Opticin Ameliorates Hypoxia-Induced Retinal Angiogenesis by Suppression of Integrin α2-I Domain-Collagen Complex Formation and RhoA/ROCK1 Signaling.

Purpose: It was previously demonstrated that opticin (OPTC) inhibits the collagen-induced promotion of bioactivities of human retinal vascular endothelial cells (hRVECs). The present in vivo study aimed to further investigate the regulatory role of opticin in vitreous collagen-mediated retinal neovascularization and to elucidate its regulatory mechanisms with regard to integrin α2-I domain-GXXGER complex formation and RhoA/ROCK1 signal change. The regulatory role of Mg2+ on integrin α2-I domain-GXXGER complex formation in the above process was also investigated. Methods: The zebrafish model of hypoxia-induced retinopathy was established, and OPTC-overexpressing plasmids were intravitreally injected to assess the antiangiogenesis effect of opticin. The regulatory role of opticin in integrin α2-I domain-GXXGER complex formation in vivo was analyzed by mass spectrometry. The mRNA and protein expression of RhoA/ROCK1 were examined. The concentration of Mg2+ as an activator of the integrin α2-I domain-GXXGER complex was measured. Solid-phase binding assays were performed to investigate the interference of opticin in integrin α2 collagen binding and the regulatory role of Mg2+ in that process. Results: Opticin and OPTC-overexpressing plasmid injection reduced retinal neovascularization in the zebrafish model of hypoxia-induced retinopathy. Mass spectrometry revealed that opticin could inhibit integrin α2-I domain-GXXGER complex formation. The Mg2+ concentration was also decreased by opticin, which was another indication of the complex activation. Injection of OPTC-overexpressing plasmids inhibited mRNA and the protein expression of RhoA/ROCK1 in the zebrafish model of hypoxia-induced retinopathy. The solid-phase binding assay revealed that opticin could block integrin α2-collagen I binding in the presence of Mg2+. Conclusions: Opticin exerts its antiangiogenesis effect by interfering in the Mg2+-modulated integrin α2-I domain-collagen complex formation and suppressing the downstream RhoA/ ROCK1 signaling pathway.

Erianin Controls Collagen-Mediated Retinal Angiogenesis via the RhoA/ROCK1 Signaling Pathway Induced by the alpha2/beta1 Integrin-Collagen Interaction.

Purpose: Erianin has been reported to inhibit tumor activity by suppressing the expression of integrins. It is hypothesized that erianin can inhibit retinal neovascularization in collagen by suppressing the expression of integrins. With an aim to test this hypothesis, the regulation of erianin on collagen-mediated retinal angiogenesis via the Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase 1 (ROCK1) signaling pathway induced by α2 and ß1 integrin-collagen interactions was investigated. Methods: The effects of erianin on human retinal vascular endothelial cells (HRVECs) were assessed in vitro using a hypoxia model in a three-dimensional cell culture induced by cobalt (II) chloride (CoCl2). A hypoxia-induced retinopathy model in adult zebrafish and zebrafish embryos was established to assess the antiangiogenic effect of erianin with and without vitreous collagen in vivo. The expression of α2 and ß1 integrin and RhoA/ROCK1 pathway in HRVECs and zebrafish retinas were analyzed. Results: In vitro, collagen improved the angiogenic potential of HRVECs, including migration, adhesion, and tube formation, in a three-dimensional cell culture model. Erianin suppressed the angiogenic processes of the CoCl2-induced hypoxia HRVEC model in a concentration-dependent manner. In vivo, erianin reduced retinal angiogenesis in the hypoxia-induced retinopathy model in adult and embryo zebrafish. Erianin inhibited the expression of α2 and ß1 integrin and RhoA/ROCK1 in a hypoxia-induced model in vitro in three-dimensional cell culture and in vivo in adult zebrafish. Conclusions: Collagen-mediated retinal angiogenesis may be regulated by erianin via the RhoA/ROCK1 signaling pathway induced by α2 and ß1 integrin-collagen interactions. These findings suggest that erianin has the therapeutic potential on intraocular collagen-mediated retinal angiogenesis.

A novel gene trap line for visualization and manipulation of erbb3b+ neural crest and glial cells in zebrafish.

Glia are a diverse and essential cell type in the vertebrate nervous system. Transgenic tools and fluorescent reporter lines are critical resources to investigate how glial subtypes develop and function. However, despite the many lines available in zebrafish, the community still lacks the ability to label all unique stages of glial development and specific subpopulations of cells. To address this issue, we screened zebrafish gene and enhancer trap lines to find a novel reporter for peripheral glial subtypes. From these, we generated the gSAIzGFFD37A transgenic line that expresses GFP in neural crest cells and central and peripheral glia. We found that the gene trap construct is located within an intron of erbb3b, a gene essential for glial development. Additionally, we confirmed that GFP+ â€‹cells express erbb3b along with sox10, a known glial marker. From our screen, we have identified the gSAIzGFFD37A line as a novel and powerful tool for studying glia in the developing zebrafish, as well as a new resource to manipulate erbb3b+ â€‹cells.

Integrated molecular analysis identifies a conserved pericyte gene signature in zebrafish.

Pericytes reside in capillary beds where they share a basement membrane with endothelial cells and regulate their function. However, little is known about embryonic pericyte development, in part, due to lack of specific molecular markers and genetic tools. Here, we applied single cell RNA-sequencing (scRNA-seq) of platelet derived growth factor beta (pdgfrb)-positive cells to molecularly characterize pericytes in zebrafish larvae. scRNA-seq revealed zebrafish cells expressing mouse pericyte gene orthologs, and comparison with bulk RNA-seq from wild-type and pdgfrb mutant larvae further refined a pericyte gene set. Subsequent integration with mouse pericyte scRNA-seq profiles revealed a core set of conserved pericyte genes. Using transgenic reporter lines, we validated pericyte expression of two genes identified in our analysis: NDUFA4 mitochondrial complex associated like 2a (ndufa4l2a), and potassium voltage-gated channel, Isk-related family, member 4 (kcne4). Both reporter lines exhibited pericyte expression in multiple anatomical locations, and kcne4 was also detected in a subset of vascular smooth muscle cells. Thus, our integrated molecular analysis revealed a molecular profile for zebrafish pericytes and allowed us to develop new tools to observe these cells in vivo.

Adequate expression of neuropeptide Y is essential for the recovery of zebrafish motor function following spinal cord injury.

In strong contrast to limited repair within the mammalian central nervous system, the spinal cord of adult zebrafish is capable of almost complete recovery following injury. Understanding the mechanism underlying neural repair and functional recovery in zebrafish may lead to innovative therapies for human spinal cord injury (SCI). Since neuropeptide Y (NPY) plays a protective role in the pathogenesis of several neurological diseases, in the present study, we evaluated the effects of NPY on neuronal repair and subsequent recovery of motor function in adult zebrafish following SCI. Real-time quantitative PCR (qRT-PCR), in situ hybridization and immunostaining for NPY revealed decreased NPY expression at 12 hours (h), 6 and 21 days (d) after SCI. Double-immunostaining for NPY and islet-1, a motoneuron marker, showed that NPY was expressed in spinal cord motoneurons. Morpholino (MO) treatment for suppressing the expression of NPY inhibited supraspinal axon regrowth and locomotor recovery, in which double-staining for proliferating cell nuclear antigen (PCNA) and islet-1 showed a reduction in motoneuron proliferation. Similarly, a downregulated mRNA level of Y1 receptor of NPY (NPY1R) was also detected at 12 h, 6 and 21 d after injury. Immunostaining for NPY and in situ hybridization for NPY1R revealed that NPY1R was co-localized with NPY. Collectively, the results suggest that NPY expression in motoneurons promotes descending axon regeneration and locomotor recovery in adult zebrafish after SCI, possibly by regulating motoneuron proliferation through activation of NPY1R.

Developmental toxicity, antioxidant, and marker enzyme assessment of swertiamarin in zebrafish (Danio rerio).

A secoiridoid glycoside called swertiamarin has been widely used as a herbal medicine for many decades. In particular, swertiamarin from the Enicostema axillare herb has been used as a multipurpose drug to treat innumerable health problems. As this medicine is consumed orally, its toxicity level should be determined. To examine the safety of this compound, toxicology work was done in zebrafish, and this is the first report to describe swertiamarin toxicity in zebrafish. Zebrafish embryos were used in this swertiamarin toxicity study, and morphological changes were observed. Further, the compound was also studied in adult zebrafish to determine the impact of the compound on the fish liver. Enzyme profiling with superoxide dismutase, glutathione peroxidase, catalase, reduced glutathione levels, glutathione S-transferase, lactate dehydrogenase, glutamic oxaloacetic transaminases, lipid peroxidation, Na+ /K+ -ATPase, and glutamic pyruvic transaminases) was evaluated (p ≤ 0.05). Results suggest that swertiamarin is a safe drug only at a low concentration (40 µM). This study also shows that even herbal medicinal compounds may be toxic to humans at higher dosages. Hence, irrespective of whether a drug is synthetic or natural, it needs to be tested for its toxicity before use in humans.

Nkx2-2 expressing taste cells in endoderm-derived taste papillae are committed to the type III lineage.

In mammals, multiple cell-signaling pathways and transcription factors regulate development of the embryonic taste system and turnover of taste cells in the adult stage. Using single-cell RNA-Seq of mouse taste cells, we found that the homeobox-containing transcription factor Nkx2-2, a target of the Sonic Hedgehog pathway and a key regulator of the development and regeneration of multiple cell types in the body, is highly expressed in type III taste cells but not in type II or taste stem cells. Using in situ hybridization and immunostaining, we confirmed that Nkx2-2 is expressed specifically in type III taste cells in the endoderm-derived circumvallate and foliate taste papillae but not in the ectoderm-derived fungiform papillae. Lineage tracing revealed that Nkx2-2-expressing cells differentiate into type III, but not type II or type I cells in circumvallate and foliate papillae. Neonatal Nkx2-2-knockout mice did not express key type III taste cell marker genes, while the expression of type II and type I taste cell marker genes were unaffected in these mice. Our findings indicate that Nkx2-2-expressing cells are committed to the type III lineage and that Nkx2-2 may be critical for the development of type III taste cells in the posterior tongue, thus illustrating a key difference in the mechanism of type III cell lineage specification between ectoderm- and endoderm-derived taste fields.

Npas4a expression in the teleost forebrain is associated with stress coping style differences in fear learning.

Learning to anticipate potentially dangerous contexts is an adaptive behavioral response to coping with stressors. An animal's stress coping style (e.g. proactive-reactive axis) is known to influence how it encodes salient events. However, the neural and molecular mechanisms underlying these stress coping style differences in learning are unknown. Further, while a number of neuroplasticity-related genes have been associated with alternative stress coping styles, it is unclear if these genes may bias the development of conditioned behavioral responses to stressful stimuli, and if so, which brain regions are involved. Here, we trained adult zebrafish to associate a naturally aversive olfactory cue with a given context. Next, we investigated if expression of two neural plasticity and neurotransmission-related genes (npas4a and gabbr1a) were associated with the contextual fear conditioning differences between proactive and reactive stress coping styles. Reactive zebrafish developed a stronger conditioned fear response and showed significantly higher npas4a expression in the medial and lateral zones of the dorsal telencephalon (Dm, Dl), and the supracommissural nucleus of the ventral telencephalon (Vs). Our findings suggest that the expression of activity-dependent genes like npas4a may be differentially expressed across several interconnected forebrain regions in response to fearful stimuli and promote biases in fear learning among different stress coping styles.

Pinocembrin-7-Methylether Protects SH-SY5Y Cells Against 6-Hydroxydopamine-Induced Neurotoxicity via Modulating Nrf2 Induction Through AKT and ERK Pathways.

The present study aimed to evaluate the neuroprotective effects and underlying mechanisms of pinocembrin-7-methylether (PME), a natural bioflavonoid, in 6-hydroxydopamine (6-OHDA)-induced models of Parkinson's disease in vivo and in vitro. First, we found that PME decreased apoptosis in 6-OHDA-intoxicated SH-SY5Y cells. PME also blocked several 6-OHDA-induced mitochondrial apoptotic cascades, including loss of mitochondrial membrane potential, caspase 3 and PARP activation, and a decrease in the Bcl-2/Bax ratio. Also, PME suppressed 6-OHDA-induced oxidative stress while increasing antioxidant enzymatic activity. Further investigations indicated that PME significantly enhanced nuclear accumulation of Nrf2, improved ARE promoter activity, and upregulated HO-1 and NQO1 expression levels. In addition, siRNA-mediated Nrf2 knockdown abolished PME-induced anti-oxidative and anti-apoptotic effects. Interestingly, we found that PME promoted phosphorylation of AKT and ERK, whereas pharmacological inhibition of AKT or ERK pathways diminished PME-induced Nrf2 activation and protective actions. Moreover, PME attenuated 6-OHDA-induced loss of dopaminergic neurons and ameliorated locomotor deficiency in zebrafish, supporting the neuroprotective actions of PME in vivo. In summary, we found that PME conferred neuroprotection against 6-OHDA-induced neurotoxicity in PD models in vivo and in vitro. Taken together, our findings suggest that activation of Nrf2/ARE/HO-1 signaling cascades contributes to PME-induced anti-oxidative and neuroprotective actions, which are at least partially mediated by AKT and ERK pathways.

The diversity in sensitivity of TRPA1 and TRPV1 of various animals to polyphenols.

The perception of tastes is sensed by the receptors that stimulate sensory cells. We previously reported that TRPA1 and TRPV1 channels expressed in the oral cavity of mammals, are activated by the auto-oxidized product of epigallocatechin gallate (oxiEGCG), a major astringent catechin in green tea. Here, we investigated and compared the sensitivity of TRPA1 and TRPV1 from various animals to astringent polyphenols. We selected three polyphenols, oxiEGCG, tannic acid and myricetin. HEK293T cells expressing TRPA1 or TRPV1 from mammal, bird, reptile, amphibian, and fish, were analyzed for their activation by the Ca2+-imaging. We found the apparent diversity in the polyphenol-sensitivity among various animals. Mammalian TRPs showed relatively higher sensitivity to polyphenols, and especially, human TRPA1 and TRPV1 could be activated by all of three polyphenols at 20 µM. Reptile TRP channels, however, were insensitive to any polyphenols examined. Moreover, the polyphenol-sensitivity of zebrafish TRPA1 and TRPV1 was quite different from that of medaka TRP channels. Since many polyphenols are present in plants and the sensing of polyphenols using TRP channels in the oral cavity might cause astringent taste, the observed diversity of the polyphenol-sensitivity of TRP channels might be involved in the divergence in the food habit of various animals.

The SARS-CoV-2 receptor and other key components of the Renin-Angiotensin-Aldosterone System related to COVID-19 are expressed in enterocytes in larval zebrafish.

People with underlying conditions, including hypertension, obesity, and diabetes, are especially susceptible to negative outcomes after infection with coronavirus SARS-CoV-2, which causes COVID-19. Hypertension and respiratory inflammation are exacerbated by the Renin-Angiotensin-Aldosterone System (RAAS), which normally protects from rapidly dropping blood pressure via Angiotensin II (Ang II) produced by the enzyme Ace. The Ace paralog Ace2 degrades Ang II, counteracting its chronic effects, and serves as the SARS-CoV-2 receptor. Ace, the coronavirus, and COVID-19 comorbidities all regulate Ace2, but we do not yet understand how. To exploit zebrafish (Danio rerio) to help understand the relationship of the RAAS to COVID-19, we must identify zebrafish orthologs and co-orthologs of human RAAS genes and understand their expression patterns. To achieve these goals, we conducted genomic and phylogenetic analyses and investigated single cell transcriptomes. Results showed that most human RAAS genes have one or more zebrafish orthologs or co-orthologs. Results identified a specific type of enterocyte as the specific site of expression of zebrafish orthologs of key RAAS components, including Ace, Ace2, Slc6a19 (SARS-CoV-2 co-receptor), and the Angiotensin-related peptide cleaving enzymes Anpep (receptor for the common cold coronavirus HCoV-229E), and Dpp4 (receptor for the Middle East Respiratory Syndrome virus, MERS-CoV). Results identified specific vascular cell subtypes expressing Ang II receptors, apelin, and apelin receptor genes. These results identify genes and cell types to exploit zebrafish as a disease model for understanding mechanisms of COVID-19.

Molecular cloning and characterization of an atypical butyrylcholinesterase-like protein in zebrafish.

Cholinesterases act as bio scavengers to clear organophosphorus (OP) compounds and prodrugs. The butyrylcholinesterase (BChE) gene has been found in several types of teleost fish but this gene has yet to be identified in cyprinid fish. Indeed, BChE homologs have not been found in the zebrafish (Danio rerio) genomic database. Here, we demonstrate that BChE activity is present in zebrafish, in line with other groups' findings. Using in-gel native-PAGE enzymatic activity staining and LC-MS/MS technique, an atypical BChE-like protein was identified in zebrafish. The si:ch211-93f2.1 gene was cloned, and His-tagged recombinant protein was expressed using the Pichia yeast system. The purified protein (molecular weight ~ 180 kDa) showed BChE activity, and degraded acetylcholinesterase (ACh) at a higher rate than BCh. However, phylogram analysis shows that this novel cholinesterase shared an evolutionary origin with carboxylic esterase rather than BChE. The zebrafish BChE-like protein shares structural characteristics with cholinesterase and carboxylesterase. The 2-arachidonoylglycerol (2-AG), nicosulfuron, and triacetin exhibited a higher binding affinity to the zebrafish BChE-like protein than BCh and ACh. With the identification of BChE-like protein in zebrafish, this study could shed light on the origin of BChE and may contribute towards the development of a BChE knockout zebrafish model for sensitive drug or toxin screening.

Lipopolysaccharide-induced TNFα factor (LITAF) promotes inflammatory responses and activates apoptosis in zebrafish Danio rerio.

Lipopolysaccharide-induced TNFα factor (LITAF) is an important transcription factor which activates the transcription of TNFα and regulates cell apoptosis and inflammatory response. In the present study, a LITAF gene homologue was identified in zebrafish (Danio rerio) and was shown to be well conserved in the protein sequence, genomic organization and synteny with human LITAF. DrLITAF was constitutively expressed in tissues, with the highest expression detected in the gills. Its expression could be modulated by LPS, poly(I:C), and infection with Edwardsiella tarda, Aeromonus hydrophila and septicemia viremia of carp virus (SVCV). DrLITAF, when overexpressed, was shown to be located on the cellular membrane and nuclear membrane of HEK293T and ZF4 cells and was associated with the endoplasmic reticulum. Stimulation with LPS resulted in rapid translocation of DrLITAF into the nucleus. In addition, DrLITAF was able to induce cell apoptosis and the expression of caspase 3. The results demonstrate that DrLITAF is involved in the immune defence against bacterial and viral infection and plays a role in regulating inflammation and apoptosis.

Systematic profiling of early regulators during tissue regeneration using zebrafish model.

Great progresses have been made in comprehension of tissue regeneration process. However, one of the central questions in regeneration research remains to be deciphered is what factors initiate regenerative process. In present study, we focused on systematic profiling of early regulators in tissue regeneration via high-throughput screening on zebrafish caudal fin model. Firstly, 53 GO-annotated regeneration-related genes, which were specifically activated upon fin amputation, were identified according to the transcriptomic analysis. Moreover, qRT-PCR analysis of a couple of randomly selected genes from the aforementioned gene list validated our sequencing results. These studies confirmed the reliability of transcriptome sequencing analysis. Fibroblast growth factor 20a (fgf20a) is a key initial factor in the regeneration of zebrafish. Through a gene expression correlation analysis, we discovered a collection of 70 genes correlating with fgf20a, whose expression increased promptly at 2 days post amputation (dpa) and went down to the basal level until the completion of fin regeneration. In addition, two genes, socs3b and nppc, were chosen to investigate their functions during the fin regeneration. Inhibition of either of those genes significantly delayed the regenerative process. Taken together, we provided a simple and effective time-saving strategy that may serve as a tool for identifying early regulators in regeneration and identified 71 genes as early regulators of fin regeneration.

Mechanisms mediating suppression of globin gene transcription in Danio rerio nonerythroid cells.

We studied the repression of adult and embryo-larval genes of the major globin gene locus in D. rerio fibroblasts. The results obtained suggest that at least some of the globin genes are repressed by Polycomb, similarly to human α-globin genes. Furthermore, within two α/ß globin gene pairs, repression of α-type and ß-type genes appears to be mediated by different mechanisms, as increasing the level of histone acetylation can activate transcription of only ß-type genes.

Quercetin mitigates ethanol-induced hepatic steatosis in zebrafish via P2X7R-mediated PI3K/ Keap1/Nrf2 signaling pathway.

Ethnopharmacological relevanceQuercetin is the active component of the higher content in PCP, which exerts various biological activities such as anti-obesity effect, anti-inflammatory and anti-oxidant activities in alcoholic liver disease (ALD). AIM OF THE STUDY: P2X7 receptor (P2X7R) plays an important role in health and disease, which can be activated by extracellular ATP to induce a variety of downstream events, including lipid metabolism, inflammatory molecule release, oxidative stress. However, whether the mechanism of quercetin on ethanol-induced hepatic steatosis via P2X7R-mediated haven't been elucidated. MATERIAL AND METHODS: Zebrafish transgenic (fabp10: EGFP) larvae were treated with 100 µM, 50 µM, 25 µM quercetin for 48 h at 3 days post fertilization (dpf), then soaked in 350 mmol/L ethanol for 32 h, treated with 1 mM ATP (P2X7R activator) for 30min. Serum lipids, liver steatosis, oxidative stress factors were respectively detected. The mRNA levels in the related pathways were measured by quantitative Real-Time PCR (RT-qPCR) to investigate the mechanisms. RESULTS: Quercetin improved the liver function via decreasing ALT, AST and γ-GT level of zebrafish with acute ethanol-induced hepatic steatosis and attenuated hepatic TG, TC accumulation. Additionally, quercetin significantly reduced the MDA content and suppressed the ethanol-induced reduction of hepatic oxidative stress biomarkers GSH, CAT and SOD and significantly down-regulated the expression of P2X7R, and up-regulated the expression of phosphatidylinositol 3-kinase (PI3K), Kelch like ECH associated protein1 (Keap1), Nuclear Factor E2 related factor 2 (Nrf2). Moreover, ATP stimulation activated P2X7R, which further mediated the mRNA expressions of PI3K, Keap1 and Nrf2. CONCLUSION: Quercetin exhibited hepatoprotective capacity in zebrafish model, via regulating P2X7R-mediated PI3K/Keap1/Nrf2 oxidative stress signaling pathway.

Hesperidin downregulates kinases lrrk2 and gsk3ß in a 6-OHDA induced Parkinson's disease model.

The depletion of dopamine in the striatum region and Lewy bodies are the hallmark characteristics of Parkinson's disease. The pathology also includes the upregulation of various Parkinson's disease (PARK) genes and kinases. Two such kinases, LRRK2 and GSK-3ß have been directly implicated in the formation of tau and alpha-synuclein proteins, causing PD. Hesperidin (HES) is a flavanone glycoside that has multiple therapeutic benefits including neuroprotective effects. In this study, we examined the neuroprotective effects of HES against 6-hydroxydopamine (6-OHDA) induced-neurotoxicity in the in-vitro and in-vivo model. Hesperidin significantly protected the SH-SY5Y cells' stress against 6-OHDA induced toxicity by downregulating biomarkers of oxidative stress. Furthermore, HES downregulated the kinases lrrk2 and gsk3ß along with casp3, casp9, and polg in the zebrafish model. The treatment with HES also improved the locomotor pattern of zebrafish that was affected by 6-OHDA. This study suggests that hesperidin could be a drug of choice in targeting kinases against a 6-OHDA model of PD.

The role of cldnh during the early retinal development in zebrafish.

Claudin-3, an integral component of tight junction, has recently been shown to be expressed in retinal ganglion cells, retinal pigment cells, and retinal vascular endothelial cells. However, the role of claudin-3 in the development of the neural retina and its vessels remains undefined. This study aimed to investigate the role of zebrafish claudin-h (cldnh), the closest ortholog of mouse and human claudin-3, in the development of the neural retina and its vessels. Cldnh levels in green fluorescent protein transgenic zebrafish were genetically manipulated by cldnh morpholino oligonucleotide (MO) and cldnh mRNA to investigate gene function. The expression of cldnh was analyzed using polymerase chain reaction and immunofluorescence staining. The altered morphological, cellular and molecular events in the cldnh MO-morphant eyes were detected using hematoxylin-eosin staining, fluorescent dye injection, confocal in vivo imaging, BrdU labeling, TUNEL assay, RNA sequencing, and Western blot. We demonstrated that the cldnh protein was expressed in the neural retina and the hyaloid vessel which is the predecessor of the retinal vessel in zebrafish. Cldnh knockdown delayed lamination of the neural retina and reduced its thickness, which might be associated with the downregulation of the retinal development-related genes of atoh7, pcdh17, crx, neurod1, insm1a, sox9b and cdh11, and the upregulation of the cell cycle and apoptosis-associated genes of tp53, cdkn1a and casp8. Cldnh knockdown also reduced the density and interrupted the lumenization of the hyaloid vessels, which might be owing to the downregulation of the vessel formation-related genes of hlx1 and myl7. In conclusion, cldnh was required for the normal development of the neural retina and its vessels in zebrafish, providing a basis for elucidating its role in the pathogenesis of retinal vascular or inflammatory diseases.

Anti-Melanogenesis Activity of 6-O-Isobutyrylbritannilactone from Inula britannica on B16F10 Melanocytes and In Vivo Zebrafish Models.

A potential natural melanogenesis inhibitor was discovered in the form of a sesquiterpene isolated from the flowers of Inula britannica, specifically 6-O-isobutyrylbritannilactone (IBL). We evaluated the antimelanogenesis effects of IBL on B16F10 melanocytes and zebrafish embryos. As a result, we found that 3-isobutyl-1-methylxanthine (IBMX)-induced melanin production was reduced in a dose-dependent manner in B16F10 cells by IBL. We also analyzed B16F10 cells that were and were not treated with IBMX, investigating the melanin concentration, tyrosinase activity, mRNA levels. We also studied the protein expressions of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related proteins (TRP1, and TRP2). Furthermore, we found that melanin synthesis and tyrosinase expression were also inhibited by IBL through the modulation of the following signaling pathways: ERK, phosphoinositide 3-kinase (PI3K)/AKT, and CREB. In addition, we studied antimelanogenic activity using zebrafish embryos and found that the embryos had significantly reduced pigmentation in the IBL-treated specimens compared to the untreated controls.

Translation of small downstream ORFs enhances translation of canonical main open reading frames.

In addition to canonical open reading frames (ORFs), thousands of translated small ORFs (containing less than 100 codons) have been identified in untranslated mRNA regions (UTRs) across eukaryotes. Small ORFs in 5' UTRs (upstream (u)ORFs) often repress translation of the canonical ORF within the same mRNA. However, the function of translated small ORFs in the 3' UTRs (downstream (d)ORFs) is unknown. Contrary to uORFs, we find that translation of dORFs enhances translation of their corresponding canonical ORFs. This translation stimulatory effect of dORFs depends on the number of dORFs, but not the length or peptide they encode. We propose that dORFs represent a new, strong, and universal translation regulatory mechanism in vertebrates.