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Vitamin C (VC) is well-known as a hydrophilic antioxidant commonly used in cosmeceutical formulations due to its protection and maintenance of youthful skin. Aminoclay (AC), a synthetic organic-nanoclay, has shown great potential for delivery of VC. However, the practical cosmeceutical applications of aminoclay for delivery of VC are severely limited due to the paucity of reported research on its cytotoxicity to human skin. Therefore, in the present study, we evaluated the biosafety of a calcium aminoclay-vitamin C (CaAC-VC) hybrid through an In-Vitro cytotoxicity assessment in HaCaT cells and an In-Vivo embryotoxicity assay in zebrafish. HaCaT cell viability and changes in the morphology and hatching rate of the zebrafish were investigated. The results indicated that the CaAC-VC hybrid showed a lower cytotoxicity relative to pure VC and that as such, it should be considered to be a promising candidate for VC-delivery applications.

Background Alpha-mangostin has potential as a chemopreventive agent but there is little information on its toxicological profile and developmental toxicity. Objective We evaluated the effects of α-mangostin on embryonic development and hepatogenesis in zebrafish. Result Exposure of embryos to 0.25–4 μM α-mangostin from 4–120 h post-fertilization (hpf) caused mortality of embryos with LC50 1.48 ± 0.29 μM. The compound also caused deformities, including head malformation, pericardial oedema, absence of swim bladder, yolk oedema, and bent tail. Exposure of zebrafish embryos to α-mangostin during early hepatogenesis (16–72 hpf) decreased the transcript expression levels of liver fatty acid-binding protein 10a (Fabp10a), but increased gene markers of inflammation, oxidative stress, and apoptosis. In Fabp10a:DsRed transgenic zebrafish, the intensity and the area of fluorescence in the liver of the treated group were decreased (non-significantly) relative to controls. Conclusion These effects were more marked during early hepatogenesis (16–72 hpf) than during post-hepatogenesis (72–120 hpf).

Human-induced pluripotent stem cells (hiPSCs) are invaluable sources for drug screening and toxicity tests because of their differentiation potential and proliferative capacity. Recently, the CRISPR-Cas9-mediated homologous recombination system has enabled reporter knock-ins at desired loci in hiPSCs, and here, we generated a hiPSC reporter line expressing mCherry-tagged cytochrome P450 1A1 (CYP1A1), which can be utilized to screen for the modulators of aryl hydrocarbon receptor (AHR) in live cells. CYP1A1-mCherry hiPSCs exhibited typical characteristics of pluripotent stem cells such as marker expression, differentiation potential, and normal karyotype. After differentiation into hepatocyte-like cells (HLCs), CYP1A1-mCherry fusion protein was expressed and localized at the endoplasmic reticulum, and induced by AHR agonists. We obtained 23 hits modulating CYP1A1 expression from high-content screening with 241 hepatotoxicity chemicals and nuclear receptor ligands, and identified three upregulating chemicals and two downregulating compounds. Responses of hiPSC-HLCs against an AHR agonist were more similar to human primary hepatocytes than of HepG2 hepatocellular carcinoma cells. This platform has the advantages of live-cell screening without sacrificing cells (unlike previously available CYP1A1 reporter cell lines), as well as an indefinite supply of cells, and can be utilized in a wide range of screening related to AHR- and CYP1A1-associated diseases in desired cell types.

3,4-Dichloroaniline (3,4-DCA) is a transformation product of herbicides that is commonly used as a reference in developmental toxicity studies (OECD TG 236) (Bonnet et al. in Environ Toxicol 22:78–91, 2007). However, the mechanisms underlying 3,4-DCA-induced hepatotoxicity are not well known. We exposed zebrafish larvae at 72 hpf to 3,4-DCA for 3 days and observed lipid accumulation in liver treated with 10-μM 3,4-DCA using oil red O staining. Subsequently, we performed qRT-PCR analysis to determine the genes involved in the observed lipid accumulation. We found that genes related to lipogenesis (srebp1, pparγ, lipin1, and scd1) and ER stress (bip, atf4, ddit3, dnajc3, and edem1) were significantly upregulated. In addition, we found that ROS generation increased in the larvae treated with 10-μM 3,4-DCA. Moreover, glutathione-S-transferase activity in these larvae was increased by 3,4-DCA in a dose-dependent manner, and the expression of the inflammation marker il-1β increased. Our results indicated that exposure to 3,4-DCA induced fatty liver in zebrafish larvae and that this, in association with additional factors such as ER stress response, can promote liver damage. We accordingly suggest that 3,4-DCA could be used to induce fatty liver in zebrafish larvae.

Biocidal disinfectants (BDs) that kill microorganisms or pathogens are widely used in hospitals and other healthcare fields. Recently, the use of BDs has rapidly increased as personal hygiene has become more apparent owing to the pandemic, namely the coronavirus outbreak. Despite frequent exposure to BDs, toxicity data of their potential neurotoxicity (NT) are lacking. In this study, a human-derived SH-SY5Y/astrocyte was used as a co-culture model to evaluate the chemical effects of BDs. Automated high-content screening was used to evaluate the potential NT of BDs through neurite growth analysis. A set of 12 BD substances classified from previous reports were tested. Our study confirms the potential NT of benzalkonium chloride (BKC) and provides the first evidence of the potential NT of poly(hexamethylenebicyanoguanide-hexamethylenediamine) hydrochloride (PHMB). BKC and PHMB showed significant NT at concentrations without cytotoxicity. This test system for analyzing the potential NT of BDs may be useful in early screening studies for NT prior to starting in vivo studies.

MicroRNAs (miRNAs) are small non-coding RNAs, which are involved in RNA silencing and post-transcriptional regulation of gene expression. Numerous studies have determined the expression of certain miRNAs in specific tissues and cell types, and their aberrant expression is associated with a variety of serious diseases such as cancers, immune-related diseases, and many infectious diseases. This suggests that miRNAs may be attractive and promising non-invasive biomarkers of diseases. In this study, we established a graphene oxide (GO)-based fluorescence/colorimetric dual sensing platform for miRNA by using a newly designed probe. The probe was designed to form a hairpin-like configuration with a fluorescent dye-labeled long tail, possessing a guanine (G)-rich DNAzyme domain in the loop region and target binding domain over the stem region and tail. By introducing this new hairpin-like probe in a conventional GO-based fluorescence platform, we observed both the miRNA-responsive color change by direct observation and sensitive fluorescence increase even below the nanomolar levels in a single solution without an additional separation step.

Acrylamide is a commonly used industrial chemical that is known to be neurotoxic to mammals. However, its developmental toxicity is rarely assessed in mammalian models because of the cost and complexity involved. We used zebrafish to assess the neurotoxicity, developmental and behavioral toxicity of acrylamide. At 6 h post fertilization, zebrafish embryos were exposed to four concentrations of acrylamide (10, 30, 100, or 300 mg/L) in a medium for 114 h. Acrylamide caused developmental toxicity characterized by yolk retention, scoliosis, swim bladder deficiency, and curvature of the body. Acrylamide also impaired locomotor activity, which was measured as swimming speed and distance traveled. In addition, treatment with 100 mg/L acrylamide shortened the width of the brain and spinal cord, indicating neuronal toxicity. In summary, acrylamide induces developmental toxicity and neurotoxicity in zebrafish. This can be used to study acrylamide neurotoxicity in a rapid and cost-efficient manner.

Two zinc-aminoclays [ZnACs] with functionalized primary amines [(-CH2)3NH2] were prepared by a simple sol-gel reaction using cationic metal precursors of ZnCl2 and Zn(NO3)2 with 3-aminopropyl triethoxysilane [APTES] under ambient conditions. Due to the facile interaction of heavy metals with primary amine sites and Zn-related intrinsic antimicrobial activity, toxicity assays of ZnACs nanoparticles (NPs) prior to their environmental and human-health applications are essential. However, such reports remain rare. Thus, in the present study, a cell viability assay of in-vitro HeLa cells comparing ZnCl2, Zn(NO3)2 salts, and ZnO (~50 nm average diameter) NPs was performed. Interestingly, compared with the ZnCl2, and Zn(NO3)2 salts, and ZnO NPs (18.73/18.12/51.49 µg/mL and 18.12/15.19/46.10 µg/mL of IC50 values for 24 and 48 h), the two ZnACs NPs exhibited the highest toxicity (IC50 values of 21.18/18.36 µg/mL and 18.37/17.09 µg/mL for 24 and 48 h, respectively), whose concentrations were calculated on Zn elemental composition. This might be due to the enhanced bioavailability and uptake into cells of ZnAC NPs themselves and their positively charged hydrophilicity by reactive oxygen species (ROS) generation, particularly as ZnACs exist in cationic NP's form, not in released Zn2+ ionic form (i.e., dissolved nanometal). However, in an in-vivo embryotoxicity assay in zebrafish, ZnACs and ZnO NPs showed toxic effects at 50–100 µg/mL (corresponding to 37.88–75.76 of Zn wt% µg/mL). The hatching rate (%) of zebrafish was lowest for the ZnO NPs, particularly where ZnAC-[(NO3)2] is slightly more toxic than ZnAC-[Cl2]. These results are all very pertinent to the issue of ZnACs’ potential applications in the environmental and biomedical fields.

Multifunctional carbon-based nanodots (C-dots) are synthesized using atmospheric plasma treatments involving reactive gases (oxygen and nitrogen). Surface design was achieved through one-step plasma treatment of C-dots (AC-paints) from polyethylene glycol used as a precursor. These AC-paints show high fluorescence, low cytotoxicity and excellent cellular imaging capability. They exhibit bright fluorescence with a quantum yield twice of traditional C-dots. The cytotoxicity of AC-paints was tested on BEAS2B, THLE2, A549 and hep3B cell lines. The in vivo experiments further demonstrated the biocompatibility of AC-paints using zebrafish as a model, and imaging tests demonstrated that the AC-paints can be used as bio-labels (at a concentration of 1 mg mL−1). AC-paints can effectively inhibit the growth of Escherichia coli (E. coli) and Acinetobacter baumannii (A. baumannii). Such remarkable performance of the AC-paints has important applications in the biomedical field and environmental systems.

Cdc2-like kinases (CLKs) play a crucial role in the alternative splicing of eukaryotic pre-mRNAs through the phosphorylation of serine/arginine-rich proteins (SR proteins). Dysregulation of this processes is linked with various diseases including cancers, neurodegenerative diseases, and many genetic diseases. Thus, CLKs have been regarded to have a potential as a therapeutic target and significant efforts have been exerted to discover an effective inhibitor. In particular, the small molecule CX-4945, originally identified as an inhibitor of casein kinase 2 (CK2), was further revealed to have a strong CLK-inhibitory activity. Four isoforms of CLKs (CLK1, CLK2, CLK3, and CLK4) can be inhibited by CX-4945, with the highest inhibitory effect on CLK2. This study aimed to elucidate the structural basis of the selective inhibitory effect of CX-4945 on different isoforms of CLKs. We determined the crystal structures of CLK1, CLK2, and CLK3 in complex with CX-4945 at resolutions of 2.4 Å, 2.8 Å, and 2.6 Å, respectively. Comparative analysis revealed that CX-4945 was bound in the same active site pocket of the CLKs with similar interacting networks. Intriguingly, the active sites of CLK/CX-4945 complex structures had different sizes and electrostatic surface charge distributions. The active site of CLK1 was somewhat narrow and contained a negatively charged patch. CLK3 had a protruded Lys248 residue in the entrance of the active site pocket. In addition, Ala319, equivalent to Val324 (CLK1) and Val326 (CLK2), contributed to the weak hydrophobic interactions with the benzonaphthyridine ring of CX-4945. In contrast, the charge distribution pattern of CLK2 was the weakest, favoring its interactions with benzonaphthyridine ring. Thus, the relatively strong binding affinities of CX-4945 with CLK2 are consistent with its strong inhibitory effect defined in the previous study. These results may provide insights into structure-based drug discovery processes.

According to the increasing concern about particulate matter (PM) pollution at subway systems, particularly its potentially severe effects on human health, this study investigated the constituents, characteristics, and toxicity of PM collected at underground subway stations in Seoul, Korea. It was found that α/γ-Fe

According to the increasing concern about particulate matter (PM) pollution at subway systems, particularly its potentially severe effects on human health, this study investigated the constituents, characteristics, and toxicity of PM collected at underground subway stations in Seoul, Korea. It was found that α/γ-Fe2O3 NPs, which are considered as thermal products derived from the brake-wheel-rail interface, were the main components of PM (57.6% and 48% of PM10 and PM2.5, respectively). In addition, hydrothermally synthesized α/γ-Fe2O3 NPs, proposing to possess similar properties to those of Fe2O3 contained in PM, were used to investigate the correlation of these oxides with PM toxicity. In particular, the synthesized γ-Fe2O3 NPs induced a negligibly toxic, while the synthesized α-Fe2O3 NPs and PM showed remarkably toxic effects on HeLa cells and zebrafish embryos, specifically in reducing cell proliferation to 85% and 72% survival, causing high apoptosis of 29.8% and 29.3%, and inhibiting the development of embryos up to 60% and 8% after prolonged exposure, respectively. It is considered that α-Fe2O3 NPs were primarily responsible for the harmful effects of PM, resulting in significant damage to DNA due to their capacity of producing high reactive oxygen species (ROS) and, thus, deleterious effects on the human body.

A novel small-molecule fluorescent probe (4f) for staining blood in live zebrafish was identified. Compound 4f turns on the fluorescent property in the presence of albumin in the blood and it mainly binds on the site II of human serum albumin. 4f efficiently visualized the blood vessels in live zebrafish, Hence, it could be used as an alternative blood vessel imaging tool. Compound 4f applied to test for anti-cancer drug screening in live zebrafish, and showed a potential as an effective in vivo drug screening agent.

Gre2 is a key enzyme in the methylglyoxal detoxification pathway, it uses NADPH or NADH as an electron donor to reduce the cytotoxic methylglyoxal to lactaldehyde. This enzyme is a member of the short-chain dehydrogenase/reductase (SDR) superfamily whose members catalyze this type of reaction with a broad range of substrates. To elucidate the structural features, we determined the crystal structures of the NADPH-dependent methylglyoxal reductase Gre2 from Candida albicans (CaGre2) for both the apo-form and NADPH-complexed form at resolutions of 2.8 and 3.02 &Aring, respectively. The CaGre2 structure is composed of two distinct domains: the N-terminal cofactor-binding domain and the C-terminal substrate-binding domain. Extensive comparison of CaGre2 with its homologous structures reveals conformational changes in &alpha, 12 and &beta, 3&prime, of the NADPH-complex forms. This study may provide insights into the structural and functional variation of SDR family proteins.

Pancreatic Langerhans islets are mainly composed of insulinsecreting beta cells and glucagon-secreting alpha cells, along with other minor cell types, and play a central role in the regulation of blood glucose levels. Because of this, imaging of viable pancreatic islets is an important component in research on diabetes both in clinical and experimental medicine. The conventional imaging technique for pancreatic islets is antibody-based immunostaining directly on pancreatic sections, or using transgenic mice with luminescent reporter genes linked to islet-specific promoters. Among small molecule probes, Newport Green and dithiazone (DTZ) have been used for ex vivo fluorescent staining of pancreatic islets, based on their Zn ion binding affinity, which are abundant in beta cells in complex with insulin. For in situ application, fluorescently labeled exendin-4 (a GLP1R binding peptide: M.W. is about 5 kDa) has been recently introduced for the measurement of the mass of pancreatic islet beta cells. However, small molecule probes for selective staining of beta cells in pancreatic islets of live animals have not yet been reported. We predicted that a diversity-oriented fluorescence library approach (DOFLA), an expedited bioimaging probe discovery method using high throughput synthesis and high contents screening, would be a powerful method to achieve this goal. Using a similar approach, we have previously elucidated probes for pluripotent stem cells (CDy1), muscle cells (CDy2), neuronal stem cells (CDr3), and pancreatic alpha cells (GY= glucagon yellow). Although the glucagon-targeting probe GY selectively stains alpha cells in isolated cell culture, it did not clearly mark mouse pancreatic islets in tissue, partially owing to the small population of alpha cells (around 15–20% in mouse islets). We expected that a fluorescent probe for pancreatic beta cells (with a larger population of 75–80% in mouse islets) would be more effective for visualizing pancreatic islets. As a first step, we synthesized fluorescent smallmolecule libraries composed of 1200 compounds, and screened them against beta TC-6 cells in comparison to alpha TC-1 cells and acinar cells (exocrine cells in the pancreas) as controls. The three cell types were compared in 384-well plates and incubated with the library compounds (1 mm) at incubation times ranging from one to 48 hours. The fluorescence live-cell images were acquired by an automated imaging microscope system, ImageXpress Micro. One compound from the BDNCA series (Scheme 1), BDNCA-325 (labs/lem= 558/585 nm, extinction cooefficient e= 58,000m 1 cm , quantum yield F= 0.06) was chosen as the most selective for the beta TC-6 cells (Figure 1a) in comparison to the two control cell types in terms of relative fluorescence intensity. The BDNCA library was prepared from a BODIPY-aniline (BDN) series by chloroacetylation. While BDN has very low fluorescence emission (less than 1% quantum yield) owing to photoinduced electron transfer (PET), by converting the amine to an amide, the fluorescence of BDNCA was moderately increased (F= 5–10%). Therefore, this amide motif is a modulator of the fluorescence intensity of the BDNCA series through interaction with the surrounding environment or binding partner. However, when we injected BDNCA-325 intravenously into a mouse, the pancreatic islets were not selectively stained at various incubation times and concentrations (data not shown). Because BDNCA-325 contains a chemically reactive chloroacetyl group, we hypothesized that the compound might have reacted with other tissues in the animal before reaching the pancreatic islets. Thus, BDNCA-325 was modified by removing the reactive alpha-chloride and also by [*] Dr. N.-Y. Kang, Dr. S.-C. Lee, Dr. S.-J. Park, Dr. S.-W. Yun, Dr. E. Kostromina, Dr. N. Gustavsson, Dr. Y. Ali, Y. Chandran, Dr. W. Han, Dr. G. K. Radda, Prof. Y.-T. Chang Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR) 138667, Singapore (Singapore) E-mail: chmcyt@nus.edu.sg Homepage: http://ytchang.science.nus.edu.sg

We have developed an efficient synthesis of LipidGreen. The conversion is achieved by selective methylation with trimethylsilyldiazomethane, selective deprotection by BBr3 and an improved microwave-assisted C-allylation procedure. Using this route, we have synthesized novel LipidGreen derivatives, and evaluated their live imaging abilities in zebrafish. In this series, Compound 7 is the most active, which is at least 10 fold more potent than LipidGreen.

Although hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) are considered a promising model for predicting hepatotoxicity, their application has been restricted because of the low activity of drug metabolizing enzymes (DMEs). Here we found that the low expression of xenobiotic receptors (constitutive androstane receptor, CAR; and pregnane X receptor, PXR) contributes to the low activity of DMEs in hPSC-HLCs. Most CAR- and PXR-regulated DMEs and transporters were transcriptionally down-regulated in hPSC-HLC. Transcriptional expression of CAR and PXR was highly repressed in hPSC-HLCs, whereas mRNA levels of aryl hydrocarbon receptor (AHR) were comparable to those of adult liver. Furthermore, ligand-induced transcriptional activation was observed only at AHR in hPSC-HLCs. Bisulfite sequencing analysis demonstrated that promoter hypermethylation of CAR and PXR was associated with diminished transcriptional activity in hPSC-HLCs. Treatment with AHR-selective ligands increased the transcription of AHR-dependent target genes by direct AHR-DNA binding at the xenobiotic response element. In addition, an antagonist of AHR significantly inhibited AHR-dependent target gene expression. Thus, AHR may function intrinsically as a xenosensor as well as a ligand-dependent transcription factor in hPSC-HLCs. Our results indicate that hPSC-HLCs can be used to screen toxic substances related to AHR signaling and to identify potential AHR-targeted therapeutics.

Autism spectrum disorder (ASD) is a serious neurodevelopmental disorder characterized by impaired or abnormal social interaction and communication and by restricted and repetitive behaviour. ASD is highly prevalent in Asia, Europe, and the United States, and the frequency of ASD is growing each year. Recent epidemiological studies have indicated that ASD may be caused or triggered by exposure to chemicals in the environment, such as those in the air or water. Thus, toxicological studies are needed to examine chemicals that might be implicated. However, the experimental efficiency of existing experimental models is limited, and many models represent challenges in terms of animal welfare. Thus, alternative ASD animal models are necessary. To address this, we examined the efficacy of the zebrafish embryo/larva as an alternative model of ASD. Specifically, we exposed zebrafish to valproic acid (0, 12.5, 25, 50, or 100 μM), which is a chemical known to induce autism-like effects. We then analysed subsequent developmental, behavioural, and transcriptomic changes. We found that 100 μM and 50 μM valproic acid decreased the hatching rate and locomotor activity of zebrafish embryos/larvae. Transcriptomic analysis revealed significant alterations in a number of genes associated with autism, such as adsl, mbd5, shank3, and tsc1b. Additionally, we found changes in gene ontology that were also reported in previous studies. Our findings indicate that zebrafish embryos/larvae and humans with ASD might have common physiological pathways, indicating that this animal model may represent an alternative tool for examining the causes of and potential treatments for this illness.

Human pluripotent stem cell-derived hepatocytes have the potential to provide in vitro model systems for drug discovery and hepatotoxicity testing. However, these cells are currently unsuitable for drug toxicity and efficacy testing because of their limited expression of genes encoding drug-metabolizing enzymes, especially cytochrome P450 (CYP) enzymes. Transcript levels of major CYP genes were much lower in human embryonic stem cell-derived hepatocytes (hESC-Hep) than in human primary hepatocytes (hPH). To verify the mechanism underlying this reduced expression of CYP genes, including CYP1A1, CYP1A2, CYP1B1, CYP2D6, and CYP2E1, we investigated their epigenetic regulation in terms of DNA methylation and histone modifications in hESC-Hep and hPH. CpG islands of CYP genes were hypermethylated in hESC-Hep, whereas they had an open chromatin structure, as represented by hypomethylation of CpG sites and permissive histone modifications, in hPH. Inhibition of DNA methyltransferases (DNMTs) during hepatic maturation induced demethylation of the CpG sites of CYP1A1 and CYP1A2, leading to the up-regulation of their transcription. Combinatorial inhibition of DNMTs and histone deacetylases (HDACs) increased the transcript levels of CYP1A1, CYP1A2, CYP1B1, and CYP2D6. Our findings suggest that limited expression of CYP genes in hESC-Hep is modulated by epigenetic regulatory factors such as DNMTs and HDACs.

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP(+)-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury.

Peroxisome proliferator-activated receptor γ (PPARγ) is a target of antidiabetic drugs. However, many PPARγ activators, including rosiglitazone, show unwanted side effects, such as weight gain. The KR-62980 [1-(trans-methylimino-N-oxy)-3-phenyl-6-(3-phenylpropoxy)-1H-indene-2-carboxylic acid ethyl ester], a novel partial agonist of PPARγ, is a new compound for diabetes with antihyperglycemic activity and weak antiadipogenic activity. This study was performed to elucidate the mechanism of the weak adipogenesis induced by KR-62980 despite its being a PPARγ agonist in zebrafish. We elucidated the mechanism of KR-62980 in lipid metabolism using adipocytes and zebrafish. Since NADPH is a critical cofactor in fat metabolism, we investigated effect of KR-62980 on NADPH-producing enzymes such as cytosolic NADP(+) isocitrate dehydrogenase (cICDH). We found that the mRNA expression of cICDH was significantly decreased by KR-62980 in 3T3-L1 cells. KR-62980 inhibited lipase activity and lipid metabolism in zebrafish. Further, KR-62980 substantially suppressed cICDH in adipocytes and zebrafish. These results suggest that cICDH may be one of the targets of KR-62980 responsible for weight gain and adipogenesis.

During normal forebrain development in vertebrates, rostral neural tissue must be protected from Wnt signals via the actions of locally expressed Wnt antagonistic factors. In zebrafish zygotic oep (Zoep) mutants, forebrain structure is severely disrupted with reduced expression of the Wnt antagonists secreted frizzled related protein1 and dickkopf1. To analyze the temporal effects of Wnt antagonism on forebrain development, we generated transgenic zebrafish that overexpressed the dominant negative form of frizzled8a (DNfz8a) in wild-type and Zoep mutants under the control of a heat-inducible promoter. This model allowed for assessment of the dynamics of Wnt antagonistic signaling during forebrain development. Our results demonstrated that overexpression of DNfz8a in Zoep embryos between 7 and 16 hpf increased putative forebrain region demarcated by anf and distal-less2 expressions. These results suggest that normal forebrain development requires continual Wnt antagonism from the early gastrula to the mid-somitogenesis stage.

Wnts have been shown to provide a posteriorizing signal that has to be repressed in the specification of vertebrate forebrain region. Previous studies have shown that Wnt activation by LiCl treatment causes an expansion of optic stalk and mid-hindbrain boundary, whereas eye and ventral diencephalon in the forebrain region were reduced. However, the molecular mechanism, by which inhibits Wnt activity in the forebrain remains poorly defined. To investigate relationship between forebrain specification and Wnt signaling, the zebrafish homologue of secreted frizzled related protein1 ( sfrp1 ) has been characterized. The transcripts of sfrp1 are detected in the presumptive forebrain at gastrula and in the ventral telencephalon, ventral diencephalon, midbrain and optic vesicles at 24 h after postfertilization (hpf). Overexpression of sfrp1 causes an anteriorization of embryo, with enlarged head and reduced posterior structure as in the embryo overexpressing dominant-negative form of Frizzled8a or Dkk1. Its overexpression restored the eye defects in the Wnt8b-overexpressing embryos, but not in the LiCl-treated embryos. These results suggest that Sfrp1 expressed in the forebrain and eye field plays a critical role in the extracellular events of antagonizing Wnt activity for the forebrain specification.

We have developed LipidGreen2, a second generation small molecule probe for lipid imaging. LipidGreen2 has a better fluorescence signal compared with the previous LipidGreen, and selectively stains neutral lipids in cells and fat deposits in live zebrafish. We also demonstrate the application of LipidGreen2 for detecting fatty liver.