Studies from human cells, rats, and zebrafish have documented that hyperglycemia (HG) induces the demethylation of specific cytosines throughout the genome. We previously documented that a subset of these changes become permanent and may provide, in part, a mechanism for the persistence of complications referred to as the metabolic memory phenomenon. In this report, we present studies aimed at elucidating the molecular machinery that is responsible for the HG-induced DNA demethylation observed. To this end, RNA expression and enzymatic activity assays indicate that the ten-eleven translocation (Tet) family of enzymes are activated by HG. Furthermore, through the detection of intermediates generated via conversion of 5-methyl-cytosine back to the unmethylated form, the data were consistent with the use of the Tet-dependent iterative oxidation pathway. In addition, evidence is provided that the activity of the poly(ADP-ribose) polymerase (Parp) enzyme is required for activation of Tet activity because the use of a Parp inhibitor prevented demethylation of specific loci and the accumulation of Tet-induced intermediates. Remarkably, this inhibition was accompanied by a complete restoration of the tissue regeneration deficit that is also induced by HG. The ultimate goal of this work is to provide potential new avenues for therapeutic discovery.

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Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al. 17 that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required. Video LinkThe video component of this article can be found at

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Introduction: A common misconception is that increased work of breathing (WOB) in hospitalized patients can be ruled-out when arterial O 2 tension (or saturation) is adequate and arterial PCO 2 is not elevated. Unrecognized WOB increase leads to respiratory muscle fatigue, cessation of respiratory function, and cardiac arrest. We previously developed a WOB Scale for bedside use adding points (maximum 7) based on respiratory rate (1 to 20 bpm = 1 point; 21 to 25 bpm = 2 points; 26 to 30 bpm = 3 points; and > 30 bpm = 4 points), nasal flaring (No = 0 points; Yes = 1 point), activation of the sternocleidomastoid muscle (No = 0 points; Yes = 1 point), and activation of abdominal muscles (No = 0 points; Yes = 1 point). A WOB Scale > 3 points typical identifies a patient in need of intervention to reduce or support WOB increase. However, widespread application of our WOB Scale - especially outside intensive care environments - would likely benefit from a simplified approach. Thus, we investigated developing a “reflex” approach whereby measurement of all four components would be contingent on the respiratory rate level. Methods: We analyzed 110 WOB Scale measurements in a mixed population of ICU and General Ward patients and assessed WOB Scale levels hypothesizing that a respiratory rate of 1 point (1 to 20 bpm) would be predictive of a low WOB Scale (i.e., not exceeding 3 points); thus, obviating the need to perform a complete WOB Scale evaluation. Results: The WOB Scale distribution showed that most patients had normal WOB with only 14 patients (12.7%) having a WOB Scale > 3 points (i.e., 1 point = 60; 2 points = 24; 3 points = 12; 4 points = 6; 5 points = 7; 6 points = 1; 7 points = 0). A respiratory rate level of 1 point (i.e., 1 to 20 bpm), occurred in 68 patients and only 3 (1.5%) had a WOB Scale > 3 points. Yet, when the respiratory rate level was 2 points (i.e., 21 to 25 bpm), which occurred in 28 patients, 3 (10.7%) had a WOB Scale > 3 points. Respiratory rate levels of 3 or 4 points (> 26 bpm) were associated with activation of at least one accessory respiratory muscle examined in 64% of the patients. Conclusions: A respiratory rate of 20 bpm or less predicted low WOB in most patients supporting a Reflex WOB Scale whereby respiratory accessory muscle activation is assessed only when the respiratory rate exceeds 20 bpm.