Identification of a Cross-reactive Epitope Widely Present in Lipopolysaccharide from Enterobacteria and Recognized by the Cross-protective Monoclonal Antibody WN1 222-5

Septic shock due to infections with Gram-negative bacteria is a severe disease with a high mortality rate. We report the identification of the antigenic determinants of an epitope that is present in enterobacterial lipopolysaccharide (LPS) and recognized by a cross-reactive monoclonal antibody (mAb WN1 222-5) regarded as a potential means of treatment. Using whole LPS and a panel of neoglycoconjugates containing purified LPS oligosaccharides obtained from Escherichia coli core types R1, R2, R3, and R4, Salmonella enterica, and the mutant strain E. coli J-5, we showed that mAb WN1 222-5 binds to the distal part of the inner core region and recognizes the structural element R1-alpha-d-Glcp-(1-->3)-[l-alpha-d-Hepp-(1-->7)]-l-alpha-d-Hepp 4P-(1-->3)-R2 (where R1 represents additional sugars of the outer core and R2 represents additional sugars of the inner core), which is common to LPS from all E. coli, Salmonella, and Shigella. WN1 222-5 binds poorly to molecules that lack the side chain heptose or lack phosphate at the branched heptose. Also molecules that are substituted with GlcpN at the side chain heptose are poorly bound. Thus, the side chain heptose and the 4-phosphate on the branched heptose are main determinants of the epitope. We have determined the binding kinetics and affinities (KD values) of the monovalent interaction of E. coli core oligosaccharides with WN1 222-5 by surface plasmon resonance and isothermal titration microcalorimetry. Affinity constants (KD values) determined by SPR were in the range of 3.6 x 10-5 to 3.2 x 10-8 m, with the highest affinity being observed for the core oligosaccharide from E. coli F576 (R2 core type) and the lowest KD values for those from E. coli J-5. Affinities of E. coli R1, R3, and R4 oligosaccharides were 5-10-fold lower, and values from the E. coli J-5 mutant were 29-fold lower than the R2 core oligosaccharide. Thus, the outer core sugars had a positive effect on binding

Intra-dance variation among waggle runs and the design of efficient protocols for honey bee dance decoding

Noise is universal in information transfer. In animal communication, this presents a challenge not only for intended signal receivers, but also to biologists studying the system. In honey bees, a forager communicates to nestmates the location of an important resource via the waggle dance. This vibrational signal is composed of repeating units (waggle runs) that are then averaged by nestmates to derive a single vector. Manual dance decoding is a powerful tool for studying bee foraging ecology, although the process is time-consuming: a forager may repeat the waggle run 1- >100 times within a dance. It is impractical to decode all of these to obtain the vector; however, intra-dance waggle runs vary, so it is important to decode enough to obtain a good average. Here we examine the variation among waggle runs made by foraging bees to devise a method of dance decoding. The first and last waggle runs within a dance are significantly more variable than the middle run. There was no trend in variation for the middle waggle runs. We recommend that any four consecutive waggle runs, not including the first and last runs, may be decoded, and we show that this methodology is suitable by demonstrating the goodness-of-fit between the decoded vectors from our subsamples with the vectors from the entire dances

Analysis by Surface Plasmon Resonance of the Influence of Valence on the Ligand Binding Affinity and Kinetics of an Anti-carbohydrate Antibody

The kinetics of ligand binding by Se155-4, an antibody specific for the Salmonella serogroup B O-polysaccharide, were studied by surface plasmon resonance. Because trace amounts of oligomers in Fab and single-chain antibody variable domain (scFv) preparations resulted in biphasic binding profiles that were difficult to analyze, all kinetic measurements were performed on purified monomeric fragments and, for certain mutant scFv, dimeric forms. Results obtained with monomeric forms indicated that the relatively low affinity of the antibody was due to rapid dissociation (koff approximately 0.25 s-1). Dimeric forms generally showed off-rates that were approximately 20-fold slower and a 5-fold increase in association rate constants to approximately 2 x 10(5) M-1 s-1. Although the association phases for scFv dimers showed good curve fitting to a one component interaction model, the dissociation phases were biphasic, presumably because the availability and accessibility of sites on the antigen always leads to some monovalent attachment. The fast off-rate for dimers was the same as the monomer off-rate. Se155-4 IgG off-rates were very similar to those observed for scFv dimer, whereas the onrate was the same as that obtained with Fab and scFv monomer

Structure of an anti-blood group A Fv and improvement of its binding affinity without loss of specificity.

The specificity of antibody recognition of the ABO blood group trisaccharide antigens has been explored by crystal structure analysis and mutation methods. The crystal structure of the Fv corresponding to the anti-blood group A antibody AC1001 has been determined to 2.2-A resolution and reveals a binding pocket that is complementary to the blood group A-trisaccharide antigen. The effect of mutating specific residues lining this pocket on binding to the A and B blood group oligosaccharide antigens was investigated through a panel of single point mutations and through a phage library of mutations in complementarity determining region H3. Both approaches gave several mutants with improved affinity for antigen. Surface plasmon resonance indicated up to 8-fold enhancement in affinity for the A-pentasaccharide with no observable binding to the blood group B antigen. This is the first example of single point mutations in a carbohydrate-binding antibody resulting in significant increases in binding affinity without loss of specificity

A mutational analysis of the globotriaosylceramide-binding sites of verotoxin VT1.

Escherichia coli verotoxin, also known as Shiga-like toxin, binds to eukaryotic cell membranes via the glycolipid Gb(3) receptors which present the P(k) trisaccharide Galalpha(1-4)Galbeta(1-4)Glcbeta. Crystallographic studies have identified three P(k) trisaccharide (P(k)-glycoside) binding sites per verotoxin 1B subunit (VT1B) monomer while NMR studies have identified binding of P(k)-glycoside only at site 2. To understand the basis for this difference, we studied binding of wild type VT1B and VT1B mutants, defective at one or more of the three sites, to P(k)-glycoside and pentavalent P(k) trisaccharide (pentaSTARFISH) in solution and Gb(3) presented on liposomal membranes using surface plasmon resonance. Site 2 was the key site in terms of free trisaccharide binding since mutants altered at sites 1 and 3 bound this ligand with wild type affinity. However, effective binding of the pentaSTARFISH molecule also required a functional site 3, suggesting that site 3 promotes pentavalent binding of linked trisaccharides at site 1 and site 2. Optimal binding to membrane-associated Gb(3) involved all three sites. Binding of all single site mutants to liposomal Gb(3) was weaker than wild type VT1B binding. Site 3 mutants behaved as if they had reduced ability to enter into high avidity interactions with Gb(3) in the membrane context. Double mutants at site 1/site 3 and site 2/site 3 were completely inactive in terms of binding to liposomal Gb(3,) even though the site 1/site 3 mutant bound trisaccharide with almost wild type affinity. Thus site 2 alone is not sufficient to confer high avidity binding to membrane-localized Gb(3). Cytotoxic activity paralleled membrane glycolipid binding. Our data show that the interaction of verotoxin with the Gb(3) trisaccharide is highly context dependent and that a membrane environment is required for biologically relevant studies of the interaction

Counter and Complicit Masculine Discourse Among Men’s Shed Members

Men’s Sheds is a growing international movement aimed at providing men with places and activities that facilitate social connectedness. Despite Men’s Sheds’ focus on males, little attention has been paid to masculinities within the specific context of these settings. The current study used a gender relations framework to explore the ways in which attendees discussed Men’s Sheds, with particular attention to discussions that were complicit and counter to traditional, hegemonic views of masculinity, and diverse positions in between these binaries. The data consisted of transcripts and field notes from four focus groups comprised of mostly older, White, retired male members of a Canadian shed (N = 22). The analysis revealed three overall themes: (1) focus on work, (2) independence, and (3) need for male-focused spaces. These themes and associated subthemes suggest that shed members ascribe to dominant masculine values and ideals, but also support more fluid and flexible views of masculinity. Implications are discussed for how working with an array of masculinities within the Men’s Sheds movement will be helpful with respect to their future growth in Canada and internationally

The Second Transmembrane Domain of P2X7 Contributes to Dilated Pore Formation

Activation of the purinergic receptor P2X7 leads to the cellular permeability of low molecular weight cations. To determine which domains of P2X7 are necessary for this permeability, we exchanged either the C-terminus or portions of the second transmembrane domain (TM2) with those in P2X1 or P2X4. Replacement of the C-terminus of P2X7 with either P2X1 or P2X4 prevented surface expression of the chimeric receptor. Similarly, chimeric P2X7 containing TM2 from P2X1 or P2X4 had reduced surface expression and no permeability to cationic dyes. Exchanging the N-terminal 10 residues or C-terminal 14 residues of the P2X7 TM2 with the corresponding region of P2X1 TM2 partially restored surface expression and limited pore permeability. To further probe TM2 structure, we replaced single residues in P2X7 TM2 with those in P2X1 or P2X4. We identified multiple substitutions that drastically changed pore permeability without altering surface expression. Three substitutions (Q332P, Y336T, and Y343L) individually reduced pore formation as indicated by decreased dye uptake and also reduced membrane blebbing in response to ATP exposure. Three others substitutions, V335T, S342G, and S342A each enhanced dye uptake, membrane blebbing and cell death. Our results demonstrate a critical role for the TM2 domain of P2X7 in receptor function, and provide a structural basis for differences between purinergic receptors. © 2013 Sun et al

Genome-wide analysis of ivermectin response by Onchocerca volvulus reveals that genetic drift and soft selective sweeps contribute to loss of drug sensitivity

Treatment of onchocerciasis using mass ivermectin administration has reduced morbidity and transmission throughout Africa and Central/South America. Mass drug administration is likely to exert selection pressure on parasites, and phenotypic and genetic changes in several Onchocerca volvulus populations from Cameroon and Ghana-exposed to more than a decade of regular ivermectin treatment-have raised concern that sub-optimal responses to ivermectin's anti-fecundity effect are becoming more frequent and may spread.Pooled next generation sequencing (Pool-seq) was used to characterise genetic diversity within and between 108 adult female worms differing in ivermectin treatment history and response. Genome-wide analyses revealed genetic variation that significantly differentiated good responder (GR) and sub-optimal responder (SOR) parasites. These variants were not randomly distributed but clustered in ~31 quantitative trait loci (QTLs), with little overlap in putative QTL position and gene content between the two countries. Published candidate ivermectin SOR genes were largely absent in these regions; QTLs differentiating GR and SOR worms were enriched for genes in molecular pathways associated with neurotransmission, development, and stress responses. Finally, single worm genotyping demonstrated that geographic isolation and genetic change over time (in the presence of drug exposure) had a significantly greater role in shaping genetic diversity than the evolution of SOR.This study is one of the first genome-wide association analyses in a parasitic nematode, and provides insight into the genomics of ivermectin response and population structure of O. volvulus. We argue that ivermectin response is a polygenically-determined quantitative trait (QT) whereby identical or related molecular pathways but not necessarily individual genes are likely to determine the extent of ivermectin response in different parasite populations. Furthermore, we propose that genetic drift rather than genetic selection of SOR is the underlying driver of population differentiation, which has significant implications for the emergence and potential spread of SOR within and between these parasite populations

A rational engineering strategy for designing protein a-binding camelid single-domain antibodies

Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species