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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]

Organisms cope with environmental fluctuations and maintain fitness in part via reversible phenotypic changes (acclimation). Aquatic animals are subject to dramatic seasonal fluctuations in water salinity, which affect osmolarity of their cells and consequently cellular function. Mechanosensory lateral line hair cells detect water motion for swimming behavior and are especially susceptible to salinity changes due to their direct contact with the environment. To maintain hair cell function when salinity decreases, neuromast (Nm)-associated ionocytes differentiate and invade lateral line neuromasts. The signals that trigger the adaptive differentiation of Nm ionocytes are unknown. We demonstrate that new Nm ionocytes are rapidly specified and selectively triggered to proliferate by low Ca2+ and Na+/Cl- levels. We further show that Nm ionocyte recruitment and induction is affected by hair cell activity. Once specified, Nm ionocyte differentiation and survival are associated with sequential activation of different Notch pathway components, a process different from other tissue-specific ionocytes. In summary, we show how environmental changes activate a signaling cascade that leads to physiological adaptation. This may prove essential for survival not only in seasonal changing environments but also in changing climates. [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]

Objective: To identify the presence of sRNA5.85 and investigate its physiological effects and regulatory mechanisms in Mycobacterium marinum (M. marinum). Methods: The presence of sRNA5.85 was validated through Northern blot analysis in M. mar- inum. Following this, the sRNA5.85 overexpression recombinant strain (referred to as the 5.85-OE strain) was constructed using the pMV261 plasmid. And M.marinum wild type strain with empty pMV261 plasmid was used as control strain (referred to as the WT-EV strain). In vitro experiments were conducted to observe the growth kinetics, sliding motility, and colony morphology of the 5.85-OE strain. To examine its behavior within human macrophages THP-1, an infection model was employed to assess the entry and intracellular proliferation of the 5.85-OE strain, with cell viability post-infection determined using LDH assays. Furthermore, a zebrafish infection model was utilized to validate the impact of sRNA5.85 on bacterial virulence. Prediction of potential target genes and interaction sites of SRNA5.85 was carried out using targetRNA2 and IntaRNA tools, with subsequent validation performed using qRT-PCR. Results: The SRNA5.85 was found to exist in the M.marinum. Overexpression of sRNA5.85 did not alter the in vitro growth, sliding ability, or colony morphology of the recombinant strain. After infecting macrophages with the 5.85-OE strain for 48 hours, THP-1 cell viability was not affected, and bacterial intracellular proliferation ability was not significantly different from the wild type strain. Overexpression of SRNA5.85 increased the bacterial load in zebrafish during long-term infection. The transcription levels of four potential target genes (MMAR 3476, MMAR_4052, MMAR_3482, MMAR_2920) predicted by targetRNA2 were significantly upregulated with sRNA5.85 overexpression. MMAR_3476 and MMAR_4052 shared the same interaction site with sRNA5.85. Conclusions: The findings reveal that sRNA5.85 enhances virulence towards zebrafish during extended infection periods by modulating the expression of low-oxygen response-related genes. This study offers crucial insights into understanding and unraveling the role of sRNA in pathogenic mycobacteria [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]

Based on recent single-cell transcriptomics and lineage tracing, sst1.1 + cell transdifferentiation and krt4 + cell differentiation have recently been recognized as the major sources of regeneration of insulin-producing cells in zebrafish. Large-scale in vivo small molecule screening has been successfully applied in zebrafish for the unbiased identification of chemicals promoting β-cell regeneration and glucose control. Multiple compounds have been validated to also promote β-cell proliferation, differentiation, or function in mammalian systems. Live imaging in zebrafish enables visualization of complex biological processes at the single-cell resolution in vivo (e.g., calcium flux in β-cells) as well as innervation and immune cell behaviors in the islet. Role of genes identified in human genetic data [e.g. genome-wide association studies (GWAS) and single-cell sequencing data] can be efficiently elucidated in zebrafish. The zebrafish has become an outstanding model for studying organ development and tissue regeneration, which is prominently leveraged for studies of pancreatic development, insulin-producing β-cells, and diabetes. Although studied for more than two decades, many aspects remain elusive and it has only recently been possible to investigate these due to technical advances in transcriptomics, chemical-genetics, genome editing, drug screening, and in vivo imaging. Here, we review recent findings on zebrafish pancreas development, β-cell regeneration, and how zebrafish can be used to provide novel insights into gene functions, disease mechanisms, and therapeutic targets in diabetes, inspiring further use of zebrafish for the development of novel therapies for diabetes. [ABSTRACT FROM AUTHOR]

Quantitative adverse outcome pathways (qAOPs) describe the response–response relationships that link the magnitude and/or duration of chemical interaction with a specific molecular target to the probability and/or severity of the resulting apical‐level toxicity of regulatory relevance. The present study developed the first qAOP for latent toxicities showing that early life exposure adversely affects health at adulthood. Specifically, a qAOP for embryonic activation of the aryl hydrocarbon receptor 2 (AHR2) of fishes by polycyclic aromatic hydrocarbons (PAHs) leading to decreased fecundity of females at adulthood was developed by building on existing qAOPs for (1) activation of the AHR leading to early life mortality in birds and fishes, and (2) inhibition of cytochrome P450 aromatase activity leading to decreased fecundity in fishes. Using zebrafish (Danio rerio) as a model species and benzo[a]pyrene as a model PAH, three linked quantitative relationships were developed: (1) plasma estrogen in adult females as a function of embryonic exposure, (2) plasma vitellogenin in adult females as a function of plasma estrogen, and (3) fecundity of adult females as a function of plasma vitellogenin. A fourth quantitative relationship was developed for early life mortality as a function of sensitivity to activation of the AHR2 in a standardized in vitro AHR transactivation assay to integrate toxic equivalence calculations that would allow prediction of effects of exposure to untested PAHs. The accuracy of the predictions from the resulting qAOP were evaluated using experimental data from zebrafish exposed as embryos to another PAH, benzo[k]fluoranthene. The qAOP developed in the present study demonstrates the potential of the AOP framework in enabling consideration of latent toxicities in quantitative ecological risk assessments and regulatory decision‐making. Environ Toxicol Chem 2024;43:2145–2156. © 2024 SETAC [ABSTRACT FROM AUTHOR]

The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen2 1-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG2 1-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG2 11 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG2 1-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications. [Display omitted] • The split-mNG2 system achieves tissue-specific protein labeling while maintaining endogenous expression levels. • mNG2 1-10 is expressed under a tissue-specific promoter using standard transgenesis. • mNG2 11 is fused to proteins of interest using CRISPR/Cas-directed gene editing. • The feasibility of the split-mNG2 approach is demonstrated for the cytoskeletal genes tubb4b and krt8. • The split-mNG2 system can also be used to direct protein localization. [ABSTRACT FROM AUTHOR]

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Paclitaxel (PCTX) is one of the most prevalently used chemotherapeutic agents. However, its use is currently beset with a host of problems: solubility issue, microplastic leaching, and drug resistance. Since drug discovery is challenging, we decided to focus on repurposing the drug itself by remedying its drawbacks and making it more effective. In this study, we have harnessed the aqueous solubility of sugars, and the high affinity of cancer cells for them, to entrap the hydrophobic PCTX within the hydrophilic shell of the carbohydrate β -cyclodextrin. We have characterized this novel drug formulation by testing its various physical and chemical parameters. Importantly, in all our in vitro assays, the conjugate performed better than the drug alone. We find that the conjugate is internalized by the cancer cells (A549) via caveolin 1-mediated endocytosis. Thereafter, it triggers apoptosis by inducing the formation of reactive oxygen species. Based on experiments on zebrafish larvae, the formulation displays lower toxicity compared to PCTX alone. Thus, our "Trojan Horse" approach, relying on minimal components and relatively faster formulation, enhances the anti-tumor potential of PCTX, while simultaneously making it more innocuous toward non-cancerous cells. The findings of this study have implications in the quest for the most cost-effective chemotherapeutic molecule. [ABSTRACT FROM AUTHOR]

Background: Alcohol use, even in moderation, causes hangover discomforts. It has been reported that alcohol-associated symptoms can be improved by either probiotics or medicinal plant extracts to some extent. However, the effects of a combination of multi-strain probiotics, prebiotic, and plant extracts have not been fully explored. Objective: Our main goal is to assess behavioral responses and alcohol metabolism in zebrafish treated with a combination of multi-strain probiotics, prebiotic, and plant extracts after ethanol administration. Methods: In this study, the zebrafish were first treated with the probiotic-prebiotic-plant extract mixture, and then was exposed to different levels of ethanol. The moving distance and activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) of zebrafish were measured after ethanol treatment. Results: This probiotic-prebiotic-plant extract mixture significantly improved total distance moved and activities of ADH and ALDH in zebrafish treated with either 1.5% or 4.0% ethanol. In particular, the mixture outperformed plant extracts (both at 250 µg/mL) in total distance moved under 1.5% ethanol administration and degrading acetaldehyde under 4.0% ethanol treatment. Conclusion: A combination of probiotics, prebiotic, and plant extracts effectively mitigated hangover symptoms in cases of moderate and heavy alcohol use and demonstrated superior performance in acetaldehyde metabolism compared to plant extracts alone after heavy drinking, based on a zebrafish model. [ABSTRACT FROM AUTHOR]

Background: Anemia is defined as a lack of erythrocytes, low hemoglobin levels, or abnormal erythrocyte morphology. Diamond‐Blackfan anemia (DBA) is a rare and severe congenital hypoplastic anemia that occurs due to the dominant inheritance of a ribosomal protein gene mutation. Even rarer is a case described as Diamond‐Blackfan anemia like (DBAL), which occurs due to a loss‐of‐function EPO mutation recessive inheritance. The effective cures for DBAL are bone marrow transfusion and treatment with erythropoiesis‐stimulating agents (ESAs). To effectively manage the condition, construction of DBAL models to identify new medical methods or screen drugs are necessary. Results: Here, an epoa‐deficient mutant zebrafish called epoaszy8 was generated to model DBAL. The epoa‐deficiency in zebrafish caused developmental defects in erythroid cells, leading to severe congenital anemia. Using the DBAL model, we validated a loss‐of‐function EPO mutation using an in vivo functional analysis and explored the ability of ESAs to alleviate congenital anemia. Conclusions: Together, our study demonstrated that epoa deficiency in zebrafish leads to a phenotype resembling DBAL. The DBAL zebrafish model was found to be beneficial for the in vivo assessment of patient‐derived EPO variants with unclear implications and for devising potential therapeutic approaches for DBAL. Key Findings: This study confirmed the potential of the epoa‐deficient zebrafish model to provide valuable insights into the study of DBAL and effects of ESAs.The epoaszy8 zebrafish model accurately portrayed the severe congenital anemia associated with DBAL and showed that ESAs like rHuEPO were able to rescue the anemia. [ABSTRACT FROM AUTHOR]

The pathological and physiological studies of Alzheimer's disease (AD) have been in-depth, and apolipoprotein E4 (ApoE4) has been proven to be highly correlated with AD, and clinical and experimental data show that ApoE4 can cause blood–brain barrier (BBB) injury, and the change of BBB permeability is an important factor affecting the development of AD. Andrographolide (Andro), as the active component of the natural plant Andrographis paniculata, has been proven to have anti-inflammatory and antioxidant effects, which have potential neuroprotective effects. To verify the protective effect of Andro on BBB in a short term, our research group used atorvastatin (Atorva)-mediated zebrafish brain injury model and the ApoE4-mediated cell co-culture model of BBB injury to explore the protective effects and mechanisms of Andro on BBB injury. Studies have shown that Andro can inhibit the activation of CypA/NF-κB/MMP-9 signaling pathway and has achieved the effect of antagonizing the inhibition of ApoE4 on intercellular tight junction proteins (occludin, claudin-5, and ZO-1). At the same time, Andro can inhibit the secretion of cell adhesion molecules (VCAM-1 and ICAM-1) in cells, thereby delaying the occurrence and progression of neuroinflammation and playing a protective role in BBB. In conclusion, Andro is a potent natural product which can protect the blood–brain barrier. [ABSTRACT FROM AUTHOR]

Traf6, an adaptor protein, exhibits non-conventional E3 ubiquitin ligase activity and was well studied as an important factor in immune systems and cancerogenesis. In mice, the traf6-null caused a perinatal death, so that the underlying pathophysiology of traf6-defeciency is still largely unclear in animals. Here, in the present study, a traf6 knockout zebrafish line (traf6−/−) was generated and could survive until adulthood, providing a unique opportunity to demonstrate the functions of traf6 gene in animals' organogenesis beyond the mouse model. The body of traf6−/− fish was found to be significantly shorter than that of the wildtype (WT). Likewise, a comparative transcriptome analysis showed that 866 transcripts were significantly altered in the traf6−/− liver, mainly involved in the immune system, metabolic pathways, and progesterone-mediated oocyte maturation. Especially, the mRNA expression of the pancreas duodenum homeobox protein 1 (pdx1), glucose-6-phosphatase (g6pcb), and the vitellogenesis genes (vtgs) were significantly decreased in the traf6−/− liver. Subsequently, the glucose was found to be accumulated in the traf6−/− liver tissues, and the meiotic germ cell was barely detected in traf6−/− testis or ovary. The findings of this study firstly implied the pivotal functions of traf6 gene in the liver and gonads' development in fish species. [ABSTRACT FROM AUTHOR]

Existing antithrombotic drugs have side effects such as bleeding, and there is an urgent need to discover antithrombotic drugs with better efficacy and fewer side effects. In this study, a zebrafish thrombosis model was used to evaluate the antithrombotic activity and mechanism of Brevianamide F, a deep-sea natural product, with transcriptome sequencing analysis, RT-qPCR analysis, and molecular docking. The results revealed that Brevianamide F significantly attenuated the degree of platelet aggregation in the thrombus model zebrafish, leading to an increase in the number of circulating platelets, an augmentation in the return of blood to the heart, an elevated heart rate, and a significant restoration of caudal blood flow velocity. Transcriptome sequencing and RT-qPCR validation revealed that Brevianamide F may exert antithrombotic effects through the modulation of the MAPK signaling pathway and the coagulation cascade reaction. Molecular docking analysis further confirmed this result. This study provides a reference for the development of therapeutic drugs for thrombosis. [ABSTRACT FROM AUTHOR]

Tin (Sn) is one of the heavy metals to which various functions in biological development are attributed in small quantities, for example, a role in cell structure, enzyme activities, and protein and carbohydrate metabolism in fish. Tributyltin (TBT) and triphenyltin (TPT) are endocrine disrupting chemicals that are widely distributed in the aquatic environment. The aim of this study was to investigate the effects of TBT, TPT, and SnCl2 on the embryonic development of zebrafish. In this study, zebrafish embryos were used to observe the acute toxicity of TBT, TPT, and SnCl2. Fish embryo toxicity analysis was performed for different TBT, TPT, and SnCl2 concentrations (1, 5, 10, 15, and 20 ng/L). Fertilized eggs in 24‐well plates (20 eggs in each concentration) were incubated at 26°C for 4 days, and embryo clotting, embryo heartbeat, and morphological abnormalities were recorded after 96 h. Coagulation increased significantly in a dose‐dependent manner, and TBT was able to induce coagulation in zebrafish embryos. Heartbeat changes significantly decreased (p < 0.05) in a dose‐dependent manner at different TBT doses (p < 0.05). The percentage of mortality was higher in embryos at trace levels of TBT, indicating that the embryos are more sensitive to the toxicity of TBT. TBT is therefore extremely toxic and can have fatal consequences for zebrafish embryos, ultimately leading to the extinction of species and a decline in biodiversity in the aquatic environment. After analyzing the images 96 h after fertilization and converting them to a teratogenic index score, it was found that the index for the two compounds TPT and TBT was 4.6 and 9.6 ng/L, respectively. [ABSTRACT FROM AUTHOR]

CHD2‐related epilepsy is characterized by early‐onset photosensitive myoclonic epilepsy with developmental delay and a high rate of pharmacoresistance. We sought to evaluate the efficacy of acetazolamide (ACZ) in CHD2‐related epilepsy, due to ACZ's unexpected efficacy in our first patient harboring a pathogenic CHD2 variant. We collected patients from different Eastern European countries with drug‐resistant CHD2‐related epilepsy who were then treated with ACZ. Patients underwent video EEG before and during ACZ treatment. In a zebrafish model of CHD2‐related epilepsy, ictal‐like events were recorded 5 days post‐fertilization after overnight ACZ exposure. Developmental delay preceded the onset of seizures in 10 of the 12 patients. Four had ataxia, and 6 exhibited autistic features. Seizures, primarily myoclonic, began at an average age of 3.4 years and were photosensitive in all 12 patients. Add‐on ACZ treatment controlled photosensitive seizures in all patients: 6 became seizure‐free, and in the remaining 6, seizure frequency decreased by over 75%. Four patients transitioned to ACZ monotherapy. The median follow‐up was 13 months. In the zebrafish model, ACZ exposure reduced ictal‐like events by 72%. ACZ, a well‐tolerated and cost‐effective medication, could be a good option for CHD2‐related epilepsy, predominantly manifesting with myoclonic seizures and photosensitivity. Plain Language Summary: Epilepsy associated with CHD2 mutations is often pharmacoresistant and associated with developmental delay and eventually ataxia. There are several generalized seizure types, including generalized tonic–clonic seizures, but the most characteristic are jerks triggered by light stimulation. We collected 12 patients who received acetazolamide, a drug usually given as a diuretic and registered as a mild antiseizure medication. All jerks triggered by light disappeared while the frequency of spontaneous seizures decreased by over 75%. Further studies are needed to confirm this promising finding and identify the mechanism by which an old compound seems to have such a specific antiseizure effect. [ABSTRACT FROM AUTHOR]

Background: Tolylfluanid is a phenylsulfamide fungicide used to mitigate enormous losses in agriculture and coastal industries. Previous studies have reported that exposure to tolylfluanid causes metabolic dysfunction, such as fat accumulation. Additionally, exposure to tolylfluanid used to be detected in strawberries at levels exceeding the maximum residue limit. However, no studies on early developmental toxicity in zebrafish models have been reported. Objectives: This study aimed to investigate the toxic effects of tolylfluanid on the early stages of zebrafish development. Results: At low tolylfluanid concentrations, survival rates, hatching rates, and yolk sac morphology remained unchanged. However, body length and eye size were slightly reduced compared to those of the control. In particular, olig2 transgenic zebrafish exposed to tolylfluanid exhibited impaired motor neuron formation, leading to reduced motor neuron length. Furthermore, tolylfluanid decreased liver size in fabp10a transgenic zebrafish larvae, inducing liver-specific red fluorescence. However, tolylfluanid exposure did not affect the vasculature of zebrafish embryos in the fli1a:eGFP transgenic model. Conclusion: Tolylfluanid disrupts organogenesis, particularly in motor neurons and the liver, during early development in zebrafish. Additionally, the results provide reference points for the concentration conditions that can be used in aquatic ecosystems. [ABSTRACT FROM AUTHOR]

Sodium hypochlorite (NaClO) has been widely utilized since the initial outbreak of coronavirus disease (COVID-19). The widespread use of NaClO means that it can directly enter aquatic ecosystems through wastewater discharge. In this study, we analyzed the expression of PPAR-γ, FAS, and ACC1, which significantly increased in larval zebrafish following exposure to 300 μg/L NaClO for 7 days. Additionally, we examined the effects of high concentrations of NaClO on zebrafish through non-targeted lipidomics and transcriptomics. A total of 44 characteristic lipid molecules were identified using non-targeted lipidomics; an absolute quantitative analysis revealed that the contents of these subclasses of lipids decreased significantly following exposure to 300 μg/L NaClO for 7 days. The levels of triglyceride (TG), phosphatidylethanolamines (PE), and diglyceride (DG) were particularly affected. Transcriptomic analysis revealed that exposure to 300 μg/L NaClO could significantly disrupt global gene transcription in larval zebrafish. Interestingly, more than 700 differentially expressed genes (DEGs) were identified, primarily associated with lipid metabolism and glycometabolism pathways. Overall, our study provided new insights into the toxicological effects of chlorine-containing disinfectants in aquatic organisms. [ABSTRACT FROM AUTHOR]

Microglia are specialized macrophages responsible for the clearance of dead neurons and pathogens by phagocytosis and degradation. The degradation requires phagosome maturation and acidification provided by the vesicular- or vacuolar-type H+-translocating adenosine triphosphatase (V-ATPase), which is composed of the cytoplasmic V1 domain and the membrane-embedded Vo domain. The V-ATPase a subunit, an integral part of the Vo domain, has four isoforms in mammals. The functions of different isoforms on phagosome maturation in different cells/species remain controversial. Here we show that mutations of both the V-ATPase Atp6v0a1 and Tcirg1b/Atp6v0a3 subunits lead to the accumulation of phagosomes in zebrafish microglia. However, their mechanisms are different. The V-ATPase Atp6v0a1 subunit is mainly distributed in early and late phagosomes. Defects of this subunit lead to a defective transition from early phagosomes to late phagosomes. In contrast, The V-ATPase Tcirg1b/Atp6v0a3 subunit is primarily located on lysosomes and regulates late phagosome-lysosomal fusion. Defective Tcirg1b/Atp6v0a3, but not Atp6v0a1 subunit leads to reduced acidification and impaired macroautophagy/autophagy in microglia. We further showed that ATP6V0A1/a1 and TCIRG1/a3 subunits in mouse macrophages preferentially located in endosomes and lysosomes, respectively. Blocking these subunits disrupted early-to-late endosome transition and endosome-to-lysosome fusion, respectively. Taken together, our results highlight the essential and conserved roles played by different V-ATPase subunits in multiple steps of phagocytosis and endocytosis across various species. Abbrevations: Apoe: apolipoprotein E; ANXA5/annexin V: annexin A5; ATP6V0A1/a1: ATPase H+-transporting V0 subunit a1; ATP6V0A2/a2: ATPase H+-transporting V0 subunit a2; ATP6V0A4/a4: ATPase H+-transporting V0 subunit a4; dpf: days post-fertilization; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; LAMP1: lysosomal associated membrane protein 1; Lcp1: lymphocyte cytosolic protein 1 (L-plastin); Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NR: neutral red; PBS: phosphate-buffered saline; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol (3,5)-bisphosphate; RAB4: RAB4, member RAS oncogene family; RAB5: RAB5, member RAS oncogene family; RAB7: RAB7, member RAS oncogene family; TCIRG1/Atp6v0a3/a3: T cell immune regulator 1, ATPase H+-transporting V0 subunit a3; V-ATPase: vacuolar-type H+-translocating adenosine triphosphatase; Xla.Tubb2b/NBT: tubulin beta 2B class IIb. [ABSTRACT FROM AUTHOR]

Background: Zebrafish have become a key model organism in neuroscience research because of their unique advantages. Their genetic, anatomical, and physiological similarities to humans, coupled with their rapid development and transparent embryos, make them an excellent tool for investigating various aspects of neurobiology. They have specifically emerged as a valuable and versatile model organism in biomedical research, including the study of neurological disorders such as multiple sclerosis. Multiple sclerosis is a chronic autoimmune disease known to cause damage to the myelin sheath that protects the nerves in the brain and spinal cord. Objective: This review emphasizes the importance of continued research in both in vitro and in vivo models to advance our understanding of MS and develop effective treatments, ultimately improving the quality of life for those affected by this debilitating disease. Conclusions: Recent studies show the significance of zebrafish as a model organism for investigating demyelination and remyelination processes, providing new insights into MS pathology and potential therapies. [ABSTRACT FROM AUTHOR]