An automated, high throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma

Ware, Sarah A; Desai, Nikita; Lopez, Mabel; Leach, Daniel; Zhang, Yingze; Giordano, Luca; Nouraie, Mehdi; Picard, Martin; Kaufman, Brett A.

doi:10.1101/2020.07.16.206987

Cited by 7 publications

(11 citation statements)

References 54 publications

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“…Quantitative polymerase chain reaction (qPCR): cf-mtDNA and cf-nDNA levels were measured simultaneously by qPCR. Taqman-based duplex qPCR reactions targeted mitochondrial-encoded ND1 and nuclear-encoded B2M sequences as described previously 112, 113 . Each gene assay contained two primers and a fluorescent probe and were assembled as a 20X working solution according to the manufacturer’s recommendations (Integrated DNA Technologies).…”

Section: Methodsmentioning

confidence: 99%

OxPhos Dysfunction Causes Hypermetabolism and Reduces Lifespan in Cells and in Patients with Mitochondrial Diseases

Sturm

Karan

Monzel

et al. 2021

Preprint

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Patients with primary mitochondrial diseases present with fatigue and multi-system disease, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. Integrating data from 17 cohorts of patients with mitochondrial diseases (n=690), we find that clinical mitochondrial disorders increase resting energy expenditure, a state termed hypermetabolism. In a longitudinal cellular model of primary patient-derived fibroblasts from multiple donors, we show that genetic and pharmacological disruptions of oxidative phosphorylation (OxPhos) similarly trigger increased energy consumption in a cell-autonomous manner, despite near-normal OxPhos coupling efficiency. Hypermetabolism was associated with mtDNA instability, activation of the integrated stress response, increased extracellular secretion of age-related cytokines and metabokines including GDF15, as well as an accelerated rate of telomere erosion and epigenetic aging, and a reduced Hayflick limit. Finally, we generate a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations to OxPhos dysfunction. Hypermetabolism, or the increased energetic cost of living in mitochondrial diseases, has important biological and clinical implications.

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Section: Methodsmentioning

confidence: 99%

OxPhos Dysfunction Causes Hypermetabolism and Reduces Lifespan in Cells and in Patients with Mitochondrial Diseases

Sturm

Karan

Monzel

et al. 2021

Preprint

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“…To ensure the absence of cell and cell debris, each saliva sample was further centrifuged at 2,000 x g for 10 minutes at 4°C prior to processing. To avoid bias in mtDNA content that may occur during DNA isolation using column-based methods (Ware et al, 2020), DNA was extracted using an isolation-free lysis method, adapted from (Picard et al, 2012). 20 ul of saliva was added to 180ul of lysis buffer (500mM Tris HCL, 1% Tween 20, dH 2 0, and 20ug/ml proteinase K), for a dilution of 1:10.…”

Section: Methodsmentioning

confidence: 99%

Dynamic behavior of cell-free mitochondrial DNA in human saliva

Trumpff

Rausser

Haahr

et al. 2021

Preprint

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Mitochondria release their genome as cell-free mitochondrial DNA (cf-mtDNA) in multiple biofluids including in the blood, where it predicts mortality and is a marker of mental and physical stress. Here we report cf-mtDNA in human saliva, an accessible biofluid used to study dynamic neuroendocrine changes. To map the natural dynamics of salivary cf-mtDNA over time, we examine cf-mtDNA and steroid hormones in a small cohort of healthy adults, and perform an intensive repeated-measures analysis of two healthy men studied at 4 daily timepoints over 53-60 consecutive days (n=412-420 observations). Salivary cf-mtDNA exhibits a robust awakening response reaching up to two orders of magnitude 30-45 minutes after awakening, varies from day-to-day, and moderately correlates with the cortisol awakening response. Moreover, we find no evidence that salivary cf-mtDNA has pro-inflammatory effects. The dynamic behavior of salivary cf-mtDNA opens the door to non-invasive studies examining the relevance of mtDNA signaling on human health.

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“…Cell-free mitochondrial (cf-mtDNA) and nuclear DNA (cf-nDNA) levels were measured simultaneously by qPCR on sampled media collected across cellular lifespan. Taqman-based duplex qPCR reactions targeted mitochondrial-encoded ND1 and nuclear-encoded B2M sequences as described previously (Belmonte et al, 2016; Trumpff et al, 2019; Ware et al, 2020). Each gene assay contained two primers and a fluorescent probe and were assembled as a 20X working solution according to the manufacturer’s recommendations (Integrated DNA Technologies).…”

Section: Methodsmentioning

confidence: 99%

Accelerating the clock: Interconnected speedup of energetic and molecular dynamics during aging in cultured human cells

Sturm

Bobba-Alves

Tumasian

et al. 2022

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The allometric theory of metabolism predicts that the rate of biological aging is proportional to an organism’s size and metabolic rate (MR). Here we test this hypothesis in humans by generating longitudinal, multi-modal signatures of aging in primary human fibroblasts. Relative to metabolic rates in the human body, isolated cells exhibit markedly elevated MR and operate closer to their maximal energy production capacity. Accordingly, per-cell division, isolated cells display accelerated telomere shortening and increased rate of DNA methylation aging. Moreover, despite a marked reduction in division rate towards the end of life, mass-specific MR increases exponentially, reflecting hypermetabolism. We develop a theoretical-mathematical model that accounts for a partitioning of energetic costs related to both growth or maintenance, quantifying the potential origins of hypermetabolism in vitro, and with advancing age. Moreover, we define genome-wide molecular rescaling factors that confirm and quantify the systematic acceleration of molecular aging kinetics in cultured fibroblasts, and use this approach to show how metabolic and pharmacological manipulations that increase or decrease MR predictably accelerate or decelerate the rates of biological aging. The interconnected speedup of energetic and molecular dynamics across the lifespan of human cells has important theoretical and clinical implications for aging biology.

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An automated, high throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma

Cited by 7 publications

References 54 publications

OxPhos Dysfunction Causes Hypermetabolism and Reduces Lifespan in Cells and in Patients with Mitochondrial Diseases

OxPhos Dysfunction Causes Hypermetabolism and Reduces Lifespan in Cells and in Patients with Mitochondrial Diseases

Dynamic behavior of cell-free mitochondrial DNA in human saliva

Accelerating the clock: Interconnected speedup of energetic and molecular dynamics during aging in cultured human cells

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