The methylome and cell-free DNA: current applications in medicine and pediatric disease.

DNA methylation is an epigenetic mechanism that contributes to cell regulation and development, and different methylation patterns allow for the identification of cell and tissue type. Cell-free DNA (cfDNA) is composed of small circulating fragments of DNA found in plasma and urine. Total cfDNA levels correlate with the presence of inflammation and tissue injury in a variety of disease states. Unfortunately, the utility of cfDNA is limited by its lack of tissue or cell-type specificity. However, methylome analysis of cfDNA allows the identification of the tissue or cell type from which cfDNA originated. Thus, methylation patterns in cfDNA from tissues isolated from direct study may provide windows into health and disease states, thereby serving as a "liquid biopsy". This review will discuss methylation and its role in establishing cellular identity, cfDNA as a biomarker and its pathophysiologic role in the inflammatory process, and the ways cfDNA and methylomics can be jointly applied in medicine. IMPACT: Cell-free DNA (cfDNA) is increasingly being used as a noninvasive diagnostic and disease-monitoring tool in pediatric medicine. However, the lack of specificity of cfDNA limits its utility. Identification of cell type-specific methylation signatures can help overcome the limited specificity of cfDNA. As knowledge of the cfDNA methylome improves, cfDNA will be more broadly applied in medicine, such that clinicians will need to understand the methods and applications of its use.

Induction of calcitonin gene-related peptide expression in rats by cortical spreading depression.

OBJECTIVE: The neuropeptide calcitonin gene-related peptide (CGRP) has now been established as a key player in migraine. However, the mechanisms underlying the reported elevation of CGRP in the serum and cerebrospinal fluid of some migraineurs are not known. A candidate mechanism is cortical spreading depression (CSD), which is associated with migraine with aura and traumatic brain injury. The aim of this study was to investigate whether CGRP gene expression may be induced by experimental CSD in the rat cerebral cortex. METHODS: CSD was induced by topical application of KCl and monitored using electrophysiological methods. Quantitative PCR and ELISA were used to measure CGRP mRNA and peptide levels in discrete ipsilateral and contralateral cortical regions of the rat brain 24 hours following CSD events and compared with sham treatments. RESULTS: The data show that multiple, but not single, CSD events significantly increase CGRP mRNA levels at 24 hours post-CSD in the ipsilateral rat cerebral cortex. Increased CGRP was observed in the ipsilateral frontal, motor, somatosensory, and visual cortices, but not the cingulate cortex, or contralateral cortices. CSD also induced CGRP peptide expression in the ipsilateral, but not contralateral, cortex. CONCLUSIONS: Repeated CSD provides a mechanism for prolonged elevation of CGRP in the cerebral cortex, which may contribute to migraine and post-traumatic headache.

End-of-life cell cycle arrest contributes to stochasticity of yeast replicative aging.

There is growing evidence that stochastic events play an important role in determining individual longevity. Studies in model organisms have demonstrated that genetically identical populations maintained under apparently equivalent environmental conditions display individual variation in life span that can be modeled by the Gompertz-Makeham law of mortality. Here, we report that within genetically identical haploid and diploid wild-type populations, shorter-lived cells tend to arrest in a budded state, while cells that arrest in an unbudded state are significantly longer-lived. This relationship is particularly notable in diploid BY4743 cells, where mother cells that arrest in a budded state have a shorter mean life span (25.6 vs. 35.6) and larger coefficient of variance with respect to individual life span (0.42 vs. 0.32) than cells that arrest in an unbudded state. Mutations that cause genomic instability tend to shorten life span and increase the proportion of the population that arrest in a budded state. These observations suggest that randomly occurring damage may contribute to stochasticity during replicative aging by causing a subset of the population to terminally arrest prematurely in the S or G2 phase of the cell cycle.

Renal Replacement Therapy in Neonates.

Acute kidney injury (AKI) is a highly prevalent disease entity in the NICU, affecting nearly one-quarter of critically ill neonates by some reports. Though medical management remains the mainstay in the treatment of AKI, renal replacement therapy (RRT) is indicated when conservative measures are unable to maintain electrolytes, fluid balance, toxins, or waste products within a safe margin. Several modalities of RRT exist for use in neonatal populations, including peritoneal dialysis, hemodialysis, and continuous RRT. It is the aim of this review to introduce each of these RRT modalities, as well as to discuss their technical considerations, benefits, indications, contraindications, and complications.

Bladder Neck Contracture Following Radical Retropubic versus Robotic-Assisted Laparoscopic Prostatectomy.

INTRODUCTION: Radical retropubic prostatectomy (RRP) and robotic-assisted laparoscopic prostatectomy (RALP) are co-standard surgical therapies for localized prostatic adenocarcinoma. These surgical modalities offer similar outcomes; however, lower rate of bladder neck contracture (BNC) is amongst the touted benefits of RALP. The differences between approaches are largely elucidated through multiple-surgeon comparisons, which can be biased by differential experience and practice patterns. We aimed to eliminate inter-surgeon bias through this single-surgeon comparison of BNC rates following RRP and RALP. MATERIALS AND METHODS: We retrospectively reviewed all RRPs and RALPs performed by one surgeon over 4 years. We compared clinical characteristics, intraoperative and postoperative outcomes. RESULTS: RRP patients had more advanced cancer and a higher biochemical recurrence rate. No significant differences were noted between groups in rates of anastomotic leakage, BNC, or 12-month postoperative pad-free continence. CONCLUSION: RRP offers similar outcomes to RALP with regard to postoperative urinary extravasation, urinary continence, and BNC.

Dietary restriction and mitochondrial function link replicative and chronological aging in Saccharomyces cerevisiae.

Chronological aging of budding yeast cells results in a reduction in subsequent replicative life span through unknown mechanisms. Here we show that dietary restriction during chronological aging delays the reduction in subsequent replicative life span up to at least 23days of chronological age. We further show that among the viable portion of the control population aged 26days, individual cells with the lowest mitochondrial membrane potential have the longest subsequent replicative lifespan. These observations demonstrate that dietary restriction modulates a common molecular mechanism linking chronological and replicative aging in yeast and indicate a critical role for mitochondrial function in this process.

Molecular mechanisms underlying genotype-dependent responses to dietary restriction.

Dietary restriction (DR) increases lifespan and attenuates age-related phenotypes in many organisms; however, the effect of DR on longevity of individuals in genetically heterogeneous populations is not well characterized. Here, we describe a large-scale effort to define molecular mechanisms that underlie genotype-specific responses to DR. The effect of DR on lifespan was determined for 166 single gene deletion strains in Saccharomyces cerevisiae. Resulting changes in mean lifespan ranged from a reduction of 79% to an increase of 103%. Vacuolar pH homeostasis, superoxide dismutase activity, and mitochondrial proteostasis were found to be strong determinants of the response to DR. Proteomic analysis of cells deficient in prohibitins revealed induction of a mitochondrial unfolded protein response (mtUPR), which has not previously been described in yeast. Mitochondrial proteotoxic stress in prohibitin mutants was suppressed by DR via reduced cytoplasmic mRNA translation. A similar relationship between prohibitins, the mtUPR, and longevity was also observed in Caenorhabditis elegans. These observations define conserved molecular processes that underlie genotype-dependent effects of DR that may be important modulators of DR in higher organisms.

pH neutralization protects against reduction in replicative lifespan following chronological aging in yeast.

Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.