Scholarly Work - Chemistry & Biochemistry
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/8714
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Item Metabolic Deficits in the Retina of a Familial Dysautonomia Mouse Model(MDPI AG, 2024-07) Costello, Stephanaan M.; Schultz, Anastasia; Smith, Donald; Horan, Danielle; Chaverra, Martha; Tripet, Brian; George, Lynn; Bothner, Brian; Lefcort, Frances; Copié, ValérieNeurodegenerative retinal diseases such as glaucoma, diabetic retinopathy, Leber’s hereditary optic neuropathy (LHON), and dominant optic atrophy (DOA) are marked by progressive death of retinal ganglion cells (RGC). This decline is promoted by structural and functional mitochondrial deficits, including electron transport chain (ETC) impairments, increased oxidative stress, and reduced energy (ATP) production. These cellular mechanisms associated with progressive optic nerve atrophy have been similarly observed in familial dysautonomia (FD) patients, who experience gradual loss of visual acuity due to the degeneration of RGCs, which is thought to be caused by a breakdown of mitochondrial structures, and a disruption in ETC function. Retinal metabolism plays a crucial role in meeting the elevated energetic demands of this tissue, and recent characterizations of FD patients’ serum and stool metabolomes have indicated alterations in central metabolic processes and potential systemic deficits of taurine, a small molecule essential for retina and overall eye health. The present study sought to elucidate metabolic alterations that contribute to the progressive degeneration of RGCs observed in FD. Additionally, a critical subpopulation of retinal interneurons, the dopaminergic amacrine cells, mediate the integration and modulation of visual information in a time-dependent manner to RGCs. As these cells have been associated with RGC loss in the neurodegenerative disease Parkinson’s, which shares hallmarks with FD, a targeted analysis of the dopaminergic amacrine cells and their product, dopamine, was also undertaken. One dimensional (1D) proton (1H) nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and retinal histology methods were employed to characterize retinae from the retina-specific Elp1 conditional knockout (CKO) FD mouse model (Pax6-Cre; Elp1LoxP/LoxP). Metabolite alterations correlated temporally with progressive RGC degeneration and were associated with reduced mitochondrial function, alterations in ATP production through the Cahill and mini-Krebs cycles, and phospholipid metabolism. Dopaminergic amacrine cell populations were reduced at timepoints P30–P90, and dopamine levels were 25–35% lower in CKO retinae compared to control retinae at P60. Overall, this study has expanded upon our current understanding of retina pathology in FD. This knowledge may apply to other retinal diseases that share hallmark features with FD and may help guide new avenues for novel non-invasive therapeutics to mitigate the progressive optic neuropathy in FD.Item Metabolomic Profiles and Pathways in Osteoarthritic Human Cartilage: A Comparative Analysis with Healthy Cartilage(MDPI AG, 2024-03) Welhaven, Hope D.; Welfley, Avery H.; Brahmachary, Priyanka; Bergstrom, Annika R.; Houske, Eden; Glimm, Matthew; Bothner, Brian; Hahn, Alyssa K.; June, Ronald K.Osteoarthritis (OA) is a chronic joint disease with heterogenous metabolic pathology. To gain insight into OA-related metabolism, metabolite extracts from healthy (n = 11) and end-stage osteoarthritic cartilage (n = 35) were analyzed using liquid chromatography–mass spectrometry metabolomic profiling. Specific metabolites and metabolic pathways, including lipid and amino acid pathways, were differentially regulated in osteoarthritis-derived and healthy cartilage. The detected alterations in amino acids and lipids highlighted key differences in bioenergetic resources, matrix homeostasis, and mitochondrial alterations in OA-derived cartilage compared to healthy cartilage. Moreover, the metabolomic profiles of osteoarthritic cartilage separated into four distinct endotypes, highlighting the heterogenous nature of OA metabolism and the diverse landscape within the joint in patients. The results of this study demonstrate that human cartilage has distinct metabolomic profiles in healthy and end-stage OA patients. By taking a comprehensive approach to assess metabolic differences between healthy and osteoarthritic cartilage and within osteoarthritic cartilage alone, several metabolic pathways with distinct regulation patterns were detected. Additional investigation may lead to the identification of metabolites that may serve as valuable indicators of disease status or potential therapeutic targets.Item Metabolic Phenotypes Reflect Patient Sex and Injury Status: A Cross-Sectional Analysis of Human Synovial Fluid(Elsevier BV, 2023-09) Welhaven, Hope D.; Welfley, Avery H.; Pershad, Prayag; Satalich, James; O'Connell, Robert; Bothner, Brian; Vap, Alexander R.; June, Ronald K.Objective. Osteoarthritis is a heterogeneous disease. The objective was to compare differences in underlying cellular mechanisms and endogenous repair pathways between synovial fluid (SF) from male and female participants with different injuries to improve the current understanding of the pathophysiology of downstream post-traumatic osteoarthritis (PTOA). Design. SF from n = 33 knee arthroscopy patients between 18 and 70 years with no prior knee injuries was obtained pre-procedure and injury pathology assigned post-procedure. SF was extracted and analyzed via liquid chromatography-mass spectrometry metabolomic profiling to examine differences in metabolism between injury pathologies (ligament, meniscal, and combined ligament and meniscal) and patient sex. Samples were pooled and underwent secondary fragmentation to identify metabolites. Results. Different knee injuries uniquely altered SF metabolites and downstream pathways including amino acid, lipid, and inflammatory-associated metabolic pathways. Notably, sexual dimorphic metabolic phenotypes were examined between males and females and within injury pathology. Cervonyl carnitine and other identified metabolites differed in concentrations between sexes. Conclusions. These results suggest that different injuries and patient sex are associated with distinct metabolic phenotypes. Considering these phenotypic associations, a greater understanding of metabolic mechanisms associated with specific injuries, sex, and PTOA development may yield data regarding how endogenous repair pathways differ between male and female injury types. Ongoing metabolomic analysis of SF in injured male and female patients can be performed to monitor PTOA development and progression.Item Distinct Metabolic States Are Observed in Hypoglycemia Induced in Mice by Ricin Toxin or by Fasting(MDPI AG, 2022-11) Kempa, Jacob; O’Shea-Stone, Galen; Moss, Corinne E.; Peters, Tami; Marcotte, Tamera K.; Tripet, Brian; Eilers, Brian; Bothner, Brian; Copié, Valérie; Pincus, Seth H.Hypoglycemia may be induced by a variety of physiologic and pathologic stimuli and can result in life-threatening consequences if untreated. However, hypoglycemia may also play a role in the purported health benefits of intermittent fasting and caloric restriction. Previously, we demonstrated that systemic administration of ricin toxin induced fatal hypoglycemia in mice. Here, we examine the metabolic landscape of the hypoglycemic state induced in the liver of mice by two different stimuli: systemic ricin administration and fasting. Each stimulus produced the same decrease in blood glucose and weight loss. The polar metabolome was studied using 1H NMR, quantifying 59 specific metabolites, and untargeted LC-MS on approximately 5000 features. Results were analyzed by multivariate analyses, using both principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), to identify global metabolic patterns, and by univariate analyses (ANOVA) to assess individual metabolites. The results demonstrated that while there were some similarities in the responses to the two stimuli including decreased glucose, ADP, and glutathione, they elicited distinct metabolic states. The metabolite showing the greatest difference was O-phosphocholine, elevated in ricin-treated animals and known to be affected by the pro-inflammatory cytokine TNF-α. Another difference was the alternative fuel source utilized, with fasting-induced hypoglycemia primarily ketotic, while the response to ricin-induced hypoglycemia involves protein and amino acid catabolism.Item NMR Hydrophilic Metabolomic Analysis of Bacterial Resistance Pathways Using Multivalent Antimicrobials with Challenged and Unchallenged Wild Type and Mutated Gram-Positive Bacteria(MDPI, 2021-12) Aries, Michelle L.; Cloninger, Mary J.Multivalent membrane disruptors are a relatively new antimicrobial scaffold that are difficult for bacteria to develop resistance to and can act on both Gram-positive and Gram-negative bacteria. Proton Nuclear Magnetic Resonance (1H NMR) metabolomics is an important method for studying resistance development in bacteria, since this is both a quantitative and qualitative method to study and identify phenotypes by changes in metabolic pathways. In this project, the metabolic differences between wild type Bacillus cereus (B. cereus) samples and B. cereus that was mutated through 33 growth cycles in a nonlethal dose of a multivalent antimicrobial agent were identified. For additional comparison, samples for analysis of the wild type and mutated strains of B. cereus were prepared in both challenged and unchallenged conditions. A C16-DABCO (1,4-diazabicyclo-2,2,2-octane) and mannose functionalized poly(amidoamine) dendrimer (DABCOMD) were used as the multivalent quaternary ammonium antimicrobial for this hydrophilic metabolic analysis. Overall, the study reported here indicates that B. cereus likely change their peptidoglycan layer to protect themselves from the highly positively charged DABCOMD. This membrane fortification most likely leads to the slow growth curve of the mutated, and especially the challenged mutant samples. The association of these sample types with metabolites associated with energy expenditure is attributed to the increased energy required for the membrane fortifications to occur as well as to the decreased diffusion of nutrients across the mutated membrane.Item NMR and Metabolomics—A Roadmap for the Future(MDPI AG, 2022-07) Wishart, David S.; Cheng, Leo L.; Copié, Valérie; Edison, Arthur S.; Eghbalnia, Hamid R.; Hoch, Jeffrey C.; Gouveia, Goncalo J.; Pathmasiri, Wimal; Powers, Robert; Schock, Tracey B.; Sumner, Lloyd W.; Uchimiya, MarioMetabolomics investigates global metabolic alterations associated with chemical, biological, physiological, or pathological processes. These metabolic changes are measured with various analytical platforms including liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). While LC-MS methods are becoming increasingly popular in the field of metabolomics (accounting for more than 70% of published metabolomics studies to date), there are considerable benefits and advantages to NMR-based methods for metabolomic studies. In fact, according to PubMed, more than 926 papers on NMR-based metabolomics were published in 2021—the most ever published in a given year. This suggests that NMR-based metabolomics continues to grow and has plenty to offer to the scientific community. This perspective outlines the growing applications of NMR in metabolomics, highlights several recent advances in NMR technologies for metabolomics, and provides a roadmap for future advancements.Item Effects of mechanical stimulation on metabolomic profiles of SW1353 chondrocytes: shear and compression(The Company of Biologists, 2022-01) Welhaven, Hope D.; McCutchen, Carley N.; June, Ronald K.Mechanotransduction is a biological phenomenon where mechanical stimuli are converted to biochemical responses. A model system for studying mechanotransduction are the chondrocytes of articular cartilage. Breakdown of this tissue results in decreased mobility, increased pain, and reduced quality of life. Either disuse or overloading can disrupt cartilage homeostasis, but physiological cyclical loading promotes cartilage homeostasis. To model this, we exposed SW1353 cells to cyclical mechanical stimuli, shear and compression, for different durations of time (15 and 30 min). By utilizing liquid chromatography-mass spectroscopy (LC-MS), metabolomic profiles were generated detailing metabolite features and biological pathways that are altered in response to mechanical stimulation. In total, 1457 metabolite features were detected. Statistical analyses identified several pathways of interest. Taken together, differences between experimental groups were associated with inflammatory pathways, lipid metabolism, beta-oxidation, central energy metabolism, and amino acid production. These findings expand our understanding of chondrocyte mechanotransduction under varying loading conditions and time periods. This article has an associated First Person interview with the first author of the paper.