Measurement of the rotational relaxation time of intracellular water in dried yeast and Jurkat cells by near infrared spectroscopy.

Molecular mobility of intracellular water is a crucial parameter in the study of the mechanism of desiccation tolerance. As one of the parameters that reflecting molecular mobility, the viscosity of intracellular water has been found intimately related with the protection of the phospholipid membrane because it quantifies the diffusion ability of water and mass in the intracellular environment. In this work we measured the intracellular water relaxation time, which can be translated into water viscosity, by using a previously established NIR-dielectric method to monitor the drying process of baker's yeast and Jurkat cells with different desiccation tolerance. We found that intracellular saccharide can significantly decrease the intracellular water viscosity. Also, the intracellular water diffusion coefficient obtained from this method were found in good agreement with other reports.

Correction: Brosch et al. Glucose and Inositol Transporters, SLC5A1 and SLC5A3, in Glioblastoma Cell Migration. Cancers 2022, 14, 5794.

Philipp Kreisz was not included as an author in the original publication [...].

Slow water dynamics in dehydrated human Jurkat T cells evaluated by dielectric spectroscopy with the Bruggeman-Hanai equation.

The picosecond orientational dynamics of intracellular water was measured by dielectric spectroscopy, with the aim of revealing the effects of cryoprotective agents (CPAs) on biological cells. As a first step, Jurkat cells (human T lymphocyte cells) suspended in aqueous sucrose solutions of different concentrations ranging from 0.3 M (isotonic) to 0.9 M (hypertonic) were examined at 25 °C with a frequency range up to 43.5 GHz. The Bruggeman-Hanai equation was employed to obtain a cellular dielectric spectrum without extracellular contributions from the measured complex permittivity of the cell suspensions. By analyzing the γ process around 1010 Hz based on the Debye relaxation function, two types of water (bulk-like water and hydration water with slower molecular dynamics) were observed. An increase in the fraction of intracellular slower water was observed in the dehydrated cells which had a highly concentrated environment of biomolecules.

Dynamic Electric Field Alignment Determines the Water Rotational Motion around Protein.

Water rotational dynamics in biomolecular solution is crucial to evaluating and controlling biomolecule stability. In this molecular dynamics simulation (MD) study on lysozyme solutions, we present how the exerted internal electric field determines water rotational dynamics. We find that the relaxation time of water rotation is equivalent to that of the reorientation of the exerted overall electric field for every single water molecule, regardless of its translation mode. Namely, water molecular rotation synchronizes with the exerted field reorientation. We also map the reorientation process of the electric field at fixed points relative to protein in the solution, which displays the local hydration dynamics commensurate with the reported time-dependent fluorescence Stokes shift (TDFSS) measurements. Comparing the spatial distribution of local field reorientation relaxation time with that of rotational relaxation time, we further suggest that water rotation dynamics are subject to the reorientation of the local overall field within the hydration layer. While outside the hydration layer, the relaxation time of the local electric field reorientation is short enough (subpicosecond) to assume the δ function, showing the electric force with randomly changing orientation is applied to each water molecule.

Contraction of the rigor actomyosin complex drives bulk hemoglobin expulsion from hemolyzing erythrocytes.

Erythrocyte ghost formation via hemolysis is a key event in the physiological clearance of senescent red blood cells (RBCs) in the spleen. The turnover rate of millions of RBCs per second necessitates a rapid efflux of hemoglobin (Hb) from RBCs by a not yet identified mechanism. Using high-speed video-microscopy of isolated RBCs, we show that electroporation-induced efflux of cytosolic ATP and other small solutes leads to transient cell shrinkage and echinocytosis, followed by osmotic swelling to the critical hemolytic volume. The onset of hemolysis coincided with a sudden self-propelled cell motion, accompanied by cell contraction and Hb-jet ejection. Our biomechanical model, which relates the Hb-jet-driven cell motion to the cytosolic pressure generation via elastic contraction of the RBC membrane, showed that the contributions of the bilayer and the bilayer-anchored spectrin cytoskeleton to the hemolytic cell motion are negligible. Consistent with the biomechanical analysis, our biochemical experiments, involving extracellular ATP and the myosin inhibitor blebbistatin, identify the low abundant non-muscle myosin 2A (NM2A) as the key contributor to the Hb-jet emission and fast hemolytic cell motion. Thus, our data reveal a rapid myosin-based mechanism of hemolysis, as opposed to a much slower diffusive Hb efflux.

Glucose and Inositol Transporters, SLC5A1 and SLC5A3, in Glioblastoma Cell Migration.

(1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.

Decarbonization in Biobanking: A Potential New Scientific Area.

Calls to reduce or entirely remove the carbon footprint of ongoing activities, collectively termed as decarbonization, have become increasingly more vocal in health care with a number of recent, high profile consensus statements. These calls encourage the biobanking field, as one of the foundational health care research infrastructures, to consider decarbonization as a potential novel research area both in terms of the molecules and the equipment used in research. The current article provides a summary of the roundtable discussion during the 2022 ISBER Annual Meeting and Exhibits, highlighting the current knowledge gaps, challenges, and opportunities in this field. In particular, technological innovation, a greater awareness of the current situation, and behavioral change are important pieces of the puzzle to improving the future of decarbonization in biobanking, even if the eventually implemented routes between resource-abundant and resource-restricted settings might be distinctly different. This article sets the foundation for raising awareness of the subject and of subsequent steps that need to be undertaken.

Stochastic Analysis of Molecular Dynamics Reveals the Rotation Dynamics Distribution of Water around Lysozyme.

Water dynamics is essential to biochemical processes by mediating all such reactions, including biomolecular degeneration in solutions. To disentangle the molecular-scale distribution of water dynamics around a solute biomolecule, we investigated here the rotational dynamics of water around lysozyme by combining molecular dynamics (MD) simulations and broadband dielectric spectroscopy (BDS). A statistical analysis using the relaxation times and trajectories of every single water molecule was proposed, and the two-dimensional probability distribution of water at a distance from the lysozyme surface with a rotational relaxation time was given. For the observed lysozyme solutions of 34-284 mg/mL, we discovered that the dielectric relaxation time obtained from this distribution agrees well with the measured γ relaxation time, which suggests that rotational self-correlation of water molecules underlies the gigahertz domain of the dielectric spectra. Regardless of protein concentration, water rotational relaxation time versus the distance from the lysozyme surface revealed that the water rotation is severely retarded within 3 Å from the lysozyme surface and is nearly comparable to pure water when farther than 10 Å. The dimension of the first hydration layer was subsequently identified in terms of the relationship between the acceleration of water rotation and the distance from the protein surface.

Effect of Relaxation Times in a Preservative Solution on Protein Deterioration Rate.

In this study, the relationship between the deterioration of the enzymatic activity of the protein in a preservative solution and the molecular mobility of the preservative solution was experimentally investigated by dielectric spectroscopy and a protein deterioration test. Dielectric spectroscopy was used to observe the molecular mobilities in the preservative solutions including various ratios of two protective agents, trehalose and ε-poly-l-lysine, at 40 °C. We also examined the enzymatic activity of l-lactate dehydrogenase (LDH) at 40 °C for 40 days to obtain the deterioration rate of LDH in the same preservative solutions. Our experimental results reveal that (1) three relaxation times of water molecular motion are detected by dielectric spectroscopy that we categorize into bulky water (relaxation time of 10 ps), weakly hydration water (relaxation time of tens to hundreds of picoseconds), and strongly hydration water (relaxation time of hundreds of picoseconds to tens of microseconds) and (2) the deterioration rate of LDH has a power-law relationship with the relaxation times of bulky and hydration water with specific power indices. The results also support the protein stabilization theory of high viscosity and the practical advantage of predicting the shelf life of proteins in the preservative solution by the relaxation time of water measured by dielectric spectroscopy.

Recrystallization and Water Absorption Properties of Vitrified Trehalose Near Room Temperature.

PURPOSE: To provide the physicochemical properties of vitrified trehalose for predicting its recrystallization. METHODS: Thin films of vitrified trehalose solutions were prepared at room temperature and exposed to various humid and temperature atmospheres. The in-situ amount of retained water in the vacuum-dried trehalose thin film during exposure was determined using its FTIR spectrum by quantifying the extremely infinitesimal amount of retained water in the trehalose solution. Recrystallization of the sample was also assessed by the FTIR spectrum of trehalose dihydrate. RESULTS: The effective water absorption coefficient, h meff , exponentially increased to the water activity of the trehalose sample, A w , at 25°C and 40°C at which the increasing rates are comparable. The surface energy of trehalose dihydrate, γ, was found to be lower than the value calculated from the reported equation, neglecting the effects of the activity of the solute and solvent water. CONCLUSIONS: The retained water in trehalose considerably increases its affinity for water vapor, and the change in this affinity with regard to the water activity is nearly independent of temperature. The dihydrate nucleation rate of trehalose-water system is maximal when trehalose weight ratio is ~0.8 at 25°C and is slightly higher (~0.85) at 40°C.

Electro-microinjection of fish eggs with an immobile capillary electrode.

Microinjection with ultra-fine glass capillaries is widely used to introduce cryoprotective agents and other foreign molecules into animal cells, oocytes, and embryos. The fragility of glass capillaries makes difficult the microinjection of fish eggs and embryos, which are usually protected by a hard outer shell, called the chorion. In this study, we introduce a new electromechanical approach, based on the electropiercing of fish eggs with a stationary needle electrode. The electropiercing setup consists of two asymmetric electrodes, including a µm-scaled nickel needle placed opposite to a mm-scaled planar counter-electrode. A fish egg is immersed in low-conductivity solution and positioned between the electrodes. Upon application of a short electric pulse of sufficient field strength, the chorion is electroporated and the egg is attracted to the needle electrode by positive dielectrophoresis. As a result, the hard chorion and the subjacent yolk membrane are impaled by the sharp electrode tip, thus providing direct access to the egg yolk plasma. Our experiments on early-stage medaka fish embryos showed the applicability of electro-microinjection to fish eggs measuring about 1 mm in diameter. We optimized the electropiercing of medaka eggs with respect to the field strength, pulse duration, and conductivity of bathing medium. We microscopically examined the injection of dye solution into egg yolk and the impact of electropiercing on embryos' viability and development. We also analyzed the mechanisms of electropiercing in comparison with the conventional mechanical microinjection. The new electropiercing method has a high potential for automation, e.g., via integration into microfluidic devices, which would allow a large-scale microinjection of fish eggs for a variety of applications in basic research and aquaculture.

[Physicochemical Environmental Change during Bio-Preservation in Cellular and Biomolecular Suspension Samples and Its Effects on the Sample Quality].

The quality of biospecimens is an important issue for clinical assays. These specimens contain various biomarkers, e.g., cells, proteins, nucleic acids, and phospholipids, most of which start to degrade just after sampling from patients. Because this degradation proceeds in a water-rich condition, under which water, as a solvent, dominates the degradation rate, the samples are often preserved at a low temperature in a frozen, lyophilized, or desiccated state to inhibit the motion of water molecules. However, frozen and/or desiccated water solutions surrounding the biomarkers markedly change the physicochemical environment, and can sometimes accelerate the degradation process. This physicochemical variation in water solutions includes dehydration by freezing or desiccation, osmotic stress by frozen-induced condensation, intra-/extracellular ice formation, and vitrification. This article provides an outline of such physicochemical variation in water solutions and its effects, especially on a fluid specimen, like a blood sample. The outline is composed of three parts after the introduction chapter: 1) general physicochemical changes in the water solution during freezing, frozen storage, and thawing, 2) damage of cells and proteins during freezing, frozen storage, and thawing, and 3) physicochemical changes of the water solution during desiccation and lyophilization and their effects on cells and proteins. As the mechanism of cellular damage is different from that of protein damage, they are discussed separately.

Hypotonic activation of the myo-inositol transporter SLC5A3 in HEK293 cells probed by cell volumetry, confocal and super-resolution microscopy.

Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol P ino [m/s] and expression/localization of SLC5A3. P ino values were determined by cell volumetry over a wide tonicity range (100-275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200-275 mOsm), P ino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼ 3 nm/s at 100-125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in P ino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200-2000 localizations/µm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80-800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.

Changes in the dielectric properties of medaka fish embryos during development, studied by electrorotation.

The Japanese medaka fish, Oryzias latipes, has become a powerful vertebrate model organism in developmental biology and genetics. The present study explores the dielectric properties of medaka embryos during pre-hatching development by means of the electrorotation (ROT) technique. Due to their layered structure, medaka eggs exhibited up to three ROT peaks in the kHz-MHz frequency range. During development from blastula to early somite stage, ROT spectra varied only slightly. But as the embryo progressed to the late-somite stage, the ROT peaks underwent significant changes in frequency and amplitude. Using morphological data obtained by light and electron microscopy, we analyzed the ROT spectra with a three-shell dielectric model that accounted for the major embryonic compartments. The analysis yielded a very high value for the ionic conductivity of the egg shell (chorion), which was confirmed by independent osmotic experiments. A relatively low capacitance of the yolk envelope was consistent with its double-membrane structure revealed by transmission electron microscopy. Yolk-free dead eggs exhibited only one co-field ROT peak, shifted markedly to lower frequencies with respect to the corresponding peak of live embryos. The dielectric data may be useful for monitoring the development and changes in fish embryos' viability/conditions in basic research and industrial aquaculture.

Effect of the grain boundary of ice crystals in a frozen gelatin solution on the dielectric properties at a subzero temperature.

The effect of the grain boundary of ice crystals in a frozen gelatin solution on the dielectric properties was investigated by the combination of a dielectric spectrometer and image analysis. A micro-slicer image processing system (MSIPS) was applied to measure the grain boundary properties as the perimeter density and number density of ice crystals. The perimeter density and number density of the ice crystals increased with increasing freezing rate. The dielectric properties of the frozen gelatin solution at various freezing rates were measured in the frequency range of 100 Hz to 100 kHz at -40 degrees C. The relaxation time did not affect the grain boundary properties. The perimeter density and number density significantly affected dielectric parameter epsilon(0)-epsilon(infinity) and electrical conductivity sigma(0). These results indicate that the dielectric spectrometer could be used to estimate the grain boundary properties in a frozen gelatin solution.

Pore size of swelling-activated channels for organic osmolytes in Jurkat lymphocytes, probed by differential polymer exclusion.

The present study explores the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200-1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions. To this end, we analyzed the cell volume responses to PEG-substituted solutions of different osmolalities (100-300 mOsm) using video microscopy. In parallel experiments, the osmotically induced changes in the membrane capacitance and cytosolic conductivity were measured by electrorotation (ROT). Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane remained impermeable to PEG300-1500, which allowed the cells to accomplish regulatory volume decrease (RVD). During RVD, lymphocytes released intracellular electrolytes through the swelling-activated pathways, as proved by a decrease of the cytosolic conductivity measured by electrorotation. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600-1500, whereas 100 mOsm solutions of PEG300-400 inhibited RVD in Jurkat cells. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300-400, but not to PEG600-1500. The swelling-activated channels conducting PEG300-400 were inserted into the plasma membrane from cytosolic vesicles via swelling-mediated exocytosis, as suggested by an increase of the whole cell capacitance. Using the hydrodynamic radii R(h) of PEGs (determined by viscosimetry), the observed size-selectivity of membrane permeation yielded an estimate of approximately 0.74 nm for the cut-off radius of the swelling-activated channel for organic osmolytes. Unlike PEG300-1500, the smallest PEG (PEG200, R(h)=0.5 nm) permeated the lymphocyte membrane under isotonic conditions thus leading to a continuous isotonic swelling. The results are of interest for biotechnology and biomedicine, where PEGs are widely used for cryopreservation of cells and tissues.

Low O2 metabolism of HepG2 cells cultured at high density in a 3D microstructured scaffold.

Among the features of in vivo liver cells that are rarely mimicked in vitro, especially in microchips, is the very high cell density. In this study, we have cultured HepG2 in a plate-type PDMS scaffold with a three-dimensional ordered microstructure optimally designed to allow cells to attach at a density of 10(8) cells/mL. After the first step of static open culture, the scaffold was sealed to simulate the in vivo oxygen supply, which is supplied only through the perfusion of medium. The oxygen consumption rate at various flow rates was measured. An average maximal cellular oxygen consumption rate of 3.4 x 10(-17) mol/s/cell was found, which is much lower than previously reported values for hepatocytes. Nevertheless, the oxygen concentration in the bulk stream was not the limiting factor. It has been further confirmed by the reported numerical model that the mass transport resistance on the surface of a cell that limits the oxygen supply to the cell. These results further emphasize that access to a sufficient quantity of oxygen, especially through the diffusion-limited layer on the surface of a cell, is very important for the metabolism of hepatocytes at such a high density.

A method for the design of 3D scaffolds for high-density cell attachment and determination of optimum perfusion culture conditions.

The application of in vitro cultured cells in tissue engineering or drug screening, aimed at complex soft tissues such as liver, requires in vivo physiological function of the cultured cells. For this purpose, the scaffold in which cells are cultured should provide a microenvironment similar to an in vivo one with a three-dimensional extracellular matrix, a high supply capacity of O(2) and nutrients, and high cell density. In this paper, we propose a method to design (1) the geometry of the scaffold, with a surface/volume ratio optimized to allow high-density (5 x 10(7)cells/mL) cell culture and (2) culture conditions that will supply optimal quantities of oxygen and nutrients. CFD modeling of mass transport was used to determine the shear stress as well as O(2) and glucose metabolism in the scaffold (20 mm width-35 mm length) for various flow rates. Validation of the model was done through comparison with flow resistance and micro-PIV experiments. CFD analysis showed the maximum metabolic rate densities for this scaffold are 6.04 x 10(-3)mol/s/m(3) for O(2) at 0.71 mL/min and 1.91 x 10(-2)mol/s/m(3) for glucose at 0.35 mL/min.