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Topic:Protein
Encapsulation of copper phenanthroline within horse spleen apoferritin: characterisation, cytotoxic activity and ability to retain temozolomide. Protein capsules are promising drug delivery vehicles for cancer research therapies. Apoferritin (AFt) is a self-assembling 12 nm diameter hollow nanocage with many desirable features for drug delivery, however, control of drug retention inside the protein cage remains challenging. Here we report the encapsulation of copper(ii)-1,10-phenanthroline (Cu(phen)) within the horse spleen AFt (HSAFt) nanocage, by diffusion of the metal through the pores between the protein subunits. Transmission electron microscopy revealed the formation of organised copper adducts inside HSAFt, without affecting pro...
Intrahepatic mucinous cholangiocarcinoma with recurrent colic in a horse: case report and literature review of cholangiocarcinoma in horses. A 17-y-old Arabian mare was presented to the Auburn Large Animal Veterinary Teaching Hospital with a long-term history of intermittent mild recurrent colic that responded to medical treatment. CBC revealed mild lymphopenia; serum biochemistry findings were of increased gamma-glutamyl transferase and creatine kinase activities, hyperferremia, hyperglycemia, hypomagnesemia, and hypokalemia. Abdominocentesis was compatible with low-protein transudate. Due to the progression and duration of clinical signs, the owner elected euthanasia. Postmortem examination and histopathology confirmed a cholangi...
De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering. To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separat...
First National Prevalence in Italian Horse Population and Phylogenesis Highlight a Fourth Sub-Type Candidate of Equine Hepacivirus. Equine hepacivirus (EqHV, , hepacivirus) is a small, enveloped RNA virus generally causing sub-clinical hepatitis with occasional fatalities. EqHV is reported in equids worldwide, but for Italy data are limited. To address this, a survey study was set up to estimate prevalence at a national level and among different production categories (equestrian; competition; work and meat; reproduction) and national macro-regions (North, Central, South, and Islands). Data obtained testing 1801 horse serum samples by Real-Time RT PCR were compared within the categories and regions. The NS3 fragment of the ...
Integrated analysis of gut metabolome, microbiome, and exfoliome data in an equine model of intestinal injury. The equine gastrointestinal (GI) microbiome has been described in the context of various diseases. The observed changes, however, have not been linked to host function and therefore it remains unclear how specific changes in the microbiome alter cellular and molecular pathways within the GI tract. Further, non-invasive techniques to examine the host gene expression profile of the GI mucosa have been described in horses but not evaluated in response to interventions. Therefore, the objectives of our study were to (1) profile gene expression and metabolomic changes in an equine model of non-ster...
Selecting potential biomarkers of plasma proteins in mares with endometritis. Endometritis is a common condition in mares that causes significant economic loss. Lacking obvious clinical signs, the clinical diagnosis of endometritis in mares relies on case-by-case clinical examinations, which can be particularly inefficient in large-scale farms. Therefore, the identification of potential biomarkers can serve as a non-invasive and efficient screening technique for endometritis in mares. Objective: To compare the blood proteome between fertile mares and mares with endometritis to identify biomarkers potentially associated with the development of endometritis and validate t...
Extracellular Vesicles Isolated from Equine Adipose-Derived Stromal Stem Cells (ASCs) Mitigate Tunicamycin-Induced ER Stress in Equine Corneal Stromal Stem Cells (CSSCs). Corneal ulcers, characterized by severe inflammation of the cornea, can lead to serious, debilitating complications and may be vision-threatening for horses. In this study, we aimed to investigate the role of endoplasmic reticulum (ER) stress in corneal stem progenitor cell (CSSC) dysfunction and explore the potential of equine adipose-derived stromal stem cell (ASC)-derived extracellular vesicles (EVs) to improve corneal wound healing. We showed that CSSCs expressed high levels of CD44, CD45, and CD90 surface markers, indicating their stemness. Supplementation of the ER-stress-inducer tunicam...
White horses – non-coding sequences drive premature hair greying and predisposition to melanoma. The allele in horses is causing premature hair greying and susceptibility to melanoma. The causal mutation is a 4.6 kb tandem duplication in intron 6 of the gene. A recent study demonstrated that the most common allele at the locus () involves three tandem copies of this sequence, whilst a more rare allele () has two tandem copies and the wild-type allele () only one copy. The allele is causing fast greying and high incidence of skin melanoma, whereas the allele is causing slow greying and no obvious increase in melanoma incidence. Further somatic copy number expansion has been documented...
Equine mesenchymal stem cell-derived extracellular vesicle productivity but not overall yield is improved via 3-D culture with chemically defined media. Mesenchymal stem cell (MSC) extracellular vesicles (EVs) have emerged as a biotherapeutic for osteoarthritis; however, manufacturing large quantities is not practical using traditional monolayer (2-D) culture. We aimed to examine the effects of 3-D and 2-D culture 2 types of media: Dulbecco modified Eagle medium and a commercially available medium (CM) on EV yield. Methods: Banked bone marrow-derived MSCs (BM-MSCs) from 6 healthy, young horses were used. Methods: 4 microcarriers (collagen-coated polystyrene, uncoated polystyrene, collagen-coated dextran, and uncoated dextran) were tested in st...
A matter of differentiation: equine enteroids as a model for the in vivo intestinal epithelium. Epithelial damage due to gastrointestinal disorders frequently causes severe disease in horses. To study the underlying pathophysiological processes, we aimed to establish equine jejunum and colon enteroids (eqJE, eqCE) mimicking the in vivo epithelium. Therefore, enteroids were cultivated in four different media for differentiation and subsequently characterized histomorphologically, on mRNA and on protein level in comparison to the native epithelium of the same donor horses to identify ideal culture conditions for an in vitro model system. With increasing enterocyte differentiation, the ente...
Nutrient Composition and Feed Hygiene of Alfalfa, Comparison of Feed Intake and Selected Metabolic Parameters in Horses Fed Alfalfa Haylage, Alfalfa Hay or Meadow Hay. The aim of this study was to examine the nutrient composition and feed hygiene of alfalfa as well as investigate the feed intake, blood, urine and faecal parameters of horses fed alfalfa haylage (AS) compared with alfalfa hay (AH) and meadow hay (MH). A total of 11 geldings were fed ad libitum (2.1% dry matter (DM) of body weight (BW)) with alfalfa haylage, alfalfa hay and meadow hay (MH) in a Latin square design. On days 0 and 21 of the feeding period, blood samples were analysed for kidney and liver parameters. Faecal samples were analysed for pH, DM and short-chain fatty acids (SCFAs). Spon...
Follicular Fluid-Derived Extracellular Vesicles Influence on In Vitro Maturation of Equine Oocyte: Impact on Cumulus Cell Viability, Expansion and Transcriptome. Cumulus cell (CC) expansion is pivotal for oocyte maturation, during which CCs release factors that initiate paracrine signaling within the follicular fluid (FF). The FF is abundant in extracellular vesicles (EVs) that facilitate intercellular communication. Although bovine and murine EVs can control cumulus expansion, these effects have not been observed in equines. This study aimed to assess the impact of FF-derived EVs (ffEVs) on equine CC expansion, viability, and transcriptome. Cumulus-oocyte complexes (COCs) that underwent in vitro maturation (IVM) in the presence (200 µg protein/mL) or...
Changes in the blood redox status of horses subjected to combat training. Combat training of police horses, involving physical activity in the presence of environmental stressors, poses a risk of oxidative stress. This study compared the oxidative imbalance after combat training in horses in the regular police service and in horses that had just been schooled. Blood collection was performed immediately after training and after 16 h rest. The activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and total antioxidant status (TAS) were determined as the markers of enzymatic antioxidant defence. At the same time, lipid peroxidation (TBAR...
Impact of Seminal Plasma Antioxidants on DNA Fragmentation and Lipid Peroxidation of Frozen-Thawed Horse Sperm. Cryopreservation is a stressful process for sperm, as it is associated with an increased production of reactive oxygen species (ROS). Elevated ROS levels, which create an imbalance with antioxidant capacity, may result in membrane lipid peroxidation (LPO), protein damage and DNA fragmentation. This study aimed to determine whether the membrane LPO and DNA fragmentation of frozen-thawed horse sperm relies upon antioxidant activity, including enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and paraoxonase type 1 (PON1)); non-enzymatic antioxidant capacity (Trolo...
Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma. Severe equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. () is a common mold species in hay and has been described as a major provoking agent of SEA. Unassigned: Aiming to identify disease-relevant antigens, we analyzed using an immunoproteomics approach on two-dimensional ...
The Effect of Repeated Blood Harvesting from Pregnant Mares on Haematological Variables. Studies have been carried out on the effect of large-volume blood harvesting from horses, but they were performed on nonpregnant horses of various breeds other than Icelandic horses. This study aimed to investigate the effect on the haematological variables indicating erythropoiesis of repeated blood harvesting from pregnant mares. To account for regional variation, two herds of mares were chosen, both kept under free-range conditions. Sequential EDTA blood samples were collected weekly from 160 mares and analysed for haematological variables in an automated analyser. Serum samples from 115 ma...
Comparative proteomic analysis of the changes in mare milk associated with different lactation stages and management systems. Mare milk has traditionally been attributed a number of health promoting properties. However, knowledge on its composition and functionality remains scarce, with particularly limited studies on mare milk proteomics. This study deeply characterized mare milk proteome accounting for both caseins and proteins in the whey fraction, also addressing the impact of lactation stage and different management systems. Milk samples from Basque Mountain Horse breed mares belonging to three different farms and three lactation stages were analysed after in-gel and in-solution digestion using nLC-MS/MS. Among ...
Single-dose nonsteroidal anti-inflammatory drugs in horses have no impact on concentrations of cytokines or growth factors in autologous protein solution and platelet-rich plasma. To determine the effects of a single dose of the NSAIDs phenylbutazone, firocoxib, flunixin meglumine, and ketoprofen on concentrations of growth factors and cytokines in autologous protein solution (APS) and platelet-rich plasma (PRP). Methods: 6 adult university-owned horses. Methods: For the first phase, 6 horses were randomized to receive ketoprofen (1,000 mg) or flunixin meglumine (500 mg) IV. Blood was obtained and processed for APS (Pro-Stride) and PRP (Restigen) before and 6 hours after administration of NSAIDs. Horses underwent a 2-week washout period, after which the protocol was rep...
Pharmacokinetic study of osilodrostat and identification of mono-hydroxylated metabolite in equine plasma for the purpose of doping control. Osilodrostat is an inhibitor of 11-beta-hydroxylase (CYP11B) and is used for the treatment of Cushing's disease but also categorized as an anabolic agent. The use of osilodrostat is prohibited in horseracing and equestrian sports. To the best of our knowledge, this is the first metabolic study of osilodrostat in equine plasma. Methods: Potential metabolites of osilodrostat were identified by differential analysis using data acquired from pre- and post-administration plasma samples after protein precipitation with liquid chromatography electrospray ionization high-resolution mass spectrometry (...
Pre-analytical stability of selected biochemical analytes in serum of horses and oxen stored at -20°C. Delays between blood collection and analysis are inevitable, and samples are always stored in the refrigerator. The current study aimed to evaluate the stability of serum total cholesterol (TC), triglycerides (TG), total protein (TP), albumin and urea (URA) in horses and oxen after storage at -20°C. Methods: Sera from apparently healthy 20 male horses and 20 oxen were obtained and aliquots of serum were divided into 3 portions. The first tube was used for baseline (T0) measurement of analyte values, whereas the other two tubes, T1 and T2, were stored at -20°C for 1 and 2 months, respectively...
The Welfare of Horses Competing in Three-Barrel Race Events Is Shown to Be Not Inhibited by Short Intervals between Starts. Equestrian sports require precise animal welfare and health evaluations. To test the hypothesis that horses maintain their welfare when subjected to two three-barrel (3TB) races with 2 min intervals, an experiment was designed to evaluate their surface temperature using infrared thermography (IRT) in regions of interest (barrel, flank, neck, jaw, corner of the mouth, and ocular caruncle) and also measure blood biomarkers (hemogram, total plasma protein, fibrinogen, urea, creatinine, GGT, CK, cortisol, IL-6, and IL-1β). Ten Quarter Horses were monitored through thermography (pre-race, +1, +4, ...
Use of quantitative mass spectrometry-based proteomics and ELISA to compare the alpha 2 macroglobulin concentration in equine blood-based products processed by three different orthobiologic devices. Alpha 2 macroglobulin (A2M), a multi-functional protein in the plasma protease inhibitor class, regulates proinflammatory cytokines and the clearance of chondrodestructive enzymes in cases of joint injury and osteoarthritis (OA). The purpose of this study was to compare A2M concentrations in equine plasma samples processed by three commercial devices developed for stall-side regenerative joint therapy. Unassigned: Plasma samples were obtained from healthy adult horses ( = 13). Mass spectrometry analysis was used to determine the concentration of protein analytes in each sample. Selected re...
Effects of β-Glucan Supplementation on LPS-Induced Endotoxemia in Horses. β-glucan is part of the cell wall of fungi and yeasts and has been known for decades to have immunomodulating effects on boosting immunity against various infections as a pathogen-associated molecular pattern that is able to modify biological responses. β-glucan has been used in rat models and in vitro studies involving sepsis and SIRS with good results, but this supplement has not been evaluated in the treatment of endotoxemia in horses. This study aims to evaluate the effects of preventive supplementation with β-glucan in horses submitted to endotoxemia by means of inflammatory response m...
Immunity transfer in mule foals fed with good IgG quality colostrum. While the passive transfer of immunity in horse and donkey foals has been extensively studied, there is limited information for mule foals. Immunoglobulin type G (IgG) and serum total protein concentration (TP) were assessed at different sampling times to evaluate the correlation between serum radial immunodiffusion (SRID) with electrophoresis, refractometry, and dry chemistry analyzer (Biuret), and to estimate serum IgG concentrations using serum TP in mule foals. We analyzed a total of 30 samples collected at birth, and at 6, 12, 24, and 48 h of life from 6 mule foals by SRID, electrophores...
Iron-related markers of inflammation in horses with colic. The aim of the study was to compare and correlate levels of ferritin, transferrin, iron and APPs in healthy horses and those surgically treated for strangulating colic. On admission, measurements of inflammatory markers related to iron and total protein, fibrinogen, albumin, haptoglobin and ceruloplasmin were made. The study comprised 22 horses, divided into a control group (CG) of healthy horses (n = 10) and horses with surgically treated acute abdomen (n = 12), obstruction group (OG). The OG was subdivided according to the affected intestinal segment (small vs. large) and according to ou...
Testicular expression of heat SHOCK proteins 60, 70, and 90 in cryptorchid horses. Heat shock proteins are the most evolutionarily conserved protein families induced by stressors including hyperthermia. In the context of pathologies of the male reproductive tract, cryptorchidism is the most common genital defect that compromises the reproductive potential of the male because it induces an increase in intratesticular temperature. In equine species, cryptorchidism affects almost 9 % of newborns and few studies have been carried out on the molecular aspects of the retained testis. In this study, the expression pattern of HSP60, 70, and 90 in abdominal and inguinal testes, in t...
Trodusquemine (MSI-1436) Restores Metabolic Flexibility and Mitochondrial Dynamics in Insulin-Resistant Equine Hepatic Progenitor Cells (HPCs). Equine metabolic syndrome (EMS) is a significant global health concern in veterinary medicine. There is increasing interest in utilizing molecular agents to modulate hepatocyte function for potential clinical applications. Recent studies have shown promising results in inhibiting protein tyrosine phosphatase (PTP1B) to maintain cell function in various models. In this study, we investigated the effects of the inhibitor Trodusquemine (MSI-1436) on equine hepatic progenitor cells (HPCs) under lipotoxic conditions. We examined proliferative activity, glucose uptake, and mitochondrial morphogenesi...
Effects of Horse Meat Hydrolysate on Oxidative Stress, Proinflammatory Cytokines, and the Ubiquitin-Proteasomal System of C2C12 Cells. Sarcopenia, the age-related muscle atrophy, is a serious concern as it is associated with frailty, reduced physical functions, and increased mortality risk. Protein supplementation is essential for preserving muscle mass, and horse meat can be an excellent source of proteins. Since sarcopenia occurs under conditions of oxidative stress, this study aimed to investigate the potential anti-muscle atrophy effect of horse meat hydrolysate using C2C12 cells. A horse meat hydrolysate less than 3 kDa (A4<3kDa) significantly increased the viability of C2C12 myoblasts against HO-induced cytotoxicity....
Comparison of differential protein expression of the marginal transitional zone in neonatal and weanling-aged foals. The marginal transitional zone (MTZ) is peripherally located within the diarthrodial joint, and represents the junction of synovium, fibrous joint capsule, articular cartilage, periosteum, and bone. The purpose of this study is to characterize age-related differences in protein expression of matrix and molecular regulators in the marginal transitional zone of neonatal and weanling foals. Several families of proteins with known roles in cartilage and bone development are investigated, including matrix molecules, members of the Wnt signaling family, apoptotic factors and paracrine cell signaling...
Procalcitonin and carbonylated protein concentrations in equine synovial fluid. Early diagnosis of joint diseases is fundamental for prompt and appropriate management, particularly in septic arthritis. Procalcitonin (PCT) and protein carbonylated content (PCC) have been investigated in both human and veterinary medicine. An increase in PCT has been shown in infectious bacterial diseases, while higher levels of PCC have been shown in inflammatory pathologies characterized by oxidative damage. This study evaluated PCT and PCC in plasma and synovial fluid (SF), in healthy and pathological equine joints, affected by different types of arthropathy. Twenty-nine joints were eval...
