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Pseudomonas aeruginosa is an opportunistic pathogen causing life-threatening infections. Previously, we showed that elevated calcium (Ca(2+)) levels increase the production of virulence factors in P. aeruginosa. In an effort to characterize the Ca(2+) regulatory network, we identified a Ca(2+)–regulated β-propeller protein, CarP, and showed that expression of the encoding gene is controlled by the Ca(2+)-regulated two-component system CarSR. Here, by using a Galleria melonella model, we showed that CarP plays a role in regulating P. aeruginosa virulence. By using transcriptome sequencing (RNA-Seq), reverse transcription (RT)-PCR, quantitative RT-PCR (RT-qPCR), and promoter fusions, we determined that carP is transcribed into at least two transcripts and regulated by several bacterial and host factors. The transcription of carP is elevated in response to Ca(2+) in P. aeruginosa cystic fibrosis isolates and PAO1 laboratory strain. Elevated Fe(2+) also induces carP. The simultaneous addition of Ca(2+) and Fe(2+) increased the carP promoter activity synergistically, which requires the presence of CarR. In silico analysis of the intergenic sequence upstream of carP predicted recognition sites of RhlR/LasR, OxyR, and LexA, suggesting regulation by quorum sensing (QS) and oxidative stress. In agreement, the carP promoter was activated in response to stationary-phase PAO1 supernatant and required the presence of elevated Ca(2+) and CarR but remained silent in the triple mutant lacking rhlI, lasI, and pqsA synthases. We also showed that carP transcription is regulated by oxidative stress and that CarP contributes to P. aeruginosa Ca(2+)-dependent H(2)O(2) tolerance. The multifactorial regulation of carP suggests that CarP plays an important role in P. aeruginosa adaptations to host environments. IMPORTANCE P. aeruginosa is a human pathogen causing life-threatening infections. It is particularly notorious for its ability to adapt to diverse environments within the host. Understanding the signals and the signaling pathways enabling P. aeruginosa adaptation is imperative for developing effective therapies to treat infections caused by this organism. One host signal of particular importance is calcium. Previously, we identified a component of the P. aeruginosa calcium-signaling network, CarP, whose expression is induced by elevated levels of calcium. Here, we show that carP plays an important role in P. aeruginosa virulence and is upregulated in P. aeruginosa strains isolated from sputa of patients with cystic fibrosis. We also identified several bacterial and host factors that regulate the transcription of carP. Such multifactorial regulation highlights the interconnectedness between regulatory circuits and, together with the pleotropic effect of CarP on virulence, suggests the importance of this protein in P. aeruginosa adaptations to the host.

Biofilms are differentiated microbial communities held together by an extracellular matrix. μCT X-ray revealed structured mineralized areas within biofilms of lung pathogens belonging to two distant phyla - the proteobacteria

In nature, bacteria reside in biofilms - multicellular differentiated communities held together by extracellular matrix. In this work, we identified a novel subpopulation essential for biofilm formation – mineral-forming cells. This subpopulation contains an intracellular calcium-accumulating niche, in which the formation of a calcium carbonate mineral is initiated. As the biofilm colony develops, this mineral grows in a controlled manner, forming a functional macrostructure that serves the entire community.The molecular mechanisms promoting calcite scaffold formation were conserved between three distant phyla – the Gram-positive Bacillus subtilis, Gram-negative Pseudomonas aeruginosa and the actinobacterium Mycobacterium abscessus. Biofilm development of all three species was similarly impaired by inhibition of calcium uptake and carbonate accumulation. Moreover, chemical inhibition and mutations targeting mineralization both significantly reduced the attachment of P. aeruginosa to the lung, as well as the subsequent damage inflicted by biofilms to lung tissues, and restored their sensitivity to antibiotics.The evolutionary conserved cellular pathway controlling the fundamental feature of biofilm development uncovered in this work offers novel druggable targets for antibiotics to combat otherwise untreatable biofilm infections.

Summary Dispersal is a key step in land plant life cycles, usually via formation of spores or seeds. Regulation of spore‐ or seed‐germination allows control over the timing of transition from one generation to the next, enabling plant dispersal. A combination of environmental and genetic factors determines when seed germination occurs. Endogenous hormones mediate this decision in response to the environment. Less is known about how spore germination is controlled in earlier‐evolving nonseed plants.Here, we present an in‐depth analysis of the environmental and hormonal regulation of spore germination in the model bryophyte Physcomitrella patens (Aphanoregma patens).Our data suggest that the environmental signals regulating germination are conserved, but also that downstream hormone integration pathways mediating these responses in seeds were acquired after the evolution of the bryophyte lineage. Moreover, the role of abscisic acid and diterpenes (gibberellins) in germination assumed much greater importance as land plant evolution progressed.We conclude that the endogenous hormone signalling networks mediating germination in response to the environment may have evolved independently in spores and seeds. This paves the way for future research about how the mechanisms of plant dispersal on land evolved.

Abstract Lockheed Martin has developed the capability to conduct AUV-based structural survey and post-hurricane platform inspection using 3D mapping and change detection with a 3D sonar. Successful validation trials for this technology were conducted with the Marlin® AUV during the summer of 2011 and the first commercial operations were conducted in the summer of 2012. Lockheed Martin Corporation, in partnership with underwater laser developer 3D at Depth LLC, and supported by funding from the Research Partnership to Secure Energy for America (RPSEA), is now extending its revolutionary 3D modeling and change detection to employ a 3D laser sensor, thereby improving model resolution and accuracy by an order of magnitude, from centimeter scale to millimeter scale. AUVs outfitted with 3D Laser imaging systems will provide new, high accuracy tools for subsea integrity management that are currently used extensively in terrestrial applications, including High Definition Scanning (HDS) for close-in inspection of problem areas, and underwater LIDAR (LIght Detection And Ranging) for 3D mapping and inspection of flowlines, risers, and other subsea infrastructure. The scope of this project is to develop and demonstrate the technology required to conduct AUV-based 3D-laser imaging utilizing 3D mapping and change detection. Objectives include demonstration of close-in, high resolution underwater structural inspection, generation of high resolution 3D models of subsea structures, and performance of change detection of flaws or damage against a priori structural models. This paper will detail the results achieved to-date and will highlight the 3D Laser performance improvements over current platform inspection methods, including significant improvements in operating efficiencies and the development of accurate 3D models for use in structural integrity management.

Abstract Lockheed Martin Corporation is conducting a multi-year technology developmentprogram to advance the state of the art of Autonomous Underwater Vehicle (AUV)inspection technologies for the offshore oil & gas industry. The scope ofthis project is to develop and demonstrate AUV technologies for conductingautonomous structural survey and inspection of subsea facilities for a widerange of applications, including pre/post-hurricane inspection of offshoreplatforms, pre/post-decommissioning structural survey, and deepwater facility /riser inspection. This paper will describe the results of Lockheed Martin'srecently completed technology demonstration project, Autonomous Inspection ofSubsea Facilities, including laboratory simulation, local offshore trials, andtechnology validation trials in the Gulf of Mexico against offshore productionplatforms. This project was jointly funded by the Research Partnership toSecure Energy for America (RPSEA), Lockheed Martin and sea trials weresupported by Chevron Energy Technology Company Capabilities demonstrated duringoffshore trials included (1) autonomous real-time three-dimensional (3D)imaging and modeling of an underwater facility, (2) detection and highlightingof changes to the facility in real time, and (3) feature-based navigation, theaiding of the AUV's navigation along its path based on feature detection andrecognition. The paper will describe the results achieved, and will highlightthe performance improvements over current platform inspection methods, including significant improvements in operating efficiencies, and thedevelopment of highly accurate 3D models for use in structural integritymanagement. Finally, the paper will outline the potential benefits of evolvingAUV and sensor technologies for applications such as structural survey, pipeline inspection, subsea facility inspection, and light intervention, including potentially game changing improvements in cost, performance, safetyand reliability that will enable more cost-effective operations in deepwaterand/or remote subsea fields. Introduction Subsea Integrity Management is defined by the Energy Institute Guidelines forthe Management of Integrity of Subsea Facilities as " the management of a subseasystem or asset to ensure that it delivers the design requirements, and doesnot harm life, health or the environment, through the required life." A keyelement in any integrity management program is regular in-service inspections. As the industry moves into deeper and harsher environments, challenges faced byoperators include the high cost of subsea inspection and the limited inspectionintervals available. Inspections provide a snapshot of the structural health ofthe system. Integrity management practices in deepwater fields rely heavily ongeneral visual inspection of subsea equipment. Remotely operated vehicles(ROVs) and divers are the primary means used today to conduct inspections -ROVs exclusively in deepwater (greater than 100-meter water depth) and diversgenerally limited to less than100-meter water depth. In both cases supportvessels larger than 70 to 100 meters with support crews numbering more than 30and with 100+ tons of equipment are required to collect the simplest visualinspection record. The quality and usefulness of the records are highlydependent on the seawater's visual clarity, illumination, camera and recordingequipment, and ROV or diver stability. An ROV inspection of a deepwaterfacility can provide visual evidence of structural degradation, impact damage, corrosion, valve damage, leaks, vibration, and other structural damage (Figure 1). Benchmarking the condition of subsea equipment following installation andtracking its status over time can provide a history of the deterioration rate. Video inspections include: well heads, valve positions, pipeline endterminations (PLETs), pipeline end manifolds (PLEMs), underwater terminationmanifolds (UTMs), flowlines, jumpers, moorings, risers, and associated cablingand equipment on the sea bed. This equipment is often spread over many squarekilometers requiring the support vessel to maneuver in DP mode for days. Inspection speed is totally dependent on the coordinated movement of the ROVand support vessel and the skill of the ROV pilot.

Abstract Integrity Management of deepwater fields requires routine general visual inspections of critical infrastructure. To date the only means of conducting general visual inspection is through the use of ROVs. Deepwater ROV spreads are large and heavy requiring large support vessels with dynamic positioning capability and a significant number of personnel at sea. The capabilities of unmanned underwater vehicles have been enhanced through developments in Autonomous technology progressing to the point that autonomous underwater vehicles can now routinely conduct general visual inspection of subsea facilities. Benefits of Autonomous inspection include:–Reduced cost of operations–Faster inspection–Automatic Change Detection–Georegistered inspection data–Simultaneous operations from a single support vessel–Large standoff distances from the facility being inspected–Increased safety of operations–Reduced environmental impact–Reduced specification requirements on support vessel○Smaller footprint○Dynamic Positioning not required