Neutron crystallography and quantum chemical analysis of bilin reductase PcyA mutants reveal substrate and catalytic residue protonation states.

PcyA, a ferredoxin-dependent bilin pigment reductase, catalyzes the site-specific reduction of the two vinyl groups of biliverdin (BV), producing phycocyanobilin. Previous neutron crystallography detected both the neutral BV and its protonated form (BVH+) in the wildtype (WT) PcyA-BV complex, and a nearby catalytic residue Asp105 was found to have two conformations (protonated and deprotonated). Semiempirical calculations have suggested that the protonation states of BV are reflected in the absorption spectrum of the WT PcyA-BV complex. In the previously determined absorption spectra of the PcyA D105N and I86D mutants, complexed with BV, a peak at 730 nm, observed in the WT, disappeared and increased, respectively. Here, we performed neutron crystallography and quantum chemical analysis of the D105N-BV and I86D-BV complexes to determine the protonation states of BV and the surrounding residues and study the correlation between the absorption spectra and protonation states around BV. Neutron structures elucidated that BV in the D105N mutant is in a neutral state, whereas that in the I86D mutant is dominantly in a protonated state. Glu76 and His88 showed different hydrogen bonding with surrounding residues compared with WT PcyA, further explaining why D105N and I86D have much lower activities for phycocyanobilin synthesis than the WT PcyA. Our quantum mechanics/molecular mechanics calculations of the absorption spectra showed that the spectral change in D105N arises from Glu76 deprotonation, consistent with the neutron structure. Collectively, our findings reveal more mechanistic details of bilin pigment biosynthesis.

In situ observation of the deterioration process of sulfide-based solid electrolytes using airtight and air-flow TEM systems.

Sulfide-based solid electrolytes (SEs) exhibiting high ionic conductivity are indispensable battery materials for next-generation all-solid-state batteries. However, sulfide-based SEs have a major drawback in their low chemical stability in air. When exposed to H2O or O2 gas, toxic H2S is generated, and their ionic conductivity considerably declines. However, their degradation mechanism caused by air exposure has not been understood yet. To clarify the degradation process, in this study, we developed a transmission electron microscope (TEM) system to evaluate the air stability of battery materials. Using a vacuum transfer double-tilt TEM holder with a gas-flow system, the in situ observation of the degradation process was conducted for a sulfide-based Li4SnS4 glass ceramic under an air-flow environment. Consequently, electron diffraction (ED) patterns and TEM images could clearly capture morphological changes and the amorphization process caused by air exposure. Moreover, based on the analysis of ED patterns, it is observed that Li4SnS4 is likely to decompose because of the reaction with H2O in air. Therefore, this airtight and air-flow TEM system should be effective in clarifying the process of the deterioration of sulfur-based SEs during exposure to air.

Enhanced sophorolipid production and effective conversion of waste frying oil using dual lipophilic substrates.

A highly efficient sophorolipid (SL) fermentation process using dual lipophilic substrates (DLS) was developed. Using DLS consisting of palm oil and oleic acid in the flask fermentation with SL-producing yeast Starmerella bombicola, the productivity and yield of SL improved 2.4 and 1.7 times, in comparison to when using palm olein alone. In the jar fermentation process with DLS, the SL productivity and yield of 127 g/L and 62.4% were achieved in 7 days, with the complete consumption of residual lipophilic substrates in the culture medium. This process was successfully applied to the effective conversion of waste frying oil to SL, maintaining a high SL productivity and yield of 142.8 g/L and 72% in 9 days. It is concluded that the newly developed fermentation process with DLS can be useful for the industrial production of SL from renewable biological sources, especially waste frying oils.

Nanoparticle size and 3D shape measurement by electron tomography: An Inter-Laboratory Comparison.

Electron tomography (ET) has been used for quantitative measurement of shape and size of objects in three dimensions (3D) for many years. However, systematic investigation of repeatability and reproducibility of ET has not been evaluated in detail. To assess the reproducibility and repeatability of a protocol for measuring size and three-dimensional (3D) shape parameters for nanoparticles (NPs) by ET, an inter-laboratory comparison (ILC) has been performed. The ILC included six laboratories and six instruments models from three instrument manufacturers following a standard measurement protocol. A technical specification describing the normative steps of the protocol is published by the International Standards Organization (ISO). Gold NPs with 30 nm nominal diameter contained within a rod-shaped carbon support were measured. The use of a rod-shaped sample support eliminated the missing wedge effect in the experimental tilt series of projected images for improved quantification. A total of 443 NPs were initially measured by NRC-NANO and then 115 out of the 443 NPs were measured by five other labs to compare measurands such as the Volume (V), maximum Feret diameter (Fmax), minimum Feret diameter (Fmin), volume-equivalent diameter (Deq) and aspect ratio (Frat) of the NPs. The results of the five labs were compared with the results obtained at NRC-NANO. The maximum disagreement in measurements of Fmin and Fmax obtained by the participating labs did not exceed 7 %. The measured Deq was between 27.5 nm and 30.3 nm in agreement with the NP manufacturer's specification (28 nm-32 nm). In addition to the above, the influence of the missing wedge effect and beam-induced NP movement was quantified based on the differences of the results between labs.

Magnetic resonance elastography of the supraspinatus muscle: A preliminary study on test-retest repeatability and wave quality with different frequencies and image filtering.

The purpose of this study was to determine an optimal condition (vibration frequency and image filtering) for stiffness estimation with high accuracy and stiffness measurement with high repeatability in magnetic resonance elastography (MRE) of the supraspinatus muscle. Nine healthy volunteers underwent two MRE exams separated by at least a 30 min break, on the same day. MRE acquisitions were performed with a gradient-echo type multi-echo MR sequence at 75, 100, and 125 Hz pneumatic vibration. Wave images were processed by a bandpass filter or filter combining bandpass and directional filters (bandpass-directional filter). An observer specified the region of interest (ROI) on clear wave propagation in the supraspinatus muscle, within which the observer measured the stiffness. This study assessed wave image quality according to two indices, as a substitute for the assessment of the accuracy of the stiffness estimation. One is the size of the clear wave propagation area (ROI size used to measure the stiffness) and the other is the qualitative stiffness resolution score in that area. These measurements made by the observer were repeated twice at least one month apart after each MRE exam. This study assessed the intra-examiner and observer repeatability of the stiffness value, ROI size and resolution score in each combination of vibration frequency and image filter. Repeatability of the data was analyzed using the intraclass correlation coefficient (ICC) and 95% limits-of-agreement (LOA) in Bland-Altman analysis. The analyses on intra-examiner and observer repeatability of stiffness indicated that the ICC and 95% LOA were not varied greatly depending on vibration frequency and image filter (intra-examiner repeatability, ICC range, 0.79 to 0.88; 95% LOA range, ±23.95 to ±32.42%, intra-observer repeatability, ICC range, 0.98 to 1.00; 95% LOA range, ±5.10 to ±10.99%). In the analyses on intra-examiner repeatability of ROI size, ICCs were rather low (ranging from: 0.03 to 0.69) while 95% LOA was large in all the combinations of vibration frequency and image filter (ranging from: ±62.66 to ±83.33%). In the analyses on intra-observer repeatability of ROI size, ICCs were sufficiently high in the total combination of vibration frequency and image filter (ranging from 0.80 to 0.87) while the 95% LOAs were better (lower) in the bandpass-directional filter than the bandpass filter (bandpass directional filter vs. bandpass filter, ±28.81 vs. ±54.83% at 75 Hz; ±25.63 vs. ±37.83% at 100 Hz; ±34.51 vs. ±43.36% at 125 Hz). In the analyses on intra-examiner and observer repeatability of resolution score, the mean difference (bias) between the two exams (or observations) was significantly low and there was almost no difference across all the combinations of vibration frequency and image filter (range of bias: -0.11-0.11 and -0.17-0.00, respectively). Additionally, effects of vibration frequency and image filter on wave image quality (ROI size and resolution score) were assessed separately in each exam. Both mean ROI size and resolution score in the bandpass-directional filter were larger than those in the bandpass filter. Among the data in the bandpass-directional filter, mean ROI size was larger at 75 and 100 Hz, and mean resolution score was larger at 100 and 125 Hz. Taking into consideration with the results of repeatability and wave image quality, the present results suggest that optimal vibration frequency and image filter for MRE of the supraspinatus muscles is 100 Hz and bandpass-directional filter, respectively.

A new technique for motion encoding gradient-less MR elastography of the psoas major muscle: A gradient-echo type multi-echo sequence.

The present study aimed to develop vibration techniques for magnetic resonance (MR) elastography (MRE) of the psoas major muscle (PM). Seven healthy volunteers were included. MRE was performed with motion-encoding gradient (MEG)-less multi-echo MRE sequence, which allows clinicians to perform MRE using conventional MR imaging. In order to transmit mechanical vibration of the pneumatic type to the PM, a long narrow vibration pad was designed using a 3D printer, and the optimum vibration techniques were verified. The vibration pad was placed under the lower back, with the volunteers in the supine position. The results indicated that the PM vibrated well through the transmitted vibration from the lumbar spine, which suggests that the placement of a narrow vibration pad under the supine body, along the lumbar spine, allows the vibration of the PM. The shear modulus of the PM (n = 7) was 1.23 ±â€¯0.09 kPa (mean ±â€¯SEM) on the right side and 1.22 ±â€¯0.15 kPa on the left side, with no significant difference (t-test, P > 0.05). Increased stiffness of the muscle due to continuous local contraction may be an important cause of non-specific low back pain (LBP). The present vibration techniques for MRE of the PM provide a quantitative diagnostic tool for changes in muscle stiffness associated with non-specific LBP.

[Influence of Vibration Waveform on MR Elastography].

The purpose of this study was to investigate an influence of vibration waveform on magnetic resonance elastography (MRE). MRE is an innovative imaging technique for the non-invasive quantification of the elasticity of soft tissues through the direct visualization of propagating shear waves in vivo using a special phase-contrast magnetic resonance imaging sequence. Since the elasticity of soft tissue calculates from the wavelength of propagating shear waves, it is necessary to propagate sine-wave-shape shear wave at the target soft tissue. However, due to the various factors; i.e. overload of vibration generator, poor contact between imaging object and vibration pad, etc.; it may be difficult to generate a simple sine wave. This work was focused on change vibration waveforms; i.e. square wave, triangle wave, saw-tooth wave; which is induced by the various factors. Phantom experimental results demonstrated that when square and saw-tooth waveforms of 25 Hz vibration frequency, into the phantom, the waveform of propagating wave was not similar to sine waveform. It may influence on the MRE that in case of the waveforms has low frequency and square or saw-tooth like waveforms.

Hemoglobin(ßC93A)-Albumin Cluster: Mutation of Cysteine-ß93 to Alanine Allows Moderate Reduction of O2 Affinity by Inositol Hexaphosphate.

Covalent wrapping of recombinant human hemoglobin (Cys-ß93→Ala) variant rHb(ßC93A) by human serum albumin (HSA) yielded the rHb(ßC93A)-HSA3 cluster as an artificial O2 carrier as a red blood cell substitute. Complexation of inositol hexaphosphate to the central rHb(ßC93A) core reduced the O2 affinity moderately, in much the same way as that of naked hemoglobin. This reduction might be attributable to the inert, small Ala-ß93 residue, which cannot be reacted with the bulky maleimide crosslinker.

Simultaneous acquisition of magnetic resonance elastography of the supraspinatus and the trapezius muscles.

We developed a Magnetic Resonance elastography (MRE) technique using a conventional magnetic resonance imaging (MRI), which allows a simultaneous elastography of the supraspinatus and trapezius muscles, by designing a new wave transducer (vibration pad) and optimizing the mechanical vibration frequency. Five healthy volunteers underwent an MRE. In order to transmit the mechanical vibration (pneumatic vibration) to the supraspinatus and trapezius muscles, a new vibration pad was designed using a three-dimensional (3D) printer. The vibration pad was placed on the skin 2 cm medial and 2 cm cephalad the deltoid tubercle. MRE acquisition was performed with a multi-slice gradient-echo type multi-echo MR sequence, which allows MREs even in a conventional MRI; two oblique axial images of the supraspinatus and trapezius muscles were obtained simultaneously. Vibration frequencies were set at 50-150 Hz, with a 25 Hz step. Wave image quality in each frequency was analyzed using a phase-to-noise ratio (PNR) and clarity of propagating wave that was assessed by two readers qualitatively. In the supraspinatus muscle, the wave images were of good quality especially at frequencies >75 Hz. In the trapezius muscle, the wave images were of better quality at low frequencies (50 and 75 Hz) compared with high frequencies (100-150 Hz). The PNR of both muscles were higher at low frequencies. The mean stiffness in the trapezius muscle (7.26 ±â€¯2.13 kPa at 75 Hz) was larger than those in the supraspinatus muscle (4.16 ±â€¯0.50 kPa at 75 Hz). The results demonstrated that our MRE technique allows simultaneous assessment of the stiffness in the supraspinatus and trapezius muscles using a conventional MRI, and that optimal vibration frequency for simultaneous MRE of these muscles is 75 Hz. This technique provides a new means for early detection of abnormality in the shoulder.

Hemoglobin-albumin cluster: physiological responses after exchange transfusion into rats and blood circulation persistence in dogs.

A core-shell protein cluster comprising hemoglobin and human serum albumins, hemoglobin-albumin cluster (Hb-HSA3), was designed and synthesized for use as an artificial O2 carrier and red blood cell (RBC) substitute. For initial preclinical safety evaluation of the Hb-HSA3 solution, we observed blood compatibility in vitro, physiological responses after exchange transfusion into rats and blood circulation lifetime in dogs. Dilution of human whole blood with Hb-HSA3 showed an appropriate decrease in blood cell number, proportional to the mixing volume ratio. Time courses in the circulation parameters and blood gas parameters after 20% exchange transfusion with Hb-HSA3 in anesthetized rats were almost identical to those observed in a sham group (without infusion) and an HSA group (with HSA administration) for 6 h. Serum biochemical tests of the withdrawn blood indicated safety of the protein cluster. Furthermore, fluorescent Hb-HSA3 was infused into beagle dogs to assess blood retention. Fluorescence measurements of the blood samples enabled us to ascertain the cluster half-life within the intravascular space. Histopathologic inspections of the vital organs imply no abnormality in tissues. All these results indicate sufficient initial preclinical safety of Hb-HSA3 as an alternative material for use in RBC transfusion.

A Gene Encoding Pentatricopeptide Repeat Protein Partially Restores Fertility in RT98-Type Cytoplasmic Male-Sterile Rice.

Cytoplasmic male sterility (CMS) lines in rice, which have the cytoplasm of a wild species and the nuclear genome of cultivated rice, are of value for the study of genetic interactions between the mitochondrial and nuclear genomes. The RT98-type CMS line RT98A and the fertility restorer line RT98C carry the cytoplasm of the wild species Oryza rufipogon and the nuclear genome of the Taichung 65 cultivar (Oryza sativa L.). Based on a classical crossing experiment, fertility is reported to be restored gametophytically by the presence of a tentative single gene, designated Rf98, which is derived from the cytoplasm donor. Fine mapping of Rf98 revealed that at least two genes, which are closely positioned, are required for complete fertility restoration in RT98A. Here, we identified seven pentatricopeptide repeat (PPR) genes that are located within a 170 kb region as candidates for Rf98 Complementation tests revealed that the introduction of one of these PPR genes, PPR762, resulted in the partial recovery of fertility with a seed setting rate up to 9.3%. We conclude that PPR762 is an essential fertility restorer gene for RT98-type CMS. The low rate of seed setting suggested that some other genes near the Rf98 locus are also necessary for the full recovery of seed setting.

Spontaneous vesicle formation from sodium salt of acidic sophorolipid and its application as a skin penetration enhancer.

In this study, spontaneous vesicle formation from the sodium salt of acidic sophorolipid (SLNa) was observed, and its potential application as a skin penetration enhancer for triterpene glycosides extracted from the fruits of Siraitia grosvenorii Swingle was then investigated. Dynamic light scattering (DLS) measurements of the SLNa assemblies prepared by the gentle mixing of SLNa with water (1%) showed their hydrodynamic radius (Rh) to be 96.2 nm, and their structure was assigned to be vesicles by freeze-fracture electron microscopy (FF-TEM). DLS and FF-TEM also revealed that the size of the vesicles increased with an increase in the concentration of the triterpene glycosides, indicating that the triterpene glycosides were incorporated into the SLNa vesicles. The results of an in vitro skin permeation assay, after loading the SLNa vesicles on a 3D cultured skin model, showed that the amount of SLNa that penetrated though the skin model increased with time. It was also found that the amount of permeated mogroside, which is the main active component of triterpene glycosides, was significantly enhanced by the SLNa vesicle formulation. These results clearly demonstrated that spontaneously formed vesicles composed of the bolaamphiphile SLNa are useful for application as penetration enhancers for active ingredients such as mogroside V.

Whole genomic sequencing of RT98 mitochondria derived from Oryza rufipogon and northern blot analysis to uncover a cytoplasmic male sterility-associated gene.

Cytoplasmic male sterility (CMS) is a maternally inherited trait resulting in the failure to produce functional pollen and is often observed when an alien cytoplasm is transferred into a cultivated species. An RT98A CMS line and an RT98C fertility restorer line were obtained by successive backcrossing between Oryza rufipogon W1109 and Oryza sativa cultivar Taichung 65. To uncover the CMS-associated mitochondrial genes, we determined the complete sequence of the RT98-CMS mitochondrial genome using next-generation pyrosequencing, and searched new open reading frames (orfs) absent in a reported mitochondrial genome of O. sativa Nipponbare. Then, six candidates were selected for the CMS-associated genes based on the criteria in which they were chimeric in structure or encoded a peptide with transmembrane domains. One of the candidates, orf113, showed different transcript sizes between RT98A and RT98C on Northern blot analysis. The orf113 gene was shown to be co-transcribed with atp4 and cox3 encoding ATP synthase F0 subunit 4 and Cyt c oxidase subunit 3, respectively, and their transcripts were distinctly processed in the presence of a fertility restorer gene. Our results indicate that orf113 is a CMS-associated gene of RT98-CMS.

Natural synergism of acid and lactone type mixed sophorolipids in interfacial activities and cytotoxicities.

Sophorolipids (SLs) naturally produced from Candida bombicola are a mixture of lactonic (SL-lactone) and acidic (SL-acid) sophorosides of 17-L-hydroxydecanoic acid with an SL-lactone:SL-acid ratio of 72:28. SLs are biodegradable low-foaming surfactants with high detergency and hardness-tolerance properties. To analyze the effect of the SL-lactone:SL-acid ratio on these properties, SL-LXs containing X% SL-lactone, in which X varied from 0 to 100, were prepared and their interfacial activities and cytotoxicities examined. The minimum surface tension values for all SLs examined were comparable. The critical micelle concentration (CMC) was 680 mg/L for SL-L0 and 62-110 mg/L for the other SLs. Interestingly, natural SL (SL-L72) had the lowest surface tension and CMC among all of the SLs examined. The foaming ability and stability of the SLs were dependent on the SL-L content. SL-L0 and L17 had higher foaming values than the other SLs examined in 0-ppm hardness water. These values greatly reduced and became constant when the SL-L content increased over 55%. The detergencies of all of the SLs examined were comparable, except for those of SL-L0 and SL-L100, which were slightly lower than those of the other SLs. These results suggest that natural synergism between SLs creates a better balance for many interfacial activities. The cytotoxicity of SL-L72 was higher than that of SL-L0, but was comparable to that of surfactin, which is commercially available for cosmetic use. The low cytotoxicities and high interfacial properties of SLs increase their usefulness as biocompatible surface active agents for many applications.

Novel characteristics of sophorolipids, yeast glycolipid biosurfactants, as biodegradable low-foaming surfactants.

Sophorolipids (SLs) are a family of glycolipid type biosurfactants, which are largely produced by the non-pathogenic yeast, Candida bombicola. In order to investigate the possibility of SLs for industrial use, here we examined the interfacial activities, cytotoxicity and biodegradability of SLs, and compared these properties with those of two lipopeptide type biosurfactants (surfactin and arthrofactin), sodium laurate (soap, SP) and four kinds of chemically synthesized surfactants including two block-copolymer nonionic surfactants (BPs), polyoxyethylene lauryl ether (AE) and sodium dodecyl sulfate (SDS). It was indicated that SLs had extremely low-foaming properties and high detergency comparable with commercially available low-foaming BPs. These interfacial activities of SLs were maintained under 100 ppm water hardness. Cytotoxicity of SLs on human keratinocytes was the same as surfactin, which has already been commercialized as cosmetic material, but higher than BPs. Moreover, biodegradability of SLs using the OECD Guidelines for Testing of Chemicals (301C, Modified MITI Test) displayed that SLs can be classified as "readily" biodegradable chemicals, which are defined as chemicals that are degraded 60% within 28 days under specified test methods. We observed 61% degradation of SLs on the eighth day of cultivation. Our results indicate that SLs are low-foaming surfactants with high detergency, which also exhibit both low cytotoxicity and readily biodegradable properties.

Effects of light intensity distribution on growth of Rhodobacter capsulatus.

For cultivation of photosynthetic cells under defined light intensity distributions, the repeated batch culture, in which a part of culture broth containing grown cells was repeatedly replaced at predetermined time intervals with a fresh medium to keep the cell concentration constant at an initial value, was employed. By use of this method the effects of the light intensity distribution on the growth characteristics of Rhodobacter capsulatus were studied. Unexpected decreases in the specific growth rate were observed in culture of R. capsulatus at high cell concentrations and a long light path length. Big differences in the light intensities of lightly and darkly illuminated portions in photobioreactors, which reflects the light intensity distribution, seemed to cause this phenomenon, which must be taken into consideration for stable growth of photosynthetic cells.

Crystalline calcium phosphate and magnetic mineral content of Daphnia resting eggs.

Daphnia is a key crustacean zooplankton of freshwater food chains. One factor that ensures successful propagation is the Daphnia resting eggs, which are able to retain structural integrity under extreme conditions. Until recently little was known about the chemical composition, microanatomy, and physical properties of the egg itself. The current study demonstrates that the resting eggs: (1) have shells that are made up of crystalline calcium phosphate and include a honeycombed structure, and (2) contain magnetic material having properties consistent with magnetite. These properties of the resting eggs may ensure Daphnia survival in harsh environments.