HCMV pUS28 initiates pro-migratory signaling via activation of Pyk2 kinase

Background: Human Cytomegalovirus (HCMV) has been implicated in the acceleration of vascular disease and chronic allograft rejection. Recently, the virus has been associated with glioblastoma and other tumors. We have previously shown that the HCMV-encoded chemokine receptor pUS28 mediates smooth muscle cell (SMC) and macrophage motility and this activity has been implicated in the acceleration of vascular disease. pUS28 induced SMC migration involves the activation of the protein tyrosine kinases (PTKs) Src and Focal adhesion kinase as well as the small GTPase RhoA. The PTK Pyk2 has been shown to play a role in cellular migration and formation of cancer, especially glioblastoma. The role of Pyk2 in pUS28 signaling and migration are unknown. Methods: In the current study, we examined the involvement of the PTK Pyk2 in pUS28-induced cellular motility. We utilized in vitro migration of SMC to determine the requirements for Pyk2 in pUS28 pro-migratory signaling. We performed biochemical analysis of Pyk2 signaling in response to pUS28 activation to determine the mechanisms involved in pUS28 migration. We performed mass spectrometric analysis of Pyk2 complexes to identify novel Pyk2 binding partners. Results: Expression of a mutant form of Pyk2 lacking the autophosphorylation site (Tyr-402) blocks pUS28-mediated SMC migration in response to CCL5, while the kinase-inactive Pyk2 mutant failed to elicit the same negative effect on migration. pUS28 stimulation with CCL5 results in ligand-dependent and calcium-dependent phosphorylation of Pyk2 Tyr-402 and induced the formation of an active Pyk2 kinase complex containing several novel Pyk2 binding proteins. Expression of the autophosphorylation null mutant Pyk2 F402Y did not abrogate the formation of an active Pyk2 kinase complex, but instead prevented pUS28-mediated activation of RhoA. Additionally, pUS28 activated RhoA via Pyk2 in the U373 glioblastoma cells. Interestingly, the Pyk2 kinase complex in U373 contained several proteins known to participate in glioma tumorigenesis. Conclusions: These findings represent the first demonstration that pUS28 signals through Pyk2 and that this PTK participates in pUS28-mediated cellular motility via activation of RhoA. Furthermore, these results provide a potential mechanistic link between HCMV-pUS28 and glioblastoma cell activation

Proteome Analyses of Strains Cyanothece ATCC 51142 and PCC 7822 of the Diazotrophic Cyanobacterium Cyanothece sp. Under Culture Conditions Resulting in Enhanced H2 Production.

Cultures of the cyanobacterial genus Cyanothece have been shown to produce high levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal cycles when grown under N2-fixing conditions in light-dark cycles. We seek to better understand the way in which proteins respond to these diurnal changes, and we performed quantitative proteome analysis of Cyanothecesp. strains ATCC 51142 and PCC 7822 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N2-fixing conditions, and in the absence of glycerol, nitrogenase gene expression was linked to the dark period. However, glycerol induced expression of nitrogenase during part of the light period, together with cytochrome c oxidase (Cox), glycogen phosphorylase (Glp), and glycolytic and pentose phosphate pathway (PPP) enzymes. This indicated that nitrogenase expression in the light was facilitated via higher levels of respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited in Cyanothece ATCC 51142 in the presence of glycerol under H2-producing conditions, suggesting a competition between these sources of carbon. However, in Cyanothece PCC 7822, the Calvin cycle still played a role in cofactor recycling during H2 production. Our data comprise the first comprehensive profiling of proteome changes in Cyanothece PCC 7822 and allow an in-depth comparative analysis of major physiological and biochemical processes that influence H2 production in both strains. Our results revealed many previously uncharacterized proteins that may play a role in nitrogenase activity and in other metabolic pathways and may provide suitable targets for genetic manipulation that would lead to improvement of large-scale H2 production

Large-Scale Spray Releases: Initial Aerosol Test Results

One of the events postulated in the hazard analysis at the Waste Treatment and Immobilization Plant (WTP) and other U.S. Department of Energy (DOE) nuclear facilities is a breach in process piping that produces aerosols with droplet sizes in the respirable range. The current approach for predicting the size and concentration of aerosols produced in a spray leak involves extrapolating from correlations reported in the literature. These correlations are based on results obtained from small engineered spray nozzles using pure liquids with Newtonian fluid behavior. The narrow ranges of physical properties on which the correlations are based do not cover the wide range of slurries and viscous materials that will be processed in the WTP and across processing facilities in the DOE complex. Two key technical areas were identified where testing results were needed to improve the technical basis by reducing the uncertainty due to extrapolating existing literature results. The first technical need was to quantify the role of slurry particles in small breaches where the slurry particles may plug and result in substantially reduced, or even negligible, respirable fraction formed by high-pressure sprays. The second technical need was to determine the aerosol droplet size distribution and volume from prototypic breaches and fluids, specifically including sprays from larger breaches with slurries where data from the literature are scarce. To address these technical areas, small- and large-scale test stands were constructed and operated with simulants to determine aerosol release fractions and generation rates from a range of breach sizes and geometries. The properties of the simulants represented the range of properties expected in the WTP process streams and included water, sodium salt solutions, slurries containing boehmite or gibbsite, and a hazardous chemical simulant. The effect of anti-foam agents was assessed with most of the simulants. Orifices included round holes and rectangular slots. The round holes ranged in size from 0.2 to 4.46 mm. The slots ranged from (width × length) 0.3 × 5 to 2.74 × 76.2 mm. Most slots were oriented longitudinally along the pipe, but some were oriented circumferentially. In addition, a limited number of multi-hole test pieces were tested in an attempt to assess the impact of a more complex breach. Much of the testing was conducted at pressures of 200 and 380 psi, but some tests were conducted at 100 psi. Testing the largest postulated breaches was deemed impractical because of the large size of some of the WTP equipment. The purpose of this report is to present the experimental results and analyses for the aerosol measurements obtained in the large-scale test stand. The report includes a description of the simulants used and their properties, equipment and operations, data analysis methodology, and test results. The results of tests investigating the role of slurry particles in plugging of small breaches are reported in Mahoney et al. 2012a. The results of the aerosol measurements in the small-scale test stand are reported in Mahoney et al. (2012b)

Large-Scale Spray Releases: Initial Aerosol Test Results

One of the events postulated in the hazard analysis at the Waste Treatment and Immobilization Plant (WTP) and other U.S. Department of Energy (DOE) nuclear facilities is a breach in process piping that produces aerosols with droplet sizes in the respirable range. The current approach for predicting the size and concentration of aerosols produced in a spray leak involves extrapolating from correlations reported in the literature. These correlations are based on results obtained from small engineered spray nozzles using pure liquids with Newtonian fluid behavior. The narrow ranges of physical properties on which the correlations are based do not cover the wide range of slurries and viscous materials that will be processed in the WTP and across processing facilities in the DOE complex. Two key technical areas were identified where testing results were needed to improve the technical basis by reducing the uncertainty due to extrapolating existing literature results. The first technical need was to quantify the role of slurry particles in small breaches where the slurry particles may plug and result in substantially reduced, or even negligible, respirable fraction formed by high-pressure sprays. The second technical need was to determine the aerosol droplet size distribution and volume from prototypic breaches and fluids, specifically including sprays from larger breaches with slurries where data from the literature are scarce. To address these technical areas, small- and large-scale test stands were constructed and operated with simulants to determine aerosol release fractions and generation rates from a range of breach sizes and geometries. The properties of the simulants represented the range of properties expected in the WTP process streams and included water, sodium salt solutions, slurries containing boehmite or gibbsite, and a hazardous chemical simulant. The effect of anti-foam agents was assessed with most of the simulants. Orifices included round holes and rectangular slots. The round holes ranged in size from 0.2 to 4.46 mm. The slots ranged from (width × length) 0.3 × 5 to 2.74 × 76.2 mm. Most slots were oriented longitudinally along the pipe, but some were oriented circumferentially. In addition, a limited number of multi-hole test pieces were tested in an attempt to assess the impact of a more complex breach. Much of the testing was conducted at pressures of 200 and 380 psi, but some tests were conducted at 100 psi. Testing the largest postulated breaches was deemed impractical because of the large size of some of the WTP equipment. The purpose of this report is to present the experimental results and analyses for the aerosol measurements obtained in the large-scale test stand. The report includes a description of the simulants used and their properties, equipment and operations, data analysis methodology, and test results. The results of tests investigating the role of slurry particles in plugging of small breaches are reported in Mahoney et al. 2012a. The results of the aerosol measurements in the small-scale test stand are reported in Mahoney et al. (2012b)