Urine proteome in animals of veterinary interest: species comparison and new biomarkers of nephropathy

Urine is considered an ideal source of biomarkers, however in veterinary medicine a complete study on the urine proteome is still lacking. The present work aimed to apply proteomic techniques to the separation of the urine proteome in dogs, cats, horses, cows and some non-conventional species. High resolution electrophoresis (HRE) was also validated for the quantification of albuminuria in dogs and cats. In healthy cats, applying SDS-PAGE and 2DE coupled to mass spectrometry (MS), was produced a reference map of the urine proteome. Moreover, 13 differentially represented urine proteins were linked with CKD, suggesting uromodulin, cauxin, CFAD, Apo-H, RBP and CYSM as candidate biomarkers to be investigated further. In dogs, applying SDS-PAGE coupled to MS, was highlighted a specific pattern in healthy animals showing important differences in patients affected by leishmaniasis. In particular, uromodulin could be a putative biomarker of tubular damage while arginine esterase and low MW proteins needs to be investigated further. In cows, applying SDS-PAGE, were highlighted different patterns between heifers and cows showing some interesting changes during pregnancy. In particular, putative alpha-fetoprotein and b-PAP needs to be further investigated. In horses, applying SDS-PAGE, was produced a reference profile characterized by 13±4 protein bands and the most represented one was the putative uromodulin. Proteinuric horses showed the decrease of the putative uromodulin band and the appearance of 2 to 4 protein bands at higher MW and a greater variability in the range of MW between 49 and 17 kDa. In felids and giraffes was quantified proteinuria reporting the first data for UTP and UPC. Moreover, by means of SDS-PAGE, were highlighted species-specific electrophoretic patterns in big felids and giraffes

antioxidant enzymes in canine mammary tumors

AbstractSpontaneous mammary tumors are very common in bitches. The involvement of oxidative stress and the function of antioxidant enzymes in cancerogenesis have been studied in depth in human medicine, while data in veterinary medicine are still fragmentary. The main aim of this study was to evaluate the activity and the expression of superoxide dismutases (Cu-ZnSOD and MnSOD) and the activity of catalase (CAT) in canine mammary tumors in comparison with the adjacent healthy tissue. Six female dogs (mean age 10.4 years) were included in this study. After surgery, fresh tumor and healthy tissue samples were immediately frozen in dry ice and stored at −80°C for biochemical analyses, while the remaining parts were used for histopathological analysis. Enzyme activity was measured by spectrophotometric assays and protein expression by western blotting. In canine mammary tumors, Cu-ZnSOD activity and expression increased significantly compared with healthy control tissues (p=0.03). MnSOD showed a significantly lower activity in tumoral tissues at stage 2 (p<0.05), while a significant increase of expression was measured in tumors. CAT activity was significantly higher in healthy tissues respect to tumors (p=0.015). These variations of antioxidant enzymes activities and expression could be related to an increase of oxidative stress in breast cancer tissues and could be considered as biomarker candidates for neoplastic transformation

Essential (Mg, Fe, Zn and Cu) and non-essential (Cd and Pb) elements in predatory insects (Vespa crabro and Vespa velutina): a molecular perspective

The recent introduction of the Asian hornet, Vespa velutina, into Europe has raised concern regarding the threat to honeybees and the competition with the European hornet, Vespa crabro. The aim of this study was to investigated essential (Mg, Fe, Zn, Cu) and non-essential (Cd and Pb) elements in these two species. Element concentrations were determined in the whole body and separately in the head, thorax and abdomen using atomic absorption spectrometry (AAS). The changes in essential element concentration and speciation during metamorphosis were also studied using gel filtration chromatography followed by AAS and proteomic analysis. In both species, the essential elements were more concentrated in the abdomen due to the presence of fat bodies. Magnesium, Fe and Zn concentrations were significantly higher in V. crabro than in V. velutina and could have been related to a higher aerobic energy demand of the former species required to sustain foraging flight at higher speeds and to cover longer distances. Low concentrations of Cd and Pb were indicative of low environmental exposure. The concentration and speciation of essential elements, particularly Fe, varied among the developmental stages, indicating a modification of ligand preferences during metamorphosis. Overall, the results in the present study provide a better understanding of the hornet metal metabolism and a foundation for additional studies

Relative abundance of heat shock proteins and clusterin transcripts in spermatozoa collected form boar routinely utilised in an artificial insemination centre: preliminary results

It is widely accepted that mature sperm contains RNA. The first hypothesis was that sperm RNAs have no functions of their own but are simply residues of spermatogenesis reflecting the events that occurred during their formation in the testes. More recently new discoveries have essentially expanded these views, showing that sperm mRNAs constitute a population of stable full-length transcripts, many of which are selectively retained during spermatogenesis and delivered to oocytes contributing to early embryo development. It is well known that semen quality can be influenced by occasional physical stress, infection, and variation in temperature and the definition of new markers for evaluation of semen could offer knowledge about the fertility potential of a semen sample. The aim of the present study was to evaluate the presence and the relative quantity of transcripts and protein of heat shock protein 70 (HSP70), 90 (HSP90) and clusterin (CLU) in Percoll-selected spermatozoa collected from seven adult boars of proven fertility routinely employed for artificial insemination. Our results showed the presence of HSP70, HSP90 and CLU transcripts with different level of expression: high for HSPs and low for CLU transcripts. The transcript level of both HSPs are similar among selected spermatozoa derived from high quality sperm with the exception of one boar that showed a reduced content of HSP70 and HSP90 mRNA together with a lower semen quality. At protein level, both HSPs were detected with similar amount among all seven boars whilst no band was evidenced for CLU protein

Identification of the most abundant proteins in equine amniotic fluid by a proteomic approach

Characterisation of the physiologic equine amniotic fluid (AF) proteome is a prerequisite to study its changes during diseases and discover new biomarkers. The aim of this study was to identify by a proteomic approach the most abundant proteins of equine AF. AF samples were collected at parturition from 24 healthy mares that delivered healthy foals. All samples were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 4\u201312% gels. A pool of the 24 samples, after SDS-PAGE, was cut in 25 slices, trypsin-digested and analysed by mass spectrometry (MS) for protein identification. Mean AF protein concentration was 1.96\ua0\ub1\ua01.12\ua0g/L. Thirty-four proteins were successfully identified by MS and subsequently categorised according to Gene Ontology (GO). Twelve proteins (e.g. fibronectin, lumican, thrombospondin and fibulin) belonged to or interacted with the extracellular matrix (ECM) playing an important role in the development of foetal tissues. Most of the remaining proteins were classified as transport (e.g. albumin, major allergen Equ c1 and alpha-fetoprotein) delivering nutrients, ions and lipids essential for foetal growth and development. Among these proteins, major allergen Equ c1 is widely studied in human medicine because it induces Ig-E mediated type I allergic reaction. The absence of immunoglobulins in equine AF was also confirmed

“Urinalysis, urinary proteome and metabolome of zoo-housed giraffes (Giraffa camelopardalis) through noninvasive sampling method”.

The study of non-domestic animals withholds more difficulties compared to the domestic counterpart, thus using noninvasive techniques to collect biological samples might play an important role in assessing the health status of wild animals. 1 The present study established the reliability of urine sampling from the ground. A preliminary study was run with 10 urine samples of 10 cows (Bos taurus) housed in a dairy farming in Northern Italy. Urine samples, collected both in sterile cups and from the ground, were analyzed and compared. Results revealed no statistical differences in the variables investigated (p &gt; 0.05, dipstick parameters and USG, protein quantification and UPC and protein electrophoresis), which proved the reliability of this noninvasive sampling method. This method was used for sampling 103 urine samples from 44 zoo-housed giraffes (Giraffa camelopardalis) of four Italian zoos. Urine samples were used to establish the urinalysis reference values in this species and to study the urinary proteome for the first time. The urinary reference values reported as median (lower and upper limit) were: urine specific gravity (USG), 1.030 (1006 - 1.049); urine total proteins (uTP), 17.58 (4.54 – 35.31) mg/dL; urine creatinine (uCr), 154.62 (39.59 – 357.95) mg/dL; urine protein: creatinine ratio (UPC), 0.11 (0.07 – 0.16). In giraffes, most urinary proteins had a low molecular mass (MM) and were present in low quantities. Proteomics disclosed fifteen different proteins, which were involved in the defense against microbes and in the ability of giraffes to concentrate urine. Albumin, lysozyme C, and ubiquitin were the most represented urinary proteins in giraffes. In addition, to define the urinary metabolome profile, 35 urine samples from 35 zoo-housed giraffes of five Italian zoos were used. Metabolomics allowed to identify and quantify 39 molecules and the most represented metabolites were hippurate, creatinine and phenylacetylglycine. This analysis provided information on physiological adaptations of giraffes. Besides, urinary metabolites were influenced by sex: urinary metabolome profile of female showed higher level of acetate, succinate, and lactate, conversely hippurate, phenylacetylglycine, and thymine were more concentrated in male urines. Similarly, the age affected the concentration of three urinary metabolites, namely formate, alanine, and valerate

Early Renal Involvement in Cats with Natural Feline Morbillivirus Infection

Feline morbillivirus (FeMV) is a newly discovered paramyxovirus infecting domestic cats and its role in the pathogenesis of feline chronic kidney disease (CKD) has been suggested, however not confirmed. The primary aim of the study was to evaluate the renal damage associated with FeMV infection in cats. In this retrospective study, clinical and clinicopathological data were compared among 14 FeMV naturally infected, 21 CKD and 22 healthy cats. FeMV positive cats had serum chemistry analytes and main urine chemistry results similar to the healthy subjects. FeMV positive cats had significantly decreased urine specific gravity (median 1054, range 1022-1065) and urine creatinine (median 227.23 mg/dL, range 83.02-489.75) when compared with healthy cats (median 1067, range 1040-1080, P &lt; 0.001; median 406.50 mg/dL, range 195.32-575.58; P &lt; 0.001, respectively). Urine protein:creatinine ratio (UPC) results of FeMV and CKD were not different (median 0.20, range 0.08-1.03; median 0.23, range 0.10-0.80, respectively), however UPC results were significantly increased in both groups, if compared with healthy cats (median 0.1, range 0.04-0.250, P &lt; 0.01). Based on clinical data, serum creatinine concentration, urine specific gravity and UPC results, CKD was suspected by clinicians in 3/14 FeMV cats. Urine protein sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 10/13 (77%) FeMV cats indicated a tubular pattern, with a decrease of uromodulin and an increase in the number and intensity of low molecular weight proteins. FeMV infection can be associated with different grades of renal dysfunction ranging from mild tubular proteinuria with less concentrated urine to azotemia in cats younger than those typically affected by CKD

Extracellular Vesicles and Epidermal Growth Factor Receptor Activation: Interplay of Drivers in Cancer Progression

Extracellular vesicles (EVs) are of great interest to study the cellular mechanisms of cancer development and to diagnose and monitor cancer progression. EVs are a highly heterogeneous population of cell derived particles, which include microvesicles (MVs) and exosomes (EXOs). EVs deliver intercellular messages transferring proteins, lipids, nucleic acids, and metabolites with implications for tumour progression, invasiveness, and metastasis. Epidermal Growth Factor Receptor (EGFR) is a major driver of cancer. Tumour cells with activated EGFR could produce EVs disseminating EGFR itself or its ligands. This review provides an overview of EVs (mainly EXOs and MVs) and their cargo, with a subsequent focus on their production and effects related to EGFR activation. In particular, in vitro studies performed in EGFR-dependent solid tumours and/or cell cultures will be explored, thus shedding light on the interplay between EGFR and EVs production in promoting cancer progression, metastases, and resistance to therapies. Finally, an overview of liquid biopsy approaches involving EGFR and EVs in the blood/plasma of EGFR-dependent tumour patients will also be discussed to evaluate their possible application as candidate biomarkers

Determining the pH in canine urine: comparing visual and automated reading variability of urine dipstick analysis within a small animal teaching hospital

Pulmonary hypertension (PH) is present in approximately 70-80% of dogs with left-sided congestive heart failure (L-CHF) and contributes to clinical signs and outcome. Anecdotal evidence suggests that PH in cats with L-CHF is much less frequent. However, data on the prevalence of PH in cats with L-CHF is not available. This study addresses the general hypothesis that PH secondary to L-CHF is uncommon in cats and that echocardiographic findings in cats with PH are different compared to dogs. This retrospective observational study included 56 healthy cats and 131 cats with L-CHF imaged between 2004 and 2016 at the Ohio State University, Veterinary Medical Center. Key diagnostic variables included tricuspid regurgitation (TR) peak velocity, right atrial (RA) size, right ventricular (RV) size and function, RV wall thickness, pulmonary artery (PA) size, Doppler-derived systolic time intervals of PA flow (STIs), presence of septal flattening, and variables characterizing left atrial size and left ventricular size and function. PH was identified if TR peak velocity was >2.7 m/s (estimated systolic PA pressure >35 mmHg). Parametric and non-parametric statistical test procedures were used to compare normal cats to cats with L-CHF, and cats with and without PH. Tricuspid regurgitation was present in 57/131 (44%) of cats with L-CHF. Pulmonary hypertension was present in 22/131 of cats with L-CHF (17%). In 15/22 cases PH was associated with cardiomyopathy, in 5/22 cases with congenital heart disease, and in 2/22 cases to other causes. All cats with PH (22/22, 100%) had subjectively-assessed right-sided enlargement, with larger RA and RV diameters (P21.5 mm; Sensitivity [Sn] 0.82, Specificity [Sp] 0.42) and RV ventricular diameter (>7.8 mm; Sn 0.91, Sp 0.64) had the most accurate cutoff to predict PH compared to other variables. Method reproducibility (measurement variability) was good, with most coefficients of variation <15%. Compared to dogs, PH is not a common finding in cats with L-CHF. Right-sided enlargement is the main finding in cats with PH, and right-heart dimensions should be used to suspect PH if TR is absent or difficult to measure. Further studies are needed to identify reasons and mechanisms for the obvious differences between dogs and cats

Aberrant MET activation impairs perinuclear actin cap organization with YAP1 cytosolic relocation

: Little is known about the signaling network responsible for the organization of the perinuclear actin cap, a recently identified structure holding unique roles in the regulation of nuclear shape and cell directionality. In cancer cells expressing a constitutively active MET, we show a rearrangement of the actin cap filaments, which crash into perinuclear patches associated with spherical nuclei, meandering cell motility and inactivation of the mechano-transducer YAP1. MET ablation is sufficient to reactivate YAP1 and restore the cap, leading to enhanced directionality and flattened nuclei. Consistently, the introduction of a hyperactive MET in normal epithelial cells, enhances nuclear height and alters the cap organization, as also confirmed by TEM analysis. Finally, the constitutively active YAP1 mutant YAP5SA is able to overcome the effects of oncogenic MET. Overall, our work describes a signaling axis empowering MET-mediated YAP1 dampening and actin cap misalignment, with implications for nuclear shape and cell motility