Uptake of borreliae by immune cells is decisive for the course of the infection, and remodelling BTK inhibitor nmr of the host actin cytoskeleton is crucial in this process.
In this study, we showed that the actin-regulatory formin Daam1 is important in Borrelia phagocytosis by primary human macrophages. Uptake of borreliae proceeds preferentially through capture by filopodia and formation of coiling pseudopods that enwrap the spirochetes. Using immunofluorescence, we localized endogenous and overexpressed Daam1 to filopodia and to F-actin-rich uptake structures. Live-cell imaging further showed that Daam1 is enriched at coiling pseudopods that arise from the macrophage surface. This filopodia-independent step was corroborated
by control experiments of phagocytic cup formation with latex beads. Moreover, siRNA-mediated knockdown of Daam1 led to a 65% reduction of borreliae-induced filopodia, and, as shown by the outside-inside staining technique, to a 50% decrease in phagocytic uptake of borreliae, as well as a 37% reduction in coiling pseudopod formation. Collectively, we showed that Daam1 plays a dual role in the phagocytic uptake of borreliae: first, as a regulator of filopodia, which are used for capturing spirochetes, and second, in the formation of the coiling pseudopod that enwraps the bacterial
cell. These data identify Daam1 LY3023414 as a novel regulator of B. burgdorferi phagocytosis. At the same time, this is the first demonstration of a role for Daam1 in phagocytic processes in general.”
“Polymeric brushes have emerged as a novel 3D material platform that provides great amounts of binding sites for biomolecules. This paper investigates the covalent immobilization mechanism of protein by spherical poly(acrylic acid) brushes (SPAABs) in the widely adopted N-hydroxysuccinimide/N-(3-dimethyl-aminopropy1)-N’-ethylcarbodiimide hydrochloride 17-AAG solubility dmso (NHS/EDC) process. It was discovered that electrostatic interaction plays a crucial role in the covalent immobilization of protein. Due to the existence of 3D architecture and “Donnan effect”, SPAABs exhibit quite different immobilization kinetics in comparison with conventional 2D materials. Under conditions favorable to electrostatic interaction, the effect of “electrostatic interaction induced covalent binding” was observed as a result of competitive immobilization by physical adsorption and chemical binding. On the basis of the mechanism study, a new “chemical conjugation after electrostatic entrapment” (CCEE) method was developed which set the chemical and physical immobilization process apart. A more effective and well-defined covalent immobilization was achieved.