Summary Background Equine squamous gastric disease (ESGD) is a very common disorder but an accurate and practical screening technique for detecting ESGD is currently lacking. Objectives To identify serum protein markers to detect ESGD using electrophoresis and mass spectrometry. Study design Proteomic analysis and bioinformatics. Methods ESGD was diagnosed using gastroscopy in 30 horses. Gastric ulceration was categorised into three groups: normal, mild/moderate and severe ESGD. Pooled sera from each group were compared using 1D electrophoresis and mass spectrometry. The candidate proteins for ESGD markers were selected based on their specifically high expression in nonglandular stomach, and their association with gastric ulceration using public gene and protein databases. Results The prevalence of ESGD in this study was 43% (with mild/moderate ESGD at 33% and severe ESGD at 10%). The proteomic study revealed that the identified serum protein markers for normal equine stomach were B4GALNT2 and XDH. The marker for mild/moderate EGSD was KRT10, while the marker for severe ESGD was KLK13. Furthermore, markers for both ulcer types were SLC4A7, PPARG, FCGBP, PKP1, ASPRV1 and KRT5‐like proteins. Main limitations The functions of the identified proteins are not well characterised in horse. Proteomics is a tool for screening protein markers, but confirmation of putative protein markers with specific antibodies is required. Conclusions In total, 10 serum proteins found in this study may be used as putative markers for ESGD. However, confirmation of candidate proteins with specific antibodies in a larger study cohort is necessary before it can be used in the veterinary clinic or on horse farms. The Summary is available in Portuguese – see Supporting Information
Gastric tissue biopsy and gene expression analysis are important tools for disease diagnosis and study of the physiology of the equine stomach. However, RNA extraction from gastric biopsy samples is a complex procedure because the samples contain low quantities of RNA and are contaminated with mucous protein and bacterial flora. The objectives of these studies were to compare the performance of RNA extraction methods and to investigate the sensitivity of commercial qPCR master mixes for gene expression analysis of gastric biopsy samples. Three commercial RNA extraction methods (TRIzol™, GENEzol™ and MiniPrep™) and four qPCR master mixes with SYBR® green (qPCRBIO, KAPA, QuantiNova, and PerfeCTa) were compared. RNA qualification and quantitation were compared. Real-time PCR was used to compare qPCR master mixes. The results revealed that TRIzol and GENEzol obtained significantly higher yield of RNA (P<0.01) but that TRIzol had the highest contamination of protein and DNA (P<0.05). Conversely, MiniPrep resulting in a significantly higher purification of RNA (P<0.05) but provided the lowest yield of RNA (P<0.01). For PCR master mixes, KAPA was significantly (P<0.05) more sensitive than other qPCR kits for all amounts of DNA template, particularly at the lowest amount of cDNA. In conclusion, GENEzol is the best method to obtain a high RNA yield and purification and it is more cost-effective than the others as well. Regarding the qPCR master mixes, KAPA SYBR qPCR Master Mix (2x) Universal is superior to the other tested master mixes for studying gene expression in equine gastric biopsies.
Equine Glandular Gastric Disease (EGGD) is a common disease in sport horses. This disease might be associated with usage of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating inflammatory diseases. Although gastroscopy has been an effective method for diagnosis, but a less invasive, and inexpensive method is preferred. This study used proteomic technology to identify candidate serum proteins that might be used as markers of NSAIDs induced EGGD. Five Thoroughbred horses were given high doses of NSAID, phenylbutazone to treat lameness. The experiment was divided into three periods: (i) Pre-EGGD period, (ii) during EGGD period, and (iii) Post-EGGD period. Gastroscopy were used to diagnose EGGD, serum was collected to perform gel electrophoresis (1D SDS-PAGE) and mass spectrometry (LC-MS) in order to identify serum proteins in each group. The candidate serum proteins were computationally predicted for the interaction between phenylbutazone and proteins, tissue specific expression, and association to gastric ulceration. After EGGD induction, all horses showed clinical signs of colic with marked congestion and erosion appearing in the mucosa of the glandular stomach whereas no change was observed in the mucosa of non-glandular stomach. Our proteomic results identified 14 proteins that might be used as EGGD markers. These proteins were highly expressed in the glandular stomach and some proteins were associated with phenylbutazone or ulcer development. However, confirmation of these candidate marker proteins is required with specific antibodies in the larger horse population before they can be considered for application in the field.
Equine melanocytic neoplasm (EMN) is a common disease in older grey horses. The purpose of this study was to examine the potential proteins throughout EMN stages from faecal proteomic outlining using functional analysis. Faecal samples were collected from the rectum of 25 grey horses divided into three groups; normal group without EMN (n = 10), mild EMN (n = 6) and severe EMN (n = 9). Based on the results, 5910 annotated proteins out of 8509 total proteins were assessed from proteomic profiling. We observed differentially expressed proteins (DEPs) between the normal group and the EMN group, and 109 significant proteins were obtained, of which 28 and 81 were involved in metabolic and non-metabolic functions, respectively. We found 10 proteins that play a key role in lipid metabolism, affecting the tumour microenvironment and, consequently, melanoma progression. Interestingly, FOSL1 (FOS like 1, AP-1 transcription factor subunit) was considered as a potential highly expressed protein in a mild EMN group involved in melanocytes cell and related melanoma. Diacylglycerol kinase (DGKB), TGc domain-containing protein (Tgm2), structural maintenance of chromosomes 4 (SMC4) and mastermind-like transcriptional coactivator 2 (MAML2) were related to lipid metabolism, facilitating melanoma development in the severe-EMN group. In conclusion, these potential proteins can be used as candidate biomarkers for the monitoring of early EMN, the development of EMN, further prevention and treatment.
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