The BARB core provides a series of molecular assays that will measure selected markers of oxidant stress, mitochondrial bioanalytical services (Figure 3) and quantification of atherosclerotic lesions (Figure 4). The.selected methods are carefully geared to the needs of the DRTC investigators, which is handled through a two-way process involving both the investigator and Core.
The following sections briefly describe these methodologies:
E.1 Atherosclerotic lesion quantification.
A basic service for aortic atherosclerotic lesion formation is provided by the BARB core, as shown in Figure 4. Both the methods for cross-sectional and en face analyses have been previously published. (8,9,12,13). This enables the association between the core's molecular measurements and resulting vascular pathology in diabetes and cardiometabolic disease.
E.2 Core Technologies and Functions.
Space limitations preclude a detailed methodological description of all the services that will be offered by the core; the director and co-directors cumulatively have expertise in all the offered core services of oxidant stress markers, mitochondrial bioanalytical services, and atherosclerotic lesion quantification, as documented in the published references.
Interaction with Other Cores.
The BARB Core interacts with other DRTC cores as well as non-DRTC cores at UAB. The interactions with other DRTC cores are critical to providing seamless services when assistance is required from multiple cores around a single project. Specifically, the BARB Core interacts with the Animal Physiology DRTC Core for tissue collection; the Human Biology core (e.g., plasma biomarker assays correlating with mtDNA damage and haplotype analyses), and Metrics (epidemiology of disease risk factors, mtDNA damage and haplotype assessment). Important interactions with cores outside the DRTC include the UAB Comprehensive Cancer Center (CCC) Proteomics/Mass Spectrometry Facility (Dr. Stephen Barnes). The BARB Core provides a unique portfolio of services that does not duplicate any other UAB core facility.
Mitochondrial Bioanalytical Services:
mtDNA Damage Analysis
Quantitative PCR Assay for DNA Damage
High Resolution Respirometry
Measure oxygen kinetics in tissues, cells, and isolated mitochondria with simultaneous multi-sensor applications including pH (as an indicator of glycolysis)
Mitochondrial Oxidative Phosphorylation Complex and other Mitochondrial Proteins' Activity Assays
As a complementary approach to measuring altered total oxygen consumption using the oxygraph-2k respirometer, in either pathological or therapeutic samples compared to controls, individual OxPhos complexes can be measured using a spectrophotometer to ascertain possible specific mechanisms and altered individual complex activities contributing to those differences.
Mitochondrial Complex I (NADH-Ubiquinone oxidoreductase) Assay
The principle of this assay is based on the consumption of NADH
Mitochondrial Complex IV (Cytochrome c oxidase) Assay
The principle of this assay is based on the oxidation of reduced cytochrome c that is followed at a spectrophotometric absorbance of 550nm.
Mitochondrial Citrate Synthase Assay
Bioenergetics' Analysis of Tissues, Mitochondria, and Cells
Using a Seahorse XF instrument, we will be able to measure energy utilization in living cells.
Mitochondria Isolation and Preparation
Aortic Lesion Assessment:
Aorta Lesion Assessment Quantitative assessment of atherosclerotic plaque, Oil Red O stain ‘En face analysis': En face assessment of lesion size will be determined after staining the aorta with oil red O and photographing it
Oxidative stress (Redox) Measures:
Elevated reactive oxygen species (ROS) levels can lead to damage of DNA/RNA, proteins and lipids which may lead to apoptosis. Cells have developed several mechanisms to counteract elevated ROS levels such as a thiol reducing buffer composed of cellular thiol levels (glutathione and thioredoxion) for the maintenance of the reduction-oxidation (Redox) state of the cell, and enzymes to remove ROS (catalase, superoxide dismutase and glutathione peroxidase).
Glutathione and Ratios of Reduced (GSH) to Oxidized (GSSG)
Using a "Tietze" recycling spectrophotometric assay, we will be able to measure oxidized and reduced levels of glutathione.
Superoxide Dismutase (SOD2; mitochondrial MnSOD) Specific Activity (expression and activity
SOD2 catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide within the mitochondria.
Oxidation of Thiols (reduced thiol levels measured)