An inter-platform repeatability study investigating real-time amplification of plasmid DNA

BACKGROUND: The wide variety of real-time amplification platforms currently available has determined that standardisation of DNA measurements is a fundamental aspect involved in the comparability of results. Statistical analysis of the data arising from three different real-time platforms was conducted in order to assess inter-platform repeatability. On three consecutive days two PCR reaction mixes were used on each of the three amplification platforms – the LightCycler(®), ABI PRISM(® )7700 and Rotor Gene 3000™. Real-time PCR amplification using a fluorogenic 5' exonuclease assay was performed in triplicate on negative controls and DNA plasmid dilutions of 10(8)–10(2 )copies to give a total of 24 reactions per PCR experiment. RESULTS: The results of the statistical analyses indicated that the platform with the most precise repeatability was the ABI PRISM(® )7700 when coupled with the FastStart PCR reaction mix. It was also found that there was no obvious relationship between plasmid copy number and repeatability. An ANOVA approach identified the factors that significantly affected the results, in descending order of magnitude, as: plasmid copy number, platform, PCR reaction mix and day (on which the experiment was performed). CONCLUSION: In order to deliver useful, informative genetic tests, standardisation of real-time PCR detection platforms to provide repeatable, reliable results is warranted. In addition, a better understanding of inter-assay and intra-assay repeatability is required

Identification of a selective G1-phase benzimidazolone inhibitor by a senescence-targeted virtual screen using artificial neural networks

Cellular senescence is a barrier to tumorigenesis in normal cells and tumour cells undergo senescence responses to genotoxic stimuli, which is a potential target phenotype for cancer therapy. However, in this setting, mixed-mode responses are common with apoptosis the dominant effect. Hence, more selective senescence inducers are required. Here we report a machine learning-based in silico screen to identify potential senescence agonists. We built profiles of differentially affected biological process networks from expression data obtained under induced telomere dysfunction conditions in colorectal cancer cells and matched these to a panel of 17 protein targets with confirmatory screening data in PubChem. We trained a neural network using 3517 compounds identified as active or inactive against these targets. The resulting classification model was used to screen a virtual library of ~2M lead-like compounds. 147 virtual hits were acquired for validation in growth inhibition and senescence-associated β-galactosidase (SA-β-gal) assays. Among the found hits a benzimidazolone compound, CB-20903630, had low micromolar IC50 for growth inhibition of HCT116 cells and selectively induced SA-β-gal activity in the entire treated cell population without cytotoxicity or apoptosis induction. Growth suppression was mediated by G1 blockade involving increased p21 expression and suppressed cyclin B1, CDK1 and CDC25C. Additionally, the compound inhibited growth of multicellular spheroids and caused severe retardation of population kinetics in long term treatments. Preliminary structure-activity and structure clustering analyses are reported and expression analysis of CB-20903630 against other cell cycle suppressor compounds suggested a PI3K/AKT-inhibitor-like profile in normal cells, with different pathways affected in cancer cells

Biomonitoring of 2,4-Dichlorophenoxyacetic Acid Exposure and Dose in Farm Families

OBJECTIVE: We estimated 2,4-dichlorophenoxyacetic acid (2,4-D) exposure and systemic dose in farm family members following an application of 2,4-D on their farm. METHODS: Farm families were recruited from licensed applicators in Minnesota and South Carolina. Eligible family members collected all urine during five 24-hr intervals, 1 day before through 3 days after an application of 2,4-D. Exposure profiles were characterized with 24-hr urine 2,4-D concentrations, which then were related to potential predictors of exposure. Systemic dose was estimated using the urine collections from the application day through the third day after application. RESULTS: Median urine 2,4-D concentrations at baseline and day after application were 2.1 and 73.1 μ g/L for applicators, below the limit of detection, and 1.2 μ g/L for spouses, and 1.5 and 2.9 μ g/L for children. The younger children (4–11 years of age) had higher median post-application concentrations than the older children (≥ 12 years of age) (6.5 vs. 1.9 μ g/L). The geometric mean systemic doses (micrograms per kilogram body weight) were 2.46 (applicators), 0.8 (spouses), 0.22 (all children), 0.32 (children 4–11 years of age), and 0.12 (children ≥ 12 years of age). Exposure to the spouses and children was primarily determined by direct contact with the application process and the number of acres treated. Multivariate models identified glove use, repairing equipment, and number of acres treated as predictors of exposure in the applicators. CONCLUSIONS: We observed considerable heterogeneity of 2,4-D exposure among farm family members, primarily attributable to level of contact with the application process. Awareness of this variability and the actual magnitude of exposures are important for developing exposure and risk characterizations in 2,4-D–exposed agricultural populations

Research Program to Investigate the Fundamental Chemistry of Technetium

Technetium (99Tc, half-life = 2.13x105 years, b-emitter) is one of the radionuclides of major concern for nuclear waste disposal. This concern is due to the long half-life of 99Tc, the ease with which pertechnetate, TcO4 -, migrates in the geosphere, and the corresponding regulatory considerations. The problem of mobility of pertechnetate in the environment is compounded by the fact that pertechnetate is the thermodynamically stable form of technetium in aerobic environments. These two factors present challenges for the safe, long term immobilization of technetium in waste forms. Because of the stability of pertechnetate, technetium has been assumed to exist as pertechnetate in the aqueous phase of nuclear waste tanks. However, recent studies indicate that a significant fraction of the technetium is in a different chemical form. This program addresses the fundamental solution chemistry of technetium in the waste tank environment, and in a second part, the stability of technetium in various waste forms. The chemistry of this element will be studied in aqueous solutions at high pH, with various added salts such as nitrate, nitrite, and organic complexants, and as a function of radiation dose, to determine whether radiolysis effects can reduce TcO4 -. A separate facet of this research is the search for chemical forms of technetium that may be thermodynamically and/or kinetically stable and may be incorporated in various waste forms for long term storage. This phase of the program will address the problem of the possible oxidation of lower valent technetium species in various waste form matrices and the subsequent leaching of the highly soluble TcO4 -

Biomonitoring Data for 2,4-Dichlorophenoxyacetic Acid in the United States and Canada: Interpretation in a Public Health Risk Assessment Context Using Biomonitoring Equivalents

Background: Several extensive studies of exposure to 2,4- dichlorophenoxyacetic acid (2,4-D) using urinary concentrations in samples from the general population, farm applicators, and farm family members are now available. Reference doses (RfDs) exist for 2,4-D, and Biomonitoring Equivalents (BEs; concentrations in urine or plasma that are consistent with those RfDs) for 2,4-D have recently been derived and published. Objective: We reviewed the available biomonitoring data for 2,4-D from the United States and Canada and compared them with BE values to draw conclusions regarding the margin of safety for 2,4-D exposures within each population group. Data sources: Data on urinary 2,4-D excretion in general and target populations from recent published studies are tabulated and the derivation of BE values for 2,4-D summarized. Data synthesis: The biomonitoring data indicate margins of safety (ratio of BE value to biomarker concentration) of approximately 200 at the central tendency and 50 at the extremes in the general population. Median exposures for applicators and their family members during periods of use appear to be well within acute exposure guidance values. Conclusions: Biomonitoring data from these studies indicate that current exposures to 2,4-D are below applicable exposure guidance values. This review demonstrates the value of biomonitoring data in assessing population exposures in the context of existing risk assessments using the BE approach. Risk managers can use this approach to integrate the available biomonitoring data into an overall assessment of current risk management practices for 2,4-D

The application of implementation science for pressure ulcer prevention best practices in an inpatient spinal cord injury rehabilitation program

To implement pressure ulcer (PU) prevention best practices in spinal cord injury (SCI) rehabilitation using implementation science frameworks

Anatomy of a Dansgaard-Oeschger warming transition: High-resolution analysis of the North Greenland Ice Core Project ice core

Large and abrupt temperature oscillations during the last glacial period, known as Dansgaard‐Oeschger (DO) events, are clearly observed in the Greenland ice core record. Here we present a new high‐resolution chemical (2 mm) and stable isotope (20 mm) record from the North Greenland Ice Core Project (NGRIP) ice core at the onset of one of the most prominent DO events of the last glacial, DO‐8, observed ∼38,000 years ago. The unique, subannual‐resolution NGRIP record provides a true sequence of change during a DO warming with detailed annual layer counting of very high depth resolution geochemical measurements used to determine the exact duration of the transition. The continental ions, indicative of long‐range atmospheric loading and dustiness from East Asia, are the first to change, followed by the snow accumulation, the moisture source conditions, and finally the atmospheric temperature in Greenland. The sequence of events shows that atmospheric and oceanic source and circulation changes preceded the DO warming by several years