Intraarticular treatment with integrin α10ß1-selected mesenchymal stem cells affects microRNA expression in experimental post-traumatic osteoarthritis in horses.

Osteoarthritis (OA) remains a major cause of lameness in horses, which leads to lost days of training and early retirement. Still, the underlying pathological processes are poorly understood. MicroRNAs (miRNAs) are small non-coding RNAs that serve as regulators of many biological processes including OA. Analysis of miRNA expression in diseased joint tissues such as cartilage and synovial membrane may help to elucidate OA pathology. Since integrin α10ß1-selected mesenchymal stem cell (integrin α10-MSC) have shown mitigating effect on equine OA we here investigated the effect of integrin α10-MSCs on miRNA expression. Cartilage and synovial membrane was harvested from the middle carpal joint of horses with experimentally induced, untreated OA, horses with experimentally induced OA treated with allogeneic adipose-derived MSCs selected for the marker integrin α10-MSCs, and from healthy control joints. miRNA expression in cartilage and synovial membrane was established by quantifying 70 pre-determined miRNAs by qPCR. Differential expression of the miRNAs was evaluated by comparing untreated OA and control, untreated OA and MSC-treated OA, and joints with high and low pathology score. A total of 60 miRNAs were successfully quantified in the cartilage samples and 55 miRNAs were quantified in the synovial membrane samples. In cartilage, miR-146a, miR-150 and miR-409 had significantly higher expression in untreated OA joints than in control joints. Expression of miR-125a-3p, miR-150, miR-200c, and miR-499-5p was significantly reduced in cartilage from MSC-treated OA joints compared to the untreated OA joints. Expression of miR-139-5p, miR-150, miR-182-5p, miR-200a, miR-378, miR-409-3p, and miR-7177b in articular cartilage reflected pathology score. Several of these miRNAs are known from research in human patients with OA and from murine OA models. Our study shows that these miRNAs are also differentially expressed in experimental equine OA, and that expression depends on OA severity. Moreover, MSC treatment, which resulted in less severe OA, also affected miRNA expression in cartilage.

Multi-Omic Temporal Landscape of Plasma and Synovial Fluid-Derived Extracellular Vesicles Using an Experimental Model of Equine Osteoarthritis.

Extracellular vesicles (EVs) contribute to osteoarthritis pathogenesis through their release into joint tissues and synovial fluid. Synovial fluid-derived EVs have the potential to be direct biomarkers in the causal pathway of disease but also enable understanding of their role in disease progression. Utilizing a temporal model of osteoarthritis, we defined the changes in matched synovial fluid and plasma-derived EV small non-coding RNA and protein cargo using sequencing and mass spectrometry. Data exploration included time series clustering, factor analysis and gene enrichment interrogation. Chondrocyte signalling was analysed using luciferase-based transcription factor activity assays. EV protein cargo appears to be more important during osteoarthritis progression than small non-coding RNAs. Cluster analysis revealed plasma-EVs represented a time-dependent response to osteoarthritis induction associated with supramolecular complexes. Clusters for synovial fluid-derived EVs were associated with initial osteoarthritis response and represented immune/inflammatory pathways. Factor analysis for plasma-derived EVs correlated with day post-induction and were primarily composed of proteins modulating lipid metabolism. Synovial fluid-derived EVs factors represented intermediate filament and supramolecular complexes reflecting tissue repair. There was a significant interaction between time and osteoarthritis for CRE, NFkB, SRE, SRF with a trend for osteoarthritis synovial fluid-derived EVs at later time points to have a more pronounced effect.

Sensitive and High-Throughput Exploration of Protein N-Termini by TMT-TAILS N-Terminomics.

Terminal amine isotopic labeling of substrates (TAILS) is a sensitive and robust quantitative mass spectrometry (MS)-based proteomics method used for the characterization of physiological or proteolytically processed protein N-termini, as well as other N-terminal posttranslational modifications (PTMs). TAILS is a well-established, high-throughput, negative enrichment workflow that enables system-wide exploration of N-terminomes independent of sample complexity. TAILS makes use of amine reactivity of free N-termini and a highly efficient aldehyde-functionalized polymer to deplete internal peptides generated after proteolytic digestion during sample preparation. Thereby, it enriches for natural N-termini, allowing for unbiased and complete investigation of differential proteolysis, protease substrate discovery, and analysis of N-terminal PTMs. In this chapter, we provide a state-of-the-art protocol, with detailed steps in all parts of the TAILS sample preparation, MS analysis, and post-processing of acquired data.

Longitudinal Evaluation of Biomarkers in Wound Fluids from Venous Leg Ulcers and Split-thickness Skin Graft Donor Site Wounds Treated with a Protease-modulating Wound Dressing.

Venous leg ulcers represent a clinical challenge and impair the quality of life of patients. This study examines impaired wound healing in venous leg ulcers at the molecular level. Protein expression patterns for biomarkers were analysed in venous leg ulcer wound fluids from 57 patients treated with a protease-modulating polyacrylate wound dressing for 12 weeks, and compared with exudates from 10 acute split-thickness wounds. Wound healing improved in the venous leg ulcer wounds: 61.4% of the 57 patients with venous leg ulcer achieved a relative wound area reduction of ≥ 40%, and 50.9% of the total 57 patients achieved a relative wound area reduction of ≥ 60%. Within the first 14 days, abundances of S100A8, S100A9, neutrophil elastase, matrix metalloproteinase-2, and fibronectin in venous leg ulcer exudates decreased significantly and remained stable, yet higher than in acute wounds. Interleukin-1ß, tumour necrosis factor alpha, and matrix metalloproteinase-9 abundance ranges were similar in venous leg ulcers and acute wound fluids. Collagen (I) α1 abundance was higher in venous leg ulcer wound fluids and was not significantly regulated. Overall, significant biomarker changes occurred in the first 14 days before a clinically robust healing response in the venous leg ulcer cohort.

Small non-coding RNA landscape of extracellular vesicles from a post-traumatic model of equine osteoarthritis.

Extracellular vesicles comprise an as yet inadequately investigated intercellular communication pathway in the field of early osteoarthritis. We hypothesised that the small non-coding RNA expression pattern in synovial fluid and plasma would change during progression of experimental osteoarthritis. In this study, we conducted small RNA sequencing to provide a comprehensive overview of the temporal expression profiles of small non-coding transcripts carried by extracellular vesicles derived from plasma and synovial fluid for the first time in a posttraumatic model of equine osteoarthritis. Additionally, we characterised synovial fluid and plasma-derived extracellular vesicles with respect to quantity, size, and surface markers. The different temporal expressions of seven microRNAs in plasma and synovial fluid-derived extracellular vesicles, eca-miR-451, eca-miR-25, eca-miR-215, eca-miR-92a, eca-miR-let-7c, eca-miR-486-5p, and eca-miR-23a, and four snoRNAs, U3, snord15, snord46, and snord58, represent potential biomarkers for early osteoarthritis. Bioinformatics analysis of the differentially expressed microRNAs in synovial fluid highlighted that in early osteoarthritis these related to the inhibition of cell cycle, cell cycle progression, DNA damage and cell proliferation as well as increased cell viability and differentiation of stem cells. Plasma and synovial fluid-derived extracellular vesicle small non-coding signatures have been established for the first time in a temporal model of osteoarthritis. These could serve as novel biomarkers for evaluation of osteoarthritis progression or act as potential therapeutic targets.

Pre-Competition Oral Findings in Danish Sport Horses and Ponies Competing at High Level.

This study addresses the presence and location of oral lesions in 342 dressage, show jumping, and eventing horses examined at an obligatory veterinary inspection before competing in the Danish National Championship in 2020. Ulcers in the lip commissures were photographed for subsequent pathological analysis. If a lesion was found at the lip commissures on one side, there was an increased risk of finding a similar lesion on the other side (ulcer: p < 0.0001; scarring/depigmentation: p < 0.0001; fissure: p = 0.002; erosion/contusion: p < 0.0001). At the lip commissures, external (cutaneous) ulcers were correlated with internal (mucosal) ulcers (p < 0.0001) and with scarring/depigmentation (p < 0.0001). Both mucosal and cutaneous ulcers were correlated with scarring/depigmentation (p < 0.0001). Erosion/contusion at the lip commissures was associated with similar lesions on the bars (p = 0.0002), and ulcers of the buccal mucosa were associated with ulcers on the bars (p = 0.003). Dental hooks or sharp enamel points on one side were associated with similar lesions on the other side (p < 0.0001). Dental findings were not related to mucosal ulcers or erosion/contusion at the lip commissures but were associated with scarring/depigmentation (p = 0.01).

Assessment of Skin and Mucosa at the Equine Oral Commissures to Assess Pathology from Bit Wear: The Oral Commissure Assessment Protocol (OCA) for Analysis and Categorisation of Oral Commissures.

This study addresses the presence and location of natural pigmentation, potentially pathological changes in pigment, interruptions of the natural lining (scars), roughness, and erosions/contusion (bruising) in and around the corners of the lips of 206 horses presented to a veterinarian for routine preventative dental treatment. After sedation, photographs were taken and later evaluated for the presence of lesions. During the photographic analysis, the Oral Commissure Assessment (OCA) protocol was developed to map precisely the areas of skin and mucosa around the corners of the lips, and the presence of lesions was recorded for each area. Potentially pathological pigment changes occurred more frequently in horses with a higher level of training (p = 0.04) and in light-coloured horses (p = 0.0004), but there was no association with the current use of a bit or the discipline that the horse participated in (p = 0.20). Scars occurred more frequently in horses competing at a higher level. Only two horses had contusions or erosions, five had ulcers, and none showed bleeding; these numbers were too low for statistical analysis. Using the OCA protocol provides a detailed method for categorizing and recording lesions in and around the corners of the lips, including natural vs. potential and/or definite pathological character.

Targeted mass spectrometry for Serum Amyloid A (SAA) isoform profiling in sequential blood samples from experimentally Staphylococcus aureus infected pigs.

Serum amyloid A (SAA) is a well-described acute phase protein induced during the acute phase response (APR) to infection. Four isoform specific genes are found in most mammals. Depending on species, SAA3 and SAA4 are generally preferentially expressed extrahepatically whereas SAA1 and SAA2 are hepatic isoforms dominating the SAA serum pool. Little is known about how specific infections affect the serum SAA isoform profile, as SAA isoform discriminating antibodies are not generally available. An antibody independent, quantitative targeted MS method (Selected Reaction Monitoring, SRM) based on available information on porcine SAA isoform genes was developed and used to profile SAA in serum samples from pigs experimentally infected with Staphylococcus aureus (Sa). While results suggest SAA2 as the main circulating porcine SAA isoform, induced around 10 times compared to non-infected controls, total SAA serum concentrations reached only around 4 µg/mL, much lower than established previously by immunoassays. This might suggest that SAA isoform variants not detected by the SRM method might be present in porcine serum. The assay allows monitoring host responses to experimental infections, infectious diseases and inflammation states in the pig at an unprecedented level of detail. It can also be used in a non-calibrated (relative quantification) format. SIGNIFICANCE: We developed an SRM MS method which for the first time allowed the specific quantification of each of the circulating porcine SAA isoforms (SAA2, SAA3, SAA4). It was found that SAA2 is the dominating circulating isoform of SAA in the pig and that, during the acute phase response to Sa infection SAA2, SAA3 and SAA4 are induced approx. 10, 15 and 2 times, respectively. Absolute levels of the isoforms as determined by SRM MS were much lower than reported previously for total SAA quantified by immunosassays, suggesting the existence of hitherto non-described SAA variants. SRM MS holds great promise for the study of the basic biology of SAA isoforms with the potential to study an even broader range of SAA variants.

Mass spectrometric analysis of the in vitro secretome from equine bone marrow-derived mesenchymal stromal cells to assess the effect of chondrogenic differentiation on response to interleukin-1ß treatment.

BACKGROUND: Similar to humans, the horse is a long-lived, athletic species. The use of mesenchymal stromal cells (MSCs) is a relatively new frontier, but has been used with promising results in treating joint diseases, e.g., osteoarthritis. It is believed that MSCs exert their main therapeutic effects through secreted trophic biomolecules. Therefore, it has been increasingly important to characterize the MSC secretome. It has been shown that the effect of the MSCs is strongly influenced by the environment in the host compartment, and it is a crucial issue when considering MSC therapy. The aim of this study was to investigate differences in the in vitro secreted protein profile between naïve and chondrogenic differentiating bone marrow-derived (BM)-MSCs when exposed to an inflammatory environment. METHODS: Equine BM-MSCs were divided into a naïve group and a chondrogenic group. Cells were treated with normal expansion media or chondrogenic media. Cells were treated with IL-1ß for a period of 5 days (stimulation), followed by 5 days without IL-1ß (recovery). Media were collected after 48 h and 10 days. The secretomes were digested and analyzed by nanoLC-MS/MS to unravel the orchestration of proteins. RESULTS: The inflammatory proteins IL6, CXCL1, CXCL6, CCL7, SEMA7A, SAA, and haptoglobin were identified in the secretome after 48 h from all cells stimulated with IL-1ß. CXCL8, OSM, TGF-ß1, the angiogenic proteins VCAM1, ICAM1, VEGFA, and VEGFC, the proteases MMP1 and MMP3, and the protease inhibitor TIMP3 were among the proteins only identified in the secretome after 48 h from cells cultured in normal expansion media. After 10-day incubation, the proteins CXCL1, CXCL6, and CCL7 were still identified in the secretome from BM-MSCs stimulated with IL-1ß, but the essential inducer of inflammation, IL6, was only identified in the secretome from cells cultured in normal expansion media. CONCLUSION: The findings in this study indicate that naïve BM-MSCs have a more extensive inflammatory response at 48 h to stimulation with IL-1ß compared to BM-MSCs undergoing chondrogenic differentiation. This extensive inflammatory response decreased after 5 days without IL-1ß (day 10), but a difference in composition of the secretome between naïve and chondrogenic BM-MSCs was still evident.

Tendon response to matrix unloading is determined by the patho-physiological niche.

Although the molecular mechanisms behind tendon disease remain obscure, aberrant stromal matrix turnover and tissue hypervascularity are known hallmarks of advanced tendinopathy. We harness a tendon explant model to unwind complex cross-talk between the stromal and vascular tissue compartments. We identify the hypervascular tendon niche as a state-switch that gates degenerative matrix remodeling within the tissue stroma. Here pathological conditions resembling hypervascular tendon disease provoke rapid cell-mediated tissue breakdown upon mechanical unloading, in contrast to unloaded tendons that remain functionally stable in physiological low-oxygen/-temperature niches. Analyses of the stromal tissue transcriptome and secretome reveal that a stromal niche with elevated tissue oxygenation and temperature drives a ROS mediated cellular stress response that leads to adoption of an immune-modulatory phenotype within the degrading stromal tissue. Degradomic analysis further reveals a surprisingly rich set of active matrix proteases behind the progressive loss of tissue mechanics. We conclude that the tendon stromal compartment responds to aberrant mechanical unloading in a manner that is highly dependent on the vascular niche, with ROS gating a complex proteolytic breakdown of the functional collagen backbone.

Mapping the N-Terminome in Tissue Biopsies by PCT-TAILS.

Proteases play pivotal roles in multiple biological processes in all living organisms and are tightly regulated under normal conditions, but alterations in the proteolytic system and uncontrolled protease activity result in multiple pathological conditions. A disease will most often be defined by an ensemble of cleavage events-a proteolytic signature, thus the system-wide study of protease substrates has gained significant attention and identification of disease specific clusters of protease substrates holds great promise as targets for diagnostics and therapy.In this chapter we describe a method that enables fast and reproducible analysis of protease substrates and proteolytic products in an amount of tissue less than the quantity obtained by a standard biopsy. The method combines tissue disruption and protein extraction by pressure cycling technology (PCT), N-terminal enrichment by tandem mass tag (TMT)-terminal amine isotopic labeling of substrates (TAILS), peptide analysis by mass spectrometry (MS), and a general pipeline for interpretation of the data.

Mapping of equine mesenchymal stromal cell surface proteomes for identification of specific markers using proteomics and gene expression analysis: an in vitro cross-sectional study.

BACKGROUND: Stem cells have great potential for tissue regeneration, but before stem cell populations can be used in the clinic, it is crucial that the stem cells have been definitely characterized by a set of specific markers. Although there have been attempts to identify a set of immunophenotypic markers to characterize equine mesenchymal stromal cells (MSCs), immunophenotyping of equine MSCs is still challenging due to the limited availability of suitable antibodies of high quality and consistent performance across different laboratories. The aim of this study was to evaluate a strategy for mapping the equine MSCs surface proteome by use of biotin-enrichment and mass spectrometry (MS) analysis and mine the proteome for potential equine MSCs surface markers belonging to the cluster of differentiation protein group. Gene expression analysis was used for verification. METHODS: Equine MSCs derived from bone marrow (BM) (n = 3) and adipose tissue (AT) (n = 3) were expanded to P3 and either used for (1) cell differentiation into mesodermal lineages (chondrogenic and osteogenic), (2) enrichment of the MSCs surface proteins by biotinylation followed by in-gel digest of the isolated proteins and nanoLC-MS/MS analysis to unravel the enriched cell surface proteome, and (3) RNA isolation and quantitative real-time reverse transcriptase PCR analysis of the CD29, CD44, CD90, CD105, CD166, CD34, CD45, and CD79a gene expression. RESULTS: A total of 1239 proteins at 1% FDR were identified by MS analysis of the enriched MSCs surface protein samples. Of these proteins, 939 were identified in all six biological samples. The identified proteins included 19 proteins appointed to the cluster of differentiation classification system as potential cell surface targets. The protein and gene expression pattern was positive for the commonly used positive MSCs markers CD29, CD44, CD90, CD105, and CD166, and lacked the negative MSCs markers CD34, CD45, and CD79a. CONCLUSIONS: The findings of this study show that enrichment of the MSCs surface proteome by biotinylation followed by MS analysis is a valuable alternative to immunophenotyping of surface markers, when suitable antibodies are not available. Further, they support gene expression analysis as a valuable control analysis to verify the data.

Evaluation of Systemic and Local Inflammatory Parameters and Manifestations of Pain in an Equine Experimental Wound Model.

In the last decades, a well-established equine wound model has been used to study fibroproliferative wound healing disorders. The aim of this study was to characterize the degree of discomfort of wounding and sampling in an equine excisional wound model by evaluating systemic and local inflammatory responses and signs of pain. A total of 12 cutaneous wounds, three on each shoulder and each metatarsus, were created in a standing surgical procedure. Wounds were biopsied on days 2, 4, 7, 14, 21, and 28 after surgery. Clinical parameters (rectal temperature, heart rate, respiratory frequency) and blood levels of white blood cell, serum amyloid A, fibrinogen, and iron were monitored to evaluate the systemic inflammatory response. Local signs of inflammation (swelling, heat, pain) were subjectively assessed, the limb circumference recorded, and temperature of the wound measured by thermometry. Pain was evaluated by a composite measure pain scale (CMPS). The results demonstrated that the wounding procedure elicits an inflammatory response. Day 1 after surgery, two horses scored 2 and 7 units (of 27 units), respectively, on the CMPS, and day 8 after surgery, one horse scored 3 units. The biopsy procedure did not elicit local or systemic signs of inflammation. Based on these findings, it appears that the equine experimental wound model causes mild discomfort and pain manifestations. This information is important for researchers, who consider using the model. To justify the use of an animal model, it should be demonstrated that the expected benefits of the research outweigh the discomfort imposed to the animal.

A selected reaction monitoring-based analysis of acute phase proteins in interstitial fluids from experimental equine wounds healing by secondary intention.

In horses, pathological healing with formation of exuberant granulation tissue (EGT) is a particular problem in limb wounds, whereas body wounds tend to heal without complications. Chronic inflammation has been proposed to be central to the pathogenesis of EGT. This study aimed to investigate levels of inflammatory acute phase proteins (APPs) in interstitial fluid from wounds in horses. A novel approach for absolute quantification of proteins, selected reaction monitoring (SRM)-based mass spectrometry in combination with a quantification concatamer (QconCAT), was used for the quantification of five established equine APPs (fibrinogen, serum amyloid A, ceruloplasmin, haptoglobin, and plasminogen) and three proposed equine APPs (prothrombin, α-2-macroglobulin, and α-1-antitrypsin). Wound interstitial fluid was recovered by large pore microdialysis from experimental body and limb wounds from five horses at days 1, 2, 7, and 14 after wounding and healing without (body) and with (limb) the formation of EGT. The QconCAT included proteotypic peptides representing each of the protein targets and was used to direct the design of a gene, which was expressed in Escherichia coli in a media supplemented with stable isotopes for metabolically labeling of standard peptides. Co-analysis of wound interstitial fluid samples with the stable isotope-labeled QconCAT tryptic peptides in known amounts enabled quantification of the APPs in absolute terms. The concentrations of fibrinogen, haptoglobin, ceruloplasmin, prothrombin, and α-1-antitrypsin in dialysate from limb wounds were significantly higher than in dialysate from body wounds. This is the first report of simultaneous analysis of a panel of APPs using the QconCAT-SRM technology. The microdialysis technique in combination with the QconCAT-SRM-based approach proved useful for quantification of the investigated proteins in the wound interstitial fluid, and the results indicated that there is a state of sustained inflammation in equine wounds healing with formation of EGT.

The Pig PeptideAtlas: A resource for systems biology in animal production and biomedicine.

Biological research of Sus scrofa, the domestic pig, is of immediate relevance for food production sciences, and for developing pig as a model organism for human biomedical research. Publicly available data repositories play a fundamental role for all biological sciences, and protein data repositories are in particular essential for the successful development of new proteomic methods. Cumulative proteome data repositories, including the PeptideAtlas, provide the means for targeted proteomics, system-wide observations, and cross-species observational studies, but pigs have so far been underrepresented in existing repositories. We here present a significantly improved build of the Pig PeptideAtlas, which includes pig proteome data from 25 tissues and three body fluid types mapped to 7139 canonical proteins. The content of the Pig PeptideAtlas reflects actively ongoing research within the veterinary proteomics domain, and this article demonstrates how the expression of isoform-unique peptides can be observed across distinct tissues and body fluids. The Pig PeptideAtlas is a unique resource for use in animal proteome research, particularly biomarker discovery and for preliminary design of SRM assays, which are equally important for progress in research that supports farm animal production and veterinary health, as for developing pig models with relevance to human health research.

The use of liquid chromatography tandem mass spectrometry to detect proteins in saliva from horses with and without systemic inflammation.

The objective of the study was to assess global expression of proteins in equine saliva using liquid chromatography tandem mass spectrometry (LC-MS/MS). Saliva was obtained from seven horses with and six horses without evidence of systemic inflammatory disease. Tryptic peptides from saliva were analysed by LC-MS/MS. Of 195 unique proteins identified, 57 were detected only in saliva samples from horses with systemic inflammation (in two to six of the seven horses). Among the differentially expressed proteins were several acute phase proteins (APPs) such as serum amyloid A, fibrinogen, haptoglobin, and alpha1-acid glycoprotein. The study is the first to describe detection of inflammatory proteins in horse saliva. The proteins detected were similar to those described in saliva from cattle, small ruminants and pigs. Detection of APPs in horses with systemic inflammation suggests that saliva may be used for non-invasive disease monitoring in horses as in humans, pigs and dogs.

Development of a method for absolute quantification of equine acute phase proteins using concatenated peptide standards and selected reaction monitoring.

The aim of this study was the development of a quantitative assay that could support future studies of a panel of acute phase proteins (APPs) in the horse. The assay was based on a quantification concatamer (QconCAT) coupled to selected reaction monitoring methodology. Thirty-two peptides, corresponding to 13 putative or confirmed APPs for the Equus caballus (equine) species were selected for the design of a QconCAT construct. The gene encoding the QconCAT was synthesized and expressed as an isotope-labeled chimaeric protein in Escherichia coli. The QconCAT tryptic peptides were analyzed on a triple-quadrupole instrument, and the quantotypic properties were assessed in equine serum, wound tissue, and wound interstitial fluid. Reasonable quantotypic performance was found for 12, 14, and 14 peptides in serum, wound tissue, and interstitial fluid, respectively. Seven proteins were quantified in absolute terms in serum collected from a horse before and after the onset of a systemic inflammatory condition, and the observed protein concentrations were in close agreement with previous data. We conclude, that this QconCAT is applicable for concurrent quantitative analysis of multiple APPs in serum and may also support future studies of these proteins in other types of tissues and body fluids from the horse.

Regional disturbances in blood flow and metabolism in equine limb wound healing with formation of exuberant granulation tissue.

As in other fibroproliferative disorders, hypoxia has been suggested to play a key role in the pathogenesis of exuberant granulation tissue (EGT). The purpose of this study was to investigate metabolism and blood flow locally in full-thickness wounds healing with (limb wounds) and without (body wounds) formation of EGT. Microdialysis was used to recover endogenous metabolites from the wounds, and laser Doppler flowmetry was used to measure blood flow. Measurements were performed before wounding and 1-28 days after wounding. Blood flow was consistently lower in limb wounds than in body wounds throughout the study period with no change over time. After wounding and throughout the study period, the glucose concentration was significantly lower in limb wounds than in body wounds, whereas the lactate level showed a significantly higher concentration in limb wounds. The lactate/glucose ratio displayed a significant difference between body and limb wounds. In conclusion, the metabolic disturbances may suggest an inadequate oxygen supply during the wound healing process in equine limb wounds healing with EGT. This may be related to the inherently decreased perfusion in the wound bed of limb wounds.

The Equine PeptideAtlas: a resource for developing proteomics-based veterinary research.

Progress in MS-based methods for veterinary research and diagnostics is lagging behind compared to the human research, and proteome data of domestic animals is still not well represented in open source data repositories. This is particularly true for the equine species. Here we present a first Equine PeptideAtlas encompassing high-resolution tandem MS analyses of 51 samples representing a selection of equine tissues and body fluids from healthy and diseased animals. The raw data were processed through the Trans-Proteomic Pipeline to yield high quality identification of proteins and peptides. The current release comprises 24 131 distinct peptides representing 2636 canonical proteins observed at false discovery rates of 0.2% at the peptide level and 1.4% at the protein level. Data from the Equine PeptideAtlas are available for experimental planning, validation of new datasets, and as a proteomic data mining resource. The advantages of the Equine PeptideAtlas are demonstrated by examples of mining the contents for information on potential and well-known equine acute phase proteins, which have extensive general interest in the veterinary clinic. The extracted information will support further analyses, and emphasizes the value of the Equine PeptideAtlas as a resource for the design of targeted quantitative proteomic studies.