G protein-coupled receptors in the neuropathophysiology of asthma

Asthma is a complex genetic disorder with environmental influences and is characterized by airway inflammation, reversible airflow obstruction, and airway hyperresponsiveness (AHR). The arachidonic acid metabolite thromboxane A2 (TXA2), is a potent lipid mediator released by platelets and inflammatory cells and is capable of inducing bronchoconstriction. In the airways, it has been postulated that TXA2 causes airway constriction by direct activation of TP receptors on airway smooth muscle (ASM) cells. Here we demonstrate that although TXA2 can mediate a dramatic increase in ASM constriction, this response is largely dependent on vagal innervation of the airways and is highly sensitive to muscarinic acetylcholine receptor (mAChR) antagonists. Further analyses demonstrate that TP-dependent ASM constriction requires M3 mAChR expression. To further define the mechanism underlying TXA2 meditated airway constriction, mice carrying a Tp receptor locus that is sensitive to disruption by cre recombinase were generated. These mice were crossed with nestin-cre transgenic mice, which express cre recombinase throughout the nervous systems. Here we demonstrate that loss of the TP receptor throughout the nervous system does not significantly affect TXA2 airway reactivity. To assess ASM TP receptor function, we crossed the floxed Tp receptor mice with SM22-cre transgenic mice, which express cre recombinase in smooth muscle. The resultant smooth muscle TP receptor deficient animals demonstrate attenuated airway responses following aerosol challenges with TXA2. Together, these findings suggest that TXA2 mediates airway reactivity and AHR through collaborations between ASM Tp receptors and the M3 mAChR. In addition to TXA2, we also evaluated the role of NPSR1 (GPRA), a newly deorphanized G protein-coupled receptor that has been shown to be a promising candidate gene associated with asthma in human populations. We report here that the change in airway resistance in response to methacholine was identical in control and NPSR1 deficient mice and the development of allergic lung disease in NPSR1 deficient mice is unaltered. In contrast to previously published data, our analyses also failed to detect expression of NPSR1 in human lung tissue or in mice with allergic lung disease. Taken together, our studies fail to support a direct contribution of NPSR1 to asthma pathogenesis

Effects of model chemistry and data biases on stratospheric ozone assimilation

The innovations or observation minus forecast (O–F) residuals produced by a data assimilation system provide a convenient metric of evaluating global analyses. In this study, O–F statistics from the Global Ozone Assimilation Testing System (GOATS) are used to examine how ozone assimilation products and their associated O–F statistics depend on input data biases and ozone photochemistry parameterizations (OPP). All the GOATS results shown are based on a 6-h forecast and analysis cycle using observations from SBUV/2 (Solar Backscatter UltraViolet instrument-2) during September–October 2002. Results show that zonal mean ozone analyses are more independent of observation biases and drifts when using an OPP, while the mean ozone O–Fs are more sensitive to observation drifts when using an OPP. In addition, SD O–Fs (standard deviations) are reduced in the upper stratosphere when using an OPP due to a reduction of forecast model noise and to increased covariance between the forecast model and the observations. Experiments that changed the OPP reference state to match the observations by using an "adaptive" OPP scheme reduced the mean ozone O–Fs at the expense of zonal mean ozone analyses being more susceptible to data biases and drifts. Additional experiments showed that the upper boundary of the ozone DAS can affect the quality of the ozone analysis and therefore should be placed well above (at least a scale height) the region of interest

Characterizing DebriSat Fragments -- Preliminary Results

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Characterizing DebriSat Fragments: So Many Fragments, So Much Data, and So Little Time

To improve prediction accuracy, the DebriSat project was conceived by NASA and DoD to update existing standard break-up models. Updating standard break-up models require detailed fragment characteristics such as physical size, material properties, bulk density, and ballistic coefficient. For the DebriSat project, a representative modern LEO spacecraft was developed and subjected to a laboratory hypervelocity impact test and all generated fragments with at least one dimension greater than 2 mm are collected, characterized and archived. Since the beginning of the characterization phase of the DebriSat project, over 130,000 fragments have been collected and approximately 250,000 fragments are expected to be collected in total, a three-fold increase over the 85,000 fragments predicted by the current break-up model. The challenge throughout the project has been to ensure the integrity and accuracy of the characteristics of each fragment. To this end, the post hypervelocity-impact test activities, which include fragment collection, extraction, and characterization, have been designed to minimize handling of the fragments. The procedures for fragment collection, extraction, and characterization were painstakingly designed and implemented to maintain the post-impact state of the fragments, thus ensuring the integrity and accuracy of the characterization data. Each process is designed to expedite the accumulation of data, however, the need for speed is restrained by the need to protect the fragments. Methods to expedite the process such as parallel processing have been explored and implemented while continuing to maintain the highest integrity and value of the data. To minimize fragment handling, automated systems have been developed and implemented. Errors due to human inputs are also minimized by the use of these automated systems. This paper discusses the processes and challenges involved in the collection, extraction, and characterization of the fragments as well as the time required to complete the processes. The objective is to provide the orbital debris community an understanding of the scale of the effort required to generate and archive high quality data and metadata for each debris fragment 2 mm or larger generated by the DebriSat project

NOGAPS-ALPHA model simulations of stratospheric ozone during the SOLVE2 campaign

This paper presents three-dimensional prognostic O₃ simulations with parameterized gas-phase photochemistry from the new NOGAPS-ALPHA middle atmosphere forecast model. We compare 5-day NOGAPS-ALPHA hindcasts of stratospheric O₃ with satellite and DC-8 aircraft measurements for two cases during the SOLVE II campaign: (1) the cold, isolated vortex during 11-16 January 2003; and (2) the rapidly developing stratospheric warming of 17-22 January 2003. In the first case we test three different photochemistry parameterizations. NOGAPS-ALPHA O₃ simulations using the NRL-CHEM2D parameterization give the best agreement with SAGE III and POAM III profile measurements. 5-day NOGAPS-ALPHA hindcasts of polar O₃ initialized with the NASA GEOS4 analyses produce better agreement with observations than do the operational ECMWF O₃ forecasts of case 1. For case 2, both NOGAPS-ALPHA and ECMWF 114-h forecasts of the split vortex structure in lower stratospheric O₃ on 21 January 2003 show comparable skill. Updated ECMWF O₃ forecasts of this event at hour 42 display marked improvement from the 114-h forecast; corresponding updated 42-hour NOGAPS-ALPHA prognostic O₃ fields initialized with the GEOS4 analyses do not improve significantly. When NOGAPS-ALPHA prognostic O₃ is initialized with the higher resolution ECMWF O₃ analyses, the NOGAPS-ALPHA 42-hour lower stratospheric O₃ fields closely match the operational 42-hour ECMWF O₃ forecast of the 21 January event. We find that stratospheric O₃ forecasts at high latitudes in winter can depend on both model initial conditions and the treatment of photochemistry over periods of 1-5 days. Overall, these results show that the new O₃ initialization, photochemistry parameterization, and spectral transport in the NOGAPS-ALPHA NWP model can provide reliable short-range stratospheric O₃ forecasts during Arctic winter

‘All hands-on deck’, working together to develop UK standards for public involvement in research

Background: Public involvement in research is an established part of the research process in the UK, however there remain questions about what good public involvement in research looks and feels like. Until now public involvement practitioners, researchers and members of the public have looked for answers in examples shared across networks, published case studies, guidance and research articles. Pulling these strands together, the UK Standards for Public Involvement provides six statements (standards) about public involvement in research. They were produced by a partnership of organisations from Scotland, Northern Ireland, Wales and England with contributions from involvement practitioners, public partners, researchers and research funders. Main body: Each standard has reflective questions, which are designed to encourage standard users to use approaches and behaviours that improve involvement, over time. The standards are designed to be used as a practical tool, and reflect the agreed hallmarks of good public involvement in research for example, flexibility in approaches used, shared learning, and mutual respect. The standards development process is described from the initial idea and scoping, via the appraisal of existing standard sets and integration of values and principles in public involvement in research. The collaborative writing process of and consultation on the draft standard set is described, together with what changed as a result of feedback. The initiation of a year-long testing programme with forty participating research organisations, the experiential feedback and the resulting changes to the standards is summarised. Conclusion: This commentary paper describes, in some detail, a process to develop a set of six standards for public involvement in research in the UK. Producing a complex, national public involvement initiative is not without its challenges, and in supplementary material partnership members reflect on and share their experiences of standards development. The next phase of integration and implementation is explored with concluding comments from those that tested and helped improve the standards

An orphaned Mce-associated membrane protein of M ycobacterium tuberculosis is a virulence factor that stabilizes Mce transporters: OmamA functions in virulence and Mce stability

Mycobacterium tuberculosis proteins that are exported out of the bacterial cytoplasm are ideally positioned to be virulence factors; however, the functions of individual exported proteins remain largely unknown. Previous studies identified Rv0199 as an exported membrane protein of unknown function. Here, we characterized the role of Rv0199 in M. tuberculosis virulence using an aerosol model of murine infection. Rv0199 appears to be a member of a Mce-associated membrane (Mam) protein family leading us to rename it OmamA, for orphaned Mce-associated membrane protein A. Consistent with a role in Mce transport, we showed OmamA is required for cholesterol import, which is a Mce4-dependent process. We further demonstrated a function for OmamA in stabilizing protein components of the Mce1 transporter complex. These results indicate a function of OmamA in multiple Mce transporters and one that may be analogous to the role of VirB8 in stabilizing Type IV secretion systems, as structural similarities between Mam proteins and VirB8 proteins are predicted by the Phyre 2 program. In this study, we provide functional information about OmamA and shed light on the function of Mam family proteins in Mce transporters

Gene expression patterns vary in clonal cell cultures from Rett syndrome females with eight different MECP2 mutations

BACKGROUND: Females with the neurological disorder Rett syndrome are heterozygous for mutations in X-linked MECP2 that encodes methyl-CpG binding protein 2 (MeCP2) thought to act as a transcriptional repressor. To identify target genes for MeCP2 modulation, we studied global gene expression in single cell-derived wild-type and mutant MECP2 expressing fibroblast clones with four common mutations (R106W, R306C, 705delG, 1155del32) and in lymphoblastoid cell lines (LCLs) that included four mutant MeCP2 (T158M, 803delG, R168X and 1159del28) expressing, and five (1159del28, R106W, R255X, 803delG, 803delG) wild-type MeCP2 expressing lines. METHODS: Clonality and mutation status were verified by androgen receptor methylation assays for X-inactivation and by sequencing MECP2 transcripts. Expression studies were done with oligonucleotide microarrays (Affymetrix U95) and verified with real-time quantitative RT-PCR using Sybr Green. RESULTS: Expression of 49 transcripts was increased, and expression of 21 transcripts was decreased, in at least 3 of 4 mutant/wild-type fibroblast comparisons. Transcript levels of 11 genes, determined by quantitative RT-PCR, were highly correlated with the microarray data. Therefore, multiple additional clones from two Rett individuals were tested by RT-PCR only. Striking expression differences were found in both mutant and wildtype MeCP2 expressing clones. Comparing expression profiles of lymphoblastoid cell lines yielded 16 differentially expressed genes. CONCLUSIONS: MeCP2 deficiency does not lead to global deregulation of gene expression. Either MeCP2's in vivo function does not involve widespread transcriptional repression, or its function is redundant in cell types that also express other methyl-CpG binding proteins. Our data suggest that clonal fibroblast strains may show substantial inter-strain variation, making them a difficult and unstable resource for genome-wide expression profiling studies

Deletion of ripA Alleviates Suppression of the Inflammasome and MAPK by Francisella tularensis

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1β, IL-18, and TNF-α by resting macrophages. IL-1β and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1β and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis