Characterisation of Schiff base and chromophore in green proteorhodopsin by solid-state NMR

The proteorhodopsin family consists of hundreds of homologous retinal containing membrane proteins found in bacteria in the photic zone of the oceans. They are colour tuned to their environment and act as light-driven proton pumps with a potential energetic and regulatory function. Precise structural details are still unknown. Here, the green proteorhodopsin variant has been selected for a chemical shift analysis of retinal and Schiff base by solid-state NMR. Our data show that the chromophore exists in mainly all-trans configuration in the proteorhodopsin ground state. The optical absorption maximum together with retinal and Schiff base chemical shifts indicate a strong interaction network between chromophore and opsin. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mark Lorch and Andreas C. Woerner contributed equally to this work.     

Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli

RNA thermometers are thermosensors that regulate gene expression by temperature-induced changes in RNA conformation. Naturally occurring RNA thermometers exhibit complex secondary structures which are believed to undergo a series of gradual structural changes in response to temperature shifts. Here, we report the de novo design of considerably simpler RNA thermometers that provide useful RNA-only tools to regulate bacterial gene expression by a shift in the growth temperature. We show that a single small stem-loop structure containing the ribosome binding site is sufficient to construct synthetic RNA thermometers that work efficiently at physiological temperatures. Our data suggest that the thermometers function by a simple melting mechanism and thus provide minimum size on/off switches to experimentally induce or repress gene expression by temperature.     

Escherichia coli produces phosphoantigens activating human gamma delta T cells.

Human Vgamma9delta2 T lymphocytes are suggested to play an important role in the immune response to various microbial pathogens. In contrast to alphabeta T cells, gammadelta T lymphocytes recognize small, non-protein, phosphate-bearing antigens (phosphoantigens) in a major histocompatibility complex-independent manner. Four different phosphoantigens termed TUBag1 to TUBag4 with a common 3-formyl-1-butyl-pyrophosphate moiety and isopentenyl-pyrophosphate have been isolated and identified from mycobacteria. However, natural occurring gammadelta T cell ligands from other bacterial species were not characterized so far. Here, we describe the structural identification of the two compounds responsible for the gammadelta T cell-stimulating capacity of Escherichia coli as similar to the mycobacterial phosphoantigens 3-formyl-1-butyl-pyrophosphate and its M(r) 275 homologue TUBag2. In addition, E. coli phosphoantigens exert bioactivities on gammadelta T cells with similar potencies to the mycobacterial phosphoantigens at 5-15 nm concentration. Furthermore, our results clearly prove that the deoxyxylulose 5-phophate pathway (also referred to as Rohmer metabolic route of isoprenoid biosynthesis) is essential for the biosynthesis of the phosphoantigens in E. coli. Because this pathway is absent from human cells, it proves an ideal target for focusing efficiently the antimicrobial selectivity of human gammadelta T lymphocytes.     

Completely serum-free and chemically defined adipocyte development and maintenance

Background aims

In vitro engineered adipose tissue is in great demand to treat lost or damaged soft tissue or to screen for new drugs, among other applications. However, today most attempts depend on the use of animal-derived sera. To pave the way for the application of adipose tissue–engineered products in clinical trials or as reliable and robust in vitro test systems, sera should be completely excluded from the production process. In this study, we aimed to develop an in vitro adipose tissue model in the absence of sera and maintain its function long-term.

Cross‐talk between monocyte invasion and astrocyte proliferation regulates scarring in brain injury

Scar formation after brain injury is still poorly understood. To further elucidate such processes, here, we examine the interplay between astrocyte proliferation taking place predominantly at the vascular interface and monocyte invasion. Using genetic mouse models that decrease or increase reactive astrocyte proliferation, we demonstrate inverse effects on monocyte numbers in the injury site. Conversely, reducing monocyte invasion using CCR2^?/? mice causes a strong increase in astrocyte proliferation, demonstrating an intriguing negative cross‐regulation between these cell types at the vascular interface. CCR2^?/? mice show reduced scar formation with less extracellular matrix deposition, smaller lesion site and increased neuronal coverage. Surprisingly, the GFAP⁺ scar area in these mice is also significantly decreased despite increased astrocyte proliferation. Proteomic analysis at the peak of increased astrocyte proliferation reveals a decrease in extracellular matrix synthesizing enzymes in the injury sites of CCR2^?/? mice, highlighting how early key aspects of scar formation are initiated. Taken together, we provide novel insights into the cross‐regulation of juxtavascular proliferating astrocytes and invading monocytes as a crucial mechanism of scar formation upon brain injury.     

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation

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Density-dependent shift from facilitation to competition in a dwarf <Emphasis Type="Italic">Avicennia germinans</Emphasis> forest

The global effort to rehabilitate and restore destroyed mangrove forests is unable to keep up with the high mangrove deforestation rates, which exceed the average pace of global deforestation. Although facilitation theory presents new possibilities for the restoration of heavily degraded mangrove sites, knowledge of tree–tree interactions in stressed mangrove forest ecosystems is too limited to utilize facilitation appropriately. The aim was to determine the mode of local interaction among stressed mangrove trees by investigating the effect of clustering on tree size and crown morphology under contrasting stand densities. The study was conducted in a dwarf Avicennia germinans forest in Northern Brazil, in which tree growth is limited by infrequent inundation and high pore-water salinity. Autoregressive regression, Voronoi tessellation and spatial point pattern statistics were used to address the spatial processes underlying tree interaction. Under low stand density (1.2 trees m^?2) dwarf trees which grew in clustered cohorts of A. germinans had a less stunted crown morphology revealing the dominance of a positive neighborhood influence among plants. In contrast, dwarf trees in the denser forest stand (2.7 trees m^?2) were interacting competitively as indicated by the more negative effect of neighbors on crown morphology and size. The shift from facilitative to competitive interactions is an important feature of mangrove forest regeneration under harsh environmental conditions. If mangrove trees are unable to regenerate naturally on severely degraded sites, intraspecific facilitation could be used to assist regeneration by planting seedlings in clusters and not evenly spaced.