Conjunctival Fungal Flora in a Tertiary Eye Hospital in Nigeria.

Objectives: The aim of this study is to determine the hospital incidence and pattern of conjunctival fungal flora in adult patients at the Guinness Eye Center Onitsha, Nigeria. Materials and Methods: New adult patients, without anterior segment disease, were randomly recruited. Using a sterile swab stick, specimen was taken from the inferior conjunctival fornix of each participant's right eye and inoculated into Sabouraud dextrose agar slant in a test tube and incubated at 27°C. The specimens were examined for fungal growth every 48 h for 4 weeks. Specimens with fungal growth were further examined under a high power microscope for fungal identification and characterization. Results: A total of 225 patients (105 males, 120 females) were examined. The age range was 18-75 years; mean age was 41 ± 17.1 years; 62 (27.6%) were culture-positive: 25 (40.3%) were males and 37 (59.7%) were females (P >0.05). Both moulds and yeasts were isolated with moulds constituting 44 (74.2%). Aspergillus [26 (41.9%)] and Candida [16 (25.8%)] were the commonest organisms. Participants >60 years had the greatest burden. Pensioners (61.5%), traders (44.0%), farmers (30.1%), and artisans (27.3%) were occupational groups with significantly higher proportions of culture-positive specimens (P < 0.05). Conclusion: Over a quarter of new adult patients without anterior segment disease harbour fungi, some of which are pathogenic, in their conjunctival fungal organisms. While Aspergillus and Candida were the commonest isolates, older participants, pensioners, traders, farmers, and artisans had significantly higher proportion of culture-positive specimens. These findings should be considered when formulating pre-operative guidelines for ocular surgery in our environment.

Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.

Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a ß-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.

Towards multiplexed protein-protein interaction analysis using protein tag-specific nanobodies.

Dynamic protein-protein interactions (PPIs) are an integral part of cellular processes. The discovery of modulators that disrupt or stabilize such interactions is highly important to understand PPIs and address correlating diseases. Bead-based protein assays analyzing PPIs between bait- and prey-proteins exemplify emerging methodologies. To date, most studies employ purified bait-proteins from bacteria. Such proteins are of limited use as they do not undergo eukaryotic folding and lack posttranslational modifications. Here, we present a novel method to generate bead-based protein arrays combining µ-scale purification of bait-proteins combined with site-directed immobilization. First, we express individual bait-proteins as GST- or GFP-fusion constructs in bacterial and mammalian cells. Next, we purify and immobilize these bait-proteins from crude lysates using high affinity tag-specific nanobodies coupled to color-coded beads. Finally, we combined those bait-coupled beads in a protein-array for miniaturized multiplexed GST- and GFP pulldown studies. In a proof-of-principle we study dynamic changes of the endogenous prey-protein ß-catenin following proteasomal inhibition or signaling pathway perturbation. Our strategy enables a fast isolation of highly pure and stable bait-proteins derived from small-scale expression cultures. We propose that this approach enables the generation of bead-based protein arrays comprising hundreds of bait-proteins from different expression systems to study complex PPIs. BIOLOGICAL SIGNIFICANCE: Protein arrays and multiplexed sandwich immunoassays, are widely applied to study protein-protein interaction or to investigate the signaling status of stimulated cells. This study describes for the first time the application of tag-specific nanobodies for site directed immobilization of bait-proteins from different expression systems to generate bead based protein arrays. The analysis of the Wnt-pathway activation by multiplexed µ-scale pulldowns demonstrated the advantages of eukaryotic expression systems regarding the stability and binding properties of individual bait proteins. This article is part of a Special Issue entitled: HUPO 2014.

Monitoring interactions and dynamics of endogenous beta-catenin with intracellular nanobodies in living cells.

ß-catenin is the key component of the canonical Wnt pathway and plays a crucial role in a multitude of developmental and homeostatic processes. The different tasks of ß-catenin are orchestrated by its subcellular localization and participation in multiprotein complexes. To gain a better understanding of ß-catenin's role in living cells we have generated a new set of single domain antibodies, referred to as nanobodies, derived from heavy chain antibodies of camelids. We selected nanobodies recognizing the N-terminal, core or C-terminal domain of ß-catenin and applied these new high-affinity binders as capture molecules in sandwich immunoassays and co-immunoprecipitations of endogenous ß-catenin complexes. In addition, we engineered intracellularly functional anti-ß-catenin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. For the first time, we were able to visualize the subcellular localization and nuclear translocation of endogenous ß-catenin in living cells using these chromobodies. Moreover, the chromobody signal allowed us to trace the accumulation of diffusible, hypo-phosphorylated ß-catenin in response to compound treatment in real time using High Content Imaging. The anti-ß-catenin nanobodies and chromobodies characterized in this study are versatile tools that enable a novel and unique approach to monitor the dynamics of subcellular ß-catenin in biochemical and cell biological assays.

Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells.

In biomedical research there is an ongoing demand for new technologies, which help to elucidate disease mechanisms and provide the basis to develop novel therapeutics. In this context a comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, posttranslational modifications and dynamic interactions of cellular components is indispensable. Besides their significant impact as therapeutic molecules, antibodies are arguably the most powerful research tools to study endogenous proteins and other cellular components. However, for cellular diagnostics their use is restricted to endpoint assays using fixed and permeabilized cells. Alternatively, live cell imaging using fluorescent protein-tagged reporters is widely used to study protein localization and dynamics in living cells. However, only artificially introduced chimeric proteins are visualized, whereas the endogenous proteins, their posttranslational modifications as well as non-protein components of the cell remain invisible and cannot be analyzed. To overcome these limitations, traceable intracellular binding molecules provide new opportunities to perform cellular diagnostics in real time. In this review we summarize recent progress in the generation of intracellular and cell penetrating antibodies and their application to target and trace cellular components in living cells. We highlight recent advances in the structural formulation of recombinant antibody formats, reliable screening protocols and sophisticated cellular targeting technologies and propose that such intrabodies will become versatile research tools for real time cell-based diagnostics including target validation and live cell imaging. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.