Improved Hydrolysis of Organophosphorus Compounds by Engineered Human Prolidases.

BACKGROUND: Human prolidase has weak hydrolytic activity for toxic organophosphorus compounds including diisopropyl fluorophosphates (DFP), chemical warfare nerve agents and pesticides. OBJECTIVES: In order to use human prolidase as a catalytic bioscavenger against toxic organophosphorus compound exposure, protein engineering is an important issue to improve the catalytic activity of human prolidase towards the hydrolysis of toxic organophosphorus compounds. METHOD: We developed two human prolidase mutants, A252R and P365R, with a single amino acid substitution using in silico analysis based on the sequence, protein structure and stability to improve the catalytic activity of human prolidase towards DFP hydrolysis. RESULTS: Our results showed that the catalytic efficiencies of A252R and P365R towards DFP hydrolysis were 1.23- and 1.36-fold increases, respectively, than that of the wild type, while the prolidase activities of A252R and P365R towards Leu-Pro hydrolysis were 0.88- and 0.78-fold decreases that of the wild type, respectively, indicating that substitution mutations of A252R and P365R in human prolidase show improved hydrolytic activity for toxic organophosphorus compounds. CONCLUSION: We report here that by introducing either the A252R or P365R substitution mutation, the structural changes affecting catalytic turnover rate and substrate binding affinity are valuable in improving the catalytic activity of human prolidase towards toxic organophosphorus compound hydrolysis.

Expression and purification of biologically active recombinant human paraoxonase 1 from a Drosophila S2 stable cell line.

Many pesticides and chemical warfare nerve agents are highly toxic organophosphorus compounds (OPs), which inhibit acetylcholinesterase activity. Human paraoxonase 1 (PON1) has demonstrated significant potential for use as a catalytic bioscavenger capable of hydrolyzing a broad range of OPs. However, there are several limitations to the use of human PON1 as a catalytic bioscavenger, including the relatively difficult purification of PON1 from human plasma and its dependence on the presence of hydrophobic binding partners to maintain stability. Therefore, research efforts to efficiently produce recombinant human PON1 are necessary. In this study, we developed a Drosophila S2 stable cell line expressing recombinant human PON1. The recombinant human PON1 was fused with the human immunoglobulin Fc domain (PON1-hFc) to improve protein stability and purification efficiency. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis compared with those of the recombinant human PON1 derived from E. coli. We observed that the recombinant human PON1-hFc is functionally more stable for OP hydrolyzing activities compared to the recombinant human PON1. The catalytic efficiency of the recombinant PON1-hFc towards diisopropyl fluorophosphate (DFP, 0.26 × 106 M-1 min-1) and paraoxon hydrolysis (0.015 × 106 M-1 min-1) was 1.63- and 1.24-fold higher, respectively, than the recombinant human PON1. Thus, we report that the recombinant PON1-hFc exerts hydrolytic activity against paraoxon and DFP.

Columnar deformation of human red blood cell by highly localized fiber optic Bessel beam stretcher.

A single human red blood cell was optically stretched along two counter-propagating fiber-optic Bessel-like beams in an integrated lab-on-a-chip structure. The beam enabled highly localized stretching of RBC, and it induced a nonlinear mechanical deformation to finally reach an irreversible columnar shape that has not been reported. We characterized and systematically quantified this optically induced mechanical deformation by the geometrical aspect ratio of stretched RBC and the irreversible stretching time. The proposed RBC mechanism can realize a versatile and compact opto-mechanical platform for optical diagnosis of biological substances in the single cell level.

Crossed fiber optic Bessel beams for curvilinear optofluidic transport of dielectric particles.

Due to its unique non-diffracting and self-reconstructing nature, Bessel beams have been successfully adopted to trap multiple particles along the beam's axial direction. However, prior bulk-optic based Bessel beams have a fundamental form-factor limitation for in situ, in-vitro, and in-vivo applications. Here we present a novel implementation of Fourier optics along a single strand of hybrid optical fiber in a monolithic manner that can generate pseudo Bessel beam arrays in two-dimensional space. We successfully demonstrate unique optofluidic transport of the trapped dielectric particles along a curvilinear optical route by multiplexing the fiber optic pseudo Bessel beams. The proposed technique can form a new building block to realize reconfigurable optofluidic transportation of particulates that can break the limitations of both prior bulk-optic Bessel beam generation techniques and conventional microfluidic channels.

Objective-free optical-resolution photoacoustic microscopy.

Optical-resolution photoacoustic microscopy (OR-PAM) becomes a premier microscopic imaging tool in biomedicine because it provides agent-free optical absorption information in tissues. By tightly focusing light to a spot, a significantly improved lateral resolution can be achieved in OR-PAM. The focal spot size is typically determined by the numerical aperture of the used objective lens. Here, we demonstrate objective-free OR-PAM using a fiber optic Bessel beam generator. In this approach, no objective lens is required and, beneficially, the complexities of conventional OR-PAM systems can be greatly relieved. We have obtained photoacoustic images of a carbon fiber with a diameter of ∼6 µm, whose lateral resolution was measured to be better than 6 to 7 µm.

Dispersion control in square lattice photonic crystal fiber using hollow ring defects.

We propose a new dispersion control scheme by introducing hollow ring defects having a central air hole and a GeO2-or F-doped silica ring with in a square lattice photonic crystal fiber. We confirmed the flexible dispersion controllability in the proposed structure in two aspects of dispersion managements: ultra-flattened near-zero dispersion in the 530 nm-bandwidth over all communication bands and dispersion compensation in C, L, and U band with a high compensation ratio of 0.96~1.0 in reference to the standard single mode fiber. The proposed SLPCFs were also estimated to have an inherently low splice loss due to the index contrast between the doped-ring and silica that kept a good guidance even along with collapsed air holes, which cannot be achieved in conventional PCFs.