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Microplastics, small pieces of plastic derived from polystyrene, have recently become an ecological hazard due to their toxicity and widespread occurrence in aquatic ecosystems. In this study, we exposed zebrafish larvae to two types of fluorescent polystyrene nanoparticles (PS-NPs) to identify their size-dependent effects. PS-NPs of 50 nm, unlike 100 nm PS-NPs, were found to circulate in the blood vessels and accumulate in the brains of zebrafish larvae. Behavioral and electroencephalogram (EEG) analysis showed that 50 nm PS-NPs induce abnormal behavioral patterns and changes in EEG power spectral densities in zebrafish larvae. In addition, the quantification of endogenous neurochemicals in zebrafish larvae showed that 50 nm PS-NPs disturb dopaminergic metabolites, whereas 100 nm PS-NPs do not. Finally, we assessed the effect of PS-NPs on the permeability of the blood–brain barrier (BBB) using a microfluidic system. The results revealed that 50 nm PS-NPs have high BBB penetration compared with 100 nm PS-NPs. Taken together, we concluded that small nanoparticles disturb the nervous system, especially dopaminergic metabolites.

Although bisphenol F (BPF), the main replacement for bisphenol A, has been commonly used in polycarbonate production, its neurotoxicity and the underlying mechanisms remain poorly understood. To address this knowledge gap, this study aimed to assess the neurotoxicity caused by chronic exposure to BPF and to identify its underlying mechanisms. We exposed adult zebrafish chronically to BPF at environmentally relevant concentrations (0.001, 0.01, and 0.1 mg/L) for 4 weeks. The results revealed that with BPF crossing the blood-brain barrier and bioaccumulating in brain tissues, chronic exposure to BPF resulted in anxiety-like behaviors and disruptions in learning and memory function in adult zebrafish. Furthermore, BPF toxicity in the zebrafish brain involved the dysregulation of metabolic pathways for choline and kynurenine in neurotransmitter systems and for 17β-estradiol, cortisol, pregnenolone-sulfate, and Dehydroepiandrosterone (DHEA)-sulfate in neurosteroid systems. RNA-seq analysis revealed that BPF exposure affected metabolic pathways, calcium signaling pathways, neuroactive ligand-receptor interactions, tight junctions, gap junctions, and the gonadotropin-releasing hormone signaling pathway. Our results indicate that chronic exposure to BPF alters the neurochemical profile of the brain and causes neurobehavioral effects, such as anxiety and cognitive decline. Overall, the multimodal approach, including behavioral and neurochemical profiling technologies, has great potential for the comprehensive assessment of potential risks posed by environmental pollutants to human and ecosystem health.

Paraquat is a polar herbicide protecting plant products against invasive species, it requires careful manipulation and restricted usage because of its harmful potentials. Exposure to paraquat triggers oxidative damage in dopaminergic neurons and subsequently causes a behavioral defect in vivo. Thereby, persistent exposure to paraquat is known to increase Parkinson's disease risk by dysregulating dopaminergic systems in humans. Therefore, most studies have focused on the dopaminergic systems to elucidate the neurotoxicological mechanism of paraquat poisoning, and more comprehensive neurochemistry including histaminergic, serotonergic, cholinergic, and GABAergic systems has remained unclear. Therefore, in this study, we investigated the toxicological potential of paraquat poisoning using a variety of approaches such as toxicokinetic profiles, behavioral effects, neural activity, and broad-spectrum neurochemistry in zebrafish larvae after short-term exposure to paraquat and we performed the molecular modeling approach. Our results showed that paraquat was slowly absorbed in the brain of zebrafish after oral administration of paraquat. In addition, paraquat toxicity resulted in behavioral impairments, namely, reduced motor activity and led to abnormal neural activities in zebrafish larvae. This locomotor deficit came with a dysregulation of dopamine synthesis induced by the inhibition of tyrosine hydroxylase activity, which was also indirectly confirmed by molecular modeling studies. Furthermore, short-term exposure to paraquat also caused simultaneous dysregulation of other neurochemistry including cholinergic and serotonergic systems in zebrafish larvae. The present study suggests that this neurotoxicological profiling could be a useful tool for understanding the brain neurochemistry of neurotoxic agents that might be a potential risk to human and environmental health.

The novel synthetic compound Di (isoquinolin-1-yl) sulfane (DIQS) was identified by zebrafish larva screening during the development of an agent to inhibit abnormal hyperpigmentation. In this study, we investigated the inhibitory effect of DIQS on melanogenesis and its underlying mechanism. DIQS inhibited melanin production and tyrosinase activity in B16F10 cells stimulated with α-melanocyte-stimulating hormone (α-MSH), as well as zebrafish embryos and reconstituted human skin tissue containing melanocytes. DIQS decreased the mRNA and protein expression of microphthalmia-associated transcription factor (MITF) and tyrosinase at a concentration of 10 μM. DIQS also inhibited the phosphorylation of cAMP response element-binding protein (CREB) and p-p38 and p-JNK stimulated by α-MSH. These results suggest that DIQS attenuates hyperpigmentation via inhibition of the cAMP/PKA/CREB/MITF/tyrosinase axis and MAPK pathways. Liquid chromatography–tandem mass spectrometry analysis revealed that DIQS blocked the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) in zebrafish embryos. Finally, we confirmed that DIQS was non-toxic in reconstituted human tissues such as the epidermis, used to test skin sensitization, and the cornea, used to test eye irritation. In summary, the results of this study suggest the potential of DIQS as a small-molecule agent for skin-whitening cosmetics and the treatment of hyperpigmentation disorders without biological toxicity.

In this study, we used the zebrafish animal model to establish a bioassay by which physiological efficacy differential of alpha-melanocyte-stimulating hormone (α-MSH) analogues could be measured by melanosome dispersion in zebrafish larvae. Brain-skin connection research has purported the interconnectedness between the nervous system and skin physiology. Accordingly, the neuropeptide α-MSH is a key regulator in several physiological processes, such as skin pigmentation in fish. In mammals, α-MSH has been found to regulate motivated behavior, appetite, and emotion, including stimulation of satiety and anxiety. Several clinical and animal model studies of autism spectrum disorder (ASD) have already demonstrated the effectiveness of α-MSH in restoring the social deficits of autism. Therefore, we sought to analyze the effect of synthetic and naturally-occurring α-MSH variants amongst different species. Our results showed that unique α-MSH derivatives from several fish species produced differential effects on the degree of melanophore dispersion. Using α-MSH human form as a standard, we could identify derivatives that induced greater physiological effects, particularly, the synthetic analogue melanotan-II (MT-II) exhibited a higher capacity for melanophore dispersion than human α-MSH. This was consistent with previous findings in an ASD mouse model demonstrating the effectiveness of MT-II in improving ASD behavioral symptoms. Thus, the melanophore assay may serve as a useful screening tool for therapeutic candidates for novel drug discovery.

Olfaction is an important neural system for survival and fundamental behaviors such as predator avoidance, food finding, memory formation, reproduction, and social communication. However, the neural circuits and pathways associated with the olfactory system in various behaviors are not fully understood. Recent advances in optogenetics, high-resolution in vivo imaging, and reconstructions of neuronal circuits have created new opportunities to understand such neural circuits. Here, we generated a transgenic zebrafish to manipulate olfactory signal optically, expressing the Channelrhodopsin (ChR2) under the control of the olfactory specific promoter, omp. We observed light-induced neuronal activity of olfactory system in the transgenic fish by examining c-fos expression, and a calcium indicator suggesting that blue light stimulation caused activation of olfactory neurons in a non-invasive manner. To examine whether the photo-activation of olfactory sensory neurons affect behavior of zebrafish larvae, we devised a behavioral choice paradigm and tested how zebrafish larvae choose between two conflicting sensory cues, an aversive odor or the naturally preferred phototaxis. We found that when the conflicting cues (the preferred light and aversive odor) were presented together simultaneously, zebrafish larvae swam away from the aversive odor. However, the transgenic fish with photo-activation were insensitive to the aversive odor and exhibited olfactory desensitization upon optical stimulation of ChR2. These results show that an aversive olfactory stimulus can override phototaxis, and that olfaction is important in decision making in zebrafish. This new transgenic model will be useful for the analysis of olfaction related behaviors and for the dissection of underlying neural circuits.

BACKGROUND The use of methyl-tertiary butyl ether (MTBE) to dissolve gallstones has been limited due to concerns over its toxicity and the widespread recognition of the safety of laparoscopic cholecystectomy. The adverse effects of MTBE are largely attributed to its low boiling point, resulting in a tendency to evaporate. Therefore, if there is a material with a higher boiling point and similar or higher dissolubility than MTBE, it is expected to be an attractive alternative to MTBE. AIM To determine whether tert-amyl ethyl ether (TAEE), an MTBE analogue with a relatively higher boiling point (102 °C), could be used as an alternative to MTBE in terms of gallstone dissolubility and toxicity. METHODS The in vitro dissolubility of MTBE and TAEE was determined by measuring the dry weights of human gallstones at predetermined time intervals after placing them in glass containers with either of the two solvents. The in vivo dissolubility was determined by comparing the weights of solvent-treated gallstones and control (dimethyl sulfoxide)-treated gallstones, after the direct infusion of each solvent into the gallbladder in both hamster models with cholesterol and pigmented gallstones. RESULTS The in vitro results demonstrated a 24 h TAEE-dissolubility of 76.7%, 56.5% and 38.75% for cholesterol, mixed, and pigmented gallstones, respectively, which represented a 1.2-, 1.4-, and 1.3-fold increase in dissolubility compared to that of MTBE. In the in vitro experiment, the 24 h-dissolubility of TAEE was 71.7% and 63.0% for cholesterol and pigmented gallstones, respectively, which represented a 1.4- and 1.9-fold increase in dissolubility compared to that of MTBE. In addition, the results of the cell viability assay and western blot analysis indicated that TAEE had a lower toxicity towards gallbladder epithelial cells than MTBE. CONCLUSION We demonstrated that TAEE has higher gallstone dissolubility properties and safety than those of MTBE. As such, TAEE could present an attractive alternative to MTBE if our findings regarding its efficacy and safety can be consistently reproduced in further subclinical and clinical studies.

Waste management is a major part of the food industry. The present study was designed to utilize the discarded byproduct of Schisandra chinensis Baillon. The antioxidant and anti-inflammatory effects of a 30% ethanol fraction (RPG-OM-30E) from the fermented hot water extraction of the Schisandra chinensis Baillon byproduct were investigated using RAW 264.7 cells and zebrafish larvae. RPG-OM-30E reduced lipopolysaccharide (LPS)-induced nitric oxide production in the RAW 264.7 cells. Additionally, RPG-OM-30E inhibited mRNA expression and protein secretion of pro-inflammatory cytokines, such as interleukin-6 (Il-6) and interleukin-1β (Il-1β). The anti-inflammatory effects of RPG-OM-30E were tested in Tg(mpx::EGFP)i114 zebrafish larvae. Neutrophil migration to a wound site was decreased by RPG-OM-30E. Neutrophil aggregation was also inhibited by RPG-OM-30E after induction of an LPS-induced immune response in the yolk. Finally, the antioxidant and hepatoprotective effects of RPG-OM-30E were examined in vivo. Mice with induced oxidative damage recovered from the stress following RPG-OM-30E treatment.

Epilepsy is one of the most common neurological disorders, and it is characterized by spontaneous seizures. In a previous study, we identified 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one (GM-90432) as a novel anti-epileptic agent in chemically- or genetically-induced epileptic zebrafish and mouse models. In this study, we investigated the anti-epileptic effects of GM-90432 through neurochemical profiling-based approach to understand the neuroprotective mechanism in a pentylenetetrazole (PTZ)-induced epileptic seizure zebrafish model. GM-90432 effectively improved PTZ-induced epileptic behaviors via upregulation of 5-hydroxytryptamine, 17-&beta, estradiol, dihydrotestosterone, progesterone, 5&alpha, dihydroprogesterone, and allopregnanolone levels, and downregulation of normetanephrine, gamma-aminobutyric acid, and cortisol levels in brain tissue. GM-90432 also had a protective effect against PTZ-induced oxidative stress and zebrafish death, suggesting that it exhibits biphasic neuroprotective effects via scavenging of reactive oxygen species and anti-epileptic activities in a zebrafish model. In conclusion, our results suggest that neurochemical profiling study could be used to better understand of anti-epileptic mechanism of GM-90432, potentially leading to new drug discovery and development of anti-seizure agents.

Osteoporosis is one of the main health problems in the world today characterized by low bone mass and deterioration in bone microarchitecture. In recent years, the use of natural products approach to treat it has been in the increase. In this study, in vitro antiosteoporosis activity and hepatotoxicity of P. jamasakura bark extracts were evaluated. Methods. Mouse bone marrow macrophage (BMM) cells were incubated with tartrate-resistant acid phosphate (TRAP) buffers and p-nitrophenyl phosphate and cultured with different P. jamasakura bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 μg/ml in the presence of the receptor activator of nuclear factor kappa-Β ligand (RANKL) for 6 days. The osteoclast TRAP activity and cell viability were measured. Nitric oxide (NO) assay was conducted using murine macrophage-like RAW 264.7 cells treated with P. jamasakura ethanolic and methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, 50, 100, and 200 μg/ml. For hepatotoxicity assessment, zebrafish larvae were exposed to P. jamasakura bark extracts, 0.05% dimethyl sulfoxide as a negative control, and 5 μM tamoxifen as a positive control. The surviving larvae were anesthetized and assessed for hepatocyte apoptosis. Results. TRAP activity was significantly inhibited (p P. jamasakura extracts compared to the control treatment. At 50 μg/ml, both ethanolic and methanolic extracts of P. jamasakura exhibited significant (p P. jamasakura ethanolic and methanolic extracts had significant inhibitory (p μg/ml and exhibited significant (p p Conclusion. The TRAP activity of P. jamasakura bark gives a foundation for further studies to enhance future development of antiosteoporosis drug.

Epilepsy is a common chronic neurological disease characterized by recurrent epileptic seizures. A seizure is an uncontrolled electrical activity in the brain that can cause different levels of behavior, emotion, and consciousness. One-third of patients fail to receive sufficient seizure control, even though more than fifty FDA-approved anti-seizure drugs (ASDs) are available. In this study, we attempted small molecule screening to identify potential therapeutic agents for the treatment of seizures using seizure-induced animal models. Through behavioral phenotype-based screening, 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one (GM-90432) was identified as a prototype. GM-90432 treatment effectively decreased seizure-like behaviors in zebrafish and mice with chemically induced seizures. These results were consistent with decreased neuronal activity through immunohistochemistry for pERK in zebrafish larvae. Additionally, electroencephalogram (EEG) analysis revealed that GM-90432 decreases seizure-specific EEG events in adult zebrafish. Moreover, we revealed the preferential binding of GM-90432 to voltage-gated Na+ channels using a whole-cell patch clamp technique. Through pharmacokinetic analysis, GM-90432 effectively penetrated the blood-brain barrier and was distributed into the brain. Taken together, we suggest that GM-90432 has the potential to be developed into a new ASD candidate.

A series of aryl sulfide derivatives was synthesized and evaluated for their anti-melanogenic activities. Several compounds, including 3e, 3i and 3q exhibited good anti-melanogenic activities. Among the derivatives, compound 3i showed good inhibitory effects against melanin synthesis and showed no toxicity in reconstituted human eye and skin tissues.

Background The demand for anti-melanogenic agents is increasing due to the unwanted side effects of current treatments. To find an effective anti-melanogenic agent, we used zebrafish as a whole animal model for phenotype-based drug and cosmetic discovery screening. Objectives The aim of this study was to identify and explore a small molecule that could be used for skin-whitening cosmetics. Methods Using zebrafish embryos, we examined the effects of 1000 compounds on zebrafish development and pigmentation. Pigmentation production was assessed by tyrosinase (TYR) enzymatic activity and melanin contents. Pigmentation marker expression in the human melanoma cell line HMV-II was analyzed by western blot. We also tested reconstituted human skin tissue and analyzed KDZ-001 with computational molecular modeling. Results We identified three compounds that affected the pigmentation of developing melanophores in zebrafish. Among them, we identified KDZ-001, a novel anti-melanogenic agent, which strongly inhibits melanin synthesis in the developing melanophores of zebrafish, HMV-II cells, and reconstituted human skin with no toxicity. We found that KDZ-001 directly inhibits TYR enzymatic activity. Notably, computational molecular modeling of KDZ-001 suggested that its interaction with copper ions in the active site of TYR is essential for melanin synthesis, further demonstrating that KDZ-001 mainly acts as a TYR inhibitor to synthesize melanin. Conclusion KDZ-001 inhibits melanin synthesis and has a potential for use in skin-whitening cosmetics.

A series of glutamic acid derivatives was synthesized and evaluated for their antioxidant activity and stability. We found several potent and stable glutamic acid derivatives. Among them, compound 12b exhibited good in vitro activity, chemical stability and cytotoxicity. A prototype compound 12b showed an anti-inflammatory effect in LPS-stimulated RAW 264.7 cell lines and in a zebrafish model.

Background DYRK1A maps to the Down syndrome critical region at 21q22. Mutations in this kinase-encoding gene have been reported to cause microcephaly associated with either intellectual disability or autism in humans. Intellectual disability accompanied by microcephaly was recapitulated in a murine model by overexpressing Dyrk1a which mimicked Down syndrome phenotypes. However, given embryonic lethality in homozygous knockout (KO) mice, no murine model studies could present sufficient evidence to link Dyrk1a dysfunction with autism. To understand the molecular mechanisms underlying microcephaly and autism spectrum disorders (ASD), we established an in vivo dyrk1aa KO model using zebrafish. Methods We identified a patient with a mutation in the DYRK1A gene using microarray analysis. Circumventing the barrier of murine model studies, we generated a dyrk1aa KO zebrafish using transcription activator-like effector nuclease (TALEN)-mediated genome editing. For social behavioral tests, we have established a social interaction test, shoaling assay, and group behavior assay. For molecular analysis, we examined the neuronal activity in specific brain regions of dyrk1aa KO zebrafish through in situ hybridization with various probes including c-fos and crh which are the molecular markers for stress response. Results Microarray detected an intragenic microdeletion of DYRK1A in an individual with microcephaly and autism. From behavioral tests of social interaction and group behavior, dyrk1aa KO zebrafish exhibited social impairments that reproduce human phenotypes of autism in a vertebrate animal model. Social impairment in dyrk1aa KO zebrafish was further confirmed by molecular analysis of c-fos and crh expression. Transcriptional expression of c-fos and crh was lower than that of wild type fish in specific hypothalamic regions, suggesting that KO fish brains are less activated by social context. Conclusions In this study, we established a zebrafish model to validate a candidate gene for autism in a vertebrate animal. These results illustrate the functional deficiency of DYRK1A as an underlying disease mechanism for autism. We also propose simple social behavioral assays as a tool for the broader study of autism candidate genes.

A new type of composite steel slab system called TUBEDECK (TD) has been proposed, which combines a cast-in-situ RC slab with circular tube voids and profiled steel decks. In order to invest...

Background Although methyl-tertiary butyl ether (MTBE) is the only clinical topical agent for gallstone dissolution, its use is limited by its side effects mostly arising from a relatively low boiling point (55 °C). In this study, we developed the gallstone-dissolving compound containing an aromatic moiety, named 2-methoxy-6-methylpyridine (MMP) with higher boiling point (156 °C), and compared its effectiveness and toxicities with MTBE. Methods The dissolubility of MTBE and MMP in vitro was determined by placing human gallstones in glass containers with either solvent and, then, measuring their dry weights. Their dissolubility in vivo was determined by comparing the weights of solvent-treated gallstones and control (dimethyl sulfoxide)-treated gallstones, after directly injecting each solvent into the gallbladder in hamster models with cholesterol and pigmented gallstones. Results In the in vitro dissolution test, MMP demonstrated statistically higher dissolubility than did MTBE for cholesterol and pigmented gallstones (88.2% vs. 65.7%, 50.8% vs. 29.0%, respectively; P

Additional file 2. Additional Materials and Methods.

Epilepsy is a neurological disease characterized by recurrent seizures. Though the exact causes for epilepsy are unknown, genetic mutations, especially altered gene functions, have been implicated as key causative components of epilepsy. We recently identified a causing gene for the Miles-Carpenter syndrome (MCS). MCS patients have intellectual disability and epilepsy. MCS knockout (KO) zebrafish also show a seizure-like phenotype with hyperactivity of pectoral fin and jaw movement, resulting from loss of GABAergic interneurons. To evaluate MCS KO zebrafish as an epilepsy model, we tested the effects of retigabine, an anticonvulsant drug, on the movement of MCS KO zebrafish.

This paper aims to validate if intrapancreatic injection of penicillin G can enhance hardness and suture holding capacity (SHC) of the pancreas through prompting the fibrosis process. Soft pancreatic texture is constantly mentioned as one of the most contributory predictors of postoperative pancreatic fistula (POPF). Soft pancreas has poor SHC and higher incidence of parenchymal tearing, frequently leading to POPF. From a library of 114 antibiotic compounds, we identified that penicillin G substantially enhanced pancreatic hardness and SHC in experimental mice. Specifically, we injected penicillin G directly into the pancreas. On determined dates, we measured the pancreatic hardness and SHC, respectively, and performed molecular and histological examinations for estimation of the degree of fibrosis. The intrapancreatic injection of penicillin G activated human pancreatic stellate cells (HPSCs) to produce various fibrotic materials such as transforming growth factor-&beta, 1 (TGF-&beta, 1) and metalloproteinases-2. The pancreatic hardness and SHC were increased to the maximum at the second day after injection and then it gradually subsided demonstrating its reversibility. Pretreatment of mice with SB431542, an inhibitor of the TGF-&beta, 1 receptor, before injecting penicillin G intrapancreatically, significantly abrogated the increase of both pancreatic hardness and SHC caused by penicillin G. This suggested that penicillin G promotes pancreatic fibrosis through the TGF-&beta, 1 signaling pathway. Intrapancreatic injection of penicillin G promotes pancreatic hardness and SHC by enhancing pancreatic fibrosis. We thus think that penicillin G could be utilized to prevent and minimize POPF, after validating its actual effectiveness and safety by further studies.

Bisphenol A (BPA) is a chemical monomer widely used in the production of hard plastics for food containers and personal items. Through improper industrial control and disposal, BPA has become a pervasive environmental contaminant, and toxicological studies have shown potent xenobiotic endocrine disruptor activity. Prenatal exposure in particular can lead to infertility and nervous system disorders characterized by behavioral aggression, depression, and cognitive impairment, thus necessitating careful hazard assessment. In this study, we evaluated BPA accumulation rate, blood-brain barrier (BBB) permeability, lethality, cardiotoxicity, behavioral effects, and impacts on multiple neurochemical pathways in zebrafish larvae. The bioconcentration factor (BCF) ranged from 1.95 to 10.0, resulting in a high rate of accumulation in the larval body. Also, high BBB permeability allowed BPA to accumulate at similar rates in both zebrafish and adult mouse (blood to brain concentration ratios of 3.2-6.7 and 1.8 to 5.5, respectively). In addition, BPA-exposed zebrafish larvae exhibited developmental deformities, reduced heart rate, and impaired behavioral patterns, including decreased total distance traveled, slower movement velocity, and altered color-preference. These impairments were associated with inhibition of the phenylalanine to dopamine synthesis pathway and an imbalance between excitatory and inhibitory neurotransmitter systems. Our results suggest that behavioral alteration in BPA-exposed zebrafish result from high accumulation and ensuing dysregulation of serotonergic, kynurenergic, dopaminergic, cholinergic, and GABAergic neurotransmitter systems. In conclusion, similarities in toxic responses to mammalian models highlight the utility of the zebrafish larva as a convenient model for screening environmental toxins.

Waste management is a major part of the food industry. The present study was designed to utilize the discarded byproduct of Schisandra chinensis Baillon. The antioxidant and anti-inflammatory effects of a 30% ethanol fraction (RPG-OM-30E) from the fermented hot water extraction of the Schisandra chinensis Baillon byproduct were investigated using RAW 264.7 cells and zebrafish larvae. RPG-OM-30E reduced lipopolysaccharide (LPS)-induced nitric oxide production in the RAW 264.7 cells. Additionally, RPG-OM-30E inhibited mRNA expression and protein secretion of pro-inflammatory cytokines, such as interleukin-6 (Il-6) and interleukin-1β (Il-1β). The anti-inflammatory effects of RPG-OM-30E were tested in Tg(mpx::EGFP)(i114) zebrafish larvae. Neutrophil migration to a wound site was decreased by RPG-OM-30E. Neutrophil aggregation was also inhibited by RPG-OM-30E after induction of an LPS-induced immune response in the yolk. Finally, the antioxidant and hepatoprotective effects of RPG-OM-30E were examined in vivo. Mice with induced oxidative damage recovered from the stress following RPG-OM-30E treatment.

The compound, 1-((4-fluorophenyl)thio)isoquinoline (FPTQ), is a synthetic isoquinoline derivative. To test the anti-inflammatory effect of FPTQ, we used neutrophil-specific transgenic zebrafish Tg(mpx::EGFP)i114 line and lipopolysaccharide (LPS)-stimulated RAW264.7 cells. We also used two different methods, involving tail transection and LPS stimulation in the zebrafish model. Neutrophils translocation in the zebrafish tail-transected model was inhibited by FPTQ. Neutrophil aggregation was also inhibited by FPTQ in the LPS-stimulated zebrafish model. Decreased mRNA expression of the pro-inflammatory cytokine genes, interleukin-1β (il-1β) and interleukin-6 (il-6), was found in zebrafish larvae injected with FPTQ. Additionally, production of nitric oxide was inhibited by FPTQ in RAW264.7 macrophage cells treated with LPS. Moreover, the mRNA expression of Il-1β and Il-6 suppressed by FPTQ treatment in RAW264.7 macrophage cells, and an enzyme immunoassay showed that FPTQ suppressed the secretion of IL-1β and IL-6 in RAW264.7 cells. These results demonstrate that FPTQ reduced inflammatory responses and, therefore, suggest that it may be effective as an anti-inflammatory agent.

Significance Emotion-related responses, such as fear and anxiety, are important behavioral phenomena in most animal species, as well as in humans. However, the underlying mechanisms of fear and anxiety in animals and in humans are still largely unknown, and anxiety disorders continue to represent a large unmet medical need in the human clinic. Animal models may speed up discovery of these mechanisms and may also lead to betterment of human health. Herein, we report the identification of a chemokine-like gene family, samdori (sam), and present functional characterization of sam2. We observed increased anxiety-related responses in both zebrafish and mouse knockout models. Taken together, these results support a crucial and evolutionarily conserved role of sam2 in regulating anxiety-like behavior., Emotional responses, such as fear and anxiety, are fundamentally important behavioral phenomena with strong fitness components in most animal species. Anxiety-related disorders continue to represent a major unmet medical need in our society, mostly because we still do not fully understand the mechanisms of these diseases. Animal models may speed up discovery of these mechanisms. The zebrafish is a highly promising model organism in this field. Here, we report the identification of a chemokine-like gene family, samdori (sam), and present functional characterization of one of its members, sam2. We show exclusive mRNA expression of sam2 in the CNS, predominantly in the dorsal habenula, telencephalon, and hypothalamus. We found knockout (KO) zebrafish to exhibit altered anxiety-related responses in the tank, scototaxis and shoaling assays, and increased crh mRNA expression in their hypothalamus compared with wild-type fish. To investigate generalizability of our findings to mammals, we developed a Sam2 KO mouse and compared it to wild-type littermates. Consistent with zebrafish findings, homozygous KO mice exhibited signs of elevated anxiety. We also found bath application of purified SAM2 protein to increase inhibitory postsynaptic transmission onto CRH neurons of the paraventricular nucleus. Finally, we identified a human homolog of SAM2, and were able to refine a candidate gene region encompassing SAM2, among 21 annotated genes, which is associated with intellectual disability and autism spectrum disorder in the 12q14.1 deletion syndrome. Taken together, these results suggest a crucial and evolutionarily conserved role of sam2 in regulating mechanisms associated with anxiety.

Cardio-facio-cutaneous (CFC) syndrome is a developmental disorder caused by constitutively active ERK signaling manifesting mainly from BRAF mutations. Little is known about the role of elevated ERK signaling in CFC syndrome during early development. Here, we show that both SMAD1 and ERK signaling pathways may contribute to the developmental defects in CFC syndrome. Induced pluripotent stem cells (iPSCs) derived from dermal fibroblasts of a CFC syndrome patient (CFC-iPSCs) revealed early developmental defects in embryoid body (EB) development, β-catenin localization, and neuronal differentiation. Both SMAD1 and ERK signalings were significantly activated in CFC-iPSCs during EB formation. Most of the β-catenin was dissociated from the membrane and preferentially localized into the nucleus in CFC-EBs. Furthermore, activation of SMAD1 signaling recapitulated early developmental defects in wild-type iPSCs. Intriguingly, inhibition of SMAD1 signaling in CFC-iPSCs rescued aberrant EB morphology, impaired neuronal differentiation, and altered β-catenin localization. These results suggest that SMAD1 signaling may be a key pathway contributing the pathogenesis of CFC syndrome during early development. Stem Cells 2015;33:1447–1455

Bone is maintained by osteoclast-mediated resorption and osteoblast-mediated formation. Recently, anti-osteoporotic activity of Saururus chinensis extract (SCE) and anti-osteoclastogenic activity of its components have been reported, but the effect of SCE on bone formation has not been studied well. Therefore, in this study, we investigated whether Saururus chinensis SCE exhibits in vitro osteogenic and in vivo bone-forming activity. extract strongly enhanced the bone morphogenetic protein (BMP)-2-stimulated induction of alkaline phosphatase, an early phase biomarker of osteoblast differentiation, in bi-potential mesenchymal progenitor C2C12 cells. In vitro osteogenic activity of SCE was accompanied by enhanced expression of BMP-2, BMP-4, BMP-7 and BMP-9 mRNA. In addition, a pharmacological inhibition study suggested the involvement of p38 activation in the osteogenic action of SCE. Moreover, the BMP dependency and the involvement of p38 activation in the osteogenic action of SCE were confirmed by the treatment of noggin, an antagonist of BMP. Saururus chinensis extract also exhibited to induce runt-related transcription factor 2 activation at the high concentration. Furthermore, the in vivo osteogenic activity of SCE was confirmed in zebrafish and mouse calvarial bone formation models, suggesting the possibility of its use for bone formation. In conclusion, we suggested that in vivo anti-osteoporotic activity of SCE could be because of its dual action in bone, anti-osteoclastogenic and anabolic activity.

Cilia are microtubule-based structures that project into the extracellular space. Ciliary defects are associated with several human diseases, including polycystic kidney disease, primary ciliary dyskinesia, left-right axis patterning, hydrocephalus and retinal degeneration. However, the genetic and cellular biological control of ciliogenesis remains poorly understood. The IFT46 is one of the highly conserved intraflagellar transport complex B proteins. In zebrafish, ift46 is expressed in various ciliated tissues such as Kupffer׳s vesicle, pronephric ducts, ears and spinal cord. We show that ift46 is localized to the basal body. Knockdown of ift46 gene results in multiple phenotypes associated with various ciliopathies including kidney cysts, pericardial edema and ventral axis curvature. In ift46 morphants, cilia in kidney and spinal canal are shortened and abnormal. Similar ciliary defects are observed in otic vesicles, lateral line hair cells, olfactory pits, but not in Kupffer׳s vesicle. To explore the functions of Ift46 during mouse development, we have generated Ift46 knock-out mice. The Ift46 mutants have developmental defects in brain, neural tube and heart. In particular Ift46(-/-) homozygotes displays randomization of the embryo heart looping, which is a hallmark of defective left-right (L/R) axis patterning. Taken together, our results demonstrated that IFT46 has an essential role in vertebrate ciliary development.

Miles–Carpenter syndrome (MCS) was described in 1991 as an XLID syndrome with fingertip arches and contractures and mapped to proximal Xq. Patients had microcephaly, short stature, mild spasticity, thoracic scoliosis, hyperextendable MCP joints, rocker-bottom feet, hyperextended elbows and knees. A mutation, p.L66H, in ZC4H2, was identified in a XLID re-sequencing project. Additional screening of linked families and next generation sequencing of XLID families identified three ZC4H2 mutations: p.R18K, p.R213W and p.V75in15aa. The families shared some relevant clinical features. In silico modeling of the mutant proteins indicated all alterations would destabilize the protein. Knockout mutations in zc4h2 were created in zebrafish and homozygous mutant larvae exhibited abnormal swimming, increased twitching, defective eye movement and pectoral fin contractures. Because several of the behavioral defects were consistent with hyperactivity, we examined the underlying neuronal defects and found that sensory neurons and motoneurons appeared normal. However, we observed a striking reduction in GABAergic interneurons. Analysis of cell-type-specific markers showed a specific loss of V2 interneurons in the brain and spinal cord, likely arising from mis-specification of neural progenitors. Injected human wt ZC4H2 rescued the mutant phenotype. Mutant zebrafish injected with human p.L66H or p.R213W mRNA failed to be rescued, while the p.R18K mRNA was able to rescue the interneuron defect. Our findings clearly support ZC4H2 as a novel XLID gene with a required function in interneuron development. Loss of function of ZC4H2 thus likely results in altered connectivity of many brain and spinal circuits.

Wnt signaling controls critical developmental processes including tissue/body patterning. Here we report the identification of a novel regulator of Wnt signaling, OTTOGI (OTG), isolated from a large-scale expression screening of human cDNAs in zebrafish embryos. Overexpression of OTG in zebrafish embryos caused dorso-anteriorized phenotype, inhibited the expression of Wnt target genes, and prevented nuclear accumulation of β-catenin. Conversely, knockdown of zebrafish otg using specific antisense morpholino promoted nuclear accumulation of β-catenin and caused ventralization. However, OTG failed to rescue headless-like phenotype induced by inhibition of GSK-3β activity, suggesting that OTG acts upstream of GSK-3β. OTG bound specifically to Frizzled8 (Fz8) receptor and caused retention of Fz8 in the endoplasmic reticulum possibly by preventing N-linked glycosylation of Fz8. Taken together, our data indicate that OTG functions as a novel negative regulator of Wnt signaling during development by the modulation of cell surface expression of Fz receptor.

Analysis of various parameters in the novel tank test. (A, B) Number of entries to the middle or top zone of the tank. (C) Freezing duration. Duration of freezing time (seconds) was measured in every minute. KO zebrafish show reduced freezing behavior, compared to that of WT zebrafish. (D, E) Total distance moved (cm) and mean velocity (cm/s). No significant difference was detected between WT and KO fish. Number of fish used in this assay: n = 8 for WT fish, n = 8 for KO fish, respectively. Data are presented as mean ± SEM. * p

Number of transit movements of tester fish at each zone boundary. (A-C) 3 cases of each transit movements were analyzed: movement “I” to “II” (A), “II” to “III” (B), and “III” to “IV” zone (C). Number of tester fish used in this assay: n = 30 for WT and n = 30 for KO. Data are presented as mean ± SEM. * p

Schematic illustration of the social interaction assay with different separation materials. (A-F) Two kinds of materials were used for the separator: metal mesh (A, B, and E) and a clear acrylic plate (C, D, and F). (B, D) Changes of duration time for tester fish, when added to a different number of fish (1–5 fish) as the social cue group, was analyzed in 4 different zones between 6 and 10 min. Multiple trials (n = 5) were performed for each group size. (E, F) Detailed information for duration time for tester fish in different zones over the course of 15 min. Data are presented as mean ± SEM. (PDF 1033 kb)

Analysis of various parameters in social interaction assay. (A, B) Video tracking of WT and dyrk1aa KO zebrafish in the social interaction assay. Dashed lines indicate position of a separator and a transparent acrylic plate was used in this experiment. Red lines show tracking of swim movement of WT and KO zebrafish. Fish were tracked every minute for 15 min during the social interaction assay. In this experiment, 3 fish were used as the social cue group in left side and 1 tester fish in right side. (C-F) Duration time for WT and dyrk1aa KO zebrafish in each zone; very close zone “I” (C), close zone “II” (D), far zone “III” (E), and very far zone “IV” (F). (G, H) Total distance moved (cm) and mean velocity (cm/s) are no different in both WT and KO fish during the test. Data were collected from video tracking for 15 min and every minute was analyzed. Number of tester fish used in the assay: n = 30 for WT and n = 30 for KO. Data are presented as mean ± SEM. * p

Cellular FLICE-inhibitory protein (cFLIP, cflara) is a regulator of death receptor (DR)-induced apoptosis and NF-κB activation. cFLIP is known to prevent activation of the caspase cascade by binding to FADD/caspase-8. Up-regulated cFLIP has been identified in many tumor types, and therefore restoring apoptosis by silencing cFLIP may be one of the more potent strategies in cancer therapeutics. The zebrafish cFLIP gene, cflara, has 2 death effector domains (DEDs) and a single caspase-like domain. Expression of cflara was detected in the zebrafish embryo by RT-PCR and whole-mount in situ hybridization. To study the in vivo function of cflara, we generated a cflara knockout mutant zebrafish using transcription activator-like effector nucleases (TALENs). Frame shift mutation is caused by a 10-bp deletion in the first DED domain. By inbreeding the F1 generation, a homozygous mutant fish was produced and confirmed by PCR. Knockout of cflara leads to abnormal heart development and embryonic lethality in mice. However, mutant zebrafish did not show any differences from wild type in heartbeat rate, survival rate or development. Zebrafish have analogues of both cflara and cflarb. Quantitative PCR showed that cflarb mRNA levels of mutant zebrafish were higher than those in the wild type. In a chemical exposure experiment, mutant zebrafish larvae showed a longer survival rate compared with wild type after CoCl2 treatment. However, no significant difference was observed from cisplatin treatment. This data suggests that cflarb may contribute to normal development and causes a difference in chemical resistance.

Characterization of early neural development and larval behavioral tests in WT and dyrk1aa KO fish. (A-F) Whole-mount in situ hybridization analysis with various molecular markers at 24 hpf (A, B, lateral view) and 48 hpf (C-F, rostral dorsal view): sox2, neural stem cell marker; neurog1, neuronal determination marker; and cyclin D1, cell proliferation marker. Anterior is to the left. Number of fish used for the analysis: 1) after sox2 staining and photography, each embryo was genotyped for WT (5/19) and KO homozygote (3/19); 2) for neurog1, it was WT (6/16) and KO homozygote (4/16); and 3) for cyclin D1, WT (3/16) and KO homozygote (5/16), respectively. (G) Locomotion response to dark flashes in WT and dyrk1aa KO larvae at 6 dpf. Movement distance was measured by video tracking analysis (cm per every 10 s). With light-on in 30 s-intervals, zebrafish larvae showed a freezing response (yellow box in the graph). However, they showed a startle response to light-off dark condition (gray box). The number of fish used for this assay: n = 14 for WT (+/+), n = 25 for heterozygote (+/−), and n = 9 for KO homozygote (−/−). (H) Circadian rhythms in WT and dyrk1aa KO larvae between 5 and 7 dpf. Circadian rhythms of locomotor activity under LD (day-night) cycles were measured. Both WT and dyrk1aa KO larvae display a similar pattern of locomotor activity in daytime or nighttime. The number of fish used for this assay: n = 11 for control heterozygote (+/−), and n = 13 for KO homozygote (−/−). Data are presented as mean ± SEM. (PDF 1219 kb)

Comparison of brain size in WT and dyrk1aa KO fish. (A) Relative size of brain compartments in KO fish brain was shown as a ratio, compared to those in WT fish brain. Also, body length (cm) of WT and KO fish used for this analysis was constant. Tel, Telencephalon; TeO, Tectum Opticum; CCe, Corpus Cerebelli. Number of dissected brains: n = 13 for WT fish and n = 13 for KO fish. (B) Percent of TeV space in the total brain. Mean value for the TeV sizes was measured in relative sections of multiple brain samples. Number of fish used for this assay: n = 6 for WT fish and n = 5 for KO fish. Data are presented as mean ± SEM. * p

Presence of neuromodulator producing cells in the brain of WT and dyrk1aa KO zebrafish. (A-H) Expression of oxt, th, vglut2.2, and gad1b in the brain of WT and dyrk1aa KO zebrafish at adult stages. In situ hybridization revealed dyrk1aa KO fish show unchanged neuromodulator-producing cells in the brain. (A, B) oxt, oxytocinergic marker. (C, D) vglut2.2, glutamatergic marker. (E, F) th1, dopaminergic marker. (G, H) gad1b, GABAergic marker. Anterior to the top and ventral view. Scale bars: 0.2Â mm. (PDF 1051 kb)

MicroRNA-122 (miRNA-122), also known as liver-specific miRNA, has recently been shown to be a potent biomarker in response to liver injury in mammals. The objective of this study was to examine its expression in response to toxicant treatment and acute liver damage, using the zebrafish system as an alternative model organism. For the hepatotoxicity assay, larval zebrafish were arrayed in 24-well plates. Adult zebrafish were also tested and arrayed in 200 mL cages. Animals were exposed to liver toxicants (tamoxifen or acetaminophen) at various doses, and miRNA-122 expression levels were analyzed using qRT-PCR in dissected liver, brain, heart, and intestine, separately. Our results showed no significant changes in miRNA-122 expression level in tamoxifen-treated larvae; however, miRNA-122 expression was highly induced in tamoxifen-treated adults in a tissue-specific manner. In addition, we observed a histological change in adult liver (0.5 μM) and cell death in larval liver (5 μM) at different doses of tamoxifen. These results indicated that miRNA-122 may be utilized as a liver-specific biomarker for acute liver toxicity in zebrafish.

Tanshinone IIA is a diterpene quinone isolated from the roots of Salvia miltiorrhiza bunge that has traditionally been used in China for the treatment of cardiovascular and cerebrovascular disorders. Although there is recent evidence showing that tanshinone IIA has an anti-obesity effect, its underlying mechanism of anti-obesity effect is poorly understood. Here, we investigated the effect of tanshinone IIA on lipid accumulation in 3T3-L1 preadipocytes and zebrafish. Notably, tanshinone IIA at 10 μM concentration greatly reduced lipid accumulation and triglyceride (TG) contents during 3T3-L1 preadipocyte differentiation, suggesting its anti-adipogenic effect. On mechanistic levels, tanshinone IIA reduced the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3/5 (STAT-3/5) in differentiating 3T3-L1 cells. In addition, tanshinone IIA strongly inhibited leptin and resistin mRNA expression in differentiating 3T3-L1 cells. Importantly, the tanshinone IIA’s lipid-reducing effect was also seen in zebrafish. In sum, these findings demonstrate that tanshinone IIA has anti-adipogenic effects on 3T3-L1 cells and zebrafish, and its anti-adipogenic effect on 3T3-L1 cells is largely attributable to the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3/5.