Background: An essential part of using real-time RT-PCR is that expression results have to be normalized before any conclusions can be drawn. This can be done by using one or multiple, validated reference genes, depending on the desired accuracy of the results. In the pig however, very little information is available on the expression stability of reference genes. The aim of this study was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in porcine backfat and longissimus dorsi muscle, both representing an economically important part of a pig's carcass. Because of its multiple functions in

The ability to identify young females with superior reproduction would have a large economic impact on commercial swine production. Previous studies have discovered SNP associated with economically important traits such as litter size, growth rate, and feed intake. The objective of this study was to test for association of candidate SNP with sow prolificacy reproductive traits in gilts of a Landrace-Duroc-Yorkshire composite population. Association analyses regressed additive (A), dominant (D), and imprinting (I) SNP effects on each trait with an animal model. A carnitine palmitoyltransferase 1A SNP and a glycogen synthase 1 SNP were associated with age at puberty (AP; D = 10 d; P = 0. 0037 and A = 3.8 d; P = 0.0078, respectively). Four IGF2 SNP were associated with AP as well, having additive or dominant effects (3.2 to 5.8 d; P < or = 0.0052). Two mannosidase 2B2 SNP and 2 prolactin receptor (PRLR) SNP were also associated with AP. Solute carrier 22, subfamily member 5 SNP was weakly associated with AP (D = 3.9 d; P < 0.10). Polymorphisms within glycogen synthase 1 and protein kinase AMP-activated, gamma 3 noncatalytic subunit had associations with ovulation rate. Estrogen receptor (ESR) 1, ESR2, PPAR gamma coactivator 1, and IGFBP3 SNP were significantly associated with weaning-to-estrus interval. Two PRLR SNP were associated with total number of piglets born (A = 0.57 piglets; P = 0.0095 and D = 0.61 piglets; P = 0.0016, respectively). A SNP within PRLR was also associated with number of piglets born alive (D = 0.61; P = 0.0016). The PPAR gamma coactivator 1 SNP was associated with total number of piglets born (D = 0.38 piglets; P = 0.0391) and number of piglets born alive (D = 0.53 piglets; P = 0.0032). The SNP within ESR1 (A = 0.65 piglets; P = 0.0950), ESR2 (A = -0.33 piglets; P = 0.0176), IGF2 SNP (A = -0.26 piglets; P = 0.0032), and IGFBP3 SNP (D = 0.35 piglets; P = 0.0683) were associated with number of piglets born dead. A leptin SNP was associated with mummified fetuses (D = 0.09 piglets; P = 0.0978). Many of the SNP analyzed in this study are from genes involved in regulation of metabolism, suggesting that there is an important link between physiological events associated with reproduction and energy utilization. Furthermore, these production and growth trait SNP may serve to assist in selection of young females for superior reproductive performance.

Long-acting injectable (LAI) drug suspensions consist of drug nano-/microcrystals suspended in an aqueous vehicle and enable prolonged therapeutic drug exposure up to several months. The examination of injection site reactions (ISRs) to the intramuscular (IM) injection of LAI suspensions is relevant not only from a safety perspective but also for the understanding of the pharmacokinetics. The aim of this study was to perform a multilevel temporal characterization of the local and lymphatic histopathological/ immunological alterations triggered by the IM injection of an LAI paliperidone palmitate suspension and an analog polystyrene suspension in rats and identify critical time points and parameters with regard to the host response. The ISRs showed a moderate to marked chronic granulomatous inflammation, which was mediated by multiple cyto-/chemokines, including interleukin-1b, monocyte Chemoattractant Protein-1, and vascular endothelial growth factor. Lymphatic uptake and lymph node retention of nano-/microparticles were observed, but the contribution to the drug absorption was negligible. A simple image analysis procedure and empirical model were proposed for the accurate evaluation of the depot geometry, cell infiltration, and vascularization. This study was designed as a reference for the evaluation and comparison of future LAIs and to support the mechanistic modeling of the formulation-physiology interplay regulating the drug absorption from LAIs.

BackgroundIn humans, adipose tissue (AT) originating from different depots shows varying gene expression profiles. In horses, the risk of certain metabolic disorders may also be influenced by the impact of specific AT depots. Macrophage infiltration in human and rat AT is considered to be a source of inflammatory changes. In horses, this relationship has not been extensively studied yet. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), a useful method to evaluate differences in mRNA expression across different tissues, can be used to evaluate differences between equine AT depots. For a correct interpretation of the RT-qPCR results, expression data have to be normalized by the use of validated reference genes. The main objectives of this study were to compare mRNA expression of inflammation-related genes, as well as adipocyte morphology and number between different equine AT depots; and in addition, to investigate the presence of antigen presenting cells in equine AT and any potential relationship with adipokine mRNA expression.ResultsIn this study, the mRNA expression of inflammation-related genes (leptin, chemokine ligand 5, interleukin 1β, interleukin 6, interleukin 10, adiponectin, matrix metalloproteinase 2, and superoxide dismutase 2) and candidate reference gene stability was investigated in 8 different AT depots collected from the nuchal, abdominal (mesenteric, retroperitoneal, and peri-renal) and subcutaneous (tail head and loin) AT region. By using GeNorm analysis, HPRT1, RPL32, and GAPDH were found to be the most stable genes in equine AT. The mRNA expression of leptin, chemokine ligand 5, interleukin 10, interleukin 1β, adiponectin, and matrix metalloproteinase 2 significantly differed across AT depots (P < 0.05). No significant AT depot effect was found for interleukin 6 and superoxide dismutase 2 (P > 0.05). Adipocyte area and number of antigen presenting cells per adipocyte significantly differed between AT depots (P < 0.05).ConclusionsAdipose tissue location was associated with differences in mRNA expression of inflammation-related genes. This depot-specific difference in mRNA expression suggests that the overall inflammatory status of horses could be partially determined by the relative proportion of the different AT depots.

Shadow of prion protein (SPRN) is an interesting candidate gene thought to be involved in prion pathogenesis. In humans, an association has already been discovered between mutations in SPRN and the incidence of variant and sporadic Creutzfeldt-Jakob disease. However, in sheep, the effect of mutations in SPRN is largely unknown. Therefore, we analysed the presence of mutations in the entire ovine SPRN gene, their association with scrapie susceptibility and their effect on SPRN promoter activity. In total, 26 mutations were found: seven in the promoter region, four in intron 1, seven in the coding sequence and eight in the 3' untranslated region. The mutations detected in the coding sequence and the promoter region were subsequently analysed in more detail. In the coding sequence, a polymorphism causing a deletion of two alanines was found to be associated with susceptibility for classical scrapie in sheep. Furthermore, a functional analysis of deletion constructs of the ovine SPRN promoter revealed that the region 464 to 230 bp upstream of exon 1 (containing a putative AP-2 and putative Sp1 binding sites) is of functional importance for SPRN transcription. Six mutations in the SPRN promoter were also found to alter the promoter activity in vitro. However, no association between any of these promoter mutations and susceptibility for classical scrapie was found.

Understanding differences in Alzheimer’s disease biomarkers before the pathology becomes evident can contribute to an improved understanding of disease pathogenesis and treatment. A decrease in amyloid-β (Aβ)42 in cerebrospinal fluid (CSF) is suggested to be a biomarker for Aβ deposition in brain. However, the relevance of CSF Aβ levels prior to deposition is not entirely known. Dogs are similar to man with respect to amyloid-β protein precursor (AβPP)-processing, age-related amyloid plaque deposition, and cognitive dysfunction. In the current study, we evaluated the relation between CSF Aβ42 levels and cognitive performance in young to middle-aged dogs (1.5–7 years old). Additionally, CSF sAβPPα and sAβPPβ were measured to evaluate AβPP processing, and CSF cytokines were measured to determine the immune status of the brain. We identified two groups of dogs showing consistently low or high CSF Aβ42 levels. Based on prior studies, it was assumed that at this age no cerebral amyloid plaques were likely to be present. The cognitive performance was evaluated in standard cognition tests. Low or high Aβ concentrations coincided with low or high sAβPPα, sAβPPβ, and CXCL-1 levels, respectively. Dogs with high Aβ concentrations showed significant learning impairments on delayed non-match to position (DNMP), object discrimination, and reversal learning compared to dogs with low Aβ concentrations. Our data support the hypothesis that high levels of CSF Aβ in dogs coincide with lower cognitive performance prior to amyloid deposition. Further experiments are needed to investigate this link, as well as the relevance with respect to Alzheimer’s disease pathology progression.

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S U M M A R Y The development and growth of renal glomeruli is regulated by specific angiogenic growth factors, including vascular endothelial growth factor (VEGF) and the angiopoietins (ANGPT1 and ANGPT2). The expression of these factors has already been studied during metanephric glomerulogenesis, but it remains to be elucidated during the development of the embryonic mesonephros, which can function as an interesting model for glomerular development and senescence. In this study, the presence of the angiogenic growth factors was studied in developing porcine mesonephroi, using IHC and real-time RT-qPCR on laser capture microdissected glomeruli. In addition, mesonephric glomerular growth was measured by using stereological methods. ANGPT2 remained upregulated during maturation of glomeruli, which may be explained by the continuous growth of the glomeruli, as observed by stereological examination. The mRNA for VEGFA was expressed in early developing and in maturing glomeruli. The VEGF receptor VEGFR1 was stably expressed during the whole lifespan of mesonephric glomeruli, whereas VEGFR2 mRNA was only upregulated in early glomerulogenesis, suggesting that VEGFR2 is important for the vascular growth but that VEGFR1 is important for the maintenance of endothelial fenestrations.