Browsing by Author "Cloninger, Mary J."

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Nanoparticles To Study Lectins in Caenorhabditis elegans: Multivalent Galactose β1-4 Fucose-Functionalized Dendrimers Provide Protection from Oxidative Stress
(American Chemical Society, 2021-11) VanKoten, Harrison W.; Moore, Rebecca S.; Cloninger, Mary J.
Galectins are galactoside-binding lectins that are functional dimers or higher-order oligomers. Multivalent binding has been shown to augment the relatively low affinity of the galectins for their galactoside-binding partners, enabling the galectins to play an important role in the global remodeling of cells that occurs during the stress conditions of disease states, including heart disease and cancer. The presence of galectins in the nematode Caenorhabditis elegans and their galactoside-binding properties have been demonstrated, but the role of multivalent interactions for C. elegans galectins is unknown. Here, we describe the synthesis of Galβ1-4Fuc-functionalized poly(amidoamine) dendrimers and their utility in studies using C. elegans during oxidative stress. C. elegans were fed Galβ1-4Fuc-functionalized dendrimers and RNA interference to knock down lectins lec-1 and lec-10 while undergoing oxidative stress. C. elegans that were pretreated with the glycodendrimers were less susceptible to oxidative stress than untreated controls. Worms that were fed fluorescently tagged glycodendrimers and imaged indicated that the dendrimers are primarily present in the digestive tract of the worms, and uptake into the vulva and proximal gonads could also be observed in some instances. This study suggests that multivalently presented Galβ1-4Fuc can protect C. elegans from oxidative stress.
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.
NMR metabolomic analysis of bacterial resistance pathways using multivalent quaternary ammonium functionalized macromolecules
(2020-07) Aries, Michelle L.; Cloninger, Mary J.
Introduction Multivalent antimicrobial dendrimers are an exciting new system that is being developed to address the growing problem of drug resistant bacteria. Nuclear Magnetic Resonance (NMR) metabolomics is a quantitative and reproducible method for the determination of bacterial response to environmental stressors and for visualization of perturbations to biochemical pathways. Objectives NMR metabolomics is used to elucidate metabolite differences between wild type and antimicrobially mutated Escherichia coli (E. coli) samples. Methods Proton (1H) NMR hydrophilic metabolite analysis was conducted on samples of E. coli after 33 growth cycles of a minimum inhibitory challenge to E. coli by poly(amidoamine) dendrimers functionalized with mannose and with C16-DABCO quaternary ammonium endgroups and compared to the metabolic profile of wild type E. coli. Results The wild type and mutated E. coli samples were separated into distinct sample sets by hierarchical clustering, principal component analysis (PCA) and sparse partial least squares discriminate analysis (sPLS-DA). Metabolite components of membrane fortification and energy related pathways had a significant p value and fold change between the wild type and mutated E. coli. Amino acids commonly associated with membrane fortification from cationic antimicrobials, such as lysine, were found to have a higher concentration in the mutated E. coli than in the wild type E. coli. N-acetylglucosamine, a major component of peptidoglycan synthesis, was found to have a 25-fold higher concentration in the mid log phase of the mutated E. coli than in the mid log phase of the wild type. Conclusion The metabolic profile suggests that E. coli change their peptidoglycan composition in order to garner protection from the highly positively charged and multivalent C16-DABCO and mannose functionalized dendrimer.
Protein aggregation nucleated by functionalized dendritic polyglycerols
(2020-06) Bernhard, Samuel P.; Fricke, Mackenzie S.; Haag, Rainer; Cloninger, Mary J.
Dendritic polyglycerols (dPGs) are emerging as important polymers for the study of biological processes due to their relatively low toxicity and excellent biocompatibility. The highly branched nature and high density of endgroups make the dPGs particularly attractive frameworks for the study of multivalent interactions such as multivalent protein–carbohydrate interactions. Here, we report the synthesis of a series of lactose functionalized dPGs with different hydrodynamic radii. A series of lactose functionalized dPGs bearing different densities of lactose functional groups was also synthesized. These lactose functionalized dPGs were used to study the templated aggregation of galectin-3, a galactoside binding protein that is overexpressed during many processes involved in cancer progression. Dynamic light scattering measurements revealed a direct correlation between the hydrodynamic radii of the lactose functionalized dPGs and the size of the galectin-3/lactose functionalized dPG aggregates formed upon mixing the lactose functionalized dPGs with galectin-3 in solution. These studies exposed the critical role of galectin-3's N-terminal domain in formation of galectin-3 multimers and also enabled comparisons of polymer templated aggregation using nonspecific interactions versus specific protein–carbohydrate binding interactions.
Time-Dependent Fluorescence Spectroscopy to Quantify Complex Binding Interactions
(2020-11) Bernhard, Samuel P.; Goodman, Candace K.; Norton, Erienne G.; Alme, Daniel G.; Lawrence, C. Martin; Cloninger, Mary J.
Measuring the binding affinity for proteins that can aggregate or undergo complex binding motifs presents a variety of challenges. In this study, fluorescence lifetime measurements using intrinsic tryptophan fluorescence were performed to address these challenges and to quantify the binding of a series of carbohydrates and carbohydrate-functionalized dendrimers to recombinant human galectin-3. Collectively, galectins represent an important target for study; in particular, galectin-3 plays a variety of roles in cancer biology. Galectin-3 binding dissociation constants (KD) were quantified: lactoside (73 ± 4 μM), methyllactoside (54 ± 10 μM), and lactoside-functionalized G(2), G(4), and G(6)-PAMAM dendrimers (120 ± 58 μM, 100 ± 45 μM, and 130 ± 25 μM, respectively). The chosen examples showcase the widespread utility of time-dependent fluorescence spectroscopy for determining binding constants, including interactions for which standard methods have significant limitations.