Enhanced growth inhibition of hepatic multicellular tumor spheroids by lactosylated poly(ethylene glycol)-siRNA conjugate formulated in PEGylated polyplexes

PEGylated polyplexes (lac-PEGylated polyplexes) composed of poly(L-lysine) and lactosylated poly(ethylene glycol)-small interfering RNA conjugate, which inhibits the RecQL1 gene product, were revealed to show an appreciable growth inhibition of multicellular HuH-7 spheroids (human hepatocarcinoma cell lines) for up to 21 days (IC(50)=6 nM); this system used as an in vitro three-dimensional (3D) model mimicking the in vivo biology of tumors. The PEGylated polyplexes thus prepared had a size of approximately 110 nm with clustered lactose moieties on their periphery as targeting ligands for the asialoglycoprotein-receptor-expressing HuH-7 cells. In contrast, OligofectAMINE/siRNA (cationic lipoplex) was observed to have almost no growth-inhibitory effect against HuH-7 spheroids, even though the lipoplex showed a stronger growth-inhibitory effect than the lac-PEGylated polyplexes on conventional monolayer-cultured HuH-7 cells. The FITC-tagged conjugate in the lac-PEGylated polyplexes showed smooth penetration into the HuH-7 spheroids compared with that in the lipoplexes, as observed by confocal fluorescence-scanning microscopy. This indicates that the small size of approximately 100 nm and the reduced nonspecific interaction due to the nonionic and hydrophilic lactosylated PEG layer contributes to the smooth penetration of the PEGylated polyplexes into the spheroid interior, eventually facilitating their uptake into the cells composing the spheroids. Cellular apoptosis indicating programmed cell death was also observed in the HuH-7 spheroids treated with the PEGylated polyplexes, revealing that the observed growth inhibition was indeed induced by the RNAi of the RecQL1 siRNA. These data suggest that the smart PEGylated polyplexes can indeed penetrate into the multiple cell layers of 3D tumor masses in vivo, exerting therapeutic effects through the RNAi.     

Simple,Mild, and Practical Esterification,Thioesterification, and Amide Formation Utilizing p‐Toluenesulfonyl Chloride and N‐Methylimidazole

We have developed an efficient method for the esterification or thioesterification of equimolar amounts of carboxylic acids and alcohols or thiols using a novel reagent, p‐toluenesulfonyl chloride (TsCl) together with N‐methylimidazole. The present method is simple, mild, and reactive, uses readily available and economical reagents. The choice of amine is critical for the present method. The amine, N‐methylimidazole, has two roles: (i) as an HCl scavenger for the initial smooth generation of mixed anhydrides between carboxylic acids and TsCl and (ii) successive formation of highly reactive ammonium intermediates from mixed anhydrides. This method could be applied to various types of carboxylic acids, alcohols, and thiols: a) several functionalities were tolerated; b) two N‐Cbz amino acids were smoothly esterified without racemization; and c) the labile 1β‐methylcarbapenem key intermediate and a pyrethroid insecticide, prallethrin, were successfully prepared. The related amide formation between carboxylic acids and primary or secondary amines was also performed. The proposed reaction mechanism involves a novel method for producing the reactive acylammonium intermediates. The production of these intermediates was rationally supported by a careful ¹H NMR monitoring study.     

Theoretical study of the temperature dependent hydrogen storage capacity of Pd and Ti nanoparticles

A hybrid simulation was carried out using discrete variational (DV) Xα molecular orbital and molecular dynamics methods to examine the storage of hydrogen in metal nanoparticles. The calculation load was light and therefore could be performed quickly on a generic personal computer. The simulation investigates the electronic states of hydrogen on Pd and Ti nanoparticles. The hydrogen dissociation behavior on the metal surface was reproduced, and the calculated bonding orbitals of Pd and hydrogen are consistent with other reports. The diffusion coefficient of hydrogen inside the metal displays the same temperature dependence as the theoretical results. This simplified calculation, which produces results in agreement with experimental/theoretical values, could lead to improved simulation methods for hydrogen storage materials.     

Switching characteristics of vacuum gap switch employing UV‐laser triggering

The switching time and arc‐ignition probability of a sustained main discharge in a laser trigger vacuum gap were measured. The third harmonic beam of an Nd:YAG pulse laser (wavelength 355 nm, energy 5 mJ/pulse, power density 4 × 10⁷ W/cm²) was used to trigger the gap. The main electrodes 85 mm in diameter were made of oxygen‐free copper. The gap length was 1.6 mm and the experimental chamber was evacuated to about 1.3 × 10⁴ Pa by a turbomolecular pump. The UV beam from the laser was focused at normal incidence onto the grounded cathode. The switching time decreased and the arc‐ignition probability increased with increasing applied voltage. The switching time was 326 ns at 15 kV with a circuit inductance of 107 μH. Its jitter was 24.8 ns. The advantageous results obtained with the UV beam are compared to the previously reported data on triggered vacuum gaps. © 2000 Scripta Technica, Electr Eng Jpn, 132(3): 8–13, 2000     

Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.     

Power distribution methods for MIMO‐OFDM systems and field experimentations

In this paper, we propose several power allocation schemes for multi‐input multi‐output (MIMO) orthogonal frequency division multiplexing (OFDM) transmission based on the minimization of an approximated bit error rate (BER) expression, and we evaluate the different solutions via field trial experimentations. The methods illustrated in this paper, serve to allocate power among the different transmit antennas and the different subcarriers which compose the MIMO OFDM transmitted signal. Several solutions are available to perform power allocation. Frequency domain power allocation, spatial domain power allocation and combined spatial and frequency power allocation are evaluated. We first review and describe the analytical solution for each power allocation scheme and then evaluate the complexity in terms of both computational operations and BER performances. Simulation results show the performance in term of BER and link the advantage of each possibility of power distribution with the associated complexity. Copyright © 2009 John Wiley & Sons, Ltd.     

Detection of pathogenic bacteria by using zinc finger protein fused with firefly luciferase

We constructed a novel bacterial genome detection system using zinc finger protein (ZF) fused with firefly luciferase (ZF-luciferase). Taking advantage of the direct recognition of double-stranded DNA (dsDNA) by ZF, we previously constructed bacterial genome detection systems that did not require dehybridization processes. To detect polymerase chain reaction (PCR) products rapidly and with a high sensitivity, we constructed two kinds of ZF-luciferase, Sp1-fused luciferase (Sp1-luciferase), and Zif268-fused luciferase (Zif268-luciferase). ZF-luciferase not only maintains luciferase activity but also shows dsDNA-binding ability and specificity. Furthermore, we succeeded in the detection of 10 copies of the genome of Legionella pneumophila and Escherichia coli O157. ZF-luciferase would be a useful tool for highly sensitive detection of pathogenic bacterial genome.     

Effects of the cultivation period of sweet potato on the sensory quality of imo‐shochu,a Japanese traditional spirit

Imo‐shochu is a Japanese traditional spirit made from sweet potatoes. Characteristic volatile compounds in imo‐shochu are mainly derived from the sweet potato and its flavour significantly depends on the quality and cultivar of sweet potato used. Thus, the effects of the cultivation period of sweet potatoes on sensory characteristics and composition of volatile compounds of imo‐shochu were investigated. Sweet potatoes (cv. Koganesengan) used in this study were harvested at 120, 150 or 180 days after planting, and each sample was used to prepare imo‐shochu. The imo‐shochu samples were evaluated by eight panellists in a blind study, who ranked them on the basis of various odour and taste attributes. Rank sums were calculated and data were analysed using the Friedman test. The compositions of the volatile compounds in the imo‐shochu samples were analysed using gas chromatography–mass spectrometry (GC‐MS). Sensory evaluations showed that a longer cultivation period of the sweet potatoes enhanced the floral aroma and characteristic taste of imo‐shochu. In addition, imo‐shochu prepared with the sample cultivated for 150 days was evaluated to have a sweeter taste than that prepared with the other samples. The GC‐MS analysis showed that imo‐shochu prepared with the sample cultivated for 180 days contained a lower concentration of monoterpene alcohols, but higher concentrations of β‐damascenone, rose oxide, and fatty acid esters than the imo‐shochu prepared with the sample cultivated for 120 days. These differences in the composition of the volatile compounds affected the sensory qualities of the imo‐shochu. Copyright © 2016 The Institute of Brewing & Distilling     

Development of a swim-type ROV for narrow space inspection

The swim-type remotely operated vehicle (ROV) for inspection of narrow spaces in nuclear power plants has been developed. Many structures are crowded in a confined space at regular intervals in the bottom area of a reactor. So, the thickness of the ROV shape is an important design point to ensure that the ROV can move in the space. The developed ROV has a three-dimensional swimming mechanism using six thrusters, three cameras for observing the position while moving and for making inspections easily, and a localization system. The localization system combines two elements: a gyroscope to detect the progression direction; and a slit laser that detects the progression distance using the optical cutting method. The localization method is called the modified inertial navigation (MIN) method and it was evaluated in a mock-up examination. The ROV was able to move smoothly using the MIN method and its position could be detected without making a mistake in the route followed.