Droplet Digital PCR Quantification of Selected Intracellular and Extracellular microRNAs Reveals Changes in Their Expression Pattern during Porcine In Vitro Adipogenesis.

Extracellular miRNAs have attracted considerable interest because of their role in intercellular communication, as well as because of their potential use as diagnostic and prognostic biomarkers for many diseases. It has been shown that miRNAs secreted by adipose tissue can contribute to the pathophysiology of obesity. Detailed knowledge of the expression of intracellular and extracellular microRNAs in adipocytes is thus urgently required. The system of in vitro differentiation of mesenchymal stem cells (MSCs) into adipocytes offers a good model for such an analysis. The aim of this study was to quantify eight intracellular and extracellular miRNAs (miR-21a, miR-26b, miR-30a, miR-92a, miR-146a, miR-148a, miR-199, and miR-383a) during porcine in vitro adipogenesis using droplet digital PCR (ddPCR), a highly sensitive method. It was found that only some miRNAs associated with the inflammatory process (miR-21a, miR-92a) were highly expressed in differentiated adipocytes and were also secreted by cells. All miRNAs associated with adipocyte differentiation were highly abundant in both the studied cells and in the cell culture medium. Those miRNAs showed a characteristic expression profile with upregulation during differentiation.

Chromosome abnormalities in dogs with disorders of sex development (DSD).

Disorders of sex development (DSD) caused by chromosome abnormalities are rarely diagnosed in dogs. In this report, there is a focus on five DSD cases in which the dogs had abnormal karyotypes. All animals were recognized by owners as females, however, these dogs had a large number of reproductive defects. Among these were abnormal external genitalia such as an enlarged clitoris, abnormal development of the labia, abnormal location of the vulva and urethral orifice, and other abnormalities were observed in four dogs. Gonadal histology assessments were conducted on three dogs and there were diagnoses of the presence of an ovary, inactive testes, and ovotestis with calcification in ovarian follicles. Results from cytogenetic analysis indicated there were the following karyotypes: (a) X trisomy in a mosaic form (79,XXX/78,XX); (b) Robertsonian translocation in a mosaic form (77,XX,rob/78,XX); (c) nonmosaic X/autosome translocation (78,X,t(X;A)); (d) X/autosome translocation in a mosaic form (78,X,t(X;A)/78,XX); and (e) leukocyte chimerism (78,XX/78,XY). The findings in the present study, emphasize that cytogenetic analysis is essential for elucidating the pathogenesis of DSD in dogs.

Alteration of active and repressive histone marks during adipogenic differentiation of porcine mesenchymal stem cells.

A characteristic spatial distribution of the main chromatin fractions is observed in most mammalian cell nuclei, with euchromatin localized in the interior and heterochromatin at the nuclear periphery. It has been shown that interactions of heterochromatin with the nuclear lamina are necessary to establish this conventional architecture. Adipocytes are specific cells in which a reduction in lamin A/C expression is observed. We hypothesize that the loss of lamin A/C during adipogenic differentiation of mesenchymal stem cells (MSCs) may be associated with the reorganization of the main classes of chromatin in the nucleus. Thus, in this study, we examine the abundance and nuclear distribution of selected heterochromatin (H3K9me3, H3K27me3 and H4K20me3) and euchromatin (H4K8ac, H3K4me3 and H3K9ac) histone marks during in vitro adipogenesis, using the pig as a model organism. We found that not only did the expression of lamin A/C decrease in our differentiation system, but so did the expression of lamin B receptor (LBR). The level of two heterochromatin marks, H3K27me3 and H4K20me3, increased during differentiation, while no changes were observed for H3K9me3. The levels of two euchromatin histone marks, H4K8ac and H3K9ac, were significantly higher in adipocytes than in undifferentiated cells, while the level of H3K4me3 did not change significantly. The spatial distribution of all the examined histone marks altered during in vitro adipogenesis. H3K27me3 and H4K20me3 moved towards the nuclear periphery and H3K9me3 localized preferentially in the intermediate part of adipocyte nuclei. The euchromatin marks H3K9ac and H3K4me3 preferentially occupied the peripheral part of the adipocyte nuclei, while H4K8ac was more evenly distributed in the nuclei of undifferentiated and differentiated cells. Analysis of the nuclear distribution of repetitive sequences has shown their clustering and relocalization toward nuclear periphery during differentiation. Our study shows that dynamic changes in the abundance and nuclear distribution of active and repressive histone marks take place during adipocyte differentiation. Nuclear reorganization of heterochromatin histone marks may allow the maintenance of the nuclear morphology of the adipocytes, in which reduced expression of lamin A/C and LBR is observed.

Polymorphisms of CSF1R and WISP1 genes are associated with severity of familial adenomatous polyposis in APC1311 pigs.

Hereditary familial adenomatous polyposis (FAP) in humans significantly increases the risk of development of colorectal cancer (CRC). Germline mutations in the APC (adenomatous polyposis coli) gene are responsible for FAP. Despite having the same causative mutation, the severity of the disease differs from patient to patient. The porcine FAP model carrying a truncating APC1311 mutation, orthologous to the dominant human mutation that leads to severe form of the disease (APC1309), mirrors the severity of polyposis. Earlier RNAseq studies have revealed the differential expression of WISP1 and CSF1R in samples derived from low-grade (LG-IEN) and more advanced high-grade (HG-IEN) colon polyps of APC1311/+ pigs. The grade of dysplasia was correlated with the severity of polyposis in APC1311/+ pigs characterized by a low (LP) and high (HP) numbers of polyps. The goal of this work was to find DNA variants that regulate the expression of CSF1R and WISP1 in LP and HP pigs. In total, 32 and 36 polymorphisms in CSF1R and WISP1 were found, respectively. Of these, the genotype frequency of four silent SNPs in the coding region of WISP1 differed significantly between LP and HP lines. In silico analysis revealed an elevated minimum free energy (MFE) for three of these SNPs, suggesting their role in mRNA structure stability. Furthermore, four polymorphisms in the promoter region of CSF1R, cosegregating as a common haplotype, were associated with polyp number in APC1311/+ pigs. A secreted alkaline phosphatase (SEAP) assay showed, however, that these variants have no direct effect on the activity of the CSF1R promoter. Concluding, our study identified polymorphisms in CSF1R and WISP1 that are potentially associated with the severity of polyposis in APC1311/+ pigs.

Expression of key genes involved in DNA methylation during in vitro differentiation of porcine mesenchymal stem cells (MSCs) into adipocytes.

The normal course of DNA methylation depends on the correct functioning of the DNA methylation machinery, which include DNA methyltransferase enzymes (DNMTs) and methyl-CpG-binding domain proteins (MBDs). So far, little is known about the activity of these components during adipogenesis in the pig. The aim of this study was to analyze the expression of ten genes (DNMT1, DNMT3a, DNMT3b, MBD1, MBD2, MBD3, MBD4, MeCP2, UHRF1, and CBX5) during in vitro differentiation into adipocytes of porcine mesenchymal stem cells derived from adipose (AD-MSC) and from bone marrow tissue (BM-MSC). We found that, in undifferentiated cells, the global methylation level was higher in BM-MSC than in AD-MSC, but had similar levels in adipocytes. The transcript level of the DNMT1 gene increased at the beginning of adipogenesis and then decreased, while DNMT3a and DNMT3b transcripts increased during differentiation. All the examined MBD genes show similar expression patterns within the studied system (AD-MSC and BM-MSC). The transcript abundance of UHRF1 and CBX5 decreased in both systems. The changes in the expression patterns of these genes points to the dynamic nature of DNA methylation during porcine adipogenesis.

The importance of the nuclear positioning of the PPARG gene for its expression during porcine in vitro adipogenesis.

Proper expression of the PPARG gene, which encodes a key transcription factor of adipogenesis, is indispensable in the formation of mature adipocytes. The positioning of a gene within the nuclear space has been implicated in gene regulation. We here report on the significance of the PPARG gene's nuclear positioning for its activity during in vitro adipogenesis in the pig. We used an established system of differentiation of mesenchymal stem cells derived from bone marrow and adipose tissue into adipocytes. The differentiation process was carried out for 7 days, and the cells were examined using the 3D DNA/immuno-FISH and RNA/DNA-FISH approaches. PPARG transcript level was measured using real-time PCR, and PPARγ activity was detected with colorimetric assay. Changes in the nuclear location of the PPARG gene were observed when we compared undifferentiated mesenchymal stem cells with mature adipocytes. The gene moved from the nuclear periphery to the nuclear center as its transcriptional activity increased. The RNA/DNA-FISH approach shows that differences in primary transcript production correlated with the allele's nuclear positioning. Transcriptionally active alleles preferentially occupy the central part of the nucleus, while inactive alleles are found on the nuclear periphery. We also show that transcription of PPARG begins with one allele, but that both alleles are active in later stages of differentiation. Our results provide evidence that functionally distinct alleles of the PPARG gene are positioned in different parts of the cell nucleus. This confirms the importance of nuclear architecture to the regulation of PPARG gene transcription, and thus to the fate of the adipose cell.

Nuclear organization during in vitro differentiation of porcine mesenchymal stem cells (MSCs) into adipocytes.

Differentiation of progenitor cells into adipocytes is accompanied by remarkable changes in cell morphology, cytoskeletal organization, and gene expression profile. Mature adipocytes are filled with a large lipid droplet and the nucleus tends to move to the cell periphery. It was hypothesized that the differentiation process is also associated with changes of nuclear organization. The aim of this study was to determine the number and distribution of selected components of nuclear architecture during porcine in vitro adipogenesis. The pig is an important animal model sharing many similarities to humans at the anatomical, physiological, and genetic levels and has been recognized as a good model for human obesity. Thus, understanding how cellular structures important for fundamental nuclear processes may be altered during adipocyte differentiation is of great importance. Mesenchymal stem cells (MSCs) were derived from bone marrow (BM-MSCs) and adipose tissue (AD-MSCs) and were cultured for 7 days in the adipogenic medium. A variable differentiation potential of these cell populations towards adipogenic lineage was observed, and for further study, a comparative characteristic of the nuclear organization in BM-MSCs and AD-MSCs was performed. Nuclear substructures were visualized by indirect immunofluorescence (nucleoli, nuclear speckles, PML bodies, lamins, and HP1α) or fluorescence in situ hybridization (telomeres) on fixed cells at 0, 3, 5, and 7 days of differentiation. Comprehensive characterization of these structures, in terms of their number, size, dynamics, and arrangement in three-dimensional space of the nucleus, was performed. It was found that during differentiation of porcine MSCs into adipocytes, changes of nuclear organization occurred and concerned: (1) the nuclear size and shape; (2) reduced lamin A/C expression; and (3) reorganization of chromocenters. Other elements of nuclear architecture such as nucleoli, SC-35 nuclear speckles, and telomeres showed no significant changes when compared to undifferentiated and mature fat cells. In addition, the presence of a low number of PML bodies was characteristic of the studied porcine mesenchymal stem cell adipogenesis system. It has been shown that the arrangement of selected components of nuclear architecture was very similar in MSCs derived from different sources, whereas adipocyte differentiation involves nuclear reorganization. This study adds new data on nuclear organization during adipogenesis using the pig as a model organism.

Variability of Potato virus Y in Tomato Crops in Poland and Development of a Reverse-Transcription Loop-Mediated Isothermal Amplification Method for Virus Detection.

A collection of 147 Potato virus Y (PVY) isolates from tomato, originating from several commercial fields and greenhouses in different regions of Poland, was tested for the presence of PVY by reverse-transcription polymerase chain reaction. However, in some cases, the results obtained were ambiguous. Therefore, a sensitive reverse-transcription loop-mediated isothermal amplification method was developed for rapid detection of PVY isolates. Phylogenetic and recombination analyses were performed based on sequences of the coat protein gene. In comparison with results obtained in 2008, the presence of other strains besides PVY(N)Wi-P was confirmed. A novel recombinant between PVY(NTN) and PVY(N)Wi-P strains was detected. Our results indicate an increasing distribution and variability of the PVY population on tomato in Poland.