Standardized data quality acceptance criteria for a rapid Escherichia coli qPCR method (Draft Method C) for water quality monitoring at recreational beaches

There is growing interest in the application of rapid quantitative polymerase chain reaction (qPCR) and other PCR-based methods for recreational water quality monitoring and management programs. This interest has strengthened given the publication of U.S. Environmental Protection Agency (EPA)-validated qPCR methods for enterococci fecal indicator bacteria (FIB) and has extended to similar methods for Escherichia coli (E. coli) FIB. Implementation of qPCR-based methods in monitoring programs can be facilitated by confidence in the quality of the data produced by these methods. Data quality can be determined through the establishment of a series of specifications that should reflect good laboratory practice. Ideally, these specifications will also account for the typical variability of data coming from multiple users of the method. This study developed proposed standardized data quality acceptance criteria that were established for important calibration model parameters and/or controls from a new qPCR method for E. coli (EPA Draft Method C) based upon data that was generated by 21 laboratories. Each laboratory followed a standardized protocol utilizing the same prescribed reagents and reference and control materials. After removal of outliers, statistical modeling based on a hierarchical Bayesian method was used to establish metrics for assay standard curve slope, intercept and lower limit of quantification that included between-laboratory, replicate testing within laboratory, and random error variability. A nested analysis of variance (ANOVA) was used to establish metrics for calibrator/positive control, negative control, and replicate sample analysis data. These data acceptance criteria should help those who may evaluate the technical quality of future findings from the method, as well as those who might use the method in the future. Furthermore, these benchmarks and the approaches described for determining them may be helpful to method users seeking to establish comparable laboratory-specific criteria if changes in the reference and/or control materials must be made

Evaluation of multiple laboratory performance and variability in analysis of recreational freshwaters by a rapid Escherichia coli qPCR method (Draft Method C)

There is interest in the application of rapid quantitative polymerase chain reaction (qPCR) methods for recreational freshwater quality monitoring of the fecal indicator bacteria Escherichia coli (E. coli). In this study we determined the performance of 21 laboratories in meeting proposed, standardized data quality acceptance (QA) criteria and the variability of target gene copy estimates from these laboratories in analyses of 18 shared surface water samples by a draft qPCR method developed by the U.S. Environmental Protection Agency (EPA) for E. coli. The participating laboratories ranged from academic and government laboratories with more extensive qPCR experience to “new” water quality and public health laboratories with relatively little previous experience in most cases. Failures to meet QA criteria for the method were observed in 24% of the total 376 test sample analyses. Of these failures, 39% came from two of the “new” laboratories. Likely factors contributing to QA failures included deviations in recommended procedures for the storage and preparation of reference and control materials. A master standard curve calibration model was also found to give lower overall variability in log10 target gene copy estimates than the delta-delta Ct (ΔΔCt) calibration model used in previous EPA qPCR methods. However, differences between the mean estimates from the two models were not significant and variability between laboratories was the greatest contributor to overall method variability in either case. Study findings demonstrate the technical feasibility of multiple laboratories implementing this or other qPCR water quality monitoring methods with similar data quality acceptance criteria but suggest that additional practice and/or assistance may be valuable, even for some more generally experienced qPCR laboratories. Special attention should be placed on providing and following explicit guidance on the preparation, storage and handling of reference and control materials

Ground and Space-Based Measurement of Rocket Engine Burns in the Ionosphere

On-orbit firings of both liquid and solid rocket motors provide localized disturbances to the plasma in the upper atmosphere. Large amounts of energy are deposited to ionosphere in the form of expanding exhaust vapors which change the composition and flow velocity. Charge exchange between the neutral exhaust molecules and the background ions (mainly O+) yields energetic ion beams. The rapidly moving pickup ions excite plasma instabilities and yield optical emissions after dissociative recombination with ambient electrons. Line-of-sight techniques for remote measurements rocket burn effects include direct observation of plume optical emissions with ground and satellite cameras, and plume scatter with UHF and higher frequency radars. Long range detection with HF radars is possible if the burns occur in the dense part of the ionosphere. The exhaust vapors initiate plasma turbulence in the ionosphere that can scatter HF radar waves launched from ground transmitters. Solid rocket motors provide particulates that become charged in the ionosphere and may excite dusty plasma instabilities. Hypersonic exhaust flow impacting the ionospheric plasma launches a low-frequency, electromagnetic pulse that is detectable using satellites with electric field booms. If the exhaust cloud itself passes over a satellite, in situ detectors measure increased ion-acoustic wave turbulence, enhanced neutral and plasma densities, elevated ion temperatures, and magnetic field perturbations. All of these techniques can be used for long range observations of plumes in the ionosphere. To demonstrate such long range measurements, several experiments were conducted by the Naval Research Laboratory including the Charged Aerosol Release Experiment, the Shuttle Ionospheric Modification with Pulsed Localized Exhaust experiments, and the Shuttle Exhaust Ionospheric Turbulence Experiments

RhoH Regulates Subcellular Localization of ZAP-70 and Lck in T Cell Receptor Signaling

RhoH is an hematopoietic-specific, GTPase-deficient Rho GTPase that plays a role in T development. We investigated the mechanisms of RhoH function in TCR signaling. We found that the association between Lck and CD3ζ was impaired in RhoH-deficient T cells, due to defective translocation of both Lck and ZAP-70 to the immunological synapse. RhoH with Lck and ZAP-70 localizes in the detergent-soluble membrane fraction where the complex is associated with CD3ζ phosphorylation. To determine if impaired translocation of ZAP-70 was a major determinant of defective T cell development, Rhoh-/- bone marrow cells were transduced with a chimeric myristoylation-tagged ZAP-70. Myr-ZAP-70 transduced cells partially reversed the in vivo defects of RhoH-associated thymic development and TCR signaling. Together, our results suggest that RhoH regulates TCR signaling via recruitment of ZAP-70 and Lck to CD3ζ in the immunological synapse. Thus, we define a new function for a RhoH GTPase as an adaptor molecule in TCR signaling pathway

Power spectrum analysis of ionospheric fluctuations with the Murchison Widefield Array

Low-frequency, wide field-of-view (FOV) radio telescopes such as the Murchison Widefield Array (MWA) enable the ionosphere to be sampled at high spatial completeness. We present the results of the first power spectrum analysis of ionospheric fluctuations in MWA data, where we examined the position offsets of radio sources appearing in two data sets. The refractive shifts in the positions of celestial sources are proportional to spatial gradients in the electron column density transverse to the line of sight. These can be used to probe plasma structures and waves in the ionosphere. The regional (10–100 km) scales probed by the MWA, determined by the size of its FOV and the spatial density of radio sources (typically thousands in a single FOV), complement the global (100–1000 km) scales of GPS studies and local (0.01–1 km) scales of radar scattering measurements. Our data exhibit a range of complex structures and waves. Some fluctuations have the characteristics of traveling ionospheric disturbances, while others take the form of narrow, slowly drifting bands aligned along the Earth's magnetic field

Digital Art, Culture and Heritage: New constructs and consciousness

Invited presentation for special symposium at EVA London 2019. This half-day Symposium explores themes of digital art, culture, and heritage, bringing together speakers from a range of disciplines to consider technology with respect to artistic and academic practice. As we increasingly see ourselves and life through a digital lens and the world communicated on digital screens, we experience altered states of being and consciousness in ways that blur the lines between digital and physical reality, while our ways of thinking and seeing become a digital stream of consciousness that flows between place and cyberspace. We have entered the postdigital world and are living, working, and thinking with machines as our computational culture driven by artificial intelligence and machine learning embeds itself in everyday life and threads across art, culture, and heritage, juxtaposing them in the digital profusion of human creativity on the Internet