Structural basis for the high specificity of a Trypanosoma congolense immunoassay targeting glycosomal aldolase

Background : Animal African trypanosomosis (AAT) is a neglected tropical disease which imposes a heavy burden on the livestock industry in Sub-Saharan Africa. Its causative agents are Trypanosoma parasites, with T. congolense and T. vivax being responsible for the majority of the cases. Recently, we identified a Nanobody (Nb474) that was employed to develop a homologous sandwich ELISA targeting T. congolense fructose-1,6-bisphosphate aldolase (TcoALD). Despite the high sequence identity between trypanosomatid aldolases, the Nb474-based immunoassay is highly specific for T. congolense detection. The results presented in this paper yield insights into the molecular principles underlying the assay's high specificity. Methodology/Principal findings : The structure of the Nb474-TcoALD complex was determined via X-ray crystallography. Together with analytical gel filtration, the structure reveals that a single TcoALD tetramer contains four binding sites for Nb474. Through a comparison with the crystal structures of two other trypanosomatid aldolases, TcoALD residues Ala77 and Leu106 were identified as hot spots for specificity. Via ELISA and surface plasmon resonance (SPR), we demonstrate that mutation of these residues does not abolish TcoALD recognition by Nb474, but does lead to a lack of detection in the Nb474-based homologous sandwich immunoassay. Conclusions/Significance : The results show that the high specificity of the Nb474-based immunoassay is not determined by the initial recognition event between Nb474 and TcoALD, but rather by its homologous sandwich design. This (i) provides insights into the optimal set-up of the assay, (ii) may be of great significance for field applications as it could explain the potential detection escape of certain T. congolense strains, and (iii) may be of general interest to those developing similar assays

Individual aggregates of amyloid beta induce temporary calcium influx through the cell membrane of neuronal cells.

Local delivery of amyloid beta oligomers from the tip of a nanopipette, controlled over the cell surface, has been used to deliver physiological picomolar oligomer concentrations to primary astrocytes or neurons. Calcium influx was observed when as few as 2000 oligomers were delivered to the cell surface. When the dosing of oligomers was stopped the intracellular calcium returned to basal levels or below. Calcium influx was prevented by the presence in the pipette of the extracellular chaperone clusterin, which is known to selectively bind oligomers, and by the presence a specific nanobody to amyloid beta. These data are consistent with individual oligomers larger than trimers inducing calcium entry as they cross the cell membrane, a result supported by imaging experiments in bilayers, and suggest that the initial molecular event that leads to neuronal damage does not involve any cellular receptors, in contrast to work performed at much higher oligomer concentrations.Herchel Smith (Postdoctoral Fellowship), Engineering and Physical Sciences Research Council (studentship), European Research Council (Advanced Grant (669237)), Augustus Newman Foundatio

Single domain antibodies targeting neuraminidase protect against an H5N1 influenza virus challenge

Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NAspecific single-domain antibodies (N1-VHHm) and evaluated their in vitro and in vivo antiviral potential. Two of them inhibited the NA activity and in vitro replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without in vitro NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity.Fil: Cardoso, Francisco Miguel. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Ibañez, Lorena Itatí. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Van Den Hoecke, Silvie. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: De Baets, Sarah. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Smet, Anouk. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Roose, Kenny. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Schepens, Bert. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Descamps, Francis J.. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Fiers, Walter. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Muyldermans, Serge. Structural Biology Research Center; Bélgica. Vrije Universiteit Brussel. Laboratory of Cellular and Molecular Immunology; BélgicaFil: Depicker, Ann. VIB. Department of Plant Systems Biology; Bélgica. Department of Biotechnology and Bioinformatics; BélgicaFil: Saelens, Xavier. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; Bélgic

The state of professionalisation of midwifery in Belgium: A discussion paper.

AIM: To describe the state of the professionalisation of midwifery in Belgium, and to formulate recommendations for advancing the midwifery profession. METHODS: A descriptive overview of maternity care in Belgium and the professionalisation of midwifery through an analysis of relevant policy and academic texts, underpinned by Greenwood's sociological criteria for a profession: (1) own body of knowledge, (2) recognised authority, (3) broader community sanctions, (4) own code of ethics and (5) professional culture sustained by formal professional associations. From these insights, recommendations for advancing the midwifery profession in Belgium are formulated. FINDINGS: Current strengths of the professionalisation of midwifery in Belgium included unified midwifery education programmes, progress in midwifery research and overarching national documents for guiding midwifery education, practice and regulation. In contrast however challenges, such as the limited recognition of midwives' roles by its clientele, limitations of midwives' competencies and autonomy, lacking development of advanced roles in maternity care practice and a lack of unity of the organisation and its members, were also identified. Based on these, recommendations are made to strengthen Belgian midwifery. CONCLUSIONS: Recommendations for advancing the midwifery profession in Belgium includes in particular increasing public awareness of midwives' roles and competencies, implementing the full scope of midwifery practice and monitoring and advancing this practice. Thus, professional autonomy over both midwifery practice and working conditions should be enhanced. United midwifery organisations, together with women's groups, other maternity care professionals and policy-makers as equal partners are key to bring about changes in the Belgian maternity care landscape

Exploiting nanobodies and Affimers for superresolution imaging in light microscopy

Antibodies have long been the main approach used for localizing proteins of interest by light microscopy. In the past 5 yr or so, and with the advent of superresolution microscopy, the diversity of tools for imaging has rapidly expanded. One main area of expansion has been in the area of nanobodies, small single-chain antibodies from camelids or sharks. The other has been the use of artificial scaffold proteins, including Affimers. The small size of nanobodies and Affimers compared with the traditional antibody provides several advantages for superresolution imaging

Soluble aggregates present in cerebrospinal fluid change in size and mechanism of toxicity during Alzheimer’s disease progression

Abstract: Soluble aggregates of amyloid-β (Aβ) have been associated with neuronal and synaptic loss in Alzheimer’s disease (AD). However, despite significant recent progress, the mechanisms by which these aggregated species contribute to disease progression are not fully determined. As the analysis of human cerebrospinal fluid (CSF) provides an accessible window into the molecular changes associated with the disease progression, we characterised soluble aggregates present in CSF samples from individuals with AD, mild cognitive impairment (MCI) and healthy controls using a range of sensitive biophysical methods. We used super-resolution imaging and atomic force microscopy to characterise the size and structure of the aggregates present in CSF and correlate this with their ability to permeabilise lipid membranes and induce an inflammatory response. We found that these aggregates are extremely heterogeneous and exist in a range of sizes, varying both structurally and in their mechanisms of toxicity during the disease progression. A higher proportion of small aggregates of Aβ that can cause membrane permeabilization are found in MCI CSF; in established AD, a higher proportion of the aggregates were larger and more prone to elicit a pro-inflammatory response in glial cells, while there was no detectable change in aggregate concentration. These results show that large aggregates, some longer than 100 nm, are present in the CSF of AD patients and suggest that different neurotoxic mechanisms are prevalent at different stages of AD

The development and optimisation of Nanobody based electrochemical immunosensors for IgG

Biosensors are increasingly heralded for their potential to create inexpensive diagnostic devices which are sensitive, selective and easy to use. One of the key categories of biosensor are immunosensors, which have historically used antibodies as bioreceptors. Though widely used, antibodies bring inherent limitations such as variability, limited stability and their reliance on animal sources. This has led to the development of alternative immuno-reagents such as non-antibody binding proteins (NABPs). These are low molecular weight proteins which largely avoid the aforementioned advantages of antibodies. They are commonly produced by bacteria enabling the use of DNA technology to manipulate bioreceptors at the molecular level. Single chain VHHs (commonly known as nanobodies) are an antibody derived NABP adapted from camelid heavy chain antibodies which are the isolated binding domain. Whilst nanobodies have been used for diagnostic and therapeutic applications, they have limited demonstration in biosensors. In this study, both antibodies and nanobodies were used to construct a biosensor. In addition nanobody performance was optimised by introducing a novel peptide spacer. The role of nanobody orientation and spacing was thus investigated and spacer length was optimised, leading to an increase in the sensitivity of the biosensor