Fluids from the Oceanic Crust Support Microbial Activities within the Deep Biosphere

The importance of crustal fluid chemical composition in driving the marine deep subseafloor biosphere was examined in northeast Pacific ridge-flank sediments. At IODP Site U1301, sulfate from crustal fluids diffuses into overlying sediments, forming a transition zone where sulfate meets in situ-produced methane. Enhanced cell counts and metabolic activity suggest that sulfate stimulates microbial respiration, specifically anaerobic methane oxidation coupled to sulfate reduction. Cell counts and activity are also elevated in basement-near layers. Owing to the worldwide expansion of the crustal aquifer, we postulate that crustal fluids may fuel the marine deep subseafloor biosphere on a global scale.     

The structure of the Shiga toxin 2a A‐subunit dictates the interactions of the toxin with blood components

Hemolytic uremic syndrome (eHUS) is a severe complication of human infections with Shiga toxins (Stxs)‐producing Escherichia coli. A key step in the pathogenesis of eHUS is the interaction of Stxs with blood components before the targeting of renal endothelial cells. Here, we show that a single proteolytic cleavage in the Stx2a A‐subunit, resulting into two fragments (A1 and A2) linked by a disulfide bridge (cleaved Stx2a), dictates different binding abilities. Uncleaved Stx2a was confirmed to bind to human neutrophils and to trigger leukocyte/platelet aggregate formation, whereas cleaved Stx2a was ineffective. Conversely, binding of complement factor H was confirmed for cleaved Stx2a and not for uncleaved Stx2a. It is worth noting that uncleaved and cleaved Stx2a showed no differences in cytotoxicity for Vero cells or Raji cells, structural conformation, and contaminating endotoxin. These results have been obtained by comparing two Stx2a batches, purified in different laboratories by using different protocols, termed Stx2a(cl; cleaved toxin, Innsbruck) and Stx2a(uncl; uncleaved toxin, Bologna). Stx2a(uncl) behaved as Stx2a(cl) after mild trypsin treatment. In this light, previous controversial results obtained with purified Stx2a has to be critically re‐evaluated; furthermore, characterisation of the structure of circulating Stx2a is mandatory to understand eHUS‐pathogenesis and to develop therapeutic approaches.     

Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.     

Identification of anammox bacteria in a full-scale deammonification plant making use of anaerobic ammonia oxidation

The existence of anaerobic ammonia-oxidizing (anammox) bacteria was postulated in the late 1970s. Approximately 20 years later, these lithotrophic members of the nitrogen cycle were identified as deep-branching members of the planctomycetes. Recently, full-scale implementation of biological deammonification was successfully achieved in the DEMON reactor at the wastewater treatment plant in Strass, Austria. The sludge of this reactor contains red granules and brownish flocs that can be physically separated. The two fractions yielded different banding patterns in denaturing gradient gel electrophoresis of PCR products obtained with primer sets targeting the 16S rRNA genes of planctomycetes. Comparative analysis of partial sequences of almost full-length 16S rRNA gene clones obtained from the granules and flocs confirms the differences in the community composition of the two fractions. The sequences retrieved from the red granules were 93% similar to those of Candidatus Brocadia anammoxidans, a bacterium known to catalyze the anaerobic ammonia oxidation.     

Early endosomal SNAREs form a structurally conserved SNARE complex and fuse liposomes with multiple topologies

SNARE proteins mediate membrane fusion in eukaryotic cells. They contain conserved SNARE motifs that are usually located adjacent to a C-terminal transmembrane domain. SNARE motifs spontaneously assemble into four helix bundles, with each helix belonging to a different subfamily. Liposomes containing SNAREs spontaneously fuse with each other, but it is debated how the SNAREs are distributed between the membranes. Here, we report that the SNAREs mediating homotypic fusion of early endosomes fuse liposomes in five out of seven possible combinations, in contrast to previously studied SNAREs involved in heterotypic fusion events. The crystal structure of the early endosomal SNARE complex resembles that of the neuronal and late endosomal complexes, but differs in surface side-chain interactions. We conclude that homotypic fusion reactions may proceed with multiple SNARE topologies, suggesting that the conserved SNARE structure allows for flexibility in the initial interactions needed for fusion.     

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism

Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.     

Factor I-mediated processing of complement fragments on HIV immune complexes targets HIV to CR2-expressing B cells and facilitates B cell-mediated transmission of opsonized HIV to T cells

Our study demonstrates that binding of complement-opsonized HIV to complement receptor type 1 on human erythrocytes (E) via C3b fragments is followed by a rapid normal human serum-mediated detachment of HIV from E. The release was dependent on the presence of factor I indicating a conversion of C3b fragments to iC3b and C3d on the viral surface. This in turn resulted in an efficient binding of opsonized HIV to CR2-expressing B cells, thus facilitating B cell-mediated transmission of HIV to T cells. These data provide a new dynamic view of complement opsonization of HIV, suggesting that association of virus with E might be a transient phenomenon and the factor I-mediated processing of C3b to iC3b and C3d on HIV targets the virus to complement receptor type 2-expressing cells. Thus, factor I in concert with CR1 on E and factor H in serum due to their cofactor activity are likely to be important contributors for the generation of C3d-opsonized infectious HIV reservoirs on follicular dendritic cells and/or B cells in HIV-infected individuals.