Novel biomarkers in cats with congestive heart failure due to primary cardiomyopathy.

The pathogenesis of feline cardiomyopathy and congestive heart failure (CHF) requires further understanding. In this study, we assessed serum proteome change in feline CHF, aiming to identify novel biomarker for both research and clinical use. The study comprised 15 cats in CHF, 5 cats in preclinical cardiomyopathy and 15 cats as healthy controls. Serum proteome profiles were obtained by tandem mass tag labelling followed by mass spectrometry. Protein concentrations in CHF cats were compared with healthy controls. Western blot was performed for proteomic validation. Correlations were assessed between the altered proteins in CHF and clinical variables in cats with cardiomyopathy to evaluate protein-cardiac association. Bioinformatic analysis was employed to identify pathophysiological pathways involved in feline CHF. Sixteen serum proteins were significantly different between CHF and healthy control cats (P < .05). These included serine protease inhibitors, apolipoproteins and other proteins associated with inflammation and coagulation. Clinical parameters from cats with cardiomyopathy significantly correlated with the altered proteins (P < .05). Bioinformatic analysis identified 13 most relevant functional profiles in feline CHF, which mostly associated with extracellular matrix organization and metabolism. Data are available via ProteomeXchange with identifier PXD017761. SIGNIFICANCE: Cardiomyopathies affect both cats and humans, and they can cause serious consequence such as congestive heart failure (CHF). To date, the pathophysiological mechanism of CHF is not fully understood. In this study, for the first time, we used a proteomic approach combined with bioinformatic analysis to evaluate serum protein change in cats with CHF. Results indicate systemic inflammation, coagulation protein changes, innate immunity and extracellular matrix remodeling are involved in feline CHF, which are largely comparable with findings in previous human studies. Our study provides new insights into CHF and cardiomyopathy in cats, and the identified novel biomarkers and pathophysiological pathways provide valuable information for future studies.

Extracellular vesicles in infectious diseases caused by protozoan parasites in buffaloes.

BACKGROUND: Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. METHODS: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. RESULTS: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. CONCLUSIONS: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.

Serum proteome profiling in canine chronic valve disease using a TMT-based quantitative proteomics approach.

Chronic valve disease (CVD) is the most common clinically significant heart disease of dogs, affecting 20 to 40% of dogs. The aim of this study was to evaluate the serum protein profile of healthy and CVD affected dogs, by means of an isobaric tandem mass tag (TMT) label-based high-resolution quantitative proteomic approach. Additionally, conventional cardiac biomarkers were measured in the serum, functional bioinformatics analysis was employed for elucidating molecular mechanisms and pathways associated with CVD, and validation of proteomic results was performed by immunoassays and Western blotting. Of 290 identified and quantified proteins, 15 proteins showed significantly different abundances (p < .05), including antithrombin-III, alpha-2-antiplasmin, tetranectin, apolipoprotein M, adiponectin, inter-alpha-trypsin inhibitor heavy chain H1, gelsolin and apolipoprotein B-100. The identified proteins with differently abundances are involved in a number of pathways, such as complement and coagulation cascades, haemostasis, regulation of actin cytoskeleton, lipid metabolism and transport. We found comparative similarities with human disease in terms of identified proteins and GO pathways, which confirmed similar pathophysiology of this disease, but also differences, mainly in lipid metabolism. SIGNIFICANCE: There have been few investigations of canine serum proteome despite the potential for biomarker discovery and comparative disease analysis. Establishing serum proteomic signatures in healthy dogs and dogs with CVD will benefit for understanding the aetiology of disease in dogs, identify putative biomarkers and provide models of comparative human disease. Circulating biomarkers are important for understanding of the mechanisms of cardiovascular disease and high incidence of CVD in dogs prioritizes the search for novel biomarkers.

Extracellular vesicles in infectious diseases caused by protozoan parasites in buffaloes

Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. Methods: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. Results: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. Conclusions: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.(AU)

LC-MS/MS analysis of the dog serum phosphoproteome reveals novel and conserved phosphorylation sites: Phosphoprotein patterns in babesiosis caused by Babesia canis, a case study.

Phosphorylation is the most commonly studied protein post-translational modification (PTM) in biological systems due to its importance in controlling cell division, survival, growth, etc. Despite the thorough research in phosphoproteomics of cells and tissues there is little information on circulating phosphoproteins. We compared serum from 10 healthy dogs and 10 dogs affected by B. canis-caused babesiosis with no organ dysfunctions by employing gel-free LC-MS/MS analysis of individual samples and tandem mass tag (TMT) label-based quantitative analyses of pools, both supported by phosphopeptide enrichment. Results showed a moderate number of phosphorylated proteins (50-55), with 89 phosphorylation sites not previously published for dogs although a number of them matched phosphorylation sites found in mammalian orthologs. Three phosphopeptides showed significant variation in babesiosis-affected dog sera compared to controls: Serum amyloid A (SAA) phosphorylated at serine 101 (up-regulation), kininogen 1 phosphorylated at threonine 326, and fibrinogen α phosphorylated at both threonine 20 and serine 22 (down-regulation). 71.9% of the detected phosphorylated sites were phosphoserine, 16.8% phosphothreonine and only 11.2% phosphotyrosine residues. TMT label-based quantitative analysis showed α-2-HS-glycoprotein / Fetuin A to be the most abundant phosphoprotein (50-70% of all phosphoproteins) followed by kininogen-1 (10-20%). The alterations of phosphorylated proteins observed in canine babesiosis caused by Babesia canis suggest new insights into the largely neglected role of extracellular protein phosphorylation in health and disease, encouraging urgent further research on this area. To the best of our knowledge the present study represents the first attempt to characterize canine serum phosphoproteome.

Effects of different n6/n3 ratios and supplementation with DHA and EPA on the testicular histology and lipogenesis in streptozotocin-treated rats.

The aim of this study was to investigate the possible influence of different dietary n6/n3 ratios and DHA/EPA addition on the testis histology, antioxidative status and lipogenesis of streptozotocin-treated rats. Diabetes was induced by a single intraperitoneal injection of streptozotocin (55 mg/kg). The rats were divided into five groups: CON (n6/n3 ratio, 7), CON-DM1 (STZ; n6/n3 ratio, 7), N3-DM1 (STZ; n6/n3 ratio, 1), N6-DM1 (STZ; n6/n3 ratio, 60) and DHA-DM1 (STZ; n6/n3 ratio, 1; added DHA/EPA). Antioxidative status was improved in the DHA-DM1 group. Seminiferous tubule diameter, testicular pathohistological scoring and total lipid content were improved in the N6-DM1 group compared to the other streptozotocin-treated groups. Streptozotocin treatment induced changes in testis fatty acid profile depending on n6/n3 ratio. The fatty acid profile of N6-DM1 group was characterised by similar or increased values for n6 fatty acids compared to the CON group, while the DHA-DM1 group had the lowest content of n6 fatty acids. The content of n3 fatty acids was increased in the N3-DM1 and DHA-DM1 groups. These results suggest that a n6/n3 ratio could significantly influence testicular antioxidative status, histology and lipogenesis and that these effects vary depending on the supplemented fatty acid.

Proteomics in Veterinary Medicine and Animal Science: Neglected Scientific Opportunities with Immediate Impact.

Animal/veterinary proteomics is an evolving field which holds a great promise not only for fundamental and applied discoveries regarding biology and pathology of domestic species, but can also be implemented in comparative applications of human diseases research. Experimental proteomics in domestic animals have advantages over use of rodents, such as multiple sampling in time series and availability of biological samples in sufficient volume for multiple analyses, such that both experimental and natural disease processes can be investigated. While there are certain technical limitations in the expansion of the field, they can currently be circumvented and in the future mastered with a greater participation of proteomic experts, which will in turn drive the accessibility of species-specific reagents, data volume expansion in bioinformatic databases, and increased funding. This Viewpoint highlights some comparative proteomics studies addressing important issues and encourages readers to expand their horizons of domestic animal proteomics research. It will hopefully inspire new fruitful collaborations between veterinary and animal scientists and proteomic specialists for research in these areas that can have immediate and direct impact on health, society, and the economy.

Serum proteome profiling in canine idiopathic dilated cardiomyopathy using TMT-based quantitative proteomics approach.

Idiopathic dilated cardiomyopathy (iDCM) is a primary myocardial disorder with an unknown aetiology, characterized by reduced contractility and ventricular dilation of the left or both ventricles. Naturally occurring canine iDCM was used herein to identify serum proteomic signature of the disease compared to the healthy state, providing an insight into underlying mechanisms and revealing proteins with biomarker potential. To achieve this, we used high-throughput label-based quantitative LC-MS/MS proteomics approach and bioinformatics analysis of the in silico inferred interactome protein network created from the initial list of differential proteins. To complement the proteomic analysis, serum biochemical parameters and levels of know biomarkers of cardiac function were measured. Several proteins with biomarker potential were identified, such as inter-alpha-trypsin inhibitor heavy chain H4, microfibril-associated glycoprotein 4 and apolipoprotein A-IV, which were validated using an independent method (Western blotting) and showed high specificity and sensitivity according to the receiver operating characteristic curve analysis. Bioinformatics analysis revealed involvement of different pathways in iDCM, such as complement cascade activation, lipoprotein particles dynamics, elastic fibre formation, GPCR signalling and respiratory electron transport chain. SIGNIFICANCE: Idiopathic dilated cardiomyopathy is a severe primary myocardial disease of unknown cause, affecting both humans and dogs. This study is a contribution to the canine heart disease research by means of proteomic and bioinformatic state of the art analyses, following similar approach in human iDCM research. Importantly, we used serum as non-invasive and easily accessible biological source of information and contributed to the scarce data on biofluid proteome research on this topic. Bioinformatics analysis revealed biological pathways modulated in canine iDCM with potential of further targeted research. Also, several proteins with biomarker potential have been identified and successfully validated.

Hepatic Lipogenesis and Brain Fatty Acid Profile in Response to Different Dietary n6/n3 Ratios and DHA/EPA Supplementation in Streptozotocin Treated Rats.

SCOPE: We investigated the interaction between streptozotocin (STZ)-induced diabetes and dietary n6/n3 ratio, and its influence on lipogenesis. METHODS AND RESULTS: The animals were treated with STZ and fed with different dietary n6/n3 ratios: 1, 7, and 60, or supplemented with DHA/EPA. Gene expression was assessed by RT-PCR and protein expression by western blotting and immunohistochemistry. Fatty acid profile was determined by GC-MS. Pancreas and liver histology were assessed by hematoxylin and eosin (H&E) staining. STZ-induced characteristic changes in all STZ treated groups, including: increased blood glucose, decreased body mass, increased lipid peroxidation and CD36 expression, decreased 16:1n7 and 18:1n7, increases in 20:3n6, decreases in phospholipid (PL) content of 20:4n6, as well as decreases in the expression of SREBP1c, Δ-9-desaturase (Δ9D), and Δ-5-desaturase (Δ5D). Additionally, other changes occurred that were dependent on the n6/n3 ratio. Among the diabetic groups, the lower n6/n3 ratio caused higher lipid peroxidation and CD36 expression, a greater decrease in 20:4n6 and decreased Δ6-desaturase (Δ6D) expression, while the higher n6/n3 ratio caused increased partitioning of 20:4n6 into hepatic neutral lipids (NL), a decrease in 20:5n3 content, and increased ß-oxidation. CONCLUSION: Presented data suggest that the n6/n3 ratio could significantly influence lipogenesis, lipid peroxidation, and ß-oxidation in STZ-induced diabetes, which could have clinical significance.

MCP-1, KC-like and IL-8 as critical mediators of pathogenesis caused by Babesia canis.

Canine babesiosis caused by the intraerythrocytic protozoan parasite Babesia canis is a tick-borne disease characterized by a host response that involves both cellular and humoral immunity. This study focuses on the secretion of cytokines Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Keratinocyte Chemotactic-like (KC-like), Interleukins (IL)-2, IL-7, IL-8, IL-10, IL-15, IL-18 and Monocyte Chemotactic Protein-1 (MCP-1) in babesiosis caused by Babesia canis upon treatment with Imizol®. We assessed time dependent changes in cytokine levels and tested whether these changes correlate with pathogenesis of the disease. Sixteen healthy dogs and 31 dogs infected with Babesia canis, of which 18 showed complications, were treated with Imizol®. One dog died during the study (3.2%). Longitudinal study was perfomed by monitoring dogs at the first day of presentation (day 1) and 6 days later (day 7). Our results show that higher MCP-1 levels on day 1 are positively associated with the occurrence of complications, (complicated vs. uncomplicated; p = 0.00016). A similar pattern was observed for KC-like on day 1 (p = 0.0326) and day 7 (p = 0.044). Moreover, babesiosis caused by B. canis produced a steady increase in IL-8 levels with a moderate to strong negative correlation with erythrocyte counts and hematocrit in uncomplicated diseased dogs only (Spearman's rank correlation coefficient rs = -0.582 and rs = -0.598 respectively). Like for MCP-1, KC-like levels also differed in complicated and uncomplicated diseased dogs on day 1 (p = 0.03236) and day 7 (p = 0.044). Furthermore, KC-like levels were strongly correlated with IL-8 levels (rs = 0.663-0.7) and non-segmented neutrophil counts (rs = 0.572-0.732) in both diseased groups. Analysis of ROC suggests the use of serum levels of MCP-1 and IL-7 as predictors of the occurrence of complications with an AUC of 0.906 and 0.896 respectively and linear combinations of MCP-1, KC-Like, IL-7 and GM-CSF with values up to AUC = 0.983. Cytokine cluster analysis presented in this study can contribute to a better understanding of the pathogenesis of babesiosis and serve as a prognostic tool for the early detection of cases with highest likelihood of developing complications. Overall, our studies show that infection by B. canis elicits a cytokine pattern that is distinct from that observed with B. rossi, and that some of the inflammatory mediators can be useful to predict complications. Our results also suggest targets for the development of novel therapeutic strategies in babesiosis caused by B. canis.

The Challenges and Advances in Diagnosis of Vector-Borne Diseases: Where Do We Stand?

Vector-borne diseases (VBD) are of major importance to human and animal health. In recent years, VBD have been emerging or re-emerging in many geographical areas, alarming new disease threats and economic losses. The precise diagnosis of many of these diseases still remains a major challenge because of the lack of comprehensive data available on accurate and reliable diagnostic methods. Here, we conducted a systematic and in-depth review of the former, current, and upcoming techniques employed for the diagnosis of VBD.

New approaches and omics tools for mining of vaccine candidates against vector-borne diseases.

Vector-borne diseases (VBDs) present a major threat to human and animal health, as well as place a substantial burden on livestock production. As a way of sustainable VBD control, focus is set on vaccine development. Advances in genomics and other "omics" over the past two decades have given rise to a "third generation" of vaccines based on technologies such as reverse vaccinology, functional genomics, immunomics, structural vaccinology and the systems biology approach. The application of omics approaches is shortening the time required to develop the vaccines and increasing the probability of discovery of potential vaccine candidates. Herein, we review the development of new generation vaccines for VBDs, and discuss technological advancement and overall challenges in the vaccine development pipeline. Special emphasis is placed on the development of anti-tick vaccines that can quell both vectors and pathogens.

Library-based display technologies: where do we stand?

Over the past two decades, library-based display technologies have been staggeringly optimized since their appearance in order to mimic the process of natural molecular evolution. Display technologies are essential for the isolation of specific high-affinity binding molecules (proteins, polypeptides, nucleic acids and others) for diagnostic and therapeutic applications in cancer, infectious diseases, autoimmune, neurodegenerative, inflammatory pathologies etc. Applications extend to other fields such as antibody and enzyme engineering, cell-free protein synthesis and the discovery of protein-protein interactions. Phage display technology is the most established of these methods but more recent fully in vitro alternatives, such as ribosome display, mRNA display, cis-activity based (CIS) display and covalent antibody display (CAD), as well as aptamer display and in vitro compartmentalization, offer advantages over phage in library size, speed and the display of unnatural amino acids and nucleotides. Altogether, they have produced several molecules currently approved or in diverse stages of clinical or preclinical testing and have provided researchers with tools to address some of the disadvantages of peptides and nucleotides such as their low affinity, low stability, high immunogenicity and difficulty to cross membranes. In this review we assess the fundamental technological features and point out some recent advances and applications of display technologies.

High-throughput proteomics and the fight against pathogens.

Pathogens pose a major threat to human and animal welfare. Understanding the interspecies host-pathogen protein-protein interactions could lead to the development of novel strategies to combat infectious diseases through the rapid development of new therapeutics. The first step in understanding the host-pathogen crosstalk is to identify interacting proteins in order to define crucial hot-spots in the host-pathogen interactome, such as the proposed pharmaceutical targets by means of high-throughput proteomic methodologies. In order to obtain holistic insight into the inter- and intra-species bimolecular interactions, apart from the proteomic approach, sophisticated in silico modeling is used to correlate the obtained large data sets with other omics data and clinical outcomes. Since the main focus in this area has been directed towards human medicine, it is time to extrapolate the existing expertise to a new emerging field: the 'systems veterinary medicine'. Therefore, this review addresses high-throughput mass spectrometry-based technology for monitoring protein-protein interactions in vitro and in vivo and discusses pathogen cultivation, model host cells and available bioinformatic tools employed in vaccine development.

Liquid chromatography-mass spectrometry-based parallel metabolic profiling of human and mouse model serum reveals putative biomarkers associated with the progression of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in most western countries. Current NAFLD diagnosis methods (e.g., liver biopsy analysis or imaging techniques) are poorly suited as tests for such a prevalent condition, from both a clinical and financial point of view. The present work aims to demonstrate the potential utility of serum metabolic profiling in defining phenotypic biomarkers that could be useful in NAFLD management. A parallel animal model/human NAFLD exploratory metabolomics approach was employed, using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) to analyze 42 serum samples collected from nondiabetic, morbidly obese, biopsy-proven NAFLD patients, and 17 animals belonging to the glycine N-methyltransferase knockout (GNMT-KO) NAFLD mouse model. Multivariate statistical analysis of the data revealed a series of common biomarkers that were significantly altered in the NAFLD (GNMT-KO) subjects in comparison to their normal liver counterparts (WT). Many of the compounds observed could be associated with biochemical perturbations associated with liver dysfunction (e.g., reduced Creatine) and inflammation (e.g., eicosanoid signaling). This differential metabolic phenotyping approach may have a future role as a supplement for clinical decision making in NAFLD and in the adaption to more individualized treatment protocols.

Salt bridges at the inter-ring interface regulate the thermostat of GroEL.

The chaperonin GroEL consists of a double-ring structure made of identical subunits and displays unusual allosteric properties caused by the interaction between its constituent subunits. Cooperative binding of ATP to a protein ring allows binding of GroES to that ring, and at the same time negative inter-ring cooperativity discharges the ligands from the opposite ring, thus driving the protein-folding cycle. Biochemical and electron microscopy analysis of wild type GroEL, a single-ring mutant (SR1), and two mutants with one inter-ring salt bridge of the chaperonin disrupted (E461K and E434K) indicate that these ion pairs form part of the interactions that allow the inter-ring allosteric signal to be transmitted. The wild type-like activities of the ion pair mutants at 25 degrees C are in contrast with their lack of inter-ring communication and folding activity at physiological temperatures. These salt bridges stabilize the inter-ring interface and maintain the inter-ring spacing so that functional communication between protein heptamers takes place. The characterization of GroEL hybrids containing different amounts of wild type and mutant subunits also indicates that as the number of inter-ring salt bridges increases the functional properties of the hybrids recover. Taken together, these results strongly suggest that inter-ring salt bridges form a stabilizing ring-shaped, ionic zipper that ensures inter-ring communication at the contact sites and therefore a functional protein-folding cycle. Furthermore, they regulate the chaperonin thermostat, allowing GroEL to distinguish physiological (37 degrees C) from stress temperatures (42 degrees C).