DNA flow cytometry for detection of genomic instability as a cancer precursor in the gastrointestinal tract.

One of the earliest applications of flow cytometry was the measurement of DNA content in cells. This method is based on the ability to stain DNA in a stoichiometric manner (i.e.

Metabolite accumulation from oral NMN supplementation drives aging-specific kidney inflammation.

The mitochondrial-rich renal tubule cells are key regulators of blood homeostasis via excretion and reabsorption of metabolic waste. With age, tubules are subject to increasing mitochondrial dysfunction and declining nicotinamide adenine dinucleotide (NAD+) levels, both hampering ATP production efficiency. We tested two mitochondrial interventions in young (6-mo) and aged (26-mo) adult male mice: elamipretide (ELAM), a tetrapeptide in clinical trials that improves mitochondrial structure and function, and nicotinamide mononucleotide (NMN), an NAD+ intermediate and commercially available oral supplement.

Mitochondrial Targeted Interventions for Aging.

Changes in mitochondrial function play a critical role in the basic biology of aging and age-related disease. Mitochondria are typically thought of in the context of ATP production and oxidant production. However, it is clear that the mitochondria sit at a nexus of cell signaling where they affect metabolite, redox, and energy status, which influence many factors that contribute to the biology of aging, including stress responses, proteostasis, epigenetics, and inflammation.

The mitochondrially targeted peptide elamipretide (SS-31) improves ADP sensitivity in aged mitochondria by increasing uptake through the adenine nucleotide translocator (ANT).

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function.

Late-life Rapamycin Treatment Enhances Cardiomyocyte Relaxation Kinetics and Reduces Myocardial Stiffness.

Diastolic dysfunction is a key feature of the aging heart. We have shown that late-life treatment with mTOR inhibitor, rapamycin, reverses age-related diastolic dysfunction in mice but the molecular mechanisms of the reversal remain unclear. To dissect the mechanisms by which rapamycin improves diastolic function in old mice, we examined the effects of rapamycin treatment at the levels of single cardiomyocyte, myofibril and multicellular cardiac muscle.

DNA content abnormality frequently develops in the right/proximal colon in patients with primary sclerosing cholangitis and inflammatory bowel disease and is highly predictive of subsequent detection of dysplasia.

Aims: Patients with primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD, termed PSC-IBD) have a higher risk of harbouring nonconventional and/or invisible dysplasias, especially in the right/proximal colon, than those with IBD alone. We postulated that DNA content abnormality may be frequently detected in the right/proximal colon in PSC-IBD patients, even in the absence of dysplasia, and that this may predispose to progression to nonconventional and/or invisible dysplasias that are often associated with increased rates of aneuploidy and advanced neoplasia.

Methods And Results: DNA flow cytometry was performed on 96 morphologically benign colon biopsies taken throughout the colon from 25 PSC-IBD patients during the surveillance colonoscopy that preceded the next procedure that detected dysplasia.

Mitochondrial proton leak in cardiac aging.

Age-associated diseases are becoming progressively more prevalent, reflecting the increased lifespan of the world's population. However, the fundamental mechanisms of physiologic aging are poorly understood, and in particular, the molecular pathways that mediate cardiac aging and its associated dysfunction are unclear. Here, we focus on certain ion flux abnormalities of the mitochondria that may contribute to cardiac aging and age-related heart failure.

Intermittent treatment with elamipretide preserves exercise tolerance in aged female mice.

The pathology of aging impacts multiple organ systems, including the kidney and skeletal and cardiac muscles. Long-term treatment with the mitochondrial-targeted peptide elamipretide has previously been shown to improve in vivo mitochondrial function in aged mice, which is associated with increased fatigue resistance and treadmill performance, improved cardiovascular diastolic function, and glomerular architecture of the kidney. However, elamipretide is a short tetrameric peptide that is not orally bioavailable, limiting its routes of administration.

Fundic Gland Polyps Lack DNA Content Abnormality Characteristic of Other Adenomatous Precursor Lesions in the Gastrointestinal Tract.

Fundic gland polyps (FGPs) develop sporadically (frequently after proton pump inhibitor therapy) or in the setting of a hereditary polyposis syndrome, such as familial adenomatous polyposis (FAP). FAP-related FGPs often demonstrate low-grade dysplasia (LGD) and are frequently associated with APC mutations, even in the absence of dysplasia. Sporadic FGPs with dysplasia are molecularly similar to FAP-related FGPs and demonstrate frequent mutations in APC gene.

The multi-tissue landscape of somatic mtDNA mutations indicates tissue-specific accumulation and removal in aging.

Accumulation of somatic mutations in the mitochondrial genome (mtDNA) has long been proposed as a possible mechanism of mitochondrial and tissue dysfunction that occurs during aging. A thorough characterization of age-associated mtDNA somatic mutations has been hampered by the limited ability to detect low-frequency mutations. Here, we used Duplex Sequencing on eight tissues of an aged mouse cohort to detect >89,000 independent somatic mtDNA mutations and show significant tissue-specific increases during aging across all tissues examined which did not correlate with mitochondrial content and tissue function.

Elamipretide Improves ADP Sensitivity in Aged Mitochondria by Increasing Uptake through the Adenine Nucleotide Translocator (ANT).

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function.

Aging Increases Susceptibility to Develop Cardiac Hypertrophy following High Sugar Consumption.

Aging and poor diet are independent risk factors for heart disease, but the impact of high-sucrose (HS) consumption in the aging heart is understudied. Aging leads to impairments in mitochondrial function that result in muscle dysfunction (e.g.

Elamipretide effects on the skeletal muscle phosphoproteome in aged female mice.

The age-related decline in skeletal muscle mass and function is known as sarcopenia. Sarcopenia progresses based on complex processes involving protein dynamics, cell signaling, oxidative stress, and repair. We have previously found that 8-week treatment with elamipretide improves skeletal muscle function, reverses redox stress, and restores protein S-glutathionylation changes in aged female mice.

Lifespan benefits for the combination of rapamycin plus acarbose and for captopril in genetically heterogeneous mice.

Mice bred in 2017 and entered into the C2017 cohort were tested for possible lifespan benefits of (R/S)-1,3-butanediol (BD), captopril (Capt), leucine (Leu), the Nrf2-activating botanical mixture PB125, sulindac, syringaresinol, or the combination of rapamycin and acarbose started at 9 or 16 months of age (RaAc9, RaAc16). In male mice, the combination of Rapa and Aca started at 9 months and led to a longer lifespan than in either of the two prior cohorts of mice treated with Rapa only, suggesting that this drug combination was more potent than either of its components used alone. In females, lifespan in mice receiving both drugs was neither higher nor lower than that seen previously in Rapa only, perhaps reflecting the limited survival benefits seen in prior cohorts of females receiving Aca alone.

TurnoveR: A Skyline External Tool for Analysis of Protein Turnover in Metabolic Labeling Studies.

In spite of its central role in biology and disease, protein turnover is a largely understudied aspect of most proteomic studies due to the complexity of computational workflows that analyze in vivo turnover rates. To address this need, we developed a new computational tool, TurnoveR, to accurately calculate protein turnover rates from mass spectrometric analysis of metabolic labeling experiments in Skyline, a free and open-source proteomics software platform. TurnoveR is a straightforward graphical interface that enables seamless integration of protein turnover analysis into a traditional proteomics workflow in Skyline, allowing users to take advantage of the advanced and flexible data visualization and curation features built into the software.

Protocol for Isolation of Cardiomyocyte from Adult Mouse and Rat.

The isolation of intact single adult cardiomyocytes from model animals, mouse and rat, is an essential tool for cardiac molecular and cellular research. While several methods are reported for adult mouse cardiomyocyte isolation, the viability and yield of the isolated cells have been variable. Here, we describe step-by-step protocols for high viability and yield cardiomyocyte isolation from mouse and rat, based on the use of a stable pressure Langendorff perfusion system.

Age-related disruption of the proteome and acetylome in mouse hearts is associated with loss of function and attenuated by elamipretide (SS-31) and nicotinamide mononucleotide (NMN) treatment.

We analyzed the effects of aging on protein abundance and acetylation, as well as the ability of the mitochondrial-targeted drugs elamipretide (SS-31) and nicotinamide mononucleotide (NMN) to reverse aging-associated changes in mouse hearts. Both drugs had a modest effect on restoring the abundance and acetylation of proteins that are altered with age, while also inducing additional changes. Age-related increases in protein acetylation were predominantly in mitochondrial pathways such as mitochondrial dysfunction, oxidative phosphorylation, and TCA cycle signaling.

A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA.

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing.

Role of DNA Flow Cytometry in the Diagnosis of Malignancy in Bile Duct Biopsies Using Paraffin-Embedded Tissue.

Objectives: Histopathologic evaluation of bile biopsies for biliary strictures is frequently challenging and is affected by interobserver disagreement. Reliable ancillary tests that can help differentiate benign from malignant are not available. This study aimed to evaluate whether DNA content abnormalities detected by flow cytometry on formalin-fixed, paraffin-embedded (FFPE) tissue can help differentiate benign/reactive, dysplastic from malignant cell populations in bile duct biopsies.

Elamipretide (SS-31) treatment attenuates age-associated post-translational modifications of heart proteins.

It has been demonstrated that elamipretide (SS-31) rescues age-related functional deficits in the heart but the full set of mechanisms behind this have yet to be determined. We investigated the hypothesis that elamipretide influences post-translational modifications to heart proteins. The S-glutathionylation and phosphorylation proteomes of mouse hearts were analyzed using shotgun proteomics to assess the effects of aging on these post-translational modifications and the ability of the mitochondria-targeted drug elamipretide to reverse age-related changes.

University of Washington Nathan Shock Center: innovation to advance aging research.

The University of Washington Nathan Shock Center of Excellence in the Basic Biology of Aging provides leadership and resources to support the geroscience community locally, nationally, and internationally. Services are provided through our Resource Cores and funds are available annually to support pilot projects by external investigators. Aging-related studies involving proteomics, metabolomics, invertebrate model organisms, and bioinformatics/artificial intelligence are supported by our Cores.

Nonampullary Duodenal Adenomas in Familial Adenomatous Polyposis and Sporadic Patients Lack the DNA Content Abnormality That Is Characteristic of the Adenoma-Carcinoma Sequence Involved in the Development of Other Gastrointestinal Malignancies.

Nonampullary duodenal adenomas (NADAs) develop sporadically or in the setting of a hereditary syndrome such as familial adenomatous polyposis (FAP). Although they are thought to progress into duodenal adenocarcinomas via an adenoma to carcinoma sequence similar to colorectal cancer, limited data suggested that they may be biologically dissimilar to colorectal adenomas. The clinicopathologic features of 71 patients diagnosed with NADAs (37 FAP and 34 sporadic) were analyzed.