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Additional file 3: Figure S3. Original blots are provided.

Additional file 1: Figure S1. TRAP IL-1 blocks proliferation and clonogenicity in CTX-resistant Caco-2 cell line. Caco-2 CXR cells (1 �� 103) were seeded in 96-well plate. The next day, Caco-2 CXR cells were treated with control medium supplemented with CTX (10 ��g/ml), only medium (FM 10% FBS), and TRAP IL-1 (20 ��g/ml). After 5 days, cell viability was assessed through AlamarBlue assay. Histograms show the average of two independent experiments quintuplicated (a). To measure clonogenicity, 3 �� 103 of Caco-2 CXR were seeded in 12-well plates in triplicate and treated after 24 h as described in a and b. After 10 days, cells were fixed in 4% PFA and stained with crystal violet for 30 min. The ability of cells to grow in a colony was determined by analyzing the covered area through ImageJ software. Percentage of covered area is shown (b). Statistical analysis was carried out using one-way ANOVA and significance calculated with Tukey���s multiple comparisons test *p

One of the main biotic threat to honey bees all over the world is the ectoparasitic mite Varroa destructor (Acari: Mesostigmata). Without proper treatment, colonies are doomed and collapse within two years. Many treatments are used by beekeepers, with well-established acaricidal efficacy; however very little is known on the impact of the above-mentioned treatments on the wellbeing of the colony. Biochemical markers represent an interesting tool to assess the animal and human welfare. Particularly promising for our purposes seems to be the evaluation of vitellogenin in the hemolymph of a pool of bees. The potential of this protein can be foreseen considering his pivotal role in the homeostasis of social insect colonies. In fact, the function of vitellogenin in these species is not limited to egg yolk constitution: trophic, antioxidant and hormonal function are also described. The aim of this research was to test the impact of two different Varroa control techniques by analyzing the vitellogenin content in the hemolymph. Brood interruption by queen caging (five colonies) and brood removal (five colonies) followed by trickling of Api-Bioxal (Oxalic acid based acaricide) were performed. The hemolymph of 30 bees per colony was sampled and pooled. Four samplings were considered: pre manipulation, after manipulation, autumn (wintering phase) and winter. All the samples underwent SDS-PAGE and quantification of vitellogenin, qualitative analysis of other relevant proteins was also performed. Vitellogenin was successfully isolated and identified by mass spectrometry. Our data are confirmatory of the abundance of this protein in the hemolymph of worker bees and interesting differences were found depending on the season. Major differences were also found between the experimental groups, suggesting the expected different impact of the techniques and stressing the need of an objective tool to better evaluate and understand the techniques for Varroa control.

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.

Urine markers are advocated to early detect kidney damage in the clinical practice, nevertheless histology remains the gold standard. The aim of this study was to evaluate quali-quantitative proteinuria and possible renal damage using different non-invasive tests in dogs affected by leishmaniasis.Based on clinical signs and serology/cytology, 26 affected dogs (Leish) were included. Fifteen healthy, non-proteinuric dogs were selected as Control. Upon admission, all dogs underwent to physical examination, Systolic Blood Pressure (SBP) measurement, clinicopathological evaluation (CBC, Urea, Creatinine, ALT, ALP, Glucose, Calcium, Phosphorous, Sodium, Potassium, Cholesterol, Triglyceride, Albumin, Total Protein), serology, urinalysis, ultrasound examination and Renal Resistive Index (RRI) determination. Urine Total Protein and Urine Albumin to Creatinine ratios (UPC; UAC), urine High Resolution agarose and Silver Staining Sodium-Dodecyl-Sulphate-PolyAcrylamide gel electrophoresis (HRE; SDS-PAGE) were performed. A cut-off of 66 kDa was selected to classify bands in High or Low Molecular Weight (HMW; LMW). Data were analyzed with non-parametric statistics and ROC curve analysis (ROC). A difference was considered significant for p0.5) was detected in 16/26 dogs (P), 5/26 were borderline proteinuric (BLP; UPC 0.2-0.5) and 5/26 were nonproteinuric (NP, UPC 0.17, 89% Sensitivity and 88% Specificity and for UAC >0.013, 94% Sensitivity and 82% Specificity. The Areas under the Curve for UPC and UAC were 0.89 and 0.92, respectively. Non-invasive methods, particularly urinary SDS-PAGE, could be useful to detect an early renal damage in canine leishmaniasis. Further studies are required to correlate these findings to renal histology

Data on proteinuria are lacking in giraffes, therefore the aim of our research was to study urine proteome with sensitive analytical methods to obtain important information regarding not only the kidney function but also the general health status. Twenty-nine samples were collected during 2013 and 2014, through non-invasive methods (from the ground by a syringe) from 4 specimens of Giraffa camelopardalis. Urinary total proteins (UTP), creatinine and the urine protein to creatinine ratio (UP/C) were measured by automated methods (Olympus AU400) routinely used in small animal practice. Urinary proteins were separated by sodium-dodecyl-sulphate polyacrilamide gel electrophoresis (SDS-PAGE) and gels were stained with silver nitrate. Mean UTP±SD was 12.1±0.6 mg/dL ranging from 2.4 to 30 mg/dL, while mean UP/C values were 0.06±0.01. SDS-PAGE yielded an overall view of the protein profile allowing the separation of 7±2 protein bands. Electropherograms showed few protein bands as compared to dog and cat and big felids. The most represented band had an apparent molecular weight (MW) of 67-69 kDa and could be identified as putative albumin. Between 65 and 14 kDa only few low abundance bands were present, while more intense bands had a MW lower than 14 kDa. Our data could be considered as representative of animals with no clinical signs of disease resulted in a useful diagnostic tool that could help clinicians in quantitative and qualitative evaluation of proteinuria also in giraffes.

Data on proteinuria in giraffes are lacking, therefore the aim of our research was to study urine proteome with sensitive analytical methods to obtain important information regarding not only the kidney function but also the general health status. Fifty urine samples were collected during 2013 and 2014, through non-invasive methods from 21 specimens of Giraffa camelopardalis. Urinary total proteins (UTP), creatinine and urine protein to creatinine ratio (UP/C) were measured by automated methods routinely used in small animal practice. Urinary proteins were separated by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gels were stained with silver nitrate. Mean UTP±SD was 16.1±8.8 mg/dL (range 2.4-30 mg/dL), while mean UP/C was 0.08±0.03 (range 0.04-0.2); these values can be considered representative of healthy animals. SDS-PAGE yielded an overall view of the urine protein profile allowing the separation of 6±2 protein bands. Electropherograms showed few protein bands as compared to dogs, cats (Ferlizza et al., 2012) and big felids (Di Cerbo et al., 2013). The most represented band had an apparent molecular weight (MW) of 67-69 kDa and could be identified as putative albumin. Between 65 and 14 kDa only few low abundance protein bands were present, while more intense bands had a MW lower than 14 kDa. This is the first study reporting UTP and UPC ranges in giraffes. 1-D-SDS-PAGE yielded an overall view of the protein profile and resulted in a useful diagnostic tool that could help clinicians in qualitative evaluation of proteinuria. References Di Cerbo P., Ferrari G., Isani G., Ferlizza E., Friedrich K.G., 2013. Urine proteome in big cats (Panthera leo, Panthera tigris, Panthera pardus). In: proceedings of the International Conference on Diseases of Zoo and Wild Animals, Vienna, A. Ferlizza E., Armuzzi E., Buono A., Carpenè E., Andreani G., Dondi F., Isani G., 2012. Urine proteome from healthy dogs, cats, horses and cows. In: proceedings of the European Society of Veterinary Clinical Pathology Conference, Ljubljana, SLO.

Introduction Urine is considered an ideal source of clinical biomarkers as it can be obtained noninvasively, repeatedly and in adequate amounts. In veterinary medicine, the application of proteomics techniques is still very limited. The aim of our work was to produce a preliminary map of the urine proteome of the healthy cats (Felis catus) and to compare it with the proteome of cats affected by chronic kidney disease (CKD). For that we have used an approach based on two-dimensional electrophoresis and protein identification using mass spectrometry (MS). Materials and Methods Urine samples were collected by cystocentesis from 4 healthy and 4 cats affected by CKD and analyzed by 2DE. The first dimension was performed by isoelectric focusing on 17 cm long IPG strips (pH 3-10); the second dimension was performed on 10% SDS-PAGE and stained with colloidal Coomassie. Spots were excised from the gel, reduced, alkylated and digested with trypsin and identified using ESI-Q-TOF MS (Campos et al., 2013). Results and Discussions 2DE allowed the separation of 66 spots in the urine proteome of healthy and CKD cats. Eighteen spots were overrepresented in CKD and nine spots were underrepresented. The 27 differentially expressed spots and the nine most abundant common spots were excised from the gels for MS identification (Figure 1; Table 1). Preliminary 2D map. 21 spots yielded significant results by MS, producing a preliminary feline urine map, including 13 proteins that may be functionally classified as transport (38%), immune and cellular response (38%), and cellular communication and growth (15%). The most abundant protein was cauxin, a serine esterase produced by healthy tubular cells, specifically excreted in urine of cats and probably involved in the synthesis of felinine pheromone (Miyazaki et al., 2007). The transport proteins, albumin, transferrin, haemopexin and haptoglobin all derive from plasma and have been identified as common components of urine also from healthy humans (Candiano et al., 2010). Among the proteins involved in immune and cellular defence response, we identified IgK light chain, protein AMBP and uromodulin. Differently from dogs (Brandt et al., 2014) and humans (Lhotta, 2010), uromodulin is not the most abundant urine-specific protein in cats. The remaining proteins, perlecan and fetuin-A, are involved in cell communication and growth. In particular, perlecan, a negatively charged proteoglycan of the glomerular filtration barrier, has also been identified in dog urine (Nabity et al., 2011). Effect of CKD. Regarding the effect of CKD on the urine proteome, seven differentially represented proteins have been identified These proteins can be indicative of tubular dysfunction when not reabsorbed (e.g. RBP) or not secreted (e.g. uromodulin and cauxin) and could be studied as putative biomarkers of nephropathy. Among the overrepresented proteins, retinol binding protein (RBP) is a 22 kDa protein freely filtered by the glomerulus and reabsorbed by the tubules. The appearance of RBP in urine is a marker of impaired tubular function in humans (Pallet et al., 2014) and it has also been reported in dogs (Nabity et al., 2011) and cats (van Hoek et al., 2008). Interesting underrepresented proteins were uromodulin and cauxin. Uromodulin is a glycoprotein produced by healthy tubular cells and its disappearance has been already proved in humans affected by CKD (Lhotta, 2010) and could be applied also in cats. Regarding cauxin, according to Miyazaki et al., (2007) this protein could be a promising biomarker for the determination of tubular damage in CKD cats. Conclusions 2DE was essential in fractionation of the complex urine proteome in cats, producing a preliminary map that included 13 proteins. In particular, uromodulin, cauxin and perlecan, specifically secreted in urine, could help in the evaluation of renal function. Seven proteins were differentially represented in CKD cats suggesting their use as a putative biomarker of nephropathy in cats and possibly in other veterinary species.

In veterinary medicine there is an increasing interest in the application of proteomic techniques to investigate protein patterns in healthy and diseased animals; however, data on urine proteome are still limited. The aims of this study were to identify a urine protein profile in healthy cats and to compare it with those obtained in patients affected by chronic kidney disease (CKD). Urine samples were collected by cystocentesis from 23 healthy and 18 CKD cats. Urinalysis was performed and urine protein to creatinine ratio (UPC) was calculated. Urine proteins were separated by SDS-PAGE on 4-12% gels, 20 different protein bands were cut, reduced, alkylated and digested by trypsin before ESIQ-TOF mass spectrometry analysis; protein identification was performed using MASCOT science search engine. CKD cats had significantly higher UPC (median 0.9, P < .01) and significantly lower urine specific gravity (USG, median 1.014, P < .01) than healthy cats (UPC 0.1; USG 1.070). SDS-PAGE allowed visualization of qualitative and quantitative differences in protein profiles. CKD cats had significantly less bands (P < .01), particular at molecular weight (MW) > 100 kDa (P < .01). Eleven different proteins were identified by mass spectrometry in cat urine. In CKD a2-macroglobulin (168 kDa) and uromodulin (90 kDa) disappeared, while albumin (64 kDa), retinol-binding protein (RBP, 22 kDa) and cystatin (16 kDa) were enhanced. Cauxin (carboxylesterase 5A, 61 kDa), transferrin (80 kDa), angiotensin-converting enzyme 2 (93 kDa), inter-a-trypsin inhibitor heavy chain H4 (103 kDa), albumin- (55 kDa) and cauxin- (40 kDa) fragments, haptoglobin (45 kDa) and immunoglobulin light chains (24 kDa) were also identified. In conclusion, proteomic techniques were successfully used to investigate urine proteins, revealing different patterns between healthy and CKD cats; some of these proteins could be considered as promising biomarkers of chronic renal damage in feline patients.

Data on urine proteome in big cats are lacking, therefore the aim of our research is to study urine proteome with sensitive analytical methods to monitor the health status in Panthera leo, Panthera tigris and Panthera pardus. Urine samples were collected 4 times from May to December 2012, by noninvasive methods from 2 tigers, 2 lions and 3 leopards from Rome’s bioparco. Urinary total proteins (UTP), creatinine and the urinary protein to creatinine ratio (UP/C) were measured by automated methods (Olympus AU400) routinely used for dogs and cats. Urine samples were analyzed by sodium-dodecyl-sulphate poliacrilamide gel electrophoresis (SDS-PAGE, Invitrogen®) to separate urinary proteins. Mean UTP values were not significantly different in lions, tigers and leopards (40, 43, 46 mg/dl respectively), while mean UP/C values were significantly lower in lions (0.11) than in tigers (0.28) but not than leopards (0.22). In absence of reference ranges, these values could be considered as representative of healthy animals. Regarding qualitative proteinuria, with the highly sensitive SDS-PAGE, 29 different protein bands were visualized in lions, 26 in tigers and 30 in leopards, including the 2 most abundant ones, which could be identified as albumin and putative cauxin, a specific feline’s protein. Noninvasive sampling was easy to apply without causing stress to animals and allowed to collect valuable samples for routine and/or more sensitive analysis.

Serum/plasma protein electrophoresis is the current standard technique in human and veterinary medicine to investigate dysproteinemias and to identify and monitor specific pathologies. Serum/plasma protein electrophoresis application in reptile medicine is limited because of lack of reference values. Aims of this study were to provide reference intervals for plasma protein fractions of 30 clinically healthy Hermann’s tortoises (Testudo hermanni) and compare the results with plasma samples collected from 10 diseased animals. 0.5-0.7 mL of blood were collected from the dorsal cervical sinus and flushed into 1.3 mL lithium heparin tubes. In the experimental conditions, at least 4 fractions were always present in all specimens: albumin, α globulins (frequently resolved in two peaks) β globulins (sometimes separated in two peaks) and γ globulins. In healthy young specimens (1-2 years old), plasma proteins were represented by 42% of albumin, 18,5 % of α globulins, 29,5 % of β globulins and 10% of γ globulins. In healthy adult specimens lower values of β (20 – 22 %) and γ (4 – 5 %) fractions were found, while values of α fractions were increased (28 – 32 %). The high amount of α globulins in adult tortoises could be related to the abundance of two specific protein components of α zone: α2 macroglobulin (α2M) and α lipoproteins (HDL). In unhealthy specimens (traumatized or subjected to surgical operations) a huge increase of β fraction was evident (40-42%) associated to a decrease of albumin fraction (21%). Changes in this fraction are also dramatic in birds with acute disease.

In veterinary medicine there is an increasing interest in the application of proteomic techniques to investigate protein patterns in healthy and diseased animals; however, data on urine proteome are still limited (1,2,3). The aims of our study were to identify a urine protein profile in healthy cats and to compare it with those obtained in patients affected by chronic kidney disease (CKD). Urine samples were collected by cystocentesis from 23 healthy and 18 CKD cats. For all samples urinalysis and urine protein to creatinine ratio (UPC) were performed. Urine proteins were further separated by SDS-PAGE and the bands were reduced, alkylated and then digested by trypsin before ESI-Q-TOF mass spectrometry analysis. Protein identification was performed using MASCOT science search engine. Healthy cats had significantly (p

In veterinary medicine there is an increasing interest in the application of proteomic techniques to investigate protein patterns in healthy and diseased animals; however, data on urine proteome are still limited. The aims of our study were to identify a urine protein profile in healthy cats and to compare it with those obtained in patients affected by chronic kidney disease (CKD). Urine samples were collected by cystocentesis from 23 healthy and 18 CKD cats. For all samples urinalysis and urine protein to creatinine ratio (UPC) were performed. Urine proteins were further separated by SDS-PAGE and the bands were reduced, alkylated and then digested by trypsin before ESI-Q-TOF mass spectrometry analysis. Protein identification was performed using MASCOT science search engine. Healthy cats had significantly (p

Background: Urinary proteome is a topic not deeply investigated in dogs. Leishmaniasis (L) and Cushing’s Syndrome (CS) are frequently associated with persistent proteinuria. Evaluation of urine proteome with sensitive methods is considered a non invasive procedure to monitor the therapy and select novel biomarkers. Objectives: Comparison of urinary electrophoretic profiles between healthy (H) and diseased dogs (L, CS) at the time of diagnosis and during the treatment monitoring. Methods: Urine samples were collected from 49 dogs (12 H, 26 L, 11 CS) and Urine Protein and Albumin to Creatinine ratios (UPC, UAC), HRE and SDS-PAGE were performed. Electrophoretic profiles were analyzed by ImageJ©; proteins were identified by ESI-Q-TOF. Results: UPC and UAC (mean±SD) were significantly higher (p

Background: Leishmaniasis is a frequent cause of nephropathy in dogs. Evaluate urine proteome with sensitive methods is a useful and non invasive method to monitor the therapy and discover novel biomarkers. Objective: Comparing different electrophoretic methods for urine protein separation and monitoring the therapy in dogs affected by leishmaniasis. Methods: Urine samples were collected from 43 leishmaniotic dogs. UP/C, UA/C and high resolution electrophoresis (HRE) were performed for all samples. Moreover, SDS-PAGE (26 samples) and a semi-automated SDS-AGE (12 samples) were tested. Results: UP/C and UA/C median (range) values were 1.45 (0.06-20.08) and 0.38 (0.01-14.8) respectively. With HRE different electrophoretic patterns were evidenced and urinary albumin concentrations (median 32 mg/dL; range 5-463 mg/dL) and percentage (mean±SD; 39±13.7) were determined. With SDS-AGE different patterns of proteinuria were found: 9 dogs presented mixed proteinuria (complete or incomplete) and 3 presented non selective glomerular proteinuria. With SDS-PAGE in most of the samples 35-40 different bands were visualized; the most abundant ones were at apparent molecular weights (expressed in kDa) of 71, 66, 55, 46, 37, 26, 22, 14. Conclusion: HRE was a useful and rapid method to monitor renal function and the therapy effectiveness, in addition urinary albumin can also be quantified. SDS-AGE, according to literature, allowed the localization of nephronal damage. SDS-PAGE, due to its high sensitivity, permitted the visualization of many urine proteins, that could be studied as novel biomarkers of nephropathy.

Background: Data on the urinary proteome of domestic animals are limited, as a consequence the study of urinary proteome of healthy domestic animals is the first step to discover new biomarkers of nephropathy. Objective: The study of the urine proteome from healthy dogs, cats, horses and cows. Methods: Urine samples were collected from clinically healthy dogs (15), cats (10), horses (37) and cows (10). For all samples, UP/C was calculated and SDS-PAGE with silver staining was performed. Results: All samples had UP/C lower than 0.2. After SDS-PAGE, all samples showed many bands, which were undetectable by routine methods. In dogs were found 15-19 bands, in cats 17-20, in horses 13-15 and in cows 15-18. On the basis of the apparent molecular weight (MW) reported in kDa and comparing with human urine proteome, we tried an identification of the most represented proteins: P90 (Tamm-Horsfall), P76 (transferrin), P64 (albumin) P54 (immunoglobulin heavy chain) P27 (immunoglobulin light chain) and P14 (lysozyme). All samples presented putative albumin, transferrin, IG heavy and light chains; Tamm-Horsfall was clearly evident in dogs, horses and cats. Dogs and cows presented proteins mainly at MW lower than albumin, cats presented proteins mainly at MW higher than albumin, while horses presented proteins at both high and low MW. Conclusion: SDS-PAGE/silver staining is a very sensitive method to detect low abundance proteins in urine of healthy domestic animals, allowing the identification of different species-specific electrophoretic profiles. Work is in progress to confirm the identity of the different proteins.