Effects of Intracellular Calcium and Actin Cytoskeleton on TCR Mobility Measured by Fluorescence Recovery

Background: The activation of T lymphocytes by specific antigen is accompanied by the formation of a specialized signaling region termed the immunological synapse, characterized by the clustering and segregation of surface molecules and, in particular, by T cell receptor (TCR) clustering. Methodology/Principal Findings: To better understand TCR motion during cellular activation, we used confocal microscopy and photo-bleaching recovery techniques to investigate the lateral mobility of TCR on the surface of human T lymphocytes under various pharmacological treatments. Using drugs that cause an increase in intracellular calcium, we observed a decrease in TCR mobility that was dependent on a functional actin cytoskeleton. In parallel experiments measurement of filamentous actin by FACS analysis showed that raising intracellular calcium also causes increased polymerization of the actin cytoskeleton. These in vitro results were analyzed using a mathematical model that revealed effective binding parameters between TCR and the actin cytoskeleton. Conclusion/Significance: We propose, based on our results, that increase in intracellular calcium levels leads to actin polymerization and increases TCR/cytoskeleton interactions that reduce the overall mobility of the TCR. In a physiological setting, this may contribute to TCR re-positioning at the immunological synapse

PDlim2 Selectively Interacts with the PDZ Binding Motif of Highly Pathogenic Avian H5N1 Influenza A Virus NS1

The multi-functional NS1 protein of influenza A virus is a viral virulence determining factor. The last four residues at the C-terminus of NS1 constitute a type I PDZ domain binding motif (PBM). Avian influenza viruses currently in circulation carry an NS1 PBM with consensus sequence ESEV, whereas human influenza viruses bear an NS1 PBM with consensus sequence RSKV or RSEV. The PBM sequence of the influenza A virus NS1 is reported to contribute to high viral pathogenicity in animal studies. Here, we report the identification of PDlim2 as a novel binding target of the highly pathogenic avian influenza virus H5N1 strain with an NS1 PBM of ESEV (A/Chicken/Henan/12/2004/H5N1, HN12-NS1) by yeast two-hybrid screening. The interaction was confirmed by in vitro GST pull-down assays, as well as by in vivo mammalian two-hybrid assays and bimolecular fluorescence complementation assays. The binding was also confirmed to be mediated by the interaction of the PDlim2 PDZ domain with the NS1 PBM motif. Interestingly, our assays showed that PDlim2 bound specifically with HN12-NS1, but exhibited no binding to NS1 from a human influenza H1N1 virus bearing an RSEV PBM (A/Puerto Rico/8/34/H1N1, PR8-NS1). A crystal structure of the PDlim2 PDZ domain fused with the C-terminal hexapeptide from HN12-NS1, together with GST pull-down assays on PDlim2 mutants, reveals that residues Arg16 and Lys31 of PDlim2 are critical for the binding between PDlim2 and HN12-NS1. The identification of a selective binding target of HN12-NS1 (ESEV), but not PR8-NS1 (RSEV), enables us to propose a structural mechanism for the interaction between NS1 PBM and PDlim2 or other PDZ-containing proteins

Biodistribution and preliminary toxicity studies of nanoparticles made of Biotransesterified β–cyclodextrins and PEGylated phospholipids

International audienceBACKGROUND:The modification of β-cyclodextrins (βCDs) by grafting alkyl chains on the primary and/or secondary face yields derivatives (βCD-C10) able to self-organize under nanoprecipitating conditions into nanoparticles (βCD-C10-NP) potentially useful for drug delivery. The co-nanoprecipitation of βCD-C10 with polyethylene glycol (PEG) chains yields PEGylated NPs (βCD-C10-PEG-NP) with potentially improved stealthiness. The objectives of the present study were to characterize the in vivo biodistribution of βCD-C10-PEG-NP with PEG chain length of 2000 and 5000Da using nuclear imaging, and to preliminarily evaluate the in vivo acute and extended acute toxicity of the most suitable system.RESEARCH DESIGN AND METHODS:The in vivo and ex vivo biodistribution features of naked and decorated nanoparticles were investigated over time following intravenous injection of 125I-radiolabeled nanoparticles to mice. The potential toxicity of PEGylated βCD-C10 nanosuspensions was evaluated in a preliminary in vivo toxicity study involving blood assays and tissue histology following repeated intraperitoneal injections of nanoparticles to healthy mice.RESULTS:The results indicated that βCD-C10-PEG5000-NP presented increased stealthiness with decreased in vivo elimination and increased blood kinetics without inducing blood, kidney, spleen, and liver acute and extended acute toxicity.CONCLUSIONS:βCD-C10-PEG5000-NPs are stealth and safe systems with potential for drug delivery

Vegetation response to climatic changes in western Amazonia over the last 7,600 years

International audienc

Upland soil charcoal in the wet tropical forests of central Guyana

A soil charcoal survey was undertaken across 60,000 ha of closed-canopy tropical forest in central Guyana to determine the occurrence, ubiquity, and age of past forest fires across a range of terra firme soil types. Samples were clustered around six centers consisting of spatially nested sample stations. Most charcoal was found between 40 and 60 cm depth with fewest samples yielding material at 0-20 cm depth. The first core yielded charcoal at most stations. Charcoal ages of a random subsample ranged from less than 200 YBP to 9500 YBP with a noticeable peak between 1000 and 1250 YBP. Results reinforce a view that most closed-canopy tropical forests in eastern Amazonia have been subject to palaeo-fire events of unknown severity with a peak in charcoal age consistently appearing between 1000 and 2000 YBP. The two samples dated to the early Holocene represent some of the oldest indicators of paleo-fire known from upland Neotropical forest soils. Ubiquitous soil charcoal in central Guyana further indicate both forest resilience to fire and the widespread propensity for regional forests to burn, particularly during anomalous periods of drought

Length Variations in the NA Stalk of an H7N1 Influenza Virus Have Opposite Effects on Viral Excretion in Chickens and Ducks

A deletion of ∼20 amino acids in the stalk of neuraminidase is frequently observed upon transmission of influenza A viruses from waterfowl to domestic poultry. A pair of recombinant H7N1 viruses bearing either a short- or long-stalk neuraminidase was genetically engineered. Inoculation of the long-stalk-neuraminidase virus resulted in a higher cloacal excretion in ducks and led conversely to lower-level oropharyngeal excretion in chickens, associated with a higher-level local immune response and better survival. Therefore, a short-stalk neuraminidase is a determinant of viral adaptation and virulence in chickens but is detrimental to virus replication and shedding in ducks