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Dravet syndrome is a severe genetic epilepsy primarily caused by de novo mutations in a voltage-activated sodium channel gene (SCN1A). Patients face life-threatening seizures that are largely resistant to available anti-seizure medications. Preclinical Dravet syndrome animal models are a valuable tool to identify candidate anti-seizure medications for these patients. Among these, scn1lab mutant zebrafish, exhibiting spontaneous seizure-like activity, are particularly amenable to large-scale drug screening. Thus far, we have screened more than 3000 drug candidates in scn1lab zebrafish mutants, identifying valproate, stiripentol, and fenfluramine e.g. Food and Drug Administration-approved drugs, with clinical application in the Dravet syndrome population. Successful phenotypic screening in scn1lab mutant zebrafish is rigorous and consists of two stages: (i) a locomotion-based assay measuring high-velocity convulsive swim behaviour and (ii) an electrophysiology-based assay, using in vivo local field potential recordings, to quantify electrographic seizure-like events. Historically, nearly 90% of drug candidates fail during translation from preclinical models to the clinic. With such a high failure rate, it becomes necessary to address issues of replication and false positive identification. Leveraging our scn1lab zebrafish assays is one approach to address these problems. Here, we curated a list of nine anti-seizure drug candidates recently identified by other groups using preclinical Dravet syndrome models: 1-Ethyl-2-benzimidazolinone, AA43279, chlorzoxazone, donepezil, lisuride, mifepristone, pargyline, soticlestat and vorinostat. First-stage locomotion-based assays in scn1lab mutant zebrafish identified only 1-Ethyl-2-benzimidazolinone, chlorzoxazone and lisuride. However, second-stage local field potential recording assays did not show significant suppression of spontaneous electrographic seizure activity for any of the nine anti-seizure drug candidates. Surprisingly, soticlestat induced frank electrographic seizure-like discharges in wild-type control zebrafish. Taken together, our results failed to replicate clear anti-seizure efficacy for these drug candidates highlighting a necessity for strict scientific standards in preclinical identification of anti-seizure medications.

Many nutrients found in saltwater fish are thought to promote amelogenesis in ameloblast cells during tooth development. The aim of the study is to determine the effect of LC50 toxicity on saltwater fish powder on zebrafish embryos. The experimental research method was a post-test-only control group design, consisting of eight groups, namely the negative control and the internal group using embryo media, the positive control using 3,4-dichloroanillin, and the treatment group 125 µg/ml, 250 µg/ml, 500 µg/ml, 1000 µg/ml, 2000 µg/ml, and 4000 µg/ml using 384 zebrafish embryos. Saltwater fish powder is diluted and observed for 96 hours, controlled every 24 hours. There were no dead zebrafish embryos from various concentrations, negative control, and internal control. The toxicity test of saltwater fish powder from the lowest concentration of 125 µg/ml to the highest concentration of 4000 µg/ml showed no signs of zebrafish embryo mortality nor developmental abnormalities. It can be concluded that saltwater fish powder is non-toxic. [ABSTRACT FROM AUTHOR]

Oral bacteria naturally secrete extracellular vesicles (EVs), which have attracted attention for their promising biomedical applications including cancer therapeutics. However, our understanding of EV impact on tumor progression is hampered by limited in vivo models. In this study, we propose a facile in vivo platform for assessing the effect of EVs isolated from different bacterial strains on oral cancer growth and dissemination using the larval zebrafish model. EVs were isolated from: wild-type Aggregatibacter actinomycetemcomitans and its mutant strains lacking the cytolethal distending toxin (CDT) or lipopolysaccharide (LPS) O-antigen; and wild-type Porphyromonas gingivalis. Cancer cells pretreated with EVs were xenotransplanted into zebrafish larvae, wherein tumor growth and metastasis were screened. We further assessed the preferential sites for the metastatic foci development. Interestingly, EVs from the CDT-lacking A. actinomycetemcomitans resulted in an increased tumor growth, whereas EVs lacking the lipopolysaccharide O-antigen reduced the metastasis rate. P. gingivalis-derived EVs showed no significant effects. Cancer cells pretreated with EVs from the mutant A. actinomycetemcomitans strains tended to metastasize less often to the head and tail compared to the controls. In sum, the proposed approach provided cost- and labor-effective yet efficient model for studying bacterial EVs in oral carcinogenesis, which can be easily extended for other cancer types. Furthermore, our results support the notion that these nanosized particles may represent promising targets in cancer therapeutics. [ABSTRACT FROM AUTHOR]

Dissolved organic matter (DOM) occurs ubiquitously in various water matrices and affects the chemical speciation and toxicity of emerging contaminants, such as cyclophosphamide (CP). However, the effects of CP in aquatic organisms with the presence of DOM have been relatively less addressed. In this study, zebrafish eggs < 4 h post fertilization (hpf) were exposed to CP (0 and 50 μg/L) and humic acid (HA, a main component of DOM, 0, 3, 10, and 30 mg-C/L) until 7 days post fertilization, and its toxicity was evaluated by behavioral approaches and transcription of nervous-related genes. An increase in swimming velocity and anxiety was noticed in zebrafish larvae exposed to CP. The related genes of neurotransmitter (drd1, mao, thp1b, and gad2), neurodevelopment (gli2b, nrd, and gfap), and neuroinflammation (thfα, casp3, and il-6) were upregulated by CP. In the presence of HA (3 mg-C/L), the behaviors and gene transcripts of zebrafish larvae were enhanced, while at 10 mg-C/L, they were mitigated. This study has demonstrated that DOM at low concentration increases the toxicity of CP and at high concentration alleviates its toxicity. This study highlights the importance of emerging contaminant exposure with the presence of DOM on their toxicities in aquatic organisms. [ABSTRACT FROM AUTHOR]

Hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG) are protoxins produced by Sapindaceae plants, particularly Acer pseudoplatanus, and are responsible for causing atypical myopathy (AM) in equids. These protoxins metabolise into toxic compounds, such as methylenecyclopropylacetyl-CoA (MCPA-CoA), which alters energy metabolism and induces severe rhabdomyolysis. Currently, no specific treatment exists for this poisoning, in vitro models fail to reproduce HGA's toxic effects on equine primary myoblasts, and mammalian models are impractical for large-scale drug screening. This study aimed to develop a zebrafish embryo model for screening therapeutic compounds against AM. Zebrafish embryos were exposed to various concentrations of HGA, MCPrG, and methylenecyclopropylacetate (MCPA) for 72 h. MCPrG did not induce toxicity, while HGA and MCPA showed median lethal concentration (LC50) values of 1.7 µM and 1 µM after 72 h, respectively. The highest levels of the conjugated metabolite MCPA–carnitine were detected 24 h after HGA exposure, and the acylcarnitines profile was highly increased 48 h post-exposure. Isovaleryl-/2- methylbutyrylcarnitine levels notably rose after 24 h, suggesting potential exposition biomarkers. Glycine and carnitine effectively reduced mortality, whereas riboflavin showed no protective effect. These findings suggest that the zebrafish embryo represents a valuable model for identifying therapeutic compounds for Sapindaceae poisoning. [ABSTRACT FROM AUTHOR]

2-Mercaptomethylbenzo[d]imidazole (2-MMBI) derivatives were designed and synthesized as tyrosinase (TYR) chelators using 2-mercaptomethylimidazole scaffolds. Seven of the ten 2-MMBI derivatives exhibited stronger inhibition of mushroom TYR activity than kojic acid. Their ability to chelate copper ions was demonstrated through experiments using the copper chelator pyrocatechol violet and assays measuring TYR activity in the presence or absence of exogenous CuSO4. The inhibition mechanisms of derivatives 1, 3, 8, and 9, which showed excellent TYR inhibitory activity, were elucidated through kinetic studies and supported by the docking simulation results. Derivatives 3, 7, 8, and 10 significantly inhibited cellular TYR activity and melanin production in B16F10 cells in a dose-dependent manner, with stronger potency than kojic acid. Furthermore, in situ, derivatives 7 and 10 showed stronger inhibitory effects on B16F10 cell TYR activity than kojic acid. Six derivatives, including 8, showed highly potent depigmentation in zebrafish larvae, outpacing kojic acid even at 200–670 times lower concentrations. Additionally, all derivatives could scavenge for reactive oxygen species without causing cytotoxicity in epidermal cells. These results suggested that 2-MMBI derivatives are promising anti-melanogenic agents. [ABSTRACT FROM AUTHOR]

Kynurenic acid (KYNA) is a metabolite of tryptophan formed on the kynurenine pathway. Its pharmacological effects are relatively well characterized in mammals, whereas its role in fish is poorly understood. Therefore, the aim of the study was to expand the knowledge of KYNA's presence inside a fish's body and its impact on fish development and function. The study was performed on zebrafish larvae and adult rainbow trout. We provide evidence that KYNA is present in the embryo, larva and mature fish and that its distribution in organs varies considerably. A study of KYNA's effect on early larval development suggests that it can accelerate larval maturation, especially under conditions that are suboptimal for fish growth. Moreover, KYNA in concentrations over 1 mM caused morphological impairment and death of larvae. However, long-lasting exposure of larvae to subtoxic concentrations of KYNA does not affect the behavior of 5-day-old larvae kept under standard optimal conditions. We also show that ingestion of KYNA-supplemented feed can lead to KYNA accumulation, particularly in the pyloric caeca of mature trout. These results shed new light on the relevance of KYNA and provide new impulse for further research on the importance of the kynurenine pathway in fish. [ABSTRACT FROM AUTHOR]

Inspired by the potent tyrosinase inhibitory activity of phenolic compounds with a 2-phenylbenzo[d]thiazole scaffold, we explored phenolic compounds 1–15 with 2-phenylbenzo[d]oxazole, which is isosterically related to 2-phenylbenzo[d]thiazole, as novel tyrosinase inhibitors. Among these, compounds 3, 8, and 13, featuring a resorcinol structure, exhibited significantly stronger mushroom tyrosinase inhibition than kojic acid, with compound 3 showing a nanomolar IC50 value of 0.51 μM. These results suggest that resorcinol plays an important role in tyrosinase inhibition. Kinetic studies using Lineweaver–Burk plots demonstrated the inhibition mechanisms of compounds 3, 8, and 13, while docking simulation results indicated that the resorcinol structure contributed to tyrosinase binding through hydrophobic and hydrogen bonding interactions. Additionally, these compounds effectively inhibited tyrosinase activity and melanin production in B16F10 cells and inhibited B16F10 tyrosinase activity in situ in a concentration-dependent manner. As these compounds showed no cytotoxicity to epidermal cells, melanocytes, or keratinocytes, they are appropriate for skin applications. Compounds 8 and 13 demonstrated substantially higher depigmentation effects on zebrafish larvae than kojic acid, even at 800- and 400-times lower concentrations than kojic acid, respectively. These findings suggest that 2-phenylbenzo[d]oxazole is a promising candidate for tyrosinase inhibition. [ABSTRACT FROM AUTHOR]

Nutritional programming is a promising concept for promoting metabolic adaptation of fish to challenging conditions, such as the increase in water temperature. The present work evaluates in ovo arginine or glutamine supplementation as enhancers of zebrafish metabolic or absorptive capacity, respectively, at optimum (28 ºC) and challenging temperatures (32 ºC) in the long-term. Growth performance, free amino acids profile, methylation index and the activity levels of digestive and intermediary metabolism enzymes were analysed to assess the metabolic plasticity induced by an early nutritional intervention. Temperature affected fish larvae growth performance. At the end of the experimental period 28 ºC-fish showed higher dry weight than 32 ºC-fish. The effects of the early supplementation were reflected in the larval free amino acids profile at the end of the experiment. Higher methylation potential was observed in the ARG-fish. In ovo amino acid supplementation modulated the metabolic response in zebrafish larvae, however, the magnitude of this effect differed according to the amino acid and the temperature. Overall, arginine supplementation enhanced carbohydrates metabolism at 32 ºC. In conclusion, the present work suggests that in ovo arginine supplementation may promote a better adaptive response to higher temperatures. [ABSTRACT FROM AUTHOR]

Pseudomonasaeruginosa (P. aeruginosa), a leading cause of hospital-acquired infections, is becoming increasingly difficult to treat due to its alarming ability to develop antibiotic resistance. Camphene (CAM), a monoterpene found in conifers and cannabis, is reported to have anti-inflammatory and antioxidant properties. Our study focuses on the synthesis of camphene conjugated with zinc oxide nanoparticles (CAM-ZnO NPs) and evaluating its bactericidal ability against P. aeruginosa. Our in vitro 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays showed that CAM-ZnO NPs possess significant antioxidant capabilities. Moreover, in vivo studies on zebrafish larvae exhibit an increase in the antioxidant enzyme levels, such as superoxide dismutase (SOD) and catalase (CAT), confirmed by the real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Furthermore, the infected zebrafish larvae showed a reduced number of localized macrophages, indicating a reduced inflammatory response at the site of injury. In conclusion, the results of our study support the CAM-ZnO NP's potential to treat P. aeruginosa infections. [ABSTRACT FROM AUTHOR]

Neurodegenerative diseases associated with gut dysbiosis are an important health concern, particularly reactive oxygen species (ROS) generation playing a critical role. However, therapeutic methods are hindered by the inadequate absorption of plant secondary metabolites into the brain. In our study, rutin trihydrate (RTH), a flavonoid, was used to synthesise rutin trihydrate-conjugated zinc oxide nanoparticles (RTH-ZnO) for addressing gut dysbiosis-generated ROS and its implications on neurodegeneration. We tested its antimicrobial properties against gut dysbiosis-associated pathogens and antioxidant activity against oxidative stress-induced zebrafish larvae. Our in vitro results indicated that the synthesised RTH-ZnO can efficiently scavenge free radicals and maintain redox homeostasis. The zebrafish developmental toxicity analysis does not reveal any lethal malformation in the RTH-ZnO-treated group with an increased survival rate. RTH-ZnO potentially regulated the intercellular reactive oxygen species (ROS) and stress-induced apoptosis in the larvae exposed to H2O2. Notably, cognitive functions were also improved with increased acetylcholinesterase (AChE) and antioxidant gene expression. In conclusion, our study supports the potential of RTH-ZnO nanoparticles to treat gut dysbiosis-generated ROS-induced neurodegeneration. [ABSTRACT FROM AUTHOR]

The zebrafish larvae/embryo model has been shown to support the replication of seven strains (G1.7[P7], GII.2[P16], GII.3[P16], GII.4[P4], GII.4[P16], GII.6[P7], and GII.17[P13]) of human norovirus (HuNoV). However, due to challenges in consistently obtaining HuNoV-positive stool samples from clinical sources, evaluating HuNoV surrogates in this model is highly valuable. This study assesses the potential of zebrafish embryos and larvae as a model for Tulane virus (TuV) replication. Three infection methods were examined: microinjection, immersion, and feeding. Droplet digital PCR was used to quantify viral RNA across all three infection methods. Microinjection of 3 nL of TuV into zebrafish embryos (< 6-h post-fertilization) resulted in significant replication, with viral RNA levels reaching 6.22 logs at 4-day post-infection. In contrast, the immersion method showed no replication after immersing 4-day post-fertilization (dpf) larvae in TuV suspension for 6 h. Similarly, no replication was observed with the feeding method, where Paramecium caudatum loaded with TuV were fed to 4 dpf larvae. The findings indicate that the zebrafish embryo model supports TuV replication through the microinjection method, suggesting that TuV may serve as a useful surrogate for studying HuNoV pathogenesis. Additionally, TuV can be utilized in place of HuNoV in method optimization studies using the zebrafish embryo model, circumventing the limited availability of HuNoV. [ABSTRACT FROM AUTHOR]

Diabetic mellitus (DM) is characterized by hyperglycaemia and defective macromolecular metabolism, arising from insulin resistance or lack of insulin production. The present study investigates the potential of artemisinin, a sesquiterpene lactone isolated from Artemisia annua, to exert anti-diabetic and antioxidant effects through modulation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signalling pathway. Our computational analyses demonstrated a high binding affinity of artemisinin with proteins belonging to the PI3K/AKT signalling cascade. α-Amylase and α-glucosidase studies revealed a notable increase in inhibition percentages with artemisinin treatment across concentrations ranging from 10 to 160 µM. A similar significant (p < 0.05) dose-dependent inhibition of free radicals was observed for the in vitro anti-oxidant assays. Further, toxicological profiling of artemisinin in the in vivo zebrafish embryo-larvae model from 4 to 96 h post-fertilization (hpf) did not exhibit any harmful repercussions. In addition, gene expression investigations confirmed artemisinin's potential mechanism in modulating hyperglycaemia and oxidative stress through the regulation of the PI3K/AKT pathway. Overall, our investigation suggests that artemisinin can be used as a therapeutic intervention for diabetes and oxidative stress, opening up opportunities for future investigation in clinical settings. [ABSTRACT FROM AUTHOR]

The effects of stress during early vertebrate development can be especially harmful. Avoiding stressors in fish larvae is essential to ensure the health of adult fish and their reproductive performance and overall production. We examined the consequences of direct exposure to successive acute stressors during early development, including their effects on miR-29a and its targets, survival, hatching and malformation rates, larval behaviour and cartilage and eye development. Our aim was to shed light on the pleiotropic effects of early-induced stress in this vertebrate model species. Our results showed that direct exposure to successive acute stressors during early development significantly upregulated miR-29a and downregulated essential collagen transcripts col2a1a, col6a2 and col11a1a, decreased survival and increased malformation rates (swim bladder, otoliths, cardiac oedema and ocular malformations), promoting higher rates of immobility in larvae. Our results revealed that stress in early stages can induce different eye tissular architecture and cranioencephalic cartilage development alterations. Our research contributes to the understanding of the impact of stressful conditions during the early stages of zebrafish development, serving as a valuable model for vertebrate research. This holds paramount significance in the fields of developmental biology and aquaculture and also highlights miR-29a as a potential molecular marker for assessing novel larval rearing programmes in teleost species. [ABSTRACT FROM AUTHOR]

Microcystin-LR (MCLR) poses a significant threat to aquatic ecosystems and public health. This study investigated the protective effects of the probiotic Lactobacillus rhamnosus against MCLR-induced developmental toxicity in zebrafish larvae. Zebrafish larvae were exposed to various concentrations of MCLR (0, 0.9, 1.8, and 3.6 mg/L) with or without L. rhamnosus from 72 to 168 h post-fertilization (hpf). Probiotic supplementation significantly improved survival, hatching, and growth rates and reduced malformation rates in MCLR-exposed larvae. L. rhamnosus alleviated MCLR-induced oxidative stress by reducing reactive oxygen species (ROS) levels and enhancing glutathione (GSH) content and catalase (CAT) activity. Probiotics also mitigated MCLR-induced lipid metabolism disorders by regulating key metabolites (triglycerides, cholesterol, bile acids, and free fatty acids) and gene expression (ppara, pparb, srebp1, and nr1h4). Moreover, 16S rRNA sequencing revealed that L. rhamnosus modulated the gut microbiome structure and diversity in MCLR-exposed larvae, promoting beneficial genera like Shewanella and Enterobacter and inhibiting potential pathogens like Vibrio. Significant correlations were found between gut microbiota composition and host antioxidant and lipid metabolism parameters. These findings suggest that L. rhamnosus exerts protective effects against MCLR toxicity in zebrafish larvae by alleviating oxidative stress, regulating lipid metabolism, and modulating the gut microbiome, providing insights into probiotic-based strategies for mitigating MCLR toxicity in aquatic organisms. [ABSTRACT FROM AUTHOR]

This study investigated the effects of environmental changes on zebrafish larval behavior, using single-factor and orthogonal experiments to assess locomotion during temperature and pH changes. In single-factor experiments, zebrafish larvae were exposed to variations in temperature (22 to 30 °C) and pH levels (6.0, 7.0, 9.0). The simultaneous temperature and pH changes were investigated by orthogonal tests. In both experiments, each zebrafish larva was recorded in three 5 min videos at different stages (before exposure, during short-term exposure (10 min), and after long-term exposure (60 min)). You Look Only Once (YOLOv5) and Deep Simple Online Real Time Tracking (DeepSORT) models were adopted to develop a zebrafish larva tracking system, and YOLOv5 was improved in two aspects of anchor clustering and network structure. The tracking accuracy of the tracking system for small targets effectively improved, reaching more than 98% MOTA (Multiple Object Tracking Accuracy). Principal Component Analysis (PCA) was employed to extract three behavioral features from 13 motion parameters, namely motion activity, edge behavior, and motion direction preference. Our findings reveal that lower temperatures and acidic conditions both led to a decrease in motion behavioral activity, and the former also increased edge behavior. Conversely, elevated temperatures and alkaline conditions had a muted impact on these behaviors. Interestingly, concurrent changes in temperature and pH significantly altered directional preference. Additionally, we observed that lower temperatures elicited distinct temporal behavioral patterns at a constant pH level. In summary, we recommend the precise control and explicit reporting of ambient temperature and pH in both breeding devices and experimental wells to minimize the environmental impact on zebrafish behavior and enhance experiment repeatability and reliability. [ABSTRACT FROM AUTHOR]

The use of novel psychoactive substances (NPSs) has dramatically increased worldwide, and among them, synthetic opioids are one of the fastest growing groups, where cinnamylpiperazines and 2‐benzylbenzimidazoles represent two of the most relevant subclasses. However, the data on their toxicity and metabolism are still limited. The aim of the present study was to evaluate the toxicity and metabolic pathways of some compounds belonging to these families, namely, AP‐237, 2‐methyl AP‐237, isotonitazene, flunitazene, etodesnitazene, metonitazene, metodesnitazene, N‐pyrrolidino etonitazene, and butonitazene. The study was performed using a zebrafish early life stages model. In fact, zebrafish (Danio rerio) embryos and larvae have recently been recognized as a suitable animal model in alternative to mammals, because they require less time and resources and do not need complex procedures for ethics approval. The cellular toxicity after a single administration was assessed at the fourth day post‐fertilization with acridine orange staining. Possible morphological defects were evaluated with a light microscope after 24 h of exposure to 1 μmol/L concentration of each drug. Subsequently, the larvae were euthanized and underwent analysis of drug metabolites using UPLC coupled to an Orbitrap high‐resolution mass spectrometer. High rates of morphological defects, as well as of cellular death, were detected, but no significant difference in mortality between treatment and control groups was observed. In addition, several metabolites, mainly produced through monohydroxylation, N‐dealkylation, and O‐dealkylation, were identified in the larvae extracts. [ABSTRACT FROM AUTHOR]

The seahorse is a traditional Chinese herbal medicine that has been utilized for the treatment of conditions such as osteoporosis, nonunion fractures, knee osteoarthritis, and herniated lumbar disks.Zebrafish is an excellent animal model to study the effects of herbal preparations on osteogenesis and bone metabolism, and bone biology is highly similar to that of mammals, so it has been used to develop multiple glucocorticoid-induced osteoporosis (GIOP) models. Our study aimed to explore whether the decoction of Hippocampus erectus (DHe) administered in zebrafish larvae promotes bone health under physiological and osteoporotic conditions. In zebrafish larvae, 25 µM prednisolone solution was successfully used to induce the zebrafish osteoporosis model, which can significantly inhibit the mineralization area and degree of bone mineralization, reduce the activity of alkaline phosphatase, and increase tartrate-resistant acid phosphatase activity. The results showed that 0.3 g/L DHe had a significant improvement effect on osteoporosis in zebrafish by enhancing the mineralization degree and phosphatase activity of zebrafish, while also promoting the expression of genes associated with bone development. It can be considered a good candidate to develop new drugs against osteoporosis.

Recently, nanomaterials including alumina have been extensively involved in aquaculture for many purposes resulting in potential hazards in fish. However, its effect on the developing metabolic organs and lipid metabolism remains unclear. This study, investigated the effects of aluminium nanoparticles (AL-NPs) on the metabolic organs of the growing zebrafish larvae including intestine, liver, and pancreas in addition to the potential ameliorative role of rosmarinic acid (RA) against AL-NPs - induced toxicity. Briefly, fertilized zebrafish eggs were divided into 4 groups. The 1st group was kept as a control. The 2nd group was exposed to RA (0.5 mg/L). The 3rd group was exposed to AL-NPs (160 mg/L). The 4th group received both RA and AL-NPs at the same concentrations from 1st day post fertilization (dpf) till the 14th dpf. Here, AL-NPs exposed group showed the highest mortality rate, external morphology defects and gastrointestinal malformations after larval-to-juvenile transition. Interestingly, mixing RA to AL-NPs significantly reduced these effects. Additionally, AL-NPs induced intestinal epithelium defects at the histological and genetic levels and abnormal lipid retention via up-regulation of the lipogenesis-related genes and down-regulation of the hepatic fatty acid β-oxidation. However, addition of RA to AL-NPs could reverse these effects. AL-NPs also induce inflammatory response and mediated endoplasmic reticulum ER and cellular oxidative stress indicated by qRT-PCR and western blot analysis which were then be reduced by RA. Collectively, AL-NPs were highly correlated with the induction of steatohepatitis and lipogenesis after the larval-to-juvenile transition and this could be relief in the presence of RA suggesting it as a promising nutrient for fish.

The recent availability of commercial platforms for behavioral analyses in zebrafish larvae based on video-tracking technologies has exponentially increased the number of studies analyzing different behaviors in this model organism to assess neurotoxicity. Among the most commonly used assays in zebrafish larvae are basal locomotor activity (BLA) and visual motor responses (VMRs). However, the effect of different intrinsic and extrinsic factors that can significantly alter the outcome of these assays is still not well understood. In this work, we have analyzed the influence of age (5–8 days post-fertilization), time of day (8:00, 10:00, 12:00, 14:00; 16:00, 18:00, and 20:00 h), and experiment (three experiments performed at different days) on BLA and VMR results (4004 analyses for each behavior) in 143 larvae. The results from both behaviors were adjusted to a random-effects linear regression model using generalized least squares (GLSs), including in the model the effect of the three variables, the second-way interactions between them, and the three-way interaction. The results presented in this manuscript show a specific effect of all three intrinsic factors and their interactions on both behaviors, supporting the view that the most stable time period for performing these behavioral assays is from 10:00 am to 04:00 pm, with some differences depending on the age of the larva and the behavioral test. [ABSTRACT FROM AUTHOR]

Silver nanoparticles (AgNPs) have a primordial role in the nanotechnology industry since they have great antimicrobial effects against diverse pathogens. AgNP production, use, and discard have been increasing; still, there is debate about the consequences of exposure to AgNPs for animals and humans. The bioaccumulation and toxicity of AgNPs depend on several factors, including AgNP concentration, physicochemical characteristics, and aggregation. So, complete activity and toxicity profiles for each silver nanoparticle synthesized must be performed. The present work aimed to characterize the behavior of specific AgNPs, then crossover of their antimicrobial capacity against Pseudomonas aeruginosa and Staphylococcus aureus with their potential in vitro cytotoxic effects in murine fibroblasts (3T3) and human keratinocytes (HaCat) cell lines, and in vivo general and organ‐specific toxicity in zebrafish embryos and larvae. A high susceptibility of P. aeruginosa and S. aureus to low concentrations of AgNPs was observed, yet a cytotoxic effect was also detected in 3T3 and HaCat cells. Moreover, these concentrations delayed embryonic development in zebrafish embryos and presented morphological abnormalities, neurotoxicity, and cardiotoxicity in zebrafish larvae. The results highlight the importance of jointly evaluating the antimicrobial activity and the toxicological profile of each nanoparticle manufactured; this study presents a suitable platform for such crossover assessment. [ABSTRACT FROM AUTHOR]

Long-wave sensitive (LWS) is a G protein-coupled receptor expressed in the retina, and zebrafish is a better model organism for studying vision, but the role of LWS1 in vision-guided behavior of larvae fish has rarely been reported. In this study, we found that zebrafish lws1 and lws2 are tandemly replicated genes, both with six exons, with lws1 being more evolutionarily conserved. The presence of Y277F in the amino acid sequence of lws2 may have contributed to the shift of λmax to green light. We established a lws1 knockout zebrafish model using CRISPR/Cas9 technology. Lws1−/− larvae showed significantly higher levels of feeding and appetite gene (agrp) expression than WT, and significantly lower levels of anorexia gene (pomc, cart) expression. In addition, green light gene compensation was observed in lws1−/− larvae with significantly increased expression levels of rh2-1. The light-dark movement test showed that lws1−/− larvae were more active under light-dark transitions or vibrational stimuli, and the expression of phototransduction-related genes was significantly up-regulated. This study reveals the important role of lws1 gene in the regulation of vision-guided behavior in larvae. [ABSTRACT FROM AUTHOR]

BPA is so ubiquitous that 27 million tons of BPA-containing plastic, including mineral water bottles and baby bottles, is produced worldwide each year. The potential toxicity of BPA to humans and aquatic organisms has been the subject of intense research. In this study, a zebrafish model system was used to assess BPA-mediated hepatotoxicity. Zebrafish larvae at 72-144 hpf were exposed to BPA at different concentrations (0,1, 3 and 5mg/L). For example, BPA-treated zebrafish larvae showed increased mortality, delayed uptake of nutrients in yolk sac, shortened body length, smaller liver area, abnormal expression of genes related to liver development, and pathological changes in the liver tissue. Mechanistically, BPA exposure induced excessive oxidative stress in the liver of zebrafish and increased the level of hepatocyte apoptosis in zebrafish larvae, and the antioxidant astaxanthin could rescue the BPA-mediated liver toxicity. [ABSTRACT FROM AUTHOR]

Microfluidic technology is unrivaled in its ability to apply soluble chemical stimuli with high spatiotemporal precision. Analogous, light-sheet microscopy is unmatched in its ability of low phototoxic but fast volumetric in vivo imaging with single cell resolution. Due to their optical translucency during the larval stages, zebrafish (Danio rerio) are an ideal model to combine both techniques; yet, thus far this required light-sheet microscopes, which were in most cases custom-built and adapted to the available softlithographic chip technology. Our aim was to use a commercial light-sheet microscope to illuminate a microfluidic chip from two opposite lateral directions and to record images with the detection objective placed orthogonally above the chip. Deep tissue penetration can be achieved by superimposing beams from opposite directions to form a single light sheet. But a microfluidic chip that allows a) targeted stimulus application in a closed microenvironment, b) interference-free incoupling of excitation light from two directions and c) outcoupling of fluorescence in the perpendicular direction through an optically perfect cover glass was not known until now. Here, we present a monolithic glass chip with the required plane-parallel sidewalls and cover slide closure at the top, constructed by advanced femtosecond laser ablation, thermal bonding and surface smoothing processes. In addition, the 3D shape of a fish fixator unit was tailored to match the body shape of a zebrafish larva to ensure stable positioning during whole-brain recording. With hydrodynamic focusing a targeted partial exposure of the larva's head to chemical stimuli and fast position switching (in less than 10 s) was possible. With the capabilities of this unique monolithic glass chip and its up-scalable wafer-level fabrication process, the new NeuroExaminer is prone to become an excellent addition to neurobiology laboratories already equipped with high-quality commercial light sheet microscopes. [ABSTRACT FROM AUTHOR]

Epilepsy ranks as the second-most prevalent neurological disease, and is characterized by seizures resulting in neurobiological and behavioral impairment. Naturally occurring in coffee beans or tea leaves, the alkaloid caffeine (CAF) is the most prevalent global stimulant. Caffeine has been observed to influence epileptic seizures and the efficacy of antiepileptic medications, with a notable impact on topiramate (TPM). This study aimed to explore the influence of CAF on TPM's anticonvulsant effects in zebrafish larvae within a PTZ-induced seizure model, concurrently determining TPM concentrations through a sophisticated analytical approach based on ultrahigh-performance liquid chromatography and subsequent mass spectrometric detection. Zebrafish larvae four days post-fertilization were incubated for 18 h with varying doses of TPM or combinations of CAF + TPM, and locomotor activity was then assessed. Seizures were induced by introducing a PTZ solution to achieve a final concentration of 20 mM. Utilizing liquid chromatography–mass spectrometry (LC–MS/MS), TPM levels in the larvae were quantified. CAF co-administration (especially in higher doses) with TPM caused a decrease in the average locomotor activity in the larvae compared to TPM alone. Moreover, CAF decreased TPM levels in the larvae at all investigated doses. In conclusion, these findings offer a novel perspective on the interplay between CAF and TPM, shedding light on previously unexplored facets. The potential impact of CAF consumption in assisting with epileptic seizure control, unless proven otherwise, suggests a noteworthy consideration for future research and clinical practices. [ABSTRACT FROM AUTHOR]

We demonstrated the effect of Ishige okamurae extract (IOE) on the receptor activator of nuclear factor-κB ligand (RANKL)-promoted osteoclastogenesis in RAW 264.7 cells and confirmed that IOE inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation. IOE inhibited protein expression of TRAP, metallopeptidase-9 (MMP-9), the calcitonin receptor (CTR), and cathepsin K (CTK). IOE treatment suppressed the expression of activated T cell cytoplasmic 1 and activator protein-1, thus controlling the expression of osteoclast-related factors. Moreover, IOE significantly reduced RANKL-phosphorylated extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). It also reduced the RANKL-induced phosphorylation of NF-κB and nuclear translocation of p65. IOE inhibited Dex-induced bone loss and osteoclast-related gene expression in zebrafish larvae. HPLC analysis shows that IOE consists of 3.13% and 3.42% DPHC and IPA, respectively. Our results show that IOE has inhibitory effects on osteoclastogenesis in vitro and in vivo and is a potential therapeutic for osteoporosis. [ABSTRACT FROM AUTHOR]

Obesity is a disease involving mechanisms of fat accumulation, low-grade inflammatory cytokine release, and mitochondrial dysfunction. The aim of the current study was to investigate the effects of abalone intestine extract on fat metabolism in 3T3-L1 adipocytes and high fat diet-induced zebrafish larvae. The total phenol content was highest in subcritical water extract at 210°C (SW210) among hot water, ethanol, and subcritical water extracts of abalone intestine. In addition, SW210 of male abalone intestine (MASW210) most effectively controlled the lipid accumulation and expression of adipogenic or lipogenic regulators (PPAR-γ, C/EBPα, SREBP-1c, and FAS) in 3T3-L1 adipocytes. Likewise, in zebrafish larvae fed high fat, MASW210 significantly suppressed body weight, glucose levels, and lipid accumulation. The mRNA expression related to adipogenesis (PPAR-γ and C/EBPα), lipogenesis (SREBP-1c and FAS), inflammatory cytokines (TNF-α and IL-6), energy m/;.etabolism (AMPK, lepr, SIRT1, and adiponectin), and mitochondrial biogenesis (PGC-1α and CPT-1) were significantly regulated by treatment with MASW210. These results suggest that abalone intestine extract such as MASW210, are useful biomaterials for improving obesity and metabolic diseases.

Background and aim: Insulin resistance (IR) is a pathological condition in which cells fail to respond normally to insulin. Loss of insulin sensitivity disrupts glucose homeostasis and elevates the risk of developing the metabolic syndrome that includes Type 2 diabetes. This study assesses the effect on subcritical-water extract of Gracilaria chorda (GC) at 210 °C (GCSW210) in IR induction models of high glucose (HG)-induced zebrafish larvae and dexamethasone (DEX)-induced L6 myotubes. Experimental procedure: The dose of HG and DEX for IR induction in zebrafish larvae and L6 myotubes was 130 mM or 0.5 μM. The capacity of glucose uptake was quantified by fluorescence staining or intensity. In addition, the activation of protein and mRNA expressions for insulin signaling (insulin-dependent or independent pathways) was measured. Results and conclusion: Exposure of zebrafish larvae to HG significantly reduced the intracellular glucose uptake with dose-dependnet manner compared to control. However, the group treated with GCSW210 significantly averted HG levels like the insulin-treated group, and significantly up- or down-regulated the mRNA expressions related to insulin production (insα) and insulin signaling pathways. Moreover, the treatment with GCSW210 effectively regulated the protein expression of PI3K/AKT, AMPK, and GLUT4 involved in the action of insulin in IR models of L6 myotubes compared to DEX-treated control. Our data indicate that GCSW210 stimulates activation of PI3K/AKT and AMPK pathways to attenuate the development of IR induced by HG in zebrafish and DEX in L6 myotubes. In conclusion, GCSW210 is a potential agent for alleviating various diseases associated with the insulin resistance.

Hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG) are protoxins produced by Sapindaceae plants, particularly Acer pseudoplatanus, and are responsible for causing atypical myopathy (AM) in equids. These protoxins metabolise into toxic compounds, such as methylenecyclopropylacetyl-CoA (MCPA-CoA), which alters energy metabolism and induces severe rhabdomyolysis. Currently, no specific treatment exists for this poisoning, in vitro models fail to reproduce HGA’s toxic effects on equine primary myoblasts, and mammalian models are impractical for large-scale drug screening. This study aimed to develop a zebrafish embryo model for screening therapeutic compounds against AM. Zebrafish embryos were exposed to various concentrations of HGA, MCPrG, and methylenecyclopropylacetate (MCPA) for 72 h. MCPrG did not induce toxicity, while HGA and MCPA showed median lethal concentration (LC50) values of 1.7 µM and 1 µM after 72 h, respectively. The highest levels of the conjugated metabolite MCPA–carnitine were detected 24 h after HGA exposure, and the acylcarnitines profile was highly increased 48 h post-exposure. Isovaleryl-/2- methylbutyrylcarnitine levels notably rose after 24 h, suggesting potential exposition biomarkers. Glycine and carnitine effectively reduced mortality, whereas riboflavin showed no protective effect. These findings suggest that the zebrafish embryo represents a valuable model for identifying therapeutic compounds for Sapindaceae poisoning.

2-Mercaptomethylbenzo[d]imidazole (2-MMBI) derivatives were designed and synthesized as tyrosinase (TYR) chelators using 2-mercaptomethylimidazole scaffolds. Seven of the ten 2-MMBI derivatives exhibited stronger inhibition of mushroom TYR activity than kojic acid. Their ability to chelate copper ions was demonstrated through experiments using the copper chelator pyrocatechol violet and assays measuring TYR activity in the presence or absence of exogenous CuSO4. The inhibition mechanisms of derivatives 1, 3, 8, and 9, which showed excellent TYR inhibitory activity, were elucidated through kinetic studies and supported by the docking simulation results. Derivatives 3, 7, 8, and 10 significantly inhibited cellular TYR activity and melanin production in B16F10 cells in a dose-dependent manner, with stronger potency than kojic acid. Furthermore, in situ, derivatives 7 and 10 showed stronger inhibitory effects on B16F10 cell TYR activity than kojic acid. Six derivatives, including 8, showed highly potent depigmentation in zebrafish larvae, outpacing kojic acid even at 200–670 times lower concentrations. Additionally, all derivatives could scavenge for reactive oxygen species without causing cytotoxicity in epidermal cells. These results suggested that 2-MMBI derivatives are promising anti-melanogenic agents.

Kynurenic acid (KYNA) is a metabolite of tryptophan formed on the kynurenine pathway. Its pharmacological effects are relatively well characterized in mammals, whereas its role in fish is poorly understood. Therefore, the aim of the study was to expand the knowledge of KYNA’s presence inside a fish’s body and its impact on fish development and function. The study was performed on zebrafish larvae and adult rainbow trout. We provide evidence that KYNA is present in the embryo, larva and mature fish and that its distribution in organs varies considerably. A study of KYNA’s effect on early larval development suggests that it can accelerate larval maturation, especially under conditions that are suboptimal for fish growth. Moreover, KYNA in concentrations over 1 mM caused morphological impairment and death of larvae. However, long-lasting exposure of larvae to subtoxic concentrations of KYNA does not affect the behavior of 5-day-old larvae kept under standard optimal conditions. We also show that ingestion of KYNA-supplemented feed can lead to KYNA accumulation, particularly in the pyloric caeca of mature trout. These results shed new light on the relevance of KYNA and provide new impulse for further research on the importance of the kynurenine pathway in fish.

Inspired by the potent tyrosinase inhibitory activity of phenolic compounds with a 2-phenylbenzo[d]thiazole scaffold, we explored phenolic compounds 1–15 with 2-phenylbenzo[d]oxazole, which is isosterically related to 2-phenylbenzo[d]thiazole, as novel tyrosinase inhibitors. Among these, compounds 3, 8, and 13, featuring a resorcinol structure, exhibited significantly stronger mushroom tyrosinase inhibition than kojic acid, with compound 3 showing a nanomolar IC50 value of 0.51 μM. These results suggest that resorcinol plays an important role in tyrosinase inhibition. Kinetic studies using Lineweaver–Burk plots demonstrated the inhibition mechanisms of compounds 3, 8, and 13, while docking simulation results indicated that the resorcinol structure contributed to tyrosinase binding through hydrophobic and hydrogen bonding interactions. Additionally, these compounds effectively inhibited tyrosinase activity and melanin production in B16F10 cells and inhibited B16F10 tyrosinase activity in situ in a concentration-dependent manner. As these compounds showed no cytotoxicity to epidermal cells, melanocytes, or keratinocytes, they are appropriate for skin applications. Compounds 8 and 13 demonstrated substantially higher depigmentation effects on zebrafish larvae than kojic acid, even at 800- and 400-times lower concentrations than kojic acid, respectively. These findings suggest that 2-phenylbenzo[d]oxazole is a promising candidate for tyrosinase inhibition.

Introduction: Alcohol consumption for a prolonged duration induces fatty liver disease such as steatosis. This research aims to examine the bioactive compounds in hydroalcoholic Moringa oleifera (MO) leaf extracts using GC-MS and their effects on alcohol-induced liver steatosis in zebrafish larvae. Methods: The bioactive compounds in hydroalcoholic MO leaf extracts were examined using GC-MS. Zebrafish larvae at 120 hpf were exposed to 2% ethanol, and co-treated with different concentrations of hydroalcoholic extracts, for 32 h. The larvae were then stained with oil red O for fatty tissues in the liver and then measured by using ImageJ. Results: GC-MS revealed the presence of bioactive compounds 8,14-Seco-3,19-epoxyandrostane-8,14-dione, 17-acetoxy-3á-methoxy-4,4-dimethyl-(73.72%), 1,2,3-Propatriol, 1-indol-4-yl(ether) (4.43%), D-mannose (2.17%) and quinic acid (2.10%) in hydromethanolic extract. For hydroethanolic extract, 9-Octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester (25.14%) and gamma-sitosterol (18.56%) are major constituents. Both hydromethanolic and hydroethanolic leaf extracts showed a reduction in the liver steatosis at the tested concentrations (0.1-0.3 mg/mL), with a significant reduction of 21-39.5% in liver injury as measured in fatty liver size. Conclusion: This finding indicates that hydroalcoholic MO leaf extracts reduce alcohol with a significant reduction of 21-39.5% in liver injury as measured in fatty liver size -induced liver steatosis, suggesting it could be a good supplement for alcohol-induced liver injury. [ABSTRACT FROM AUTHOR]

Microcystin-LR (MCLR) poses a significant threat to aquatic ecosystems and public health. This study investigated the protective effects of the probiotic Lactobacillus rhamnosus against MCLR-induced developmental toxicity in zebrafish larvae. Zebrafish larvae were exposed to various concentrations of MCLR (0, 0.9, 1.8, and 3.6 mg/L) with or without L. rhamnosus from 72 to 168 h post-fertilization (hpf). Probiotic supplementation significantly improved survival, hatching, and growth rates and reduced malformation rates in MCLR-exposed larvae. L. rhamnosus alleviated MCLR-induced oxidative stress by reducing reactive oxygen species (ROS) levels and enhancing glutathione (GSH) content and catalase (CAT) activity. Probiotics also mitigated MCLR-induced lipid metabolism disorders by regulating key metabolites (triglycerides, cholesterol, bile acids, and free fatty acids) and gene expression (ppara, pparb, srebp1, and nr1h4). Moreover, 16S rRNA sequencing revealed that L. rhamnosus modulated the gut microbiome structure and diversity in MCLR-exposed larvae, promoting beneficial genera like Shewanella and Enterobacter and inhibiting potential pathogens like Vibrio. Significant correlations were found between gut microbiota composition and host antioxidant and lipid metabolism parameters. These findings suggest that L. rhamnosus exerts protective effects against MCLR toxicity in zebrafish larvae by alleviating oxidative stress, regulating lipid metabolism, and modulating the gut microbiome, providing insights into probiotic-based strategies for mitigating MCLR toxicity in aquatic organisms.

Jiuzao, the residue from Baijiu production, has shown radical scavenging properties in prior investigations, suggesting its potential as a hepatoprotective agent against acute liver damage. This study reveals that Jiuzao polysaccharides ameliorated liver morphological damage in zebrafish larvae afflicted with alcoholic fatty liver disease (AFLD), as evidenced by Oil red O, H&E, and Nile red staining. These polysaccharides notably modulated antioxidant enzyme levels and lipid peroxidation components. The real-time quantitative polymerase chain reactions analyses illustrated the significant impact of Jiuzao polysaccharides on genes integral to ethanol and lipid metabolism. The 16 S rRNA results showed that Jiuzao polysaccharides could improve the intestinal flora in zebrafish larvae exposed to ethanol. In summary, Jiuzao polysaccharides efficaciously mitigate liver lipid accumulation, enhance ethanol metabolism, and reduce oxidative stress by downregulating genes involved in AFLD development. They also regulate the changes in gut microbiota, providing further protection against acute alcoholic liver insult in zebrafish larvae.

Ras-related C3 botulinum toxin substrate 1 (Rac1) is a GTPase implicated in cell migration and homing of hematopoietic cells to the hematopoietic niche, and is commonly overexpressed in acute myeloid leukemia (AML). This can lead to quiescence of leukemic blasts in the niche and reduced response to therapy. We investigated the Rac1 inhibitor EHop-016 on AML by assessing its effects on MOLM-13 cells in vitro and in zebrafish larvae, regarding cell motility and therapeutic potential in combination with daunorubicin (DNR). In vitro assessment of proliferation and viability was by measurement of 3H-thymidine incorporation and detection of Annexin V/PI positive cells. Cell motility was evaluated by measurement of migration in a transwell system. Fluorescently stained MOLM-13 cells were injected into zebrafish larvae, and individual cells followed by confocal microscopy. Cell accumulation in the caudal hematopoietic tissue (CHT) was studied using a 12-hour timelapse, while in vivo efficacy of DNR, EHop-016 or a combination was investigated over 24 h.The in vitro results showed that EHop-016 acted synergistically in combination with DNR in reducing the viability of MOLM-13 cells (Bliss synergy score above 10 %). Non-toxic concentrations of EHop-016 reduced cell migration. These findings were reproduced in zebrafish larvae: larvae receiving both DNR and EHop-016 had significantly reduced tumor burden compared to the untreated control or single treatments. The accumulation of MOLM-13 cells in the CHT was reduced in larvae receiving EHop-016 treatment.Our findings demonstrate that targeting Rac1 in AML holds promise as a complementary treatment to established chemotherapy and should be further investigated.

Zebrafish embryo-based assays are a promising alternative for animal testing to screen new compounds for developmental toxicity. However, recent studies in zebrafish embryos showed an immature intrinsic cytochrome P450 (CYP)-mediated biotransformation capacity, as most CYPs were only active at the end of the organogenesis period. Data on other phase I enzymes involved in the biotransformation of xenobiotics in zebrafish embryos is limited. This information is pivotal for proteratogens needing bioactivation to exert their teratogenic potential. Therefore, this study aimed to investigate whether carbamazepine (CBZ) and levetiracetam (LTC), two anti-epileptic drugs that require bioactivation to exert their teratogenic potential, are biotransformed into non-CYP mediated metabolites in the zebrafish embryo and whether one or more of these metabolites cause developmental toxicity in this species. In the first step, zebrafish embryos were exposed to LTC and CBZ and their non-CYP mediated human metabolites, etiracetam carboxylic acid (ECA) and 9-acridine carboxaldehyde (9ACA), acridine (AI), and acridone (AO), respectively, from 5.25 to 120 hpf and morphologically evaluated. Next, the uptake of all compounds and the formation of the metabolites were assessed using LC-MS methods. As LTC and ECA were, respectively, poorly or not taken up by zebrafish larvae during the exposure experiments, we could not determine if LTC and ECA are teratogenic. However, biotransformation of LTC into ECA was observed at 24 hpf and 120 hpf, which indicates that the special type of B-esterase is already active at 24 hpf. CBZ and its three metabolites were teratogenic, as a significant increase in malformed embryos was observed for all of them. All three metabolites were more potent teratogens than CBZ, with AI being the most potent, followed by 9ACA and AO. The myeloperoxidase (MPO) homologue is already active at 24 hpf, as CBZ was biotransformed into 9ACA and AO in 24 hpf zebrafish embryos, and into 9ACA in 120 hpf larvae. Moreover, 9ACA was also found to be biotransformed into AI and AO, and AI into AO. As such, one or more of these metabolites probably contribute to the teratogenic effects observed in zebrafish larvae after exposure to CBZ.

Background: Eurycomanone (EN) is a diterpenoid compound isolated from the roots of Eurycoma longifolia (E. longifolia). Previous studies have confirmed that E. longifolia can enhance bone regeneration and bone strength. We previously isolated and identified ten quassinoids from E. longifolia, and the result displayed that five aqueous extracts have the effects on promotion of bone formation, among whom EN showed the strongest activity. However, the molecular mechanism of EN on bone formation was unknown, and we further investigated in this study. Methods: After the verification of purity of extracted EN, following experiments were conducted. Firstly, the pharmacologic action of EN on normal bone mineralization and the therapeutic effect of EN on Dex-induced bone loss using zebrafish larvae. The mineralization area and integral optical density (IOD) were evaluated using alizarin red staining. Then the vital signaling pathways of EN relevant to OP was identified through network pharmacology analysis. Eventually in vitro, the effect of EN on cell viability, osteogenesis activities were investigated in human bone marrow mesenchymal stem cells (hMSCs) and C3H10 cells, and the molecular mechanisms by which applying AKT inhibitor A-443654 in hMSCs. Results: In zebrafish larvae, the administration in medium of EN (0.2, 1, and 5 μM) dramatically enhanced the skull mineralization area and integral optical density (IOD), and increased mRNA expressions of osteoblast formation genes (ALP, RUNX2a, SP7, OCN). Meanwhile, exposure of EN remarkably alleviated the inhibition of bone formation induced by dexamethasone (Dex), prominently improved the mineralization, up-regulated osteoblast-specific genes and down-regulated osteoclast-related genes (CTSK, RANKL, NFATc1, TRAF6) in Dex-treated bone loss zebrafish larvae. Network pharmacology outcomes showed the MAPK and PI3K-AKT signaling pathways are closely associated with 10 hub genes (especially AKT1), and AKT/GSK-3β/β-catenin was selected as the candidate analysis pathway. In hMSCs and C3H10 cells, results showed that EN at appropriate concentrations of 0.008–5 μM effectively increased the cell proliferation. In addition, EN (0.04, 0.2, and 1 μM) significantly stimulated osteogenic differentiation and mineralization as well as significantly increased the protein phosphorylation of AKT and GSK-3β, and expression of β-catenin, evidencing by the results of ALP and ARS staining, qPCR and western blotting. Whereas opposite results were presented in hMSCs when treated with AKT inhibitor A-443654, which effectively inhibited the pro-osteogenesis effect induced by EN, suggesting EN represent powerful potential in promoting osteogenesis of hMSCs, which may be closely related to the AKT/GSK-3β/β-catenin signaling pathway. Conclusions: Altogether, our findings indicate that EN possesses remarkable effect on bone formation via activating AKT/GSK-3β/β-catenin signaling pathway in most tested concentrations. The translational potential of this article: This study demonstrates EN is a new effective monomer in promoting bone formation, which may be a promising anabolic agent for osteoporosis (OP) treatment.

This study investigated the effects of environmental changes on zebrafish larval behavior, using single-factor and orthogonal experiments to assess locomotion during temperature and pH changes. In single-factor experiments, zebrafish larvae were exposed to variations in temperature (22 to 30 °C) and pH levels (6.0, 7.0, 9.0). The simultaneous temperature and pH changes were investigated by orthogonal tests. In both experiments, each zebrafish larva was recorded in three 5 min videos at different stages (before exposure, during short-term exposure (10 min), and after long-term exposure (60 min)). You Look Only Once (YOLOv5) and Deep Simple Online Real Time Tracking (DeepSORT) models were adopted to develop a zebrafish larva tracking system, and YOLOv5 was improved in two aspects of anchor clustering and network structure. The tracking accuracy of the tracking system for small targets effectively improved, reaching more than 98% MOTA (Multiple Object Tracking Accuracy). Principal Component Analysis (PCA) was employed to extract three behavioral features from 13 motion parameters, namely motion activity, edge behavior, and motion direction preference. Our findings reveal that lower temperatures and acidic conditions both led to a decrease in motion behavioral activity, and the former also increased edge behavior. Conversely, elevated temperatures and alkaline conditions had a muted impact on these behaviors. Interestingly, concurrent changes in temperature and pH significantly altered directional preference. Additionally, we observed that lower temperatures elicited distinct temporal behavioral patterns at a constant pH level. In summary, we recommend the precise control and explicit reporting of ambient temperature and pH in both breeding devices and experimental wells to minimize the environmental impact on zebrafish behavior and enhance experiment repeatability and reliability.

The recent availability of commercial platforms for behavioral analyses in zebrafish larvae based on video-tracking technologies has exponentially increased the number of studies analyzing different behaviors in this model organism to assess neurotoxicity. Among the most commonly used assays in zebrafish larvae are basal locomotor activity (BLA) and visual motor responses (VMRs). However, the effect of different intrinsic and extrinsic factors that can significantly alter the outcome of these assays is still not well understood. In this work, we have analyzed the influence of age (5–8 days post-fertilization), time of day (8:00, 10:00, 12:00, 14:00; 16:00, 18:00, and 20:00 h), and experiment (three experiments performed at different days) on BLA and VMR results (4004 analyses for each behavior) in 143 larvae. The results from both behaviors were adjusted to a random-effects linear regression model using generalized least squares (GLSs), including in the model the effect of the three variables, the second-way interactions between them, and the three-way interaction. The results presented in this manuscript show a specific effect of all three intrinsic factors and their interactions on both behaviors, supporting the view that the most stable time period for performing these behavioral assays is from 10:00 am to 04:00 pm, with some differences depending on the age of the larva and the behavioral test.

We demonstrated the effect of Ishige okamurae extract (IOE) on the receptor activator of nuclear factor-κB ligand (RANKL)-promoted osteoclastogenesis in RAW 264.7 cells and confirmed that IOE inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation. IOE inhibited protein expression of TRAP, metallopeptidase-9 (MMP-9), the calcitonin receptor (CTR), and cathepsin K (CTK). IOE treatment suppressed the expression of activated T cell cytoplasmic 1 and activator protein-1, thus controlling the expression of osteoclast-related factors. Moreover, IOE significantly reduced RANKL-phosphorylated extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). It also reduced the RANKL-induced phosphorylation of NF-κB and nuclear translocation of p65. IOE inhibited Dex-induced bone loss and osteoclast-related gene expression in zebrafish larvae. HPLC analysis shows that IOE consists of 3.13% and 3.42% DPHC and IPA, respectively. Our results show that IOE has inhibitory effects on osteoclastogenesis in vitro and in vivo and is a potential therapeutic for osteoporosis.

Polybrominated diphenyl ether flame retardants are known to have adverse effects on the development of organisms. We investigated the molecular mechanisms associated with the developmental hazards of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) in zebrafish, as well as the behavioral and morphological alterations involved, focusing on endoplasmic reticulum stress (ERS), oxidative stress, and apoptosis. Our study revealed behavioral alterations in zebrafish exposed to BDE-47, including impaired motor activity, reduced exploration, and abnormal swimming patterns. In addition, we observed malformations in craniofacial regions and other developmental abnormalities that may be associated with ERS-induced cellular dysfunction. BDE-47 exposure showed apparent changes in ERS, oxidative stress, and apoptosis biomarkers at different developmental stages in zebrafish through gene expression analysis and enzyme activity assays. The study indicated that exposure to BDE-47 results in ERS, as supported by the upregulation of ERS-related genes and increased activity of ERS markers. In addition, oxidative stress-related genes showed different expression patterns, suggesting that oxidative stress is involved in the BDE-47 toxic effects. Moreover, an assessment of apoptotic biomarkers revealed an imbalance in the expression levels of pro- and anti-apoptotic genes, suggesting that BDE-47 exposure activated the apoptotic pathway. These results highlight the complex interactions between ERS, oxidative stress, apoptosis, behavioral alterations, and morphological malformations following BDE-47 exposure in zebrafish. Understanding the mechanisms of toxicity of developmental hazards is essential to elucidate the toxicological effects of environmental contaminants. The knowledge can help develop strategies to mitigate their adverse effects on the health of ecosystems and humans.

Background: The etiology of autism spectrum disorder (ASD) is multifactorial, involving genetic and environmental contributors such as endocrine-disrupting chemicals (EDCs). Objective: To evaluate the association between perinatal exposure to 27 potential EDCs and ASD among Norwegian children, and to further examine the neurodevelopmental toxicity of associated chemicals using zebrafish embryos and larvae. Method: 1,199 mothers enrolled in the prospective birth-cohort (HUMIS, 2002–2009) study. Breastmilk levels of 27 chemicals were measured: polychlorinated biphenyls, organochlorine pesticides, polybrominated diphenyl ethers, and perfluoroalkyl substances as a proxy for perinatal exposure. We employed multivariable logistic regression to determine association, utilized elastic net logistic regression as variable selection method, and conducted an in vivo study with zebrafish larvae to confirm the neurodevelopmental effect. Results: A total of 20 children had specialist confirmed diagnosis of autism among 1,199 mother–child pairs in this study. β-Hexachlorocyclohexane (β-HCH) was the only chemical associated with ASD, after adjusting for 26 other chemicals. Mothers with the highest levels of β-HCH in their milk had a significant increased risk of having a child with ASD (OR = 1.82, 95 % CI: 1.20, 2.77 for an interquartile range increase in ln-transformed β-HCH concentration). The median concentration of β-HCH in breast milk was 4.37 ng/g lipid (interquartile range: 2.92–6.47), and the estimated daily intake (EDI) for Norwegian children through breastfeeding was 0.03 µg/kg of body weight. The neurodevelopmental and social behavioral effects of β-HCH were established in zebrafish embryos and larvae across various concentrations, with further analysis suggesting that perturbation of dopaminergic neuron development may underlie the neurotoxicity associated with β-HCH. Conclusions: Prenatal exposure to β-HCH was associated with an increased risk of specialist-confirmed diagnoses of ASD among Norwegian children, and the EDI surpasses the established threshold. Zebrafish experiments confirm β-HCH neurotoxicity, suggesting dopaminergic neuron disruption as a potential underlying mechanism.

The interaction between metals is ubiquitous, but there is still a lack of quantitative models considering the interaction between metals, which leads to the deviations in predicting the joint toxicity of metals. The present study estimated the uptake rate constants (kin) and elimination rate constants (kout) and elucidated how the presence of one metal (Cu or Cd) affects the absorption and excretion of another metal (Cd or Cu) in zebrafish larvae. The results showed that Cd and Cu inhibited each other in the process of absorption and excretion by comparing separately kin and kout of Cd or Cu with the other metal Cu or Cd mixed concentrations increased, thereby affecting the Cd and Cu bioaccumulation in the zebrafish larvae. Then the interactions between Cd and Cu in the uptake and elimination processes were quantified to obtain a refined toxicokinetic model. Verification with independent experiment data showed that the refined toxicokinetic model could significantly improve the prediction of the Cd or Cu bioaccumulation in the zebrafish larvae. This study contributes to understand the toxicokinetic process of the Cd-Cu mixture in the zebrafish larvae, and the developed model could be used to predict the toxicity of the metal mixtures.

Epilepsy is a common neurological disorder characterized by seizures that cause neurobiological and behavioral impairment. Caffeine (CAF), which is the most widely consumed stimulant in the world, is reported to influence epileptic seizures and antiepileptic drugs, especially topiramate (TPM). The aim of the study was to optimize the zebrafish larvae pentylenetetrazol-induced seizure model for the study of CAF and TPM interactions, which include the determination of dose space, and the delivery of an analytical method for monitoring CAF, TPM, and CAF metabolite paraxanthine (PAR) in Zebrafish larvae. Methods: The zebrafish larvae, 4 days post-fertilization, were incubated for 18 h with CAF, TPM, or CAF + TPM, with subsequent locomotor activity assessment. Seizures were evoked by adding PTZ solution to obtain a final concentration of 20 mM. Subsequently, the liquid chromatography–mass spectrometry (LC–MS/MS) analytical method was used to simultaneously assess the levels of both CAF and TPM in the larvae. CAF (50 mg/L) and TPM (75 μM) given separately decreased the average larvae locomotor activity compared to the PTZ group but, however, were not able to lower it to the control level. Co-administration of 25 mg/L CAF and 50 μM TPM suppressed the activity to the same level. Adding 25 μM TPM to 50 mg/L CAF decrease the measured CAF level in the larvae. Until proven otherwise, CAF consumption should be regarded as a potential determinant in the modulation of TPM's efficacy in the management of epileptic seizures. The optimized model will contribute to the standardization of studying CAF and TPM interactions and building the understanding of the molecular bases of the interaction. [ABSTRACT FROM AUTHOR]

To identify potent plant extracts with strong antioxidant activity, we evaluated the free radical scavenging activity of 184 plant extracts obtained from the Freshwater Bioresources Culture Collection (FBCC) of Nakdonggang National Institute of Biological Resources (Republic of Korea), as various plant extracts have been used therapeutically to prevent chronic diseases associated with oxidative stress. From them, three plant extracts (FBCC-EP858 from Ammannia multiflora, FBCC-EP920 from Ammannia coccinea, and FBCC-EP1014 from Salix gracilistyla) were selected based on their abilities to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical with more than 80% efficiency. We found that these extracts had in vitro half maximal inhibitory concentration (IC50) values ranging from 11.89 to 14.26 μg/mL and strong total antioxidant activity (corresponding to approximately 0.18, 0.22, and 0.23 mM Trolox, respectively). We also studied the effect of these extracts on RAW 264.7 macrophages and found that FBCC-EP920 significantly downregulated relative cell viability at a concentration of 100 μg/mL. However, the other two extracts, FBCC-EP858 and FBCC-EP1014, did not affect cell viability at the same concentration. Additionally, all three extracts inhibited hydrogen peroxide (H2O2)-induced reactive oxygen species (ROS) production and depolarization of mitochondrial membrane potential in RAW 264.7 macrophages. An additional experiment in zebrafish larvae showed that the three extracts reduced 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent intensity induced by H2O2. The extracts also upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression, and an HO-1 inhibitor, zinc protoporphyrin (ZnPP), attenuated the extract-induced antioxidant activity both in vivo and in vitro. Taken together, these findings suggest that the extracts from A. multiflora, A. coccinea, and S. gracilistyla have potential free radical scavenging and antioxidant capacities both in vivo and in vitro by activating the Nrf2/HO-1 signaling pathway. These results could be useful for the prevention and treatment of various oxidative stress-mediated human diseases. [ABSTRACT FROM AUTHOR]

Wolfram syndrome (WS) is a rare neurodegenerative disease resulting in deafness, optic atrophy, diabetes, and neurological disorders. Currently, no treatment is available for patients. The mutated gene, WFS1, encodes an endoplasmic reticulum (ER) protein, Wolframin. We previously reported that Wolframin regulated the ER-mitochondria Ca2+ transfer and mitochondrial activity by protecting NCS1 from degradation in patients’ fibroblasts. We relied on a zebrafish model of WS, the wfs1abKO line, to analyze the functional and behavioral impact of NCS1 overexpression as a novel therapeutic strategy. The wfs1abKO line showed an increased locomotion in the visual motor and touch-escape responses. The absence of wfs1 did not impair the cellular unfolded protein response, in basal or tunicamycin-induced ER stress conditions. In contrast, metabolic analysis showed an increase in mitochondrial respiration in wfs1abKO larvae. Interestingly, overexpression of NCS1 using mRNA injection restored the alteration of mitochondrial respiration and hyperlocomotion. Taken together, these data validated the wfs1abKO zebrafish line as a pertinent experimental model of WS and confirmed the therapeutic potential of NCS1. The wfs1abKO line therefore appeared as an efficient model to identify novel therapeutic strategies, such as gene or pharmacological therapies targeting NCS1 that will correct or block WS symptoms.