Scholarly Works, School of Biomedical Engineering and Sciences
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Browsing Scholarly Works, School of Biomedical Engineering and Sciences by Department "Biological Sciences"
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- A microchip platform for structural oncology applications.Winton, Carly E.; Gilmore, Brian L.; Demmert, Andrew C.; Karageorge, Vasilea; Sheng, Zhi; Kelly, Deborah F. (2016)Recent advances in the development of functional materials offer new tools to dissect human health and disease mechanisms. The use of tunable surfaces is especially appealing as substrates can be tailored to fit applications involving specific cell types or tissues. Here we use tunable materials to facilitate the three-dimensional (3D) analysis of BRCA1 gene regulatory complexes derived from human cancer cells. We employed a recently developed microchip platform to isolate BRCA1 protein assemblies natively formed in breast cancer cells with and without BRCA1 mutations. The captured assemblies proved amenable to cryo-electron microscopy (EM) imaging and downstream computational analysis. Resulting 3D structures reveal the manner in which wild-type BRCA1 engages the RNA polymerase II (RNAP II) core complex that contained K63-linked ubiquitin moieties-a putative signal for DNA repair. Importantly, we also determined that molecular assemblies harboring the BRCA1(5382insC) mutation exhibited altered protein interactions and ubiquitination patterns compared to wild-type complexes. Overall, our analyses proved optimal for developing new structural oncology applications involving patient-derived cancer cells, while expanding our knowledge of BRCA1's role in gene regulatory events.
- Molecular Analysis of BRCA1 in Human Breast Cancer Cells Under Oxidative StressGilmore, Brian L.; Liang, Yanping; Winton, Carly E.; Patel, Kaya; Karageorge, Vasilea; Varano, A. Cameron; Dearnaley, William J.; Sheng, Zhi; Kelly, Deborah F. (Nature Publishing Group, 2017-03-06)The precise manner in which physical changes to the breast cancer susceptibility protein (BRCA1) affect its role in DNA repair events remain unclear. Indeed, cancer cells harboring mutations in BRCA1 suffer from genomic instability and increased DNA lesions. Here, we used a combination of molecular imaging and biochemical tools to study the properties of the BRCA1 in human cancer cells. Our results reveal new information for the manner in which full-length BRCA1 engages its binding partner, the BRCA1-associated Ring Domain protein (BARD1) under oxidative stress conditions. We also show how physical differences between wild type and mutated BRCA15382insC impact the cell’s response to oxidative damage. Overall, we demonstrate how clinically relevant changes to BRCA1 affect its structure-function relationship in hereditary breast cancer.
- A Molecular Toolkit to Visualize Native Protein Assemblies in the Context of Human DiseaseGilmore, Brian L.; Winton, Carly E.; Demmert, Andrew C.; Tanner, Justin R.; Bowman, Sam; Karageorge, Vasilea; Patel, Kaya; Sheng, Zhi; Kelly, Deborah F. (Springer Nature, 2015-09-23)We present a new molecular toolkit to investigate protein assemblies natively formed in the context of human disease. The system employs tunable microchips that can be decorated with switchable adaptor molecules to select for target proteins of interest and analyze them using molecular microscopy. Implementing our new streamlined microchip approach, we could directly visualize BRCA1 gene regulatory complexes from patient-derived cancer cells for the first time.
- Optimizing the restored chemotactic behavior of anticancer agent Salmonella enterica serovar Typhimurium VNP20009Broadway, Katherine M.; Suh, SeungBeum; Behkam, Bahareh; Scharf, Birgit E. (Elsevier, 2017-06-10)Bacteria, including strains of Salmonella, have been researched and applied as therapeutic cancer agents for centuries. Salmonella are particularly of interest due to their facultative anaerobic nature, facilitating colonization of differentially oxygenated tumor regions. Additionally, Salmonella can be manipulated with relative ease, resulting in the ability to attenuate the pathogen or engineer vectors for drug delivery. It was recently discovered that the anti-cancer Salmonella enterica serovar Typhimurium strain VNP20009 is lacking in chemotactic ability, due to a non-synonymous single nucleotide polymorphism in cheY. Replacing the mutated copy of cheY with the wild-type sequence restored chemotaxis to 70% of the parental strain. We aimed to investigate further if chemotaxis of VNP20009 can be optimized. By restoring the gene msbB in VNP20009 cheY+, which confers attenuation by lipid A modification, we observed a 9% increase in swimming speed, 13% increase in swim plate performance, 19% increase in microfluidic device partitioning towards the attractant at the optimum concentration gradient, and mitigation of a non-motile cell subpopulation. We conclude that chemotaxis can be enhanced further but at the cost of changing one defining characteristic of VNP20009. A less compromised strain might be needed to employ for investigating bacterial chemotaxis in tumor interactions.
- Structural analysis of BRCA1 reveals modification hotspotLiang, Yanping; Dearnaley, William J.; Varano, A. Cameron; Winton, Carly E.; Gilmore, Brian L.; Alden, Nick A.; Sheng, Zhi; Kelly, Deborah F. (American Association for the Advancement of Science, 2017-09-01)
- TNF alpha Modulates Cardiac Conduction by Altering Electrical Coupling between MyocytesGeorge, Sharon A.; Calhoun, Patrick J.; Gourdie, Robert G.; Smyth, James W.; Poelzing, Steven (Frontiers, 2017-05-23)Background: Tumor Necrosis Factor alpha (TNF alpha) upregulation during acute inflammatory response has been associated with numerous cardiac effects including modulating Connexin43 and vascular permeability. This may in turn alter cardiac gap junctional (GJ) coupling and extracellular volume (ephaptic coupling) respectively. We hypothesized that acute exposure to pathophysiological TNF alpha levels can modulate conduction velocity (CV) in the heart by altering electrical coupling: GJ and ephaptic. Methods and Results: Hearts were optically mapped to determine CV from control, TNF alpha and TNF alpha + high calcium(2.5 vs. 1.25 mM) treated guinea pig hearts over 90 mins. Transmission electron microscopy was performed to measure changes in intercellular separation in the gap junction-adjacent extracellular nanodomain-perinexus (W-P). Cx43 expression and phosphorylation were determined by Western blotting and Cx43 distribution by confocal immunofluorescence. At 90 mins, longitudinal and transverse CV (CVL and CVT, respectively) increased with control Tyrode perfusion but TNF alpha slowed CVT alone relative to control and anisotropy of conduction increased, but not significantly. TNF alpha increased W-P relative to control at 90 mins, without significantly changing GJ coupling. Increasing extracellular calcium after 30 mins of just TNF alpha exposure increased CVT within 15 mins. TNF alpha + high calcium also restored CVT at 90 mins and reduced W-P to control values. Interestingly, TNF alpha + high calcium also improved GJ coupling at 90 mins, which along with reduced W-P may have contributed to increasing CV. Conclusions: Elevating extracellular calcium during acute TNF alpha exposure reduces perinexal expansion, increases ephaptic, and GJ coupling, improves CV and may be a novel method for preventing inflammation induced CV slowing.