Mitral valve transcriptome analysis in thirty-four age-matched Cavalier King Charles Spaniels with or without congestive heart failure caused by myxomatous mitral valve disease.

We here report the results of a mitral valve transcriptome study designed to identify genes and molecular pathways involved in development of congestive heart failure (CHF) following myxomatous mitral valve disease (MMVD) in dogs. The study is focused on a cohort of elderly age-matched dogs (n = 34, age ~ 10 years) from a single breed-Cavalier King Charles Spaniels (CKCS)-with a high incidence of MMVD. The cohort comprises 19 dogs (10♀, 9♂) without MMVD-associated CHF, and 15 dogs (6♀, 9♂) with CHF caused by MMVD; i.e., we compare gene expression in breed and age-matched groups of dogs, which only differ with respect to CHF status. We identify 56 genes, which are differentially expressed between the two groups. In this list of genes, we confirm an enrichment of genes related to the TNFß-signaling pathway, extracellular matrix organization, vascular development, and endothelium damage, which also have been identified in previous studies. However, the genes with the greatest difference in expression between the two groups are CNTN3 and MYH1. Both genes encode proteins, which are predicted to have an effect on the contractile activity of myocardial cells, which in turn may have an effect on valvular performance and hemodynamics across the mitral valve. This may result in shear forces with impact on MMVD progression.

Association of serum and fecal microRNA profiles in cats with gastrointestinal cancer and chronic inflammatory enteropathy.

BACKGROUND: Differentiation of gastrointestinal cancer (GIC) from chronic inflammatory enteropathies (CIE) in cats can be challenging and often requires extensive diagnostic testing. MicroRNAs (miRNAs) have promise as non-invasive biomarkers in serum and feces for diagnosis of GIC. HYPOTHESIS/OBJECTIVES: Cats with GIC will have serum and fecal miRNA profiles that differ significantly from healthy cats and cats with CIE. Identify serum and fecal miRNAs with diagnostic potential for differentiation between cats with GIC and CIE as compared to healthy cats. ANIMALS: Ten healthy cats, 9 cats with CIE, and 10 cats with GIC; all client-owned. METHODS: Cats were recruited for an international multicenter observational prospective case-control study. Serum and feces were screened using small RNA sequencing for miRNAs that differed in abundance between cats with GIC and CIE, and healthy cats. Diagnostic biomarker potential of relevant miRNAs from small RNA sequencing and the literature was confirmed using reverse transcription quantitative real-time PCR (RT-qPCR). RESULTS: Serum miR-223-3p was found to distinguish between cats with GIC and CIE with an area under the curve (AUC) of 0.9 (95% confidence interval [CI], 0.760-1.0), sensitivity of 90% (95% CI, 59.6-99.5%), and specificity of 77.8% (95% CI, 45.3-96.1%). Serum miR-223-3p likewise showed promise in differentiating a subgroup of cats with small cell lymphoma (SCL) from those with CIE. No fecal miRNAs could distinguish between cats with GIC and CIE. CONCLUSION AND CLINICAL IMPORTANCE: Serum miR-223-3p potentially may serve as a noninvasive diagnostic biomarker of GIC in cats, in addition to providing a much needed tool for the differentiation of CIE and SCL.

Association of fecal and serum microRNA profiles with gastrointestinal cancer and chronic inflammatory enteropathy in dogs.

BACKGROUND: Reliable biomarkers to differentiate gastrointestinal cancer (GIC) from chronic inflammatory enteropathy (CIE) in dogs are needed. Fecal and serum microRNAs (miRNAs) have been proposed as diagnostic and prognostic markers of GI disease in humans and dogs. HYPOTHESIS/OBJECTIVES: Dogs with GIC have fecal and serum miRNA profiles that differ from those of dogs with CIE. AIMS: (a) identify miRNAs that differentiate GIC from CIE, (b) use high-throughput reverse transcription quantitative real-time PCR (RT-qPCR) to establish fecal and serum miRNA panels to distinguish GIC from CIE in dogs. ANIMALS: Twenty-four dogs with GIC, 10 dogs with CIE, and 10 healthy dogs, all client-owned. METHODS: An international multicenter observational prospective case-control study. Small RNA sequencing was used to identify fecal and serum miRNAs, and RT-qPCR was used to establish fecal and serum miRNA panels with the potential to distinguish GIC from CIE. RESULTS: The best diagnostic performance for distinguishing GIC from CIE was fecal miR-451 (AUC: 0.955, sensitivity: 86.4%, specificity: 100%), miR-223 (AUC: 0.918, sensitivity: 90.9%, specificity: 80%), and miR-27a (AUC: 0.868, sensitivity: 81.8%, specificity: 90%) and serum miR-20b (AUC: 0.905, sensitivity: 90.5%, specificity: 90%), miR-148a-3p (AUC: 0.924, sensitivity: 85.7%, specificity: 90%), and miR-652 (AUC: 0.943, sensitivity: 90.5%, specificity: 90%). Slightly improved diagnostic performance was achieved when combining fecal miR-451 and miR-223 (AUC: 0.973, sensitivity: 95.5%, specificity: 90%). CONCLUSIONS AND CLINICAL IMPORTANCE: When used as part of a diagnostic RT-qPCR panel, the abovementioned miRNAs have the potential to function as noninvasive biomarkers for the differentiation of GIC and CIE in dogs.

Urinary microRNAome in healthy cats and cats with pyelonephritis or other urological conditions.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at the post-transcriptional level. miRNAs have been found in urine and have shown diagnostic potential in human nephropathies. Here, we aimed to characterize, for the first time, the feline urinary miRNAome and explore the use of urinary miRNA profiles as non-invasive biomarkers for feline pyelonephritis (PN). Thirty-eight cats were included in a prospective case-control study and classified in five groups: healthy Control cats (n = 11), cats with PN (n = 10), cats with subclinical bacteriuria or cystitis (SB/C, n = 5), cats with ureteral obstruction (n = 7) and cats with chronic kidney disease (n = 5). By small RNA sequencing we identified 212 miRNAs in cat urine, including annotated (n = 137) and putative novel (n = 75) miRNAs. The 15 most highly abundant urinary miRNAs accounted for nearly 71% of all detected miRNAs, most of which were previously identified in feline kidney. Ninety-nine differentially abundant (DA) miRNAs were identified when comparing Control cats to cats with urological conditions and 102 DA miRNAs when comparing PN to other urological conditions. Tissue clustering analysis revealed that the majority of urine samples clustered close to kidney, which confirm the likely cellular origin of the secreted urinary miRNAs. Relevant DA miRNAs were verified by quantitative real-time PCR (qPCR). Eighteen miRNAs discriminated Control cats from cats with a urological condition. Of those, seven miRNAs were DA by both RNAseq and qPCR methods between Control and PN cats (miR-125b-5p, miR-27a-3p, miR-21-5p, miR-27b-3p, miR-125a-5p, miR-17-5p and miR-23a-3p) or DA between Control and SB/C cats (miR-125b-5p). Six additional miRNAs (miR-30b-5p, miR-30c, miR-30e-5p, miR-27a-3p, miR-27b-39 and miR-222) relevant for discriminating PN from other urological conditions were identified by qPCR alone (n = 4) or by both methods (n = 2) (P<0.05). This panel of 13 miRNAs has potential as non-invasive urinary biomarkers for diagnostic of PN and other urological conditions in cats.

Evaluation of fecal microRNA stability in healthy cats.

BACKGROUND: Gastrointestinal (GI) cancer accounts for 14% of feline malignancies. There is a great need for reliable noninvasive diagnostic biomarkers to reach a timely diagnosis and initiate treatment. Fecal microRNAs (miRNAs) could be such a biomarker and have shown great potential in colorectal screening in people but have yet to be investigated in cats. OBJECTIVES: We aimed to evaluate the presence and stability of feline fecal miRNA under different storage conditions (room temperature [RT], 4, and -20°C) and to evaluate the expression levels of specific fecal miRNAs collected on three separate days (days 1, 4, and 7) in healthy cats. METHODS: Healthy cats were prospectively recruited. Fecal samples were collected, aliquoted, and stored for 24 hours at RT and then transferred to -20°C, stored for 24 hours at 4°C and then transferred to -20°C, or were immediately placed at -20°C on day 1 or at -20°C on days 4 and 7 postcollection. Expression of 22 miRNAs was investigated using quantitative real-time PCR. RESULTS: Ten miRNA assays worked well, and one, let-7b, was used for normalization. No differences in miRNA expression were seen between the three storage temperatures for the nine miRNAs investigated. Only miR-26a showed a significant increase in expression between samples of days 1 and 7. The rest of the miRNAs levels were stable over time. CONCLUSIONS: Fecal miRNA can be isolated from healthy cats. The expression was stable at different temperatures and for most of the miRNAs over time. Prospective studies evaluating fecal miRNA as biomarkers in cats with GI neoplasia are warranted.

Evaluation of microRNA stability in feces from healthy dogs.

BACKGROUND: Gastrointestinal cancer accounts for approximately 8% of all canine malignancies. Early detection of cancer may have a tremendous impact on both treatment options and prognosis. MicroRNAs (miRNAs), a class of noncoding RNAs that can be found stably expressed in body fluids and feces, have been suggested as valuable human cancer biomarkers. OBJECTIVES: The purpose of the study was to investigate the feasibility of detecting miRNAs in canine feces and to determine the miRNA stability in fecal samples stored at different temperatures for different duration. METHODS: The levels of 4 Canine familiaris (cfa) miRNAs (cfa-miR-16, cfa-miR-20a, cfa-miR-21, and cfa-miR-92a) were investigated by quantitative real-time PCR(qPCR) in fecal samples from 10 healthy dogs. Fecal samples were collected at 3 different time points and samples from the first time point were stored at different temperatures and for a different duration. RESULTS: A statistically significant difference was found in miRNA levels from samples stored at room temperature compared with samples stored at -20°C for cfa-miR-16 and cfa-miR-21. No significant difference was found in the level of the investigated miRNAs over time. CONCLUSIONS: Overall, miRNAs are present in dog feces at measurable levels. Some miRNAs seem to be subject to a higher degree of degradation in samples stored at room temperature for 24 hours compared with samples frozen after collection at -20°C. The investigated miRNAs were stably expressed over time. This study provides the basis for further research on miRNA expression profiles as biomarkers for gastrointestinal cancer in dogs.

Systematic in vitro and in vivo characterization of Leukemia-inhibiting factor- and Fibroblast growth factor-derived porcine induced pluripotent stem cells.

Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to shed more light on the underlying biological mechanisms of porcine pluripotency. LIF-derived piPSCs were more successful than their FGF-derived counterparts in the generation of in vitro chimeras and in teratoma formation. When LIF piPSCs chimeras were transferred into surrogate sows and allowed to develop, only their prescence within the embryonic membranes could be detected. Whole-transcriptome analysis of the piPSCs and porcine neonatal fibroblasts showed that they clustered together, but apart from the two pluripotent cell populations of early porcine embryos, indicating incomplete reprogramming. Indeed, bioinformatic analysis of the pluripotency-related gene network of the LIF- versus FGF-derived piPSCs revealed that ZFP42 (REX1) expression was absent in both piPSC-like cells, whereas it was expressed in the porcine inner cell mass at Day 7/8. A second striking difference was the expression of ATOH1 in piPSC-like cells, which was absent in the inner cell mass. Moreover, our gene expression analyses plus correlation analyses of known pluripotency genes identified unique relationships between pluripotency genes in the inner cell mass, which are to some extent, in the piPSC-like cells. This deficiency in downstream gene activation and divergent gene expression may be underlie the inability to derive germ line-transmitting piPSCs, and provides unique insight into which genes are necessary to achieve fully reprogrammed piPSCs. 84: 229-245, 2017. © 2016 Wiley Periodicals, Inc.

Evaluation of microRNA Stability in Plasma and Serum from Healthy Dogs.

BACKGROUND: Early and specific detection of cancer is of great importance for successful treatment of the disease. New biomarkers, such as microRNAs, could improve treatment efficiency and survival ratio. In human medicine, deregulation of microRNA profiles in circulation has shown great potential as a new type of biomarker for cancer diagnostics. There are, however, few studies of circulating microRNAs in dogs. Extracellular circulating microRNAs have shown a high level of stability in human blood and other body fluids. Nevertheless, there are still important issues to be solved before microRNAs can be applied routinely as a clinical tool, one of them being their stability over time in media commonly used for blood sampling. OBJECTIVE: Evaluation of the stability of microRNA levels in plasma and serum from healthy dogs after storage at room temperature for different time points before being processed. METHODS: The levels of four microRNAs (cfa-let-7a, cfa-miR-16, cfa-miR-23a and cfa-miR-26a) known to be stably expressed from other canine studies, have been measured by quantitative real-time PCR (qPCR). RESULTS: MicroRNA levels were found sufficiently stable for gene profiling in serum- and plasma stored at room temperature for 1 hour but not for samples stored at room temperature for 24 hours. CONCLUSION: Storage at room temperature of serum and plasma samples intended for microRNA profiling should be kept for a minimum period of time before proceeding with RNA isolation. For the four microRNAs investigated in the present study, we did not find significant differences in microRNA levels between serum and plasma samples from the same time point.

Comparison of American mink embryonic stem and induced pluripotent stem cell transcriptomes.

BACKGROUND: Recently fibroblasts of many mammalian species have been reprogrammed to pluripotent state using overexpression of several transcription factors. This technology allows production of induced pluripotent stem (iPS) cells with properties similar to embryonic stem (ES) cells. The completeness of reprogramming process is well studied in such species as mouse and human but there is not enough data on other species. We produced American mink (Neovison vison) ES and iPS cells and compared these cells using transcriptome analysis. RESULTS: We report the generation of 10 mink ES and 22 iPS cell lines. The majority of the analyzed cell lines had normal diploid chromosome number. The only ES cell line with XX chromosome set had both X-chromosomes in active state that is characteristic of pluripotent cells. The pluripotency of ES and iPS cell lines was confirmed by formation of teratomas with cell types representing all three germ layers. Transcriptome analysis of mink embryonic fibroblasts (EF), two ES and two iPS cell lines allowed us to identify 11831 assembled contigs which were annotated. These led to a number of 6891 unique genes. Of these 3201 were differentially expressed between mink EF and ES cells. We analyzed expression levels of these genes in iPS cell lines. This allowed us to show that 80% of genes were correctly reprogrammed in iPS cells, whereas approximately 6% had an intermediate expression pattern, about 7% were not reprogrammed and about 5% had a "novel" expression pattern. We observed expression of pluripotency marker genes such as Oct4, Sox2 and Rex1 in ES and iPS cell lines with notable exception of Nanog. CONCLUSIONS: We had produced and characterized American mink ES and iPS cells. These cells were pluripotent by a number of criteria and iPS cells exhibited effective reprogramming. Interestingly, we had showed lack of Nanog expression and consider it as a species-specific feature.

Expression study of GLUT4 translocation-related genes in a porcine pre-diabetic model.

Obesity is a world-wide exponentially growing health problem that increases the risk of co-morbidities including metabolic syndrome, pre-diabetes, Type 2 Diabetes Mellitus (T2DM), and cancer. These co-morbidities are all complex conditions constituting a big challenge when searching for susceptibility genes. Identification of relevant genes, which could contribute to an earlier identification of individuals prone to develop diabetes, is urgently needed as many long-term complications can be avoided by preventive measures. Pre-diabetes is mainly associated with hyperglycemia; thus studying this phenotype might provide knowledge on relevant genes implicated in molecular mechanisms underlying pre-diabetes, and contributing to the development of T2DM. In the present study, two groups of pigs with high (HGG, N=6) and low (NGG, N=6) fasting plasma glucose level respectively were selected from a large pig population. Transcriptional levels of seven genes involved in the glucose transporter 4 (GLUT4) translocation pathway were studied by quantitative real-time PCR (qPCR) in diabetes relevant tissues (pancreas, adipose tissue, skeletal muscle, liver and kidney). Three of the genes, TBC (Tre-2, BUB2, CDC16) 1 Domain Family Member 4 (TBC1D4), insulin receptor and GLUT4 showed altered expression in some of the tissues. The expression pattern observed is in agreement with what has previously been reported in pre-diabetic humans confirming the pre-diabetic status of our pigs. Moreover, a novel isoform of TBC1D4 was detected by Western blotting using protein extracted from pancreas. The expression level of this novel isoform was further verified by qPCR in all tissues, showing the highest expression in the pancreas.

Plasma proANP and SDMA and microRNAs are associated with chronic mitral regurgitation in a pig model.

OBJECTIVE: NON-ISCHEMIC MITRAL REGURGITATION (MR) IS PRIMARILY CAUSED BY MYXOMATOUS MITRAL VALVE (MV) DISEASE LEADING TO ADAPTIVE REMODELING, ENLARGEMENT, AND DYSFUNCTION OF THE LEFT VENTRICLE. THE AIM OF THIS STUDY WAS TO EXAMINE THE REGULATION OF PLASMA MARKERS AND SEVERAL CARDIAC KEY GENES IN A NOVEL PORCINE MODEL OF NON-ISCHEMIC MR. METHODS AND RESULTS: Twenty-eight production pigs (Sus scrofa) were randomized to experimental MR or sham surgery controls. MR was induced by external suture(s) through the posterior MV leaflet and quantified using echocardiography. The experimental group was subdivided into mild MR (mMR, MR=20-50%, n=10) and moderate/severe MR (sMR, MR >50%, n=6) and compared with controls (CON, MR ≤10%, n=12). Eight weeks postoperatively, follow-up examinations were performed followed by killing. Circulating concentrations of pro-atrial natriuretic peptide (proANP), l-arginine, asymmetric dimethylarginine, and symmetric dimethylarginine (SDMA) were measured. MV, anterior papillary muscle, and left ventricular free wall tissues were collected to quantify mRNA expression of eNOS (NOS3), iNOS (NOS2), MMP9, MMP14, ANP (NPPA), BNP (NPPB), and TGFB1, 2, and 3 and five microRNAs by quantitative real-time PCR. Pigs with sMR displayed markedly increased plasma proANP and SDMA concentrations compared with both controls and mMR (P<0.05). The expression of all genes examined differed significantly between the three localizations in the heart. miR-21 and miR-133a were differently expressed among the experimental groups (P<0.05). CONCLUSIONS: Plasma proANP and SDMA levels and tissue expression of miR-21 and miR-133a are associated with severity of chronic MR in an experimental porcine model.

Functional characterization of a porcine emphysema model.

BACKGROUND: Lung emphysema is a central feature of chronic obstructive pulmonary disease (COPD), a frequent human disease worldwide. Cigarette smoking is the major cause of COPD, but genetic predisposition seems to be an important factor. Mutations in surfactant protein genes have been linked to COPD phenotypes in humans. Also, the catalytic activities of metalloproteinases (MMPs) are central in the pathogenesis of emphysema/COPD. Especially MMP9, but also MMP2, MMP7, and MMP12 seem to be involved in human emphysema. MMP12-/- mice are protected from smoke-induced emphysema. ITGB6-/- mice spontaneously develop age-related lung emphysema due to lack of ITGB6-TGF-ß1 regulation of the MMP12 expression. METHODS: A mutated pig phenotype characterized by age-related lung emphysema and resembling the ITGB6-/- mouse has been described previously. To investigate the emphysema pathogenesis in this pig model, we examined the expression of MMP2, MMP7, MMP9, MMP12, and TGF-ß1 by quantitative PCR (qPCR). In addition, immunohistochemical stainings of the lungs with SP-B, SP-C, MMP9, and MMP12 antibodies were performed. The haematologic/immunologic status of the pigs also was studied. RESULTS: The qPCR study showed no difference between pigs with and without emphysema, and no systemic differences were indicated by the haematologic and immunologic studies. However, the immunohistochemical stainings showed an increased expression of MMP9 and MMP12 in older, mutated pigs (with emphysema) compared with normal and young mutated pigs (without emphysema). CONCLUSIONS: The pig model is comparable to human emphysema patients and the ITGB6-/- mouse model with respect to both morphology and functionality.

New insights into the melanophilin (MLPH) gene controlling coat color phenotypes in American mink.

The mutation causing the Silverblue color type (pp) is one of the most used recessive mutations within American mink (Neovison vison) fur farming, since it is involved in some of the popular color types such as Violet and Saphire which originate from a combination of recessive mutations. In the present study, the genomic and mRNA sequences of the melanophilin (MLPH) gene were studied in Violet, Silverblue and wild-type (wt) mink animals. Although breeding schemes and previous literature indicates that the Violet (aammpp) phenotype is a triple recessive color type involving the same locus as the Silverblue (pp) color type, our findings indicate different genotypes at the MLPH locus. Upon comparison at genomic level, we identified two deletions of the entire intron 7 and of the 5' end of intron 8 in the sequence of the Silverblue MLPH gene. When investigating the mRNA, the Silverblue animals completely lack exon 8, which encodes 65 residues, of which 47 define the Myosin Va (MYO5A) binding domain. This may cause the incorrect anchoring of the MLPH protein to MYO5A in Silverblue animals, resulting in an improper pigmentation as seen in diluted phenotypes. Additionally, in the MLPH mRNA of wt, Violet and Silverblue phenotypes, part of intron 8 is retained resulting in a truncated MLPH protein, which is 359 residues long in wt and Violet and 284 residues long in Silverblue. Subsequently, our findings point out that the missing actin-binding domain, in neither of the 3 analyzed phenotypes affects the transport of melanosomes or the consequent final pigmentation. Moreover, the loss of the major part of the MYO5A domain in the Silverblue MLPH protein seems to be the responsible for the dilute phenotype. Based on our genomic DNA data, genetic tests for selecting Silverblue and Violet carrier animals can be performed in American mink.

A Gly98Val mutation in the N-Myc downstream regulated gene 1 (NDRG1) in Alaskan Malamutes with polyneuropathy.

The first cases of early-onset progressive polyneuropathy appeared in the Alaskan Malamute population in Norway in the late 1970s. Affected dogs were of both sexes and were ambulatory paraparetic, progressing to non-ambulatory tetraparesis. On neurologic examination, affected dogs displayed predominantly laryngeal paresis, decreased postural reactions, decreased spinal reflexes and muscle atrophy. The disease was considered eradicated through breeding programmes but recently new cases have occurred in the Nordic countries and the USA. The N-myc downstream-regulated gene (NDRG1) is implicated in neuropathies with comparable symptoms or clinical signs both in humans and in Greyhound dogs. This gene was therefore considered a candidate gene for the polyneuropathy in Alaskan Malamutes. The coding sequence of the NDRG1 gene derived from one healthy and one affected Alaskan Malamute revealed a non-synonymous G>T mutation in exon 4 in the affected dog that causes a Gly98Val amino acid substitution. This substitution was categorized to be "probably damaging" to the protein function by PolyPhen2 (score: 1.000). Subsequently, 102 Alaskan Malamutes from the Nordic countries and the USA known to be either affected (n = 22), obligate carriers (n = 7) or healthy (n = 73) were genotyped for the SNP using TaqMan. All affected dogs had the T/T genotype, the obligate carriers had the G/T genotype and the healthy dogs had the G/G genotype except for 13 who had the G/T genotype. A protein alignment showed that residue 98 is conserved in mammals and also that the entire NDRG1 protein is highly conserved (94.7%) in mammals. We conclude that the G>T substitution is most likely the mutation that causes polyneuropathy in Alaskan Malamutes. Our characterization of a novel candidate causative mutation for polyneuropathy offers a new canine model that can provide further insight into pathobiology and therapy of human polyneuropathy. Furthermore, selection against this mutation can now be used to eliminate the disease in Alaskan Malamutes.

Expression of innate immune genes, proteins and microRNAs in lung tissue of pigs infected experimentally with influenza virus (H1N2).

This study aimed at providing a better understanding of the involvement of innate immune factors, including miRNA, in the local host response to influenza virus infection. Twenty pigs were challenged by influenza A virus subtype H1N2. Expression of microRNA (miRNA), mRNA and proteins were quantified in lung tissue at different time points after challenge (24 h, 72 h and 14 d post-infection (p.i.). Several groups of genes were significantly regulated according to time point and infection status including pattern recognition receptors (TLR2, TLR3, TLR7, retinoic acid-inducible gene I, melanoma differentiation associated protein-5), IFN and IFN-induced genes (IFN-ß, IFN-γ, IRF7, STAT1, ISG15 and OASL), cytokines (IL-1 ß, IL-1RN, IL-6, IL-7, IL-10, IL-12A, TNF-α, CCL2, CCL3 and CXCL10) and several acute phase proteins. Likewise, the following miRNAs were differentially expressed in one or more time groups compared with the control pigs: miR-15a, miR-21, miR-146, miR-206, miR-223 and miR-451. At d 1 p.i. lung tissue protein levels of IL-6, IL-12 and IFN-α were significantly increased compared with the control group, and haptoglobin and C-reactive protein were significantly increased at d 3 p.i. Our results suggest that, in addition to a wide range of innate immune factors, miRNAs may also be involved in controlling acute influenza infection in pigs.

MicroRNA expression profiles associated with development of drug resistance in Ehrlich ascites tumor cells.

Multidrug resistance (MDR) poses a major obstacle to successful chemotherapeutic treatment of cancer, and often involves multiple genes, which may be regulated post-transcriptionally by microRNAs (miRNAs). The purpose of the present study was therefore to identify any resistance-associated changes in miRNA expression in a sensitive and five increasingly drug-resistant Ehrlich ascites tumor (EAT) cell lines, representing different steps in the development of resistance. We used an LNA-enhanced microarray platform to study the global miRNA expression profiles in the six murine EAT cell lines, and identified growth-, hypoxia-, and resistance-specific miRNA patterns. Among the differentially expressed miRNAs, we found the two clusters miR-183∼miR-96∼miR-182 and miR-200b∼miR-200a∼miR-429 as well as miR-141 to be consistently upregulated in the MDR cell lines, while miR-125b-5p and the two clusters miR-30d∼miR-30b and miR-23b∼miR-27b∼miR-24-1 were downregulated in most of the resistant EAT cells. Several of the target genes for these miRNAs-including Zeb1/Zeb2 and members of the Fox gene family-could contribute to the drug-resistant phenotype, although we did not find that the degree of resistance was directly correlated to any specific changes in miRNA expression. Probably, the observed miRNA expression patterns reflect the underlying genomic instability of the tumor cells, and further studies are needed to explore how the highly complex regulatory miRNA networks contribute to the development of MDR.

Identification of 2 loci associated with development of myxomatous mitral valve disease in Cavalier King Charles Spaniels.

Myxomatous mitral valve disease (MMVD) is the most common heart disease in dogs. It is characterized by chronic progressive degenerative lesions of the mitral valve. The valve leaflets become thickened and prolapse into the left atrium resulting in mitral regurgitation (MR). MMVD is most prevalent in small to medium sized dog breeds, Cavalier King Charles Spaniels (CKCS) in particular. The onset of MMVD is highly age dependent, and at the age of 10 years, nearly all CKCS are affected. The incidence of a similar disease in humans-mitral valve prolapse-is 1-5%. By defining CKCSs with an early onset of MMVD as cases and old dogs with no or mild signs of MMVD as controls, we conducted a genome-wide association study (GWAS) to identify loci associated with development of MMVD. We have identified a 1.58 Mb region on CFA13 (P(genome) = 4.0 × 10(-5)) and a 1.68 Mb region on CFA14 (P(genome) = 7.9 × 10(-4)) associated with development of MMVD. This confirms the power of using the dog as a model to uncover potential candidate regions involved in the molecular mechanisms behind complex traits.

Global microRNA analysis of the NCI-60 cancer cell panel.

MicroRNAs (miRNA) are a group of short noncoding RNAs that regulate gene expression at the posttranscriptional level. They are involved in many biological processes, including development, differentiation, apoptosis, and carcinogenesis. Because miRNAs may play a role in the initiation and progression of cancer, they comprise a novel class of promising diagnostic and prognostic molecular markers and potential drug targets. By applying an LNA-enhanced microarray platform, we studied the expression profiles of 955 miRNAs in the NCI-60 cancer cell lines and identified tissue- and cell-type-specific miRNA patterns by unsupervised hierarchical clustering and statistical analysis. A comparison of our data to three previously published miRNA expression studies on the NCI-60 panel showed a remarkably high correlation between the different technical platforms. In addition, the current work contributes expression data for 369 miRNAs that have not previously been profiled. Finally, by matching drug sensitivity data for the NCI-60 cells to their miRNA expression profiles, we found numerous drug-miRNAs pairs, for which the miRNA expression and drug sensitivity profiles were highly correlated and thus represent potential candidates for further investigation of drug resistance and sensitivity mechanisms.

Phenotypic and genetic characterization of a novel phenotype in pigs characterized by juvenile hairlessness and age dependent emphysema.

BACKGROUND: A pig phenotype characterized by juvenile hairlessness, thin skin and age dependent lung emphysema has been discovered in a Danish pig herd. The trait shows autosomal co-dominant inheritance with all three genotypes distinguishable. Since the phenotype shows resemblance to the integrin beta6 -/- knockout phenotype seen in mice, the two genes encoding the two subunits of integrin alphavbeta6, i.e. ITGB6 and ITGAV, were considered candidate genes for this trait. RESULTS: The mutated pig phenotype is characterized by hairlessness until puberty, thin skin with few hair follicles and absence of musculi arrectores pili, and at puberty or later localized areas of emphysema are seen in the lungs. Comparative mapping predicted that the porcine ITGB6 andITGAV orthologs map to SSC15. In an experimental family (n = 113), showing segregation of the trait, the candidate region was confirmed by linkage analysis with four microsatellite markers. Mapping of the porcine ITGB6 and ITGAV in the IMpRH radiation hybrid panel confirmed the comparative mapping information. Sequencing of the ITGB6 and ITGAV coding sequences from affected and normal pigs revealed no evidence of a causative mutation, but alternative splicing of the ITGB6 pre-mRNA was detected. For both ITGB6 and ITGAV quantitative PCR revealed no significant difference in the expression levels in normal and affected animals. In a western blot, ITGB6 was detected in lung protein samples of all three genotypes. This result was supported by flow cytometric analyses which showed comparable reactions of kidney cells from affected and normal pigs with an integrin alphavbeta6 monoclonal antibody. Also, immunohistochemical staining of lung tissue with an integrin beta6 antibody showed immunoreaction in both normal and affected pigs. CONCLUSION: A phenotype resembling the integrin beta6 -/- knockout phenotype seen in mice has been characterized in the pig. The candidate region on SSC15 has been confirmed by linkage analysis but molecular and functional analyses have excluded that the mutated phenotype is caused by structural mutations in or ablation of any of the two candidate genes.

Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR.

BACKGROUND: Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription. Internal standards such as reference genes are used to normalise mRNA levels between different samples for an exact comparison of mRNA transcription level. Selection of high quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR. RESULTS: In this study nine commonly used reference genes were investigated in 17 different pig tissues using real-time qPCR with SYBR green. The genes included beta-actin (ACTB), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L4 (RPL4), succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TPB)and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes in different pig tissues was investigated using the geNorm application. The range of expression stability in the genes analysed was (from the most stable to the least stable): ACTB/RPL4, TBP, HPRT, HMBS, YWHAZ, SDHA, B2M and GAPDH. CONCLUSION: Expression stability varies greatly between genes. ACTB, RPL4, TPB and HPRT1 were found to have the highest stability across tissues. Based on both expression stability and expression level, our data suggest that ACTB and RPL4 are good reference genes for high abundant transcripts while TPB and HPRT1 are good reference genes for low abundant transcripts in expression studies across different pig tissues.