Silintaphin-1--interaction with silicatein during structure-guiding bio-silica formation

Silicateins are unique enzymes of sponges (phylum Porifera) that template and catalyze the polymerization of nanoscale silicate to siliceous skeletal elements. These multifunctional spicules are often elaborately shaped, with complex symmetries. They carry an axial proteinaceous filament, consisting of silicatein and the scaffold protein silintaphin-1, which guides silica deposition and subsequent spicular morphogenesis. In vivo, the synthesis of the axial filament very likely proceeds in three steps: (a) assembly of silicatein monomers to form one pentamer; (b) assembly of pentamers to form fractal-like structures; and finally (c) assembly of fractal-like structures to form filaments. The present study was aimed at exploring the effect of self-assembled complexes of silicatein and silintaphin-1 on biosilica synthesis in vitro. Hence, in a comparative approach, recombinant silicatein and recombinant silintaphin-1 were used at different stoichiometric ratios to form axial filaments and to synthesize biosilica. Whereas recombinant silicatein-α reaggregates to randomly organized structures, coincubation of silicatein-α and silintaphin-1 (molecular ratio 4 : 1) resulted in synthetic filaments via fractal-like patterned self-assemblies, as observed by electron microscopy. Concurrently, owing to the concerted action of both proteins, the enzymatic activity of silicatein-α strongly increased by 5.3-fold (with the substrate tetraethyl orthosilicate), leading to significantly enhanced synthesis of biosilica. These results indicate that silicatein-α-mediated biosilicification depends on the concomitant presence of silicatein-α and silintaphin-1. Accordingly, silintaphin-1 might not only enhance the enzymatic activity of silicatein-α, but also accelerate the nonenzymatic polycondensation of the silica product before releasing the fully synthesized biosiliceous polymer.     

High-resolution peptide mapping of cerebrospinal fluid: a novel concept for diagnosis and research in central nervous system diseases

Peptides, such as many hormones, cytokines and growth factors play a central role in biological processes. Furthermore, as degradation products and processed forms of larger proteins they are part of the protein turnover. Thus, they can reflect disease-related changes in an organism's homeostasis in several ways. Since two-dimensional gel electrophoresis is restricted to analysis and display of proteins with relative molecular masses above 5000, we developed Differential Peptide Display (DPD), a new technology for analysis and visualization of peptides. Here we describe its application to cerebrospinal fluid of three subjects without a disease of the central nervous system (CNS) undergoing routine myelography and of two patients suffering from a primary CNS lymphoma. Peptides with a relative molecular mass below 20000 were extracted and analysed by a combination of chromatography and mass spectrometry. The peptide pattern of a sample was depicted as a multi-dimensional peptide mass fingerprint with each peptide's position being characterized by its molecular mass and chromatographic behaviour. Such a fingerprint of a CNS sample consists of more than 6000 different signals. Data analysis of peptide patterns from patients with CNS lymphoma compared to controls revealed obvious differences regarding the peptide content of the samples. By analysing peptides within a mass range of 750-20000, DPD extends 2D gel electrophoresis, thus offering the chance to investigate CNS diseases on the level of peptides. This represents a new approach for diagnosis and possible therapy.     

Launch of a new report: “Road testing organizational life cycle assessment around the world: applications,experiences and lessons learned”

Purpose

Organizational life cycle assessment (O-LCA) is still a rather young proposal, but moving towards becoming more broadly accepted as a scientifically mature and practical method. The UNEP/SETAC flagship project “LCA of organizations” concluded its “road-testing” phase and is glad to announce the publication of the final report “Road testing organizational life cycle assessment around the world: applications, experiences and lessons learned.” The full report can be accessed at http://www.lifecycleinitiative.org/download/6060. This article shortly summarizes the flagship project phases and main outcomes, particularly the report recently launched, and pinpoints future actions.

Methods

In 2015, the “Guidance on Organizational Life Cycle Assessment” was published. During the following 2 years, the flagship project accompanied 12 organizations in the road testing of that O-LCA Guidance. They represent four world regions, different sectors and sizes. The road testers’ case studies and their feedback are the basis of the Road-testing Report.

Results and discussion

The Road-testing Report aims to complement the O-LCA Guidance through the road testers’ experience, thus delivering advice for future practitioners and inspiration to method developers. It includes executive summaries of the O-LCA road testers’ case studies and the main results of a comprehensive survey through which the road testers share their experience, feedback, and lessons learned. The road testing confirmed the application potential of the O-LCA method and the positive outcomes of the road testing have shown that no immediate updates to the O-LCA Guidance are needed, but some priority actions were identified in order to further ease the application of O-LCA.

Conclusions

Three main tasks for the coming years are identified by the authors: firstly, the challenges highlighted during the road testing should be addressed in the future by the LCA community; specific methodological difficulties of certain kinds of organizations, like the service sector, should be targeted; and finally, the potential revealed by the organizational perspective can be deployed in adjacent LCA fields. The flagship project team hopes that this second publication, together with the great acceptance of the O-LCA Guidance and the contribution of third parties, will pave the way to make O-LCA a mainstream tool.

     

Chlamydia pneumoniae infection acts as an endothelial stressor with the potential to initiate the earliest heat shock protein 60-dependent inflammatory stage of atherosclerosis

We identified increased expression and redistribution of the intracellular protein 60-kDa human heat shock protein (hHSP60) (HSPD1) to the cell surface in human endothelial cells subjected to classical atherosclerosis risk factors and subsequent immunologic cross-reactivity against this highly conserved molecule, as key events occurring early in the process of atherosclerosis. The present study aimed at investigating the role of infectious pathogens as stress factors for vascular endothelial cells and, as such, contributors to early atherosclerotic lesion formation. Using primary donor-matched arterial and venous human endothelial cells, we show that infection with Chlamydia pneumoniae leads to marked upregulation and surface expression of hHSP60 and adhesion molecules. Moreover, we provide evidence for an increased susceptibility of arterial endothelial cells for redistribution of hHSP60 to the cellular membrane in response to C. pneumoniae infection as compared to autologous venous endothelial cells. We also show that oxidative stress has a central role to play in endothelial cell activation in response to chlamydial infection. These data provide evidence for a role of C. pneumoniae as a potent primary endothelial stressor for arterial endothelial cells leading to enrichment of hHSP60 on the cellular membrane and, as such, a potential initiator of atherosclerosis.     

Upregulation of miR-24 is associated with a decreased DNA damage response upon etoposide treatment in highly differentiated CD8(+) T cells sensitizing them to apoptotic cell death

The life-long homeostasis of memory CD8(+) T cells as well as persistent viral infections have been shown to facilitate the accumulation of highly differentiated CD8(+) CD28(-) T cells, a phenomenon that has been associated with an impaired immune function in humans. However, the molecular mechanisms regulating homeostasis of CD8(+) CD28(-) T cells have not yet been elucidated. In this study, we demonstrate that the miR-23～24～27 cluster is up-regulated during post-thymic CD8(+) T-cell differentiation in humans. The increased expression of miR-24 in CD8(+) CD28(-) T cells is associated with decreased expression of the histone variant H2AX, a protein that plays a key role in the DNA damage response (DDR). Following treatment with the classic chemotherapeutic agent etoposide, a topoisomerase II inhibitor, apoptosis was increased in CD8(+) CD28(-) when compared to CD8(+) CD28(+) T cells and correlated with an impaired DDR in this cell type. The reduced capacity of CD8(+) CD28(-) T cell to repair DNA was characterized by the automated fluorimetric analysis of DNA unwinding (FADU) assay as well as by decreased phosphorylation of H2AX at Ser139, of ATM at Ser1981, and of p53 at Ser15. Interleukin (IL)-15 could prevent etoposide-mediated apoptosis of CD8(+) CD28(-) T cells, suggesting a role for IL-15 in the survival and the age-dependent accumulation of CD8(+) CD28(-) T cells in humans.     

Cancer as an overhealing wound: an old hypothesis revisited

What is the relationship between the wound-healing process and the development of cancer? Malignant tumours often develop at sites of chronic injury, and tissue injury has an important role in the pathogenesis of malignant disease, with chronic inflammation being the most important risk factor. The development and functional characterization of genetically modified mice that lack or overexpress genes that are involved in repair, combined with gene-expression analysis in wounds and tumours, have highlighted remarkable similarities between wound repair and cancer. However, a few crucial differences were also observed, which could account for the altered metabolism, impaired differentiation capacity and invasive growth of malignant tumours.     

Tyrosine phosphoproteomics of fibroblast growth factor signaling: a role for insulin receptor substrate-4

Signal transduction by receptor tyrosine kinases is initiated by recruitment of a variety of signaling proteins to tyrosine-phosphorylated motifs in the activated receptors. Several signaling pathways are thus activated in parallel, the combination of which decides the cellular response. Here, we present a dual strategy for extensive mapping of tyrosine-phosphorylated proteins and probing of signal-dependent protein interactions of a signaling cascade. The approach relies on labeling of cells with "heavy" and "light" isotopic forms of Arg to distinguish two cell populations. First, tyrosine-phosphorylated proteins from stimulated ("heavy"-labeled) and control samples ("normal"-labeled) are isolated and subjected to high sensitivity Fourier transform ion cyclotron resonance mass spectrometry analysis. Next, phosphopeptides corresponding to tyrosine phosphorylation sites identified during the tyrosine phosphoproteomic analysis are used as baits to isolate phosphospecific protein binding partners, which are subsequently identified by mass spectrometry. We used this approach to identify 28 components of the signaling cascade induced by stimulation with the basic fibroblast growth factor. Insulin receptor substrate-4 was identified as a novel candidate in fibroblast growth factor receptor signaling, and we defined phosphorylation-dependent interactions with other components, such as adaptor protein Grb2, of the signaling cascade. Finally, we present evidence for a complex containing insulin receptor substrate-4 and ShcA in signaling by the fibroblast growth factor receptor.     

Soluble M1 protein of Streptococcus pyogenes triggers potent T cell activation

Streptococcus pyogenes of the M1 serotype is commonly associated with large outbreaks of invasive streptococcal infections and development of streptococcal toxic shock syndrome (STSS). The pathogenesis behind these infections is believed to involve bacterial superantigens that induce potent inflammatory responses, but the reason why strains of the M1 serotype are over-represented in STSS is still not understood. In the present investigation, we show that a highly purified soluble form of the M1 protein from S. pyogenes , which lacks the membrane-spanning region, is a potent inducer of T cell proliferation and release of Th1 type cytokines. M1 protein-evoked T cell proliferation was HLA class II-dependent but not MHC-restricted, did not require intracellular processing and was Vβ-restricted. Extensive mass spectrometry studies indicated that there were no other detectable proteins in the preparation. Taken together, our data demonstrate that soluble M1 protein is a novel streptococcal superantigen, which likely contributes to the excessive T cell activation and hyperinflammatory response seen in severe invasive streptococcal infections.