM AUTHOR]

Detecting the toxic effects of chemicals on reproduction and development without using mammalian animal models is crucial in the exploitation of pharmaceuticals for human use. Zebrafish are a promising animal model for investigating pharmacological effects and toxicity during vertebrate development. Several studies have suggested the use of zebrafish embryos for the assessment of malformations or embryo-fetal lethality (MEFL). However, a reproducible protocol as a standard for the zebrafish MEFL test method that fulfills global requests has not been established based on the International Council of Harmonisation (ICH) S5 (R3) guidelines. To establish such a toxicity test method, we developed a new and easy protocol to detect MEFL caused by chemicals, especially those with teratogenic potential, using fertilized zebrafish eggs (embryos) within 5 days of development. Our toxicity test trials using the same protocol in two to four different laboratories corroborated the high inter-laboratory reproducibility. Our test method enabled the detection of 18 out of 22 test compounds that induced rat MEFL. Thus, the prediction rate of our zebrafish test method for MEFL was almost 82% compared with that of rat MEFL. Collectively, our study proposes the establishment of an easy and reproducible protocol for the zebrafish MEFL test method for reproductive and developmental toxicity that meets ICH guideline S5 (R3), which can be further considered in combination with information from other sources for regulatory use. [ABSTRACT FROM AUTHOR]

Chronic exposure to heavy metals has been widely demonstrated to induce pathological features in different tissues and, in particular, in the central nervous system. Specific neurons, including dopaminergic neurons, were observed to be more susceptible to toxic agents. Several previous studies performed on zebrafish (Danio rerio) models observed that exposure to nickel (one of the most abundant heavy metals) induces impairment of memory and anxious-like behaviors. Nevertheless, this phenotypical evidence has not been associated with dopaminergic system damage, and no reports showing the effects of nickel on dopaminergic neurons are available. In this study, we aim to analyze the precise distribution and variation in dopaminergic neurons in adult zebrafish after chronic (96 h) exposure to nickel ions dissolved in water at different sub-lethal doses (0.4 mg L−1; 2 mg L−1 and 4 mg L−1). The effects of treatment on dopaminergic neurons were evaluated by measuring transcript and protein levels of tyrosine hydroxylase (TH), described as a dopaminergic neuron marker. As shown, the expression of the th1 and th2 genes was reduced in the entire brain of zebrafish treated with nickel. Immunostaining analysis allowed us to localize TH-expressing neurons mainly in the posterior tuberculum, where they were observed to be reduced after nickel treatment in a dose-dependent fashion. Consistently, the TUNEL assay revealed a significant increase in apoptosis of TH-expressing cells after treatment with 2 mg L−1 and 4 mg L−1 of nickel. Our findings represent the first evidence of the effect of nickel on the dopaminergic system. [ABSTRACT FROM AUTHOR]

Animals with different life‐history types vary in their stress‐coping styles, which can affect their fitness and survival in changing environments. We studied how chronic exposure to manganese sulfate (MnSO4), a common aquatic pollutant, affects life‐history traits, physiology, and behavior of zebrafish (Danio rerio) with two life‐history types: fast (previously selected for fast juvenile growth, early maturation, and small adult body size) and slow life histories (selected for slow juvenile growth, late maturation, and large adult body size). We found that MnSO4 had negative effects on growth and condition factors, but the magnitude of these effects depended on the life‐history type. Individuals with fast life histories were more susceptible to MnSO4 than fish with slow life histories as they had lower growth rate, condition factor and feeding probability in high MnSO4 concentrations. Our results demonstrate that MnSO4 can impair fish performance, and life‐history variation can modulate the stress‐coping ability of individuals. Animals with different life‐history types vary in their stress‐coping styles, which can affect their fitness and survival in changing environments. We found that MnSO4 had negative effects on growth and condition factors, but the magnitude of these effects depended on the life‐history type. Individuals with fast life histories were more susceptible to MnSO4 than fish with slow life histories as they had lower growth rate, condition factor and feeding probability in high MnSO4 concentrations. [ABSTRACT FROM AUTHOR]

Death of mechanosensory hair cells in the inner ear is a common cause of auditory and vestibular impairment in mammals, which have a limited ability to regrow these cells after damage. In contrast, nonmammalian vertebrates, including zebrafish, can robustly regenerate hair cells after severe organ damage. The zebrafish inner ear provides an understudied model system for understanding hair cell regeneration in organs that are highly conserved with their mammalian counterparts. Here, we quantitatively examine hair cell addition during growth and regeneration of the larval zebrafish inner ear. We used a genetically encoded ablation method to induce hair cell death and we observed gradual regeneration with correct spatial patterning over a 2-week period following ablation. Supporting cells, which surround and are a source of new hair cells, divide in response to hair cell ablation, expanding the possible progenitor pool. In parallel, nascent hair cells arise from direct transdifferentiation of progenitor pool cells temporally uncoupled from supporting cell division. These findings reveal a previously unrecognized mechanism of hair cell regeneration with implications for how hair cells may be encouraged to regenerate in the mammalian ear. [ABSTRACT FROM AUTHOR]

Cohesin, a chromatin-associated protein complex with four core subunits (Smc1a, Smc3, Rad21 and either Stag1 or 2), has a central role in cell proliferation and gene expression in metazoans. Human developmental disorders termed 'cohesinopathies' are characterized by germline variants of cohesin or its regulators that do not entirely eliminate cohesin function. However, it is not clear whether mutations in individual cohesin subunits have independent developmental consequences. Here, we show that zebrafish rad21 or stag2b mutants independently influence embryonic tailbud development. Both mutants have altered mesoderm induction, but only homozygous or heterozygous rad21 mutation affects cell cycle gene expression. stag2b mutants have narrower notochords and reduced Wnt signaling in neuromesodermal progenitors as revealed by single-cell RNA sequencing. Stimulation of Wnt signaling rescues transcription and morphology in stag2b, but not rad21, mutants. Our results suggest that mutations altering the quantity versus composition of cohesin have independent developmental consequences, with implications for the understanding and management of cohesinopathies. [ABSTRACT FROM AUTHOR]

• Neutrophils modulate the spinal cord injury microenvironment. • Neutrophil immune profile determines the regenerative outcome. • Cxcr4 inhibition at the injury site enhances spinal cord regeneration. • Cxcr2 inhibition at the site of injury impairs spinal cord regeneration. • Neutrophils disperse from the injury site by reverse migration. Spinal cord injury triggers a strong innate inflammatory response in both non-regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet, their role in the repair process, particularly in a regenerative context, remains largely unknown. Here, we show that, following rapid recruitment to the injured spinal cord, neutrophils mostly reverse migrate throughout the zebrafish body. In addition, promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil-specific RNA-seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf-α expression in macrophage/microglia populations. Conversely, blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether, these findings provide new insights into the role of neutrophils in spinal cord regeneration, emphasizing the significant impact of their immune profile on the outcome of the repair process. [ABSTRACT FROM AUTHOR]

Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested these 24 mutations affect 24 different genes. We applied unsupervised clustering algorithms to unbiasedly classify the recovered mutants into three classes. Further computational analysis revealed that each of the recovered mutations in these three classes has a unique phenotype on node-subtype composition and distribution within the intrahepatic biliary network. In addition, we found most of the recovered mutations are viable. In those mutant fish, which are already good animal models to study chronic cholestatic liver diseases, the biliary network phenotypes persist into adulthood. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases. [Display omitted] • 24 zebrafish mutants with intrahepatic biliary network defects were identified. • Unsupervised classification algorithms sorted the mutants into subgroups. • Computational analysis revealed the detailed network structures in the mutants. • This work provides computational and biological toolsets for biliary system biology. [ABSTRACT FROM AUTHOR]

In the zebrafish lateral line, non-sensory supporting cells readily re-enter the cell cycle to generate new hair cells and supporting cells during homeostatic maintenance and following damage to hair cells. This contrasts with supporting cells from mammalian vestibular and auditory sensory epithelia which rarely re-enter the cell cycle, and hence loss of hair cells results in permanent sensory deficit. Lateral line supporting cells are derived from multipotent progenitor cells that migrate down the trunk midline as a primordium and are deposited to differentiate into a neuromast. We have found that we can revert zebrafish support cells back to a migratory progenitor state by pharmacologically altering the signaling environment to mimic that of the migratory primordium, with active Wnt signaling and repressed FGF signaling. The reverted supporting cells migrate anteriorly and posteriorly along the horizontal myoseptum and will re-epithelialize to form an increased number of neuromasts along the midline when the pharmacological agents are removed. These data demonstrate that supporting cells can be readily reprogrammed to a migratory multipotent progenitor state that can form new sensory neuromasts, which has important implications for our understanding of how the lateral line system matures and expands in fish and also suggest avenues for returning mammalian supporting cells back to a proliferative state. [Display omitted] • Zebrafish neuromasts can be pharmacologically reprogrammed to a migratory state. • Reprogrammed progenitors migrate along midline. • Migratory progenitors reaggregate into sensory organs following washout. • Reconstituted sensory organs form functional sensory cells and are innervated. [ABSTRACT FROM AUTHOR]

Green-synthesis of biodegradable polymeric curcumin-nanoparticles using affordable biodegradable polymers to enhance curcumin's solubility and anti-oxidative potential. The curcumin-nanoparticle was prepared based on the ionic-interaction method without using any chemical surfactants, and the particle-size, zeta-potential, surface-morphology, entrapmentefficiency, and in-vitro drug release study were used to optimise the formulation. The antioxidant activity was investigated using H2DCFDA staining in the zebrafish (Danio rerio) model. The mean-diameter of blank nanoparticles was 178.2 nm (±4.69), and that of curcuminnanoparticles was about 227.7 nm (±10.4), with a PDI value of 0.312 (±0.023) and 0.360 (±0.02). The encapsulation-efficacy was found to be 34% (±1.8), with significantly reduced oxidative-stress and toxicity (∼5 times) in the zebrafish model compared to standard curcumin. The results suggested that the current way of encapsulating curcumin using affordable, biodegradable, natural polymers could be a better approach to enhancing curcumin's water solubility and bioactivity, which could further be translated into potential therapeutics. [ABSTRACT FROM AUTHOR]

In recent decades, developmental biologists have come to view vascular development as a series of progressive transitions. Mesoderm differentiates into endothelial cells; arteries, veins and lymphatic endothelial cells are specified from early endothelial cells; and vascular networks diversify and invade developing tissues and organs. Our understanding of this elaborate developmental process has benefitted from detailed studies using the zebrafish as a model system. Here, we review a number of key developmental transitions that occur in zebrafish during the formation of the blood and lymphatic vessel networks. [ABSTRACT FROM AUTHOR]

Immune-mediated inflammatory disease (IMID) prevalence is estimated at 3–7% for Westernised populations, with annual incidence reported at almost 1 in 100 people globally. More recently, drug discovery approaches have been evolving towards more targeted therapies with an improved long-term safety profile, while the requirement for individualisation of medicine in complex conditions such as IMIDs, is acknowledged. However, existing preclinical models—such as cellular and in vivo mammalian models—are not ideal for modern drug discovery model requirements, such as real-time in vivo visualisation of drug effects, logistically feasible safety assessment over the course of a lifetime, or dynamic assessment of physiological changes during disease development. Zebrafish share high homology with humans in terms of proteins and disease-causing genes, with high conservation of physiological processes at organ, tissue, cellular and molecular level. These and other unique attributes, such as high fecundity, relative transparency and ease of genetic manipulation, positions zebrafish as the next major role player in IMID drug discovery. This review provides a brief overview of the suitability of this organism as model for human inflammatory disease and summarises the range of approaches used in zebrafish-based drug discovery research. Strengths and limitations of zebrafish as model organism, as well as important considerations in research study design, are discussed. Finally, under-utilised avenues for investigation in the IMID context are highlighted. [ABSTRACT FROM AUTHOR]

In order to clarify the combined acute toxic effects of the fungicide fenbuconazole and copper to zebrafish, the acute toxicity of fenbuconazole and copper alone, as well as their combination at equaltoxicity ratio or at different mass concentration ratios to zebrafish during different life stages were determined. The combined acute toxic effects were evaluated using the additional index (AI) method and co-toxicity coefficient (CTC) method. The results showed that the 96 h-LC50 values of fenbuconazole for adults, embryos, and larvae of zebrafish were 2.615, 4.121 and 4.140 mg/L, respectively, while that of copper were 0.235, 0.353, and 0.429 mg/L, respectively. The order of the acute toxicity was adult > embryo > larvae. The toxic effects of the mixtures on both zebrafish larvae and embryos showed an enhancement tendency. At equitoxicity ratio, the 96 h-LC50 of the mixed fenbuconazole and copper for larvae and embryos was 2.051 and 2.207 mg/L, respectively, and the evaluation results with AI and CTC methods were both additive; while the 96 h-LC50 of the mixture for adults was 1.755 mg/L, and the evaluation results of the two methods were different, which were antagonistic and additive effects, respectively. At different mass concentration ratios, the 96 h-LC50 values of fenbuconazole and copper mixed at 2 : 8, 5 : 5, and 8 : 2 were 0.272, 0.378 and 0.785 mg/L for embryos, respectively, which showed synergistic effects; the 96 h-LC50 values were 0.532, 0.621 and 0.934 mg/L for larvae, respectively, which showed either synergistic or additive effects. The results of the two methods were consistent for the embryos and larvae. As for the adults zebrafish, the 96 h-LC50 values were 0.434、0.653 and 1.053 mg/L, respectively, and the mixtures showed antagonistic effects except that it showed antagonistic and additive effects at 8:2 ratio with the two different methods. The results suggested that the acute toxicity of the mixture of fenbuconazole and copper was stronger to zebrafish at early life stages. This finding will help us to better evaluate the impact of contamination of fenbuconazole and copper on aquatic organisms. [ABSTRACT FROM AUTHOR]

Agriculture has gained increasing importance in response to the continuous growth of the world population and constant need for food. To avoid production losses, farmers commonly use pesticides. Mancozeb is a fungicide used in agriculture as this compound is effective in combating fungi that harm crops. However, this fungicide may also produce damage to non-target organisms present in soil and water. Therefore, this study aimed to investigate the influence of exposure to mancozeb on survival rate, locomotor activity, behavior, and oxidative status utilizing adult zebrafish (Danio rerio) as a model following exposure to environmentally relevant concentrations of this pesticide. The experimental groups were negative control, positive control, and mancozeb (0.3; 1.02; 3.47; 11.8 or 40 μg/L). Zebrafish were exposed to the respective treatments for 96 hr. Exposure to mancozeb did not markedly alter survival rate and oxidative status of Danio rerio. At a concentration of 11.8 μg/L, the fungicide initiated changes in locomotor pattern of the animals. The results obtained suggest that the presence of mancozeb in the environment might produce locomotor alterations in adult zebrafish, which subsequently disrupt the animals' innate defense mechanisms. In nature, this effect attributed to mancozeb on non-target organisms might result in adverse population impacts and ecological imbalance. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila, an opportunistic warm water pathogen, has always been a threat to aquaculture, leading to substantial economic losses. Vaccination of the cultured fish would effectively prevent Aeromoniasis, and recent advancements in nanotechnology show promise for efficacious vaccines. Oral delivery would be the most practical and convenient method of vaccine delivery in a grow-out pond. This study studied the immunogenicity and protective efficacy of a nanoparticle-loaded outer membrane protein A from A. hydrophila in the zebrafish model. The protein was over-expressed, purified, and encapsulated using poly lactic-co-glycolic acid (PLGA) nanoparticles via the double emulsion method. The PLGA nanoparticles loaded with recombinant OmpA (rOmpA) exhibited a size of 295 ± 15.1 nm, an encapsulation efficiency of 72.52%, and a polydispersity index of 0.292 ± 0.07. Scanning electron microscopy confirmed the spherical and isolated nature of the PLGA-rOmpA nanoparticles. The protective efficacy in A. hydrophila-infected zebrafish after oral administration of the nanovaccine resulted in relative percentage survival of 77.7. Gene expression studies showed significant upregulation of immune genes in the vaccinated fish. The results demonstrate the usefulness of oral administration of nanovaccine-loaded rOmpA as a potential vaccine since it induced a robust immune response and conferred adequate protection against A. hydrophila in zebrafish, Danio rerio. [ABSTRACT FROM AUTHOR]

Zebrafish are considered as model organisms in biological and medical research because of their high degree of homology with human genes. Automatic behavioral analysis of multiple zebrafish based on visual tracking is expected to improve research efficiency. However, vision-based multi-object tracking algorithms often suffer from data loss owing to mutual occlusion. In addition, simply tracking zebrafish as points is not sufficient-more detailed information, which is required for research on zebrafish behavior. In this paper, we propose Zebrafishtracker3D, which utilizes a skeleton stability strategy to reduce detection error caused by frequent overlapping of multiple zebrafish effectively and estimates zebrafish skeletons using head coordinates in the top view. Further, we transform the front- and top-view matching task into an optimization problem and propose a particle-matching method to perform 3D tracking. The robustness of the algorithm with respect to occlusion is estimated on the dataset comprising two and three zebrafish. Experimental results demonstrate that the proposed algorithm exhibits a multiple object tracking accuracy (MOTA) exceeding 90% in the top view and a 3D tracking matching accuracy exceeding 90% in the complex videos with frequent overlapping. It is noteworthy that each instance in the trace saves its skeleton. In addition, Zebrafishtracker3D is applied in the zebrafish courtship experiment, establishes the stability of the method in applications of life science, and proves that the data can be used for behavioral analysis. Zebrafishtracker3D is the first algorithm that realizes 3D skeleton tracking of multiple zebrafish simultaneously. • The first 3D multi-object zebrafish skeleton-level tracking method is proposed. • It can effectively solve the problem of data loss caused by cross occlusion. • A general method of bone spur removal based on morphology is proposed. • The algorithm has been successfully applied to life science experiments. [ABSTRACT FROM AUTHOR]

Plants are an immense source of drugs, and 50% of modern pharmacopeia has a plant origin. With increasing life expectancy in humans, many age-related degenerative diseases converge on oxidative cellular stress pathways. This provides an opportunity to develop broad treatments by targeting the cause of common pathologic cell degeneration. Toxicological effects can be readily assessed in a live animal model system to establish potential fauna for clinical use. Here, we characterized and evaluated the antioxidant potential and toxicological effects of anise myrtle (Syzygium anisatum) and lemon myrtle (Backhousia citriodora) leaves. Using zebrafish larvae, a model for high-throughput pre-clinical in vivo toxicology screening, we identified safe levels of extract exposures for development of future therapeutics. The antioxidant capacity and toxicity were very similar in these two myrtles. The LC50-96h for anise myrtle was 284 mg/L, and for lemon myrtle, it was 270 mg/L. These measurements are comparable to ongoing studies we are performing using the same criteria in zebrafish, which allow for robust testing and prioritization of natural fauna for drug development. [ABSTRACT FROM AUTHOR]

Developmental toxicity studies help to understand the impact of a pollutant on this crucial phase of the living organisms which can affect their population dynamics. Zebrafish has become an ideal model for studying environmental and embryo toxicity. The present study was carried out using zebrafish embryos for assessing the environmental toxicity of Di(2-ethylhexyl) phthalate (DEHP) which is universally considered to be an omnipresent environmental contaminant as it is the most widely used plasticizer. The embryos were exposed to DEHP for a range of five concentrations of 0.2, 20, 80, 140 and 200 μg/L for the duration of 96 hours. The treatment resulted in increased mortality and decreased hatch rate, hatchability and heartrate. It also induced teratogenic endpoints like yolk sac edema, pericardial edema and spinal deformity in the embryos which increased in dose and time dependent manner. [ABSTRACT FROM AUTHOR]

Total suspended solids (TSS) are a major contributor of anthropogenic impacts to aquatic systems. TSS exposure have been shown to affect the function of gills, but the mode of action is unclear. Zebrafish (Danio rerio) is emerging as an excellent model for mechanistic toxicology, and as there are no baseline studies on TSS effects in zebrafish gills, we tested the hypothesis that environmental concentrations of TSS damages gill structure and function in this species. Adult zebrafish were exposed to either 0, 10, 100, 500, 1000, or 2000 mg/L TSS for 4 days to assess the gill morphology. The minimal concentration that affected the gill structure was further tested for the distribution of key ion transporters, including Na+/K+- ATPase (NKA) and vacuolar-type H+-ATPase (VHA), using confocal microscopy. Our results reveal that TSS concentration as low as 100 mg/L alters the morphology of gills, including greater filament thickness, lamellae thickness, and epithelial lifting. This was also associated with a reduction in NKA immunoreactive (IR) cell count and intensity in the 100 mg/L TSS group, while there was neither a change in the VHA-IR cell count or expression nor the transcript abundance of atp6v1a and atp1a1a4 in the gills. Markers of stress response in these animals, including levels of cortisol, glucose, lactate, and glycogen were not altered after 4 days of TSS exposure. Overall, environmentally relevant concentrations of TSS can damage the gill structure and function in zebrafish and has the potential to enhance the toxicity of contaminants acting via the gills. [ABSTRACT FROM AUTHOR]

Vasoactive-intestinal peptide (Vip) is a pleiotropic peptide with a wide range of distribution and functions. Zebrafish possess 2 isoforms of Vip (a and b), in which Vipa is most homologous to the mammalian form. In female zebrafish, Vipa can stimulate LH secretion from the pituitary but is not essential for female reproduction, as vipa −/− females display normal reproduction. In contrast, we have found that vipa −/− males are severely subfertile and sex ratio of offspring is female-biased. By analyzing all aspects of male reproduction with wild-type (WT) males, we show that the testes of vipa −/− are underdeveloped and contain ∼70% less spermatids compared to WT counterparts. The sperm of vipa −/− males displayed reduced potency in terms of fertilization (by ∼80%) and motility span and duration (by ∼50%). In addition, vipa −/− male attraction to WT females was largely nonexistent, indicating decreased sexual motivation. We show that vipa mRNA and protein is present in Leydig cells and in developing germ cells in the testis of WT, raising the possibility that endogenous Vipa contributes to testicular function. Absence of Vipa in vipa −/− males resulted in downregulation of 3 key genes in the androgen synthesis chain in the testis, 3β-hsd, 17β-hsd1 , and cyp11c1 (11β-hydrogenase), associated with a pronounced decrease in 11-ketotestosterone production and, in turn, compromised reproductive fitness. Altogether, this study establishes a crucial role for Vipa in the regulation of male reproduction in zebrafish, like in mammals, with the exception that Vipa is also expressed in zebrafish testis. [ABSTRACT FROM AUTHOR]

Intracerebral hemorrhage (ICH), for which there are currently no effective preventive or treatment methods, has a very high fatality rate. Statins, such as atorvastatin (ATV), are the first‐line drugs for regulating blood lipids and treating hyperlipidemia‐related cardiovascular diseases. However, ATV‐associated ICH has been reported, although its incidence is rare. In this study, we aimed to investigate the protective action and mechanisms of berberine (BBR) against ATV‐induced brain hemorrhage. We established an ICH model in zebrafish induced by ATV (2 μM) and demonstrated the effects of BBR (10, 50, and 100 μM) on ICH via protecting the vascular network using hemocyte staining and three transgenic zebrafish. BBR was found to reduce brain inflammation and locomotion injury in ICH‐zebrafish. Mechanism research showed that ATV increased the levels of VE‐cadherin and occludin proteins but disturbed their localization at the cell membrane by abnormal phosphorylation, which decreased the number of intercellular junctions between vascular endothelial cells (VECs), disrupting the integrity of vascular walls. BBR reversed the effects of ATV by promoting autophagic degradation of phosphorylated VE‐cadherin and occludin in ATV‐induced VECs examined by co‐immunoprecipitation (co‐IP). These findings provide crucial insights into understanding the BBR mechanisms involved in the maintenance of vascular integrity and in mitigating adverse reactions to ATV. This study investigated the effect of berberine on atorvastatin‐induced cerebral hemorrhage in zebrafish. Our results indicate that BBR enhances the survival rate of zebrafish with brain hemorrhage, reduces hemorrhage and inflammation, and improves the locomotion function of ICH‐zebrafish by stabilizing vascular integrity. In vitro experiments using HUVEC cells show that BBR counteracts the adverse effects of ATV on endothelial cell connections by promoting the autophagic degradation of intracellularly phosphorylated connexins. These findings suggest the potential of BBR as a treatment for hemorrhagic stroke. [ABSTRACT FROM AUTHOR]

Isobavachalcone (IBC) is a flavonoid component of the traditional Chinese medicine Psoraleae Fructus, with a range of pharmacological properties. However, IBC causes some hepatotoxicity, and the mechanism of toxicity is unclear. The purpose of this paper was to investigate the possible mechanism of toxicity of IBC on HepG2 cells and zebrafish embryos. The results showed that exposure to IBC increased zebrafish embryo mortality and decreased hatchability. Meanwhile, IBC induced liver injury and increased expression of ALT and AST activity. Further studies showed that IBC caused the increase of ROS and MDA the decrease of CAT, GSH, and GSH‐Px; the increase of Fe2+ content; and the changes of ferroptosis related genes (acsl4, gpx4, and xct) and iron storage related genes (tf, fth, and fpn) in zebrafish embryos. Through in vitro verification, it was found that IBC also caused oxidative stress and increased Fe2+ content in HepG2 cells. IBC caused depolarization of mitochondrial membrane potential (MMP) and reduction of mitochondrial ATP, as well as altered expression of ACSl4, SLC7A11, GPX4, and FTH1 proteins. Treatment of HepG2 cells with ferrostatin‐1 could reverse the effect of IBC. Targeting the System Xc−‐GSH‐GPX4 pathway of ferroptosis and preventing oxidative stress damage might offer a theoretical foundation for practical therapy and prevention of IBC‐induced hepatotoxicity. Isobavachalcone (IBC) is a flavonoid component in the traditional Chinese medicine Psoraleae Fructus, which is widely used. However, IBC has toxicity and requires further in‐depth research. This study found that IBC was hepatotoxic to zebrafish embryos and HepG2 cells, while causing an imbalance in antioxidant capacity, an increase in Fe2+ content, and ferroptosis through the System Xc−‐ GSH‐GPX4 pathway. This provides a theoretical basis for the application and detoxification of IBC. [ABSTRACT FROM AUTHOR]

Although hyperactivity is associated with a wide variety of neurodevelopmental disorders, the early embryonic origins of locomotion have hindered investigation of pathogenesis of these debilitating behaviors. The earliest motor output in vertebrate animals is generated by clusters of early-born motor neurons (MNs) that occupy distinct regions of the spinal cord, innervating stereotyped muscle groups. Gap junction electrical synapses drive early spontaneous behavior in zebrafish, prior to the emergence of chemical neurotransmitter networks. We use a genetic model of hyperactivity to gain critical insight into the consequences of errors in motor circuit formation and function, finding that Fragile X syndrome model mutant zebrafish are hyperexcitable from the earliest phases of spontaneous behavior, show altered sensitivity to blockade of electrical gap junctions, and have increased expression of the gap junction protein Connexin 34/35. We further show that this hyperexcitable behavior can be rescued by pharmacological inhibition of electrical synapses. We also use functional imaging to examine MN and interneuron (IN) activity in early embryogenesis, finding genetic disruption of electrical gap junctions uncouples activity between mnx1+ MNs and INs. Taken together, our work highlights the importance of electrical synapses in motor development and suggests that the origins of hyperactivity in neurodevelopmental disorders may be established during the initial formation of locomotive circuits. [ABSTRACT FROM AUTHOR]

Background: Energy allocation between growth and reproduction determines puberty onset and fertility. In mammals, peripheral hormones such as leptin, insulin and ghrelin signal metabolic information to the higher centres controlling gonadotrophin-releasing hormone neurone activity. However, these observations could not be confirmed in lower vertebrates, suggesting that other factors may mediate the energetic trade-off between growth and reproduction. A bioinformatic and experimental study suggested co-regulation of the circadian clock, reproductive axis and growth-regulating genes in zebrafish. While loss-of-function of most of the identified co-regulated genes had no effect or only had mild effects on reproduction, no such information existed about the co-regulated somatostatin, well-known for its actions on growth and metabolism. Results: We show that somatostatin signalling is pivotal in regulating fecundity and metabolism. Knock-out of zebrafish somatostatin 1.1 (sst1.1) and somatostatin 1.2 (sst1.2) caused a 20–30% increase in embryonic primordial germ cells, and sst1.2−/− adults laid 40% more eggs than their wild-type siblings. The sst1.1−/− and sst1.2−/− mutants had divergent metabolic phenotypes: the former had 25% more pancreatic α-cells, were hyperglycaemic and glucose intolerant, and had increased adipocyte mass; the latter had 25% more pancreatic β-cells, improved glucose clearance and reduced adipocyte mass. Conclusions: We conclude that somatostatin signalling regulates energy metabolism and fecundity through anti-proliferative and modulatory actions on primordial germ cells, pancreatic insulin and glucagon cells and the hypothalamus. The ancient origin of the somatostatin system suggests it could act as a switch linking metabolism and reproduction across vertebrates. The results raise the possibility of applications in human and animal fertility. [ABSTRACT FROM AUTHOR]

Objective Methods Results Interpretation Intellectual disability is often the outcome of neurodevelopmental disorders and is characterized by significant impairments in intellectual and adaptive functioning. X‐linked intellectual disability (XLID) is a subset of these disorders caused by genetic defects on the X chromosome, affecting about 2 out of 1,000 males. In syndromic form, it leads to a broad range of cognitive, behavioral, ocular, and physical disabilities.Employing exome or genome sequencing, here we identified 4 missense variants (c.475C > G; p.H159D, c.1373C > A; p.T458N, and c.1585G > A; p.E529K, c.953C > T; p.S318L) and a putative truncating variant (c.1413_1414del; p.Y471*) in the SRPK3 gene in 9 XLID patients from 5 unrelated families. To validate SRPK3 as a novel XLID gene, we established a knockout (KO) model of the SRPK3 orthologue in zebrafish.The 8 patients ascertained postnatally shared common clinical features including intellectual disability, agenesis of the corpus callosum, abnormal eye movement, and ataxia. A ninth case, ascertained prenatally, had a complex structural brain phenotype. Together, these data indicate a pathological role of SRPK3 in neurodevelopmental disorders. In post‐fertilization day 5 larvae (free swimming stage), KO zebrafish exhibited severe deficits in eye movement and swim bladder inflation, mimicking uncontrolled ocular movement and physical clumsiness observed in human patients. In adult KO zebrafish, cerebellar agenesis and behavioral abnormalities were observed, recapitulating human phenotypes of cerebellar atrophy and intellectual disability.Overall, these results suggest a crucial role of SRPK3 in the pathogenesis of syndromic X‐linked intellectual disability and provide new insights into brain development, cognitive and ocular dysfunction in both humans and zebrafish. ANN NEUROL 2024 [ABSTRACT FROM AUTHOR]

The emergence of drug-resistant Mycobacterium tuberculosis (M.tb) has led to the development of novel anti-tuberculosis (anti-TB) drugs. Common methods for testing the efficacy of new drugs, including two-dimensional cell culture models or animal models, have several limitations. Therefore, an appropriate model representative of the human organism is required. Here, we developed an M.tb infection model using human lung organoids (hLOs) and demonstrated that M.tb H37Rv can infect lung epithelial cells and human macrophages (hMφs) in hLOs. This novel M.tb infection model can be cultured long-term and split several times while maintaining a similar number of M.tb H37Rv inside the hLOs. Anti-TB drugs reduced the intracellular survival of M.tb in hLOs. Notably, M.tb growth in hLOs was effectively suppressed at each passage by rifampicin and bedaquiline. Furthermore, a reduction in inflammatory cytokine production and intracellular survival of M.tb were observed upon knockdown of MFN2 and HERPUD1 (host-directed therapeutic targets for TB) in our M.tb H37Rv-infected hLO model. Thus, the incorporation of hMφs and M.tb into hLOs provides a powerful strategy for generating an M.tb infection model. This model can effectively reflect host-pathogen interactions and be utilized to test the efficacy of anti-TB drugs and host-directed therapies. Author summary: Establishment of M.tb infection model is imperative to develop new anti-TB drugs based on the pathogenesis of TB. Various animal models, including mice, rats, guinea pigs, non-human primates, rabbits, cattle, and zebrafish, are commonly used in TB research to mimic TB symptoms and study immune responses to M.tb infection. In vitro models, such as agent-based models allow examination of host-pathogen interactions, early granuloma formation and drug screening, providing cellular-level insights. However, these models may not fully represent human immunopathology owing to differences in immune cell distributions. Lung organoids mimic human lung dynamics and functions, providing crucial insights into immune responses to TB. In this study, an M.tb infection model developed using hLOs demonstrated infection of lung epithelial cells and human macrophages, reflecting host-pathogen interactions. This model is attractive for evaluating the efficacy of anti-TB drugs and host-directed therapies. [ABSTRACT FROM AUTHOR]

Many organisms exhibit social behaviors and are part of some scheme of social structure. Zebrafish are highly social, shoaling fish and therefore, social isolation may have notable impacts on their physiology and behavior. The objective of this study was to evaluate the effects of social isolation on feed intake, monoaminergic system related gene expression, and intestinal health of juvenile zebrafish fed a high-inclusion soybean meal based diet. At 20 days post-fertilization zebrafish were randomly assigned to chronic isolation (1 fish per 1.5 L tank) or social housing (6 fish per 9 L tank) with 18 tanks per treatment group (n = 18). Dividers were placed between all tanks to prevent visual cues between fish. Zebrafish were fed a commercial fishmeal based diet until 35 days post-fertilization and then fed the experimental high-inclusion soybean meal based diet until 50 days post-fertilization. At the end of the experiment (51 days post-fertilization), the mean total length, weight, and weight gain were not significantly different between treatment groups. Feed intake and feed conversion ratio were significantly higher in chronic isolation fish than in social housing fish. Expression of monoaminergic and appetite-related genes were not significantly different between groups. The chronic isolation group showed higher expression of the inflammatory gene il-1b, however, average intestinal villi width was significantly smaller and average length-to-width ratio was significantly higher in chronic isolation fish, suggesting morphological signs of inflammation were not present at the time of sampling. These results indicate that chronic isolation positively affects feed intake of juvenile zebrafish and suggest that isolation may be useful in promoting feed intake of less-palatable diets such as those based on soybean meal. [ABSTRACT FROM AUTHOR]

Gephyrin is thought to play a critical role in clustering glycine receptors at synapses within the central nervous system (CNS). The main in vivo evidence for this comes from Gephyrin (Gphn)-null mice, where glycine receptors are depleted from synaptic regions. However, these mice die at birth, possibly due to impaired molybdenum cofactor (MoCo) synthesis, an essential role Gephyrin assumes throughout an animal. This complicates the interpretation of synaptic phenotypes in Gphn-null mice and raises the question whether the synaptic and enzymatic functions of Gephyrin can be investigated separately. Here, we generated a gephyrinb zebrafish mutant, vo84, that almost entirely lacks Gephyrin staining in the spinal cord. gephyrinbvo84 mutants exhibit normal gross morphology at both larval and adult stages. In contrast to Gphn-null mice, gephyrinbvo84 mutants exhibit normal motor activity and MoCo-dependent enzyme activity. Instead, gephyrinbvo84 mutants display impaired rheotaxis and increased mortality in late development. To investigate what may mediate these defects in gephyrinbvo84 mutants, we examined the cell density of neurons and myelin in the spinal cord and found no obvious changes. Surprisingly, in gephyrinbvo84 mutants, glycine receptors are still present in the synaptic regions. However, their abundance is reduced, potentially contributing to the observed defects. These findings challenge the notion that Gephyrin is absolutely required to cluster glycine receptors at synapses and reveals a new role of Gephyrin in regulating glycine receptor abundance and rheotaxis. They also establish a powerful new model for studying the mechanisms underlying synaptic, rather than enzymatic, functions of Gephyrin. [ABSTRACT FROM AUTHOR]

Despite their young age, zebrafish larvae have a well-developed visual system and can distinguish between different visual stimuli. First, we investigated if the first visual surroundings the larvae experience during the first days after hatching shape their habitat preference. Indeed, these animals seem to "imprint" on the first surroundings they see and select visual stimuli accordingly at 7 days post fertilization (dpf). In particular, if zebrafish larvae experience a bar background just after hatching, they later on prefer bars over white stimuli, and vice versa. We then used this acquired preference for bars to investigate innate numerical abilities. We wanted to specifically test if the zebrafish larvae show real numerical abilities or if they rely on a lower-level mechanism—i.e. spatial frequency—to discriminate between two different numerosities. When we matched the spatial frequency in stimuli with different numbers of bars, the larvae reliably selected the higher numerosity. A previous study has ruled out that 7 dpf zebrafish larvae use convex hull, cumulative surface area and density to choose between two numerosities. Therefore, our results indicate that zebrafish larvae rely on real numerical abilities rather than other cues, including spatial frequency, when spontaneously comparing two sets with different numbers of bars. [ABSTRACT FROM AUTHOR]