Body-wide changes in bioenergetics, i.e., energy metabolism, occur in normal aging and disturbed bioenergetics may be an important contributing mechanism underlying late-onset Alzheimer's disease ...(LOAD). We investigated the bioenergetic profiles of fibroblasts from LOAD patients and healthy controls, as a function of age and disease. LOAD cells exhibited an impaired mitochondrial metabolic potential and an abnormal redox potential, associated with reduced nicotinamide adenine dinucleotide metabolism and altered citric acid cycle activity, but not with disease-specific changes in mitochondrial mass, production of reactive oxygen species, transmembrane instability, or DNA deletions. LOAD fibroblasts demonstrated a shift in energy production to glycolysis, despite an inability to increase glucose uptake in response to IGF-1. The increase of glycolysis and the abnormal mitochondrial metabolic potential in LOAD appeared to be inherent, as they were disease- and not age-specific. Our findings support the hypothesis that impairment in multiple interacting components of bioenergetic metabolism may be a key mechanism contributing to the risk and pathophysiology of LOAD.
Extraction of genomic DNA (gDNA) from fresh whole blood is the first step in multiple translational research and molecular diagnostics applications, such as next-generation sequencing (NGS), ...multiplex PCR, qPCR, and droplet digital PCR (ddPCR). For example, DNA from whole blood is used as a matched-control for solid tumor somatic mutation profiling and for the detection of clinically-relevant variants in hematological malignancies, such as leukemia and myelodysplastic syndrome. An increasing number of clinical studies demonstrate the value of detecting disease-specific biomarkers from blood for early detection, diagnosis, monitoring treatment efficacy, and cohort study recruitment. Scaling down fresh blood volumes while scaling up processing capabilities is desirable to maximize laboratory throughput. At present, most DNA extraction methods require high volumes of blood and are challenging to automate because centrifugation or vacuum equipment are necessary. Additionally, conventional column and magnetic-based workflows need larger volumes for wash and elution steps, which require the use of deep well plates and other specialized consumables. Larger volumes are also needed to avoid excessive viscosity of the lysate, which can interfere with magnetic bead separation. To circumvent sample and process-related challenges, Covaris has adapted the oneTUBE-10 Plate for streamlining high-throughput nucleic acid extractions from whole blood using Adaptive Focused Acoustics (AFA) technology. Here, we performed 12 DNA extractions from healthy donors to evaluate recoveries, sample quality, and purity. Our results show recoveries in the range between 376 to 809 ng with an average fragment size of >1 kb. We also show that the extracted and purified DNA is devoid of any detectable PCR inhibiting contaminants. Taken together, this less than 90-minute AFA-enabled workflow significantly improves blood cell lysis and reduces hands-on time. The workflow can be performed on a liquid handler using the integrated Covaris R230 Focused-ultrasonicator, or off-deck using the LE220-plus Focused-ultrasonicator.
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