A double blind randomized placebo control crossover trial on the effect of dietary nitrate supplementation on exercise tolerance in stable moderate chronic obstructive pulmonary disease.

BACKGROUND: Dietary nitrate supplementation has been shown to decrease the oxygen cost of exercise and prolong exercise tolerance, as measured by sub-maximal exercise endurance distance and time at 85% V̇O2max, in both elite athletes and normal healthy subjects. Patients with chronic obstructive pulmonary disease (COPD) have reduced quality of life and ability to perform activities of daily living attributable to diminished exercise tolerance, and dietary nitrate may be able to ameliorate this. METHODS: We performed a double-blind, computer-randomized placebo control crossover trial at a tertiary Australian hospital to investigate whether dietary nitrate supplementation as beetroot juice (BR) would augment submaximal exercise endurance in individuals with spirometrically confirmed stable moderate COPD. Volunteers underwent an incremental shuttle walk test to determine V̇O2max followed by a test dose of BR to establish safety in the study population. Participants performed an endurance shuttle walk test (ESWT) at 85% V̇O2max after randomization to either a 3 day wash-in of BR (4.8 mmol twice a day) or placebo (nitrate deplete BR), with a final dose on the morning of testing. They then crossed over after 4 day washout. Repeated measures two sided paired t-tests were employed. RESULTS: 35 participants were recruited with 19 completing the trial. In the initial safety phase, we measured systolic blood pressure over four hours post first dose of BR, and found a mean 10 mmHg decrement maximal at 1 hour. One individual developed symptomatic postural hypotension and was excluded. The primary outcomes of ESWT distance and time to fatigue improved by 11% and 6% respectively; however these differences did not achieve statistical significance (p = 0.494 and 0.693 respectively). CONCLUSIONS: Our study does not support a role for routine dietary nitrate supplementation for enhancement of exercise endurance in COPD. TRIAL REGISTRATION: Australia and New Zealand Clinical Trial Register: ACTRN12611001088932.

Pathways activated during human asthma exacerbation as revealed by gene expression patterns in blood.

BACKGROUND: Asthma exacerbations remain a major unmet clinical need. The difficulty in obtaining airway tissue and bronchoalveolar lavage samples during exacerbations has greatly hampered study of naturally occurring exacerbations. This study was conducted to determine if mRNA profiling of peripheral blood mononuclear cells (PBMCs) could provide information on the systemic molecular pathways involved during asthma exacerbations. METHODOLOGY/PRINCIPAL FINDINGS: Over the course of one year, gene expression levels during stable asthma, exacerbation, and two weeks after an exacerbation were compared using oligonucleotide arrays. For each of 118 subjects who experienced at least one asthma exacerbation, the gene expression patterns in a sample of peripheral blood mononuclear cells collected during an exacerbation episode were compared to patterns observed in multiple samples from the same subject collected during quiescent asthma. Analysis of covariance identified genes whose levels of expression changed during exacerbations and returned to quiescent levels by two weeks. Heterogeneity among visits in expression profiles was examined using K-means clustering. Three distinct exacerbation-associated gene expression signatures were identified. One signature indicated that, even among patients without symptoms of respiratory infection, genes of innate immunity were activated. Antigen-independent T cell activation mediated by IL15 was also indicated by this signature. A second signature revealed strong evidence of lymphocyte activation through antigen receptors and subsequent downstream events of adaptive immunity. The number of genes identified in the third signature was too few to draw conclusions on the mechanisms driving those exacerbations. CONCLUSIONS/SIGNIFICANCE: This study has shown that analysis of PBMCs reveals systemic changes accompanying asthma exacerbation and has laid the foundation for future comparative studies using PBMCs.

Rhinovirus 3C protease can localize in the nucleus and alter active and passive nucleocytoplasmic transport.

The degradation of nuclear pore components and disruption of nucleocytoplasmic trafficking during rhinovirus infection have been attributed to viral 2A protease. Here we show for the first time that rhinovirus 3C protease may also have a role. Specifically, we show that 3C and its precursor, 3CD, can target green fluorescent protein to the nucleus of living cells, leading to degradation of nuclear pore components, and that incubation with recombinant 3C disrupts active and passive nucleocytoplasmic transport in a semi-intact cell nuclear transport system dependent on 3C protease activity. 3C may thus contribute to host cell shutoff in infected cells by localizing in the nucleus and facilitating nuclear pore breakdown.

The respiratory syncytial virus matrix protein possesses a Crm1-mediated nuclear export mechanism.

The respiratory syncytial virus (RSV) matrix (M) protein is localized in the nucleus of infected cells early in infection but is mostly cytoplasmic late in infection. We have previously shown that M localizes in the nucleus through the action of the importin beta1 nuclear import receptor. Here, we establish for the first time that M's ability to shuttle to the cytoplasm is due to the action of the nuclear export receptor Crm1, as shown in infected cells, and in cells transfected to express green fluorescent protein (GFP)-M fusion proteins. Specific inhibition of Crm1-mediated nuclear export by leptomycin B increased M nuclear accumulation. Analysis of truncated and point-mutated M derivatives indicated that Crm1-dependent nuclear export of M is attributable to a nuclear export signal (NES) within residues 194 to 206. Importantly, inhibition of M nuclear export resulted in reduced virus production, and a recombinant RSV carrying a mutated NES could not be rescued by reverse genetics. That this is likely to be due to the inability of a nuclear export deficient M to localize to regions of virus assembly is indicated by the fact that a nuclear-export-deficient GFP-M fails to localize to regions of virus assembly when expressed in cells infected with wild-type RSV. Together, our data suggest that Crm1-dependent nuclear export of M is central to RSV infection, representing the first report of such a mechanism for a paramyxovirus M protein and with important implications for related paramyxoviruses.

Association of respiratory syncytial virus M protein with viral nucleocapsids is mediated by the M2-1 protein.

Cytoplasmic inclusions in respiratory syncytial virus-infected cells comprising viral nucleocapsid proteins (L, N, P, and M2-1) and the viral genome are sites of viral transcription. Although not believed to be necessary for transcription, the matrix (M) protein is also present in these inclusions, and we have previously shown that M inhibits viral transcription. In this study, we have investigated the mechanisms for the association of the M protein with cytoplasmic inclusions. Our data demonstrate for the first time that the M protein associates with cytoplasmic inclusions via an interaction with the M2-1 protein. The M protein colocalizes with M2-1 in the cytoplasm of cells expressing only the M and M2-1 proteins and directly interacts with M2-1 in a cell-free binding assay. Using a cotransfection system, we confirmed that the N and P proteins are sufficient to form cytoplasmic inclusions and that M2-1 localizes to these inclusions; additionally, we show that M associates with cytoplasmic inclusions only in the presence of the M2-1 protein. Using truncated mutants, we show that the N-terminal 110 amino acids of M mediate the interaction with M2-1 and the subsequent association with nucleocapsids. The interaction of M2-1 with M and, in particular, the N-terminal region of M may represent a target for novel antivirals that block the association of M with nucleocapsids, thereby inhibiting virus assembly.

Nuclear localization of dengue virus nonstructural protein 5 through its importin alpha/beta-recognized nuclear localization sequences is integral to viral infection.

Dengue virus nonstructural protein 5 (NS5) is a large multifunctional protein with a central role in viral replication. We previously identified two nuclear localization sequences (NLSs) within the central region of dengue virus type-2 (DENV-2) NS5 ('aNLS' and 'bNLS') that are recognized by the importin alpha/beta and importin beta1 nuclear transporters, respectively. Here, we demonstrate the importance of the kinetics of NS5 nuclear localization to virus production for the first time and show that the aNLS is responsible. Site-specific mutations in the bipartite-type aNLS or bNLS region were introduced into a reporter plasmid encoding green fluorescent protein fused to the N-terminus of DENV-2 NS5, as well as into DENV-2 genomic length complementary DNA. Mutation of basic residues in the highly conserved region of the bNLS did not affect nuclear import of NS5. In contrast, mutations in either basic cluster of the aNLS decreased NS5 nuclear accumulation and reduced virus production, with the greatest reduction observed for mutation of the second cluster (K(387)K(388)K(389)); mutagenesis of both clusters abolished NS5 nuclear import and DENV-2 virus production completely. The latter appeared to relate to the impaired ability of virus lacking nuclear-localizing NS5, as compared with wild-type virus expressing nuclear-localizing NS5, to reduce interleukin-8 production as part of the antiviral response. The results overall indicate that NS5 nuclear localization through the aNLS is integral to viral infection, with significant implications for other flaviviruses of medical importance, such as yellow fever and West Nile viruses.

Interaction between the respiratory syncytial virus G glycoprotein cytoplasmic domain and the matrix protein.

Paramyxovirus assembly at the cell membrane requires the movement of viral components to budding sites and envelopment of nucleocapsids by cellular membranes containing viral glycoproteins, facilitated by interactions with the matrix protein. The specific protein interactions during assembly of respiratory syncytial virus (RSV) are unknown. Here, the postulated interaction between the RSV matrix protein (M) and G glycoprotein (G) was investigated. Partial co-localization of M with G was demonstrated, but not with a truncated variant lacking the cytoplasmic domain and one-third of the transmembrane domain, in cells infected with recombinant RSV or transfected to express G and M. A series of G mutants was constructed with progressively truncated or modified cytoplasmic domains. Data from co-expression in cells and a cell-free binding assay showed that the N-terminal aa 2-6 of G play a key role in G-M interaction, with serine at position 2 and aspartate at position 6 playing key roles.