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Abstract Background In dairy cows circulating non-esterified fatty acids (NEFA) increase early post-partum while liver and other tissues undergo adaptation to greater lipid metabolism, mainly regulated by peroxisome proliferator-activated receptors (PPAR). PPAR are activated by fatty acids (FA), but it remains to be demonstrated that circulating NEFA or dietary FA activate bovine PPAR. We hypothesized that circulating NEFA and dietary FA activate PPAR in dairy cows. Methods The dose-response activation of PPAR by NEFA or dietary FA was assessed using HP300e digital dispenser and luciferase reporter in several bovine cell types. Cells were treated with blood plasma isolated from Jersey cows before and after parturition, NEFA isolated from the blood plasma, FA released from lipoproteins using milk lipoprotein lipase (LPL), and palmitic acid (C16:0). Effect on each PPAR isotype was assessed using specific synthetic inhibitors. Results NEFA isolated from blood serum activate PPAR linearly up to ~ 4-fold at 400 μmol/L in MAC-T cells but had cytotoxic effect. Addition of albumin to the culture media decreases cytotoxic effects of NEFA but also PPAR activation by ~ 2-fold. Treating cells with serum from peripartum cows reveals that much of the PPAR activation can be explained by the amount of NEFA in the serum (R2 = 0.91) and that the response to serum NEFA follows a quadratic tendency, with peak activation around 1.4 mmol/L. Analysis of PPAR activation by serum in MAC-T, BFH-12 and BPAEC cells revealed that most of the activation is explained by the activity of PPARδ and PPARγ, but not PPARα. Palmitic acid activated PPAR when added in culture media or blood serum but the activation was limited to PPARδ and PPARα and the response was nil in serum from post-partum cows. The addition of LPL to the serum increased > 1.5-fold PPAR activation. Conclusion Our results support dose-dependent activation of PPAR by circulating NEFA in bovine, specifically δ and γ isotypes. Data also support the possibility of increasing PPAR activation by dietary FA; however, this nutrigenomics approach maybe only effective in pre-partum but not post-partum cows.

RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potencial for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid containing the yellow fever 17D strain replicon (pBSC-repYFV-17D), previously characterized, was handled to heterologous expression of the green fluorescent protein (repYFV-17D-GFP) and firefly luciferase (repYFV-17D-Luc) reporter genes. Both replicons were constructed by homologous recombination between the linearized vector pBSC-repYFV-17D and the PCR product containing homologous 25 nucleotides ends incorporated into PCR primers. The genomic organization of these constructs is similar to repYFV-17D, but with insertion of the reporter gene between the remaining 63 N-terminal nucleotides of the capsid protein and 72 C-terminal nucleotides of the E protein. The replicons repYFV-17D-GFP and repYFV-17D-Luc showed efficient replication and expression of the reporter genes. The yeast-based homologous recombination technique used in this study proved to be applicable for manipulation of the yellow fever virus genome in order to construct subgenomic replicons.

RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potencial for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid containing the yellow fever 17D strain replicon (pBSC-repYFV-17D), previously characterized, was handled to heterologous expression of the green fluorescent protein (repYFV-17D-GFP) and firefly luciferase (repYFV-17D-Luc) reporter genes. Both replicons were constructed by homologous recombination between the linearized vector pBSC-repYFV-17D and the PCR product containing homologous 25 nucleotides ends incorporated into PCR primers. The genomic organization of these constructs is similar to repYFV-17D, but with insertion of the reporter gene between the remaining 63 N-terminal nucleotides of the capsid protein and 72 C-terminal nucleotides of the E protein. The replicons repYFV-17D-GFP and repYFV-17D-Luc showed efficient replication and expression of the reporter genes. The yeast-based homologous recombination technique used in this study proved to be applicable for manipulation of the yellow fever virus genome in order to construct subgenomic replicons.O replicon de RNA derivado do genoma de Flavivirus é uma ferramenta valiosa para o estudo de replicação viral independente da montagem e da maturação do virion, além de possuir um grande potencial para expressão de genes heterólogos. Neste estudo nós descrevemos a construção de replicons subgenômicos do vírus da febre amarela utilizando a técnica de recombinação homóloga em levedura. O plasmídeo contendo o replicon do vírus febre amarela cepa 17D (pBSC-repYFV-17D), caracterizado anteriormente, foi manipulado para a expressão heteróloga dos genes repórteres green fluorescent protein (repYFV-17D-GFP) e firelly luciferase (repYFV-17D-Luc). Ambos os replicons foram construídos por recombinação homóloga entre o vetor pBSC-repYFV-17D linearizado e o produto de PCR contendo 25 nucleotídeos terminais homólogos incorporados aos oligonucleotídeos iniciadores. A organização genômica dos construídos é semelhante ao repYFV-17D, com inserção de um gene repórter entre os restantes 63 nucleotídeos N-terminais da proteína do capsídeo e 72 nucleotídeos C-terminais da proteína E. Os replicons repYFV-17D-GFP e repYFV-17D-Luc mostraram uma eficiente replicação e expressão dos genes repórteres. A técnica de recombinação homóloga em levedura usada neste estudo demonstrou ser aplicável à manipulação do genoma do vírus da febre amarela para a construção de replicons subgenômicos.

The phenylacetic acid (PA) degradative pathway is the central pathway by which complex aromatic compounds (e.g. styrene) get degraded. Upper pathways for different aromatic compounds converge at common intermediate phenylacetyl-CoA, which is then metabolized to succinyl-CoA and acetyl-CoA. We previously made a link in Burkholderia cenocepacia between phenylacetic acid (PA) degradation and virulence by showing that insertional mutagenesis of paaA and paaE results in PA-conditional growth and an attenuated killing phenotype in the Caenorhabditis elegans model of infection. Insertional mutagenesis of paaK1, which encodes a phenylacetyl-CoA ligase, did not result in a PA-conditional growth probably due to the presence of a second similar gene (paaK2) encoding another phenylacetyl-CoA ligase (PaaK2). Recently published crystallographic and enzyme kinetics studies comparing the two ligases show that PaaK1 is better able to bind hydroxylated PA derived molecules such as 3-hydroxyphenylacetic (3-OHPA) acid and 4-hydroxyphenylacetic acid (4-OHPA) due to its having a larger binding pocket than PaaK2. However, the biological significance of this finding remains unanswered. In this study, we demonstrate that 3-OHPA but not 4-OHPA, induces the phenylacetic acid degradative pathway in Burkholderia cenocepacia K56-2. Using reporter activity assays, we show paaK1-dependent induction of the PA degradative pathway when 3-OHPA is added to the culture medium. To shed light on a possible relevance of this finding to pathogenicity, we compared the pathogenicity of the paaK1 deletion mutant with that of the wild type in C. elegans. Results show that the loss of PaaK1 function does not equal to a loss of pathogenicity. Taken together our results suggest that 3-OHPA but not 4-OHPA may be degraded through the PA degradative pathway but that 3-OHPA-related intermediates may not be relevant to pathogenesis as previously thought.

Transformação genética estável de plantas é geralmente um processo prolongado, envolvendo em muitos casos mais de um ano de regeneração, propagação e seleção de transgénicos. Análises de expressão transiente são úteis para economizar tempo em estudos de função de genes, particularmente em genes que expressam em fruto. Sistema de expressão transiente de um gene repórter em frutos de banana foi então desenvolvido. Em frutos maduros, Agrobacterium tumefaciens, cujo plasmídio contem o gene de gusintron sob o controle do promotor de CaMV 35S, foi infiltrada nos frutos por meio de uma seringa. Em frutos imaturos, as Agrobacteria foram infiltradas por vácuo nos frutos fatiados e foram co-cultivados. Ensaio histoquímico de GUS foi realizado a 3 dias depois da infiltração ou co-cultivo. A manipulação dos frutos maduros na análise histoquímica era difícil e baixa expressão de GUS foi obtida. Por outro lado, os frutos imaturos fatiados eram fáceis de manipular e alto nível de expressão foi observado. O sistema de expressão transiente desenvolvido neste estudo pode ser útil para análises rotineiras de validação de função de genes em frutos.