Quantification of resistant starch content in rice after hydrothermal treatments using terahertz spectroscopy.

Since hydrothermal treatments can enhance resistant starch (RS) content in rice and provide health benefits when consumed, a less laborious and non-destructive method to determine RS content is needed. Terahertz (THz) spectroscopy is hypothesized as a suitable method to quantify RS content in rice after hydrothermal treatment with its sensitivity for the intermolecular forces increase in the formation of RS. In this study, we first used the traditional in vitro hydrolysis method to determine the content of RS in rice. Then, the potential of starch absorbance peaks to quantify RS content after three commonly used hydrothermal methods, soaking, mild heat-moisture treatment, and parboiling, was investigated. The second derivative intensities of the peak at 9.0, 10.5, 12.1, and 13.1 THz were confirmed as being correlated with RS content and showed the high accuracy to predict RS content in samples (R2 > 0.96). Our results indicate the RS content of hydrothermally treated rice can be accurately quantified using these peaks.

Determining changes in crystallinity of rice starch after heat-moisture treatment using terahertz spectroscopy.

To investigate the potential of Fourier-transform terahertz (FT-THz) spectroscopy to follow crystalline structure changes in rice starch after heat-moisture treatment (HMT), we measured the crystallinity by X-ray diffraction (XRD) spectra and found its correlation with THz spectra. According to A-type crystal structure and Vh-type crystalline structure of amylose-lipid complex (ALC) in rice starch, crystallinity is divided into A-type and Vh-type. The intensity of second derivative spectra peak at 9.0 THz was highly correlated with both A-type and Vh-type crystallinity. Additionally, other three peaks at 10.5 THz, 12.2 THz, and 13.1 THz were also sensitive to Vh-type crystalline structure. These results indicate that after HMT, the crystallinity of ALC (Vh-type) and A-type starch can be quantified using THz peaks.

Hydrogen-Bond Configurations of Hydration Water around Glycerol Investigated by HOH Bending and OH Stretching Analysis.

Toward a comprehensive understanding of the mechanism of glycerol as a moisturizer, studies on the hydrogen-bond (HB) structure of hydration water, which is known to be disordered by glycerol, are insufficient. To this aim, we evaluated the HB configurations based on the HOH bending and OH stretching spectra of the hydration water from those of glycerol/water mixtures by subtracting the contributions of bulk water and glycerol using dielectric relaxation spectroscopy. Analysis of the HOH bending band showed that hydration water-donating HBs lose the intermolecular bending coupling with increasing glycerol by replacing the water-water HBs with water-glycerol HBs. The OH stretching band provided more detailed insight into the HB configuration, indicating that the double-donor double-acceptor and double-donor single-acceptor configurations in bulk water change to a predominantly double-donor single-acceptor configuration in hydration water around glycerol. The formation of more donor HBs than acceptor HBs may be due to the steric constrains by glycerol and/or differences in the partial charge on the oxygen atom between water and glycerol.

Increase in the Intracellular Bulk Water Content in the Early Phase of Cell Death of Keratinocytes, Corneoptosis, as Revealed by 65 GHz Near-Field CMOS Dielectric Sensor.

While bulk water and hydration water coexist in cells to support the expression of biological macromolecules, how the dynamics of water molecules, which have long been only a minor role in molecular biology research, relate to changes in cellular states such as cell death has hardly been explored so far due to the lack of evaluation techniques. In this study, we developed a high-precision measurement system that can discriminate bulk water content changes of ±0.02% (0.2 mg/cm3) with single-cell-level spatial resolution based on a near-field CMOS dielectric sensor operating at 65 GHz. We applied this system to evaluate the temporal changes in the bulk water content during the cell death process of keratinocytes, called corneoptosis, using isolated SG1 (first layer of stratum granulosum) cells in vitro. A significant irreversible increase in the bulk water content was observed approximately 1 h before membrane disruption during corneoptosis, which starts with cytoplasmic high Ca2+ signal. These findings suggest that the calcium flux may have a role in triggering the increase in the bulk water content in SG1 cells. Thus, our near-field CMOS dielectric sensor provides a valuable tool to dissect the involvement of water molecules in the various events that occur in the cell.

Japanese dace (Tribolodon hakonensis) fish freshness estimation using front-face fluorescence spectroscopy coupled with chemometric analysis.

Although fish and its related products are good sources of protein and unsaturated fatty acids, like omega-3 in the human diet, their shelf-life is limited by biochemical and microbial changes. In this study, a front-face fluorescence spectroscopy technique was used to acquire Excitation-emission matrices (EEM) to monitor Japanese dace (Tribolodon hakonensis) fish freshness degradation during storage. EEM of Japanese dace fish parts (intact eyeball and surface-containing scales), excitation from 220 to 585 nm and emissions from 250 to 600 nm, were measured at different times during storage. To simplify the acquired complex spectra datasets from each fish part, the variables were reduced to those that were only significant/important (those with higher positive or negative correlation) for K value prediction, and as an index of freshness. Partial least square regression (PLSR) results demonstrated that combining the fluorescence EEM of the eyeball and surface-containing scales the best monitoring of fish freshness; excitation at 280 and 350 nm for both the eyeball and surface-containing scales, with 2.84 and 0.96 as RMSE and R2, respectively. These findings demonstrate that multiple excitation fluorescence approaches can be convenient for the freshness evaluation of fish.

A unique mode of keratinocyte death requires intracellular acidification.

The stratum corneum (SC), the outermost epidermal layer, consists of nonviable anuclear keratinocytes, called corneocytes, which function as a protective barrier. The exact modes of cell death executed by keratinocytes of the upper stratum granulosum (SG1 cells) remain largely unknown. Here, using intravital imaging combined with intracellular Ca2+- and pH-responsive fluorescent probes, we aimed to dissect the SG1 death process in vivo. We found that SG1 cell death was preceded by prolonged (∼60 min) Ca2+ elevation and rapid induction of intracellular acidification. Once such intracellular ionic changes were initiated, they became sustained, irreversibly committing the SG1 cells to corneocyte conversion. Time-lapse imaging of isolated murine SG1 cells revealed that intracellular acidification was essential for the degradation of keratohyalin granules and nuclear DNA, phenomena specific to SC corneocyte formation. Furthermore, intravital imaging showed that the number of SG1 cells exhibiting Ca2+ elevation and the timing of intracellular acidification were both tightly regulated by the transient receptor potential cation channel V3. The functional activity of this protein was confirmed in isolated SG1 cells using whole-cell patch-clamp analysis. These findings provide a theoretical framework for improved understanding of the unique molecular mechanisms underlying keratinocyte-specific death mode, namely corneoptosis.

Dynamical Collectivity and Nuclear Quantum Effects on the Intermolecular Stretching Mode of Liquid Water.

This study investigated the broadband terahertz and low-frequency Raman spectroscopy of liquid water (H2O, D2O, and H218O) over 2 decades of frequency to address long-standing challenges regarding the interpretation of the intermolecular stretching mode at around 5 THz. We experimentally demonstrated that the intermolecular stretching mode of liquid water obtained via terahertz spectroscopy is significantly redshifted and broadened compared with that via Raman. This result was rationalized by the enhanced dynamical collectivity probed by terahertz spectroscopy, although both have a common origin in the kinetic motion. Their temperature and isotope dependences emphasize the significance of oscillation mass in determining the intermolecular stretching lineshape, while quantum effects cannot be overlooked in both terahertz and low-frequency Raman spectra.

Near-field sensor array with 65-GHz CMOS oscillators for rapid detection of viable Escherichia coli.

In this study, the growth of Escherichia coli was monitored using a complementary metal-oxide-semiconductor (CMOS) near-field sensor array. Each of the 1488 integrated elements, arranged in a 3 mm square, has a resonator that oscillates at 65 GHz. The effective capacitance of the resonator is altered by changes in the dielectric properties of the sensor surface, which shifts the resonance frequency. Growth curves of E. coli at different initial concentrations (OD600 = 0.01, 0.03, and 0.05) were monitored. A suspension with initial turbidity of OD600 = 0.05 was cultured in a medium, and the sensor successfully distinguished between viable E. coli and heat-treated dead E. coli in 20 min. Moreover, the apparent suppression of growth was observed in the presence of 500 µg/mL streptomycin. As the sensor is composed of arrayed elements, and the area of sensitivity distribution of the element is larger than the size of one bacteria, the variation in the output value of each element may reflect the number and movement of bacteria. This study revealed that the presence of viable E. coli could be rapidly confirmed by using the change in permittivity caused by the displacement of media by E. coli near the sensor surface.

Highly precise characterization of the hydration state upon thermal denaturation of human serum albumin using a 65 GHz dielectric sensor.

The biological functions of proteins depend on harmonization with hydration water surrounding them. Indeed, the dynamical transition of proteins, such as thermal denaturation, is dependent on the changes in the mobility of hydration water. However, the role of hydration water during dynamical transition is yet to be fully understood due to technical limitations in precisely characterizing the amount of hydration water. A state-of-the-art CMOS dielectric sensor consisting of 65 GHz LC resonators addressed this issue by utilizing the feature that oscillation frequency sensitively shifts in response to the complex dielectric constant at 65 GHz with extremely high precision. This study aimed to establish an analytical algorithm to derive the hydration number from the measured frequency shift and to demonstrate the transition of hydration number upon the thermal denaturation of human serum albumin. The determined hydration number in the native state drew a "global" hydration picture beyond the first solvation shell, with substantially reduced uncertainty of the hydration number (about ±1%). This allowed the detection of a rapid increase in the hydration number at about 55 °C during the heating process, which was in excellent phase with the irreversible rupture of the α-helical structure into solvent-exposed extended chains, whereas the hydration number did not trace the forward path in the subsequent cooling process. Our result indicates that the weakening of water hydrogen bonds trigger the unfolding of the protein structure first, followed by the changes in the number of hydration water as a consequence of thermal denaturation.

Quantification of starch content in germinating mung bean seedlings by terahertz spectroscopy.

To investigate the potential of terahertz spectroscopy to monitor and quantify starch in plants, terahertz spectra (3.0-13.5 THz) of mung bean plants 1-7 days after germination were examined and compared to those of starch and its constituent saccharides (standard reagents). Day 1 seedlings showed similar spectral features with standard starch, and absorption peaks gradually disappeared in the subsequent 6 day growth period. To interpret this result and identify useful peaks for starch quantification, standard starch and day 1 seedlings were hydrolyzed by α-amylase in vitro. Since both standard starch and seedlings showed that absorption peak at 9.0 THz disappeared after amylase hydrolysis, this peak is sensitive to changes in starch. Additionally, intensity of this peak was correlated with starch content as quantified by chemical analysis (r = 0.98). Our results indicate terahertz spectra of seedlings can provide an identifiable peak that is attributed to starch and not affected by the constituent saccharides.

Reconsideration of the relaxational and vibrational line shapes of liquid water based on ultrabroadband dielectric spectroscopy.

The Debye relaxation function is widely used to describe the large dielectric dispersion of ambient water around 20 GHz. However, from a theoretical point of view, this function is supposed to give incorrect predictions at high frequencies owing to the inappropriate assumption that inertial effects and intermolecular interactions do not affect the relaxation dynamics. Our ultrabroadband spectroscopy investigation of liquid water ranging from 500 MHz to 400 THz did demonstrate that the Debye function is inaccurate far above the microwave region. As an alternative, we tried a stochastic frequency modulation (SFM) model assuming instantaneous modification of the line shapes by the correlation with the surrounding system. The SFM relaxation model reproduced the experimental dielectric spectra up to 400 THz, showing that the hydrogen-bond dynamics are associated with the inertial effect that causes the non-exponential relaxation behaviour in a very short time (typically 25 fs). Within the framework of this relaxation model, the hindered translation modes are able to be approximated as fast modulation (homogeneous) line shapes because the interaction time with frequency modulation is too short. Compared with them, the libation mode is found to have a relatively slow modulation (inhomogeneous) origin, where disturbance of water hydrogen bonds induced by the hindered translations leads to fluctuations in the libration frequency.

Coexistence of Kosmotropic and Chaotropic Impacts of Urea on Water As Revealed by Terahertz Spectroscopy.

Whether urea can serve as a kosmotrope or chaotrope has long been a topic of debate. In this study, broad-band THz spectroscopy (0.2-12 THz) of aqueous solutions of urea was used to characterize the hydration state and the hydrogen bond structure of water around urea. Three low-frequency vibration modes of urea were found around 2, 4, and above 12 THz. After eliminating the contribution of these modes, the "urea-vibration-free" complex dielectric constant was decomposed into the relaxation modes of bulk water and the oscillation modes of water. When hydration water is defined to be reorientationally retarded relative to bulk, our analysis revealed that the hydration number is 1.9 independent of urea concentrations up to 5 M, and this number is in close agreement with that of water constrained by strong acceptor hydrogen bonds of urea oxygen. Regarding the hydrogen bond structure, it was found that the tetrahedral-like water structure is mostly preserved (though the hydrogen bond lifetime is significantly shortened) but the population of non-hydrogen-bonded water molecules fragmented from the network is markedly increased, presumably due to urea's NH2 inversion. These experimental results point to the coexistence of apparently two contradictory aspects of urea: dynamical retardation (the kosmotropic aspect) by the -CO group and slight structural disturbance (the chaotropic aspect) by the -NH2 group.

The Effect of Lactoferrin and Pepsin-Treated Lactoferrin on IEC-6 Cell Damage Induced by Clostridium Difficile Toxin B.

Clostridium difficile infections (CDI) have recently increased worldwide. Some CDI progress to fulminant and recurrent CDI and are associated with high mortality and morbidity. CD produces toxins A and B, which cause intestinal mucosal damage, although toxin B exhibits greater cytotoxicity. Pepsin-treated lactoferrin (PLF) is the decomposed product of lactoferrin (LF), a multifunctional glycoprotein with anti-inflammatory properties. Here, we investigate the effects of LF and PLF in toxin B-stimulated rat intestinal epithelial (IEC-6) cells. Different toxin B concentrations were added to IEC-6 cells with or without LF or PLF. Mitochondrial function and cell cytotoxicity were assessed by measuring WST-1 and LDH levels, respectively. WST-1 levels were higher in IEC-6 cells treated with toxin B and LF or PLF than in the toxin B-only control (P < 0.05). Compared with the toxin B-only control, LDH levels significantly decreased after toxin B and LF or PLF addition (P < 0.05). Wound restitution measurement using microscopy demonstrated significantly greater levels of wound restitution in cells treated with toxin B and LF or PLF than in those treated with toxin B alone after 12 h (P < 0.001). Furthermore, changes in IEC-6 cell tight junctions (TJs) were evaluated by immunofluorescence microscopy and zonula occludens-1 (ZO-1) protein expression. When LF or PLF were added to IEC-6 cells, TJ structures were maintained, and ZO-1 and occludin expression was upregulated. Taken together, these results demonstrate that LF and PLF prevent the cytotoxicity of toxin B and might have the potential to control CDI.

Characterization of the hydrogen-bond network of water around sucrose and trehalose: Microwave and terahertz spectroscopic study.

Modification of the water hydrogen bond network imposed by disaccharides is known to serve as a bioprotective agent in living organisms, though its comprehensive understanding is still yet to be reached. In this study, aiming to characterize the dynamical slowing down and destructuring effect of disaccharides, we performed broadband dielectric spectroscopy, ranging from 0.5 GHz to 12 THz, of sucrose and trehalose aqueous solutions. The destructuring effect was examined in two ways (the hydrogen bond fragmentation and disordering) and our result showed that both sucrose and trehalose exhibit an obvious destructuring effect with a similar strength, by fragmenting hydrogen bonds and distorting the tetrahedral-like structure of water. This observation strongly supports a chaotropic (structure-breaking) aspect of disaccharides on the water structure. At the same time, hydration water was found to exhibit slower dynamics and a greater reorientational cooperativity than bulk water because of the strengthened hydrogen bonds. These results lead to the conclusion that strong disaccharide-water hydrogen bonds structurally incompatible with native water-water bonds lead to the rigid but destructured hydrogen bond network around disaccharides. Another important finding in this study is that the greater dynamical slowing down of trehalose was found compared with that of sucrose, at variance with the destructuring effect where no solute dependent difference was observed. This discovery suggests that the exceptionally greater bioprotective impact especially of trehalose among disaccharides is mainly associated with the dynamical slowing down (rather than the destructuring effect).

Hydrogen Bond Network of Water around Protein Investigated with Terahertz and Infrared Spectroscopy.

The dynamical and structural properties of water at protein interfaces were characterized on the basis of the broadband complex dielectric constant (0.25 to 400 THz) of albumin aqueous solutions. Our analysis of the dielectric responses between 0.25 and 12 THz first revealed hydration water with retarded reorientational dynamics extending ∼8.5 Å (corresponding to three to four layers) out from the albumin surface. Second, the number of nonhydrogen-bonded water was decreased in the presence of the albumin solute, indicating protein inhibits the fragmentation of the water hydrogen-bond network. Finally, water molecules at the albumin interface were found to form a distorted hydrogen-bond structure due to topological and energetic disorder of the protein surface. In addition, the intramolecular O-H stretching vibration of water (∼100 THz), which is sensitive to hydrogen-bond environment, pointed to a trend that hydration water has a larger population of strongly hydrogen-bonded water molecules compared with that of bulk water. From these experimental results, we concluded that the "strengthened" water hydrogen bonds at the protein interface dynamically slow down the reorientational motion of water and form the less-defective hydrogen-bond network by inhibiting the fragmentation of water-water hydrogen bonds. Nevertheless, such a strengthened water hydrogen-bond network is composed of heterogeneous hydrogen-bond distances and angles, and thus characterized as structurally "distorted."

Broadband dielectric spectroscopy of glucose aqueous solution: Analysis of the hydration state and the hydrogen bond network.

Recent studies of saccharides' peculiar anti-freezing and anti-dehydration properties point to a close association with their strong hydration capability and destructuring effect on the hydrogen bond (HB) network of bulk water. The underlying mechanisms are, however, not well understood. In this respect, examination of the complex dielectric constants of saccharide aqueous solutions, especially over a broadband frequency region, should provide interesting insights into these properties, since the dielectric responses reflect corresponding dynamics over the time scales measured. In order to do this, the complex dielectric constants of glucose solutions between 0.5 GHz and 12 THz (from the microwave to the far-infrared region) were measured. We then performed analysis procedures on this broadband spectrum by decomposing it into four Debye and two Lorentz functions, with particular attention being paid to the ß relaxation (glucose tumbling), δ relaxation (rotational polarization of the hydrated water), slow relaxation (reorientation of the HB network water), fast relaxation (rotation of the non-HB water), and intermolecular stretching vibration (hindered translation of water). On the basis of this analysis, we revealed that the hydrated water surrounding the glucose molecules exhibits a mono-modal relaxational dispersion with 2-3 times slower relaxation times than unperturbed bulk water and with a hydration number of around 20. Furthermore, other species of water with distorted tetrahedral HB water structures, as well as increases in the relative proportion of non-HB water molecules which have a faster relaxation time and are not a part of the surrounding bulk water HB network, was found in the vicinity of the glucose molecules. These clearly point to the HB destructuring effect of saccharide solutes in aqueous solution. The results, as a whole, provide a detailed picture of glucose-water and water-water interactions in the vicinity of the glucose molecules at various time scales from sub-picosecond to hundreds of picoseconds.

Hydration and Hydrogen Bond Network of Water during the Coil-to-Globule Transition in Poly(N-isopropylacrylamide) Aqueous Solution at Cloud Point Temperature.

Aqueous solutions of poly(N-isopropylacrylamide), P-NIPAAm, exhibit a noticeable temperature responsive change in molecular conformation at a cloud point temperature (Tcp). As the temperature rises above Tcp, the extended coil-like P-NIPAAm structure changes into a swollen globule-like conformation as hydration levels decrease and hydrophobic interactions increase. Though water plays an important role in this coil-to-globule transition of P-NIPAAm, the behavior of water molecules and the associated hydrogen-bond (HB) network of the surrounding bulk water are still veiled in uncertainty. In this study, we elucidate changes in the hydration state and the dynamical structure of the water HB network of P-NIPAAm aqueous solutions during the coil-to-globule transition by analyzing the complex dielectric constant in the terahertz region (0.25-12 THz), where bulk water reorientations and intermolecular vibrations of water can be selectively probed. The structural properties of the water HB network were examined in terms of the population of the non-HB water molecules (not directly engaged in the HB network or hydrated to P-NIPAAm) and the tetrahedral coordination of the water molecules engaged in the HB network. We found the hydration number below Tcp (≈10) was decreased to approximately 6.5 as temperature increased, in line with previous studies. The HB network of bulk water becomes more structured as the coil-to-globule phase transition takes place, via decreases in non-HB water and reduction in the orderliness of the tetrahedral HB architecture. Together these results indicate that the coil-to-globule transition is associated with a shift to hydrophobic-dominated interactions that drive thermoresponsive structural changes in the surrounding water molecules.

Evaluation of the hydration state of saccharides using terahertz time-domain attenuated total reflection spectroscopy.

Despite intensive studies regarding the hydration state, experimental investigations have not fully explained the global hydration state. Terahertz (THz) spectroscopy is an emerging technology that has the potential to evaluate global hydration. This is because THz waves are very sensitive to picosecond water dynamics and, as such, effectively measures the state of water that is weakly bound to solute molecules by observing slowed down water dynamics. THz time-domain attenuated total reflection (THz TD-ATR) spectroscopy allowed to determine the complex refractive index of saccharide solutions and to experimentally characterize the global hydration state. Our result indicates the global hydration state is closely related to the number of hydrophilic groups and steric configuration of hydroxyl groups in saccharide molecules. This new tool to investigate the global hydration state will provide new knowledge about water dynamics around solutes that couldn't have been elucidated with the conventional techniques.