Functional expression of a plant hydroxynitrile lyase in Escherichia coli by directed evolution: creation and characterization of highly in vivo soluble mutants

Low protein solubility of recombinantly expressed proteins in Escherichia coli is a major factor hindering their application and analysis. We generated highly in vivo soluble mutants of a hydroxynitrile lyase in E.coli using protein engineering. Structure-guided saturation mutagenesis caused high solubility of single Lys-Pro mutations at positions 176, 199 and 224 of this low soluble wild-type enzyme. The triple Lys-Pro mutant generated at these surface conserved residues showed up to 8-fold increase in specific activity in the cell-free extract. Random mutagenesis also created a mutant of His103Met with 18.5-fold increase. The main expression form was reversed from insoluble to the soluble fraction following both types of above-mentioned mutations in E.coli at 37°C. The findings challenge the rationale of producing recombinant proteins in this host at 37°C. Formerly wild type low soluble protein was then present as soluble protein by these mutations, which also elevated the total soluble protein fraction in E.coli. Saturation mutagenesis of His103 provided other highly soluble mutants with hydrophobic substitutions. These mutations caused only minor secondary structural changes as determined by circular dichroism and Fourier-transform infrared spectroscopy and affected catalytic efficiency slightly for the purified mutants (0.82-1.6-fold for benzaldehyde and 0.9-1.9-fold for mandelonitrile). The stability of the mutants was differed from that of the wild type at high temperatures and at pH >8. Exchanging the buried basic-polar residue His103 with hydrophobic amino acids is in line with the overall structure of the enzyme, i.e. having hydrophilic residues in solvent-exposed areas and hydrophobic residues in the core.     

A comparative study and phage typing of silage-making Lactobacillus bacteriophages

To investigate basic characteristics of 10 virulent phages active on silage-making lactobacilli, morphological properties, host ranges, protein composition and genome characterization were separated into five groups based on host ranges and basic properties. The seven phages of groups I, II and V were active on Lactobacillus plantarum and Lactobacillus pentosus. Phage phiPY4 (group III) infected both L. casei and Lactobacillus rhamnosus. Phage phiPY5 (group IV) specifically infected Lactobacillus casei. Morphologically, three phages of groups I belonged to the Myoviridae family, while seven other phages of groups II, III and V belonged to the Siphoviridae family. SDS-PAGE profiles, restriction analysis, G + C contents of DNA and Dot blot hybridization revealed a high degree of homology in each group. Clustering derived from host range analysis was closely related to results of DNA and protein analyses. These phages may be applicable to phage typing for silage-making lactobacilli.     

Gating electrical transport through DNA molecules that bridge between silicon nanogaps

DNA electronic devices were prepared on silicon-based three-terminal electrodes. Both ends of DNA molecules (400 bp long, mixed sequences) were bridged via chemical bonds between the source-drain nanogap (120 nm) electrodes. S-Shaped I-V curves were obtained and the electric current can be modulated by the gate voltage. The DNA molecules act as semiconducting p-type nanowires in the three-terminal device.     

Effects of alkali or acid treatment on the isomerization of amino acids

The effect of alkali treatment on the isomerization of amino acids was investigated. The 100×D/(D+L) values of amino acids from peptide increased with increase in the number of constituent amino acid residues. Furthermore, the N-terminal amino acid of a dipeptide was isomerized to a greater extent than the C-terminal residue.     

Thin titanium oxide films deposited by e-beam evaporation with additional rapid thermal oxidation and annealing for ISFET applications

Titanium oxide (TiO₂) has been extensively applied in the medical area due to its proved biocompatibility with human cells [1]. This work presents the characterization of titanium oxide thin films as a potential dielectric to be applied in ion sensitive field-effect transistors. The films were obtained by rapid thermal oxidation and annealing (at 300, 600, 960 and 1200 °C) of thin titanium films of different thicknesses (5 nm, 10 nm and 20 nm) deposited by e-beam evaporation on silicon wafers. These films were analyzed as-deposited and after annealing in forming gas for 25 min by Ellipsometry, Fourier Transform Infrared Spectroscopy (FTIR), Raman Spectroscopy (RAMAN), Atomic Force Microscopy (AFM), Rutherford Backscattering Spectroscopy (RBS) and Ti-K edge X-ray Absorption Near Edge Structure (XANES). Thin film thickness, roughness, surface grain sizes, refractive indexes and oxygen concentration depend on the oxidation and annealing temperature. Structural characterization showed mainly presence of the crystalline rutile phase, however, other oxides such Ti₂O₃, an interfacial SiO₂ layer between the dielectric and the substrate and the anatase crystalline phase of TiO₂ films were also identified. Electrical characteristics were obtained by means of I-V and C-V measured curves of Al/Si/TiO_x/Al capacitors. These curves showed that the films had high dielectric constants between 12 and 33, interface charge density of about 10¹⁰/cm² and leakage current density between 1 and 10⁻⁴ A/cm². Field-effect transistors were fabricated in order to analyze I_D x V_DS and log I_D × Bias curves. Early voltage value of −1629 V, R_OUT value of 215 MΩ and slope of 100 mV/dec were determined for the 20 nm TiO_x film thermally treated at 960 °C.     

Equivocal Colonic Carcinogenicity of Aloe arborescens Miller var. natalensis Berger at High‐Dose Level in a Wistar Hannover Rat 2‐y Study

ABSTRACT: A 2‐y carcinogenicity study of Aloe, Aloe arborescens Miller var. natalensis Berger, a food additive, was conducted for assessment of toxicity and carcinogenic potential in the diet at doses of 4% or 0.8% in groups of male and female Wistar Hannover rats. Both sexes receiving 4% showed diarrhea, with loss of body weight gain. The survival rate in the 4% female group was significantly increased compared with control females after 2 y. Hematological and biochemical examination showed increase of RBC, Hb, and Alb in the 4% males. The cause of these increases could conceivably have been dehydration through diarrhea. AST and Na were significantly decreased in the males receiving 4%, and Cl was significantly decreased in both 4% and 0.8% males. A/G was significantly increased in the 4% females, and Cl was significantly decreased (0.8%) in the female group. Histopathologically, both sexes receiving 4% showed severe sinus dilatation of ileocecal lymph nodes, and yellowish pigmentation of ileocecal lymph nodes and renal tubules. Adenomas or adenocarcinomas in the cecum, colon, and rectum were observed in 4% males but not in the 0.8% and control male groups. Similarly, in females, adenomas in the colon were also observed in the 4% but not 0.8% and control groups. In conclusion, Aloe, used as a food additive, exerted equivocal carcinogenic potential at 4% high‐dose level on colon in the 2‐y carcinogenicity study in rats. Aloe is not carcinogenic at nontoxic‐dose levels and that carcinogenic potential in at 4% high‐dose level on colon is probably due to irritation of the intestinal tract by diarrhea.     

In Vivo Study of the Efficacy and Safety of 5-Aminolevulinic Radiodynamic Therapy for Glioblastoma Fractionated Radiotherapy

To treat malignant glioma, standard fractionated radiotherapy (RT; 60 Gy/30 fractions over 6 weeks) was performed post-surgery in combination with temozolomide to improve overall survival. Malignant glioblastoma recurrence rate is extremely high, and most recurrent tumors originate from the excision cavity in the high-dose irradiation region. In our previous study, protoporphyrin IX physicochemically enhanced reactive oxygen species generation by ionizing radiation and combined treatment with 5-aminolevulinic acid (5-ALA) and ionizing radiation, while radiodynamic therapy (RDT) improved tumor growth suppression in vivo in a melanoma mouse model. We examined the effect of 5-ALA RDT on the standard fractionated RT protocol using U251MG- or U87MG-bearing mice. 5-ALA was orally administered at 60 or 120 mg/kg, 4 h prior to irradiation. In both models, combined treatment with 5-ALA slowed tumor progression and promoted regression compared to treatment with ionizing radiation alone. The standard fractionated RT protocol of 60 Gy in 30 fractions with oral administration of 120 and 240 mg/kg 5-ALA, the human equivalent dose of photodynamic diagnosis, revealed no significant increase in toxicity to normal skin or brain tissue compared to ionizing radiation alone. Thus, RDT is expected to enhance RT treatment of glioblastoma without severe toxicity under clinically feasible conditions.     

High Level Oscillations With Narrow Linewidth in Magnetic Nano-Contact Spin Torque Oscillator With Synthetic AF Spin-Valve Structure

We investigated the current induced magnetization dynamics, so called spin torque oscillation, in magnetic nano-contact MR element with a synthetic antiferromagnetic type spin-valve structure under high in-plane applied magnetic field of 0.9-1 kOe. Very high level oscillation of 18 nVHz^-1/2 with narrow FWHM of 12 MHz was observed in the condition of negative applied current where electron-spin is injected from free layer to reference one. Applied current dependency on an oscillation frequency is a blueshift with good linearlity, and same dependency on level and FWHM is almost constant in the range of -10 to -14 mA, while applied field dependency on frequency shows redshift with two different slopes, -2.5 MHz/Oe in less than 1 kOe and -8.7 MHz/Oe in more than 1 kOe which field is smaller than the spin-flop field for the synthetic antiferromagnet, besides very clear and reasonable peak and bottom for oscillation level and FWHM are observed, respectively. It is thought that these magnetization dynamics are originated from synthetic antiferromagnet.     

Electrical characterization and morphological properties of AlN films prepared by dc reactive magnetron sputtering

Aluminum nitride (AlN) films were deposited by dc reactive magnetron sputtering on p-Si-(1 0 0) substrate in Ar-N₂ gas mixtures. The effects of nitrogen concentration and sputtering power on AlN films deposition rate, crystallographic orientation, refractive index, and surface morphology are investigated by means of several characterization techniques. The results show that AlN films reasonably textured in (0 0 2) orientation with low surface roughness can be obtained with the deposition rate as high as 70 nm/min by the control of either target power or N₂ concentration in the gas mixture. Increasing the dc discharge power, Al atoms are not completely nitridized and the Al phases appear, as well as the AlN phases. MIS (Metal-Insulator-Semiconductor) structures were fabricated and electrically evaluated by I-V (current-voltage) and C-V (capacitance-voltage) measurements at high frequency (1 MHz). The results obtained from C-V curves indicate that charges at the dielectric/semiconductor interface occur, and the dielectric constant values (extracted under strong accumulation region) are compatible with those found in literature.