Novel polymorphisms in the prion protein gene (PRNP) and stability of the resultant prion protein in different horse breeds.

Prion diseases are fatal neurodegenerative disorders in which the main pathogenic event is the conversion of the cellular prion protein (PrPC) into an abnormal and misfolded isoform known as PrPSc. Most prion diseases and their susceptibility and pathogenesis are mainly modulated by the PRNP gene that codes for PrP. Mutations and polymorphisms in the PRNP gene can alter PrPC amino acid sequence, leading to a change in transmission efficiency depending on the place where it occurs. Horses are animals that are considered to be highly resistant to prions. Several studies have attempted to identify polymorphisms in the PRNP gene that explain the reason for this high resistance. In this study, we have analysed 207 horses from 20 different breeds, discovering 3 novel PRNP polymorphisms. By using computer programmes such as PolyPhen-2, PROVEAN, PANTHER, Meta-SNP and PredictSNP, we have predicted the possible impact that these new polymorphisms would have on the horse prion protein. In addition, we measured the propensity for amyloid aggregation using AMYCO and analysed the lack of hydrogen bridges that these changes would entail together with their electrostatic potentials using Swiss-PdbViewer software, showing that an increased amyloid propensity could be due to changes at the level of electrostatic potentials.

5-Methylcytosine and 5-Hydroxymethylcytosine in Scrapie-Infected Sheep and Mouse Brain Tissues.

Scrapie is a neurodegenerative disorder belonging to the group of transmissible spongiform encephalopathies or prion diseases, which are caused by an infectious isoform of the innocuous cellular prion protein (PrPC) known as PrPSc. DNA methylation, one of the most studied epigenetic mechanisms, is essential for the proper functioning of the central nervous system. Recent findings point to possible involvement of DNA methylation in the pathogenesis of prion diseases, but there is still a lack of knowledge about the behavior of this epigenetic mechanism in such neurodegenerative disorders. Here, we evaluated by immunohistochemistry the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels in sheep and mouse brain tissues infected with scrapie. Expression analysis of different gene coding for epigenetic regulatory enzymes (DNMT1, DNMT3A, DNMT3B, HDAC1, HDAC2, TET1, and TET2) was also carried out. A decrease in 5mC levels was observed in scrapie-affected sheep and mice compared to healthy animals, whereas 5hmC displayed opposite patterns between the two models, demonstrating a decrease in 5hmC in scrapie-infected sheep and an increase in preclinical mice. 5mC correlated with prion-related lesions in mice and sheep, but 5hmC was associated with prion lesions only in sheep. Differences in the expression changes of epigenetic regulatory genes were found between both disease models, being differentially expressed Dnmt3b, Hdac1, and Tet1 in mice and HDAC2 in sheep. Our results support the evidence that DNA methylation in both forms, 5mC and 5hmC, and its associated epigenetic enzymes, take part in the neurodegenerative course of prion diseases.

Epigenetic Changes in Prion and Prion-like Neurodegenerative Diseases: Recent Advances, Potential as Biomarkers, and Future Perspectives.

Prion diseases are transmissible spongiform encephalopathies (TSEs) caused by a conformational conversion of the native cellular prion protein (PrPC) to an abnormal, infectious isoform called PrPSc. Amyotrophic lateral sclerosis, Alzheimer's, Parkinson's, and Huntington's diseases are also known as prion-like diseases because they share common features with prion diseases, including protein misfolding and aggregation, as well as the spread of these misfolded proteins into different brain regions. Increasing evidence proposes the involvement of epigenetic mechanisms, namely DNA methylation, post-translational modifications of histones, and microRNA-mediated post-transcriptional gene regulation in the pathogenesis of prion-like diseases. Little is known about the role of epigenetic modifications in prion diseases, but recent findings also point to a potential regulatory role of epigenetic mechanisms in the pathology of these diseases. This review highlights recent findings on epigenetic modifications in TSEs and prion-like diseases and discusses the potential role of such mechanisms in disease pathology and their use as potential biomarkers.

Neurogranin and Neurofilament Light Chain as Preclinical Biomarkers in Scrapie.

Prion diseases are diagnosed in the symptomatic stage, when the neuronal damage is spread throughout the central nervous system (CNS). The assessment of biological features that allow the detection of asymptomatic cases is needed, and, in this context, scrapie, where pre-symptomatic infected animals can be detected through rectal biopsy, becomes a good study model. Neurogranin (Ng) and neurofilament light chain (NfL) are proteins that reflect synaptic and axonal damage and have been studied as cerebrospinal fluid (CSF) biomarkers in different neurodegenerative disorders. In this study, we evaluated Ng and NfL both at the protein and transcript levels in the CNS of preclinical and clinical scrapie-affected sheep compared with healthy controls and assessed their levels in ovine CSF. The correlation between these proteins and the main neuropathological events in prion diseases, PrPSc deposition and spongiosis, was also assessed. The results show a decrease in Ng and NfL at the protein and gene expression levels as the disease progresses, and significant changes between the control and preclinical animals. On the contrary, the CSF levels of NfL increased throughout the progression of the disease. Negative correlations between neuropathological markers of prion disease and the concentration of the studied proteins were also found. Although further research is needed, these results suggest that Ng and NfL could act as biomarkers for neurodegeneration onset and intensity in preclinical cases of scrapie.

Evidence of p75 Neurotrophin Receptor Involvement in the Central Nervous System Pathogenesis of Classical Scrapie in Sheep and a Transgenic Mouse Model.

Neurotrophins constitute a group of growth factor that exerts important functions in the nervous system of vertebrates. They act through two classes of transmembrane receptors: tyrosine-kinase receptors and the p75 neurotrophin receptor (p75NTR). The activation of p75NTR can favor cell survival or apoptosis depending on diverse factors. Several studies evidenced a link between p75NTR and the pathogenesis of prion diseases. In this study, we investigated the distribution of several neurotrophins and their receptors, including p75NTR, in the brain of naturally scrapie-affected sheep and experimentally infected ovinized transgenic mice and its correlation with other markers of prion disease. No evident changes in infected mice or sheep were observed regarding neurotrophins and their receptors except for the immunohistochemistry against p75NTR. Infected mice showed higher abundance of p75NTR immunostained cells than their non-infected counterparts. The astrocytic labeling correlated with other neuropathological alterations of prion disease. Confocal microscopy demonstrated the co-localization of p75NTR and the astrocytic marker GFAP, suggesting an involvement of astrocytes in p75NTR-mediated neurodegeneration. In contrast, p75NTR staining in sheep lacked astrocytic labeling. However, digital image analyses revealed increased labeling intensities in preclinical sheep compared with non-infected and terminal sheep in several brain nuclei. This suggests that this receptor is overexpressed in early stages of prion-related neurodegeneration in sheep. Our results confirm a role of p75NTR in the pathogenesis of classical ovine scrapie in both the natural host and in an experimental transgenic mouse model.

Cerebrospinal Fluid and Plasma Small Extracellular Vesicles and miRNAs as Biomarkers for Prion Diseases.

Diagnosis of transmissible spongiform encephalopathies (TSEs), or prion diseases, is based on the detection of proteinase K (PK)-resistant PrPSc in post-mortem tissues as indication of infection and disease. Since PrPSc detection is not considered a reliable method for in vivo diagnosis in most TSEs, it is of crucial importance to identify an alternative source of biomarkers to provide useful alternatives for current diagnostic methodology. Ovine scrapie is the prototype of TSEs and has been known for a long time. Using this natural model of TSE, we investigated the presence of PrPSc in exosomes derived from plasma and cerebrospinal fluid (CSF) by protein misfolding cyclic amplification (PMCA) and the levels of candidate microRNAs (miRNAs) by quantitative PCR (qPCR). Significant scrapie-associated increase was found for miR-21-5p in plasma-derived but not in CSF-derived exosomes. However, miR-342-3p, miR-146a-5p, miR-128-3p and miR-21-5p displayed higher levels in total CSF from scrapie-infected sheep. The analysis of overexpressed miRNAs in this biofluid, together with plasma exosomal miR-21-5p, could help in scrapie diagnosis once the presence of the disease is suspected. In addition, we found the presence of PrPSc in most CSF-derived exosomes from clinically affected sheep, which may facilitate in vivo diagnosis of prion diseases, at least during the clinical stage.

Impairment of autophagy in scrapie-infected transgenic mice at the clinical stage.

Autophagy appears to play a role in the etiology and progress of misfolded protein disorders. Although this process is dysregulated in prion diseases, it is unknown whether this impairment is a cause or a consequence of prion neuropathology. The study of autophagy during the progress of the disease could elucidate its role. For this purpose, we have investigated its regulation at different stages of the disease in Tg338 mice, a transgenic murine model that overexpresses the highly susceptible ovine VRQ prion protein allele. Mice were intracerebrally inoculated with mouse-adapted classical scrapie and euthanized at the preclinical and clinical stages of the disease. Regulation of autophagy was investigated analyzing the distribution of LC3-B and p62 proteins by immunohistochemistry. Moreover, the expression of genes involved in autophagy regulation was quantified by real-time PCR. LC3-B and p62 proteins were downregulated and upregulated, respectively, in the central nervous system of infected mice with clinical signs of scrapie. Accumulation of p62 correlated with scrapie-related lesions, suggesting an impairment of autophagy in highly prion-affected areas. In addition, Gas5 (growth arrest-specific 5), Atg5 (autophagy-related 5), and Fbxw7 (F-box and WD repeat domain containing 7) transcripts were downregulated in mesencephalon and cervical spinal cord of the same group of animals. The impairment of autophagic machinery seems to be part of the pathological process of scrapie, but only during the late stage of prion infection. Similarities between Tg338 mice and the natural ovine disease make them a reliable in vivo model to study prion infection and autophagy side by side.

Forkhead Box P3 Methylation and Expression in Men with Obstructive Sleep Apnea.

BACKGROUND: Epigenetic changes in obstructive sleep apnea (OSA) have been proposed as a mechanism for end-organ vulnerability. In children with OSA, Forkhead Box P3 (FOXP3) DNA methylation were associated with inflammatory biomarkers; however, the methylation pattern and its effect in the expression of this gene have not been tested in adults with OSA. METHODS: Plasma samples from subjects without comorbid conditions other than OSA were analyzed (the Epigenetics Status and Subclinical Atherosclerosis in Obstructive Sleep Apnea (EPIOSA) Study: NCT02131610). In 16 patients with severe OSA (Apnea-Hypopnea Index-AHI- > 30 events/h) and seven matched controls (AHI < 5), methylation of FOXP3 gen was evaluated by PCR of the promoter and by pyrosequencing of the intron 1 Treg-specific demethylated region (TSDR). In another 74 patients with OSA (AHI > 10) and 31 controls, we quantified FOXP3 protein expression by ELISA and gene expression by quantitative real-time PCR. C-reactive protein (CRP) and plasma Treg cells were also evaluated. RESULTS: Neither the levels of the promoter nor the TSDR demethylated region were different between controls and patients with OSA, whether they were grouped by normal or high CRP. FOXP3 protein and mRNA expression did not differ between groups. CONCLUSIONS: FOXP3 methylation or its expression is not altered in adults with OSA, whatever their inflammatory status.

Antimicrobial resistance among canine enteric Escherichia coli isolates and prevalence of attaching-effacing and extraintestinal pathogenic virulence factors in Spain.

The aim of this study was to estimate the prevalence of antimicrobial resistance (AMR) in Escherichia coli from a dog population in Spain and assess specific virulence factors. Susceptibility to 22 antimicrobials was tested along with the production of extended-spectrum ß-lactamases (ESBLs) and AmpC in faecal isolates from 100 dogs. Virulence-related genes associated with attaching and effacing E. coli (eae, Stx1, Stx2) and extraintestinal pathogenic E. coli - ExPEC - (papC, hlyA and cnf1) were detected by PCR. At least one kind of AMR was observed in 73% of the isolates. The highest prevalences corresponded to penicillin (45%), aminoglycoside (40%) and non-extended spectrum cephalosporin (39%) classes. Multidrug resistance (MDR) was observed in 53.4% of the resistant isolates. No resistance to colistin was found. Production of ESBL/AmpC enzymes was detected in 5% of E. coli. Shiga toxin-producing E. coli were not observed, enteropathogenic E. coli were identified in only 12% of them, and ExPEC were found in 25%. Dog faeces can be a source of E. coli strains potentially presenting a threat to humans through their virulence factors or AMR. The non-hygienic keeping of animals may increase the risk of colonisation of such pathogens in humans.

Determining the Relative Susceptibility of Four Prion Protein Genotypes to Atypical Scrapie.

Atypical scrapie is a sheep prion (PrPSc) disease whose epidemiology is consistent with a sporadic origin and is associated with specific polymorphisms of the normal cellular prion protein (PrPC). To determine the relative amounts of PrP polymorphisms present in atypical scrapie, total PrP was digested with chymotrypsin to generate characteristic peptides spanning relevant polymorphisms at positions 136, 141, 154, 171, and 172 of sheep PrPC. A multiple reaction monitoring method (MRM), employing 15N-labeled internal standards, was used to detect and quantify these polymorphisms present in both the PrPSc and PrPC from heterozygous (ALRRY and ALHQY or ALRQD or AFRQY) atypical scrapie-infected or uninfected control sheep. Both polymorphisms of the full length and truncated (C1) natively expressed PrPC are produced in equal amounts. The overall amount of PrPC present in the infected or uninfected animals was similar. PrPSc isolated from heterozygotes was composed of significant amounts of both PrP polymorphisms, including the ALRRY polymorphism which is highly resistant to classical scrapie. Thus, an atypical scrapie infection does not result from an overexpression of sheep PrPC. The replication of all atypical scrapie prions occurs at comparable rates, despite polymorphisms at positions 141, 154, 171, or 172.

Experimental transmission to a calf of an isolate of Spanish classical scrapie.

Multiple theories exist regarding the origin of bovine spongiform encephalopathy (BSE). An early and prominent theory proposed that BSE was the result of the adaptation of sheep scrapie to cattle. The reports to date indicate that the distribution of the pathological prion protein (PrPSc) in experimental bovine scrapie is largely restricted to the central nervous system (CNS). Here, we describe pathological findings in a calf intracerebrally inoculated with a Spanish classical scrapie isolate. While clinical disease was observed 30 months after inoculation and PrPSc was detected in the CNS, the corresponding phenotype differed from that of BSE. Immunohistochemistry and PMCA also revealed the presence of PrPSc in the peripheral nerves, lymphoid tissues, skeletal muscle and gastrointestinal tract, suggesting centrifugal spread of the scrapie agent from the brain. To the best of our knowledge, this is the first report describing the detection of PrPSc in tissues other than the CNS after experimental transmission of scrapie to cattle.

Increased circulating microRNAs miR-342-3p and miR-21-5p in natural sheep prion disease.

Scrapie is a transmissible spongiform encephalopathy (TSE), or prion disease, of sheep and goats. As no simple diagnostic tests are yet available to detect TSEs in vivo, easily accessible biomarkers could facilitate the eradication of scrapie agents from the food chain. To this end, we analysed by quantitative reverse transcription PCR a selected set of candidate microRNAs (miRNAs) from circulating blood plasma of naturally infected, classical scrapie sheep that demonstrated clear scrapie symptoms and pathology. Significant scrapie-associated increase was repeatedly found for miR-342-3p and miR-21-5p. This is the first demonstration, to our knowledge, of circulating miRNA alterations in any animal suffering from TSE. Genome-wide expression studies are warranted to investigate the true depth of miRNA alterations in naturally occurring TSEs, especially in presymptomatic animals, as the presented study demonstrates the potential feasibility of miRNAs as circulating TSE biomarkers.

Analysis of microsatellite markers in a Cuban water buffalo breed.

The aim of this Regional Research Communication was to validate a panel of 30 microsatellite markers recommended by FAO/ISAG for studies of biodiversity in cattle to improve the characterisation of Cuban buffalo populations. The water buffalo (Bubalus bubalis) is an economically important livestock species. Therefore, research focused on the study of the genetic relationships among water buffalo populations is useful to support conservation decisions and to design breeding schemes. Twenty-eight of the 30 tested regions were amplified, one of which (ETH10) turned out to be monomorphic. A total of 143 alleles were observed in the Cuban water buffalo population. The average number of alleles per locus was 5·04. The number of alleles per polymorphic locus ranged from two (INRA 63 and MM12) to nine (ETH185). The observed and expected heterozygosity ranged from 0·108 (HAUT24) to 0·851 (CSSM66) and 0·104 (MM12) to 0·829(INRA32), respectively. The polymorphic information content (PIC) ranged from 0·097 (MM12) to 0·806 (INRA32), and the overall value for these markers was 0·482. Within the population, inbreeding estimates (F IS) was positive in 14 of the 30 loci analysed. This study thus highlights the usefulness of heterologous bovine microsatellite markers to assess the genetic variability in Cuban water buffalo breeds. Furthermore, the results can be utilised for future breeding strategies and conservation.

Characterization of mesenchymal stem cells in sheep naturally infected with scrapie.

Mesenchymal stem cells (MSCs) can be infected with prions and have been proposed as in vitro cell-based models for prion replication. In addition, autologous MSCs are of interest for cell therapy in neurodegenerative diseases. To the best of our knowledge, the effect of prion diseases on the characteristics of these cells has never been investigated. Here, we analysed the properties of MSCs obtained from bone marrow (BM-MSCs) and peripheral blood (PB-MSCs) of sheep naturally infected with scrapie ­ a large mammal model for the study of prion diseases. After three passages of expansion, MSCs derived from scrapie animals displayed similar adipogenic, chondrogenic and osteogenic differentiation ability as cells from healthy controls, although a subtle decrease in the proliferation potential was observed. Exceptionally, mesenchymal markers such as CD29 were significantly upregulated at the transcript level compared with controls. Scrapie MSCs were able to transdifferentiate into neuron-like cells, but displayed lower levels of neurogenic markers at basal conditions, which could limit this potential .The expression levels of cellular prion protein (PrPC) were highly variable between cultures, and no significant differences were observed between control and scrapie-derived MSCs. However, during neurogenic differentiation the expression of PrPC was upregulated in MSCs. This characteristic could be useful for developing in vitro models for prion replication. Despite the infectivity reported for MSCs obtained from scrapie-infected mice and Creutzfeldt­Jakob disease patients, protein misfolding cyclic amplification did not detect PrPSc in BM- or PB-MSCs from scrapie-infected sheep, which limits their use for in vivo diagnosis for scrapie.

Gene expression profiling of mesenteric lymph nodes from sheep with natural scrapie.

BACKGROUND: Prion diseases are characterized by the accumulation of the pathogenic PrPSc protein, mainly in the brain and the lymphoreticular system. Although prions multiply/accumulate in the lymph nodes without any detectable pathology, transcriptional changes in this tissue may reflect biological processes that contribute to the molecular pathogenesis of prion diseases. Little is known about the molecular processes that occur in the lymphoreticular system in early and late stages of prion disease. We performed a microarray-based study to identify genes that are differentially expressed at different disease stages in the mesenteric lymph node of sheep naturally infected with scrapie. Oligo DNA microarrays were used to identify gene-expression profiles in the early/middle (preclinical) and late (clinical) stages of the disease. RESULTS: In the clinical stage of the disease, we detected 105 genes that were differentially expressed (≥2-fold change in expression). Of these, 43 were upregulated and 62 downregulated as compared with age-matched negative controls. Fewer genes (50) were differentially expressed in the preclinical stage of the disease. Gene Ontology enrichment analysis revealed that the differentially expressed genes were largely associated with the following terms: glycoprotein, extracellular region, disulfide bond, cell cycle and extracellular matrix. Moreover, some of the annotated genes could be grouped into 3 specific signaling pathways: focal adhesion, PPAR signaling and ECM-receptor interaction. We discuss the relationship between the observed gene expression profiles and PrPSc deposition and the potential involvement in the pathogenesis of scrapie of 7 specific differentially expressed genes whose expression levels were confirmed by real time-PCR. CONCLUSIONS: The present findings identify new genes that may be involved in the pathogenesis of natural scrapie infection in the lymphoreticular system, and confirm previous reports describing scrapie-induced alterations in the expression of genes involved in protein misfolding, angiogenesis and the oxidative stress response. Further studies will be necessary to determine the role of these genes in prion replication, dissemination and in the response of the organism to this disease.

Epigenetics modifications and Subclinical Atherosclerosis in Obstructive Sleep Apnea: The EPIOSA study.

BACKGROUND: Obstructive sleep apnea (OSA) is associated with increased risk for cardiovascular morbidity and mortality. Epidemiological and animal models studies generate hypotheses for innovative strategies in OSA management by interfering intermediates mechanisms associated with cardiovascular complications. We have thus initiated the Epigenetics modification in Obstructive Sleep Apnea (EPIOSA) study (ClinicalTrials.gov identifier: NCT02131610). METHODS/DESIGN: EPIOSA is a prospective cohort study aiming to recruit 350 participants of caucasian ethnicity and free of other chronic or inflammatory diseases: 300 patients with prevalent OSA and 50 non-OSA subjects. All of them will be follow-up for at least 5 years. Recruitment and study visits are performed in single University-based sleep clinic using standard operating procedures. At baseline and at each one year follow-up examination, patients are subjected to a core phenotyping protocol. This includes a standardized questionnaire and physical examination to determine incident comorbidities and health resources utilization, with a primary focus on cardiovascular events. Confirmatory outcomes information is requested from patient records and the regional Department of Health Services. Every year, OSA status will be assessed by full sleep study and blood samples will be obtained for immediate standard biochemistry, hematology, inflammatory cytokines and cytometry analysis. For biobanking, aliquots of serum, plasma, urine, mRNA and DNA are also obtained. Bilateral carotid echography will be performed to assess subclinical atherosclerosis and atherosclerosis progression. OSA patients are treated according with national guidelines. DISCUSSION: EPIOSA will enable the prospective evaluation of inflammatory and epigenetics mechanism involved in cardiovascular complication of treated and non-treated patients with OSA compared with non OSA subjects.

Multidrug resistance in pathogenic Escherichia coli isolates from urinary tract infections in dogs, Spain.

Escherichia coli (E. coli) is a pathogen frequently isolated in cases of urinary tract infections (UTIs) in both humans and dogs and evidence exists that dogs are reservoirs for human infections. In addition, E. coli is associated to increasing antimicrobial resistance rates. This study focuses on the analysis of antimicrobial resistance and the presence of selected virulence genes in E. coli isolates from a Spanish dog population suffering from UTI. This collection of isolates showed an extremely high level of phenotypic resistance to 1st-3rd generation cephalosporins, followed by penicillins, fluoroquinolones and amphenicols. Apart from that, 13.46% of them were considered extended-spectrum beta-lactamase producers. An alarmingly high percentage (71.15%) of multidrug resistant isolates were also detected. There was a good correlation between the antimicrobial resistance genes found and the phenotypic resistance expressed. Most of the isolates were classified as extraintestinal pathogenic E. coli, and two others harbored virulence factors related to diarrheagenic pathotypes. A significant relationship between low antibiotic resistance and high virulence factor carriage was found, but the mechanisms behind it are still poorly understood. The detection of high antimicrobial resistance rates to first-choice treatments highlights the need of constant antimicrobial resistance surveillance, as well as continuous revision of therapeutic guidelines for canine UTI to adapt them to changes in antimicrobial resistance patterns.

Analysis of Plasma-Derived Exosomal MicroRNAs as Potential Biomarkers for Canine Idiopathic Epilepsy.

Epilepsy is one of the most prevalent complex neurological diseases in both the canine and human species, with the idiopathic form as its most common diagnosis. MicroRNAs (miRNAs) are small, noncoding RNA molecules that play a role in gene regulation processes and appear to be a promising biological target for convulsion control. These molecules have been reported as constituents of the internal content of exosomes, which are small extracellular vesicles released by cells. In this study, exosome samples were isolated from the plasma of 23 dogs, including 9 dogs with epilepsy responsive to treatment, 6 dogs with drug-resistant epilepsy, and 8 control dogs. Plasma exosomes were then characterized by electron transmission microscopy, nanoparticle tracking analysis, and dot blotting. Afterwards, the microRNA-enriched RNA content of exosomes was isolated, and miRNA quantification was performed by quantitative real-time PCR. Seven circulating miRNAs that have been previously described in the literature as potential diagnostic or prognostic biomarkers for epilepsy were evaluated. We observed significant differences in miR-16 (p < 0.001), miR-93-5p (p < 0.001), miR-142 (p < 0.001), miR-574 (p < 0.01), and miR-27 (p < 0.05) levels in dogs with refractory epilepsy compared to the control group. In drug-sensitive epileptic dogs, miR-142 (p < 0.01) showed significant differences compared to healthy dogs. Moreover, distinct levels of miR-16 (p < 0.05), miR-93-5p (p < 0.01), miR-132 (p < 0.05), and miR-574 (p < 0.05) were also found between drug-sensitive and drug-resistant epileptic dogs. Our results present plasma-circulating exosomes as an advantageous source of epileptic biomarkers, highlighting the potential of miRNAs as prognostic and diagnostic biomarkers of canine idiopathic epilepsy.

Gene and protein patterns of potential prion-related markers in the central nervous system of clinical and preclinical infected sheep.

The molecular pathogenic mechanisms of prion diseases are far from clear. Genomic analyses have revealed genetic biomarkers potentially involved in prion neuropathology in naturally scrapie-infected sheep, a good animal model of infectious prionopathies. However, these biomarkers must be validated in independent studies at different stages of the disease. The gene and protein expression profiles and protein distribution of six potential genetic biomarkers (i.e., CAPN6, COL1A2, COL3A1, GALA1, MT2A and MTNR1B) are presented here for both the early and terminal stages of scrapie in five different brain regions. Gene transcription changes were confirmed in the medulla oblongata, and the expression profiles were generally similar in other central nervous system regions. The changes were more substantial in clinical animals compared to preclinical animals. The expression of the CAPN6 protein increased in the spinal cord and cerebellum of the clinical and preclinical brains. The distribution of the GALA1 was identified in glial cells from the cerebellum of scrapie-infected animals, GALA1 protein expression was increased in clinical animals in the majority of regions, and the increase of MT2A was in agreement with previous reports. The downregulation of MTNR1B was especially marked in the Purkinje cells. Finally, although collagen genes were downregulated the protein immunostaining did not reveal significant changes between the scrapie-infected and control animals. In conclusion, this study of gene transcription and protein expression and distribution confirm CAPN6, GALA1, MTNR1B and MT2A as potential targets for further prion disease research.

Analysis of conservation priorities of Iberoamerican cattle based on autosomal microsatellite markers.

BACKGROUND: Determining the value of livestock breeds is essential to define conservation priorities, manage genetic diversity and allocate funds. Within- and between-breed genetic diversity need to be assessed to preserve the highest intra-specific variability. Information on genetic diversity and risk status is still lacking for many Creole cattle breeds from the Americas, despite their distinct evolutionary trajectories and adaptation to extreme environmental conditions. METHODS: A comprehensive genetic analysis of 67 Iberoamerican cattle breeds was carried out with 19 FAO-recommended microsatellites to assess conservation priorities. Contributions to global diversity were investigated using alternative methods, with different weights given to the within- and between-breed components of genetic diversity. Information on Iberoamerican plus 15 worldwide cattle breeds was used to investigate the contribution of geographical breed groups to global genetic diversity. RESULTS: Overall, Creole cattle breeds showed a high level of genetic diversity with the highest level found in breeds admixed with zebu cattle, which were clearly differentiated from all other breeds. Within-breed kinships revealed seven highly inbred Creole breeds for which measures are needed to avoid further genetic erosion. However, if contribution to heterozygosity was the only criterion considered, some of these breeds had the lowest priority for conservation decisions. The Weitzman approach prioritized highly differentiated breeds, such as Guabalá, Romosinuano, Cr. Patagonico, Siboney and Caracú, while kinship-based methods prioritized mainly zebu-related breeds. With the combined approaches, breed ranking depended on the weights given to the within- and between-breed components of diversity. Overall, the Creole groups of breeds were generally assigned a higher priority for conservation than the European groups of breeds. CONCLUSIONS: Conservation priorities differed significantly according to the weight given to within- and between-breed genetic diversity. Thus, when establishing conservation programs, it is necessary to also take into account other features. Creole cattle and local isolated breeds retain a high level of genetic diversity. The development of sustainable breeding and crossbreeding programs for Creole breeds, and the added value resulting from their products should be taken into consideration to ensure their long-term survival.