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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.

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.

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.

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.

Background and Aim Mitochondrial dysfunction contributes to the pathophysiology of diabetic cardiomyopathy. The aim of this study was to investigate the acute changes in the mitochondrial proteome in response to insulin stimulation. Methods and Results Cardiac mitochondria from C57BL/6 mice after insulin stimulation were analyzed using two-dimensional fluorescence difference gel electrophoresis. MALDI-TOF MS/MS was utilized to identify differences. Two enzymes involved in metabolism and four structural proteins were identified. Succinyl-CoA ligase [ADP forming] subunit beta was identified as one of the differentially regulated proteins. Upon insulin stimulation, a relatively more acidic isoform of this protein was increased by 53% and its functional activity was decreased by ∼32%. Conclusions This proteomic remodeling in response to insulin stimulation may play an important role in the normal and diabetic heart.

Molecular profiling of tumor mutations has expedited molecular targeted therapy for cancer patients. Additionally, tumor mutation load and DNA microsatellite instability (MSI) status can help predict patient’s response to immunotherapy. We analytically validated a gene test targeting clinically actionable cancer mutations of all 4 major types: single nucleotide variations (SNVs); insertion/deletions (INDELs); whole gene copy number variations (CNVs); and structural rearrangements (translocations) as well as the MSI status. This test interrogates all coding exons from 49 core cancer genes, introns for a subset of genes selected for detection of prevalent gene rearrangements, and the TERT (telomerase reverse transcriptase) promoter region. MSI status is determined from a set of 5 intronic mono-nucleotide repeats collectively associated with microsatellite instability. Targeted DNA regions are captured by in-solution hybridization with complementary biotinylated RNA baits and sequenced on the Illumina NextSeq®500 platform. Paired formalin-fixed, paraffin-embedded (FFPE) tumor samples and whole blood samples are analyzed simultaneously for the detection of SNV, INDEL, CNV, translocation and MSI status. FFPE samples can also be analyzed alone if the whole blood sample is not available, eliminating the reporting of MSI status. A minimum of 50 ng FFPE DNA and 100 ng of whole blood DNA are required for this test. A total of 123 FFPE, 2 FFPE FNA samples, 19 paired FFPE and whole blood paired samples covering lung cancer, colorectal cancer, melanoma and breast cancer were included in this validation study. Analytical validation of assay performance demonstrated that, on average, 700-fold read depth was achieved across all targeted regions with >95% of these regions covered by a minimum of 300 unique reads. Analytical sensitivity was ≥5% mutation frequency for SNV and INDEL and ≥20% for translocation and CNV. Thirty-eight unique variants were confirmed between this test and orthogonal methodologies: 22 SNVs, 6 INDELs, 5 translocations, and 5 CNV. Fifty-eight out of 59 (98% concordance) paired FFPE/blood samples achieved the same MSI status result with this targeted sequencing approach compared to the reference National Comprehensive Cancer Network (NCCN) Bethesda PCR test. MSI-positive samples, on average, were determined to have 5-fold higher mutation count compared to MSI-negative samples, consistent with the previously reported higher mutation burden. In conclusion, we have developed and analytically validated a core cancer gene test employing NGS technology with demonstrated high analytical sensitivity and specificity. Coupled with clinical interpretation, this test will facilitate clinical decision-making for molecular targeted therapy and immunotherapy. Citation Format: Lin Ma, Michael Hua, Steven Rivera, Anna Gerasimova, Quoclinh Nguyen, Sirisha Sunkara, Robert Lagier, Alla Smolgovsky, David Wolfson, Jared F. Taylor, Frederick Racke, Charles Strom, Andrew Grupe, Joseph Catanese, Feras Hantash. Validation of a clinically actionable cancer core gene test for solid tumors facilitating targeted molecular therapy and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5356. doi:10.1158/1538-7445.AM2017-5356