Effect of Fermentation Scale on Microbiota Dynamics and Metabolic Functions for Indigo Reduction.

During indigo dyeing fermentation, indigo reduction for the solubilization of indigo particles occurs through the action of microbiota under anaerobic alkaline conditions. The original microbiota in the raw material (sukumo: composted indigo plant) should be appropriately converged toward the extracellular electron transfer (EET)-occurring microbiota by adjusting environmental factors for indigo reduction. The convergence mechanisms of microbiota, microbial physiological basis for indigo reduction, and microbiota led by different velocities in the decrease in redox potential (ORP) at different fermentation scales were analyzed. A rapid ORP decrease was realized in the big batch, excluding Actinomycetota effectively and dominating Alkalibacterium, which largely contributed to the effective indigo reduction. Functional analyses of the microbiota related to strong indigo reduction on approximately day 30 indicated that the carbohydrate metabolism, prokaryotic defense system, and gene regulatory functions are important. Because the major constituent in the big batch was Alkalibacterium pelagium, we attempted to identify genes related to EET in its genome. Each set of genes for flavin adenine dinucleotide (FAD) transportation to modify the flavin mononucleotide (FMN)-associated family, electron transfer from NADH to the FMN-associated family, and demethylmenaquinone (DMK) synthesis were identified in the genome sequence. The correlation between indigo intensity reduction and metabolic functions suggests that V/A-type H+/Na+-transporting ATPase and NAD(P)H-producing enzymes drive membrane transportations and energization in the EET system, respectively.

Indigofera tinctoria leaf powder as a promising additive to improve indigo fermentation prepared with sukumo (composted Polygonum tinctorium leaves).

Being insoluble in the oxidize form, indigo dye must be solubilized by reduction for it to penetrate textile. One of the procedures is the reduction by natural bacterial fermentation. Sukumo, composted leaves of Polygonum tinctorium, is a natural source of indigo in Japan. Although sukumo has an intrinsic bacterial seed, the onset of indigo reduction with this material may vary greatly. Certain additives improve indigo fermentation. Here, we studied the effects of Indigofera tinctoria leaf powder (LP) on the initiation of indigo reduction, bacterial community, redox potential (ORP), and dyeing intensity in the initial stages and in aged fermentation fluids prepared with sukumo. I. tinctoria LP markedly decreased ORP at day 1 and stabilised it during early fermentation. These effects could be explained by the phytochemicals present in I. tinctoria LP that act as oxygen scavengers and electron mediators. Using next generation sequencing results, we observed differences in the bacterial community in sukumo fermentation treated with I. tinctoria LP, which was not influenced by the bacterial community in I. tinctoria LP per se. The concomitant decrease in Bacillaceae and increase in Proteinivoraceae at the onset of fermentation, increase in the ratio of facultative to obligate anaerobes (F/O ratio), or the total abundance of facultative anaerobes (F) or obligate anaerobes (O) (designated F + O) are vital for the initiation and maintenance of indigo reduction. Hence, I. tinctoria LP improved early indigo reduction by decreasing the ORP and hasten the appropriate transitions in the bacterial community in sukumo fermentation.

Performance of five ultrasonic transducers modified for efficient atomization.

Performance of five ultrasonic transducers modified with different shapes of electrodes is investigated for efficient atomization. A circular silver electrode, which is conventionally used as standard on a transducer, is chemically replaced with five shapes of silver electrodes (circular, toroidal, singlet, doublet, and cross). Each electrode reflects its precise shape on the water surface and statically forms characteristic three-dimensional geometry of a water column. The modified electrode also affects the dynamics of this water column, generating two types of wobbling on the column and inducing three types of atomization depending on the shape of the electrodes. Statistical analysis indicates that the shape of the electrode on an ultrasonic transducer affects the speed of atomization, showing that the singlet electrode exhibits the highest speed of atomization (4 mg/s). The mechanisms of atomization are analyzed from the viewpoint of energy transformation with reference to mass transformation of the oscillating liquid, indicating that the vibration energy of the transducer is transferred to the water film through resonance, consuming this vibration energy with four kinds of energy such as kinetic energy of atomized mists and work function of atomization, which are defined in this study. This analysis clarifies why the speed of atomization increases with a decreasing amount of water on the transducer. Application of the appropriate shape of an electrode will greatly contribute to the fields of engineering, medicine, and biology where various types of atomization are highly desired as the situation demands.

Super-resolution technique for high-resolution multichannel Fourier transform spectrometer.

We propose a super-resolution technique for multichannel Fourier transform spectrometers, which is advantageous for feeble-light spectroscopy. The spectral resolution of an area sensor is limited by the number of lateral pixels. Our method fills the signals with vertical pixels, which is practically equivalent to increasing the number of lateral pixels. When applying our proposed technique, the resolution of an Ar-lamp spectrum, which is obtained by using an area sensor with 659 lateral pixels, becomes comparable to that of an area sensor with 1,626 lateral pixels. The spectral resolution is improved at least twice. Thus, using our method, a spectrometer with an area sensor can overcome the Nyquist frequency limitations.

Feature-preserving noise reduction by using time-domain Gaussian-weighted multiple noise reduction filters for real-time bioluminescence measurement.

This paper proposes a time-domain Gaussian-weighted noise reduction filter for bioluminescence measurement with low signal-to-noise ratio through photon counting. The filter was used for estimating the true fold-change signal from noisy gene expression data obtained through real-time dual-color luciferase assay. Furthermore, not only was the higher harmonics noise of the measurement system confirmed to reduce from the gene expression data but rapid and slow changes were also preserved in the estimated signal. In addition, the probability value of Pearson's chi-squared test was improved 257 times at most and 1.5 times on average without impairing the noise reduction ratio.

Environmental factors contributing to the convergence of bacterial community structure during indigo reduction.

Indigo is solubilized through the reducing action of the microbiota that occurs during alkaline fermentation of composted leaves of Polygonum tinctorium L. (sukumo). However, the environmental effects on the microbiota during this treatment, as well as the mechanisms underlying the microbial succession toward stable state remain unknown. In this study, physicochemical analyses and Illumina metagenomic sequencing was used to determine the impact pretreatment conditions on the subsequent initiation of bacterial community transition and their convergence, dyeing capacity and the environmental factors critical for indigo reducing state during aging of sukumo. The initial pretreatment conditions analyzed included 60°C tap water (heat treatment: batch 1), 25°C tap water (control; batch 2), 25°C wood ash extract (high pH; batch 3) and hot wood ash extract (heat and high pH; batch 4), coupled with successive addition of wheat bran from days 5 to 194. High pH had larger impact than heat treatment on the microbiota, producing more rapid transitional changes from days 1 to 2. Although the initial bacterial community composition and dyeing intensity differed during days 2-5, the microbiota appropriately converged to facilitate indigo reduction from day 7 in all the batches, with Alkaliphilus oremalandii, Amphibacillus, Alkalicella caledoniensis, Atopostipes suicloalis and Tissierellaceae core taxa contributing to the improvement of when the dyeing intensity. This convergence is attributed to the continuous maintenance of high pH (day 1 ~) and low redox potential (day 2~), along with the introduction of wheat bran at day 5 (day 5~). PICRUSt2 predictive function profiling revealed the enrichment of phosphotransferease system (PTS) and starch and sucrose metabolism subpathways key toward indigo reduction. Seven NAD(P)-dependent oxidoreductases KEGG orthologs correlating to the dyeing intensity was also identified, with Alkalihalobacillus macyae, Alkalicella caledoniensis, and Atopostipes suicloalis contributing significantly toward the initiation of indigo reduction in batch 3. During the ripening period, the staining intensity was maintained by continuous addition of wheat bran and the successive emergence of indigo-reducing bacteria that also contributed to material circulation in the system. The above results provide insight into the interaction of microbial system and environmental factors in sukumo fermentation.

Evaluation of near-infrared spectroscopy as a contactless method for health monitoring of resin-based coating materials applied to concrete surfaces.

The surfaces of concrete structures are often coated with protective materials to minimize corrosion and weathering-based deterioration. Therefore, it is important to monitor the aging of the coating materials and their overall condition to extend the service lifetime of the structure effectively. Near-infrared spectroscopy (NIRS) is a contactless, nondestructive, rapid, and convenient method for material characterization; therefore, it is useful for onsite inspection of coating materials. Hence, in this study, we attempt to determine whether NIRS can be used for simple inspection for health monitoring of organic resin-based coating materials. In addition to identifying different severities of peeling damage, we characterize the ultraviolet-induced deterioration of coating materials with different thicknesses using diffuse reflection spectra acquired in the near-infrared wavelength region. For independent comparison with the NIR spectra, the state of the coating materials on the mortar specimens was analyzed using a combination of Fourier-transform infrared spectroscopy and scanning electron microscopy, while the state of the underlying mortar specimens was analyzed using permeability and salt-water immersion tests. The results confirm that the NIRS could detect the degradation of coating materials at early stages of deterioration before their permeability had been affected. NIRS offers the possibility of intermittent monitoring of coating deterioration. In addition, because the NIR spectrometer is portable, it can help in inspecting high-rise areas and areas that are difficult to reach. Therefore, we believe that NIRS is a simple, safe, and inexpensive method for inspection of surface coating materials.

Prokaryote or eukaryote? A unique microorganism from the deep sea.

There are only two kinds of organisms on the Earth: prokaryotes and eukaryotes. Although eukaryotes are considered to have evolved from prokaryotes, there were no previously known intermediate forms between them. The differences in their cellular structures are so vast that the problem of how eukaryotes could have evolved from prokaryotes is one of the greatest enigmas in biology. Here, we report a unique organism with cellular structures appearing to have intermediate features between prokaryotes and eukaryotes, which was discovered in the deep sea off the coast of Japan using electron microscopy and structome analysis. The organism was 10 µm long and 3 µm in diameter, having >100 times the volume of Escherichia coli. It had a large 'nucleoid', consisting of naked DNA fibers, with a single nucleoid membrane and endosymbionts that resemble bacteria, but no mitochondria. Because this organism appears to be a life form distinct from both prokaryotes and eukaryotes but similar to eukaryotes, we named this unique microorganism the 'Myojin parakaryote' with the scientific name of Parakaryon myojinensis ('next to (eu)karyote from Myojin') after the discovery location and its intermediate morphology. The existence of this organism is an indication of a potential evolutionary path between prokaryotes and eukaryotes.

Structural evidence of glycoprotein assembly in cellular membrane compartments prior to Alphavirus budding.

Membrane glycoproteins of alphavirus play a critical role in the assembly and budding of progeny virions. However, knowledge regarding transport of viral glycoproteins to the plasma membrane is obscure. In this study, we investigated the role of cytopathic vacuole type II (CPV-II) through in situ electron tomography of alphavirus-infected cells. The results revealed that CPV-II contains viral glycoproteins arranged in helical tubular arrays resembling the basic organization of glycoprotein trimers on the envelope of the mature virions. The location of CPV-II adjacent to the site of viral budding suggests a model for the transport of structural components to the site of budding. Thus, the structural characteristics of CPV-II can be used in evaluating the design of a packaging cell line for replicon production.

Structome of Saccharomyces cerevisiae determined by freeze-substitution and serial ultrathin-sectioning electron microscopy.

The cell structure has been studied using light and electron microscopies for centuries, and it is assumed that the whole structure is clarified by now. Little quantitative and three-dimensional analysis of cell structure, however, has been undertaken. We have coined a new word, 'structome', by combining 'structure' and '-ome', and defined it as the 'quantitative and three-dimensional structural information of a whole cell at the electron microscopic level'. In the present study, we performed structome analysis of Saccharomyces cerevisiae, one of the most widely researched biological materials, by using freeze-substitution and serial ultrathin-sectioning electron microscopy. Our analysis revealed that there were one to three mitochondria, ~220 000 ribosomes in a cell, and 13-28 endoplasmic reticula/Golgi apparatus which do not form networks in the cytoplasm in the G1 phase. Nucleus occupied ~10.5% of the cell volume; cell wall occupied ~17%; vacuole occupied ~5.8%; cytoplasm occupied ~64%; and mitochondria occupied only ~1.7% in the G1 phase. Structome analysis of cells would form a base for the post-genome research.

Comparison of the envelope architecture of E. coli using two methods: CEMOVIS and cryo-electron tomography.

Cryo-electron microscopy of vitreous sections (CEMOVIS) and cryo-electron tomography (cryo-ET) of vitrified specimens are gradually gaining popularity. However, similar to the conventional methods, these techniques tend to produce different images of the same sample. In CEMOVIS, the mechanical stress caused by sectioning may cause inaccuracies smaller than those caused by crevasses. Therefore, we examined Escherichia coli cells by using CEMOVIS and cryo-ET to determine the differences in the computed sizes of the envelope layers, which are smaller than crevasses. We found that the width of the periplasmic space in vitreous sections and tomograms was 12 and 14 nm, respectively; furthermore, while the distance between the outer membrane (OM) and the peptidoglycan (PG) layer was almost equal (11 nm) in the two techniques, that between the plasma membrane (PM) and PG was clearly different. Thus, the observed size difference can be mainly attributed to the PM-PG distance. Since our data were obtained from images acquired using the same microscope in the same conditions, the size differences cannot be attributed to microscope-related factors. One possible factor is the angle of the cutting plane against the long axis of the cell body in CEMOVIS. However, the same PG-OM distance in both methods may exclude the variations caused by this factor. Furthermore, the mechanical stress caused by vitreous sectioning or high-pressure freezing may result in shrinkage. If this shrinkage is responsible for the nanometre-scale deformation in CEMOVIS, this factor will have to be considered in determining the molecular resolution obtained by CEMOVIS.

Electron tomography imaging methods with diffraction contrast for materials research.

Transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) enable the visualization of three-dimensional (3D) microstructures ranging from atomic to micrometer scales using 3D reconstruction techniques based on computed tomography algorithms. This 3D microscopy method is called electron tomography (ET) and has been utilized in the fields of materials science and engineering for more than two decades. Although atomic resolution is one of the current topics in ET research, the development and deployment of intermediate-resolution (non-atomic-resolution) ET imaging methods have garnered considerable attention from researchers. This research trend is probably not irrelevant due to the fact that the spatial resolution and functionality of 3D imaging methods of scanning electron microscopy (SEM) and X-ray microscopy have come to overlap with those of ET. In other words, there may be multiple ways to carry out 3D visualization using different microscopy methods for nanometer-scale objects in materials. From the above standpoint, this review paper aims to (i) describe the current status and issues of intermediate-resolution ET with regard to enhancing the effectiveness of TEM/STEM imaging and (ii) discuss promising applications of state-of-the-art intermediate-resolution ET for materials research with a particular focus on diffraction contrast ET for crystalline microstructures (superlattice domains and dislocations) including a demonstration of in situ dislocation tomography.

Analysis of the electromeniscus phenomenon using a different interpretation of the Maxwell model applied to three-dimensional molecular orientations.

An electrohydrodynamic phenomenon called the electromeniscus is analyzed. This phenomenon is closely related to the oscillation of a microscale liquid and is analyzed using an interpretation of the Maxwell model coupled with a variational principle. The analysis clearly shows that the electromeniscus phenomenon is generated as a result of mass transformation of the oscillating liquid in order to transform the electric energy at the electrode to kinetic energy of the liquid most efficiently through their resonance. Mass transformation is a characteristic phenomenon of a liquid, and control of this phenomenon demonstrates great potential for self-aligning nanoscale materials and production of functional polymers characterized by specific molecular orientations.

High-voltage electron microscopy tomography and structome analysis of unique spiral bacteria from the deep sea.

Structome analysis is a useful tool for identification of unknown microorganisms that cannot be cultured. In 2012, we discovered a unique deep-sea microorganism with a cell structure intermediate between those of prokaryotes and eukaryotes and described its features using freeze-substitution electron microscopy and structome analysis (quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level). We named it Myojin parakaryote Here we describe, using serial ultrathin sectioning and high-voltage electron microscopy tomography of freeze-substituted specimens, the structome analysis and 3D reconstruction of another unique spiral bacteria, found in the deep sea off the coast of Japan. The bacteria, which is named as 'Myojin spiral bacteria' after the discovery location and their morphology, had a total length of 1.768 ± 0.478 µm and a total diameter of 0.445 ± 0.050 µm, and showed either clockwise or counter-clockwise spiral. The cells had a cell surface membrane, thick fibrous layer, ribosomes and inner fibrous structures (most likely DNA). They had no flagella. The bacteria had 322 ± 119 ribosomes per cell. This ribosome number is only 1.2% of that of Escherichia coli and 19.3% of Mycobacterium tuberculosis and may reflect a very slow growth rate of this organism in the deep sea.

The spindle pole body of the pathogenic yeast Exophiala dermatitidis: variation in morphology and positional relationship to the nucleolus and the bud in interphase cells.

The spindle pole body (SPB) in the interphase cell of the pathogenic yeast Exophiala dermatitidis was studied in detail. The SPB was located on the outer nuclear envelope and was 342 +/- 86 nm long in a haploid strain. It consisted of two disk elements that measured 151 +/- 43 nm in diameter and 103 +/- 17 nm in thickness, connected by a rod-shaped midpiece that measured 56 +/- 20 nm in length and 37 +/- 9 nm in diameter. There were considerable variations in size and morphology of interphase SPB. Some disk elements appeared spherical but others were more flattened, and there was variation in electron density. A few SPBs did not have the midpiece. The SPB of a diploid strain was 486 +/- 118 nm long, thus significantly bigger than that of the haploid strain. The SPB tended to be localized away from the nucleolus (110 +/- 48 degrees), but close to the bud (78 +/- 45 degrees). The present study highlights the necessity of observing a large number of micrographs in three-dimensions to describe accurately the ultrastructure of the SPB in yeast.

Three-dimensional reconstruction of a pathogenic yeast Exophiala dermatitidis cell by freeze-substitution and serial sectioning electron microscopy.

The structure of a budding cell of the pathogenic yeast Exophiala dermatitidis was observed in three dimensions after freeze-substitution, serial ultrathin sectioning and computer reconstruction. The nucleus occupied about 10% of the cell volume. The spindle pole body was composed of two disk elements connected by an intervening midpiece, and occupied about 0.01% of the cell volume. The cell wall consisted of an inner transparent layer, a middle electron-opaque layer, and an outer fibrous layer. The mitochondria occupied about 10% of the cell volume. There were numerous mitochondria in the mother cell and the bud, but no 'giant mitochondrion' was seen. The ratio of mitochondrial volume within the bud to the mitochondrial volume of the cell was close to the ratio of bud:cell cytoplasmic volume. The results emphasize the importance of good cryofixation for 'perfect' preservation of yeast cell structure.

In vivo absorption spectroscopy for absolute measurement.

In in vivo spectroscopy, there are differences between individual subjects in parameters such as tissue scattering and sample concentration. We propose a method that can provide the absolute value of a particular substance concentration, independent of these individual differences. Thus, it is not necessary to use the typical statistical calibration curve, which assumes an average level of scattering and an averaged concentration over individual subjects. This method is expected to greatly reduce the difficulties encountered during in vivo measurements. As an example, for in vivo absorption spectroscopy, the method was applied to the reflectance measurement in retinal vessels to monitor their oxygen saturation levels. This method was then validated by applying it to the tissue phantom under a variety of absorbance values and scattering efficiencies.

Oximetry of retinal capillaries by multicomponent analysis.

Retinal oximetry of capillaries was performed for early detection of retinal vascular abnormalities, which are caused predominantly by complications of systemic circulatory diseases. As the conventional method for determining absorbance is not applicable to capillaries, multicomponent analysis was used to estimate the absorbance spectra of the retinal blood vessels. In this analysis, the capillary spectrum was classified as intermediate between those of the retinal arteries and veins, enabling relative estimation of oxygen saturation in the capillaries. This method could be useful for early recognition of disturbances in the peripheral circulation. Furthermore, a spectroscopic ophthalmoscope system based on the proposed method was developed to examine the human retina. A clinical trial of this system demonstrated that oximetry of the retinal capillaries may be an improvement over the present diagnosis for patients of malignant hypertension.

Retinal blood oxygen saturation mapping by multispectral imaging and morphological angiography.

We report on an experiment for mapping two-dimensional blood oxygen saturation distribution by measuring multispectral images in the wavelength range from 500 to 650 nm with the resolution of 7 nm. The multispectral images of the retina are acquired with the originally designed imaging system equipped with the tunable spectral filter. To separate retinal blood vessels from other tissue area, morphological image processing is adopted. The small flick motion is also compensated. After prepossessing, the partial least squares regression model for the oxygen saturation is built by sampling typical spectra reflected from artery and vein. Applying the regression model for all the point extracted by the morphological processing allows the two-dimensional oxygen saturation map.

The spindle pole body duplicates in early G1 phase in the pathogenic yeast Exophiala dermatitidis: an ultrastructural study.

The spindle pole body of the pathogenic yeast Exophiala dermatitidis was observed during the cell cycle using freeze-substitution and serial ultrathin sectioning electron microscopy. The spindle pole body was located on the outer membrane of the nuclear envelope and consisted of two disk elements connected by an intervening midpiece in G1 through G2 phases. Each disk element was composed of filamentous materials and measured 150 nm in diameter and 100 nm in thickness. The midpiece had higher electron density and measured 60 nm in length and 40 nm in thickness. At the beginning of prophase, each disk element of the spindle pole body enlarged to more than double in size. They were separated on the nuclear envelope, and associated with numerous cytoplasmic microtubules. At mitosis, the spindle pole body entered the nuclear envelope, associated with numerous nuclear microtubules, and was located at the spindle poles. At the end of telophase, it was extruded back into the cytoplasm from the nuclear envelope. Three-dimensional analysis of cells in different cell cycles suggested that duplication of the spindle pole body took place in early G1 phase. Thus, the location, structure, and duplication cycle of the E. dermatitidis spindle pole body were different from those of Saccharomyces cerevisiae.