Effects of oral ß-cryptoxanthin administration on the transcriptomes of peripheral neutrophil and liver tissue using microarray analysis in post-weaned Holstein calves.

We investigated the effects of oral administration of ß-cryptoxanthin (ß-CRX), a precursor of vitamin A synthesis, on the transcriptomes of peripheral neutrophils and liver tissue in post-weaned Holstein calves with immature immunity. A single oral administration of ß-CRX (0.2 mg/kg body weight) was performed in eight Holstein calves (4.0 ± 0.8 months of age; 117 ± 10 kg) on Day 0. Peripheral neutrophils (n = 4) and liver tissue (n = 4) were collected on Days 0 and 7. Neutrophils were isolated by density gradient centrifugation and treated with the TRIzol reagent. mRNA expression profiles were examined by microarray and differentially expressed genes were investigated using the Ingenuity Pathway Analysis software. The differentially expressed candidate genes identified in neutrophils (COL3A1, DCN, and CCL2) and liver tissue (ACTA1) were involved in enhanced bacterial killing and maintenance of cellular homoeostasis respectively. The changes in the expression of six of the eight common genes encoding enzymes (ADH5 and SQLE) and transcription regulators (RARRES1, COBLL1, RTKN, and HES1) were in the same direction in neutrophils and liver tissue. ADH5 and SQLE are involved in the maintenance of cellular homoeostasis by increasing the availability of substrates, and RARRES1, COBLL1, RTKN, and HES1 are associated with the suppression of apoptosis and carcinogenesis. An in silico analysis revealed that MYC, which is related to the regulation of cellular differentiation and apoptosis, was the most significant upstream regulator in neutrophils and liver tissue. Transcription regulators such as CDKN2A (cell growth suppressor) and SP1 (cell apoptosis enhancer) were significantly inhibited and activated, respectively, in neutrophils and liver tissue. These results suggest that oral administration of ß-CRX promotes the expression of candidate genes related to bactericidal ability and regulation of cellular processes in peripheral neutrophils and liver cells in response to the immune-enhancing function of ß-CRX in post-weaned Holstein calves.

Expression pattern of microRNAs in ovine endometrium during the peri-implantation.

MicroRNA (miRNA), acting as the transcriptional regulator of gene expression, has been widely demonstrated to be involved in many biological functions, including embryo implantation and development. The objective of the current study was to illuminate the expression pattern of microRNAs (miRNAs) in the endometrium during the peri-implantation in ewes. Intercaruncular endometrial samples was obtained from a total of 24 ewes on days of 12 (pre-implantation, n = 4), 16 (implantation, n = 4) and 22 (post-implantation, n = 4) of pregnancy following mating, and on their corresponding days of 12 (n = 4), 16 (n = 4) and 22 (n = 4) of the estrous cycle. The miRNA profiles were examined in the endometrium by microarray technology. We detected 116 ovine specifics miRNAs in the endometrium. Of these, nineteen were differentially expressed in early pregnancy. Four miRNAs (oar-miR-370-3p, oar-miR-411b-5p, oar-miR-379-3p and oar-miR-411a-3p) that had the most differential fold change were confirmed by RT-qPCR in ovine endometrium. The differentially expressed miRNAs targeted a total of 315 genes, resulting in 39 GO terms in molecular function, 353 in biological process, and 17 in the cellular component. The construction of the PPI network of target genes established two functional modules mostly enriched in the innate immune system, toll receptor cascades in module 1, whereas genes in module 2 were associated with GMCSF-mediated signaling events, insulin pathway, and mTOR signaling pathway. Based on the results, we may imply that miRNAs modulate ovine endometrium during the peri-implantation.

Estradiol-17ß-Induced Changes in the Porcine Endometrial Transcriptome In Vivo.

Estradiol-17ß (E2) is a key hormone regulating reproductive functions in females. In pigs, E2, as the main conceptus signal, initiates processes resulting in prolonged corpus luteum function, embryo development, and implantation. During early pregnancy the endometrium undergoes morphological and physiological transitions that are tightly related to transcriptome changes. Recently, however, the importance of E2 as a primary conceptus signal in the pig has been questionable. Thus, the aim of the present study was to determine the effects of E2 on the porcine endometrial transcriptome in vivo and to compare these effects with transcriptome profiles on day 12 of pregnancy. Microarray analysis revealed differentially expressed genes (DEGs) in response to E2 with overrepresented functional terms related to secretive functions, extracellular vesicles, cell adhesion, proliferation and differentiation, tissue rearrangements, immune response, lipid metabolism, and many others. Numerous common DEGs and processes for the endometrium on day 12 of pregnancy and E2-treated endometrium were identified. In summary, the present study is the first evidence for the effect of E2 on transcriptome profiles in porcine endometrium in vivo in the period corresponding to the maternal recognition of pregnancy. The presented results provide a valuable resource for further targeted studies considering genes and pathways regulated by conceptus-derived estrogens and their role in pregnancy establishment.

Transcriptional analysis of RstA/RstB in avian pathogenic Escherichia coli identifies its role in the regulation of hdeD-mediated virulence and survival in chicken macrophages.

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.

Piglet weight gain during the first two weeks of lactation influences the immune system development.

The aim of this study was to investigate the effect of the piglet growth during the first week of life on ileal expression of genes and on development of the immune system. Eight litters adjusted to 12 piglets were used. Within each litter, the piglet that showed the lowest weight gain (LWG; n = 8) and the one that showed the highest weight gain (HWG; n = 8) in their first week of life were enrolled. Peripheral blood mononuclear cells (PBMC) were isolated on days 8 and 16 to characterize cellular population profiles and to assess ex-vivo secretion of interleukin-10 (IL-10), IL-6 and tumor necrosis factor-α (TNF-α). On day 16, piglets were euthanized and ileum samples were collected to extract RNA for microarray analysis and gene expression by qPCR. As expected, growth performance of LWG piglet was impaired compared to HWG piglets (P < 0.05). From day 8 to 16, the percentage of CD21+ B cells significantly increased in blood of heavier HWG piglets while the percentage remained constant in smaller LWG piglets (P weight x day = 0.01). For the CD4+CD8α- Th cells, a marked increase was observed in LWG piglets from 8 to 16 days of age (P = 0.002) whereas no significant change occurred in HWG piglets. Percentages of CD14+ monocytes and other MHC-II+ cells were respectively higher and lower on day 8 compared to day 16 for both groups of piglets (P < 0.01). On day 8, LPS-activated PBMC from LWG piglets produced less IL-6 compared to HWG piglets (P < 0.05). Microarray analysis of gene expression in piglets' ileum tissue indicated that several genes involed in defense response and response to oxidative stress were modulated differently in LWG compared to HWG. Gene analysis by Q-PCR confirmed microarray results and revealed that IL-10, SOD1, NOS2, NOD2, TLR4, TLR9, CD40 and CD74 expressions were significantly decreased (P < 0.05) in LWG in comparison to HWG piglets, while MYD88 and NFkBiA showed a tendency to decrease (0.05 ≤ P < 0.07). These results suggest that birth weight and milk intake affect the growth performances and the development of immunity by modulating the expression of genes associated with immunity and oxidative stress in piglets' intestinal tissue, and by affecting the leukocyte populations involved in innate and cell-mediated immunity in nursing piglets. Therefore, impaired development of immune system in LWG piglets might have an impact on their resistance to infections later in life.

A multiplex xTAG assay for the simultaneous detection of five chicken immunosuppressive viruses.

BACKGROUND: Chicken anemia virus (CAV), avian reovirus (ARV), infectious bursal disease virus (IBDV), Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) all cause immunosuppressive disease in birds through vertical or horizontal transmission. Mixed infections with these immunosuppressive pathogens lead to atypical clinical signs and obstruct accurate diagnoses and epidemiological investigations. Therefore, it is essential to develop a high-throughput assay for the simultaneous detection of these immunosuppressive viruses with high specificity and sensitivity. The aim of this study was to establish a novel method using a RT-PCR assay combined with fluorescence labeled polystyrene bead microarray (multiplex xTAG assay) to detect single or mixed viral infections. RESULTS: The results showed that the established xTAG assay had no nonspecific reactions with avian influenza virus (AIV), infectious bronchitis virus (IBV), newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). The limit of detection was 1.0 × 103 copies/µL for IBDV and 1.0 × 102copies/µL for the other four viruses. Ninety field samples were tested and the results were confirmed using conventional RT-PCR methods. The detection results of these two methods were 100% consistent. The established multiplex xTAG assay allows a high throughput and simultaneous detection of five chicken immunosuppressive viruses. CONCLUSION: The multiplex xTAG assay has been showed to be an additional tool for molecular epidemiology studies of five chicken immunosuppressive viruses in the poultry industry.

Tumourigenic canine osteosarcoma cell lines associated with frizzled-6 up-regulation and enhanced side population cell frequency.

An increased serum alkaline phosphatase concentration is known to be associated with a negative prognosis in canine and human osteosarcoma. To expand upon previous studies regarding the biological relevance of increased serum alkaline phosphatase as a negative prognostic factor, xenogeneic heterotopic transplants were performed using six canine primary osteosarcoma cell lines generated from patients with differing serum alkaline phosphatase concentrations (three normal and three increased). Three of the six cell lines were capable of generating tumours and tumour formation was independent of the serum alkaline phosphatase status of the cell line. Microarray analysis identified 379 genes as being differentially expressed between the tumourigenic and non-tumourigenic cell lines. Frizzled-6 was upregulated to the greatest extent (7.78-fold) in tumourigenic cell lines compared with non-tumourigenic cell lines. Frizzled-6, a co-receptor for Wnt ligands has been associated with enhanced tumour-initiating cells and poor prognosis for other tumours. The increased expression of frizzled-6 was confirmed by quantitative reverse transcription polymerase chain reaction (QPCR) and Western blot analysis. Additionally, the tumourigenic cell lines also had an increase in the percentage of side population cells compared with non-tumourigenic cell lines (5.89% versus 1.58%, respectively). There were no differences in tumourigenicity, frizzled-6 or percentage of side population cells noted between osteosarcoma cell lines generated from patients of differing serum alkaline phosphatase concentration. However, to our knowledge this is the first study to identified frizzled-6 as a possible marker of osteosarcoma cell populations with enhanced tumourigenicity and side population cells. Future work will focus on defining the role of frizzled-6 in osteosarcoma tumourigenesis and tumour-initiating cells.

A miRNA target network putatively involved in follicular atresia.

In a previous microarray study, we identified a subset of micro RNAS (miRNAs), which expression was distinctly higher in atretic than healthy follicles of cattle. In the present study, we investigated the involvement of those miRNAs in granulosa and theca cells during atresia. Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) confirmed that miR-21-5p/-3p, miR-150, miR-409a, miR-142-5p, miR-378, miR-222, miR-155, and miR-199a-5p were expressed at higher levels in atretic than healthy follicles (9-17 mm, classified based on steroidogenic capacity). All miRNAs except miR-21-3p and miR-378 were expressed at higher levels in theca than granulosa cells. The expression of 13 predicted miRNA targets was determined in follicular cells by RT-qPCR, revealing downregulation of HIF1A, ETS1, JAG1, VEGFA, and MSH2 in either or both cell types during atresia. Based on increases in miRNA levels simultaneous with decreases in target levels in follicular cells, several predicted miRNA target interactions were confirmed that are putatively involved in follicular atresia, namely miR-199a-5p/miR-155-HIF1A in granulosa cells, miR-155/miR-222-ETS1 in theca cells, miR-199a-5p-JAG1 in theca cells, miR-199a-5p/miR-150/miR-378-VEGFA in granulosa and theca cells, and miR-155-MSH2 in theca cells. These results offer novel insight on the involvement of miRNAs in follicle development by identifying a miRNA target network that is putatively involved in follicle atresia.

Characterisation of Chlamydia pneumoniae and other novel chlamydial infections in captive snakes.

Chlamydiosis has been described in both free-ranging and captive reptiles. The infection usually manifests as granulomatous inflammation in inner organs such as spleen, heart, lung and liver but might also occur in asymptomatic reptiles. The aim of this study was to investigate and characterise Chlamydia pneumoniae and potential other novel chlamydial infections in the choana and cloaca samples of 137 clinically healthy captive snakes from six private collections. Forty eight samples from 29 animals were found to be positive by a Chlamydiaceae family-specific qPCR. By Chlamydia species-specific ArrayTube Microarray, 43 samples were positive, with 36 of these being identified as C. pneumoniae. The prevalence of Chlamydia ranged from 5 to 33%. PCR and sequencing of the Chlamydiales 16S rRNA signature sequence of 21 Chlamydia positive samples revealed the presence of seven novel 16S rRNA genotypes. BLAST-n and phylogenetic analysis of the near-full length 16S rRNA gene sequence of each of these novel 16S rRNA sequences revealed that five genotypes share closest sequence identity to 16S rRNA sequences from C. pneumoniae (98.6-99.2%), suggesting that these sequences are novel C. pneumoniae strains. One genotype is 96.9% similar to C. pneumoniae strains suggesting it may originate from a yet undescribed chlamydial species within the genus Chlamydia. This study further highlights the broad host range for C. pneumoniae and suggests that reptiles may still contain a significant and largely uncharacterised level of chlamydial genetic diversity that requires further investigation.

Genome-wide expression analysis of hereditary hyperplastic gingivitis in silver foxes (Vulpes vulpes) using canine microarrays.

Hereditary hyperplastic gingivitis (HHG) is an autosomal recessive condition found predominantly in farmed silver foxes, first documented in Europe in the 1940s. Hereditary gingival fibromatosis (HGF) is an analogous condition occurring in humans. HGF has a heterogeneous aetiology with emphasis placed on the autosomal dominant forms of inheritance for which there are three known loci: HGF1, HGF2, and HGF3. Among these, only one causative mutation has been determined, in the Son of sevenless homolog 1 (SOS1) gene. The goal of this study was to explore potential molecular or cellular mechanisms underlying HHG by analysis of global gene expression patterns from Affymetrix Canine 2.0 microarrays cross-referenced against candidate genes within the human loci. We conclude that the SOS1 gene involved in HGF1 is not significantly up-regulated in HHG. However, the structurally and functionally similar SOS2 gene is up-regulated in affected foxes, and we propose this as a candidate gene for HHG. At HGF2 we identify RASA1 (rat sarcoma viral p21 protein activator 1) as a candidate gene for HHG, as it is up-regulated in affected foxes and is involved in MAPK signalling. From comparison to the genes within the HGF3 locus, we find evidence for a role of androgens in HHG phenotype severity by differential up-regulation of SRD5A2 in HHG-affected foxes. We hypothesize that the putative mutation occurs upstream of RAS in the extracellular signal-regulated kinase component of MAPK signalling.

Transcriptome analysis of CNS immediately before and after the detection of PrP(Sc) in SSBP/1 sheep scrapie.

Sheep scrapie is a transmissible spongiform encephalopathy (TSE), progressive and fatal neurodegenerative diseases of the central nervous system (CNS) linked to the accumulation of misfolded prion protein, PrP(Sc). New Zealand Cheviot sheep, homozygous for the VRQ genotype of the PRNP gene are most susceptible with an incubation period of 193 days with SSBP/1 scrapie. However, the earliest time point that PrP(Sc) can be detected in the CNS is 125 days (D125). The aim of this study was to quantify changes to the transcriptome of the thalamus and obex (medulla) at times immediately before (D75) and after (D125) PrP(Sc) was detected. Affymetrix gene arrays were used to quantify gene expression in the thalamus and Illumina DGE-tag profiling for obex. Ingenuity Pathway Analysis was used to help describe the biological processes of scrapie pathology. Neurological disease and Cancer were common Bio Functions in each tissue at D75; inflammation and cell death were major processes at D125. Several neurological receptors were significantly increased at D75 (e.g. CHRNA6, GRM1, HCN2), which might be clues to the molecular basis of psychiatric changes associated with TSEs. No genes were significantly differentially expressed at both D75 and D125 and there was no progression of events from earlier to later time points. This implies that there is no simple linear progression of pathological or molecular events. There seems to be a step-change between D75 and D125, correlating with the detection of PrP(Sc), resulting in the involvement of different pathological processes in later TSE disease.

Effects of capsicum oleoresin, garlic botanical, and turmeric oleoresin on gene expression profile of ileal mucosa in weaned pigs.

This study was conducted to characterize the effects of feeding 3 plant extracts on gene expression in ileal mucosa of weaned pigs. Weaned pigs (n = 32, 6.3 ± 0.2 kg BW, and 21 d old) were housed in individual pens for 9 d and fed 4 different diets: a nursery basal diet as control diet, basal diet supplemented with 10 mg/kg of capsicum oleoresin, garlic botanical, or turmeric oleoresin. Results reported elsewhere showed that the plant extracts reduced diarrhea and increased growth rate of weaning pigs. Total RNA (4 pigs/treatment) was extracted from ileal mucosa of pigs at d 9. Double-stranded cDNA was amplified, labeled, and further hybridized to the microarray. Microarray data were analyzed in R using packages from the Bioconductor project. Differential gene expression was tested by fitting a mixed linear model equivalent to ANOVA using the limma package. Bioinformatics analysis was conducted by DAVID Bioinformatics Resources. Three pairwise comparisons were used to compare each plant extract diet with the control diet. Quantitative real time PCR was applied to verify the mRNA expression detected by microarray. Compared with the control diet, feeding capsicum oleoresin altered (P < 0.05) the expression of 490 genes (280 up, 210 down), and feeding garlic botanical altered (P < 0.05) the expression of 64 genes (33 up, 31 down), while feeding turmeric oleoresin altered (P < 0.05) the expression of 327 genes (232 up, 95 down). Compared with the control diet, feeding capsicum oleoresin and turmeric oleoresin increased [Expression Analysis Systematic Explorer (EASE) < 0.05] the expression of genes related to integrity of membranes and tight junctions, indicating enhanced gut mucosa health, but decreased (EASE < 0.05) the cell cycle pathway. Feeding each of the 3 plant extracts enhanced (EASE < 0.05) the expression of genes associated with immune responses, indicating that feeding these plant extracts may stimulate the immune responses of pigs in the normal conditions. In conclusion, plant extracts regulated the expression of genes in ileal mucosa of pigs, perhaps providing benefits by enhancing the gut mucosa health and stimulating the immune system.

Subcutaneous and gonadal adipose tissue transcriptome differences in lean and obese female dogs.

Canine obesity leads to shortened life span and increased disease incidence. Adipose tissue depots are known to have unique metabolic and gene expression profiles in rodents and humans, but few comparisons of depot gene expression have been performed in the dog. Using microarray technology, our objective was to identify differentially expressed genes and enriched functional pathways between subcutaneous and gonadal adipose of lean and obese dogs to better understand the pathogenesis of obesity in the dog. Because no depot × body weight status interactions were identified in the microarray data, depot differences were the primary focus. A total of 946 and 703 transcripts were differentially expressed (FDR P < 0.05) between gonadal and subcutaneous adipose tissue in obese and lean dogs respectively. Of the adipose depot-specific differences in gene expression, 162 were present in both lean and obese dogs, with the majority (85%) expressed in the same direction. Both lean and obese dog gene lists had enrichment of the complement and coagulation cascade and systemic lupus erythematosus pathways. Obese dogs had enrichment of lysosome, extracellular matrix-receptor interaction, renin-angiotensin system and hematopoietic cell lineage pathways. Lean dogs had enrichment of glutathione metabolism and synthesis and degradation of ketone bodies. We have identified a core set of genes differentially expressed between subcutaneous and gonadal adipose tissue in dogs regardless of body weight. These genes contribute to depot-specific differences in immune function, extracellular matrix remodeling and lysosomal function and may contribute to the physiological differences noted between depots.

Early host response in the mammary gland after experimental Streptococcus uberis challenge in heifers.

Streptococcus uberis is a highly prevalent causative agent of bovine mastitis, which leads to large economic losses in the dairy industry. The aim of this study was to examine the host response during acute inflammation after experimental challenge with capsulated Strep. uberis. Gene expression in response to Strep. uberis was compared between infected and control quarters in 3 animals. All quarters (n=16) were sampled at 16 different locations. Microarray data showed that 239 genes were differentially expressed between infected and control quarters. No differences in gene expression were observed between the different locations. Microarray data were confirmed for several genes using quantitative PCR analysis. Genes differentially expressed due to early Strep. uberis mastitis represented several stages of the process of infection: (1) pathogen recognition; (2) chemoattraction of neutrophils; (3) tissue repair mechanisms; and (4) bactericidal activity. Three different pathogen recognition genes were induced: ficolins, lipopolysaccharide binding protein, and toll-like receptor 2. Calgranulins were found to be the most strongly upregulated genes during early inflammation. By histology and immunohistochemistry, we demonstrated that changes in gene expression in response to Strep. uberis were induced both in infiltrating somatic milk cells and in mammary epithelial cells, demonstrating that the latter cell type plays a role in milk production as well as immune responsiveness. Given the rapid development of inflammation or mastitis after infection, early diagnosis of (Strep. uberis) mastitis is required for prevention of disease and spread of the pathogen. Insight into host responses could help to design immunomodulatory therapies to dampen inflammation after (early) diagnosis of Strep. uberis mastitis. Future research should focus on development of these early diagnostics and immunomodulatory components for mastitis treatment.

Immunohistochemical expression study of proapoptotic BH3-only protein bad in canine nonneoplastic tissues and canine lymphomas.

The BH3-only protein Bad is a proapoptotic Bcl-2 family member that acts as a sensitizer in intrinsic apoptosis by inactivating antiapoptotic members through heterodimer formation. Bad has been shown to contribute to tumorigenesis, including lymphoma formation in humans and mice, through alteration in expression or functional status. Here, its immunohistochemical expression was analyzed in canine nonneoplastic and lymphoma tissues using tissue microarrays. Bad was expressed in the cytoplasm of a wide range of nonneoplastic tissues, especially epithelial cells. Nonneoplastic lymph nodes displayed weak immunostaining in the follicular germinal centers only. Immunoblotting supported these observations but also revealed presence of nonspecific labeling in some organs. Of 81 lymphomas, 29 (35.8%) displayed moderate to strong immunohistochemical Bad labeling, and a significant expression increase was found in lymphomas (especially B cell and double negative) compared to nonneoplastic lymph nodes. These findings warrant further investigations of the functional status, the involvement of partner proteins, and a possible impact of Bad on prognosis in canine lymphoma.

Global gene expression in the bovine corpus luteum is altered after stimulatory and superovulatory treatments.

Equine chorionic gonadotrophin (eCG) has been widely used in superovulation and artificial insemination programmes and usually promotes an increase in corpus luteum (CL) volume and stimulates progesterone production. Therefore, to identify eCG-regulated genes in the bovine CL, the transcriptome was evaluated by microarray analysis and the expression of selected genes was validated by qPCR and western blot. Eighteen Nelore crossbred cows were divided into control (n=5), stimulated (n=6) and superovulated groups (n=7). Ovulation was synchronised using a progesterone device-based protocol. Stimulated animals received 400 IU of eCG at device removal and superovulated animals received 2000 IU of eCG 4 days prior. Corpora lutea were collected 7 days after gonadotrophin-releasing hormone administration. Overall, 242 transcripts were upregulated and 111 transcripts were downregulated in stimulated cows (P ≤ 0.05) and 111 were upregulated and 113 downregulated in superovulated cows compared to the control animals (1.5-fold, P ≤ 0.05). Among the differentially expressed genes, many were involved in lipid biosynthesis and progesterone production, such as PPARG, STAR, prolactin receptors and follistatin. In conclusion, eCG modulates gene expression differently depending on the treatment, i.e. stimulatory or superovulatory. Our data contribute to the understanding of the pathways involved in increased progesterone levels observed after eCG treatment.

Sequencing of the first ayu (Plecoglossus altivelis) macrophage transcriptome and microarray development for investigation the effect of LECT2 on macrophages.

Macrophages play an important role in first-line host defense of innate immune in fishes. However, it is difficult to investigate cellular mechanism of immune response in fish species with little genomic information available. Here we present the first use of RNA-Sequencing to study the macrophage transcriptome of ayu, Plecoglossus altivelis, which is an economically important fish in East Asia. De novo assembly generated 49,808 non-redundant consensus sequences, among which 23,490 transcripts found respective coding sequences. 15,707 transcripts are predicted to be involved in known metabolic or signaling pathways. The sequences were then used to develop a microarray for measurement the effect of recombinant LECT2 on ayu macrophages. LECT2 altered expression of a variety of genes mainly implicated in actin cytoskeleton, pattern recognition receptors and cytokines. Meanwhile, LECT2 enhanced phagocytosis, bacterial killing, and respiratory burst in ayu macrophages, which supported the thought derived from the microarray data that LECT2 activates macrophages. In conclusion, our results contribute to understanding the specific regulation mechanism of LECT2 in macrophage activation, and the combination of transcriptome analysis and microarray assay is a good method for screening a special tissue or cell response to a stimulus or pathogen in non-model fish species.

Estrogen modulates hepatic gene expression and survival of rainbow trout infected with pathogenic bacteria Yersinia ruckeri.

In the aquatic environment, fish are exposed to various stimuli at once and have developed different response mechanisms to deal with these multiple stimuli. The current study assessed the combined impacts of estrogens and bacterial infection on the physiological status of fish. Juvenile rainbow trout were exposed to two different concentrations of 17ß-estradiol (E2) (2 or 20 mg/kg feed) and then infected with three concentrations of Yersinia ruckeri, a bacterial pathogen causing massive losses in wild and farmed salmonid populations. Organism-level endpoints to assess the impact of the single and combined treatments included hepatic vitellogenin transcript expression to evaluate the E2 exposure efficiency and survival rate of pathogen-challenged fish. The two E2 doses increased vitellogenin levels within the physiological range. Infection with Y. ruckeri caused mortality of trout, and this effect was significantly enhanced by a simultaneous exposure to high E2 dose. The hormone reduced survival at intermediate and high (10(4) and 10(6) colony forming units, cfu) bacterial concentrations, but not for a low one (10(2) cfu). Analysis of hepatic gene expression profiles by a salmonid 2 k cDNA microarray chip revealed complex regulations of pathways involved in immune responses, stress responses, and detoxicification pathways. E2 markedly reduced the expression of several genes implicated in xenobiotic metabolism. The results suggest that the interaction between pathogen and E2 interfered with the fish's capability of clearing toxic compounds. The findings of the current study add to our understanding of multiple exposure responses in fish.

Comparative analysis of gene expression profiles in ruminal tissue from Holstein dairy cows fed high or low concentrate diets.

Cattle are often fed high concentrate (HC) diets to increase productivity, although HC diets cause changes in ruminal environment such as pH reduction. Despite those well-documented changes in cattle fed HC diets, there is currently a paucity of data describing the molecular events regulating the ruminal environment. Our objective was to gain an understanding of which genes are differentially expressed in ruminal tissue from Holstein cows fed a HC comparing to low concentrate (LC) diet using microarray analysis using a bovine 24 k microarray. A total of 5,200 differentially expressed genes (DEG) were detected for cows fed HC relative to LC. The DEG were firstly annotated with gene ontology (GO) and Kyoto encyclopedia of Genes and Genomes (KEGG), indicating that the DEG were associated with catalytic activity and MAPK pathway, respectively. Further characterization using GeneCodis identified patterns of interrelated annotations for the DEG to elucidate the relationships among annotation groups revealed that a cAMP-dependent protein kinase A catalytic subunit beta (PRKACB), may be associated with ruminal tissue maintenance. The results contributed to understanding of the regulatory mechanisms at the mRNA level for Holstein cows fed at different concentrate ratio diets.

Arrhythmogenic right ventricular cardiomyopathy in Boxer dogs is associated with calstabin2 deficiency.

OBJECTIVE: To examine the presence and effect of calstabin2-deficiency in Boxer dogs with arrhythmogenic right ventricular cardiomyopathy (ARVC). ANIMALS: Thirteen Boxer dogs with ARVC. MATERIALS AND METHODS: Tissue samples were collected for histopathology, oligonucleotide microarray, PCR, immunoelectrophoresis, ryanodine channel immunoprecipitation and single-channel recordings, and calstabin2 DNA sequencing. RESULTS: In cardiomyopathic Boxer dogs, myocardial calstabin2 mRNA and protein were significantly decreased as compared to healthy control dogs (calstabin2 protein normalized to tetrameric cardiac ryanodine receptor (RyR2) complex: affected, 0.51+/-0.04; control, 3.81+/-0.22; P<0.0001). Calstabin2 deficiency in diseased dog hearts was associated with a significantly increased open probability of single RyR2 channels indicating intracellular Ca(2+) leak. PCR-based sequencing of the promoter, exonic and splice site regions of the canine calstabin2 gene did not identify any causative mutations. CONCLUSIONS: Calstabin2 deficiency is a potential mechanism of Ca(2+) leak-induced ventricular arrhythmias and heart disease in Boxer dogs with ARVC.