MITF in melanoma progression, pathology and survival in vivo

MITF is the master melanocyte transcription factor and has a complex role in melanoma. Both gain- and loss-of function mutations in MITF have been identified in melanoma, although its’ role in melanoma development and the effects of targeting MITF are unknown. Using a temperature-sensitive mitf zebrafish mutant I show that low levels of MITF are oncogenic with BRAFV600E in melanoma progression. By pathology and MITF target gene expression, BRAFV600Emitfavc7 tumours are distinct from BRAFV600Ep53M214K tumours, and represent two melanoma subtypes. Melanomagenesis can also be driven independently of BRAFV600E, in a transgenic zebrafish with mutations in mitf and p53, representing a new melanoma model. Abrogating MITF activity in BRAFV600Emitfavc7 melanoma leads to regression of the tumour, characterised by macrophage infiltration and increased apoptosis. This result confirms the dependence on MITF activity in BRAFV600Emitfavc7 melanomas and highlights the role of MITF as a therapeutic target for melanoma. Exome and transcriptome sequencing has been carried out to gain insight into the expression and genomic mutational landscape that is driven by these melanoma transgenic models and results in these genotype-phenotype specific subtypes observed

Cobalt ions recruit inflammatory cells in vitro through human Toll-like receptor 4

AbstractMetal-on-metal (MoM) hip replacements, often manufactured from a cobalt-chrome alloy, are associated with adverse reactions including soft tissue necrosis and osteolysis. Histopathological analysis of MoM peri-implant tissues reveals an inflammatory cell infiltrate that includes macrophages, monocytes and neutrophils.Toll-like receptor 4 (TLR4) is an innate immune receptor activated by bacterial lipopolysaccharide. Recent studies have demonstrated that cobalt ions from metal-on-metal joints also activate human TLR4, increasing cellular secretion of inflammatory chemokines including interleukin-8 (IL-8, CXCL8) and CCL2. Chemokines recruit immune cells to the site of inflammation, and their overall effect depends on the chemokine profile produced.This study investigated the effect of cobalt on the secretion of inflammatory cytokines CCL20 and IL-6. The chemotactic potential of conditioned media from a cobalt-stimulated human monocyte cell line on primary monocytes and neutrophils was investigated using an in vitro transwell migration assay. The role of TLR4 in observed effects was studied using a small molecule TLR4-specific antagonist.Cobalt ions significantly increased release of CCL2 and IL-6 by MonoMac 6 cells (P<0.001). Conditioned media from cobalt-stimulated cells significantly increased monocyte and neutrophil chemotaxis in vitro (P<0.001). These effects were abrogated by the TLR4 antagonist (P<0.001) suggesting that they occur through cobalt activation of TLR4.This study demonstrates the role of TLR4 in cobalt-mediated immune cell chemotaxis and provides a potential mechanism by which cobalt ions may contribute to the immune cell infiltrate surrounding failed metal hip replacements. It also highlights the TLR4 signalling pathway as a potential therapeutic target in preventing cobalt-mediated inflammation

A Conditional Zebrafish MITF Mutation Reveals MITF Levels Are Critical for Melanoma Promotion vs. Regression In Vivo

The microphthalmia-associated transcription factor (MITF) is the “master melanocyte transcription factor” with a complex role in melanoma. MITF protein levels vary between and within clinical specimens, and amplifications and gain- and loss-of-function mutations have been identified in melanoma. How MITF functions in melanoma development and the effects of targeting MITF in vivo are unknown because MITF levels have not been directly tested in a genetic animal model. Here, we use a temperature-sensitive mitf zebrafish mutant to conditionally control endogenous MITF activity. We show that low levels of endogenous MITF activity are oncogenic with BRAFV600E to promote melanoma that reflects the pathology of the human disease. Remarkably, abrogating MITF activity in BRAFV600Emitf melanoma leads to dramatic tumor regression marked by melanophage infiltration and increased apoptosis. These studies are significant because they show that targeting MITF activity is a potent antitumor mechanism, but also show that caution is required because low levels of wild-type MITF activity are oncogenic

Dhx34 and Nbas function in the NMD pathway and are required for embryonic development in zebrafish

The nonsense-mediated mRNA decay (NMD) pathway is a highly conserved surveillance mechanism that is present in all eukaryotes. It prevents the synthesis of truncated proteins by selectively degrading mRNAs harbouring premature termination codons (PTCs). The core NMD effectors were originally identified in genetic screens in Saccharomyces cerevisae and in the nematode Caenorhabditis elegans, and subsequently by homology searches in other metazoans. A genome-wide RNAi screen in C. elegans resulted in the identification of two novel NMD genes that are essential for proper embryonic development. Their human orthologues, DHX34 and NAG/NBAS, are required for NMD in human cells. Here, we find that the zebrafish genome encodes orthologues of DHX34 and NAG/NBAS. We show that the morpholino-induced depletion of zebrafish Dhx34 and Nbas proteins results in severe developmental defects and reduced embryonic viability. We also found that Dhx34 and Nbas are required for degradation of PTC-containing mRNAs in zebrafish embryos. The phenotypes observed in both Dhx34 and Nbas morphants are similar to defects in Upf1, Smg-5- or Smg-6- depleted embryos, suggesting that these factors affect the same pathway and confirming that zebrafish embryogenesis requires an active NMD pathway

The genetic heterogeneity and mutational burden of engineered melanomas in zebrafish models.

BACKGROUND: Melanoma is the most deadly form of skin cancer. Expression of oncogenic BRAF or NRAS, which are frequently mutated in human melanomas, promote the formation of nevi but are not sufficient for tumorigenesis. Even with germline mutated p53, these engineered melanomas present with variable onset and pathology, implicating additional somatic mutations in a multi-hit tumorigenic process. RESULTS: To decipher the genetics of these melanomas, we sequence the protein coding exons of 53 primary melanomas generated from several BRAF(V600E) or NRAS(Q61K) driven transgenic zebrafish lines. We find that engineered zebrafish melanomas show an overall low mutation burden, which has a strong, inverse association with the number of initiating germline drivers. Although tumors reveal distinct mutation spectrums, they show mostly C > T transitions without UV light exposure, and enrichment of mutations in melanogenesis, p53 and MAPK signaling. Importantly, a recurrent amplification occurring with pre-configured drivers BRAF(V600E) and p53-/- suggests a novel path of BRAF cooperativity through the protein kinase A pathway. CONCLUSION: This is the first analysis of a melanoma mutational landscape in the absence of UV light, where tumors manifest with remarkably low mutation burden and high heterogeneity. Genotype specific amplification of protein kinase A in cooperation with BRAF and p53 mutation suggests the involvement of melanogenesis in these tumors. This work is important for defining the spectrum of events in BRAF or NRAS driven melanoma in the absence of UV light, and for informed exploitation of models such as transgenic zebrafish to better understand mechanisms leading to human melanoma formation

Palatability and chemical defences of benthic cyanobacteria to a suite of herbivores

Nuisance blooms of toxic cyanobacteria are a common occurrence in many tropical and subtropical locations. Benthic marine cyanobacteria of the genera Lyngbya, Okeania, and Moorea are frequently observed in both Florida and throughout the Caribbean, sometimes forming large mats, and are prolific producers of bioactive secondary metabolites that often act as feeding deterrents to generalist herbivores. Little is known regarding the ecological roles of the secondary metabolite chemistry and the palatability of benthic cyanobacteria to grazers. This study examines the palatability of benthic cyanobacterial species from Florida (IRL1, IRL2, IRL3 and Okeania erythroflocculosa) and Belize (BEL1, BEL2) to a range of macro- and mesograzers in Florida and Belize. Pair-wise feeding assays using artificial diets of Gracilaria tikvahiae or fish food coated with cyanobacterial extracts and a control were used to determine palatability of extracts to Floridian and Belizean generalist grazers. The extracts of IRL1, IRL2, IRL3 and O. erythroflocculosa from Florida did not deter feeding by invertebrate grazers. Reef fish, however, were deterred by the non-polar extracts of IRL1, IRL3 and O. erythroflocculosa. Stylocheilus striatus was stimulated to feed on IRL2 extracts and non-polar extracts from IRL3. Non-polar extracts of BEL1 stimulated feeding in S. striatus; however, no significant difference was observed between BEL2 extracts and the control. Most generalist invertebrate grazers, sympatric and non-sympatric, appear indifferent to cyanobacteria extracts whilst reef fish are more likely to be deterred by cyanobacterial extracts, which may affect species interaction within communities with fluctuating or dominating benthic cyanobacterial blooms.This research was funded by a Smithsonian Marine Station Fellowship to A.C. and Grant Number NA05 NOS 4781194 from the U.S. Department of Commerce (DoC, NOAA, ECOHAB programme), through UNC-CH.Peer Reviewe

Palatability and chemical defences of benthic cyanobacteria to a suite of herbivores

Nuisance blooms of toxic cyanobacteria are a common occurrence in many tropical and subtropical locations. Benthic marine cyanobacteria of the genera Lyngbya, Okeania, and Moorea are frequently observed in both Florida and throughout the Caribbean, sometimes forming large mats, and are prolific producers of bioactive secondary metabolites that often act as feeding deterrents to generalist herbivores. Little is known regarding the ecological roles of the secondary metabolite chemistry and the palatability of benthic cyanobacteria to grazers. This study examines the palatability of benthic cyanobacterial species from Florida (IRL1, IRL2, IRL3 and Okeania erythroflocculosa) and Belize (BEL1 BEL2) to a range of macro- and mesograzers in Florida and Belize. Pair-wise feeding assays using artificial diets of Gracilaria tikvahiae or fish food coated with cyanobacterial extracts and a control were used to determine palatability of extracts to Floridian and Belizean generalist grazers. The extracts of IRL1, IRL2, IRL3 and O. erythroflocculosa from Florida did not deter feeding by invertebrate grazers. Reef fish, however, were deterred by the non-polar extracts of IRL1, IRL3 and O. etythroflocculosa. Stylocheilus striatus was stimulated to feed on IRL2 extracts and non-polar extracts from IRL3. Non-polar extracts of BEL1 stimulated feeding in S. striatus; however, no significant difference was observed between BEL2 extracts and the control. Most generalist invertebrate grazers, sympatric and non-sympatric, appear indifferent to cyanobacteria extracts whilst reef fish are more likely to be deterred by cyanobacterial extracts, which may affect species interaction within communities with fluctuating or dominating benthic cyanobacterial blooms

A pilot study investigating reactive oxygen species production in capillary blood after a marathon and the influence of an antioxidant-rich beetroot juice

We report that reactive oxygen species (ROS), as measured in capillary blood taken from the finger-tip, increased after a marathon (+128% P < 0.01; effect size = 1.17), indicating that this collection method might be useful for measuring ROS in field settings. However, mitochondrial DNA damage remained unchanged. Beetroot juice, taken before and after exercise, was unable to mitigate exercise-induced ROS production, questioning its use an antioxidant-rich food