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Abstract The paralogs 9–13 Hox genes in mouse HoxA and HoxD clusters are critical for limb development. When both HoxA and HoxD clusters are deleted in mice, significant limb truncation is observed compared to the phenotypes of single and compound mutants of Hox9-13 genes in these clusters. In zebrafish, mutations in hox13 genes in HoxA- and HoxD-related clusters result in abnormal morphology of pectoral fins, homologous to forelimbs. However, the effect of the simultaneous deletions of entire HoxA- and HoxD-related clusters on pectoral fin development remains unknown. Here, we generated mutants with several combinations of hoxaa, hoxab, and hoxda cluster deletions and analyzed the pectoral fin development. In hoxaa −/− ;hoxab −/− ;hoxda −/− larvae, the endoskeletal disc and the fin-fold are significantly shortened in developing pectoral fins. In addition, we show that this anomaly is due to defects in the pectoral fin growth after the fin bud formation. Furthermore, in the surviving adult mutants, micro-CT scanning reveals defects in the posterior portion of the pectoral fin which is thought to represent latent regions of the limb. Our results further support that the functional role of HoxA and HoxD clusters is conserved in the paired appendage formation in bony fishes.

Abstract 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 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.

Although the concept of endocrine disruptors first appeared almost 30 years ago, the relatively recent involvement of these substances in the etiology of metabolic pathologies (obesity, diabetes, hepatic steatosis, etc.) has given rise to the concept of Metabolic Disrupting Chemicals (MDCs). Organs such as the liver and adipose tissue have been well studied in the context of metabolic disruption by these substances. The intestine, however, has been relatively unexplored despite its close link with these organs. In vivo models are useful for the study of the effects of MDCs in the intestine and, in addition, allow investigations into interactions with the rest of the organism. In the latter respect, the zebrafish is an animal model which is used increasingly for the characterization of endocrine disruptors and its use as a model for assessing effects on the intestine will, no doubt, expand. This review aims to highlight the importance of the intestine in metabolism and present the zebrafish as a relevant alternative model for investigating the effect of pollutants in the intestine by focusing, in particular, on cytochrome P450 3A (CYP3A), one of the major molecular players in endogenous and MDCs metabolism in the gut.

Abstract Background Zebrafish are widely used in various research fields and to fulfil the diverse research needs, numerous zebrafish lines are available, each with a unique domestication background, potentially resulting in intraspecies differences in specific biological functions. Few studies have compared multiple zebrafish lines under identical conditions to investigate both inter- and intra-line variability related to different functions. However, such variability could pose a challenge for the reproducibility of results in studies utilising zebrafish, particularly when the line used is not clearly specified. This study assessed growth, stress status (cortisol, serotonin) and reproductive capabilities (maturity, fecundity, fertilisation rate, sperm quality) of four commonly used wild-type zebrafish lines (AB, SJD, TU, WIK) using standardized protocols. Results The stress markers levels were found to be similar across the lines, indicating that the endocrine stress status is robust to diverse domestication histories. Variations were observed in the growth and reproductive parameters. The lines exhibited differences in the timing of puberty (86 dpf for AB and SJD lines vs. 107 dpf for the WIK line) despite achieving similar sizes, suggesting that there are line-specific variations in the induction of maturation. Additionally, the AB line demonstrated higher sperm quality than did the other lines and higher fecundity and fertilization rates than did the SJD line. The AB line also exhibiting a smaller adult size but a heavier brain relative to its body weight. Conclusion These findings emphasize the importance of line selection for zebrafish research, indicating that researchers should consider line-specific traits to ensure the biological relevance and reproducibility of the results.

Abstract To elucidate the lipidomic and metabolomic alterations associated with hypertrophic cardiomyopathy (HCM) pathogenesis, we utilized cmybpc3-/- zebrafish model. Fatty acid profiling revealed variability of 10 fatty acids profiles, with heterozygous (HT) and homozygous (HM) groups exhibiting distinct patterns. Hierarchical cluster analysis and multivariate analyses demonstrated a clear separation of HM from HT and control (CO) groups related to cardiac remodeling. Lipidomic profiling identified 257 annotated lipids, with two significantly dysregulated between CO and HT, and 59 between HM and CO. Acylcarnitines and phosphatidylcholines were identified as key contributors to group differentiation, suggesting a shift in energy source. Untargeted metabolomics revealed 110 and 53 significantly dysregulated metabolites. Pathway enrichment analysis highlighted perturbations in multiple metabolic pathways in the HM group, including nicotinate, nicotinamide, purine, glyoxylate, dicarboxylate, glycerophospholipid, pyrimidine, and amino acid metabolism. Our study provides comprehensive insights into the lipidomic and metabolomic unique signatures associated with cmybpc3-/- induced HCM in zebrafish. The identified biomarkers and dysregulated pathways shed light on the metabolic perturbations underlying HCM pathology, offering potential targets for further investigation and potential new therapeutic interventions.

Metal oxide nanoparticles (MONPs) are widely used in medicine and environmental remediation because of their unique properties. However, their size, surface area, and reactivity can cause toxicity, potentially leading to oxidative stress, inflammation, and cellular or DNA damage. In this study, a nano-quantitative structure–toxicity relationship (nano-QSTR) model was initially developed to assess zebrafish toxicity for 24 MONPs. Previously established 23 first- and second-generation periodic table descriptors, along with five newly proposed third-generation descriptors derived from the periodic table, were employed. Subsequently, to enhance the quality and predictive capability of the nano-QSTR model, a nano-quantitative read across structure–toxicity relationship (nano-qRASTR) model was created. This model integrated read-across descriptors with modeled descriptors from the nano-QSTR approach. The nano-qRASTR model, featuring three attributes, outperformed the previously reported simple QSTR model, despite having one less MONP. This study highlights the effective utilization of the nano-qRASTR algorithm in situations with limited data for modeling, demonstrating superior goodness-of-fit, robustness, and predictability (R2 = 0.81, Q2LOO = 0.70, Q2F1/R2PRED = 0.76) compared to simple QSTR models. Finally, the developed nano-qRASTR model was applied to predict toxicity data for an external dataset comprising 35 MONPs, addressing gaps in zebrafish toxicity assessment.

Abstract DHPS deficiency syndrome is an ultra-rare neurodevelopmental disorder (NDD) which results from biallelic mutations in the gene encoding the enzyme deoxyhypusine synthase (DHPS). DHPS is essential to synthesize hypusine, a rare amino acid formed by post-translational modification of a conserved lysine in eukaryotic initiation factor 5 A (eIF5A). DHPS deficiency syndrome causes epilepsy, cognitive and motor impairments, and mild facial dysmorphology. In mice, a brain-specific genetic deletion of Dhps at birth impairs eIF5AHYP-dependent mRNA translation. This alters expression of proteins required for neuronal development and function, and phenotypically models features of human DHPS deficiency. We studied the role of DHPS in early brain development using a zebrafish loss-of-function model generated by knockdown of dhps expression with an antisense morpholino oligomer (MO) targeting the exon 2/intron 2 (E2I2) splice site of the dhps pre-mRNA. dhps knockdown embryos exhibited dose-dependent developmental delay and dysmorphology, including microcephaly, axis truncation, and body curvature. In dhps knockdown larvae, electrophysiological analysis showed increased epileptiform activity, and confocal microscopy analysis revealed reduced arborisation of GABAergic neurons. Our findings confirm that hypusination of eIF5A by DHPS is needed for early brain development, and zebrafish with an antisense knockdown of dhps model features of DHPS deficiency syndrome.

ABSTRACTVirtually every major medical advance of the last century and at still has depended upon research with animals. Zebrafish's journey from the ocean to the laboratory leads to major scientific breakthroughs. Transparency structure of zebrafish helps in monitoring their internal structures and are permitting scientist to see effectes of nano particles in fish. Their organs share the same main features as humans and so can be used to study human developmental processes. Zebrafish congruence 70% of their genes with humans, and 84% of ailment-depended genes have zebrafish congruence. The zebrafish embryos can also genetically modified. Certain fishes like zebrafish are able to regenerate damaged retinal nerve cells. Müller galia cells in retina of zebrafish can transform in response to injury and act like stem cells to regrow the retina and replace all damaged neurons. Though humans have the same exact Müller galia cell, they don’t respond to damaged in the same way. Zebrafish are also very responsive to having their genomes edited. Zebrafish regenerate some tissue such as heart in during larval stage. In additionaly zebrafish are used as an animal model to study pharmocology – how drugs work and what they do to an organism’s body. Aim of this review, here, we review current knowledge of how these specialized structures and model organism by focusing on cellular behaviors and molecular mechanisms, highlighting findings from in vivo models and briefly discussing the recent advances in tissue cell culture and organoids. Review discusses the applications of human organoids models of disease on model organism and outlines the ailment treatments.

Abstract Background The quest for candidate probiotics and prebiotics to develop novel synbiotics for sustainable and profitable fish farming remains a major focus for various stakeholders. In this study, we examined the effects of combining two fungal probiotics, Saccharomyces cerevisiae and Aspergillus niger with extracts of Jerusalem artichoke and white button mushroom to develop a synbiotic formulation to improve the growth and health status of zebrafish (Danio rerio). An initial in vitro study determined the most effective synbiotic combination, which was then tested in a 60-day in vivo nutritional trial using zebrafish (80 ± 1.0 mg) as a model animal. Four experimental diets were prepared: a control diet (basal diet), a prebiotic diet with 100% selected mushroom extract, a probiotic diet with 107 CFU of S. cerevisiae/g of diet, and a synbiotic diet with 107 CFU of S. cerevisiae/g of diet and 100% mushroom extract. As readouts, growth performance, survival, digestive enzyme activity and innate immune responses were evaluated. Results In vitro results showed that the S. cerevisiae cultured in a medium containing 100% mushroom extract exhibited the maximum specific growth rate and shortest doubling time. In the in vivo test with zebrafish, feeding them with a synbiotic diet, developed with S. cerevisiae and mushroom extract, led to a significant improvement in the growth performance of zebrafish (P

Abstract Fish domestication progresses through five levels: from the initial acclimatization to captivity (Level 1), to the life cycle completion in captivity (Level 4), and even to the implementation of selective breeding programs (Level 5). Domestication leads to phenotypic changes over generations, sometimes from the very first generation. Behavioral traits are among the first to change. However, in fish, potential behavioral changes during early domestication have been little studied. Therefore, we studied potential behavioral changes among early and advanced levels of domestication in a model species, the zebrafish (Danio rerio), using a mirror test experiment, commonly used to assess traits involved in activity, aggressiveness, and stress in this species. We compared these traits between wild zebrafish in captivity (F0; Level 1), the first generation of their captive-born offspring (F1; Level 4), and three laboratory strains (AB, TU, and WIK; Level 5). Each fish was individually filmed and tracked using an automated procedure for 5 min. Nine behavioral traits and one activity-related trait were characterized for each individual based on the movements and positioning of the fish. We applied a principal component analysis (PCA) and tested the significance of potential differences between groups using an analysis of similarities (ANOSIM). We applied an indicator value analysis (IndVal) to determine which traits were most expressed by each group. We detected differences between groups and across domestication levels. More specifically, we highlighted differentiations between different levels of domestication (e.g. between F1, AB, TU, and WIK) as early as the beginning of the domestication process (i.e. F0 vs. F1), but also within the same level of domestication (i.e. AB vs. TU). Based on PCA and IndVal, (i) F0 and F1 tended to show stronger expression of stress-related traits than the other groups, (ii) F0 was more active than others, and (iii) TU was more aggressive than AB. Our results confirmed that domestication can change fish behavior, even in the first generation born in captivity, although these modifications remain limited. In contrast, we did not observe any general trends correlated with domestication levels, given that AB and TU diverged in their aggressiveness levels, and WIK differed only from F1. This result needs to be generalized to other species but also considered for domestication for aquaculture. If future studies confirm that the changes observed at the beginning of the domestication process remain limited and that there is no consistent evolutionary trend across generations in fish, this would highlight the crucial importance of selecting the right species from the outset of domestication. It would also emphasize the need to design selective breeding programs that shape fish stocks with the most desirable characteristics. To our knowledge, this study is one of the few to examine the behavior of wild zebrafish alongside laboratory strains, offering a unique insight into the early stages of domestication.

Chunjiao Lu,* Xuewei Wu,* Xin Meng,* Yi Liu, Ting Yang, Yan Zeng, Yang Chen, Yishan Huang, Zhou Fang, Xiaojun Yang, Juanjuan Luo Engineering Research Center of Key Technique for Biotherapy of Guangdong Province, Shantou University Medical College, Shantou, 515041, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaojun Yang; Juanjuan Luo, Shantou University Medical College, 22 Xinling Road, Shantou, 515041, People’s Republic of China, Email yangx@stu.edu.cn; 15jjluo1@stu.edu.cnIntroduction: The potential toxic effects of wastewater discharges containing silver nanoparticles (AgNPs) and their release into aquatic ecosystems on aquatic organisms are becoming a major concern for environmental and human health. However, the potential risks of AgNPs to aquatic organisms, especially for cardiac development by Focal adhesion pathway, are still poorly understood.Methods: The cardiac development of various concentrations of AgNPs in zebrafish were examined using stereoscopic microscope. The expression levels of cardiac development-related genes were analyzed by qRT-PCR and Whole-mount in situ hybridization (WISH). In addition, Illumina high-throughput global transcriptome analysis was performed to explore the potential signaling pathway involved in the treatment of zebrafish embryos by AgNPs after 72 h.Results: We systematically investigated the cardiac developing toxicity of AgNPs on the embryos of zebrafish. The results demonstrated that 2 or 4 mg/L AgNPs exposure induces cardiac developmental malformations, such as the appearance of pericardial edema phenotype. In addition, after 72 h of exposure, the mRNA levels of cardiac development-related genes, such as myh7, myh6, tpm1, nppa, tbx5, tbx20, myl7 and cmlc1, were significantly lower in AgNPs-treated zebrafish embryos than in control zebrafish embryos. Moreover, RNA sequencing, KEGG (Kyoto Encyclopedia of Genes) and Genomes and GSEA (gene set enrichment analysis) of the DEGs (differentially expressed genes) between the AgNPs-exposed and control groups indicated that the downregulated DEGs were mainly enriched in focal adhesion pathways. Further investigations demonstrated that the mRNA levels of focal adhesion pathway-related genes, such as igf1ra, shc3, grb2b, ptk2aa, akt1, itga4, parvaa, akt3b and vcla, were significantly decreased after AgNPs treatment in zebrafish.Conclusion: Thus, our findings illustrated that AgNPs could impair cardiac development by regulating the focal adhesion pathway in zebrafish.Keywords: AgNPs, focal adhesion, cardiac development, zebrafish

BMP15 is a conserved regulator of ovarian development and maintenance in vertebrates. In humans, premature ovarian insufficiency is caused by autoimmunity and genetic factors, including mutation of BMP15. The cellular mechanisms underlying ovarian failure caused by BMP15 mutation and immune contributions are not understood. Using zebrafish, we established a causal link between macrophage activation and ovarian failure, which, in zebrafish, causes sex reversal. We define a germline-soma signaling axis that activates macrophages and drives ovarian failure and female-to-male sex reversal. Germline loss of zebrafish Bmp15 impairs oogenesis and initiates this cascade. Single-cell RNA sequencing and genetic analyses implicate ovarian somatic cells that express conserved macrophage-activating ligands as mediators of ovarian failure and sex reversal. Genetic ablation of macrophages or elimination of Csf1Rb ligands, Il34 or Csf1a, delays or blocks premature oocyte loss and sex reversal. The axis identified here provides insight into the cells and pathways governing oocyte and ovary maintenance and potential therapeutic targets to preserve female fertility.

Abnormal expression of the transcriptional regulator and hedgehog (Hh) signaling pathway effector Gli3 is known to trigger congenital disease, most frequently affecting the central nervous system (CNS) and the limbs. Accurate delineation of the genomic cis-regulatory landscape controlling Gli3 transcription during embryonic development is critical for the interpretation of noncoding variants associated with congenital defects. Here, we employed a comparative genomic analysis on fish species with a slow rate of molecular evolution to identify seven previously unknown conserved noncoding elements (CNEs) in Gli3 intronic intervals (CNE15-21). Transgenic assays in zebrafish revealed that most of these elements drive activities in Gli3 expressing tissues, predominantly the fins, CNS, and the heart. Intersection of these CNEs with human disease associated SNPs identified CNE15 as a putative mammalian craniofacial enhancer, with conserved activity in vertebrates and potentially affected by mutation associated with human craniofacial morphology. Finally, comparative functional dissection of an appendage-specific CNE conserved in slowly evolving fish (elephant shark), but not in teleost (CNE14/hs1586) indicates co-option of limb specificity from other tissues prior to the divergence of amniotes and lobe-finned fish. These results uncover a novel subset of intronic Gli3 enhancers that arose in the common ancestor of gnathostomes and whose sequence components were likely gradually modified in other species during the process of evolutionary diversification.

Many neurons in the central nervous system produce a single primary cilium that serves as a specialized signaling organelle. Several neuromodulatory G-protein-coupled receptors (GPCRs) localize to primary cilia in neurons, although it is not understood how GPCR signaling from the cilium impacts circuit function and behavior. We find that the vertebrate ancient long opsin A (VALopA), a Gi-coupled GPCR extraretinal opsin, targets to cilia of zebrafish spinal neurons. In the developing 1-d-old zebrafish, brief light activation of VALopA in neurons of the central pattern generator circuit for locomotion leads to sustained inhibition of coiling, the earliest form of locomotion. We find that a related extraretinal opsin, VALopB, is also Gi-coupled, but is not targeted to cilia. Light-induced activation of VALopB also suppresses coiling, but with faster kinetics. We identify the ciliary targeting domains of VALopA. Retargeting of both opsins shows that the locomotory response is prolonged and amplified when signaling occurs in the cilium. We propose that ciliary localization provides a mechanism for enhancing GPCR signaling in central neurons.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

Triphenyl phosphate (TPHP) - a widely used organophosphate-based flame retardant - blocks cardiac looping during zebrafish development in a concentration-dependent manner, a phenotype that is dependent on disruption of embryonic osmoregulation and pericardial edema formation. However, its currently unclear whether (1) TPHP-induced effects on osmoregulation are driven by direct TPHP-induced injury to the embryonic epidermis and (2) whether TPHP-induced pericardial edema is reversible or irreversible following cessation of exposure. Therefore, the objectives of this study were to determine whether TPHP-induced pericardial edema is reversible and whether TPHP causes injury to the embryonic epidermis by quantifying the number of DAPI-positive epidermal cells and analyzing the morphology of the yolk sac epithelium using scanning electron microscopy. First, we found that exposure to 5 μM TPHP from 24-72 h post-fertilization (hpf) did not increase prolactin - a hormone that regulates ions and water levels - in embryonic zebrafish, whereas high ionic strength exposure media was associated with elevated levels of prolactin. Second, we found that exposure to 5 μM TPHP from 24-72 hpf did not decrease DAPI-positive epidermal cells within the embryonic epithelium, and that co-exposure with 2.14 μM fenretinide - a synthetic retinoid that promotes epithelial wound repair - from 24-72 hpf did not mitigate the prevalence of TPHP-induced epidermal folds within the yolk sac epithelium when embryos were exposed within high ionic strength exposure media. Finally, we found that the pericardial area and body length of embryos exposed to 5 μM TPHP from 24-72 hpf were similar to vehicle-treated embryos at 120 hpf following transfer to clean water and depuration of TPHP from 72-120 hpf. Overall, our findings suggest that (1) the ionic strength of exposure media may influence the baseline physiology of zebrafish embryos; (2) TPHP does not cause direct injury to the embryonic epidermis; and (3) TPHP-induced effects on pericardial area and body length are reversible 48 h after transferring embryos to clean water.

Dae Seok Eom spoke with Cell Reports about his scientific journey, inspirations to become a scientist, and his work on cellular protrusions called "airinemes" that are involved in long-range intercellular signal transmission in zebrafish; in particular, he discussed recent work regarding characterization of a macrophage population that plays a critical role in this process.

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder. Transgenic and pharmacological AD models are extensively studied to understand AD mechanisms and drug discovery. However, they are time-consuming and relatively costly, which hinders the discovery of potential anti-AD therapeutics. Here, we established a new model of AD in larval zebrafish by co-treatment with aluminum chloride (AlCl3) and D-galactose (D-gal) for 72 h. In particular, exposure to 150 μM AlCl3 + 40 mg/mL D-gal, 200 μM AlCl3 + 30 mg/mL D-gal, or 200 μM AlCl3 + 40 mg/mL D-gal successfully induced AD-like symptoms and aging features. Co-treatment with AlCl3 and D-gal caused significant learning and memory deficits, as well as impaired response ability and locomotor capacity in the plus-maze and light/dark test. Moreover, increased acetylcholinesterase and β-galactosidase activities, β-amyloid 1–42 deposition, reduced telomerase activity, elevated interleukin 1 beta mRNA expression, and enhanced reactive oxygen species production were also observed. In conclusion, our zebrafish model is simple, rapid, effective and affordable, incorporating key features of AD and aging, thus may become a unique and powerful tool for high-throughput screening of anti-AD compounds in vivo.

The zebrafish (Danio rerio) has emerged as a model organism for investigating lncRNAs-driven fundamental biological processes, such as circadian rhythms, physiology, metabolism, and various diseases. While state-of-the-art sequencing technologies have identified an increasing number of lncRNAs in zebrafish, their annotations are far from complete. In this study, we collect 28,925 lncRNAs from both the published studies and our own RNA-seq analyses and establish a novel webserver-based database called SUDAZFLNC (https://sudarna.website/). The database, containing 28,925 lncRNAs, 25,432 mRNAs, and 368 miRNAs, provides several crucial features and annotations for the zebrafish RNAs, such as sequence identifiers (IDs), sequence length, hexamer score, coding probabilities, GO and KEGG annotations, and micropeptides. SUDAZFLNC also includes time-course expression profiles of 3288 lncRNAs, 25,432 mRNAs, and 342 miRNAs generated from our RNA-seq experiments, and 149, 4407, and 43 rhythmically expressed lncRNAs, mRNAs, and miRNAs, respectively. Based on the peak expression patterns, we classified these RNAs into morning RNAs, evening RNAs, and night RNAs. Users of the database can access the RNA sequences and their expression profiles by searching the corresponding IDs from the Graphical User Interface (GUI) of the database. The database supports several features to investigate RNA sequences and expression profiles, including BLAST, search of sequence and data, ID conversion, and RNA-RNA interaction prediction. This is the largest curated database of zebrafish RNAs and their expression profiles to date.

Krabbe disease is a sphingolipidosis characterized by the genetic deficiency of the acid hydrolase β-galactosylceramidase (GALC). Most of the studies concerning the biological role of GALC performed on Krabbe patients and Galc-deficient twitcher mice (an authentic animal model of the disease) indicate that the pathogenesis of this disorder is the consequence of the accumulation of the neurotoxic GALC substrate β-galactosylsphingosine (psychosine), ignoring the possibility that this enzyme may exert a wider biological impact. Indeed, limited information is available about the effect of GALC downregulation on the cell lipidome in adult and developing organisms. The teleost zebrafish (Danio rerio) has emerged as a useful platform to model human genetic diseases, including sphingolipidoses, and two GALC co-orthologs have been identified in zebrafish (galca and galcb). Here, we investigated the effect of the competitive and irreversible GALC inhibitor β-galactose-cyclophellitol (GCP) on the lipid profile of zebrafish embryos. Molecular modelling indicates that GCP can be sequestered in the catalytic site of the enzyme and covalently binds human GALC, and the zebrafish Galca and Galcb proteins in a similar manner. Accordingly, GCP inhibits the β-galactosylceramide hydrolase activity of zebrafish in vitro and in vivo, leading to significant alterations of the lipidome of zebrafish embryos. These results indicate that the lack of GALC activity deeply affects the lipidome during the early stages of embryonic development, and thereby provide insights into the pathogenesis of Krabbe disease.

ABSTRACTAortic aneurysm/dissection (AAD) poses a life-threatening cardiovascular emergency with complex mechanisms and a notably high mortality rate. Zebrafish (Danio rerio) serve as valuable models for AAD due to the conservation of their three-layered arterial structure and genome with that of humans. However, the existing studies have predominantly focused on larval zebrafish, leaving a gap in our understanding of adult zebrafish. In this study, we utilized β-Aminopropionic Nitrile (BAPN) impregnation to induce AAD in both larval and adult zebrafish. Following induction, larval zebrafish exhibited a 28% widening of the dorsal aortic diameter (p

Purpose: The presence of microplastics (MPs) and nanoplastics (NPs) in aquatic ecosystems has raised serious environmental and health concerns. Polystyrene is one of the most abundant plastic polymers found in the environment. Polystyrene MPs/NPs have harmful implications for human health and their removal from the environment has become a serious challenge. Methods: In this study, we investigated the adsorptive uptake of polystyrene nanoplastics (PS NPs) from aqueous solutions using fly ash-loaded magnetic nanoparticles (FAMNPs) as the magnetic nano-adsorbent. During the factor screening study, the adsorption process was studied as a function of four variables namely pH (5–10), adsorption time (30–120 min), amount of FAMNPs (0.01–0.04 g), and stirring speed (50–200 rpm). Central composite design (CCD) and response surface methodology (RSM) were employed to establish the relationship between the variables. Furthermore, toxicity assessments of PS NPs were checked on a zebrafish model, shedding light on its potential ecological effects. Results: Two variables namely the pH and amount of FAMNPs significantly influenced the adsorption capacity of FAMNPs and were further optimized for subsequent analysis. The optimum operational readings proposed by the model were pH (8.5), and the amount of FAMNPs (0.03 g), resulting in a good adsorption capacity of 29.12 mg/g for PS NPs. The adequacy of the proposed model was evaluated by analysis of variance (ANOVA). Zebrafish embryos exposed to PS NPs revealed physical deformations such as pericardial edema and malformed notochord. Conclusion: The study demonstrates the effectiveness of FAMNPs in the adsorption of PS NPs from aqueous solutions, with optimal conditions identified at pH 8.5 and 0.03 g of FAMNPs using RSM. The adequacy of the model was confirmed through ANOVA analysis. Toxicity assessments on zebrafish embryos exposed to PS NPs revealed significant mortality and physical deformations, highlighting the importance of PS NPs removal for environmental health. [ABSTRACT FROM AUTHOR]

This study was conducted to assess the toxicological potential of synthesized pure and Sn-doped TiO2 NPs (Sn-TiO2 NPs) in zebrafish after acute and chronic exposure. The pure TiO2 NPs, 4%, and 8% Sn-TiO2 NPs were synthesized and characterized using X-ray diffraction, Scanning Electron Microscope, diffuse reflectance spectra, dynamic light scattering, and zeta potential analyses. The pure TiO2 NPs, 4%, and 8% Sn-TiO2 NPs were spherical with average sizes of about 40, 28, and 21 nm, respectively, indicating significant size reduction of TiO2 NPs following Sn doping. According to our results, the LC50-96h increased in the order of 8% Sn-TiO2 NPs (45 mg L−1) < 4% Sn-TiO2 NPs (80.14 mg L−1) < pure TiO2 NPs (105.47 mg L−1), respectively. Exposure of fish to Sn-TiO2 NPs after 30 days resulted in more severe histopathological alterations in gills, liver, intestine, and kidneys than pure TiO2 NPs. Furthermore, Sn-doping significantly elevated malondialdehyde levels and micronuclei frequency, indicating increased oxidative stress and genotoxicity. Expression analysis revealed altered expression of various genes, including upregulation of pro-apoptotic Bax gene and downregulation of anti-apoptotic Bcl-2 gene, suggesting potential induction of apoptosis in response to Sn-doped NPs. Additionally, antioxidant genes (Gpx, Sod, Cat, and Ucp-2) and stress response gene (Hsp70) showed altered expression, suggesting complex cellular responses to mitigate the toxic effects. Overall, this study highlights the concerning impact of Sn-doping on the toxicity of TiO2 NPs in zebrafish and emphasizes the need for further research to elucidate the exact mechanisms underlying this enhanced toxicity. [ABSTRACT FROM AUTHOR]

Chemotherapy-induced cognitive impairment (CICI), also known as "chemobrain," is a common side effect of breast cancer therapy which causes oxidative stress and generation of reactive oxygen species (ROS). Ferulic acid (FA), a natural polyphenol, belongs to BCS class II is confirmed to have nootropic, neuroprotective and antioxidant effects. Here, we have developed FA solid dispersion (SD) in order to enhance its therapeutic potential against chemobrain. An amorphous ferulic acid loaded leucin solid dispersion (FA-Leu SD) was prepared by utilizing amino acid through spray-drying technique. The solid-state characterization was carried out via Fourier-transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and field emission scanning electron microscopy (FE-SEM). Additionally, in-vitro release studies and antioxidant assay were also performed along with in-vivo locomotor, biochemical and histopathological analysis. The physical properties showed that FA-Leu SD so formed exhibited spherical, irregular surface hollow cavity of along with broad melting endotherm as observed from FE-SEM and DSC results. The XRD spectra demonstrated absence of sharp and intense peaks in FA-Leu SD which evidenced for complete encapsulation of drug into carrier. Moreover, in-vitro drug release studies over a period of 5 h in PBS (pH 7.4) displayed a significant enhanced release in the first hr (68. 49 ± 5.39%) and in-vitro DPPH assay displayed greater antioxidant potential of FA in FA-Leu SD. Furthermore, the in-vivo behavioral findings of FA-Leu SD (equivalent to 150 mg/kg of free FA) exhibited positive results accompanied by in-vivo biochemical and molecular TNF-α showed a significant difference (p < 0.001) vis-à-vis DOX treated group upon DOX + FA-Leu SD. Additionally, histopathological analysis revealed neuroprotective effects of FA-Leu SD together with declined oxidative stress due to antioxidant potential of FA which was induced by anticancer drug doxorubicin (DOX). Overall, the above findings concluded that spray-dried FA-Leu SD could be useful for the treatment of chemotherapy induced cognitive impairment. [ABSTRACT FROM AUTHOR]

Cardiovascular disease (CVD) is a common cardiac disorder that leads to heart attacks, strokes, and heart failure. It is primarily characterized by conditions that impact the heart and blood arteries, including peripheral artery disease, arrhythmias, atherosclerosis, myocardial ischemia, congenital heart abnormalities, heart failure, rheumatic heart disease, hypertension, and cardiomyopathies. These conditions are mainly effect the heart and blood vessels, causing blockages or weakened pumping, due to severe hereditary and environmental factors. The frequency of CVD is rising significantly as life expectancy increases. Despite this, no effective treatment or management for its symptoms has been found. One of the most difficult obstacles to overcome, is finding a suitable animal model for drug screening and drug development. Although rodents, mice, swine, and mammals serve as the basis for most animal models of cardiovascular disease, no model accurately captures the epidemiology of the condition. Zebrafish (Danio rerio) have drawn the interest of the international scientific community due to certain shortcomings of the previously discussed animal models because they are smaller, less costly, and have an incredibly high rate of reproduction. This review article emphasizes the significance of using zebrafish as an animal model to investigate the possible facets of cardiovascular disease. Moreover, the ultimate purpose of this review article is to establish the advantages of employing zebrafish over other animal models and to investigate the boundaries of using zebrafish to study human disease. Furthermore, the mechanisms of cardiovascular diseases induction in zebrafish were covered to improve understanding for readers. Finally, the analysis of cardiotoxicity using Zebra fish model, is also explained. In order to stop the health index from deteriorating, the current study also covers some innovative, effective, and relatively safer treatments for treatment and management of cardiotoxicity. [ABSTRACT FROM AUTHOR]

This study aimed to assess the neurotoxic effects of the novel psychoactive substance 4-hydroxy-N-methyl-N-isopropyltryptamine (4-HO-MiPT) on brain. By employing zebrafish as a model organism, the study applied a multifaceted approach, including the recording and analysis of spontaneous behavioral activities, identification of brain metabolic products via ultra-high performance liquid chromatography coupled with quadrupole-Orbitrap high resolution mass spectrometry (UHPLC-Q/Orbitrap HRMS), and examination of changes in the transcriptional level of target genes. Following the injection of 4-HO-MiPT, zebrafish exhibites a pronounced reduction in movement speed and range, indicating a significant alteration in locomotor activity. The subsequent analysis of brain metabolic products finds 37 differential metabolites, including 7 elevated and 30 reduced ones, alongside notable alterations in 6 metabolic pathways. Moreover, the examination of transcriptional level of key genes in zebrafish brain underscores a discernible decrease in transcriptional level of genes associated with the nervous system, implying a substantial impact on neuronal function and synaptic transmission. The experimental findings substantiate the neurotoxic effects of 4-HO-MiPT on the zebrafish brain, shedding light on its deleterious repercussions on both the nervous and immune systems. Furthermore, the perturbations observed in metabolic processes and behavioral responses emphasize the profound impact of 4-HO-MiPT exposure on fundamental physiological functions. The identification of potential carcinogenicity associated with 4-HO-MiPT further underscores the urgent need for comprehensive risk assessment and regulatory scrutiny. This study represents a crucial step towards unraveling the intricate neurotoxic mechanisms underlying 4-HO-MiPT exposure. The research has far-reaching implications, not only in advancing our understanding of the adverse health effects associated with novel psychoactive substances, but also in guiding the development of targeted interventions and regulatory strategies aiming at safeguarding public health and well-being. Continued exploration into the long-term consequences of 4-HO-MiPT exposure, alongside comprehensive toxicological evaluations, holds promise in mitigating its potential hazards and ensuring the safety of individuals and communities exposed to this emerging threat.

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

Abstract Objective Inhibition and knockdown of GPR68 negatively affects glioblastoma cell survival in vitro by inducing ferroptosis. Herein, we aimed to demonstrate that inhibition of GPR68 reduces the survival of glioblastoma cells in vivo using two orthotopic larval xenograft models in Danio rerio, using GBM cell lines U87-MG and U138-MG. In vivo survival of the cancer cells was assessed in the setting of GPR68 inhibition or knockdown. Results In vitro, shRNA-mediated knockdown of GPR68 inhibition demonstrated potent cytotoxic effects against U87 and U138 glioblastoma cell lines. This effect was associated with increased intracellular lipid peroxidation, suggesting ferroptosis as the underlying mechanism of cell death. Translating these findings in vivo, we established a novel xenograft model in zebrafish by successfully grafting fluorescently labeled human glioblastoma cells, which were previously shown to overexpress GPR68. shRNA knockdown of GPR68 significantly reduced the viability of grafted GBM cells within this model. Additionally, treatment with ogremorphin (OGM), a highly specific small molecule inhibitor of GPR68, also reduced the viability of grafted GBM cells with limited toxicity to the developing zebrafish embryos. This study suggests that therapeutic targeting of GPR68 with small molecules like OGM represents a promising approach for the treatment of GBM.

Abstract Data scarcity is one of the most critical issues impeding the development of prediction models for chemical effects. Multitask learning algorithms leveraging knowledge from relevant tasks showed potential for dealing with tasks with limited data. However, current multitask methods mainly focus on learning from datasets whose task labels are available for most of the training samples. Since datasets were generated for different purposes with distinct chemical spaces, the conventional multitask learning methods may not be suitable. This study presents a novel multitask learning method MTForestNet that can deal with data scarcity problems and learn from tasks with distinct chemical space. The MTForestNet consists of nodes of random forest classifiers organized in the form of a progressive network, where each node represents a random forest model learned from a specific task. To demonstrate the effectiveness of the MTForestNet, 48 zebrafish toxicity datasets were collected and utilized as an example. Among them, two tasks are very different from other tasks with only 1.3% common chemicals shared with other tasks. In an independent test, MTForestNet with a high area under the receiver operating characteristic curve (AUC) value of 0.911 provided superior performance over compared single-task and multitask methods. The overall toxicity derived from the developed models of zebrafish toxicity is well correlated with the experimentally determined overall toxicity. In addition, the outputs from the developed models of zebrafish toxicity can be utilized as features to boost the prediction of developmental toxicity. The developed models are effective for predicting zebrafish toxicity and the proposed MTForestNet is expected to be useful for tasks with distinct chemical space that can be applied in other tasks. Scieific contribution A novel multitask learning algorithm MTForestNet was proposed to address the challenges of developing models using datasets with distinct chemical space that is a common issue of cheminformatics tasks. As an example, zebrafish toxicity prediction models were developed using the proposed MTForestNet which provide superior performance over conventional single-task and multitask learning methods. In addition, the developed zebrafish toxicity prediction models can reduce animal testing.

Water pollution remains a major environmental concern, with increased toxic by-products being released into water bodies. Many of these chemical contaminants persist in the environment and bio-accumulate in aquatic organisms. At present, toxicological tests are mostly based on laboratory tests, and effective methods for monitoring wild aquatic environments remain lacking. In the present study, we used a well-characterized toxic chemical, 3,3′,4,4′,5-polychlorinated biphenyl (PCB126), as an example to try to identify common biomarker genes to be used for predictive toxicity of this toxic substance. First, we used two laboratory fish models, the zebrafish (Danio rerio) and medaka (Oryzias latipes), to expose PCB126 to obtain liver transcriptomic data by RNA-seq. Comparative transcriptomic analyses indicated generally conserved and concerted changes from the two species, thus validating the transcriptomic data for biomarker gene selection. Based on the common up- and downregulated genes in the two species, we selected nine biomarker genes to further test in other fish species. The first validation experiment was carried out using the third fish species, Mozambique tilapia (Oreochromis mossambicus), and essentially, all these biomarker genes were validated for consistent responses with the two laboratory fish models. Finally, to develop universal PCR primers suitable for potentially all teleost fish species, we designed degenerate primers and tested them in the three fish species as well as in another fish species without a genomic sequence available: guppy (Poecilia reticulata). We found all the biomarker genes showed consistent response to PCB126 exposure in at least 50% of the species. Thus, our study provides a promising strategy to identify common biomarker genes to be used for teleost fish analyses. By using degenerate PCR primers and analyzing multiple biomarker genes, it is possible to develop diagnostic PCR arrays to predict water contamination from any wild fish species sampled in different water bodies.

Background and Aim: Aeromonas hydrophila infections in fish result in significant financial losses within aquaculture. Previous research indicates black soldier fly (BSF) prepupae provide immunomodulatory benefits through their fatty acids, chitin, and proteins. The study evaluated the impact of hexane extract from black soldier fly prepupae (HEBP) on interleukin (IL)-4 and IL-10 cytokine expression in zebrafish, both infected and uninfected with A. hydrophila. Materials and Methods: Adult zebrafish (aged 4–5 months) was assigned to a negative control group (fed commercial feed), a positive control group (commercial feed + A. hydrophila infection at 107 colony-forming unit/mL), and three treatment groups (T1, T2, T3) that received HEBP at doses of 1000; 2000 and 4000 mg/kg feed for 30 days, respectively. A. hydrophila infection was introduced on day 31 through immersion. Analysis of IL-4 and IL-10 expression in the head kidney trunk region (body without head and tail) through quantitative polymerase chain reaction was conducted on day 33. Results: The HEBP modulated the immune response to A. hydrophila infection at a concentration of 1000 mg/kg feed, as evidenced by an increase in IL-4 and IL-10 expression in the groups not infected with the bacteria. However, these cytokines were decreased in the infected groups. Conclusion: A feed concentration of 1000 mg/kg HEBP was identified as optimal for cytokine modulation. This discovery marks a significant advancement in the development and benefit of a natural extract-based immunomodulator in a zebrafish model, which is potentially immunotherapeutic against bacterial infections in fish for the aquaculture industry.

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

BackgroundNano-alumina (nano-Al2O3) is a widely utilized nanomaterial. Its impacts on the environment and biological systems have garnered significant attention. Zebrafish serves as a common model organism in scientific research due to its high homology with the human genome and is extensively used in toxicity studies. ObjectiveTo investigate the developmental toxicity and neurotoxicity of nano-Al2O3 exposure in zebrafish and the corresponding mechanisms of action. MethodZebrafish embryos at 6 h post-fertilization (hpf) were randomly assigned to a control group and five dose groups exposed to nano-Al2O3 at concentrations of 200, 400, 600, 800, and 1000 μg·mL−1, respectively. Thirty embryos were included in each group, and the culture medium was replaced every 24 h until 144 hpf. The hatching rates at 48 and 72 hpf and the cumulative malformation rates up to 144 hpf were calculated. At 144 hpf, a zebrafish behavior analyzer was used to record the movement trajectories of the zebrafish, and the total distance traveled and average speed were analyzed for each group. At 144 hpf, the development of dopaminergic neurons in transgenic zebrafish expressing vmat2: GFP, brain vessels in those expressing vegf: GFP, and central nervous system neurons in those expressing elavl3: EGFP were observed under a fluorescence microscope, and statistical analysis was conducted using Image Pro Plus. Real-time quantitative PCR was employed to detect the expression levels of neuron development-related genes (syn2α, gap43, dat), Lewy body formation-related gene (α-syn), and autophagy-related genes (pink1, parkin) at 144 hpf. ResultsCompared to the control group, the nano-Al2O3 exposed groups exhibited reduced hatching rates at 48 hpf and increased cumulative malformation rates (P

Rui Shen,1,2,&ast; Kebin Cheng,3,&ast; Guanyi Li,1 Zhendong Pan,4 Xijier Qiaolongbatu,1 Yuting Wang,1 Cui Ma,1 Xucong Huang,1 Li Wang,1 Wenjing Li,1 Yuanyuan Wang,2 Lili Jing,1 Guorong Fan,2 Zhenghua Wu2 1School of Pharmaceutical Sciences, Shanghai Jiao Tong University, Shanghai, 200240, People’s Republic of China; 2Department of Clinical Pharmacy, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, People’s Republic of China; 3Department of Respiratory and Critical Care Medicine, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, 200433, People’s Republic of China; 4Department of Clinical Pharmacy, Eye and ENT Hospital, Fudan University, Shanghai, 200031, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Zhenghua Wu, Department of Clinical Pharmacy, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, No. 85 Wujin Road, Shanghai, 200080, People’s Republic of China, Tel +86-159-0161-7923, Email wuzhenghua@sjtu.edu.cnPurpose: Alisma orientale (AO, Alisma orientale (Sam). Juzep) has been widely employed for the treatment of macular edema (ME) in traditional Chinese medicine due to its renowned water-relief properties. Nonetheless, the comprehensive investigation of AO in alleviating ME remained unexplored. This study aims to identify the active components of AO that target the eye and investigate its pharmacological effects and mechanisms on ME.Methods: The study commenced with UPLC-Triple-TOF/MS analysis to identify the primary constituents of AO. Zebrafish eye tissues were then analyzed after a five-day administration of AO to detect absorbed components and metabolites. Subsequently, network pharmacology, molecular docking, and molecular dynamics simulations were employed to predict the mechanisms of ME treatment via biological target pathways. In vivo experiments were conducted to corroborate the pharmacological actions and mechanisms.Results: A total of 7 compounds, consisting of 2 prototype ingredients and 5 metabolites (including isomers), were found to traverse the blood-eye barrier and localized within eye tissues. Network pharmacology results showed that AO played a role in the treatment of ME mainly by regulating the pathway network of PI3K-AKT and MAPK with TNF-α centered. Computational analyses suggested that 11-dehydro-16-oxo-24-deoxy-alisol A, a metabolite of alisol A, mitigates edema through TNF-α inhibition. Furthermore, zebrafish fundus confocal experiments and HE staining of eyes confirmed the attenuating effects of alisol A on fundus angiogenesis and ocular edema, representing the first report of AO’s ME-inhibitory effects.Conclusion: In this study, computational analyses with experimental validation were used to understand the biological activity and mechanism of alisol A in the treatment of ME. The findings shed light on the bioactive constituents and pharmacological actions of AO, offering valuable insights and a theoretical foundation for its clinical application in managing ME. Keywords: Alisma orientale, macular edema, network pharmacology, zebrafish, TNF-&#x03B1

Abstract Hypertrophic cardiomyopathy (HCM) may lead to cardiac dysfunction and sudden death. This study was designed to develop a HCM signature applying bioinformatics and machine learning methods. Data of HCM and normal tissues were obtained from public databases to screen differentially expressed genes (DEGs) using the R software limma package. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed for enrichment analysis of HCM-associated DEGs. Hub genes for HCM were determined using weighted gene co-expression network analysis (WGCNA) together with two machine learning algorithms (SVM-RFE and LASSO). Finally, we introduced a zebrafish model to simulate changes in the hub genes in the HCM and to observe their effects on cardiac disease development. The mRNA expression data from a total of 106 HCM tissues and 39 normal samples were collected and we screened 157 DEGs. Enrichment analysis showed that immune pathways played an important role in the pathogenesis of HCM. Three hub genes (FCN3, MYH6 and RASD1) were identified using WGCNA, SVM-RFE, and LASSO analysis. In a zebrafish model, knockdown of MYH6 and RASD1 resulted in cardiac malformations with reduced ventricular capacity and heart rate, which validated the clinical significance of these genes in the diagnosis of HCM. Based on machine learning algorithms, our study created a signature with potential impact on cardiac function and cardiac quality index for HCM. The current findings had important implications for the early diagnosis and treatment of HCM.

Abstract Non-small cell lung cancer (NSCLC) constitutes one of the deadliest and most common malignancies. The LKB1/STK11 tumour suppressor is mutated in ∼ 30% of NSCLCs, typically lung adenocarcinomas (LUAD). We implemented zebrafish and human lung organoids as synergistic platforms to pre-clinically screen for metabolic compounds selectively targeting LKB1-deficient tumours. Interestingly, two kinase inhibitors, Piceatannol and Tyrphostin 23, appeared to exert synthetic lethality with LKB1 mutations. Although LKB1 loss alone accelerates energy expenditure, unexpectedly we find that it additionally alters regulation of the key energy homeostasis maintenance player leptin (LEP), further increasing the energetic burden and exposing a vulnerable point; acquired sensitivity to the identified compounds. We show that compound treatment stabilises Hypoxia-inducible factor 1-alpha (HIF1A) by antagonising Von Hippel-Lindau (VHL)-mediated HIF1A ubiquitination, driving LEP hyperactivation. Importantly, we demonstrate that sensitivity to piceatannol/tyrphostin 23 epistatically relies on a HIF1A-LEP-Uncoupling Protein 2 (UCP2) signaling axis lowering cellular energy beyond survival, in already challenged LKB1-deficient cells. Thus, we uncover a pivotal metabolic vulnerability of LKB1-deficient tumours, which may be therapeutically exploited using our identified compounds as mitochondrial uncouplers.

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

Abstract Visual systems have evolved to discriminate between different wavelengths of light. The ability to perceive color, or specific light wavelengths, is important as color conveys crucial information about both biotic and abiotic features in the environment. Indeed, different wavelengths of light can drive distinct patterns of activity in the vertebrate brain, yet what remains incompletely understood is whether distinct wavelengths can invoke etiologically relevant behavioral changes. To address how specific wavelengths in the visible spectrum modulate behavioral performance, we use larval zebrafish and a stereotypic light-search behavior. Prior work has shown that the cessation of light triggers a transitional light-search behavior, which we use to interrogate wavelength-dependent behavioral modulation. Using 8 narrow spectrum light sources in the visible range, we demonstrate that all wavelengths induce motor parameters consistent with search behavior, yet the magnitude of search behavior is spectrum sensitive and the underlying motor parameters are modulated in distinct patterns across short, medium, and long wavelengths. However, our data also establishes that not all motor features of search are impacted by wavelength. To define how wavelength modulates search performance, we performed additional assays with alternative wavelengths, dual wavelengths, and variable intensity. Last, we also tested blind larvae to resolve which components of wavelength dependent behavioral changes potentially include signaling from non-retinal photoreception. These findings have important implications as organisms can be exposed to varying wavelengths in laboratory and natural settings and therefore impose unique behavioral outputs.

Abstract Background Spring viremia of carp virus (SVCV) infects a wide range of fish species and causes high mortality rates in aquaculture. This viral infection is characterized by seasonal outbreaks that are temperature-dependent. However, the specific mechanism behind temperature-dependent SVCV infectivity and pathogenicity remains unclear. Given the high sensitivity of the composition of intestinal microbiota to temperature changes, it would be interesting to investigate if the intestinal microbiota of fish could play a role in modulating the infectivity of SVCV at different temperatures. Results Our study found that significantly higher infectivity and pathogenicity of SVCV infection in zebrafish occurred at relatively lower temperature. Comparative analysis of the intestinal microbiota in zebrafish exposed to high- and low-temperature conditions revealed that temperature influenced the abundance and diversity of the intestinal microbiota in zebrafish. A significantly higher abundance of Parabacteroides distasonis and its metabolite secondary bile acid (deoxycholic acid, DCA) was detected in the intestine of zebrafish exposed to high temperature. Both colonization of Parabacteroides distasonis and feeding of DCA to zebrafish at low temperature significantly reduced the mortality caused by SVCV. An in vitro assay demonstrated that DCA could inhibit the assembly and release of SVCV. Notably, DCA also showed an inhibitory effect on the infectious hematopoietic necrosis virus, another Rhabdoviridae member known to be more infectious at low temperature. Conclusions This study provides evidence that temperature can be an important factor to influence the composition of intestinal microbiota in zebrafish, consequently impacting the infectivity and pathogenicity of SVCV. The findings highlight the enrichment of Parabacteroides distasonis and its derivative, DCA, in the intestines of zebrafish raised at high temperature, and they possess an important role in preventing the infection of SVCV and other Rhabdoviridae members in host fish. Video Abstract

Abstract Background Salvia miltiorrhiza (Danshen, DS) and Radix Paeoniae Rubra (Chishao, CS) herbal pair (DS-CS) is a famous traditional Chinese combination which has been used as antithrombotic formular for centuries. However, there is still lack of sufficient scientific evidence to illustrate its underlying mechanisms. The purpose of this study is to investigate the antithrombotic effects of DS-CS extract in zebrafish and explore its possible mechanism of action. Methods The quality of traditional Chinese medicines DS and CS granules was evaluated using High Performance Liquid Chromatography (HPLC). Subsequently, the therapeutic effect of the DS-CS combination and its components, Salvianolic Acid A (SAA) and Paeoniflorin (PF), in various concentrations on thrombosis was experimentally validated. Moreover, the interaction between DS-CS and the thrombosis disease targets was analyzed through network pharmacology, predicting the potential antithrombotic mechanism of DS-CS. Molecular docking and in vivo zebrafish experiments were conducted to validate the predicted targets, with qRT-PCR utilized for target validation. Results DS-CS exhibited anti-thrombotic effect in zebrafish with concentrations ranging from 25 to 300 μg/mL. The co-administration of PF and SAA at 25 μg/mL each revealed a synergistic antithrombotic effect exceeding that of individual components when contrasted with PHZ treatment. Protein–protein interaction (PPI) analysis identified key genes, including Albumin (ALB), Proto-oncogene tyro-sine-protein kinase Src (SRC), Matrix metalloproteinase-9 (MMP9), Caspase-3 (CASP3), Epidermal growth factor receptor (EGFR), Fibroblast growth factor 2 (FGF2), Vascular endothelial growth factor receptor 2 (KDR), Matrix metalloprotein-ase-2(MMP2), Thrombin (F2), and Coagulation factor Xa (F10), associated with the antithrombotic action of PF and SAA. Furthermore, KEGG pathway analysis indicated involvement of lipid metabolism and atherosclerosis pathways. Molecular docking revealed strong binding of PF and SAA to pivotal hub genes, such as SRC, EGFR, and F10. The experimental findings demonstrated that DS-CS could upregulate the mRNA expression levels of EGFR while inhibiting F10 and SRC mRNA levels, thereby ameliorating thrombotic conditions. Conclusion This research provided valuable insights into the potential mechanisms underlying the antithrombotic activity of DS-CS. Our findings suggested that PF and SAA could be the key active ingredients responsible for this activity. The antithrombotic effects of DS-CS appeared to be mediated through the regulation of mRNA expression of SRC, EGFR, and F10. These results enhanced our understanding of DS-CS's therapeutic potential and lay the groundwork for future studies to further elucidate its mechanisms of action.

Kaiyue Zhang, Chunnan Li, Peitong Wu, Xiaochen Gao, Xueqin Feng, Jiaming Shen, Nanxi Zhang, Xuesheng Hu, Shuo Wang, Hui Zhang, Jingwei Lv, Jiaming Sun Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun, Jilin, People’s Republic of ChinaCorrespondence: Jiaming Sun; Jingwei Lv, Changchun University of Chinese Medicine, No. 1035, Boshuo Road, Nanguan District, Changchun, 130117, People’s Republic of China, Tel +0431-86763809, Fax + 8676 3968, Email Sun_jiaming2000@163.com; jingwei-lv@hotmail.comPurpose: Zhixiao Tang (ZXT), a traditional Chinese compound prescription, has been used clinically to treat pneumonia in China. However, the underlying mechanism of ZXT treatment in pneumonia is still unclear. The present study aimed to reveal the potential mechanism of ZXT in pneumonia using a strategy combining metabolomics and network pharmacology.Methods: Initially, the chemical compositions were identified by UPLC-QE-Orbitrap-MS, while the prediction of potential signal pathways was performed through network pharmacology. To assess the anti-inflammatory properties of ZXT in the context of pneumonia, models of 16HBE cells induced by LPS and zebrafish induced by CuSO4 were established to measure levels of inflammatory markers and apoptosis. Subsequently, the differential changes of endogenous metabolites in cells caused by ZXT were examined using metabolomics technology, and the molecular docking analysis of key targets was carried out using Autodock Vina software. Ultimately, the validation of the primary pathways and targets was conducted through quantitative RT-PCR and Western blot techniques.Results: A total of 75 compounds were identified through UPLC-QE-Orbitrap-MS analyses. Network pharmacological analysis shows that it plays an anti-inflammatory role in C-type lectin receptor signaling pathway. After ZXT intervention, the inflammatory factors and apoptosis in cells were significantly reduced. Metabonomics analysis showed that 18 metabolites changed significantly. Four key genes were identified, which exhibited partial compatibility with the findings of network pharmacology. Molecular docking analysis confirmed the substantial affinity of the primary targets for ZXT. Furthermore, ZXT exerted a suppressive effect on neutrophil migration, down-regulated the expression of pro-inflammatory cytokine genes, and inhibited the up-regulation of the Dectin-1/SYK/NF-κB signaling pathway. In vivo cell experiments also yielded consistent experimental outcomes.Conclusion: This study enhances comprehension of the pharmacological mechanism underlying ZXT’s efficacy in pneumonia treatment, thereby establishing a scholarly basis for future research and clinical utilization of ZXT in pneumonia management. Keywords: Zhixiao Tang, pneumonia, network pharmacology, integrated metabolomics, 16HBE, Zebrafish

Lycopene, a potent antioxidant predominantly found in red-hued fruits and vegetables such as tomatoes, has been incorporated into embryonic maturation media in vitro, demonstrating enhancements in embryo quality. Despite these advancements, the specific effects of lycopene on Zebrafish embryo quality remain unexplored. This study aims to investigate the impact of lycopene (0.65 ppm) on the development of Zebrafish embryos, focusing on hatchability (HA), and heart rate (HR) under conditions of heat stress (HS). Fertile Zebrafish embryos at the gastrula stage were induced HS (30oC; 1 hour). The embryos will be segregated into two groups: those exposed to HS and nonHS (28oC). Each group will receive one of three treatments (four replicates): control / without lycopene (P1), 2μL lycopene (P2), and 4μL lycopene (P3). Observations using an inverted microscope was done every three up to 48 hours post-fertilization (hpf), and subsequently every five hours up to 96 hpf. Quantitative data obtained for HA and HR were analyzed with SPSS, One-Way ANNOVA (p

ABSTRACT: Tributyltin (TBT), an organotin-endocrine-disrupting substance, is recognized as one of the most important toxic environmental pollutants. The present study was carried out to investigate the developmental toxicity of TBT on zebrafish embryos. Eggs of zebrafish were exposed to 2.5, 5, and 10 µg of TBT per liter of E3 medium for 72 hours to evaluate various parameters of developmental toxicity. The TBT exposure resulted in a dose-dependent negative effect on the hatching rate and increased larval mortality. Different abnormalities in larvae, following exposure to various concentrations of TBT, have also been observed. The tail and spinal deformities, along with dwarfism, were important abnormalities observed in TBT-exposed zebrafish embryos. It has been concluded that tributyltin produced deleterious effects on embryos and larvae during developmental stages, along with multiple deformities that ultimately affected the survival and growth of fish

Objective To investigate the developmental defects caused by knockdown of best1 gene in zebrafish as a model for a subtype of craniovertebral junction (CVJ) malformation. Methods Two antisense morpholinos (MOs) were designed targeting zebrafish best1 to block translation (ATG-MO) or to disrupt splicing (I3E4-MO). MOs were microinjected into fertilized one-cell embryos. Efficacy of splicing MO was confirmed by reverse transcription-polymerase chain reaction. Phenotypes were analyzed and quantified by microscopy at multiple developmental stages. Neuronal outgrowth was assessed in transgenic zebrafish expressing green fluorescent protein in neurons. Skeletal ossification was visualized by Calcein staining. Results Knockdown of best1 resulted in zebrafish embryos with shorter body length, curved axis, low survival rate, microcephaly, reduced eye size, smaller head and brain, impaired neuronal outgrowth, and reduced ossification of craniofacial and vertebral bone. Conclusion Best1 gene plays critical roles in ophthalmologic, neurological and skeletal development in zebrafish. A patient with a premature stop codon in BEST1 gene exhibited similar phenotypes, implying a subtype of CVJ malformation.

Abstract Hydrogen peroxide is considered deleterious molecule that cause cellular damage integrity and function. Its key redox signaling molecule in oxidative stress and exerts toxicity on a wide range of organisms. Thus, to understand whether oxidative stress alters visual development, zebrafish embryos were exposed to H2O2 at concentration of 0.02 to 62.5 mM for 7 days. Eye to body length ratio (EBR) and apoptosis in retina at 48 hpf, and optomotor response (OMR) at 7 dpf were all measured. To investigate whether hydrogen peroxide-induced effects were mediated by oxidative stress, embryos were co-incubated with the antioxidant, glutathione (GSH) at 50 μM. Results revealed that concentrations of H2O2 at or above 0.1 mM induced developmental toxicity, leading to increased mortality and hatching delay. Furthermore, exposure to 0.1 mM H2O2 decreased EBR at 48 hpf and impaired OMR visual behavior at 7 dpf. Additionally, exposure increased the area of apoptotic cells in the retina at 48 hpf. The addition of GSH reversed the effects of H2O2, suggesting the involvement of oxidative stress. H2O2 decreased the expression of eye development-related genes, pax6α and pax6β. The expression of apoptosis-related genes, tp53, casp3 and bax, significantly increased, while bcl2α expression decreased. Antioxidant-related genes sod1, cat and gpx1a showed decreased expression. Expression levels of estrogen receptors (ERs) (esr1, esr2α, and esr2β) and ovarian and brain aromatase genes (cyp19a1a and cyp19a1b, respectively) were also significantly reduced. Interestingly, co-incubation of GSH effectivity reversed the impact of H2O2 on most parameters. Overall, these results demonstrate that H2O2 induces adverse effects on visual development via oxidative stress, which leads to alter apoptosis, diminished antioxidant defenses and reduced estrogen production.

Abstract Early-life exposure to environmental toxicants like Benzo[a]pyrene (BaP) is associated with several health consequences in vertebrates (i.e., impaired or altered neurophysiological and behavioral development). Although toxicant impacts were initially studied relative to host physiology, recent studies suggest that the gut microbiome is a possible target and/or mediator of behavioral responses to chemical exposure in organisms, via the gut-brain axis. However, the connection between BaP exposure, gut microbiota, and developmental neurotoxicity remains understudied. Using a zebrafish model, we determined whether the gut microbiome influences BaP impacts on behavior development. Embryonic zebrafish were treated with increasing concentrations of BaP and allowed to grow to the larval life stage, during which they underwent behavioral testing and intestinal dissection for gut microbiome profiling via high-throughput sequencing. We found that exposure affected larval zebrafish microbiome diversity and composition in a manner tied to behavioral development: increasing concentrations of BaP were associated with increased taxonomic diversity, exposure was associated with unweighted UniFrac distance, and microbiome diversity and exposure predicted larval behavior. Further, a gnotobiotic zebrafish experiment clarified whether microbiome presence was associated with BaP exposure response and behavioral changes. We found that gut microbiome state altered the relationship between BaP exposure concentration and behavioral response. These results support the idea that the zebrafish gut microbiome is a determinant of the developmental neurotoxicity that results from chemical exposure.

Abstract In electroretinographic (ERG) recordings of zebrafish, the light stimulus is usually delivered by a fiber optic cable. The purpose of this study was to determine whether the angle of incidence of the stimulus light from the fiber optic cable will affect the amplitudes and implicit times of the ERGs of zebrafish larvae. The larvae were positioned on their side with the right eye pointed upward. The light stimuli were delivered by a fiber optic cable from three directions of the larvae: frontal 0° (F0°), dorsal 30°(D30°), and ventral 30°(V30°). Photopic ERGs were recorded from 16 larvae at age 5–6 days post-fertilization. Our results showed that the mean amplitude of the b-wave elicited at D30° and V30° stimulation was significantly smaller than that elicited at F0° stimulation (P = 0.014 and P = 0.019, respectively). In addition, the mean amplitude of the d-wave elicited at D30° and V30° stimulation was significantly smaller than that elicited at F0° stimulation (P

Abstract Background Recent reports suggest that amyloid beta (Aβ) peptides can exhibit prion-like pathogenic properties. Transmission of Aβ peptide and the development of associated pathologies after surgeries with contaminated instruments and intravenous or intracerebral inoculations have now been reported across fish, rodents, primates, and humans. This raises a worrying prospect of Aβ peptides also having other characteristics typical of prions, such as evasion of the digestive process. We asked if such transmission of Aβ aggregates via ingestion was possible. Methods We made use of a transgenic Drosophila melanogaster line expressing human Aβ peptide prone to aggregation. Fly larvae were fed to adult zebrafish under two feeding schemes. The first was a short-term, high-intensity scheme over 48 h to determine transmission and retention in the gut. The second, long-term scheme specifically examined retention and accumulation in the brain. The gut and brain tissues were examined by histology, western blotting, and mass spectrometric analyses. Results None of the analyses could detect Aβ aggregates in the guts of zebrafish following ingestion, despite being easily detectable in the feed. Additionally, there was no detectable accumulation of Aβ in the brain tissue or development of associated pathologies after prolonged feeding. Conclusions While human Aβ aggregates do not appear to be readily transmissible by ingestion across species, two prospects remain open. First, this mode of transmission, if occurring, may stay below a detectable threshold and may take much longer to manifest. A second possibility is that the human Aβ peptide is not able to trigger self-propagation or aggregation in other species. Either possibility requires further investigation, taking into account the possibility of such transmission from agricultural species used in the food industry.