Cutting edge: myeloid differentiation factor 88 deficiency improves resistance against sepsis caused by polymicrobial infection

Toll-like receptors (TLRs) are important for the activation of innate immune cells upon encounter of microbial pathogens. The present study investigated the potential roles of TLR2, TLR4, and the signaling protein myeloid differentiation factor 88 (MyD88) in polymicrobial septic peritonitis. Whereas both TLR2 and TLR4 were dispensable for host defense against septic peritonitis, MyD88-deficient mice were protected in this infection model. Recruitment of neutrophils to the septic focus and bacterial clearance were normal in MyD88-deficient mice. In contrast, the systemic inflammatory response was strongly attenuated in the absence of MyD88. Surprisingly, MyD88 deficiency did not alter cytokine and chemokine production in spleen, but markedly reduced the inflammatory response in liver and lung. Production of monocyte chemoattractant protein-1 and macrophage-inflammatory protein-1alpha was entirely independent of MyD88. These results imply a central role of MyD88 for the systemic immune pathology of polymicrobial sepsis and show that cytokine production in spleen and induction of certain chemokines are MyD88 independent.     

Capturing single cell genomes of active polysaccharide degraders: an unexpected contribution of Verrucomicrobia

Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution to polysaccharide degradation, although they constituted only a minor fraction of the total microbial community. Genomic sequencing of five cells, representing the most predominant, polysaccharide-active Verrucomicrobia phylotype, revealed significant enrichment in genes encoding a wide spectrum of glycoside hydrolases, sulfatases, peptidases, carbohydrate lyases and esterases, confirming that these organisms were well equipped for the hydrolysis of diverse polysaccharides. Remarkably, this enrichment was on average higher than in the sequenced representatives of Bacteroidetes, which are frequently regarded as highly efficient biopolymer degraders. These findings shed light on the ecological roles of uncultured Verrucomicrobia and suggest specific taxa as promising bioprospecting targets. The employed method offers a powerful tool to rapidly identify and recover discrete genomes of active players in polysaccharide degradation, without the need for cultivation.     

Prevention of cardiovascular complications in manifest arteriosclerosis by the use of Micristine]

A long-term clinical trial in micristin-treated patients suffering from organic arterial circulatory disturbances is reported. Problems of therapy monitoring by determination of the ASA level in plasma and of control of platelet aggregation are discussed. Acute cardiovascular complications (myocardial infarction, stroke, acute vascular occlusion, amputation and angiographically demonstrated progression) were observed. The observation time did not suffice to establish statistically significant differences between micristin therapy anticoagulant treatment and basic cardiovascular therapy. The results are suggestive of a more beneficial effect of anticoagulant treatment.     

Single-cell genomics shedding light on marine Thaumarchaeota diversification

Previous studies based on analysis of amoA, 16S ribosomal RNA or accA gene sequences have established that marine Thaumarchaeota fall into two phylogenetically distinct groups corresponding to shallow- and deep-water clades, but it is not clear how water depth interacts with other environmental factors, including light, temperature and location, to affect this pattern of diversification. Earlier studies focused on single-gene distributions were not able to link phylogenetic structure to other aspects of functional adaptation. Here, we analyzed the genome content of 46 uncultivated single Thaumarchaeota cells sampled from epi- and mesopelagic waters of subtropical, temperate and polar oceans. Phylogenomic analysis showed that populations diverged by depth, as expected, and that mesopelagic populations from different locations were well mixed. Functional analysis showed that some traits, including putative DNA photolyase and catalase genes that may be related to adaptive mechanisms to reduce light-induced damage, were found exclusively in members of the epipelagic clade. Our analysis of partial genomes has thus confirmed the depth differentiation of Thaumarchaeota populations observed previously, consistent with the distribution of putative mechanisms to reduce light-induced damage in shallow- and deep-water populations.     

In vivo behavior of copper-64-labeled methanephosphonate tetraaza macrocyclic ligands

Copper-64 ( T(1/2)=12.7 h; beta(+): 0.653 MeV, 17.4%; beta(-): 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. In this paper, we evaluated three tetraaza macrocyclic ligands with two, three, and four pendant methanephosphonate functional groups. DO2P [1,4,7,10-tetraazacyclododecane-1,7-di(methanephosphonic acid)], DO3P [1,4,7,10-tetraazacyclododecane-1,4,7-tri(methanephosphonic acid)], and DOTP [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methanephosphonic acid)] were all radiolabeled with (64)Cu in high radiochemical yields. Copper-64-labeled DO2P and DOTP were highly stable in rat serum out to 24 h, while (64)Cu-DO3P remained 73% intact, with the remainder possibly forming a (64)Cu(.)2DO3P dimer by 24 h. The biodistribution experiments were performed in normal Sprague-Dawley rats. Of the three complexes, (64)Cu-DO2P demonstrated the most optimal clearance through the blood and liver. Copper-64-DO3P and (64)Cu-DOTP exhibited higher liver uptake and longer retention of liver activity, possibly because of the large negative charge of the complexes under physiological conditions. All three (64)Cu-labeled complexes showed high accumulation in bone, likely due to the binding of the methanephosphonate groups to hydroxyapatite. These results suggest that this series of methanephosphonate macrocyclic ligands may be useful as potential bone-imaging agents. The thermodynamic stability constants of the Cu(II) complexes with these three ligands were determined, and were found to be significantly higher than those of their acetate analogues. The Cu(II)-DO2P complex exhibited the highest stability constant among divalent transition metal ion DO2P complexes. Metabolism studies of (64)Cu-DO2P in rat liver suggest that the DO2P ligand may be used as a bifunctional chelator for copper radionuclides in radiodiagnostic or radiotherapeutic studies.     

Single-cell enabled comparative genomics of a deep ocean SAR11 bathytype

Bacterioplankton of the SAR11 clade are the most abundant microorganisms in marine systems, usually representing 25% or more of the total bacterial cells in seawater worldwide. SAR11 is divided into subclades with distinct spatiotemporal distributions (ecotypes), some of which appear to be specific to deep water. Here we examine the genomic basis for deep ocean distribution of one SAR11 bathytype (depth-specific ecotype), subclade Ic. Four single-cell Ic genomes, with estimated completeness of 55%–86%, were isolated from 770 m at station ALOHA and compared with eight SAR11 surface genomes and metagenomic datasets. Subclade Ic genomes dominated metagenomic fragment recruitment below the euphotic zone. They had similar COG distributions, high local synteny and shared a large number (69%) of orthologous clusters with SAR11 surface genomes, yet were distinct at the 16S rRNA gene and amino-acid level, and formed a separate, monophyletic group in phylogenetic trees. Subclade Ic genomes were enriched in genes associated with membrane/cell wall/envelope biosynthesis and showed evidence of unique phage defenses. The majority of subclade Ic-specfic genes were hypothetical, and some were highly abundant in deep ocean metagenomic data, potentially masking mechanisms for niche differentiation. However, the evidence suggests these organisms have a similar metabolism to their surface counterparts, and that subclade Ic adaptations to the deep ocean do not involve large variations in gene content, but rather more subtle differences previously observed deep ocean genomic data, like preferential amino-acid substitutions, larger coding regions among SAR11 clade orthologs, larger intergenic regions and larger estimated average genome size.     

Genome sequencing of a single cell of the widely distributed marine subsurface Dehalococcoidia,phylum Chloroflexi

Bacteria of the class Dehalococcoidia (DEH), phylum Chloroflexi, are widely distributed in the marine subsurface, yet metabolic properties of the many uncultivated lineages are completely unknown. This study therefore analysed genomic content from a single DEH cell designated ‘DEH-J10'' obtained from the sediments of Aarhus Bay, Denmark. Real-time PCR showed the DEH-J10 phylotype was abundant in upper sediments but was absent below 160 cm below sea floor. A 1.44 Mbp assembly was obtained and was estimated to represent up to 60.8% of the full genome. The predicted genome is much larger than genomes of cultivated DEH and appears to confer metabolic versatility. Numerous genes encoding enzymes of core and auxiliary beta-oxidation pathways were identified, suggesting that this organism is capable of oxidising various fatty acids and/or structurally related substrates. Additional substrate versatility was indicated by genes, which may enable the bacterium to oxidise aromatic compounds. Genes encoding enzymes of the reductive acetyl-CoA pathway were identified, which may also enable the fixation of CO₂ or oxidation of organics completely to CO₂. Genes encoding a putative dimethylsulphoxide reductase were the only evidence for a respiratory terminal reductase. No evidence for reductive dehalogenase genes was found. Genetic evidence also suggests that the organism could synthesise ATP by converting acetyl-CoA to acetate by substrate-level phosphorylation. Other encoded enzymes putatively conferring marine adaptations such as salt tolerance and organo-sulphate sulfohydrolysis were identified. Together, these analyses provide the first insights into the potential metabolic traits that may enable members of the DEH to occupy an ecological niche in marine sediments.     

Comparative single-cell genomics reveals potential ecological niches for the freshwater acI Actinobacteria lineage

Members of the acI lineage of Actinobacteria are the most abundant microorganisms in most freshwater lakes; however, our understanding of the keys to their success and their role in carbon and nutrient cycling in freshwater systems has been hampered by the lack of pure cultures and genomes. We obtained draft genome assemblies from 11 single cells representing three acI tribes (acI-A1, acI-A7, acI-B1) from four temperate lakes in the United States and Europe. Comparative analysis of acI SAGs and other available freshwater bacterial genomes showed that acI has more gene content directed toward carbohydrate acquisition as compared to Polynucleobacter and LD12 Alphaproteobacteria, which seem to specialize more on carboxylic acids. The acI genomes contain actinorhodopsin as well as some genes involved in anaplerotic carbon fixation indicating the capacity to supplement their known heterotrophic lifestyle. Genome-level differences between the acI-A and acI-B clades suggest specialization at the clade level for carbon substrate acquisition. Overall, the acI genomes appear to be highly streamlined versions of Actinobacteria that include some genes allowing it to take advantage of sunlight and N-rich organic compounds such as polyamines, di- and oligopeptides, branched-chain amino acids and cyanophycin. This work significantly expands the known metabolic potential of the cosmopolitan freshwater acI lineage and its ecological and genetic traits.