Lenalidomide binding proteins identified by SPIDER assay
ABSTRACT: To assess whether SPIDER could capture protein-small molecule interaction in a complex environment, here we carried out the SPIDER assay by incubating biotin-Lenalidomide or a biotin control with HEK293T total lysate, followed by the use of biotin conjugated beads coupled with mass spectrometry to identify the interacting proteins
Project description:To assess whether SPIDER could identify modified nucleic acids binding proteins within a complex environment, such as the cellular milieu, we sought to identify N6-methyladenosine (m6A) binding proteins within a total cell lysate. We performed SPIDER assay by incubating biotin-ssRNAs（with and without m6A modification）with the total lysate of YTDHFs (YTHDF1,YTHDF2 and YTHDF3)-overexpressed HEK293T cells
Project description:Orb web spiders use sticky capture spiral silk to retain prey in webs. Capture spiral silk is composed of an axial fibre of flagelliform silk covered with glue droplets that are arranged in a beads-on-a-string morphology that allows multiple droplets to simultaneously extend and resist pull off. Previous studies showed that the adhesion of capture silk is responsive to environmental humidity, increasing up to an optimum humidity that varied among different spider species. The maximum adhesion was hypothesized to occur when the viscoelasticity of the glue optimized contributions from glue spreading and bulk cohesion. In this study, we show how glue droplet shape during peeling contributes significantly to capture silk adhesion. Both overspreading and underspreading of glue droplets reduces adhesion through changes in crack propagation and failure regime. Understanding the mechanism of stimuli-responsive adhesion of spider capture silk will lead to new designs for smarter adhesives.
Project description:To assess whether SPIDER could detect transient interactions such as enzymes and their substrates, we examined the interactome of the E. coli protein deacetylase CobB. As the only member of the Sir2 family of deacetylases in E. coli, CobB is known to play a role in many different pathways but their interactors is still incompletely known. Here we applied SPIDER assay and a SILAC-based mass spectrometry strategy to capture and identify CobB Substrates. Biotinylated CobB was incubated with E. coli total lysate labeled with heavy stable isotope. As a control, free biotin was incubated with E. coli total lysate labeled with light stable isotope.
Project description:We describe a proof-of-principle, immune sandwich assay in which immune complexes link micron-size beads via DNA tethers to a sensor surface. The number of tethered beads, counted using low-magnification microscopy, provides a measure of the concentration of analyte. The prototype assay was sensitive to pM concentration of analyte. In theory, the assay could be sensitive to sub-fM analyte because beads attached via single-immune complexes and DNA strands form tethers, and tether formation in the absence of analyte is extremely rare. The limiting step at present is binding of streptavidin at the end of DNA to biotin on capture beads. Potential advantages of this type of sensor are discussed.
Project description:It is well known that capture silk, the main sticky component of the orb web of a spider, plays an important role in the spider's ability to capture prey via adhesion. However, the detailed mechanism with which the spider achieves its unparalleled high-adhesion performance remains elusive. In this work, we combine experiments and theoretical analysis to investigate the adhesion mechanisms of spider silk. In addition to the widely recognized adhesion effect of the sticky glue, we reveal a synergistic enhancement mechanism due to the elasticity of silk fibres. A balance between silk stiffness, strength and glue stickiness is crucial to endow the silk with superior adhesion, as well as outstanding energy absorption capacity and structural robustness. The revealed mechanisms deepen our understanding of the working principles of spider silk and suggest guidelines for biomimetic designs of spider-inspired adhesion and capture devices.
Project description:Spider silk fibers were produced through an alternative processing route that differs widely from natural spinning. The process follows a procedure traditionally used to obtain fibers directly from the glands of silkworms and requires exposure to an acid environment and subsequent stretching. The microstructure and mechanical behavior of the so-called spider silk gut fibers can be tailored to concur with those observed in naturally spun spider silk, except for effects related with the much larger cross-sectional area of the former. In particular spider silk gut has a proper ground state to which the material can revert independently from its previous loading history by supercontraction. A larger cross-sectional area implies that spider silk gut outperforms the natural material in terms of the loads that the fiber can sustain. This property suggests that it could substitute conventional spider silk fibers in some intended uses, such as sutures and scaffolds in tissue engineering.
Project description:Deciphering the mechanisms involved in shaping social structure is key to a deeper understanding of the evolutionary processes leading to sociality. Individual specialization within groups can increase colony efficiency and consequently productivity. Here, we test the hypothesis that within-group variation in individual personalities (i.e. boldness and aggression) can shape task differentiation. The social spider Stegodyphus sarasinorum (Eresidae) showed task differentiation (significant unequal participation) in simulated prey capture events across 10-day behavioural assays in the field, independent of developmental stage (level of maturation), eliminating age polyethism. Participation in prey capture was positively associated with level of boldness but not with aggression. Body size positively correlated with being the first spider to emerge from the colony as a response to prey capture but not with being the first to attack, and dispersal distance from experimental colonies correlated with attacking but not with emerging. This suggests that different behavioural responses to prey capture result from a complex set of individual characteristics. Boldness and aggression correlated positively, but neither was associated with body size, developmental stage or dispersal distance. Hence, we show that personalities shape task differentiation in a social spider independent of age and maturation. Our results suggest that personality measures obtained in solitary, standardized laboratory settings can be reliable predictors of behaviour in a social context in the field. Given the wealth of organisms that show consistent individual behavioural differences, animal personality could play a role in social organization in a diversity of animals.
Project description:This study reports the development of a sensitive magnetic bead-based enzyme-linked immunoassay (MELISA) for the pan-reactive detection of the Influenza A virus. The assay combines immunomagnetic beads and biotin-nanoparticle-based detection to quantify a highly conserved viral nucleoprotein in virus lysates. At the capture step, monoclonal antibody-coated magnetic microbeads were used to bind and concentrate the nucleoprotein in samples. The colorimetric detection signal was amplified using biotinylated silica nanoparticles (NP). These nanoparticles were functionalized on the surface with short DNA spacers bearing biotin groups by an automated supported synthesis method performed on nano-on-micro assemblies with a DNA/RNA synthesizer. A biotin-nanoparticle and immunomagnetic bead-based assay was developed. We succeeded in detecting Influenza A viruses directly in the lysis buffer supplemented with 10% saliva to simulate the clinical context. The biotin-nanoparticle amplification step enabled detection limits as low as 3?×?103 PFU mL-1 and 4?×?104 PFU mL-1 to be achieved for the H1N1 and H3N2 strains respectively. In contrast, a classical ELISA test based on the same antibody sandwich showed detection limit of 1.2?×?107 PFU mL-1 for H1N1. The new enhanced MELISA proved to be specific, as no cross-reactivity was found with a porcine respiratory virus (PRRSV). Graphical abstract.
Project description:This work establishes a tensegrity model of spider dragline silk. Tensegrity systems are ubiquitous in nature, being able to capture the mechanics of biological shapes through simple and effective modes of deformation via extension and contraction. Guided by quantitative microstructural characterization via air plasma etching and low voltage scanning electron microscopy, we report that this model is able to capture experimentally observed phenomena such as the Poisson effect, tensile stress-strain response, and fibre toughness. This is achieved by accounting for spider silks' hierarchical organization into microfibrils with radially variable properties. Each fibril is described as a chain of polypeptide tensegrity units formed by crystalline granules operating under compression, which are connected to each other by amorphous links acting under tension. Our results demonstrate, for the first time, that a radial variability in the ductility of tensegrity chains is responsible for high fibre toughness, a defining and desirable feature of spider silk. Based on this model, a discussion about the use of graded tensegrity structures for the optimal design of next-generation biomimetic fibres is presented.
Project description:The aim of the project is to identify proteins the ustilize S-adenosyl methionine as cofactor for performing methylation of target molecules in D. melanogaster S2 cells. For this purpose, we applied a small-molecule capturing protocol using a SAH-capture compound with UV-activated crosslinker and biotin immobilization groups. We tested the protein affinity with and without crosslinking for two different photo-activatable crosslinkers benzophenone and nitrobenzene, respectively, as well as different concentrations of the capture compound. As control we incubated the cell lysate with the streptavidin beads without adding the capture compound. Two specific capture compounds containing a S-adenosyl homocysteine as capturing functionality and either benzophenone or azo-benzene as reactive crosslinkers as well as a biotin group for affinity purification were tested in this protocol. One sample representing a HeLa cell lysate (Waters) under equal conditions was added to the dataset.