Energy-optimized structure of antifreeze protein and its binding mechanism.

A combination of Monte Carlo simulated annealing and energy minimization was utilized to determine the conformation of the antifreeze protein from the fish winter flounder. It was found from the energy-optimized structure that the hydroxyl groups of its four threonine residues, i.e. Thr2, Thr13, Thr24, Thr35, are aligned on almost the same line parallel to the helix axis and separated successively by 16.1, 16.0 and 16.2 A, respectively, very close to the 16.6 A repeat spacing along [0112] in ice. Based on such a space match, a zipper-like model is proposed to elucidate the binding mechanism of the antifreeze protein to ice crystals. According to the current model, the antifreeze protein may bind to an ice nucleation structure in a zipper-like fashion through hydrogen bonding of the hydroxyl groups of these four Thr residues to the oxygen atoms along the [0112] direction in ice lattice, subsequently stopping or retarding the growth of ice pyramidal planes so as to depress the freeze point. The calculated results and the binding mechanism thus derived accord with recent experimental observations. The mechanistic implications derived from such a special antifreeze molecule might be generally applied to elucidate the structure-function relationship of other antifreeze proteins with the following two common features: (1) recurrence of a Thr residue (or any other polar amino acid residue whose side-chain can form a hydrogen bond with water) in an 11-amino-acid period along the sequence concerned; and (2) a high percentage of Ala residue component therein. Further experiments are suggested to test the ice binding model.     

A new method to model change in cutaneous blood flow due to mechanical skin irritation part II: parameter identification procedure

Mechanical skin irritation, for example a light scratch with a needle, induces histamine and neuropeptide release on the line of stroke and in the surrounding tissue. Both histamine and neuropeptides are vasodilators. They cause vasodilation by changing the contraction state of the vascular smooth muscles and hence vessel compliance. Smooth muscle contraction state is very difficult to measure in vivo. For that reason we propose in this article an identification procedure to establish an irritation law. The law gives change in vessel compliance as a function of space, time and the intensity of the stroke. We have showed that vessel compliance increases immediately after the stroke not only on the line of stroke, but also in the surrounding tissue. Then, after a short delay, vessel compliance starts decreasing in the surrounding tissue, whereas vessel compliance on the line of stroke keeps increasing. Hence, blood is transported from the surrounding tissue to the line of stroke. In this way, higher blood volume on the line of stroke can be obtained than by only changing vessel compliance locally.     

Mirror image technique for the thermal analysis in cryoablation: Experimental setup and validation

The paper presents a set of experiments that were performed to characterize the freezing front propagation in water first, and in an agar-gel solution afterwards. The experimental setup made of Peltier devices, to emulate the cryogenic effect, and a copper cold finger, to mimic the cold probe interface, are described. We claim that by monitoring some temperatures at the generating cryodevice, several pieces of information can be derived through the cold interface to assess the outside thermodynamic changes. The employed technique, known as mirror image, allows determining the occurrence of the initial ice formation outside the cryo-probe and in the surrounding material, also with different magnitudes of the thermal contact resistance at the cold interface. For both water and agar the ice penetration was found to be non linear versus time, and proportional to the square root of time in the performed experiments. The ice drift velocity decreases according to its penetration inside the tested materials. At the beginning of ice formation, the measured drift velocities are approximately 0.11 mm/s and 0.06 mm/s for water and agar, respectively, and after the ice penetrates 2 mm, they become approximately 0.03 mm/s for both materials.     

Sol-gel processing of actin to obtain homogeneous glasses at low temperatures

The lethal effects of freezing on cells are currently attributed to the crystallization of extracellular water which leaves concentrated solutions of salts, macromolecules and so forth in the extracellular space. This concentrated fluid establishes a strong osmotic gradient which draws water from the cells. Thus, a cell surrounded by ice can survive only if means can be found for reducing the osmotically driven outflow of cellular water. This is usually attempted through vitrification of the extracellular space, but may also be attained through suitable modifications of cellular plasms. Starting from microscopic observations on early rabbit embryos and related cryotolerance, we investigated purified actin solutions under similar conditions, and found that sol-gel processing could result in the formation of homogeneous glass, and through drying, give rise to monolithic solids, glasses and composites. The first process may be at least partially responsible for the induced cryotolerance of cells, while the second may be representative of new and useful biomaterials.     

Liquid-like water confined in stacks of biological membranes at 200 k and its relation to protein dynamics

Confined water is of considerable current interest owing to its biophysical importance and relevance to cryopreservation. It can be studied in its amorphous or supercooled state in the "no-man's land", i.e., in the temperature range between 150 and 235 K, in which bulk water is always crystalline. Amorphous deuterium oxide (D(2)O) was obtained in the intermembrane spaces of a stack of purple membranes from Halobacterium salinarum by flash cooling to 77 K. Neutron diffraction showed that upon heating to 200 K the intermembrane water space decreased sharply with an associated strengthening of ice diffraction, indicating that water beyond the first membrane hydration layer flowed out of the intermembrane space to form crystalline ice. It was concluded that the confined water undergoes a glass transition at or below 200 K to adopt an ultraviscous liquid state from which it crystallizes to form ice as soon as it finds itself in an unconfined, bulk-water environment. Our results provide model-free evidence for translational diffusion of confined water in the no-man's land. Potential effects of the confined-water glass transition on nanosecond membrane dynamics were investigated by incoherent elastic neutron scattering experiments. These revealed no differences between flash-cooled and slow-cooled samples (in the latter, the intermembrane space at temperatures <250 K is occupied only by the first membrane hydration layers), with dynamical transitions at 150 and 260 K, but not at 200 K, suggesting that nanosecond membrane dynamics are not sensitive to the state of the water beyond the first hydration shell at cryotemperatures.     

The North East Water polynya (Greenland Sea)

Physical observations of the North East Water (NEW) polynya, located near the north-eastern corner of Greenland, are presented. Data were collected in June 1991 by RV Polarstern. An idea is put forward to explain how the NEW is generated. A northward coastal current interacts with a persistent shelf ice barrier under which water can flow but that retains ice floes and therefore protects the NEW area from ice advection. Since in summer, the combination of currents, barrier and air-sea heat balance gives rise to a polynya. The distribution of upper water column vertical stability in the NEW is also influenced by its generation process. Surface melt water is retained by the shelf ice barrier, causing neutral vertical stability in its lee. Sea ice melting and land runoff then act as two distinct sources of vertical stability enabling the development of plankton blooms, especially in the northern part of the NEW.     

Large geographical differences in the sensitivity of ice‐covered lakes and rivers in the Northern Hemisphere to temperature changes

Based on a unique dataset of more than 50 000 observations of ice phenology from 1213 lakes and 236 rivers in 12 different countries, we show that interannual variations in the timing of ice‐on and ice‐off on lakes and rivers are not equally pronounced over the entire Northern Hemisphere, but increase strongly towards geographical regions that experience only short periods during which the air temperature falls below 0 °C. We explain our observations by interannual fluctuation patterns of air temperature and suggest that lake and river ecosystems in such geographical regions are particularly vulnerable to global warming, as high interannual variability is known to have important ramifications for ecosystem structure and functioning. We estimate that the standard deviation of the duration of ice cover, viewed as a measure of interannual variability, exceeds 25 days for lakes and rivers located on 7% of the land area of the Northern Hemisphere. Such high variability might be an early warning signal for a critical transition from strictly dimictic, ice‐covered systems to monomictic, open‐water systems. Using the Global Lake and Wetland Database, we suggest that 3.7% of the world's lakes larger than 0.1 km² are at high risk of becoming open‐water systems in the near future, which will have immediate consequences for global biogeochemical cycles.     

Blood Vessels and Related Structures in the Gill Bars of Glossobalanus minutus (Enteropneusta)

The branchial circulatory system of Glossobalanus minutus (Enteropneusta) is investigated by means of transmission electron microscopy. Primary gill bars, or septa, have a single blood vessel longitudinally located along the outer edge of the bar. Secondary gill bars, or tongue bars, show a vessel in their inner, pharyngeal edge. The walls of both vessels are made up of the basement membranes of surrounding epithelia, lacking an endothelium. No definite limits between the vessel lumen and the skeletal rods inside the bars can be seen. Furthermore, the blood seems to penetrate into the rods of both primary and secondary gill bars. In the secondary bars such a phenomenon gives rise to the so-called 'lateral vessels' reported in the light microscopical literature. The significance of these observations is discussed, with special reference to the gill circulatory system of amphioxus, which seems to be strongly similar from a morphological and ultrastructural point of view.     

Notes on the biology of sea ice in the Arctic and Antarctic

The sea ice which covers large areas of the polar regions plays a major role in the marine ecosystem of both the Arctic and Southern Oceans. Not only do warmblooded animals depend on sea ice as a platform, but the sympagic organisms living internally within the sea ice or at the interfaces ice/snow and ice/water provide a substantial part of the total primary production of the ice covered regions. In addition sea ice organisms are an important food source for a variety of pelagic animals and may initiate phytoplankton spring blooms after ice melt by seeding effects.Sea ice organisms often are enriched by some orders of magnitude if the same volume of melted ice is compared to that of the underlying water column. Three hypotheses try to explain this discrepancy and are discussed. Investigations on the nutrient chemistry within the sea ice system and in-situ observations still are rare. Intense growth of sympagic organisms can result in nutrient deficiencies, at least in selected habitats. Advances in endoscopie methods may lead to a better understanding of the life within the sea ice.Paper presented at the Symposium on Polar regions: the challenge for biological and ecological research organised by the Swiss Committee for Polar Research, Basel on 2 October 1992     

The origin, ultrastructure, and microbiology of the sediment accumulating in liquid nitrogen storage vessels

During long-term cryopreservation, ice sediment accumulates in storage Dewars and poses a risk of microbial contamination to stored samples. Ice accumulates in liquid nitrogen via two general processes: (1) ice forming in the atmosphere above an open Dewar falls into the vessel; and (2) ice forming on cold surfaces of the Dewar or inventory system enters the liquid nitrogen. These ice crystals aggregate and entrap other materials, such as bacteria, fungal spores, and general laboratory debris present within the liquid nitrogen. Measured changes in the ultrastructure of ice aggregates following long-term storage are consistent with transient warming events to temperatures of -100 degrees C. Bacteria were identified in all samples and filamentous fungi in 9 out of 10 samples. These micro-organisms are commonly found in the environment and would not be expected to have been derived from IVF samples. Some of the bacteria identified are associated with nosocomial infections in humans. The implications that the association of microbial contamination with ice crystals has on cryopreservation procedures are discussed.     

Home ranges in moving habitats: polar bears and sea ice

Home range size estimates are often used to assess the amount of space required for animals to perform the activities essential for their survival and reproduction. However, in moving environments, traditional home range estimates may be ill suited to this task. In particular, traditional home range estimates are inaccurate representations of the space required by polar bears Ursus maritimus. The sea ice is the prime foraging platform of polar bears, and estimating the amount of ice encountered by bears may provide a better approximation of space use. We develop a technique to make these estimates. Our results confirm that polar bears use more space than terrestrial carnivores to find the resources and conditions they require. We also show that the traditional geographic home range can underestimate both the movement of bears and the amount of space encountered. Moreover, area of ice encountered increased with ice drift, indicating that bears living on highly mobile ice might be exposed to higher energetic costs, and potentially larger energetic gains, than bears inhabiting more stable ice. The methods and concepts presented here can serve as a foundation for new approaches to study the space use of the many species living in moving environments.     

Relationships between ice crystal size, water content and proton NMR relaxation times in cells

Biological specimens were frozen under controlled conditions. We questioned how the size of ice crystals, as measured in cryosectioned and cryoadsorbed sections of these biological specimens, relates to the water content and to the proton NMR relaxation times (T1 and T2) of the unfrozen specimens. The results permit the following conclusions: After rapid freezing in liquid propane cooled in a liquid nitrogen bath, the average size of ice crystals at distances of 150 microns or more from the surface of a particular tissue was always the same. Thus, the average size of the ice crystals was found to be characteristic of the type of biological tissue studied. Linear regression analysis showed average ice crystal size to have a significant correlation coefficient to T1 relaxation time and to water content. Specifically ice crystal size increased with T1 relaxation time and with water content. Multiple regression and path analysis demonstrated a positive correlation between the T1 relaxation time and the ice crystal size variation. Path analysis showed that both water content and T2 relaxation time were less directly correlated with ice crystal size. The findings from the path analysis and other observations show that the average size of ice crystals in subcellular compartments is best predicted by the proton T1 relaxation time. A working model is put forth to explain differences in ice crystal size observed between specimens enriched in globular or in parallel filamentous proteins.     

Refilling of a hydraulically isolated embolized xylem vessel: model calculations

When they are hydraulically isolated, embolized xylem vessels can be refilled, while adjacent vessels remain under tension. This implies that the pressure of water in the refilling vessel must be equal to the bubble gas pressure, which sets physical constraints for recovery. A model of water exudation into the cylindrical vessel and of bubble dissolution based on the assumption of hydraulic isolation is developed. Refilling is made possible by the turgor of the living cells adjacent to the refilling vessel, and by a reflection coefficient below 1 for the exchange of solutes across the interface between the vessel and the adjacent cells. No active transport of solutes is assumed. Living cells are also capable of importing water from the water-conducting vessels. The most limiting factors were found to be the osmotic potential of living cells and the ratio of the volume of the adjacent living cells to that of the embolized vessel. With values for these of 1.5 MPa and 1, respectively, refilling times were in the order of hours for a broad range of possible values of water conductivity coefficients and effective diffusion distances for dissolved air, when the xylem water tension was below 0.6 MPa and constant. Inclusion of the daily pattern for xylem tension improved the simulations. The simulated gas pressure within the refilling vessel was in accordance with recent experimental results. The study shows that the refilling process is physically possible under hydraulic isolation, while water in surrounding vessels is under negative pressure. However, the osmotic potentials in the refilling vessel tend to be large (in the order of 1 MPa). Only if the xylem water tension is, at most, twice atmospheric pressure, the reflection coefficient remains close to 1 (0.95) and the ratio of the volume of the adjacent living cells to that of the embolized vessel is about 2, does the osmotic potential stay below 0.4 MPa.     

Desmin-positive stellate cells associated with angiogenesis in a tumour and non-tumour system

The angiogenesis induced after implantation of fragments of the Walker 256 carcinoma was compared with the angiogenesis following implantation of different amounts of Indian ink. Morphologically and chronologically the tumour system showed no difference from the Indian ink system, provided sufficient amounts of ink were implanted. Both systems were characterized by significant macrophage infiltration. The vascular development, which was clearly concentrated in a dense rim around the tumour, remained present when the tumour enlarged, suggesting an acquisition of vasculature by the tumour through vessel incorporation and not vessel ingrowth. Initially, scattered desmin-positive cells, in contact or encircled by collagen IV, were found in the developing angiogenic rim. Later many desmin-positive cells were found around vessels and could be identified by electron microscopy as pericytes. They exhibited close local contacts with endothelial cells. After incorporation of the peritumour vascular rim into the tumour the number of pericytes decreased and their shape became flattened and elongated.     

Wood anatomy of Rauvolfioideae (Apocynaceae): a search for meaningful non-DNA characters at the tribal level

Wood anatomical studies in the economically important Apocynaceae or dogbane family are fragmentary. This study represents a first attempt to unravel the phylogenetic significance and major evolutionary trends in the wood of the family, using existing and new microscopic wood observations within the large subfamily Rauvolfioideae. On the basis of LM and SEM observations of 91 species representing all 10 currently recognized tribes, we found that most of the tribes are characterized by a unique combination of wood characters, such as vessel grouping, vessel element length, fiber type, frequency of uniseriate rays, and fused multiseriate rays. Climbing rauvolfioid taxa can generally be distinguished from erect species by their wider vessels, tendency to form paratracheal axial parenchyma, presence of tracheids, and occurrence of laticifers in rays. With respect to the entire family, there is a general phylogenetic trend toward shorter vessel elements, a higher proportion of vessels in multiples and more vessels per multiple, higher tracheid abundance, more paratracheal parenchyma, and fewer cells per axial parenchyma strand in the more derived Apocynaceae. Most of these evolutionary trends are likely to be triggered by drier environmental conditions and/or shifts from an erect to a climbing habit.     

The application of convolution neural network based cell segmentation during cryopreservation

For most of the cells, water permeability and plasma membrane properties play a vital role in the optimal protocol for successful cryopreservation. Measuring the water permeability of cells during subzero temperature is essential. So far, there is no perfect segmentation technique to be used for the image processing task on subzero temperature accurately. The ice formation and variable background during freezing posed a significant challenge for most of the conventional segmentation algorithms. Thus, a robust and accurate segmentation approach that can accurately extract cells from extracellular ice that surrounding the cell boundary is needed. Therefore, we propose a convolutional neural network (CNN) architecture similar to U-Net but differs from those conventionally used in computer vision to extract all the cell boundaries as they shrank in the engulfing ice. The images used was obtained from the cryo-stage microscope, and the data was validated using the Hausdorff distance, means ± standard deviation for different methods of segmentation result using the CNN model. The experimental results prove that the typical CNN model extracts cell borders contour from the background in its subzero state more coherent and effective as compared to other traditional segmentation approaches.     

Prediction of ice content in biological model solutions when frozen under high pressure

High pressure is, at least, as effective as cryoprotective agents (CPAs) and are used for decreasing both homogenous nucleation and freezing temperatures. This fact gives rise to a great variety of possible cryopreservation processes under high pressure. They have not been optimized yet, since they are relatively recent and are mainly based on the pressure–temperature phase diagram of pure water. Very few phase diagrams of biological material are available under pressure. This is owing to the lack of suitable equipment and to the difficulties encountered in carrying out the measurements. Different aqueous solutions of salt and CPAs as biological models are studied in the range of 0°C down to ‐35°C, 0.1 up to 250 MPa, and 0–20% w/w total solute concentration. The phase transition curves of glycerol and of sodium chloride with either glycerol or sucrose in aqueous solutions are determined in a high hydrostatic pressure vessel. The experimental phase diagrams of binary solutions were well described by a third‐degree polynomial equation. It was also shown that Robinson and Stokes' equation at high pressure succeeds in predicting the phase diagrams of both binary and ternary solutions. The solute cryoconcentration and the ice content were calculated as a function of temperature and pressure conditions during the freezing of a binary solution. This information should provide a basis upon which high‐pressure cryopreservation processes may be performed and the damages derived from ice formation evaluated. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Raman spectra of a solid antifreeze glycoprotein and its liquid and frozen aqueous solutions.

Raman spectroscopy was used to study the anomalous decrease in the freezing temperature of water produced by an antifreeze glycoprotein obtained from the sera of an Antarctic fish. An active fraction of this glycoprotein has a molecular weight of approximately 18,000 by equilibrium sedimentation compared to an apparent weight of 20 by freezing temperature depression. The Raman spectra of water present in a 1% antifreeze glycoprotein solution and of ice frozen from this solution were indistinguishable from the spectra of pure water and ice, respectively. These results indicate that the bulk properties of water and ice are unaffected by the presence of the antifreeze glycoprotein. Raman measurements on ice grown slowly, using as seed an oriented single crystal of ice in contact with 1% glycoprotein solutions, showed that the active glycoprotein was not excluded from the ice phase. On the other hand, we found that a smaller, inactive glycoprotein was excluded. Comparison of the Raman spectra of active and inactive glycoprotein components as solids, in 5% solutions, and rapidly frozen 5% solutions, showed that the two components differ in conformation and possibly in the environment of their carbohydrate hydroxyls. These observations suggest that hydrogen bonding of the carbohydrate hydroxyls of the active glycoprotein at the ice-solution interface may physically prevent growth of the ice lattice.     

Growth of an ice phase in frozen tissue studied by proton NMR-spectroscopy

Development of an ice phase in frozen tissue is a severe problem in cryo-ultramicrotomy. It is assumed that the growth-rate of such an ice phase is controlled by the mobility of the water molecules in the tissue. In order to verify this, wide-line nmr-spectroscopy has been performed on the protons of frozen whole blood, with and without protective agent, in the temperature range −100 °C- −40 °C. The molecular motion may be characterized by a correlation time, τ, which can be thought of as roughly the mean time between proton jumps. The value of τ can be approximately evaluated from the width of the proton resonance line. It is shown that τ varies rapidly with temperature, in a similar manner to maximum storage time for frozen blood in blood banks. Addition of glycerol increases τ at low temperature, which is consistent with its use as a protective agent in cryobiology. At high temperature, addition of glycerol reduces τ, which is in agreement with reported light-microscope observations on the growth-rate of an ice phase in the same temperature range, nmr-spectroscopy seems to be a useful tool in the further development of cryo-ultramicrotomy. However, to estimate the value in full, more data is required than is presented here.     

Reaction of Harbor Seals to Cruise Ships

Abstract: The largest aggregations of harbor seals (Phoca vitulina) in Alaska, USA, haul out on floating ice in tidewater glacial fjords. Seals use these fjords in peak numbers during the critical periods of pupping, breeding, and molting when visits by tour ships also peak. Documented and suspected declines of harbor seals in fjords with rising vessel traffic underscore the need to better understand possible impacts, particularly in areas where ship visits have risen substantially in the past 2 decades. We examined the interruption of haul-out bouts of harbor seals due to approaching cruise ships in Disenchantment Bay, Alaska. We conducted observations from cruise ships and focused on disturbance of seals as evidenced by seals flushing into the water from the floating ice on which they rested. We investigated rate of flushing in relation to vessel distance, approach angle, group size, and seal type (mother, pup, or other). Using a survival-regression analysis, we found that the risk of disturbing harbor seals increased when ships approached within 500 m; seals approached as close as 100 m were 25 times more likely to enter the water than seals 500 m from a ship. Seals were 4 times more prone to enter the water when ships were approaching directly rather than passing abeam. Seals responded similarly regardless of group size or seal type. Energetic models indicated a potential to disrupt energy balance and cause thermal stress in disturbed pups if they spent >50% of their time in ice-chilled water. Studies at non-glacial sites suggest that pups spend 40–70% of their time in the water. Voluntary guidelines for approaching seals in Alaska recommend that cruise ships approach ≥91 m (100 yards), a distance at which we show 90% of seals would flush into the water. Our findings indicate a need to develop regulations to maintain a 500-m separation between cruise ships and seals in all Alaskan glacial fjords.