A novel DSP zebrafish model reveals training- and drug-induced modulation of arrhythmogenic cardiomyopathy phenotypes.

Arrhythmogenic cardiomyopathy (AC) is an inherited disorder characterized by progressive loss of the ventricular myocardium causing life-threatening ventricular arrhythmias, syncope and sudden cardiac death in young and athletes. About 40% of AC cases carry one or more mutations in genes encoding for desmosomal proteins, including Desmoplakin (Dsp). We present here the first stable Dsp knock-out (KO) zebrafish line able to model cardiac alterations and cell signalling dysregulation, characteristic of the AC disease, on which environmental factors and candidate drugs can be tested. Our stable Dsp knock-out (KO) zebrafish line was characterized by cardiac alterations, oedema and bradycardia at larval stages. Histological analysis of mutated adult hearts showed reduced contractile structures and abnormal shape of the ventricle, with thinning of the myocardial layer, vessels dilation and presence of adipocytes within the myocardium. Moreover, TEM analysis revealed "pale", disorganized and delocalized desmosomes. Intensive physical training protocol caused a global worsening of the cardiac phenotype, accelerating the progression of the disease. Of note, we detected a decrease of Wnt/ß-catenin signalling, recently associated with AC pathogenesis, as well as Hippo/YAP-TAZ and TGF-ß pathway dysregulation. Pharmacological treatment of mutated larvae with SB216763, a Wnt/ß-catenin agonist, rescued pathway expression and cardiac abnormalities, stabilizing the heart rhythm. Overall, our Dsp KO zebrafish line recapitulates many AC features observed in human patients, pointing at zebrafish as a suitable system for in vivo analysis of environmental modulators, such as the physical exercise, and the screening of pathway-targeted drugs, especially related to the Wnt/ß-catenin signalling cascade.

Antitumor Effect of Berberine Analogs in a Canine Mammary Tumor Cell Line and in Zebrafish Reporters via Wnt/ß-Catenin and Hippo Pathways.

The heterogeneous nature of human breast cancer (HBC) can still lead to therapy inefficacy and high lethality, and new therapeutics as well as new spontaneous animal models are needed to benefit translational HBC research. Dogs are primarily investigated since they spontaneously develop tumors that share many features with human cancers. In recent years, different natural phytochemicals including berberine, a plant alkaloid, have been reported to have antiproliferative activity in vitro in human cancers and rodent animal models. In this study, we report the antiproliferative activity and mechanism of action of berberine, its active metabolite berberrubine, and eight analogs, on a canine mammary carcinoma cell line and in transgenic zebrafish models. We demonstrate both in vitro and in vivo the significant effects of specific analogs on cell viability via the induction of apoptosis, also identifying their role in inhibiting the Wnt/ß-catenin pathway and activating the Hippo signals with a downstream reduction in CTGF expression. In particular, the berberine analogs NAX035 and NAX057 show the highest therapeutic efficacy, deserving further analyses to elucidate their mechanism of action more in detail, and in vivo studies on spontaneous neoplastic diseases are needed, aiming at improving veterinary treatments of cancer as well as translational cancer research.

Epithelial-to-Mesenchymal Transition and Phenotypic Marker Evaluation in Human, Canine, and Feline Mammary Gland Tumors.

Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties. EMT has been closely associated with cancer cell aggressiveness. The aim of this study was to evaluate the mRNA and protein expression of EMT-associated markers in mammary tumors of humans (HBC), dogs (CMT), and cats (FMT). Real-time qPCR for SNAIL, TWIST, and ZEB, and immunohistochemistry for E-cadherin, vimentin, CD44, estrogen receptor (ER), progesterone receptor (PR), ERBB2, Ki-67, cytokeratin (CK) 8/18, CK5/6, and CK14 were performed. Overall, SNAIL, TWIST, and ZEB mRNA was lower in tumors than in healthy tissues. Vimentin was higher in triple-negative HBC (TNBC) and FMTs than in ER+ HBC and CMTs (p < 0.001). Membranous E-cadherin was higher in ER+ than in TNBCs (p < 0.001), whereas cytoplasmic E-cadherin was higher in TNBCs when compared with ER+ HBC (p < 0.001). A negative correlation between membranous and cytoplasmic E-cadherin was found in all three species. Ki-67 was higher in FMTs than in CMTs (p < 0.001), whereas CD44 was higher in CMTs than in FMTs (p < 0.001). These results confirmed a potential role of some markers as indicators of EMT, and suggested similarities between ER+ HBC and CMTs, and between TNBC and FMTs.

Hyperactivation of Wnt/ß-catenin and Jak/Stat3 pathways in human and zebrafish foetal growth restriction models: Implications for pharmacological rescue.

Foetal Growth Restriction (FGR), previously known as Intrauterine Growth Restriction (IUGR), is an obstetrical condition due to placental insufficiency, affecting yearly about 30 million newborns worldwide. In this work, we aimed to identify and pharmacologically target signalling pathways specifically involved in the FGR condition, focusing on FGR-related cardiovascular phenotypes. The transcriptional profile of human umbilical cords from FGR and control cases was compared with the response to hypoxia of zebrafish (Danio rerio) transgenic lines reporting in vivo the activity of twelve signalling pathways involved in embryonic development. Wnt/ß-catenin and Jak/Stat3 were found as key pathways significantly dysregulated in both human and zebrafish samples. This information was used in a chemical-genetic analysis to test drugs targeting Wnt/ß-catenin and Jak/Stat3 pathways to rescue a set of FGR phenotypes, including growth restriction and cardiovascular modifications. Treatments with the Wnt/ß-catenin agonist SB216763 successfully rescued body dimensions, cardiac shape, and vessel organization in zebrafish FGR models. Our data support the Wnt/ß-catenin pathway as a key FGR marker and a promising target for pharmacological intervention in the FGR condition.

Evolving Diagnostic Criteria for Arrhythmogenic Cardiomyopathy.

Criteria for diagnosis of arrhythmogenic cardiomyopathy (ACM) were first proposed in 1994 and revised in 2010 by a Task Force. Although the Task Force criteria demonstrated a good accuracy for diagnosis of the original right ventricular phenotype (arrhythmogenic right ventricular cardiomyopathy), they lacked sensitivity for identification of the expanding phenotypic spectrum of ACM, which includes left-sided variants and did not incorporate late-gadolinium enhancement findings by cardiac magnetic resonance. The 2020 International criteria ("Padua criteria") have been developed by International experts with the aim to improve the diagnosis of ACM by providing new criteria for the diagnosis of left ventricular phenotypic features. The key upgrade was the incorporation of tissue characterization findings by cardiac magnetic resonance for noninvasive detection of late-gadolinium enhancement/myocardial fibrosis that are determinants for characterization of arrhythmogenic biventricular and left ventricular cardiomyopathy. The 2020 International criteria are heavily dependent on cardiac magnetic resonance, which has become mandatory to characterize the ACM phenotype and to exclude other diagnoses. New criteria regarding left ventricular depolarization and repolarization ECG abnormalities and ventricular arrhythmias of left ventricular origin were also provided. This article reviews the evolving approach to diagnosis of ACM, going back to the 1994 and 2010 International Task Force criteria and then grapple with the modern 2020 International criteria.

Novel pathogenic role for galectin-3 in early disease stages of arrhythmogenic cardiomyopathy.

BACKGROUND: Arrhythmogenic cardiomyopathy (AC) is a myocardial disease due to desmosomal mutations whose pathogenesis is incompletely understood. OBJECTIVE: The purpose of this study was to identify molecular pathways underlying early AC by gene expression profiling in both humans and animal models. METHODS: RNA sequencing for differentially expressed genes (DEGs) was performed on the myocardium of transgenic mice overexpressing the Desmoglein2-N271S mutation before phenotype onset. Zebrafish signaling reporters were used for in vivo validation. Whole exome sequencing was undertaken in 10 genotype-negative AC patients and subsequent direct sequencing in 140 AC index cases. RESULTS: Among 29 DEGs identified at early disease stages, Lgals3/GAL3 (lectin, galactoside-binding, soluble, 3) showed reduced cardiac expression in transgenic mice and in 3 AC patients who suffered sudden cardiac death without overt structural remodeling. Four rare missense variants of LGALS3 were identified in 5 human AC probands. Pharmacologic inhibition of Lgals3 in zebrafish reduced Wnt and transforming growth factor-ß signaling, increased Hippo/YAP-TAZ signaling, and induced alterations in desmoplakin membrane localization, desmosome integrity and stability. Increased LGALS3 plasma expression in genotype-positive AC patients and CD98 activation supported the galectin-3 (GAL3) release by circulating macrophages pointing toward the stabilization of desmosomal assembly at the injured regions. CONCLUSION: GAL3 plays a crucial role in early AC onset through regulation of Wnt/ß-catenin signaling and intercellular adhesion.

Efficient clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish.

The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders.

Mechanotransduction and Adrenergic Stimulation in Arrhythmogenic Cardiomyopathy: An Overview of in vitro and in vivo Models.

Arrhythmogenic Cardiomyopathy (AC) is a rare inherited heart disease, manifesting with progressive myocardium degeneration and dysfunction, and life-threatening arrhythmic events that lead to sudden cardiac death. Despite genetic determinants, most of AC patients admitted to hospital are athletes or very physically active people, implying the existence of other disease-causing factors. It is recognized that AC phenotypes are enhanced and triggered by strenuous physical activity, while excessive mechanical stretch and load, and repetitive adrenergic stimulation are mechanisms influencing disease penetrance. Different approaches have been undertaken to recapitulate and study both mechanotransduction and adrenergic signaling in AC, including the use of in vitro cellular and tissue models, and the development of in vivo models (particularly rodents but more recently also zebrafish). However, it remains challenging to reproduce mechanical load stimuli and physical activity in laboratory experimental settings. Thus, more work to drive the innovation of advanced AC models is needed to recapitulate these subtle physiological influences. Here, we review the state-of-the-art in this field both in clinical and laboratory-based modeling scenarios. Specific attention will be focused on highlighting gaps in the knowledge and how they may be resolved by utilizing novel research methodology.

Wnt/ß-Catenin and Hippo Pathway Deregulation in Mammary Tumors of Humans, Dogs, and Cats.

Mammary cancer is a common neoplasm in women, dogs, and cats that still represents a therapeutic challenge. Wnt/ß-catenin and Hippo pathways are involved in tumor progression, cell differentiation, and metastasis. The aim of this study was to evaluate mRNA and protein expression of molecules involved in these pathways in human (HBC), canine (CMT), and feline mammary tumors (FMT). Real-time quantitative polymerase chain reaction (qPCR) for ß-catenin, CCND1, YAP, TAZ, CTGF, and ANKRD1, western blotting for YAP, TAZ, and ß-catenin, and immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), ERBB2, ß-catenin, and YAP/TAZ were performed on mammary tumor tissues. The protein expression of active ß-catenin was higher in tumors than in healthy tissues in all 3 species. The mRNA expression of the downstream gene CCND1 was increased in HBC ER+ and CMTs compared to healthy tissues. Membranous and cytoplasmic protein expression of ß-catenin were strongly negatively correlated in all 3 species. Tumors showed an increased protein expression of YAP/TAZ when compared to healthy tissues. Notably, YAP/TAZ expression was higher in triple negative breast cancers when compared to HBC ER+ and in FMTs when compared to CMTs. The mRNA expression of ß-catenin, YAP, TAZ, CTGF, and ANKRD1 was not different between tumors and healthy mammary gland in the 3 species. This study demonstrates deregulation of Wnt/ß-catenin and Hippo pathways in mammary tumors, which was more evident at the protein rather than the mRNA level. Wnt/ß-catenin and Hippo pathways seem to be involved in mammary carcinogenesis and therefore represent interesting therapeutic targets that should be further investigated.

Diagnosis of arrhythmogenic cardiomyopathy: The Padua criteria.

The original designation of "Arrhythmogenic right ventricular (dysplasia/) cardiomyopathy"(ARVC) was used by the scientists who first discovered the disease, in the pre-genetic and pre-cardiac magnetic resonance era, to describe a new heart muscle disease predominantly affecting the right ventricle, whose cardinal clinical manifestation was the occurrence of malignant ventricular arrhythmias. Subsequently, autopsy investigations, genotype-phenotype correlations studies and the increasing use of contrast-enhancement cardiac magnetic resonance showed that the fibro-fatty replacement of the myocardium represents the distinctive phenotypic feature of the disease that affects the myocardium of both ventricles, with left ventricular involvement which may parallel or exceed the severity of right ventricular involvement. This has led to the new designation of "Arrhythmogenic Cardiomyopathy" (ACM), that represents the evolution of the original term of ARVC. The present International Expert Consensus document proposes an upgrade of the criteria for diagnosis of the entire spectrum of the phenotypic variants of ACM. The proposed "Padua criteria" derive from the diagnostic approach to ACM, which has been developed over 30 years by the multidisciplinary team of basic researchers and clinical cardiologists of the Medical School of the University of Padua. The Padua criteria are a working framework to improve the diagnosis of ACM by introducing new diagnostic criteria regarding tissue characterization findings by contrast-enhanced cardiac magnetic resonance, depolarization/repolarization ECG abnormalities and ventricular arrhythmia features for diagnosis of the left ventricular phenotype. The proposed diagnostic criteria need to be further validated by future clinical studies in large cohorts of patients.

miR-7 Controls the Dopaminergic/Oligodendroglial Fate through Wnt/ß-catenin Signaling Regulation.

During the development of the central nervous system, the proliferation of neural progenitors and differentiation of neurons and glia are tightly regulated by different transcription factors and signaling cascades, such as the Wnt and Shh pathways. This process takes place in cooperation with several microRNAs, some of which evolutionarily conserved in vertebrates, from teleosts to mammals. We focused our attention on miR-7, as its role in the regulation of cell signaling during neural development is still unclear. Specifically, we used human stem cell cultures and whole zebrafish embryos to study, in vitro and in vivo, the role of miR-7 in the development of dopaminergic (DA) neurons, a cell type primarily affected in Parkinson's disease. We demonstrated that the zebrafish homologue of miR-7 (miR-7a) is expressed in the forebrain during the development of DA neurons. Moreover, we identified 143 target genes downregulated by miR-7, including the neural fate markers TCF4 and TCF12, as well as the Wnt pathway effector TCF7L2. We then demonstrated that miR-7 negatively regulates the proliferation of DA-progenitors by inhibiting Wnt/ß-catenin signaling in zebrafish embryos. In parallel, miR-7 positively regulates Shh signaling, thus controlling the balance between oligodendroglial and DA neuronal cell fates. In summary, this study identifies a new molecular cross-talk between Wnt and Shh signaling pathways during the development of DA-neurons. Being mediated by a microRNA, this mechanism represents a promising target in cell differentiation therapies for Parkinson's disease.

Zebrafish as a Smart Model to Understand Regeneration After Heart Injury: How Fish Could Help Humans.

Myocardial infarction (MI) in humans is a common cause of cardiac injury and results in irreversible loss of myocardial cells and formation of fibrotic scar tissue. This fibrotic tissue preserves the integrity of the ventricular wall but undermines pump function, leading to congestive heart failure. Unfortunately, the mammalian heart is unable to replace cardiomyocytes, so the life expectancy for patients after an episode of MI is lower than for most common types of cancers. Whereas, humans cannot efficiently regenerate their heart after injury, the teleost zebrafish have the capability to repair a "broken" heart. The zebrafish is probably one of the most important models for developmental and regenerative biology of the heart. In the last decades, the zebrafish has become increasingly important for scientific research: it has many characteristics that make it a smart model for studying human disease. Moreover, adult zebrafish efficiently regenerate their hearts following different forms of injury. Due to these characteristics, and to the availability of genetic approaches, and biosensor zebrafish lines, it has been established useful for studying molecular mechanisms of heart regeneration. Regeneration of cardiomyocytes in zebrafish is not based on stem cells or transdifferentiation of other cells but on the proliferation of preexisting cardiomyocytes. For this reason, future studies into the zebrafish cardiac regenerative mechanisms could identify specific molecules able to regulate the proliferation of preexisting cardiomyocytes; these factors may be studied in order to understand regulation of myocardial plasticity in cardiac repair processes after injury and, in particular, after MI in humans.

A novel murine model for arrhythmogenic cardiomyopathy points to a pathogenic role of Wnt signalling and miRNA dysregulation.

AIMS: Arrhythmogenic cardiomyopathy (AC) is one of the most common inherited cardiomyopathies, characterized by progressive fibro-fatty replacement in the myocardium. Clinically, AC manifests itself with ventricular arrhythmias, syncope, and sudden death and shows wide inter- and intra-familial variability. Among the causative genes identified so far, those encoding for the desmosomal proteins plakophilin-2 (PKP2), desmoplakin (DSP), and desmoglein-2 (DSG2) are the most commonly mutated. So far, little is known about the molecular mechanism(s) behind such a varied spectrum of phenotypes, although it has been shown that the causative mutations not only lead to structural abnormalities but also affect the miRNA profiling of cardiac tissue. Here, we aimed at studying the pathogenic effects of a nonsense mutation of the desmoglein-2 gene, both at the structural level and in terms of miRNA expression pattern. METHODS AND RESULTS: We generated transgenic mice with cardiomyocyte-specific overexpression of a FLAG-tagged human desmoglein-2 harbouring the Q558* nonsense mutation found in an AC patient. The hearts of these mice showed signs of fibrosis, decrease in desmosomal size and number, and reduction of the Wnt/ß-catenin signalling. Genome-wide RNA-Seq performed in Tg-hQ hearts and non-transgenic hearts revealed that 24 miRNAs were dysregulated in transgenic animals. Further bioinformatic analyses for selected miRNAs suggested that miR-217-5p, miR-499-5p, and miR-708-5p might be involved in the pathogenesis of the disease. CONCLUSION: Down-regulation of the canonical Wnt/ß-catenin signalling might be considered a common key event in the AC pathogenesis. We identified the miRNA signature in AC hearts, with miR-708-5p and miR-217-5p being the most up-regulated and miR-499-5p the most down-regulated miRNAs. All of them were predicted to be involved in the regulation of the Wnt/ß-catenin pathway and might reveal the potential pathophysiology mechanisms of AC, as well as be useful as therapeutic targets for the disease.

Loss of cardiac Wnt/ß-catenin signalling in desmoplakin-deficient AC8 zebrafish models is rescuable by genetic and pharmacological intervention.

Aims: Arrhythmogenic cardiomyopathy (AC) is an inherited heart disease characterized by life-threatening ventricular arrhythmias and fibro-fatty replacement of the myocardium. More than 60% of AC patients show pathogenic mutations in genes encoding for desmosomal proteins. By focusing our attention on the AC8 form, linked to the junctional protein desmoplakin (DSP), we present here a zebrafish model of DSP deficiency, exploited to identify early changes of cell signalling in the cardiac region. Methods and results: To obtain an embryonic model of Dsp deficiency, we first confirmed the orthologous correspondence of zebrafish Dsp genes (dspa and dspb) to the human DSP counterpart. Then, we verified their cardiac expression, at embryonic and adult stages, and subsequently we targeted them by antisense morpholino strategy, confirming specific and disruptive effects on desmosomes, like those identified in AC patients. Finally, we exploited our Dsp-deficient models for an in vivo cell signalling screen, using pathway-specific reporter transgenes. Out of nine considered, three pathways (Wnt/ß-catenin, TGFß/Smad3, and Hippo/YAP-TAZ) were significantly altered, with Wnt as the most dramatically affected. Interestingly, under persistent Dsp deficiency, Wnt signalling is rescuable both by a genetic and a pharmacological approach. Conclusion: Our data point to Wnt/ß-catenin as the final common pathway underlying different desmosomal AC forms and support the zebrafish as a suitable model for detecting early signalling pathways involved in the pathogenesis of DSP-associated diseases, possibly responsive to pharmacological or genetic rescue.

Mutations and polymorphism in bottlenose dolphin (Tursiops truncatus, Montagu 1821) albumin gene: First identification of mutations responsible for inherited bisalbuminemia.

Hereditary bisalbuminemia is an asymptomatic and heterozygous condition in a range of species characterized by the presence of two serum albumin fractions with different electrophoretic mobility resulting in a bicuspid pattern on serum electrophoresis. Bisalbuminemia has been diagnosed by electrophoresis in two bottlenose dolphin (Tursiops truncatus) families, but causative mutations and the inheritance pattern have not been identified. The aims of this work are: to investigate polymorphisms of the bottlenose dolphin albumin gene and to identify mutations causative of bisalbuminemia; to identify the inheritance pattern in two bottlenose dolphin families. Coding regions of the albumin gene were screened for mutations in 15 bottlenose dolphins kept under human care from two distinct families. Eighteen albumin mutations (three synonymous and 15 non-synonymous) were identified. Two non-synonymous variations co-segregated with bisalbuminemic phenotype: p.Phe146Leu in exon 4 and p.Tyr163His in exon 5. The amino acid change in exon 5 was associated with the secondary and/or tertiary structure variation of the protein and has been reported as causative of bisalbuminemia in humans. Pedigree analysis of the dolphin families showed an autosomal codominant inheritance pattern. In this work, the mutations potentially responsible for bisalbuminemia were identified and confirmed the autosomal codominant trait in bottlenose dolphins.

Dolphin Morbillivirus in a Cuvier's Beaked Whale (Ziphius cavirostris), Italy.

Dolphin morbillivirus (DMV) has caused several mortality events in Mediterranean striped (Stenella coeruleoalba) and bottlenose (Tursiops truncatus) dolphins populations since 19; in the last 5 years, the virus was reported to infect new hosts in this basin, such as fin whales (Balaenoptera physalus), sperm whales (Physeter macrocephalus), and even a harbor seal (Phoca vitulina). Very recently, a calf Cuvier's beaked whale (Ziphius cavirostris) calf stranded on the Southern Italian coastline with mild pathological findings suggestive of morbilliviral infection, received the first confirmation of DMV infection in this species by biomolecular evidences on lung tissue. This new cross-species infection report, along with 19% of the cetaceans specimens examined by the Italian Stranding Network being found positive to DMV, support the hypothesis of an endemic circulation of this virus among Mediterranean cetaceans.

Circulating Cell-Free DNA in Dogs with Mammary Tumors: Short and Long Fragments and Integrity Index.

Circulating cell-free DNA (cfDNA) has been considered an interesting diagnostic/prognostic plasma biomarker in tumor-bearing subjects. In cancer patients, cfDNA can hypothetically derive from tumor necrosis/apoptosis, lysed circulating cells, and some yet unrevealed mechanisms of active release. This study aimed to preliminarily analyze cfDNA in dogs with canine mammary tumors (CMTs). Forty-four neoplastic, 17 non-neoplastic disease-bearing, and 15 healthy dogs were recruited. Necrosis and apoptosis were also assessed as potential source of cfDNA on 78 CMTs diagnosed from the 44 dogs. The cfDNA fragments and integrity index significantly differentiated neoplastic versus non-neoplastic dogs (P<0.05), and allowed the distinction between benign and malignant lesions (P<0.05). Even if without statistical significance, the amount of cfDNA was also affected by tumor necrosis and correlated with tumor size and apoptotic markers expression. A significant (P<0.01) increase of Bcl-2 in malignant tumors was observed, and in metastatic CMTs the evasion of apoptosis was also suggested. This study, therefore, provides evidence that cfDNA could be a diagnostic marker in dogs carrying mammary nodules suggesting that its potential application in early diagnostic procedures should be further investigated.

Genomic and structural investigation on dolphin morbillivirus (DMV) in Mediterranean fin whales (Balaenoptera physalus).

Dolphin morbillivirus (DMV) has been deemed as one of the most relevant threats for fin whales (Balaenoptera physalus) being responsible for a mortality outbreak in the Mediterranean Sea in the last years. Knowledge of the complete viral genome is essential to understand any structural changes that could modify virus pathogenesis and viral tissue tropism. We report the complete DMV sequence of N, P/V/C, M, F and H genes identified from a fin whale and the comparison of primary to quaternary structure of proteins between this fin whale strain and some of those isolated during the 1990-'92 and the 2006-'08 epidemics. Some relevant substitutions were detected, particularly Asn52Ser located on F protein and Ile21Thr on N protein. Comparing mutations found in the fin whale DMV with those occurring in viral strains of other cetacean species, some of them were proven to be the result of diversifying selection, thus allowing to speculate on their role in host adaptation and on the way they could affect the interaction between the viral attachment and fusion with the target host cells.

Detection of hereditary bisalbuminemia in bottlenose dolphins (Tursiops truncatus, Montagu 1821): comparison between capillary zone and agarose gel electrophoresis.

BACKGROUND: Hereditary bisalbuminemia is a relatively rare anomaly characterized by the occurrence of two albumin fractions on serum protein separation by electrophoresis. In human medicine, it is usually revealed by chance, is not been clearly associated with a specific disease and the causative genetic alteration is a point mutation of human serum albumin gene inherited in an autosomal codominant pattern. This type of alteration is well recognizable by capillary zone electrophoresis (CZE), whilst agarose gel electrophoresis (AGE) not always produces a clear separation of albumin fractions. The aims of this study is to report the presence of this abnormality in two separate groups of related bottlenose dolphins and to compare the results obtained with capillary zone and agarose gel electrophoresis. RESULTS: Serum samples from 40 bottlenose dolphins kept under human care were analyzed. In 9 samples a double albumin peak was evident in CZE electrophoresis while no double peak was noted in AGE profile. Since only an apparently wider albumin peaks were noted in some AGE electrophoretic profiles, the ratio between base and height (b/h) of the albumin peak was calculated and each point-value recorded in the whole set of data was used to calculate a receiver operating characteristic curve: when the b/h ratio of albumin peak was equal or higher than 0.25, the sensitivity and specificity of AGE to detect bisalbuminemic samples were 87 and 63 %, respectively. The bisalbuminemic dolphins belong to two distinct families: in the first family, all the siblings derived from the same normal sire were bisalbuminemic, whereas in the second family bisalbuminemia was present in a sire and in two out of three siblings. CONCLUSIONS: We report for the first time the presence of hereditary bisalbuminemia in two groups of related bottlenose dolphins identified by means of CZE and we confirm that AGE could fail in the identification of this alteration.