Search

Formaldehyde fixation is worldwide the most used system for histopathological examination. However, its toxicity is well known, and preservation of proteins and nucleic acids is not optimal. Alternative fixatives warranting similar morphological quality of tissues and costs, but lacking toxicity and allowing better preservation of proteins and nucleic acids would therefore increase both safety of operators and quality of molecular analysis in pathology.This multi-institutional study aimed to compare the morphological, histochemical, immunohistochemical (IHC), and molecular analyses outcomes of a newly patented, non-toxic, acid-free Glyoxal (GAF) fixative with neutral buffered formaldehyde (NBF). Tissues from a total of 73 subjects were analyzed, including 13 necropsies.Gross features were preserved after GAF fixation, with no tissue hardening or discoloration. Cellular ultrastructure was also better preserved with GAF and histology and histochemistry on GAF-fixed samples showed good results when compared to NBF-fixed samples, with the exception of loss of tinctorial affinity of erythrocytes and mast cell granules. IHC analyses also showed comparable results with only slight and rare protocol adjustment. DNA and RNA yields were higher from GAF-fixed samples (Pp53andCOX1) were better amplified. RNA scope showed positive results forc-KITexpression in GAF-fixed mast cell tumors.Based on these data, the non-toxic GAF fixative allows good macroscopical, histological and immunohistochemical analyses of tissue samples, including on-field application, and better molecular analyses when compared to NBF. This represents a promising possibility for teaching, diagnostic, and research in veterinary pathology.

Chronic exposure to pollutants affects natural populations, creating specific molecular and biochemical signatures. In the present study, we tested the hypothesis that chronic exposure to pollutants might have substantial effects on the Manila clam hologenome long after removal from contaminated sites. To reach this goal, a highly integrative approach was implemented, combining transcriptome, genetic and microbiota analyses with the evaluation of biochemical and histological profiles of the edible Manila clam Ruditapes philippinarum, as it was transplanted for 6 months from the polluted area of Porto Marghera (PM) to the clean area of Chioggia (Venice lagoon, Italy). One month post‐transplantation, PM clams showed several modifications to its resident microbiota, including an overrepresentation of the opportunistic pathogen Arcobacter spp. This may be related to the upregulation of several immune genes in the PM clams, potentially representing a host response to the increased abundance of deleterious bacteria. Six months after transplantation, PM clams demonstrated a lower ability to respond to environmental/physiological stressors related to the summer season, and the hepatopancreas‐associated microbiota still showed different compositions among PM and CH clams. This study confirms that different stressors have predictable effects in clams at different biological levels and demonstrates that chronic exposure to pollutants leads to long‐lasting effects on the animal hologenome. In addition, no genetic differentiation between samples from the two areas was detected, confirming that PM and CH clams belong to a single population. Overall, the obtained responses were largely reversible and potentially related to phenotypic plasticity rather than genetic adaptation. The results here presented will be functional for the assessment of the environmental risk imposed by chemicals on an economically important bivalve species.

In recent years recurrent bivalve mass mortalities considerably increased around the world, causing the collapse of natural and farmed populations. Venice Lagoon has historically represented one of the major production areas of the Manila clam Ruditapes philippinarum in Europe. However, in the last 20 years a 75 % decrease in the annual production has been experienced. While climate change and anthropogenic interventions may have played a key role in natural and farmed stocks reductions, to date no studies investigated at multiple levels the environmental stressors affecting farmed Manila clam. In this work we carried out a long-term monitoring campaign on Manila clam reared in four farming sites located at different distances from the southern Venice Lagoon inlet, integrating (meta)genomic approaches (i.e. RNA-seq; microbiota characterization), biometric measurements and chemical-physical parameters. Our study allowed to characterize the molecular mechanisms adopted by this species to cope with the different environmental conditions characterizing farming sites and to propose hypotheses to explain mortality events observed in recent years. Among the most important findings, the disruption of clam's immune response, the spread of Vibrio spp., and the up-regulation of molecular pathways involved in xenobiotic metabolism suggested major environmental stressors affecting clams farmed in sites placed close to Chioggia's inlet, where highest mortality was also observed. Overall, our study provides knowledge-based tools for managing Manila clam farming on-growing areas. In addition, the collected data is a snapshot of the time immediately before the commissioning of MoSE, a system of mobile barriers aimed at protecting Venice from high tides, and will represent a baseline for future studies on the effects of MoSE on clams farming and more in general on the ecology of the Venice lagoon.

The analysis of different samples from the purification lines in WWTPs provides valuable data on SARS-CoV-2 prevalence, the predominant variants, and decontamination efficacy.

Background Susceptibility of European sea bass (Dicentrarchus labrax L.) to viral nervous necrosis (VNN) is well-known. Interest towards selective breeding as a tool to enhance genetic resistance in this species has increased sharply due to the major threat represented by VNN for farmed sea bass and limitations concerning specific therapeutical measures. A sea bass experimental population (N = 650) was challenged with nervous necrosis virus (NNV) to investigate genetic variation in VNN mortality. In addition, relationships of this trait with serum cortisol concentration after stress exposure, antibody titer against NNV antigens, and body weight at a fixed age were studied to identify potential indicator traits of VNN resistance. Results The estimate of heritability for VNN mortality was moderate and ranged from 0.15 (HPD95%, 95% highest posterior density interval: 0.02, 0.31) to 0.23 (HPD95%: 0.06, 0.47). Heritability estimates for cortisol concentration, antibody titer, and body weight were 0.19 (HPD95%: 0.07, 0.34), 0.36 (HPD95%: 0.16, 0.59) and 0.57 (HPD95%: 0.33, 0.84), respectively. Phenotypic relationships between traits were trivial and not statistically significant, except for the estimated correlation between antibody titer and body weight (0.24). Genetic correlations of mortality with body weight or antibody titer (− 0.39) exhibited a 0.89 probability of being negative. A negligible genetic correlation between mortality and cortisol concentration was detected. Antibody titer was estimated to be positively correlated with body weight (0.49). Conclusions Antibody titer against NNV offers the opportunity to use indirect selection to enhance resistance, while the use of cortisol concentration as an indicator trait in breeding programs for VNN resistance is questionable. The estimate of heritability for VNN mortality indicates the feasibility of selective breeding to enhance resistance to NNV and raises attention to the development of genomic prediction tools to simplify testing procedures for selection candidates.

C6O4 (difluoro{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetic acid) is a new surfactant and emulsifier used as a substitute of perfluorooctanoic acid (PFOA). Recently, C6O4 has been detected in aquatic environments, but, at present, no information concerning the effects of C6O4 on aquatic species, such as bivalves, are available in the literature. Therefore, in this study we evaluated for the first time the effects of C6O4 (0.1 and 1 µg/L) and PFOA (1 µg/L) to the clam Ruditapes philippinarum. Short-term (7 days) and long-term (21 days) exposures of clams to the two compounds were carried out and numerous biomarkers were measured in haemocytes/haemolymph, as well as in gills and digestive gland. The MANOVA analysis demonstrated statistically significant effects of the independent variables “treatment”, “time” and “treatment-time interaction” on the whole dataset of biomarker responses. The two-way ANOVA analysis performed for each biomarker response indicated that the two compounds affected most of the cellular and tissue parameters measured. Despite preliminary, the results obtained suggested that C6O4 - similarly to PFOA - can affect both cellular and biochemical parameters of clams.

Dolphinfish is an important fish species for both commercial and sport fishing, but so far limited information is available on genetic variability and pattern of differentiation of dolphinfish populations in the Mediterranean basin. Recently developed techniques allow genome-wide identification of genetic markers for better understanding of population structure in species with limited genome information. Using restriction-site associated DNA analysis we successfully genotyped 140 individuals of dolphinfish from eight locations in the Mediterranean Sea at 3324 SNP loci. We identified 311 sex-related loci that were used to assess sex-ratio in dolphinfish populations. In addition, we identified a weak signature of genetic differentiation of the population closer to Gibraltar Strait in comparison to other Mediterranean populations, which might be related to introgression of individuals from Atlantic. No further genetic differentiation could be detected in the other populations sampled, as expected considering the known highly mobility of the species. The results obtained improve our knowledge of the species and can help managing dolphinfish stock in the future.

WOS:000487521600001; Mass mortalities due to disease outbreaks have recently affected a number of major taxa in marine ecosystems. Climate- and pollution-induced stress may compromise host immune defenses, increasing the risk of opportunistic diseases. Despite growing evidence that mass mortality events affecting marine species worldwide are strongly influenced by the interplay of numerous environmental factors, the reductionist approaches most frequently used to investigate these factors hindered the interpretation of these multifactorial pathologies. In this study, we propose a broader approach based on the combination of RNA-sequencing and 16S microbiota analyses to decipher the factors underlying mass mortality in the striped venus clam, Chamelea gallina, along the Adriatic coast. On one hand, gene expression profiling and functional analyses of microbial communities showed the over-expression of several genes and molecular pathways involved in xenobiotic metabolism, suggesting potential chemical contamination in mortality sites. On the other hand, the down-regulation of several genes involved in immune and stress response, and the over-representation of opportunistic pathogens such as Vibrio and Photobacterium spp. indicates that these microbial species may take advantage of compromised host immune pathways and defense mechanisms that are potentially affected by chemical exposure, resulting in periodic mortality events. We propose the application of our approach to interpret and anticipate the risks inherent in the combined effects of pollutants and microbes on marine animals in today's rapidly changing environment.

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.

Developing reliable tools to trace food origin represents a major goal for producers and control authorities. Here, we test the hypothesis whether NGS-generated data could provide a reliable tool to ensure seafood traceability. As a test case, we used the Manila clam Ruditapes philippinarum, a bivalve mollusk of high commercial interest with worldwide distribution, collected in the Venice lagoon sites subjected to prohibition of clam harvesting because of chemical contamination as well as in authorized clam harvesting areas. The results obtained demonstrated that the geographic origin of Manila clam may be more accurately determined basing on microbiome data than single nucleotide polymorphisms. In particular, combining microbiome data with machine-learning techniques, we provide the experimental evidence that it is possible to trace the clam place of origin at high spatial resolution. Considering its low cost and portability, NGS-analysis of microbiome data might represent a cost-effective, high-resolution tool for reliable food traceability.