A potential antitumor drug (arginine deiminase) reengineered for efficient operation under physiological conditions

Arginine deiminase (ADI, EC 3.5.3.6) is a potential antitumor drug for the treatment of arginine‐auxotrophic tumors such as hepatocellular carcinomas (HCCs) and melanomas, and studies on human lymphatic leukemia cell lines have confirmed that ADI has antiangiogenic activity. Recent studies showed that a combination of taxane and ADI‐PEG20, which induces caspase‐independent apoptosis, is more effective than taxane monotherapy for prostate cancer. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) and of many other ADI enzymes lies in their pH‐dependent activity profile. PpADI has a pH optimum at 6.5 and a pH shift from 6.5 to 7.5 results in an ～80 % activity drop (the pH of human plasma is 7.35 to 7.45). In 2010, we reported a proof of concept for ADI engineering by directed evolution that resulted in variant M2 (K5T/D44E/H404R). M2 has a pH optimum of pH 7.0, a fourfold higher k_cat value than the wild‐type PpADI (pH 7.4, 0.5 M phosphate buffer), and an increased K_m value for substrate arginine. In our latest work, variants M5 (K5T/D38H/D44E/A128T/H404R) and M6 (K5T/D38H/D44E/A128T/E296K/H404R) were generated by directed evolution by employing PBS buffer (pH 7.4), which mimics physiological conditions. The S_0.5 value of parent M3 (K5T/D44E/A128T/H404R) decreased from 2.01 to 1.48 mM (M5) and 0.81 mM (M6). The S_0.5 value of M6 (0.81 mM ) is lower than that of wild‐type PpADI (1.30 mM ); the k_cat values improved from 0.18 s^?1 (wild‐type PpADI) to 17.56 s^?1 (M5, 97.6‐fold) and 11.64 s^?1 (M6, 64.7‐fold).     

Directed Evolution of an Antitumor Drug (Arginine Deiminase PpADI) for Increased Activity at Physiological pH

Arginine deiminase (ADI; EC 3.5.3.6) has been studied as a potential antitumor drug for the treatment of arginine‐auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. Studies with human lymphatic leukemia cell lines confirmed that ADI is an antiangiogenic agent for treating leukemia. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) lies in its pH‐dependent activity profile, its pH optimum is at 6.5. A pH shift from 6.5 to 7.5 results in an approximately 80 % drop in activity. (The pH of human plasma is 7.35 to 7.45.) In order to shift the PpADI pH optimum, a directed‐evolution protocol based on an adapted citrulline‐screening protocol in microtiter‐plate format was developed and validated. A proof of concept for ADI engineering resulted in a pH optimum of pH 7.0 and increased resistance under physiological and slightly alkaline conditions. At pH 7.4, variant M2 (K5T/D44E/H404R) is four times faster than the wild‐type PpADI and retains ～50 % of its activity relative to its pH optimum, compared to ～10 % in the case of the wild‐type PpADI.     

Atomic layer deposition on biological macromolecules: metal oxide coating of tobacco mosaic virus and ferritin

Decoration of nanoparticles, in particular biomolecules, gathered high attention in recent years.(1-7) Of special interest is the potential use of biomolecules as templates for the fabrication of semiconducting or metallic nanostructures.(1-7,26) In this work we show the application of atomic layer deposition, a gas-phase thin film deposition process, to biological macromolecules, which are frequently used as templates in nanoscale science, and the possibility to fabricate metal oxide nanotubes and thin films with embedded biomolecules.(1-13).     

Control of Cell Adhesion and Neurite Outgrowth by Patterned Gold Nanoparticles with Tunable Attractive or Repulsive Surface Properties

Guiding of neuronal cells on surfaces is required for the investigation of fundamental aspects of neurobiology, for tissue engineering, and for numerous bioelectronic applications. A modular method to establish nanostructured chemical templates for local deposition of gold nanoparticles is presented. A process comprising nanoimprint lithography, silanization, lift‐off, and gold nanoparticle immobilization is used to fabricate the particle patterns. The chemical composition of the surface can be modified by in situ adsorption of cell‐binding ligands to locally addressed particles. The versatility of this approach is demonstrated by inverting the binding affinity between rat cortical neurons and nanopatterned surfaces via wet‐chemical means and thereby reversing the pattern of guided neurons.     

A finite element analysis in polar co-ordinates of the Saint Venant torsion problem

A characteristic feature of the variational functionals for several boundary value problems in polar co-ordinates is the fact that one independent variable occurs explicitly in the denominator. Therefore, the coefficients of the finite element equations for sectors of circular ring shaped elements are not constants but functions of the distance of the elements from the origin of co-ordinates.¹ We name them coefficient functions. In order to show the particular aspects of the calculations in terms of polar co-ordinates we deal here with the solution of the torsion problem by bilinear and bicubic Hermitian interpolation. The finite element equations are arranged according to Schaefer² in the form of block which can easily be transformed into ‘stars’³ or molecules^4,5 similar to those used in finite difference methods. The origin of co-ordinates requires a special consideration, firstly because of the coincidence of several nodes at that point and secondly because of the divergent behaviour of some coefficient functions. It turns out to be advantageous for the numerical calculations to expand the coefficient functions in power series. Besides, the expansions are required to deduce the equations for rectangular elements by limiting processes. The twisting moments and shearing stresses calculated for several cross sections illustrate the numerical suitability of the method. The finite element values are compared partly with exact solutions and partly with experimental results obtained by a moiré method using Prandtl's soap film analogy.⁶ Finally it is shown how the accuracy of the finite element values can be improved by the Richardson extrapolation⁷.     

Modeling and physical analysis of an out-of-plane capacitive MEMS transducer with dynamically coupled electrodes

Microsystem Technologies - This paper presents a physics-based system-level compact model of a novel out-of-plane capacitive MEMS transducer for detection of mechanical forces, pressure variations...