Measurement of grouped intracellular solute osmotic virial coefficients

Models of cellular osmotic behaviour depend on thermodynamic solution theories to calculate chemical potentials in the solutions inside and outside the cell. These solutions are generally thermodynamically non-ideal under cryobiological conditions. The molality-based Elliott et al. form of the multi-solute osmotic virial equation is a solution theory which has been demonstrated to provide accurate predictions in cryobiological solutions, accounting for the non-ideality of these solutions using solute-specific thermodynamic parameters called osmotic virial coefficients. However, this solution theory requires as inputs the exact concentration of every solute in the solution being modeled, which poses a problem for the cytoplasm, where such detailed information is rarely available. This problem can be overcome by using a grouped solute approach for modeling the cytoplasm, where all the non-permeating intracellular solutes are treated as a single non-permeating “grouped” intracellular solute. We have recently shown (Zielinski et al., J Physical Chemistry B, 2017) that such a grouped solute approach is theoretically valid when used with the Elliott et al. model, and Ross-Rodriguez et al. (Biopreservation and Biobanking, 2012) have previously developed a method for measuring the cell type-specific osmotic virial coefficients of the grouped intracellular solute. However, the Ross-Rodriguez et al. method suffers from a lack of precision, which—as we demonstrate in this work—can severely impact the accuracy of osmotic model predictions under certain conditions. Thus, we herein develop a novel method for measuring grouped intracellular solute osmotic virial coefficients which yields more precise values than the existing method and then apply this new method to measure these coefficients for human umbilical vein endothelial cells.     

Estimation of Water Diffusion Coefficients in Freeze-Concentrated Matrices of Sugar Solutions Using Molecular Dynamics: Correlation Between Estimated Diffusion Coefficients and Measured Ice-Crystal Recrystallization Rates

The diffusion coefficient of the water component in a freeze-concentrated matrix is a useful parameter for predicting and controlling the recrystallization rate of ice crystals in sugar solutions relevant to frozen desserts. Herein, application of molecular dynamics (MD) for estimating the water diffusion coefficient in a freeze-concentrated matrix of sugar solutions is described. Diffusion coefficients evaluated using MD with the optimized potentials for liquid simulations all atom force field and water models of three types (simple point charge, simple point charge extended, and transferable intermolecular potential-4 point) show a good positive linear relation with measured values, indicating that the MD methods used in this study are useful for predicting differences in water diffusion coefficients in a sugar freeze-concentrated matrix. Furthermore, similarly to measured values, the estimated diffusion coefficients show a good positive correlation with recrystallization rates of ice crystals, which suggests that MD is useful to predict differences in recrystallization rates of ice crystals in frozen sugar solutions.     

Effects of solution composition on the theoretical prediction of ice nucleation kinetics and thermodynamics

Predictions by various mathematical models of intracellular ice formation (proposed by Mazur, Pitt, Toner, and Karlsson, respectively) were compared to the known thermodynamic and kinetic behavior of ice formation in supercooled aqueous systems. The older models (Mazur, Pitt, and Toner) significantly underestimated the magnitude of colligative nonequilibrium freezing point depression in response to increased concentration of solutes, such as salts or cryoprotectants. Furthermore, kinetics predicted using phenomenological models (by Mazur and Pitt) exhibited implausible temperature-dependence, with the probability of intracellular ice formation being allowed to increase even at temperatures below the glass transition point. The Toner model, on the other hand, produced invalid results at temperatures below −48 °C. The Karlsson model was the only model that consistently yielded realistic predictions over a wide range of temperatures and solute concentrations, especially in the presence of cryoprotectant additives. To facilitate adoption of the Karlsson model of intracellular ice nucleation, the complete set of model equations has been collected and described in detail.     

Some ways of looking at compensatory kosmotropes and different water environments

Hydration of macromolecular structures determines biological activity. Stabilizing solutes are kosmotropic (increase order of water) rather than chaotropic (decrease order). Preferential hydration of surfaces is a thermodynamic consequence of the solution behavior of kosmotropic solutes, but inconsistencies imply interactions such as the hydration of specific sites within macromolecules. Thermodynamic measures require bulk pure solutes; here simpler measures of the effects on bulk water, water at surfaces and hydration water of probes have been applied to solutes including natural stabilizers, analogues and example chaotropes. Changes in the near-infrared spectra, water proton NMR chemical shifts and relaxation times measure changes in the bulk liquid; HPLC-column retention of solutes indicate interactions with hydration water at different surfaces, and fluorescence probes detect effects on functional group hydration water. Ab initio calculations and Monte-Carlo simulations of the solutes in water measure the energetics of the solute-water interactions, the dipole moments of these molecules, their charge distributions and the effect of the solute molecules on the structure of water. The rankings of the test solutes by these measures are not consistent. Thus, stabilizing solutes are not interchangeable in biological systems and the intracellular replacement of one by another could affect the integration of cell metabolism.     

Evidence for Ideal and Non-Ideal Equilibrium Freezing of Leaf Water in Frosthardy Ivy (Hedera helix) and Winter Barley (Hordeum vulgare)

Temperature dependences of leaf water potentials (ψ_leaf) of frozen leaves of frosthardy ivy and winter barley were determined psychrometrically and found to coincide with the respective water potentials of ice which were obtained using the same technique. The water potentials of ice showed good agreement with theoretically established data. Analysis of the components of ψ of frozen leaves of Hedera helix revealed ideal equilibrium freezing, i.e. the governing of the relative content of liquid (or frozen) water solely by the osmotic potential. In winter barley, by contrast, a negative pressure potential was demonstrated to contribute to ψ_leaf. even under conditions of moderate frost. This reduced the degree of protoplast dehydration and the extent to which the concentrations of the cellular solutes rose. Such a freezing behavior is termed non-ideal equilibrium freezing. Depending on the original content of leaf water, the volume increments of liquid water due to the negative pressure potential amounted up to 10% at ?6 °C and even more at a lower temperature. In addition to the experimental data, a theoretical treatment of psychrometry at subzero temperatures is presented.     

Effects of a type I antifreeze protein (AFP) on the melting of frozen AFP and AFP+solute aqueous solutions studied by NMR microimaging experiment

The effects of a type I AFP on the bulk melting of frozen AFP solutions and frozen AFP+solute solutions were studied through an NMR microimaging experiment. The solutes studied include sodium chloride and glucose and the amino acids alanine, threonine, arginine, and aspartic acid. We found that the AFP is able to induce the bulk melting of the frozen AFP solutions at temperatures lower than 0 °C and can also keep the ice melted at higher temperatures in the AFP+solute solutions than those in the corresponding solute solutions. The latter shows that the ice phases were in super-heated states in the frozen AFP+solute solutions. We have tried to understand the first experimental phenomenon via the recent theoretical prediction that type I AFP can induce the local melting of ice upon adsorption to ice surfaces. The latter experimental phenomenon was explained with the hypothesis that the adsorption of AFP to ice surfaces introduces a less hydrophilic water-AFP-ice interfacial region, which repels the ionic/hydrophilic solutes. Thus, this interfacial region formed an intermediate chemical potential layer between the water phase and the ice phase, which prevented the transfer of water from the ice phase to the water phase. We have also attempted to understand the significance of the observed melting phenomena to the survival of organisms that express AFPs over cold winters.     

Osmotic relations of the drought-tolerant shrub Artemisia tridentata in response to water stress

Turgor maintenance, solute content and recovery from water stress were examined in the drought-tolerant shrub Artemisia tridentata. Predawn water potentials of shrubs receiving supplemental water remained above ?2 MPa throughout summer, while predawn water potentials of untreated shrubs decreased to ?5 MPa. Osmotic potentials decreased in conjunction with water potentials maintaining turgor pressures above 0 MPa. The decreases in osmotic potentials were not the result of osmotic adjustment (i.e. solute accumulation). Leaf solute contents decreased during drought, but leaf water volumes decreased more than 75% from spring to summer, thereby passively concentrating solutes within the leaves. The maintenance of positive turgor pressures despite decreases in leaf water volumes is consistent with other studies of species with elastic cell walls. Inorganic ion, organic acid, and carbohydrate contents of leaves declined during drought. The only solutes accumulating in leaves of A. tridentata with water stress were proline and a cyclitol, both considered compatible solutes. Total and osmotic potentials recovered rapidly following rewatering of shrubs; solute contents did not change except for a decrease in proline. Maintaining turgor through the passive concentration of solutes may be advantageous compared to synthesis of new solutes for osmotic adjustment in arid environments.     

Exclusion and retention of compensatory kosmotropes by HPLC columns

With water as the elution solvent, zwitterionic solutes and polyols were retained on HPLC columns, more than was water, by totally hydrophobic packing materials. Relative retentions were systematically affected by oxygen functional groups in the packing material, explicable as specific retention of water. Reproducible elution sequences of 20 solutes at a variety of hydrophobic surfaces (aromatic and both long- and short-alkyl aliphatic surfaces) showed there is a general process, consistent with interactions with hydration water at the surface having solvent properties distinct from bulk water. Early eluting solutes included glycine, sarcosine and taurine. Glycine betaine followed both these and N,N-dimethylglycine. The natural betaines propionobetaine and dimethylsulfoniopropionate also preceded glycine betaine. Dimethylsulfoxide was strongly retained, as (to a lesser extent) was proline betaine. Polyols eluted in the sequence sorbitol, trehalose, glycerol. Changes in the chemical nature of the surface or base material affected relative retentions of water and solutes. The presence of hydrogen-bonding functions increased retention of polyols, as well as water, relative to zwitterionic solutes. Specific effects with some solutes may be related to inconsistencies seen in biological systems. Pressures up to 8 MPa did not affect relative retention, constraining models based on the formation of low-density water.     

Carbon-hydrogen and carbon-heteroatom bond activation using iridium(I) complexes

In this research, the B3LYP density functional and Stevens effective core potentials are used to compare carbon-hydrogen and carbon-heteroatom bond activation by an iridium(I) complex. Of particular importance is to address the kinetic (transition state) and thermodynamic (ground state) selectivity. The complex Ir(PH₃)₂(H) with CH₃-X (X=F, Cl, OH, SH, NH₂, PH₂) as the substrate has been used as a model. Good agreement in geometries is obtained between the target molecules and experimental models. The resultant products of C-H and C-X oxidative addition are Y-shaped minima (i.e., a distorted trigonal bipyramid with one acute and two obtuse angles among the equatorial ligands). Oxidative addition of the C-X bond to the substrate is exothermic for groups 16 and 17, but endothermic for group 15. A significant thermodynamic preference for C-X activation over C-H activation is observed for these Ir(I) complexes. However, analysis of the transition states for oxidative addition suggests that there is a kinetic preference for C-H activation.     

Recrystallization of Ice Crystals in Trehalose Solution at Isothermal Condition

An isothermal ice recrystallization behavior in trehalose solution was investigated. The isothermal recrystallization rate constants of ice crystals in trehalose solution were obtained at ?5 °C, ?7 °C, and ?10 °C. Then the results were compared to those of a sucrose solution used as a control sample. Simultaneous estimation of water mobility in the freeze-concentrated matrix was conducted by ¹H spin–spin relaxation time T₂ to investigate mechanisms causing the different ice crystal recrystallization behaviors of sucrose and trehalose. At lower temperatures, lower recrystallization rates were obtained for both trehalose and sucrose solutions. The ice crystallization rate constants in trahalose solution tended to be smaller than those in sucrose solution at the same temperature. Although different ice contents (less than 3.6%) were observed between trehalose and sucrose solutions at the same temperature, the recrystallization behaviors of ice crystals were not markedly different. The ¹H spin–spin relaxation time T₂ of water components in a freeze-concentrated matrix for trehalose solution was shorter than in a sucrose solution at the same temperature. Results show that the water mobility of trehalose solutions in freeze-concentrated matrix was less than that of sucrose solutions, which was suggested as the reason for retarded ice crystal growth in a trehalose solution. Results of this study suggest that the replacement of sucrose with trehalose will not negatively affect deterioration caused by ice crystal recrystallization in frozen foods and cryobiological materials.     

The importance of heat flow direction for reproducible and homogeneous freezing of bulk protein solutions

Freezing is an important operation in biotherapeutics industry. However, water crystallization in solution, containing electrolytes, sugars and proteins, is difficult to control and usually leads to substantial spatial solute heterogeneity. Herein, we address the influence of the geometry of freezing direction (axial or radial) on the heterogeneity of the frozen matrix, in terms of local concentration of solutes and thermal history. Solutions of hemoglobin were frozen radially and axially using small‐scale and pilot‐scale freezing systems. Concentration of hemoglobin, sucrose and pH values were measured by ice‐core sampling and temperature profiles were measured at several locations. The results showed that natural convection is the major source for the cryoconcentration heterogeneity of solutes over the geometry of the container. A significant improvement in this spatial heterogeneity was observed when the freezing geometry was nonconvective, i.e., the freezing front progression was unidirectional from bottom to top. Using this geometry, less than 10% variation in solutes concentration was obtained throughout the frozen solutions. This result was reproducible, even when the volume was increased by two orders of magnitude (from 30 mL to 3 L). The temperature profiles obtained for the nonconvective freezing geometry were predicted using a relatively simple computational fluid dynamics model. The reproducible solutes distribution, predictable temperature profiles, and scalability demonstrate that the bottom to top freezing geometry enables an extended control over the freezing process. This geometry has therefore shown the potential to contribute to a better understanding and control of the risks inherent to frozen storage. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1212–1221, 2013     

Counteracting effects of renal solutes on amyloid fibril formation by immunoglobulin light chains

In primary (light chain-associated) amyloidosis, immunoglobulin light chains deposit as amyloid fibrils in vital organs, especially the kidney. Because the kidney contains high concentrations of urea that can destabilize light chains as well as solutes such as betaine and sorbitol that serve as protein stabilizers, we investigated the effects of these solutes on in vitro amyloid fibril formation and thermodynamic stability of light chains. Two recombinant light chain proteins, one amyloidogenic and the other nonamyloidogenic, were used as models. For both light chains, urea enhanced fibril formation by reducing the nucleation lag time and diminished protein thermodynamic stability. Conversely, betaine or sorbitol increased thermodynamic stability of the proteins and partially inhibited fibril formation. These solutes also counteracted urea-induced reduction in protein thermodynamic stability and accelerated fibril formation. Betaine was more effective than sorbitol. A model is presented to explain how the thermodynamic effects of the solutes on protein state equilibria can alter nucleation lag time and, hence, fibril formation kinetics. Our results provide evidence that renal solutes control thermodynamic and kinetic stability of light chains and thus may modulate amyloid fibril formation in the kidney.     

Cohesive sediment transport: emerging issues for toxic chemical management

The association of many environmentally sensitive chemicals and their transformation products with mineral and organic substrates is of considerable importance for environmental monitoring, prediction and management purposes in rivers and their basins. Our understanding of these relationships is poor. This paper reviews processes of particular concern, including the physical behaviour of fine-grained (< 63 µm) sediment in freshwater; the role of flocculation as a transport vector; the processes that control freshwater flocculation including microbiological factors; the uncertainty in conventional sediment transport models for predicting pathways of sediment-associated chemistry; the relationship between suspended sediment and toxicity in the water column; and the partitioning of chemicals between the sediment, organic and water phase, including the significance of these in predicting chemical transport on suspended matter.     

Relationship between intracellular ice formation in oocytes of the mouse and Xenopus and the physical state of the external medium--a revisit

We have previously reported that intracellular ice formation (IIF) in mouse oocytes suspended in glycerol/PBS solutions or ethylene glycol (EG)/PBS solutions and rapidly cooled to −50 °C or below occurs at temperatures where a critical fraction of the external water remains unfrozen [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29-53; P. Mazur, I.L. Pinn, F.W. Kleinhans, The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature, Cryobiology 54 (2007) 223-233]. For mouse oocytes in PBS or glycerol/PBS that fraction is 0.06; for oocytes in EG that fraction was calculated to be 0.13, more than double. The fractions unfrozen are computed from ternary phase diagrams. In the previous publication, we used the EG data of Woods et al. [E.J. Woods, M.A.J. Zieger, D.Y. Gao, J.K. Critser, Equations for obtaining melting points for the ternary system ethylene glycol/sodium chloride/Water and their application to cryopreservation., Cryobiology 38 (1999) 403-407]. Since then, we have determined that ternary phase diagrams for EG/NaCl/water synthesized by summing binary phase data for EG/water NaCl/water gives substantially different curves, which seem more realistic [F.W. Kleinhans, P. Mazur, Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest, Cryobiology 54 (2007) 212-222]. Unfrozen fractions at the temperatures of IIF computed from these synthesized phase diagrams are about half of those calculated from the Woods et al. data, and are in close agreement with the computations for glycerol; i.e., IIF occurs when about 92-94% of the external water is frozen. A parallel paper was published by Guenther et al. [J.F. Guenther, S. Seki, F.W. Kleinhans, K. Edashige, D.M. Roberts, P. Mazur, Extra-and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes, Cryobiology 52 (2006) 401-416] on IIF in oocytes of the frog Xenopus. It too examined whether the temperatures of IIF were related to the unfrozen fractions at those temperatures. It also used the Woods et al. ternary phase data to calculate the unfrozen fractions for EG solutions. As reported here, once again the values of these unfrozen fractions are substantially different from those calculated using synthesized phase diagrams. With the latter, the unfrozen fractions at IIF become very similar for EG and glycerol.     

Identification of trehalose as a compatible solute in different species of acidophilic bacteria

The major industrial heap bioleaching processes are located in desert regions (mainly Chile and Australia) where fresh water is scarce and the use of resources with low water activity becomes an attractive alternative. However, in spite of the importance of the microbial populations involved in these processes, little is known about their response or adaptation to osmotic stress. In order to investigate the response to osmotic stress in these microorganisms, six species of acidophilic bacteria were grown at elevated osmotic strength in liquid media, and the compatible solutes synthesised were identified using ion chromatography and MALDI-TOF mass spectrometry. Trehalose was identified as one of, or the sole, compatible solute in all species and strains, apart from Acidithiobacillus thiooxidans where glucose and proline levels increased at elevated osmotic potentials. Several other potential compatible solutes were tentatively identified by MALDITOF analysis. The same compatible solutes were produced by these bacteria regardless of the salt used to produce the osmotic stress. The results correlate with data from sequenced genomes which confirm that many chemolithotrophic and heterotrophic acidophiles possess genes for trehalose synthesis. This is the first report to identify and quantify compatible solutes in acidophilic bacteria that have important roles in biomining technologies.     

Phenomena at the advancing ice-liquid interface: solutes, particles and biological cells

Ice formation in aqueous solutions and suspensions involves a number of significant changes and processes in the residual liquid. The resulting effects were described concerning the redistribution of dissolved salts, the behaviour of gaseous solutes and bubble formation, the rejection and entrapment of second-phase particles. This set of conditions is also experienced by biological cells subjected to freezing. The influences of ice formation in that respect and their relevance for cryopreservation were considered as well. A model of transient heat conduction and solute diffusion with a planar ice front, propagating through a system of finite length was found to be in good agreement with measured salt concentration profiles. The spacing of the subsequently developing columnar solidification pattern was of the same order of magnitude as the pertubation wavelengths predicted from the stability criterion. Non-planar solidification of binary salt solutions was described by a pure heat transfer model under the assumption of local thermodynamic equilibrium. The rejection of gaseous solutes and the resulting gas concentration profile ahead of a planar ice front has been estimated by means of a test bubble method, yielding a distribution coefficient of 0.05 for oxygen. The nucleation of gas bubbles has been observed to occur at slightly less than 20-fold supersaturation. The subsequent radial growth of the bubbles obeys a square-root time dependence as expected from a diffusion controlled model until the still expanding bubbles become engulfed by the advancing ice-liquid interface. The maximum bubble radii decrease for increasing ice front velocities. The transition between repulsion and entrapment of spherical latex particles by an advancing planar ice-front has been characterized by a critical value of the velocity of the solidification interface. The critical velocity is inversely proportional to the particle radius as suggested by models assuming an undisturbed ice front. The increase of the critical velocity for increasing thermal gradients shows good agreement with a theoretically predicted square-root type of dependence. Critical velocities have also been measured for yeast and red blood cells. The effect of freezing on biological cells has been analyzed for human lymphocytes and erythrocytes. The reduction of cell volume observed during non-planar freezing agrees reasonably well with shrinkage curves calculated from a water transport model. The probability of intracellular ice formation has been characterized by threshold cooling rates above which the amount of water remaining within the cell is sufficient for crystallization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Foundations of modeling in cryobiology—I: Concentration,Gibbs energy,and chemical potential relationships

Mathematical modeling plays an enormously important role in understanding the behavior of cells, tissues, and organs undergoing cryopreservation. Uses of these models range from explanation of phenomena, exploration of potential theories of damage or success, development of equipment, and refinement of optimal cryopreservation/cryoablation strategies. Over the last half century there has been a considerable amount of work in bio-heat and mass-transport, and these models and theories have been readily and repeatedly applied to cryobiology with much success. However, there are significant gaps between experimental and theoretical results that suggest missing links in models. One source for these potential gaps is that cryobiology is at the intersection of several very challenging aspects of transport theory: it couples multi-component, moving boundary, multiphase solutions that interact through a semipermeable elastic membrane with multicomponent solutions in a second time-varying domain, during a two-hundred Kelvin temperature change with multi-molar concentration gradients and multi-atmosphere pressure changes. In order to better identify potential sources of error, and to point to future directions in modeling and experimental research, we present a three part series to build from first principles a theory of coupled heat and mass transport in cryobiological systems accounting for all of these effects. The hope of this series is that by presenting and justifying all steps, conclusions may be made about the importance of key assumptions, perhaps pointing to areas of future research or model development, but importantly, lending weight to standard simplification arguments that are often made in heat and mass transport. In this first part, we review concentration variable relationships, their impact on choices for Gibbs energy models, and their impact on chemical potentials.     

Application of the local-bulk partitioning and competitive binding models to interpret preferential interactions of glycine betaine and urea with protein surface

Two thermodynamic models have been developed to interpret the preferential accumulation or exclusion of solutes in the vicinity of biopolymer surface and the effects of these solutes on protein processes. The local-bulk partitioning model treats solute (and water) as partitioning between the region at/or near the protein surface (the local domain) and the bulk solution. The solvent exchange model analyzes a 1:1 competition between water and solute molecules for independent surface sites. Here we apply each of these models to interpret thermodynamic data for the interactions of urea and the osmoprotectant glycine betaine (N,N,N-trimethylglycine; GB) with the surface exposed in unfolding the marginally stable lacI HTH DNA binding domain. The partition coefficient K(P) quantifying accumulation of urea at this protein surface (K(P) approximately equal 1.1) is only weakly dependent on urea concentration up to 6 M urea. However, K(P) quantifying exclusion of GB from the vicinity of this protein surface increases from 0.83 (extrapolated to 0 M GB) to 1.0 (indicating that local and bulk GB concentrations are equal) at 4 M GB (activity > 40 M). We interpret the significant concentration dependence of K(P) for GB, predicted to be general for excluded, nonideal solutes such as GB, as a modest (8%) attenuation of the GB concentration dependence of solute nonideality in the local domain relative to that in the bulk solution. Above 4 M, K(P) for the interaction of GB with the surface exposed in protein unfolding is predicted to exceed unity, which explains the maximum in thermal stability observed for RNase and lysozyme at 4 M GB (Santoro, M. M., Liu, Y. F., Khan, S. M. A., Hou, L. X., and Bolen, D. W. (1992) Biochemistry 31, 5278-5283). Both thermodynamic models provide good two-parameter fits to GB and urea data for lacI HTH unfolding over a wide concentration range. The solute partitioning model allows for a full spectrum of attenuation effects in the local domain, encompasses the cases treated by the competitive binding model, and provides a somewhat better two-parameter fit of effects of high GB concentration on lacI HTH stability. Parameters of this fit should be applicable to isothermal and thermal unfolding data for all proteins with similar compositions of surface exposed in unfolding.