Effect of Benzene and Ethylbenzene on the Transcription of methyl-\u3cem\u3etert\u3c/em\u3e-butyl Ether Degradation Genes of \u3cem\u3eMethylibium petroleiphilum\u3c/em\u3e PM1

Methyl-tert-butyl ether (MTBE) and its degradation by-product, tert-butyl alcohol (TBA), are widespread contaminants detected frequently in groundwater in California. Since MTBE was used as a fuel oxygenate for almost two decades, leaking underground fuel storage tanks are an important source of contamination. Gasoline components such as BTEX (benzene, toluene, ethylbenzene and xylenes) are often present in mixtures with MTBE and TBA. Investigations of interactions between BTEX and MTBE degradation have not yielded consistent trends, and the molecular mechanisms of BTEX compounds’ impact on MTBE degradation are not well understood. We investigated trends in transcription of biodegradation genes in the MTBE-degrading bacterium, Methylibium petroleiphilum PM1 upon exposure to MTBE, TBA, ethylbenzene and benzene as individual compounds or in mixtures. We designed real-time quantitative PCR assays to target functional genes of strain PM1 and provide evidence for induction of genes mdpA (MTBE monooxygenase), mdpJ (TBA hydroxylase) and bmoA (benzene monooxygenase) in response to MTBE, TBA and benzene, respectively. Delayed induction of mdpA and mdpJ transcription occurred with mixtures of benzene and MTBE or TBA, respectively. bmoA transcription was similar in the presence of MTBE or TBA with benzene as in their absence. Our results also indicate that ethylbenzene, previously proposed as an inhibitor of MTBE degradation in some bacteria, inhibits transcription of mdpA, mdpJ and bmoAgenes in strain PM1

Gene \u3cem\u3emdpC\u3c/em\u3e Plays a Regulatory Role in the Methyl-\u3cem\u3etert\u3c/em\u3e-butyl Ether Degradation Pathway of \u3cem\u3eMethylibium petroleiphilum\u3c/em\u3e Strain PM1

Among the few bacteria known to utilize methyl tert-butyl ether (MTBE) as a sole carbon source, Methylibium petroleiphilum PM1 is a well-characterized organism with a sequenced genome; however, knowledge of the genetic regulation of its MTBE degradation pathway is limited. We investigated the role of a putative transcriptional activator gene, mdpC, in the induction of MTBE-degradation genes mdpA (encoding MTBE monooxygenase) and mdpJ (encoding tert-butyl alcohol hydroxylase) of strain PM1 in a gene-knockout mutant mdpC−. We also utilized quantitative reverse transcriptase PCR assays targeting genes mdpA, mdpJ and mdpC to determine the effects of the mutation on transcription of these genes. Our results indicate that gene mdpC is involved in the induction of both mdpA and mdpJ in response to MTBE and tert-butyl alcohol (TBA) exposure in PM1. An additional independent mechanism may be involved in the induction of mdpJ in the presence of TBA

On the Effect of Rigid Swept Surface Waves on Turbulent Drag

Passive turbulent drag reduction techniques are of interest as a cost effective means to improve air vehicle fuel consumption. In the past, rigid surface waves slanted at an angle from the streamwise direction were deemed ineffective to reduce skin friction drag due to the pressure drag that they generate. A recent analysis seeking similarities to the spanwise shear stress generated by spatial Stokes layers suggested that there may be a range of wavelength, amplitude, and orientation in which the wavy surface would reduce turbulent drag. The present work explores, by experiments and Direct Numerical Simulations (DNS), the effect of swept wavy surfaces on skin friction and pressure drag. Plates with shallow and deep wave patterns were rapid-prototyped and tested using a drag balance in the 7x11 inch Low-Speed Wind Tunnel at the NASA LaRC Research Center. The measured drag o set between the wavy plates and the reference at plate is found to be within the experimental repeatability limit. Oil vapor flow measurements indicate a mean spanwise flow over the deep waves. The turbulent flow in channels with at walls, swept wavy walls and spatial Stokes spanwise velocity forcing was simulated at a friction Reynolds number of two hundred. The time-averaged and dynamic turbulent flow characteristics of the three channel types are compared. The drag obtained for the channel with shallow waves is slightly larger than for the at channel, within the range of the experiments. In the case of the large waves, the simulation over predicts the drag. The shortcomings of the Stokes layer analogy model for the estimation of the spanwise shear stress and drag are discussed

Comparative analysis of homology models of the Ah receptor ligand binding domain: Verification of structure-function predictions by site-directed mutagenesis of a nonfunctional receptor

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the biological and toxic effects of a wide variety of structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While significant interspecies differences in AHR ligand binding specificity, selectivity, and response have been observed, the structural determinants responsible for those differences have not been determined, and homology models of the AHR ligand-binding domain (LBD) are available for only a few species. Here we describe the development and comparative analysis of homology models of the LBD of 16 AHRs from 12 mammalian and nonmammalian species and identify the specific residues contained within their ligand binding cavities. The ligand-binding cavity of the fish AHR exhibits differences from those of mammalian and avian AHRs, suggesting a slightly different TCDD binding mode. Comparison of the internal cavity in the LBD model of zebrafish (zf) AHR2, which binds TCDD with high affinity, to that of zfAHR1a, which does not bind TCDD, revealed that the latter has a dramatically shortened binding cavity due to the side chains of three residues (Tyr296, Thr386, and His388) that reduce the amount of internal space available to TCDD. Mutagenesis of two of these residues in zfAHR1a to those present in zfAHR2 (Y296H and T386A) restored the ability of zfAHR1a to bind TCDD and to exhibit TCDD-dependent binding to DNA. These results demonstrate the importance of these two amino acids and highlight the predictive potential of comparative analysis of homology models from diverse species. The availability of these AHR LBD homology models will facilitate in-depth comparative studies of AHR ligand binding and ligand-dependent AHR activation and provide a novel avenue for examining species-specific differences in AHR responsiveness. © 2013 American Chemical Society

Distributed leadership, trust and online communities

This paper analyses the role of distributed leadership and trust in online communities. The team-based informal ethos of online collaboration requires a different kind of leadership from that in formal positional hierarchies. Such leadership may be more flexible and sophisticated, capable of encompassing ambiguity and rapid change. Online leaders need to be partially invisible, delegating power and distributing tasks. Yet, simultaneously, online communities are facilitated by the high visibility and subtle control of expert leaders. This paradox: that leaders need to be both highly visible and invisible as appropriate, was derived from prior research and tested in the analysis of online community discussions using a pattern-matching process. It is argued that both leader visibility and invisibility are important for the facilitation of trusting collaboration via distributed leadership. Advanced leadership responses to complex situations in online communities foster positive group interaction and decision-making, facilitated through active distribution of specific tasks

Adapting the Finetech-Brindley Sacral Anterior Root Stimulator for Bioelectronic Medicine*

The Finetech-Brindley Sacral Anterior Root Stimulator (SARS) is a low cost and reliable system. The architecture has been used for various bioelectric treatments, including several thousand implanted systems for restoring bladder function following spinal cord injury (SCI). Extending the operational frequency range would expand the capability of the system; enabling, for example, the exploration of eliminating the rhizotomy through an electrical nerve block. The distributed architecture of the SARS system enables stimulation parameters to be adjusted without modifying the implant design or manufacturing. To explore the design degrees-of-freedom, a circuit simulation was created and validated using a modified SARS system that supported stimulation frequencies up to 600 Hz. The simulation was also used to explore high frequency (up to 30kHz) behaviour, and to determine the constraints on charge delivered at the higher rates. A key constraint found was the DC blocking capacitors, designed originally for low frequency operation, not fully discharging within a shortened stimulation period. Within these current implant constraints, we demonstrate the potential capability for higher frequency operation that is consistent with presynaptic stimulation block, and also define targeted circuit improvements for future extension of stimulation capability

A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease

Live-attenuated RNA virus vaccines are efficacious but subject to reversion to virulence. Among RNA viruses, replication fidelity is recognized as a key determinant of virulence and escape from antiviral therapy; increased fidelity is attenuating for some viruses. Coronavirus replication fidelity is approximately 20-fold greater than that of other RNA viruses and is mediated by a 3′-5′ exonuclease activity (ExoN) that likely functions in RNA proofreading. In this study, we demonstrate that engineered inactivation of SARS-CoV ExoN activity results in a stable mutator phenotype with profoundly decreased fidelity in vivo and attenuation of pathogenesis in young, aged, and immunocompromised mouse models of human SARS. The ExoN inactivation genotype and mutator phenotype are stable and do not revert to virulence, even after serial passage or long-term persistent infection in vivo. Our approach represents a strategy with potential for broad applications for the stable attenuation of coronaviruses and possibly other RNA viruses