Production of Human Antibody Fragments Binding to Melittin and Phospholipase A2 in Africanised Bee Venom: Minimising Venom Toxicity

The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals.

Anisotropic Brownian motion in ordered phases of DNA fragments

Using Fluorescence Recovery After Photobleaching, we investigate the Brownian motion of DNA rod-like fragments in two distinct anisotropic phases with a local nematic symmetry. The height of the measurement volume ensures the averaging of the anisotropy of the in-plane diffusive motion parallel or perpendicular to the local nematic director in aligned domains. Still, as shown in using a model specifically designed to handle such a situation and predicting a non-Gaussian shape for the bleached spot as fluorescence recovery proceeds, the two distinct diffusion coefficients of the DNA particles can be retrieved from data analysis. In the first system investigated (a ternary DNA-lipid lamellar complex), the magnitude and anisotropy of the diffusion coefficient of the DNA fragments confined by the lipid bilayers are obtained for the first time. In the second, binary DNA-solvent system, the magnitude of the diffusion coefficient is found to decrease markedly as DNA concentration is increased from isotropic to cholesteric phase. In addition, the diffusion coefficient anisotropy measured within cholesteric domains in the phase coexistence region increases with concentration, and eventually reaches a high value in the cholesteric phase.

Stable assemblies of cationic bilayer fragments and CpG oligonucleotide with enhanced immunoadjuvant activity in vivo

The cationic lipid dioctadecyldimethylammonium bromide (DODAB) and the CpG oligonucleotide (CpG) have been separately used as potent immunoadjuvants driving Th1 responses. Here DODAB bilayer fragments (BF) and CpG (5 -TTGACGTTCG-3) assemblies have their physical properties and immunoadjuvant activity determined using ovalbumin (OVA) as a model antigen. At 0.1 mg/mL OVA, the dependence of DODAB BF/OVA size and zeta-potential on time and [DODAB] establishes 0.1 mMDODAB as suitable for obtaining stable and cationic DODAB BF/OVA assemblies. At 0.1 mMDODAB, 0.1 mg/mL OVA and 0.006 mMCpG, the zeta-potential is zero. At [CpG]>0.006 mM, good colloidal stability for the anionic assemblies is due to charge overcompensation. At 0.020 mM CpG, these DODAB BF/OVA/CpG assemblies are highly effective in vivo generating responses similar to those elicited by the stable and cationic DODAB BF/OVA. The anti-OVA DTH reaction and the secretion of IFN-gamma and IL-12 are 6, 42 and 9 times larger for the DODAB BF/OVA/CpG-immunized mice than the same responses by OVA-immunized mice, respectively. This work shows for the first time that charge of small assemblies is not important to determine the immune response. (C) 2011 Elsevier B. V. All rights reserved.

HELMINTH PARASITES OF HYPSIBOAS PRASINUS (ANURA: HYLIDAE) FROM TWO ATLANTIC FOREST FRAGMENTS, SAO PAULO STATE, BRAZIL

Seventy-seven males of Hypsiboas prasinus from 2 Atlantic forest fragments in the municipalities of Botucatu and Jundiai, sao Paulo State, Brazil, were examined for endoparasites. The frogs were captured in summer (January until March) and winter (July/August) of 2008 and 2009. Thirty-three males (75%) from Botucatu were infected with Rhabdias cf. fuelleborni, cosmocerciid nematodes, and Cylindrotaenia americana. Twenty-five tree frogs (78.5%) from Jundiai were infected by Rhabdias cf. fuelleborni, Physaloptera sp., and cosmocerciid nematodes. Only cosmocerciid nematodes presented a statistically significance difference in prevalence (z = 4.345; P < 0.001) and mean abundance (t = 562.0; P < 0.001) between Botucatu and Jundiai during the winter. Also, the cosmocerciids exhibited higher mean abundance (t = 196.0; P = 0.034) in winter when compared with summer at the Jundiai site. Moreover, to our knowledge, this is the first report of C. americana in the Brazilian Hylidae. This study presents 4 new records of nematodes in H. prasinus.

The importance of landscape structure for seed dispersal in rain forest fragments

Questions What are the main features of the seed rain in a fragmented Atlantic forest landscape? Can seed rain species attributes (life form, dispersal mode, successional status) relate to the spatial arrangement (size and number of fragments, edge density and presence of corridor) of forest fragments in the landscape? How does the rain forest landscape structure affect the seed rain? Location Atlantic rainforest, Sao Paulo State, Southeastern Brazil. Methods Seed rain samples were collected monthly throughout 1yr, counted, identified and classified according to species dispersal mode, successional status and life form. Seed rain composition was compared with woody species near the seed traps. Relationships between seed rain composition and landscape spatial arrangement (fragment area, presence of corridor, number of fragments in the surroundings, proximity of fragments, and edge density) were tested using canonical correspondence analysis (CCA). Results We collected 20142 seeds belonging to 115 taxa, most of them early successional and anemochorous trees. In general, the seed rain had a species composition distinct from that of the nearby forest tree community. Small isolated fragments contained more seeds, mainly of anemochorous, epiphytic and early-successional species; large fragments showed higher association with zoochorous and late-successional species compared to small fragments. The CCA significantly distinguished the species dispersal mode according to fragment size and isolation, anemochorous species being associated to small and isolated fragments, and zoochorous species to larger areas and fragment aggregation. Nevertheless, a gradient driven by proximity (PROX) and edge density (ED) segregated lianas (in the positive extremity), early successional and epiphyte species (in the negative end); large fragments were positively associated to PROX and ED. Conclusions The results highlight the importance of the size and spatial arrangement of forest patches to promote habitat connectivity and improve the flux of animal-dispersed seeds. Landscape structure controls seed fluxes and affects plant dispersal capacity, potentially influencing the composition and structure of forest fragments. The seed rain composition may be used to assess the effects of landscape spatial structure on plant assemblages, and provide relevant information for biodiversity conservation.

Interaction of gramicidin with DPPC/DODAB bilayer fragments

The interaction between the antimicrobial peptide gramicidin (Gr) and dipalmitoylphosphatidylcholine (DPPC)/dioctadecyldimethylammonium bromide (DODAB) 1:1 large unilamellar vesicles (LVs) or bilayer fragments (BFs) was evaluated by means of several techniques. The major methods were: 1) Gr intrinsic fluorescence and circular dichroism (CD) spectroscopy; 2) dynamic light scattering for sizing and zeta-potential analysis; 3) determination of the bilayer phase transition from extrinsic fluorescence of bilayer probes; 4) pictures of the dispersions for evaluation of coloidal stability over a range of time and NaCl concentration. For Gr in LVs, the Gr dimeric channel conformation is suggested from: 1) CD and intrinsic fluorescence spectra similar to those in trifluoroethanol (TFE); 2) KCl or glucose permeation through the LVs/Gr bilayer. For Gr in BFs, the intertwined dimeric, non-channel Gr conformation is evidenced by CD and intrinsic fluorescence spectra similar to those in ethanol. Both LVs and BFs shield Gr tryptophans against quenching by acrylamide but the Stern-Volmer quenching constant was slightly higher for Gr in BFs confirming that the peptide is more exposed to the water phase in BFs than in LVs. The DPPC/DODAB/Gr supramolecular assemblies may predict the behavior of other antimicrobial peptides in assemblies with lipids. (C) 2012 Elsevier B.V. All rights reserved.

A multimedia system to produce and deliver video fragments on demand on parliamentary websites

[EN]Parliamentary websites have become one of the most important windows for citizens and media to follow the activities of their legislatures and to hold parliaments to account. Therefore, most parliamentary institutions aim to provide new multimedia solutions capable of displaying video fragments on demand on plenary activities. This paper presents a multimedia system for parliamentary institutions to produce video fragments on demand through a website with linked information and public feedback that helps to explain the content shown in these fragments. A prototype implementation has been developed for the Canary Islands Parliament (Spain) and shows how traditional parliamentary streaming systems can be enhanced by the use of semantics and computer vision for video analytics...

Stab or throw? Biomechanical studies on the injuring potential of glass fragments

During a Christmas party, two male guests started fighting. The perpetrator was allegedly pushed onto a glass table by the victim or fell into the table together with that man so that the glass top broke and caused a cut wound on the perpetrator's back. According to his statement he then threw a fragment of the broken glass table in the direction of the other man hitting him accidentally in a way so that the subclavian artery was severed and he died from exsanguination. Tests on the breaking characteristics of the glass table, the flying behaviour and the kinetics of thrown glass fragments conducted on various models supported the conclusion that the fatal injury on the victim's neck could not have been caused by a thrown glass fragment. It was much more likely that a stab with a blade-shaped glass fragment was the cause of the fatal injuries.

Anti-idiotypic Fab Fragments Image a Conserved N-terminal Epitope Patch of Grass Pollen Allergen Phl p 1

BACKGROUND AND AIMS: Naturally occurring anti-idiotypic antibodies structurally mimic the original antibody epitope. Anti-idiotypes, therefore, are interesting tools for the portrayal of conformational B-cell epitopes of allergens. In this study we used this strategy particularly for major timothy grass pollen (Phleum pratense) allergen Phl p 1. METHODS AND RESULTS: We used a combinatorial phage display library constructed from the peripheral IgG repertoire of a grass pollen allergic patient which was supposed to contain anti-idiotypic Fab specificities. Using purified anti-Phl p 1 IgG for biopanning, several Fab displaying phage clones could be isolated. 100 amplified colonies were screened for their binding capacity to anti-Phl p 1-specific antibodies, finally resulting in four distinct Fab clones according to sequence analysis. Interestingly, heavy chains of all clones derived from the same germ line sequence and showed high homology in their CDRs. Projecting their sequence information on the surface of the natural allergen Phl p 1 (PDB ID: 1N10) indicated matches on the N-terminal domain of the homo-dimeric allergen, including the bridging region between the two monomers. The resulting epitope patches were formed by spatially distant sections of the primary allergen sequence. CONCLUSION: In this study we report that anti-idiotypic specificities towards anti-Phl p 1 IgG, selected from a Fab library of a grass pollen allergic patient, mimic a conformational epitope patch being distinct from a previously reported IgE epitope area.