Serum Proteomic Profiles Reflect the Stages of Myxomatous Mitral Valve Disease in Dogs.

Canine myxomatous mitral valve disease (MMVD) is similar to Barlow's form of MMVD in humans. These valvulopathies are complex, with varying speeds of progression. We hypothesized that the relative abundances of serum proteins would help identify the consecutive MMVD stages and discover new disease pathways on a systemic level. To identify distinction-contributing protein panels for disease onset and progression, we compared the proteomic profiles of serum from healthy dogs and dogs with different stages of naturally occurring MMVD. Dogs were divided into experimental groups on the basis of the left-atrium-to-aorta ratio and normalized left ventricular internal dimension in diastole values. Serum was collected from healthy (N = 12) dogs, dogs diagnosed with MMVD in stages B1 (N = 13) and B2 (N = 12) (asymptomatic), and dogs diagnosed with MMVD in chronic stage C (N = 13) (symptomatic). Serum biochemistry and selected ELISAs (galectin-3, suppression of tumorigenicity, and asymmetric dimethylarginine) were performed. Liquid chromatography-mass spectrometry (LC-MS), tandem mass tag (TMT) quantitative proteomics, and statistical and bioinformatics analysis were employed. Most of the 21 serum proteins with significantly different abundances between experimental groups (p < 0.05, FDR ˂ 0.05) were classified as matrix metalloproteinases, protease inhibitors, scaffold/adaptor proteins, complement components, anticoagulants, cytokine, and chaperone. LC-MS TMT proteomics results obtained for haptoglobin, clusterin, and peptidase D were further validated analytically. Canine MMVD stages, including, for the first time, asymptomatic B1 and B2 stages, were successfully distinguished in dogs with the disease and healthy dogs on the basis of the relative abundances of a panel of specific serum proteins. Most proteins with significantly different abundances were involved in immune and inflammatory pathways. Their role in structural remodeling and progression of canine MMVD must be further investigated. Further research is needed to confirm the resemblance/difference with human MMVD. Proteomics data are available via ProteomeXchange with the unique dataset identifier PXD038475.

Revealing the Changes in Saliva and Serum Proteins of Pigs with Meningitis Caused by Streptococcus Suis: A Proteomic Approach.

Meningitis due to Streptococcus suis causes high mortality and morbidity on pig farms and has increasing zoonotic potential worldwide. Saliva proteome analysis would potentially be useful in elucidating pathophysiological changes and mining for new biomarkers to diagnose and monitor S. suis infection. The objective of this study was to investigate the changes in the salivary and serum proteome profile of piglets with meningitis. The LC-MS/MS TMT proteomic approach was used to analyze saliva and serum samples from 20 male piglets: 10 with meningitis and 10 healthy. In saliva, 11 proteins had higher and 10 had lower relative abundance in piglets with meningitis. The proteins with the highest relative abundance were metavinculin (VCL) and desmocollin-2 (DSC2). Adenosine deaminase (ADA) was selected for validation using a spectrophotometric assay and demonstrated excellent performance in the differentiation between healthy and pigs with meningitis due to S. suis. In serum, the most protruding changes occurred for one SERPIN and haptoglobin (HP). In saliva and serum, the highest number of proteins with altered abundance were linked, via the enrichment analysis, with platelet and neutrophil pathways. Overall, meningitis caused by S. suis resulted in specific proteome changes in saliva and serum, reflecting different pathophysiological mechanisms, and marking new potential biomarkers for this infection.

Saliva changes in composition associated to COVID-19: a preliminary study.

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2), is usually associated with a wide variety of clinical presentations from asymptomatic to severe cases. The use of saliva as a diagnostic and monitoring fluid has gained importance since it can be used to investigate the immune response and to direct quantification of antibodies against COVID-19. Additionally, the use of proteomics in saliva has allowed to increase  our understanding of the underlying pathophysiology of diseases, bringing new perspectives on diagnostics, monitoring, and treatment. In this work, we compared the salivary proteome of 10 patients with COVID-19, (five patients with mild and five patients with severe COVID-19) and ten control healthy patients. Through the application of proteomics, we have identified 30 proteins whose abundance levels differed between the COVID-19 groups and the control group. Two of these proteins (TGM3 and carbonic anhydrase-CA6) were validated by the measurement of gGT and TEA respectively, in 98 additional saliva samples separated into two groups: (1) COVID-19 group, integrated by 66 patients who tested positive for COVID-19 (2) control group, composed of 32 healthy individuals who did not show any sign of disease for at least four weeks and were negative for COVID-19 in RT-PCR. In the proteomic study there were observed upregulations in CAZA1, ACTN4, and ANXA4, which are proteins related to the protective response against the virus disturbance, and the upregulation of TGM3, that is correlated to the oxidative damage in pulmonary tissue. We also showed the downregulation in cystatins and CA6 that can be involved in the sensory response to stimulus and possibly related to the presence of anosmia and dysgeusia during the COVID-19. Additionally, the presence of FGB in patients with severe COVID-19 but not in mild COVID-19 patients could indicate a higher viral aggregation and activation in these cases. In conclusion, the salivary proteome in patients with COVID-19 showed changes in proteins related to the protective response to viral infection, and the altered sensory taste perception that occur during the disease. Moreover, gGT and TEA could be potential biomarkers of respiratory complications that can occurs during COVID 19 although further larger studies should be made to corroborate this.

Multi Platforms Strategies and Metabolomics Approaches for the Investigation of Comprehensive Metabolite Profile in Dogs with Babesia canis Infection.

Canine babesiosis is an important tick-borne disease worldwide, caused by parasites of the Babesia genus. Although the disease process primarily affects erythrocytes, it may also have multisystemic consequences. The goal of this study was to explore and characterize the serum metabolome, by identifying potential metabolites and metabolic pathways in dogs naturally infected with Babesia canis using liquid and gas chromatography coupled to mass spectrometry. The study included 12 dogs naturally infected with B. canis and 12 healthy dogs. By combining three different analytical platforms using untargeted and targeted approaches, 295 metabolites were detected. The untargeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) metabolomics approach identified 64 metabolites, the targeted UHPLC-MS/MS metabolomics approach identified 205 metabolites, and the GC-MS metabolomics approach identified 26 metabolites. Biological functions of differentially abundant metabolites indicate the involvement of various pathways in canine babesiosis including the following: glutathione metabolism; alanine, aspartate, and glutamate metabolism; glyoxylate and dicarboxylate metabolism; cysteine and methionine metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis. This study confirmed that host-pathogen interactions could be studied by metabolomics to assess chemical changes in the host, such that the differences in serum metabolome between dogs with B. canis infection and healthy dogs can be detected with liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) methods. Our study provides novel insight into pathophysiological mechanisms of B. canis infection.

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)

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.