Identification of SYS1 as a Host Factor Required for Shiga Toxin-Mediated Cytotoxicity in Vero Cells

Shiga toxin (STx) or Vero toxin is a virulence factor produced by enterohemorrhagic Escherichia coli. The toxin binds to the glycosphingolipid globotriaosylceramide (Gb3) for its entry, and causes cell death by inhibiting ribosome function. Previously, we performed a loss-of-function screen in HeLa cells using a human CRISPR knockout (KO) library and identified various host genes required for STx-induced cell death. To determine whether this library targeted to the human genome is applicable to non-human primate cells and to identify previously unrecognized factors crucial for STx-induced cell death, we herein performed a similar screen in the African green monkey kidney-derived Vero C1008 subline. Many genes relevant to metabolic enzymes and membrane trafficking were enriched, although the number of enriched genes was less than that obtained in the screening for HeLa cells. Of note, several genes that had not been enriched in the previous screening were enriched: one of these genes was SYS1, which encodes a multi-spanning membrane protein in the Golgi apparatus. In SYS1 KO Vero cells, expression of Gb3 and sphingomyelin was decreased, while that of glucosylceramide and lactosylceramide was increased. In addition, loss of SYS1 inhibited the biosynthesis of protein glycans, deformed the Golgi apparatus, and perturbed the localization of trans-Golgi network protein (TGN) 46. These results indicate that the human CRISPR KO library is applicable to Vero cell lines, and SYS1 has a widespread effect on glycan biosynthesis via regulation of intra-Golgi and endosome–TGN retrograde transports.     

Chemical Modification of Phage-Displayed Helix-Loop-Helix Peptides to Construct Kinase-Focused Libraries

Conformationally constrained peptides hold promise as molecular tools in chemical biology and as a new modality in drug discovery. The construction and screening of a target-focused library could be a promising approach for the generation of de novo ligands or inhibitors against target proteins. Here, we have prepared a protein kinase-focused library by chemically modifying helix-loop-helix (HLH) peptides displayed on phage and subsequently tethered to adenosine. The library was screened against aurora kinase A (AurA). The selected HLH peptide Bip - 3 retained the α-helical structure and bound to AurA with a K_D value of 13.7 μM. Bip - 3 and the adenosine-tethered peptide Bip - 3 - Adc provided IC₅₀ values of 103 μM and 7.7 μM, respectively, suggesting that Bip - 3 - Adc bivalently inhibited AurA. In addition, the selectivity of Bip - 3 - Adc to several protein kinases was tested, and was highest against AurA. These results demonstrate that chemical modification can enable the construction of a kinase-focused library of phage-displayed HLH peptides.     

Temperature-stable low-noise ferrite memory cores

Temperature-stable low-noise memory cores made from the substituted lithium and nickel ferrites were studied. Ferrite compositions which are suitable for miniature cores have been developed by studying magnetic properties of the ferrite in the system Li-M-M' and Ni-M-M' ferrite (M:Mn, Fe, Co, Ni, Cu, and Zn). Technological approaches for fabricating small memory cores were made by studying the effect of ferrite-powder parameters and sintering conditions on magnetic properties of the substituted ferrites. Typical pulse characteristics taken with coincident current mode for cores of 0.48, 0.32, and 0.27 mm φ OD are presented. It was found that the Ni-Mn-F(II) ferrites are suitable for a low-drive high signal-to-noise ratio (dV1/dVz) temperature-stable memory core.     

Measurement of internal stresses in coatings using time resolved fluorescence

This paper describes a new nondestructive technique for detecting internal stresses in coatings using time resolved fluorescence. The measurement principle is based upon an experimental result that the decay time of fluorescence from poly(3-octylthiophene), P3OT, dispersed in uniaxially-stretched polymer films decreases with increasing tensile stress acting on the films. Internal stresses in the clear coat and in the base coat of a multilayer structure, which was composed of electrodeposited coat, surface coat, base coat, and clear coat, were estimated from the decay time of fluorescence from P3OT in both coats. The order of internal stresses in the clear coat and base coat of the multilayer system was 1 MPa. When the coatings were piled up, the internal stress decreased as the distance from a metal substrate was increased. It was found that moisture and temperature influenced internal stresses in the clear coat rather than in the base coat. Internal stress in the clear coat, which was one layer coated on a metal substrate, was measured by the traditional bimetallic method or by the time resolved fluorescence technique. Comparing both methods, it was concluded that the time resolved fluorescence technique gave reliable values for internal stresses in coatings.     

Adsorption properties of oxygen and methane on Ga-ZSM-5; the origin of the selectivity of NOx reduction using methane

The adsorption properties of oxygen and methane on Ga-ZSM-5 and Cu-ZSM-5 catalysts were examined by a TPD method to clarify the extraordinary selectivity of HC-SCR using methane on Ga-ZSM-5. It was observed that Ga-ZSM-5 did not adsorb oxygen but adsorbed methane dissociatively, while on Cu-ZSM-5 oxygen was dissociatively adsorbed and reacted with adsorbed ethylene.     

Characterization of Silicon Carbide–Silicon Nitride Composite Ultrafine Particles Synthesized Using a CO2 Laser by Silicon-29 Magic Angle Spinning NMR and ESR

The structure of silicon carbide–silicon nitride (SiC–Si₃N₄) composite particles synthesized using a CO₂ laser was studied by magic angle spinning nuclear magnetic resonance (MAS-NMR) and electron spin resonance (ESR). The structure around Si atoms changed by introducing N. C atoms around Si were substituted by N atoms, and N-rich configurations around Si atoms increased stepwise as the N content increased. The low N content composite particles consisted of mainly SiC phase containing dissolved N. N atoms were partly present in β-SiC microcrystal and partly in the grain boundary layer in the particle. N atoms were tetrahedrally surrounded by four Si atoms in β-SiC microcrystal and were trivalent state bonded to three Si atoms in the grain boundary layer. The high N content particles consisted of SiC, Si₃N₄, and amorphous phases, whose amount depended on N content.     

Effect of Silicon Dioxide on the Thermal Diffusivity of Aluminum Nitride Ceramics

The thermal diffusivity of AlN ceramics was significantly decreased by the addition of SiO₂. The AlN ceramics with 4 wt% SiO₂ could not be densified by pressureless sintering in the temperature range 1400° to 1800°C. The thermal diffusivity of these samples was very low because of their porous structure. The AlN ceramics containing 2, 4, and 8 wt% SiO₂ were densified by hot-pressing and also had low thermal diffusivity. In these samples, the grains of the 27R polytype that resulted from the reaction between AlN and SiO₂ were dispersed, obstructing the conduction of heat. The relation between the amount of 27R polytype and the thermal diffusivity of the AlN ceramics was determined.     

Reverse osmosis separation of hydrocarbons in aqueous solutions using porous cellulose acetate membranes

A physicochemical parameter, represented by the symbol Σs^*, based on molar solubility in water and molar attraction constants of Small, has been developed to express quantitatively the relative hydrophobicity, or nonpolar character, of the hydrocarbon molecule. The value of Σs^* can be calculated for a hydrocarbon from its chemical structure. The scale of Σs^* is consistent within each group of aromatic, cyclic, and noncyclic hydrocarbons. Reverse osmosis data have been obtained at 250 psig for single-solute aqueous feed solution systems involving low concentrations of 39 different hydrocarbons (including 13 aromatics, 10 cyclic, and 16 noncyclic compounds) and several samples of cellulose acetate membranes of different surface porosities. The effect of operating pressure on membrane performance has also been studied for two aromatic hydrocarbon solutes. The values of Σs^* for the solutes used were in the range of 425 to 924 for aromatic hydrocarbons, 521 to 931 for cyclic hydrocarbons, and 369 to 960 for noncyclic hydrocarbons. The reverse osmosis data have been correlated with Σs^* for each group of hydrocarbons studied. In all cases, positive solute separations were obtained, and the ratio [PR]/[PWP] was less than 1. With respect to each film, solute separation increased with increase in Σs^*, and decreased with increase in operating pressure. Also, solute separation decreased in the order aromatic hydrocarbon > cyclic hydrocarbon > noncyclic hydrocarbon at any given value of Σs^*. At a given operating pressure, for low values of Σs^* (～500 or less) solute separation increased with progressive decrease in average pore size on the membrane surface. For high values of Σs^* (～800 or more), solute separation initially increased with decrease in average pore size, then passed through a maximum and minimum with further decrease in average pore size, and again increased with still further decrease in average pore size. The results are discussed on the basis of preferential sorption of solute at the membrane–solution interface under the experimental conditions studied.     

Reverse osmosis separations of polyethylene glycols in dilute aqueous solutions using porous cellulose acetate membranes

Reverse osmosis separations of eight polyethylene glycol (PEG) solutes in the average molecular weight range of 200 to 6750 in single-solute dilute aqueous solutions have been studied using porous cellulose acetate membranes at the operating pressures of 50, 75, and 100 psig. Diffusivity data for the above PEG solutes have also been obtained from experimental data on intrinsic viscosities. From an analysis of all experimental data, numerical values for the parameters representing the polar (?ΔΔG/RT), steric (δ^*ΣE_s), and nonpolar (ω^*Σs^*) forces governing reverse osmosis separations of PEG solutes have been generated. These numerical values are useful for precise characterization of cellulose acetate membranes for whose specifications sodium chloride is not the appropriate reference solute because of its low or practically negligible separation under reverse osmosis operating conditions. This work also illustrates that solute separation in reverse osmosis can predictably increase or decrease with increase in operating pressure depending on experimental conditions.     

Prolonged degradation of end-capped polyelectrolyte multilayer films

Cationic chitosan (CT) and anionic dextran sulfate (Dex) were layer-by-layer (LbL) assembled from aqueous solutions containing 1 M NaCl on a quartz crystal microbalance (QCM) substrate, and the original films ((CT-Dex)₃-CT)) were end-capped with LbL assembly from CT solutions containing 1 M NaCl and Dex solutions without NaCl. The enzymatic degradation of films by chitosanase was quantitatively analyzed by QCM in terms of numbers of end-capping steps. The degradation of films end-capped with (Dex-CT)₃ was considerably prolonged when compared to those end-capped with other end-capping steps. A mechanism for the prolonged degradation was proposed by quantitative QCM data and zeta potential results.