A meta-analysis and a functional study support the influence of mtDNA variant m.16519C on the risk of rapid progression of knee osteoarthritis.

OBJECTIVES: To identify mitochondrial DNA (mtDNA) genetic variants associated with the risk of rapid progression of knee osteoarthritis (OA) and to characterise their functional significance using a cellular model of transmitochondrial cybrids. METHODS: Three prospective cohorts contributed participants. The osteoarthritis initiative (OAI) included 1095 subjects, the Cohort Hip and Cohort Knee included 373 and 326 came from the PROspective Cohort of Osteoarthritis from A Coruña. mtDNA variants were screened in an initial subset of 450 subjects from the OAI by in-depth sequencing of mtDNA. A meta-analysis of the three cohorts was performed. A model of cybrids was constructed to study the functional consequences of harbouring the risk mtDNA variant by assessing: mtDNA copy number, mitochondrial biosynthesis, mitochondrial fission and fusion, mitochondrial reactive oxygen species (ROS), oxidative stress, autophagy and a whole transcriptome analysis by RNA-sequencing. RESULTS: mtDNA variant m.16519C is over-represented in rapid progressors (combined OR 1.546; 95% CI 1.163 to 2.054; p=0.0027). Cybrids with this variant show increased mtDNA copy number and decreased mitochondrial biosynthesis; they produce higher amounts of mitochondrial ROS, are less resistant to oxidative stress, show a lower expression of the mitochondrial fission-related gene fission mitochondrial 1 and an impairment of autophagic flux. In addition, its presence modulates the transcriptome of cybrids, especially in terms of inflammation, where interleukin 6 emerges as one of the most differentially expressed genes. CONCLUSIONS: The presence of the mtDNA variant m.16519C increases the risk of rapid progression of knee OA. Among the most modulated biological processes associated with this variant, inflammation and negative regulation of cellular process stand out. The design of therapies based on the maintenance of mitochondrial function is recommended.

Bivalve omics: state of the art and potential applications for the biomonitoring of harmful marine compounds.

The extraordinary progress experienced by sequencing technologies and bioinformatics has made the development of omic studies virtually ubiquitous in all fields of life sciences nowadays. However, scientific attention has been quite unevenly distributed throughout the different branches of the tree of life, leaving molluscs, one of the most diverse animal groups, relatively unexplored and without representation within the narrow collection of well established model organisms. Within this Phylum, bivalve molluscs play a fundamental role in the functioning of the marine ecosystem, constitute very valuable commercial resources in aquaculture, and have been widely used as sentinel organisms in the biomonitoring of marine pollution. Yet, it has only been very recently that this complex group of organisms became a preferential subject for omic studies, posing new challenges for their integrative characterization. The present contribution aims to give a detailed insight into the state of the art of the omic studies and functional information analysis of bivalve molluscs, providing a timely perspective on the available data resources and on the current and prospective applications for the biomonitoring of harmful marine compounds.

The CHROMEVALOA database: a resource for the evaluation of Okadaic Acid contamination in the marine environment based on the chromatin-associated transcriptome of the mussel Mytilus galloprovincialis.

Okadaic Acid (OA) constitutes the main active principle in Diarrhetic Shellfish Poisoning (DSP) toxins produced during Harmful Algal Blooms (HABs), representing a serious threat for human consumers of edible shellfish. Furthermore, OA conveys critical deleterious effects for marine organisms due to its genotoxic potential. Many efforts have been dedicated to OA biomonitoring during the last three decades. However, it is only now with the current availability of detailed molecular information on DNA organization and the mechanisms involved in the maintenance of genome integrity, that a new arena starts opening up for the study of OA contamination. In the present work we address the links between OA genotoxicity and chromatin by combining Next Generation Sequencing (NGS) technologies and bioinformatics. To this end, we introduce CHROMEVALOAdb, a public database containing the chromatin-associated transcriptome of the mussel Mytilus galloprovincialis (a sentinel model organism) in response to OA exposure. This resource constitutes a leap forward for the development of chromatin-based biomarkers, paving the road towards the generation of powerful and sensitive tests for the detection and evaluation of the genotoxic effects of OA in coastal areas.

Genome-wide DNA methylation of the liver reveals delayed effects of early-life exposure to 17-α-ethinylestradiol in the self-fertilizing mangrove rivulus.

Organisms exposed to endocrine disruptors in early life can show altered phenotype later in adulthood. Although the mechanisms underlying these long-term effects remain poorly understood, an increasing body of evidence points towards the potential role of epigenetic processes. In the present study, we exposed hatchlings of an isogenic lineage of the self-fertilizing fish mangrove rivulus for 28 days to 4 and 120 ng/L of 17-α-ethinylestradiol. After a recovery period of 140 days, reduced representation bisulphite sequencing (RRBS) was performed on the liver in order to assess the hepatic genome-wide methylation landscape. Across all treatment comparisons, a total of 146 differentially methylated fragments (DMFs) were reported, mostly for the group exposed to 4 ng/L, suggesting a non-monotonic effect of EE2 exposure. Gene ontology analysis revealed networks involved in lipid metabolism, cellular processes, connective tissue function, molecular transport and inflammation. The highest effect was reported for nipped-B-like protein B (NIPBL) promoter region after exposure to 4 ng/L EE2 (+ 21.9%), suggesting that NIPBL could be an important regulator for long-term effects of EE2. Our results also suggest a significant role of DNA methylation in intergenic regions and potentially in transposable elements. These results support the ability of early exposure to endocrine disruptors of inducing epigenetic alterations during adulthood, providing plausible mechanistic explanations for long-term phenotypic alteration. Additionally, this work demonstrates the usefulness of isogenic lineages of the self-fertilizing mangrove rivulus to better understand the biological significance of long-term alterations of DNA methylation by diminishing the confounding factor of genetic variability.

Behavior and gene expression in the brain of adult self-fertilizing mangrove rivulus fish (Kryptolebias marmoratus) after early life exposure to the neurotoxin ß-N-methylamino-l-alanine (BMAA).

ß-N-Methylamino-l-alanine (BMAA), a neurotoxin naturally produced by cyanobacteria, diatoms and dinoflagellates, constitutes a serious environmental and health threat especially during acute blooms, which are becoming more frequent. This neurotoxin is implicated in several neurodegenerative diseases (ND) in humans through contaminated water or food consumption. Even low doses of neurotoxic compounds (NCs) can have lasting effects later in life. In this sense, early stages of development constitute a period of high sensitivity to environmental influence, particularly for the central nervous system. To understand the mechanisms underlying the delayed effects of NCs, newly hatched larvae of the mangrove rivulus fish, Kryptolebias marmoratus, were exposed to two sub-lethal doses of BMAA (20 µg/L and 15 mg/L) for 14 days. This fish naturally produces isogenic lineages due to its self-fertilizing reproduction, which is unique case among vertebrates. It thus provides genetic characteristics that allow scientists to study organisms' true reaction norm, minimizing genetic variability and focusing exclusively on the effects of the environment. Effect assessment was performed at different levels of biological organization to detect inconspicuous effects of BMAA, since this molecule displays long retention in organisms. BMAA effects on life history traits as well as behavioral traits such as boldness and aggressiveness were assessed more than 100 days after exposure. In addition, the relative expression of 7 potential BMAA target genes was studied, given their involvement in neurotransmission or their association with individual variation in boldness and aggressiveness. Selected genes code for reticulon 4 (RTN4), glutamate vesicular transporter 1 (Slc17a7), glutamine synthetase a (Glula), dopamine receptor D4 (DRD4), monoamine oxidase A (MAOA), calmodulin (CaM) and epedymine (Epd). Despite observing no effects of BMAA on growth, reproduction and behavioral traits, BMAA induced a significant increase of the expression of CaM and MAOA genes at 20 µg/L BMAA compared to the control group. A significant decrease of expression was observed between this lowest BMAA dose and 15 mg/L for DRD4, MAOA and CaM genes. Our results suggest disruption of glutamate turnover, intracellular dopamine depletion and activation of astrocyte protective mechanisms, indicating that BMAA might be excitotoxic. Our study revealed that BMAA can have long-lasting effects on the brain that are suspected to affect phenotypic traits with aging. Furthermore, it highlights the importance of studying delayed effects in ecotoxicological studies.

Behavioral effects of the neurotoxin -N-methylamino- L-alanine on the mangrove rivulus (Kryptolebias marmoratus) larvae.

Mangrove rivulus, Kryptolebias marmoratus, is a hermaphrodite fish capable of self-fertilization. This particularity allows to naturally produce highly homozygous and isogenic individuals. Despite the low genetic diversity, rivulus can live in extremely variable environments and adjust its phenotype accordingly. This species represents a unique opportunity to clearly distinguish the genetic and non-genetic factors implicated in adaptation and evolution, such as epigenetic mechanisms. It is thus a great model in aquatic ecotoxicology to investigate the effects of xenobiotics on the epigenome, and their potential long-term impacts. In the present study, we used the mangrove rivulus to investigate the effects of the neurotoxin b-N-methylamino-L-alanine (BMAA) on larvae behaviors after 7 days exposure to two sub-lethal concentrations. Results show that BMAA can affect the maximal speed and prey capture (trials and failures), suggesting potential impacts on the organism's fitness.

Effects of Florida Red Tides on histone variant expression and DNA methylation in the Eastern oyster Crassostrea virginica.

Massive algal proliferations known as Harmful Algal Blooms (HABs) represent one of the most important threats to coastal areas. Among them, the so-called Florida Red Tides (FRTs, caused by blooms of the dinoflagellate Karenia brevis and associated brevetoxins) are particularly detrimental in the southeastern U.S., causing high mortality rates and annual losses in excess of $40 million. The ability of marine organisms to cope with environmental stressors (including those produced during HABs) is influenced by genetic and epigenetic mechanisms, the latter resulting in phenotypic changes caused by heritable modifications in gene expression, without involving changes in the genetic (DNA) sequence. Yet, studies examining cause-effect relationships between environmental stressors, specific epigenetic mechanisms and subsequent responses are still lacking. The present work contributes to increase this knowledge by investigating the effects of Florida Red Tides on two types of mechanisms participating in the epigenetic memory of Eastern oysters: histone variants and DNA methylation. For that purpose, a HAB simulation was conducted in laboratory conditions, exposing oysters to increasing concentrations of K. brevis. The obtained results revealed, for the first time, the existence of H2A.X, H2A.Z and macroH2A genes in this organism, encoding histone variants potentially involved in the maintenance of genome integrity during responses to the genotoxic effect of brevetoxins. Additionally, an increase in H2A.X phosphorylation (γH2A.X, a marker of DNA damage) and a decrease in global DNA methylation were observed as the HAB simulation progressed. Overall, the present work provides a basis to better understand how epigenetic mechanisms participate in responses to environmental stress in marine invertebrates, opening new avenues to incorporate environmental epigenetics approaches into management and conservation programs.

Metagenomics, Metatranscriptomics, and Metabolomics Approaches for Microbiome Analysis.

Microbiomes are ubiquitous and are found in the ocean, the soil, and in/on other living organisms. Changes in the microbiome can impact the health of the environmental niche in which they reside. In order to learn more about these communities, different approaches based on data from multiple omics have been pursued. Metagenomics produces a taxonomical profile of the sample, metatranscriptomics helps us to obtain a functional profile, and metabolomics completes the picture by determining which byproducts are being released into the environment. Although each approach provides valuable information separately, we show that, when combined, they paint a more comprehensive picture. We conclude with a review of network-based approaches as applied to integrative studies, which we believe holds the key to in-depth understanding of microbiomes.

Environmental epigenetics: A promising venue for developing next-generation pollution biomonitoring tools in marine invertebrates.

Environmental epigenetics investigates the cause-effect relationships between specific environmental factors and the subsequent epigenetic modifications triggering adaptive responses in the cell. Given the dynamic and potentially reversible nature of the different types of epigenetic marks, environmental epigenetics constitutes a promising venue for developing fast and sensible biomonitoring programs. Indeed, several epigenetic biomarkers have been successfully developed and applied in traditional model organisms (e.g., human and mouse). Nevertheless, the lack of epigenetic knowledge in other ecologically and environmentally relevant organisms has hampered the application of these tools in a broader range of ecosystems, most notably in the marine environment. Fortunately, that scenario is now changing thanks to the growing availability of complete reference genome sequences along with the development of high-throughput DNA sequencing and bioinformatic methods. Altogether, these resources make the epigenetic study of marine organisms (and more specifically marine invertebrates) a reality. By building on this knowledge, the present work provides a timely perspective highlighting the extraordinary potential of environmental epigenetic analyses as a promising source of rapid and sensible tools for pollution biomonitoring, using marine invertebrates as sentinel organisms. This strategy represents an innovative, groundbreaking approach, improving the conservation and management of natural resources in the oceans.

Unbiased high-throughput characterization of mussel transcriptomic responses to sublethal concentrations of the biotoxin okadaic acid.

Background. Harmful Algal Blooms (HABs) responsible for Diarrhetic Shellfish Poisoning (DSP) represent a major threat for human consumers of shellfish. The biotoxin Okadaic Acid (OA), a well-known phosphatase inhibitor and tumor promoter, is the primary cause of acute DSP intoxications. Although several studies have described the molecular effects of high OA concentrations on sentinel organisms (e.g., bivalve molluscs), the effect of prolonged exposures to low (sublethal) OA concentrations is still unknown. In order to fill this gap, this work combines Next-Generation sequencing and custom-made microarray technologies to develop an unbiased characterization of the transcriptomic response of mussels during early stages of a DSP bloom. Methods. Mussel specimens were exposed to a HAB episode simulating an early stage DSP bloom (200 cells/L of the dinoflagellate Prorocentrum lima for 24 h). The unbiased characterization of the transcriptomic responses triggered by OA was carried out using two complementary methods of cDNA library preparation: normalized and Suppression Subtractive Hybridization (SSH). Libraries were sequenced and read datasets were mapped to Gene Ontology and KEGG databases. A custom-made oligonucleotide microarray was developed based on these data, completing the expression analysis of digestive gland and gill tissues. Results. Our findings show that exposure to sublethal concentrations of OA is enough to induce gene expression modifications in the mussel Mytilus. Transcriptomic analyses revealed an increase in proteasomal activity, molecular transport, cell cycle regulation, energy production and immune activity in mussels. Oppositely, a number of transcripts hypothesized to be responsive to OA (notably the Serine/Threonine phosphatases PP1 and PP2A) failed to show substantial modifications. Both digestive gland and gill tissues responded similarly to OA, although expression modifications were more dramatic in the former, supporting the choice of this tissue for future biomonitoring studies. Discussion. Exposure to OA concentrations within legal limits for safe consumption of shellfish is enough to disrupt important cellular processes in mussels, eliciting sharp transcriptional changes as a result. By combining the study of cDNA libraries and a custom-made OA-specific microarray, our work provides a comprehensive characterization of the OA-specific transcriptome, improving the accuracy of the analysis of expresion profiles compared to single-replicated RNA-seq methods. The combination of our data with related studies helps understanding the molecular mechanisms underlying molecular responses to DSP episodes in marine organisms, providing useful information to develop a new generation of tools for the monitoring of OA pollution.