RESUMEN
Osteoarthritis (OA) is a chronic joint disease characterized by pain and stiffness. Recently, there has been great interest in the use of plant-derived compounds and supplements in managing the symptoms of OA. Arthrocen is a plant-based supplement consisting of avocado and soy unsaponifiable extracts in a 1 : 2 ratio. In an effort to unravel the potential mechanisms of its action on the cellular level, we utilized an in vitro assay to study its effects on cultured human chondrocytes. By pairing this assay with protein arrays on inflammatory markers, RNA-Seq with downstream pathway analysis, and lipidomics on eicosanoids, we were able to further define its action at the molecular level. Specifically, we found a role for Arthrocen in attenuating the inflammatory response both at the protein and mRNA level. Furthermore, we discovered that Arthrocen diminished prostaglandin E2 (PGE2) levels in response to an inflammatory trigger. Additionally, unlike traditional COX-2 inhibitors, this response rather specifically attenuated PGE2 levels in the presence of inflammation and without lowering levels of other eicosanoids. This implies that Arthrocen could potentially bring about the reduced pain produced by COX-2 inhibitors without the known side effects of COX-2 inhibition.
RESUMEN
Osteoarthritis (OA) is the most common form of arthritis. Symptomatically characterized by stiffness and pain, OA is a chronic degenerative disease of joints. Of note, there is growing interest in the potential use of plant-based compounds for symptomatic treatment of OA. Arthrocen is a plant-derived agent consisting of a one to two ratio of avocado and soy unsaponifiable extracts. In order to decipher the potential mechanisms of Arthrocen's action at the molecular level, we employed an in vitro assay using cultured human THP-1 cells (a model cell line for monocytes) to study its effects. By pairing protein arrays enriched for inflammatory markers, transcriptomic pathway analysis using RNA-Sequencing, and eicosanoid specific lipidomics, we have begun to unravel its potential mechanism of action. Specifically, we found that Arthrocen can attenuate the inflammatory response at the transcript level while inducing significant changes in numerous cytokines. Furthermore, we discovered that while Arthrocen alone did not increase IL-8 or MCP-1 levels, its presence had a synergistic effect on the observed increase in response to LPS stimulation. Additionally, this synergistic effect of Arthrocen on LPS stimulation of IL-8 and MCP-1 protein levels was also observed at the mRNA level and suggests a regulatory mechanism at the transcriptional level. Interestingly, Arthrocen induced no changes in any of the eicosanoids studied. This multi-omics approach implies that Arthrocen functions at the level of gene transcription to dampen inflammation mediated by monocytes in OA.
RESUMEN
OBJECTIVE: The aim of this study was to identify liver proteome changes in a mouse model of severe insulin resistance and markedly decreased leptin levels. METHODS: Two-dimensional differential gel electrophoresis was utilized to identify liver proteome changes in AKT1(+/-)/AKT2(-/-) mice. Proteins with altered levels were identified with tandem mass spectrometry. Ingenuity Pathway Analysis was performed for the interpretation of the biological significance of the observed proteomic changes. RESULTS: 11 proteins were identified from 2 biological replicates to be differentially expressed by a ratio of at least 1.3 between age-matched insulin resistant (Akt1(+/-)/Akt2(-/-)) and wild type mice. Albumin and mitochondrial ornithine aminotransferase were detected from multiple spots, which suggest post-translational modifications. Enzymes of the urea cycle were common members of top regulated pathways. CONCLUSION: Our results help to unveil the regulation of the liver proteome underlying altered metabolism in an animal model of severe insulin resistance.
Asunto(s)
Resistencia a la Insulina , Hígado/metabolismo , Proteómica/métodos , Proteínas Proto-Oncogénicas c-akt/fisiología , Animales , Anexinas/fisiología , Anhidrasas Carbónicas/fisiología , Femenino , Glucosa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ornitina-Oxo-Ácido Transaminasa/fisiologíaRESUMEN
Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently, chromatin has been shown to play a part in this process. To elucidate this relationship further, we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed, that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally, cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp) and end (115 bp) of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts, and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed, our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer.
Asunto(s)
Cromatina/genética , Reparación del ADN/genética , ADN/genética , Genoma Humano , Neoplasias/genética , Nucleosomas/genética , Células Cultivadas , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Genómica , Histonas/genética , Humanos , Tasa de Mutación , Regiones Promotoras Genéticas/genéticaRESUMEN
The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a "ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this "ground state" by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states) are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational analysis of nucleosome organization alone is sufficient to elucidate much of the circuitry of pluripotency. Our results, suggest that nucleosome organization is associated with numerous genomic and epigenomic processes and can be used to elucidate cellular identity.