Osmotic response of mammalian cells: Effects of permeating cryoprotectants on nonsolvent volume

The nonsolvent volume, b, of a cell permits calculation of cell water volume from measurements of total cell volume, and, consequently, it is used extensively in the determination of membrane permeability coefficients for water and solutes and also in simulations of water and solute fluxes during freezing of cells. The nonsolvent volume is most commonly determined from the ordinate intercept of plots of cell volume as a function of the reciprocal of extracellular nonpermeating solute concentration (so-called Boyle-van't Hoff plots). Once derived, b is often assumed to be constant even under conditions that may differ markedly from those under which it was determined. Our aim was to investigate whether this assumption was valid when cells were exposed to the cryoprotectants glycerol, dimethyl sulphoxide (Me₂SO), or propane-1,2-diol. Rabbit corneal keratocytes, a fibroblastic cell type, were exposed to 10% (v/v) cryoprotectant for 30 min at 22°C in solutions containing a range of nonpermeating solute concentrations. Cell volumes were determined by an electronic particle sizer and mode volume plotted as an inverse function of the concentration of nonpermeating solute. The cells behaved as osmometers under all conditions studied, but we found no evidence to suggest that the nonsolvent volume of cells was altered by Me₂SO or propane-1,2-diol. Glycerol, however, reduced the slope of the Boyle-van't Hoff plot, but this could be ascribed to the failure of the cells to equilibrate fully with the glycerol over the 30 min exposure time; thus, b was unaffected by glycerol. It may be assumed, therefore, that the nonsolvent volume was not influenced by the presence inside cells of any of these nonelectrolyte cryoprotectants. © 1996 Wiley-Liss, Inc.     

Theory of Diffusion in Gels

It has been shown that when concentration of solute is expressed as amount per unit volume of gel—solvent—solute system, Fick's laws for diffusion in a gel take the same form as for diffusion in solvent alone, except that the usual coefficient must be replaced by a new coefficient, D′, equal to D(1 - α)/(1 - ), where is the effective volume fraction of the gel substance and α is a coefficient of obstruction equal to 5/3 if the gel substance can be considered to be made up of randomly oriented rods. An equation was derived for the total amount of solute entering the gel, which is analogous to but not identical with the equation for the total amount of solute crossing the initial boundary in free diffusion. The effect of slice thickness was investigated by a mathematical procedure involving the solutions of approximate differential equations. It was shown that even for slices so thick that 95 per cent of the solute in the gel is contained in the first two, a correction factor equal to the square of the slice thickness divided by 48D't permits one to obtain accurate measurement of D′ from the mean concentration and the position of the midplane of the slice.     

Thermodynamic factors determining the permeability barrier properties of lipid bilayers

The permeability barrier properties of lipid bilayers are usually determined by the rate of swelling of multilamellar liposomes or by the exchange of radioactively labeled molecules in sonicated vesicles. The values reported in the literature for the permeability of water and non electrolytes differ according to which method is applied in their determination. In addition, drastic assumptions (i.e. homogeneity of the membrane) are commonly introduced for the interpretation of the phenomenological permeability coefficients. This paper discusses the permeability coefficient considering the departures from the ideality of the membrane system. The non ideal terms can be put in function of measurable quantities such as the excluded volume of the membrane and the hydration degree of the lipid molecules. By means of this formalism it is possible to explain quantitatively the experimental values found for the permeability coefficient of water in sonicated vesicles below and above the phase transition temperature. In addition, different magnitudes of the energies of activation for the permeation of non electrolytes have been found depending on if the liposomes are dispersed in isotonic or hipertonic solutions of a permeant. The formalism described allows to explain such differences in terms of the influence of the solute concentration on the density of the lipid membrane. The reasons for which the simple formalism for homogeneous membranes can not be applied to lipid membranes are discussed in detail.     

Nonsolvent water in human erythrocytes

From the ability of a concentrated suspension of human erythrocytes to regulate the pH of unbuffered, anisotonic, external media it is possible to calculate the fractional cell volume in which chloride is dissolved. The difference between this volume and the total cell water gives the nonsolvent water (for chloride) of the cell. Nonsolvent water is less than 3% of the isotonic cell volume. The quantity of nonsolvent water per cell may increase as the cells shrink in hypertonic solutions.     

An Equilibrium Model for the Combined Effect of Macromolecular Crowding and Surface Adsorption on the Formation of Linear Protein Fibrils

The formation of linear protein fibrils has previously been shown to be enhanced by volume exclusion or crowding in the presence of a high concentration of chemically inert protein or polymer, and by adsorption to membrane surfaces. An equilibrium mesoscopic model for the combined effect of both crowding and adsorption upon the fibrillation of a dilute tracer protein is presented. The model exhibits behavior that differs qualitatively from that observed in the presence of crowding or adsorption alone. The model predicts that in a crowded solution, at critical values of the volume fraction of crowder or intrinsic energy of the tracer-wall interaction, the tracer protein will undergo an extremely cooperative transition—approaching a step function—from existence as a slightly self-associated species in solution to existence as a highly self-associated and completely adsorbed species. Criteria for a valid experimental test of these predictions are presented.     

A near-infrared analysis of water-macromolecule interactions: Hydration and the spectra of aqueous solutions of intact proteins

A procedure utilizing a variable path length absorption cell has made possible the recording of what appear to be the first well-resolved, compensated near-infrared spectra of intact proteins in aqueous solution. Individual spectra, corresponding to (1) absorbance by the protein plus bound water, and (2) the solvent volume excluded by the hydrated protein, were obtained using the same experimental sample. Calculations of bound water and excluded volume from these spectra were compared to other results in the literature. The validity of this spectral method was supported by comparisons with the spectra of proteins in films, where there is no excluded volume effect and where the amount of water present has been determined independently by gravimetric measurements. The results indicate that the bound water detected by the near-infrared spectra has an absence or deficiency of molecules with quasi-free OH groups (relative to bulk water), and that in conjunction with results of other methods, these water molecules may represent those that are most firmly or more completely bonded to the protein surface.     

Pathogenesis of Cardiac Oedema

When the heart ceases to meet the requirements of the body for oxygen, the sympathetic-adrenal system is activated. This occurs in people with a healthy heart when the demands for oxygen are excessive—for example, in heavy muscular work—and in subjects with a failing heart when the demands are normal or small. Eventually, when the heart is unable to meet even the ordinary requirements of everyday life, the sympathetic activity becomes more or less continuous. It may lessen during rest at night, but with a further failing of the heart its output may become inadequate even in complete rest.The sympathetically-medicated renal vasoconstriction, with reduction of the glomerular sodium load, redistribution of the blood flow in the renal cortex to the juxtamedullary glomeruli, and the mobilization of the renin-angiotensin-aldosterone system, is responsible for the salt and water retention which will ultimately become clinically manifest as oedema—especially when it is no longer counteracted by the tidal output of water and sodium at night. A by-product of this continuing dehydration reaction is a cumulative potassium loss which may lead to disastrous consequences if untreated.     

Analysis of time hierarchy in plant cell volume changes

A simple model of plant cell volume changes is presented. It is based on Kedem-Katchalsky equations for water and solute transport and on linear approximation of the dependence of intracellular hydrostatic pressure on the cell volume. Active transport of solute is also included. The time hierarchy within the system is analyzed by appropriate normalization of variables and by the assessment of the numerical values of model coefficients. The dynamics of the system comprises a slow process of solute exchange and a fast process of water transport. This explains the wellknown biphasic response of the cell volume to a sudden change in external conditions. An approximation of equations describing the system behaviour on the basis of the Tikhonov's theorem is proposed. The approximative solution is compared with the exact numerical solution of the original equations. The approximation is very good under physiological conditions, but it ceases to hold when the solute permeability of the cell membrane increases causing the breakdown of the entire time hierarchy within the system.     

Membrane Permeability: Generalization of the Reflection Coefficient Method of Describing Volume and Solute Flows

The reflection coefficient method for describing volume and solute fluxes through membranes is generalized to take into account the nonideality of the solutions bathing the membrane and/or multicomponent systems. The reflection coefficient of the impermeable species in these systems is less than unity by a coefficient γ. The reflection coefficient obtained solely from the volume flow equation, σ^v, will always be less than the reflection coefficient obtained from the solute flow equation, σ^8v. These two coefficients are related by σ^8v = σ^v + γ.     

On the mechanism of chromatin loss induced by the B chromosome of maize

Knobbed regions of the regular maize complement frequently are eliminated at the second microspore division in spores which have two or more B chromosomes. Evidence is presented that no or little loss occurs in spores with one B and that the rate is not increased in spores with more than two B's.—The B chromosomes from an unrelated strain proved as effective in inducing loss as did the B's of the original high loss stock.—Chromatin loss induced by B's is restricted to knobbed A chromosomes and occurs only at the second microspore division. Knobbed chromosomes 3, 5, and 9 have been tested and all interact with B's to give loss. Chromosomes with large knobs are more frequently broken than are those with smaller knobs and knobless chromosomes show negligible loss.—Although knobs and B's are essential for chromatin elimination, modifying genes can markedly affect the rate of loss.——Two knobbed heterologous chromosomes undergo simultaneous loss more frequently than expected from independent events. The data indicate that joint loss occurs in competent cells and that preferential assortment of the two deficient chromosomes to specific poles is unlikely.—B chromosomes and deficient chromosomes assort independently at the second microspore anaphase.—Genetic data from crosses with marker genes in both arms of chromosome 3 show that breakage of the postulated dicentric bridge does not occur solely at the centric region since a variety of deficient chromosomes were recovered.—Nondisjunction of B chromosomes and elimination of knobbed chromatin take place during the second microspore mitosis. The argument is advanced that the two phenomena result from faulty replication of heterochromatic segments. The position of the nonreplicating segment in the two kinds of chromosomes determines whether nondisjunction or breakage takes place.—Finally, it is suggested that all of the reported effects of the B chromosome can be accounted for if the B is a parasitic entity having no genetic function other than controlling the replication of its proximal heterochromatic knob and increasing the ability of B-containing sperm cells to compete successfully for fertilization of the egg.     

Effects of macromolecular crowding agents on protein folding in vitro and in silico

Proteins fold and function inside cells which are environments very different from that of dilute buffer solutions most often used in traditional experiments. The crowded milieu results in excluded-volume effects, increased bulk viscosity and amplified chances for inter-molecular interactions. These environmental factors have not been accounted for in most mechanistic studies of protein folding executed during the last decades. The question thus arises as to how these effects—present when polypeptides normally fold in vivo—modulate protein biophysics. To address excluded volume effects, we use synthetic macromolecular crowding agents, which take up significant volume but do not interact with proteins, in combination with strategically selected proteins and a range of equilibrium and time-resolved biophysical (spectroscopic and computational) methods. In this review, we describe key observations on macromolecular crowding effects on protein stability, folding and structure drawn from combined in vitro and in silico studies. As expected based on Minton’s early predictions, many proteins (apoflavodoxin, VlsE, cytochrome c, and S16) became more thermodynamically stable (magnitude depends inversely on protein stability in buffer) and, unexpectedly, for apoflavodoxin and VlsE, the folded states changed both secondary structure content and, for VlsE, overall shape in the presence of macromolecular crowding. For apoflavodoxin and cytochrome c, which have complex kinetic folding mechanisms, excluded volume effects made the folding energy landscapes smoother (i.e., less misfolding and/or kinetic heterogeneity) than in buffer.     

Entropic stabilization of the folded states of RNA due to macromolecular crowding

We review the effects of macromolecular crowding on the folding of RNA by considering the simplest scenario when excluded volume interactions between crowding particles and RNA dominate. Using human telomerase enzyme as an example, we discuss how crowding can alter the equilibrium between pseudoknot and hairpin states of the same RNA molecule—a key aspect of crowder–RNA interactions. We summarize data showing that the crowding effect is significant only if the size of the spherical crowding particle is smaller than the radius of gyration of the RNA in the absence of crowding particles. The implication for function of the wild type and mutants of human telomerase is outlined by using a relationship between enzyme activity and its conformational equilibrium. In addition, we discuss the interplay between macromolecular crowding and ionic strength of the RNA buffer. Finally, we briefly review recent experiments which illustrate the connection between excluded volume due to macromolecular crowding and the thermodynamics of RNA folding.     

Water-membrane partition thermodynamics of an amphiphilic lipopeptide: an enthalpy-driven hydrophobic effect

To shed light on the driving force for the hydrophobic effect that partitions amphiphilic lipoproteins between water and membrane, we carried out an atomically detailed thermodynamic analysis of a triply lipid modified H-ras heptapeptide anchor (ANCH) in water and in a DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayer. Combining molecular mechanical and continuum solvent approaches with an improved technique for solute entropy calculation, we obtained an overall transfer free energy of ~−13 kcal mol⁻¹. This value is in qualitative agreement with free energy changes derived from a potential of mean force calculation and indirect experimental observations. Changes in free energies of solvation and ANCH conformational reorganization are unfavorable, whereas ANCH-DMPC interactions—especially van der Waals—favor insertion. These results are consistent with an enthalpy-driven hydrophobic effect, in accord with earlier calorimetric data on the membrane partition of other amphiphiles. Furthermore, structural and entropic analysis of molecular dynamics-generated ensembles suggests that conformational selection may play a hitherto unappreciated role in membrane insertion of lipid-modified peptides and proteins.     

Clinical Trial of Mefruside,a New Diuretic

A controlled clinical trial of a new diuretic—mefruside—is reported, in which it was compared with frusemide in 15 normal subjects and 15 patients with fluid retention. It was found to be an effective diuretic which, in the patients, produced a significantly greater excretion of water and electrolytes than an equal dose of frusemide. Its smooth prolonged action, maximal in the first 12 hours, made it of particular value for maintenance therapy. In a short-term trial on a further 15 hypertensive patients mefruside was shown to have a useful hypotensive action. The drug was well tolerated with minimal side effects.     

The osmotic behavior of corn mitochondria

The volume changes undergone by corn (Zea mays L.) mitochondria suspended in solutions of constant or varying osmolarity were studied. Within the range of osmotic pressure from 1.8 to 8.4 atmospheres, corn mitochondria behave as osmometers, if allowance is made for an osmotic “dead space” of about 6.9 μl/mg protein. The final equilibrium volume of mitochondria swollen in solutions containing both ribose and sucrose were shown to depend upon the concentration of impermeable solute (sucrose) present and not upon the concentration of ribose present. Osmotic reversibility was found for mitochondria swollen in isotonic solutions of KCI or ribose. The passive swelling of corn mitochondria may be due to the osmotic flow of water coupled to the diffusion of a permeable solute.     

Reconciling competing mechanisms posited to underlie auditory verbal hallucinations

Perception is not the passive registration of incoming sensory data. Rather, it involves some analysis by synthesis, based on past experiences and context. One adaptive consequence of this arrangement is imagination—the ability to richly simulate sensory experiences, interrogate and manipulate those simulations, in service of action and decision making. In this paper, we will discuss one possible cost of this adaptation, namely hallucinations—perceptions without sensory stimulation, which characterize serious mental illnesses like schizophrenia, but which also occur in neurological illnesses, and—crucially for the present piece—are common also in the non-treatment-seeking population. We will draw upon a framework for imagination that distinguishes voluntary from non-voluntary experiences and explore the extent to which the varieties and features of hallucinations map onto this distinction, with a focus on auditory-verbal hallucinations (AVHs)—colloquially, hearing voices. We will propose that sense of agency for the act of imagining is key to meaningfully dissecting different forms and features of AVHs, and we will outline the neural, cognitive and phenomenological sequelae of this sense. We will conclude that a compelling unifying framework for action, perception and belief—predictive processing—can incorporate observations regarding sense of agency, imagination and hallucination.This article is part of the theme issue ‘Offline perception: voluntary and spontaneous perceptual experiences without matching external stimulation’.     

Systematically assessing microbiome–disease associations identifies drivers of inconsistency in metagenomic research

Evaluating the relationship between the human gut microbiome and disease requires computing reliable statistical associations. Here, using millions of different association modeling strategies, we evaluated the consistency—or robustness—of microbiome-based disease indicators for 6 prevalent and well-studied phenotypes (across 15 public cohorts and 2,343 individuals). We were able to discriminate between analytically robust versus nonrobust results. In many cases, different models yielded contradictory associations for the same taxon–disease pairing, some showing positive correlations and others negative. When querying a subset of 581 microbe–disease associations that have been previously reported in the literature, 1 out of 3 taxa demonstrated substantial inconsistency in association sign. Notably, >90% of published findings for type 1 diabetes (T1D) and type 2 diabetes (T2D) were particularly nonrobust in this regard. We additionally quantified how potential confounders—sequencing depth, glucose levels, cholesterol, and body mass index, for example—influenced associations, analyzing how these variables affect the ostensible correlation between Faecalibacterium prausnitzii abundance and a healthy gut. Overall, we propose our approach as a method to maximize confidence when prioritizing findings that emerge from microbiome association studies.

The human microbiome has been associated with many aspects of our health, but how many of these associations are truly reproducible? This study attempts to address this question by systematically testing the robustness of 581 microbial features that have been reported as being disease-associated.     

Economic and Disease Burden of Dengue in Southeast Asia

Background

Dengue poses a substantial economic and disease burden in Southeast Asia (SEA). Quantifying this burden is critical to set policy priorities and disease-control strategies.

Methods and Findings

We estimated the economic and disease burden of dengue in 12 countries in SEA: Bhutan, Brunei, Cambodia, East-Timor, Indonesia, Laos, Malaysia, Myanmar, Philippines, Singapore, Thailand, and Viet Nam. We obtained reported cases from multiple sources—surveillance data, World Health Organization (WHO), and published studies—and adjusted for underreporting using expansion factors from previous literature. We obtained unit costs per episode through a systematic literature review, and completed missing data using linear regressions. We excluded costs such as prevention and vector control, and long-term sequelae of dengue. Over the decade of 2001–2010, we obtained an annual average of 2.9 million (m) dengue episodes and 5,906 deaths. The annual economic burden (with 95% certainty levels) was US$950m (US$610m–US$1,384m) or about US$1.65 (US$1.06–US$2.41) per capita. The annual number of disability-adjusted life years (DALYs), based on the original 1994 definition, was 214,000 (120,000–299,000), which is equivalent to 372 (210–520) DALYs per million inhabitants.

Conclusion

Dengue poses a substantial economic and disease burden in SEA with a DALY burden per million inhabitants in the region. This burden is higher than that of 17 other conditions, including Japanese encephalitis, upper respiratory infections, and hepatitis B.     

USE OF THE ROCKING BED TO AUGMENT VENTILATION IN PATIENTS WITH POLIOMYELITIS

As the first step in an attempt to clarify criteria for use of the rocking bed rather than the respirator as an aid to breathing for patients with weakness of respiratory muscle function caused by poliomyelitis, ventilation studies were done on seven patients with pronounced weakness or paralysis of the respiratory muscles. Average tidal air volume was considerably less when the patient was on the rocking bed than when he was in the respirator. Since the tidal air volume with the patient on the rocking bed represents the maximum that can be produced with the apparatus, whereas the volume in the respirator represents the patient''s usual tidal air and the respirator is capable of a greater volume if necessary, it is apparent that in cases of complete paralysis of the respiratory muscles the respirator has a large margin of safety, the rocking bed none.From clinical observations made on 51 patients who were put upon the rocking bed—23 of them early in the course of the disease and 28 after they had been ill three months or more—it was concluded that the rocking bed is contraindicated for patients who are febrile and in whom the disease is progressing rapidly, and for those with atelectasis or urinary or pulmonary infection. It must be used with extreme care in the case of patients early in the course of the disease who are not tracheotomized, because of a tendency toward increased accumulation of mucus and the danger of atelectasis.General guides were developed with regard to use of the rocking bed for patients with post-acute poliomyelitis, and somewhat different rules were drawn for use of the apparatus in cases in which there is a chronic respiratory problem.The rocking bed will give artificial respiration in cases of respiratory weakness, but will not provide enough tidal air for the patient with paralysis of the muscles of respiration.     

Joint Estimation of Contamination,Error and Demography for Nuclear DNA from Ancient Humans

When sequencing an ancient DNA sample from a hominin fossil, DNA from present-day humans involved in excavation and extraction will be sequenced along with the endogenous material. This type of contamination is problematic for downstream analyses as it will introduce a bias towards the population of the contaminating individual(s). Quantifying the extent of contamination is a crucial step as it allows researchers to account for possible biases that may arise in downstream genetic analyses. Here, we present an MCMC algorithm to co-estimate the contamination rate, sequencing error rate and demographic parameters—including drift times and admixture rates—for an ancient nuclear genome obtained from human remains, when the putative contaminating DNA comes from present-day humans. We assume we have a large panel representing the putative contaminant population (e.g. European, East Asian or African). The method is implemented in a C++ program called ‘Demographic Inference with Contamination and Error’ (DICE). We applied it to simulations and genome data from ancient Neanderthals and modern humans. With reasonable levels of genome sequence coverage (>3X), we find we can recover accurate estimates of all these parameters, even when the contamination rate is as high as 50%.