In vivo detection and replication studies of α-anomeric lesions of 2'-deoxyribonucleosides.

DNA damage, arising from endogenous metabolism or exposure to environmental agents, may perturb the transmission of genetic information by blocking DNA replication and/or inducing mutations, which contribute to the development of cancer and likely other human diseases. Hydroxyl radical attack on the C1', C3' and C4' of 2-deoxyribose can give rise to epimeric 2-deoxyribose lesions, for which the in vivo occurrence and biological consequences remain largely unexplored. Through independent chemical syntheses of all three epimeric lesions of 2'-deoxyguanosine (dG) and liquid chromatography-tandem mass spectrometry analysis, we demonstrated unambiguously the presence of substantial levels of the α-anomer of dG (α-dG) in calf thymus DNA and in DNA isolated from mouse pancreatic tissues. We further assessed quantitatively the impact of all four α-dN lesions on DNA replication in Escherichia coli by employing a shuttle-vector method. We found that, without SOS induction, all α-dN lesions except α-dA strongly blocked DNA replication and, while replication across α-dA was error-free, replicative bypass of α-dC and α-dG yielded mainly C→A and G→A mutations. In addition, SOS induction could lead to markedly elevated bypass efficiencies for the four α-dN lesions, abolished the G→A mutation for α-dG, pronouncedly reduced the C→A mutation for α-dC and triggered T→A mutation for α-dT. The preferential misincorporation of dTMP opposite the α-dNs could be attributed to the unique base-pairing properties of the nucleobases elicited by the inversion of the configuration of the N-glycosidic linkage. Our results also revealed that Pol V played a major role in bypassing α-dC, α-dG and α-dT in vivo. The abundance of α-dG in mammalian tissue and the impact of the α-dNs on DNA replication demonstrate for the first time the biological significance of this family of DNA lesions.

Pediatric Sleep Survey Instrument--a screening tool for sleep disordered breathing.

PURPOSE: The aim of this study was to assess the construct validity and clinical application of the Pediatric Sleep Survey Instrument (PSSI) as a tool to screen for sleep disordered breathing (SDB) in children. METHODS: Polysomnography (PSG) outcomes and PSSI subscale scores were compared between a clinical cohort (N = 87, 5-10 years, 62 M/25 F) and a nonsnoring community sample (N = 55, 5-10 years, 28 M/27 F). Group comparisons assessed the ability of the PSSI subscales to discriminate between the clinical and community cohorts. Receiver operating characteristic (ROC) curves assessed construct validity, with the Apnea/Hypopnea Index (AHI) >5 events/h, OSA-18 score >60, and Pediatric Daytime Sleepiness Scale (PDSS) above the 70th percentile as the target references. RESULTS: The clinical group had more respiratory events, respiratory-related arousals, fragmented sleep, and lower oxygen saturation nadir than the community group (p < 0.001 for all). PSSI subscale scores of Morning Tiredness, Night Arousals, SDB, and Restless Sleep were higher (p < 0.001 for all) in the clinical cohort, confirming the tool's ability to identify clinically relevant sleep problems. ROC curves confirmed the diagnostic accuracy of the SDB subscale against an AHI > 5 events/h (area under the curve (AUC) = 0.7), an OSA-18 score >60 (AUC = 0.7), and a PDSS score in the 70th percentile (AUC = 0.8). The Morning Tiredness subscale accurately predicted a PDSS score in the 70th percentile (AUC = 0.8). A cutoff score of 5 on the SDB subscale showed a sensitivity of 0.94 and a specificity of 0.76, correctly identifying 77 and 100 % of the clinical and community cohorts, respectively. CONCLUSION: The PSSI Sleep Disordered Breathing subscale is a valid tool for screening SDB and daytime sleepiness in children aged 5-10 years.

The Achilles' heel of senescent cells: from transcriptome to senolytic drugs.

The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1(-/Δ) mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1(-/∆) mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.