Phylogenetic and functional diverse ANME-1 thrive in Arctic hydrothermal vents.

The methane-rich areas, the Loki's Castle vent field and the Jan Mayen vent field at the Arctic Mid Ocean Ridge (AMOR), host abundant niches for anaerobic methane-oxidizers, which are predominantly filled by members of the ANME-1. In this study, we used a metagenomic-based approach that revealed the presence of phylogenetic and functional different ANME-1 subgroups at AMOR, with heterogeneous distribution. Based on a common analysis of ANME-1 genomes from AMOR and other geographic locations, we observed that AMOR subgroups clustered with a vent-specific ANME-1 group that occurs solely at vents, and with a generalist ANME-1 group, with a mixed environmental origin. Generalist ANME-1 are enriched in genes coding for stress response and defense strategies, suggesting functional diversity among AMOR subgroups. ANME-1 encode a conserved energy metabolism, indicating strong adaptation to sulfate-methane-rich sediments in marine systems, which does not however prevent global dispersion. A deep branching family named Ca. Veteromethanophagaceae was identified. The basal position of vent-related ANME-1 in phylogenomic trees suggests that ANME-1 originated at hydrothermal vents. The heterogeneous and variable physicochemical conditions present in diffuse venting areas of hydrothermal fields could have favored the diversification of ANME-1 into lineages that can tolerate geochemical and environmental variations.

A specialized method of sputum collection and processing for therapeutic interventions in cystic fibrosis.

Cystic fibrosis (CF) lung disease is characterized by aggressive neutrophil-dominated inflammation mediated in large part by neutrophil elastase (NE), an omnivorous protease released by activated or disintegrating neutrophils and a key therapeutic target. To date, several short-term studies have shown that anti-NE compounds can inhibit NE and have anti-inflammatory effects. However, progression to large-scale or multicenter clinical trials has been hampered by the fact that the current gold standard methodology of evaluating airway NE inhibition, bronchoalveolar lavage (BAL), is invasive, difficult to standardize across sites and excludes those with severe lung disease. Attempts to utilize sputum that is either spontaneously expectorated (SS) or induced (IS) have been hindered by poor reproducibility, often due to the various processing methods employed. In this study, we evaluate TEmperature-controlled Two-step Rapid Isolation of Sputum (TETRIS), a specialized method for the acquisition and processing of SS and IS. Using TETRIS, we show for the first time that NE activity and cytokine levels are comparable in BAL, SS and IS samples taken from the same people with CF (PWCF) on the same day once this protocol is used. We correlate biomarkers in TETRIS-processed IS and clinical outcome measures including FEV1, and show stability and reproducible inhibition of NE over time in IS processed by TETRIS. The data offer a tremendous opportunity to evaluate prognosis and therapeutic interventions in CF and to study the full spectrum of people with PWCF, many of whom have been excluded from previous studies due to being unfit for BAL or unable to expectorate sputum.

Demodex-associated bacterial proteins induce neutrophil activation.

BACKGROUND: Patients with rosacea demonstrate a higher density of Demodex mites in their skin than do controls. A bacterium isolated from a Demodex mite from a patient with papulopustular rosacea (PPR) was previously shown to provoke an immune response in patients with PPR or ocular rosacea, thus suggesting a possible role for bacterial proteins in the aetiology of this condition. OBJECTIVES: To examine the response of neutrophils to proteins derived from a bacterium isolated from a Demodex mite. METHODS: Bacterial cells were lysed and proteins were partially purified by ÄKTA fast protein liquid chromatography. Isolated neutrophils were exposed to bacterial proteins and monitored for alterations in migration, degranulation and cytokine production. RESULTS: Neutrophils exposed to proteins from Bacillus cells demonstrated increased levels of migration and elevated release of matrix metalloprotease 9, an enzyme known to degrade collagen, and cathelicidin, an antimicrobial peptide. In addition, neutrophils exposed to the bacterial proteins demonstrated elevated rates of interleukin 8 and tumour necrosis factor-α production. CONCLUSIONS: Proteins produced by a bacterium isolated from a Demodex mite have the ability to increase the migration, degranulation and cytokine production abilities of neutrophils. These results suggest that bacteria may play a role in the inflammatory erythema associated with rosacea.

The NADPH oxidase components p47(phox) and p40(phox) bind to moesin through their PX domain.

The NADPH oxidase of phagocytes is a membrane-bound heterodimeric flavocytochrome which catalyses the transfer of electrons from NADPH in the cytoplasm to oxygen in the phagosome. A number of cytosolic proteins are involved in its activation/deactivation: p47phox, p67phox, p40phox and the small GTP-binding protein, rac. The cytosolic phox proteins interact with the cytoskeleton in human neutrophils and, in particular, an interaction with coronin has been reported (Grogan A., Reeves, E., Keep, N. H., Wientjes, F., Totty, N., Burlingame, N. L., Hsuan, J., and Segal, A. W. (1997) J. Cell Sci. 110, 3071-3081). Here, we report on the interaction of another cytoskeletal protein, moesin, with the phox proteins. Moesin belongs to the ezrin-radixin-moesin family of F-actin-binding proteins and we show that it binds to p47phox and p40phox in a phosphoinositide-dependent manner. Furthermore, we show that its N-terminal part binds to the PX domain of p47phox and p40phox.

Direct interaction between p47phox and protein kinase C: evidence for targeting of protein kinase C by p47phox in neutrophils.

p47(phox) is an essential component of the NADPH oxidase, and phosphorylation of p47(phox) is associated with activation of the enzyme. Here we have used p47(phox) affinity chromatography to extract a p47(phox) kinase from neutrophil cytosol. The kinase activity was purified by gel filtration and Mini Q chromatography and shown to be indistinguishable from the catalytic fragments of protein kinase C (PKC)-beta(I), -beta(II) and -delta. The C-terminus of p47(phox) represented the site of interaction with PKC. Co-immunoprecipitation experiments revealed that the interaction between PKC isotypes and p47(phox) takes place in intact cells. However PKC-beta and -delta showed different time courses of co-immunoprecipitation, suggesting that the interactions may serve different functions for the various PKC isotypes. Using cells lacking p47(phox), we investigated the functional relevance of the interaction between PKC and p47(phox). Subcellular fractionation revealed an abnormal recruitment of PKC-beta(I) and -beta(II), but not PKC-delta, to particulate fractions in p47(phox)-deficient cells. Phosphorylation of cytosolic proteins was generally increased in stimulated p47(phox)-deficient neutrophils as compared with normal neutrophils. Furthermore, the cytoskeletal protein coronin was not phosphorylated upon stimulation of p47(phox)-deficient neutrophils. These findings were confirmed in an in vitro-reconstituted system using rat brain cytosol in which addition of p47(phox) affected phosphorylation by PKC/PKM (PKM is the catalytic fragment of PKC). These results indicate that p47(phox) can act as a regulator of PKC in neutrophils.