Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo

Understanding AGN-host connection in partially obscured active galactic nuclei. Part III: Properties of ROSAT-selected SDSS AGNs

As the third paper of our serial studies that are aim at examining the AGN-host coevolution by using partially obscured AGNs, we extend the broad-line composite galaxies (composite AGNs) into ROSAT-selected Seyfert 1.8/1.9 galaxies basing upon the RASS/SDSS-DR5 catalog given by Anderson et al.. The SDSS spectra of in total 92 objects are analyzed by the same method used in our previous studies, after requiring the signal-to-noise ratio in the SDSS r' band is larger than 20. Combing the ROSAT-selected Seyfert galaxies with the composite AGNs reinforces the tight correlation between the line ratio [OI]/H\alpha vs. D_n(4000), and establishes a new tight correlation between [SII]/H\alpha vs. D_n(4000). Both correlations suggest the two line ratios are plausible age indicators of the circumnuclear stellar population for typical type I AGNs in which the stellar populations are difficult to be derived from their optical spectra. The ROSAT-selected Seyfert galaxies show that the two correlations depend on the soft X-ray spectral slope \alpha_X that is roughly estimated from the hardness ratios by requiring the X-ray count rates within 0.1-2.4 keV are larger than 0.02 counts s^-1. However, we fail to establish a relationship between \alpha_X and D_n(4000), which is likely caused by the relatively large uncertainties of both parameters (especially for \alpha_X because of the AGN intrinsic obscuration). The previously established L/L_Edd-D_n(4000) evolutionary sequence is reinforced again by the extension to the ROSAT-selected Seyfert galaxies. These X-ray-selected Seyfert galaxies are, however, biased against the two ends of the sequence, which implies that the X-ray Seyfert galaxies present a population at middle evolutionary stage.Comment: 31 pages, 9 figures, 2 tables, to be published in Ap

Role of NADPH Oxidase versus Neutrophil Proteases in Antimicrobial Host Defense

NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47phox−/−) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)−/−×cathepsin G (CG)−/− mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47phox−/− mice, whereas NE−/−×CG−/− mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens

ARTEFACTS: How do we want to deal with the future of our one and only planet?

The European Commission’s Science and Knowledge Service, the Joint Research Centre (JRC), decided to try working hand-in-hand with leading European science centres and museums. Behind this decision was the idea that the JRC could better support EU Institutions in engaging with the European public. The fact that European Union policies are firmly based on scientific evidence is a strong message which the JRC is uniquely able to illustrate. Such a collaboration would not only provide a platform to explain the benefits of EU policies to our daily lives but also provide an opportunity for European citizens to engage by taking a more active part in the EU policy making process for the future. A PILOT PROGRAMME To test the idea, the JRC launched an experimental programme to work with science museums: a perfect partner for three compelling reasons. Firstly, they attract a large and growing number of visitors. Leading science museums in Europe have typically 500 000 visitors per year. Furthermore, they are based in large European cities and attract local visitors as well as tourists from across Europe and beyond. The second reason for working with museums is that they have mastered the art of how to communicate key elements of sophisticated arguments across to the public and making complex topics of public interest readily accessible. That is a high-value added skill and a crucial part of the valorisation of public-funded research, never to be underestimated. Finally museums are, at present, undergoing something of a renaissance. Museums today are vibrant environments offering new techniques and technologies to both inform and entertain, and attract visitors of all demographics.JRC.H.2-Knowledge Management Methodologies, Communities and Disseminatio

Cross Talk between Transition Metal Cathode and Li Metal Anode: Unraveling Its Influence on the Deposition/Dissolution Behavior and Morphology of Lithium

Lithium metal batteries (LMBs) combining a Li metal anode with a transition metal (TM) cathode can achieve higher practical energy densities (Wh L−1) than Li/S or Li/O2 cells. Research for improving the electrochemical behavior of the Li metal anode by, for example, modifying the liquid electrolyte is often conducted in symmetrical Li/Li or Li/Cu cells. This study now demonstrates the influence of the TM cathode on the Li metal anode, thus full cell behavior is analyzed in a way not considered so far in research with LMBs. Therefore, the deposition/dissolution behavior of Li metal and the resulting morphology is investigated with three different cathode materials (LiNi0.5Mn1.5O4, LiNi0.6Mn0.2Co0.2O2, and LiFePO4) by post mortem analysis with a scanning electron microscope. The observed large differences of the Li metal morphology are ascribed to the dissolution and crossover of TMs found deposited on Li metal and in the electrolyte by X‐ray photoelectron spectroscopy, energy‐dispersive X‐ray spectroscopy, and total reflection X‐ray fluorescence analysis. To support this correlation, the TM dissolution is simulated by adding Mn salt to the electrolyte. This study offers new insights into the cross talk between the Li metal anodes and TM cathodes, which is essential, when investigating Li metal electrodes for LMB full cells

Cross Talk between Transition Metal Cathode and Li Metal Anode: Unraveling Its Influence on the Deposition/Dissolution Behavior and Morphology of Lithium

Lithium metal batteries (LMBs) combining a Li metal anode with a transition metal (TM) cathode can achieve higher practical energy densities (Wh L−1) than Li/S or Li/O2 cells. Research for improving the electrochemical behavior of the Li metal anode by, for example, modifying the liquid electrolyte is often conducted in symmetrical Li/Li or Li/Cu cells. This study now demonstrates the influence of the TM cathode on the Li metal anode, thus full cell behavior is analyzed in a way not considered so far in research with LMBs. Therefore, the deposition/dissolution behavior of Li metal and the resulting morphology is investigated with three different cathode materials (LiNi0.5Mn1.5O4, LiNi0.6Mn0.2Co0.2O2, and LiFePO4) by post mortem analysis with a scanning electron microscope. The observed large differences of the Li metal morphology are ascribed to the dissolution and crossover of TMs found deposited on Li metal and in the electrolyte by X‐ray photoelectron spectroscopy, energy‐dispersive X‐ray spectroscopy, and total reflection X‐ray fluorescence analysis. To support this correlation, the TM dissolution is simulated by adding Mn salt to the electrolyte. This study offers new insights into the cross talk between the Li metal anodes and TM cathodes, which is essential, when investigating Li metal electrodes for LMB full cells

Mutagenesis of the GXXXG motif in the transmembrane domain of VSV-G.

<p>Schematic representation of the VSV* genome in which the authentic viral open reading frames (ORFs, VSV-N, -P, -M, -G and -L; light grey) are surrounded by non-translated regulatory elements (white). The additional ORF for eGFP (dark grey) was inserted between the ORFs for VSV-G and -L (restriction sites used for cloning and further modification of the genome are highlighted). The transmembrane domain (underlined amino acids) of VSV-G wt contains a GXXXG motif (amino acid residues 473–477) that has been mutated to LXXXL.</p

The glycoprotein of vesicular stomatitis virus promotes release of virus-like particles from tetherin-positive cells

<div><p>Vesicular stomatitis virus (VSV) release from infected cells is inhibited by the interferon (IFN)-inducible antiviral host cell factor tetherin (BST-2, CD317). However, several viruses encode tetherin antagonists and it is at present unknown whether residual VSV spread in tetherin-positive cells is also promoted by a virus-encoded tetherin antagonist. Here, we show that the viral glycoprotein (VSV-G) antagonizes tetherin in transfected cells, although with reduced efficiency as compared to the HIV-1 Vpu protein. Tetherin antagonism did not involve alteration of tetherin expression and was partially dependent on a GXXXG motif in the transmembrane domain of VSV-G. However, mutation of the GXXXG motif did not modulate tetherin sensitivity of infectious VSV. These results identify VSV-G as a tetherin antagonist in transfected cells but fail to provide evidence for a contribution of tetherin antagonism to viral spread.</p></div

Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner

Summary: The Ebola virus glycoprotein (EBOV-GP) forms GP-containing microvesicles, so-called virosomes, which are secreted from GP-expressing cells. However, determinants of GP-virosome release and their functionality are poorly understood. We characterized GP-mediated virosome formation and delineated the role of the antiviral factor tetherin (BST2, CD317) in this process. Residues in the EBOV-GP receptor-binding domain (RBD) promote GP-virosome secretion, while tetherin suppresses GP-virosomes by interactions involving the GP-transmembrane domain. Tetherin from multiple species interfered with GP-virosome release, and tetherin from the natural fruit bat reservoir showed the highest inhibitory activity. Moreover, analyses of GP from various ebolavirus strains, including the EBOV responsible for the West African epidemic, revealed the most efficient GP-virosome formation by highly pathogenic ebolaviruses. Finally, EBOV-GP-virosomes were immunomodulatory and acted as decoys for EBOV-neutralizing antibodies. Our results indicate that GP-virosome formation might be a determinant of EBOV immune evasion and pathogenicity, which is suppressed by tetherin. : Nehls et al. demonstrate that the glycoprotein of the highly pathogenic Ebola virus is incorporated into secretory vesicles, called GP-virosomes, to dampen the immune response and capture neutralizing antibodies. The lack of replication competence and the incorporation of antigenically intact GP might qualify GP-virosomes as safe vaccine candidates. Keywords: Ebola virus, glycoprotein, microvesicles, virosome, exosome, tetherin, immune modulation, immune evasion, antiviral immune response, neutralizing antibod

The GXXXG motif is dispensable for viral spread in tetherin-positive cells.

<p>(A) HeLa cells were transfected with the indicated siRNAs and subsequently infected with VSV wt or LXXXL mutant at an MOI of 0.005 for 1 h. Virus titers in culture supernatants were determined at the indicated time points post infection. The results of a single representative experiment carried out with triplicate samples are shown and were confirmed in two separate experiments. (B) The experiment was carried out as described for panel (A) but relative titers measured at 12 h post infection are shown. The results represent the average of three independent experiments performed with triplicate samples. Titers measured for untransfected control cells were set to 1. Error bars indicate standard error of the mean (SEM). (C) Vero cells stably expressing human tetherin (Vero-Tetherin) or stably containing empty vector (Vero) were infected with VSV wt or mutant LXXXL. Viral titers in culture supernatants were determined at the indicated time points post infection. The results of a single representative experiment carried out with triplicate samples are shown and were confirmed in two separate experiments. (D) The experiment was carried out as described for panel (C) but relative titers measured at 12 h post infection are shown. The results represent the average of three independent experiments performed with triplicate samples. Titers obtained from the respective control Vero cells were set to 1. Error bars indicate SEM. One-way ANOVA with Bonferroni post-test analyses were performed (B and D) to test statistical significance between selected groups (***, p ≤ 0.001).</p