Project description:Background: Clenbuterol, a beta2-adrenergic receptor agonist, is used therapeutically to treat respiratory conditions in the horse. However, by virtue of its mechanism of action it has been suggested that clenbuterol may also have repartitioning affects in horses and as such the potential to affect performance. Clenbuterol decreases the percent fat and increases fat-free mass following high dose administration in combination with intense exercise in horses. In the current study, microarray analysis and real-time PCR were used to study the temporal effects of low and high dose chronic clenbuterol administration on differential gene expression of several skeletal muscle myosin heavy chains, genes involved in lipid metabolism and the β2-adrenergic receptor. The effect of clenbuterol administration on differential gene expression has not been previously reported in the horse, therefore the primary objective of the current study was to describe clenbuterol-induced temporal changes in gene expression following chronic oral administration of clenbuterol at both high and low doses. Steady state clenbuterol concentrations were achieved at approximately 50 hours post administration of the first dose for the low dose regimen and at approximately 18-19 days (10 days post administration of 3.2 μg/kg) for the escalating dosing regimen. Following chronic administration of the low dose (0.8 µg/kg BID) of clenbuterol, a total of 114 genes were differentially expressed, however, none of these changes were found to be significant following FDR adjustment of the p-values. A total of 7,093 genes were differentially expressed with 3,623 genes up regulated and 3,470 genes down regulated following chronic high dose administration. Of the genes selected for further study by real-time PCR, down-regulation of genes encoding myosin heavy chains 2 and 7, steroyl CoA desaturase and the β2-adrenergic receptor were noted. For most genes, expression levels returned towards baseline levels following cessation of drug administration. Conclusion: This study showed no evidence of modified gene expression following chronic low dose administration of clenbuterol to horses. However, following chronic administration of high doses of clenbuterol alterations were noted in transcripts encoding various myosin heavy chains, lipid metabolizing enzymes and the β2-adrenergic receptor. Five healthy horses were studied. Twenty-two of the horses received 0.8 µg/kg clenbuterol PO BID for 30 days and an additional 4 horses received 0.8 µg/kg, BID x 3 days; 1.6 µg/kg, BID x 3 days; 2.4 µg/kg, BID x 3 days; 3.2 µg/kg, BID for 21 days. Muscle biopsy samples were collected one day prior to administration of the first dose of clenbuterol and at a number of times post drug administration. A final sample was collected one-week post administration of the final dose (35 days post administration of the first dose). The tissue was transferred to a cryovial containing RNAlater (Qiagen Inc, Valencia, CA) and stored at -20° C until processed. Total RNA was purified using a miRNeasy Mini kit (RNeasy Mini, Qiagen Inc, Valencia, CA) and following the manufacturer’s instructions. Total RNA integrity was assessed using the Experion Automated Electrophoresis System (Bio-rad, Hercules, CA). Only RNA samples with RIN ≥ 8 and 260/280 ratios between 1.7 and 2.1 were used. Equine specific microarrays (EquGene-1.0-st; Affymetrix, Santa Clara, CA), containing expression profiling of 30,559 well-characterized genes using 504,603 probes were utilized. To reduce biological noise as a result of genetic variability, each horse was analyzed separately and served as their own control for comparison of baseline samples to day 14 (low dose administration) or day 28 (escalating dose regimen). Five biological replicates per time point were tested. Purified total RNA (5 µg) was used for cDNA synthesis in accordance with the Ambion® WT Expression assay kit (Affymetrix, Santa Clara, CA) manufacturer’s protocol. In vitro transcription was used to incorporate biotin labels using the GeneChip® WT Terminal Labeling system (Affymetrix, Santa Clara, CA) and samples hybridized to the Equine microarray. Arrays were washed and stained on a Fluidics Station 450 (Affymetrix, Santa Clara, CA) and scanned on a GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA) in accordance with manufacturer’s protocols. The microarrays were evaluated for differential gene expression using Transcriptome Analysis Console (TAC) and for hybridization quality control using Expression Console Software (Affymetrix, Santa Clara, CA). In brief, a total of five Cell Intensity Files were generated per time point, uploaded and normalized under the following conditions: PM (perfect match)-only as a PM intensity adjustment and the Robust Multichip Analysis (RMA) quantification method. For evaluation of the assays performance the number of differentially expressed genes detected between baseline and day 14 (low dose administration) or day 28 (escalating dose regimen) were assessed. Based on the TAC software user’s manual, genes with mean transformed ratios significantly less than -2 and larger than +2 were considered significantly regulated. A number of the significant genes were selected by filtering the genes using an ANOVA (p value < 0.05). A Pearson's correlation coefficient was used to calculate linear dependence between time point and baseline samples to evaluate the correlation coefficient, where 1 was a positive correlation and 0 was no correlation. For each probeset, expression at day 14 (low dose administration) or day 28 (escalating dose regimen) was compared to expression at baseline in the same horse using a paired t-test. Fold changes and their confidence intervals were calculated by exponentiating (base 2) the mean within-horse difference in expression for each gene and the associated t confidence intervals. P-values were adjusted for multiple testing using the False Discovery Rate (FDR) method. Analyses were conducted using the statistical software environment R, version 3.0.2 (R Core Team, 2013).
Project description:The purpose of this experiment was to further our understanding of gene expression in the central nervous system (thalamus and cerebrum) after exposure to West Nile virus. To that end, three different analyses were performed. The first examined differences in gene expression between horses not vaccinated and exposed to WNV and normal control horses (exposure). The second examined differences in gene expression between horses not vaccinated and exposed to WNV and horses vaccinated and exposed to WNV (survival). And the third examined differences between the nonvaccinated cerebrum and nonvaccinated thalamus of horses exposed to WNV (location). Six conditions- Gene expression in the thalamus and cerebrum of three different groups of horses (Non-vaccinated horses exposed to West Nile virus, Vaccinated horses exposed to West Nile virus, normal horses not exposed to West Nile virus). Biological replicates- 6 normal cerebrums, 6 normal thalamus, 6 vaccinated and exposed cerebrums, 6 vaccinated and exposed thalamus, 6 non-vaccinated and exposed cerebrum, 6 non-vaccinated and exposed thalamus.
Project description:Purpose: Next-generation sequencing (NGS) was used to select genes potentially associated with exercise adaptation in Arabian horses. Methods: Whole transcriptome profiling of blood was performed for untrained horses and horses from which samples were collected during at 3 different periods of training procedure (T1-during intense training period - March, T2- before starts - May and T3 -after flat racing season - October). The muscle transcriptome sequencing was performed for 37 blood samples using Illumina HiScan SQ in 75 single-end cycles. The quantifying transcript abundances was made using the RSEM supported by STAR aligner. The raw reads were aligned to the Equus caballus reference genome. Differentially expressed genes in blood tissue were detected by DESeq2. The RNA-seq results were validated using by qPCR. Results: The increase of the number of DEGs between subsequent training periods has been observed and the highest amount of DEGs was detected between untrained horses (T0) and horses at the end of the racing season (T3) – 440. The comparison of transcriptome of T2 vs T3 and T0 vs T3 showed a significant advantage of up-regulated genes during long-term exercise (up-regulation of 266 and 389 DEGs in T3 period compared T2 and T0; respectively). Our results showed that the largest number of identified genes encoded transcription factors, nucleic acid binding proteins and G-protein modulators, which mainly were transcriptional activated at the last training phase (T3) . Moreover, in the T3 period the identified DEGs represented genes coded for cytoskeletal proteins including actin cytoskeletal proteins and kinases. The most abundant exercise-upregulated genes were involved in pathways important in regulating the cell cycle (PI3K-Akt signaling pathway), cell communication (cAMP-dependent pathway), proliferation, differentiation and apoptosis as well as immunity processes (Jak-STAT signaling pathway). We also observed exercise induced expression of genes related in regulation of actin cytoskeleton, gluconeogenesis (FoxO signaling pathway; Insulin signaling pathway), glycerophospholipid metabolism and calcium signaling. Conclusions: TOur results allow to identify changes in genes expression profile following training schedule in Arabian horses. Based on comparison analysis of blood transcriptomes, several exercise-regulated pathways and genes most affected by exercise were detected. We pinpointed overrepresented molecular pathways and genes essential for exercise adaptive response via maintaining of body homeostasis. The observed transcriptional activation of such gene as LPGAT1, AGPAT5, PIK3CG, GPD2, FOXN2, FOXO3, ACVR1B and ACVR2A can be a base for further research in order to identify genes potentially associated with race performance in Arabian horses. Such markers will be essential to choice the training type, and could result in differences in racing performance specific to various breeds. The blood transcriptome sequencing was performed for 37 samples collected form Arabian horses using Illumina HiScan SQ in75 single-end cycles and in 3-4 technical repetitions.repetitions.
Project description:The unprecedented magnitude of the 2013-2016 Makona Ebola virus (M-EBOV) epidemic likely resulted from multiple epidemiologic factors that set it apart from previous outbreaks. Nonetheless, genetic adaptations that distinguish M-EBOV from previous isolates may also have contributed to the scale of the epidemic. Of particular interest is a M-EBOV glycoprotein (GP) variant, GP-A82V, that was first detected at the inflection point of the 2013-2016 outbreak - when the number of cases increased exponentially - and which completely supplanted the earlier M-EBOV sequence. We found that, as compared with the earlier strain, GP-A82V increased the ability of M-EBOV to fuse with and infect cells of primate origin, including human blood dendritic cells, without altering innate immune signaling in target cells. Residue 82 is located at the NPC1-binding site on M-EBOV GP and the increased infectivity of GP-A82V was restricted to cells from species in which the NPC1 orthologue bears primate-defining residues at the critical interface. We utilized HIV-derived lentiviral vectors pseudotyped with founder and A82V containing M-EBOV GPs to explore the potential that this modification alters how human monocyte-derived dendritic cells (MDDCs) respond to EBOV GP stimulation. Overall design: We generated stocks of lentiviral vector bearing one the following three M-EBOV GPs: founder, A82V, and A82V/T230A. These viral stocks were used to challenge MDDCs from two healthy, anonymous human donors. Stimulated MDDCs were harvested at 1, 2, 4, and 6 hours after viral addition. Gene expression in M-EBOV GP challenged MDDCs was compared to a unstimulated control.
Project description:We undertook gene expression microarray experiments to identify genes that are differentially expressed in heaves-affected horses versus matched controls. Mediastinal (pulmonary-draining) lymph nodes were sterilely obtained from affected and control horses, dissected, and frozen at -80oC. RNA was extracted from these tissues for downstream applications. These experiments utilized a commercially available Agilent horse array that featured >43,000 probes on a 4x44k array format. Mediastinal lymph node RNA from seven heaves-affected horses was compared to matching RNA from healthy, normal control horses.
Project description:Capacity of exercise and performance is the most valuable in the horses. They have been selected for strength, speed, and indurance trait. Athletic pheno types are influenced markedly by environment, management, and training. However, it has long been accepted that there are underlying genetic factors. To determine altered mRNA expression in circulating leukocytes of horses induced by exercise. Healthy neutered male warmblood horses were subjected to indoor exercise (trotting with alternative cantering for 6o minutes). Peripheral blood was collected from the jugular vein before and after the exercise, and subsequently buffy coat leukocytes were isolated by centrifugation. Total RNAs was isolated. Cyanine 3-labeled cRNA (complementary RNA) was generated from Agilent’s Low RNA Input Linear Amplification kit with 500 ng total RNA. Labeled cRNA was applied microarray (Agilent technologies, 8x60K) using Agilent’s Gene Expression Hybridization Kit. The present study revealed a subset of mRNAs in equine peripheral blood leukocytes affected by exercise, providing background information for genes associated with exercise in warm-blood horses. Three healthy, gelding warmblood horses between 9 and 17 yr were selected. 6 samples were collected containing 3 samples before exercise and 3 samples after exercise
Project description:Purpose: RNA-seq method was used to select genes expressed in muscle tissue and are potentially associated with exercise adaptation in Arabian horses. Methods: Whole transcriptomes between three time points of muscle tissue collection were compared: T0 (untrained horses), T1 (horses after intense gallop phase) and T2 (at the end of the racing season), in total 23 samples. The biopsy of gluteus medius muscle was performed by using minimally invasive ProMag™ Ultra Automatic Biopsy Instrument with a 2 mm diameter biopsy needle. The total RNA was isolated using by TriReagent and 300ng was used to cDNA libraries preparation. The NGS sequencing was performed on HiScan SQ (Illumina). The quantifying transcript abundances was made using the RSEM supported by STAR aligner. The raw reads were aligned to the Equus caballus reference genome. Differentially expressed genes were detected by DESeq2. The RNA-seq results were validated using by qPCR. Results: To detected differentially expressed genes during training preparing to the flat racing, whole transcriptomes between three time points of muscle tissue collection were compared: T0 (untrained horses), T1 (horses after intense gallop phase) and T2 (at the end of the racing season). We identified 1168 DEGs between T0 vs T1; 1593 between T1 vs T2 and 763 between T2 vs T0. The analysis for all DEGs allow to detect 11 pathways which ale significant over represented between at last two training periods. The numerous group of exercise-regulated DEGs was related with muscle cell structure and signaling (‘focal adhesion’, ‘adherens juntion’ and ‘PI3-ATK signaling’) and included insulin-like growth factor 1 receptor (IGF1R); insulin receptor (INSR); transforming growth factor beta receptors 1 and 2 (TGFBR1; TGFBR2); vascular endothelial growth factor B (VEGFB); epidermal growth factor (EGF); hepatocyte growth factor (HGF) and vascular endothelial growth factor D (FIGF). Our results showed that in Arabian horses exercise modified the expression of genes belonging to the ‘PPAR signaling pathway’ (e.g. PPARA; PPARD; PLIN2); ‘calcium signaling pathway’ (e.g. PLN; PLCD1; TNNC1; TNNC2) as well as pathways associated with metabolism processes - ‘oxidative phosphorylation’; ‘fatty acid metabolism’; ‘glycolysis/gluconeogenesis’ and ‘citrate cycle’. Conclusions: Our research allowed to identify the group of exercise-regulated genes which was related with muscle cell structure as well as signaling and pinpointed the significant metabolic processes critical for adaptive response during training. We confirmed that in Arabians, the exercise switch energy generation towards fatty acid utilization, enhance glycogen transport and calcium signaling. The sequencing of skeletal muscle transcriptome allowed to propose the panel of new candidate genes (such as SLC16A1; ME3; ACTN3; PPARα; SH3RF2; TPM3; TNNC1; TNNI3; TGFBR1; TGFBR2; FABP3) potentially related with body homeostasis maintenance and race performance in Arabian horse. Overall design: The muscle (gluteus medius) transcriptome sequencing was performed for 23 samples collected form Arabian horses , using Illumina HiScan SQ in50 single-end cycles and in 6 technical repetitions repetitions.
Project description:In the current study, we use gene expression approach to assess the effects and duration of action of IPA on the expression levels of genes encoding pro and anti-inflammatory genes and structural proteins in an exercised horse model.Equine specific microarrays containing expression profiling of 25,923 genes were used. Baseline samples collected the day prior to IPA administration were compared to those collected at 24 hours and on day 7 and samples from day 7 compared to 24 hours. The volcano plots of differentially expressed abundant transcripts generated from microarray analysis are depicted in Figure 2. Comparison of baseline samples to 24 hours revealed 855 differentially expressed genes with 632 genes up-regulated and 223 genes down regulated. Comparison of baseline samples to those collected on day 7 showed 23,358 genes differentially expressed with 23,318 genes up-regulated and 40 down regulated. When samples from day 7 were compared to 24 hours, 26,411 genes were differentially expressed with 26,409 genes up-regulated and 2 genes down regulated. Based on the results of the microarray analysis (greatest fold change and statistical significance), 20 genes were chosen for further analysis to quantitate the change in expression levels, relative to baseline, at several time points post drug administration. Overall design: Twelve university owned exercised adult Thoroughbred horses including 6 geldings and 6 mares age: 4-8 years; weight: 492-600 kg) were studied. Horses did not receive corticosteroids or any other intra-articular medications for at least one year and any other medications for at least two weeks prior to commencement of the study. This study was approved by the Institutional Animal Care and Use Committee of the University of California, Davis. Each horse was weighed the morning of drug administration and a 14- gauge catheter placed in the external jugular vein for blood sampling. Immediately prior to drug administration, the area over the right antebrachiocarpal joint was scrubbed with betadine solutionc and 70% isopropyl alcohol, the joint flexed and a total dose of 8 mg of IPA (Predef® 2X)d or 0.9% saline administered aseptically into the right joint. Two days after synovial fluid collection, horses were allowed to freely exercise in a round pen. Horses returned to their regular exercise regimen on day 3 post drug administration. The IPA dose chosen for this study was based upon a published survey of the most commonly used dose by equine practitioners [ Ferris, D.J, 2009].
Project description:Epidemiologic information is key when interpreting whole genome sequence data – lessons learned from the genomic analysis of the largest German Legionella pneumophila outbreak (Warstein, 2013)
Project description:Purpose: RNA-seq method was used to identify differentially expressed genes in whole blood involved in bone remodelling during racing training in young Arabian horses. Methods: The comparisons of transcriptomes of whole blood between GI (6 untrained horses) and GII (4 horses after 24 weeks of flat racing training) has been performed. The RNA was isolated using MagMAX™-96 Total RNA Isolation Kit and 400ng were directed to cDNA libraries construction Illumina deep sequencing (75 single-end cycles on Illumina HiScan SQ platform). The bioinformatics analysis include the RSEM and STAR aligner. The raw reads were aligned to the Equus caballus reference genome. Differentially expressed genes were detected by DESeq2.The validation of RNA-seq results were performed by qPCR. Results: After comparison of whole blood transcriptomes from control and trained horses, we identified 1290 training induced genes. Among significant deregulated molecules we recognized twelve genes potentially involved in metabolism of bone (BGLAP, CTSK, TYROBP, PDLIM7, SLC9B2, TWSG1, NOTCH2, IL6ST, VAV3, NFATC1, CLEC5A, TXLNG). Within significantly deregulated pathways one from the most overrepresented was associated with osteoclast differentiation. Conclusions: In the presented study, we identified a panel of DEGs which should be evaluated as candidate biomarkers for bone homeostasis indicators in Arabians performed racetrack. In our results, we pinpointed that intense training has itself effect on immature skeletal system. Thus, further studies are essential to establish biomarkers which could be used in assessment of bone remodelling state during training for race track competition. Overall design: The samples of whole blood from 10 Arabian horses were sequenced in 75 single-end cycles on Illumina HiScan SQ platform in 6 technical repetitions.