Exploring the Pathogenic and Drug Resistance Mechanisms of Staphylococcus aureus

We have previously identified σS, an ECF sigma factor that is important in the virulence and stress response of S. aureus. Transcriptional profiling of sigS revealed that it is differentially regulated in a variety of laboratory and clinical strains of S. aureus, suggesting that there exists a regulatory network that modulates its expression. In order to identify direct regulators of sigS expression, we performed a biotin pull down assay in tandem with mass spectrometry. We identified CymR as a direct regulator and observed that sigS expression is increased in cells lacking cymR. In addition, transposon mutagenesis was performed to identify regulators of sigS expression. We identified insertions in genes that are transcriptional regulators, and elements involved in amino acid biosynthesis and DNA replication, recombination and repair as influencing sigS expression. Finally, methyl nitro-nitrosoguanidine mutagenesis in conjunction with whole genome sequencing was employed and revealed mutations in the lactose repressor, lacR, and the membrane sensor histidine kinase, kdpD, as negatively effecting sigS expression. EMSAs revealed that LacR is an indirect regulator of sigS expression, while the response regulator KdpE is a direct repressor. These results indicate that a complex regulatory network is in place for sigS that modulates its expression. In a continuation of studies on σS regulation, we next explored interplay with the products of genes conserved within the sigS locus. We determined that this region is conserved amongst all the sequenced staphylococci, and includes four genes: SAUSA300_1721 (a conserved hypothetical protein), as well as sigS, ecfX, and ecfY. In order to investigate the relationship between EcfX and σS we performed protein pull down assays and observed that these two protein interact. Further to this, transcriptional analysis of sigS in an ecfX mutant reveal that expression of sigS is decreased, indicating that it is an activator. Architectural analysis of the sigS locus via RNAseq revealed that the majority of transcription in this region comes from ecfY, a gene that is downstream and divergent to sigS. We demonstrate that inactivation of ecfY leads to a significant increase in sigS expression, and that ecfY null strains are more resistant to DNA damaging agents such as UV, H2O2, MMS, and ethidium bromide, which we have previously demonstrated that a sigS mutant is highly sensitive to. Our studies also revealed that an ecfY null strain is better able to survive intracellularly following phagocytosis by RAW 264.7 cell and demonstrates increased survival in whole-human blood, which is again opposed to that previously observed for sigS deficient strains. Because the ecfY null strain overexpresses sigS, we investigated the regulon of this sigma factor using this mutant in conjunction with RNAseq analysis. We identified that genes putatively under the control of σS are involved in DNA damage and repair, virulence, amino acid starvation and nucleic acid biosynthesis. Collectively, our results indicate that σS is regulated via a unique mechanism: positively through an apparent need for an activator protein (EcfX) and negatively via RNA-RNA interaction (the 3’ UTR of ecfY). We suggest that the evidence presented here greatly adds not only to our understanding of the regulatory circuits extant within S. aureus, but also to alternative sigma factor biology in general. Finally, we evaluated the efficacy of a novel library of quinazoline-based compounds against a highly drug resistant strain of S. aureus. We performed structure activity and structure property relationship assays in order to identify lead compounds. These methods lead to the identification of N2,N4-disubstituted quinazoline-2,4-diamines that had low minimum inhibitory concentrations, along with favorable physiochemical properties. Evaluation of their biological activity demonstrated limited potential for resistance of to our quinazoline based compounds, low toxicity to human epithelial cells, and strong efficacy in vivo. Taken together, our findings support the use of quinazoline derivatives as potential new antimicrobials against multidrug resistant S. aureus

Large-eddy simulatoin of flow field and pollutant dispession in urban street canyons under unstable atmospheric

Thermal stratification plays an important role in the air flow and pollutant dispersion processes. This study employed a large-eddy simulation (LES) code based on a one-equation subgrid-scale (SGS) model to investigate the flow field and pollutant dispersion characteristics inside urban street canyons. The unstable thermal stratification was simulated by heating the ground level of the street canyons. The thermal buoyancy forces were, using the Boussinesq assumption, taken into account in both the Navier-Stokes equations and the transport equation for SGS turbulent kinetic energy (TKE). The LES had been validated against experimental data obtained in wind tunnel studies before it was applied to study the detailed turbulence and pollutant dispersion characteristics in urban street canyons. The effects of different bulk Richardson number (Rb) were investigated. Several typical temperature differences between the street bottom and ambient air were configured to simulate the scenarios occurring at different times during the day.postprintThe 7th International Conference of Urban Climate (ICUC-7), Yokohama, Japan, 29 June-3 July 2009

05/18/1993 - Eastern\u27s 1993 All Student Show Winner Laura Brahos.pdf

This paper reports on the stages forming a model evaluation protocol for urban flow and dispersion models proposed within the COST Action 732 on "Quality Assurance and Improvement of Micro-Scale Meteorological Models". It discusses the different components forming model evaluation with emphasis on validation and implementation of the protocol for the test case Mock Urban Setting Test (MUST). The protocol was proposed with building-resolving models in mind, but integral models have also been included. The suggested approach can be used for further micro-scale model evaluation and for the standardisation of their applications

“Exposure Track”—The Impact of Mobile-Device-Based Mobility Patterns on Quantifying Population Exposure to Air Pollution

Air pollution is now recognized as the world’s single largest environmental and human health threat. Indeed, a large number of environmental epidemiological studies have quantified the health impacts of population exposure to pollution. In previous studies, exposure estimates at the population level have not considered spatially- and temporally varying populations present in study regions. Therefore, in the first study of it is kind, we use measured population activity patterns representing several million people to evaluate population-weighted exposure to air pollution on a city-wide scale. Mobile and wireless devices yield information about where and when people are present, thus collective activity patterns were determined using counts of connections to the cellular network. Population-weighted exposure to PM2.5 in New York City (NYC), herein termed “Active Population Exposure” was evaluated using population activity patterns and spatiotemporal PM2.5 concentration levels, and compared to “Home Population Exposure”, which assumed a static population distribution as per Census data. Areas of relatively higher population-weighted exposures were concentrated in different districts within NYC in both scenarios. These were more centralized for the “Active Population Exposure” scenario. Population-weighted exposure computed in each district of NYC for the “Active” scenario were found to be statistically significantly (p < 0.05) different to the “Home” scenario for most districts. In investigating the temporal variability of the “Active” population-weighted exposures determined in districts, these were found to be significantly different (p < 0.05) during the daytime and the nighttime. Evaluating population exposure to air pollution using spatiotemporal population mobility patterns warrants consideration in future environmental epidemiological studies linking air quality and human health

Longitudinal flow evolution and turbulence structure of dynamically similar, sustained, saline density and turbidity currents

Experimental results are presented concerning flow evolution and turbulence structure of sustained saline and turbidity flows generated on 0°, 3°, 6°, and 9° sloping ramps that terminate abruptly onto a horizontal floor. Two-component velocity and current density were measured with an ultrasonic Doppler velocity profiler and siphon sampler on the slope, just beyond the slope break and downstream on the horizontal floor. Three main factors influence longitudinal flow evolution and turbulence structure: sediment transport and sedimentation, slope angle, and the presence of a slope break. These controls interact differently depending on flow type. Sediment transport is accompanied by an inertial fluid reaction that enhances Reynolds stresses in turbidity flows. Thus turbidity flows mix more vigorously than equivalent saline density flows. For saline flows, turbulent kinetic energy is dependent on slope, and rapid deceleration occurs on the horizontal floor. For turbidity flows, normalized turbulent kinetic energy increases downstream, and mean streamwise deceleration is reduced compared with saline flows. The slope break causes mean bed-normal velocity of turbidity flows to become negative and have a gentler gradient compared with other locations. A reduction of peak Reynolds normal stress in the bed-normal direction is accompanied by an increase in turbulent accelerations across the rest of the flow thickness. Thus the presence of particles acts to increase Reynolds normal stresses independently of gradients of mean velocity, and sediment transport increases across the break in slope. The experiments illustrate that saline density currents may not be good dynamic analogues for natural turbidity currents

In-street wind direction variability in the vicinity of a busy intersection in central London

We present results from fast-response wind measurements within and above a busy intersection between two street canyons (Marylebone Road and Gloucester Place) in Westminster, London taken as part of the DAPPLE (Dispersion of Air Pollution and Penetration into the Local Environment; www.dapple.org.uk) 2007 field campaign. The data reported here were collected using ultrasonic anemometers on the roof-top of a building adjacent to the intersection and at two heights on a pair of lamp-posts on opposite sides of the intersection. Site characteristics, data analysis and the variation of intersection flow with the above-roof wind direction (θref) are discussed. Evidence of both flow channelling and recirculation was identified within the canyon, only a few metres from the intersection for along-street and across-street roof-top winds respectively. Results also indicate that for oblique rooftop flows, the intersection flow is a complex combination of bifurcated channelled flows, recirculation and corner vortices. Asymmetries in local building geometry around the intersection and small changes in the background wind direction (changes in 15-min mean θref of 5–10 degrees) were also observed to have profound influences on the behaviour of intersection flow patterns. Consequently, short time-scale variability in the background flow direction can lead to highly scattered in-street mean flow angles masking the true multi-modal features of the flow and thus further complicating modelling challenges

Evaluation of urban local-scale aerodynamic parameters: implications for the vertical profile of wind speed and for source areas

Nine methods to determine local-scale aerodynamic roughness length (z0) and zero-plane displacement (zd) are compared at three sites (within 60 m of each other) in London, UK. Methods include three anemometric (single-level high frequency observations), six morphometric (surface geometry) and one reference-based approach (look-up tables). A footprint model is used with the morphometric methods in an iterative procedure. The results are insensitive to the initial zd and z0 estimates. Across the three sites, zd varies between 5 – 45 m depending upon the method used. Morphometric methods that incorporate roughness-element height variability agree better with anemometric methods, indicating zd is consistently greater than the local mean building height. Depending upon method and wind direction, z0 varies between 0.1 and 5 m with morphometric z0 consistently being 2 – 3 m larger than the anemometric z0. No morphometric method consistently resembles the anemometric methods. Wind-speed profiles observed with Doppler lidar provide additional data with which to assess the methods. Locally determined roughness parameters are used to extrapolate wind-speed profiles to a height roughly 200 m above the canopy. Wind-speed profiles extrapolated based on morphometric methods that account for roughness-element height variability are most similar to observations. The extent of the modelled source area for measurements varies by up to a factor of three, depending upon the morphometric method used to determine zd and z0

Evaluation of fast atmospheric dispersion models in a regular street network

The need to balance computational speed and simulation accuracy is a key challenge in designing atmospheric dispersion models that can be used in scenarios where near real-time hazard predictions are needed. This challenge is aggravated in cities, where models need to have some degree of building-awareness, alongside the ability to capture effects of dominant urban flow processes. We use a combination of high-resolution large-eddy simulation (LES) and wind-tunnel data of flow and dispersion in an idealised, equal-height urban canopy to highlight important dispersion processes and evaluate how these are reproduced by representatives of the most prevalent modelling approaches: (i) a Gaussian plume model, (ii) a Lagrangian stochastic model and (iii) street-network dispersion models. Concentration data from the LES, validated against the wind-tunnel data, were averaged over the volumes of streets in order to provide a high-fidelity reference suitable for evaluating the different models on the same footing. For the particular combination of forcing wind direction and source location studied here, the strongest deviations from the LES reference were associated with mean over-predictions of concentrations by approximately a factor of 2 and with a relative scatter larger than a factor of 4 of the mean, corresponding to cases where the mean plume centreline also deviated significantly from the LES. This was linked to low accuracy of the underlying flow models/parameters that resulted in a misrepresentation of pollutant channelling along streets and of the uneven plume branching observed in intersections. The agreement of model predictions with the LES (which explicitly resolves the turbulent flow and dispersion processes) greatly improved by increasing the accuracy of building-induced modifications of the driving flow field. When provided with a limited set of representative velocity parameters, the comparatively simple street-network models performed equally well or better compared to the Lagrangian model run on full 3D wind fields. The study showed that street-network models capture the dominant building-induced dispersion processes in the canopy layer through parametrisations of horizontal advection and vertical exchange processes at scales of practical interest. At the same time, computational costs and computing times associated with the network approach are ideally suited for emergency-response applications