Epitranscriptomics m6A analyses reveal distinct m6A marks under tumor necrosis factor α (TNF-α)-induced apoptotic conditions in HeLa cells.

Tumor necrosis factor-α (TNF-α) is a ligand that induces both intrinsic and extrinsic apoptotic pathways in HeLa cells by modulating complex gene regulatory mechanisms. However, the full spectrum of TNF-α-modulated epitranscriptomic m6A marks is unknown. We employed a genomewide approach to examine the extent of m6A RNA modifications under TNF-α-modulated apoptotic conditions in HeLa cells. miCLIP-seq analyses revealed a plethora of m6A marks on 632 target mRNAs with an enrichment on 99 mRNAs associated with apoptosis. Interestingly, the m6A RNA modification patterns were quite different under cisplatin- and TNF-α-mediated apoptotic conditions. We then examined the abundance and translational efficiencies of several mRNAs under METTL3 knockdown and/or TNF-α treatment conditions. Our analyses showed changes in the translational efficiency of TP53INP1 mRNA based on the polysome profile analyses. Additionally, TP53INP1 protein amount was modulated by METTL3 knockdown upon TNF-α treatment but not CP treatment, suggesting the existence of a pathway-specific METTL3-TP53INP1 axis. Congruently, METLL3 knockdown sensitized HeLa cells to TNF-α-mediated apoptosis, which was also validated in a zebrafish larval xenograft model. These results suggest that apoptotic pathway-specific m6A methylation marks exist in cells and TNF-α-METTL3-TP53INP1 axis modulates TNF-α-mediated apoptosis in HeLa cells.

Canonical Wnt and TGF-ß/BMP signaling enhance melanocyte regeneration but suppress invasiveness, migration, and proliferation of melanoma cells.

Melanoma is the deadliest form of skin cancer and develops from the melanocytes that are responsible for the pigmentation of the skin. The skin is also a highly regenerative organ, harboring a pool of undifferentiated melanocyte stem cells that proliferate and differentiate into mature melanocytes during regenerative processes in the adult. Melanoma and melanocyte regeneration share remarkable cellular features, including activation of cell proliferation and migration. Yet, melanoma considerably differs from the regenerating melanocytes with respect to abnormal proliferation, invasive growth, and metastasis. Thus, it is likely that at the cellular level, melanoma resembles early stages of melanocyte regeneration with increased proliferation but separates from the later melanocyte regeneration stages due to reduced proliferation and enhanced differentiation. Here, by exploiting the zebrafish melanocytes that can efficiently regenerate and be induced to undergo malignant melanoma, we unravel the transcriptome profiles of the regenerating melanocytes during early and late regeneration and the melanocytic nevi and malignant melanoma. Our global comparison of the gene expression profiles of melanocyte regeneration and nevi/melanoma uncovers the opposite regulation of a substantial number of genes related to Wnt signaling and transforming growth factor beta (TGF-ß)/(bone morphogenetic protein) BMP signaling pathways between regeneration and cancer. Functional activation of canonical Wnt or TGF-ß/BMP pathways during melanocyte regeneration promoted melanocyte regeneration but potently suppressed the invasiveness, migration, and proliferation of human melanoma cells in vitro and in vivo. Therefore, the opposite regulation of signaling mechanisms between melanocyte regeneration and melanoma can be exploited to stop tumor growth and develop new anti-cancer therapies.

Addition of exogenous diacylglycerol enhances Wnt/ß-catenin signaling through stimulation of macropinocytosis.

Activation of Wnt signaling triggers macropinocytosis and drives many tumors. We now report that the exogenous addition of the second messenger lipid sn-1,2 DAG to the culture medium rapidly induces macropinocytosis. This is accompanied by potentiation of the effects of added Wnt3a recombinant protein or the glycogen synthase kinase 3 (GSK3) inhibitor lithium chloride (LiCl, which mimics Wnt signaling) in luciferase transcriptional reporter assays. In a colorectal carcinoma cell line in which mutation of adenomatous polyposis coli (APC) causes constitutive Wnt signaling, DAG addition increased levels of nuclear ß-catenin, and this increase was partially inhibited by an inhibitor of macropinocytosis. DAG also expanded multivesicular bodies marked by the tetraspan protein CD63. In an in vivo situation, microinjection of DAG induced Wnt-like twinned body axes when co-injected with small amounts of LiCl into Xenopus embryos. These results suggest that the DAG second messenger plays a role in Wnt-driven cancer progression.

Comparative membrane lipidomics of hepatocellular carcinoma cells reveals diacylglycerol and ceramide as key regulators of Wnt/ß-catenin signaling and tumor growth.

Hepatocellular carcinoma (HCC) is largely associated with aberrant activation of Wnt/ß-catenin signaling. Nevertheless, how membrane lipid composition is altered in HCC cells with abnormal Wnt signaling remains elusive. Here, by exploiting comprehensive lipidome profiling, we unravel the membrane lipid composition of six different HCC cell lines with mutations in components of Wnt/ß-catenin signaling, leading to differences in their endogenous signaling activity. Among the differentially regulated lipids are diacylglycerol (DAG) and ceramide, which were downregulated at the membrane of HCC cells after Wnt3a treatment. DAG and ceramide enhanced Wnt/ß-catenin signaling by inducing caveolin-mediated endocytosis of the canonical Wnt-receptor complex, while their depletion suppressed the signaling activity along with a reduction of caveolin-mediated endocytosis in SNU475 and HepG2 cells. Moreover, depletion of DAG and ceramide significantly impeded the proliferation, tumor growth, and in vivo migration capacity of SNU475 and HepG2 cells. This study, by pioneering plasma membrane lipidome profiling in HCC cells, exhibits the remarkable potential of lipids to correct dysregulated signaling pathways in cancer and stop abnormal tumor growth.

Mitigation potential of zingerone and rutin on toxicity mechanisms of nickel to zebrafish based on morphological, DNA damage and apoptosis outcome analysis.

Although nickel (Ni) is an important cofactor for various enzymes in biological systems, it can cause serious problems when insufficient or excessive in an organism. Therefore, it is very important to investigate Ni in biological systems, especially in cells with its related pathogenic mechanism. This study was carried out to demonstrate the effects of zingerone (ZO) and rutin (RN) administration against nickel chloride (NiCl2) toxicity on neurobehavioral performance and brain oxidative status in zebrafish (Danio rerio) embryos/larvae on histological perspective. The experimental design of the study, which included twenty groups of fish, each containing 10 embryos, was prepared as semi-static and the trial continued for 96 hpf. In the obtained findings, it was determined that ZO and RN had a mitigating effect in this toxicity table where Ni caused oxidative stress in zebrafish larvae, induced DNA damage and apoptosis. A similar picture is valid for malformation processes as well as survival and hatching rates. These results showed that nickel is toxic to developing embryos via acting different mechanisms. In conclusion, we observed that ZO and RN have a greater effect on physiology, DNA damage and apoptosis than gross morphology, with a significant ameliorative effect.

High-fat diet feeding triggers a regenerative response in the adult zebrafish brain.

Non-alcoholic fatty liver disease (NAFLD) includes a range of liver conditions ranging from excess fat accumulation to liver failure. NAFLD is strongly associated with high-fat diet (HFD) consumption that constitutes a metabolic risk factor. While HFD has been elucidated concerning its several systemic effects, there is little information about its influence on the brain at the molecular level. Here, by using a high-fat diet (HFD)-feeding of adult zebrafish, we first reveal that excess fat uptake results in weight gain and fatty liver. Prolonged exposure to HFD induces a significant increase in the expression of pro-inflammation, apoptosis, and proliferation markers in the liver and brain tissues. Immunofluorescence analyses of the brain tissues disclose stimulation of apoptosis and widespread activation of glial cell response. Moreover, glial activation is accompanied by an initial decrease in the number of neurons and their subsequent replacement in the olfactory bulb and the telencephalon. Long-term consumption of HFD causes activation of Wnt/ß-catenin signaling in the brain tissues. Finally, fish fed an HFD induces anxiety, and aggressiveness and increases locomotor activity. Thus, HFD feeding leads to a non-traumatic brain injury and stimulates a regenerative response. The activation mechanisms of a regeneration response in the brain can be exploited to fight obesity and recover from non-traumatic injuries.

A Novel Missense Variant in Actin Binding Domain of MYH7 Is Associated With Left Ventricular Noncompaction.

Cardiomyopathies are a group of common heart disorders that affect numerous people worldwide. Left ventricular non-compaction (LVNC) is a structural disorder of the ventricular wall, categorized as a type of cardiomyopathy that mostly caused by genetic disorders. Genetic variations are underlying causes of developmental deformation of the heart wall and the resultant contractile insufficiency. Here, we investigated a family with several affected members exhibiting LVNC phenotype. By whole-exome sequencing (WES) of three affected members, we identified a novel heterozygous missense variant (c.1963C>A:p.Leu655Met) in the gene encoding myosin heavy chain 7 (MYH7). This gene is evolutionary conserved among different organisms. We identified MYH7 as a highly enriched myosin, compared to other types of myosin heavy chains, in skeletal and cardiac muscles. Furthermore, MYH7 was among a few classes of MYH in mouse heart that highly expresses from early embryonic to adult stages. In silico predictions showed an altered actin-myosin binding, resulting in weaker binding energy that can cause LVNC. Moreover, CRISPR/Cas9 mediated MYH7 knockout in zebrafish caused impaired cardiovascular development. Altogether, these findings provide the first evidence for involvement of p.Leu655Met missense variant in the incidence of LVNC, most probably through actin-myosin binding defects during ventricular wall morphogenesis.

Brain Regeneration Resembles Brain Cancer at Its Early Wound Healing Stage and Diverges From Cancer Later at Its Proliferation and Differentiation Stages.

Gliomas are the most frequent type of brain cancers and characterized by continuous proliferation, inflammation, angiogenesis, invasion and dedifferentiation, which are also among the initiator and sustaining factors of brain regeneration during restoration of tissue integrity and function. Thus, brain regeneration and brain cancer should share more molecular mechanisms at early stages of regeneration where cell proliferation dominates. However, the mechanisms could diverge later when the regenerative response terminates, while cancer cells sustain proliferation. To test this hypothesis, we exploited the adult zebrafish that, in contrast to the mammals, can efficiently regenerate the brain in response to injury. By comparing transcriptome profiles of the regenerating zebrafish telencephalon at its three different stages, i.e., 1 day post-lesion (dpl)-early wound healing stage, 3 dpl-early proliferative stage and 14 dpl-differentiation stage, to those of two brain cancers, i.e., low-grade glioma (LGG) and glioblastoma (GBM), we reveal the common and distinct molecular mechanisms of brain regeneration and brain cancer. While the transcriptomes of 1 dpl and 3 dpl harbor unique gene modules and gene expression profiles that are more divergent from the control, the transcriptome of 14 dpl converges to that of the control. Next, by functional analysis of the transcriptomes of brain regeneration stages to LGG and GBM, we reveal the common and distinct molecular pathways in regeneration and cancer. 1 dpl and LGG and GBM resemble with regard to signaling pathways related to metabolism and neurogenesis, while 3 dpl and LGG and GBM share pathways that control cell proliferation and differentiation. On the other hand, 14 dpl and LGG and GBM converge with respect to developmental and morphogenetic processes. Finally, our global comparison of gene expression profiles of three brain regeneration stages, LGG and GBM exhibit that 1 dpl is the most similar stage to LGG and GBM while 14 dpl is the most distant stage to both brain cancers. Therefore, early convergence and later divergence of brain regeneration and brain cancer constitutes a key starting point in comparative understanding of cellular and molecular events between the two phenomena and development of relevant targeted therapies for brain cancers.

Investigation of the Oxidative Stress Response of a Green Synthesis Nanoparticle (RP-Ag/ACNPs) in Zebrafish.

Silver nanoparticles (AgNPs) are prominent nanomaterials that are efficiently used in different industries including medical products, water treatment, and cosmetics. However, AgNPs are known to cause adverse effects on the ecosystem and human health. In this study, aqueous extract of Rumex patientia (RP) was used as a reducing and stabilizing agent in AgNP biosynthesis. The obtained activated carbon (AC) from Chenopodium album (CA) plant was combined with RP-AgNPs to synthesize RP-Ag/AC NPs. Next, the effects of these green synthesis RP-Ag/AC NPs on zebrafish (Danio rerio) embryos and larvae were investigated. First, we characterized the RP-Ag/AC NPs by using X-ray diffraction (XRD) and transmission electron microscopy (TEM) and determined LC50 value as 217.23 mg/L at 96 h. Next, the alterations in survival rate, hatching rate, and morphology of the larvae at 96 h were monitored. The survival rates decreased in a dose-dependent manner. Morphological defects such as yolk sac edema, pericardial edema, spinal curvature, and tail malformation in the NP-treated larvae were observed. RP-Ag/AC NPs stimulated the production of neuronal NOS (nNOS) and 8-OHdG in zebrafish brain tissues in a dose-dependent manner and enhanced neutrophil degeneration and necrosis at concentrations of 50 and 100 mg/L. Thus, the obtained data suggest that the green synthesis process is not sufficient to reduce the effect of oxidative stress caused by AgNPs on oxidative signaling.

Misregulation of Wnt Signaling Pathways at the Plasma Membrane in Brain and Metabolic Diseases.

Wnt signaling pathways constitute a group of signal transduction pathways that direct many physiological processes, such as development, growth, and differentiation. Dysregulation of these pathways is thus associated with many pathological processes, including neurodegenerative diseases, metabolic disorders, and cancer. At the same time, alterations are observed in plasma membrane compositions, lipid organizations, and ordered membrane domains in brain and metabolic diseases that are associated with Wnt signaling pathway activation. Here, we discuss the relationships between plasma membrane components-specifically ligands, (co) receptors, and extracellular or membrane-associated modulators-to activate Wnt pathways in several brain and metabolic diseases. Thus, the Wnt-receptor complex can be targeted based on the composition and organization of the plasma membrane, in order to develop effective targeted therapy drugs.

Changes in Wnt and TGF-ß Signaling Mediate the Development of Regorafenib Resistance in Hepatocellular Carcinoma Cell Line HuH7.

Hepatocellular carcinoma (HCC) is an aggressive, chemo resistant neoplasm with poor prognosis and limited treatment options. Exploring activated pathways upon drug treatment can be used to discover more effective anticancer agents to overcome therapy resistance and enhance therapeutic outcomes for patients with advanced HCC. Human tumor-derived cell lines recapitulate HCC diversity and are widely used for studying mechanisms that drive drug resistance in HCC. In this study, we show that regorafenib treatment activates Wnt/ß-catenin signaling only in hepatoblast-like HCC cell lines and induces enrichment of markers associated with hepatic stem/progenitor cells. Moreover, activation of Wnt/ß-catenin signaling via Wnt3a/R-Spo1 treatment protects these cells from regorafenib induced apoptosis. On the other hand, regorafenib resistant cells established by long-term regorafenib treatment demonstrate diminished Wnt/ß-catenin signaling activity while TGF-ß signaling activity of these cells is significantly enhanced. Regorafenib resistant cells (RRCs) also show increased expression of several mesenchymal genes along with an induction of CD24 and CD133 cancer stem cell markers. Moreover, regorafenib resistant cells also exhibit significantly augmented in vitro and in vivo migration capacity which could be reversed by TGF-ß type 1 receptor (TGFb -R1) inhibition. When combined with regorafenib treatment, TGFß-R1 inhibition also significantly decreased colony formation ability and augmented cell death in resistant spheroids. Importantly, when we knocked down TGFß-R1 using a lentiviral plasmid, regorafenib resistant cells entered senescence indicating that this pathway is important for their survival. Treatment of RRCs with TGFß-R1 inhibitor and regorafenib significantly abolished pSTAT3, pSMAD2 and pERK (44/42) expression suggesting the involvement of both canonical and non-canonical pathways. In conclusion, our data suggest that HCC tumors with aberrant activation in the Wnt/ß-catenin pathway, might have higher intrinsic regorafenib resistance and the inhibition of this pathway along with regorafenib administration might increase regorafenib-induced cell death in combinational therapies. However, to resolve acquired regorafenib resistance developed in HCC patients, the combined use of TGF-ß pathway inhibitors and Regorafenib constitute a promising approach that can increase regorafenib sensitization and prevent tumor recurrence.

Wnt3 Is Lipidated at Conserved Cysteine and Serine Residues in Zebrafish Neural Tissue.

Wnt proteins are a family of hydrophobic cysteine-rich secreted glycoproteins that regulate a gamut of physiological processes involved in embryonic development and tissue homeostasis. Wnt ligands are post-translationally lipidated in the endoplasmic reticulum (ER), a step essential for its membrane targeting, association with lipid domains, secretion and interaction with receptors. However, at which residue(s) Wnts are lipidated remains an open question. Initially it was proposed that Wnts are lipid-modified at their conserved cysteine and serine residues (C77 and S209 in mWnt3a), and mutations in either residue impedes its secretion and activity. Conversely, some studies suggested that serine is the only lipidated residue in Wnts, and substitution of serine with alanine leads to retention of Wnts in the ER. In this work, we investigate whether in zebrafish neural tissues Wnt3 is lipidated at one or both conserved residues. To this end, we substitute the homologous cysteine and serine residues of zebrafish Wnt3 with alanine (C80A and S212A) and investigate their influence on Wnt3 membrane organization, secretion, interaction and signaling activity. Collectively, our results indicate that Wnt3 is lipid modified at its C80 and S212 residues. Further, we find that lipid addition at either C80 or S212 is sufficient for its secretion and membrane organization, while the lipid modification at S212 is indispensable for receptor interaction and signaling.

LGR5/R-Spo1/Wnt3a axis promotes stemness and aggressive phenotype in hepatoblast-like hepatocellular carcinoma cell lines.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a newly defined stem cell marker in endoderm-derived organs such as the small intestine, colon and pancreas. Recently, LGR5 was demonstrated to be an important factor in liver regeneration and stem cell maintenance. Moreover, LGR5 expression is highly up-regulated in various cancers including hepatocellular carcinoma. Herein, we demonstrate that LGR5 expression is specifically observed in certain subset of HCC cell lines with "hepatoblast-like" appearance, characterized by the expression of liver fetal/progenitor markers. Notably, the activation of the canonical Wnt pathway significantly increases the expression of LGR5 in this subset of cell lines, whereas it does not cause any induction of LGR5 expression in mesenchymal like cell lines SNU-475 and SNU-449. Furthermore, we showed that treatment of the hepatoblast-like HCC cell lines HuH-7 and Hep3B with LGR5 ligand R-Spo1 significantly amplifies the induction of LGR5 expression, the phosphorylation of LRP6 and ß-catenin resulting in enhanced TCF/LEF activity either alone or in combination with Wnt3a. Consistently, the silencing of the LGR5 gene attenuates the co-stimulatory effect of R-Spo1/Wnt3a on TCF/LEF activity while overexpression of LGR5 enhances it. On the contrary, overexpression of LGR5 does not change TCF/LEF activity induced by R-Spo1/Wnt3a in mesenchymal-like HCC line, SNU-449. Importantly, LGR5-overexpressing cells have increased expression of several Wnt target genes and stemness-related genes including EpCAM and CK19 upon R-Spo1/Wnt3a treatment. LGR5-overexpressing cells also have increased spheroid forming, migration and invasion abilities and stimulation with R-Spo1/Wnt3a augments these abilities of LGR5-overexpressing cells. In addition, ectopic overexpression of LGR5 significantly increases cell proliferation rate independent of R-Spo1/Wnt3a stimulation. Moreover, in vitro tubulogenesis assay demonstrates that treatment with R-Spo1/Wnt3a enhances the sprouting of capillary tubules in only LGR5-overexpressing cells. Finally, R-Spo1/Wnt3a significantly promotes dissemination of LGR5-overexpressing cells in vivo in a zebrafish xenograft model. Our study unravels a tumor-promoting role for LGR5 through activation of canonical Wnt/ß-catenin signaling in the hepatoblast-like HCCs. In conclusion, our results suggest that LGR5/R-Spo1/Wnt3a generates an axis that mediates the acquisition of aggressive phenotype specifically in hepatoblast-like subset of HCCs and might represent a valuable target for treatment of HCC tumors with aberrant activation of Wnt/ß-catenin pathway.

TAp73ß Can Promote Hepatocellular Carcinoma Dedifferentiation.

Hepatocyte dedifferentiation is a major source of hepatocellular carcinoma (HCC), but its mechanisms are unknown. We explored the p73 expression in HCC tumors and studied the effects of transcriptionally active p73ß (TAp73ß) in HCC cells. Expression profiles of p73 and patient clinical data were collected from the Genomic Data Commons (GDC) data portal and the TSVdb database, respectively. Global gene expression profiles were determined by pan-genomic 54K microarrays. The Gene Set Enrichment Analysis method was used to identify TAp73ß-regulated gene sets. The effects of TAp73 isoforms were analyzed in monolayer cell culture, 3D-cell culture and xenograft models in zebrafish using western blot, flow cytometry, fluorescence imaging, real-time polymerase chain reaction (RT-PCR), immunohistochemistry and morphological examination. TAp73 isoforms were significantly upregulated in HCC, and high p73 expression correlated with poor patient survival. The induced expression of TAp73ß caused landscape expression changes in genes involved in growth signaling, cell cycle, stress response, immunity, metabolism and development. Hep3B cells overexpressing TAp73ß had lost hepatocyte lineage biomarkers including ALB, CYP3A4, AFP, HNF4α. In contrast, TAp73ß upregulated genes promoting cholangiocyte lineage such as YAP, JAG1 and ZO-1, accompanied with an increase in metastatic ability. Our findings suggest that TAp73ß may promote malignant dedifferentiation of HCC cells.

Regulation of Wnt Signaling Pathways at the Plasma Membrane and Their Misregulation in Cancer.

Wnt signaling is one of the key signaling pathways that govern numerous physiological activities such as growth, differentiation and migration during development and homeostasis. As pathway misregulation has been extensively linked to pathological processes including malignant tumors, a thorough understanding of pathway regulation is essential for development of effective therapeutic approaches. A prominent feature of cancer cells is that they significantly differ from healthy cells with respect to their plasma membrane composition and lipid organization. Here, we review the key role of membrane composition and lipid order in activation of Wnt signaling pathway by tightly regulating formation and interactions of the Wnt-receptor complex. We also discuss in detail how plasma membrane components, in particular the ligands, (co)receptors and extracellular or membrane-bound modulators, of Wnt pathways are affected in lung, colorectal, liver and breast cancers that have been associated with abnormal activation of Wnt signaling. Wnt-receptor complex components and their modulators are frequently misexpressed in these cancers and this appears to correlate with metastasis and cancer progression. Thus, composition and organization of the plasma membrane can be exploited to develop new anticancer drugs that are targeted in a highly specific manner to the Wnt-receptor complex, rendering a more effective therapeutic outcome possible.

Nradd Acts as a Negative Feedback Regulator of Wnt/ß-Catenin Signaling and Promotes Apoptosis.

Wnt/ß-catenin signaling controls many biological processes for the generation and sustainability of proper tissue size, organization and function during development and homeostasis. Consequently, mutations in the Wnt pathway components and modulators cause diseases, including genetic disorders and cancers. Targeted treatment of pathway-associated diseases entails detailed understanding of the regulatory mechanisms that fine-tune Wnt signaling. Here, we identify the neurotrophin receptor-associated death domain (Nradd), a homolog of p75 neurotrophin receptor (p75NTR), as a negative regulator of Wnt/ß-catenin signaling in zebrafish embryos and in mammalian cells. Nradd significantly suppresses Wnt8-mediated patterning of the mesoderm and neuroectoderm during zebrafish gastrulation. Nradd is localized at the plasma membrane, physically interacts with the Wnt receptor complex and enhances apoptosis in cooperation with Wnt/ß-catenin signaling. Our functional analyses indicate that the N-glycosylated N-terminus and the death domain-containing C-terminus regions are necessary for both the inhibition of Wnt signaling and apoptosis. Finally, Nradd can induce apoptosis in mammalian cells. Thus, Nradd regulates cell death as a modifier of Wnt/ß-catenin signaling during development.

Textile dyes Maxilon blue 5G and Reactive blue 203 induce acute toxicity and DNA damage during embryonic development of Danio rerio.

Common textile dyes used in various industrial sectors are organic compounds and considered for the aquatic environment as pollutants. The textile dye industry is one of the main sectors that have serious impacts on the environment due to a large amount of wastewater released into the ecosystem. Maxilon blue 5G (MB-5G) and Reactive Blue 203 (RB-203) are widely used textile dyes. However, their potential toxicity on living organisms remains to be elucidated. Here, we investigate the acute toxicity and genotoxicity of MB-5G and RB-203 dyes using the zebrafish embryos/larvae. Embryos treated with each dye for 96 h revealed LC50 values of acute toxicity as 166.04 mg L-1 and 278.32 mg L-1 for MB-5G and RB 203, respectively. When exposed to MB-5G and RB-203 at different concentrations (1, 10, and 100 mg L-1) for 96 h, the expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, significantly increased in brain tissues as compared to control. MB-5G and RB-203 resulted in common developmental abnormalities including tail malformation, microphthalmia, pericardial edema, curved body axis, and yolk sac/pericardial edemas. Moreover, at its highest dose (100 mg L-1), RB-203 caused premature hatching after 48 h, while MG-5G did not. Our results collectively reveal that the textile dyes MB-5G and RB-203 cause genotoxicity and teratogenicity during embryonic and larval development of zebrafish. Thus, it is necessary to eliminate these compounds from wastewater or reduce their concentrations to safe levels before discharging the textile industry wastewater into the environment.

Comparative Transcriptome Analysis of the Regenerating Zebrafish Telencephalon Unravels a Resource With Key Pathways During Two Early Stages and Activation of Wnt/ß-Catenin Signaling at the Early Wound Healing Stage.

Owing to its pronounced regenerative capacity in many tissues and organs, the zebrafish brain represents an ideal platform to understand the endogenous regeneration mechanisms that restore tissue integrity and function upon injury or disease. Although radial glial and neuronal cell populations have been characterized with respect to specific marker genes, comprehensive transcriptomic profiling of the regenerating telencephalon has not been conducted so far. Here, by processing the lesioned and unlesioned hemispheres of the telencephalon separately, we reveal the differentially expressed genes (DEGs) at the early wound healing and early proliferative stages of regeneration, i.e., 20 h post-lesion (hpl) and 3 days post-lesion (dpl), respectively. At 20 hpl, we detect a far higher number of DEGs in the lesioned hemisphere than in the unlesioned half and only 7% of all DEGs in both halves. However, this difference disappears at 3 dpl, where the lesioned and unlesioned hemispheres share 40% of all DEGs. By performing an extensive comparison of the gene expression profiles in these stages, we unravel that the lesioned hemispheres at 20 hpl and 3 dpl exhibit distinct transcriptional profiles. We further unveil a prominent activation of Wnt/ß-catenin signaling at 20 hpl, returning to control level in the lesioned site at 3 dpl. Wnt/ß-catenin signaling indeed appears to control a large number of genes associated primarily with the p53, apoptosis, forkhead box O (FoxO), mitogen-activated protein kinase (MAPK), and mammalian target of rapamycin (mTOR) signaling pathways specifically at 20 hpl. Based on these results, we propose that the lesioned and unlesioned hemispheres react to injury dynamically during telencephalon regeneration and that the activation of Wnt/ß-catenin signaling at the early wound healing stage plays a key role in the regulation of cellular and molecular events.

Agonist of RORA Attenuates Nonalcoholic Fatty Liver Progression in Mice via Up-regulation of MicroRNA 122.

BACKGROUND & AIMS: Development of nonalcoholic steatohepatitis (NASH) is associated with reductions in hepatic microRNA122 (MIR122); the RAR related orphan receptor A (RORA) promotes expression of MIR122. Increasing expression of RORA in livers of mice increases expression of MIR122 and reduces lipotoxicity. We investigated the effects of a RORA agonist in mouse models of NASH. METHODS: We screened a chemical library to identify agonists of RORA and tested their effects on a human hepatocellular carcinoma cell line (Huh7). C57BL/6 mice were fed a chow or high-fat diet (HFD) for 4 weeks to induce fatty liver. Mice were given hydrodynamic tail vein injections of a MIR122 antagonist (antagomiR-122) or a control antagomiR once each week for 3 weeks while still on the HFD or chow diet, or intraperitoneal injections of the RORA agonist RS-2982 or vehicle, twice each week for 3 weeks. Livers, gonad white adipose, and skeletal muscle were collected and analyzed by reverse-transcription polymerase chain reaction, histology, and immunohistochemistry. A separate group of mice were fed an atherogenic diet, with or without injections of RS-2982 for 3 weeks; livers were analyzed by immunohistochemistry, and plasma was analyzed for levels of aminotransferases. We analyzed data from liver tissues from patients with NASH included in the RNA-sequencing databases GSE33814 and GSE89632. RESULTS: Injection of mice with antagomiR-122 significantly reduced levels of MIR122 in plasma, liver, and white adipose tissue; in mice on an HFD, antagomiR-122 injections increased fat droplets and total triglyceride content in liver and reduced ß-oxidation and energy expenditure, resulting in significantly more weight gain than in mice given the control microRNA. We identified RS-2982 as an agonist of RORA and found it to increase expression of MIR122 promoter activity in Huh7 cells. In mice fed an HFD or atherogenic diet, injections of RS-2982 increased hepatic levels of MIR122 precursors and reduced hepatic synthesis of triglycerides by reducing expression of biosynthesis enzymes. In these mice, RS-2982 significantly reduced hepatic lipotoxicity, reduced liver fibrosis, increased insulin resistance, and reduced body weight compared with mice injected with vehicle. Patients who underwent cardiovascular surgery had increased levels of plasma MIR122 compared to its levels before surgery; increased expression of plasma MIR122 was associated with increased levels of plasma free fatty acids and levels of RORA. CONCLUSIONS: We identified the compound RS-2982 as an agonist of RORA that increases expression of MIR122 in cell lines and livers of mice. Mice fed an HFD or atherogenic diet given injections of RS-2982 had reduced hepatic lipotoxicity, liver fibrosis, and body weight compared with mice given the vehicle. Agonists of RORA might be developed for treatment of NASH.