Evaluation of quantity and purity of miRNAs extracted from different matrices collected from dogs with Mast Cell Tumours.

MicroRNAs (miRNAs) are a class of short non-coding RNA, which interact with the 3’ UTR region of complementary mRNA to decrease or inhibit the translation of proteins (Lai, 2002). MiRNAs regulate pathways in various pathophysiological status, and are regarded as biomarkers for early diagnosis of several diseases, including cancer (Di Leva et al., 2014).The study aims to evaluate the quality and purity of miRNAs extracted from a) 11 archival Formalin Fixed and Paraffin Embedded (FFPE) samples of Mast Cell Tumour (MCT) at stage I, II, III and IV, and 8 intra-patient healthy controls; b) samples collected during surgery, including 6 samples of saliva, primary tumour biopsy and serum/plasma. The quality of miRNA largely influence the downstream experiments, and must be carefully evaluated before performing for examples, the sequencing reaction. MiRNA extraction was carried out using commercial kits (Qiagen) and quantify using Small RNA Kit (Agilent) on Agilent 2100 Bioanalyzer. The results showed that the concentration of miRNAs from FFPE, saliva,  primary tumor biopsy and serum was acceptable with a Median (Me)= 56,91 ng/ml, Me=10,30 ng/ml, Me=3,44 ng/ml and  Me=0,71 ng/ml, and a miRNA/Small RNA ratio of 48%, 61%, 17% and 76%, respectively. The concentration of miRNAs from plasma was not detectable. Studies reveal that plasma ranks as the first choice source for diagnostic purpose, much more than serum (Aung et al., 2014), but the debate remains open and subsequent analyses are needed.The concentration of miRNAs from FFPE and saliva samples is higher than that from other matrices. Possible explanations include a) different quantity and quality of starting materials; b) nucleic acids fragmentation, due to the formalin fixation and paraffin embedded procedure; c) presence of nucleases in saliva, which produce small fragments recognized as miRNAs or smallRNAs.In conclusion, the quantity and the purity of miRNAs, obtained using Qiagen commercial kits, are reliable for further NGS analysis

Expression Analysis of MicroRNAs in FFPE samples of canine cutaneous and oral melanoma by RT-qPCR

MicroRNA (miRNA), a class of small, non-coding RNA - regulating post-transcriptionally protein expression - are emerging as clinical biomarkers in many pathologies, including cancer [1]. Since miRNA are supposed to represent fundamental key regulators, better understanding of melanoma biology is essential to improve staging and therapy. The aim of the study was to investigate whether miRNA expression can vary in canine melanoma samples derived from formalin-fixed-paraffin-embedded (FFPE) tissues. Experimental design of the study included three groups, each one composed of 7 animals: i) control healthy skin group ii) oral melanoma group iii) skin melanoma group. The histhopatology and immunoistochemistry details of dogs included in the study are previously reported  [2]. Two tissue slides were used for miRNA extraction. The expression levels of seven miRNA - miR-145, miR-146a, miR-425-5p, miR-223, miR-365, miR-155 and miR-134 - were detected and assessed by qPCR using TaqMan® probes [3-5]. Five miRNA were significantly up-(n=3) or down-(n=2) regulated. In details, miR-146a and miR-155 abundance was increased as compared with control in both oral and skin melanoma (Fig 1 B,E) (p = 0.004 and 0.014 and p = 0.043 and 0.035 respectively), while the levels of miR-145 and miR-365 were lower (Fig 1 A,D) (p = 0.018 and 0.008 and p = 0.01 and 0.028, respectively). MiR-425-5p was upregulated (p = 0.039) only in skin melanoma (Fig. 1 C). Furthermore, functional analysis, carried out using miRNet web-based tool, showed that 76 genes related to cancer-associated pathways were possible target of these five microRNA (p = 6.95E-9); in particular, 21 target genes were associated with melanoma (p = 1.47E-5), including BRAF and CDK, E2F, FGF and PIK3 families. In conclusion, miR-145, miR-146a, miR-425-5p, miR-365 and miR-155 are differentially expressed in melanoma and healthy FFPE samples, suggesting that they may play a role in canine melanoma pathogenesis and/or progression

MicroARNs como biomarcadores de salud y bienestar animal en cerdos

Los microARNs (miARNs) son pequeñas moléculas de ARN no codificante muy conservadas que intervienen en una amplia gama de procesos biológicos mediante la regulación postranscripcional de la expresión génica. Un aspecto intrigante en la identificación de estas moléculas como biomarcadores se deriva de su papel en la comunicación entre células, su secreción activa desde las células al medio extracelular, su alta estabilidad en los fluidos corporales y su facilidad de obtención.Todas estas características confieren a los miARNs el potencial para convertirse en una herramienta no invasiva que permita evaluar el bienestar animal en condiciones de estrés metabólico, ambiental y de manejo. Esta revisión ofrece una visión general de los conocimientos actuales sobre el uso potencial de miARNs tisulares y/o circulantes como biomarcadores para la evaluación del estado de salud y bienestar en el porcino

Widespread extrahepatic expression of acute-phase proteins in chicken (Gallus gallus) tissues

Acute Phase Proteins (APP) are plasma proteins that can modify their expression in response toinflammation caused by tissue injury, infections, immunological disorders, or stress. Although APP areproduced mainly in liver, extrahepatic production has been described (Marques et al., 2016; Lecchi etal., 2012). The aim of this work was to study the extrahepatic gene expression of five APP, namely α1-acid glycoprotein (AGP), Serum amyloid A (SAA), Haptoglobin-like protein (PIT54), C-rective protein(CRP) and Ovotransferrin (OVT) (O'Reilly and Eckersall, 2014) in different healthy chicken (Gallus gallus)tissues by quantitative real time PCR (qPCR) and immunohistochemistry to detect the precise locationof the proteins.APP gene expression was higher in liver compared with other tissues. mRNA coding for CRP, OVT andSAA was detected in all tissues involved in this study with a higher expression in gastrointestinal tract,respiratory system and lymphatic system. SAA expression was particularly high in cecal tonsil, lung,spleen and meckel’s diverticulum, whereas OVT showed a high expression in lung, bursa of Fabricius,pancreas, brain and adipose tissue. AGP and PIT54 was also detected in pericardial adipose tissue,spleen, kidney, lung, mucosa of proventriculus, mucosa of gizzard and pancreas but, oppositely to SAA,their mRNA was not detected in meckel’s diverticulum, cecal tonsil and bursa of Fabricius. These resultssuggest that each tissue is able to express different amount of APP even in healthy conditions andmount a local acute phase reaction. Immunohistochemistry to detect the precise location for AGP, OVTand SAA using available antibodies is ongoing

Serum miRNA disregulation during transport-related stress in turkey (Meleagris gallopavo)

MicroRNAs (miRNAs) are small 21-25 nucleotide regulatory non-coding RNAs that modulate gene expression in eukaryotic organisms. miRNAs are complementary to the 3′-untranslated regions of mRNA and act as post-transcriptional regulators of gene expression, exhibiting remarkable stability in extracellular fluids such as blood. Turkey (Meleagris gallopavo) farming is a species economically relevant but the lack of efficient protocols for the evaluation of commercial turkeys prevents to measure the impact of industry practices on birds productivity and welfare. In order to identify potential molecular biomarkers for monitoring stress in turkey’s handling, we investigated by TaqMan qPCR the abundance of five circulating miRNA, namely miR-22, miR-155, miR-181a, miR-204 and miR-365, previously demonstrated to be involved in stress in chicken due to feed deprivation. Road transportation related procedures were selected as stressful model for this study. The serum of twenty healthy animals was collected before and after 2h transportation. Our results demonstrated that miR-22, miR-155 and miR-365 are statistically more expressed after road transportation. Receiver-operator characteristics (ROC) analysis was used to estimate the diagnostic value of these miRNAs to evaluate the stress in animals. The serum level of miR-22, miR-155 and miR-365 can discriminate stressed from non-stressed animals with an AUC=0.763, 0.710 and 0.704, respectively, and the average expression of their combination has the same specificity (AUC=0.745). miR-22, miR-155 and miR-365 are stress-specific markers and can be considered as suitable biomarkers to identify turkeys stressed by road transportation

Short communication:Intra- and inter-individual milk microbiota variability in healthy and infected water buffalo udder quarters

The concept that ruminant mammary gland quarters are anatomically and physiologically unrelated has been recently challenged by immunological evidence. How this interdependence reflects on individual quarter milk microbiota is unknown. The aim of the present study was to cover this gap by investigating the interdependence of quarters among the same mammary gland at the milk microbiota level using next-generation sequencing of the V4\u201316S rRNA gene. A total of 52 samples were included in this study and classified as healthy or affected by subclinical mastitis. Extraction of DNA, amplification of the V4\u201316S rRNA gene, and sequencing using Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, Waltham, MA) were carried out. We found that the intra-individual variability was lower than the inter-individual one. The present findings further support at milk microbiota level the hypothesis of the interdependence of quarters, as previously demonstrated following immunological studies, suggesting that individual factors (e.g., immunity, genetics) may have a role in modulating milk microbiota

Characterization of skin surface and dermal microbiota in dogs with mast cell tumor

The skin microbiota interacts with the host immune response to maintain the homeostasis. Changes in the skin microbiota are linked to the onset and the progression of several diseases, including tumors. We characterized the skin surface and dermal microbiota of 11 dogs affected by spontaneous mast cell tumor (MCT), using skin contralateral sites as intra-animal healthy controls. The microbial profile differed between healthy and tumor skin surfaces and dermis, demonstrating that the change in microbiota composition is related to the presence of MCT. The number of observed taxa between MCT and healthy skin surfaces was detected, showing a decrease in number and heterogeneity of taxa over the skin surface of MCT, at both inter- and intra-individual level. Preliminary data on bacterial population of MCT dermis, obtained only on three dogs, demonstrated an intra-individual reduction of taxa number when compared to the skin surface. Taxonomy reveals an increase of Firmicutes phylum and Corynebacteriaceae family in MCT skin surface when compared to the healthy contralateral. In conclusion, we demonstrate that microbial population of skin surface and dermis is related to mast cell tumor. Our study provides the basis for future investigations aiming to better define the interaction between mast cell tumors, microbiota and host immune response

Identification of Altered miRNAs in Cerumen of Dogs Affected by Otitis Externa

Otitis externa is one of the most common diseases in dogs. It is associated with bacteria and yeast, which are regarded as secondary causes. Cerumen is a biological substance playing an important role in the protection of ear skin. The involvement of cerumen in immune defense is poorly understood. MicroRNAs can modulate the host immune response and can provide promising biomarkers for several inflammatory and infectious disorder diagnosis. The aims of this study were to profile the cerumen miRNA signature associated with otitis externa in dogs, integrate miRNAs to their target genes related to immune functions, and investigate their potential use as biomarkers. Cerumen was collected from healthy and otitis affected dogs and the expression of miRNAs was profiled by Next Generation Sequencing; the validation of the altered miRNAs was performed using RT-qPCR. The potential ability of miRNAs to modulate immune-related genes was investigated using bioinformatics tools. The results pointed out that 32 miRNAs, of which 14 were up- and 18 down-regulated, were differentially expressed in healthy vs. otitis-affected dogs. These results were verified by RT-qPCR. To assess the diagnostic value of miRNAs, ROC analysis was carried out, highlighting that 4 miRNAs are potential biomarkers to discriminate otitis-affected dogs. Bioinformatics showed that cerumen miRNAs may be involved in the modulation of host immune response. In conclusion, we have demonstrated for the first time that miRNAs can be efficiently extracted and quantified from cerumen, that their profile changes between healthy and otitis affected dogs, and that they may serve as potential biomarkers. Further studies are necessary to confirm their diagnostic value and to investigate their interaction with immune-related genes

Effect of HLA-matching recipients to donor non-inherited maternal antigens on outcomes after mismatched umbilical cord blood transplantation for hematologic malignancy

peer reviewe

Circulating MiR-30b-5p is upregulated in Cavalier King Charles Spaniels affected by early myxomatous mitral valve disease.

There is a growing interest in developing new molecular markers of heart disease in young dogs affected by myxomatous mitral valve disease. The study aimed to measure 3 circulating microRNAs and their application as potential biomarkers in the plasma of Cavalier King Charles Spaniels with early asymptomatic myxomatous mitral valve disease. The hypothesis is that healthy Cavalier King Charles Spaniels have different microRNA expression profiles than affected dogs in American College of Veterinary Internal Medicine (ACVIM) stage B1. The profiles can differ within the same class among subjects of different ages. This is a prospective cross-sectional study. Thirty-three Cavalier King Charles Spaniels in ACVIM stage B1 were divided into three groups (11 younger than 3 years, 11 older than 3 years and younger than 7 years, and 11 older than 7 years), and 11 healthy (ACVIM stage A) dogs of the same breed were included as the control group. Three circulating microRNAs (miR-1-3p, miR30b-5p, and miR-128-3p) were measured by quantitative real-time PCR using TaqMan® probes. Diagnostic performance was evaluated by calculating the area under the receiver operating curve (AUC). MiR-30b-5p was significantly higher in ACVIM B1 dogs than in ACVIM A subjects, and the area under the receiver operating curve was 0.79. According to the age of dogs, the amount of miR-30b-5p was statistically significantly higher in group B17y (2.7 folds, P = 0.018) than in group A. The area under the receiver operating curves were fair in discriminating between group B17y and A (AUC 0.822). Identifying dogs with early asymptomatic myxomatous mitral valve disease through the evaluation of miR-30b-5p represents an intriguing possibility that certainly merits further research. Studies enrolling a larger number of dogs with preclinical stages of myxomatous mitral valve disease are needed to expand further and validate conclusively the preliminary findings from this report