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Canine oral melanoma (COM) is the most frequent tumour with oral localization in dogs. Copy number gains and amplifications of CCND1, a gene coding for Cyclin D1, are the most frequent chromosomal aberrations described in human non-UV induced melanomas. Twenty-eight cases of COM were retrieved from paraffin-blocks archives. A total of 4 μm thick sections were immunostained with an antibody against human Cyclin D1 and Ki-67. Cyclin D1 and Ki-67 expressions were scored through two counting methods. DNA was extracted from 20 μm thick sections of formalin-fixed paraffin-embedded blocks. Pathological and surrounding healthy tissue was extracted independently. Cyclin D1 immunolabelling was detected in 69% (18/26) while Ki-67 was present in 88.5% (23/26) of cases. Statistical analysis revealed correlation between two counting methods for Cyclin D1 (r = 0.54; P = .004) and Ki-67 (r = 0.56; P = .003). The correlation found between Ki-67 and Cyclin D1 indexes in 16/26 cases labelled by both antibodies (r = 0.7947; P = .0002) suggests a possible use of Cyclin D1 index as prognostic marker. Polymerase chain reaction analysis on CCND1 coding sequence revealed the presence of nine somatic mutations in seven samples producing synonymous, missense and stop codons. Since none of the single-nucleotide polymorphisms was found to be recurrent, it is suggested that overexpression of Cyclin D1 may be the consequence of alterations of CCND1 upstream regions or other genetic aberrations not detectable with the methodology used in this study. Future studies are needed to verify the potential use of Cyclin D1 index as prognostic indicator and to highlight the molecular events responsible for Cyclin D1 overexpression in COMs.

Human Mucosal Melanoma (hMM) is an aggressive neoplasm of neuroectodermal origin with distinctive features from the more common cutaneous form of malignant melanoma (cMM). At the molecular level, hMMs are characterized by large chromosomal aberrations rather than single-nucleotide mutations, typically observed in cMM. Given the scarcity of available cases, there have been many attempts to establish a reliable animal model. In pet dogs, Canine Oral Melanoma (COM) is the most common malignant tumor of the oral cavity, sharing clinical and histological aspects with hMM. To improve the knowledge about COM's genomic DNA alterations, in the present work, formalin-fixed, paraffin-embedded (FFPE) samples of COM from different European archives were collected to set up an array Comparative Genomic Hybridization (aCGH) analysis to estimate recurrent Copy Number Aberrations (CNAs). DNA was extracted in parallel from tumor and healthy fractions and 19 specimens were successfully submitted to labeling and competitive hybridization. Data were statistically analyzed through GISTIC2.0 and a pathway-enrichment analysis was performed with ClueGO. Recurrent gained regions were detected, affecting chromosomes CFA 10, 13 and 30, while lost regions involved chromosomes CFA 10, 11, 22, and 30. In particular, CFA 13 showed a whole-chromosome gain in 37% of the samples, while CFA 22 showed a whole-chromosome loss in 25%. A distinctive sigmoidal trend was observed in CFA 10 and 30 in 25 and 30% of the samples, respectively. Comparative analysis revealed that COM and hMM share common chromosomal changes in 32 regions. MAPK- and PI3K-related genes were the most frequently involved, while pathway analysis revealed statistically significant perturbation of cancer-related biological processes such as immune response, drug metabolism, melanocytes homeostasis, and neo-angiogenesis. The latter is a new evidence of a significant involvement of neovascularization-related pathways in COMs and can provide the rationale for future application in anti-cancer targeted therapies.

Background: Diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma (MZL) and follicular lymphoma (FL) are the most common B-cell lymphomas (BCL) in dogs. Recent investigations have demonstrated overlaps of these histotypes with the human counterparts, including clinical presentation, biologic behavior, tumor genetics, and treatment response. The molecular mechanisms that underlie canine BCL are still unknown and new studies to improve diagnosis, therapy, and the utilization of canine species as spontaneous animal tumor models are undeniably needed. Recent work using human DLBCL transcriptomes has suggested that long non-coding RNAs (lncRNAs) play a key role in lymphoma pathogenesis and pinpointed a restricted number of lncRNAs as potential targets for further studies. Results: To expand the knowledge of non-coding molecules involved in canine BCL, we used transcriptomes obtained from a cohort of 62 dogs with newly-diagnosed multicentric DLBCL, MZL and FL that had undergone complete staging work-up and were treated with chemotherapy or chemo-immunotherapy. We developed a customized R pipeline performing a transcriptome assembly by multiple algorithms to uncover novel lncRNAs, and delineate genome-wide expression of unannotated and annotated lncRNAs. Our pipeline also included a new package for high performance system biology analysis, which detects high-scoring network biological neighborhoods to identify functional modules. Moreover, our customized pipeline quantified the expression of novel and annotated lncRNAs, allowing us to subtype DLBCLs into two main groups. The DLBCL subtypes showed statistically different survivals, indicating the potential use of lncRNAs as prognostic biomarkers in future studies. Conclusions: In this manuscript, we describe the methodology used to identify lncRNAs that differentiate B-cell lymphoma subtypes and we interpreted the biological and clinical values of the results. We inferred the potential functions of lncRNAs to obtain a comprehensive and integrative insight that highlights their impact in this neoplasm.

Marginal zone lymphoma (MZL) and follicular lymphoma (FL) are classified as indolent B-cell lymphomas in dogs. Aside from the clinical and histopathological similarities with the human counterpart, the molecular pathogenesis remains unclear. We integrated transcriptome, genome-wide DNA methylation and copy number aberration analysis to provide insights on the pathogenesis of canine MZL (n = 5) and FL (n = 7), also comparing them with diffuse large B-cell lymphoma (DLBCL). Transcriptome profiling highlighted the presence of similar biological processes affecting both histotypes, including BCR and TLR signalling pathways. However, FLs showed an enrichment of E2F targets, whereas MZLs were characterized by MYC-driven transcriptional activation signatures. FLs showed a distinctive loss on chr1 containing CEACAM23 and 24, conversely MZLs presented multiple recurrent gains on chr13, where MYC is located. The distribution of methylation peaks was similar between the two histotypes. Integrating data from the three omics, FLs resulted clearly separated from MZLs and DLBCL dataset. MZLs showed the enrichment of FoxM1 network and TLR associated TICAM1-dependent IRFs activation pathway. However, no specific signatures differentiated MZLs from DLBCLs. In conclusion, our study presents the first comprehensive analysis of molecular and epigenetic pathogenesis of canine FL and MZL.

Background. Diffuse large B-cell lymphoma (DLBCL) is the commonest lymphoma in both humans and dogs. Canine DLBCL (cDLBCL) is considered an ideal comparative model for drug development, but a complete genomic characterization of this tumor is still lacking. In this study, we report an integrated analysis to comprehensively define the molecular mechanisms of cDLBCL and possible associations with clinical outcome. Methods. Fifty cDLBCLs were analyzed by RNA-Seq, methyl-CpG-binding sequencing and array comparative genomic hybridization. Normal B-cells derived from lymph nodes of 11 healthy dogs were used as controls.Additionally, immunohistochemistry, in vitroand in vivoexperiments were performed as validation analyses. Results.Compared to normal B-cells, cDLBCL showed a marked up-regulation of genes involved in the PI3K/mTOR and NF-κB pathways, including several TLRs in association with MYD88, indicating mechanisms similar to the human activated B cell-like subtype DLBCL. Both RNA-Seq and methylation sequencing led to the identification of two groups of cDLBCLs bearing different clinical outcome. The two groups did not overlap with the human germinal center B-cell (GCB) and the activated B-cell-like (ABC) DLBCL subtypes or the human DLBCL consensus clusters. The dogs with the poorest outcome presented a signature largely defined by markers of T-cell-mediated immune responses, with a high expression of PDL-1, PD-1 and CTLA-4, also validated in an independent cohort of cDLBCL by immunohistochemistry. These data provide a strong rationale for the use of cDLBCL to study immune checkpoint modulators. The observed high expression of PI3K/mTOR pathway genes was confirmed and validated achieving a clear anti-tumor activity with the use of the PI3K-delta inhibitor idelalisib and of the novel dual PI3K/mTOR inhibitor bimiralisib in the cDLBCL cell line CLBL-1. The cDLBCLs showed an up-regulation of MYC and of its targets, sustained by recurrent gains in the chromosome 13, where the oncogene is located, in approximately half of the cases. Thus, we have exposed the cDLBCL cell line CLBL-1 to the BET inhibitor birabresib (OTX015) and to the BRD4 degrader MZ1. Both compounds caused a significant reduction in the proliferation of tumor cells, and this effect was stronger especially with the second compound. Exposure to MZ1 determined an important downregulation of MYC and also of LIN28B, the most overexpressed transcript in cDLBCL when compared to controls. While LIN28B does not seem to be a relevant gene for human DLBCL, its overexpression causes murine T-cell lymphomas (Beachy et al, Blood 2011), and there is a direct association of MYC with LIN28B promoter resulting in transcriptional transactivation (Chang et al, PNAS 2009). Here, LIN28B genetic silencing in the CLBL-1 lead to a reduction in cell growth, opening new therapeutic target perspectives in canine lymphoma. Conclusions. We have reported the first large next generation sequencing study investigating the cDLBCL transcriptome, methylome and the genome-wide CNVs. We identified deregulated pathways and individual transcripts providing therapeutic targets, including an immune-related signature affecting the outcome of a subgroup of cDLBCL. Our data sustain the use of cDLBCL as comparative models for human DLBCL but also highlight differences that must be kept in consideration. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Wymann:PIQUR Therapeutics AG: Employment, Equity Ownership, Patents & Royalties.

Canine malignant melanoma is a highly aggressive tumor with a low survival rate and represents an ideal spontaneous model for the human counterpart. Considerable progress has been recently obtained, but the therapeutic success for canine melanoma is still challenging. Little is known about the mechanisms beyond pathogenesis and melanoma development, and the molecular response to radiotherapy has never been explored before. A faster and deeper understanding of cancer mutational processes and developing mechanisms are now possible through next generation sequencing technologies. In this study, we matched whole exome and transcriptome sequencing in four dogs affected by malignant melanoma at diagnosis and at disease progression to identify possible genetic mechanisms associated with therapy failure. According to previous studies, a genetic similarity between canine malignant melanoma and its human counterpart was observed. Several somatic mutations were functionally related to MAPK, PI3K/AKT and p53 signaling pathways, but located in genes other than BRAF, RAS and KIT. At disease progression, several mutations were related to therapy effects. Natural killer cell-mediated cytotoxicity and several immune-system-related pathways resulted activated opening a new scenario on the microenvironment in this tumor. In conclusion, this study suggests a potential role of the immune system associated to radiotherapy in canine melanoma, but a larger sample size associated with functional studies are needed. This article is protected by copyright. All rights reserved.

The genomic landscape in human B-cell lymphoma has revealed several somatic mutations and potentially relevant germline alterations affecting therapy and prognosis. Also, mutations originally described as somatic aberrations have been shown to confer cancer predisposition when occurring in the germline. The relevance of mutations in canine B-cell lymphoma is scarcely known and gene expression profiling has shown similar molecular signatures among different B-cell histotypes, suggesting other biological mechanisms underlining differences. Here, we present a highly accurate approach to identify single nucleotide variants (SNVs) in RNA-seq data obtained from 62 completely staged canine B-cell lymphomas and 11 normal B-cells used as controls. A customized variant discovery pipeline was applied and SNVs were found in tumors and differentiated for histotype. A number of known and not previously identified SNVs were significantly associated to MAPK signaling pathway, negative regulation of apoptotic process and cell death, B-cell activation, NF-kB and JAK-STAT signaling. Interestingly, no significant genetic fingerprints were found separating diffuse large B-cell lymphoma from indolent lymphomas suggesting that differences of genetic landscape are not the pivotal causative factor of indolent behavior. We also detected several variants in expressed regions of canine B-cell lymphoma and identified SNVs having a direct impact on genes. Using this brand-new approach the consequence of a gene variant is directly associated to expression. Further investigations are in progress to deeply elucidate the mechanisms by which altered genes pathways may drive lymphomagenesis and a higher number of cases is also demanded to confirm this evidence.

Posttransplant lymphoproliferative disorders (PTLDs) are a heterogeneous group of lymphoid proliferations that occur in the setting of depressed T-cell function due to immunosuppressive therapy used following solid organ transplantation, hematopoietic stem cell transplantation, and also xenotransplantation. In the present study, 28 immunosuppressed parkinsonian Macaca fascicularis were intracerebrally injected with wild-type or CTLA4-Ig transgenic porcine xenografts to identify a suitable strategy to enable long-term cell survival, maturation, and differentiation. Nine of 28 (32%) immunosuppressed primates developed masses compatible with PTLD, located mainly in the gastrointestinal tract and/or nasal cavity. The masses were classified as monomorphic PTLD according to the World Health Organization classification. Immunohistochemistry and polymerase chain reaction (PCR) analyses revealed that the PTLDs were associated with macaca lymphocryptovirus as confirmed by double-labeling immunohistochemistry for CD20 and Epstein-Barr nuclear antigen 2 (EBNA-2), where the viral protein was located within the CD20+ neoplastic B cells. In sera from 3 distinct phases of the experimental life of the primates, testing by quantitative PCR revealed a progression of the viral load that paralleled the PTLD progression and no evidence of zoonotic transmission of porcine lymphotropic herpesvirus through xenoneuronal grafts. These data suggest that monitoring the variation of macaca lymphocryptovirus DNA in primates could be used as a possible early diagnostic tool for PTLD progression, allowing preemptive treatment such as immunosuppression therapy reduction.

The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single-step, high-sensitivity real-time PCR assay for simultaneous detection and quantification of P. damselae was designed targeting partial of the sequence of the bamB gene and tested for specificity and sensitivity on laboratory-generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non-target bacterial species proved the assay was able to discriminate Phdd-Phdp subspecies from diverse hosts/geographical origins and between non-target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between Phdp-Phdd. This is the first time that a molecular method for P. damselae diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease.

Background Xenotransplantation is a potential answer to the current organ shortage, but the risk of infections related to overimmunosuppression is an important parameter that may predict the recipient's long-term survival. Cytomegalovirus (CMV) in xenotransplanted and immunosuppressed primates is a well-known cause of disease particularly affecting the gastrointestinal (GI) tract and a zoonotic concern. Methods Post-mortem sera and tissues from 45 immunosuppressed and xenografted Macaca fascicularis were evaluated for CMV using antisera specific for the immediate early 1 (IE1), anti-RhCMV, and QPCR for virus. Results Serological analysis showed 100% positivity for the presence of CMV antibodies following the application of a specific test designed for RhCMV. Five of 45 primates showed typical lesions of CMV infection in the GI tract, including neutrophilic enteritis and inclusion bodies. Molecular analysis confirmed the presence of recipient's CMV in the tissues with CMV histopathology. Porcine CMV from the donor animals was not found in any of the CMV-specific IHC-positive recipients. Conclusion The presence of active CMV infection in animals intended for xenograft experiments can lead to severe gastrointestinal lesions that could impact the overall aims of the study. In such cases, the animals should be investigated using appropriate (non-human primate-specific) diagnostic tools prior to use and treated aggressively with state-of-the-art antiviral therapy.

Background Water temperature greatly influences the physiology and behaviour of teleost fish as other aquatic organisms. While fish are able to cope with seasonal temperature variations, thermal excursions outside their normal thermal range might exceed their ability to respond leading to severe diseases and death. Profound differences exist in thermal tolerance across fish species living in the same geographical areas, promoting for investigating the molecular mechanisms involved in susceptibility and resistance to low and high temperatures toward a better understanding of adaptation to environmental challenges. The gilthead sea bream, Sparus aurata, is particularly sensitive to cold and the prolonged exposure to low temperatures may lead to the "winter disease", a metabolic disorder that significantly affects the aquaculture productions along the Northern Mediterranean coasts during winter-spring season. While sea bream susceptibility to low temperatures has been extensively investigated, the cascade of molecular events under such stressful condition is not fully elucidated. Results In the present study two groups of wild sea bream were exposed for 21 days to two temperature regimes: 16 ± 0.3°C (control group) and 6.8 ± 0.3°C (cold-exposed group) and DNA microarray analysis of liver transcriptome was carried out at different time points during cold exposure. A large set of genes was found to be differentially expressed upon cold-exposure with increasingly relevant effects being observed after three weeks at low temperature. All major known responses to cold (i.e. anti-oxidant response, increased mitochondrial function, membrane compositional changes) were found to be conserved in the gilthead sea bream, while, evidence for a key role of unfolded protein response (UPR) to endoplasmic reticulum (ER) stress, during short- and long-term exposure to cold is reported here for the first time. Conclusions Transcriptome data suggest a scenario where oxidative stress, altered lipid metabolism, ATP depletion and protein denaturation converge to induce ER stress. The resulting UPR activation further promotes conditions for cell damage, and the inability to resolve ER stress leads to severe liver dysfunction and potentially to death. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-765) contains supplementary material, which is available to authorized users.

Platelet-derived growth factors (PDGFs) belong to a family of polypeptide growth factors that signal through cell surface tyrosine kinase receptors to stimulate growth, proliferation and differentiation. Platelet-derived growth factor receptors (PDGFRs) are also considered important targets for specific kinase inhibitors in the treatment of several human tumours. The aim of this study was to investigate the role of PDGF-A, PDGFB, PDGFR-alpha and PDGFR-beta in canine lymphoma by determining gene and protein expression in lymph nodes of dogs with diffuse large B-cell lymphoma (DLBCL), peripheral T-cell lymphoma (PTCL), T-Iymphoblastic lymphoma (T-LBL) and in healthy control dogs. One lymph node was also studied at the end of therapy in a subset of dogs in remission for DLBCL. In controls, PDGF-A, PDGFR-alpha and PDGFR-beta mRNA levels were significantly higher than in DLBCLs, PTCLs and T-LBLs. However, PDGFR-alpha and PDGFR-beta were minimally expressed by lymphocytes and plasma cells in normal lymph nodes as determined by immunohistochemistry, while neoplastic B and T cells showed the highest score (P

Canine Diffuse Large B-cell Lymphoma (cDLBCL) is an aggressive cancer with variable clinical response. Despite recent attempts by gene expression profiling to identify the dog as a potential animal model for human DLBCL, this tumor remains biologically heterogeneous with no prognostic biomarkers to predict prognosis. The aim of this work was to identify copy number aberrations (CNAs) by high-resolution array comparative genomic hybridization (aCGH) in 12 dogs with newly diagnosed DLBCL. In a subset of these dogs, the genetic profiles at the end of therapy and at relapse were also assessed. In primary DLBCLs, 90 different genomic imbalances were counted, consisting of 46 gains and 44 losses. Two gains in chr13 were significantly correlated with clinical stage. In addition, specific regions of gains and losses were significantly associated to duration of remission. In primary DLBCLs, individual variability was found, however 14 recurrent CNAs (>30%) were identified. Losses involving IGK, IGL and IGH were always found, and gains along the length of chr13 and chr31 were often observed (>41%). In these segments, MYC, LDHB, HSF1, KIT and PDGFR alpha are annotated. At the end of therapy, dogs in remission showed four new CNAs, whereas three new CNAs were observed in dogs at relapse compared with the previous profiles. One ex novo CNA, involving TCR, was present in dogs in remission after therapy, possibly induced by the autologous vaccine. Overall, aCGH identified small CNAs associated with outcome, which, along with future expression studies, may reveal target genes relevant to cDLBCL.

Epigenetic modifications are important early events during carcinogenesis. In particular, hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a well-known mechanism of gene silencing that contributes to cancer development and progression. Tissue factor pathway inhibitor 2 (TFPI-2) is a tumor suppressor involved in invasiveness inhibition. Although TFPI-2 transcriptional silencing, through promoter hypermethylation, has been widely reported in several human malignancies, it has never been explored in lymphoma. In the present study TFPI-2 methylation and gene expression have been investigated in canine Diffuse Large B-cell lymphomas (cDLBCL). The methylation level of 23 CpGs located within the TFPI-2 promoter was investigated by bisulfite-specific PCR and next generation amplicon deep sequencing (GS Junior 454, Roche) in 22 cDLBCLs and 9 controls. For the same specimens, TFPI-2 gene expression was assessed by means of Real-time RT-PCR. Sequence analysis clearly demonstrated that TFPI2 is frequently hypermethylated in cDLBCL. Hypermethylation of the TFPI-2 promoter was found in 77% of DLBCLs ( 17 out of 22) and in one normal lymph node. Globally, dogs with DLBCL showed a mean methylation level significantly increased compared to controls (p

The most promising techniques for detecting minimal residual disease (MRD) in canine lymphoma are flow cytometry (FC) and polymerase chain reaction amplification of antigen receptor genes (PARR). However, the agreement between these methods has not been established. MRD was monitored by FC and PARR following treatment of dogs affected with diffuse large B-cell lymphoma (DLBCL), comparing results in lymph node (LN), peripheral blood (PB) and bone marrow (BM) samples. The prognostic impact of MRD on time to relapse (TTR) and lymphoma-specific survival (LSS) was also assessed.Fourteen dogs with previously untreated DLBCL were enrolled into the study; 10 dogs eventually relapsed, while four dogs with undetectable MRD were still in remission at the end of the study. At diagnosis, the concordance rate between FC and PARR was 100%, 78.6%, and 643% for LN, PB and BM, respectively. At the end of treatment, the agreement rates were 35.7%, 50%, and 57.1% for LN, PB and BM, respectively. At least one of the follow-up samples from dogs experiencing relapse was PARR.; conversely, FC was not able to detect MRD in seven of the dogs that relapsed. PARR was more sensitive than FC in predicting TTR, whereas the combination of PARR and FC was more sensitive than either technique alone in predicting LSS using PB samples. The results suggest that immunological and molecular techniques should be used in combination when monitoring for MRD in canine DLBCL. (C) 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t The goal of the present study was to investigate mRNA expression levels of several cytokines and inflammatory mediators in broncho-alveolar lavage (BAL) fluid and respi- ratory epithelium in recurrent airway obstruction (RAO)-affected horses. RAO, also called heaves, is a common, performance-limiting, equine respiratory disease with clinical signs and pathophysiological similarities to human asthma, and characterized by bronchospasm, neutrophilic infiltration and increased mucus in the airways. Six RAO-affected horses were examined twice within 15 days and seven clinically healthy horses were examined for comparison. Quantitative real-time RT-PCR was used to assess mRNA expression of the inflammatory mediators IL-1, IL-6, IL-8, IL-13, IL-17, TNF, INF, TGF1, NF- and TRL4 in bronchial biopsies and in BAL fluid. Gene expression levels were then compared with clinical signs, endoscopic examination, complete blood cell count, cytology of BAL fluid, histological examination of bronchial tissue and bacteriological and mycological examinations. Expression of IL1, IL8, TLR4, TNF, TGF1 and NFk transcripts was significantly up- regulated in RAO-affected compared to healthy horses. A similar trend, albeit not significant, was showed for IL17 and INF. A highly significant correlation was observed among IL-1, IL8, TGF1, NFk, TRL4, and INF expression patterns as well as between expression levels of these genes and clinical parameters. In the present study, the comparison between clinically healthy and RAO-affected horses gave new insights on the cytokine expression in equine health and disease status. The identification of cytokines implicated in the pathogenesis of RAO may contribute to the diagnosis and treatment of this disease.

The assessment of marine environmental health is a complex but fundamental task both for ecosystem conservation and food safety related to the human consumption of marine products. Manila clams inhabiting the Venice Lagoon constitute an excellent case study for evaluating the effects of complex mixtures of industrial and urban effluents on aquatic organisms. Clams were collected in different seasons at four locations within the Venice Lagoon. The sampling sites were characterized by a range of pollutant concentrations and included Porto Marghera, a highly polluted industrial area where clam harvesting for human consumption is strictly forbidden. Pooled soft tissues were subjected to mass spectroscopy analysis to measure the concentrations of PCDDs/PCDFs/PCBs-DL, PCBs, PBDEs, HCB and PAHs, and pooled digestive gland samples were used for gene expression profiling. While seasonal variation was found to be responsible for the largest proportion of transcriptional changes, significance analysis of microarrays quantitative correlation analysis identified 162 transcripts that were correlated with at least one class of chemicals measured in the samples from the four different sampling sites. Prediction Analysis of Microarrays (PAM) identified a minimal set of seven genes that correctly assigned samples collected in the restricted polluted area (Porto Marghera), independent of the season in which they were collected. An integrated approach combining transcriptomics and chemical analyses of the Manila clam provided a global picture of how Manila clams respond to complex mixtures of xenobiotics and their interplay with other biotic and abiotic factors. We were also able to identify gene expression signatures for different classes of chemicals and a set of robust biomarkers of exposure to these chemicals.

Dexamethasone (Dex), alone or in association with estrogens, is often illegally administered per os at very low dosage as a growth promoter in beef cattle, with effects that are opposite to the muscle wasting and atrophy induced by repeated administration at therapeutic dosages. In vitro and in vivo studies have investigated the catabolic effects of Dex at therapeutic doses on skeletal muscle, demonstrating an increase in the expression of GDF8 (myostatin) gene, a well-known negative regulator of skeletal muscle mass, in a dose-dependent way. This suggested a direct role of myostatin in Dex-induced muscle wasting. In the present study, an oligonucleotide microarray platform was used to compare expression profiles of beef cattle muscle in animals treated with either Dex or Dex plus 17-β estradiol (Estr) administered at subtherapeutic dosage, against untreated controls. Data analysis demonstrates that the expression profiles were strongly affected by Dex treatment with hundreds of genes upregulated with relevant fold-change, whereas seven genes were downregulated including the myostatin gene. On the contrary, the number of differentially regulated genes was lower in response to the addition of Estr to the Dex treatment. Differentially regulated genes were analyzed to describe the effects of these treatments on muscle physiology, highlighting the importance of specific pathways (e.g., Wnt or cytokine signaling) and cellular processes (e.g., cell shape and motility). Finally, the observed differences in the expression profile will allow the development of indirect bio-markers to detect illegal Dex treatments in beef cattle using quantitative RT-PCR.