The Catalytic Activity of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 3 Is Required To Sustain CD4+ CD8+ Thymocyte Survival

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family whose function is largely unknown. Given the central role of MAPKs in T cell development, we hypothesized that ERK3 may regulate thymocyte development. Here we have shown that ERK3 deficiency leads to a 50% reduction in CD4⁺ CD8⁺ (DP) thymocyte number. Analysis of hematopoietic chimeras revealed that the reduction in DP thymocytes is intrinsic to hematopoietic cells. We found that early thymic progenitors seed the Erk3^−/− thymus and can properly differentiate and proliferate to generate DP thymocytes. However, ERK3 deficiency results in a decrease in the DP thymocyte half-life, associated with a higher level of apoptosis. As a consequence, ERK3-deficient DP thymocytes are impaired in their ability to make successful secondary T cell receptor alpha (TCRα) gene rearrangement. Introduction of an already rearranged TCR transgene restores thymic cell number. We further show that knock-in of a catalytically inactive allele of Erk3 fails to rescue the loss of DP thymocytes. Our results uncover a unique role for ERK3, dependent on its kinase activity, during T cell development and show that this atypical MAPK is essential to sustain DP survival during RAG-mediated rearrangements.     

Investigating the Role of Free-living Amoebae as a Reservoir for Mycobacterium ulcerans

Background

The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, still remain a mystery. It has been suggested that M. ulcerans persists with difficulty as a free-living organism due to its natural fragility and inability to withstand exposure to direct sunlight, and thus probably persists within a protective host environment.

Methodology/Principal Findings

We investigated the role of free-living amoebae as a reservoir of M. ulcerans by screening the bacterium in free-living amoebae (FLA) cultures isolated from environmental specimens using real-time PCR. We also followed the survival of M. ulcerans expressing green fluorescence protein (GFP) in Acanthameoba castellanii by flow cytometry and observed the infected cells using confocal and transmission electron microscopy for four weeks in vitro. IS2404 was detected by quantitative PCR in 4.64% of FLA cultures isolated from water, biofilms, detritus and aerosols. While we could not isolate M. ulcerans, 23 other species of mycobacteria were cultivated from inside FLA and/or other phagocytic microorganisms. Laboratory experiments with GFP-expressing M. ulcerans in A. castellani trophozoites for 28 days indicated the bacteria did not replicate inside amoebae, but they could remain viable at low levels in cysts. Transmission electron microscopy of infected A. castellani confirmed the presence of bacteria within both trophozoite vacuoles and cysts. There was no correlation of BU notification rate with detection of the IS2404 in FLA (r = 0.07, n = 539, p = 0.127).

Conclusion/Significance

This study shows that FLA in the environment are positive for the M. ulcerans insertion sequence IS2404. However, the detection frequency and signal strength of IS2404 positive amoabae was low and no link with the occurrence of BU was observed. We conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans.     

Establishment of the comet assay in the freshwater snail Biomphalaria glabrata (Say, 1818)

The single cell gel electrophoresis or the comet assay was established in the freshwater snail Biomphalaria glabrata. For detecting DNA damage in circulating hemocytes, adult snails were irradiated with single doses of 2.5, 5, 10 and 20 Gy of (60)Co gamma radiation. Genotoxic effect of ionizing radiation was detected at all doses as a dose-related increase in DNA migration. Comet assay in B. glabrata demonstrated to be a simple, fast and reliable tool in the evaluation of genotoxic effects of environmental mutagens.     

Draft genome sequence for Microbacterium laevaniformans strain OR221, a bacterium tolerant to metals, nitrate, and low pH

Microbacterium laevaniformans strain OR221 was isolated from subsurface sediments obtained from the Field Research Center (FRC) in Oak Ridge, TN. It was characterized as a bacterium tolerant to heavy metals, such as uranium, nickel, cobalt, and cadmium, as well as nitrate and low pH. We present its draft genome sequence.     

In Polytomella sp. mitochondria, biogenesis of the heterodimeric COX2 subunit of cytochrome c oxidase requires two different import pathways

In the vast majority of eukaryotic organisms, the mitochondrial cox2 gene encodes subunit II of cytochrome c oxidase (COX2). However, in some lineages including legumes and chlorophycean algae, the cox2 gene migrated to the nucleus. Furthermore, in chlorophycean algae, this gene was split in two different units. Thereby the COX2 subunit is encoded by two independent nuclear genes, cox2a and cox2b, and mitochondria have to import the cytosol-synthesized COX2A and COX2B subunits and assemble them into the cytochrome c oxidase complex. In the chlorophycean algae Chlamydomonas reinhardtii and Polytomella sp., the COX2A precursor exhibits a long (130-140 residues), cleavable mitochondrial targeting sequence (MTS). In contrast, COX2B lacks an MTS, suggesting that mitochondria use different mechanisms to import each subunit. Here, we explored the in vitro import processes of both, the Polytomella sp. COX2A precursor and the COX2B protein. We used isolated, import-competent mitochondria from this colorless alga. Our results suggest that COX2B is imported directly into the intermembrane space, while COX2A seems to follow an energy-dependent import pathway, through which it finally integrates into the inner mitochondrial membrane. In addition, the MTS of the COX2A precursor is eliminated. This is the first time that the in vitro import of split COX2 subunits into mitochondria has been achieved.     

Spontaneous remodeling of HDL particles at acidic pH enhances their capacity to induce cholesterol efflux from human macrophage foam cells

HDL particles may enter atherosclerotic lesions having an acidic intimal fluid. Therefore, we investigated whether acidic pH would affect their structural and functional properties. For this purpose, HDL(2) and HDL(3) subfractions were incubated for various periods of time at different pH values ranging from 5.5 to 7.5, after which their protein and lipid compositions, size, structure, and cholesterol efflux capacity were analyzed. Incubation of either subfraction at acidic pH induced unfolding of apolipoproteins, which was followed by release of lipid-poor apoA-I and ensuing fusion of the HDL particles. The acidic pH-modified HDL particles exhibited an enhanced ability to promote cholesterol efflux from cholesterol-laden primary human macrophages. Importantly, treatment of the acidic pH-modified HDL with the mast cell-derived protease chymase completely depleted the newly generated lipid-poor apoA-I, and prevented the acidic pH-dependent increase in cholesterol efflux. The above-found pH-dependent structural and functional changes were stronger in HDL(3) than in HDL(2). Spontaneous acidic pH-induced remodeling of mature spherical HDL particles increases HDL-induced cholesterol efflux from macrophage foam cells, and therefore may have atheroprotective effects.     

Airway Microbiota in Severe Asthma and Relationship to Asthma Severity and Phenotypes

BackgroundThe lower airways harbor a community of bacterial species which is altered in asthma.ObjectivesWe examined whether the lower airway microbiota were related to measures of asthma severity.MethodsWe prospectively recruited 26 severe asthma, 18 non-severe asthma and 12 healthy subjects. DNA was extracted from induced sputum and PCR amplification of the V3-V5 region of bacterial 16S rRNA gene was performed.ResultsWe obtained 138,218 high quality sequences which were rarefied at 133 sequences/sample. Twenty OTUs had sequences ≥1% of total. There were marked differences in the distribution of Phyla between groups (P = 2.8x10^-118). Bacteroidetes and Fusobacteria were reduced in non-severe and severe asthmatic groups. Proteobacteria were more common in non-severe asthmatics compared to controls (OR = 2.26; 95% CI = 1.94–2.64) and Firmicutes were increased in severe asthmatics compared to controls (OR = 2.15; 95%CI = 1.89–2.45). Streptococcal OTUs amongst the Firmicutes were associated with recent onset asthma, rhinosinusitis and sputum eosinophilia.ConclusionsSputum microbiota in severe asthma differs from healthy controls and non-severe asthmatics, and is characterized by the presence of Streptococcus spp with eosinophilia. Whether these organisms are causative for the pathophysiology of asthma remains to be determined.     

Heteroalicyclic carboxamidines as inhibitors of inducible nitric oxide synthase; the identification of (2R)-2-pyrrolidinecarboxamidine as a potent and selective haem-co-ordinating inhibitor

Heteroalicyclic carboxamidines were synthesised and evaluated as inhibitors of nitric oxide synthases. (2R)-2-Pyrrolidinecarboxamidine, in particular, was shown to be a highly potent in vitro (IC₅₀ = 0.12 μM) and selective iNOS inhibitor (>100-fold vs both eNOS and nNOS), with probable binding to the key anchoring glutamate residue and co-ordination to the haem iron.