Identification and characterization of nanobodies targeting the EphA4 receptor

The ephrin receptor A4 (EphA4) is one of the receptors in the ephrin system that plays a pivotal role in a variety of cell-cell interactions, mostly studied during development. In addition, EphA4 has been found to play a role in cancer biology as well as in the pathogenesis of several neurological disorders such as stroke, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Pharmacological blocking of EphA4 has been suggested to be a therapeutic strategy for these disorders. Therefore, the aim of our study was to generate potent and selective Nanobodies against the ligand-binding domain of the human EphA4 receptor. Weidentified two Nanobodies, Nb 39 and Nb 53, that bind EphA4 with affinities in the nanomolar range. These Nanobodies were most selective for EphA4, with residual binding to EphA7 only. Using Alphascreen technology, we found that both Nanobodies displaced all known EphA4-binding ephrins from the receptor. Furthermore, Nb39 andNb53 inhibited ephrin-induced phosphorylationoftheEphA4proteininacell-basedassay. Finally, in a cortical neuron primary culture, both Nanobodies were able to inhibit endogenous EphA4-mediated growth-cone collapse induced by ephrin-B3. Our results demonstrate the potential of Nanobodies to target the ligand-binding domain of EphA4. These Nanobodiesmaydeservefurtherevaluationaspotentialtherapeutics in disorders in which EphA4-mediated signaling plays a role

A nanobody modulates the p53 transcriptional program without perturbing its functional architecture

The p53 transcription factor plays an important role in genome integrity. To perform this task, p53 regulates the transcription of genes promoting various cellular outcomes including cell cycle arrest, apoptosis or senescence. The precise regulation of this activity remains elusive as numerous mechanisms, e.g. posttranslational modifications of p53 and (non-)covalent p53 binding partners, influence the p53 transcriptional program. We developed a novel, non-invasive tool to manipulate endogenous p53. Nanobodies (Nb), raised against the DNA-binding domain of p53, allow us to distinctively target both wild type and mutant p53 with great specificity. Nb3 preferentially binds ‘structural’ mutant p53, i.e. R175H and R282W, while a second but distinct nanobody, Nb139, binds both mutant and wild type p53. The co-crystal structure of the p53 DNA-binding domain in complex with Nb139 (1.9 Å resolution) reveals that Nb139 binds opposite the DNA-binding surface. Furthermore, we demonstrate that Nb139 does not disturb the functional architecture of the p53 DNA-binding domain using conformation-specific p53 antibody immunoprecipitations, glutaraldehyde crosslinking assays and chromatin immunoprecipitation. Functionally, the binding of Nb139 to p53 allows us to perturb the transactivation of p53 target genes. We propose that reduced recruitment of transcriptional co-activators or modulation of selected post-transcriptional modifications account for these observations

In planta expression of nanobody-based designer chicken antibodies targeting Campylobacter

Campylobacteriosis is a widespread infectious disease, leading to a major health and economic burden. Chickens are considered as the most common infection source for humans. Campylobacter mainly multiplies in the mucus layer of their caeca. No effective control measures are currently available, but passive immunisation of chickens with pathogen-specific maternal IgY antibodies, present in egg yolk of immunised chickens, reduces Campylobacter colonisation. To explore this strategy further, anti-Campylobacter nanobodies, directed against the flagella and major outer membrane proteins, were fused to the constant domains of chicken IgA and IgY, combining the benefits of nanobodies and the effector functions of the Fc-domains. The designer chimeric antibodies were effectively produced in leaves of Nicotiana benthamiana and seeds of Arabidopsis thaliana. Stable expression of the chimeric antibodies in seeds resulted in production levels between 1% and 8% of the total soluble protein. These in planta produced antibodies do not only bind to their purified antigens but also to Campylobacter bacterial cells. In addition, the anti-flagellin chimeric antibodies are reducing the motility of Campylobacter bacteria. These antibody-containing Arabidopsis seeds can be tested for oral passive immunisation of chickens and, if effective, the chimeric antibodies can be produced in crop seeds

Удосконалення комерційної діяльності як фактор підвищення конкурентоспроможності підприємства

Additional file 5. ELISA to assess the interaction between Campylobacter -specific nanobodies and purified MOMP. The saturation binding curve of the interaction between coated MOMP (1 µg/mL) and a His-tagged nanobody (1 × 10−6 to 1 × 102 µg/mL) was obtained via ELISA. The dose-dependent inhibitory effect of a strep-tagged nanobody (1 × 10−6 to 1 × 102 µg/mL) on the interaction between His-tagged Nb84 (5.10−2 µg/mL) and MOMP (1 µg/mL), is demonstrated in the competition binding curve. Inhibition by strep-tagged (A) Nb5, (B) Nb22, (C) Nb23, (D) Nb24, (E) Nb49, (F) 84, (G) Nb15, (H) Nb32, (I) Nb34, (J) Nb45, (K) Nb48 and (L) Nb63, was assessed. The ELISA was developed with mouse anti-Histidine tag monoclonal antibody and goat anti-mouse IgG conjugated to alkaline phosphatase. The error bars represent the standard deviations

Meta-KANSEI modeling with Valence-Arousal fMRI dataset of brain

Background: Traditional KANSEI methodology is an important tool in the field of psychology to comprehend the concepts and meanings; it mainly focusses on semantic differential methods. Valence-Arousal is regarded as a reflection of the KANSEI adjectives, which is the core concept in the theory of effective dimensions for brain recognition. From previous studies, it has been found that brain fMRI datasets can contain significant information related to Valence and Arousal. Methods: In this current work, a Valence-Arousal based meta-KANSEI modeling method is proposed to improve the traditional KANSEI presentation. Functional Magnetic Resonance Imaging (fMRI) was used to acquire the response dataset of Valence-Arousal of the brain in the amygdala and orbital frontal cortex respectively. In order to validate the feasibility of the proposed modeling method, the dataset was processed under dimension reduction by using Kernel Density Estimation (KDE) based segmentation and Mean Shift (MS) clustering. Furthermore, Affective Norm English Words (ANEW) by IAPS (International Affective Picture System) were used for comparison and analysis. The data sets from fMRI and ANEW under four KANSEI adjectives of angry, happy, sad and pleasant were processed by the Fuzzy C-Means (FCM) algorithm. Finally, a defined distance based on similarity computing was adopted for these two data sets. Results: The results illustrate that the proposed model is feasible and has better stability per the normal distribution plotting of the distance. The effectiveness of the experimental methods proposed in the current work was higher than in the literature. Conclusions: mean shift can be used to cluster and central points based meta-KANSEI model combining with the advantages of a variety of existing intelligent processing methods are expected to shift the KANSEI Engineering (KE) research into the medical imaging field

Entropies from Markov Models as Complexity Measures of Embedded Attractors

ABSTRACT: This paper addresses the problem of measuring complexity from embedded attractors as a way to characterize changes in the dynamical behavior of different types of systems with a quasi-periodic behavior by observing their outputs. With the aim of measuring the stability of the trajectories of the attractor along time, this paper proposes three new estimations of entropy that are derived from a Markov model of the embedded attractor. The proposed estimators are compared with traditional nonparametric entropy measures, such as approximate entropy, sample entropy and fuzzy entropy, which only take into account the spatial dimension of the trajectory. The method proposes the use of an unsupervised algorithm to find the principal curve, which is considered as the “profile trajectory”, that will serve to adjust the Markov model. The new entropy measures are evaluated using three synthetic experiments and three datasets of physiological signals. In terms of consistency and discrimination capabilities, the results show that the proposed measures perform better than the other entropy measures used for comparison purposes

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

Interfacing Polymers and Tissues: Quantitative Local Assessment of the Foreign Body Reaction of Mononuclear Phagocytes to Polymeric Materials

A quantitative method to assess the in vitro foreign body reaction (FBR) of mononuclear phagocytes (MP) to polymers relevant in implants for prosthetics, advanced therapies, and regenerative medicine is presented. It integrates single‐cell force spectroscopy (SCFS) with immunogenic profiles of the MPs. In cell force spectroscopy experiments a single phagocyte, linked at the end of an atomic force microscopy cantilever, probes the adhesion forces between the cell and the polymer surface. SCFS measures adhesion forces in a range from 10 pN to 100 nN and with spatial resolution from cell size down to nanometers, accessing the early adhesion events established at contact times between milliseconds and minutes. The time evolution within the first 60 s of the adhesion force between the phagocyte and the polymer surface before and after the treatment with an immunosuppressive drug, viz. Minocycline, a Federal Drug Administration (FDA)‐approved third generation tetracycline with anti‐inflammatory effects, is then studied. The adhesion force values measured at the single cell level is shown to correlate to the immunogenic profiles obtained by analysis of biomarkers and morphology of the MPs in culture. Also, Minocycline causes a decrease of both proinflammatory gene expression profiles and adhesive forces of single cell