Accelerated Discovery of Potent Bioactive anti-TNFα Aptamers by Microbead-Assisted Capillary Electrophoresis (MACE)-SELEX

Dysregulation of tumor necrosis factor-α (TNFα), a pro-inflammatory cytokine, causes several diseases, making it an important therapeutic target. Here, we identified a novel DNA aptamer against human TNFα using in vitro selection, which included a high exclusion pressure process against non-binding and weak binders through microbead-assisted capillary electrophoresis (MACE) in only three rounds. Among the 15 most enriched aptamers, Apt14 exhibited the highest inhibitory activity for the interaction between TNFα and its cognate receptor in mouse L929 cells. For further improving the bioactivity of the aptamer, dimerization programed by hybridization was evaluated, resulting in the Apt14 dimer exhibited a twofold higher binding affinity and stronger inhibition compared to the monomer counterpart. Rapid identification of bioactive aptamers using MACE in combination with facile dimerization by hybridization accelerates the discovery of novel bioactive aptamers, paving the way toward replacing current monoclonal antibody therapy with the less expensive and non-immunogenic aptamer therapy.     

A Metal-Free,Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

Superoxide dismutase 1 (SOD1) is a metalloenzyme with high structural stability, but a lack of Cu and Zn ions decreases its stability and enhances the likelihood of misfolding, which is a pathological hallmark of amyotrophic lateral sclerosis (ALS). A growing body of evidence has demonstrated that misfolded SOD1 has prion-like properties such as transmissibility between cells and intracellular propagation of misfolding of natively folded SOD1. Recently, we found that SOD1 is misfolded in the cerebrospinal fluid of sporadic ALS patients, providing a route by which misfolded SOD1 spreads via the extracellular environment of the central nervous system. Unlike intracellular misfolded SOD1, it is unknown which extracellular misfolded species is most relevant to prion-like properties. Here, we determined a conformational feature of extracellular misfolded SOD1 that is linked to prion-like properties. Using culture media from motor neuron-like cells, NSC-34, extracellular misfolded wild-type, and four ALS-causing SOD1 mutants were characterized as a metal-free, disulfide oxidized form of SOD1 (apo-SOD1^S-S). Extracellular misfolded apo-SOD1^S-S exhibited cell-to-cell transmission from the culture medium to recipient cells as well as intracellular propagation of SOD1 misfolding in recipient cells. Furthermore, culture medium containing misfolded apo-SOD1^S-S exerted cytotoxicity to motor neuron-like cells, which was blocked by removal of misfolded apo-SOD1^S-S from the medium. We conclude that misfolded apo-SOD1^S-S is a primary extracellular species that is linked to prion-like properties.     

In Vivo Electrophysiology of Peptidergic Neurons in Deep Layers of the Lumbar Spinal Cord after Optogenetic Stimulation of Hypothalamic Paraventricular Oxytocin Neurons in Rats

The spinal ejaculation generator (SEG) is located in the central gray (lamina X) of the rat lumbar spinal cord and plays a pivotal role in the ejaculatory reflex. We recently reported that SEG neurons express the oxytocin receptor and are activated by oxytocin projections from the paraventricular nucleus of hypothalamus (PVH). However, it is unknown whether the SEG responds to oxytocin in vivo. In this study, we analyzed the characteristics of the brain–spinal cord neural circuit that controls male sexual function using a newly developed in vivo electrophysiological technique. Optogenetic stimulation of the PVH of rats expressing channel rhodopsin under the oxytocin receptor promoter increased the spontaneous firing of most lamina X SEG neurons. This is the first demonstration of the in vivo electrical response from the deeper (lamina X) neurons in the spinal cord. Furthermore, we succeeded in the in vivo whole-cell recordings of lamina X neurons. In vivo whole-cell recordings may reveal the features of lamina X SEG neurons, including differences in neurotransmitters and response to stimulation. Taken together, these results suggest that in vivo electrophysiological stimulation can elucidate the neurophysiological response of a variety of spinal neurons during male sexual behavior.     

Measurement of vertical and horizontal vibrations of a probe for acoustic evaluation of food texture

Two‐dimensional (vertical and horizontal) vibrations of a wedge‐type probe upon food rupture were evaluated separately using two accelerometers placed perpendicular to a guide rod of a swing‐arm device for texture evaluation of the flesh of three varieties of apples and three types of potato chips. Voltage signals from the accelerometers were filtered using a half‐octave band‐pass filter. The energy texture index (ETI), based on kinetic energy of the vertical or horizontal probe vibrations, was calculated over low to high frequency bands (no. 1–21). The spectra for the flesh of the three varieties of apples included two common peaks for vertical ETI at band no. 11 (1,120–1,600 Hz) and 19 (17,920–25,600 Hz) and horizontal ETI at band no. 1 (0–10 Hz) and 15 (4,480–6,400 Hz). The spectra for the three types of potato chips had a common ETI peak at band no. 11 (1,120–1,600 Hz) for horizontal ETI and at no. 15 (4,480–6,400 Hz) for vertical ETI. The three apple varieties gave rise to different intensities of vertical and horizontal ETIs while the two peaks were maintained. The thick potato chip type had higher vertical and horizontal ETIs than the thin and soft varieties in most bands; however, the thin type had the highest vertical ETIs only in lower bands (0–200 Hz). The soft type had the lowest vertical and horizontal ETI. The above results suggest that different food textures can be distinguished by two‐dimensional vibration analyses of probe insertion into a food sample based on frequency bands.     

Hydrogen isotope permeation from cooling water through various metal piping

In order to investigate the behavior of hydrogen isotope on the water–metal boundary, deuterium permeation experiments from heavy water vessel through various metal piping, such as pure iron (Fe), nickel (Ni), stainless steel (SS304), and pure iron with 10 μm gold plating, were performed at 573 K and at 15 MPa. During the experiment, surfaces of metal piping except gold plating one were oxidized at the heavy water boundary and then deuterium would generate by the oxidation reactions. This deuterium could be detected by mass spectrometer, which monitored the inside gases of the piping under continuous evacuation. The result showed clearly that the deuterium permeated through Fe, Ni, and SS304 piping was detected as mainly deuterium gas (D₂) under continuous evacuation, though that through gold plating one could not be detected effectively. The D₂ permeation rate through Fe, Ni, and SS304 piping reached equilibrium conditions with oxide generation at D₂O–metal boundary, although concluding the transfer mechanism will require further testing and modeling activities.     

Improvement of lysozyme recovery method from the precipitate of protein-anionic surfactant complexes

A new protein separation process using a surfactant and a polar organic solvent consists of a precipitation step and a recovery step. In the precipitation step, a protein-surfactant complex is precipitated from an aqueous solution, when an ionic surfactant, sodium di(2-ethylhexyl) sulfosuccinate (AOT), is added to an aqueous solution, including protein (lysozyme). In the recovery step, the precipitate is dissolved in a polar organic solvent, such as acetone, and the protein is recovered as precipitates when a very small amount of salt solution was added to remove surfactants from the protein-surfactant complex. However, the details of the protein recovery step from precipitate have not been studied yet. In this study, the improvement of the protein recovery step was examined from the viewpoint of a recovery ratio of protein and a remaining ratio of surfactant. The optimum NaCl concentration in the feed for the protein recovery was in the range of 0.05–0.2 kmol/m³. As the NaCl concentration in the feed increased to more than 0.2 kmol/m³, the precipitation ratio decreased due to the electrostatic screening effect of NaCl. It was found that the addition of a very small amount of NaCl solution to acetone was unnecessary when NaCl was included in the feed lysozyme solution. On the other hand, as the NaCl concentration decreased to less than 0.05 kmol/m³, the precipitation ratio was decreased due to the low re-precipitation of protein by the addition of a small amount of NaCl solution in acetone. In the case of the feed containing no salt, the desired NaCl concentration added to acetone was in the range above 0.2 kmol/m³. In addition, the most suitable volume ratio of acetone to feed was found to be 0.2.     

Fabrication of conducting poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) thin films by ultrasonic spray-assisted mist deposition method

Transparent conducting polymer, poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) thin films were fabricated by a vapor-deposition technique, ultrasonic spray-assisted mist deposition method. The thickness was well controlled from 40 to 600 nm, keeping reasonable conductivity of 300-450 S/cm. The films with thickness less than 180 nm have high (> 80%) transmission over a wide (270-800 nm) spectral region. In addition, formation of ring-dot electrode pattern with a hard-mask was demonstrated, achieving lithography-less patterning. The results encourage that this deposition method is developed as an actual process technology of transparent electrodes in devices.     

Synthesis of Nanohole‐Structured Single‐Crystalline Platinum Nanosheets Using Surfactant‐Liquid‐Crystals and their Electrochemical Characterization

Nanohole‐structured single‐crystalline Pt nanosheets have been synthesized by the borohydride reduction of Na₂PtCl₆ confined to the lyotropic liquid crystals (LLCs) of polyoxyethylene (20) sorbitan monooleate (Tween 80) with or without nonaethylene‐glycol (C₁₂EO₉). The Pt nanosheets of around 4–10 nm in central thickness and up to 500 nm or above in diameter have a number of hexagonal‐shaped nanoholes ∼1.8 nm wide. High‐resolution electron microscope images of the nanosheets showed atomic fringes with a spacing of 0.22 nm indicating that the nanosheets are crystallographically continuous through the nanoholed and non‐holed areas. The inner‐angle distributions for the hexagonal nanoholes indicate that the six sides of the nanoholes are walled with each two Pt (111), Pt (1 1) and Pt (010) planes. The formation mechanism of nanoholed Pt nanosheets is discussed on the basis of structural and compositional data for the resulting solids and their precursory LLCs, with the aid of similar nanohole growth observed for a Tween 80 free but oleic acid‐incorporated system. It is also demonstrated that the nanoholed Pt nanostructures loaded on carbon exhibit fairly high electrocatalytic activity for oxygen reduction reaction and a high performance as a cathode material for polymer‐electrolyte fuel cells, along with their extremely high thermostability revealed through the effect of electron‐irradiation.     

Solidification of Al2O3–YAG eutectic composites with off-metastable eutectic composition from undercooled melt produced by melting Al2O3–YAP eutectics

It has been reported that solidification of the Al₂O₃–YAG equilibrium eutectic structure follows melting of the Al₂O₃–YAP metastable eutectic structure. Since the exothermic heat due to solidification was consumed by the endothermic heat due to melting, a fine and uniform eutectic structure was obtained. However, the composition of the Al₂O₃–YAG eutectic structure is restricted to the metastable eutectic composition. In this paper, Al₂O₃–YAG eutectic compacts with an off-metastable eutectic composition were prepared by the addition of Al₂O₃ particles to Al₂O₃–YAP eutectic particles with diameters less than 20 μm. In compositions ranging from 18.5 mol%Y₂O₃ to 13.5 mol%Y₂O₃, dense Al₂O₃–YAG eutectic compacts were formed without any Al₂O₃ segregation. The flexural strength and the fracture toughness remained almost unchanged with the increase in the Al₂O₃ phase. The addition of Al₂O₃ particles to the Al₂O₃–YAP eutectic particles enabled the matrix phase to change from the YAG phase to the Al₂O₃ phase.