Keiichiro Shiraga

Quantitative characterization of hydration state and destructuring effect of monosaccharides and disaccharides on water hydrogen bond network

Terahertz time-domain attenuated total reflection measurements of monosaccharide (glucose and fructose) and disaccharide (sucrose and trehalose) solutions from 0.146 M to 1.462 M were performed to evaluate (1) the hydration state and (2) the destructuring effect of saccharide solutes on the hydrogen bond (HB) network. Firstly, the extent of hydration water was determined by the decreased amount of bulk water with picosecond relaxation time that was replaced by that with much longer orientational relaxation time. As a result, we found glucose and trehalose exhibits stronger hydration capacity than fructose and sucrose, respectively, despite of the same number of the hydroxyl groups. For each saccharide, the hydration number tended to decrease with solute concentration. Secondly, the destructuring effect of these saccharide solutes on the HB network of the surrounding bulk water was discussed from the perspective of the fraction of non-hydrogen-bonded (NHB) water isolated from the HB network. We found the fraction of NHB water molecules that are not engaged in the HB network monotonously increased with saccharide concentration, indicating saccharide solutes promote the disruption of the water HB network. However, no noticeable differences were confirmed in the fraction of NHB water between glucose and fructose or between sucrose and trehalose. In contrast to hydration number, the number of NHB water produced by a single saccharide solute was less dependent on solute concentration, and three monosaccharide/disaccharide solutes were found to produce one/two NHB water molecules.

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.

Hydration and hydrogen bond network of water around hydrophobic surface investigated by terahertz spectroscopy

Water conformation around hydrophobic side chains of four amino acids (glycine, L-alanine, L-aminobutyric acid, and L-norvaline) was investigated via changes in complex dielectric constant in the terahertz (THz) region. Each of these amino acids has the same hydrophilic backbone, with successive additions of hydrophobic straight methylene groups (-CH2-) to the side chain. Changes in the degree of hydration (number of dynamically retarded water molecules relative to bulk water) and the structural conformation of the water hydrogen bond (HB) network related to the number of methylene groups were quantitatively measured. Since dielectric responses in the THz region represent water relaxations and water HB vibrations at a sub-picosecond and picosecond timescale, these measurements characterized the water relaxations and HB vibrations perturbed by the methylene apolar groups. We found each successive straight -CH2- group on the side chain restrained approximately two hydrophobic hydration water molecules. Additionally, the number of non-hydrogen-bonded (NHB) water molecules increased slightly around these hydrophobic side chains. The latter result seems to contradict the iceberg model proposed by Frank and Evans, where water molecules are said to be more ordered around apolar surfaces. Furthermore, we compared the water-hydrophilic interactions of the hydrophilic amino acid backbone with those with the water-hydrophobic interactions around the side chains. As the hydrophobicity of the side chain increased, the ordering of the surrounding water HB network was altered from that surrounding the hydrophilic amino acid backbone, thereby diminishing the fraction of NHB water and ordering the surrounding tetrahedral water HB network.

Determination of the complex dielectric constant of an epithelial cell monolayer in the terahertz region

We present a method to determine the complex dielectric constant of a cell monolayer using terahertz time-domain attenuated total reflection spectroscopy combined with a two-interface model. The imaginary part of the dielectric constant of the cell monolayer shows a lower absorption of slow relaxation mode than that of the liquid medium. This result allows us to estimate the intracellular water dynamics on a picosecond time scale, and the existence of weakly hydrated water molecules inside the cell monolayer was indicated. This method will provide a perspective to investigate the intracellular water dynamics in detail.

Hydration state inside HeLa cell monolayer investigated with terahertz spectroscopy

The hydration state in living cells is believed to be associated with various cellular activities. Nevertheless, in vivo characterization of intracellular hydration state under physiological condition has not been well documented to date. In this study, the hydration state of an intact HeLa cell monolayer was investigated by terahertz time-domain attenuated total reflection spectroscopy. Combined with the extended theory of Onsager, we found 23.8 ± 7.4% of HeLa intracellular water was hydrated to biomolecules (corresponding to 1.25 g H2O/g solute); exhibiting slower relaxation dynamics than bulk water.

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).

Inspection of milk components by terahertz attenuated total reflectance (THz-ATR) spectrometer equipped temperature controller

It was confirmed that THz spectroscopy has a potential for quantitative analysis of milk fat, total solid, lactose, milk protein, casein and somatic cell. We investigated the correlation between the absorption spectrum of raw milk in the THz region and the measured milk contents by the Partial Least Square (PLS) regression and full cross validation as test for the calibration models. It was difficult to conduct transmission measurements because the absorption of water in the THz range is large. Thus, we used the attenuated total reflection (ATR) method with a high resistance silicon ATR-prism. This prism equipped a sample temperature controller to reduce the error due to temperature dependent changes of spectra. As the result of this experimentation, the R2 of validation of the best regression model to predict milk fat, total solid, lactose, milk protein, casein and somatic cell were 0.72, 0.80, 0.38, 0.37, 0.41 and 0.67. It was inferred that THz spectroscopy is suitable to predict several milk contents such as milk fat, total solid and somatic cell counts.

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

ABSTRACT 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.

Double-beam CW THz system with photonic phase modulator for sub-THz glucose hydration sensing

We present a double-beam dielectric spectroscopic system using an integrated photonic phase modulator for glucose hydration sensing from 0.25 to 1.0 THz. Compact phase modulators with a photonic circuit and a double-beam scheme using a broadband beam splitter enable us to improve signal stability in sub-THz generation and detection. Using the system, we carried out fast vector measurement on glucose solutions with effective signal stabilization. By incorporating a simple calibration procedure for the extraction of dielectric properties, the Fabry-Perot effect in liquid cell window was mitigated. The system demonstrated accurate detection of glucose dielectric spectra for hydration analysis of glucose-water interaction in sub-THz region.

Sensing living cells using a terahertz split-ring resonator with reflection spectroscopy

Studies with THz split ring resonators (THz-SRRs) have revealed the possibility of using them as sensors to monitor changes in the complex dielectric constant of samples. In this regard, THz reflection spectroscopy in combination with a SRR could be used to measure even target samples in aqueous solution since the interference of waters excessive absorption is minimized with this technique. In this letter, we demonstrate that reflectance intensity at the resonant frequency is more sensitive than frequency shift to changes in the imaginary part of the dielectric constant associated with the absorbance of the sample. Moreover, we distinguished significant changes in reflectance between a cell monolayer in a medium cultured directly onto the SRR and the SRR with medium alone. We confirmed that THz-SRR can be a highly sensitive biosensor to monitor changes in the complex dielectric constant of dielectric samples, such as living cells where abundant extracellular water is present.