Decay of Skin-Specific Gene Modules in Pangolins.

The mammalian skin exhibits a rich spectrum of evolutionary adaptations. The pilosebaceous unit, composed of the hair shaft, follicle, and the sebaceous gland, is the most striking synapomorphy. The evolutionary diversification of mammals across different ecological niches was paralleled by the appearance of an ample variety of skin modifications. Pangolins, order Pholidota, exhibit keratin-derived scales, one of the most iconic skin appendages. This formidable armor is intended to serve as a deterrent against predators. Surprisingly, while pangolins have hair on their abdomens, the occurrence of sebaceous and sweat glands is contentious. Here, we explore various molecular modules of skin physiology in four pangolin genomes, including that of sebum production. We show that genes driving wax monoester formation, Awat1/2, show patterns of inactivation in the stem pangolin branch, while the triacylglycerol synthesis gene Dgat2l6 seems independently eroded in the African and Asian clades. In contrast, Elovl3 implicated in the formation of specific neutral lipids required for skin barrier function is intact and expressed in the pangolin skin. An extended comparative analysis shows that genes involved in skin pathogen defense and structural integrity of keratinocyte layers also show inactivating mutations: associated with both ancestral and independent pseudogenization events. Finally, we deduce that the suggested absence of sweat glands is not paralleled by the inactivation of the ATP-binding cassette transporter Abcc11, as previously described in Cetacea. Our findings reveal the sophisticated and complex history of gene retention and loss as key mechanisms in the evolution of the highly modified mammalian skin phenotypes.

A Multiplex Molecular Cell-Based Sensor to Detect Ligands of PPARs: An Optimized Tool for Drug Discovery in Cyanobacteria.

Cyanobacteria produce a wealth of secondary metabolites. Since these organisms attach fatty acids into molecules in unprecedented ways, cyanobacteria can serve as a novel source for bioactive compounds acting as ligands for Peroxisome Proliferator-Activated Receptors (PPAR). PPARs (PPARα, PPARß/δ and PPARγ) are ligand-activated nuclear receptors, involved in the regulation of various metabolic and cellular processes, thus serving as potential drug targets for a variety of pathologies. Yet, given that PPARs' agonists can have pan-, dual- or isoform-specific action, some controversy has been raised over currently approved drugs and their side effects, highlighting the need for novel molecules. Here, we expand and validate a cell-based PPAR transactivation activity biosensor, and test it in a screening campaign to guide drug discovery. Biosensor upgrades included the use of different reporter genes to increase signal intensity and stability, a different promoter to modulate reporter gene expression, and multiplexing to improve efficiency. Sensor's limit of detection (LOD) ranged from 0.36-0.89 nM in uniplex and 0.89-1.35 nM in multiplex mode. In triplex mode, the sensor's feature screening, a total of 848 fractions of 96 cyanobacteria extracts were screened. Hits were confirmed in multiplex mode and in uniplex mode, yielding one strain detected to have action on PPARα and three strains to have dual action on PPARα and -ß.

PseudoChecker: an integrated online platform for gene inactivation inference.

The rapid expansion of high-quality genome assemblies, exemplified by ongoing initiatives such as the Genome-10K and i5k, demands novel automated methods to approach comparative genomics. Of these, the study of inactivating mutations in the coding region of genes, or pseudogenization, as a source of evolutionary novelty is mostly overlooked. Thus, to address such evolutionary/genomic events, a systematic, accurate and computationally automated approach is required. Here, we present PseudoChecker, the first integrated online platform for gene inactivation inference. Unlike the few existing methods, our comparative genomics-based approach displays full automation, a built-in graphical user interface and a novel index, PseudoIndex, for an empirical evaluation of the gene coding status. As a multi-platform online service, PseudoChecker simplifies access and usability, allowing a fast identification of disruptive mutations. An analysis of 30 genes previously reported to be eroded in mammals, and 30 viable genes from the same lineages, demonstrated that PseudoChecker was able to correctly infer 97% of loss events and 95% of functional genes, confirming its reliability. PseudoChecker is freely available, without login required, at http://pseudochecker.ciimar.up.pt.

Antifouling Marine Coatings with a Potentially Safer and Sustainable Synthetic Polyphenolic Derivative.

The development of harmless substances to replace biocide-based coatings used to prevent or manage marine biofouling and its unwanted consequences is urgent. The formation of biofilms on submerged marine surfaces is one of the first steps in the marine biofouling process, which facilitates the further settlement of macrofoulers. Anti-biofilm properties of a synthetic polyphenolic compound, with previously described anti-settlement activity against macrofoulers, were explored in this work. In solution this new compound was able to prevent biofilm formation and reduce a pre-formed biofilm produced by the marine bacterium, Pseudoalteromonas tunicata. Then, this compound was applied to a marine coating and the formation of P. tunicata biofilms was assessed under hydrodynamic conditions to mimic the marine environment. For this purpose, polyurethane (PU)-based coating formulations containing 1 and 2 wt.% of the compound were prepared based on a prior developed methodology. The most effective formulation in reducing the biofilm cell number, biovolume, and thickness was the PU-based coating containing an aziridine-based crosslinker and 2 wt.% of the compound. To assess the marine ecotoxicity impact of this compound, its potential to disrupt endocrine processes was evaluated through the modulation of two nuclear receptors (NRs), peroxisome proliferator-activated receptor γ (PPARγ), and pregnane X receptor (PXR). Transcriptional activation of the selected NRs upon exposure to the polyphenolic compound (10 µM) was not observed, thus highlighting the eco-friendliness towards the addressed NRs of this new dual-acting anti-macro- and anti-microfouling agent towards the addressed NRs.

Convergent Cortistatin losses parallel modifications in circadian rhythmicity and energy homeostasis in Cetacea and other mammalian lineages.

The ancestors of Cetacea underwent profound morpho-physiological alterations. By displaying an exclusive aquatic existence, cetaceans evolved unique patterns of locomotor activity, vigilant behaviour, thermoregulation and circadian rhythmicity. Deciphering the molecular landscape governing many of these adaptations is key to understand the evolution of phenotypes. Here, we investigate Cortistatin (CORT), a neuropeptide displaying an important role in mammalian biorhythm regulation. This neuropeptide is a known neuroendocrine factor, stimulating slow-wave sleep, but also involved in the regulation of energy metabolism and hypomotility inducement. We assessed the functional status of CORT in 359 mammalian genomes (25 orders), including 30 species of Cetacea. Our findings indicate that cetaceans and other mammals with atypical biorhythms, thermal constraints and/or energy metabolism, have accumulated deleterious mutations in CORT. In light of the pleiotropic action of this neuropeptide, we suggest that this inactivation contributed to a plethora of phenotypic adjustments to accommodate adaptive solutions to specific ecological niches.

Functional or Vestigial? The Genomics of the Pineal Gland in Xenarthra.

Vestigial organs are historical echoes of past phenotypes. Determining whether a specific organ constitutes a functional or vestigial structure can be a challenging task, given that distinct levels of atrophy may arise between and within lineages. The mammalian pineal gland, an endocrine organ involved in melatonin biorhythmicity, represents a classic example, often yielding contradicting anatomical observations. In Xenarthra (sloths, anteaters, and armadillos), a peculiar mammalian order, the presence of a distinct pineal organ was clearly observed in some species (i.e., Linnaeus's two-toed sloth), but undetected in other closely related species (i.e., brown-throated sloth). In the nine-banded armadillo, contradicting evidence supports either functional or vestigial scenarios. Thus, to untangle the physiological status of the pineal gland in Xenarthra, we used a genomic approach to investigate the evolution of the gene hub responsible for melatonin synthesis and signaling. We show that both synthesis and signaling compartments are eroded and were probably lost independently among Xenarthra orders. Additionally, by expanding our analysis to 157 mammal genomes, we offer a comprehensive view showing that species with very distinctive habitats and lifestyles have convergently evolved a similar phenotype: Cetacea, Pholidota, Dermoptera, Sirenia, and Xenarthra. Our findings suggest that the recurrent inactivation of melatonin genes correlates with pineal atrophy and endorses the use of genomic analyses to ascertain the physiological status of suspected vestigial structures.

Complete Inactivation of Sebum-Producing Genes Parallels the Loss of Sebaceous Glands in Cetacea.

Genomes are dynamic biological units, with processes of gene duplication and loss triggering evolutionary novelty. The mammalian skin provides a remarkable case study on the occurrence of adaptive morphological innovations. Skin sebaceous glands (SGs), for instance, emerged in the ancestor of mammals serving pivotal roles, such as lubrication, waterproofing, immunity, and thermoregulation, through the secretion of sebum, a complex mixture of various neutral lipids such as triacylglycerol, free fatty acids, wax esters, cholesterol, and squalene. Remarkably, SGs are absent in a few mammalian lineages, including the iconic Cetacea. We investigated the evolution of the key molecular components responsible for skin sebum production: Dgat2l6, Awat1, Awat2, Elovl3, Mogat3, and Fabp9. We show that all analyzed genes have been rendered nonfunctional in Cetacea species (toothed and baleen whales). Transcriptomic analysis, including a novel skin transcriptome from blue whale, supports gene inactivation. The conserved mutational pattern found in most analyzed genes, indicates that pseudogenization events took place prior to the diversification of modern Cetacea lineages. Genome and skin transcriptome analysis of the common hippopotamus highlighted the convergent loss of a subset of sebum-producing genes, notably Awat1 and Mogat3. Partial loss profiles were also detected in non-Cetacea aquatic mammals, such as the Florida manatee, and in terrestrial mammals displaying specialized skin phenotypes such as the African elephant, white rhinoceros and pig. Our findings reveal a unique landscape of "gene vestiges" in the Cetacea sebum-producing compartment, with limited gene loss observed in other mammalian lineages: suggestive of specific adaptations or specializations of skin lipids.

Cartilaginous fishes offer unique insights into the evolution of the nuclear receptor gene repertoire in gnathostomes.

Nuclear receptors (NRs) are key transcription factors that originated in the common ancestor of metazoans. The vast majority of NRs are triggered by binding to either endogenous (e.g. retinoic acid) or exogenous (e.g. xenobiotics) ligands, and their evolution and expansion is tightly linked to the function of endocrine systems. Importantly, they represent classic targets of physiological exploitation by endocrine disrupting chemicals. The NR gene repertoire in different lineages has been shaped by gene loss, duplication and mutation, denoting a dynamic evolutionary route. As the earliest diverging class of gnathostomes (jawed vertebrates), cartilaginous fishes offer an exceptional opportunity to address the early diversification of NR gene families and the evolution of the endocrine system in jawed vertebrates. Here we provide an exhaustive analysis into the NR gene composition in five elasmobranch (sharks and rays) and two holocephalan (chimaeras) species. For this purpose, we generated also a low coverage draft genome assembly of the chimaera small-eyed rabbitfish, Hydrolagus affinis. We show that cartilaginous fish retain an archetypal NR gene repertoire, similar to that of mammals and coincident with the two rounds of whole genome duplication that occurred in the gnathostome ancestor. Furthermore, novel gene members of the non-canonical NR0B receptors were found in the genomes of this lineage. Our findings provide an essential view into the early diversification of NRs in gnathostomes, paving the way for functional studies.

Convergent inactivation of the skin-specific C-C motif chemokine ligand 27 in mammalian evolution.

The appearance of mammalian-specific skin features was a key evolutionary event contributing for the elaboration of physiological processes such as thermoregulation, adequate hydration, locomotion, and inflammation. Skin inflammatory and autoimmune processes engage a population of skin-infiltrating T cells expressing a specific C-C chemokine receptor (CCR10) which interacts with an epidermal CC chemokine, the skin-specific C-C motif chemokine ligand 27 (CCL27). CCL27 is selectively produced in the skin by keratinocytes, particularly upon inflammation, mediating the adhesion and homing of skin-infiltrating T cells. Here, we examined the evolution and coding condition of Ccl27 in 112 placental mammalian species. Our findings reveal that a number of open reading frame inactivation events such as insertions, deletions, and start and stop codon mutations independently occurred in Cetacea, Pholidota, Sirenia, Chiroptera, and Rodentia, totalizing 18 species. The diverse habitat settings and lifestyles of Ccl27-eroded lineages probably implied distinct evolutionary triggers rendering this gene unessential. For example, in Cetacea, the rapid renewal of skin layers minimizes the need for an elaborate inflammatory mechanism, mirrored by the absence of epidermal scabs. Our findings suggest that the convergent and independent loss of Ccl27 in mammalian evolution concurred with unique adaptive roads for skin physiology.

Evolutionary Plasticity in Detoxification Gene Modules: The Preservation and Loss of the Pregnane X Receptor in Chondrichthyes Lineages.

To appraise how evolutionary processes, such as gene duplication and loss, influence an organism's xenobiotic sensitivity is a critical question in toxicology. Of particular importance are gene families involved in the mediation of detoxification responses, such as members of the nuclear receptor subfamily 1 group I (NR1I), the pregnane X receptor (PXR), and the constitutive androstane receptor (CAR). While documented in multiple vertebrate genomes, PXR and CAR display an intriguing gene distribution. PXR is absent in birds and reptiles, while CAR shows a tetrapod-specific occurrence. More elusive is the presence of PXR and CAR gene orthologs in early branching and ecologically-important Chondrichthyes (chimaeras, sharks and rays). Therefore, we investigated various genome projects and use them to provide the first identification and functional characterization of a Chondrichthyan PXR from the chimaera elephant shark (Callorhinchus milii, Holocephali). Additionally, we substantiate the targeted PXR gene loss in Elasmobranchii (sharks and rays). Compared to other vertebrate groups, the chimaera PXR ortholog displays a diverse expression pattern (skin and gills) and a unique activation profile by classical xenobiotic ligands. Our findings provide insights into the molecular landscape of detoxification mechanisms and suggest lineage-specific adaptations in response to xenobiotics in gnathostome evolution.

Retention of fatty acyl desaturase 1 (fads1) in Elopomorpha and Cyclostomata provides novel insights into the evolution of long-chain polyunsaturated fatty acid biosynthesis in vertebrates.

BACKGROUND: Provision of long-chain polyunsaturated fatty acids (LC-PUFA) in vertebrates occurs through the diet or via endogenous production from C18 precursors through consecutive elongations and desaturations. It has been postulated that the abundance of LC-PUFA in the marine environment has remarkably modulated the gene complement and function of Fads in marine teleosts. In vertebrates two fatty acyl desaturases, namely Fads1 and Fads2, encode ∆5 and ∆6 desaturases, respectively. To fully clarify the evolutionary history of LC-PUFA biosynthesis in vertebrates, we investigated the gene repertoire and function of Fads from species placed at key evolutionary nodes. RESULTS: We demonstrate that functional Fads1Δ5 and Fads2∆6 arose from a tandem gene duplication in the ancestor of vertebrates, since they are present in the Arctic lamprey. Additionally, we show that a similar condition was retained in ray-finned fish such as the Senegal bichir and spotted gar, with the identification of fads1 genes in these lineages. Functional characterisation of the isolated desaturases reveals the first case of a Fads1 enzyme with ∆5 desaturase activity in the Teleostei lineage, the Elopomorpha. In contrast, in Osteoglossomorpha genomes, while no fads1 was identified, two separate fads2 duplicates with ∆6 and ∆5 desaturase activities respectively were uncovered. CONCLUSIONS: We conclude that, while the essential genetic components involved LC-PUFA biosynthesis evolved in the vertebrate ancestor, the full completion of the LC-PUFA biosynthesis pathway arose uniquely in gnathostomes.

Cetacea are natural knockouts for IL20.

The Cetacea infraorder comprises a very unique group within the mammalian lineage. While sharing common ancestors with terrestrial mammals, their exclusive dependence on aquatic environments makes them attractive models to explore the landscape of molecular shifts in radical habitat transitions. Among their diverse anatomical and physiological solutions, we find detectable genetic remodeling of the immune system. In agreement, here we show that the gene sequence of interleukin-20 (IL20) displays unambiguous signs of inactivation with several disruptive mutations, including stop codons, insertions, and a conserved trans-species mutation abolishing a canonical splice site, in nine analyzed cetacean genomes. Considering the suggested role of IL20 in skin immunity processes, including inflammation, epithelization, and remodeling, we propose that gene inactivation follows specific adaptations of cetacean skin to the aquatic environment, in frame with the less-is-more hypothesis.

Evolutionary Exploitation of Vertebrate Peroxisome Proliferator-Activated Receptor γ by Organotins.

Globally persistent man-made chemicals display ever-growing ecosystemic consequences, a hallmark of the Anthropocene epoch. In this context, the assessment of how lineage-specific gene repertoires influence organism sensitivity toward endocrine disruptors is a central question in toxicology. A striking example highlights the role of a group of compounds known as obesogens. In mammals, most examples involve the modulation of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). To address the structural and biological determinants of PPARγ exploitation by a model obesogen, tributyltin (TBT), in chordates, we employed comparative genomics, transactivation and ligand binding assays, homology modeling, and site-directed-mutagenesis. We show that the emergence of multiple PPARs (α, ß and γ) in vertebrate ancestry coincides with the acquisition of TBT agonist affinity, as can be deduced from the conserved transactivation and binding affinity of the chondrichthyan and mammalian PPARγ. The amphioxus single-copy PPAR is irresponsive to TBT; as well as the investigated teleosts, this is a probable consequence of a specific mutational remodeling of the ligand binding pocket. Our findings endorse the modulatory ability of man-made chemicals and suggest an evolutionarily diverse setting, with impacts for environmental risk assessment.

Unusual loss of chymosin in mammalian lineages parallels neo-natal immune transfer strategies.

Gene duplication and loss are powerful drivers of evolutionary change. The role of loss in phenotypic diversification is notably illustrated by the variable enzymatic repertoire involved in vertebrate protein digestion. Among these we find the pepsin family of aspartic proteinases, including chymosin (Cmy). Previous studies demonstrated that Cmy, a neo-natal digestive pepsin, is inactivated in some primates, including humans. This pseudogenization event was hypothesized to result from the acquisition of maternal immune immunoglobulin G (IgG) transfer. By investigating 94 mammalian subgenomes we reveal an unprecedented level of Cmy erosion in placental mammals, with numerous independent events of gene loss taking place in Primates, Dermoptera, Rodentia, Cetacea and Perissodactyla. Our findings strongly suggest that the recurrent inactivation of Cmy correlates with the evolution of the passive transfer of IgG and uncovers a noteworthy case of evolutionary cross-talk between the digestive and the immune system, modulated by gene loss.

Mechanism of proton/substrate coupling in the heptahelical lysosomal transporter cystinosin.

Secondary active transporters use electrochemical gradients provided by primary ion pumps to translocate metabolites or drugs "uphill" across membranes. Here we report the ion-coupling mechanism of cystinosin, an unusual eukaryotic, proton-driven transporter distantly related to the proton pump bacteriorhodopsin. In humans, cystinosin exports the proteolysis-derived dimeric amino acid cystine from lysosomes and is impaired in cystinosis. Using voltage-dependence analysis of steady-state and transient currents elicited by cystine and neutralization-scanning mutagenesis of conserved protonatable residues, we show that cystine binding is coupled to protonation of a clinically relevant aspartate buried in the membrane. Deuterium isotope substitution experiments are consistent with an access of this aspartate from the lysosomal lumen through a deep proton channel. This aspartate lies in one of the two PQ-loop motifs shared by cystinosin with a set of eukaryotic membrane proteins of unknown function and is conserved in about half of them, thus suggesting that other PQ-loop proteins may translocate protons.

An historical "wreck": A transcriptome assembly of the naval shipworm, Teredo navalis Linnaeus, 1978.

Historically famous for their negative impact on human-built marine wood structures, mollusc shipworms play a central ecological role in marine ecosystems. Their association with bacterial symbionts, providing cellulolytic and nitrogen-fixing activities, underscores their exceptional wood-eating and wood-boring behaviours, improving energy transfer and the recycling of essential nutrients locked in the wood cellulose. Importantly, from a molecular standpoint, a minute of omic resources are available from this lineage of Bivalvia. Here, we produced and assembled a transcriptome from the globally distributed naval shipworm, Teredo navalis (family Teredinidae). The transcriptome was obtained by sequencing the total RNA from five equidistant segments of the whole body of a T. navalis specimen. The quality of the produced assembly was accessed with several statistics, revealing a highly contiguous (1194 N50) and complete (over 90% BUSCO scores for Eukaryote and Metazoan databases) transcriptome, with nearly 38,000 predicted ORF, more than half being functionally annotated. Our findings pave the way to investigate the unique evolutionary biology of these highly modified bivalves and lay the foundation for an adequate gene annotation of a full genome sequence of the species.

Preliminary hazard assessment of a new nature-inspired antifouling (NIAF) agent.

A recently synthesized aminated 3,4-dioxygenated xanthone (Xantifoul2) was found to have promising antifouling (AF) effects against the settlement of the macrofouler Mytilus galloprovincialis larvae. Preliminary assessment indicated that Xantifoul2 has reduced ecotoxicological impacts: e.g., being non-toxic to the marine crustacea Artemia salina (<10 % mortality at 50 µM) and showing low bioconcentration factor in marine organisms. In order to meet the EU Biocidal Product Regulation, a preliminary hazard assessment of this new nature-inspired antifouling (NIAF) agent was conducted in this work. Xantifoul2 did not affect the swimming ability of the planktonic crustacean Daphnia magna, the growth of the diatom Phaeodactylum tricornutum, and the cellular respiration of luminescent Gram-negative bacteria Vibrio fischeri, supporting the low toxicity towards several non-target marine species. Regarding human cytotoxicity, Xantifoul2 did not affect the cell viability of retinal human cells (hTERT-RPE-1) and lipidomic studies revealed depletion of lipids involved in cell death, membrane modeling, lipid storage, and oxidative stress only at a high concentration (10 µM). Accelerated degradation studies in water were conducted under simulated sunlight to allow the understanding of putative transformation products (TPs) that could be generated in the aquatic ecosystems. Both Xantifoul2 and photolytic-treated Xantifoul2 in the aqueous matrix were therefore evaluated on several nuclear receptors (NRs). The results of this preliminary hazard assessment of Xantifoul2, combined with the high degradation rates in water, provide strong evidence of the safety of this AF agent under the evaluated conditions, and provide the support for future validation studies before this compound can be introduced in the market.

A whole-body transcriptome assembly of the annelid worm Hediste diversicolor.

The Annelida phylum is composed of a myriad of species exhibiting key phenotypic adaptations. They occupy key ecological niches in a variety of marine, freshwater and terrestrial ecosystems. Importantly, the increment of omic resources is rapidly modifying the taxonomic landscape and knowledge of species belonging to this phylum. Here, we comprehensively characterised and annotated a transcriptome of the common ragworm, Hediste diversicolor (OF Müller). This species belongs to the family Nereididae and inhabits estuarine and lagoon areas on the Atlantic coasts of Europe and North America. Ecologically, H. diversicolor plays an important role in benthic food webs. Given its commercial value, H. diversicolor is a promising candidate for aquaculture development and production in farming facilities, under a circular economy framework. We used Illumina next-generation sequencing technology, to produce a total of 105 million (M) paired-end (PE) raw reads and generate the first whole-body transcriptome assembly of H. diversicolor species. This high-quality transcriptome contains 69,335 transcripts with an N50 transcript length of 2313 bp and achieved a BUSCO gene completeness of 97.7% and 96% in Eukaryota and Metazoa lineage-specific profile libraries. Our findings offer a valuable resource for multiple biological applications using this species.

Successful prediction of substrate-binding pocket in SLC17 transporter sialin.

Secondary active transporters from the SLC17 protein family are required for excitatory and purinergic synaptic transmission, sialic acid metabolism, and renal function, and several members are associated with inherited neurological or metabolic diseases. However, molecular tools to investigate their function or correct their genetic defects are limited or absent. Using structure-activity, homology modeling, molecular docking, and mutagenesis studies, we have located the substrate-binding site of sialin (SLC17A5), a lysosomal sialic acid exporter also recently implicated in exocytotic release of aspartate. Human sialin is defective in two inherited sialic acid storage diseases and is responsible for metabolic incorporation of the dietary nonhuman sialic acid N-glycolylneuraminic acid. We built cytosol-open and lumen-open three-dimensional models of sialin based on weak, but significant, sequence similarity with the glycerol-3-phosphate and fucose permeases from Escherichia coli, respectively. Molecular docking of 31 synthetic sialic acid analogues to both models was consistent with inhibition studies. Narrowing the sialic acid-binding site in the cytosol-open state by two phenylalanine to tyrosine mutations abrogated recognition of the most active analogue without impairing neuraminic acid transport. Moreover, a pilot virtual high-throughput screening of the cytosol-open model could identify a pseudopeptide competitive inhibitor showing >100-fold higher affinity than the natural substrate. This validated model of human sialin and sialin-guided models of other SLC17 transporters should pave the way for the identification of inhibitors, glycoengineering tools, pharmacological chaperones, and fluorescent false neurotransmitters targeted to these proteins.

Using zebrafish embryo bioassays to identify chemicals modulating the regulation of the epigenome: a case study with simvastatin.

Contaminants of emerging concern have been increasingly associated with the modulation of the epigenome, leading to potentially inherited and persistent impacts on apical endpoints. Here, we address the performance of the OECD Test No. 236 FET (fish embryo acute toxicity) in the identification of chemicals able to modulate the epigenome. Using zebrafish (Danio rerio) embryos, acute and chronic exposures were performed with the pharmaceutical, simvastatin (SIM), a widely prescribed hypocholesterolemic drug reported to induce inter and transgenerational effects. In the present study, the epigenetic effects of environmentally relevant concentrations of SIM (from 8 ng/L to 2000 ng/L) were addressed following (1) an acute embryo assay based on OECD Test No. 236 FET, (2) a chronic partial life-cycle exposure using adult zebrafish (90 days), and (3) F1 embryos obtained from parental exposed animals. Simvastatin induced significant effects in gene expression of key epigenetic biomarkers (DNA methylation and histone acetylation/deacetylation) in the gonads of exposed adult zebrafish and in 80 hpf zebrafish embryos (acute and chronic parental intergenerational exposure), albeit with distinct effect profiles between biological samples. In the chronic exposure, SIM impacted particularly DNA methyltransferase genes in males and female gonads, whereas in F1 embryos SIM affected mostly genes associated with histone acetylation/deacetylation. In the embryo acute direct exposure, SIM modulated the expression of both genes involved in DNA methylation and histone deacetylase. These findings further support the use of epigenetic biomarkers in zebrafish embryos in a high throughput approach to identify and prioritize epigenome-modulating chemicals.