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Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour.

We have examined p53 protein levels in cell lines selected for resistance to the chemotherapeutic drug cis-diamminedichloroplatinum (II), cisplatin. The majority of the independent cisplatin-resistant clones isolated by a single selection with cisplatin from the ovarian tumour cell line A2780 showed increased levels of p53 protein compared to the parental cell line. Elevated p53 protein levels were also observed in cisplatin-resistant ovarian human tumour lines isolated after multiple exposures to cisplatin (A2780/cp70 and OVIP/DDP). Direct PCR sequencing of p53 cDNAs showed that both the A2780/cp70 and the parental A2780 cell lines had a wild-type p53 gene sequence. The OVIP and OVIP/DDP lines both had a heterozygous mutation at codon 126. Cell-cycle analysis after gamma-irradiation or cisplatin treatment showed evidence of a G1/S and G2/M cell-cycle checkpoint in both A2780/cp70 and the sensitive parental cell lines. However, the resistant cell line A2780/cp70 showed less inhibition of DNA synthesis after gamma-irradiation than the sensitive cell line. Transfection of a mutant p53 gene construct (containing a mutation at codon 143, val to ala) into the A2780/cp70 resistant cells conferred a significantly increased sensitivity to cisplatin, suggesting that p53 is a direct determinant of cisplatin resistance in these cells. However, expression of this mutant p53 in the A2780 cells did not affect sensitivity.

BackgroundFluorine-18–sodium fluoride (18F-NaF) uptake is a marker of active vascular calcification associated with high-risk atherosclerotic plaque.ObjectivesIn patients with abdominal aortic aneurysm (AAA), the authors assessed whether 18F-NaF positron emission tomography (PET) and computed tomography (CT) predicts AAA growth and clinical outcomes.MethodsIn prospective case-control (n = 20 per group) and longitudinal cohort (n = 72) studies, patients with AAA (aortic diameter >40 mm) and control subjects (aortic diameter <30 mm) underwent abdominal ultrasound, 18F-NaF PET-CT, CT angiography, and calcium scoring. Clinical endpoints were aneurysm expansion and the composite of AAA repair or rupture.ResultsFluorine-18-NaF uptake was increased in AAA compared with nonaneurysmal regions within the same aorta (p = 0.004) and aortas of control subjects (p = 0.023). Histology and micro-PET-CT demonstrated that 18F-NaF uptake localized to areas of aneurysm disease and active calcification. In 72 patients within the longitudinal cohort study (mean age 73 ± 7 years, 85% men, baseline aneurysm diameter 48.8 ± 7.7 mm), there were 19 aneurysm repairs (26.4%) and 3 ruptures (4.2%) after 510 ± 196 days. Aneurysms in the highest tertile of 18F-NaF uptake expanded 2.5× more rapidly than those in the lowest tertile (3.10 [interquartile range (IQR): 2.34 to 5.92 mm/year] vs. 1.24 [IQR: 0.52 to 2.92 mm/year]; p = 0.008) and were nearly 3× as likely to experience AAA repair or rupture (15.3% vs. 5.6%; log-rank p = 0.043).ConclusionsFluorine-18-NaF PET-CT is a novel and promising approach to the identification of disease activity in patients with AAA and is an additive predictor of aneurysm growth and future clinical events. (Sodium Fluoride Imaging of Abdominal Aortic Aneurysms [SoFIA3]; NCT02229006; Magnetic Resonance Imaging [MRI] for Abdominal Aortic Aneurysms to Predict Rupture or Surgery: The MA3RS Trial; ISRCTN76413758)

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Nitrosated glycine derivatives react with DNA to form O6-carboxymethyl-2'-deoxyguanosine (O6-CMdG) and O6-methyl-2'-deoxyguanosine (O6-MedG) adducts concurrently. O6-CMdG is not repaired by O6-alkylguanine alkyltransferases and might be expected to lead to mutations via a similar mechanism to O6-MedG. Potassium diazoacetate (KDA) is a stable form of nitrosated glycine and its ability to induce mutations in the p53 gene in a functional yeast assay was studied. Treatment of a plasmid containing the human p53 cDNA sequence with KDA afforded readily detectable levels of O6-CMdG and O6-MedG. The treated plasmid was used to transform yeast cells and coloured colonies harbouring a p53 sequence with functional mutations were detected. Recovery of the mutated plasmids followed by DNA sequencing enabled the mutation spectrum of KDA to be characterised. The most common mutations induced by KDA were substitutions with >50% occurring at GC base pairs. In contrast to the methylating agent methylnitrosourea which gives predominantly (>80%) GC-->AT transitions, KDA produced almost equal amounts of transitions (GC-->AT) and transversions (GC-->TA and AT-->TA). This difference is probably due to a different mode of base mispairing for O6-CMdG compared with O6-MedG. The pattern of mutations induced by KDA was very similar to the patterns observed in mutated p53 in human gastrointestinal tract tumours. These results are consistent with the hypothesis that nitrosation of glycine (or glycine derivatives) may contribute to characteristic human p53 mutation profiles. This conclusion is borne out by recent observations that O6-CMdG is present in human DNA both from blood and exfoliated colorectal cells and is consistent with recent epidemiological studies that have concluded that endogenous nitrosation arising from red meat consumption is related to an increased risk of colorectal cancer.

Alkylating treatments predominantly induce G: C = greater than A:T transitions, consistent with the predicted significance of the miscoding potential of the O6-alG lesion. However, the frequency and distribution of these events induced by any one compound may be diagnostic. SN1 agents that act via an alkyldiazonium cation, such as the N-nitroso compounds, preferentially generate G: C = greater than A:T transitions at 5'-RG-3' sites, while the more SN2 alkylsulfates and alkylalkane-sulfonates do not. The precise nature of this site bias and the possibility of strand bias are target dependent. The extent of this site bias and the contribution of other base substitutions are substituent size dependent. A similar 5'-RT-3' effect is seen for A:T = greater than G:C transitions, presumably directed by O4-alT lesions. The 5'-RG-3' effect, at least, likely reflects a deposition specificity arising from some aspect of helix geometry, although it may be further exaggerated by alkylation-specific repair. Excision repair appears to preferentially reduce the occurrence of ethylation-induced G:C = greater than A:T and A:T = greater than G:C transitions at sites flanked by A:T base pairs. This may be due to an enhancement of the helical distortion imposed by damage at such positions. A similar effect is not seen for methylation-induced mutations and in the case of propyl adducts, the influence of excision repair on the ultimate distribution of mutation cannot be as easily defined with respect to neighbouring sequence.

Patients with CVU have a 41% prevalence rate of thrombophilia. This rate is two to 30 times higher than the rate of the general population but is similar to that reported for patients with previous DVT. However, in patients with CVU, thrombophilia does not appear to be related to a history of DVT, a pattern of reflux, or severity of disease. Many patients with CVU may have unsuspected postthrombotic disease.