Observation of water droplets in microporous layers for polymer electrolyte fuel cells by X-ray computed nano-tomography.

An X-ray computed nano-tomography (nano-CT) system has been established at the BL33XU beamline of SPring-8. The optical system consists of pseudo-Köhler illumination with a sector condenser zone plate, an apodization Fresnel zone plate as the objective lens, and a Zernike phase plate. The imaging detector is a fiber-coupling type X-ray camera. The performance of the X-ray nano-CT system was confirmed by imaging an X-ray test chart. The system was subsequently applied to the observation of a microporous layer for polymer electrolyte fuel cells and a simulated microporous layer including liquid water. The nano-CT system, which can perform a computed tomography measurement in less than 4 min, allowed visualization of a spherical water droplet produced in the microporous layer. In the present study, the shape of water droplets in a nanoscale porous structure is investigated.

Effective physical therapy activities to improve the supine-to-seated transfer time in stroke patients: an observational pilot study.

[Purpose] This study aimed to examine the effective time allocation for physical therapy activities in patients with stroke. The primary outcome measure was the improvement in the time required to transition from the supine to the sitting position. [Participants and Methods] This study enrolled 19 inpatients with stroke. The activities performed during physical therapy were classified as nontherapeutic activities, minimal therapeutic activities, moderate therapeutic activities, high therapeutic activities, and other activities. We determined the relationship between the activities and the relative shortening ratio of the time required to sit up from the supine position for up to 13 weeks of physical therapy. We also considered the following background factors: patient information, functional independence measure, and Brunnstrom recovery stage. [Results] The Brunnstrom recovery stage for the lower extremity was identified as the confounding factor, and the participants were stratified into the Brunnstrom recovery stage 6 group, in which moderate therapeutic activities and other activities were significantly related to the relative shortening ratio. [Conclusion] The results suggested that other activities exerted a similar effect as moderate therapeutic activities in the Brunnstrom recovery stage 6 group and were more effective than high therapeutic activities in reducing the time required to sit up from the supine position.

Raman Study on Lipid Droplets in Hepatic Cells Co-Cultured with Fatty Acids.

The purpose of the present study was to investigate molecular compositions of lipid droplets changing in live hepatic cells stimulated with major fatty acids in the human body, i.e., palmitic, stearic, oleic, and linoleic acids. HepG2 cells were used as the model hepatic cells. Morphological changes of lipid droplets were observed by optical microscopy and transmission electron microscopy (TEM) during co-cultivation with fatty acids up to 5 days. The compositional changes in the fatty chains included in the lipid droplets were analyzed via Raman spectroscopy and chemometrics. The growth curves of the cells indicated that palmitic, stearic, and linoleic acids induced cell death in HepG2 cells, but oleic acid did not. Microscopic observations suggested that the rates of fat accumulation were high for oleic and linoleic acids, but low for palmitic and stearic acids. Raman analysis indicated that linoleic fatty chains taken into the cells are modified into oleic fatty chains. These results suggest that the signaling pathway of cell death is independent of fat stimulations. Moreover, these results suggest that hepatic cells have a high affinity for linoleic acid, but linoleic acid induces cell death in these cells. This may be one of the causes of inflammation in nonalcoholic fatty liver disease (NAFLD).

Genes regulated by branched-chain polyamine in the hyperthermophilic archaeon Thermococcus kodakarensis.

Branched-chain polyamine (BCPA) synthase (BpsA), encoded by the bpsA gene, is responsible for the biosynthesis of BCPA in the hyperthermophilic archaeon Thermococcus kodakarensis, which produces N4-bis(aminopropyl)spermidine and spermidine. Here, next-generation DNA sequencing and liquid chromatography-mass spectrometry (LC-MS) were used to perform transcriptomic and proteomic analyses of a T. kodakarensis strain (DBP1) lacking bpsA. Subsequently, the contributions of BCPA to gene transcription (or transcript stabilization) and translation (or protein stabilization) were analyzed. Compared with those in the wild-type strain (KU216) cultivated at 90 °C, the transcript levels of 424 and 21 genes were up- and downregulated in the DBP1 strain, respectively. The expression levels of 12 frequently-used tRNAs were lower in DBP1 cells than KU216 cells, suggesting that BCPA affects translation efficiency in T. kodakarensis. LC-MS analyses of cells grown at 90 °C detected 50 proteins in KU216 cells only, 109 proteins in DBP1 cells only, and 499 proteins in both strains. Notably, the transcript levels of some genes did not correlate with those of the proteins. RNA-seq and RT-qPCR analyses of ten proteins that were detected in KU216 cells only, including three flagellin-related proteins (FlaB2-4) and cytosolic NiFe-hydrogenase subunit alpha (HyhL), revealed that the corresponding transcripts were expressed at higher levels in DBP1 cells than KU216 cells. Electron microscopy analyses showed that flagella formation was disrupted in DBP1 cells at 90 °C, and western blotting confirmed that HyhL expression was eliminated in the DBP1 strain. These results suggest that BCPA plays a regulatory role in gene expression in T. kodakarensis.

Fabrication of a laser cavity mirror in a large mode area fiber by an ultrashort pulse laser.

We present herein a method to fabricate a higher-order fiber grating (HOFG) for use as a fiber-cavity mirror in a fiber laser. The HOFG was fabricated by irradiating the Yb-doped large core of a double-clad fiber by a femtosecond pulsed laser. The HOFG served as a laser cavity mirror with a reflectance of 13.2% and yielded a laser line with a spectral full width at half-maximum of 0.56 nm.

Alternative Splicing for Activation of Coagulation Factor XIII-A in the Fish Retina After Optic Nerve Injury.

Factor XIII-A (FXIII-A), which has become known as cellular transglutaminase, plays important roles in mediating cross-linking reactions in various tissues. FXIII-A acts as one of the regeneration molecules in the fish retina and optic nerve after optic nerve injury and becomes activated at the site of injury within a few hours. Previous research has shown that activated FXIII-A induces neurite outgrowth from injured retinal ganglion cells and supports elongation of the regenerating optic nerve. However, the activation mechanism of FXIII-A remains unknown. Furthermore, the injured tissues do not express thrombin, a known activator of plasma FXIII. Here, we investigated the mRNA expression of FXIII-A based on two different regions, one encoding the activation peptide and the other encoding the enzymatic active site. We found that expression of the region encoding the activation peptide was markedly suppressed compared with the region encoding the active site. An overexpression study with a short-type FXIII-A cDNA lacking the activation peptide revealed induction of long neurite outgrowth in fish retinal explant cultures compared with full-length FXIII-A cDNA. The present findings suggest that alternative splicing may occur in the FXIII-A gene, resulting in deletion of the region encoding the activation peptide and thus allowing direct production of activated FXIII-A protein in the fish retina and optic nerve after optic nerve injury.

Effect of Solvent Dielectric Constant on the Formation of Large Flat Bilayer Stacks in a Lecithin/Hexadecanol Hydrogel.

We investigated the effect of dielectric properties of the aqueous medium on the novel type of hydrogel composed of a crude lecithin mixture (PC70) and hexadecanol (HD), in which charged sheet-like bilayers are kept far apart due to interbilayer repulsive interaction. We used dipropylene glycol (DPG) as a modifier of the dielectric properties and examined its effect on the hydrogel by synchrotron X-ray diffraction, differential scanning calorimetry (DSC), polarized optical microscopy, and freeze-fracture electron microscopy. We found that at a DPG weight fraction in the aqueous medium WDPG ≈ 0.4, the bilayer organization is transformed into unusually large flat bilayer stacks with a regular lamellar spacing of 6.25 nm and consequently disintegration of the hydrogel takes place. Semiquantitative calculation of the interbilayer interaction energy based on the Deyaguin-Landau-Verwey-Overbeek (DLVO) theory suggested that the reduction of the aqueous medium dielectric constant ε by DPG may lower the energy barrier preventing flat bilayers from coming closer together. We inferred that the size of the bilayer sheet increases because the reduction of ε promotes protonation of acidic lipids that work as edge-capping molecules.

Geranylgeranylacetone Suppresses N-Methyl-N-nitrosourea-Induced Photoreceptor Cell Loss in Mice.

Retinitis pigmentosa is a disease characterized by the loss of photoreceptor cells. The N-methyl-N-nitrosourea (MNU)-induced retinal degeneration model is widely used to study the mechanism of these retinal degenerative disorders because of its selective photoreceptor cell death. As for the cell death mechanism of MNU, calcium-calpain activation and lipid peroxidation processes are involved in the initiation of this cell death. Although such molecular mechanisms of the MNU-induced cell death have been described, the total image of the cell death is still obscure. Heat shock protein 70 (HSP70) has been shown to function as a chaperon molecule to protect cells against environmental and physiological stresses. In this study, we investigated the effect of geranylgeranylacetone (GGA), an accylic polyisoprenoid, on MNU-induced photoreceptor cell loss. HSP70 induction by GGA was effective against MNU-induced photoreceptor cell loss as a result of its ability to prevent HSP70 degradation. The data indicate that GGA may help to suppress the onset and progression of retinitis pigmentosa.

Nitric Oxide Synthase Activation as a Trigger of N-methyl-N-nitrosourea-Induced Photoreceptor Cell Death.

Retinal degeneration (RD) such as retinitis pigmentosa and age-related macular degeneration are major causes of blindness in adulthood. As one of the model for RD, intraperitoneal injection of N-methyl-N-nitrosourea (MNU) is widely used because of its selective photoreceptor cell death. It has been reported that MNU increases intracellular calcium ions in the retina and induces photoreceptor cell death. Although calcium ion influx triggers the neuronal nitric oxide synthase (nNOS) activation, the role of nNOS on photoreceptor cell death by MNU has not been reported yet. In this study, we investigated the contribution of nNOS on photoreceptor cell death induced by MNU in mice. MNU significantly increased NOS activation at 3 day after treatment. Then, we evaluated the effect of nNOS specific inhibitor, ethyl[4-(trifluoromethyl) phenyl]carbamimidothioate (ETPI) on the MNU-induced photoreceptor cell death. At 3 days, ETPI clearly inhibited the MNU-induced cell death in the ONL. These data indicate that nNOS is a key molecule for pathogenesis of MNU-induced photoreceptor cell death.

Cell Fate of Müller Cells During Photoreceptor Regeneration in an N-Methyl-N-nitrosourea-Induced Retinal Degeneration Model of Zebrafish.

Zebrafish can regenerate several organs such as the tail fin, heart, central nervous system, and photoreceptors. Very recently, a study has demonstrated the photoreceptor regeneration in the alkylating agent N-methyl-N-nitrosourea (MNU)-induced retinal degeneration (RD) zebrafish model, in which whole photoreceptors are lost within a week after MNU treatment and then regenerated within a month. The research has also shown massive proliferation of Müller cells within a week. To address the question of whether proliferating Müller cells are the source of regenerating photoreceptors, which remains unknown in the MNU-induced zebrafish RD model, we employed a BrdU pulse-chase technique to label the proliferating cells within a week after MNU treatment. As a result of the BrdU pulse-chase technique, a number of BrdU(+) cells were observed in the outer nuclear layer as well as the inner nuclear layer. This implies that regenerating photoreceptors are derived from proliferating Müller cells in the zebrafish MNU-induced RD model.

A Possible Role of Neuroglobin in the Retina After Optic Nerve Injury: A Comparative Study of Zebrafish and Mouse Retina.

Neuroglobin (Ngb) is a new member of the family of heme proteins and is specifically expressed in neurons of the central and peripheral nervous systems in all vertebrates. In particular, the retina has a 100-fold higher concentration of Ngb than do other nervous tissues. The role of Ngb in the retina is yet to be clarified. Therefore, to understand the functional role of Ngb in the retina after optic nerve injury (ONI), we used two types of retina, from zebrafish and mice, which have permissible and non-permissible capacity for nerve regeneration after ONI, respectively. After ONI, the Ngb protein in zebrafish was upregulated in the amacrine cells within 3 days, whereas in the mouse retina, Ngb was downregulated in the retinal ganglion cells (RGCs) within 3 days. Zebrafish Ngb (z-Ngb) significantly enhanced neurite outgrowth in retinal explant culture. According to these results, we designed an overexpression experiment with the mouse Ngb (m-Ngb) gene in RGC-5 cells (retinal precursor cells). The excess of m-Ngb actually rescued RGC-5 cells under hypoxic conditions and significantly enhanced neurite outgrowth in cell culture. These data suggest that mammalian Ngb has positive neuroprotective and neuritogenic effects that induce nerve regeneration after ONI.

A detailed analysis of partial molecular volumes in DPPC/cholesterol binary bilayers.

We examined the volumetric behavior of the dipalmitoylphosphatidylcholine (DPPC)/cholesterol binary bilayer system with high accuracy and more cholesterol concentrations to reveal the detailed molecular states in the liquid-disordered (Ld) phase, the liquid-ordered (Lo) phase and the gel phase. We measured the average specific volume of the binary bilayer at several temperatures by the neutral flotation method and calculated the average volume per molecule to estimate the partial molecular volumes of DPPC and cholesterol in each phase. As a result, we found that the region with intermediate cholesterol concentrations showed a more complicated behavior than expected from simple coexistence of Ld and Lo domains. We also measured fluorescence decay of trans-parinaric acid (tPA) added into the binary bilayer with more cholesterol concentrations to get further insight into the cholesterol-induced formation of the Lo phase. On the basis of these results we discuss the molecular interaction between DPPC and cholesterol molecule in the Lo phase and the manner of Ld/Lo phase coexistence.

Detailed Analysis of the Surface Area and Elasticity in the Saturated 1,2-Diacylphosphatidylcholine/Cholesterol Binary Monolayer System.

The surface pressure-area (π-A) isotherms of DMPC, DPPC, and DSPC/cholesterol binary monolayers were systematically measured with great care to gain insight into the lateral molecular packing in these binary monolayer systems. The average molecular area A and the area elastic modulus C(s)⁻¹ at a given surface pressure were calculated as a function of cholesterol mole fraction x(chol). As a result, data reliable enough for the analysis of detailed phase behavior were obtained. We identified several characteristic phase regions and assigned the phase state in each region on the basis of the deviation of A(x(chol)) and C(s)⁻¹(x(chol)) from ideal additivity. We also estimated the partial molecular areas of DMPC, DPPC, DSPC, and cholesterol in the single-phase regions, where C(s)⁻¹(x(chol)) values fell on an ideal additivity curve. We found that the addition of cholesterol induces the formation of a highly condensed phase where the diacylphosphatidylcholine (diacyl PC) molecule has a surface area even smaller than that in the solid phase, irrespective of the surface pressure and the chain length of diacyl PC. Here, we call the cholesterol-induced condensed phase the CC phase. Furthermore, we demonstrated that the basic features of A(x(chol)) and C(s)⁻¹(x(chol)) profiles can be explained semiquantitatively by assuming the state of vicinity lipids surrounding sparsely distributed cholesterol molecules in the low x(chol) region as a third state of the diacyl PC molecule in addition to the states in the pure diacyl PC monolayer and in the CC phase.

Involvement of neuronal nitric oxide synthase in N-methyl-N-nitrosourea-induced retinal degeneration in mice.

N-methyl-N-nitrosourea (MNU) is widely used to study the mechanism of retinal degenerative diseases (RDs) because of its selectivity of photoreceptor cell death. Many reports suggest that excessive nitric oxide (NO) plays a crucial role in neuronal cell death. We hypothesized that nitric oxide synthase (NOS)/NO are involved in photoreceptor cell death by MNU. We found that the levels of NO increased after MNU treatment. Furthermore, we demonstrated that neuronal NOS specific inhibitor attenuated photoreceptor cell death by MNU in mice. We believe that our findings might be a new target for the treatment of RDs.

Low-flux electron diffraction study for the intercellular lipid organization on a human corneocyte.

Human skin stratum corneum (SC) structures were investigated by electron diffraction (ED) with a very low-flux electron beam with the help of high-sensitivity detectors, the imaging plate and the CCD camera. This low-flux electron diffraction (LFED) method made it possible to minimize the unfavorable effect of electron beam damage and to give a reliable diffraction pattern from a small selected area (0.2µm(2)) on a corneocyte. Dependence of the 2-dimensional ED pattern on the size of the selected area showed that orientational correlation between lipid packing domains can persist over the area much larger than their domain size. The LFED method also allowed us to trace the detailed structural change induced by the electron beam damage. The ED diffraction peak for the lattice constant of about 4.1nm decayed in three steps. The detailed analysis of these three steps suggested that a different type of orthorhombic structure exists interacted with the well-described hexagonal and orthorhombic structures, in the process of decay resulting from electron beam damage.

Functional characterization of fish neuroglobin: zebrafish neuroglobin is highly expressed in amacrine cells after optic nerve injury and can translocate into ZF4 cells.

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection under conditions of oxidative stress, such as ischemia and reperfusion. We previously found that zebrafish Ngb can penetrate the mammalian cell membrane. In the present study, we investigated the functional characteristics of fish Ngb by using the zebrafish cell line ZF4 and zebrafish retina. We found that zebrafish Ngb translocates into ZF4 cells, but cannot protect ZF4 cells against cell death induced by hydrogen peroxide. Furthermore, we demonstrated that a chimeric ZHHH Ngb protein, in which module M1 of human Ngb is replaced by that of zebrafish, is a cell-membrane-penetrating protein that can protect ZF4 cells against hydrogen peroxide exposure. Moreover, we investigated the localization of Ngb mRNA and protein in zebrafish retina and found that Ngb mRNA is expressed in amacrine cells in the inner nuclear layer and is significantly increased in amacrine cells 3days after optic nerve injury. Immunohistochemical studies clarified that Ngb protein levels were increased in both amacrine cells and presynaptic regions in the inner plexiform layer after nerve injury. Taken together, we hypothesize that fish Ngb, whose expression is upregulated in amacrine cells after optic nerve injury, might be released from amacrine cells, translocate into neighboring ganglion cells, and function in the early stage of optic nerve regeneration. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Heat shock protein 70 induction by valproic acid delays photoreceptor cell death by N-methyl-N-nitrosourea in mice.

Retinal degenerative diseases (RDs) are a group of inherited diseases characterized by the loss of photoreceptor cells. Selective photoreceptor loss can be induced in mice by an intraperitoneal injection of N-methyl-N-nitrosourea (MNU) and, because of its selectivity, this model is widely used to study the mechanism of RDs. Although it is known that calcium-calpain activation and lipid peroxidation are involved in the initiation of cell death, the precise mechanisms of this process remain unknown. Heat shock protein 70 (HSP70) has been shown to function as a chaperone molecule to protect cells against environmental and physiological stresses. In this study, we investigated the role of HSP70 on photoreceptor cell death in mice. HSP70 induction by valproic acid, a histone deacetylase inhibitor, attenuated the photoreceptor cell death by MNU through inhibition of apoptotic caspase signals. Furthermore, HSP70 itself was rapidly and calpain-dependently cleaved after MNU treatment. Therefore, HSP70 induction by valproic acid was dually effective against MNU-induced photoreceptor cell loss as a result of its anti-apoptotic actions and its ability to prevent HSP70 degradation. These findings might help lead us to a better understanding of the pathogenic mechanism of RDs. Retinal degenerative diseases are characterized by the loss of photoreceptor cells. We proposed the following cascade for N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell death: MNU gives rise to cleavage of heat shock protein 70 (HSP70); HSP70 induction by valproic acid (VPA) is dually effective against MNU-induced photoreceptor cell loss because of its anti-apoptotic actions and its ability to prevent HSP70 degradation. We hope that the present study heralds a new era in developing therapeutic tools against retinal degenerative diseases.

Different roles of two transcription factor B proteins in the hyperthermophilic archaeon Thermococcus kodakarensis.

Two genes, TK1280 and TK2287, encode orthologous transcription factor B proteins (TFB1 and TFB2, respectively) in the hyperthermophilic archaeon Thermococcus kodakarensis. The functional difference between their TFBs remains unknown. While TFB1 and TFB2 displayed equivalent thermostability, mRNA levels of tfb1 at 93 °C were eightfold higher than those at 60 or 85 °C, and were 4- to 10-fold greater than those of tfb2 at all temperatures. This suggests that TFB1 is the abundant TFB in T. kodakarensis and is heat-inducible. By contrast, the mRNA level of tfb2 increased at 93 °C, but the levels were less than twofold of those at 60 or 85 °C. No significant differences in growth were observed among the DTF1 (∆tfb1, ∆pyrF), DTF2 (∆tfb2 ∆pyrF), and parental host strain KU216 (∆pyrF) at 60 °C. However, DTF2 showed a decrease in cell yield at 85 °C, and both DTF1 and DTF2 showed growth defects at 93 °C. Comparative transcriptome analysis between KU216 and DTF1 or DTF2 indicated that TFB1 apparently controls the expression of genes essential for motility/adhesion, whereas TFB2 regulates genes involved in mevalonate/lipid biosynthesis. In DTF1, the ratio of cells with flagella decreased at 85 and 93 °C, and reporter studies indicated that flaB1 transcription is dependent on TFB1 at 85 °C but not at 60 °C.

Neuritogenic activity of trichostatin A in adult rat retinal ganglion cells through acetylation of histone H3 lysine 9 and RARß induction.

Like other CNS neurons, mature retinal ganglion cells (RGCs) cannot regenerate their axons after nerve injury due to loss of regenerative capacity. One of the reasons why they lose their capacity seems to be a dramatic shift in gene expression of RGCs under epigenetic modulation. In here, we found that levels of histone H3 lysine 9 acetylation decreased after birth in RGCs. This decrease showed good correlation with restriction of retinoic acid receptor ß (RARß) expression in RGCs after birth. Furthermore, we demonstrated that a histone deacetylase inhibitor, trichostatin A, induced axonal regeneration of adult rat RGCs through RARß induction.

Comparative study of novel ultradeformable liposomes: menthosomes, transfersomes and liposomes for enhancing skin permeation of meloxicam.

In the present study, novel ultradeformable liposomes (menthosomes; MTS), deformable liposomes (transfersomes; TFS) and conventional liposomes (CLP) were compared in their potential for transdermal delivery of meloxicam (MX). MTS, TFS and CLP were investigated for size, size distribution, zeta potential, elasticity, entrapment efficiency and stability. In vitro skin permeation using hairless mice skin was evaluated. Vesicular morphology was observed under freeze-fractured transmission electron microscopy (FF-TEM). Intrinsic thermal properties were performed using differential scanning calorimetry (DSC) and X-ray diffraction. The skin permeation mechanism was characterized using confocal laser scanning microscopy (CLSM). The results indicated that the difference in physicochemical characteristics of MTS, TFS and CLP affected the skin permeability. MTS and TFS showed higher flux of MX than CLP. CLSM image showed deformable vesicles mechanism for delivery of MX across the hairless mice skin. Our study suggested that ultradeformable and deformable liposomes (MTS and TFS) had a potential to use as transdermal drug delivery carriers for MX.