Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41

The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.     

Black Petrels (Procellaria parkinsoni) Patrol the Ocean Shelf-Break: GPS Tracking of a Vulnerable Procellariiform Seabird

Background

Determining the foraging movements of pelagic seabirds is fundamental for their conservation. However, the vulnerability and elusive lifestyles of these animals have made them notoriously difficult to study. Recent developments in satellite telemetry have enabled tracking of smaller seabirds during foraging excursions.

Methodology/Principal Findings

Here, we report the first successful precision tracking of a c. 700 g seabird, the vulnerable Black Petrel, Procellaria parkinsoni, foraging at sea during the breeding season, using miniature GPS-logging technology. Employing a combination of high-resolution fixes and low-power duty-cycles, we present data from nine individual foraging excursions tracked during the chick-rearing period in February 2006.

Conclusions/Significance

We provide a snapshot of the species'' foraging range and behaviour in relation to detailed underlying bathymetry off the coast of New Zealand, finding a significant relationship between foraging movements and regions of the shelf-break. We also highlight the potential of more sophisticated analyses to identify behavioural phenomena from position data alone.     

Understanding stem cell-mediated brain repair through neuroimaging

Transplantation of stem cells into the damaged brain can lead to behavioral recovery. However, at present, the mechanisms by which these cells exert their beneficial effects are still poorly understood. Survival, migration and differentiation are but a few of the factors that are thought to be involved in stem cell-mediated brain repair. It is hoped that neuroimaging, by MRI and PET, will provide serial in vivo assessments of transplanted cells that can lead to a greater understanding of the mechanisms involved in brain repair.     

Proteomic response of Schizosaccharomyces pombe to static and oscillating extremely low-frequency electromagnetic fields

There is considerable public concern regarding the health effects of exposure to low-frequency electromagnetic fields. In addition, the association between exposure and disease incidence or the possible biological effects of exposure are unclear. Using 2D-DIGE and MS in a blind study, we have investigated the effects of static and oscillating extremely low-frequency electromagnetic fields (ELF EMFs) on the proteomes of wild type Schizosaccharomyces pombe and a Sty1p deletion mutant which displays increased sensitivity to a variety of cellular stresses. Whilst this study identifies a number of protein isoforms that display significant differential expression across experimental conditions, there was no correlation between their patterns of expression and the ELF EMF exposure regimen. We conclude that there are no significant effects of either static or oscillating EMF on the yeast proteome at the sensitivity afforded by 2D-DIGE. We hypothesise that the proteins identified must be sensitive to subtle changes in culture and/or handling conditions, and that the identification of these proteins in other proteomic studies should be treated with some caution when the results of such studies are interpreted in a biological context.     

The effect of the single substitution of arginine within the RGD tripeptide motif of a modified neurotoxin dendroaspin on its activity of platelet aggregation and cell adhesion

The Arg-Gly-Asp (RGD) tripeptide unit is a cell-cell and cell-extracellular matrix recognition sequence of some integrins that is found within several extracellular matrix glycoproteins and dendroaspin, a disintegrin-like venom protein isolated from the snake venom of the Dendroaspis jamsonii. In the present study, the RGD motif in dendroaspin was substituted by Lys-Gly-Asp (KGD), His-Gly-Asp (HGD), Gln-Gly-Asp (QGD) and Ala-Gly-Asp (AGD) denoted as KGD-den, HGD-den, QGD-den and AGD-den, respectively. Each of the mutants exhibited activity as inhibitor of ADP-induced platelet aggregation with IC50 values of 0.26, 2.5, 6, and 17 microM for KGD-den, HGD-den, QGD-den, and AGD-den, respectively, as compared with RGD-den (IC50 = 0.18 microM). Interestingly, HGD-den was approx. two-fold more potent and a more selective inhibitor than either the KGD-den or QGD-den counterpart at blocking A375-SM human melanoma cell adhesion to fibrinogen (beta3-mediated). KGD-den, HGD-den, and QGD-den were preferentially antagonists of A375-SM human melanoma cell adhesion to fibrinogen rather than to fibronectin (alpha5beta1-, beta3-mediated). Both HGD-den and KGD-den were equipotent as inhibitors of human erythroleukaemia (HEL) cell adhesion to fibrinogen (IC50 = 0.15 microM) and also preferential inhibitors of HEL cell adhesion to fibrinogen (beta3 and beta1-mediated) rather than to fibronectin. These findings show that the presence of the arginine within the RGD motif of dendroaspin is not obligatory and substitution of this residue can modulate inhibitory potency and integrin binding selectivity.     

A genetic module regulates the melanization response of Anopheles to Plasmodium

Two modes of refractoriness to Plasmodium, ookinete lysis and melanization, are known in the malaria vector, Anopheles gambiae. Melanization, a potent insect immune response, is manifested in a genetically selected refractory strain and in susceptible mosquitoes that are depleted of specific C-type lectins (CTLs). Here we use a systematic in vivo RNA interference-mediated reverse genetic screen and other recent results to define a melanization-regulating genetic module or network. It encompasses at least 14 genes, including those that encode five Easter-like clip domain serine proteases and four Masquerade-like serine protease homologues of the mosquito CLIPB and CLIPA subfamilies respectively. We show that several but not all CLIPB genes promote Plasmodium melanization, exhibiting partial functional overlap and synergy. We also report that several CLIPA genes have contrasting roles: CLIPA8 is essential for parasite melanization, while three other CLIPAs are novel synergistic inhibitors of this response. Importantly, the roles of certain CLIPAs and CLIPBs are strain specific, indicating that this network may differ between strains. Finally, we provide evidence that in susceptible mosquitoes melanization induced by knockdown of either CTL4 or CLIPA2/CLIPA5 directly kills ookinetes, in contrast to refractory mosquitoes where it merely disposes of dead parasites.     

Engineering human IL-18 with increased bioactivity and bioavailability

Cytokines in plasmid form can act as potent adjuvants when co-administered with DNA vaccines, resulting in an enhanced immune response to the DNA-encoded antigen. This is true of interleukin-18 (IL-18), which has been shown to serve as an adjuvant in conjunction with certain DNA vaccines. To determine if the properties of IL-18 could be optimized for use as a DNA vaccine adjuvant, a model of IL-18/IL-18R binding was developed to identify variants of human IL-18 that were predicted to improve receptor interactions and potentially bioactivity. The linkage of mature IL-18 to a secretion signal sequence provided improved protein expression from mammalian cells and signal peptidase cleavage of this protein produced the authentic N-terminus. The IL-18 variant proteins secreted this way were bioactive, as demonstrated by their ability to induce interferon gamma (IFNgamma) expression by human peripheral blood mononuclear cells (PBMCs) and to bind to IL-18R, as demonstrated by BIAcore analysis. The IL-18 variants were inhibited by IL-18 binding protein (IL-18BP), the soluble inhibitor of IL-18, as measured by neutralization of the IFNgamma response in PBMCs. One variant, V11I/T63A, demonstrated increases both in bioactivity and mammalian cell expression as compared to native IL-18, indicating that this molecule may be particularly well suited for use as a DNA-encoded vaccine adjuvant.     

Site-specific 3D imaging of cells and tissues with a dual beam microscope

Current approaches to 3D imaging at subcellular resolution using confocal microscopy and electron tomography, while powerful, are limited to relatively thin and transparent specimens. Here we report on the use of a new generation of dual beam electron microscopes capable of site-specific imaging of the interior of cellular and tissue specimens at spatial resolutions about an order of magnitude better than those currently achieved with optical microscopy. The principle of imaging is based on using a focused ion beam to create a cut at a designated site in the specimen, followed by viewing the newly generated surface with a scanning electron beam. Iteration of these two steps several times thus results in the generation of a series of surface maps of the specimen at regularly spaced intervals, which can be converted into a three-dimensional map of the specimen. We have explored the potential of this sequential "slice-and-view" strategy for site-specific 3D imaging of frozen yeast cells and tumor tissue, and establish that this approach can identify the locations of intracellular features such as the 100 nm-wide yeast nuclear pore complex. We also show that 200 nm thick sections can be generated in situ by "milling" of resin-embedded specimens using the ion beam, providing a valuable alternative to manual sectioning of cells and tissues using an ultramicrotome. Our results demonstrate that dual beam imaging is a powerful new tool for cellular and subcellular imaging in 3D for both basic biomedical and clinical applications.