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Understanding the effect of uncertainties of Core Disruptive Accident (CDA) scenarios on debris bed coolability on a core catcher is required for decision making on design options to mitigate a CDA consequence. For the understanding, a huge number of calculations are required but are extremely difficult to perform because a huge number of calculations require much calculation time to solve non-steady equations in the coolability calculation model. Thus, we applied Artificial Neural Network (ANN), which is one of models for machine learning, to debris bed coolability calculations. The application of ANN is expected to exponentially improve the calculation speed of debris bed coolability because ANN provides results from experimental rules learned through training without solving non-steady equations. The application is in three steps. Firstly, we created many data for training ANN and validating the trained ANN through coolability calculations parameterizing main dominant inputs (particle diameter of debris bed, porosity of debris bed, etc.) by using Latin hypercube sampling. Secondly, ANN was trained and validated with the created data. The accuracy rate of the results by the ANN to the validation data exceeded 99%. In addition, the calculation time using ANN was micro seconds order. Finally, through demonstration calculations, it was confirmed that we can easily understand the effect of uncertainties of CDA scenarios on debris bed coolability owing to results visualization based on a huge number of parametric calculations using ANN. Thus, the application of ANN to debris bed coolability calculations should contribute to the decision making on design options to mitigate a CDA consequence.

The published literature on the association of circulatory branched-chain amino acids (BCAAs) and aromatic amino acids (AAAs) with reduced kidney function is inconsistent or conflicting. Clarification of it might help to better understand the underlying pathophysiology and to determine potential biomarkers for early detection and evaluation of kidney function decline. Our main purpose was to explore and clarify the potential relationships of individual BCAAs and AAAs with estimated glomerular filtration rate (eGFR) decline. We included the data from 2804 healthy subjects and categorized them into three groups based on eGFR tertiles. The associations between individual amino acids and eGFR were explored by covariate-adjusted logistic regression models. There was a progressive increase in the concentrations of BCAAs and AAAs from the upper to the lower tertiles. We revealed significant positive associations of isoleucine, leucine, and phenylalanine with lower tertiles of eGFR in the adjusted models (p &lt, 0.01–0.001). The findings hold a promising potential of using plasma isoleucine, leucine, and phenylalanine levels for evaluation of kidney function decline. Future longitudinal studies should investigate the causal association between altered levels of these amino acids and impaired kidney function and also the utility of the former as potential biomarkers for evaluating the risk and early detection of the latter.

The N-terminal (1–83) fragment of the major constituent of plasma high-density lipoprotein, apolipoprotein A-I (apoA-I), strongly tends to form amyloid fibrils, leading to systemic amyloidosis. Here, using a series of deletion variants, we examined the roles of two major amyloidogenic segments (residues 14–22 and 50–58) in the aggregation and fibril formation of an amyloidogenic G26R variant of the apoA-I 1–83 fragment (apoA-I 1–83/G26R). Thioflavin T fluorescence assays and atomic force microscopy revealed that elimination of residues 14–22 completely inhibits fibril formation of apoA-I 1–83/G26R, whereas Δ32–40 and Δ50–58 variants formed fibrils with markedly reduced nucleation and fibril growth rates. CD measurements revealed structural transitions from random coil to β-sheet structures in all deletion variants except for the Δ14–22 variant, indicating that residues 14–22 are critical for the β-transition and fibril formation. Thermodynamic analysis of the kinetics of fibril formation by apoA-I 1–83/G26R indicated that both nucleation and fibril growth are enthalpically unfavorable, whereas entropically, nucleation is favorable, but fibril growth is unfavorable. Interestingly, the nucleation of the Δ50–58 variant was entropically unfavorable, indicating that residues 50–58 entropically promote the nucleation step in fibril formation of apoA-I 1–83/G26R. Moreover, a residue-level structural investigation of apoA-I 1–83/G26R fibrils with site-specific pyrene labeling indicated that the two amyloidogenic segments are in close proximity to form an amyloid core structure, whereas the N- and C-terminal tail regions are excluded from the amyloid core. These results provide critical insights into the aggregation mechanism and fibril structure of the amyloidogenic N-terminal fragment of apoA-I.

Circular dichroism (CD) is a technique used for conformational studies of peptides and proteins. We studied the specific calibration procedures of CD spectrometers based on procedures specified in the European Pharmacopoeia. We aimed to develop procedures to improve the usability of CD, in addition to reducing adverse effects on users' health. The use of ethanol instead of 1,4-dioxane as the solvent for isoandrosterone was examined. Both solvents yielded the same maximum value of +3.3 for molar CD. We also studied a two-point calibration method using (1S)-(+)-ammonium 10-camphorsulfonate instead of (1S)-(+)-10-camphorsulfonic acid, which is a hygroscopic compound. Both compounds yielded similar results and the values for (1S)-(+)-ammonium 10-camphorsulfonate of 2.39 ± 0.04 and -4.92 ± 0.06 at 290.5 and 192.5 nm, respectively, were within the criteria defined in the European Pharmacopoeia. The inter-laboratory repeatability was also acceptable. These studies provide specific procedures for calibrating CD spectrometers for drug development.

Amyloid fibrils represent a special type of protein aggregates that are currently receiving enormous attention due to their strong implication in molecular etiology of a wide range of human disorders. Amyloid fibrils represent highly ordered self-assemblies sharing a core cross-β-sheet structure. Such organization of the fibrils is responsible for amyloid insolubility and exceptional mechanical properties. The remarkable rigidity of the protein fibrillar aggregates is due to intra- and interstrand hydrogen bonds which stabilize the β-strand scaffold of amyloid fibrils. Increasing evidence indicates that physical properties of amyloid assemblies, especially their mechanical characteristics, play essential role in determining their cytotoxic action. This highlights the necessity of deciphering the correlation between the elastic properties of amyloid aggregates and their cytotoxicity. In the present paper we utilized the atomic force microscopy (AFM) to visualize and analyze the amyloid fibrils of G26R/W@8 mutant of N-terminal fragment of human apolipoprotein A-I (apoA-I). The examination of AFM images revealed the existence of two polymorphic forms of apoA-I fibrils – twisted ribbon and helical ribbon. The quantitative analysis of apoA-I elastic properties was performed within the framework of worm-like model of polymer chain using the Easyworm software. The Easyworm package analyzes the images of individual polymer chains obtained by the atomic force microscopy and allows calculation of the persistent length of a chain in three regimes depending on the ratio between the contour and persistent lengths of the polymer. The set of evaluated parameters included the Young’s modulus, persistent length, bending rigidity and the second moment of inertia. All parameters calculated for the helical ribbon conformation were higher than those of the twisted ribbon. These findings suggest that helical ribbon represents a more rigid and mechanically stable configuration. The results obtained may prove of importance for a deeper understanding the mechanics-driven pathological activities of amyloid fibrils.

The effects of one of the amyloidogenic mutations of apolipoprotein A-I (apoA-I), G26R, on the thermal stability, structural dynamics and lipid-associating properties of the 1-83 N-terminal fragment of apoA-I (A83) have been investigated using the Förster resonance energy transfer (FRET) and molecular dynamics (MD) simulation. The measurements of FRET between the tryptophan residues of the single Trp variants of A83 as donors and the membrane-incorporated fluorescent probe 4-dimethylaminochalcone as an acceptor provided evidence for a less depth of A83/G26R penetration into phosphatidylcholine (PC) bilayer compared to WT counterpart. The unfolding MD simulations showed that G26R mutation destabilizes the overall structure of A83, with individual alpha-helices differing in their thermal stability. The MD simulations performed at physiological temperature revealed that A83 and A83/G26R differ in their conformational behavior in an aqueous solution, PC and PC/Cholesterol bilayers. These findings may prove of importance for deeper understanding of the key determinants of apoA-I amyloidogenesis.

Arginine-rich cell penetrating peptides (CPPs) are very promising drug carriers to deliver membrane-impermeable pharmaceuticals, such as siRNA, bioactive peptides and proteins. CPPs directly penetrate into cells across cell membranes via a spontaneous energy-independent process, in which CPPs appear to interact with acidic lipids in the outer leaflet of the cell membrane. However, acidic lipids represent only 10 to 20% of the total membrane lipid content and in mammalian cell membranes they are predominantly located in the inner leaflet. Alternatively, CPPs favorably bind in a charge density- dependent manner to negatively charged, sulfated glycosaminoglycans (GAGs), such as heparan sulfate and chondroitin sulfate, which are abundant on the cell surface and are involved in many biological functions. We have recently demonstrated that the interaction of CPPs with sulfated GAGs plays a critical role in their direct cell membrane penetration: the favorable enthalpy contribution drives the high-affinity binding of arginine-rich CPPs to sulfated GAGs, initiating an efficient cell membrane penetration. The favorable enthalpy gain is presumably mainly derived from a unique property of the guanidino group of arginine residues forming multidentate hydrogen bonding with sulfate and carboxylate groups in GAGs. Such interactions can be accompanied with charge neutralization of arginine-rich CPPs, promoting their partition into cell membranes. This review summarizes the current understanding of the physicochemical mechanism for lipid membrane penetration of CPPs, and discusses the role of the GAG interactions on the cell membrane penetration of CPPs.

Here, we examined the effects of phosphatidylserine (PS) and cholesterol on the fibril-forming properties of the N-terminal 1‒83 fragment of an amyloidogenic G26R variant of apoA-I bound to small unilamellar vesicles. A thioflavin T fluorescence assay together with microscopic observations showed that PS significantly retards the nucleation step in fibril formation by apoA-I 1‒83/G26R, whereas cholesterol slightly enhances fibril formation. Circular dichroism analyses demonstrated that PS facilitates a structural transition from random coil to α-helix in apoA-I 1‒83/G26R with great stabilization of the α-helical structure upon lipid binding. Isothermal titration calorimetry measurements revealed that PS induces a marked increase in capacity for binding of apoA-I 1‒83/G26R to the membrane surface, perhaps due to electrostatic interactions of positively charged amino acids in apoA-I with PS. Such effects of PS to enhance lipid interactions and inhibit fibril formation of apoA-I were also observed for the amyloidogenic region-containing apoA-I 8‒33/G26R peptide. Fluorescence measurements using environment-sensitive probes indicated that PS induces a more solvent-exposed, membrane-bound conformation in the amyloidogenic region of apoA-I without affecting membrane fluidity. Since cell membranes have highly heterogeneous lipid compositions, our findings may provide a molecular basis for the preferential deposition of apoA-I amyloid fibrils in tissues and organs.

A model which predict the field life of electronic modules corroded by S8 gas in vehicle engine rooms is constructed. The in-vehicle module field life is predicted from the accelerated test results of module alone and the measured results of in-vehicle module field environment conditions. In the modules, a circuit substrate with Ag wiring is placed on a baseplate within a housing and sealed with silicone. The S8 gas is produced by the release of the free sulphur of rubber ducts in engine room at the maximum temperature holding time after engine stop. This S8 gas permeates the silicone seal and corrodes the Ag wiring. The ratio of the corroded part thickness to the initial thickness of the Ag wiring increases with the increase of the number of engine start/stop cycles. The calculated results coincide well with the observed results of the corrosion thickness in an actual vehicle.

The number of complaints regarding offensive odors from service industries, such as restaurants and garages, has recently increased. In this study, we aimed to develop an adsorbent for hydrogen sulfide gas derived from domestic wastewater and reveal the mechanisms of its removal. The adsorbent used for hydrogen sulfide gas removal was prepared by mixing coal fly ash and blast furnace cement with a mixing ratio of 87:13 by mass percentage. The optimum calcination temperature of the adsorbent was 700 °C to achieve a high removal performance for both dry and humid hydrogen sulfide gas. The X-ray absorption fine structure analysis revealed that hydrogen sulfide was removed on the adsorbent by oxidizing to sulfate. A pilot-scale experiment was conducted to evaluate the removal performance of hydrogen sulfide gas derived from domestic wastewater using the developed adsorbent. For a week, the average removal percentage of hydrogen sulfide gas derived from domestic wastewater remained at 99.1%. Therefore, the developed adsorbent for hydrogen sulfide gas is promising and cost-effective for promoting the recycling of coal fly ash. HIGHLIGHTS Adsorbent for H2S gas was prepared by mixing coal fly ash and blast furnace cement.; Adsorbent could remove both dry and humid H2S gas.; H2S was oxidized to sulfate and removed from the adsorbent.; Average removal percentage of H2S gas remained at 99.1%.; This study contributes to sustainable development goals in terms of coal fly ash recycling.

The effects of the oxidized phospholipids (oxPLs) on amyloid fibril formation by the apolipoprotein A-I variant 1-83/G26R have been investigated using Thioflavin T fluorescence assay. All types of the PoxnoPC assemblies (dispersions, micelles and lipid bilayer vesicles) induced retardation of amyloid nucleation and elongation and the enhancement of the 1-83/G26R fibrillization, although PazePC micelles completely prevented protein aggregation at low protein-to-lipid molar ratios. The ability of PazePC to inhibit 1-83/G26R aggregation was explained by the protein-lipid electrostatic interactions, which either stabilize the a-helical structure of the membrane-associated 1-83/G26R or facilitate the protein solubilization by the detergent micelles. (C) 2017 Elsevier B.V. All rights reserved.

Apolipoprotein A-I (apoA-I) undergoes a large conformational reorganization during remodeling of high-density lipoprotein (HDL) particles. To detect structural transition of apoA-I upon HDL formation, we developed novel monoclonal antibodies (mAbs). Splenocytes from BALB/c mice immunized with a recombinant human apoA-I, with or without conjugation with keyhole limpet hemocyanin, were fused with P3/NS1/1-Ag4-1 myeloma cells. After the HAT-selection and cloning, we established nine hybridoma clones secreting anti-apoA-I mAbs in which four mAbs recognize epitopes on the N-terminal half of apoA-I while the other five mAbs recognize the central region. ELISA and bio-layer interferometry measurements demonstrated that mAbs whose epitopes are within residues 1–43 or 44–65 obviously discriminate discoidal and spherical reconstituted HDL particles despite their great reactivities to lipid-free apoA-I and plasma HDL, suggesting the possibility of these mAbs to detect structural transition of apoA-I on HDL. Importantly, a helix-disrupting mutation of W50R into residues 44–65 restored the immunoreactivity of mAbs whose epitope being within residues 44–65 against reconstituted HDL particles, indicating that these mAbs specifically recognize the epitope region in a random coil state. These results encourage us to develop mAbs targeting epitopes in the N-terminal residues of apoA-I as useful probes for monitoring formation and remodeling of HDL particles.

The changes in inorganic and organic gases retained in shales and metapelites with increasing heating temperature are not fully understood. The compositional and isotopic changes of residual gases such as H-2, CH4, and CO2 in marine shales and metapelites during burial diagenesis and metamorphism were investigated in the present study. Shale rocks and metapelites which experienced paleo-temperatures in the range of 100-600 degrees C were collected from the borehole cores in Niigata sedimentary basin and outcrops exposed in Kochi district, Japan. Gases released from shale or metapelite fragments during pulverization in the laboratory were analyzed as the residual gas. The major residual gas in shales and metapelites changes from CO2, CH4, to H-2 in this order with increasing temperature. Drastic decrease of CO2 derived from decarboxylation of sedimentary organic matter is due to the expulsion of pore fluids with dissolved CO2. The CH4 concentration in residual gas subsequently increases with organic maturation and reaches the maximum at paleo-temperature of ca. 250 degrees C. The CH4 concentration decreases during late metagenesis to metamorphism probably due to the formation of H-2 gas and graphite. The H2 starts to increase at paleo-temperature of ca. 200 degrees C. The H-2 gas is the most abundant gas in residual gas of metapelite. The delta H-2 value of H-2 in shales and metapelites is quite low to be in the range of ca.-850 to-650%.. The change of major residual gas from CH4 to H-2 with increasing paleo-temperature and the significantly low delta H-2 values of H-2 suggest that H-2 gas in shales and metapelites is largely derived from liberation of hydrogen in organic matter. (C) 2017 Elsevier B.V. All rights reserved.

Membrane lipid composition is known to influence aggregation and fibril formation of many amyloidogenic proteins. Here, we found that phosphatidylethanolamine (PE) accelerates aggregation of the N-terminal 1-83 fragment of an amyloidogenic G26R variant of apoA-I on lipid membranes. Circular dichroism and isothermal titration calorimetry measurements demonstrated that PE does not affect the α-helical structure and lipid binding property of apoA-I 1-83/G26R. Rather, fluorescence measurements indicated that PE induces more ordered lipid packing at the interfacial and acyl chain regions, providing more hydrophobic environments especially around the highly amyloidogenic regions in apoA-I on the membrane surface. These results suggest that PE promotes aggregation of the amyloidogenic N-terminal fragment of apoA-I on lipid membranes by inducing hydrophobic membrane environments.

Human norovirus is one of the major causes of foodborne gastroenteritis, and it can be easily transmitted from infected person, virus-contaminated foods and environmental surfaces. Effective disinfection method is needed to stop the transmission of human norovirus. CAC-717 is a new disinfectant consisting of calcium hydrogen carbonate mesoscopic crystals. We aimed to evaluate the efficacy of CAC-717 against human norovirus. This study used human norovirus derived from fecal specimens and cultured murine norovirus, which is one of the surrogate viruses for human norovirus. The disinfection effect against murine norovirus was estimated by infectivity assay and transmission electron microscopy. The inactivation effect against human norovirus was assessed by reverse transcription polymerase chain reaction. Disinfection effect of CAC-717 against the infectivity of murine norovirus was shown within 100 s after the CAC-717 treatment, presenting the destruction of viral capsids. The treatment of CAC-717 significantly reduced human norovirus genomic RNA (3.25-log reduction) by the presence of the mesoscopic structure of calcium hydrogen carbonate. CAC-717 stably inactivated human norovirus in stool suspensions. The inactivation effect of CAC-717 against human norovirus was less susceptible to organic substances than sodium hypochlorite. CAC-717 would be a useful alternative for disinfecting human norovirus in contaminated environmental surfaces.

We investigated the association between the period of exposure and changes in urinary excretion of chromium and nickel among rotating shift workers in a stainless-steel plant. The study participants were composed of two groups: the workers who were occupationally exposed to metals ("exposed group") and those who were not occupationally exposed to metals ("unexposed group"). The exposed and unexposed groups consisted of 56 and 40 male rotating shift workers, respectively. Urine samples were collected immediately before and immediately after the day shift, evening shift, and night shift. Urinary chromium and nickel were measured using inductively coupled plasma mass spectrometry. To correct for variations in urine dilution, urinary metal concentrations were expressed as a ratio to urinary creatinine concentration. In the exposed group, post-shift urinary excretion of chromium was significantly higher than pre-shift excretion. However, although urinary chromium excretion clearly increased after the day and night shift [63% (p < .0001) and 87% (p < .0001), respectively], urinary chromium excretion after the evening shift was only slightly higher than that measured before the evening shift (8%, p = .028). Similar patterns were found for urinary nickel excretion (p = .0001, 0.20, and 0.18 for the day, evening, and night shifts, respectively). Non-uniform urinary excretion of metals between the day shift, evening shift, and night shift were observed in the exposed group; specifically, urinary metal excretion increased only slightly during the evening shift. In the unexposed group, no significant increase or decrease was found in median urinary chromium or nickel excretion (p= .63-0.87). Work shift-specific permissible exposure level would be necessary.

International audience; In the direct cell membrane penetration, arginine-rich cell-penetrating peptides are thought to penetrate into cells across the hydrophobic lipid membranes. To investigate the effect of the amphipathic property of argininerich peptide on the cell-penetrating ability, we designed a novel amphipathic cell-penetrating peptide, A2-17, and its derivative, A2-17KR, in which all lysine residues are substituted with arginine residues, based on the glycosaminoglycan binding region in the N-terminal α-helix bundle of human apolipoprotein E. Isothermal titration calorimetry showed that A2-17 variants have a strong ability to bind to heparin with high affinity. Circular dichroism and tryptophan fluorescence measurements demonstrated that A2-17 variants bind to lipid vesicles with a structural change from random coil to amphipathic α-helix, being inserted into the hydrophobic membrane interiors. Flow cytometric analysis and confocal laser scanning microscopy demonstrated the great cell penetration efficiency of A2-17 variants into CHO-K1 cells when incubated at low peptide concentrations (2 μM or less), suggesting that the increased amphipathicity with α-helix formation enhances the cell membrane penetration ability of arginine-rich peptides. Interestingly, A2-17KR exhibited lower efficiency of cell membrane penetration compared to A2-17 despite of their similar binding affinity to lipid membranes. Since high peptide concentrations (typically > 10 μM) are usually prerequisite for efficient cell penetration of arginine-rich peptides, A2-17 is a unique amphipathic cell-penetrating peptide that exhibits an efficient cell penetration ability even at low peptide concentrations.

The highly sulfated domains of heparan sulfate (HS), alias HS S-domains, are made up of repeated trisulfated disaccharide units [iduronic acid (2S)-glucosamine (NS, 6S)] and are selectively remodeled by extracellular endoglucosamine 6-sulfatases (Sulfs). Although HS S-domains are critical for signal transduction of several growth factors, their roles in amyloidoses are not yet fully understood. Herein, we found HS S-domains in the kidney of a patient with transthyretin amyloidosis. In in vitro assays with cells stably expressing human Sulfs, heparin, a structural analog of HS S-domains, promoted aggregation of transthyretin in an HS S-domain–dependent manner. Interactions of cells with transthyretin fibrils and cytotoxicity of these fibrils also depended on HS S-domains at the cell surface. Furthermore, glypican-5, encoded by the susceptibility gene for nephrotic syndrome GPC5, was found to be accumulated in the transthyretin amyloidosis kidney. Our study, thus, provides a novel insight into the pathologic roles of HS S-domains in amyloidoses, and we propose that enzymatic remodeling of HS chains by Sulfs may offer an effective approach to inhibiting formation and cytotoxicity of amyloid fibrils. 189;2

We develop a high-performance adsorbent for phosphate removal from water or wastewater by impregnating lanthanum (La) on a coal fly ash—blast furnace cement composite (La-FACC). The optimized impregnation percentage of La and calcination conditions of the La-FACC were 1% and 800 ℃ for 2 h, respectively. The adsorption kinetics of phosphate onto the La-FACC was well fit by the intra-particle diffusion model, indicating that film and intra-particle diffusion were the rate-controlling step in the adsorption process of phosphate onto the La-FACC. The pseudo second-order kinetic model could also describe the adsorption kinetics of phosphate. Hence, adsorption of phosphate onto the La-FACC occurred mainly via chemisorption. The Langmuir isotherm was appropriate for describing the phosphate adsorption behavior onto the La-FACC. The monolayer maximum adsorption capacity was 24.9 mg-P g−1. The La-FACC showed high adsorption capacity and selectivity for phosphate with a wide range of pH, and with high concentrations of coexisting ions attributed to both formation of inner sphere complexes and electrostatic interaction. Magnesium ion slightly inhibited the adsorption of phosphate. Hence, the La-FACC developed in this study is a promising adsorbent for water treatment with a wide pH range and high ion strength.

The mutants of the tumor suppressor protein p53 form protein aggregates. It has been proposed that these aggregates propagate like prions, albeit the detailed mechanism of the propagation is unclear. Our recent study revealed that sulfated glycosaminoglycans, especially highly sulfated domains of heparan sulfate (heparan sulfate S-domains), participate in cancer pathology by mediating transcellular propagation of p53 aggregates. 8;2

The effect of carrier morphology on the intestinal absorption of insulin was investigated using a morphology-tunable polymeric carrier, β-cyclodextrin-grafted chitosan (BCC). The insulin-BCC complexes were prepared in either acetate or citrate buffer solutions, followed by dilution with phosphate buffer for the administration. The complex had a molecular network structure in the acetate buffer, whereas nanoparticles formed in the citrate buffer. The network structure in the acetate buffer was maintained even after dilution with a phosphate buffer, but the nanoparticles in the citrate buffer caused aggregation after dilution. Both complexes enhanced the intestinal absorption of insulin. Interestingly, their absorption profiles were totally different; prompt absorption was observed for the complex prepared in acetate buffer, whereas sustained absorption was observed for the complex prepared in citrate buffer. The difference in the absorption patterns was attributed to the difference in the complex morphology. Next, penetratin, a cell-penetrating peptide, was grafted to BCC to find further improvement in the absorption behavior. A simple mixture of penetratin and BCC was also effective. An oral administration study was also conducted in mice to observe effective suppression of glucose levels, which was further enhanced by coadministration of penetratin. Thus, BCC was proven to be an effective carrier for enhancing oral absorption of peptide drugs, and it is suggested that the carrier morphology is also an important factor that influences the absorption profile.

Glycosaminoglycans are known to be associated with extracellular amyloid deposits of various amyloidogenic proteins. In this study, we found that the glycosaminoglycan heparin greatly accelerates the elongation step in fibril formation by the N-terminal 1-83 fragment of human apolipoprotein A-I (apoA-I), especially in the amyloidogenic W50R variant. Using fragment peptides, we demonstrate that heparin significantly promotes β-transition and fibril formation of the highly amyloidogenic region spanning residues 44-65 and co-localizes with fibrils formed by the W50R variant. These results suggest the possible role of glycosaminoglycans in fibril formation by amyloidogenic apoA-I variants. This article is protected by copyright. All rights reserved.

An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers. Here, we examined fET of cells stably expressing luciferase in the presence of iRed encoding anti-luciferase shRNA. Transfection of lipofectamine 2000 (LFN)/iRed lipoplexes showed an RNAi effect, but fET-mediated iRed transfection did not, likely because of the endosomal localization of iRed after delivery. However, fET in the presence of lysosomotropic agent chloroquine significantly improved the RNAi effect of iRed/fET to levels that were higher than those for the LFN/iRed lipoplexes. Furthermore, the amount of lipid droplets in adipocytes significantly decreased following fET with iRed against resistin in the presence of chloroquine. Thus, iRed could be a useful tool to regulate target genes following fET-mediated cytoplasmic delivery with endosomal escape devices.

The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides.

Nanodiscs are composed of scaffold protein or peptide such as apolipoprotein A-I (apoA-I) and phospholipids. Although peptide-based nanodiscs have an advantage to modulate the size of nanodiscs by changing phospholipid/peptide ratios, they are usually less stable than apoA-I-based nanodiscs. In this study, we designed a novel nanodisc scaffold peptide (NSP) that has proline-punctuated bihelical amphipathic structure based on apoA-I mimetic peptides. NSP formed α-helical structure on 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) nanodiscs prepared by cholate dialysis method. Dynamic light scattering measurements demonstrated that diameters of NSP nanodiscs vary depending upon POPC/NSP ratios. Comparison of thermal unfolding of nanodiscs monitored by circular dichroism measurements demonstrated that NSP forms much more stable nanodiscs with POPC than monohelical peptide, 4F, exhibiting comparable stability to apoA-I-POPC nanodiscs. Intrinsic Trp fluorescence measurements showed that Trp residues of NSP exhibit more hydrophobic environment than that of 4 F on nanodiscs, suggesting the stronger interaction of NSP with phospholipids. Thus, the bihelical structure of NSP appears to increase the stability of nanodiscs because of the enhanced interaction of peptides with phospholipids. In addition, NSP as well as 4F spontaneously solubilized POPC vesicles into nanodiscs without using detergent. These results indicate that bihelical NSP forms nanodiscs with comparable stability to apoA-I and has an ability to control the size of nanodiscs simply by changing phospholipid/peptide ratios.

Findings of the available studies regarding the roles of branched-chain amino acids (BCAAs) and aromatic amino acids (AAAs) in hypertension are inconsistent, conflicting and inconclusive. The purpose of this study was to explore and clarify the existence of any relationships of individual BCAAs and AAAs with hypertension with adjustments for potential relevant confounders. A total of 2805 healthy controls and 2736 hypertensive patients were included in the current analysis. The associations between individual amino acids and hypertension were explored by logistic regression analyses adjusted for potential confounding variables. Among the investigated amino acids, only the BCAAs showed consistently significant positive associations with hypertension in the adjusted models (p-trend &lt, 0.05 to 0.001). However, compared with the corresponding lowest quartile of individual BCAAs, the positive association with hypertension remained significant only in the highest quartile (p &lt, 0.01 to 0.001). We confirmed in a relatively large cohort of subjects that BCAAs, not AAAs, demonstrated consistent positive associations with hypertension. The results display the promising potential for the use of BCAAs as relevant and accessible biomarkers, and provide perspectives on interventions directed towards the reduction in plasma BCAA levels in the prevention and management of hypertension.

In Japan, conduits buried underground protect telecommunication cables, and it is desirable to continue to use these conduits as much as possible. Hence, it is important to grasp the deterioration in considering the use limit. There are several kinds of conduits, and carbon steel conduits are mainly utilized especially. Though the carbon steel conduits outer surface contacting with the soil is coated with asphalt jute or polyethylene, the inner surface is protected only with synthetic resin paint. Since the conduit inner surface is connected to the manhole, it may be filled with stagnant water such as groundwater or rainwater. Therefore, it is necessary to predict the long-term corrosion rate of carbon steel conduits in fresh water. We carried out a test to measure the average corrosion depth converted from the weight loss by immersing test pieces made from a carbon steel conduit in distilled water at 60 °C and in a manhole stagnant water as a sample. The test pieces were obtained by cutting the steel pipe into 50 mm, removing the coating film on the inner and outer surfaces, shaping it to a thickness of 3 mm, and applying a urethane coating film on the outer surface so that bare steel was exposed only on the inner surface. This test continued for about 1500 days, and test pieces were brought out every 45 days for the specimens immersed in distilled water and every 90 days for the specimens immersed in manhole. The corrosion products were removed using 10% ammonium dihydrogen citrate heated to 80 °C, and weight loss of each test piece was measured. Using this result, the applicability of the model equation proposed by Ozawa et al. in 2017 was considered. Ozawa et al. developed a model to predict the corrosion depth of carbon steel pipes in neutral static water based on temperature, salinity, and Larson Skold Index (LSI). The model assumes that the corrosion depth with time is determined by the initial corrosion rate, time and film resistance coefficient. Salinity and temperature determine the initial corrosion rate, and LSI and temperature determine the film resistance coefficient. In order to apply this model, the pH, chloride ion and sulfate ion of manhole stagnant water were measured by ion chromatography. The pH was 7.8, Cl- was 2.9 mg/l, and SO42- was 5.3 mg/l. The value of hydrogen carbonate ion has not been measured yet, and 207.5 mg/l, which is the average value measured in 1960 in Tokyo 45 manholes, is used. At this time, the film resistance coefficient becomes 2909.3. The temperature and salinity of the manhole reservoir water were 20 ° C and 0.05 ‰, respectively. The model was applied to distilled water with salinity of 0.01 ‰ and LSI with 1. The results of observed corrosion depth is shown on the vertical axis, and the results are shown in the log diagram with the predicted values obtained from the model equation of Ozawa et al. on the horizontal axis. The corrosion depth on day 45 after immersion in distilled water and the corrosion amount on day 90 after immersion in manhole reservoir water agree with the measured value predicted from the model equation, but the observed corrosion depth tends to be larger than the value derived from the model equation after that. The original model equation showed good agreement when compared with the test results at approximately 500 hours. It may be considered that this model equation is applicable in 45 days (1080 hours), but it is highly possible that the corrosion depth becomes larger than the model equation in the long term. In addition, the corrosion rate of the specimen immersed in the manhole deviates more from the model equation. Figure shows a photograph in which corrosion products were observed with an electron microscope on the test specimen as of 1321 days, and it can be seen that cracks of several μm in width were generated just above the base metal and at 150 μm to 300 μm positions, respectively. There is a possibility that the corrosion rate becomes faster than expected due to the influence of the cracks. In addition, though the stagnant water is presumed as still water in the usual time, the flow with the inflow of the water may be generated depending on the season. Considering these factors, it is possible to improve the long-term prediction accuracy of carbon steel conduits. Figure 1

The left-handed, extended polyproline II (PPII) helix is a unique secondary structure which potently modulates peptide/protein functions through its constraint conformation. To investigate the effect of PPII helix on the direct cell membrane penetration of arginine-rich peptides, we designed a polyproline-containing arginine-rich peptide P9R7W (PPPPPPPPPRRRRRRRW) by introducing nine proline residues into a linear R7W (RRRRRRRW) peptide. Circular dichroism spectroscopy showed that P9R7W has the PPII helix structure in solution whereas R7W is predominantly in random coil structure. Tryptophan fluorescence measurements demonstrated that P9R7W binds to negatively charged lipid vesicles with similar affinity to R7W, in which there was no significant change in the PPII helix structure. Flow cytometry and confocal laser scanning microscopy analyses showed that P9R7W has an ability to penetrate into CHO-K1 cells more efficiently compared to R7W with no cytotoxicity. Consistently, a channel current analysis unveiled that P9R7W penetrates planar lipid bilayer membranes more efficiently than R7W without significant membrane perturbation. Our results indicate that the PPII helix structure can enhance the membrane penetration efficiency of arginine-rich peptides without lipid membrane perturbation.

The single amino acid mutation G26R in human apolipoprotein A-I (apoA-I) is associated with familial amyloid polyneuropathy III. ApoA-I carrying this mutation (apoA-IIowa) forms amyloid fibrils in vitro. Heparan sulfate (HS) is a glycosaminoglycan that is abundant at the cell surface and in the extracellular matrix. Although HS and its highly sulfated domains are involved in aggregation of amyloid-β and accumulate in cerebral amyloid plaques of patients with Alzheimer disease and mouse models of this disease, the role of HS in familial amyloid polyneuropathy III has never been addressed. Here, we used cell models to investigate the possible role of HS in the cytotoxicity of apoA-IIowa amyloid. Wild-type CHO cells, but not pgsD-677 cells, an HS-deficient CHO mutant, demonstrated uptake of apoA-IIowa amyloid after incubation with the amyloid. Addition of sulfated glycosaminoglycans to culture media prevented interaction with and cytotoxicity of apoA-IIowa amyloid to CHO cells. Elimination of cell surface HS or inhibition of HS sulfation with chemical reagents interfered with interaction of apoA-IIowa amyloid with CHO cells. We also found that cellular interaction and cytotoxicity of apoA-IIowa amyloid were significantly attenuated in CHO cells that stably expressed the human extracellular endoglucosamine 6-sulfatases HSulf-1 and HSulf-2. Our results thus suggest that cell surface HS mediates cytotoxicity of apoA-IIowa amyloid and that enzymatic remodeling of HS mitigates the cytotoxicity.

The N-terminal amino acid 1-83 fragment of apolipoprotein A-I (apoA-I) has a strong propensity to form amyloid fibrils at physiological neutral pH. Because apoA-I has an ability to bind to lipid membranes, we examined the effects of the lipid environment on fibril-forming properties of the N-terminal fragment of apoA-I variants. Thioflavin T fluorescence assay as well as fluorescence and transmission microscopies revealed that upon lipid binding, fibril formation by apoA-I 1-83 is strongly inhibited, whereas the G26R mutant still retains the ability to form fibrils. Such distinct effects of lipid binding on fibril formation were also observed for the amyloidogenic prone region-containing peptides, apoA-I 8-33 and 8-33/G26R. This amyloidogenic region shifts from random coil to α-helical structure upon lipid binding. The G26R mutation appears to prevent this helix transition because lower helical propensity and more solvent-exposed conformation of the G26R variant upon lipid binding were observed in the apoA-I 1-83 fragment and 8-33 peptide. With a partially α-helical conformation induced by the presence of 2,2,2-trifluoroethanol, fibril formation by apoA-I 1-83 was strongly inhibited, whereas the G26R variant can form amyloid fibrils. These findings suggest a new possible pathway for amyloid fibril formation by the N-terminal fragment of apoA-I variants: the amyloidogenic mutations partially destabilize the α-helical structure formed upon association with lipid membranes, resulting in physiologically relevant conformations that allow fibril formation.

Excess of cholesterol in peripheral cells is known to lead to atherosclerosis. In this study, a molecular complex composed of β-cyclodextrin-grafted chitosan (BCC) and cellular cholesterol efflux enhancing peptide (CEEP), synthesized by modifying pH sensitive amphipathic GALA peptide, is introduced with the eventual aim of treating atherosclerosis. BCC has a markedly enhanced ability to induce cholesterol efflux from cell membranes compared to β-cyclodextrin, and the BCC-CEEP complex exhibited a 2-fold increase in cellular cholesterol efflux compared to BCC alone under weakly acidic conditions. Isothermal titration calorimetry and fluorescence spectroscopy measurements demonstrated that the random coil structure of CEEP at neutral pH converted to the α-helical structure at acidic pH, resulting in a three-order larger binding constant to BCC (K = 3.7 × 10(7) at pH 5.5) compared to that at pH 7.4 (K = 7.9 × 10(4)). Such high-affinity binding of CEEP to BCC at acidic pH leads to the formation of 100-nm-sized aggregate with positive surface charge, which would efficiently interact with cell membranes and induce cholesterol efflux. Since the cholesterol efflux ability of HDL is thought to be impaired under acidic environments in advanced atherosclerotic lesions, the BCC-CEEP complex might serve as a novel nanomaterial for treating atherosclerosis.

Formaldehyde is widely used to improve textile quality, but it is categorized as a potential carcinogen in humans. The concentration of formaldehyde emitted from textile products therefore has implications for public health. The standard method uses acetylacetone for determining formaldehyde extracted from textile products, but requires a water solution and the use of an analyzer. In this study, we present a method for screening textile-emitted formaldehyde via a porous glass sensor that we previously developed. Our method is suitable for on-site screening, whereas the commonly used method is not. The glass sensor was used to measure formaldehyde concentrations emitted from several kinds of textile products. The results showed good agreement (coefficient 0.92) with those of the acetylacetone method. Moreover, we estimate the effects of temperature, and suggest a formula to calculate concentration including temperature correction.

The binding of monomeric and aggregated variants of 1-83 N-terminal fragment of apolipoprotein A-I with substitution mutations G26R, G26R/W@8, G26R/W@50 and G26R/W@72 to the model lipid membranes composed of phosphatidylcholine and its mixture with cholesterol has been investigated using fluorescent probes pyrene and Laurdan. Examination of pyrene spectral behavior did not reveal any marked influence of apoA-I mutants on the hydrocarbon region of lipid bilayer. In contrast, probing the membrane effects by Laurdan revealed decrease in the probe generalized polarization in the presence of aggregated proteins. suggesting that oligomeric and fibrillar apoA-I species induce increase in hydration degree and reduction of lipid packing density in the membrane interfacial region. These findings may shed light on molecular details of amyloid cytotoxicity.

Apolipoprotein A-I (apoA-I) is an amyloid-forming protein whose amyloidogenic properties are attributed mainly to its N-terminal fragment. Cell membranes are thought to be the primary target for the toxic amyloid aggregates. In the present study Förster resonance energy transfer (FRET) between the membrane fluorescent probe Laurdan as a donor and amyloid-specific dye Thioflavin T (ThT) as an acceptor was employed to explore the interactions of amyloid fibrils from apoA-I variants 1-83/G26R and 1-83/G26R/W@8 with the model membranes composed of phosphatidylcholine and its mixture with cholesterol. The changes in FRET efficiency upon fibril-lipid binding were found to correlate with the extent of protein fibrillization. AFM imaging revealed the presence of two polymorphic states of fibrillar 1-83/G26R/W@8 with the helical and twisted ribbon morphologies. The simulation-based analysis of the experimental FRET profiles provided the arguments in favor of untwisting of fibrillar assemblies upon their interaction with the model membranes. Evidence for the face-on orientation and superficial bilayer location of the membrane-bound fragments of 1-83/G26R/W@8 fibrils was obtained.

As an alternative method to slow strain rate technique (SSRT) under high-pressure hydrogen gas evaluation, SSRT was performed with a cathodically charged specimen. Cr-Mo low alloy steel with a tensile strength of 1000 MPa grade was selected as a test material. Cathodic charging was performed in 3% NaCl solution and at a current density in the range of 50–600 A/m2. The effect of specimen size on the hydrogen embrittlement properties was evaluated. Relative reduction of area (RRA) values obtained by tests at a cathode current density of 400 A/m2 were equivalent to those performed in hydrogen gas at pressures of 10 to 35 MPa. Fracture surface observations were also performed using scanning electron microscopy (SEM). The quasi-cleavage fracture surface was observed only after rupture of small specimens that were subjected to hydrogen charged tests. It was also necessary for the diameter of the specimen to be small to form the quasi-cleavage fracture surface. The results indicated that to simulate the high-pressure hydrogen gas test, a specimen with a smaller parallel section diameter that is continuously charged until rupture is preferable.

Fluorescence represents one of the most powerful tools for the detection and structural characterization of the pathogenic protein aggregates, amyloid fibrils. The traditional approaches to the identification and quantification of amyloid fibrils are based on monitoring the fluorescence changes of the benzothiazole dye thioflavin T (ThT) and absorbance changes of the azo dye Congo red (CR). In routine screening it is usually sufficient to perform only the ThT and CR assays, but both of them, when used separately, could give false results. Moreover, fibrillization kinetics can be measured only by ThT fluorescence, while the characteristic absorption spectra and birefringence of CR represent more rigid criteria for the presence of amyloid fibrils. Therefore, it seemed reasonable to use both these dyes simultaneously, combining the advantages of each technique. To this end, we undertook a detailed analysis of the fluorescence spectral behavior of these unique amyloid tracers upon their binding to amyloid fibrils from lysozyme, insulin and an N-terminal fragment of apolipoprotein A-I with Iowa mutation. The fluorescence measurements revealed several criteria for distinguishing between fibrillar and monomeric protein states: (i) a common drastic increase in ThT fluorescence intensity; (ii) a sharp decrease in ThT fluorescence upon addition of CR; (iii) an appearance of the maximum at 535-540 nm in the CR excitation spectra; (iv) increase in CR fluorescence intensity at 610 nm. Based on these findings we designed a novel combined ThT-CR fluorescence assay for amyloid identification. Such an approach not only strengthens the reliability of the ThT assay, but also provides new opportunities for structural characterization of amyloid fibrils.

ABCA1 mediates the efflux of cholesterol and phospholipids into apoA-I to form HDL, which is important in the prevention of atherosclerosis. To develop a novel method for the evaluation of HDL formation, we prepared an apoA-I-POLARIC by labeling the specific residue of an apoA-I variant with a hydrophobicity-sensitive fluorescence probe that detects the environmental change around apoA-I during HDL formation. apoA-I-POLARIC possesses the intact ABCA1-dependent HDL formation activity and shows 4.0-fold higher fluorescence intensity in HDL particles than in the lipid-free state. Incubation of apoA-I-POLARIC with ABCA1-expressing cells, but not ABCA1-non-expressing cells, caused a 1.7-fold increase in fluorescence intensity. Gel filtration analysis demonstrated that the increase in fluorescence intensity of apoA-I-POLARIC represents the amount of apoA-I incorporated into the discoidal HDL particles rather than the amount of secreted cholesterol. THP-1 macrophage-mediated HDL formation and inhibition of HDL formation by cyclosporine A could also be measured using apoA-I-POLARIC. Furthermore, HDL formation-independent lipid release induced by microparticle formation or cell death was not detected by apoA-I-POLARIC. These results demonstrate that HDL formation by ABCA1-expressing cells can be specifically detected by sensing hydrophobicity change in apoA-I, thus providing a novel method for assessing HDL formation and screening of the HDL formation modulator.

For the samples taken from the void region of the CZ silicon crystal grown with the same solidification condition and different thermal histories after the solidification, we measure the magnitude S of the elastic softening which is proportional to the concentration of the single vacancies [V]. For these samples, we also measure the size distribution of the void density by using the infrared light-scattering tomography, to evaluate the concentration [Vc] of the vacancies consumed for the void formation. From these two experiments, we find a sum rule [Vc] + a S= C, where C depends only on the solidification condition and is independent of the thermal history after the solidification. This enables us to find the conservation rule of the vacancies [Vc] + [V] = C. The value of the proportionality constant a in the relation [V] = a S is determined. Demonstration of determining the absolute values of [V] from the measured S is given. An estimate is made for the value of the quadrupole-strain coupling constant.

Gases generated by laboratory anhydrous closed-system pyrolysis of hydrogen-rich Eocene Yubari subbituminous coal were distinguished as expelled gas, desorbed gas, residual free gas, and residual adsorbed gas, and the compositional and isotopic changes in these gases during laboratory maturation were investigated. The relative abundances of methane and H-2 in the expelled gas were higher than those in the residual free and adsorbed gases, showing compositional fractionation during gas expulsion from the coal fragments. The delta C-13 and delta H-2 values of ethane, propane, and CO2 in all the gas fractions were nearly the same. However, the delta C-13 and delta H-2 values of methane in the residual free gas were significantly higher than those of methane in the expelled and desorbed gases, demonstrating a large isotopic fractionation associated with the expulsion of methane from the coal fragments. The carbon and hydrogen isotopic fractionations of ethane, propane, and CO2 during their expulsion were almost negligible. The evolution paths of the differential delta C-13 values of hydrocarbon gases generated from hydrogen-rich Eocene Yubari coal during laboratory maturation were rather close to those of hydrocarbon gases from Type II kerogen in the early stage of maturation (VRr% < 1.2), but these shifted closer to those of hydrocarbon gases from Type III kerogen in the higher maturation stage (VRr% > 1.6). The similar delta C-13 values of hydrocarbon gases from Eocene Yubari coal and Type II kerogen are partly due to the abundant and C-13 depleted aliphatic structures in the Eocene Yubari coal. The delta H-2 of methane in the expelled gas from Yubari coal also increased systematically with laboratory maturation, although it can be influenced by various factors. Estimation of the maturity level and type of source organic matter of thermogenic methane based on isotopic composition requires caution, especially in the Cenozoic sedimentary basins.

Muscarinic M2, M4, and M2-M4 chimera receptors were transiently expressed in HEK-293 tsA201 cells, and agonist-dependent internalization of these receptors and recycling of internalized receptors were examined by measuring the amount of cell-surface receptors as [3H]N-methylscopolamine (NMS) binding activity. Coexpression of a dominant negative form of dynamin (DN-dynamin,dynamin K44A) greatly reduced the agonist-dependent internalization of M4 receptors but not of M2 receptors, as was reported by Vögler et al. (J Biol Chem 273, 12155-12160, 1998).The agonist-dependent internalization of M2/M4-i3/M2 chimera receptors (M2 receptors with the i3 loop replaced by that of M4 receptors) was greatly reduced by co-expression of DN-dynamin as was the case for M4 receptors, whereas the agonist-dependent internalization of M4/M2-i3/M4 chimera receptors was hardly affected by co-expression of DN-dynamin as was the case for M2 receptors.Internalized M2/M4-i3/M2 receptors as well as internalized M4 receptors were shown to be recycled back to the cell surface after removal of agonists, whereas no recycling was observed for M4/M2-i3/M4 receptors as well as M2 receptors. These results indicate that the i3 loops of M2 and M4 receptors take a major role in their agonist-dependent internalization and recycling.

Amyloid fibrils, a special class of protein aggregates with a core β-sheet structure, are currently associated with a number of human disorders. The fluorescence-based techniques play essential role in detection and structural characterization of amyloid assemblies. The amyloid-sensing potential of the two-step Forster resonance energy transfer has been assessed using the three-chromophore system containing a benzothiazole dye Thioflavin T, a phosphonium dye TDV and a squaraine dye SQ1. The WT and amyloidogenic variants of the N-terminal fragment of apolipoprotein A-I with varying degrees of fibrillization have served as a scaffold for the cascade energy transfer ThT → TDV → SQ1. The FRET efficiencies in the donor-acceptor pairs ThT-TDV and TDV-SQ1 have been found to correlate with the extent of amyloid formation. It has been demonstrated that the two-step FRET format has the advantages relative to the conventional one-step FRET, among which are the large Stokes shift enabling the acquisition of the well-resolved 3D fluorescence patterns and a higher amyloid detection sensitivity.