Equine melanocytic neoplasm (EMN) is a cutaneous neoplasm and is mostly observed in aged grey horses. This preliminary study aimed to identify potential proteins to differentiate normal, mild and severe EMN from serum proteomic profiling. Serum samples were collected from 25 grey horses assigned to three groups: normal (free of EMN; n = 10), mild (n = 6) and severe EMN (n = 9). To explore the differences in proteins between groups, proteomic profiling and analysis were employed. Accordingly, 8241 annotated proteins out of 8725 total proteins were compared between normal and EMN groups and inspected based on differentially expressed proteins (DEPs). Through DEP analysis, 95 significant DEPs differed between normal and EMN groups. Among these DEPs, 41 significant proteins were categorised according to protein functions. Based on 41 significant proteins, 10 were involved in metabolism and 31 in non-metabolism. Interestingly, phospholipid phosphatase6 (PLPP6) and ATPase subunit alpha (Na+/K+-ATPase) were considered as potential proteins uniquely expressed in mild EMN and related to lipid and energy metabolism, respectively. Non-metabolism-related proteins (BRCA1, phosphorylase B kinase regulatory subunit: PHKA1, tyrosine-protein kinase receptor: ALK and rho-associated protein kinase: ROCK1) correlated to melanoma development differed among all groups. The results of our study provide a foundation for early EMN biomonitoring and prevention.

Phenylbutazone (PBZ) is a widely used nonsteroidal anti-inflammatory drug for horses. However, because of its gastrointestinal side effects, its administration requires careful attention in veterinary practice. Malondialdehyde (MDA) is a serum biomarker associated with increased damage to the equine gastrointestinal system. This study investigated the hematological effects and alterations in the gastrointestinal tract and assessed serum MDA concentrations following repeated oral PBZ administration at clinical doses. Fourteen horses were randomly divided into control and treatment groups. All horses in the treatment group were administered 4.4 milligrams per kilogram of body weight of PBZ syrup orally twice a day for 7 days, whereas the control group received syrup as a placebo. The development of gastrointestinal side effects was investigated using gastroscopy, abdominal ultrasound, and fecal pH; serum MDA concentrations were assessed using a commercially available enzyme-linked immunosorbent assay kit. Data were compared between PBZ-treated and control horses before and after the treatment period. The treatment group exhibited decreased albumin and total protein concentrations. Moreover, this group exhibited a higher thickness of the right dorsal colon wall (p = 0.03) and had higher scores for squamous gastric ulcers (p = 0.01). Fecal pH was lower in the treatment group than in the control group after PBZ administration (p < 0.01). Although MDA concentrations were higher in the treatment group after PBZ administration, they did not differ significantly from those of the control group. This study highlighted the changes in hematological and gastrointestinal lesions resulting from PBZ administration in horses at clinical doses, even without clinical signs. However, MDA may not be an optimal biomarker for the early detection of gastrointestinal damage due to PBZ treatment in horses.

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.

Equine melanocytic neoplasm (EMN) represents a form of skin tumor observed predominantly in grey horses aged over 15 years. Despite its prevalence, current therapeutic and preventive strategies for EMN have been subject to limited investigation. This study endeavors to shed light on potential phosphoproteins present in equine serum and fecal samples, potentially linked to EMN, with a specific focus on functional interactions in EMN pathogenesis. We examined 50 samples (25 serum, 25 feces), divided into three groups based on EMN severity: normal (n = 16), mild (n = 18), and severe EMN (n = 16). Equine phosphoproteome analysis identified 2,359 annotated serum phosphoproteins and 2002 annotated fecal phosphoproteins through differentially expressed proteins (DEPs). KEGG analysis emphasized the role of environmental information processing. Notably, the integrin NF-kappaB binding P-TEFb to stimulate transcriptional elongation signaling pathway, involving SNW1 protein, was implicated in early stage of EMN development in both serum and fecal samples. This highlights SNW1’s potential role in mediating transcriptional processes, offering a novel marker within environmental information processing. This study enhances understanding of EMN mechanisms in horses, suggesting early detection through non-invasive methods and identifying a functional pathway involving SNW1, which could inform future treatment and prevention strategies.

Background: Phenylbutazone (PBZ) can potentially induce gastrointestinal ulceration, and early detection of PBZ‐induced gastroenteropathy will be useful for the diagnosis, treatment, and prevention of PBZ toxicity. Objectives: To identify putative proteins associated with equine gastric ulcer syndrome after clinical dose (4.4 mg/kg) administration of PBZ by proteomic study. Study Design: In vivo experiments. Methods: Proteomic analysis using LC–MS/MS compared protein expression in serum and faeces of seven PBZ‐treated horses with seven placebo‐treated controls, and a novel putative biomarker was validated via enzyme‐linked immunosorbent assay. Results: Differentially expressed proteins (DEPs) analysis on 5298 serum annotated proteins and 3538 faecal annotated proteins using the DESeq2 were performed between the control and treatment of EGUS groups. The results showed a list of 226 and 181 significant proteins in serum and faecal samples, respectively with a p adjust value <0.05. The proteomic serum and faeces samples were integrated into STITCH to illustrate PBZ interaction with bile acid homeostasis. FABP6 was significantly increased in PBZ‐treated horses. The serum FABP6 concentration in the treatment group on Day 8 (1.80 ± 0.37 ng/mL) was higher than on Day 0 (1.15 ± 0.33 ng/mL, p = 0.01, 95% CI [−1.07, −0.25]). On Day 8, the serum FABP6 concentration in the treatment group was also higher than the control group (1.20 ± 0.48 ng/mL; p = 0.02, 95% CI [−1.10, −0.11]). Main Limitations: Validation of all expressed proteins is a main limitation. Conclusions: Administration of PBZ at a clinical dose of 4.4 mg/kg twice daily for 7 days may cause gastric mucosal damage. PBZ treatment increased the expression of SLC10A1 and FABP6, suggesting that early gastric mucosal injury may be linked to the bile acid pathway. Bile acids could potentially exacerbate PBZ‐induced EGUS.

Equine melanocytic neoplasms (EMN) are aggressive tumours characterised by high metastatic potential and limited therapeutic options available. However, the molecular mechanisms underlying their progression remain poorly understood. This study therefore presents the integrative phosphoproteomic analysis of EMN tissue, with the aim of elucidating stage‐specific alterations in signalling pathways and metabolism. Nineteen tissue samples from grey horses were categorised as normal‐stage ( n = 6), early‐stage EMN ( n = 7), and severe‐stage EMN ( n = 6) and subjected to in‐depth analysis using liquid chromatography–tandem mass spectrometry (LC–MS/MS). A total of 2035 phosphoproteins were identified, of which 219 were differentially expressed across the disease stages. Interestingly, early‐stage EMN showed dysregulation of inositol phosphate metabolism and activation of the PI3K‐Akt pathway which involved INPP5F and PKN2. In severe‐stage EMN, upregulation of SYNJ1, STRN4 and VIM indicated enhanced membrane trafficking, cytoskeletal remodelling, and MAPK signalling. Additionally, ASPM and GNAO1 upregulation reflected heightened proliferation and altered Rap1 signalling, while UBR5 dysregulation suggested aberrant protein homeostasis. Metabolic reprogramming was also noticed, with elevated TKT and GAPDH expression supporting glycolysis and NADPH production. Observably, the severe‐stage EMN exhibited a higher expression of Dickkopf‐3 (DKK3) which suggests a role in aberrant Wnt/β‐catenin activation and tumour progression. These findings reveal stage‐specific molecular mechanisms in EMN pathogenesis and highlight potential biomarkers and therapeutic targets for equine melanoma.