Development of sensitive antigen-detection system using photoactivatable antibody-Fc binding protein capable introducing oriented antibody

The photoactivatable antibody Fc-binding protein (PFcBP) allows oriented antibody (Ab) immobilization on various surfaces. Previously, we developed a system for producing the PFcBP containing photo-methionine (pMet) in E. coli, which can induce the covalent crosslinking to the Ab-Fc region, by engineering of methionyl-tRNA synthetase and FcBP derived from Protein G (1). In this study, we improved the photo-crosslinking efficiency by multipoint mutagenesis of PFcBP. and optimized the Ab immobilization process. The mutant PFcBP with 7-point substitutions showed the 25-30% enhanced photo-crosslinking efficiency as compared that with 4-point substitutions. The PFcBPs were immobilized onto the solid surfaces using the bifunctional crosslinkers with NHS and maleimide groups, and the Abs were then photo-crosslinked to the PFcBPs upon UV irradiation. The longer spacer arm length of the crosslinker was critical for immobilization of 1xPFcBP with a single Ab-Fc binding domain, but less critical for immobilization of 2xPFcBP with two domains. We also conjugated the PFcBP to the fluorescent beads, and subsequently photo-crosslinked detection Abs upon UV irradiation. Finally, we developed a cassette system capable of introducing capture and detection Abs with orientation onto the PFcBP immobilized chips and fluorescent beads, respectively, and demonstrated the effectiveness of the system in the detection of antigens in sera (Figure 1). We also first prepared the Ab-FcBP conjugates by direct photo-crosslinking of Abs and PFcBPs. After removing free PFcBP by gel filtration, the conjugates were immobilized onto the maleimide-activated surface. This process allowed more sensitive antigen detection than the sequential Ab-immobilization process. Please click Additional Files below to see the full abstract

Identification and smart design of peptide binders recognizing target biomarkers

There is an urgent need for novel diagnostic methods capable of non-invasive, sensitive and feasible early prediction of colorectal adenoma-to-carcinoma progression over conventional techniques. In this paper, we describe for the first time the use of phage display for the identification of novel peptide motifs that specifically recognize colorectal cancer (CRC) biomarkers for the prediction of colorectal adenoma-to-carcinoma progression. We performed a biopanning of phage displayed peptide library to identify novel peptide sequences specific for promising CRC biomarker, LRG1 and TTR. The peptides specific for LRG1 that is upregulated proteins in carcinoma had an amino acid sequence with QHIMHLPHINTL, while the peptides specific for TTR that is downregulated proteins in adenoma had an amino acid sequence with VHDDFRQDWQPS. ELISA assays were used to evaluate binding affinity for their targets. As a consequence, both phage-displayed peptides were found to be sub-picomolar binding affinities for their proteins. A quartz crystal microbalance (QCM) is used as a diagnosis tool during biosensor development, and electrochemical techniques (cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS)) and surface-enhanced Raman scattering (SERS) are used as the detection methods in the biosensor. Overall these results demonstrate a simple platform for developing sensitive peptide-based biosensors for almost any desired protein target. Please click Additional Files below to see the full abstract

Implementation of the submarine diving simulation in a distributed environment

ABSTRACTTo implement a combined discrete event and discrete time simulation such as submarine diving simulation in a distributed environment, e.g., in the High Level Architecture (HLA)/Run-Time Infrastructure (RTI), a HLA interface, which can easily connect combined models with the HLA/RTI, was developed in this study. To verify the function and performance of the HLA interface, it was applied to the submarine dive scenario in a distributed environment, and the distributed simulation shows the same results as the stand-alone simulation. Finally, by adding a visualization model to the simulation and by editing this model, we can confirm that the HLA interface can provide user-friendly functions such as adding new model and editing a model

Automatic generation of equations of motion for multibody system in discrete event simulation framework

AbstractIn this paper, the development of a simulation program that can automatically generate equations of motion for mutibody systems in the discrete event simulation framework is presented. The need to analyze the dynamic response of mechanical systems that are under event triggered conditions is increasing. General mechanical systems can be defined as multibody systems that are collections of interconnected rigid bodies, consistent with various types of joints that limit the relative motion of pairs of bodies. For complex multibody systems, a systematic approach is required to efficiently set up the mathematical models. Therefore, a dynamics kernel was developed to automatically generate the equations of motion for multibody systems based on multibody dynamics. The developed dynamics kernel also provides the numerical solver for the dynamic analysis of multibody systems. The general multibody dynamics kernel cannot deal with discontinuous state variables, event triggered conditions, and state triggered conditions, though. To enable it to deal with multibody systems in discontinuous environments, the multibody dynamics kernel was integrated into a discrete event simulation framework, which was developed based on the discrete event system specification (DEVS) formalism. DEVS formalism is a modular and hierarchical formalism for modeling and analyzing systems under event triggered conditions, which are described by discontinuous state variables. To verify the developed program, it was applied to an block-lifting and transport simulation, and dynamic analysis of the system is carried out

The Effect of UVA and UVB on DNA Synthesis and Unscheduled DNA Synthesis in Mouse Skin

The effects of ultraviolet light A (UVA) and ultraviolet light B (UVB) on DNA synthesis and unscheduled DNA synthesis (UDS) were studied in mouse skin by microautoradiography. The mice were exposed to 50mJ / em" UVB by fluorescent sunlamp or 50J / ern" UVA by metal halide mercury lamp. Time course studies were performed immediately, 6 hours, 24 hours, and 48 hours after UVA and UVB exposure. There was no decreased number of heavily labeled cells (HLC) representing DNA synthesis immediately after UVA exposure and up to 48 hours postirradiation. However, immediately after and at 6 hours after UVA irradiation there was an increased number of sparsely labeled cells (SLC) representing UDS. Recovery was noticed 24 hours after irradiation and it was maintained after 48 hours postirradiation. These results clearly demonstrate that UVA induces considerable DNA damage and repair. DNA synthesis decreased immediately and at 6 hours and at 24 hours after UVB exposure. It recovered at 48 hours after UVB exposure. UDS increased immediately and at 6 hours after UVB exposure. Repair synthesis was completed at 24 hours after UVB exposure