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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.

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.

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Colorectal cancer (CRC) develops over several years, thus, early diagnosis and prevention are fundamental to reduce CRC burden. A new non-invasive blood test based on 4 mRNAs encompassing adhesion molecules such as CEACAM6, as well as a member of the collagen I superfamily, namely COL1A2, combined with LGALS4 and TSPAN8, was recently discovered and referred to as CELTIC panel [1]. The expression of the CELTiC panel was measured by quantitative PCR. The panel of putative biomarkers was subsequently evaluated in 101 subjects resulted positive to fecal immunochemical screening test (FIT) highlighting the potential to distinguish colonoscopy negative-FIT positive patients (NFIT, n=36), patients with low risk lesions (LR, n=36) and with high risk lesions or CRC (HR/CRC, n=92) [2]. In this study 174 healthy FIT negative subjects (FITN) were analysed [3] and compared to previous groups, evaluating also the influence of age and sex. Within the FITN group, CEACAM6 and COL1A2 display significantly lower expression in female than in male, confirming this sex difference of CEACAM6, a differentiation marker in normal colonocytes, also in the older groups (60–70 y.o.) and supporting current data on the importance of gender- and age-specific reference intervals for the early diagnosis of CRC [4,5]. The four genes showed significantly lower expression in FITN than in HR/CRC. Interestingly, TSPAN8 and COL1A2 were significantly lower expressed in FITN also than in NFIT and LR patients. TSPAN8, an integral membrane protein which upregulation promotes metastasis [6], was confirmed by logistic model as able to discriminate FITN from NFIT, LR and HR/CRC. Interestingly, also LGALS4, involved in cell-cell interaction and studied in CRC patients [7,8], was able to differentiate FITN from NFIT (false FIT positive). TSPAN8 and galectins have been described also in exosomes [9] suggesting the origin of the mRNAs of the CELTiC panel. Finally, the CELTIC panel showed good sensitivity (84-90%), specificity (76-81%) and AUC (0.87-0.89) to discriminate FITN from the other groups. The CELTiC panel was confirmed as a useful tool to diagnose CRC highlighting the importance of sex and age. A multicenter cross-sectional study will enroll 800 FIT positive subject at the Universities of Amsterdam and Bologna to better estimates the performances of the CELTiC panel and to further validate its robustness and usefulness in the early diagnosis of CRC and to exclude false FIT positive subjects.

Background EGFR targeting is currently the main treatment strategy for metastatic colorectal cancer (mCRC). Results of different clinical trials show that patients with wild-type KRAS and BRAF benefit from anti-EGFR monoclonal antibodies (moAbs) cetuximab (CTX) or panitumumab. Unfortunately, despite initial response, patients soon became refractory. Tumor heterogeneity and multiple escaping routes have been addressed as the main culprit, and, behind genomic alterations already described, changes in signaling pathways induced by drug pressure are emerging as mechanisms of acquired resistance. We previously reported an association between reduced sensitivity to CTX and increased expression of IL-1. However, how IL-1 mediates CTX resistance in mCRC is still unclear. Methods Under CTX treatment, the upregulation of IL-1R1 expression and a senescence program in sensitive colorectal cancer (CRC) cell lines is examined over time using qPCR, immunoblotting, and immunofluorescence. Results In sensitive CRC cells, IL-1 appeared responsible for a CTX-mediated G0 phase arrest. On the contrary, CTX-resistant CRC cells (CXR) maintained high mRNA levels of IL-1R1 and a post-senescence reprogramming, as indicated by increased SNAIL expression. Interestingly, treatment of CXR cells with a recombinant decoy, able to sequester the soluble form of IL-1, pushed CTX-resistant CRC cells back into a stage of senescence, thus blocking their proliferation. Our model suggests a trans-regulatory mechanism mediated by IL-1 on EGFR signaling. By establishing senescence and regulating EGFR activity and expression, IL-1 exposure ultimately bestows resistance. Conclusions To sum up, our findings point to the combined blockage of IL-1R and EGFR as a promising therapeutical approach to restore sensitivity to EGFR-targeting monoclonal antibodies.

The matrix metalloproteinases (MMPs) are a family of enzymes involved in extracellular matrix remodeling. MMPs are secreted in a latent form and activated by local and infiltrating cells. MMP-2 and -9 are the most studied in reproduction and have been detected in bovine, ovine, equine and human placenta. There is only one study on MMPs in the equine amniotic fluid (AF) reporting a decrease in the activity of MMP-2 in case of premature delivery. The aim of this study was focused on MMP-2 and -9 activity in AF collected at parturition from mares with normal or high-risk pregnancy. High-risk pregnancy was defined as a history of premature udder development/lactation, increase of combined thickness of the uterus and placenta, vulvar discharge and/or mare's systemic illness. The diagnosis of placental insufficiency was confirmed retrospectively after macroscopic and histopatologic examination of the placenta. AF was collected by needle puncture of the amnion within 5 min after its appearance through the vulva. The activity of MMP-2 and -9 was analyzed by in-gel zymography allowing the evaluation of both latent and active forms. Twenty mares with normal pregnancy (group 1) and 8 mares with high-risk pregnancy (group 2) were included. All mares in group 2 had a high-risk pregnancy with a diagnosis of placental insufficiency associated with placental villous hypoplasia, placentitis or placental edema. The bands relative to latent and active forms of MMP-2 were clearly visible in both groups and the activity of latent (P = 0.010) and active (P = 0.004) forms was lower in the AF samples of group 2. The band of the latent form of MMP-9 was visible in 17/20 samples of group 1, while it was completely absent in all samples of group 2. In contrast, the band of the active form was clearly visible and with a greater activity in AF samples of group 2 (P = 0.002). Placental dysfunction seems to induce a lower MMP-2 activity and a higher MMP-9 activity through the release of pro-inflammatory cytokines. Because fetal pulmonary secretions are a likely source of gelatinases in AF during late gestation, the increased MMP-9 activity could be related to fetal distress. These data provide a starting point to better understand the role of MMPs in equine pregnancy, although it should be confirmed in a larger and more homogeneous population of mares with high-risk pregnancy.

The recent introduction of the Asian yellow-legged 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 size exclusion 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 the higher aerobic energy demand of the former species required to sustain foraging flight. 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.

Urinalysis could be helpful to investigate the health status of giraffes held in captivity using noninvasive methods to avoid animal handling or anesthesia. We collected 52 voided urine samples from 20 giraffes of different ages, sexes, and subspecies from the ground. To evaluate potential interference by soil contaminants, a pilot study was performed using 20 urine samples obtained from 10 cows. All bovine and 29 giraffe samples were subjected to routine urinalysis including urine specific gravity (USG). All samples were analyzed for urine total protein (uTP), urine creatinine (uCrea) concentration, and urine protein-to-urine creatinine ratio (UPC). Urinary proteins were separated by SDS-PAGE electrophoresis. No significant differences were determined between free-catch and urine sampled from the ground in cows. Giraffe urine was pale-yellow, with alkaline pH (>8.0) and a mean USG of 1.035 ± 0.013. The uTP, uCrea, and UPC expressed as median (range) were 0.20 (0.08–0.47) g/L, 2.36 (0.62–5.2) g/L, and 0.08 (0.05–0.15), respectively. SDS-PAGE allowed the separation of protein bands with different molecular masses, including putative uromodulin at 90 kD, putative albumin at 64 kD, and putative immunoglobulin heavy and light chains at 49 kD and 25 kD, respectively. Urine collection from the ground appears to be a reliable technique for urinalysis and urine electrophoresis in giraffes.

Simple Summary Honeybees are vital pollinators for the human food chain. Colony depopulation is a serious threat to Apis mellifera populations and unfortunately it is also one of the most elusive and difficult to study. This research deals with the problem at its foundation: population imbalances. The proposed method allows to discriminate, with remarkably good performances, precocious foragers from proper aged ones using SDS-PAGE patterns of haemolymph proteins. Implications and future perspectives are discussed. Abstract Honeybees, as social insects, live in highly organised colonies where tasks reflect the age of individuals. As is widely known, in this context, emergent properties arise from interactions between them. The accelerated maturation of nurses into foragers, stimulated by many negative factors, may disrupt this complex equilibrium. This complexity needs a paradigm shift: from the study of a single stressor to the study of the effects exerted by multiple stressors on colony homeostasis. The aim of this research is, therefore, to study colony population disturbances by discriminating overaged nurses from proper aged nurses and precocious foragers from proper aged foragers using SDS-PAGE patterns of haemolymph proteins and a machine-learning algorithm. The KNN (K Nearest Neighbours) model fitted on the forager dataset showed remarkably good performances (accuracy 0.93, sensitivity 0.88, specificity 1.00) in discriminating precocious foragers from proper aged ones. The main strength of this innovative approach lies in the possibility of it being deployed as a preventive tool. Depopulation is an elusive syndrome in bee pathology and early detection with the method described could shed more light on the phenomenon. In addition, it enables countermeasures to revert this vicious circle.

Wild animals have been used as food since ancient times and, currently, the consumption of unconventional animals is increasing worldwide. The process of cooking meat using traditional recipes includes a variety of ingredients, which can influence the total metal intake from the diet. In this study, the concentrations of eight essential (Fe, Zn, Cu, Mn, Se, Ni, Mo, and Co) and six non-essential (Pb, Cd, Hg, Al, As, and Cr) trace elements were determined in home-processed food obtained from snails and from three common species of game animals (woodcock, pheasant, and hare), seasoned with anchovies, mushrooms, and different vegetables using inductively coupled plasma mass spectrometry (ICP-MS). In general, Fe was the most abundant trace element, ranging from 18 &plusmn, 8 &micro, g/g in pheasant to 99 &plusmn, 76 &micro, g/g in snail, and Co was the least abundant, ranging from 0.007 &plusmn, 0.003 &micro, g/g in hare to 0.093 &plusmn, 0.048 &micro, g/g in snail. Regarding the non-essential trace elements, Pb concentrations showed wide variations, reaching a concentration of 17.30 &micro, g/g in hare, while Cd concentrations were higher in snail, ranging from 0.18 to 0.46 &micro, g/g. These alternative food sources can offer an important contribution to the human nutritional requirements of essential trace elements, in particular of Fe. The high concentrations of Pb and Cd present in some samples should be considered as potentially dangerous for the consumers.

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.

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.

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

Chronic kidney disease (CKD) is a progressive and irreversible disease. Although urine is an ideal biological sample for proteomics and metabolomics studies, sensitive and specific biomarkers are currently lacking in dogs. This study characterised dog urine proteome and metabolome aiming to identify and possibly quantify putative biomarkers of CKD in dogs. Twenty-two healthy dogs and 28 dogs with spontaneous CKD were selected and urine samples were collected. Urinary proteome was separated by SDS-PAGE and analysed by mass spectrometry, while urinary metabolome was analysed in protein-depleted samples by 1D 1H NMR spectra. The most abundant proteins in urine samples from healthy dogs were uromodulin, albumin and, in entire male dogs, arginine esterase. In urine samples from CKD dogs, the concentrations of uromodulin and albumin were significantly lower and higher, respectively, than in healthy dogs. In addition, these samples were characterised by a more complex protein pattern indicating mixed glomerular (protein bands ≥65 kDa) and tubular (protein bands

Urinalysis is widely recognized to be a useful tool in routine health investigations, since it can diagnose numerous pathologies. Considering the paucity of knowledge concerning giraffes, urine from 44 giraffes (Giraffa camelopardalis) (18 males and 26 females, from 3 months of age to 21 years of age) underwent routine urinalysis, 1D-electrophoresis, and protein identification using mass spectrometry, with the aim of identifying the urinary reference values and the urine proteome. The urine specific gravity (USG), urine total proteins (uTP), urine creatinine (uCr), and urine protein:creatinine ratio (UPC) reference values, reported as the median, and lower limit (LL) and upper limit (UL), were 1.030 (1006&ndash, 1.049), 17.58 (4.54&ndash, 35.31) mg/dL, 154.62 (39.59&ndash, 357.95) mg/dL, and 0.11 (0.07&ndash, 0.16), respectively. Mass spectrometry, together with electrophoresis, revealed a pattern of common urinary proteins, albumin, lysozyme C, and ubiquitin were the most represented proteins in the giraffe urine. It has been hypothesized that these proteins could act as a defense against microbes. Moreover, in giraffes, urinalysis could be a valid tool for gauging renal function and physiological status changes.

Urinalysis offers important clinical information regarding not only the kidney function but also the general health status of an organism.The aims of this research were to obtain preliminary data on urine chemistry and electrophoretic protein profile from cows and heifers,to compare electrophoretic profiles of not pregnantwith pregnant animals and to evaluate their changes as the pregnancy progresses. Eight heifers and six cows were included in the study and 97 urine samples were collected. Complete urinalysis was performed and urinary proteins were separated by electrophoresis. Considering the pregnancy as a source of variability, significant differences were reported between pregnant and not pregnantheifers for the urine specific gravity (P=0.005), urine total proteins (P=0.009) and urine total proteins to urine creatinine ratio (UPC) (P=0.008). The majority of urine samples analysed in this study showed common protein bands. A mean of protein bands of 17±3 was detected in heifers, while a mean of 13±3 protein bands was recorded in cows. The putative proteins were uromodulin, transferrin, albumin, heavy and light chains of immunoglobulins. The comparison between pregnant and not pregnantanimals showed qualitative differences, with the absence of threebands in not pregnantcows including the putative alpha-fetoprotein. In conclusion, urinalysis is an economical and a non-invasive diagnostic protocol, which should be routinely used for the clinical evaluation of large animals. The data reported in the present study could be considered suggestive of healthy animals and they confirmed those previously reported in the literature.

Graphical abstract, 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.

Nei mammiferi, l’elettroforesi zonale delle proteine sieriche (SPE) su gel di agarosio premette la loro separazione in 6 frazioni, che solitamente corrispondono ad albumina, α1-, α2-, β1-, β2- e γ-globuline. L’elettroforesi è una tecnica analitica che presenta molti vantaggi: è semi-automatica, necessita di pochi microlitri di campione, è rapida, riproducibile ed economica. La SPE è utilizzata di routine per valutare disproteinemie nel cane e nel gatto, ma è ancora poco utilizzata per la maggior parte degli animali “non convenzionali”. In questo capitolo sono presentati due esempi di applicazioni di elettroforesi: - in un mammifero, il furetto (Mustela putorius furo), per il quale vengono riportati i ferogrammi di riferimento e i valori medi per ciascuna frazione ottenuti da 12 soggetti clinicamente sani, e, per confronto, i ferogrammi di due soggetti affetti uno da linfoma e uno da cimurro; - in un rettile, la testuggine Testudo hermanni, riportando i ferogrammi ed i valori medi ottenuti da esemplari campionati in due periodi dell’anno, agli inizi dell’estate e dell’autunno, ed il confronto con due soggetti affetti uno da tumore cervicale e uno da anoressia.

none 3 si Urine is an ideal biological sample. It is an important source of clinical biomarkers of systemic and renal diseases and can be obtained repeatedly without causing stress. Different analytical protocols for the study of the urine proteome have been used, from the simplest electrophoresis on agarose gels to the most sensitive and complex 2DE. As with urine, a range of bodily fluids (saliva, cerebrospinal, bronchoalveolar, amniotic fluids) are amenable to the tools of proteomics. The identification and characterization of the protein content of such fluids are being used to provide novel insights into the evolutionary adaptations of farm and domestic animal species and to characterize their normal physiological state. A comprehensive review on the application of proteomics to these biological samples is presented. mixed Gloria Isani, Enea Ferlizza, Jarlath E. Nally Gloria Isani, Enea Ferlizza, Jarlath E. Nally

Objective To assess the agreement between point-of-care and laboratory analysers in measuring biochemical and blood gas analytes in venous samples from tortoises and to define preliminary reference intervals for venous blood gas analysis in Hermann's tortoises (Testudo hermanni). Materials and methods Jugular venous blood samples from 47 Hermann's tortoises underwent paired analysis with a portable gas analyser (i-STAT 1, Abaxis), a portable chemical analyser (VetScan VS2, Abaxis), and with the respective reference analysers. Agarose gel electrophoresis was used to determine albumin concentrations on 12 specimens. Agreement was evaluated with Bland-Altman plots and regression analysis using the Passing-Bablok method. Results Point-of-care analysers had variable agreement with the reference analysers, presenting constant or proportional bias depending on the analyte. Relevant analytes in reptiles, such as ionised and total calcium, had acceptable agreement. The method for determining albumin concentration currently available in both point-of-care and laboratory analysers significantly overestimated albumin concentrations as compared to protein electrophoresis. Clinical significance While the use of POC analysers is extremely advantageous in small animal primary care facilities, agreement between point-of-care and laboratory analysers varies depending on the analyte. For certain analytes, interchangeability of results is limited and specific reference intervals for point-of-care analysers are required. Veterinarians should be aware of the size and the direction of the bias of each analyte.

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.

Chronic kidney disease (CKD) is a major cause of mortality in cats, but sensitive and specific biomarkers for early prediction and monitoring of CKD are currently lacking. The present study aimed to apply proteomic techniques to map the urine proteome of the healthy cat and compare it with the proteome of cats with CKD. Urine samples were collected by cystocentesis from 23 healthy young cats and 17 cats with CKD. One-dimensional sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) was conducted on 4-12% gels. Two-dimensional electrophoresis (2DE) was applied to pooled urine samples from healthy cats (n = 4) and cats with CKD (n = 4), respectively. Sixteen protein bands and 36 spots were cut, trypsin-digested and identified by mass spectrometry. 1D-SDS-PAGE yielded an overall view of the protein profile and the separation of 32 ± 6 protein bands in the urine of healthy cats, while CKD cats showed significantly fewer bands (P

Background Hermann's tortoise, Testudo hermanni, is currently on the International Union for Conservation of Nature (IUCN) red list of endangered species. Reptile medicine relies also on laboratory analyses to evaluate health status, but reference ranges for hematology and biochemistry variables and protein electrophoresis in plasma of healthy tortoises are not available. Objective The purposes of this study were to establish reference ranges for select hematologic and biochemical variables in clinically healthy Hermann's tortoises, and evaluate the impact of sex and season. Methods Blood samples were collected from 34 healthy tortoises at the end of September and beginning of July. Blood smears, HCT, concentrations of HGB and select plasma biochemical analytes, select enzyme activities, and plasma protein fractions were evaluated. Reference ranges were determined and checked for influence of sex and sampling time point. Results Typical reptilian RBC and WBC were observed in blood smears. HCT and concentrations of HGB, uric acid and urea, and ALT and AST activities were significantly higher in males than in females. Concentrations of glucose, uric acid, and phosphate, and AST activity were significantly higher at the beginning of July, whereas concentrations of urea and Cl were higher at the end of September prior to hibernation. The electrophoretic protein fractions included albumin, and α, β, and γ globulins. Conclusions The reference ranges defined in the present study are useful for clinical tortoise medicine and conservation. Sex and seasonal sampling were identified as factors significantly affecting hematology and blood chemistry analytes; they should be taken into consideration when assessing tortoise health status.

Summary Alkaline phosphatase (AP) catalyses the detachment of phosphate residues from different substrates. Its activity has been demonstrated in seminal plasma and spermatozoa from porcine and other mammalian species; anyway, the role of AP in male reproduction has not been clarified yet and the aim of this study was to determine AP function in boar sperm capacitation and in vitro fertilization (IVF). AP activity was assayed in seminal plasma and in uncapacitated and in vitro capacitated (IVC) spermatozoa; in addition, capacitation was studied in presence of different doses of AP (1.2 and 2.5 IU/mL). The effect of different doses of AP (1.2 and 2.5 IU/mL) on several sperm parameters after IVC (viability, acrosome integrity with FITC-PSA, capacitation status with CTC staining, tyrosine phosphorylation) and on fertilizing ability during IVF were also evaluated. High AP activity was detected in seminal plasma, in particular in sperm-rich fraction; a lower activity was detected in uncapacitated spermatozoa while a significant decrease was evidenced after IVC. Viability was not changed by AP supplementation of the capacitating medium, whereas acrosome integrity and capacitation status were significantly affected by 1.2 and 2.5 doses, with a dose-dependent decrease in acrosome-reacted cells as well as in CTC B pattern displaying cells. As for sperm head protein phosphorylation, a decrease in relative fluorescence was detected in AP 2.5 group, if compared with capacitated one. After IVF, a dose-dependent decrease in penetrated oocytes was recorded, with an increase in monospermic zygote rate. In conclusion, we demonstrated that AP activity decreases under capacitating condition and that addition of AP to spermatozoa during capacitation results in a depression of the capacitating process and IVF. We can infer that AP plays a role in keeping spermatozoa quiescent until they are ejaculated and in modulating the acquisition of the fertilizing ability.

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.

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: 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.