Browsing by Author "Rawle, Rachel A."

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Characterization of synovial fluid metabolomic phenotypes of cartilage morphological changes associated with osteoarthritis
(2019-08) Carlson, Alyssa K.; Rawle, Rachel A.; Wallace, Cameron W.; Brooks, Ellen G.; Adams, Erik; Greenwood, Mark C.; Olmer, Merissa; Lotz, Martin K.; Bothner, Brian; June, Ronald K.
"Objective Osteoarthritis (OA) is a multifactorial disease with etiological heterogeneity. The objective of this study was to classify OA subgroups by generating metabolomic phenotypes from human synovial fluid. Design: Post mortem synovial fluids (n = 75) were analyzed by high performance-liquid chromatography mass spectrometry (LC-MS) to measure changes in the global metabolome. Comparisons of healthy (grade 0), early OA (grades I-II), and late OA (grades III-IV) donor populations were considered to reveal phenotypes throughout disease progression. Results: Global metabolomic profiles in synovial fluid were distinct between healthy, early OA, and late OA donors. Pathways differentially activated among these groups included structural deterioration, glycerophospholipid metabolism, inflammation, central energy metabolism, oxidative stress, and vitamin metabolism. Within disease states (early and late OA), subgroups of donors revealed distinct phenotypes. Synovial fluid metabolomic phenotypes exhibited increased inflammation (early and late OA), oxidative stress (late OA), or structural deterioration (early and late OA) in the synovial fluid. Conclusion: These results revealed distinct metabolic phenotypes in human synovial fluid, provide insight into pathogenesis, represent novel biomarkers, and can move toward developing personalized interventions for subgroups of OA patients.
In vivo mechanotransduction: Effect of acute exercise on the metabolomic profiles of mouse synovial fluid
(Elsevier BV, 2022-03) Hahn, Alyssa K.; Rawle, Rachel A.; Bothner, Brian; Prado Lopes, Erika Barboza; Griffin, Timothy M.; June, Ronald K.
Objective. Exercise is known to induce beneficial effects in synovial joints. However, the mechanisms underlying these are unclear. Synovial joints experience repeated mechanical loading during exercise. These mechanical stimuli are transduced into biological responses through cellular mechanotransduction. Mechanotransduction in synovial joints is typically studied in tissues. However, synovial fluid directly contacts all components of the joint, and thus may produce a whole-joint picture of the mechanotransduction response to loading. The objective of this study was to determine if metabolic phenotypes are present in the synovial fluid after acute exercise as a first step to understanding the beneficial effects of exercise on the joint. Material and methods. Mice underwent a single night of voluntary wheel running or standard housing and synovial fluid was harvested for global metabolomic profiling by LC-MS. Hierarchical unsupervised clustering, partial least squares discriminant, and pathway analysis provided insight into exercise-induced mechanotransduction. Results. Acute exercise produced a distinct metabolic phenotype in synovial fluid. Mechanosensitive metabolites included coenzyme A derivatives, prostaglandin derivatives, phospholipid species, tryptophan, methionine, vitamin D3, fatty acids, and thiocholesterol. Enrichment analysis identified several pathways previously linked to exercise including amino acid metabolism, inflammatory pathways, citrulline-nitric oxide cycle, catecholamine biosynthesis, ubiquinol biosynthesis, and phospholipid metabolism. Conclusion. To our knowledge, this is the first study to investigate metabolomic profiles of synovial fluid during in vivo mechanotransduction. These profiles indicate that exercise induced stress-response processes including both pro- and anti-inflammatory pathways. Further research will expand these results and define the relationship between the synovial fluid and the serum.
Metabolic Implications of Using BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT) for Tracking Protein Synthesis
(2020-02) Steward, Katherine F.; Eilers, Brian J.; Tripet, Brian P.; Fuchs, Amanda; Dorle, Michael; Rawle, Rachel A.; Soriano, Berliza; Balasubramanian, Narayanaganesh; Copie, Valerie; Bothner, Brian; Hatzenpichler, Roland
BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) is a powerful tool for tracking protein synthesis on the level of single cells within communities and whole organisms. A basic premise of BONCAT is that the non-canonical amino acids (NCAA) used to track translational activity do not significantly alter cellular physiology. If the NCAA would induce changes in the metabolic state of cells, interpretation of BONCAT studies could be challenging. To address this knowledge-gap, we have used a global metabolomics analyses to assess the intracellular effects of NCAA incorporation. Two NCAA were tested: L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG); L-methionine (MET) was used as a minimal stress baseline control. Liquid Chromatography Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used to characterize intracellular metabolite profiles of Escherichia coli cultures, with multivariate statistical analysis using XCMS and MetaboAnalyst. Results show that doping with NCAA induces metabolic changes, however, the metabolic impact was not dramatic. A second set of experiments in which cultures were placed under mild stress to simulate real-world environmental conditions showed a more consistent and more robust perturbation. Pathways that changed include amino acid and protein synthesis, choline and betaine, and the TCA cycle. Globally, these changes were statistically minor, indicating that NCAA are unlikely to exert a significant impact on cells during incorporation. Our results are consistent with previous reports of NCAA doping under replete conditions and extend these results to bacterial growth under environmentally relevant conditions. Our work highlights the power of metabolomics studies in detecting cellular response to growth conditions and the complementarity of NMR and LCMS as omics tools.
Metabolic Responses to Arsenite Exposure Regulated through Histidine Kinases PhoR and AioS in Agrobacterium tumefaciens 5A
(MDPI, 2020-09) Rawle, Rachel A.; Tokmina-Lukaszewska, Monika; Shi, Zunji; Kang, Yoon-Suk; Tripet, Brian P.; Dang, Fang; Wang, Gejiao; McDermott, Timothy R.; Copie, Valerie; Bothner, Brian
Arsenite (AsIII) oxidation is a microbially-catalyzed transformation that directly impacts arsenic toxicity, bioaccumulation, and bioavailability in environmental systems. The genes for AsIII oxidation (aio) encode a periplasmic AsIII sensor AioX, transmembrane histidine kinase AioS, and cognate regulatory partner AioR, which control expression of the AsIII oxidase AioBA. The aio genes are under ultimate control of the phosphate stress response via histidine kinase PhoR. To better understand the cell-wide impacts exerted by these key histidine kinases, we employed 1H nuclear magnetic resonance (1H NMR) and liquid chromatography-coupled mass spectrometry (LC-MS) metabolomics to characterize the metabolic profiles of ΔphoR and ΔaioS mutants of Agrobacterium tumefaciens 5A during AsIII oxidation. The data reveals a smaller group of metabolites impacted by the ΔaioS mutation, including hypoxanthine and various maltose derivatives, while a larger impact is observed for the ΔphoR mutation, influencing betaine, glutamate, and different sugars. The metabolomics data were integrated with previously published transcriptomics analyses to detail pathways perturbed during AsIII oxidation and those modulated by PhoR and/or AioS. The results highlight considerable disruptions in central carbon metabolism in the ΔphoR mutant. These data provide a detailed map of the metabolic impacts of AsIII, PhoR, and/or AioS, and inform current paradigms concerning arsenic–microbe interactions and nutrient cycling in contaminated environments.
Multi-level Omics Analysis Provides Insight to the Ignicoccus hospitalis-Nanoarchaeum equitans Association
(2017-09) Rawle, Rachel A.; Hamerly, Timothy; Tripet, Brian P.; Giannone, Richard J.; Wurch, Louie; Hettich, Robert L.; Podar, Mircea; Copie, Valerie; Bothner, Brian
BACKGROUND Studies of interspecies interactions are inherently difficult due to the complex mechanisms which enable these relationships. A model system for studying interspecies interactions is the marine hyperthermophiles Ignicoccus hospitalis and Nanoarchaeum equitans. Recent independently-conducted 'omics' analyses have generated insights into the molecular factors modulating this association. However, significant questions remain about the nature of the interactions between these archaea. METHODS We jointly analyzed multiple levels of omics datasets obtained from published, independent transcriptomics, proteomics, and metabolomics analyses. DAVID identified functionally-related groups enriched when I. hospitalis is grown alone or in co-culture with N. equitans. Enriched molecular pathways were subsequently visualized using interaction maps generated using STRING. RESULTS Key findings of our multi-level omics analysis indicated that I. hospitalis provides precursors to N. equitans for energy metabolism. Analysis indicated an overall reduction in diversity of metabolic precursors in the I. hospitalis-N. equitans co-culture, which has been connected to the differential use of ribosomal subunits and was previously unnoticed. We also identified differences in precursors linked to amino acid metabolism, NADH metabolism, and carbon fixation, providing new insights into the metabolic adaptions of I. hospitalis enabling the growth of N. equitans. CONCLUSIONS This multi-omics analysis builds upon previously identified cellular patterns while offering new insights into mechanisms that enable the I. hospitalis-N. equitans association. GENERAL SIGNIFICANCE Our study applies statistical and visualization techniques to a mixed-source omics data set to yield a more global insight into a complex system, that was not readily discernable from separate omics studies.