The role of receptor MAS in microglia-driven retinal vascular development

Objective: The receptor MAS, encoded by Mas1, is expressed in microglia and its activation has been linked to anti-inflammatory actions. However, microglia are involved in several different processes in the central nervous system, including the promotion of angiogenesis. We therefore hypothesized that the receptor MAS also plays a role in angiogenesis via microglia. Approach and results: To assess the role of MAS on vascular network development, flat-mounted retinas from 3-day-old wild-type (WT) and Mas1−/− mice were subjected to Isolectin B4 staining. The progression of the vascular front was reduced (− 24%, p < 0.0001) and vascular density decreased (− 38%, p < 0.001) in Mas1−/− compared to WT mice with no change in the junction density. The number of filopodia and filopodia bursts were decreased in Mas1−/− mice at the vascular front (− 21%, p < 0.05; − 29%, p < 0.0001, respectively). This was associated with a decreased number of vascular loops and decreased microglial density at the vascular front in Mas1−/− mice (-32%, p < 0.001; − 26%, p < 0.05, respectively). As the front of the developing vasculature is characterized by reduced oxygen levels, we determined the expression of Mas1 following hypoxia in primary microglia from 3-day-old WT mice. Hypoxia induced a 14-fold increase of Mas1 mRNA expression (p < 0.01). Moreover, stimulation of primary microglia with a MAS agonist induced expression of Notch1 (+ 57%, p < 0.05), Dll4 (+ 220%, p  < 0.001) and Jag1 (+ 137%, p < 0.001), genes previously described to mediate microglia/endothelial cell interaction during angiogenesis. Conclusions: Our study demonstrates that the activation of MAS is important for microglia recruitment and vascular growth in the developing retina

Domain Organization of Long Signal Peptides of Single-Pass Integral Membrane Proteins Reveals Multiple Functional Capacity

Targeting signals direct proteins to their extra - or intracellular destination such as the plasma membrane or cellular organelles. Here we investigated the structure and function of exceptionally long signal peptides encompassing at least 40 amino acid residues. We discovered a two-domain organization (“NtraC model”) in many long signals from vertebrate precursor proteins. Accordingly, long signal peptides may contain an N-terminal domain (N-domain) and a C-terminal domain (C-domain) with different signal or targeting capabilities, separable by a presumably turn-rich transition area (tra). Individual domain functions were probed by cellular targeting experiments with fusion proteins containing parts of the long signal peptide of human membrane protein shrew-1 and secreted alkaline phosphatase as a reporter protein. As predicted, the N-domain of the fusion protein alone was shown to act as a mitochondrial targeting signal, whereas the C-domain alone functions as an export signal. Selective disruption of the transition area in the signal peptide impairs the export efficiency of the reporter protein. Altogether, the results of cellular targeting studies provide a proof-of-principle for our NtraC model and highlight the particular functional importance of the predicted transition area, which critically affects the rate of protein export. In conclusion, the NtraC approach enables the systematic detection and prediction of cryptic targeting signals present in one coherent sequence, and provides a structurally motivated basis for decoding the functional complexity of long protein targeting signals

Intervening with Urinary Tract Infections Using Anti-Adhesives Based on the Crystal Structure of the FimH–Oligomannose-3 Complex

Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-binding FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-Ia and alpha3beta1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infection of neighbouring cells, being the major cause of the troublesome recurrent urinary tract infections.We demonstrate that alpha-D-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl alpha-D-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl alpha-D-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl alpha-D-mannosides was elucidated in the crystal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH interacts with Man alpha1,3Man beta1,4GlcNAc beta1,4GlcNAc in an extended binding site. The interactions along the alpha1,3 glycosidic bond and the first beta1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl alpha-D-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group.The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extends into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection

Simulating rewetting events in intermittent rivers and ephemeral streams: A global analysis of leached nutrients and organic matter

Climate change and human pressures are changing the global distribution and the ex‐ tent of intermittent rivers and ephemeral streams (IRES), which comprise half of the global river network area. IRES are characterized by periods of flow cessation, during which channel substrates accumulate and undergo physico‐chemical changes (precon‐ ditioning), and periods of flow resumption, when these substrates are rewetted and release pulses of dissolved nutrients and organic matter (OM). However, there are no estimates of the amounts and quality of leached substances, nor is there information on the underlying environmental constraints operating at the global scale. We experi‐ mentally simulated, under standard laboratory conditions, rewetting of leaves, river‐ bed sediments, and epilithic biofilms collected during the dry phase across 205 IRES from five major climate zones. We determined the amounts and qualitative character‐ istics of the leached nutrients and OM, and estimated their areal fluxes from riverbeds. In addition, we evaluated the variance in leachate characteristics in relation to selected environmental variables and substrate characteristics. We found that sediments, due to their large quantities within riverbeds, contribute most to the overall flux of dis‐ solved substances during rewetting events (56%–98%), and that flux rates distinctly differ among climate zones. Dissolved organic carbon, phenolics, and nitrate contrib‐ uted most to the areal fluxes. The largest amounts of leached substances were found in the continental climate zone, coinciding with the lowest potential bioavailability of the leached OM. The opposite pattern was found in the arid zone. Environmental vari‐ ables expected to be modified under climate change (i.e. potential evapotranspiration, aridity, dry period duration, land use) were correlated with the amount of leached sub‐ stances, with the strongest relationship found for sediments. These results show that the role of IRES should be accounted for in global biogeochemical cycles, especially because prevalence of IRES will increase due to increasing severity of drying event

A global analysis of terrestrial plant litter dynamics in non-perennial waterways

Perennial rivers and streams make a disproportionate contribution to global carbon (C) cycling. However, the contribution of intermittent rivers and ephemeral streams (IRES), which sometimes cease to flow and can dry completely, is largely ignored although they represent over half the global river network. Substantial amounts of terrestrial plant litter (TPL) accumulate in dry riverbeds and, upon rewetting, this material can undergo rapid microbial processing. We present the results of a global research collaboration that collected and analysed TPL from 212 dry riverbeds across major environmental gradients and climate zones. We assessed litter decomposability by quantifying the litter carbon-to-nitrogen ratio and oxygen (O2) consumption in standardized assays and estimated the potential short-term CO2 emissions during rewetting events. Aridity, cover of riparian vegetation, channel width and dry-phase duration explained most variability in the quantity and decomposability of plant litter in IRES. Our estimates indicate that a single pulse of CO2 emission upon litter rewetting contributes up to 10% of the daily CO2 emission from perennial rivers and stream, particularly in temperate climates. This indicates that the contributions of IRES should be included in global C-cycling assessments

Sediment respiration pulses in intermittent rivers and ephemeral streams

Intermittent rivers and ephemeral streams (IRES) may represent over half the global stream network, but their contribution to respiration and carbon dioxide (CO2) emissions is largely undetermined. In particular, little is known about the variability and drivers of respiration in IRES sediments upon rewetting, which could result in large pulses of CO2. We present a global study examining sediments from 200 dry IRES reaches spanning multiple biomes. Results from standardized assays show that mean respiration increased 32–66‐fold upon sediment rewetting. Structural equation modelling indicates that this response was driven by sediment texture and organic matter quantity and quality, which, in turn, were influenced by climate, land use and riparian plant cover. Our estimates suggest that respiration pulses resulting from rewetting of IRES sediments could contribute significantly to annual CO2 emissions from the global stream network, with a single respiration pulse potentially increasing emission by 0.2–0.7%. As the spatial and temporal extent of IRES increases globally, our results highlight the importance of recognizing the influence of wetting‐drying cycles on respiration and CO2 emissions in stream networks

A Novel TGF-β-like Gene,fugacin,Specifically Expressed in the Spemann Organizer ofXenopus

AbstractUsing a differential screening strategy, we have cloned a novelXenopusgene,fugacin,related to the transforming growth factor β superfamily. Transcripts were detected primarily in the dorsal marginal zone of late blastula. Thereafter, they became highly localized to the blastopore lip of early gastrula and were not observed at later stages. This gene, which is most homologous to the mouse genenodal,displays a new pattern of cysteine residues. These findings highlight the potential role of these growth factors during early vertebrate development