Evaluating Contingency Construction Methods: A Delphi Study

The purpose of this research was to evaluate which existing RED HORSE vertical construction system was “best” suited for contingency operations and the relative pros/cons/challenges associated with each. RED HORSE units, having existed for over 50 years, currently have 4 active duty squadrons currently employed overseas in multiple military contingency environments. Based on site visits, it was established that the Ultimate Building Machine (UBM)/K-Span, Professionally Engineered Buildings (PEB), and the FrameCAD system are the three most prevalent vertical construction systems/methods in use by RED HORSE. Fourteen Subject Matter Experts (SME) from across the Air Force participated in a Delphi study to evaluate these construction methods and answer multiple rounds of questions. While no one system/method could be conclusively declared “the best”, the Delphi study did generate consensus around the pros, cons, and challenges of each of these contingency construction methods/systems. The results of this study will serve to inform RED HORSE commanders/planner decisions regarding the use of vertical construction methods/systems for current and future contingency operations

Engineering microbes and biology for communicating with electronics

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Repurposing E. coli by Engineering Quorum Sensing and Redox Genetic Circuits

Because cells have the extraordinary ability to sense and respond to even subtle environmental changes by intricately regulating their gene expression patterns, their behaviors can be intentionally “tuned” by altering the state of their environments in a prescribed or rational manner. Rational control of both external and internal molecular stimuli provides a basis for many biotechnological applications including the expression of foreign protein products. This is done by coordinately controlling product synthesis while retaining the cell in a productive state. Quorum sensing (QS), a molecular signaling modality that mediates cell-cell communication, autonomously facilitates both inter- and intra-species gene regulation. This process can be rewired to enable autonomously actuated, but molecularly programmed, genetic control. Recently, even electrical signals, which have long been used to control the most sophisticated of man-made devices, are now employed to alter cell signaling processes enabling computer programmed behavior, particularly in cells suitably engineered to accommodate electrical signals. By minimally engineering these genetic circuits, new applications have emerged for the repurposing of Escherichia coli, from creating innovative sensor concepts to stimulating the emerging field of electrogenetics

Microarray analysis of Pseudomonas aeruginosa reveals induction of pyocin genes in response to hydrogen peroxide

BACKGROUND: Pseudomonas aeruginosa, a pathogen infecting those with cystic fibrosis, encounters toxicity from phagocyte-derived reactive oxidants including hydrogen peroxide during active infection. P. aeruginosa responds with adaptive and protective strategies against these toxic species to effectively infect humans. Despite advances in our understanding of the responses to oxidative stress in many specific cases, the connectivity between targeted protective genes and the rest of cell metabolism remains obscure. RESULTS: Herein, we performed a genome-wide transcriptome analysis of the cellular responses to hydrogen peroxide in order to determine a more complete picture of how oxidative stress-induced genes are related and regulated. Our data reinforce the previous conclusion that DNA repair proteins and catalases may be among the most vital antioxidant defense systems of P. aeruginosa. Our results also suggest that sublethal oxidative damage reduces active and/or facilitated transport and that intracellular iron might be a key factor for a relationship between oxidative stress and iron regulation. Perhaps most intriguingly, we revealed that the transcription of all F-, R-, and S-type pyocins was upregulated by oxidative stress and at the same time, a cell immunity protein (pyocin S2 immunity protein) was downregulated, possibly leading to self-killing activity. CONCLUSION: This finding proposes that pyocin production might be another novel defensive scheme against oxidative attack by host cells

Microarray analysis of toxicogenomic effects of Ortho-phenylphenol in Staphylococcus aureus

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>(<it>S. aureus</it>), is responsible for many infectious diseases, ranging from benign skin infections to life-threatening endocarditis and toxic shock syndrome. Ortho-phenylphenol (OPP) is an antimicrobial agent and an active ingredient of EPA-registered disinfectants with wide human exposure in various agricultural, hospital and veterinary disinfectant products. Despite many uses, an understanding of a cellular response to OPP and it's mechanism of action, targeted genes, and the connectivity between targeted genes and the rest of cell metabolism remains obscure.</p> <p>Results</p> <p>Herein, we performed a genome-wide transcriptome analysis of the cellular responses of <it>S. aureus </it>when exposed to 0.82 mM of OPP for 20 and 60 min. Our data indicated that OPP downregulated the biosynthesis of many amino acids, which are required for protein synthesis. In particular, the genes encoding the enzymes of the diaminopimelate (DAP) pathway which results in lysine biosynthesis were significantly downregualted. Intriguingly, we revealed that the transcription of genes encoding ribosomal proteins was upregulated by OPP and at the same time, the genes encoding iron acquisition and transport were downregulated. The genes encoding virulence factors were upregulated and genes encoding phospholipids were downregulated upon 20 min exposure to OPP.</p> <p>Conclusion</p> <p>By using microarray analysis that enables us to simultaneously and globally examine the complete transcriptome during cellular responses, we have revealed novel information regarding the mode of action of OPP on <it>Staphylococcus</it>: OPP inhibits anabolism of many amino acids and highly downregulates the genes that encode the enzymes involved in the DAP pathway. Lysine and DAP are essential for building up the peptidoglycan cell wall. It was concluded that the mode of action of OPP is similar to the mechanism of action of some antibiotics. The discovery of this phenomenon provides useful information that will benefit further antimicrobial research on <it>S. aureus</it>.</p

Effects of Different Lifting Cadences on Ground Reaction Forces during the Squat Exercise

The purpose of this investigation was to determine the effect of different cadences on the ground reaction force (GRF(sub R)) during the squat exercise. It is known that squats performed with greater acceleration will produce greater inertial forces; however, it is not well understood how different squat cadences affect GRF(sub R). It was hypothesized that faster squat cadences will result in greater peak GRF(sub R). METHODS: Six male subjects (30.8+/-4.4 y, 179.5+/-8.9 cm, 88.8+/-13.3 kg) with previous squat experience performed three sets of three squats using three different cadences (FC = 1 sec descent/1 sec ascent; MC = 3 sec descent/1 sec ascent; SC = 4 sec descent/2 sec ascent) with barbell mass equal to body mass. Ground reaction force was used to calculate inertial force trajectories of the body plus barbell (FI(sub system)). Forces were normalized to body mass. RESULTS: Peak GRF(sub R) and peak FI(sub system) were significantly higher in FC squats compared to MC (p=0.0002) and SC (p=0.0002). Range of GRF(sub R) and FI(sub system) were also significantly higher in FC compared to MC (p<0.05), and MC were significantly higher than SC (p<0.05). DISCUSSION: Faster squat cadences result in significantly greater peak GRF(sub R) due to the inertia of the system. GRF(sub R) was more dependent upon decent cadence than on ascent cadence. PRACTICAL APPLICATION: This study demonstrates that faster squat cadences produce greater ground reaction forces. Therefore, the use of faster squat cadences might enhance strength and power adaptations to long-term resistance exercise training. Key Words: velocity, weight training, resistive exercis

Toxicogenomic response of Pseudomonas aeruginosa to ortho-phenylphenol

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas aeruginosa </it>(<it>P. aeruginosa</it>) is the most common opportunistic pathogen implicated in nosocomial infections and in chronic lung infections in cystic fibrosis patients. Ortho-phenylphenol (OPP) is an antimicrobial agent used as an active ingredient in several EPA registered disinfectants. Despite its widespread use, there is a paucity of information on its target molecular pathways and the cellular responses that it elucidates in bacteria in general and in <it>P. aeruginosa </it>in particular. An understanding of the OPP-driven gene regulation and cellular response it elicits will facilitate more effective utilization of this antimicrobial and possibly lead to the development of more effective disinfectant treatments.</p> <p>Results</p> <p>Herein, we performed a genome-wide transcriptome analysis of the cellular responses of <it>P. aeruginosa </it>exposed to 0.82 mM OPP for 20 and 60 minutes. Our data indicated that OPP upregulated the transcription of genes encoding ribosomal, virulence and membrane transport proteins after both treatment times. After 20 minutes of exposure to 0.82 mM OPP, genes involved in the exhibition of swarming motility and anaerobic respiration were upregulated. After 60 minutes of OPP treatment, the transcription of genes involved in amino acid and lipopolysaccharide biosynthesis were upregulated. Further, the transcription of the ribosome modulation factor (<it>rmf</it>) and an alternative sigma factor (<it>rpo</it>S) of RNA polymerase were downregulated after both treatment times.</p> <p>Conclusion</p> <p>Results from this study indicate that after 20 minutes of exposure to OPP, genes that have been linked to the exhibition of anaerobic respiration and swarming motility were upregulated. This study also suggests that the downregulation of the <it>rmf </it>and <it>rpoS </it>genes may be indicative of the mechanism by which OPP causes decreases in cell viability in <it>P. aeruginosa</it>. Consequently, a protective response involving the upregulation of translation leading to the increased synthesis of membrane related proteins and virulence proteins is possibly induced after both treatment times. In addition, cell wall modification may occur due to the increased synthesis of lipopolysaccharide after 60 minutes exposure to OPP. This gene expression profile can now be utilized for a better understanding of the target cellular pathways of OPP in <it>P. aeruginosa </it>and how this organism develops resistance to OPP.</p

Microbial nar-GFP cell sensors reveal oxygen limitations in highly agitated and aerated laboratory-scale fermentors

Background: Small-scale microbial fermentations are often assumed to be homogeneous, and oxygen limitation due to inadequate micromixing is often overlooked as a potential problem. To assess the relative degree of micromixing, and hence propensity for oxygen limitation, a new cellular oxygen sensor has been developed. The oxygen responsive E. coli nitrate reductase (nar) promoter was used to construct an oxygen reporter plasmid (pNar-GFPuv) which allows cell-based reporting of oxygen limitation. Because there are greater than 109 cells in a fermentor, one can outfit a vessel with more than 109 sensors. Our concept was tested in high density, lab-scale ( 5 L), fed-batch, E. coli fermentations operated with varied mixing efficiency - one verses four impellers. Results: In both cases, bioreactors were maintained identically at greater than 80% dissolved oxygen ( DO) during batch phase and at approximately 20% DO during fed-batch phase. Trends for glucose consumption, biomass and DO showed nearly identical behavior. However, fermentations with only one impeller showed significantly higher GFPuv expression than those with four, indicating a higher degree of fluid segregation sufficient for cellular oxygen deprivation. As the characteristic time for GFPuv expression (approx 90 min.) is much larger than that for mixing (approx 10 s), increased specific fluorescence represents an averaged effect of oxygen limitation over time and by natural extension, over space. Conclusion: Thus, the pNar-GFPuv plasmid enabled bioreactor-wide oxygen sensing in that bacterial cells served as individual recirculating sensors integrating their responses over space and time. We envision cell-based oxygen sensors may find utility in a wide variety of bioprocessing applications.open1123sciescopu

Diversification of bacterial genome content through distinct mechanisms over different timescales

Bacterial populations often consist of multiple co-circulating lineages. Determining how such population structures arise requires understanding what drives bacterial diversification. Using 616 systematically sampled genomes, we show that Streptococcus pneumoniae lineages are typically characterized by combinations of infrequently transferred stable genomic islands: those moving primarily through transformation, along with integrative and conjugative elements and phage-related chromosomal islands. The only lineage containing extensive unique sequence corresponds to a set of atypical unencapsulated isolates that may represent a distinct species. However, prophage content is highly variable even within lineages, suggesting frequent horizontal transmission that would necessitate rapidly diversifying anti-phage mechanisms to prevent these viruses sweeping through populations. Correspondingly, two loci encoding Type I restriction-modification systems able to change their specificity over short timescales through intragenomic recombination are ubiquitous across the collection. Hence short-term pneumococcal variation is characterized by movement of phage and intragenomic rearrangements, with the slower transfer of stable loci distinguishing lineages