Juan Fernández-Tajes

Genome-wide DNA methylation analysis of articular chondrocytes reveals a cluster of osteoarthritic patients

Objective Alterations in DNA methylation patterns have been found to correlate with several diseases including osteoarthritis (OA). The aim of this study was to identify, for the first time, the genome-wide DNA methylation profiles of human articular chondrocytes from OA cartilage and healthy control cartilage samples. Methods DNA methylation profiling was performed using Illumina Infinium HumanMethylation27 in 25 patients with OA and 20 healthy controls. Subsequent validation was performed by genome-wide expression analysis using the Affymetrix Human Gene 1.1 ST array in an independent cohort of 24 patients with OA. Finally, the most consistent genes in both assays were amplified by quantitative reverse transcriptase PCR in a validation cohort of 48 patients using microfluidic real-time quantitative PCR. Appropriate bioinformatics analyses were carried out using R bioconductor software packages and qBase plus software from Biogazelle. Results We found 91 differentially methylated (DM) probes, which permitted us to separate patients with OA from healthy controls. Among the patients with OA, we detected 1357 DM probes that identified a tight cluster of seven patients who were different from the rest. This cluster was also identified by genome-wide expression in which 450 genes were differentially expressed. Further validation of the most consistent genes in an independent cohort of patients with OA permitted us to identify this cluster, which was characterised by increased inflammatory processes. Conclusions We were able to identify a tight subgroup of patients with OA, characterised by an increased inflammatory response that could be regulated by epigenetics. The identification and isolation of this subgroup may be critical for the development of effective treatment and disease prevention.

Use of three bivalve species for biomonitoring a polluted estuarine environment

Estuaries are marine areas at great contamination risk due to their hydrodynamic features. PAH are wide and ubiquitous contaminants with a high presence in these marine environments. Chemical analysis of sediments can provide information, although it does not give a direct measure of the toxicological effect of such contaminants in the biota. Samples of Venerupis pullastra, Cerastoderma edule, and Mytilus galloprovincialis were collected from two locations in Corcubión estuary (Norhwest of Spain). The level of PAH in sediment and biota, and its possible origin were assessed. A moderate level of contamination was observed with a predominance of PAH of a pyrogenic origin. Genotoxic damage, measured as single-strand DNA breaks with the comet assay, was evaluated in gill tissue and in hemolymph. The values of DNA damage obtained showed a higher sensitivity of clams and cockles to the pollution load level. These differences among species make us suggest the use of some other species coupled with mussels as an optimal tool for biomonitoring estuarine environments.

Quantitative proteomic profiling of human articular cartilage degradation in osteoarthritis.

Osteoarthritis (OA) is the most common rheumatic pathology and is characterized primarily by articular cartilage degradation. Despite its high prevalence, there is no effective therapy to slow disease progression or regenerate the damaged tissue. Therefore, new diagnostic and monitoring tests for OA are urgently needed, which would also promote the development of alternative therapeutic strategies. In the present study, we have performed an iTRAQ-based quantitative proteomic analysis of secretomes from healthy human articular cartilage explants, comparing their protein profile to those from unwounded (early disease) and wounded (advanced disease) zones of osteoarthritic tissue. This strategy allowed us to identify a panel of 76 proteins that are distinctively released by the diseased tissue. Clustering analysis allowed the classification of proteins according to their different profile of release from cartilage. Among these proteins, the altered release of osteoprotegerin (decreased in OA) and periostin (increased in OA), both involved in bone remodelling processes, was verified in further analyses. Moreover, periostin was also increased in the synovial fluid of OA patients. Altogether, the present work provides a novel insight into the mechanisms of human cartilage degradation and a number of new cartilage-characteristic proteins with possible biomarker value for early diagnosis and prognosis of OA.

Effect of okadaic acid on carpet shell clam (Ruditapes decussatus) haemocytes by in vitro exposure and harmful algal bloom simulation assays

Okadaic acid (OA), produced by dinoflagellates during harmful algal blooms (HAB), belongs to the Diarrheic Shellfish Poisoning toxins that cause gastrointestinal symptoms in humans after consumption. In the present work, Ruditapes decussatus haemocytes were selected to evaluate the effect of OA on cell viability, enzymatic status and immune capacity through the measure by flow cytometry of apoptosis–cell death, non-specific esterase activity and phagocytosis. In order to compare different exposure conditions, two experiments were developed: in vitro exposure to OA and HAB simulation by feeding clams with the OA producer, Prorocentrum lima. Apoptosis was not OA dose-dependent and cell death increased in both assays. Phagocytosis of latex beads and esterase activity decreased in haemocytes incubated with OA. In contrast, esterases increased during the feeding with P. lima. Our results showed that OA and the simulated HAB caused damages on haemocyte functions and viability.

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.

Identification of differentially expressed genes in SHSY5Y cells exposed to okadaic acid by suppression subtractive hybridization

BackgroundOkadaic acid (OA), a toxin produced by several dinoflagellate species is responsible for frequent food poisonings associated to shellfish consumption. Although several studies have documented the OA effects on different processes such as cell transformation, apoptosis, DNA repair or embryogenesis, the molecular mechanistic basis for these and other effects is not completely understood and the number of controversial data on OA is increasing in the literature.ResultsIn this study, we used suppression subtractive hybridization in SHSY5Y cells to identify genes that are differentially expressed after OA exposure for different times (3, 24 and 48 h). A total of 247 subtracted clones which shared high homology with known genes were isolated. Among these, 5 specific genes associated with cytoskeleton and neurotransmission processes (NEFM, TUBB, SEPT7, SYT4 and NPY) were selected to confirm their expression levels by real-time PCR. Significant down-regulation of these genes was obtained at the short term (3 and 24 h OA exposure), excepting for NEFM, but their expression was similar to the controls at 48 h.ConclusionsFrom all the obtained genes, 114 genes were up-regulated and 133 were down-regulated. Based on the NCBI GenBank and Gene Ontology databases, most of these genes are involved in relevant cell functions such as metabolism, transport, translation, signal transduction and cell cycle. After quantitative PCR analysis, the observed underexpression of the selected genes could underlie the previously reported OA-induced cytoskeleton disruption, neurotransmission alterations and in vivo neurotoxic effects. The basal expression levels obtained at 48 h suggested that surviving cells were able to recover from OA-caused gene expression alterations.

Chromatin specialization in bivalve molluscs: A leap forward for the evaluation of Okadaic Acid genotoxicity in the marine environment

Marine biotoxins synthesized by Harmful Algal Blooms (HABs) represent one of the most important sources of contamination in marine environments as well as a serious threat to fisheries and aquaculture-based industries in coastal areas. Among these biotoxins Okadaic Acid (OA) is of critical interest as it represents the most predominant Diarrhetic Shellfish Poisoning biotoxin in the European coasts. Furthermore, OA is a potent tumor promoter with aneugenic and clastogenic effects on the hereditary material, most notably DNA breaks and alterations in DNA repair mechanisms. Therefore, a great effort has been devoted to the biomonitoring of OA in the marine environment during the last two decades, mainly based on physicochemical and physiological parameters using mussels as sentinel organisms. However, the molecular genotoxic effects of this biotoxin make chromatin structure a good candidate for an alternative strategy for toxicity assessment with faster and more sensitive evaluation. To date, the development of chromatin-based studies to this purpose has been hampered by the complete lack of information on chromatin of invertebrate marine organisms, especially in bivalve molluscs. Our preliminary results have revealed the presence of histone variants involved in DNA repair and chromatin specialization in mussels and clams. In this work we use this information to put forward a proposal focused on the development of chromatin-based tests for OA genotoxicity in the marine environment. The implementation of such tests in natural populations has the potential to provide an important leap in the biomonitoring of this biotoxin. The outcome of such monitoring may have critical implications for the evaluation of DNA damage in these marine organisms. They will provide as well important tools for the optimization of their harvesting and for the elaboration of additional tests designed to evaluate the safety of their consumption and potential implications for consumers health.

Evaluation of Genotoxicity in Gills and Hemolymph of Clam Ruditapes decussatus Fed with the Toxic Dinoflagellate Prorocentrum lima

Diarrheic shellfish poisoning (DSP) is a gastrointestinal (GIT) disease that appears a few hours after ingesting okadaic acid (OA)-contaminated mollusks; okadaic acid is present in dinoflagellates of the genera Dinophysis and Prorocentrum. Toxic manifestations occur all year round at a higher or lesser intensity, and as a consequence, extractive production factories need to be closed during these periods which affects the economy of aquaculture industries. Although the concentration of harmful algae is usually found at high levels in clam digestive gland, bivalve mortality was not increased. In this study, the genotoxic effects produced by OA in clam Ruditapes decussatus were determined using the comet assay. In vitro (exposing hemocytes to different concentrations of OA) and in vivo (feeding clams with toxic dinoflagellate Prorocentrum lima) experiments were conducted in order to determine the genotoxic effects of OA on bivalve cells. Hemocytes and gill cells were analyzed by in vivo and in vitro approaches. While the in vitro study showed a rapid effect of OA on hemocytes, data obtained in the in vivo experiment reflected contradictory results dependent upon the concentration of OA and cell type evaluated. An increase in DNA damage was observed at the lower concentration and only in gill tissue. The results obtained may contribute to a better understanding of the mechanisms underlying genotoxic effects induced by OA on bivalves.

Comparison Between Two Bivalve Species as Tools for the Assessment of Pollution Levels in an Estuarian Environment

Estuaries are semi-enclosed marine areas with water short residence times. Estuary ecosystems show a higher susceptibility to contamination, as historically these sites are linked to urban and industrial development. Polycyclic aromatic hydrocarbons (PAH) are ubiquitous contaminants present in high quantities in these marine environments. Chemical analyses of sediments provides information regarding PAH pollution levels but not a direct measure of the toxicological effects attributed to these contaminants. Samples of sediments and of two bivalve species, Cerastoderma edule and Mytilus galloprovincialis, were collected from two locations (Corcubión and A Concha) in an estuary from northwestern Spain. The PAH levels in sediment and bivalve species and possible sources were determined. A moderate level and a low level of pyrogenic PAH contamination were observed in Corcubión and in A Concha, respectively. Genotoxic damage was evaluated in gills and hemocytes from mussels and cockles by means of the comet assay. DNA damage measured as DNAt values showed a reliable relationship with pollution load levels of the two sampling sites. The higher sensitivity of C. edule compared to M. galloprovincialis enables one to recommend including another species coupled with mussels for biomonitoring estuarine environments.

Identification of razor clams Ensis arcuatus and Ensis siliqua by PCR–RFLP analysis of ITS1 region

Ensis arcuatus and Ensis siliqua are the most economically important species of razor clams in the European Union. Due to similarities between their shell morphology, and the differing retail value, these species are often misidentified. Therefore, it is necessary to develop an appropriate protocol to allow accurate differentiation between these species of razor clam in order to protect consumer rights, avoid commercial fraud (whether intentional or unintentional), and to enforce labeling and safety regulations. With the aim of developing a rapid and reliable method of differentiation, individuals of E. arcuatus and of E. siliqua were examined by polymerase chain reaction restriction fragment polymorphism (PCR-RFLP) using the internal transcribed spacer region 1 (ITS1). A species-specific restriction endonuclease pattern was found with the enzyme Kspl for both species, allowing their exact identification. Thus, this work provides a simple, reliable and rapid protocol for accurate discrimination between E. arcuatus and E. siliqua, which proves useful for traceability and enabling the enforcement of labeling regulations.