Influence of Cell Type in In Vitro Induced Reprogramming in Cattle.

Induced pluripotent stem cells (iPSCs) have been considered an essential tool in stem cell research due to their potential to develop new therapies and technologies and answer essential questions about mammalian early development. An important step in generating iPSCs is selecting their precursor cell type, influencing the reprogramming efficiency and maintenance in culture. In this study, we aim to characterize bovine mesenchymal cells from adipose tissue (bAdMSCs) and fetal fibroblasts (bFFs) and to compare the reprogramming efficiency of these cells when induced to pluripotency. The cells were characterized by immunostaining (CD90, SSEA1, SSEA3, and SSEA4), induced differentiation in vitro, proliferation rates, and were subjected to cell reprogramming using the murine OSKM transcription factors. The bFFs presented morphological changes resembling pluripotent cells after reprogramming and culture with different supplementation, and putative iPSCs were characterized by immunostaining (OCT4, SOX2, NANOG, and AP). In the present study, we demonstrated that cell line origin and cellular proliferation rate are determining factors for reprogramming cells into pluripotency. The generation of biPSCs is a valuable tool to improve both translational medicine and animal production and to study the different supplements required to maintain the pluripotency of bovine cells in vitro.

Prenatal Supplementation in Beef Cattle and Its Effects on Plasma Metabolome of Dams and Calves.

This study investigated the effect of different prenatal nutrition on the plasma metabolome of Nellore dams and their offspring. For that purpose, three nutritional treatments were used in 126 cows during pregnancy: NP­(control) only mineral supplementation; PP­protein-energy supplementation in the final third; and FP­protein-energy supplementation during the entire pregnancy. Targeted metabolomics were analyzed in plasma at the beginning of pregnancy and in pre-delivery of cows (n = 27) as well as in calves (n = 27, 30 ± 9.6 days of age). Data were analyzed by the analysis of variance, partial least squares discriminant analysis, and the principal component analysis (PCA). The PCA showed a clear clustering in the periods investigated only in cows (early gestation and pre-delivery). We found significant metabolites in both supervised analyses (p < 0.05 and VIP score > 1) for cows (Taurine, Glutamic acid, Histidine, and PC aa C42:2) and for calves (Carnosine, Alanine, and PC aa C26:0). The enrichment analysis revealed biological processes (p < 0.1) common among cows and calves (histidine metabolism and beta-alanine metabolism), which may be indicative of transgenerational epigenetic changes. In general, fetal programming affected mainly the metabolism of amino acids.

Oncolytic effect of Newcastle disease virus is attributed to interferon regulation in canine mammary cancer cell lines.

Canine mammary carcinoma (CMC) is one of the major health threats in dogs. The oncolytic virotherapy is a promising strategy to treat canine as well as human cancer patients with non-pathogenic replicating viruses. Here, we evaluated the antitumor activity of one lentogenic, non-lytic Newcastle disease virus (NDV) LaSota strain expressing GFP (NDV-GFP) on five different CMCs and one non-tumorigenic cell line, regarding cell viability, cell death, selectivity index, morphology, global and target gene expression analysis. As evidenced by the selectivity index, all CMC cell lines were more susceptible to NDV-GFP in comparison with the non-tumorigenic cells (~3.1× to ~78.7×). In addition, the oncolytic effect of NDV-GFP was more evident in more malignant CMC cells. Also, we observed an inverse association of the IFN pathway expression and the susceptibility to NDV. The downregulated genes in NDV-GFP-sensitive cells were functionally enriched for antiviral mechanisms by interferon and immune system pathways, demonstrating that these mechanisms are the most prominent for oncolysis by NDV. To our knowledge, this is the first description of oncolysis by an NDV strain in canine mammary cancer cells. We also demonstrated specific molecular pathways related to NDV susceptibility in these cancer cells, opening the possibility to use NDV as a therapeutic-targeted option for more malignant CMCs. Therefore, these results urge for more studies using oncolytic NDVs, especially considering genetic editing to improve efficacy in dogs.

Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues.

INTRODUCTION: Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. OBJECTIVES: We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. METHODS: Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. RESULTS: Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. CONCLUSIONS: We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

Identification of a metabolomic signature associated with feed efficiency in beef cattle.

BACKGROUND: Ruminants play a great role in sustainable livestock since they transform pastures, silage, and crop residues into high-quality human food (i.e. milk and beef). Animals with better ability to convert food into animal protein, measured as a trait called feed efficiency (FE), also produce less manure and greenhouse gas per kilogram of produced meat. Thus, the identification of high feed efficiency cattle is important for sustainable nutritional management. Our aim was to evaluate the potential of serum metabolites to identify FE of beef cattle before they enter the feedlot. RESULTS: A total of 3598 and 4210 m/z features was detected in negative and positive ionization modes via liquid chromatography-mass spectrometry. A single feature was different between high and low FE groups. Network analysis (WGCNA) yielded the detection of 19 and 20 network modules of highly correlated features in negative and positive mode respectively, and 1 module of each acquisition mode was associated with RFI (r = 0.55, P < 0.05). Pathway enrichment analysis (Mummichog) yielded the Retinol metabolism pathway associated with feed efficiency in beef cattle in our conditions. CONCLUSION: Altogether, these findings demonstrate the existence of a serum-based metabolomic signature associated with feed efficiency in beef cattle before they enter the feedlot. We are now working to validate the use of metabolites for identification of feed efficient animals for sustainable nutritional management.

Caffeine increases Nr1i3 expression and potentiates the effects of its ligand, TCPOBOP, in mice liver / A cafeína aumenta a expressão Nr1i3 e potencializa os efeitos do seu ligando, TCPOBOP, em fígado de camundongos

Caffeine is one of the world's most consumed substances. It is present in coffee, green tea and guarana, among others. The xenobiotic-sensing nuclear receptor subfamily 1, group I, member 3 (Nr1i3), also known as the Constitutive Androstane Receptor (Car) is a key regulator of drug metabolism and excretion. No consistent description of caffeine effects on this receptor has been described. Thus, to unravel the effects of caffeine on this receptor, we performed experiments in mice. First, C57Bl/6 mice that were treated daily with caffeine (50 mg/kg) for 15 days presented a slight but significant increase in Nr1i3 and Cyp2b10 gene expression. A second experiment was then performed to verify the effects of caffeine on TCPOBOP (1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene, 3,3′,5,5′-tetrachloro-1,4-bis(pyridyloxy)benzene), the most potent agonist known for mice Nr1i3. Interestingly, caffeine potentiated TCPOBOP pleiotropic effects in mice liver, such as hepatomegaly, hepatotoxicity, hepatocyte proliferation and loss of cell-to-cell communication through gap junctions. In addition, caffeine plus TCPOBOP treatment increased liver gene expression of Nr1i3 and Cyp2b10 comparing with only caffeine or TCPOBOP treatments. Together, these results indicate that caffeine increases the expression of Nr1i3 in mice liver, although at this point it is not possible to determine if Nr1i3 directly or indirectly mediates this effect...

A cafeína é uma das substâncias mais consumidas mundialmente, estando presente no café, chá-verde e guaraná, entre outros. O receptor sensor de xenobióticos Receptor Nuclear subfamília 1, grupo I, membro 3 (Nr1i3, mais conhecido como Androstano Consititutivo - Car) é um regulador chave da biotransformação e excreção de substâncias e nenhuma descrição consistente dos efeitos da cafeína sobre este receptor foi feita. Então, para avaliar os efeitos da cafeína sobre este receptor, realizamos experimentos em camundongos. Primeiramente, camundongos C57/Bl/6 foram tratados diariamente com cafeína (50 mg/kg) por 15 dias e apresentaram um leve, mas significativo, aumento na expressão do Car e do seu gene alvo Cyp2b10. Assim, um segundo experimento foi realizado para verificar os efeitos da cafeína sobre o TCPOBOP (1,4-bis-[2-(3,5-dicloropiridiloxi)]benzeno,3,3′,5,5′-tetracloro-1,4-bis(piridiloxi)benzeno), o mais potente agonista do Nr1i3 de camundongos conhecido. Interessantemente, a cafeína potencializou os efeitos pleiotrópicos do TCPOBOP no fígado dos camundongos, como hepatomegalia, hepatotoxicidade, proliferação celular e perda da comunicação intercelular por junções do tipo gap. Os camundongos tratados com cafeína e TCPOBOP apresentaram maior expressão gênica de Nr1i3 e Cyp2b10, quando comparados aos camundongos tratados apenas com cafeína ou TCPOBOP. Juntos, nossos resultados indicam que a cafeína aumenta a expressão do receptor CAR em fígados de camundongos C57/Bl/6, porém nesta etapa ainda não é possível afirmar se estes efeitos são direta ou indiretamente mediados pelo Nr1i3...

Prematuration of bovine oocytes with butyrolactone I: effects on meiosis progression, cytoskeleton, organelle distribution and embryo development.

The effects of prematuration (PM) of bovine oocytes with butyrolactone I (BLI) for 24h on meiosis progression, cell structures and embryo development were assessed. Germinal vesicle (GV) rates decreased (97.4-65.1%, P<0.05) with decreasing BLI concentrations (100-25microM). Without BSA in PM medium, GV rates were similar (98.7-97.2, P>0.05) with low BLI (10-25microM). After in vitro maturation (IVM) for 24h, metaphase II (MII) rates for controls (IVM only) were similar (91.1%, P>0.05) to PM with 10microM BLI in BSA-free medium (B10=91.5%) and 100microM BLI in medium with BSA (B100=92.4%). Meiosis resumption occurred earlier in treated oocytes (71.4-74.3% in GV for B10 and B100, respectively, after 6h IVM compared with 97.3% in controls, P<0.05). By 18h of IVM, most oocytes reached MII (72.0-78.9%, P>0.05). Microtubules and microfilaments were unaffected by BLI. Cortical granules (CG) migration was reversibly blocked by BLI. Mitochondria translocation was partially blocked by PM culture and after IVM more oocytes in B10 and B100 (95.2 and 98.2%, respectively) had mitochondria translocated to a mature pattern (all cytoplasm) than controls (81.5%, P<0.05). Cleavage rates were similar (81-87%, P>0.05), but blastocysts (day 7) decreased in B100 (33.0%, P<0.05) compared with controls and B10 (38.3 and 41.6%, respectively). Day 8 hatching rates (11.0-19.2%) and mean total cell numbers (136-150) were similar (P>0.05). PM did not improve oocyte competence but also did not cause major structural alterations, suggesting that PM may be improved and used to study the mechanisms involved in oocyte differentiation.