Modifying the antigen-immunization schedule improves the variety of monoclonal antibodies obtained from immune-phage antibody libraries against HIV-1 Nef and Vif

Immune phage antibody libraries are an attractive technology for isolating antigen-specific monoclonal antibodies (mAbs). Here we show that the immunization schedule affects the immune phage antibody library properties. We subcutaneously (s.c.) administered HIV-1 Nef and Vif antigens with different schedules (25 μg × 2 s.c. and 10 μg × 3 s.c.). The variety of isolated mAbs in 25 μg × 2 s.c. groups (Nef: 11 clones, Vif: 9 clones) was superior to that in the 10 μg × 3 s.c. groups (Nef: 2 clones, Vif: 1 clone). This finding suggests that it is important to optimize the immunization schedule for isolating a wide variety of mAbs.     

Effects of finite mass ratio and inelastic reflection on the interaction between graphene and laminar flow

A modified boundary condition for the distribution function in the Lattice Boltzmann method at the interface between solid and fluid that takes into account a finite mass ratio between two phases and an inelastic reflection is proposed. The new boundary condition is built into the immersed boundary method to compute the interaction between graphene and laminar flow. Numerical simulations are carried out at a Reynolds number of 40, and drag and lift acting on the graphene and its deformation are examined by changing the mass ratio and the coefficient of restitution. It is found that the amplitude of the oscillating motion of the graphene is enhanced when compared with the case of infinite mass ratio with perfect collision.     

Fabrication of dense material having homogeneous GdAlO3–Al2O3 eutectic-like microstructure with off-eutectic composition by consolidation of the amorphous

Off-eutectic microstructures generally have both coarse crystals of rich component and ordinary eutectic microstructures. This paper shows a new method to form a dense bulk material having homogeneous eutectic-like microstructure with off-eutectic compositions. Mixture of Gd₂O₃ and Al₂O₃ powders with the off-eutectic composition was melted and quenched rapidly to form the amorphous phase. The amorphous film was pulverized. The dense bulk material could be fabricated by the consolidation of the amorphous powder using spark plasma sintering method. Field emission scanning electron microscope (FE-SEM) observation of the material showed homogeneous fine eutectic-like microstructure without coarse crystals. This is the first case that such material was successfully prepared.     

Carrier‐envelope phase‐stabilized chirped‐pulse amplification system

We demonstrated a carrier‐envelope phase (CEP) stabilized chirped‐pulse amplifier system. This amplifier system is composed of grating based pulse‐stretcher and compressor, a regenerative amplifier and a multi‐pass amplifier. We employed a new pulse‐pick‐up method to select CEP stabilized seed pulses. This pulse selection method is different from established practice which is based on pulse train timing, but is based on CEP of seed pulse. We measured amplitude‐to‐phase noise conversion coefficient of microstructure fiber and evaluated the additional out‐of‐loop error of carrier‐envelope offset (CEO) control. We also investigated the effect of beam pointing of the measured fringe shift in self‐referencing spectral interference method. © 2006 Wiley Periodicals, Inc. Electr Eng Jpn, 157(3): 35–42, 2006; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/eej.20334 Copyright © 2006 Wiley Periodicals, Inc.     

Effect of ion motion on zeta-potential distribution at microchannel wall obtained from nanoscale laser-induced fluorescence

The present study has experimentally investigated the two-dimensional distribution of zeta-potential at the wall, which dominates electroosmotic microchannel flow. Nanoscale laser-induced fluorescence imaging using fluorescent dye and the evanescent wave with total internal reflection was developed for the zeta-potential measurement. The fluorescent dye in the vicinity of the wall is excited by the evanescent wave, which decays exponentially from the wall. The zeta-potential is obtained from the fluorescent intensity because the distribution of fluorescent dye near the wall is related to the zeta-potential by the Boltzman distribution. Two kinds of solution at different Na+ concentrations were mixed in a T-shaped microchannel composed of PDMS and silica glass. The zeta-potential distribution at the silica glass wall was measured with the uncertainty of 4.7 mV. The motion of Na+ in the microchannel was estimated by the numerical analysis using the velocity information obtained by micrometer-resolution particle image velocimetry. It is concluded that electroosmotic flow was generated by the zeta-potential distribution at the silica glass and PDMS wall, which was dependent on the Na+ transport in the flow field.     

A Computational Drug Metabolite Detection Using the Stable Isotopic Mass-Shift Filtering with High Resolution Mass Spectrometry in Pioglitazone and Flurbiprofen

The identification of metabolites in drug discovery is important. At present, radioisotopes and mass spectrometry are both widely used. However, rapid and comprehensive identification is still laborious and difficult. In this study, we developed new analytical software and employed a stable isotope as a tool to identify drug metabolites using mass spectrometry. A deuterium-labeled compound and non-labeled compound were both metabolized in human liver microsomes and analyzed by liquid chromatography/time-of-flight mass spectrometry (LC-TOF-MS). We computationally aligned two different MS data sets and filtered ions having a specific mass-shift equal to masses of labeled isotopes between those data using our own software. For pioglitazone and flurbiprofen, eight and four metabolites, respectively, were identified with calculations of mass and formulas and chemical structural fragmentation analysis. With high resolution MS, the approach became more accurate. The approach detected two unexpected metabolites in pioglitazone, i.e., the hydroxypropanamide form and the aldehyde hydrolysis form, which other approaches such as metabolite-biotransformation list matching and mass defect filtering could not detect. We demonstrated that the approach using computational alignment and stable isotopic mass-shift filtering has the ability to identify drug metabolites and is useful in drug discovery.     

4‐Hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐one Synthesis by a Type III Polyketide Synthase from Rhodospirillum centenum

The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III polyketide synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl‐CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C_10–14 fatty acyl‐CoA unit, one malonyl‐CoA unit and two methylmalonyl‐CoA units. We identified these products as 4‐hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of methylmalonyl‐CoA as the extender substrate. In addition, in vitro reactions with ¹³C‐labeled malonyl‐CoA revealed that RpsA produced tetraketide 6‐alkyl‐4‐hydroxy‐1,5‐dimethyl‐2‐oxocyclohexa‐3,5‐diene‐1‐carboxylic acids from C_14–20 fatty acyl‐CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III polyketide synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III polyketide synthase family.     

Biosynthetic Gene Cluster for Surugamide A Encompasses an Unrelated Decapeptide,Surugamide F

Genome mining is a powerful method for finding novel secondary metabolites. In our study on the biosynthetic gene cluster for the cyclic octapeptides surugamides A–E (inhibitors of cathepsin B), we found a putative gene cluster consisting of four successive non‐ribosomal peptide synthetase (NRPS) genes, surA, surB, surC, and surD. Prediction of amino acid sequence based on the NRPSs and gene inactivation revealed that surugamides A–E are produced by two NRPS genes, surA and surD, which were separated by two NRPS genes, surB and surC. The latter genes are responsible for the biosynthesis of an unrelated peptide, surugamide F. The pattern of intercalation observed in the sur genes is unprecedented. The structure of surugamide F, a linear decapeptide containing one 3‐amino‐2‐methylpropionic acid (AMPA) residue, was determined by spectroscopic methods and was confirmed by solid‐phase peptide synthesis.     

Pemafibrate Prevents Retinal Dysfunction in a Mouse Model of Unilateral Common Carotid Artery Occlusion

Cardiovascular diseases lead to retinal ischemia, one of the leading causes of blindness. Retinal ischemia triggers pathological retinal glial responses and functional deficits. Therefore, maintaining retinal neuronal activities and modulating pathological gliosis may prevent loss of vision. Previously, pemafibrate, a selective peroxisome proliferator-activated receptor alpha modulator, was nominated as a promising drug in retinal ischemia. However, a protective role of pemafibrate remains untouched in cardiovascular diseases-mediated retinal ischemia. Therefore, we aimed to unravel systemic and retinal alterations by treating pemafibrate in a new murine model of retinal ischemia caused by cardiovascular diseases. Adult C57BL/6 mice were orally administered pemafibrate (0.5 mg/kg) for 4 days, followed by unilateral common carotid artery occlusion (UCCAO). After UCCAO, pemafibrate was continuously supplied to mice until the end of experiments. Retinal function (a-and b-waves and the oscillatory potentials) was measured using electroretinography on day 5 and 12 after UCCAO. Moreover, the retina, liver, and serum were subjected to qPCR, immunohistochemistry, or ELISA analysis. We found that pemafibrate enhanced liver function, elevated serum levels of fibroblast growth factor 21 (FGF21), one of the neuroprotective molecules in the eye, and protected against UCCAO-induced retinal dysfunction, observed with modulation of retinal gliosis and preservation of oscillatory potentials. Our current data suggest a promising pemafibrate therapy for the suppression of retinal dysfunction in cardiovascular diseases.