The Influence of Repair Pathways on the Cytotoxicity and Mutagenicity Induced by the Pyridyloxobutylation Pathway of Tobacco-Specific Nitrosamines

Tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN), are considered to be human carcinogens. Both compounds are metabolized to pyridyloxobutylating intermediates that react with DNA to form adducts such as 7-[4-(3-pyridyl)-4-oxobut-1-yl]-guanine (7-pobG), O2-[4-(3-pyridyl)-4-oxobut-1-yl]-cytosine (O2-pobC), O2-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxythymidine (O2-pobdT), O6-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine (O6-pobdG) and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing adducts. The role of specific DNA adducts in the overall genotoxic activity of the pyridyloxobutylation pathway is not known. One adduct, O6-pobdG, is mutagenic. To characterize the mutagenic and cytotoxic properties of pyridyloxobutyl DNA adducts, the impact of DNA repair pathways on the cytotoxic and mutagenic properties of the model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) was investigated in Chinese hamster ovary (CHO) cell lines proficient or deficient in O6-alkylguanine DNA alkyltransferase (AGT), deficient in both AGT and base excision repair (BER), or deficient in both AGT and nucleotide excision repair (NER). The repair of the four pyridyloxobutyl DNA adducts was determined in the same cell lines via sensitive LC-MS/MS methods. NNKOAc was more cytotoxic in the cell lines lacking AGT, BER and NER repair pathways. It also induced more mutations in the hprt gene in the BER and NER deficient cell lines. However, AGT expression did not influence NNKOAc’s mutagenicity despite efficient repair of O6-pobdG. Analysis of the hprt mutational spectra indicated that NNKOAc primarily caused point mutations at AT base pairs. GC to AT transition mutations were a minor contributor to the overall mutation spectrum, providing a rationale for the observation that AGT does not protect against the overall mutagenic properties of NNKOAc in this model system. The only adduct affected by the absence of effective NER was O2-pobdT. Slower repair of O2-pobdT in NER deficient cells was associated with increased AT to TA transversion mutations, supporting the hypothesis that these mutations are caused by O2-pobdT. Together, these data support a hypothesis that the pyridyloxobutylation pathway generates multiple mutagenic and toxic adducts

Genome-wide association study identifies a variant in HDAC9 associated with large vessel ischemic stroke

Genetic factors have been implicated in stroke risk but few replicated associations have been reported. We conducted a genome-wide association study (GWAS) in ischemic stroke and its subtypes in 3,548 cases and 5,972 controls, all of European ancestry. Replication of potential signals was performed in 5,859 cases and 6,281 controls. We replicated reported associations between variants close to PITX2 and ZFHX3 with cardioembolic stroke, and a 9p21 locus with large vessel stroke. We identified a novel association for a SNP within the histone deacetylase 9(HDAC9) gene on chromosome 7p21.1 which was associated with large vessel stroke including additional replication in a further 735 cases and 28583 controls (rs11984041, combined P = 1.87×10−11, OR=1.42 (95% CI) 1.28-1.57). All four loci exhibit evidence for heterogeneity of effect across the stroke subtypes, with some, and possibly all, affecting risk for only one subtype. This suggests differing genetic architectures for different stroke subtypes

Homozygous microdeletion of exon 5 in ZNF277 in a girl with specific language impairment

Peer reviewedPublisher PD

Measurement of the Lifetime Difference Between B_s Mass Eigenstates

We present measurements of the lifetimes and polarization amplitudes for B_s --> J/psi phi and B_d --> J/psi K*0 decays. Lifetimes of the heavy (H) and light (L) mass eigenstates in the B_s system are separately measured for the first time by determining the relative contributions of amplitudes with definite CP as a function of the decay time. Using 203 +/- 15 B_s decays, we obtain tau_L = (1.05 +{0.16}/-{0.13} +/- 0.02) ps and tau_H = (2.07 +{0.58}/-{0.46} +/- 0.03) ps. Expressed in terms of the difference DeltaGamma_s and average Gamma_s, of the decay rates of the two eigenstates, the results are DeltaGamma_s/Gamma_s = (65 +{25}/-{33} +/- 1)%, and DeltaGamma_s = (0.47 +{0.19}/-{0.24} +/- 0.01) inverse ps.Comment: 8 pages, 3 figures, 2 tables; as published in Physical Review Letters on 16 March 2005; revisions are for length and typesetting only, no changes in results or conclusion

Fluorescent N-arylaminonaphthalene sulfonate probes for amyloid aggregation of α-synuclein.

The deposition of fibrillar structures (amyloids) is characteristic of pathological conditions including Alzheimer's and Parkinson's diseases. The detection of protein deposits and the evaluation of their kinetics of aggregation are generally based on fluorescent probes such as thioflavin T and Congo red. In a search for improved fluorescence tools for studying amyloid formation, we explored the ability of N-arylaminonaphthalene sulfonate (NAS) derivatives to act as noncovalent probes of α-synuclein (AS) fibrillation, a process linked to Parkinson's disease and other neurodegenerative disorders. The compounds bound to fibrillar AS with micromolar K(d)s, and exhibited fluorescence enhancement, hyperchromism, and high anisotropy. We conclude that the probes experience a hydrophobic environment and/or restricted motion in a polar region. Time- and spectrally resolved emission intensity and anisotropy provided further information regarding structural features of the protein and the dynamics of solvent relaxation. The steady-state and time-resolved parameters changed during the course of aggregation. Compared with thioflavin T, NAS derivatives constitute more sensitive and versatile probes for AS aggregation, and in the case of bis-NAS detect oligomeric as well as fibrillar species. They can function in convenient, continuous assays, thereby providing useful tools for studying the mechanisms of amyloid formation and for high-throughput screening of factors inhibiting and/or reversing protein aggregation in neurodegenerative diseases

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array "waves", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance

Hundreds of variants clustered in genomic loci and biological pathways affect human height

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits, but these typically explain small fractions of phenotypic variation, raising questions about the use of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways (P = 0.016) and that underlie skeletal growth defects (P < 0.001). Second, the likely causal gene is often located near the most strongly associated variant: in 13 of 21 loci containing a known skeletal growth gene, that gene was closest to the associated variant. Third, at least 19 loci have multiple independently associated variants, suggesting that allelic heterogeneity is a frequent feature of polygenic traits, that comprehensive explorations of already-discovered loci should discover additional variants and that an appreciable fraction of associated loci may have been identified. Fourth, associated variants are enriched for likely functional effects on genes, being over-represented among variants that alter amino-acid structure of proteins and expression levels of nearby genes. Our data explain approximately 10% of the phenotypic variation in height, and we estimate that unidentified common variants of similar effect sizes would increase this figure to approximately 16% of phenotypic variation (approximately 20% of heritable variation). Although additional approaches are needed to dissect the genetic architecture of polygenic human traits fully, our findings indicate that GWA studies can identify large numbers of loci that implicate biologically relevant genes and pathways.

Cost-Effectiveness of a Telephone-Delivered Intervention for Physical Activity and Diet

Background: Given escalating rates of chronic disease, broad-reach and cost-effective interventions to increase physical activity and improve dietary intake are needed. The cost-effectiveness of a Telephone Counselling intervention to improve physical activity and diet, targeting adults with established chronic diseases in a low socio-economic area of a major Australian city was examined. Methodology/Principal Findings: A cost-effectiveness modelling study using data collected between February 2005 and November 2007 from a cluster-randomised trial that compared Telephone Counselling with a “Usual Care” (brief intervention) alternative. Economic outcomes were assessed using a state-transition Markov model, which predicted the progress of participants through five health states relating to physical activity and dietary improvement, for ten years after recruitment. The costs and health benefits of Telephone Counselling, Usual Care and an existing practice (Real Control) group were compared. Telephone Counselling compared to Usual Care was not cost-effective ($78,489 per quality adjusted life year gained). However, the Usual Care group did not represent existing practice and is not a useful comparator for decision making. Comparing Telephone Counselling outcomes to existing practice (Real Control), the intervention was found to be cost-effective ($29,375 per quality adjusted life year gained). Usual Care (brief intervention) compared to existing practice (Real Control) was also cost-effective ($12,153 per quality adjusted life year gained). Conclusions/Significance: This modelling study shows that a decision to adopt a Telephone Counselling program over existing practice (Real Control) is likely to be cost-effective. Choosing the ‘Usual Care’ brief intervention over existing practice (Real Control) shows a lower cost per quality adjusted life year, but the lack of supporting evidence for efficacy or sustainability is an important consideration for decision makers. The economics of behavioural approaches to improving health must be made explicit if decision makers are to be convinced that allocating resources toward such programs is worthwhile

A call for new approaches to quantifying biases in observations of sea-surface temperature

Global surface-temperature changes are a fundamental expression of climate change. Recent, much-debated, variations in the observed rate of surface-temperature change have highlighted the importance of uncertainty in adjustments applied to sea-surface temperature (SST) measurements. These adjustments are applied to compensate for systematic biases and changes in observing protocol. Better quantification of the adjustments and their uncertainties would increase confidence in estimated surface-temperature change and provide higher- quality gridded SST fields for use in many applications. Bias adjustments have been based either on physical models of the observing processes or on the assumption of an unchanging relationship between SST and a reference data set such as night marine air temperature. These approaches produce similar estimates of SST bias on the largest space and timescales, but regional differences can exceed the estimated uncertainty. We describe challenges to improving our understanding of SST biases. Overcoming these will require clarification of past observational methods, improved modeling of biases associated with each observing method, and the development of statistical bias estimates that are less sensitive to the absence of metadata regarding the observing method. New approaches are required that embed bias models, specific to each type of observation, within a robust statistical framework. Mobile platforms and rapid changes in observation type require biases to be assessed for individual historic and present-day platforms (i.e., ships or buoys) or groups of platforms. Lack of observational metadata and of high-quality observations for validation and bias model development are likely to remain major challenges