Analysis of infected human mononuclear cells by atomic force microscopy

The surfaces of the human lymphoid cells of the line H9 chronically infected with the Human Immunodeficiency Virus HIV-1, and of human monocytes acutely infected in vitro with Mycobacterium Tuberculosis (MTB) were dried, fixed and imaged with atomic force microscopy (AFM). These images were compared with those of non-infected samples. Dried and fixed samples of infected cells can be distinguished from non-infected ones by AFM technology due to their different surface structures and by the presence of pathogenic (viz al or mycobacterial) agents on the cell surface

Photoresponse from noble metal nanoparticles-multi walled carbon nanotube composites

In this Letter, we investigated the photo-response of multi wall carbon nanotube-based composites obtained from in situ thermal evaporation of noble metals (Au, Ag, and Cu) on the nanotube films. The metal deposition process produced discrete nanoparticles on the nanotube outer walls. The nanoparticle-carbon nanotube films were characterized by photo-electrochemical measurements in a standard three electrode cell. The photocurrent from the decorated carbon nanotubes remarkably increased with respect to that of bare multiwall tubes. With the aid of first-principle calculations, these results are discussed in terms of metal nanoparticle–nanotube interactions and electronic charge transfer at the interface.VC 2012 American Institute of Physics

Applications of three-dimensional carbon nanotube

In this paper, we show that it is possible to synthesize carbon-based three-dimensional networks by adding sulfur, as growth enhancer, during the synthesis process. The obtained material is self-supporting and consists of curved and interconnected carbon nanotubes and to lesser extent of carbon fibers. Studies on the microstructure indicate that the assembly presents a marked variability in the tube external diameter and in the inner structure. We study the relationship between the observed microscopic properties and some potential applications. In particular, we show that the porous nature of the network is directly responsible for the hydrophobic and the lipophilic behavior. Moreover, we used a cut piece of the produced carbon material as working electrode in a standard electrochemical cell and, thus, demonstrating the capability of the system to respond to incident light in the visible and near-ultraviolet region and to generate a photocurrent

The factor H binding protein of Neisseria meningitidis interacts with xenosiderophores in vitro.

The factor H binding protein (fHbp) is a key virulence factor of Neisseria meningitidis that confers to the bacterium the ability to resist killing by human serum. The determination of its three-dimensional structure revealed that the carboxyl terminus of the protein folds into an eight-stranded ߠbarrel. The structural similarity of this part of the protein to lipocalins provided the rationale for exploring the ability of fHbp to bind siderophores. We found that fHbp was able to bind in vitro siderophores belonging to the cathecolate family and mapped the interaction site by nuclear magnetic resonance. Our results indicated that the enterobactin binding site was distinct from the site involved in binding to human factor H and stimulates new hypotheses about possible multiple activities of fHbp.Full Tex

Identification of a novel zinc metalloprotease through a global analysis of clostridium difficile extracellular proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis

Carbon nanotube-Cu hybrids enhanced catalytic activity in aqueous media

V. González, O. Martín and J. Baselga wish to thank Ministerio de Economía y Competitividad for funding through Grant MAT2010-17091. Authors also wish to thank to Sofía M. Vega for XPS measurements, Juan P. Fernández for TGA measurements and Lakshmy Pulickal Rajukumar for EDX measurements. C. Martín-Alberca thanks the University of Alcalá for his pre-doctoral Grant

Cell Invasion by Neisseria meningitidis Requires a Functional Interplay between the Focal Adhesion Kinase, Src and Cortactin

Entry of Neisseria meningitidis (the meningococcus) into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin or vitronectin bound to the surface protein Opc forming a bridge to the respective integrins. This interaction leads to cytoskeletal rearrangement and uptake of meningococci. In this study, we determined that the focal adhesion kinase (FAK), which directly associates with integrins, is involved in integrin-mediated internalization of N. meningitidis in HBMEC. Inhibition of FAK activity by the specific FAK inhibitor PF 573882 reduced Opc-mediated invasion of HBMEC more than 90%. Moreover, overexpression of FAK mutants that were either impaired in the kinase activity or were not capable of autophosphorylation or overexpression of the dominant-negative version of FAK (FRNK) blocked integrin-mediated internalization of N. meningitidis. Importantly, FAK-deficient fibroblasts were significantly less invaded by N. meningitidis. Furthermore, N. meningitidis induced tyrosine phosphorylation of several host proteins including the FAK/Src complex substrate cortactin. Inhibition of cortactin expression by siRNA silencing and mutation of critical amino acid residues within cortactin, that encompass Arp2/3 association and dynamin binding, significantly reduced meningococcal invasion into eukaryotic cells suggesting that both domains are critical for efficient uptake of N. meningitidis into eukaryotic cells. Together, these results indicate that N. meningitidis exploits the integrin signal pathway for its entry and that FAK mediates the transfer of signals from activated integrins to the cytoskeleton. A cooperative interplay between FAK, Src and cortactin then enables endocytosis of N. meningitidis into host cells

Receptor Complementation and Mutagenesis Reveal SR-BI as an Essential HCV Entry Factor and Functionally Imply Its Intra- and Extra-Cellular Domains

HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection