Alzheimer's disease (AD) is a neurodegenerative olfactory disorder affecting millions of people worldwide. Alterations in the hexosamine or glucose-related pathways have been described through AD progression. Specifically, an alteration in glucosamine 6 phosphate isomerase 2 (GNPDA2) protein levels has been observed in olfactory areas from AD subjects. However, the biological role of GNPDA2 in neurodegeneration remains unknown. Using mass-spectrometry, multiple GNPDA2 interactors were identified in human nasal epithelial cells (NECs) mainly involved in intraciliary transport. Moreover, GNPDA2 overexpression induced an increment in NEC proliferation rates, accompanied by transcriptomic alterations in Type II interferon signalling or cellular stress responses. In contrast, the presence of beta-amyloid or mutated Tau-P301L in GNPDA2-overexpressing NECs induced a slow-down in the proliferative capacity in parallel with a disruption in protein processing. The proteomic characterization of Tau-P301L transgenic zebrafish embryos demonstrated that GNPDA2 overexpression interfered with collagen biosynthesis and RNA/protein processing, without inducing additional changes in axonal outgrowth defects nor neuronal cell death. In humans, a significant increase in serum GNPDA2 levels was observed across multiple neurological proteinopathies (AD, Lewy body dementia, progressive supranuclear palsy, mixed-dementia, and amyotrophic lateral sclerosis) (n=187). These data shed new light on GNPDA2-dependent mechanisms associated to the neurodegenerative process beyond the hexosamine route.

Dual specificity phosphatase 2 (DUSP2) regulates the activation of members in the mitogen-activated protein kinase (MAPK) family, which is involved in a variety of cellular processes like cell proliferation, differentiation, apoptosis and migration. DUSP2 also regulates the expression of inflammatory mediators in macrophages, however, it remains unknown whether DUSP2 participates in macrophage migration. Here, using the tail fin injury model in zebrafish larvae, we found that deletion of DUSP2 inhibited expression of pro-inflammatory cytokines and macrophage chemokines. Moreover, live imaging results showed that migration of macrophages to the injury site was inhibited after DUSP2 deletion. This inhibitory effect was mediated through reduced activation of extracellular regulated protein kinases (ERK) in DUSP2 knockout zebrafish.

The industry is increasingly relying on fish for toxicity assessment. However, current guidelines for toxicity assessment focus on teratogenicity and mortality. From an ecotoxicological point of view, however, these endpoints may not reflect the “full picture” of possible deleterious effects that can nonetheless result in decreased fitness and/or inability to adapt to a changing environment, affecting whole populations. Therefore, assessing sublethal effects add relevant data covering different aspects of toxicity at different levels of analysis. Impacts of toxicants on neurobehavioral function have the potential to affect many different life-history traits, and are easier to assess in the laboratory than in the wild. We propose that carefully-controlled laboratory experiments on different behavioral domains – including anxiety, aggression, and exploration – can increase our understanding of the ecotoxicological impacts of contaminants, since these domains are related to traits such as defense, sociality, and reproduction, directly impacting life-history traits. The effects of selected contaminants on these tests are reviewed, focusing on larval and adult zebrafish, showing that these behavioral domains are highly sensitive to small concentrations of these substances. These strategies suggest a way forward on ecotoxicological research using fish.

Ozonated sunflower oil (OSO) is well known functional oil with its antioxidant, antimi-crobial, anti-allergic, and skin-moisturizing properties. However, studies on the effects of OSO on high-cholesterol diet (HCD)-induced metabolic disorders have been scarce. In the current study, we aimed to determine the anti-inflammatory effects of OSO on lipid me-tabolism in adult hypercholesterolemic zebrafish and its embryo. Microinjection of OSO (final 2%, 10 nL) into zebrafish embryos under presence of carboxymethyllysine (CML, 500 ng) protected acute embryo death upto 61% survival, while sunflower oil (final 2%) showed much less protection around 42% survival. The microinjection of OSO was more effective than SO to inhibit reactive oxygen species (ROS) production and apoptosis in the CML induced embryo toxicity. Intraperitoneal injection of OSO under presence of CML protected acute death from CML-induced neurotoxicity with improved hepatic inflamma-tion, less detection of ROS and interleukin (IL)-6, and lowering blood total cholesterol (TC) and triglyceride (TG), while SO-injected group did not protect the CML-toxicity. Long-term supplementation of OSO (final 20%, wt/wt) with HCD for 6 months resulted higher sur-vivability than HCD alone group or HCD+SO group (final 20%, wt/wt) with significant lowering of plasma TC and TG levels. The HCD+OSO group showed the least hepatic in-flammation, fatty liver change, ROS, and IL-6 production. In conclusion, short-term treat-ment of OSO by injection exhibited potent anti-inflammatory activity against acute neuro-toxicity of CML in zebrafish and its embryo. Long-term supplementation of OSO in diet also revealed the highest survivability and blood lipid-lowering effect through potent an-ti-oxidant and anti-inflammatory activity.

Currently, there is an increase in the aging of the population, which represents a risk factor for many diseases, including sarcopenia. Sarcopenia involves progressive loss of mass, strength, and function of the skeletal muscle. Some mechanisms include alterations in muscle structure, reduced regenerative capacity, oxidative stress, mitochondrial dysfunction, and inflammation. The zebrafish has emerged as a new model for studying skeletal muscle aging because of its numerous advantages, including histological and molecular similarity to human skeletal muscle. In this study, we used fish of 2, 10, 30, and 60 months of age. The older fish showed a higher frailty index because of reduced locomotor activity and alterations in biometric measurements. We observed changes in muscle structure with a decreased number of myocytes and an increase in collagen with aging, corresponding to alterations in the synthesis, degradation, and differentiation pathways. These changes were accompanied by mitochondrial alterations, such as a decrease in intermyofibrillar mitochondria, increased mitochondrial damage, and reduced mitochondrial dynamics. Overall, we demonstrated a similarity in the aging processes of muscle aging between zebrafish and mammals.

Microplastic pollution represents an emerging problem of great interest in the public domain in the last decade; in addition, it overlaps with another delicate problem - pollution with pharmaceutical products that can have negative effects on the environment and people, even in small amounts. The main purpose of this study was to assess the biochemical and behavioral effects of exposure of adult zebrafish (Danio rerio) to polyethylene (PE), polypropylene (PP) and valproic acid (VPA), respectively to their mixtures - possible situations in natural aquatic environments. In terms of behavioral responses, sociability appears to be more impaired in the PP group after 5 days of exposure. The mechanisms affected are more those of swimming performance than of sociability. Even more, VPA increases presence in the arm with conspecifics but decreases mobility and locomotion, indicating a possible anxiety mechanism. The mixtures decrease the aggressiveness, especially in the case of the PE+VPA group, where it reaches a super low level compared to the control, which could endanger the species in nature. Regarding the anxiogenic effect, PP and PE act differently, if PE has an anxiogenic effect, on the opposite side is the PP group, which shows a bolder and more agitated behavior. All four variants showed behavioral changes indicative of toxicity from the first dose.

This work is based on identifying the analysis techniques used to evaluate the antioxidant and anti-inflammatory effects using the zebrafish model. In this context, a literature review was performed with the Web of Science database. We used the terms zebra fish, antioxidant, anti-inflammatory, model, and Danio rerio. Fifty articles were reviewed, of which thirty-three were chosen to perform this review and were classified according to the source of plant extracts, compounds extracted from plants, chemical compounds, and other sources. This paper is an effort to provide a literature review on zebrafish models and elucidate their pros and cons to evaluate anti-inflamatory and antioxidant activity.

There is much concern about disruption of endocrine physiology regulated by steroid hormones in humans, other terrestrial vertebrates and fish by industrial chemicals, such as bisphenol A, and pesticides, such as DDT. These endocrine-disrupting chemicals influence steroid-mediated physiology in humans and other vertebrates by competing with steroids for receptor binding sites, disrupting diverse responses involved in reproduction, development and differentiation. Here I discuss that due to evolution of the progesterone receptor (PR) and mineralocorticoid receptor (MR) after ray-finned fish and terrestrial vertebrates diverged from a common ancestor, each receptor evolved to respond to different steroids in ray-finned fish and terrestrial vertebrates. In elephant shark, a cartilaginous fish, ancestral to ray-finned fish and terrestrial vertebrates, both progesterone and 17,20-beta-dihydroxy-progesterone activate the PR. During the evolution of ray-finned fish and terrestrial vertebrates, the PR in terrestrial vertebrates continued responding to progesterone and evolved to weakly respond to 17,20-beta-dihydroxy-progesterone. In contrast, the physiological progestin for the PR in zebrafish and other ray-finned fish is 17,20-beta-dihydroxy-progesterone, and ray-finned fish PR responds weakly to progesterone. The MR in fish and terrestrial vertebrates also diverged to have different responses to progesterone. Progesterone is a potent agonist for elephant shark MR, zebrafish MR and other fish MRs, in contrast to progesterone’s opposite activity as an antagonist for aldosterone, the physiological mineralocorticoid for human MR. These different physiological ligands for fish and terrestrial vertebrate PR and MR need to be considered in applying data for their disruption by chemicals in fish and terrestrial vertebrates to each other.

Cardiovascular disease (CVD) is the number one cause of death worldwide. This condition resulted in huge research on CVD increasing the need for animal models suitable for the in vivo research. Daphnia and Zebrafish are good animal models for cardiovascular research due to their relative body transparency and easy culture property. Several methods have been developed to conduct cardiac performance measurement in Daphnia and Zebrafish. However, most of the methods only able to obtain heartbeat rate. The other important cardiac endpoints like stroke volume, ejection fraction, fraction shortening, cardiac output and heartbeat regularity must use other programs for measurement. To overcome this limitation, in this study, we successfully developed a one-stop ImageJ-based method using kymograph macros language that able to obtain multiple cardiac performance endpoints simultaneously for the first time. To validate its utility, we incubate Daphnia magna at different ambient temperatures and exposed zebrafish with astemizole to detect the corresponding cardiac performance alterations. In summary, the kymograph method reported in this study provides a new, easy to use, and inexpensive one-stop method on obtaining multiple cardiac performance endpoints with high accuracy and convenience.

Donepezil (DPZ) is an acetylcholinesterase inhibitor used for the clinical treatment of mild cognitive impairment. However, DPZ has been reported to have adverse effects, including abnormal cardiac rhythm, insomnia, vomiting, and muscle cramps. However, the existence of these effects in subjects without Dementia is unknown. In this study, we use zebrafish to conduct a deeper analysis of the potential adverse effects of DPZ on the short-term memory and behaviors of normal zebrafish by performing multiple behavioral and biochemical assays. Adult zebrafish were exposed to 1 ppm and 2.5 ppm of DPZ. From the results, DPZ caused a slight improvement in the short-term memory of zebrafish and induced significant elevation in aggressiveness, while the novel tank and shoaling tests revealed anxiolytic-like behavior to be caused by DPZ. Furthermore, zebrafish circadian locomotor activity displayed a higher reduction of locomotion and abnormal movement orientation in both low- and high-dose groups, compared to the control group. Biomarker assays revealed that these alterations were associated with an elevation of oxytocin and a reduction of cortisol levels in the brain. Moreover, the significant increases of reactive oxygen species (ROS) and malondialdehyde (MDA) levels in muscle tissue suggest DPZ exposure induced muscle tissue oxidative stress and muscle weakness, which may underlie the locomotor activity impairment. In conclusion, we show, for the first time, that the chronic waterborne exposure of DPZ can severely induce adverse effects on normal zebrafish in a dose-dependent manner. These unexpected adverse effects on behavioral alteration should be carefully addressed in future studies considering DPZ conducted on zebrafish or other animals.

To investigate the effect of montmorillonite on nonylphenol enrichment in a zebrafish model. The AB strain zebrafish were used as the animal subjects and three concentration gradients were set for both nonylphenol and montmorillonite, according to their actual concentrations in aquaculture water in Huzhou city. A group treated with nonylphenol alone was also set, adding up to 12 experimental groups. Concentrations of nonylphenol enriched in the liver, muscle, and gill of zebrafish were detected by solid phase microextraction–high performance liquid chromatography at Day 7, 15, and 30, respectively. Besides, the relative enzymatic activity of Superoxide dismutase (SOD) and the Glutathione S-transferase (GST) were also been detected, and the data were statistically analyzed. The results showed that the concentrations of nonylphenol in zebrafish peaked at Day 7 and gradually decreased afterwards for all the experimental groups. And the montmorillonite reduces short-term accumulation of nonylphenol in gills, and the high concentration of nonylphenol facilitates its enrichment in liver and muscle while the low concentration of nonylphenol doesn’t. Meanwhile, the low concentration of nonylphenol in liver exerts an influence on the inductive effect of SOD and GST while the high concentration of nonylphenol shows the inhibiting effect of SOD and GST.

Currently, drug screening is primarily based on human cell culture for initial high-throughput screening, and subsequently, rodent model to confirm the biological effects. However, the mammalian system is known for time-consuming and highly-cost to be difficult to perform high-throughput drug screening, which exists a critical gap between in vitro cell-based models and the in vivo mammalian models. Therefore, the zebrafish could bridge this gap in preclinical toxicity screening along the drug development pipeline because of its efficiency. We aimed to develop an in vivo zebrafish platform for rapid drug screening. Zebrafish, due to its high genomic conservation with mammals and rapid development and differentiation, it has many advantages, such as short life span, large number of offspring and low cost, easy manipulation for generating transgenic species, to serve as animal model for disease-based research. In 96-well microplates, zebrafish embryos were incubated with small molecular compounds that affected bone mineralization. The level of osteogenic mineralization was evaluated by fluorescent dye staining and quantified by image analysis software. Quantitative real time-PCR (qRT-PCR) was performed to evaluate the biological pathways involved in bone metabolism at the molecular level. The system was validated by demonstrating that response to alendronate and Dorsomorphin in zebrafish. In our study, we screened for 24 compounds within the CYCU-1120~1152 chemical library and identified 3 compounds, pentamidine (CYCU-1140), BML-267 (CYCU-1147), and alendronate (CYCU-1152), increased embryonic mineralization; while 6 compounds, RWJ-60475 (CYCU-1126), levamisole HCL (CYCU-1128), tetramisole HCL (CYCU-1129), fenvalerate (CYCU-1132), NSC-663284 (CYCU-1138), and BML-267ester (CYCU-1148), were inhibitory to bone mineralization. We also found that alendronate enhanced the level of bone mineralization by inhibiting osteoclast-related genes. To sum up, our research showed that zebrafish may have potential to be a drug-screening and mechanism-analysis platform for bone mineralization.

Embryonic and larval Danio rerio is increasingly used as a toxicological model to conduct rapid in vivo tests and developmental toxicity assays; the zebrafish features as high genetic homology to mammals; robust phenotypes; and its value in high-throughput genetic and chemical screening have made it a powerful tool to evaluate in vivo toxicity. New methodologies of genome editing as CRISPR/Cas9; ZFN or Talen make it a suitable model to perform studies to pair human genetic diseases as well. This review surveys recent studies; employing zebrafish as experimental model; comparing it with other in vivo and in vitro models; presenting zebrafish as a potent vertebrate tool to evaluate drug toxicity to facilitate more extensive; easy and comprehensive knowledge of new generation drugs.

Cholera is a diarrheal disease prevalent in populations without access to clean water. Cholera is caused by Vibrio cholerae, which colonizes the upper small intestine in humans once ingested. A growing number of studies suggests the gut microbiome composition modulates animal behavior. Zebrafish are an established cholera model that can maintain a complex, mature gut microbiome during infection. Larval zebrafish, which have immature gut microbiomes, provide the advantage of high-throughput analyses for established behavioral models. We identified the effects of V. cholerae O1 El Tor C6706 colonization at 5 days post-fertilization (dpf) on larval zebrafish behavior by tracking startle responses at 10 dpf. We also characterized the larval gut microbiome using 16S rRNA sequencing. V. cholerae-infected or uninfected control groups were exposed to either an alternating light/dark stimuli or a single-tap stimulus, and average distance and velocity were tracked. While there was no significant difference in the light/dark trial, we report a significant decrease in distance moved for C6706-colonized larvae during the single-tap trial. This suggests that early V. cholerae colonization of the larval gut microbiome has a dampening effect on sensorimotor function, supporting the idea of a link between the gut microbiome and behavior.

Arrhythmogenic cardiomyopathy (AC) is a hereditary cardiac disorder characterized by the gradual replacement of cardiomyocytes with fibrous and adipose tissue, leading to ventricular wall thinning, chamber dilation, arrhythmias, and sudden cardiac death. Despite advances in treatment, disease management remains challenging. Animal models, particularly mice and zebrafish, have become invaluable tools for understanding AC's pathophysiology and testing potential therapies. Mice models, although useful for scientific research, cannot fully replicate the complexity of the human AC. However, they have provided valuable insights into gene involvement, signalling pathways, and disease progression. Zebrafish offer a promising alternative to mammalian models, despite the phylogenetic distance, due to their economic and genetic advantages. By combining animal models with in vitro studies, researchers can comprehensively understand AC, paving the way for more effective treatments and interventions for patients and improving their quality of life and prognosis.

Anxiety is a multifactorial pathology associated with oxidative stress and changes in different neurotransmitters in the central nervous system (CNS). Sarcomphalus joazeiro (Rhamnaceae) has a predicted effect on the CNS, being considered an alternative for pre-clinical investigations of anxiolytic drugs. The objective of this study is to evaluate the chemical composition, analyze the antioxidant capacity and the anxiolytic effect of the ethanolic extract of S. joazeiro leaves (EEFSJ) in zebrafish. The chemical profile was analyzed by liquid chromatography coupled to mass spectrometry (UPLC-PDA-ESI-QDA). Antioxidant action by DPPH• elimination and ABTS•⁺ capture assays. The 96 h acute toxicity and open field and light and dark tests, for sedative and anxiolytic evaluation, respectively, were applied in vivo, using zebrafish. It was possible to identify 11 compounds from the flavonoid and saponin class. EEFSJ exhibited a mean inhibitory concentration (IC50) of 185.2 ± 2.2 µg/mL and 74.17 ± 1.5 µg/mL against the DPPH• and ABTS•⁺ radicals, respectively. In vivo, EEFSJ demonstrated reduced locomotor activity and an anxiolytic effect similar to diazepam via GABAergic systems, without presenting toxicity within 96 h. These results suggest the safety of using EEFSJ for the development of clinical trials for the production of new anxiolytic drugs.

In this study, we present data on the effects of condensed (CTs) and hydrolysable (HTs) tannins, polyphenols extracted from plants, at different concentrations on zebrafish development to identify the range of concentrations with toxic effects. Zebrafish embryos were exposed to CTs and HTs at two different concentration ranges (5.0, 10.0, 20.0 μgL-1 and 5.0, 10.0, 20.0 mgL-1) for 72 h. The toxicity parameters were observed up to 72 h of treatment. The uptake of CTs and HTs by zebrafish larvae was assessed by HPLC analysis. The qRT-PCR analysis was performed to evaluate gene expression of cd63, zhe1 and klf4, involved in the hatching process of zebrafish. CTs and HTs at 5.0, 10.0, and 20.0 μgL-1 were not toxic. On the contrary, at 5.0, 10.0, and 20.0 mgL-1 HTs induced a delayed in hatching starting from 48 h of treatment, while CTs showed a delayed in hatching mainly at 48h. The analysis of gene expression showed a down-regulation in the group exposed to HTs confirming the hatching data. We believe that this study is important to define the optimal doses of CTs and HTs, to be employed in different application fields such as chemical and animal feed industry, and medical science.

Inflammation is natural protective response of the body tissue caused by various stimuli. Macrophages start and control the inflammatory response. Microalgae have high value of ingredients including carotenoids which are well known for anti-inflammatory properties and used for various products in functional food industries. A green microalgal, Tetraselmis species contains a various type of carotenoids such as lutein, violaxanthin, astaxanthin, fucoxanthin, α-carotene and β-carotene. Therefore, we studied whether carotenoids isolated from Tetraselmis sp. with different culture conditions in Raw 264.7 cells and zebrafish (Danio rerio) have an anti -inflammatory effect. Carotenoid extracts of natural seawater (NS) and magma seawater (MS) showed inhibitory activity of nitric oxide (NO) overproduction without toxicity. In addition, carotenoid extracts of NS and MS inhibited pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)- 1β and IL-6. Inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, which are inflammatory mediators also decreased by pretreatment of NS and MS. Carotenoid extracts of NS and MS pretreatment also reduced phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kB (NF-kB). In conclusion, the carotenoid extract of Tetraselmis sp. has an anti -inflammatory effect and can be potential industrial ingredient.

Background: Lately, the high incidence of pesticide usage has attracted everyone's interest due to the serious effects produced. Fipronil (FIP) is a phenylpyrazole compound that acts on the insect’s GABA neurotransmitter by inhibiting its activity. Moreover, literature reports highlight its impli-cation in neurodevelopmental abnormalities and oxidative stress production in different organisms. Similarly, pyriproxyfen (PYR) is known to affect insects’ activity by mimicking the natural hormones involved in the maturation of the young insects. The aim of the present study was to investigate the impact of these pesticides’ mixture on zebrafish tissues and behavior. Methods: To assess the in-fluence of this cocktail on zebrafish, three groups of animals were randomly selected and exposed to 0, 0.05 and 0.1 mg L-1 FIP and PYR mixture for 5 days. The fish were evaluated daily by the T-maze tests as locomotor activity and dark-light test and recorded for 4 minutes. The data was quantified using the EthoVision software. Results: Our results indicated significant changes in locomotor ac-tivity parameters showing increased levels following exposure to the mixture of FIP and PYR. On the other hand, the mixture also triggered anxiety in the zebrafish, which spent more time in the light area than in the dark. In addition, mixture-induced histological changes were observed in the form of numerous hemosiderin deposits found in various zebrafish tissues. Conclusions: The cur-rent findings indicate that the mixture of FIP and PYR can have considerable consequences on adult zebrafish and may promote or cause functional neurological changes in addition to histological ones.

Artificial sweeteners are widely used food ingredients in beverages and drinks to lower calorie intake which in turn prevent lifestyle diseases such as obesity. Epidemiological evidences suggest that an overdose of artificial sweeteners could result to adverse effects after consumption. Thus, our study aims to systematically explore the potential adverse effects of eight commercial artificial sweeteners, including acesulfame-K, alitame, aspartame, sodium cyclamate, dulcin, neotame, saccharin and sucralose on cardiac performances of zebrafish (Danio rerio) and Daphnia as model animals. Embryonic zebrafish and Daphnia were exposed to eight artificial sweeteners at 100 ppb concentrations and their cardiac performance (heart rate, ejection fraction, fractional shortening, stroke volume, cardiac output and heartbeat regularity) were measured and compared. Saccharin significantly increased the heart rate of zebrafish larvae while a significant decrease was observed in Daphnia. Significant increase was also noted in zebrafish heart rate variability after incubation in acesulfame K, dulcin, sodium cyclamate, and sucralose. However, a significant increase in Daphnia was only observed after incubation in dulcin. Based on Principal Component Analysis (PCA) and hierarchical clustering results, several artificial sweetener samples were species-specific to zebrafish and Daphnia. Our study demonstrates the potential adverse physiological effects of artificial sweeteners in cardiovascular systems of zebrafish larvae and Daphnia.

Nitric oxide has been implicated in symptoms of ethanol withdrawal in animal models. Zebrafish have been used as models to study neurobehavioral effects of ethanol (EtOH) withdrawal, but the mechanisms associated with these effects are not yet clear. Adult zebrafish were treated with 1% EtOH for 20 min per day for 8 days, injected with the nitric oxide synthase 2 (NOS-2) inhibitor aminoguanidine (50 mg/kg), and allowed to experience withdrawal (WD) in their hometanks for 7 days. EtOH WD increased anxiety-like behavior in the novel tank test, an effect that was blocked by aminoguanidine. EtOH WD also increased brain levels of nitrite, an effect that was partially blocked by aminoguanidine. These results underline a novel mechanism by which NOS-2 controls anxiety-like responses to ethanol withdrawal, with implications for the mechanistic study of symptoms associated with chronic ethanol abuse.

Fear can sometimes paralyze us, and it can sometimes be exciting; for some people, fear is so crippling it can significantly mix up their lifes! We understand a little bit about how the brain acts when we are afraid, mainly by studying the brains of animals. Recently, surprising findings were made using a humble animal, the zebrafish – a small aquarium fish that in the past has helped scientists figure out how our organs develop. Zebrafish are useful because they develop quickly, reproduce richly, and have brains which are similar to ours. They also produce what we call an “alarm substance” that alerts shoalmates when one of them has been injured; when they smell this substance in the water they act as if they are very scared. When this happens, they release serotonin in their brains, a neurotransmitter that acts as a light switch, making them less afraid but more cautious – as if trying to figure out if a predator is there or not. Hopefully, finding more about how the zebrafish brains process this serotonin signal can help scientists develop better treatments for mental disorders that are associated with fear.

Though the cancer research community has used mouse xenografts for decades more than zebrafish xenografts, zebrafish have much to offer: they are cheap, easy to work with, and the embryonic model is relatively easy to use in high-throughput assays. Zebrafish can be imaged live, allowing us to observe cellular and molecular processes in vivo in real time. Opponents dismiss the zebrafish model due to the evolutionary distance between zebrafish and humans, as compared to mice, but proponents argue for the zebrafish xenograft’s superiority to cell culture systems and its advantages in imaging. This review places the zebrafish xenograft in the context of current views on cancer and gives an overview of how several aspects of this evolutionary disease can be addressed in the zebrafish model. Zebrafish are missing homologs of some human proteins and (of particular interest) several members of the matrix metalloproteinase (MMP) family of proteases, which are known for their importance in tumour biology. This review draws attention to the implicit evolutionary experiment taking place when the molecular ecology of the xenograft host is significantly different than that of the donor.

MFGE8 is a major exosome (EV) protein known to mediate inflammation and atherosclerosis in type 2 diabetes mellitus (T2DM) in animal studies. However, its role in the pathophysiology of human T2DM, obesity, and cardiovascular disease has been controversial and less explored. Earlier, we reported a rare Asian Indian population-specific missense variant (rs371227978; Arg148His) in the MFGE8 associated with increased circulating Mfge8 and T2DM. Here, we have further investigated the role of Mfge8 with T2DM risk in additional Asian Indians (n=4917) and Europeans and other multiethnic cohorts from UK Biobank (UKBB) (n= 455,808) and the US (n=1150). We also evaluated the exposure of Mfge8-enriched human EVs (with and without mutation) in zebrafish (ZF) for their impact on the cardiometabolic organ system. Most individual carriers of Arg148His variant not only had high circulating Mfge8 but also showed a positive significant correlation with glucose (r= 0.42; p = 4.9x10-04), while the non-carriers showed a negative correlation of Mfge8 with glucose (r = -0.38; p = 0.001) in Asian Indians. However, the same variant was monomorphic in Europeans and other ethnic groups of UKBB and US cohorts. Even in the absence of Arg148His variant, serum Mfge8 correlated significantly with blood glucose both in Caucasians (r = 0.56; p = 8.4x10-10) and individuals from other ethnicities (African Americans, Hispanics, and others) (r = 0.31; p = 5.8x10-4). Since Mfge8 is an EV marker, the exposure of Mfge8-enriched human EVs to ZF larvae rapidly developed fatty liver disease and heart hypertrophy and exhibited redundant growth with poor muscular architecture with and without the high-fat diet (HFD), while the control group fishes developed fatty liver disease and heart hypertrophy only after the HFD feeding. With strong evidence from animal studies supporting the role of Mfge8 in obesity, insulin resistance, and the development of atherosclerosis in T2DM, the current research suggests that circulating Mfge8 may be a potential marker for preventing or reducing the risk of T2DM and cardiovascular disease.

Oleanolic acid (OA) being the ubiquitous compound in the plant kingdom is studied for both the neuroprotective and neurotoxic properties. The mechanism of acetylcholinesterase (AChE) inhibitory potential of OA is investigated using the molecular dynamic simulations (MD) and docking as well as membrane-like and in vitro tests. There are also applied SH-SY5Y human neuroblastoma cells as well as in vivo on the zebrafish tests. The inhibitory potential towards the AChE enzyme is examined using the TLC-bioautography assay (the IC50 value is 9.22 μM). The CH- interactions between the central fragment of the ligand molecule and the aromatic cluster created by the His440, Phe288, Phe290, Phe330, Phe331, Tyr121, Tyr334, Trp84 and Trp279 side chains are observed. The in vitro tests carried out on the SH-SY5Y cells indicates that the viability rate is reduced to 71.5%, 61%, and 43% at the concentrations of 100 µg/mL, 300 µg/mL, and 1000 µg/mL, respectively after 48 hours of incubation whereas cytotoxicity against the tested cell line with the IC50 value is 714.32 ± 32.40 µg/mL. The in vivo tests on the zebrafish prove that there is no difference between the control and experimental groups as regards the mortality rate and morphology (P>0.05).

Endophytic fungi are those that present part of their life cycle in healthy tissues of different plant hosts in symbiosis without causing harm. At the same time, fungus-plant symbiosis makes it possible for microorganisms to synthesize their own bioactive secondary metabolites while in stationary stage. Such microorganism-derived secondary metabolites contribute. Therefore, this work aimed to. To accomplish this, the endophytic fungus Trichoderma asperellum was isolated from Bertholletia excelsa (Brazil nut) almonds. The fungus was cultivated and extracted with ethyl acetate, obtaining AM07Ac. Then, using HPTLC (High-performance thin-layer chromatography) and Nuclear Magnetic Resonance (1H NMR), β-amyrin, kaempferol, and brucine were identified as major compounds. Further in vivo assays in Zebrafish demonstrated the activity of AM07Ac on melanogenesis by producing a concentration-response inhibitory effect, which, through an in silico study, proved to be related to the noted major compounds known to inhibit tyrosinase activity. The inhibition of tyrosinase prevents melanin accumulation in skin. Therefore, these results imply the importance of investigating microorganisms and their pharmacological activities, in particular the endophytic fungus Trichoderma asperellum as a generator of active metabolites for melanogenesis modulation.

5G technology is evolving to satisfy several service requirements favoring high data-rate connections and lower latency times than current ones (< 1ms). 5G systems use different frequency bands of the radio wave spectrum, taking advantage of higher frequencies than previous mobile radio generations. In order to guarantee a capillary coverage of the territory for high reliability applications, it will be necessary to install a large number of repeaters because higher frequencies waves have a lower capacity to propagate in free space. Following the introduction of this new technology, there has been a growing concern about possible harmful effects on human health. The aim of this study is investigating possible short term effects induced by 5G-millimeter waves on embryonic development of Danio rerio. We have exposed fertilized eggs to 27 GHz frequency, 9.7 mW/cm2 incident power density, 23 dbm and have measured several endpoints every 24 hours. The exposure to electromagnetic fields at 27 GHz (5G) caused no significant impacts on mortality nor on morphology because the exposed larvae showed a normal detachment of the tail, presence of heart-beat and well-organised somites. A weak positivity on exposed larvae has been highlighted by immunohistochemical analysis.

Folic acid, one of the 13 essential vitamins, plays an important role in cardiovascular development. Mutations in folic acid synthesis gene 5,10-methylenetetrahydrofolate reductase (MTHFR) is significantly associated with the occurrence of congenital heart disease. However, the mechanisms underlying the regulation of cardiac development by mthfr gene are poorly understood. Here, we exposed zebrafish embryos to excessive folate or folate metabolism inhibitors. And we established a knock-out mutant of mthfr gene in zebrafish by using CRISPR/Cas9. The zebrafish embryos of insufficient or excessive folic acid, and mthfr-/- mutant all gave rise to early pericardial edema and cardiac defect at 3 days after fertilization(dpf). Furthermore, the folic acid treated embryos showed abnormal movement at 5dpf. The expression levels of cardiac marker genes hand2, gata4 and nppa changed in the abnormality of folate metabolism embryos and mthfr-/- mutant, and there is evidence that they are related to the change of methylation level caused by the change of folate metabolism. In conclusion, our study provides a novel model for the in-depth study of MTHFR gene and folate metabolism. And our results reveal that folic acid has a dose-dependent biphasic effect on early cardiac development.

Zebrafish is an excellent model for observing human genetic disorders. Hearing impairment is the most common genetic disorder including syndromic & non-syndromic hearing loss. Stem cell therapies are considered a new hope in case of hearing impairment. Stem cells are the master cells of the human body and having the capability to differentiate into any other form of cells in more than 200 types of cells (regeneration). This review article describes zebrafish as a biomedical model for stem cell research in hearing impairment, which revolutionized the biomedical arena to compete for the challenges.

CRISPR/Cas genome editing is a widely used research technology. Its simplest variant is gene knockout resulting from reparation errors after introduction of dsDNA breaks by Cas nuclease. We compared the outcomes of the break repair by two commonly used nucleases (SpCas9 and LbCas12a) in zebrafish embryos to reveal if application of one nuclease is advantageous in comparison to the other. To address this question, we injected ribonucleoprotein complexes of nucleases and corresponding guide RNAs in zebrafish zygotes and three days later sequenced the target gene regions. We found that LbCas12a breaks resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies of both nucleases were the same. On the other hand, overlapping protospacers were shown to lead to similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence.

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

Gestational Diabetes Mellitus (GDMs) is worldly wide pregnancy complication. Gestational Diabetes can significantly impact the fetus development. However, the effects of high glucose on embryological development post-fertilization are yet to be researched. Danio rerio embryos are great model to study the embryonic development. In this study, the effects on embryological (morphological and genetic) development were examined in the presence of a high glucose environment that mimic the developing fetus in pregnant women with GMD. Fertilized Zebrafish embryos were treated with normal media and high glucose for 5 days from 3 hours post-fertilization(hpf) to 96hpf respectively as control and experimental groups. Morphological changes are recorded with Microscope images. Hatch rate and heart rate are compared between groups in set up time points. RNA-Seq is performed to examine the gene changes in experimental group. Glucose delayed the zebrafish embryo development by slowing the hatch rate about 24 hours. The brain, heart, and tail started showing morphology smaller in the glucose group compared to the control group at 24 hpf. Heart rate is faster in the glucose group compared to the control group on day 2 and 3 with a statistically significant difference. Among zebrafish whole genome, the significantly changed genes are 556 genes up-regulated and 1118 genes down-regulated respectively in high glucose group. The metabolic and Wnt pathways are altered under high glucose condition. These conditions contribute to significant physiological differences that may provide insight on functionality post-embryological development.

Environmental chemicals, including PFAS (per- and polyfluoroalkyl substances), pesticides, industrial chemicals, and consumer products, commonly exist as mixtures. These substances are frequently exposed or co-exposed in varying concentrations, leading to potentially hazardous health effects such as cancer in humans. Thus, understanding the dose-dependent toxicity of chemical mixtures is important for assessing health risks. In this context, comprehensive methods for assessing the toxicity and identifying the mechanisms of harmful chemical mixtures are currently lacking. Here, the dose-dependent toxicity assessments of chemical mixtures are performed in three methodologically distinct phases. In the first phase, we evaluated our machine learning method (AI-HNN) and pathophysiology method (CPTM) for predicting toxicity. In the second phase, we integrated AI-HNN and CPTM to establish a comprehensive new approach method (NAM) framework called AI-CPTM, targeted at refining prediction accuracy and providing a comprehensive understanding of toxicity mechanisms. The third phase involved experimental validations of the AI-CPTM predictions. Initially, we developed binary, multiclass classification, and regression models to predict binary, categorical toxicity, and toxic potencies, using nearly a thousand experimental mixtures. This empirical dataset was expanded with virtual mixtures compensating the lack of experimental data and broadening the scope of the dataset. For comparison, we also developed additional machine learning models based on RF, Bagging, AdaBoost, SVR, GB, KR, DT, KN, and Consensus methods. The models achieved overall accuracies of over 80% with AUC values exceeding 90%. The regression models achieved an R2 >0.88. In the second phase, we innovatively integrated HNN-derived toxicity predictions with Z-scores from CPTM, resulting method called AI-CPTM. In the final phase, we demonstrated the superior performance of AI-CPTM through rigorous literature and statistical performance validations. Additionally, the predictive capability of AI-CPTM, including for PFAS mixtures and their interaction effects, was demonstrated by experimental validations using dose-response zebrafish embryo toxicity assays. Overall, the AI-CPTM approach significantly improves upon the limitations of standalone models and has shown extensive enrichments in the identification of toxic chemicals and mixtures. Further experimental studies involving human cell models, patient-derived xenografts, and investigations into the toxicity of multiple mixtures are currently underway.

The etiology of Autism Spectrum Disorder (ASD) has been linked to both genetic and epigenetic factors. Among the epigenetic factors, exposure to valproic acid (VPA), an antiepileptic and mood-modulating drug, has been shown to induce characteristic traits of ASD when exposed during embryogenesis. Conversely, in animal models, Enriched Environment (EE) has demonstrated positive behavioral and neural effects, suggesting its potential as a complementary treatment to pharmacological approaches in central nervous system disorders. In this study, we utilized zebrafish to model ASD characteristics induced by VPA and hypothesized that sensory stimulation through EE could ameliorate the behavioral and neuroanatomical features associated with ASD. To test this hypothesis, we assessed social behavior, cerebellar volume, and Purkinje cell populations via histology and immunohistochemistry after exposing the fish to EE. The results revealed that zebrafish exposed to VPA exhibited social deficits, reduced cerebellar cortex volume, and a decrease in c-Fos-positive cells in the Purkinje layer. In contrast, VPA-exposed fish treated with EE showed increased socialization, augmented cerebellar cortex volume, and an elevation in c-Fos-positive Purkinje cells. These findings suggest that alterations induced by VPA may be ameliorated through EE treatment, highlighting the potential therapeutic impact of sensory stimulation in conditions related to ASD.

Bone Morphogenetic Proteins (BMPs) are a group of structurally and functionally related signaling molecules that comprise a subfamily, belonging to the TGF-β superfamily. Most BMPs play roles in the regulation of embryonic development, stem cell differentiation, tumor growth and some cardiovascular and cerebrovascular diseases. Although evidences are emerging for the antiviral immunity of a few BMPs, more BMPs are needed to determine whether this function is universal. Here we identified the zebrafish bmp4 ortholog, whose expression is up-regulated by challenge with virus or its mimic poly(I:C). Overexpression of bmp4 in EPC cells significantly decreased the viral titer of GCRV-infected cells. Moreover, compared to wild type zebrafish, viral load and mortality were significantly increased in both larvae and adults of bmp4-/- mutant zebrafish infected with GCRV virus. We further demonstrated that Bmp4 promotes the phosphorylation of Tbk1 and Irf3 through p38 MAPK pathway, thereby inducing the production of type I IFNs in response to virus infection. These data suggest that Bmp4 runs an important role in the host defense against virus infection. Our study expands the understanding of BMP protein functions and opens up new targets for the control of viral infection.

Idiopathic scoliosis (IS) is an unexplained dysplasia of the spine that can occur at any age, with idiopathic scoliosis making up the largest proportion of the total population worldwide, approximately 2-3%. Scoliosis is not just a cosmetic defect, but the development of the spinal deformity can lead to cardiovascular and respiratory lesions, often resulting in serious health problems. Treatment of the condition usually involves major surgery, which is both a physical and financial burden for patients. In order to clarify the aetiology of IS and provide a theoretical basis for new diagnostic and treatment methods IS, it is important to use different animal models for experiments. Zebrafish is an emerging model animal with a short reproductive cycle, minimal breeding expenses, and other beneficial traits including in vitro fertilisation, in vitro development, and embryo transparency. As a result, genetic alteration and observation are simpler than with traditional model animals. This study examines the history of animal models for IS research, focuses on the benefits and drawbacks of zebrafish as an IS model and the advances it provides to IS research, and anticipates zebrafish application prospects in IS research.

Rice bran (RB) is a promising food ingredient that can improve biological function. In this study, we investigated the effects of RB, both unfermented (RB30) and fermented (RBF30) with five different microorganisms on the neurobehavioral activity in zebrafish larvae. Analytical methods such as UV spectrum and MS scan were performed. Interestingly, niacin content, which is known to improve brain functions such as cognition and emotion, was found to be higher in RBF30 than in RB30. Furthermore, niacin content was highly increased in the RBF30-exposed fish, compared to those in control fish. Therefore, we profiled behavioral patterns and various neurochemistry in zebrafish larvae following supplementation with RBF30 as well as performed calcium imaging on Tg (huC:GAL4-VP16);(UAS:GCaMP7a) zebrafish larvae to determine the correlation of neural activity. RBF30 revealed greater stimulation of locomotor activity without negatively affecting decision-making behavior in zebrafish larvae, as compared to RB30 or niacin. Its behavioral activation is mainly linked with the elevations of neural activity and several neurochemicals such as serotonergic and dopaminergic systems that are implicated in the control of anxiety and stress. Taken together, these results suggest that RBF30 could be a food material that improve the behavioral health by modulating neural activity and brain neurochemistry.

Melanoma is a type of highly malignant and metastatic skin cancer. In situ molecular imaging of endogenous levels of the melanoma biomarker tyrosinase (TYR) may decrease the likelihood of mortality. In this study we proposed the weakly fluorescent probe 1-(4-(2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)phenyl)-3-(4-hydroxybenzyl)urea (DCM-HBU), which releases a strong red-shifted fluorescent signal after a TYR-mediated oxidation followed by hydrolysis of the urea linkage. The large Stokes shift of the dye is owed to the recovery of the intramolecular charge transfer (ICT) effect. The resulting probe derivate shows a highly ratiometric fluorescence output. Furthermore, the simultaneous excitation by two near-infrared (NIR) photons of the released DCM-NH2 fluorophore could avoid the usual drawbacks, such as cellular absorption, autofluorescence and light scattering, due to an usually short wavelength of the excitation light on biological systems, resulting in images with deeper tissue penetration. In addition, the probe is useful for the quantitative sensing of TYR activity in vivo, as demonstrated in zebrafish larvae. This new ratiometric two photon NIR fluorescent probe is expected to be useful for the accurate detection of TYR in complex biosystems at greater depths than other one-photon excited fluorescent probes.

Recently, fasting has been spotlighted from a healthcare perspective. However, the de-tailed biological mechanisms and significance by which the effects of fasting confer health benefits are not yet clear. Due to certain advantages of zebrafish, as a vertebrate model widely utilized in biological studies, we used mRNA-sequencing and bioinformatics analysis to examine comprehensive gene expression changes in skeletal muscle tissues during fasting-refeeding. Our results produced a novel set of nutrition-related genes under a fasting-refeeding protocol. We found five dramatically upregulated genes in each fasting (for 24 hours) and refeeding (after 3 hours), exhibiting a rapid response to the provided conditional changes. The assessment of the gene length revealed, the gene set whose expression was elevated only after 3 hours of refeeding had a shorter length, suggesting that nutrition-related gene function is associated with gene length. Taken together, our results from bioinformatics analyses provide new insights into biological mechanisms induced by fasting-refeeding conditions within zebrafish skeletal muscle.

Hearing dysfunctions can be classified by type, degree, configuration, time of onset, aetiology, and finally, consequences on speech development. They can be divided into conductive, mixed, central types and sensorineural. Conductive hearing loss (CHL) results from interference with the mechanical transmission of sound through the external and middle ear; it can be congenital, as a consequence of anatomic abnormalities, but it can commonly be acquired following middle ear inflammatory pathologies. Sensorineural hearing loss (SNHL) results from failure to transduce vibrations to neural impulses in the cochlean and is a consequence of an irreversible damage to the differentiated cells which make up the organ of hearing and the acoustic paths at various levels. Mixed hearing loss involves a combination of these two types in the same ear. Studies in neuroscience field have shown that the prevention of cell degeneration is only possible if all the factors taken at the different stages of stem cells’ multiplication and differentiation are administered together. We have demonstrated this in a recent study on the ability of SCDSFs to prevent neurodegeneration in hippocampal cells of the CA1 zone in mice. This study confirms previous findings demonstrating that early developmental zebrafish embryo extracts could act as a modulator of senescence in human mesenchymal stem cells (hMSC) isolated from many adult tissues. These findings have open a promising way for the approaches promoting the rejuvenation and regeneration of different tissues, by-passing stem cell transplantation. In the present clinical trial we have used SCDSFs to study the possible reversion of neurosensory hearing loss, until now considered an irreversible condition.

Background: Zebrafish (Danio rerio) is a model organism emerged for the study of human cancer. Compared with the murine model, the zebrafish model has several properties ideal for personalized therapies. The transparency of the zebrafish embryos and the development of the pigment-deficient “casper” zebrafish line give the capacity to directly observe cancer formation and progression in the living animal. Automatic quantification of cellular proliferation in vivo is critical to the development of personalized medicine. Methods: A new methodology was defined to automatically quantify the cancer cellular evolution. ZFTool was developed to establish a base threshold that eliminates the embryo auto-fluorescence and automatically measures the area and intensity of GFP (green-fluorescent protein) marked cells and define a proliferation index. Results: Proliferation index automatically computed on different targets demonstrates the efficiency of ZFTool to provide a good automatic quantification of cancer cell evolution and dissemination. Conclusion: Our results demonstrate that ZFTool is a reliable tool for the automatic quantification of the proliferation index, being a measure of cancer mass evolution in zebrafish eliminating the influence of its autofluorescence.

CYP1B1 loss-of-function (LoF) is the main known genetic alteration present in recessive primary congenital glaucoma (PCG), an infrequent disease characterized by delayed embryonic development of the ocular iridocorneal angle and caused by poorly understood molecular mechanisms. To model CYP1B1 LoF underlying PCG, we developed a cyp1b1 knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries the c.535_667del frameshift mutation that results in a 72% mRNA reduction with residual mRNA predicted to produce an inactive truncated protein (p.(His179Glyfs*6)). Craniofacial defects and jaw maldevelopment were observed in 23% of somatic mosaic F0 crispant larvae (144 hpf). These early phenotypes were not detected in KO F3 larvae (144 hpf) but 27% of adult fishes (4 months) showed uni or bilateral craniofacial alterations, indicating the existence of incomplete penetrance and variable expressivity. These phenotypes increased to 86% in the adult offspring of inbred progenitors with craniofacial defects. No glaucoma-related phenotypes were observed in the cyp1b1 mutants. Transcriptomic analyses of the offspring (7dpf) of KO cyp1b1 progenitors with adult-onset craniofacial defects revealed that differentially expressed genes were functionally enriched in groups related with extracellular matrix and cell adhesion, cell growth and proliferation, lipid metabolism (retinoids, steroids, and fatty acids, and oxidation-reduction processes which included several cytochrome P450 genes) and inflammation. In summary, this study shows the complexity of phenotypes and molecular pathways associated with cyp1b1 LoF, with species-dependency, and provides evidence for dysregulation of extracellular matrix gene expression as one of the mechanisms underlaying pathogenicity associated with cyp1b1 disruption.

The capacity of 3D printing (3DP) technologies to initiate speedy polymerization of solvent free resins accounts for their utility in the manufacturing of medical devices. Nonetheless, independent biological evaluation of 3D printed materials is recommended due to the unique parameters of the manufacturing process, which can influence their physical, chemical, and biological properties. In this study, E-Shell 450 material indicated for 3DP of hearing aid shells and inner ear devices was examined for biological safety using zebrafish bioassays adapted to OECD fish embryo test. In addition, the proprietary material was characterized for composition using headspace gas-chromatography mass spectrometry (GC-MS). To initiate test, newly fertilized zebrafish eggs were cultured on non-treated and ethanol-treated materials in glass petri dishes with ultrapure water, incubated at 28.5°C and assessed for developmental endpoints of toxicity at 24h interval until 96h. Data confirmed non-treated material was extremely toxic in bioassays within 24h whereas ethanol-treated material showed a relative lower toxicity possibly due to ethanoic-aqueous interactions as observed by GC-MS. With the current influx of 3D printing materials, users are urged to exercise caution. Operators must also take cognizance of the potential toxicity of the chemicals used in 3DP and implement safety measures to limit their exposure.

Ozonated sunflower oil (OSO) is renowned for its diverse therapeutic benefits. Nonetheless, the consequences of extended dietary intake of OSO have yet to be thoroughly investigated. Herein, the effect of 2-year dietary supplementation of OSO was examined on the zebrafish survivability, obesity, skeletal deformities, swimming behavior, and liver, kidney, ovary, and testis function. Results showed that the zebrafish feed supplemented with 20% (wt/wt) OSO for 2 years emerged with higher survivability and body weight management compared to sunflower oil (SO) and normal diet (ND) supplemented zebrafish. Radio imaging (X-ray) based analysis revealed 2.6o and 15.2o lower spinal curvature in the OSO-supplemented groups than in SO and ND-supplemented groups; consistently, OSO-supplemented zebrafish showed better swimming behavior. The histology analysis of the liver revealed a least fatty liver change and interleukin 6 (IL-6) generation in the OSO-supplemented group. Additionally, a significantly lower level of reactive oxygen species (ROS), apoptotic, and senescent cells were observed in the liver of the OSO-supplemented zebrafish. Also, no adverse effect on the kidney, testis, and ovary morphology was detected during 2 years of OSO consumption. Moreover, lower senescence with diminished ROS and apoptosis was noticed in the kidney and ovary in response to OSO consumption. The OSO supplementation was found to be effective in countering age-associated dyslipidemia by alleviating total cholesterol (TC), triglycerides (TG), low-density lipoproteins (LDL-C) and elevating high-density lipoproteins (HDL-C)/TC levels. Conclusively, prolonged OSO consumption showed no adverse effect on the morphology and functionality of vital organs; in fact, OSO supplementation displayed a protective effect against age-associated detrimental effects on spinal deformities, vital organ functionality, cell senescence, and survivability of zebrafish.

The heterogeneous nature of human breast cancer (HBC) can still lead to therapy inefficacy and high le-thality and new therapeutics are needed as well as new spontaneous animal models to benefit translational HBC research. Dogs are primarily investigated since they spontaneously develop tumors which share many features with human cancers. In recent years, different natural phytochemicals including berberine, a plant alkaloid, have been reported to have antiproliferative activity in vitro in human cancers and in rodent animal models. In this study, we report the antiproliferative activity and mechanism of action of berberine, of its active metabolite berberrubine, and of eight analogues, on a canine mammary carcinoma cell line and in transgenic zebrafish models. We demonstrate both in vitro and in vivo the significant effects of specific analogues on cell viability, via induction of apoptosis, also identifying their role in inhibiting the Wnt/β-catenin pathway and activating the Hippo signals with a downstream reduction of CTGF expres-sion. Particularly the berberine analogues NAX035 and NAX057 show the highest therapeutic efficacy, de-serving further analyses to elucidate their mechanism of action more in detail, and in vivo studies on spon-taneous neoplastic diseases aiming at improving veterinary treatments of cancer as well as translational cancer research.

Heart regeneration after myocardial infarction remains challenging in reconstruction of blood resupply system. Here, we find that in zebrafish heart after resection of the ventricular apex, the local myocardial cells and the clotted blood cells undergo cell remodeling process via cytoplasmic exocytosis and nuclear reorganization within revascularization-based blastema. The regenerative processes are visualized by spatiotemporal expression of three blastema representative factors (alpha-SMA- which marks for fibrogenesis, Flk1for angiogenesis/hematopoiesis, and Pax3a for remusculogensis)，and two histone modifications (H3K9Ac and H3K9Me3 mark for chromatin remodeling). Using the cultured zebrafish embryonic fibroblasts we identify blastema fraction components and show that Krt5 peptide could link cytoskeleton network and BMP4 signaling pathway to regulate the transcription and chromatin accessibility at the blastema representative genes and bmp4 genes. Our study provides new mechanistic insights into the epithelial-dependent and revascularization-based blastema regeneration for potential myocardial infarction therapy.

The zebrafish Danio rerio is suitable to study gametes as a model organism. There were &gt; 70% of zebrafish spermatozoa activated, because they were contaminated with urine or excrement. The movement of spermatozoon in water was propagated along the flagellum at 16 s after sperm activation, then damped from the end of the flagellum for 35 s and fully disappear at 61 s after activation. For artificial fertilization, milt must be added to an extender, which stops the movement of sperm and keeps the sperm motionless until fertilization. E400 was shown to be the most suitable extender as it allows to store sperm for fertilization for 6 to 12 h at 0-2oC. Sperm motility decreased only to 36% at 12 h post stripping (HPS) for E400 extender and to 19% for Kurokura extender. To achieve an optimal level of fertilization and hatching, a test tube with a well-defined amount of 6,000,000 spermatozoa in E400 extender per 100 eggs and 100 µl of activation solution has proved to be more successful than using a Petri dish. The highest fertilization and hatching rates reached 80% and 40-60%, respectively, with milt stored for 1.5 h in E400 extender at 0-2oC.

Primary congenital glaucoma (PCG) is a heterogeneous, inherited, and severe optical neuropathy caused by apoptotic degeneration of the retinal ganglion cell layer. Whole-exome sequencing analysis of one PCG family identified two affected siblings who carried a low-frequency homozygous nonsense GUCA1C variant (c.52G>T/p.Glu18Ter/rs143174402). This gene encodes GCAP3, a member of the guanylate cyclase activating protein family, involved in phototransduction and with a potential role in intraocular pressure regulation. Segregation analysis supported the notion that the variant was coinherited with the disease in an autosomal recessive fashion. GCAP3 was detected immunohistochemically in the adult human ocular ciliary epithelium and retina. To evaluate the ocular effect of GUCA1C loss-of-function, a guca1c knockout zebrafish line was generated by CRISPR/Cas9 genome editing. Immunohistochemistry demonstrated the presence of GCAP3 in the non-pigmented ciliary epithelium and retina of adult wild-type fishes. Knockout animals presented up-regulation of the glial fibrillary acidic protein in Müller cells and evidence of retinal ganglion cell apoptosis, indicating the existence of gliosis and glaucoma-like retinal damage. In summary, our data provide evidence for the role of GUCA1C as a candidate gene in PCG and offer new insights into the function of this gene in the ocular anterior segment and the retina.

Recent research has revealed surprisingly important connections between animals’ microbiome and social behaviour. Social interactions can affect the composition and function of the microbiome; conversely, the microbiome affects social communication by influencing the hosts’ central nervous system and peripheral chemical communication. These discoveries set the stage for novel research venues focusing on the evolution and physiology of animal social behaviour in relation to microbial transmission strategies. Here, we discuss the emerging roles of teleost fish model candidates and their key potential for advancing research fields linked to sociality and microbial regulation. We argue that fish models, such as the zebrafish, sticklebacks, guppies and cleaner-client dyads, will provide valuable insights into the roles of microbiome in shaping social behaviour and vice versa, while also being of direct relevance to the food and ornamental fish trades.

Evodiamine is a bioactive alkaloid that is specified as a biomarker for the quality assessment of Evodia rutaecarpa and for traditional Chinese medicines containing this plant. We previously reported that quantitative structure-activity modeling indicated that evodiamine may cause cardiotoxicity. However, previous investigations have indicated that evodiamine has beneficial effects in patients with cardiovascular diseases and there are no previous in vitro or in vivo reports of evodiamine-induced cardiotoxicity. The present study investigated the effects of evodiamine on primary cultured neonatal rat cardiomyocytes in vitro, and on zebrafish in vivo. Cell viability was reduced in vitro, where evodiamine had a 24-h 50% inhibitory concentration of 28.44 µg/mL. Cells exposed to evodiamine also showed increased lactate dehydrogenase release and maleic dialdehyde levels, and reduced superoxide dismutase activity. In vivo, evodiamine had a 10% lethal concentration of 354 ng/mL and induced cardiac malfunction, as evidenced by changes in heart rate and circulation, and pericardial malformations. This study indicated that evodiamine could cause cardiovascular side effects involving oxidative stress. These findings suggest that cardiac function should be monitored in patients receiving preparations containing evodiamine.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interaction and communication, anxiety, hyperactivity, and restricted interest for specific subjects. In addition to the genetic factors, multiple environmental factors have been related to the development of ASD. Animal models can serve as crucial tools for understanding the complexity of ASD and identifying potential therapeutic strategies. In this study, a chemical model of ASD has been developed in zebrafish by exposing embryos to valproic acid (VPA) from 4 to 48 hours post-fertilization. After the exposure, embryos were transferred to fish water and the effects on behavior and neurotransmitters profile were analyzed at both larval and adult stages. Larvae from VPA-treated embryos showed hyperactivity and decreased visual and vibrational escape responses, as well as an altered neurotransmitters profile, with increased glutamate and decreased acetylcholine and norepinephrine levels. Adults from VPA-treated embryos exhibited impaired social behavior characterized by larger shoal sizes and a decreased interest for their conspecifics. Neurotransmitter analysis revealed a significant decrease in dopamine and GABA levels in the brain. These results support the interest of this model in the study of ASD.

This study unveils verapamil's compelling cytoprotective and proliferative effects on pancreatic β-cells amidst diabetic stressors, spotlighting its unforeseen role in augmenting cholecystokinin (CCK) expression. Through rigorous investigations employing MIN6 β-cells and zebrafish models under type 1 and type 2 diabetic conditions, we demonstrate verapamil's capacity to significantly boost β-cell proliferation, enhance glucose-stimulated insulin secretion, and fortify cellular resil-ience. A pivotal revelation of our research is verapamil's induction of CCK, a peptide hormone known for its role in nutrient digestion and insulin secretion, which signifies a novel pathway through which verapamil exerts its therapeutic effects. Furthermore, our mechanistic insights reveal that verapamil orchestrates a broad spectrum of gene and protein expressions pivotal for β-cell survival and adaptation to immune-metabolic challenges. In vivo validation in a zebrafish larvae model confirms verapamil's efficacy in fostering β-cell recovery post-metronidazole inflic-tion. Collectively, our findings advocate for verapamil's reevaluation as a multifaceted agent in diabetes therapy, highlighting its novel function in CCK upregulation alongside enhancing β-cell proliferation, glucose sensing, and oxidative respiration. This research enriches the therapeutic landscape, proposing verapamil not only as a cytoprotector but also as a promoter of β-cell re-generation, thereby offering fresh avenues for diabetes management strategies aimed at pre-serving and augmenting β-cell functionality.

Ovarian cancer (OC) is an umbrella term for cancerous malignancies affecting the ovaries, yet treatment options for all subtypes are predominantly derived from high-grade serous ovarian cancer, the largest subgroup. The concept of "functional precision medicine" involves gaining personalized insights on therapy choice, based on direct exposure of patient tissues to drugs. This especially holds promise for rare subtypes like low-grade serous ovarian cancer (LGSOC). This study aims to establish an in vivo model for LGSOC using zebrafish embryos, comparing treatment responses previously observed in mouse PDX models, cell lines, and 3D tumor models. To address this goal, a well characterized patient-derived LGSOC cell line with the KRAS mutation c.35 G > T (p.(Gly12Val)) was used. Fluorescently labelled tumor cells were injected into the perivitelline space of 2 days post fertilization zebrafish embryos. At 1 day post-injection xenografts were as-sessed for tumor size, followed by random allocation into treatment groups with trametinib, luminespib, and trametinib + luminespib. Subsequently xenografts were euthanized and analysed for apoptosis and proliferation by confocal microscopy. Tumor cells formed compact tumor masses (n=84) in vivo and with clear Ki67 staining, indicating proliferation. Zebrafish xenografts exhibited sensitivity to trametinib and luminespib, individually or combined, within a two-week period, establishing them as a rapid and complementary tool to existing in vitro and in vivo models for evaluating targeted therapies in LGSOC.

Mutations of PKD1 coding for polycystin-1 (PC1) account for most of the autosomal dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many evolutionarily conserved domains for ligand interactions. Among these is the leucine-rich repeats (LRR) in the far N-terminus of PC1. Using zebrafish (Danio rerio) as an in vivo model system, we explored the role of LRR in the function of PC1. Zebrafish expresses two human PKD1 paralogs, pkd1a and pkd1b. Knockdown of both genes in zebrafish by morpholino antisense oligonucleotides produced phenotypes of dorsal axis curvature and pronephric cyst formation. We found that overexpression of LRR suppressed both phenotypes in pkd1-morphant zebrafish. Purified recombinant LRR domain inhibited proliferation of HEK cells in culture and interacted with the heterotrimeric basement membrane protein laminin-511 (α5β1γ1) in vitro. Mutations of amino acid residues in LRR structurally predicted to bind laminin-511 disrupted LRR-laminin interaction in vitro and neutralized the ability of LRR to inhibit cell proliferation and cystogenesis. Our data support the hypothesis that the extracellular region of PC1 plays a role in modulating PC1 interaction with the extracellular matrix and contributes to cystogenesis of PC1-deficiency.

Triclopyr is an auxin-like herbicide extensively employed to manage weeds in food crops and pastures. Triclopyr has been detected in environmental matrices, with a notable presence within aquatic ecosystems. Given the limited understanding of its fate in the environment, potential implications for environmental and human health, and the lack of adequate monitoring in various environmental compartments, triclopyr is now considered a contaminant of emerging concern (CEC). The present study was undertaken to investigate the impact of triclopyr on zebrafish development, with a specific focus on assessing its embryotoxic, behavioral, and neurotoxic effects at environmentally realistic concentrations ranging from 0.5 to 50 µM on zebrafish embryos. Our results revealed that triclopyr caused sublethal effects on zebrafish embryos, such as delayed hatchability at 50 µM, yolk sac retention at 0.5, 1, 5, and 10 µM, and uninflated swim bladder at 50 µM concentration of triclopyr. Moreover, a subtle alteration in the swimming activity of larvae was observed in the 10 µM triclopyr after 144 hours of exposure. However, the activity of acetylcholinesterase, remained unchanged after triclopyr exposure. Taken together, these comprehensive findings suggest that triclopyr can induce adverse effects on the early development of zebrafish, even at concentrations detected in aquatic environments.

Abstract: Sarcopenia poses a significant challenge to public health and can severely impact the quality of life of aging populations. Despite extensive efforts to study muscle degeneration using traditional animal models, there is still a lack of effective diagnostic tools, precise biomarkers, and treatments for sarcopenia. Zebrafish models have emerged as powerful tools in biomedical research, providing unique insights into age-related muscle disorders like sarcopenia. The advantages of using zebrafish models include their rapid growth outside of the embryo, optical transparency during early developmental stages, high reproductive potential, ease of husbandry, compact size, and genetic tractability. By deepening our understanding of the molecular processes underlying sarcopenia, we may develop novel diagnostic tools and effective treatments that can improve the lives of aging individuals affected by this condition. This review aims to explore the unique advantages of zebrafish as a model for sarcopenia research, highlight recent breakthroughs, outline potential avenues for future investigations, and emphasize the distinctive contributions that zebrafish models offer. Our research endeavors to contribute significantly to addressing the urgent need for practical solutions to the impact of sarcopenia on aging populations, ultimately striving to enhance the quality of life for individuals affected by this condition.

Background: Long QT syndrome (LQTS) can lead to ventricular arrhythmia and sudden cardiac death. The most common congenital cause of LQTS is mutations in the channel subunits generating the cardiac potassium current IKs. Zebrafish (Danio rerio) have been proposed as a powerful system to model human cardiac diseases due to the similar electrical properties of the zebrafish heart and the human heart. Methods: We used high resolution all-optical electrophysiology on ex-vivo zebrafish hearts to assess the effects of IKs analogues on the cardiac action potential. Findings: We found that chromanol 293B (an IKs inhibitor) prolonged the action potential duration (APD) in the presence of E4031 (an IKr inhibitor applied to drug-induced LQT2) and to a lesser extent in the absence of E4031. Moreover, we show that PUFA analogues slightly shorten the APD of the zebrafish heart. However, PUFA analogues failed to reverse the APD prolongation in drug-induced LQT2. However, a more potent IKs activator, ML-277, partially reversed the APD prolongation in drug-induced LQT2 zebrafish hearts. Interpretation: Our results suggest that IKs plays a limited role in ventricular repolarizations in the zebrafish heart under resting conditions but plays a more important role when IKr is compromised, as if IKs in zebrafish serves as a repolarization reserve as in human hearts. This study shows that potent IKs activators can restore the action potential duration in drug-induced LQT2 in zebrafish heart.

Introduction: The combination of Virtual Screening (VS) techniques with in vivo screening in the zebrafish model is currently being used in tandem for drug development in a faster and more efficient way. Areas covered: We review the different virtual screening techniques, the use of zebrafish as a vertebrate model for drug discovery and the synergy that exists between them. Expert opinion: We highlight the advantages of combining virtual and zebrafish larvae screening for drug discovery. On the one hand, VS is a faster and cheaper tool for searching active compounds and possible candidates for therapy than in vivo screening when processing large compound libraries. On the other hand, zebrafish larvae form a vertebrate model which allows in vivo screening of large amounts of the compounds. Importantly physiology and chemical response are mostly conserved between zebrafish and mammals. The availability of the transgenic and mutant zebrafish lines allows an analysis of a specific phenotype upon treatment along with toxicity, off-target effect, side effects, and dosage. Advantages of VS, in vivo whole animal approach screening, and the screening combinations are also reviewed.

Adult neurogenesis is an evolutionary conserved process occurring in all vertebrates. However, striking differences are observed between the taxa, considering the number of neurogenic niches, the neural stem cell (NSC) identity and brain plasticity under constitutive and injury-induced conditions. Zebrafish has become a popular model for the investigation of the molecular and cellular mechanisms involved in adult neurogenesis. Compared to mammals, the adult zebrafish displays a high number of neurogenic niches distributed throughout the brain. Furthermore, it exhibits a strong regenerative capacity without scar formation or any obvious disabilities. In this review, we will first discuss the similarities and differences regarding (i) the distribution of neurogenic niches in the brain of adult zebrafish and mammals (mainly mouse) and (ii) the nature of the neural stem cells within the main telencephalic niches. In the second part, we will describe the cascade of cellular events occurring after telencephalic injury in zebrafish and mouse. Our study clearly shows that most early events happening right after the brain injury are shared between zebrafish and mouse including cell death, microglia and oligodendrocyte recruitment, as well as injury-induced neurogenesis. In mammals one of the consequences following an injury is the formation of a glial scar that is persistent. This is not the case in zebrafish, which may be one of the main reasons that zebrafish display a higher regenerative capacity.

Andean blueberries are wild berries grown and consumed in Ecuador which contain high values of bioactive compounds, mainly anthocyanins, with powerful antioxidant activity. The aim of this study was to evaluate the profile and contents of (poly)phenols and carotenoids in Andean blueberry by HPLC-DAD-MSn and determine a wide range of its biological activities. The antioxidant capacity of this fruit was evaluated in vitro by three different methods and in vivo using the zebrafish animal model, also the toxicity effect was determined by the zebrafish embryogenesis test. Besides, the antimicrobial activity and the capacity of Andean blueberry to produce hemagglutination in blood cells were evaluated. Finally, the bioaccessibility of (poly)phenols and related antioxidant capacity were determined in the different phases of an in vitro digestion. The global results indicated no toxicity of Andean blueberry, weakly bacteriostatic activity, and high contents of anthocyanins and antioxidant capacity, which were partially bioaccesible in vitro (~ 50 % at the final intestinal step), contributing to the knowledge of its health benefits for consumers and its potential use in the food and pharmaceutical industry as functional ingredient.

Dietary contaminants are often an over-looked factor in the health of zebrafish. Typically, water is considered to be the source for most contaminants, especially within an aquatic environment. For this reason, source water for zebrafish recirculating systems is highly regulated and monitored daily. Most facilities use reverse osmosis or de-ionized water filtration systems to purify incoming water to ensure that contaminants, as well as pathogens, do not enter their zebrafish housing units. However, diets are rarely tested for contaminants and, in the case of manufactured zebrafish feeds, since the product is marketed for aquaculture or aquarium use it is assumed that the feed is acceptable for animals used for research. The following provides examples as to how contaminants could lead to negative effects on development and behavior of developing zebrafish.

Social plasticity, defined as the ability to adaptively change the expression of social behavior according to previous experience and to social context, is a key ecological performance trait that should be viewed as crucial for Darwinian fitness. The neural mechanisms for social plasticity are poorly understood, in part due to skewed reliance on rodent models. Fish model organisms are relevant in the field of social plasticity for at least two reasons: first, the diversity of social organization among fish species is staggering, increasing the breadth of evolutionary relevant questions that can be asked. Second, that diversity also suggests translational relevance, since it is more likely that “core” mechanisms of social plasticity are discovered by analyzing a wider variety of social arrangements than relying on a single species. We analyze examples of social plasticity across fish species with different social organizations, concluding that a “core” mechanism is the initiation of behavioral shifts through the modulation of a conserved “social decision-making network”, along with other relevant brain regions, by monoamines, neuropeptides, and steroid hormones. The consolidation of these shifts may be mediated via neurogenomic adjustments and regulation of the expression of plasticity-related molecules (transcription factors, cell cycle regulators, and plasticity products).

Marine microalgae are a rich reservoir of natural compounds, including bioactives. Nonetheless, these organisms remain fairly unexplored despite their potential biotechnological applications. Culture collections with diverse taxonomic groups and lifestyles are a good source to unlock this potential and discover new molecules for multiple applications such as the treatment of human pathologies or the production of aquaculture species. In the present work extracts from 33 strains (including 20 dinoflagellates, 4 diatoms and 9 strains from other 7 algal classes), cultivated under identical conditions were examined for antiviral, antibacterial, anti-inflammatory and anti-cancer activities. Among these, antiviral and anti-inflammatory activities were detected in a few strains while antibacterial tests showed positive results in most assays. In turn, most trials did not show any anti-cancer activity. Wide differences were observed between species within the same class, in particular dinoflagellates which were better represented in this study. These preliminary findings pave the way for in-depth characterization of extracts with highest signals in each test, identification of compounds responsible of the biological activities found, and a further screening of the CCVIEO culture collection.

The objective of the present study is to isolate phages targeting multidrug resistant (MDR), extended spectrum beta lactamase (ESBL) positive clinical isolate of E. coli ( U1007), sequence and analyze the phage genome and use machine learning tools to predict host cell surface receptor and finally evaluate the efficiency of monophage and phage cocktail in vitro and in vivo in a zebrafish model. Phage specific for E. coli U1007 was isolated from Ganges River (designated as U1G), Cuoom River (designated as CR) and Hospital waste water (designated as M phage). The obtained phages were triple purified and enriched. U1G phages had a greater burst size of 124 PFU/cell and a latent time of 25 min.M phage had a burst size of 150 PFU/cell with a shortlatent time of 20min. Similarly CR phage has a short latent 20 min and a burst size of 115 PFU/cell. Based on capsid architecture, U1G phage resembles Podoviridae, CR phage is structurally similar to Myoviridae and M phage has morphology that resembles Siphoviridae. Genome sequencing and analysis revealed that the size of the U1G phage genome is 73275 bp Whereas that of CR phage and M phage are 45236 bp and 45294 bp, respectively. All three genomes were marked by the absence of genes encoding tRNA sequence, antibiotic resistant or virulent genes. A machine learning (ML) based multi-class classification model using algorithms such as Random Forest, Logistic Regression, and Decision Tree were employed to predict the host receptor targeted by all 3 phages and the best performing algorithm Random Forest predicted LPS O antigen , LamB or OmpC for U1G FhuA, OmpC for CR phage and FhuA, LamB, TonB or OmpF for the M phage as the host receptor targeted by the receptor binding protein (RBP) of the phages. In vitro time kill assay showed that treatment with monophages alone and along with colistin resulted in regrowth whereas phage combinations significantly reduced the regrowth and by 24h the phage cocktail along with colistin produced a significant 3 log declinein cell counts relative to the untreated control. In vivo intramuscular infection study in zebrafish showed that phages were non toxic and a cocktail of dual (U1G +M) phage along with colsitin resulted in a significant 3.5 log decline in cell counts whereas triple phage combination along with colistin resulted in 3 log decline in cell counts probably due to host receptor competition. Our study highlights the potential of phage cocktail therapy in mitigating MDR clinical isolate of E. coli in vitro and in vivo.

Four previously undescribed highly oxygenated diterpenoids (1–4), zeylleucapenoids A–D, characterized by halimane and labdane skeletons, were isolated from the aerial parts of Leucas zeylanica. Their structures were elucidated primarily by NMR experiments. The absolute configuration of 1 was established by theoretical ECD calculations and X-ray crystallographic analysis, whereas those for 2−4 were assigned by theoretical ORD calculations. Zeylleucapenoid D (4), with an IC₅₀ value of 38.45 μM in RAW264.7 macrophages and nontoxic activity for zebrafish embryo, and obviously inhibited pro-inflammatory cytokines TNF-α and IL-6 in a dose-dependent manner. Compound 4 inhibited of the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, molecular docking analysis indicated the possible mechanism of action for 4 may be bind to targets by hydrogen- and hydrophobic- bond interactions.

The receptor tyrosine kinase Ret plays a critical role in regulating enteric nervous system (ENS) development. Ret is important for proliferation, migration, and survival of enteric progenitor cells (EPCs). Ret also promotes neuronal fate, but its role during neuronal differentiation and in the adult ENS is less well understood. Inactivating RET mutations are associated with ENS diseases, e.g. Hirschsprung Disease, in which distal bowel lacks ENS cells. Zebrafish is an established model system for studying ENS development and modeling human ENS diseases. One advantage of the zebrafish model system is that their embryos are transparent allowing visualization of developmental phenotypes in live animals. However, we lack tools to monitor Ret expression in live zebrafish. Here, we developed a new BAC transgenic line that expresses GFP under the ret promoter. We find that EPCs and the majority of ENS neurons express ret:GFP during ENS development. In the adult ENS, GFP+ neurons are equally present in female and male. In homozygous mutants of ret and sox10 – another important ENS developmental regulator gene – GFP+ ENS cells are absent. In summary, we characterize a ret:GFP transgenic line as a new tool to visualize and study the Ret signaling pathway from early development through adulthood.

The neural-crest derived enteric nervous system (ENS) is the intrinsic nervous system of the gastrointestinal (GI) tract and controls all gut functions, including motility. Lack of ENS neurons causes various ENS disorders such as Hirschsprung Disease. One treatment option for ENS dis-orders includes the activation of resident stem cells to regenerate ENS neurons. Regeneration in the ENS has mainly been studied in mammalian species using surgical or chemically-induced injury methods. These mammalian studies showed a variety of regenerative responses with generally limited regeneration of ENS neurons, but (partial) regrowth and functional recovery of nerve fibers. Several aspects might contribute to the variety in regenerative responses, including observation time after injury, species, and gut region targeted. Zebrafish have recently emerged as a promising model system to study ENS regeneration as larvae possess the ability to generate new neurons after ablation. As the next steps in ENS regeneration research, we need a detailed under-standing of how regeneration is regulated on a cellular and molecular level both in animal models with high and low regenerative capacity. Understanding the regulatory programs necessary for robust ENS regeneration will pave the way for using neural regeneration as a therapeutic approach to treating ENS disorders.

The antioxidant and anti-inflammatory abilities of beeswax alcohol (BWA) are well reported in animal and human clinical studies, with a significant decrease in malondialdehyde (MDA) in blood, reduction of liver steatosis, and decrease in insulin. On the other hand, there is insufficient information to explain the in vitro antioxidant activity of BWA because it is extremely insoluble in aqueous buffer system. The BWA mixture was incorporated into reconstituted HDL (rHDL) with apoA-I, and the physiological functions of BWA in a water system were evaluated. After synthesis of rHDL with palmitoyloleoyl phosphatidylcholine (POPC), cholesterol, apolipoprotein A-I (apoA-I), and the BWA at molar ratios of 95:5:1:0 (rHDL-0), 95:5:1:0.5 (rHDL-0.5), and 95:5:1:1 (rHDL-1) for POPC:FC:apoA-I:BWA, the particle size of rHDL-1 was increased 15% compared to rHDL-0. rHDL-1 exhibited the strongest anti-glycation activity, up to 18% less glycation than rHDL-0 treated HDL3, with the highest protection of apoA-I from proteolytic degradation: a 28% larger band intensity than that of rHDL-0 treated HDL3 in the presence of fructose (final 250 mM). The antioxidant ability to inhibit cupric ion-mediated LDL oxidation increased as the BWA content in rHDL increased. The rHDL-1-treated LDL exhibited the smallest oxidation extent in electromobility with the least quantification of oxidized species (MDA). The antioxidant activities of HDL, ferric ion reduction ability (FRA), and paraoxonase (PON) were enhanced by the BWA in rHDL treatment with a concomitant increase in the molar ratio. rHDL-1-treated HDL showed 20–22% higher FRA and PON activities than rHDL-0-treated HDL. A microinjection of each rHDL into zebrafish embryos was performed in the presence of carboxymethyllysine (CML). The rHDL-1 injected embryo exhibited the highest survivability (~63%), whereas the CML alone group showed 28% survivability. A higher BWA content in rHDL helped neutralize the CML toxicity, resulting in higher survivability and normal developmental morphology. In contrast, the CML alone injected embryo showed severe retardation of the developmental speed and morphological defect. The CML-alone group showed the highest extent of reactive oxygen species (ROS) production and cellular apoptosis in embryos, but a co-injection of rHDL-1 resulted in a remarkable decrease in ROS and apoptosis. The dermal application of rHDL containing BWA resulted in higher potent wound-healing activity in a dose-dependent manner with decreased reactive oxygen species and cellular apoptosis in the cutaneous wound area in the presence of CML. Conclusively, incorporating BWA in rHDL significantly enhanced the anti-glycation and antioxidant activities in rHDL via more stabilization of apoA-I with a larger particle size. The rHDL containing BWA facilitated enforced inherent antioxidant ability of HDL and anti-inflammatory activity to suppress the CML toxicities in zebrafish embryos and to ameliorate CML-aggravated chronic wounds in adult zebrafish.

Elevation of the intermediate amino acid metabolite Homocysteine (Hcy) causes Hyperhomocysteinemia (HHcy), a metabolic disorder frequently associated with mutations in the methionine-cysteine metabolic cycle as well as with nutritional deficiency and aging. Previous literature suggest that HHcy is a strong risk factor for cardiovascular diseases. Severe HHcy is well established to correlate with vascular pathologies primarily via endothelial cell death. Though moderate HHcy is more prevalent and associated with an increased risk of cardiovascular abnormalities in later part of life, its precise role in endothelial physiology is largely unknown. In this study, we report that moderate elevation of Hcy causes endothelial dysfunction through impairment of their migration and proliferation. We established that unlike severe elevation of Hcy, moderate HHcy is not associated with suppressed endothelial VEGF/VEGFR signaling and oxidative stress induction. We further showed that moderate HHcy induces a sub-lethal ER stress that causes defective endothelial migration through abnormal actin cytoskeletal remodeling. We also found that sub-lethal increase of Hcy causes endothelial proliferation defect by suppressing mitochondrial respiration and concomitantly increases glycolysis to compensate the consequential ATP loss and maintain overall energy homeostasis. Finally, analyzing an available microarray dataset, we confirmed that these hallmarks of moderate HHcy are conserved in adult endothelial cells as well. Thus, we identified adaptive UPR and metabolic rewiring as two key mechanistic signatures in moderate HHcy-associated endothelial dysfunction. As HHcy is clinically associated with enhanced vascular inflammation and hypercoagulability, identifying these mechanistic pathways may serve as future targets to regulate endothelial function and health.

Myocilin is an enigmatic glaucoma-associated glycoprotein whose biological role remains incompletely understood. To gain novel insight into its normal function, we used transposon-mediated transgenesis to generate the first zebrafish line stably overexpressing myocilin [Tg(actb1:myoc-2A-mCherry)]. qPCR showed an approximately four-fold increased myocilin expression in transgenic zebrafish embryos (144 hpf). Adult (13 months old) transgenic animals displayed variable and age-dependent ocular anterior segment alterations. Almost 60% of two-years old male, but not female, transgenic zebrafish developed enlarged eyes with severe asymmetrical and variable abnormalities in the anterior segment, characterized by corneal limbus hypertrophy, and thickening of the cornea, iris, annular ligament and lens capsule. The most severe phenotype presented small or absent ocular anterior chamber and pupils, due to iris overgrowth along with dysplastic retinal growth and optic nerve hypertrophy. Immunohistochemistry revealed increased presence of myocilin in most altered ocular tissues of adult transgenic animals, as well as signs of retinal gliosis, and expanded ganglion cells and nerve fibers. The preliminary results indicate that these cells contributed to retinal dysplasia. Visual impairment was demonstrated in all old male transgenic zebrafish. Transcriptomic analysis of the abnormal transgenic eyes identified disrupted expression of genes involved in lens, muscular and extracellular matrix activities, among other processes. In summary, the developed transgenic zebrafish provides a new tool to investigate this puzzling protein and provides evidence for the role of zebrafish myocilin in ocular anterior segment and retinal biology, through the influence of extracellular matrix organization and cellular proliferation.

Acute chemical ablation of lateral line hair cells is an important tool to understand lateral line-mediated behaviors in free-swimming fish larvae and adults. However, lateral line-mediated behaviors have not been described in fish larvae prior to swim bladder inflation, possibly because single doses of ototoxin do not effectively silence lateral line function at early developmental stages. To determine if ototoxins can effectively silence the lateral line during early development, we repeatedly expose zebrafish larvae to the ototoxin neomycin during a 36-hour period from 3-4 days post-fertilization (dpf). We use simultaneous transgenic and vital dye labeling of hair cells to compare 6- hour and 12-hour repeated treatment timelines and neomycin concentrations between 0–400 µM in terms of larval survival, hair cell death, regeneration, and functional recovery. Following exposure to neomycin, we find that the emergence of newly functional hair cells outpaces cellular regeneration, likely due to the maturation of ototoxin-resistant hair cells that survive treatment. Furthermore, hair cells of 4 dpf larvae exhibit faster recovery compared to 3 dpf larvae. Our data suggest that the rapid functional maturation of ototoxin-resistant hair cells limits the effectiveness of chemical-based methods to disrupt lateral line function. Furthermore, we show that repeated neomycin treatments can continually ablate lateral line hair cells between 3–4 dpf in larval zebrafish.

Symbioses between animals and bacteria are ubiquitous. To better understand these relationships, it is essential to unravel how bacteria evolve to colonize hosts. Previously, we serially passaged the free-living bacterium, Shewanella oneidensis, through the digestive tracts of germ-free larval zebrafish (Danio rerio) to uncover the evolutionary changes involved in the initiation of a novel symbiosis with a vertebrate host. After 20 passages, we discovered an adaptive missense mutation in the mshL gene of the msh pilus operon, which improved host colonization, increased swimming motility, and reduced surface adhesion. In the present study, we have determined that this mutation was a loss-of-function mutation and found that it improved zebrafish colonization by augmenting S. oneidensis representation in the water column outside larvae through a reduced association with environmental surfaces. Additionally, we found that strains containing the mshL mutation were able to immigrate into host digestive tracts at higher rates per capita. However, mutant and evolved strains exhibited no evidence of a competitive advantage after colonizing hosts. Our results demonstrate that bacterial behaviors outside the host can play a dominant role in facilitating the onset of novel host associations.

The acoustic startle response (ASR) is an explosive motor response leaded by a sudden and intense acoustic stimulus. ASR has several clinically relevant forms of plasticity, including habituation and sensorimotor gating. Although ASR and its plasticity have been intensively studied in different animal models, including zebrafish (Danio rerio) larvae, information in adult zebrafish is still very scarce. In this manuscript we present Zebra_K, a new automated high-content kinematic analysis platform for assessing ASR, its habituation and prepulse inhibition (PPI), a quantitative measure of sensorimotor gating, in adult zebrafish. The analysis of the kinematic parameters in 1,875 ASR in adult zebrafish has shown a single response wave consistent with the short-latency C-bend described in zebrafish larvae. Moreover, protocols have been designed and validated in Zebra_K for the analysis of sensitivity, habituation and PPI of this response. Then, this platform has been used to determine the effect of pharmacological and physiological modulators of ASR plasticity. As described in other animal models, the NMDA-receptor antagonist ketamine increased the responsiveness to the acoustic stimuli, decreasing habituation and leading to complete abolition of PPI. Moreover, the effect of the gender on zebrafish ASR plasticity has been analyzed for the first time. Females exhibited higher responsiveness and a lower habituation and PPI than males, a result consistent with the gender effect described in other animal models and in humans. These results enhance the interest of using adult zebrafish to assess the potential effect of chemicals or genetic manipulations on ASR plasticity.

The zebrafish is now an important model organism for cancer biology studies and provides some unique and complementary opportunities in comparison to the mammalian equivalent. The translucency of zebrafish has allowed in vivo live imaging studies of tumour initiation and progression at the cellular level thus providing novel insights into our understanding of cancer. Here we summarise and discuss available transgenic zebrafish tumour models and what we have gleaned from them with respect to cancer inflammation. In particular, we focus on the host inflammatory response toward transformed cells during the pre-neoplastic stage of tumour development. We discuss features of tumour associated macrophages and neutrophils in mammalian models and present evidence which supports the idea that these inflammatory cells promote early stage tumour development and progression. Direct live imaging of tumour initiation in zebrafish models has shown that the intrinsic inflammation induced by pre-neoplastic cells is tumour promoting. Signals mediating leukocyte recruitment to pre-neoplastic cells in zebrafish correspond to signals mediating leukocyte recruitment in mammalian tumours. The activation state of macrophages and neutrophils recruited to pre-neoplastic cells appears to be heterogenous, as seen in mammalian models, which provides an opportunity to study the plasticity of innate immune cells during tumour initiation. Although several potential mechanisms are described that might mediate the trophic function of innate immune cells during tumour initiation in zebrafish, there are several unknowns that are yet to be resolved. Rapid advancement of genetic tools and imaging technologies for zebrafish will facilitate research into the mechanisms that modulate leukocyte function during tumour initiation and identify targets for cancer prevention.

Oxidative stress is one of the primary instigators of the onset of various human ailments, including cancers, cardiovascular diseases, and dementia. Particularly, oxidative stress has a severe effect on low-density lipoprotein (LDL) oxidation, leading to several detrimental health effects. Thereof, in this study, the effect of beeswax alcohol (BWA) was evaluated to prevent LDL oxidation, enhancement of paraoxonase 1 (PON-1) activity of high-density lipoprotein (HDL), and zebrafish embryo survivability. Furthermore, the implication of BWA consumption on the oxidative plasma variables was assessed by a preliminary clinical study on middle and older human subjects (n=50). Results support BWA augmentation of PON-1 activity in a dose-dependent manner (10-30 μM), which was significantly better them the effect exerted by coenzyme Q10 (CoQ10). Moreover, BWA significantly curtails CuSO4 (final 0.5 μM) evoked LDL/apo-B oxidation and a marked reduction of lipid peroxidation in LDL. The transmission electron microscopy (TEM) analysis revealed a healing effect of BWA towards the restoration of LDL morphology and size impaired by the exposure of Cu2+ ions (final 0.5 μM). Additionally, BWA counter carboxymethyllysine (CML, 500 ng) induced toxicity and rescued zebrafish embryos from development deformities and apoptotic cell death. A completely randomized, double-blind, placebo-controlled preliminary clinical study on middle and older-aged human subjects (n=50) exhibited that 12 weeks of BWA (100 mg/day) supplementation efficiently diminished serum malondialdehyde (MDA), total hydroperoxides, and enhanced total antioxidant status by 25%, 27%, and 22%, respectively, compared to the placebo control and baseline values. Furthermore, the consumption of BWA did not exhibit any noteworthy changes in anthropometric profile, lipid profile, glucose levels, and biomarkers pertinent to kidney and liver function, thus confirming the safety of BWA for consumption. Conclusively, BWA prevents LDL oxidation, enhances PON-1 activity in HDL, and has a positive impact on the oxidative variables of human subjects.

During the last decades, zebrafish (Danio rerio) has become one of the most important model organisms to study different physiological and biological phenomena. Research field of carbonic anhydrases (CAs) and carbonic anhydrase related proteins (CARPs) is not an exception in this. The best-known known function of CAs is the regulation of acid-base balance. However, studies performed with zebrafish, among others, have revealed important roles for these proteins in many other physiological processes, some of which have been unpredicted in the light of the previous studies and suggestions. Examples are roles in zebrafish pigmentation as well as motor coordination. Disruption of the function of these proteins may generate even lethal outcomes. In this review, we summarize the current knowledge of CA-related studies performed in zebrafish.

Kaposi’s sarcoma associated-herpesvirus (KSHV, also known as human herpesvirus-8) is a gammaherpesvirus that establishes life-long infection in human B lymphocytes. KSHV infection is typically asymptomatic but immunosuppression can predispose KSHV-infected individuals to primary effusion lymphoma (PEL); a malignancy driven by aberrant proliferation of latently infected B lymphocytes, and supported by pro-inflammatory cytokines and angiogenic factors produced by cells that succumb to lytic viral replication. Here, we report the development of the first in vivo model for a virally-induced lymphoma in zebrafish, whereby KSHV-infected PEL tumour cells engraft and proliferate in the yolk sac of zebrafish larvae. Using a PEL cell line engineered to produce the viral lytic switch protein RTA in the presence of doxycycline, we demonstrate drug-inducible reactivation from KSHV latency in vivo, which enabled real-time observation and evaluation of latent and lytic phases of KSHV infection. In addition, we developed a sensitive droplet digital PCR method to monitor latent and lytic viral gene expression and host cell gene expression in xenografts. The zebrafish yolk sac is not well-vascularized and using fluorogenic assays we confirmed that this site provides a hypoxic environment that may mimic the microenvironment of some human tumors. We found that PEL cell proliferation in xenografts was dependent on the host hypoxia-dependent translation initiation factor, eukaryotic initiation factor 4E2 (eIF4E2). This demonstrates that the zebrafish yolk sac is a functionally hypoxic environment and xenografted cells must switch to dedicated hypoxic gene expression machinery to survive and proliferate. The establishment of the PEL xenograft model enables future studies that exploit the innate advantages of the zebrafish as a model for genetic and pharmacologic screens.

The recent availability of commercial platforms for behavioral analysis 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 response (VMR). 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 in 143 larvae 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 (4,004 analyses for each behavior). Results from both behaviors were adjusted to a Random-Effects linear regression model using generalized least squares (GLS), 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 age of the larva and the behavioral test.

Directed movement towards a target requires spatial working memory, including processing of sensory inputs and motivational drive. In a stimulus-driven, operant conditioning paradigm designed to train zebrafish, we present a pulse of light via LED’s and/or sounds via an underwater transducer. A webcam placed below a glass tank records fish swimming behavior. During operant conditioning, a fish must interrupt an infrared beam at one location to obtain a small food reward at the same or different location. A timing-gated interrupt activates robotic-arm and feeder stepper motors via custom software controlling a microprocessor (Arduino). “Ardulink”, a JAVA facility, implements Arduino-computer communication protocols. In this way, full automation of stimulus-conditioned directional swimming is achieved. Precise multi­day scheduling of training, including timing, location and intensity of stimulus parameters, and feeder control is accomplished via a user-friendly interface. Our training paradigm permits tracking of learning by monitoring, turning, location, response times and directional swimming of individual fish. This facilitates comparison of performance within and across a cohort of animals. We demonstrate the ability to train and test zebrafish using visual and auditory stimuli. Current methods used for associative conditioning often involve human intervention, which is labor intensive, stressful to animals, and introduces noise in the data. Our relatively simple yet flexible paradigm requires a simple apparatus and minimal human intervention. Our scheduling and control software and apparatus (NemoTrainer) can be used to screen neurologic drugs and test the effects of CRISPR­-based and optogenetic modification of neural circuits on sensation, locomotion, learning and memory.

Dietary vitamin A/all-trans retinol/ROL plays a critical role in human vision. ROL circulates bound to the plasma retinol-binding protein (RBP4) as RBP4-ROL. In the eye, the STRA6 membrane receptor binds to circulatory RBP4 and internalizes ROL. STRA6 is however not expressed in systemic tissues, where there is high-affinity RBP4 binding and ROL uptake. We tested the hypothesis, that the second retinol-binding protein 4 receptor 2 (Rbpr2) which is highly expressed in systemic tissues of zebrafish and mouse, contains a functional RBP4 binding domain, critical for ROL transport. As for STRA6, modeling and docking studies confirmed three conserved RBP4 binding residues in zebrafish Rbpr2. In cell culture studies, disruption of the RBP4 binding residues on Rbpr2 almost completely abolished uptake of exogenous vitamin A. CRISPR generated rbpr2-RBP4 domain zebrafish mutants showed microphthalmia, shorter photoreceptor outer segments, and decreased opsins, that were attributed to impaired ocular retinoid content. Injection of WT-Rbpr2 mRNA into rbpr2 mutant or all-trans retinoic acid treatments rescued the mutant eye phenotypes. In conclusion, zebrafish Rbpr2 contains a putative extracellular RBP4-ROL ligand-binding domain, critical for yolk vitamin A transport to the eye for ocular retinoid production and homeostasis, for photoreceptor cell survival.

Two-photon imaging of light stimulus-evoked neuronal activity has been used to study all neuron classes in the vertebrate retina, from the photoreceptors to the retinal ganglion cells. Clearly, the ability to study retinal circuits down to the level of single synapses or zoomed out at the level of complete populations of neurons, has been a major asset in our understanding of this beautiful circuit. In this chapter, we discuss the possibilities and pitfalls of using an all-optical approach in this highly light-sensitive part of the brain.

Metalloproteinases are zinc-dependent endopeptidases that function as primary effectors of tissue remodelling, cell-signalling, and many other roles. Their regulation is ferociously complex, and is exquisitely sensitive to their molecular milieu, making in vivo studies challenging. Phenanthroline (PhN) is an inexpensive, broad-spectrum inhibitor of metalloproteinases that functions by chelating the catalytic zinc ion, however its use in vivo has been limited due to suspected off-target effects. PhN is very similar in structure to phenanthrene (Phe), a well-studied poly aromatic hydrocarbon (PAH) known to cause toxicity in aquatic animals by activating the aryl hydrocarbon receptor (AhR). We show that zebrafish are more sensitive to PhN than Phe, and that PhN causes a superset of the effects caused by Phe. Morpholino knock-down of the AhR rescues the effects of PhN that are shared with Phe, suggesting these are due to PAH toxicity. The effects of PhN that are not shared with Phe (specifically disruption of neural crest development and angiogenesis) involve processes known to depend on metalloproteinase activity. Furthermore these PhN-specific effects are not rescued by AhR knock-down, suggesting that these are bona fide effects of metalloproteinase inhibition, and that PhN can be used as a broad spectrum metalloproteinase inhibitor for studies with zebrafish in vivo.

New technologies have resulted in a better understanding of blood and lymphatic vascular heterogeneity at the cellular and molecular levels. Meanwhile, we still need to learn more about the heterogeneity of the cardiovascular and lymphatic systems between different species at the anatomical and functional levels. Even the deceptively simple question of the functions of fish lymphatic vessels has yet to be conclusively answered. The most common interpretation assumes a similar dual setup of the vasculature in zebrafish and mammals: on the one hand, there is the cardiovascular, circulatory system, and on the other hand, the lymphatic vascular system, in which the unidirectional flow is derived from surplus interstitial fluid and returned into the cardiovascular system. A competing interpretation questions the identity of the lymphatic vessels in fish as at least some of them receive their flow from arteries via specialised anastomoses, neither requiring an interstitial source for the lymphatic flow nor stipulating unidirectionality. In this alternative view, the “fish lymphatics” are a specialised subcompartment of the cardiovascular system, called the “secondary vascular system” (SVS). Many of the contradictions found in the literature appear to stem from the fact that the SVS develops in part or its entirety from an embryonic lymphatic vascular system by transdifferentiation. Future research needs to establish the extent of embryonic transdifferentiation of lymphatics into SVS blood vessels. Similarly, more insight is needed into the molecular regulation of vascular development in fish. Most fish do feature more than the five VEGF genes and three VEGF receptor genes that we know from mice or humans, and the relative tolerance of fish to whole genome and gene duplications could underlie the evolutionary diversification of the vasculature. This review discusses the key elements of the fish lymphatics versus the SVS and attempts to draw a picture coherent with the existing data, including the phylogenetic knowledge.

Zebrafish have been found to be the premier model organism in biological and biomedical research, specifically offering many advantages in developmental biology and genetics. This unique aquatic species has been found to have the capacity to regenerate their spinal cord after injury. However, the complete molecular and cellular mechanisms behind glial bridge formation in the central and peripheral nervous systems upon glial cell injury remains unclear. This review paper focuses on the molecular mechanisms and cellular processes that underlie spinal cord regeneration in four initial phases: proliferation and initial migration; migration and differentiation; glial bridge formation; and remodeling. We propose that within these four phases the cellular mechanisms that underlie spinal cord regeneration each express a terminating signal that aborts one step of the process and initiates the next. Specifically, future studies would be devoted to investigate transmitting signals in the spinal cord injury micro-environment in hope to contribute to the understanding of underlying cellular mechanisms by connecting each process of spinal cord regeneration in zebrafish.