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1.  Assessment of Heat Shock Protein 70 Induction by Heat in Alfalfa Varieties and Constitutive Overexpression in Transgenic Plants 
PLoS ONE  2015;10(5):e0126051.
Heat shock proteins (HSPs) are molecular chaperones involved in many cellular functions. It has been shown that mammalian cytosolic HSP70 binds antigenic peptides mediating the activation of the immune system, and that it plays a determining role in tumour immunogenicity. This suggests that HSP70 may be used for the production of conjugated vaccines. Human and plant HSPs share high sequence similarity and some important biological functions in vitro. In addition, plant HSPs have no endotoxic side effects. Extraction of HSP70 from plants for use as vaccine adjuvant requires enhancing its concentration in plant tissues. In this work, we explored the possibility to produce HSP70 in both transgenic and non-transgenic plants, using alfalfa as a model species. First, a transcriptional analysis of a constitutive and an inducible HSP70 genes was conducted in Arabidopsis thaliana. Then the coding sequence of the inducible form was cloned and introduced into alfalfa by Agrobacterium-mediated transformation, and the accumulation of the protein in leaf tissue of transgenic plants was demonstrated. We also tested diverse alfalfa varieties for heat-inducible expression of endogenous HSP70, revealing variety-specific responses to heat shock.
PMCID: PMC4423914  PMID: 25951604
2.  First report of junctional epidermolysis bullosa (JEB) in the Italian draft horse 
Epitheliogenesis imperfecta in horses was first recognized at the beginning of the 20th century when it was proposed that the disease could have a genetic cause and an autosomal recessive inheritance pattern. Electron microscopy studies confirmed that the lesions were characterized by a defect in the lamina propria and the disease was therefore reclassified as epidermolysis bullosa. Molecular studies targeted two mutations affecting genes involved in dermal–epidermal junction: an insertion in LAMC2 in Belgians and other draft breeds and one large deletion in LAMA3 in American Saddlebred.
Case presentation
A mechanobullous disease was suspected in a newborn, Italian draft horse foal, which presented with multifocal to coalescing erosions and ulceration on the distal extremities. Histological examination of skin biopsies revealed a subepidermal cleft formation and transmission electron microscopy demonstrated that the lamina densa of the basement membrane remained attached to the dermis. According to clinical, histological and ultrastructural findings, a diagnosis of junctional epidermolysis bullosa (JEB) was made. Genetic tests confirmed the presence of 1368insC in LAMC2 in the foal and its relatives.
This is the first report of JEB in Italy. The disease was characterized by typical macroscopic, histologic and ultrastructural findings. Genetic tests confirmed the presence of the 1368insC in LAMC2 in this case: further investigations are required to assess if the mutation could be present at a low frequency in the Italian draft horse population. Atypical breeding practices are responsible in this case and played a role as odds enhancer for unfavourable alleles. Identification of carriers is fundamental in order to prevent economic loss for the horse industry.
PMCID: PMC4372232  PMID: 25889423
Junctional epidermolysis bullosa; Horse; Mechanobullous disease; Electron microscopy; Lamina densa; LAMC2; Italian draft horse; Inbreeding
3.  RNA Sequencing of the Exercise Transcriptome in Equine Athletes 
PLoS ONE  2013;8(12):e83504.
The horse is an optimal model organism for studying the genomic response to exercise-induced stress, due to its natural aptitude for athletic performance and the relative homogeneity of its genetic and environmental backgrounds. Here, we applied RNA-sequencing analysis through the use of SOLiD technology in an experimental framework centered on exercise-induced stress during endurance races in equine athletes. We monitored the transcriptional landscape by comparing gene expression levels between animals at rest and after competition. Overall, we observed a shift from coding to non-coding regions, suggesting that the stress response involves the differential expression of not annotated regions. Notably, we observed significant post-race increases of reads that correspond to repeats, especially the intergenic and intronic L1 and L2 transposable elements. We also observed increased expression of the antisense strands compared to the sense strands in intronic and regulatory regions (1 kb up- and downstream) of the genes, suggesting that antisense transcription could be one of the main mechanisms for transposon regulation in the horse under stress conditions. We identified a large number of transcripts corresponding to intergenic and intronic regions putatively associated with new transcriptional elements. Gene expression and pathway analysis allowed us to identify several biological processes and molecular functions that may be involved with exercise-induced stress. Ontology clustering reflected mechanisms that are already known to be stress activated (e.g., chemokine-type cytokines, Toll-like receptors, and kinases), as well as “nucleic acid binding” and “signal transduction activity” functions. There was also a general and transient decrease in the global rates of protein synthesis, which would be expected after strenuous global stress. In sum, our network analysis points toward the involvement of specific gene clusters in equine exercise-induced stress, including those involved in inflammation, cell signaling, and immune interactions.
PMCID: PMC3877044  PMID: 24391776
4.  Genome-Wide Analysis Reveals Selection for Important Traits in Domestic Horse Breeds 
PLoS Genetics  2013;9(1):e1003211.
Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an FST-based statistic calculated in 500-kb windows across the genome. A 5.5-Mb region of ECA18, in which the myostatin (MSTN) gene was centered, contained the highest signature of selection in both the Paint and Quarter Horse. Gene sequencing and histological analysis of gluteal muscle biopsies showed a promoter variant and intronic SNP of MSTN were each significantly associated with higher Type 2B and lower Type 1 muscle fiber proportions in the Quarter Horse, demonstrating a functional consequence of selection at this locus. Signatures of selection on ECA23 in all gaited breeds in the sample led to the identification of a shared, 186-kb haplotype including two doublesex related mab transcription factor genes (DMRT2 and 3). The recent identification of a DMRT3 mutation within this haplotype, which appears necessary for the ability to perform alternative gaits, provides further evidence for selection at this locus. Finally, putative loci for the determination of size were identified in the draft breeds and the Miniature horse on ECA11, as well as when signatures of selection surrounding candidate genes at other loci were examined. This work provides further evidence of the importance of MSTN in racing breeds, provides strong evidence for selection upon gait and size, and illustrates the potential for population-based techniques to find genomic regions driving important phenotypes in the modern horse.
Author Summary
A breed of the horse typically consists of individuals sharing very similar aesthetic and performance traits. However, a great deal of variation in traits exists between breeds. The range of variation observed among breeds can be illustrated by the size difference between the Miniature horse (0.74 m and 100 kg) and draft horse (1.8 m and 900 kg), or by comparing the optimum racing distance of the Quarter Horse (1/4 mile) to that of the Arabian (100 miles or more). In this study, we exploited the breed structure of the horse to identify regions of the genome that are significantly different between breeds and therefore may harbor genes and genetic variants targeted by selective breeding. This work resulted in the identification of variants in the Paint and Quarter Horse significantly associated with altered muscle fiber type proportions favorable for increased sprinting ability. A strong signature of selection was also identified in breeds that perform alternative gaits, and several genomic regions identified are hypothesized to be involved in the determination of size. This study has demonstrated the utility of this approach for studying the equine genome and is the first to show a functional consequence of selective breeding in the horse.
PMCID: PMC3547851  PMID: 23349635
5.  Molecular Detection, Epidemiology, and Genetic Characterization of Novel European Field Isolates of Equine Infectious Anemia Virus▿  
The application of molecular diagnostic techniques along with nucleotide sequence determination to permit contemporary phylogenetic analysis of European field isolates of equine infectious anemia virus (EIAV) has not been widely reported. As a result, of extensive testing instigated following the 2006 outbreak of equine infectious anemia in Italy, 24 farms with a history of exposure to this disease were included in this study. New PCR-based methods were developed, which, especially in the case of DNA preparations from peripheral blood cells, showed excellent correlation with OIE-approved agar gel immunodiffusion (AGID) tests for identifying EIAV-infected animals. In contrast, the OIE-recommended oligonucleotide primers for EIAV failed to react with any of the Italian isolates. Similar results were also obtained with samples from four Romanian farms. In addition, for the first time complete characterization of gag genes from five Italian isolates and one Romanian isolate has been achieved, along with acquisition of extensive sequence information (86% of the total gag gene) from four additional EIAV isolates (one Italian and three Romanian). Furthermore, in another 23 cases we accomplished partial characterization of gag gene sequences in the region encoding the viral matrix protein. Analysis of this information suggested that most Italian isolates were geographically restricted, somewhat reminiscent of the “clades” described for human immunodeficiency virus type 1 (HIV-1). Collectively this represents the most comprehensive genetic study of European EIAV isolates conducted to date.
PMCID: PMC3020406  PMID: 21084503
6.  Athletic humans and horses: Comparative analysis of interleukin-6 (IL-6) and IL-6 receptor (IL-6R) expression in peripheral blood mononuclear cells in trained and untrained subjects at rest 
BMC Physiology  2011;11:3.
Horses and humans share a natural proclivity for athletic performance. In this respect, horses can be considered a reference species in studies designed to optimize physical training and disease prevention. In both species, interleukin-6 (IL-6) plays a major role in regulating the inflammatory process induced during exercise as part of an integrated metabolic regulatory network. The aim of this study was to compare IL-6 and IL-6 receptor (IL-6R) mRNA expression in peripheral blood mononuclear cells (PBMCs) in trained and untrained humans and horses.
Nine highly trained male swimmers (training volume: 21.6 ± 1.7 h/wk in 10-12 sessions) were compared with two age-matched control groups represented by eight lightly trained runners (training volume: 6.4 ± 2.6 h/wk in 3-5 sessions) and nine untrained subjects. In addition, eight trained horses (training volume: 8.0 ± 2.1 h/wk in 3-4 sessions) were compared with eight age-matched sedentary mares. In humans, IL-6 mRNA levels in PBMCs determined by quantitative reverse transcription-polymerase chain reaction were significantly higher in highly trained subjects, whereas IL-6R expression did not differ among groups. In horses, transcripts of both IL-6 and IL-6R were significantly up-regulated in the trained group.
Up-regulation of IL-6R expression in PBMCs in horses could reflect a mechanism that maintains an adequate anti-inflammatory environment at rest through ubiquitous production of anti-inflammatory cytokines throughout the body. These findings suggest that the system that controls the inflammatory response in horses is better adapted to respond to exercise than that in humans.
PMCID: PMC3036646  PMID: 21255427
7.  Exercise-induced up-regulation of MMP-1 and IL-8 genes in endurance horses 
BMC Physiology  2009;9:12.
The stress response is a critical factor in the training of equine athletes; it is important for performance and for protection of the animal against physio-pathological disorders.
In this study, the molecular mechanisms involved in the response to acute and strenuous exercise were investigated using peripheral blood mononuclear cells (PBMCs).
Quantitative real-time PCR (qRT-PCR) was used to detect modifications in transcription levels of the genes for matrix metalloproteinase-1 (MMP-1) and interleukin 8 (IL-8), which were derived from previous genome-wide expression analysis. Significant up-regulation of these two genes was found in 10 horses that had completed a race of 90–120 km in a time-course experimental design.
These results suggest that MMP-1 and IL-8 are both involved in the exercise-induced stress response, and this represents a starting point from which to understand the adaptive responses to this phenomenon.
PMCID: PMC2705340  PMID: 19552796
8.  Exercise induced stress in horses: Selection of the most stable reference genes for quantitative RT-PCR normalization 
Adequate stress response is a critical factor during athlete horses' training and is central to our capacity to obtain better performances while safeguarding animal welfare.
In order to investigate the molecular mechanisms underlying this process, several studies have been conducted that take advantage of microarray and quantitative real-time PCR (qRT-PCR) technologies to analyse the expression of candidate genes involved in the cellular stress response.
Appropriate application of qRT-PCR, however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability.
The expression of nine potential reference genes was evaluated in lymphocytes of ten endurance horses during strenuous exercise. These genes were tested by qRT-PCR and ranked according to the stability of their expression using three different methods (implemented in geNorm, NormFinder and BestKeeper). Succinate dehydrogenase complex subunit A (SDHA) and hypoxanthine phosphoribosyltransferase (HPRT) always ranked as the two most stably expressed genes. On the other hand, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), transferrin receptor (TFRC) and ribosomal protein L32 (RPL32) were constantly classified as the less reliable controls.
This study underlines the importance of a careful selection of reference genes for qRT-PCR studies of exercise induced stress in horses. Our results, based on different algorithms and analytical procedures, clearly indicate SDHA and HPRT as the most stable reference genes of our pool.
PMCID: PMC2412902  PMID: 18489742

Results 1-8 (8)