Pyrosequencing: applicability for studying DNA damage‐induced mutagenesis

Site‐specifically modified DNAs are routinely used in the study of DNA damage‐induced mutagenesis. These analyses involve the creation of DNA vectors containing a lesion at a pre‐determined position, DNA replication, and detection of mutations at the target site. The final step has previously required the isolation of individual DNA clones, hybridization with radioactively labeled probes, and verification of mutations by Sanger sequencing. In the search for an alternative procedure that would allow direct quantification of sequence variants in a mixed population of DNA molecules, we evaluated the applicability of pyrosequencing to site‐specific mutagenesis assays. The progeny DNAs were analyzed that originated from replication of N⁶‐(deoxy‐D‐erythro‐pentofuranosyl)‐2,6‐diamino‐3,4‐dihydro‐4‐oxo‐5‐N‐methylformamidopyrimidine (MeFapy‐dG)‐containing vectors in primate cells, with the lesion being positioned in the 5′‐GCNGG‐3′ sequence context. Pyrosequencing detected ～8% G to T transversions and ～3.5% G to A transitions, a result that was in excellent agreement with frequencies previously measured by the standard procedure (Earley LF et al. [2013]: Chem Res Toxicol 26:1108–1114). However, ～3.5% G to C transversions and ～2.0% deletions could not be detected by pyrosequencing. Consistent with these observations, the sensitivity of pyrosequencing for measuring the single deoxynucleotide variants differed depending on the deoxynucleotide identity, and in the given sequence contexts, was determined to be ～1–2% for A and T and ～5% for C. Pyrosequencing of other DNA isolates that were obtained following replication of MeFapy‐dG‐containing vectors in primate cells or Escherichia coli, identified several additional limitations. Collectively, our data demonstrated that pyrosequencing can be used for studying DNA damage‐induced mutagenesis as an effective complementary experimental approach to current protocols. Environ. Mol. Mutagen. 55:601–608, 2014. © 2014 Wiley Periodicals, Inc.     

Consequences of redefining Alzheimer's disease in terms of amyloid burden without regard to cognitive decline

正Alzheimer’s disease(AD)redefined:For the past century,AD has been defined as a disease of progressive cognitive decline paired with a burden of amyloid-β(Aβ)plaques and pathologic tau tangles in the hippocampus and forebrain.However,a recent Framework paper jointly sponsored by the National Institute on Aging and the Alzheimer’s Association(Jack et al.,2018)proposes new classification guidelines for AD,which,if adopted,will have profound     

Flow cytometric analysis of Pig‐a gene mutation and chromosomal damage induced by procarbazine hydrochloride in CD‐1 mice

Procarbazine is a genotoxic carcinogen whose DNA‐damaging activities are not reliably detected in vitro. We evaluated the in vivo genotoxic effects of procarbazine on hematopoietic cells of male CD‐1 mice using a multi‐endpoint study design that scored micronucleated reticulocyte (MN‐RET) frequency and gene mutation at the Pig‐a locus. CD‐1 mice were treated for 3 days with procarbazine, up to 150 mg/kg/day. Blood samples collected on Day 3 exhibited robust induction of MN‐RETs, with the high dose group exhibiting a mean 29‐fold increase. Blood collected 15 and 30 days after treatment began was analyzed for Pig‐a mutation with a dual labeling method that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. Procarbazine significantly increased mutant reticulocyte frequencies by Day 15. Mutant erythrocyte responses were also apparent, with a peak incidence observed for the high dose group on Day 30. These results demonstrate that the complex metabolism and resulting genotoxicity of procarbazine is best evaluated in intact animal models, and show that the flow cytometric methods employed offer a means to efficiently monitor both in vivo chromosomal damage and mutation. Environ. Mol. Mutagen. 54:294–298, 2013. © 2013 Wiley Periodicals, Inc.     

Postoperative Remote Automated Monitoring: Need for and State of the Science

Worldwide, more than 230 million adults have major noncardiac surgery each year. Although surgery can improve quality and duration of life, it can also precipitate major complications. Moreover, a substantial proportion of deaths occur after discharge. Current systems for monitoring patients postoperatively, on surgical wards and after transition to home, are inadequate. On the surgical ward, vital signs evaluation usually occurs only every 4-8 hours. Reduced in-hospital ward monitoring, followed by no vital signs monitoring at home, leads to thousands of cases of undetected/delayed detection of hemodynamic compromise. In this article we review work to date on postoperative remote automated monitoring on surgical wards and strategy for advancing this field. Key considerations for overcoming current barriers to implementing remote automated monitoring in Canada are also presented.     

Could the PI3K canonical pathway be a common link between chronic inflammatory conditions and oral carcinogenesis?

The association between chronic inflammatory disorders and oral carcinogenesis has been both a source of interest and contention. Based upon its central importance in oral carcinogenesis, the finding that the PI3k/Akt/mTOR pathway is activated in oral lichen planus, chronic graft‐versus‐host disease, and chronic oral candidiasis suggests that it may provide a link between benign and malignant oral conditions. Here, we discuss a possible mechanistic rationale that addresses the activation of this important signaling pathway and its downstream events, while correlating it with the carcinogenic potential of chronic oral disorders.     

Effects of Middle-Ear Disease and Cleft Palate on High-Frequency Hearing in Children

High-frequency hearing loss in children with cleft palate has been documented recently. The present study was designed to investigate whether hearing loss can result solely as a consequence of middle-ear disease in early life or as a result of cleft palate and its sequelae which include middle-ear disease. Our results demonstrate that auditory functions for test frequencies 250-6 000 Hz were not significantly different among the two investigational groups of children with high incidence of middle-ear disease, and a control group of children with virtually no middle-ear disease. However, for high-frequency thresholds (8 000-20 000 Hz), both groups of children with high incidence of middle-ear disease were statistically different from the control group. Moreover, the children with cleft palate had high-frequency hearing that was statistically similar to that of children with normal orofacial structures and high incidence of middle-ear disease. Middle-ear disease alone, then, is a sufficient condition for loss of high-frequency sensitivity.

La perte auditive des hautes fréquences chez les enfants porteurs d'une fissure palatine a fait l'objet d'une documentation récente. Cette étude a été organisée de façon à établir si la perte auditive pouvait résulter uniquement d'une maladie de l'oreille moyenne dans la petite enfance, ou d'une fissure palatine et de ses séquelles qui comprennent les otites moyennes. Entre les deux groupes d'enfants prédisposés aux otites moyennes, et un groupe de contro?le constitué d'enfants rarement atteints d'otites moyennes, nos résultats n'indiquent pas de différences significatives quant aux fonctions auditives pour des fréquences d'essai de 250 à 6 000 Hz. Néanmoins, en ce qui concerne les seuils aux hautes fréquences (de 8 000 à 20 000 Hz), les deux groupes d'enfants prédisposés aux otites moyennes étaient statistiquement différents du groupe de contro?le. En outre, les enfants porteurs d'une fissure palatine avaient une audition des hautes fréquences qui était statistiquement semblable à celle des enfants porteurs de structures orofaciales normales et qui étaient prédisposés aux otites moyennes. A elle seule, une atteinte de l'oreille moyenne semble done ětre une condition suffisante pour l'apparition d'une perte auditive sur les hautes fréquences.     

<Emphasis Type="Bold">9</Emphasis><Superscript><Emphasis Type="Bold">th</Emphasis></Superscript><Emphasis Type="Bold">International Conference On Homocysteine and One-Carbon Metabolism - HCY2013</Emphasis>

Canavan disease (CD) is a fatal neurological disorder caused by defects in the gene that encodes for a critical metabolic enzyme. The enzyme aspartoacylase catalyzes the deacetylation of N-acetylaspartate to produce acetate required for fatty acid biosynthesis in the brain. The loss of aspartoacylase activity leads to the demyelination and disrupted brain development that is found in CD patients. Sixteen different clinical mutants of aspartoacylase have been cloned, expressed and purified to examine their properties and the relationship between enzyme properties and disease phenotype. In contrast to numerous cell culture studies that reported virtually complete loss of function, each of these purified mutant enzymes was found to have measureable catalytic activity. However, the activities of these mutants are diminished, by as little as three-fold to greater than 100-fold when compared to the native enzyme. Many of these mutated enzyme forms show decreased thermal stability and an increased propensity for denaturation upon exposure to urea, but only four of the 16 mutants examined showed both diminished thermal and diminished conformational stability. Significantly, each of these lower stability mutants are responsible for the more severe phenotypes of CD, while patients with milder forms of CD have aspartoacylase mutants with generally high catalytic activity and with either good thermal or good conformational stability. These results suggest that the loss of catalytic function and the accumulation of N-acetylaspartate in Canavan disease is at least partially a consequence of the decreased protein stability caused by these mutations.