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Tracking long-term environmental change is important, particularly for freshwater ecosystems, often with high rates of decline. Waterbirds are key indicators of freshwater ecosystem change, with groups reflecting food availability (e.g. piscivores and fish). We store waterbird (species abundance, numbers of nests and broods) and wetland area data from aerial surveys of waterbirds across Australia, mostly at the species' level (∼100 species) from three aerial survey programs: Eastern Australian Waterbird Survey, National Survey and Murray-Darling Basin wetlands. Across eastern Australia, we survey up to 2,000 wetlands annually (October, since 1983), along 10 survey bands (30 km wide), east to west across about one third of Australia. In 2008, we surveyed 4,858 wetlands across Australia and each year (since 2010) we survey the major wetlands in the Murray-Darling Basin. These data inform regulation of hunting seasons in Victoria and South Australia, Game bird culling in NSW, State of the Environment Reporting, environmental assessments, river and wetland management, the status of individual species and identification of high conservation sites.

The genus Nocardia contains >50 human pathogenic species that cause a range of illnesses from skin and soft tissue infections to lung and brain infections. However, despite their membership in the most prominent family of secondary metabolite producers (the Actinomycetes), the ability of Nocardia species, especially those that cause human infections, to produce secondary metabolites has not been as well studied. Using genome mining, we have investigated cryptic secondary metabolite biosynthesis gene clusters from Nocardia species and identified a conserved locus within human pathogenic strains of Nocardia brasiliensis and Nocardia vulneris . Direct capture and heterologous expression in a Streptomyces host activated the biosynthetic locus, revealing it to be the source of the brasiliquinones, benz[ a ]anthraquinone antibiotics whose biosynthetic pathway has remained hidden for over two decades, until now. Our findings highlight these hitherto neglected human pathogenic Nocardia as a source of diverse and important natural products.

Ultrafast x-ray imagers developed at Sandia National Laboratories are a transformative diagnostic tool in inertial confinement fusion and high energy density physics experiments. The nanosecond time scales on which these devices operate are a regime with little precedent, and applicable characterization procedures are still developing. This paper presents pulsed x-ray characterization of the Icarus imager under a variety of illumination levels and timing modes. Results are presented for linearity of response, absolute sensitivity, variation of response with gate width, and image quality.

The neglected tropical disease Buruli ulcer (BU) is an infection of subcutaneous tissue with Mycobacterium ulcerans There is no effective vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl reductase (ER) domain of the M. ulcerans mycolactone polyketide synthases electrostatically coupled with a previously described Toll-like receptor 2 (TLR-2) agonist-based lipopeptide adjuvant, R 4 Pam 2 Cys. Mice were vaccinated and then challenged via tail inoculation with 14 to 20 CFU of a bioluminescent strain of M. ulcerans Mice receiving either the experimental ER vaccine or Mycobacterium bovis bacillus Calmette-Guérin (BCG) were equally protected, with both groups faring significantly better than nonvaccinated animals ( P <  0.05). To explore potential correlates of protection, a suite of 29 immune parameters were assessed in the mice at the end of the experimental period. Multivariate statistical approaches were used to interrogate the immune response data to develop disease-prognostic models. High levels of interleukin 2 (IL-2) and low gamma interferon (IFN-γ) produced in the spleen best predicted control of infection across all vaccine groups. Univariate logistic regression revealed vaccine-specific profiles of protection. High titers of ER-specific IgG serum antibodies together with IL-2 and IL-4 in the draining lymph node (DLN) were associated with protection induced by the ER vaccine. In contrast, high titers of IL-6, tumor necrosis factor alpha (TNF-α), IFN-γ, and IL-10 in the DLN and low IFN-γ titers in the spleen were associated with protection following BCG vaccination. This study suggests that an effective BU vaccine must induce localized, tissue-specific immune profiles with controlled inflammatory responses at the site of infection., (Copyright © 2020 American Society for Microbiology.)

Fast x-ray detectors are critical tools in pulsed power and fusion applications, where detector impulse response of a nanosecond or better is often required. Semiconductor detectors can create fast, sensitive devices with extensive operational flexibility. There is typically a trade-off between detector sensitivity and speed, but higher atomic number absorbers can increase hard x-ray absorption without increasing the charge collection time, provided carriers achieve high velocity. This paper presents x-ray pulse characterization conducted at the Advanced Photon Source of x-ray absorption efficiency and temporal impulse response of current-mode semiconductor x-ray detectors composed of Si, GaAs, and CdTe.

Buruli ulcer (BU) is a neglected tropical disease caused by infection with Mycobacterium ulcerans . Unclear transmission, no available vaccine, and suboptimal treatment regimens hamper the control of this disease. Carefully designed preclinical research is needed to address these shortcomings. In vivo imaging (IVIS ® , Perkin Elmer, Waltham, MA) of infection is an emerging tool that permits monitoring of disease progression and reduces the need to using large numbers of mice at different time-points during the experiment, as individual mice can be imaged at multiple time-points. We aimed to further describe the use of in vivo imaging (IVIS) in BU. We studied the detection of M. ulcerans in experimentally infected BALB/c mouse tails and the subsequent histopathology and immune response in this pilot study. IVIS-monitoring was performed weekly in ten infected BALB/c mice to measure light emitted as a proxy for bacterial load. Nine of 10 (90%) BALB/c mice infected subcutaneously with 3.3 × 10 5 M. ulcerans JKD8049 (containing pMV306 hsp16+luxG13) exhibited light emission from the site of infection, indicating M. ulcerans growth in vivo, whereas only five of 10 (50%) animals developed clinical signs of the disease. Specific antibody titers were detected within 2 weeks of the infection. Interferon (IFN)-γ and interleukin (IL)-10 were elevated in animals with pathology . Histopathology revealed clusters of acid-fast bacilli in the subcutaneous tissue, with macrophage infiltration and granuloma formation resembling human BU. Our study successfully showed the utility of M. ulcerans IVIS monitoring and lays a foundation for further research.

An enzyme must be soluble, stable, active and easy to produce to be useful in industrial applications. Not all enzymes possess these attributes. We set out to determine how many changes are required to convert an enzyme with poor properties into one that has useful properties. Lipase Lip3 from Drosophila melanogaster had been previously optimised for expression in Escherichia coli. The expression levels were good, but Lip3 was mainly insoluble with poor activity. Directed evolution was used to identify variants with enhanced activity along with improved solubility. Five variants and the wild-type (wt) enzyme were purified and characterised. The yield of the wt enzyme was just 2.2 mg/L of culture, while a variant, produced under the same conditions, gave 351 mg. The improvement of activity of the best variant was 200 times higher than that of the wt when the crude lysates were analysed using pNP-C8, but with purified protein, the improvement observed was 1.5 times higher. This means that most of the increase of activity is due to increase in solubility and stability. All the purified variants showed increased thermal stability compared with the wt enzyme that had a T1/2 of 37°C, while the mutant with P291L of 42.2°C and the mutant R7&#95;47D with five mutations had a value of 52.9°C, corresponding to an improvement of 16°C. The improved variants had between five and nine changes compared with the wt enzyme. There were four changes that were found in all 30 final round variants for which sequences were obtained; three of these changes were found in the substrate-binding domain., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The conversion of saturated fatty acids to high value chiral hydroxy-acids and lactones poses a number of synthetic challenges: the activation of unreactive C-H bonds and the need for regio- and stereoselectivity. Here the first example of a wild-type cytochrome P450 monooxygenase (CYP116B46 from Tepidiphilus thermophilus) capable of enantio- and regioselective C5 hydroxylation of decanoic acid 1 to (S)-5-hydroxydecanoic acid 2 is reported. Subsequent lactonization yields (S)-δ-decalactone 3, a high value fragrance compound, with greater than 90 % ee. Docking studies provide a rationale for the high regio- and enantioselectivity of the reaction., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The nargenicin family of antibiotics are macrolides containing a rare ether-bridged cis-decalin motif. Several of these compounds are highly active against multi-drug resistant organisms. Despite the identification of the first members of this family almost 40 years ago, the genetic basis for the production of these molecules and the enzyme responsible for formation of the oxa bridge, remain unknown. Here, the 85 kb nargenicin biosynthetic gene cluster was identified from a human pathogenic Nocardia arthritidis isolate and this locus is solely responsible for nargenicin production. Further investigation of this locus revealed a putative iron-α-ketoglutarate-dependent dioxygenase, which was found to be responsible for the formation of the ether bridge from the newly identified deoxygenated precursor, 8,13-deoxynargenicin. Uncovering the nargenicin biosynthetic locus provides a molecular basis for the rational bioengineering of these interesting antibiotic macrolides., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Digital dental technology is increasingly becoming an integral part of the modern orthodontic practice. The accuracy of digitally articulated models is critical when developing orthodontic treatment plans., Objective: to determine the accuracy of model articulation generated by extraoral and intraoral scanners., Design: One extraoral scanner with a wax (EO W ) or vinyl polysiloxane bite registration (EO VPS ), and three intraoral digital scanners utilizing confocal static (IO CS ), confocal continuous (IO CC ), and blue LED light technologies (IO LED ) were used., Methods: On each scanned image (n = 25 per group), measurements between the maxillary and mandibular molars and canines were performed and then compared to the gold standard values. A deviation of ± 0.5 mm from the gold standard value was considered acceptable. The significance level was kept at 0.05., Results: IO CS and IO CC were accurate for all six interarch measurements. IO LED and EO VPS groups produced the next most accurate articulation of the digital models. EO W group resulted in the least accurate articulation. Also, of the software platforms used, the OrthoCAD™ was found to be the most accurate system for making measurements on digital casts., Conclusions: Only the scanners with the confocal imaging technology produced accurately articulated models. Differences between the scanners may be related to measurement errors inherent to the technologies employed and the software systems used to process the images.

Amplification of the transverse scattered component of stimulated Brillouin scattering (SBS) can contribute to optical damage in the large aperture optics of multi-kJ lasers. Because increased laser bandwidth from optical phase modulation (PM) can suppress SBS, high energy laser amplifiers are injected with PM light. Phase modulation distributes the single-frequency spectrum of a master oscillator laser among individual PM sidebands, so a sufficiently high modulation index β can maintain the fluence for all spectral components below the SBS threshold. To avoid injection of single frequency light in the event of a PM failure, a high-speed PM failsafe system (PMFS) must be employed. Because PM is easily converted to AM, essentially all PM failsafes detect AM, with the one described here employing a novel configuration where optical heterodyne detection converts PM to AM, followed by passive AM power detection. Although the PMFS is currently configured for continuous monitoring, it can also detect PM for pulse durations ≥2 ns and could be modified to accommodate shorter pulses. This PMFS was deployed on the Z-Beamlet Laser (ZBL) at Sandia National Laboratories, as required by an energy upgrade to support programs at Sandia's Z Facility such as magnetized liner inertial fusion. Depending on the origin of a PM failure, the PMFS responds in as little as 7 ns. In the event of an instantaneous failure during initiation of a laser shot, this response time translates to a 30-50 ns margin of safety by blocking a pulse from leaving ZBL's regenerative amplifier, which prevents injection of single frequency light into the main amplification chain. The performance of the PMFS, without the need for operator interaction, conforms to the principles of engineered safety.

X-ray diffraction measurements to characterize phase transitions of dynamically compressed high-Z matter at Mbar pressures require both sufficient photon energy and fluence to create data with high fidelity in a single shot. Large-scale laser systems can be used to generate x-ray sources above 10 keV utilizing line radiation of mid-Z elements. However, the laser-to-x-ray energy conversion efficiency at these energies is low, and thermal x-rays or hot electrons result in unwanted background. We employ polycapillary x-ray lenses in powder x-ray diffraction measurements using solid target x-ray emission from either the Z-Beamlet long-pulse or the Z-Petawatt (ZPW) short-pulse laser systems at Sandia National Laboratories. Polycapillary lenses allow for a 100-fold fluence increase compared to a conventional pinhole aperture while simultaneously reducing the background significantly. This enables diffraction measurements up to 16 keV at the few-photon signal level as well as diffraction experiments with ZPW at full intensity.

Crystal x-ray imaging is frequently used in inertial confinement fusion and laser-plasma interaction applications as it has advantages compared to pinhole imaging, such as higher signal throughput, better achievable spatial resolution, and chromatic selection. However, currently used x-ray detectors are only able to obtain a single time resolved image per crystal. The dilation aided single-line-of-sight x-ray camera described here was designed for the National Ignition Facility (NIF) and combines two recent diagnostic developments, the pulse dilation principle used in the dilation x-ray imager and a ns-scale multi-frame camera that uses a hold and readout circuit for each pixel. This enables multiple images to be taken from a single-line-of-sight with high spatial and temporal resolution. At the moment, the instrument can record two single-line-of-sight images with spatial and temporal resolution of 35 μ m and down to 35 ps, respectively, with a planned upgrade doubling the number of images to four. Here we present the dilation aided single-line-of-sight camera for the NIF, including the x-ray characterization measurements obtained at the COMET laser, as well as the results from the initial timing shot on the NIF.

The first crystal structure of a class VII P450, CYP116B46 from Tepidiphilus thermophilus, has been solved at 1.9 Å resolution. The structure reveals overall conservation of the P450-fold and a water conduit around the I-helix. Active site residues have been identified and sequence comparisons have been made with other class VII enzymes. A structure similarity search demonstrated that the P450-TT structure is similar to enzymes capable of oxy-functionalization of fatty acids, terpenes, macrolides, steroids and statins. The insight gained from solving this structure will provide a guideline for future engineering and modelling studies on this catalytically promiscuous class of enzymes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cytochrome P450 monooxygenases are able to catalyse a range of synthetically challenging reactions ranging from hydroxylation and demethylation to sulfoxidation and epoxidation. As such they have great potential for biocatalytic applications but are underutilised due to often-poor expression, stability and solubility in recombinant bacterial hosts. The use of self-sufficient P450 s with fused haem and reductase domains has already contributed heavily to improving catalytic efficiency and simplifying an otherwise more complex multi-component system of P450 and redox partners. Herein, we present a new addition to the class VII family with the cloning, sequencing and characterisation of the self-sufficient CYP116B62 Hal1 from Halomonas sp. NCIMB 172, the genome of which has not yet been sequenced. Hal1 exhibits high levels of expression in a recombinant E. coli host and can be utilised from cell lysate or used in purified form. Hal1 favours NADPH as electron donor and displays a diverse range of activities including hydroxylation, demethylation and sulfoxidation. These properties make Hal1 suitable for future biocatalytic applications or as a template for optimisation through engineering., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Plasmid vectors based on bacteriophage integrases are important tools in molecular microbiology for the introduction of foreign DNA, especially into bacterial species where other systems for genetic manipulation are limited. Site specific integrases catalyze recombination between phage and bacterial attachment sites ( attP and attB , respectively) and the best studied integrases in the actinomycetes are the serine integrases from the Streptomyces bacteriophages ΦC31 and ΦBT1. As this reaction is unidirectional and highly stable, vectors containing phage integrase systems have been used in a number of genetic engineering applications. Plasmids bearing the ΦBT1 integrase have been used to introduce DNA into Streptomyces and Amycolatopsis strains; however, they have not been widely studied in other actinobacterial genera. Here, we show that vectors based on ΦBT1 integrase can stably integrate into the chromosomes of a range of Nocardia species, and that this integration occurs despite the absence of canonical attB sites in these genomes. Furthermore, we show that a ΦBT1 integrase-based vector can insert at multiple pseudo- attB sites within a single strain and we determine the sequence of a pseudo- attB motif. These data suggest that ΦBT1 integrase-based vectors can be used to readily and semi-randomly introduce foreign DNA into the genomes of a range of Nocardia species. However, the precise site of insertion will likely require empirical determination in each species to avoid unexpected off-target effects., Competing Interests: Timothy P Stinear is an Academic Editor for PeerJ.

Since 2000, cases of the neglected tropical disease Buruli ulcer, caused by infection with Mycobacterium ulcerans , have increased 100-fold around Melbourne (population 4.4 million), the capital of Victoria, in temperate southeastern Australia. The reasons for this increase are unclear. Here, we used whole-genome sequence comparisons of 178 M. ulcerans isolates obtained primarily from human clinical specimens, spanning 70 years, to model the population dynamics of this pathogen from this region. Using phylogeographic and advanced Bayesian phylogenetic approaches, we found that there has been a migration of the pathogen from the east end of the state, beginning in the 1980s, 300 km west to the major human population center around Melbourne. This move was then followed by a significant increase in M. ulcerans population size. These analyses inform our thinking around Buruli ulcer transmission and control, indicating that M. ulcerans is introduced to a new environment and then expands, rather than it being from the awakening of a quiescent pathogen reservoir. IMPORTANCE Buruli ulcer is a destructive skin and soft tissue infection caused by Mycobacterium ulcerans and is characterized by progressive skin ulceration, which can lead to permanent disfigurement and long-term disability. Despite the majority of disease burden occurring in regions of West and central Africa, Buruli ulcer is also becoming increasingly common in southeastern Australia. Major impediments to controlling disease spread are incomplete understandings of the environmental reservoirs and modes of transmission of M. ulcerans The significance of our research is that we used genomics to assess the population structure of this pathogen at the Australian continental scale. We have then reconstructed a historical bacterial spread and modeled demographic dynamics to reveal bacterial population expansion across southeastern Australia. These findings provide explanations for the observed epidemiological trends with Buruli ulcer and suggest possible management to control disease spread., (Copyright © 2018 American Society for Microbiology.)

Cytochrome P450 monooxygenases are highly desired biocatalysts owing to their ability to catalyse a wide variety of chemically challenging C-H activation reactions. The CYP102A subfamily of enzymes are natural catalytically self-sufficient proteins consisting of a haem and FMN-FAD reductase domain fused in a single-component system. They catalyse the oxygenation of saturated and unsaturated fatty acids to produce primarily ω-1, ω-2 and ω-3 hydroxy acids. These monooxygenases have potential applications in biotechnology; however, their substrate range is still limited and there is a continued need to add diversity to this class of biocatalysts. Herein, we present the characterisation of two new members of this class of enzymes, CYP102A25 (BMar) from Bacillus marmarensis and CYP102A26 (PHal) from Pontibacillus halophilus, both of which express readily in a recombinant bacterial host. BMar exhibits the highest activity toward myristic acid and shows moderate activity towards unsaturated fatty acids. PHal exhibits broader activity towards mid-chain-saturated (C 14 -C 18 ) and unsaturated fatty acids. Furthermore, PHal shows good regioselectivity for the hydroxylation of myristic acid, targeting the ω-2 position for C-H activation., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This exploratory study examines the impact of auditory status, gender, and prior sexual victimization on attitudes and behaviors related to date rape among undergraduate college students (n = 3,352) at a private university in the northeast. An abbreviated version of the College Date Rape Attitude and Behavior Survey (Lanier & Elliot, 1997) and the Sexual Experiences Survey (Koss, Gidycz, & Wisniewski, 1987) were employed. Findings indicated that Deaf and hard of hearing (DHH) students were more likely to experience negative sexual experiences than hearing students. No differences were found in rape-supportive attitudes by auditory status. The analysis also found that DHH students were more likely to engage in sexual behaviors that increase the risk of date rape than were hearing students. Finally, significant differences were found regarding gender and prior sexual victimization on rape attitudes and behaviors. Implications and directions for future research are discussed.

The world's freshwater biotas are declining in diversity, range and abundance, more than in other realms, with human appropriation of water. Despite considerable data on the distribution of dams and their hydrological effects on river systems, there are few expansive and long analyses of impacts on freshwater biota. We investigated trends in waterbird communities over 32 years, (1983-2014), at three spatial scales in two similarly sized large river basins, with contrasting levels of water resource development, representing almost a third (29%) of Australia: the Murray-Darling Basin and the Lake Eyre Basin. The Murray-Darling Basin is Australia's most developed river basin (240 dams storing 29,893 GL) while the Lake Eyre Basin is one of the less developed basins (1 dam storing 14 GL). We compared the long-term responses of waterbird communities in the two river basins at river basin, catchment and major wetland scales. Waterbird abundances were strongly related to river flows and rainfall. For the developed Murray-Darling Basin, we identified significant long-term declines in total abundances, functional response groups (e.g., piscivores) and individual species of waterbird (n = 50), associated with reductions in cumulative annual flow. These trends indicated ecosystem level changes. Contrastingly, we found no evidence of waterbird declines in the undeveloped Lake Eyre Basin. We also modelled the effects of the Australian Government buying up water rights and returning these to the riverine environment, at a substantial cost (>3.1 AUD billion) which were projected to partly (18% improvement) restore waterbird abundances, but projected climate change effects could reduce these benefits considerably to only a 1% or 4% improvement, with respective annual recovery of environmental flows of 2,800 GL or 3,200 GL. Our unique large temporal and spatial scale analyses demonstrated severe long-term ecological impact of water resource development on prominent freshwater animals, with implications for global management of water resources., (© 2017 John Wiley & Sons Ltd.)

Many experiments on Sandia National Laboratories' Z Pulsed Power Facility-a 30 MA, 100 ns rise-time, pulsed-power driver-use a monochromatic quartz crystal backlighter system at 1.865 keV (Si He α ) or 6.151 keV (Mn He α ) x-ray energy to radiograph an imploding liner (cylindrical tube) or wire array z-pinch. The x-ray source is generated by the Z-Beamlet laser, which provides two 527-nm, 1 kJ, 1-ns laser pulses. Radiographs of imploding, thick-walled beryllium liners at convergence ratios C R above 15 [C R =r i (0)/r i (t)] using the 6.151-keV backlighter system were too opaque to identify the inner radius r i of the liner with high confidence, demonstrating the need for a higher-energy x-ray radiography system. Here, we present a 7.242 keV backlighter system using a Ge(335) spherical crystal with the Co He α resonance line. This system operates at a similar Bragg angle as the existing 1.865 keV and 6.151 keV backlighters, enhancing our capabilities for two-color, two-frame radiography without modifying the system integration at Z. The first data taken at Z include 6.2-keV and 7.2-keV two-color radiographs as well as radiographs of low-convergence (C R about 4-5), high-areal-density liner implosions.

Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium - M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico -predicted specificity for M. chimaera Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera ., (Copyright © 2017 American Society for Microbiology.)

Addressing the transmission enigma of the neglected disease Buruli ulcer (BU) is a World Health Organization priority. In Australia, we have observed an association between mosquitoes harboring the causative agent, Mycobacterium ulcerans, and BU. Here we tested a contaminated skin model of BU transmission by dipping the tails from healthy mice in cultures of the causative agent, Mycobacterium ulcerans. Tails were exposed to mosquito (Aedes notoscriptus and Aedes aegypti) blood feeding or punctured with sterile needles. Two of 12 of mice with M. ulcerans contaminated tails exposed to feeding A. notoscriptus mosquitoes developed BU. There were no mice exposed to A. aegypti that developed BU. Eighty-eight percent of mice (21/24) subjected to contaminated tail needle puncture developed BU. Mouse tails coated only in bacteria did not develop disease. A median incubation time of 12 weeks, consistent with data from human infections, was noted. We then specifically tested the M. ulcerans infectious dose-50 (ID50) in this contaminated skin surface infection model with needle puncture and observed an ID50 of 2.6 colony-forming units. We have uncovered a biologically plausible mechanical transmission mode of BU via natural or anthropogenic skin punctures.

P450 monooxygenases are able to catalyze the highly regio- and stereoselective oxidations of many organic molecules. However, the scale-up of such bio-oxidations remains challenging due to the often-low activity, level of expression and stability of P450 biocatalysts. Despite these challenges they are increasingly desirable as recombinant biocatalysts, particularly for the production of drug metabolites. Diclofenac is a widely used anti-inflammatory drug that is persistent in the environment along with the 4'- and 5-hydroxy metabolites. Here we have used the self-sufficient P450 RhF (CYP116B2) from Rhodococcus sp. in a whole cell system to reproducibly catalyze the highly regioselective oxidation of diclofenac to 5-hydroxydiclofenac. The product is a human metabolite and as such is an important standard for environmental and toxicological analysis. Furthermore, access to significant quantities of 5-hydroxydiclofenac has allowed us to demonstrate further oxidative degradation to the toxic quinoneimine product. Our studies demonstrate the potential for gram-scale production of human drug metabolites through recombinant whole cell biocatalysis., (Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A novel x-ray imager, which takes time-resolved gated images along a single line-of-sight, has been successfully implemented at the National Ignition Facility (NIF). This Gated Laser Entrance Hole diagnostic, G-LEH, incorporates a high-speed multi-frame CMOS x-ray imager developed by Sandia National Laboratories to upgrade the existing Static X-ray Imager diagnostic at NIF. The new diagnostic is capable of capturing two laser-entrance-hole images per shot on its 1024 × 448 pixels photo-detector array, with integration times as short as 1.6 ns per frame. Since its implementation on NIF, the G-LEH diagnostic has successfully acquired images from various experimental campaigns, providing critical new information for understanding the hohlraum performance in inertial confinement fusion (ICF) experiments, such as the size of the laser entrance hole vs. time, the growth of the laser-heated gold plasma bubble, the change in brightness of inner beam spots due to time-varying cross beam energy transfer, and plasma instability growth near the hohlraum wall.

Enzymes have the potential to catalyse a wide variety of chemical reactions. They are increasingly being sought as environmentally friendly and cost-effective alternatives to conventional catalysts used in industries ranging from bioremediation to applications in medicine and pharmaceutics. Despite the benefits, they are not without their limitations. Many naturally occurring enzymes are not suitable for use outside of their native cellular environments. However, protein engineering can be used to generate enzymes tailored for specific industrial applications. Directed evolution is particularly useful and can be employed even when lack of structural information impedes the use of rational design. The aim of this review is to provide an overview of current industrial applications of enzyme technology and to show how directed evolution can be used to modify and to enhance enzyme properties. This includes a brief discussion on library generation and a more detailed focus on library screening methods, which are critical to any directed evolution experiment., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Conductive and electroactive polymers have the potential to enhance engineered cardiac tissue function. In this study, an interpenetrating network of the electrically-conductive polymer polypyrrole (PPy) was grown within a matrix of flexible polycaprolactone (PCL) and evaluated as a platform for directing the formation of functional cardiac cell sheets. PCL films were either treated with sodium hydroxide to render them more hydrophilic and enhance cell adhesion or rendered electroactive with PPy grown via chemical polymerization yielding PPy-PCL that had a resistivity of 1.0 ± 0.4 kΩ cm, which is similar to native cardiac tissue. Both PCL and PPy-PCL films supported cardiomyocyte attachment; increasing the duration of PCL pre-treatment with NaOH resulted in higher numbers of adherent cardiomyocytes per unit area, generating cell densities which were more similar to those on PPy-PCL films (1568 ± 126 cells mm(-2), 2880 ± 439 cells mm(-2), 3623 ± 456 cells mm(-2) for PCL with 0, 24, 48 h of NaOH pretreatment, respectively; 2434 ± 166 cells mm(-2) for PPy-PCL). When cardiomyocytes were cultured on the electrically-conductive PPy-PCL, more cells were observed to have peripheral localization of the gap junction protein connexin-43 (Cx43) as compared to cells on NaOH-treated PCL (60.3 ± 4.3% vs. 46.6 ± 5.7%). Cx43 gene expression remained unchanged between materials. Importantly, the velocity of calcium wave propagation was faster and calcium transient duration was shorter for cardiomyocyte monolayers on PPy-PCL (1612 ± 143 μm/s, 910 ± 63 ms) relative to cells on PCL (1129 ± 247 μm/s, 1130 ± 20 ms). In summary, PPy-PCL has demonstrated suitability as an electrically-conductive substrate for culture of cardiomyocytes, yielding enhanced functional properties; results encourage further development of conductive substrates for use in differentiation of stem cell-derived cardiomyocytes and cardiac tissue engineering applications., Statement of Significance: Current conductive materials for use in cardiac regeneration are limited by cytotoxicity or cost in implementation. In this manuscript, we demonstrate for the first time the application of a biocompatible, conductive polypyrrole-polycaprolactone film as a platform for culturing cardiomyocytes for cardiac regeneration. This study shows that the novel conductive film is capable of enhancing cell-cell communication through the formation of connexin-43, leading to higher velocities for calcium wave propagation and reduced calcium transient durations among cultured cardiomyocyte monolayers. Furthermore, it was demonstrated that chemical modification of polycaprolactone through alkaline-mediated hydrolysis increased overall cardiomyocyte adhesion. The results of this study provide insight into how cardiomyocytes interact with conductive substrates and will inform future research efforts to enhance the functional properties of cardiomyocytes, which is critical for their use in pharmaceutical testing and cell therapy., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes., Methodology: We compared the number and location of single nucleotide polymorphisms (SNPs) identified by genomic screening between four pairs of Mycobacterium ulcerans isolates collected at the time of first diagnosis and at recurrence, derived from a collection of almost 5000 well characterized clinical samples from one BU treatment center in Benin., Principal Findings: The findings suggest that after surgical treatment-without antibiotics-the second episodes were due to relapse rather than reinfection. Since specific antibiotics were introduced for the treatment of BU, the one patient with a culture available from both disease episodes had M. ulcerans isolates with a genomic distance of 20 SNPs, suggesting the patient was most likely reinfected rather than having a relapse., Conclusions: To our knowledge, this study is the first to study recurrences in M. ulcerans using NGS, and to identify exogenous reinfection as causing a recurrence of BU. The occurrence of reinfection highlights the contribution of ongoing exposure to M. ulcerans to disease recurrence, and has implications for vaccine development.

We report a case of chronic pulmonary multi-drug-resistant tuberculosis. Despite 14 years of treatment, Mycobacterium tuberculosis was persistently isolated from sputum. Following treatment cessation the patient remained well, although M. tuberculosis was isolated from sputum for a further 8 years. Genome sequencing of eight serial M. tuberculosis isolates cultured between 1991 and 2011 revealed 17 mutations (0.8 mutations per genome year - 1 ). Eight of these were persisting mutations and only two mutations were detected in the 7 years following cessation of treatment in 2004. In four isolates there were mixed alleles, suggesting the likely presence of bacterial subpopulations. The initial 1991 isolate demonstrated genotypic resistance to isoniazid ( katG W91R), rifampicin ( rpoB S531L), ethambutol ( embB M306V), streptomycin ( gidB L16R), quinolones ( gyrA S95T) and P-aminosalicylic acid ( thyA T202A). Subsequent resistance mutations developed for pyrazinamide ( pncA I31F) and ethionamide ( ethA frameshift). Such information might have been instructive when developing a treatment regimen. In retrospect and with the benefit of high-resolution genomic hindsight we were able to determine that the patient received only one or two active anti-tuberculous agents for most of their treatment. Additionally, mutations in bacA and Rv2326c were detected, which may have contributed to the persistent but mild disease course. BacA is likely to be associated with maintenance of chronic infection and Rv2326c with a decreased bacterial metabolic state. These results expand our understanding of M. tuberculosis evolution during human infection and underline the link between antibiotic resistance and clinical persistence.

Imaging systems that include a specific source, imaging concept, geometry, and detector have unique properties such as signal-to-noise ratio, dynamic range, spatial resolution, distortions, and contrast. Some of these properties are inherently connected, particularly dynamic range and spatial resolution. It must be emphasized that spatial resolution is not a single number but must be seen in the context of dynamic range and consequently is better described by a function or distribution. We introduce the "dynamic granularity" G(dyn) as a standardized, objective relation between a detector's spatial resolution (granularity) and dynamic range for complex imaging systems in a given environment rather than the widely found characterization of detectors such as cameras or films by themselves. This relation can partly be explained through consideration of the signal's photon statistics, background noise, and detector sensitivity, but a comprehensive description including some unpredictable data such as dust, damages, or an unknown spectral distribution will ultimately have to be based on measurements. Measured dynamic granularities can be objectively used to assess the limits of an imaging system's performance including all contributing noise sources and to qualify the influence of alternative components within an imaging system. This article explains the construction criteria to formulate a dynamic granularity and compares measured dynamic granularities for different detectors used in the X-ray backlighting scheme employed at Sandia's Z-Backlighter facility.

Partner violence is a pervasive public health concern that has received significant attention over the past three decades. Although a number of studies have reported that college students who are Deaf or hard of hearing are at an increased risk of experiencing partner violence compared with their hearing counterparts, little is known about partner violence perpetration among college students who are Deaf or hard of hearing. Furthermore, beyond disability, studies examining partner violence among students with disabilities tend to ignore other potential risk factors that may increase the risk of partner violence as a victim and/or a perpetrator. This exploratory study examines the extent of partner violence among male and female college students by auditory status and the relationship between experiencing and perpetrating partner abuse (i.e., physical abuse and psychological abuse) and child maltreatment (i.e., witnessing abuse and experiencing child physical abuse). The study also examines gender differences in the relationship between child maltreatment and physical and psychological abuse victimization and perpetration. Data were collected from a sample of approximately 680 college students at a northeastern university. Findings indicate that having witnessed interparental abuse as a child was only significant for being an adult victim of physical abuse. Having been a child victim of parental abuse was not significant for any of the abuse measures. Gender was only significant for being an adult victim of physical abuse. Deaf students were significantly more likely to report all abuse measures. Implications and directions for further research are discussed., (© The Author(s) 2014.)

We present calculations for the field of view (FOV), image fluence, image monochromaticity, spectral acceptance, and image aberrations for spherical crystal microscopes, which are used as self-emission imaging or backlighter systems at large-scale high energy density physics facilities. Our analytic results are benchmarked with ray-tracing calculations as well as with experimental measurements from the 6.151 keV backlighter system at Sandia National Laboratories. The analytic expressions can be used for x-ray source positions anywhere between the Rowland circle and object plane. This enables quick optimization of the performance of proposed but untested, bent-crystal microscope systems to find the best compromise between FOV, image fluence, and spatial resolution for a particular application.

Efforts to control the spread of Buruli ulcer--an emerging ulcerative skin infection caused by Mycobacterium ulcerans--have been hampered by our poor understanding of reservoirs and transmission. To help address this issue, we compared whole genomes from 18 clinical M. ulcerans isolates from a 30 km2 region within the Asante Akim North District, Ashanti region, Ghana, with 15 other M. ulcerans isolates from elsewhere in Ghana and the surrounding countries of Ivory Coast, Togo, Benin and Nigeria. Contrary to our expectations of finding minor DNA sequence variations among isolates representing a single M. ulcerans circulating genotype, we found instead two distinct genotypes. One genotype was closely related to isolates from neighbouring regions of Amansie West and Densu, consistent with the predicted local endemic clone, but the second genotype (separated by 138 single nucleotide polymorphisms [SNPs] from other Ghanaian strains) most closely matched M. ulcerans from Nigeria, suggesting another introduction of M. ulcerans to Ghana, perhaps from that country. Both the exotic genotype and the local Ghanaian genotype displayed highly restricted intra-strain genetic variation, with less than 50 SNP differences across a 5.2 Mbp core genome within each genotype. Interestingly, there was no discernible spatial clustering of genotypes at the local village scale. Interviews revealed no obvious epidemiological links among BU patients who had been infected with identical M. ulcerans genotypes but lived in geographically separate villages. We conclude that M. ulcerans is spread widely across the region, with multiple genotypes present in any one area. These data give us new perspectives on the behaviour of possible reservoirs and subsequent transmission mechanisms of M. ulcerans. These observations also show for the first time that M. ulcerans can be mobilized, introduced to a new area and then spread within a population. Potential reservoirs of M. ulcerans thus might include humans, or perhaps M. ulcerans-infected animals such as livestock that move regularly between countries.

Mycobacterium ulcerans causes Buruli ulcer (BU), a debilitating infection of subcutaneous tissue. There is a WHO-recommended antibiotic treatment requiring an 8-week course of streptomycin and rifampicin. This regime has revolutionized the treatment of BU but there are problems that include reliance on daily streptomycin injections and side effects such as ototoxicity. Trials of all-oral treatments for BU show promise but additional drug combinations that make BU treatment safer and shorter would be welcome. Following on from reports that avermectins have activity against Mycobacterium tuberculosis, we tested the in-vitro efficacy of ivermectin and moxidectin on M. ulcerans. We observed minimum inhibitory concentrations of 4-8 μg/ml and time-kill assays using wild type and bioluminescent M. ulcerans showed a significant dose-dependent reduction in M. ulcerans viability over 8-weeks. A synergistic killing-effect with rifampicin was also observed. Avermectins are well tolerated, widely available and inexpensive. Based on our in vitro findings we suggest that avermectins should be further evaluated for the treatment of BU.

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

Directed evolution is a common tool employed to generate enzymes suitable for industrial use. High thermal stability is often advantageous or even a requirement for biocatalysts, as such the evolution of protein stability is of practical as well as academic interest. Even when evolving enzymes for new or improved catalytic functions, stability is an important factor since it can limit the accumulation rate and number of desired active site mutations. Dienelactone hydrolase, a small monomeric protein, has been previously evolved via a three-stage process to possess enhanced activity and specificity toward non-physiological substrates. In addition to seven active site mutations there were three surface mutations that were thought to increase the stability of the enzyme and compensate for the destabilizing active site mutations. Here, the individual influence of the three surface mutations--Q110L, Y137C and N154D--on the thermal and chemical stability of DLH has been assessed. While the Q110L and N154D mutations improved the thermal stability, the influence of the Y137C mutation was more complex. Individually it was destabilizing both thermally and chemically, but when in the presence of the Q110L and N154D mutations its effect was neutralized in relation to thermal but not chemical stability. In the context of a directed evolution experiment, these compensatory surface mutations play important roles. However, our results show that detrimental mutations can arise, thus the simultaneous monitoring of stability changes while evolving enzymes for enhanced catalytic properties can be beneficial.

This Letter presents results from the first fully integrated experiments testing the magnetized liner inertial fusion concept [S. A. Slutz et al., Phys. Plasmas 17, 056303 (2010)], in which a cylinder of deuterium gas with a preimposed 10 Taxial magnetic field is heated by Z beamlet, a 2.5 kJ, 1 TW laser, and magnetically imploded by a 19 MA, 100 ns rise time current on the Z facility. Despite a predicted peak implosion velocity of only 70 km = s, the fuel reaches a stagnation temperature of approximately 3 keV, with T(e) ≈ T(i), and produces up to 2 x 10(12) thermonuclear deuterium-deuterium neutrons. X-ray emission indicates a hot fuel region with full width at half maximum ranging from 60 to 120 μm over a 6 mm height and lasting approximately 2 ns. Greater than 10(10) secondary deuterium-tritium neutrons were observed, indicating significant fuel magnetization given that the estimated radial areal density of the plasma is only 2 mg = cm(2).

Magnetizing the fuel in inertial confinement fusion relaxes ignition requirements by reducing thermal conductivity and changing the physics of burn product confinement. Diagnosing the level of fuel magnetization during burn is critical to understanding target performance in magneto-inertial fusion (MIF) implosions. In pure deuterium fusion plasma, 1.01 MeV tritons are emitted during deuterium-deuterium fusion and can undergo secondary deuterium-tritium reactions before exiting the fuel. Increasing the fuel magnetization elongates the path lengths through the fuel of some of the tritons, enhancing their probability of reaction. Based on this feature, a method to diagnose fuel magnetization using the ratio of overall deuterium-tritium to deuterium-deuterium neutron yields is developed. Analysis of anisotropies in the secondary neutron energy spectra further constrain the measurement. Secondary reactions also are shown to provide an upper bound for the volumetric fuel-pusher mix in MIF. The analysis is applied to recent MIF experiments [M. R. Gomez et al., Phys. Rev. Lett. 113, 155003 (2014)] on the Z Pulsed Power Facility, indicating that significant magnetic confinement of charged burn products was achieved and suggesting a relatively low-mix environment. Both of these are essential features of future ignition-scale MIF designs.

Advancements have been made in the diagnostic techniques to measure accurately the total radiated x-ray yield and power from z-pinch implosion experiments at the Z machine with high accuracy. The Z machine is capable of outputting 2 MJ and 330 TW of x-ray yield and power, and accurately measuring these quantities is imperative. We will describe work over the past several years which include the development of new diagnostics, improvements to existing diagnostics, and implementation of automated data analysis routines. A set of experiments on the Z machine were conducted in which the load and machine configuration were held constant. During this shot series, it was observed that the total z-pinch x-ray emission power determined from the two common techniques for inferring the x-ray power, a Kimfol filtered x-ray diode diagnostic and the total power and energy diagnostic, gave 449 TW and 323 TW, respectively. Our analysis shows the latter to be the more accurate interpretation. More broadly, the comparison demonstrates the necessity to consider spectral response and field of view when inferring x-ray powers from z-pinch sources.

Dienelactone hydrolase (DLH) is a monomeric protein with a simple α/β-hydrolase fold structure. It readily crystallizes in space group P2₁2₁2₁ from either a phosphate or ammonium sulfate precipitation buffer. Here, the structure of DLH at 1.85 Å resolution crystallized in space group C2 with two molecules in the asymmetric unit is reported. When crystallized in space group P2₁2₁2₁ DLH has either phosphates or sulfates bound to the protein in crucial locations, one of which is located in the active site, preventing substrate/inhibitor binding. Another is located on the surface of the enzyme coordinated by side chains from two different molecules. Crystallization in space group C2 from a sodium citrate buffer results in new crystallographic protein-protein interfaces. The protein backbone is highly similar, but new crystal contacts cause changes in side-chain orientations and in loop positioning. In regions not involved in crystal contacts, there is little change in backbone or side-chain configuration. The flexibility of surface loops and the adaptability of side chains are important factors enabling DLH to adapt and form different crystal lattices.

In non-small cell lung carcinoma (NSCLC), the immune system fails to eradicate established tumors partly due to the induction of immune tolerance within the tumor microenvironment. Plasmacytoid dendritic cells (pDCs) play critical roles in regulating the immune system. In this study, we investigated pDCs in the peripheral blood of NSCLC. CD4 + CD123 + BDCA2+ pDCs were tested from peripheral blood mononuclear cells in 52 NSCLC patients and 52 healthy controls by flow cytometry. Results revealed that proportion of pDCs was significantly increased in cases than in controls (0.52 ± 0.07% versus 0.21 ± 0.02%, p < 0.001), whereas myeloid dendritic cells (mDCs) did not present any obvious difference between patients and healthy donors (0.25 ± 0.04 % versus 0.18 ± 0.02%, p = 0.120). We further studied pDCs in NSCLC patients with different clinical stages. Data showed that cases with higher stages (III/IV) had elevated level of pDCs than those with lower stages (I/II) (0.65 ± 0.09% versus 0.25 ± 0.07%, p = 0.006). In addition, the amount of pDCs was identified to be associated with squamous cell carcinoma, one of the major subtypes of NSCLC. Interestingly, we observed that smoking patients presented significantly elevated pDCs than those non-smokers (0.63 ± 0.09% versus 0.22 ± 0.05%, p = 0.008). These data suggested that pDCs may be closely involved in the pathogenesis of NSCLC and may predict the progression of the disease.

Background: The community-associated methicillin-resistant S. aureus (CA-MRSA) ST93 clone is becoming dominant in Australia and is clinically highly virulent. In addition, sepsis and skin infection models demonstrate that ST93 CA-MRSA is the most virulent global clone of S. aureus tested to date. While the determinants of virulence have been studied in other clones of CA-MRSA, the basis for hypervirulence in ST93 CA-MRSA has not been defined., Results: Here, using a geographically and temporally dispersed collection of ST93 isolates we demonstrate that the ST93 population hyperexpresses key CA-MRSA exotoxins, in particular α-hemolysin, in comparison to other global clones. Gene deletion and complementation studies, and virulence comparisons in a murine skin infection model, showed unequivocally that increased expression of α-hemolysin is the key staphylococcal virulence determinant for this clone. Genome sequencing and comparative genomics of strains with divergent exotoxin profiles demonstrated that, like other S. aureus clones, the quorum sensing agr system is the master regulator of toxin expression and virulence in ST93 CA-MRSA. However, we also identified a previously uncharacterized AraC/XylS family regulator (AryK) that potentiates toxin expression and virulence in S. aureus., Conclusions: These data demonstrate that hyperexpression of α-hemolysin mediates enhanced virulence in ST93 CA-MRSA, and additional control of exotoxin production, in particular α-hemolysin, mediated by regulatory systems other than agr have the potential to fine-tune virulence in CA-MRSA.

Epstein-Barr Virus (EBV) causes infectious mononucleosis and establishes lifelong infection associated with cancer and autoimmune disease. To better understand immunity to EBV, we performed a prospective study of natural infection in healthy humans. Transcriptome analysis defined a striking and reproducible expression profile during acute infection but no lasting gene changes were apparent during latent infection. Comparing the EBV response profile to multiple other acute viral infections, including influenza A (influenza), respiratory syncytial virus (RSV), human rhinovirus (HRV), attenuated yellow fever virus (YFV), and Dengue fever virus (DENV), revealed similarity only to DENV. The signature shared by EBV and DENV was also present in patients with hemophagocytic syndromes, suggesting these two viruses cause uncontrolled inflammatory responses. Interestingly, while EBV induced a strong type I interferon response, a subset of interferon induced genes, including MX1, HERC5, and OAS1, were not upregulated, suggesting a mechanism by which viral antagonism of immunity results in a profound inflammatory response. These data provide an important first description of the response to a natural herpesvirus infection in humans.

Purpose: The University of Missouri School of Medicine developed the Summer Community Program through which rising second-year medical students work alongside rural, community-based physician preceptors. This program is part of a comprehensive, longitudinal pipeline designed to increase student interest in rural practice. The authors describe the Summer Community Program, explain changes in students' perceptions of rural practice and rural lifestyle post program, and report participants' specialty choices and first practice locations., Method: The authors analyzed 229 participant responses (1996-2010) to pre- and postexperience questionnaires focused on perceptions of rural practice and lifestyle. The authors calculated the likelihood of participants matching into primary care compared with nonparticipants and analyzed participants' first practice locations., Results: After the experience, participants' perceptions toward rural practice and lifestyle changed favorably, and 72% (n=208) reported more interest in rural practice. Compared with nonparticipants, summer participants were more likely to enter a primary care residency (relative risk [RR]=1.31; 95% confidence interval [CI]: 1.12-1.50) and twice as likely to choose specifically family medicine (RR=2.21; 95% CI: 1.68-2.88). Forty-six percent (n=78) of participants chose rural locations for their first practices., Conclusions: This program has positively influenced students' perceptions of rural practice and lifestyle and increased their interest in rural practice. Participants entered primary care and family medicine residencies at higher rates than nonparticipants, and nearly half started their medical practices in rural locations. Replicating this program may increase interest in rural medicine and address rural physician workforce needs.

Mycolactones are polyketide-derived lipid virulence factors made by the slow-growing human pathogen, Mycobacterium ulcerans. Three unusually large and homologous plasmid-borne genes (mlsA1: 51 kb, mlsB: 42 kb and mlsA2: 7 kb) encode the mycolactone type I polyketide synthases (PKS). The extreme size and low sequence diversity of these genes has posed significant barriers for exploration of the genetic and biochemical basis of mycolactone synthesis. Here, we have developed a truncated, more tractable 3-module version of the 18-module mycolactone PKS and we show that this engineered PKS functions as expected in the natural host M. ulcerans to produce an additional polyketide; a triketide lactone (TKL). Cell fractionation experiments indicated that this 3-module PKS and the putative accessory enzymes encoded by mup045 and mup038 associated with the mycobacterial cell wall, a finding supported by confocal microscopy. We then assessed the capacity of the faster growing, Mycobacterium marinum to harbor and express the 3-module Mls PKS and accessory enzymes encoded by mup045 and mup038. RT-PCR, immunoblotting, and cell fractionation experiments confirmed that the truncated Mls PKS multienzymes were expressed and also partitioned with the cell wall material in M. marinum. However, this heterologous host failed to produce TKL. The systematic deconstruction of the mycolactone PKS presented here suggests that the Mls multienzymes are necessary but not sufficient for mycolactone synthesis and that synthesis is likely to occur (at least in part) within the mycobacterial cell wall. This research is also the first proof-of-principle demonstration of the potential of this enzyme complex to produce tailored small molecules through genetically engineered rearrangements of the Mls modules.

Detailed spectroscopic diagnostics of the stagnating plasma in two disparate z pinches allow, for the first time, the examination of the plasma properties within a 1D shock wave picture, demonstrating a good agreement with this picture. The conclusion is that for a wide range of imploding-plasma masses and current amplitudes, in experiments optimizing non-Planckian hard radiation yields, contrary to previous descriptions the stagnating plasma pressure is balanced by the implosion pressure, and the radiation energy is provided by the imploding-plasma kinetic energy, rather than by the magnetic-field pressure and magnetic-field-energy dissipation, respectively.

Vancomycin-intermediate Staphylococcus aureus (VISA) strains often arise by mutations in the essential two-component regulator walKR; however their impact on walKR function has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptible S. aureus (VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS256 insertions in the walKR 5' untranslated region (5' UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction in walKR gene expression in the IS256 mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS256 insertions had replaced two SigA-like walKR promoters with weaker, hybrid promoters. Removal of IS256 reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation of ssaA but no change in expression of sak and sceD in both IS256 mutants. Whole-genome sequencing of the two mutants revealed an additional IS256 insertion within agrC for one mutant, and we confirmed that this mutation abolished agr function. These data provide the first substantial analysis of walKR promoter function and show that prolonged vancomycin exposure can result in VISA through an IS256-mediated reduction in walKR expression; however, the mechanisms by which this occurs remain to be determined.

Next-generation sequencing (NGS) of bacterial genomes has recently become more accessible and is now available to the routine diagnostic microbiology laboratory. However, questions remain regarding its feasibility, particularly with respect to data analysis in nonspecialist centers. To test the applicability of NGS to outbreak investigations, Ion Torrent sequencing was used to investigate a putative multidrug-resistant Escherichia coli outbreak in the neonatal unit of the Mercy Hospital for Women, Melbourne, Australia. Four suspected outbreak strains and a comparator strain were sequenced. Genome-wide single nucleotide polymorphism (SNP) analysis demonstrated that the four neonatal intensive care unit (NICU) strains were identical and easily differentiated from the comparator strain. Genome sequence data also determined that the NICU strains belonged to multilocus sequence type 131 and carried the bla(CTX-M-15) extended-spectrum beta-lactamase. Comparison of the outbreak strains to all publicly available complete E. coli genome sequences showed that they clustered with neonatal meningitis and uropathogenic isolates. The turnaround time from a positive culture to the completion of sequencing (prior to data analysis) was 5 days, and the cost was approximately $300 per strain (for the reagents only). The main obstacles to a mainstream adoption of NGS technologies in diagnostic microbiology laboratories are currently cost (although this is decreasing), a paucity of user-friendly and clinically focused bioinformatics platforms, and a lack of genomics expertise outside the research environment. Despite these hurdles, NGS technologies provide unparalleled high-resolution genotyping in a short time frame and are likely to be widely implemented in the field of diagnostic microbiology in the next few years, particularly for epidemiological investigations (replacing current typing methods) and the characterization of resistance determinants. Clinical microbiologists need to familiarize themselves with these technologies and their applications.

The implosions of initially solid beryllium liners (tubes) have been imaged with penetrating radiography through to stagnation. These novel radiographic data reveal a high degree of azimuthal correlation in the evolving magneto-Rayleigh-Taylor structure at times just prior to (and during) stagnation, providing stringent constraints on the simulation tools used by the broader high energy density physics and inertial confinement fusion communities. To emphasize this point, comparisons to 2D and 3D radiation magnetohydrodynamics simulations are also presented. Both agreement and substantial disagreement have been found, depending on how the liner's initial outer surface finish was modeled. The various models tested, and the physical implications of these models are discussed. These comparisons exemplify the importance of the experimental data obtained.