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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

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

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.

Cyanobacteria are characterized by high iron content. In this research, we collected ten commercial samples of Arthrospira spp. sold as food supplement to determine iron content and assess whether iron speciation showed variability among samples and changed respect to A. platensis grown in controlled conditions. Particular attention was also paid to phycocyanin, as an iron-binding protein. In six of the ten samples, 14 essential and non-essential trace elements were analysed using ICP-MS. Iron content measured in samples using atomic absorption spectrometry (AAS) varied from 353 (sample S5) to 1459 (sample S7) µg g−1 dry weight and was in the range of those reported by other authors in commercial supplements. Iron speciation was studied using size exclusion chromatography followed by the analysis of the collected fraction for the determination of iron by AAS and for protein separation using SDS-PAGE. Overlapping chromatographic profiles were obtained for total proteins, phycocyanin and iron, although quantitative differences were evidenced among the samples analysed. In most samples, iron was mainly bound to ligands with high molecular mass; however, in four samples iron was also bound to ligands with low molecular mass. In fractions containing the most relevant iron burden, the principal protein was phycocyanin, confirming its role as an iron-binding protein in commercial samples.

Prevention is essential to reduce Colorectal Cancer (CRC) mortality. We previously reported a panel of four genes: CEACAM6, LGALS4, TSPAN8, COL1A2 (CELTiC) able to discriminate patients with CRC. Here, we assessed the CELTiC panel by quantitative polymerase chain reaction, in the blood of 174 healthy subjects, who resulted negative to the faecal immunochemical test (FITN). Using non-parametric statistic and multinomial logistic models, the FITN were compared to previously analysed subjects: 36 false positive FIT (NFIT), who were negative at colonoscopy, 36 patients with low risk lesions (LR) and 92 patients with high risk lesions or CRC (HR/CRC). FITN showed a significantly lower expression of the four genes when compared to HR/CRC. Moreover, FITN showed a significantly lower expression of TSPAN8 and COL1A2 compared to NFIT and LR patients. The multinomial logistic model confirmed that TSPAN8 alone specifically discriminated FITN from NFIT, LR and HR/CRC, while LGALS4 was able to differentiate FITN from false positive FIT. Finally, ROC curves analysis of the comparisons between FITN and HR/CRC, LR or NFIT reported AUC greater than 0.87, with a sensitivity and specificity of 83% and 76%, respectively. The CELTiC panel was confirmed a useful tool to identify CRC patients and to discriminate false FIT positive subjects.

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&ndash, 1065) and urine creatinine (median 227.23 mg/dL, range 83.02&ndash, 489.75) when compared with healthy cats (median 1067, range 1040&ndash, 1080, P &lt, 0.001, median 406.50 mg/dL, range 195.32&ndash, 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&ndash, 1.03, median 0.23, range 0.10&ndash, 0.80, respectively), however UPC results were significantly increased in both groups, if compared with healthy cats (median 0.1, range 0.04&ndash, 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.

Spontaneous 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

Diabetes mellitus (DM) is a common disorder in middle-aged to older dogs. Treatment options, similar to those for humans, include insulin injection, dietary changes and exercise. Since some diabetic dogs may develop humoral immune responses to exogenous insulin resulting in treatment failure, the use of alternative medicine could represent an interesting new therapeutic strategy for DM in addition to the traditional insulin therapy. The main objective of this report was to evaluate a new therapeutic strategy for DM, based on the association of insulin injections with an orally administered extract of Boswellia serrata to improve glycemic control in a diabetic dog. A nine year old female neutered mixed breed dog diagnosed with DM was treated with increasing doses of a porcine insulin zinc suspension starting from 0.2 U/kg up to 0.6 U/kg q 12 hours. Twenty weeks after the start of insulin therapy the duration of effect and glucose nadir were ideal and clinical symptoms had disappeared but hyperglycemia, although less severe, persisted. Supplementation with a dry extract from the gum- resin of Boswellia serrata was initiated, at a dosage of 15 mg/kg q 12 hours, to improve insulin sensitivity or possible remaining endogenous insulin secretion. B. serrata supplementation led to good glycemic control. No side-effect or adverse reaction were observed during the study. The present case report provides the first evidence in veterinary medicine of a positive effect of dietary supplementation with boswellic acids associated with traditional insulin therapy on glycemic control in a diabetic dog. A major pitfall of the study is the lack of a control. More extensive clinical trials are required to provide definitive evidence of B. serrata efficacy. Key words: diabetes mellitus; dog; boswellic acids; glycemic control  VPLIV DODAJANJA BOSVELIJE ( Boswellia serrata ) OB ZDRAVLJENJU Z INZULINOM NA UREJANJE RAVNI GLUKOZE V KRVI PRI PSU S SLADKORNO BOLEZNIJO Sladkorna bolezen oz. diabetes mellitus (DM) je pogosta motnja pri psih v srednjih letih in starejAiih psih. MoA¾nosti zdravljenja, ki so podobne kot pri ljudeh, vkljuAujejo dodajanje inzulina, spremembe v prehrani in telesno aktivnost. Ker lahko nekateri psi s sladkorno boleznijo razvijejo imunski odziv na dodajanje inzulina, kar lahko vodi do neuspeAinega zdravljenja, lahko uporaba alternativne medicine predstavlja zanimivo novo metodo zdravljenja DM poleg tradicionalnega zdravljenja z inzulinom. Glavni cilj raziskave je bil ovrednotiti novo metodo zdravljenja DM, ki temelji na hkratnem dodajanju inzulina in peroralnem dodajanju izvleAka bosvelije ( Boswellia serrata ) za izboljAianje urejanja ravni glukoze v krvi pri psu s sladkorno boleznijo. Devet let stara sterilizirana samica meAiane pasme, ki je bila diagnosticirana s sladkorno boleznijo, je bila zdravljena z naraAiAajoAimi odmerki suspenzije praAiiAjega inzulina in cinka (od 0,2 E/kg do 0,6 U/kg), ki jih je dobivala vsakih 12 ur. Dvajset tednov po zaAetku zdravljenja z inzulinom so bili uAinki in najniA¾ja vrednost izmerjene glukoze v normalnih mejah vrednosti, kliniAni simptomi so izginili, le hiperglikemija je bila Aie vedno prisotna, vendar pa je bila manj resna. Dodatek suhega izvleAka iz gumijeve smole bosvelije Boswellia serrata v odmerku 15 mg/kg vsakih 12 ur je poveAal obAutljivost na inzulin oz. morebitne preostanke endogenega izloAanja inzulina. Dodatek B. serrata je pozitivno vplival na nadzorovanje sladkorne bolezni. Med zdravljenjem niso bili opaA¾eni stranski ali nezaA¾eleni uAinki. PoroAilo o opisanem primeru vsebuje prve dokaze o pozitivnem uAinku prehranskega dopolnila z bosveliAnimi kislinami v veterinarski medicini, povezanimi z obiAajnim zdravljenjem z inzulinom, pri nadzoru glukoze v krvi pri psu s sladkorno boleznijo. Najbolj neugoden del te Aitudije je odsotnost kontrole. Za zagotovitev dokonAnega dokaza o uAinkovitosti B. serrata so potrebni obseA¾nejAii kliniAni poskusi. KljuAne besede: diabetes mellitus ; pes; bosveliAna kislina; nadzor sladkorne bolezn

Objectives The aims of this study were to validate a semi-automated high-resolution electrophoretic technique to quantify urinary albumin in healthy and diseased cats, and to evaluate its diagnostic performance in cases of proteinuria and renal diseases. Methods Urine samples were collected from 88 cats (healthy; chronic kidney disease [CKD]; lower urinary tract disease [LUTD]; non-urinary tract diseases [OTHER]). Urine samples were routinely analysed and high-resolution electrophoresis (HRE) was performed. Within-assay and between-assay variability, linearity, accuracy, recovery and the lowest detectable and quantifiable bands were calculated. Receiver operating curve (ROC) analysis was also performed. Results All coefficients of variation were Conclusions and relevance HRE is an accurate and precise method that could be used to measure albuminuria in cats. UAC was useful to correctly classify proteinuria and to discriminate between healthy and diseased cats. HRE might also provide additional information on urine proteins with a profile of all proteins (albumin and globulins) to aid clinicians in the diagnosis of diseases characterised by proteinuria.

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 4–12% 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±1.12g/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.

Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer's incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15-20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss.