Tracking the Molecular Scenarios for Tumorigenic Remodeling of Extracellular Matrix Based on Gene Expression Profiling in Equine Skin Neoplasia Models.

An important component of tissues is the extracellular matrix (ECM), which not only forms a tissue scaffold, but also provides the environment for numerous biochemical reactions. Its composition is strictly regulated, and any irregularities can result in the development of many diseases, including cancer. Sarcoid is the most common skin cancer in equids. Its formation results from the presence of the genetic material of the bovine papillomavirus (BPV). In addition, it is assumed that sarcoid-dependent oncogenic transformation arises from a disturbed wound healing process, which may be due to the incorrect functioning of the ECM. Moreover, sarcoid is characterized by a failure to metastasize. Therefore, in this study we decided to investigate the differences in the expression profiles of genes related not only to ECM remodeling, but also to the cell adhesion pathway, in order to estimate the influence of disturbances within the ECM on the sarcoid formation process. Furthermore, we conducted comparative research not only between equine sarcoid tissue bioptates and healthy skin-derived explants, but also between dermal fibroblast cell lines transfected and non-transfected with a construct encoding the E4 protein of the BP virus, in order to determine its effect on ECM disorders. The obtained results strongly support the hypothesis that ECM-related genes are correlated with sarcoid formation. The deregulated expression of selected genes was shown in both equine sarcoid tissue bioptates and adult cutaneous fibroblast cell (ACFC) lines neoplastically transformed by nucleofection with gene constructs encoding BPV1-E1^E4 protein. The identified genes (CD99, ITGB1, JAM3 and CADM1) were up- or down-regulated, which pinpointed the phenotypic differences from the backgrounds noticed for adequate expression profiles in other cancerous or noncancerous tumors as reported in the available literature data. Unravelling the molecular pathways of ECM remodeling and cell adhesion in the in vivo and ex vivo models of epidermal/dermal sarcoid-related cancerogenesis might provide powerful tools for further investigations of genetic and epigenetic biomarkers for both silencing and re-initiating the processes of sarcoid-dependent neoplasia. Recognizing those biomarkers might insightfully explain the relatively high capacity of sarcoid-descended cancerous cell derivatives to epigenomically reprogram their nonmalignant neoplastic status in domestic horse cloned embryos produced by somatic cell nuclear transfer (SCNT).

Assessment of BPV-1 Mediated Matrix Metalloproteinase Genes Deregulation in the In Vivo and In Vitro Models Designed to Explore Molecular Nature of Equine Sarcoids.

Matrix metalloproteinases (MMPs) represent a family of enzymes capable of biocatalytically breaking down the structural and functional proteins responsible for extracellular matrix (ECM) integrity. This capability is widely used in physiological processes; however, imbalanced MMP activity can trigger the onset and progression of various pathological changes, including the neoplasmic transformation of different cell types. We sought to uncover molecular mechanisms underlying alterations in transcriptional profiles of genes coding for MMPs, which were comprehensively identified in equine adult dermal tissue bioptates, sarcoid-derived explants, and ex vivo expanded adult cutaneous fibroblast cell (ACFC) lines subjected to inducible oncogenic transformation into sarcoid-like cells. The results strongly support the hypothesis that the transcriptional activity of MMP genes correlates with molecular modifications arising in equine dermal cells during their conversion into sarcoid cells. The alterations in MMP transcription signatures occurs in both sarcoid tissues and experimentally transformed equine ACFC lines expressing BPV1-E4^E1 transgene, which were characterized by gene up- and down-regulation patterns.

Bacterial aerosol, particulate matter, and microclimatic parameters in the horse stables in Poland.

Keeping horses in good condition requires providing them with living conditions that meet welfare requirements. These animals should be accommodated with suitable space, access to high nutritional fodder and water, and a suitable microclimate in their shelters. When it comes to the environment in the stables, a serious problem is created by particulate matter (PM), which consists of bacteria. PM concentration may be responsible for developing multiple lower respiratory tract diseases in horses, including allergies and recurrent airway obstruction (RAO). In turn, these ailments may lead to decreasing equine physical and mental fitness. Additionally, people who spend time in the stables are exposed to the same harmful factors. The study was conducted in Udórz Stud Farm located in the southern region of Poland. The study was carried out in 2 different types of stables: 3 runners (a type of stable where horses are housed together and occasionally linked up, e.g., for feeding or grooming) and 2 box stables. The research continued for 2 years and the samples were collected in each season. The bioaerosol samples were collected using a six-stage Andersen-Graseby cascade impactor to assess size distribution and concentrations of airborne bacteria. PM concentration was analyzed using the DustTrak™ II Aerosol Monitor 8530, while microclimate parameters were measured using the Kestrel 5000 Weather Meter. There are almost no studies concerning size distribution of airborne bacteria, individual PM fractions, and the impact of everyday handling on the changes in the bioaerosol and PM concentration. This preliminary study provided basic information on this subject. We have revealed a strong correlation between high PM and bacterial aerosol concentrations. Higher contamination levels were recorded in runners, as compared to box stables. The highest bacterial aerosol level was detected in the spring. The analysis of the fractions of the bacterial aerosol in the stables indicated the highest share of ultra-fine fraction (0.65-2.1 µm), while respirable fraction (below 4.7 µm) exceeded 75%. It was established that the concentration of the bacterial aerosol inside the stables was many times higher than outside. It depended significantly on everyday activities undertaken in the stables, like feeding or cleaning. Taking the above into account, a different cleaning system should be developed (a wet cleaning system, with the use of water) and excrement should be removed more frequently.

Characteristics of diluted-stored and post-thawed semen of Hutsul stallions.

The use of frozen semen lowers the risk of disease transmission, eliminates geographical limitations and supports the implementation of genetic resource protection programs. However, due to the very rare use of frozen semen from Hutsul stallions, their genetic material is not secured in sperm banks, and very little information is available about their semen, including its suitability for cryopreservation, and sperm survival rates after thawing. The aim of this study was to analyse basic parameters such as sperm motility, vitality and morphology in diluted-stored and post-thawed Hutsul semen, using a CASA system. There were no differences in sperm motility (P = 0.3372) or morphology between the groups, although the progressive motility was higher in thawed semen (P = 0.0151), while the sperm vitality was higher in diluted-stored semen (P = 0.00517). This study demonstrates that semen from Hutsul horses is suitable for cryopreservation, thus supporting the creation of a sperm bank as a genetic reserve for representatives of this breed.

Identification of a new haplotype within the promoter region of the MSTN gene in horses from five of the most common breeds in Poland.

Myostatin (GDF-8) encoded by the MSTN gene is a negative regulator of muscle growth and development and belongs to the TGF-ß superfamily of secreted growth and differentiation factors. In Thoroughbred horses, an MSTN sequence polymorphism (g.66493737C>T) is associated with optimum race distance. In the present study, a genetic polymorphism of a predicted promoter of the MSTN gene was investigated in 451 horses belonging to five different breeds: Arabian, Thoroughbred, Polish Konik, Hucul and Polish Heavy Draft. Two SNPs located at g.66495826T>C and g.66495696T>C (chr;18 EquCab 2.0) showed three haplotypes previously described: [g.66495826:T, g.66495696:T], [g.66495826:T, g.66495696:C], [g.66495826:C, g.66495696:T] with frequencies 0.877; 0.101; 0.005; respectively. Analysis performed on Polish Heavy Draft indicated the occurrence of a new haplotype [g.6649582626:C, g.66495696:C] with frequency 0.016.