Isolation, functional characterization and crystallization of Aq_1259, an outer membrane protein with porin features, from Aquifex aeolicus

The "hypothetical protein" Aq_1259 was identified by mass spectrometry and purified from native membranes of Aquifex aeolicus. It is a 49.4kDa protein, highly homologous (>52% identity) to several conserved hypothetical proteins from other bacteria. However, none of these proteins has been characterized using biochemical or electrophysiological techniques. Based on the sequence and circular dichroism spectroscopy, the structure of Aq_1259 is predicted to be a β-barrel with 16 β-strands. The strands with loops and turns are distributed evenly through the entire sequence. The function of Aq_1259 was analyzed after incorporation into a lipid bilayer. Electrophysiological measurements revealed a pore that has a basic stationary conductance of 0.48±0.038nS in a buffer with 0.5M NaH(2)PO(4) at pH 6.5 and 0.2±0.015nS in a buffer with 0.5M NaCl at pH 6.5. Superimposed on this is a fluctuating conductance of similar amplitude. Aq_1259 could be crystallized. The crystals diffract to a resolution of 3.4? and belong to space group I222 with cell dimensions of a=138.3?, b=144.6?, c=151.8?.     

Anti-peptidoglycan antibodies and fcγ receptors are the key mediators of inflammation in gram-positive sepsis

Gram-positive bacteria are an important public health problem, but it is unclear how they cause systemic inflammation in sepsis. Our previous work showed that peptidoglycan (PGN) induced proinflammatory cytokines in human cells by binding to an unknown extracellular receptor, followed by phagocytosis leading to the generation of NOD ligands. In this study, we used flow cytometry to identify host factors that supported PGN binding to immune cells. PGN binding required plasma, and plasma from all tested healthy donors contained IgG recognizing PGN. Plasma depleted of IgG or of anti-PGN Abs did not support PGN binding or PGN-triggered cytokine production. Adding back intact but not F(ab')(2) IgG restored binding and cytokine production. Transfection of HEK293 cells with FcγRIIA enabled PGN binding and phagocytosis. These data establish a key role for anti-PGN IgG and FcγRs in supporting inflammation to a major structural element of Gram-positive bacteria and suggest that anti-PGN IgG contributes to human pathology in Gram-positive sepsis.     

Mitochondrial quality control: a matter of life and death for neurons

Neuronal survival critically depends on the integrity and functionality of mitochondria. A hierarchical system of cellular surveillance mechanisms protects mitochondria against stress, monitors mitochondrial damage and ensures the selective removal of dysfunctional mitochondrial proteins or organelles. Mitochondrial proteases emerge as central regulators that coordinate different quality control (QC) pathways within an interconnected network of mechanisms. A failure of this system causes neuronal loss in a steadily increasing number of neurodegenerative disorders, which include Parkinson's disease, spinocerebellar ataxia, spastic paraplegia and peripheral neuropathies. Here, we will discuss the role of the mitochondrial QC network for neuronal survival and neurodegeneration.     

Polymer microarrays for high throughput discovery of biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format (1). Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique (2). This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion (3-5). Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction (6). HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials (5,3).     

Expression Profiling of Stem Cell-Related Genes in Neoadjuvant-Treated Gastric Cancer: A NOTCH2, GSK3B and β-catenin Gene Signature Predicts Survival

Cancer stem cell (CSC) based gene expression signatures are associated with prognosis in various tumour types and CSCs are suggested to be particularly drug resistant. The aim of our study was first, to determine the prognostic significance of CSC-related gene expression in residual tumour cells of neoadjuvant-treated gastric cancer (GC) patients. Second, we wished to examine, whether expression alterations between pre- and post-therapeutic tumour samples exist, consistent with an enrichment of drug resistant tumour cells. The expression of 44 genes was analysed in 63 formalin-fixed, paraffin embedded tumour specimens with partial tumour regression (10-50% residual tumour) after neoadjuvant chemotherapy by quantitative real time PCR low-density arrays. A signature of combined GSK3B(high), β-catenin (CTNNB1)(high) and NOTCH2(low) expression was strongly correlated with better patient survival (p<0.001). A prognostic relevance of these genes was also found analysing publically available gene expression data. The expression of 9 genes was compared between pre-therapeutic biopsies and post-therapeutic resected specimens. A significant post-therapeutic increase in NOTCH2, LGR5 and POU5F1 expression was found in tumours with different tumour regression grades. No significant alterations were observed for GSK3B and CTNNB1. Immunohistochemical analysis demonstrated a chemotherapy-associated increase in the intensity of NOTCH2 staining, but not in the percentage of NOTCH2. Taken together, the GSK3B, CTNNB1 and NOTCH2 expression signature is a novel, promising prognostic parameter for GC. The results of the differential expression analysis indicate a prominent role for NOTCH2 and chemotherapy resistance in GC, which seems to be related to an effect of the drugs on NOTCH2 expression rather than to an enrichment of NOTCH2 expressing tumour cells.     

X-Ray Phase Nanotomography Resolves the 3D Human Bone Ultrastructure

Bone strength and failure are increasingly thought to be due to ultrastructural properties, such as the morphology of the lacuno-canalicular network, the collagen fiber orientation and the mineralization on the nanoscale. However, these properties have not been studied in 3D so far. Here we report the investigation of the human bone ultrastructure with X-ray phase nanotomography, which now provides the required sensitivity, spatial resolution and field of view. The 3D organization of the lacuno-canalicular network is studied in detail over several cells in osteonal and interstitial tissue. Nanoscale density variations are revealed and show that the cement line separating these tissues is hypermineralized. Finally, we show that the collagen fibers are organized as a twisted plywood structure in 3D.     

Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and Leads to Tau Hyperphosphorylation and Neurodegeneration

Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegeneration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds. In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the dynamin-like GTPase OPA1 (optic atrophy 1), which mediates mitochondrial inner membrane fusion and cristae morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus, demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1, affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability, mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins. Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for tau pathologies caused by a dysfunction of mitochondria and raise the possibility that tau pathologies are associated with other neurodegenerative disorders caused by deficiencies in mitochondrial dynamics.     

Insights into the binding and activation sites of the receptors for cholecystokinin and gastrin

CCK receptors represent potential targets in a number of diseases. Knowledge of CCK receptor binding sites is a prerequisite for the understanding of the molecular basis for their ligand recognition, partial agonism, ligand-induced trafficking of signalling. In the current paper, we report studies from our laboratory and others which have provided new data on the molecular basis of the pharmacology and functioning of CCK1 and CCK2 receptors. It has been shown that: 1) homologous regions of the two receptors are involved in the binding site of CCK, however, positioning of CCK slightly differs in agreement with distinct pharmacophores of CCK toward the two receptors and receptor sequence variations; 2) Binding sites of most of non-peptide agonists/ antagonist are buried in the pocket formed by transmembrane helices and overlap that of CCK; Aromatic amino acids within and near the binding site, especially in helix VI, are involved in receptor activation; 4) Like for other members of family A of G-protein coupled receptors, residues of the binding sites as well as of conserved motifs such as E/DRY, NPXXY are crucial for receptor activation.