SDS-PAGE-Based Quantitative Assay of Hemolymph Proteins in Honeybees: Progress and Prospects for Field Application.

In human and veterinary medicine, serum proteins are considered to be useful biomarkers for assessing the health and nutritional status of the organism. Honeybee hemolymph has a unique proteome that could represent a source of valuable biomarkers. Therefore, the aims of this study were to separate and identify the most abundant proteins in the hemolymph of worker honeybees to suggest a panel of these proteins that could represent useful biomarkers for assessing the nutritional and health status of the colonies and, finally, to analyze them in different periods of the year. Four apiaries were selected in the province of Bologna, and the bees were analyzed in April, May, July, and November. Thirty specimens from three hives of each apiary were sampled and their hemolymph was collected. The most represented bands obtained after 1D sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were cut from the gel, and the proteins were identified using an LC-ESI-Q-MS/MS System. A total of twelve proteins were unmistakably identified; the two most abundant proteins were apolipophorin and vitellogenin, which are known biomarkers of bee trophic and health status. The two other proteins identified were transferrin and hexamerin 70a, the first being involved in iron homeostasis and the second being a storage protein. Most of these proteins showed an increase from April to November, mirroring the physiological changes of honeybees during the productive season. The current study suggests a panel of biomarkers from honeybee hemolymph worth testing under different physiological and pathological field conditions.

Senescence-associated reprogramming induced by interleukin-1 impairs response to EGFR neutralization.

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.

Iron Content, Iron Speciation and Phycocyanin in Commercial Samples of Arthrospira spp.

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.

Essential (Mg, Fe, Zn and Cu) and Non-Essential (Cd and Pb) Elements in Predatory Insects (Vespa crabro and Vespa velutina): A Molecular Perspective.

The recent introduction of the Asian 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.

Biochemical responses to cadmium exposure in Oncorhynchus mykiss erythrocytes.

Cd is known for its carcinogenic effects, however its mechanism of toxicity and in particular its ability to promote oxidative stress is debated. In fact, although it is considered a redox-inactive metal, at high concentration Cd was shown to promote indirectly oxidative stress. In this study we investigated metal accumulation in ex vivo exposed trout (Oncorhynchus mykiss) erythrocytes and Cd dose-dependent effect in terms of RBC viability, cytosolic and mitochondrial ROS levels as well as its effects on mitochondrial membrane depolarization, hemoglobin stability and precipitation. In the concentration range used, Cd did not affect cell viability. However, metal accumulation was associated with an increase in all oxidative indexes evaluated, except mitochondrial superoxide anion production that, on the contrary, was significantly decreased, probably due to a lowered respiration rate associated with interference of Cd with complex I, II and III, as suggested by the observed Cd-dependent mitochondrial membrane depolarization. On the other hand, hemoglobin destabilisation seems to be the major trigger of oxidative stress in this cell type.

Effects of exercise on urinary biochemical parameters and proteins in a group of well-trained military working dogs.

Exercise-induced proteinuria has been widely investigated in humans, also in relation to intensity and duration of activity. Instead, there are only limited publications regarding urinary biochemical parameters and urinary proteins before and after physical activity in dogs. This paper aimed to investigate the effects of exercise on urinary biochemistry and proteins in military dogs. Twenty-four dogs were enrolled in this study. All the dogs were clinically sound, and they were examined before and after activity. Pulse rates (PR) and respiratory rate (RR) were monitored. Urine was sampled before and after a training session of search activity. Standard urinalysis was carried out, urine total proteins and creatinine were measured and the urinary protein:creatinine ratio was calculated; finally, the urinary proteins were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Clinical examination before and after activity did not reveal any pathological finding. After activity, the PR was slightly increased, while the RR was notably increased (p < 0.05). Total proteins, albumin, and their ratio with creatinine were significantly higher after exercise when considering all the dogs included or only the females while, when considering only the males no significant difference was detected. The clinical relevance of this study was related to the possibility of using urine as a non-invasive sample for monitoring health status after training activity and exercise in dogs. An increase in microalbuminuria after search activity, measured using SDS-PAGE could be considered an early biomarker of renal function during training sessions.

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

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

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

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

Supplementation of Boswellia serrata and Salix alba Extracts during the Early Laying Phase: Effects on Serum and Albumen Proteins, Trace Elements, and Yolk Cholesterol.

Extracts from Boswellia serrata (Bs) and Salix alba (Sa) are used as supplements in poultry feed. The aims of this research were to study the possible effects of dietary supplementation with Bs and Sa extracts on serum and albumen proteins, zinc and iron, and yolk cholesterol content in Leghorn hens during the critical phase of the onset of laying. A total of 120 pullets, 17 weeks of age, were assigned to two groups (control (C) and treated (T), n = 60 each). The T group received a supplement containing Bs (5%) and Sa (5%) for 12 weeks. The study lasted 19 weeks. Serum proteins were fractionated using agarose gel electrophoresis (AGE) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Trace elements were determined in serum using atomic absorption spectrometry, and yolk cholesterol was determined using a colorimetric test. No significant differences were observed between control and supplemented hens for the analyzed biochemical indices. Moreover, the supplementation with phytoextracts did not negatively affect the physiological variations in serum proteins; therefore, it can be safely used as a treatment to prevent inflammatory states at onset and during the early laying phase.

The Roadmap of Colorectal Cancer Screening.

Colorectal cancer (CRC) is the third most common form of cancer in terms of incidence and the second in terms of mortality worldwide. CRC develops over several years, thus highlighting the importance of early diagnosis. National screening programs based on fecal occult blood tests and subsequent colonoscopy have reduced the incidence and mortality, however improvements are needed since the participation rate remains low and the tests present a high number of false positive results. This review provides an overview of the CRC screening globally and the state of the art in approaches aimed at improving accuracy and participation in CRC screening, also considering the need for gender and age differentiation. New fecal tests and biomarkers such as DNA methylation, mutation or integrity, proteins and microRNAs are explored, including recent investigations into fecal microbiota. Liquid biopsy approaches, involving novel biomarkers and panels, such as circulating mRNA, micro- and long-non-coding RNA, DNA, proteins and extracellular vesicles are discussed. The approaches reported are based on quantitative PCR methods that could be easily applied to routine screening, or arrays and sequencing assays that should be better exploited to describe and identify candidate biomarkers in blood samples.

A Machine Learning Approach to Study Demographic Alterations in Honeybee Colonies Using SDS-PAGE Fingerprinting.

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.

Activities of matrix metalloproteinase-2 and -9 in amniotic fluid at parturition in mares with normal and high-risk pregnancy.

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.

Noninvasive sampling method for urinalysis and urine protein profile in captive giraffes.

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.

Urinary Reference Values and First Insight into the Urinary Proteome of Captive Giraffes.

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 (1006-1.049), 17.58 (4.54-35.31) mg/dL, 154.62 (39.59-357.95) mg/dL, and 0.11 (0.07-0.16), 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.

Trace Elements in Home-Processed Food Obtained from Unconventional Animals.

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 ± 8 µg/g in pheasant to 99 ± 76 µg/g in snail, and Co was the least abundant, ranging from 0.007 ± 0.003 µg/g in hare to 0.093 ± 0.048 µg/g in snail. Regarding the non-essential trace elements, Pb concentrations showed wide variations, reaching a concentration of 17.30 µg/g in hare, while Cd concentrations were higher in snail, ranging from 0.18 to 0.46 µg/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.

Colorectal cancer screening: Assessment of CEACAM6, LGALS4, TSPAN8 and COL1A2 as blood markers in faecal immunochemical test negative subjects.

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.

Urinary proteome and metabolome in dogs (Canis lupus familiaris): The effect of chronic kidney disease.

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 <65 kDa) proteinuria. Urine spectra acquired by NMR allowed the identification of 86 metabolites in healthy dogs, belonging to 49 different pathways mainly involved in amino acid metabolism, purine and aminoacyl-tRNA biosynthesis or tricarboxylic acid cycle. Seventeen metabolites showed significantly different concentrations when comparing healthy and CKD dogs. In particular, carnosine, trigonelline, and cis-aconitate, might be suggested as putative biomarkers of CKD in dogs. SIGNIFICANCE: Urine is an ideal biological sample, however few proteomics and metabolomics studies investigated this fluid in dogs and in the context of CKD (chronic kidney disease). In this research, applying a multi-omics approach, new insights were gained regarding the molecular changes triggered by this disease in canine urinary proteome and metabolome. In particular, the involvement of the tubular component was highlighted, suggesting uromodulin, trigonelline and carnosine as possible biomarkers of CKD in dogs.

Early Renal Involvement in Cats with Natural Feline Morbillivirus Infection.

Feline morbillivirus (FeMV) is a newly discovered paramyxovirus infecting domestic cats and its role in the pathogenesis of feline chronic kidney disease (CKD) has been suggested, however not confirmed. The primary aim of the study was to evaluate the renal damage associated with FeMV infection in cats. In this retrospective study, clinical and clinicopathological data were compared among 14 FeMV naturally infected, 21 CKD and 22 healthy cats. FeMV positive cats had serum chemistry analytes and main urine chemistry results similar to the healthy subjects. FeMV positive cats had significantly decreased urine specific gravity (median 1054, range 1022-1065) and urine creatinine (median 227.23 mg/dL, range 83.02-489.75) when compared with healthy cats (median 1067, range 1040-1080, P < 0.001; median 406.50 mg/dL, range 195.32-575.58; P < 0.001, respectively). Urine protein:creatinine ratio (UPC) results of FeMV and CKD were not different (median 0.20, range 0.08-1.03; median 0.23, range 0.10-0.80, respectively), however UPC results were significantly increased in both groups, if compared with healthy cats (median 0.1, range 0.04-0.250, P < 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.

Validation of an electrophoretic method to detect albuminuria in cats.

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 <10%, percentage recovery was between 97% and 109% with a high linearity (r = 0.99). HRE allowed the visualisation of a faint band of albumin and a diffused band between alpha and beta zones in healthy cats, while profiles from diseased cats were variable. Albumin (mg/dl) and urine albumin:creatinine ratio (UAC) were significantly ( P <0.05) different between healthy and diseased cats. After ROC analysis, UAC values of 0.035 and 0.074 had a high sensitivity and high specificity, respectively, to classify proteinuria and identify borderline proteinuric cats. Moreover, a UAC of 0.017 had a high sensitivity in distinguishing between healthy and diseased cats. However, UAC was not able to distinguish between renal (CKD) and non-renal diseases (LUTD/OTHER), probably owing to the pathophysiology of CKD in cats, which is characterised by low-grade proteinuria and less glomerular involvement than in dogs. 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.

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

Characterisation of the physiologic equine amniotic fluid (AF) proteome is a prerequisite to study its changes during diseases and discover new biomarkers. The aim of this study was to identify by a proteomic approach the most abundant proteins of equine AF. AF samples were collected at parturition from 24 healthy mares that delivered healthy foals. All samples were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 4-12% 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.