Non-POU Domain-Containing Octomer-Binding (NONO) protein expression and stability promotes the tumorigenicity and activation of Akt/MAPK/ß-catenin pathways in human breast cancer cells.

Breast cancer is one of the most common cancers with a high mortality rate, underscoring the need to identify new therapeutic targets. Here we report that non-POU domain-containing octamer-binding (NONO) protein is overexpressed in breast cancer and validated the interaction of the WW domain of PIN1 with c-terminal threonine-proline (thr-pro) motifs of NONO. The interaction of NONO with PIN1 increases the stability of NONO by inhibiting its proteasomal degradation, and this identifies PIN1 as a positive regulator of NONO in promoting breast tumor development. Functionally, silencing of NONO inhibits the growth, survival, migration, invasion, epithelial to mesenchymal transition (EMT), and stemness of breast cancer cells in vitro. A human metastatic breast cancer cell xenograft was established in transparent zebrafish (Danio rerio) embryos to study the metastatic inability of NONO-silenced breast cancer cells in vivo. Mechanistically, NONO depletion promotes the expression of the PDL1 cell-surface protein in breast cancer cells. The identification of novel interactions of NONO with c-Jun and ß-catenin proteins and activation of the Akt/MAPK/ß-catenin signaling suggests that NONO is a novel regulator of Akt/MAPK/ß-catenin signaling pathways. Taken together, our results indicated an essential role of NONO in the tumorigenicity of breast cancer and could be a potential target for anti-cancerous drugs. Video Abstract.

BML-257, a Small Molecule that Protects against Drug-Induced Liver Injury in Zebrafish.

The use of many essential drugs is restricted due to their deleterious effects on the liver. Molecules that can prevent or protect the liver from drug-induced liver injury (DILI) would be invaluable in such situations. We used a transgenic line in zebrafish with a hepatocyte-specific expression of bacterial nitroreductase to cause temporally controlled liver damage. A whole organism-based chemical screen using the transgenic line identified BML-257, a potent small molecule AKT inhibitor, that protected the liver against metronidazole-induced liver injury. BML-257 also showed potent prophylactic and pro-regenerative activity in this liver damage model. BML-257 was tested in two independent toxicological models of liver injury caused by acetaminophen and isoniazid and was found to be protective against damage. This suggests that BML-257 has the potential to protect against multiple kinds of DILI.

Rescue of neural crest-derived phenotypes in a zebrafish CHARGE model by Sox10 downregulation.

CHD7 mutations are implicated in a majority of cases of the congenital disorder, CHARGE syndrome. CHARGE, an autosomal dominant syndrome, is known to affect multiple tissues including eye, heart, ear, craniofacial nerves and skeleton and genital organs. Using a morpholino-antisense-oligonucleotide-based zebrafish model for CHARGE syndrome, we uncover a complex spectrum of abnormalities in the neural crest and the crest-derived cell types. We report for the first time, defects in myelinating Schwann cells, enteric neurons and pigment cells in a CHARGE model. We also observe defects in the specification of peripheral neurons and the craniofacial skeleton as previously reported. Chd7 morphants have impaired migration of neural crest cells and deregulation of sox10 expression from the early stages. Knocking down Sox10 in the zebrafish CHARGE model rescued the defects in Schwann cells and craniofacial cartilage. Our zebrafish CHARGE model thus reveals important regulatory roles for Chd7 at multiple points of neural crest development viz., migration, fate choice and differentiation and we suggest that sox10 deregulation is an important driver of the neural crest-derived aspects of Chd7 dependent CHARGE syndrome.

Chemical and genetic rescue of an ep300 knockdown model for Rubinstein Taybi Syndrome in zebrafish.

EP300 is a member of the EP300/CBP family of lysine acetyltransferases (KATs) with multiple roles in development and physiology. Loss of EP300/CBP activity in humans causes a very rare congenital disorder called Rubinstein Taybi Syndrome (RSTS). The zebrafish genome has two co-orthologs of lysine acetyltransferase EP300 (KAT3B) in zebrafish viz. ep300a and ep300b. Chemical inhibition of Ep300 with C646, a competitive inhibitor and morpholino-based genetic knockdown of ep300a and ep300b cause defects in embryonic development reminiscent of the human RSTS syndrome. Remarkably, overexpression of Ep300a KAT domain results in near complete rescue of the jaw development defects, a characteristic feature of RSTS in human suggesting the dispensability of the protein-interaction and DNA-binding domains for at least some developmental roles of Ep300. We also perform a chemical screen and identify two inhibitors of deacetylases, CHIC35 and HDACi III, that can partially rescue the RSTS-like phenotypes. Thus, modeling rare human genetic disorders in zebrafish allows for functional understanding of the genes involved and can also yield small molecule candidates towards therapeutic goals.

Identification of noreremophilane-based inhibitors of angiogenesis using zebrafish assays.

Noreremophilanes are a rare class of cis-hydrindanes produced by genus Ligularia herbaceous plants which are known to exhibit interesting biological activities. We synthesized cis-hydrindanes based on a naturally occurring noreremophilane scaffold using a Diels-Alder/aldol sequence and screened them for multiple biological activities using high-content zebrafish embryonic development assays. We discovered a noreremophilane that has strong anti-angiogenic effects on the developing zebrafish embryos as well as on tumor-induced angiogenesis in a zebrafish xenograft model. We synthesized several derivatives of this class of noreremophilanes and performed structure-activity relationship studies in zebrafish to identify more potent and less toxic analogs of the original structure.

Generation of an induced pluripotent stem cell line IGIBi18-A from an Indian patient with Rubinstein Taybi Syndrome.

Rubinstein Taybi Syndrome (RSTS) is a rare genetic disorder which is caused by mutations in either CREBBP or EP300. RSTS with mutations in CREBBP is known as RSTS-1. We have generated an induced pluripotent stem cell (iPSC) line, IGIBi018-A from an Indian RSTS-patient using the episomal reprogramming method. The CREBBP gene in the patient harbours a nonsense mutation at position NM_004380.3(c.6876 del C). IGIBi018-A iPSC showed expression of pluripotent stem cell markers, has a normal karyotype and could be differentiated into three germ layers. This iPSC line will help to explore the role of CREBBP in RSTS associated developmental defects.

Inhibition of bone morphogenetic protein signaling attenuates anemia associated with inflammation.

Anemia of inflammation develops in settings of chronic inflammatory, infectious, or neoplastic disease. In this highly prevalent form of anemia, inflammatory cytokines, including IL-6, stimulate hepatic expression of hepcidin, which negatively regulates iron bioavailability by inactivating ferroportin. Hepcidin is transcriptionally regulated by IL-6 and bone morphogenetic protein (BMP) signaling. We hypothesized that inhibiting BMP signaling can reduce hepcidin expression and ameliorate hypoferremia and anemia associated with inflammation. In human hepatoma cells, IL-6-induced hepcidin expression, an effect that was inhibited by treatment with a BMP type I receptor inhibitor, LDN-193189, or BMP ligand antagonists noggin and ALK3-Fc. In zebrafish, the induction of hepcidin expression by transgenic expression of IL-6 was also reduced by LDN-193189. In mice, treatment with IL-6 or turpentine increased hepcidin expression and reduced serum iron, effects that were inhibited by LDN-193189 or ALK3-Fc. Chronic turpentine treatment led to microcytic anemia, which was prevented by concurrent administration of LDN-193189 or attenuated when LDN-193189 was administered after anemia was established. Our studies support the concept that BMP and IL-6 act together to regulate iron homeostasis and suggest that inhibition of BMP signaling may be an effective strategy for the treatment of anemia of inflammation.

A diet-independent zebrafish model for NAFLD recapitulates patient lipid profiles and offers a system for small molecule screening.

Non-alcoholic Fatty Liver Disease (NAFLD) or pathological hepatic lipid overload, is considered to affect obese individuals. However, NAFLD in lean individuals is prevalent, especially in South Asian population. The pathophysiology of lean NAFLD is not well understood and most animal models of NAFLD use the high-fat diet paradigm. To bridge this gap, we have developed a diet-independent model of NAFLD in zebrafish. We have previously shown that chronic systemic inflammation causes metabolic changes in the liver leading to hepatic fat accumulation in an IL6 overexpressing (IL6-OE) zebrafish model. In the present study, we compared the hepatic lipid composition of adult IL6-OE zebrafish to the controls and found an accumulation of saturated triacylglycerols and a reduction in the unsaturated triacylglycerol species reminiscent of NAFLD patients. Zebrafish is an ideal system for chemical genetic screens. We tested whether the hepatic lipid accumulation in the IL6-OE is responsive to chemical treatment. We found that PPAR-gamma agonist Rosiglitazone, known to reduce lipid overload in the high-fat diet models of NAFLD, could ameliorate the fatty liver phenotype of the IL6-OE fish. Rosiglitazone treatment reduced the accumulation of saturated lipids and showed a concomitant increase in unsaturated TAG species in our inflammation-induced NAFLD model. Our observations suggest that the IL6-OE model can be effective for small molecule screening to identify compounds that can reverse hepatic lipid accumulation, especially relevant to lean NAFLD.

Chronic systemic exposure to IL6 leads to deregulation of glycolysis and fat accumulation in the zebrafish liver.

Inflammation is a constant in Non-Alcoholic Fatty Liver Disease (NAFLD), although their relationship is unclear. In a transgenic zebrafish system with chronic systemic overexpression of human IL6 (IL6-OE) we show that inflammation can cause intra-hepatic accumulation of triglycerides. Transcriptomics and proteomics analysis of the IL6-OE liver revealed a deregulation of glycolysis/gluconeogenesis pathway, especially a striking down regulation of the glycolytic enzyme aldolase b. Metabolomics analysis by mass spectrometry showed accumulation of hexose monophosphates and their derivatives, which can act as precursors for triglyceride synthesis. Our results suggest that IL6-driven repression of glycolysis/gluconeogenesis, specifically aldolase b, may be a novel mechanism for fatty liver. This mechanism may be relevant for NAFLD in lean individuals, an emerging class of NAFLD prevalent more in Asian Indian populations.

Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism.

Bone morphogenetic protein (BMP) signals coordinate developmental patterning and have essential physiological roles in mature organisms. Here we describe the first known small-molecule inhibitor of BMP signaling-dorsomorphin, which we identified in a screen for compounds that perturb dorsoventral axis formation in zebrafish. We found that dorsomorphin selectively inhibits the BMP type I receptors ALK2, ALK3 and ALK6 and thus blocks BMP-mediated SMAD1/5/8 phosphorylation, target gene transcription and osteogenic differentiation. Using dorsomorphin, we examined the role of BMP signaling in iron homeostasis. In vitro, dorsomorphin inhibited BMP-, hemojuvelin- and interleukin 6-stimulated expression of the systemic iron regulator hepcidin, which suggests that BMP receptors regulate hepcidin induction by all of these stimuli. In vivo, systemic challenge with iron rapidly induced SMAD1/5/8 phosphorylation and hepcidin expression in the liver, whereas treatment with dorsomorphin blocked SMAD1/5/8 phosphorylation, normalized hepcidin expression and increased serum iron levels. These findings suggest an essential physiological role for hepatic BMP signaling in iron-hepcidin homeostasis.

Chemical screens in a zebrafish model of CHARGE syndrome identifies small molecules that ameliorate disease-like phenotypes in embryo.

CHARGE syndrome is an autosomal dominant congenital disorder caused primarily by mutations in the CHD7 gene. Using a small molecule screen in a zebrafish model of CHARGE syndrome, we identified 4 compounds that rescue embryos from disease-like phenotypes. Our screen yielded DAPT, a Notch signaling inhibitor that could ameliorate the craniofacial, cranial neuronal and myelination defects in chd7 morphant zebrafish embryos. We discovered that Procainamide, an inhibitor of DNA methyltransferase 1, was able to recover the pattern of expression of isl2a, a cranial neuronal marker while also reducing the effect on craniofacial cartilage and myelination. M344, an inhibitor of Histone deacetylases had a strong recovery effect on craniofacial cartilage defects and could also modestly revert the myelination defects in zebrafish embryos. CHIC-35, a SIRT1 inhibitor partially restored the expression of isl2a in cranial neurons while causing a partial reversion of myelination and craniofacial cartilage defects. Our results suggest that a modular approach to phenotypic rescue in multi-organ syndromes might be a more successful approach to treat these disorders. Our findings also open up the possibility of using these compounds for other disorders with shared phenotypes.

A Temporal Map of Gene Expression Pattern During Zebrafish Liver Regeneration.

Zebrafish is increasingly being used to study liver injury and regeneration. However, very little is known about molecular players that respond to injury and those important for liver regeneration. We use a metronidazole nitroreductase (MTZ-nfsb)-based system to selectively ablate hepatocytes in adult zebrafish to create a model for liver injury and regeneration. In this study, we generate a comprehensive temporal map of gene expression changes during regeneration through RNA sequencing of liver samples at various stages of injury and regeneration. Analyzing these data, we find that soon after injury the immediate early transcription factor MYC induces a battery of genes that respond to the MTZ-induced ROS by activating oxido-reductase pathways and apoptosis machinery. Immediately after injury, liver cells downregulate many functional genes, including complement protein synthesis, bile acid, and lipid biosynthesis, in a concerted manner. At 6 days postinjury, we find a dramatic induction of cholesterol biosynthesis and protein folding machinery, with expression levels returning to predamage levels by 8 days, suggesting an important role for these pathways in liver regeneration. This chronological transcriptomic map of liver regeneration in zebrafish would serve as a framework for further studies in understanding, and for screening for compounds that augment liver regeneration.

Structure-activity relationship study of bone morphogenetic protein (BMP) signaling inhibitors.

A structure-activity relationship study of dorsomorphin, a previously identified inhibitor of SMAD 1/5/8 phosphorylation by bone morphogenetic protein (BMP) type 1 receptors ALK2, 3, and 6, revealed that increased inhibitory activity could be accomplished by replacing the pendent 4-pyridine ring with 4-quinoline. The activity contributions of various nitrogen atoms in the core pyrazolo[1,5-a]pyrimidine ring were also examined by preparing and evaluating pyrrolo[1,2-a]pyrimidine and pyrazolo[1,5-a]pyridine derivatives. In addition, increased mouse liver microsome stability was achieved by replacing the ether substituent on the pendent phenyl ring with piperazine. Finally, an optimized compound 13 (LDN-193189 or DM-3189) demonstrated moderate pharmacokinetic characteristics (e.g., plasma t(1/2)=1.6h) following intraperitoneal administration in mice. These studies provide useful molecular probes for examining the in vivo pharmacology of BMP signaling inhibition.

Small Molecule Inhibitors of NFkB Reverse Iron Overload and Hepcidin Deregulation in a Zebrafish Model for Hereditary Hemochromatosis Type 3.

Hereditary hemochromatosis (HH) is one of the most common genetic disorders in Caucasian populations, with no viable therapeutic options except phlebotomy. We describe a zebrafish model of human HH (HH) created by targeted mutagenesis of the gene encoding transferrin receptor 2 ( tfr2). TFR2 mutations in humans lead to HH Type 3, a rare but severe form of the disease. The tfr2 mutant model in zebrafish recapitulates the defining features of HH3: iron overload and suppression of hepcidin, the iron regulatory hormone. Using in vivo chemical screens in zebrafish embryos, we identify a new small molecule inducer of hepcidin: SC-514, a specific chemical inhibitor of NFkB signaling. Using independent small molecule inhibitors of the NFkB pathway, we demonstrate that inhibition of NFkB signaling causes induction of hepcidin transcription and reduction of iron overload in the HH3 model. This first successful chemical intervention for hereditary hemochromatosis may also have relevance in treatment of other very prevalent iron regulatory iron overload disorders such as thalassemia.

Deciphering the role of hydrophobic and hydrophilic bile acids in angiogenesis using in vitro and in vivo model systems.

Bile acids have emerged as strong signaling molecules capable of influencing various biological processes like inflammation, apoptosis, cancer progression and atherosclerosis depending on their chemistry. In the present study, we investigated the effect of major hydrophobic bile acids lithocholic acid (LCA) and deoxycholic acid (DCA) and hydrophilic bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) on angiogenesis. We employed human umbilical vein endothelial cells (HUVECs) and zebrafish embryos as model systems for studying the role of bile acids in angiogenesis. Our studies revealed that the hydrophilic CDCA enhanced ectopic vessel formation as observed by the increase in the number of sub-intestinal vessels (SIVs) in the zebrafish embryos. The pro-angiogenic role of CDCA was further corroborated by in vitro vessel formation studies performed with human umbilical vein endothelial cells (HUVECs), whereas the hydrophobic LCA reduced tubulogenesis and was toxic to the zebrafish embryos. We validated that CDCA enhances angiogenesis by increasing the expression of vascular growth factor receptors (VEGFR1 and VEGFR2) and matrix metalloproteinases (MMP9) and by decreasing the expression of adhesion protein vascular endothelial cadherin (VE-cadherin). Our work implicates that the nature of bile acids plays a critical role in dictating their biological functions and in regulating angiogenesis.

Sustainable, Rapid Synthesis of Bright-Luminescent CuInS2-ZnS Alloyed Nanocrystals: Multistage Nano-xenotoxicity Assessment and Intravital Fluorescence Bioimaging in Zebrafish-Embryos.

Near-infrared (NIR) luminescent CuInS2-ZnS alloyed nanocrystals (CIZS-NCs) for highly fluorescence bioimaging have received considerable interest in recent years. Owing, they became a desirable alternative to heavy-metal based-NCs and organic dyes with unique optical properties and low-toxicity for bioimaging and optoelectronic applications. In the present study, bright and robust CIZS-NCs have been synthesized within 5 min, as-high-as 230 °C without requiring any inert-gas atmosphere via microwave-solvothermal (MW-ST) method. Subsequently, the in vitro and in vivo nano-xenotoxicity and cellular uptake of the MUA-functionalized CIZS-NCs were investigated in L929, Vero, MCF7 cell lines and zebrafish-embryos. We observed minimal toxicity and acute teratogenic consequences upto 62.5 µg/ml of the CIZS-NCs in zebrafish-embryos. We also observed spontaneous uptake of the MUA-functionalized CIZS-NCs by 3 dpf older zebrafish-embryos that are evident through bright red fluorescence-emission at a low concentration of 7.8 µg/mL. Hence, we propose that the rapid, low-cost, large-scale "sustainable" MW-ST synthesis of CIZS-NCs, is an ideal bio-nanoprobe with good temporal and spatial resolution for rapid labeling, long-term in vivo tracking and intravital-fluorescence-bioimaging (IVBI).

The Light Intermediate Chain 2 Subpopulation of Dynein Regulates Mitotic Spindle Orientation.

Cytoplasmic dynein 1 is a multi-protein intracellular motor essential for mediating several mitotic functions, including the establishment of proper spindle orientation. The functional relevance and mechanistic distinctions between two discrete dynein subpopulations distinguished only by Light Intermediate Chain (LIC) homologues, LIC1 and LIC2 is unknown during mitosis. Here, we identify LIC2-dynein as the major mediator of proper spindle orientation and uncover its underlying molecular mechanism. Cortically localized dynein, essential for maintaining correct spindle orientation, consists majorly of LIC2-dynein, which interacts with cortical 14-3-3 ε- ζ and Par3, conserved proteins required for orienting the spindle. LIC2-dynein is also responsible for the majority of dynein-mediated asymmetric poleward transport of NuMA, helping focus microtubule minus ends. In addition, LIC2-dynein dominates in equatorially aligning chromosomes at metaphase and in regulating mitotic spindle length. Key mitotic functions of LIC2 were remarkably conserved in and essential for early embryonic divisions and development in zebrafish. Thus LIC2-dynein exclusively engages with two major cortical pathways to govern spindle orientation. Overall, we identify a novel selectivity of molecular interactions between the two LICs in mitosis as the underlying basis for their uneven distribution of labour in ensuring proper spindle orientation.

The Zebrafish GenomeWiki: a crowdsourcing approach to connect the long tail for zebrafish gene annotation.

A large repertoire of gene-centric data has been generated in the field of zebrafish biology. Although the bulk of these data are available in the public domain, most of them are not readily accessible or available in nonstandard formats. One major challenge is to unify and integrate these widely scattered data sources. We tested the hypothesis that active community participation could be a viable option to address this challenge. We present here our approach to create standards for assimilation and sharing of information and a system of open standards for database intercommunication. We have attempted to address this challenge by creating a community-centric solution for zebrafish gene annotation. The Zebrafish GenomeWiki is a 'wiki'-based resource, which aims to provide an altruistic shared environment for collective annotation of the zebrafish genes. The Zebrafish GenomeWiki has features that enable users to comment, annotate, edit and rate this gene-centric information. The credits for contributions can be tracked through a transparent microattribution system. In contrast to other wikis, the Zebrafish GenomeWiki is a 'structured wiki' or rather a 'semantic wiki'. The Zebrafish GenomeWiki implements a semantically linked data structure, which in the future would be amenable to semantic search. Database URL: http://genome.igib.res.in/twiki.

Time for the zebrafish ENCODE.

Genomics research in recent years, especially the human ENCODE project, have made great strides in understanding the genomic and epigenomic structure and organization of humans. These advances promise a new era of precision medicine, through a better understanding of the genomic correlates of human physiology and promise to offer precise and personalized preventive and therapeutic options. The translation of genome-scale maps of genomic and epigenomic markers to clinically relevant information and further to medical practice await functional validation of the genomic features identified through these large-scale efforts. Such studies must essentially be done in model systems where it is possible to model physiological and pathological processes and enquire how they could be modulated by genomic elements and epigenomic signatures. The availability of large number of personal genomes and maps of genomic variations at population scale has created an acute necessity for model systems to model phenotypic and molecular effects of variations, especially in regulatory regions. Efforts to create orthologous maps have been underway in other model systems including Caenorhabditis elegans and Drosophila through the modENCODE programe and in Mus musculus through the mouse ENCODE. We propose that the enormous wealth of disease models and excellent tools to engineer genomes in zebrafish could be effectively capitalized towards making it an effective and widely used model system for precision medicine. This would be possible only through a concerted and systematic effort to create orthologous genomic and epigenomic maps for zebrafish.We discuss how the present understanding and genome-scale methodologies available in this model organism could be effectively used towards realizing this goal.