Biological Applications of Synthetic Binders Isolated from a Conceptually New Adhiron Library.

Background: Adhirons are small (10 kDa) synthetic ligands that might represent an alternative to antibody fragments and to alternative scaffolds such as DARPins or affibodies.

Methods: We prepared a conceptionally new adhiron phage display library that allows the presence of cysteines in the hypervariable loops and successfully panned it against antigens possessing different characteristics.

Results: We recovered binders specific for membrane epitopes of plant cells by panning the library directly against pea protoplasts and against soluble C-Reactive Protein and SpyCatcher, a small protein domain for which we failed to isolate binders using pre-immune nanobody libraries.

Nanobodies Selectively Binding to the Idiotype of a Dengue Virus Neutralizing Antibody Do Not Necessarily Mimic the Viral Epitope.

Vaccination against dengue virus is challenged by the fact that a generic immune response can induce antibody-dependent-enhancement (ADE) in secondary infections. Only some antibodies targeting a quaternary epitope formed by the dimerization of the virus protein E possess sufficient neutralizing capacity. Therefore, the immunization with anti-idiotypic antibodies of neutralizing antibodies might represent a safe vaccination strategy.

A framework for evaluating the performance of SMLM cluster analysis algorithms.

Single-molecule localization microscopy (SMLM) generates data in the form of coordinates of localized fluorophores. Cluster analysis is an attractive route for extracting biologically meaningful information from such data and has been widely applied. Despite a range of cluster analysis algorithms, there exists no consensus framework for the evaluation of their performance.

Native llama Nanobody Library Panning Performed by Phage and Yeast Display Provides Binders Suitable for C-Reactive Protein Detection.

C-reactive protein (CRP) is an inflammation biomarker that should be quantified accurately during infections and healing processes. Nanobodies are good candidates to replace conventional antibodies in immunodiagnostics due to their inexpensive production, simple engineering, and the possibility to obtain higher binder density on capture surfaces. Starting from the same pre-immune library, we compared the selection output resulting from two independent panning strategies, one exclusively exploiting the phage display and another in which a first round of phage display was followed by a second round of yeast display.

CDR1 Composition Can Affect Nanobody Recombinant Expression Yields.

The isolation of nanobodies from pre-immune libraries by means of biopanning is a straightforward process. Nevertheless, the recovered candidates often require optimization to improve some of their biophysical characteristics. In principle, CDRs are not mutated because they are likely to be part of the antibody paratope, but in this work, we describe a mutagenesis strategy that specifically addresses CDR1.

Nanobody-Dependent Detection of Microcystis aeruginosa by ELISA and Thermal Lens Spectrometry.

Nanobodies against cell surface antigens of toxic cyanobacteria Microcystis aeruginosa were recovered by whole-cell biopanning of a naïve phage display library of nanobodies. Six unique sequences were identified and three sub-cloned and purified as fusion immunoreagents together with either green fluorescent protein or AviTag to be used for diagnostics. The yields of nanobody constructs were in the range of 5-10 mg/l and their specificity and sensitivity was initially evaluated by immunofluorescence and by fluorescent enzyme-linked immunosorbent assay (ELISA) using fluorescent nanobodies.

An anti-HER2 nanobody binds to its antigen HER2 via two independent paratopes.

High-resolution structural data of complexes between antibodies and membrane receptors still represent a demanding task. In this study, we used complementary sets of experimental data to obtain a structural model of the complex formed by the human epidermal growth factor receptor 2 (HER2) and its specific nanobody A10. First we identified by NMR the residues that bind or rearrange as a consequence of the complex formation.

Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region.

In vivo clinical applications of nanobodies (VHHs) require molecules that induce minimal immunoresponse and therefore possess sequences as similar as possible to the human VH domain. Although the relative sequence variability in llama nanobodies has been used to identify scaffolds with partially humanized signature, the transformation of the hallmarks in the framework2 still represents a major problem. We assessed a set of mutants in silico and experimentally to elucidate what is the contribution of single residues to the VHH stability and how their combinations affect the mutant nanobody stability.

Molecules involved in motility regulation in cells: a review.

The initial colonization of the host organism by commensal, probiotic, and pathogenic strains is an important step in the development of infections and biofilms. Sensing and colonization of host cell surfaces are governed by flagellar and fimbriae/pili appendages, respectively. Biofilm formation confers great advantages on pathogenic cells such as protection against the host immune system, antimicrobial agents, and several environmental stress factors.

Treatment strategies targeting persister cell formation in bacterial pathogens.

Persister cells are transiently antibiotic-tolerant and dormant subpopulations that are produced to escape the effects of antibiotics within biofilms or planktonic cell populations. Persister cells are of high clinical importance due to their tolerance to antimicrobial agents and subsequent failure in antibiotic treatments. Understanding persister cell formation mechanisms is therefore highly important for developing effective therapeutic strategies against pathogenic bacterial persisters.

Peroxidase zymograms obtained by agarose native gel electrophoresis have unmet resolution and completeness.

A recently published method to separate protein isoforms by means of a flat-bed agarose native gel was adapted for identifying simultaneously both acid and basic isoforms of plant peroxidases. These were evidenced by in situ activity staining using alternative substrates for which the isoforms showed specific preference. Such approach allowed the detection of a significantly higher number of horseradish peroxidases than the conventional methods based on sample separation by acrylamide gel and the single bands were clearer to observe.

Electrochemical immunosensor functionalized with nanobodies for the detection of the toxic microalgae Alexandrium minutum using glassy carbon electrode modified with gold nanoparticles.

In this work an electrochemical immunosensor for the toxic microalgae Alexandrium minutum (A. minutum AL9T) detection is described. A glassy carbon electrode (GCE) was modified by depositing gold nanoparticles followed by L-cysteine for obtaining a self-assembled monolayer.

Regulation and controlling the motility properties of Pseudomonas aeruginosa.

Chronic infections caused by Pseudomonas aeruginosa have been a major concern as their spread and mortality continue to be on the rise. These infections are majorly attributed to biofilm formation via sequential steps where motility plays an essential role in initial attachment of bacterial cells onto biotic and abiotic surfaces, thereby contributing to multi-drug resistance among pathogens. Therefore, attenuating motility properties can be considered as highly potential for controlling P.

Chitosan and their derivatives: Antibiofilm drugs against pathogenic bacteria.

Biofilm formed by several pathogenic bacteria results in the development of resistance against antimicrobial compounds. The polymeric materials present in the biofilm architecture hinder the entry of antimicrobial compounds through the surface of bacterial cells which are embedded as well as enclosed beneath the biofilm matrix. Recent and past studies explored the alternative approaches to inhibit the formation of biofilm by different agents isolated from plants, animals, and microbes.

Antibiotics Application Strategies to Control Biofilm Formation in Pathogenic Bacteria.

Background: The establishment of a biofilm by most pathogenic bacteria has been known as one of the resistance mechanisms against antibiotics. A biofilm is a structural component where the bacterial community adheres to the biotic or abiotic surfaces by the help of Extracellular Polymeric Substances (EPS) produced by bacterial cells. The biofilm matrix possesses the ability to resist several adverse environmental factors, including the effect of antibiotics.

A compact nanobody-DNAzyme conjugate enables antigen detection and signal amplification.

Hemin-utilizing G-quadruplex DNAzymes with peroxidase-like (POX) activity are widely used as signal reporters in biosensing technology. However, their application to protein detection has been mostly limited to sandwich-type assays involving streptavidin or nanoparticles as indirect bridging platforms between DNAzymes and antibodies. Herein, we describe the generation of a compact, covalently DNAzyme-labeled nanobody which was successfully tested in a direct enzyme-linked immunosorbent assay (ELISA).

Diversity of Bacteria and Bacterial Products as Antibiofilm and Antiquorum Sensing Drugs Against Pathogenic Bacteria.

The increase in antibiotic resistance of pathogenic bacteria has led to the development of new therapeutic approaches to inhibit biofilm formation as well as interfere quorum sensing (QS) signaling systems. The QS system is a phenomenon in which pathogenic bacteria produce signaling molecules that are involved in cell to cell communication, production of virulence factors, biofilm maturation, and several other functions. In the natural environment, several non-pathogenic bacteria are present as mixed population along with pathogenic bacteria and they control the behavior of microbial community by producing secondary metabolites.

Purification-independent immunoreagents obtained by displaying nanobodies on bacteria surface.

The availability of preimmune libraries of antibody fragments allows for the fast generation of binders which can be expressed in both eukaryotic and prokaryotic systems. We exploited the recombinant nature of antibody fragments to demonstrate the possibility of expressing them as functional proteins displayed on the surface of Escherichia coli and by such a way to generate living reagents ready-to-use for diagnostics. Such immunoreagents were effectively exploited without the necessity of any purification step to prepare immunocapture surfaces suitable for the diagnostic of both cancer cells and toxic microalgae.

Bacteria and bacterial products: Foe and friends to Caenorhabditis elegans.

Caenorhabditis elegans is a model organism for the study of different molecular, biochemical, microbial and immunity-related mechanisms. In its natural habitat, C. elegans survives by feeding microorganisms (mainly bacteria), though majorly on Escherichia coli OP50 when grown in the laboratory.

Strategies for Biofilm Inhibition and Virulence Attenuation of Foodborne Pathogen-Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (E. coli) O157:H7, a gram-negative bacteria identified as a foodborne pathogen causing severe disease is of great concern worldwide. The pathogenicity of E.

Inhibition strategies of Listeria monocytogenes biofilms-current knowledge and future outlooks.

There is an increasing trend in the food industry on the Listeria monocytogenes biofilm formation and inhibition. This is attributed to its easy survival on contact surfaces, resistance to disinfectants or antibiotics and growth under the stringent condition used for food processing and preservation thereby leading to food contamination products by direct or indirect exposure. Though, there is a lack of conclusive evidences about the mechanism of biofilm formation, in this review, the concept of biofilm formation and various chemical, physical, and green technology approaches to prevent or control the biofilm formed is discussed.