Active control of the nucleation temperature enhances freezing survival of multipotent mesenchymal stromal cells

Cryopreservation is a technique that has been extensively used for storage of multipotent mesenchymal stromal cells (MSCs) in regenerative medicine. Therefore, improving current cryopreservation procedures in terms of increasing cell viability and functionality is important. In this study, we optimized the cryopreservation protocol of MSCs derived from the common marmoset Callithrix jacchus (cj), which can be used as a non-human primate model in various pathological and transplantation studies and have a great potential for regenerative medicine. We have investigated the effect of the active control of the nucleation temperature using induced nucleation at a broad range of temperatures and two different dimethylsulfoxide concentrations (Me₂SO, 5% (v/v) and 10%, (v/v)) to evaluate the overall effect on the viability, metabolic activity and recovery of cells after thawing. Survival rate and metabolic activity displayed an optimum when ice formation was induced at −10 °C. Cryomicroscopy studies indicated differences in ice crystal morphologies as well as differences in intracellular ice formation with different nucleation temperatures. High subzero nucleation temperatures resulted in larger extracellular ice crystals and cellular dehydration, whereas low subzero nucleation temperatures resulted in smaller ice crystals and intracellular ice formation.     

Components of ice nucleation structures of bacteria

Nonprotein components attached to the known protein product of the inaZ gene of Pseudomonas syringae have been identified and shown to be necessary for the most efficient ice nucleation of supercooled H2O. Previous studies have shown that cultures of Ina+ bacteria have cells with three major classes of ice-nucleating structures with readily differentiated activities. Further, some cells in the culture have nucleating activities intermediate between those of the different classes and presumably have structures that are biosynthetic intermediates between those of the different classes. Since these structures cannot be readily isolated and analyzed, their components have been identified by the use of specific enzymes or chemical probes, by direct incorporation of labeled precursors, and by stimulation of the formation of specific classes of freezing structures by selective additions to the growth medium. From these preliminary studies it appears that the most active ice nucleation structure (class A) contains the ice nucleation protein linked to phosphatidylinositol and mannose, probably as a complex mannan, and possibly glucosamine. These nonprotein components are characteristic of those used to anchor external proteins to cell membranes of eucaryotic cells and suggest that a similar but not identical anchoring mechanism is required for efficient ice nucleation structure. The class B structure has been found to contain protein presumably linked to the mannan and glucosamine moieties but definitely not to the phosphatidylinositol. The class C structure, which has the poorest ice nucleation activity, appears to be the ice nucleation protein linked to a few mannose residues and to be partially imbedded in the outer cell membrane.     

Effect of molecular structure on thermodynamic properties of carbohydrates. A calorimetric study of aqueous di- and oligosaccharides at subzero temperatures

For aqueous solutions of di- and oligosaccharides thermodynamic properties have been investigated at subzero temperatures using differential scanning calorimetry. The amount of unfrozen water observed is found to increase linearly with the glass transition temperatures of anhydrous carbohydrates. Furthermore, the amount of unfrozen water shows a linear relationship with known solution properties of aqueous carbohydrates, such as partial molar compressibility and heat of solution. The different effectiveness among various di- and oligosaccharides to avoid ice formation is associated with the combination of constitutive monosaccharides and attendant molecular structure features including the position and type of the glycosidic linkage between the constituent units. More unfrozen water is induced in the presence of a carbohydrate having a poorer compatibility with the three-dimensional hydrogen-bond network of water. A series of these results obtained imply that there is a common key of carbohydrate stereochemistry governing several different thermodynamic amounts of a given system involving carbohydrates. In this context, a modified stereospecific-hydration model can be used to interpret the present results in terms of stereochemical effects of carbohydrates.     

The biota of Antarctic pack ice in the Weddell sea and Antarctic Peninsula regions

Summary Pack ice surrounding Antarctica supports rich and varied populations of microbial organisms. As part of the Antarctic Marine Ecosystem Research in the Ice Edge Zone (AMERIEZ) studies, we have examined this community during the late spring, autumn, and winter. Although organisms are found throughout the ice, the richest concentrations often occur in the surface layer. The ice flora consists of diatoms and flagellates. Chrysophyte cysts (archaeomonads) of unknown affinity and dinoflagellate cysts are abundant and may serve as overwintering stages in ice. The ice fauna includes a variety of heterotrophic flagellates, ciliates, and micrometazoa. The abundance of heterotrophs indicates an active food web within the ice community. Ice may serve as a temporary habitat or refuge for many of the microbial forms and some of these appear to provide an inoculum for planktonic populations when ice melts. Larger consumers, such as copepods and the Antarctic krill, Euphausia superba are often found on the underside of ice floes and within weathered floes. The importance of the ice biota as a food resource for these pelagic consumers is unknown.     

Comparaison des performances de deux systèmes TEP/TDM corps entier selon la norme NEMA NU 2-2001

The goal of work is the characterization of two PET/CT units that are based on different technologies, using the methodology proposed in the NEMA NU 2-2001 standard. The two systems were qualified in the 3D acquisition mode by means of the National Electric Manufacturers Association (NEMA) phantoms. The results obtained showed that the NEMA standard allows to highlight differences in terms of spatial resolution, sensitivity, scatter fraction and counting rate performances between the two systems ; differences that can be explained by the geometry of the units and the materials of the detectors used. Thus, the use of the standard makes it possible to benchmark PET units and establish reference values that can be used to follow the stability of the system over the time. The tests of image quality, obtained under conditions closer to the clinical applications, showed nevertheless that the two units give, in spite of different technologies, images with sufficient contrast, within short acquisition times, allowing the detection of small lesions. This test should be part of the constancy tests to enable the comparison of examinations performed on different units when the quantitative aspects are of importance.     

Plant Viability as a Function of Temperature Stress (The Richards Function Applied to Data from Freezing Tests of Growing Shoots)

Frost resistance of growing Salix viminalis L. shoots was determined by rating mortality percentage under two commonly used freezing conditions: a condition in which plants were encased in crushed ice and another in which plants were moistened with tap water prior to freezing. The mortality-temperature data were fitted with a logistic function (having a fixed inflection point halfway between the asymptotes) and with a Richards function, which is a double asymptotic sigmoid function with a variable inflection point. Different frost resistance curves were obtained, depending on the freezing conditions used. However, conditions were inadequate for efficient ice nucleation under either condition. This implies that the applied freezing conditions are not suitable when the purpose is to induce and duplicate early ice crystal formation conditions. The Richards derivatives were negatively skewed in the one case and positively skewed in the other case, giving inflection points, as a function of the upper asymptote, situated at 0.37 when shoots were frosted in the presence of ice and at 0.81 when shoots were frozen in the presence of added moisture. These values differed significantly from 0.50, through which the logistic function would have forced the curves. Because of the significant asymmetry in these frost-resistance curves, the Richards function led to a more accurate reflection of the temperature-mortality course of growing Salix stems than the logistic function. The Richards function possesses the flexibility needed to describe plant injury response in terms of physical and plant physiological mechanisms. Therefore, the Richards function is recommended rather than the logistic function for the assessment of frost resistance.     

Antifreeze proteins at the ice/water interface: three calculated discriminating properties for orientation of type I proteins

Antifreeze proteins (AFPs) protect many plants and organisms from freezing in low temperatures. Of the different AFPs, the most studied AFP Type I from winter flounder is used in the current computational studies to gain molecular insight into its adsorption at the ice/water interface. Employing molecular dynamics simulations, we calculate the free energy difference between the hydrophilic and hydrophobic faces of the protein interacting with ice. Furthermore, we identify three properties of Type I "antifreeze" proteins that discriminate among these two orientations of the protein at the ice/water interface. The three properties are: the "surface area" of the protein; a measure of the interaction of the protein with neighboring water molecules as determined by the number of hydrogen bond count, for example; and the side-chain orientation angles of the threonine residues. All three discriminants are consistent with our free energy results, which clearly show that the hydrophilic protein face orientations toward the ice/water interface, as hypothesized from experimental and ice/vacuum simulations, are incorrect and support the hypothesis that the hydrophobic face is oriented toward the ice/water interface. The adsorption free energy is calculated to be 2-3 kJ/mol.     

Nuclear and chloroplast DNA phylogeography reveals vicariance among European populations of the model species for the study of metal tolerance, Arabidopsis halleri (Brassicaceae)

Arabidopsis halleri is a pseudometallophyte involved in numerous molecular studies of the adaptation to anthropogenic metal stress. In order to test the representativeness of genetic accessions commonly used in these studies, we investigated the A. halleri population genetic structure in Europe. Microsatellite and nucleotide polymorphisms from the nuclear and chloroplast genomes, respectively, were used to genotype 65 populations scattered over Europe. The large-scale population structure was characterized by a significant phylogeographic signal between two major genetic units. The localization of the phylogeographic break was assumed to result from vicariance between large populations isolated in southern and central Europe, on either side of ice sheets covering the Alps during the Quaternary ice ages. Genetic isolation was shown to be maintained in western Europe by the high summits of the Alps, whereas admixture was detected in the Carpathians. Considering the phylogeographic literature, our results suggest a distinct phylogeographic pattern for European species occurring in both mountain and lowland habitats. Considering the evolution of metal adaptation in A. halleri, it appears that recent adaptations to anthropogenic metal stress that have occurred within either phylogeographic unit should be regarded as independent events that potentially have involved the evolution of a variety of genetic mechanisms.     

Inhibiting ice recrystallization and optimization of cell viability after cryopreservation

The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220?mM solutions of disaccharides; however, the best cell viability was obtained when a 200?mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.     

Type I 'antifreeze' proteins. Structure-activity studies and mechanisms of ice growth inhibition.

The type I 'antifreeze' proteins, found in the body fluids of fish inhabiting polar oceans, are alanine-rich alpha-helical proteins that are able to inhibit the growth of ice. Within this class there are two distinct subclasses of proteins: those related to the winter flounder sequence HPLC6 and which contain 11-residue repeat units commencing with threonine; and those from the sculpins that are unique in the N-terminal region that contains established helix breakers and lacks the 11-residue repeat structure present in the rest of the protein. Although 14 type I proteins have been isolated, almost all research has focused on HPLC6, the 37-residue protein from the winter flounder Pseudopleuronectes americanus. This protein modifies both the rate and shape (or 'habit') of ice crystal growth, displays hysteresis and accumulates specifically at the {2 0 2; 1} ice plane. Until very recently, all models to explain the mechanism for this specific interaction have relied on the interaction of the four threonine hydroxyls, which are spaced equally apart on one face of the helix, with the ice lattice. In contrast, proteins belonging to the sculpin family accumulate specifically at the {2 1; 1; 0} plane. The molecular origin of this difference in specificity between the flounder and sculpin proteins is not understood. This review will summarize the structure-activity and molecular modelling and dynamics studies on HPLC6, with an emphasis on recent studies in which the threonine residues have been mutated. These studies have identified important hydrophobic contributions to the ice growth inhibition mechanism. Some 50 mutants of HPLC6 have been reported and the data is consistent with the following requirements for ice growth inhibition: (a) a minimum length of approx. 25 residues; (b) an alanine-rich sequence in order to induce a highly helical conformation; (c) a hydrophobic face; (d) a number of charged/polar residues which are involved in solubility and/or interaction with the ice surface. The emerging picture, that requires further dynamics studies including accurate modelling of the ice/water interface, suggests that a hydrophobic interaction between the surface of the protein and ice is the key to explaining accumulation at specific ice planes, and thus the molecular level mechanism for ice growth inhibition.     

Quench cooling and ice crystal formation in biological tissues

The rates of cooling of tissue packages quenched in liquid nitrogen were investigated using microthermocouples. By assembling tissue packages from a standard 200-μm tissue slice a microthermocouple could be positioned at different depths within the package. Results showed that for a given mass of tissue the rates of cooling at different depths were the same. When the tissue mass was varied the rates of cooling at a fixed depth decreased with increasing tissue mass.The ice crystal formations produced when tissues are quenched in liquid nitrogen were investigated using freeze substitution. Assemblies of rabbit cornea of different thicknesses were quenched in liquid nitrogen and freeze substituted. The size of the ice crystal cavities produced during the quenching increased with increasing tissue mass, exhibiting a saturation size for the larger tissue masses. There was no obvious size distribution of the ice crystal cavities across the thickness of the corneas.The results suggest an “isotherm” model for the quenching conditions used in these experiments, there being small or negligible temperature gradients through the tissue which uniformly cools at a fixed rate.     

Change of supercooling capability in solutions containing different kinds of ice nucleators by flavonol glycosides from deep supercooling xylem parenchyma cells in trees

Deep supercooling xylem parenchyma cells (XPCs) in Katsura tree contain flavonol glycosides with high supercooling-facilitating capability in solutions containing the ice nucleation bacterium (INB) Erwinia ananas, which is thought to have an important role in deep supercooling of XPCs. The present study, in order to further clarify the roles of these flavonol glycosides in deep supercooling of XPCs, the effects of these supercooling-facilitating (anti-ice nucleating) flavonol glycosides, kaempferol 3-O-β-d-glucopyranoside (K3Glc), kaempferol 7-O-β-d-glucopyranoside (K7Glc) and quercetin 3-O-β-d-glucopyranoside (Q3Glc), in buffered Milli-Q water (BMQW) containing different kinds of ice nucleators, including INB Xanthomonas campestris, silver iodide and phloroglucinol, were examined by a droplet freezing assay. The results showed that all of the flavonol glycosides promoted supercooling in all solutions containing different kinds of ice nucleators, although the magnitudes of supercooling capability of each flavonol glycoside changed in solutions containing different kinds of ice nucleators. On the other hand, these flavonol glycosides exhibited complicated nucleating reactions in BMQW, which did not contain identified ice nucleators but contained only unidentified airborne impurities. Q3Glc exhibited both supercooling-facilitating and ice nucleating capabilities depending on the concentrations in such water. Both K3Glc and K7Glc exhibited only ice nucleation capability in such water. It was also shown by an emulsion freezing assay in BMQW that K3Glc and Q3Glc had no effect on homogeneous ice nucleation temperature, whereas K7Glc increased ice nucleation temperature. The results indicated that each flavonol glycoside affected ice nucleation by very complicated and varied reactions. More studies are necessary to determine the exact roles of these flavonol glycosides in deep supercooling of XPCs in which unidentified heterogeneous ice nucleators may exist.     

Prokaryotic diversity of arctic ice shelf microbial mats

The prokaryotic diversity and respiratory activity of microbial mat communities on the Markham Ice Shelf and Ward Hunt Ice Shelf in the Canadian high Arctic were analysed. All heterotrophic isolates and > 95% of bacterial 16S rRNA gene clone library sequences from both ice shelves grouped within the phyla Bacteroidetes , Proteobacteria and Actinobacteria . Clone library analyses showed that the bacterial communities were diverse and varied significantly between the two ice shelves, with the Markham library having a higher estimated diversity (Chao1 = 243; 105 operational taxonomic units observed in 189 clones) than the Ward Hunt library (Chao1 = 106; 52 operational taxonomic units observed in 128 clones). Archaeal 16S rRNA gene clone libraries from both ice shelves were dominated by a single Euryarchaeota sequence, which appears to represent a novel phylotype. Analyses of community activity by radiorespiration assays detected metabolism in mat samples from both ice shelves at temperatures as low as −10°C. These findings provide the first insight into the prokaryotic biodiversity of Arctic ice shelf communities and underscore the importance of these cryo-ecosystems as a rich source of microbiota that are adapted to extreme cold.     

Theoretical analysis of the ice crystal size distribution in frozen aqueous specimens.

To estimate theoretically how suited different freezing techniques are for freezing of freeze-etch specimens, it is necessary to know the relationship between specimen cooling rate and the resulting average ice crystal size. Using a somewhat simplified theoretical analysis, we have derived the approximate ice crystal size distribution of nonvitrified frozen aqueous specimens frozen at different cooling rates. The derived size distribution was used to calculate the relationship between relative change in average ice crystal size, (delta l/l), and relative change in specimen cooling rate delta (dT/dt)/(dT/dt). We found this relationship to be (delta l/l) = -k X delta (dT/dt)/(dT/dt) where k = 1.0 when specimen solidification takes place at about -6 degrees C, and k congruent to 1.3 when it takes place at about -40 degrees C.     

Economic evaluation and health care. What does it mean?

Ever since the concept of value for money in health care was introduced into the NHS, economic terms and jargon have become part of our everyday lives--but do we understand what the different types of economic evaluation all mean, particularly those that sound similar to the uninitiated? This article introduces readers to the purpose of economic evaluation, and briefly explains the differences between cost-minimisation analysis (used when the outcomes of the procedures being compared are the same); cost-effectiveness analysis (used when the outcomes may vary, but can be expressed in common natural units, such as mm Hg for treatments of hypertension); cost-utility analysis (used when outcomes do vary--for example, quality of life scales); and cost-benefit analysis (used when a monetary value is being placed on services received). Further articles will deal with each one in more detail.     

Retention of ice-associated amphipods: possible consequences for an ice-free Arctic Ocean

Recent studies predict that the Arctic Ocean will have ice-free summers within the next 30 years. This poses a significant challenge for the marine organisms associated with the Arctic sea ice, such as marine mammals and, not least, the ice-associated crustaceans generally considered to spend their entire life on the underside of the Arctic sea ice. Based upon unique samples collected within the Arctic Ocean during the polar night, we provide a new conceptual understanding of an intimate connection between these under-ice crustaceans and the deep Arctic Ocean currents. We suggest that downwards vertical migrations, followed by polewards transport in deep ocean currents, are an adaptive trait of ice fauna that both increases survival during ice-free periods of the year and enables re-colonization of sea ice when they ascend within the Arctic Ocean. From an evolutionary perspective, this may have been an adaptation allowing success in a seasonally ice-covered Arctic. Our findings may ultimately change the perception of ice fauna as a biota imminently threatened by the predicted disappearance of perennial sea ice.     

Cesare Emiliani (1922–1995), pioneer of Ice Age studies and oxygen isotope stratigraphy

Cesare Emiliani established that the ice ages of the last half million years or so are cyclic phenomena, which gave strong support to the hypothesis of Milankovitch. He discovered the cycles when analyzing foraminifers from long deep-sea cores for their oxygen isotope composition (cores were from the Swedish Deep-Sea Expedition, from the Lamont collection and later from collections made at Miami). Emiliani’s method has become the standard procedure for interpreting the deep-sea record in terms of ocean and climate history. Emiliani introduced a time scale suggesting that the cycles are typically 40 000 years in duration, and he defended this scale for almost 20 years. He also thought that temperature was a more important influence on oxygen isotope variations of the ocean than the buildup and decay of northern hemisphere ice sheets. Both these notions proved incorrect. However, his insistence that the Milankovitch mechanism, in conjunction with ice dynamics and crustal response to loading, is the driving force behind the climate cycles of the Quaternary proved well founded.     

Ice nucleation and freezing tolerance in New Zealand alpine and lowland weta, Hemideina spp. (Orthoptera; Stenopelmatidae)

Abstract.The alpine tree weta Hemidiena maori Pictet et Saussure (Orthoptera: Stenopelmatidae) is a large, flightless insect found above the treeline on many of the mountain ranges of the South Island of New Zealand. The population found on the Rock and Pillar Range, Central Otago has been identified as freezing tolerant with a haemolymph ice nucleating agent. The ability of H. maori to survive freezing is compared to the lowland weta Hemideina thoracica Walker and H. crassidens Blanchard, both of which are able to survive the formation of some ice in their bodies. Mortality is associated with time spent frozen in H. thoracica , and it is hypothesized that this species is killed when a critical proportion of its body water is frozen. All five subalpine and alpine populations of H. maori surveyed were found to be freezing tolerant.
Comparison of temperatures of first nucleation and mean supercooling point of haemolymph droplets suggest that haemolymph ice nucleating activity varies between populations of H. maori. Hemideina maori collected from the Mt Cook region appear to lack a haemolymph ice nucleator. This population is nevertheless freezing tolerant, suggesting that the haemolymph ice nucleating agent described in H. maori is not essential for freezing tolerance. Hemideina crassidens and H. ricta Hutton, both of which are found in lowland habitats, also had high mean supercooling point and temperatures of first nucleation of haemolymph droplets, suggesting that these species also have a haemolymph ice nucleator.
Comparison of ice nucleation characteristics of haemolymph and faecal material (representing gut contents) suggests that gut nucleators in H. maori may be at least as efficient as the haemolymph nucleator. It is concluded that freezing tolerance is probably not an adaptation to the alpine environment. This highlights the need for inter- and intraspecific comparative studies if physiological data are to be used to draw evolutionary conclusions.     

Characterization of Protistan Assemblages in the Ross Sea, Antarctica, by Denaturing Gradient Gel Electrophoresis

The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes.