Characterizing the secondary hydration shell on hydrated myoglobin, hemoglobin, and lysozyme powders by its vitrification behavior on cooling and its calorimetric glass-->liquid transition and crystallization behavior on reheating.

For hydrated metmyoglobin, methemoglobin, and lysozyme powders, the freezable water fraction of between approximately 0.3-0.4 g water/g protein up to approximately 0.7-0.8 g water/g protein has been fully vitrified by cooling at rates up to approximately 1500 K min-1 and the influence of cooling rate characterized by x-ray diffractograms. This vitreous but freezable water fraction started to crystallize at approximately 210 K to cubic ice and at approximately 240 K to hexagonal ice. Measurements by differential scanning calorimetry have shown that this vitreous but freezable water fraction undergoes, on reheating at a rate of 30 K min-1, a glass-->liquid transition with an onset temperature of between approximately 164 and approximately 174 K, with a width of between approximately 9 and approximately 16 degrees and an increase in heat capacity of between approximately 20 and approximately 40 J K-1 (mol of freezable water)-1 but that the glass transition disappears upon crystallization of the freezable water. These calorimetric features are similar to those of water imbibed in the pores of a synthetic hydrogel but very different from those of glassy bulk water. The difference to glassy bulk water's properties is attributed to hydrophilic interaction and H-bonding of the macromolecules' segments with the freezable water fraction, which thereby becomes dynamically modified. Abrupt increase in minimal or critical cooling rate necessary for complete vitrification is observed at approximately 0.7-0.8 g water/g protein, which is attributed to an abrupt increase of water's mobility, and it is remarkably close to the threshold value of water's mobility on a hydrated protein reported by Kimmich et al. (1990, Biophys. J. 58:1183). The hydration level of approximately 0.7-0.8 g water/g protein is approximately that necessary for completing the secondary hydration shell.     

Calorimetric study of crystal growth of ice in hydrated methemoglobin and of redistribution of the water clusters formed on melting the ice.

Calorimetric studies of the melting patterns of ice in hydrated methemoglobin powders containing between 0.43 and 0.58 (g water)/(g protein), and of their dependence on annealing at subzero temperatures and on isothermal treatment at ambient temperature are reported. Cooling rates were varied between approximately 1500 and 5 K min-1 and heating rate was 30 K min-1. Recrystallization of ice during annealing is observed at T > 228 K. The melting patterns of annealed samples are characteristically different from those of unannealed samples by the shifting of the melting temperature of the recrystallized ice fraction to higher temperatures toward the value of "bulk" ice. The "large" ice crystals formed during recrystallization melt on heating into "large" clusters of water whose redistribution and apparent equilibration is followed as a function of time and/or temperature by comparison with melting endotherms. We have also studied the effect of cooling rate on the melting pattern of ice with a methemoglobin sample containing 0.50 (g water)/(g protein), and we surmise that for this hydration cooling at rates of > or = approximately 150 K min-1 preserves on the whole the distribution of water molecules present at ambient temperature.     

Fluorescence spectra of chlorella in the 295–77°K range

A relation of fluorescence spectra of Chlorella (in the 295–77°K range) to the changes in the phase of the ice is suggested in this paper.

When Chlorella cells are first cooled to 77°K and then warmed slowly, the change of the fluorescence intensity as a function of temperature is different for different bands as earlier reported in spinach chloroplasts. In the 80–150°K range, the F698 decreases rapidly, F687 remains almost unchanged, and f680 increases; above 150°K (the temperature at which ice changes from vitreous to cubic phase), F687 decreases more rapidly than any other band. The F725, that decreases smoothly in the 77–295°K range, is composed of several bands (F717 and F725).

If Chlorella cells are first cooled to 77°K, and then warmed to 260°K, and recooled to 77°K before melting, f685, f698 and f730 — but not f717 — are lowered; this fluorescence decrease is not due to changes in the reabsorption of fluorescence or to major changes in the cellular structure due to freezing.

The F698 band is greatly influenced by the aqueous environment; the phase of the ice and the addition of polar solvents (10% dimethylsulfoxide) greatly influence it. These data are consistent with the hypothesis that F698 is from an energy trap (at low temperatures) of System II.     

Safety zone as a barrier to root-shoot ice propagation

Winter wheat, rye and triticale were sown at a depth of 12 cm to promote internode elongation. The root-shoot junction was separated by an internode about 4.5 cm long. The rate of ice propagation was measured separately across the root-internode and crown-internode at –4°C. Ice nucleation in seminal roots was experimentally induced by ice nucleating bacteria. The rate of movement of the ice front was measured by differential thermal analysis. The highest rate of ice propagation occurred in the internode. The rate of ice propagation across the root-internode section was slower than in the internode alone, but differences exist in the various genotypes. The rate of ice propagation between the seminal roots and the internode did not correlate with the number of roots with unsafe vessels, as detected by the pressurized air method. The rate of ice propagation between the internode and the crown was approximately one third of the rate in the internode. The discontinuity in internode-crown section reached nearly 100% as was observed by the pressurized air method. Anatomical studies supported the discontinuity of vessels evaluated by the pressurized air method. Experimental data support the view that the barriers in root-internode and in internode-crown section reduced the rate of ice propagation but these barriers are not correlated with either cold hardiness or number of safe roots.Abbreviations AN adventitious nodal roots - AS adventitious seminal roots - SS secondary seminal roots     

Quantitation of molecular densities by cryo-electron microscopy. Determination of the radial density distribution of tobacco mosaic virus.

We have determined the absolute mass and radial scattering density distribution of tobacco mosaic virus in the frozen-hydrated state by energy-filtered low-dose bright-field transmission electron microscopy. The absolute magnitude of electron scattering from tobacco mosaic virus in 150 nm of ice was within 3.0% of that predicted, with inelastic scattering accounting for approximately 80% of the scattering contrast. In order to test the accuracy of the radial reconstruction, a computer model of tobacco mosaic virus was built from the atomic co-ordinates assuming uniform solvent density. The validity of the model was confirmed by comparison of X-ray scattering and predictions of the model (R factor = 0.05). First-order corrections for the microscope contrast transfer function were necessary and sufficient for conversion of the cryo-electron microscopy images into accurate representations of the mass density. At 1.9 nm resolution the compensated reconstruction and model had density peaks of similar magnitude at 2.4, 4.2, 6.0 and 7.8 nm radius and a central hole of 2 nm radius. Equatorial Fourier transforms of the corrected electron images were in excellent agreement with predictions of the model (R factor = 0.12). Thus, the uniform solvent approximation was adequate at 1.9 nm resolution to describe quantitatively X-ray scattering in liquid water and electron imaging in vitreous ice. This is the first demonstration that cryo-electron microscopy images can be used to quantitate the absolute mass, mass per unit length and internal density distributions of proteins and nucleic acids.     

A comparison of liquid nitrogen and liquid helium as cryogens for electron cryotomography

The principal resolution limitation in electron cryomicroscopy of frozen-hydrated biological samples is radiation damage. It has long been hoped that cooling such samples to just a few kelvins with liquid helium would slow this damage and allow statistically better-defined images to be recorded. A new "G2 Polara" microscope from FEI Company was used to image various biological samples cooled by either liquid nitrogen or liquid helium to approximately 82 or approximately 12 K, respectively, and the results were compared with particular interest in the doses (10-200 e-/A2) and resolutions (3-8 nm) typical for electron cryotomography. Simple dose series revealed a gradual loss of contrast at approximately 12K through the first several tens of e-/A2, after which small bubbles appeared. Single particle reconstructions from each image in a dose series showed no difference in the preservation of medium-resolution (3-5 nm) structural detail at the two temperatures. Tomographic reconstructions produced with total doses between 10 and 350 e-/A2 showed better results at approximately 82 K than approximately 12 K for every dose tested. Thus disappointingly, cooling with liquid helium is actually disadvantageous for cryotomography.     

Response of nematode communities after large‐scale ice‐shelf collapse events in the Antarctic Larsen area

Owing to large‐scale ice‐shelf disintegration events, the Antarctic Larsen A and B areas recently became ice‐free. During the ANT‐XXIII/8 Polarstern campaign, this region was sampled for the first time. Our study is the first to investigate benthic communities in this area and their response to the collapse of ice shelves in the Antarctic. The nematofauna appears to be strongly influenced by the sudden ice‐cover removal, although its response differs from that of the macro‐ and megabenthos. Our results indicate that precollapse, sub‐ice communities were impoverished and characterized by low densities, low diversity and high dominance of a few taxa. This might still be visible at a station located deep inside the Larsen B embayment, where Halomonhystera was dominant. Post‐collapse recolonization of the ‘inner’ stations, i.e. those located furthermost from the former ice‐shelf edge, is believed to be a long‐time process. At the time of sampling, community structure at the inner stations was not or only slightly influenced by colonization, and might be structured by local environmental conditions. Our results indicate that a locally increased food supply after ice‐cover removal could provoke a faster, local response of the nematode assemblages compared with the response due to recolonization. Thalassomonhystera is recognized as an opportunist, taking advantage of increased food supply at inner stations A_South and B_North. Communities living close to the former ice‐shelf edge are believed to be at an intermediate or late stage of succession, with a dominance of Microlaimus, a common Antarctic genus and quick colonizer. Densities here were comparable with those at other Antarctic stations, whereas they were considerably decreased at the inner stations. In general, the collapse of the Larsen ice shelves initially has a positive effect on the shelf nematode fauna in the area, both in terms of abundance and diversity.     

Collapse temperature of solutions important for lyopreservation of living cells at ambient temperature

In this study, the collapse temperature was determined using the freeze‐drying microscopy (FDM) method for a variety of cell culture medium‐based solutions (with 0.05–0.8 M trehalose) that are important for long‐term stabilization of living cells in the dry state at ambient temperature (lyopreservation) by freeze‐drying. Being consistent with what has been reported in the literature, the collapse temperature of binary water‐trehalose solutions was found to be similar to the glass transition temperature (T′_g ～ ?30°C) of the maximally freeze‐concentrated trehalose solution (～80 wt% trehalose) during the freezing step of freeze‐drying, regardless of the initial concentration of trehalose. However, the effect of the initial trehalose concentration on the collapse temperature of the cell culture medium‐based trehalose solutions was identified to be much more significant, particularly when the trehalose concentration is less than 0.2 M (the collapse temperature can be as low as ?65°C). We also determined that cell density from 1 to 10 million cells/mL and ice seeding at high subzero temperatures (?4 and ?7°C) have negligible impact on the solution collapse temperature. However, ice seeding does significantly affect the ice crystal morphology formed during the freezing step and therefore the drying rate. Finally, bulking agents (mannitol) could significantly affect the collapse temperature only when trehalose concentration is low (<0.2 M). However, improving the collapse temperature by using a high concentration of trehalose might be preferred to the addition of bulking agents in the solutions for freeze‐drying of living cells. We further confirmed the applicability of the collapse temperature measured with small‐scale (2 µL) samples using the FDM system to freeze‐drying of large‐scale (1 mL) samples using scanning electron microscopy (SEM) data. Taken together, the results reported in this study should provide useful guidance to the development of optimal freeze‐drying protocols for lyopreservation of living cells at ambient temperature for easy maintenance and convenient wide distribution to end users, which is important to the eventual success of modern cell‐based medicine. Biotechnol. Bioeng. 2010;106: 247–259. © 2010 Wiley Periodicals, Inc.     

Visualization of beta-sheets and side-chain clusters in two-dimensional periodic arrays of streptavidin on phospholipid monolayers by electron crystallography.

The biotin-binding protein streptavidin was crystallized as two-dimensional periodic arrays on biotinylated phospholipid monolayers. Electron diffraction patterns and images of the arrays embedded in vitreous ice were recorded to near-atomic resolution. Amplitudes and phases of structure factors were computed and combined to produce a 3 A projection density map. The reliability of the map was verified by comparing it to the available x-ray atomic model of the molecule. Projection densities from beta-strands and some amino acid side chains were identified from the electron cryomicroscopy map. These results demonstrate the first near-atomic image of this type of protein periodic array by electron crystallography, which has a great potential to aid in the structural characterization of molecular arrays engineered on a monolayer for various basic or biotechnological applications.     

Contribution of sea ice microbial production to Antarctic benthic communities is driven by sea ice dynamics and composition of functional guilds

Organic matter produced by the sea ice microbial community (SIMCo) is an important link between sea ice dynamics and secondary production in near‐shore food webs of Antarctica. Sea ice conditions in McMurdo Sound were quantified from time series of MODIS satellite images for Sept. 1 through Feb. 28 of 2007–2015. A predictable sea ice persistence gradient along the length of the Sound and evidence for a distinct change in sea ice dynamics in 2011 were observed. We used stable isotope analysis (δ¹³C and δ¹⁵N) of SIMCo, suspended particulate organic matter (SPOM) and shallow water (10–20 m) macroinvertebrates to reveal patterns in trophic structure of, and incorporation of organic matter from SIMCo into, benthic communities at eight sites distributed along the sea ice persistence gradient. Mass‐balance analysis revealed distinct trophic architecture among communities and large fluxes of SIMCo into the near‐shore food web, with the estimates ranging from 2 to 84% of organic matter derived from SIMCo for individual species. Analysis of patterns in density, and biomass of macroinvertebrate communities among sites allowed us to model net incorporation of organic matter from SIMCo, in terms of biomass per unit area (g/m²), into benthic communities. Here, organic matter derived from SIMCo supported 39 to 71 per cent of total biomass. Furthermore, for six species, we observed declines in contribution of SIMCo between years with persistent sea ice (2008–2009) and years with extensive sea ice breakout (2012–2015). Our data demonstrate the vital role of SIMCo in ecosystem function in Antarctica and strong linkages between sea ice dynamics and near‐shore secondary productivity. These results have important implications for our understanding of how benthic communities will respond to changes in sea ice dynamics associated with climate change and highlight the important role of shallow water macroinvertebrate communities as sentinels of change for the Antarctic marine ecosystem.     

Bearded seal (Erignathus barbatus) birth mass and pup growth in periods with contrasting ice conditions in Svalbard,Norway

Global climate warming has caused major reductions in sea ice in the Arctic, posing a serious threat to ice-associated marine mammals. Herein, novel data on birth mass and pup growth rates over a 15-year period (1993–2007; 10 years with growth data) are reported for bearded seals, as well as initial behavioral responses by this species to major, local declines in sea-ice in Svalbard, Norway. In total, 205 pups were captured; 64 of which were recaptured, some repeatedly, producing 85 growth intervals for nursing pups. Average birth mass of pups was 37.1 ± 3.8 (SD) kg (range 33–47 kg, n = 25); birth mass before vs. after the sea ice collapse (2006 onward) were not found to differ. Pups grew at a rate of 3.0 ± 0.7 kg/day (1.8–4.8 kg/day, n = 64) during the nursing period. LME models suggest that ice concentration did not affect the growth rate of pups. Most females shifted from traditional first-year ice floes to glacier-ice pieces for birthing and nursing their young, following the regional sea ice collapse. However, retraction of tidal glaciers will likely eliminate this replacement birthing and nursing habitat for bearded seals in Svalbard in the coming decades.     

南极海冰耐盐细菌NJ82的分子鉴定及其耐盐性初步研究

从129株南极海冰细菌筛选到1株耐盐细菌NJ82,该菌最适生长盐度是12%,能够耐受最高盐度为18%.对该菌株进行 16S rRNA 基因序列的同源性和系统发育分析,结果表明:菌株NJ82 属于 Pseudoalteromonas 属.从总蛋白质、脯氨酸、丙二醛(MDA)含量及膜透性变化等方面对高盐的适应性进行初步探讨.结果表明,当盐度介于3.3%～12.0%时,随着盐度升高,菌株的蛋白质和脯氨酸含量呈快速增加趋势,而 MDA 含量和膜透性变化幅度不大;随着盐度进一步升高,蛋白质含量呈下降趋势,脯氨酸变化幅度不大;而 MDA 含量升高和膜透性变化都达到极显著水平(P＜0.01).这些重要生理参数的变化将有助于了解海冰细菌在高盐环境下的适应机制.     

Location of the 100 kd-50 kd accessory proteins in clathrin coats.

We present a three-dimensional map of the clathrin coat of coated vesicles, generated from tilt series of electron micrographs of unstained specimens embedded in vitreous ice. We have examined native placental coated vesicles and coats reassembled from their purified constituents, namely clathrin triskelions and accessory proteins of approximate mol. wts 100 kd and 50 kd. Our results show that the accessory proteins contribute a further shell of density within the double shell of the clathrin cage, extending from the terminal domains of the clathrin to the membrane of the vesicle. The thickness of the complete coat is approximately 22 nm.     

南极海冰耐盐细菌NJ82的分子鉴定及其耐盐性初步研究

从129株南极海冰细菌筛选到1株耐盐细菌NJ82, 该菌最适生长盐度是12%, 能够耐受最高盐度为18%。对该菌株进行16S rRNA基因序列的同源性和系统发育分析, 结果表明：菌株NJ82属于Pseudoalteromonas属。从总蛋白质、脯氨酸、丙二醛(MDA)含量及膜透性变化等方面对高盐的适应性进行初步探讨。结果表明, 当盐度介于3.3%~12.0％时, 随着盐度升高, 菌株的蛋白质和脯氨酸含量呈快速增加趋势, 而MDA含量和膜透性变化幅度不大; 随着盐度进一步升高, 蛋白质含量呈下降趋势, 脯氨酸变化幅度不大; 而MDA含量升高和膜透性变化都达到极显著水平(P<0.01)。这些重要生理参数的变化将有助于了解海冰细菌在高盐环境下的适应机制。     

An ice-binding protein from an Antarctic sea ice bacterium

An Antarctic sea ice bacterium of the Gram-negative genus Colwellia, strain SLW05, produces an extracellular substance that changes the morphology of growing ice. The active substance was identified as a approximately 25-kDa protein that was purified through its affinity for ice. The full gene sequence was determined and was found to encode a 253-amino acid protein with a calculated molecular mass of 26,350 Da. The predicted amino acid sequence is similar to predicted sequences of ice-binding proteins recently found in two species of sea ice diatoms and a species of snow mold. A recombinant ice-binding protein showed ice-binding activity and ice recrystallization inhibition activity. The protein is much smaller than bacterial ice-nucleating proteins and antifreeze proteins that have been previously described. The function of the protein is unknown but it may act as an ice recrystallization inhibitor to protect membranes in the frozen state.     

The influence of hydroxyethyl starch on ice formation in aqueous solutions

Differential scanning calorimetry, and, in some supplementary experiments, X-ray diffractometry and cryomicroscopy, were applied to study the influence of concentration (< 70 wt%) and cooling/warming rates (< 320 K/min) on ice formation in aqueous solutions of HES. The calorimetric measurements of the quantity of crystallizing water indicated that a mass fraction ? = 0.522 (i.e., grams water per gram HES) remained unfrozen. These results are in good agreement with our earlier extrapolations from ternary phase diagram data and tend to support the proposed cryoprotective mechanism. The value of ? determined during warming was essentially independent of composition up to the corresponding saturation concentration. It was observed that solutions containing 60 wt% HES or more remained wholly amorphous during cooling even at rates as low as 2.5 K/min (down to 120 K). Such glassy solutions are subject to devitrification at temperatures T_d which depend on the warming rate. The concentrations close to 55 wt% HES mark a transitional range exhibiting two crystallization peaks, probably due to different mechanisms of nucleation, the portion of ice formed during cooling being related to the imposed cooling rate. All samples showed a recrystallization transition at 257.5 K which was also observed cryomicroscopically. Glass transitions, however, could not be detected by the methods applied in this study. The X-ray diffraction patterns contained the structure of only one solid phase, namely hexagonal ice. A comparison of various modifications of HES, PEG, and PVP involving bound water and melting temperature did not reveal marked differences. Minimum initial HES concentrations preventing lethal salt enrichment were computed for both binary and ternary mass fractions of NaCl as biologically relevant parameters, yielding 24.1 and 10.8 wt% HES, respectively.     

X-ray diffraction evidence for the existence of 102.0- and 230.0-nm transverse periodicities in striated muscle

Synchrotron radiation techniques have enabled us to record meridional x-ray diffraction patterns from frog sartorius muscle at resolutions ranging from approximately 2,800 to 38 nm (i.e., overlapping with the optical microscope and the region normally accessible with low angle diffraction cameras). These diffraction patterns represent the transform of the low resolution structure of muscle projected on the sarcomere axis and sampled by its repeat. Altering the sarcomere length results in the sampling of different parts of this transform, which induces changes in the positions and the integrated intensities of the diffraction maxima. This effect has been used to determine the transform of the mass projection on the muscle axis in a quasicontinuous fashion. The results reveal the existence of maxima arising from long-range periodicities in the structure. Determination of the zeroes in the transforms has been used to obtain phase information from which electron density maps have been calculated. The x-ray diffraction diagrams and the resulting electron density maps show the existence of a series of mass bands, disposed transversely to the sarcomere axis and distributed at regular intervals. A set of these transverse structures is associated with thin filaments, and their 102.0-nm repeat suggests a close structural relationship with their known molecular components. A second set, spaced by approximately 230.0 nm, is also present; from diffraction theory one has to conclude that this repeat simultaneously exists in thick and thin filament regions.     

Cryoelectron microscopy of low density lipoprotein in vitreous ice.

In this report, images of low density lipoprotein (LDL) in vitreous ice at approximately 30 A resolution are presented. These images show that LDL is a quasi-spherical particle, approximately 220-240 A in diameter, with a region of low density (lipid) surrounded by a ring (in projection) of high density believed to represent apolipoprotein B-100. This ring is seen to be composed of four or five (depending on view) large regions of high density material that may represent protein superdomains. Analysis of LDL images obtained at slightly higher magnification reveals that areas of somewhat lower density connect these regions, in some cases crossing the projectional interiors of the LDL particles. Preliminary image analysis of LDL covalently labeled at Cys3734 and Cys4190 with 1.4-nm Nanogold clusters demonstrates that this methodology will provide an important site-specific marker in studies designed to map the organization of apoB at the surface of LDL.     

Protists in Arctic drift and land‐fast sea ice

Global climate change is having profound impacts on polar ice with changes in the duration and extent of both land‐fast ice and drift ice, which is part of the polar ice pack. Sea ice is a distinct habitat and the morphologically identifiable sympagic community living within sea ice can be readily distinguished from pelagic species. Sympagic metazoa and diatoms have been studied extensively since they can be identified using microscopy techniques. However, non‐diatom eukaryotic cells living in ice have received much less attention despite taxa such as the dinoflagellate Polarella and the cercozoan Cryothecomonas being isolated from sea ice. Other small flagellates have also been reported, suggesting complex microbial food webs. Since smaller flagellates are fragile, often poorly preserved, and are difficult for non‐experts to identify, we applied high throughput tag sequencing of the V4 region of the 18S rRNA gene to investigate the eukaryotic microbiome within the ice. The sea ice communities were diverse (190 taxa) and included many heterotrophic and mixotrophic species. Dinoflagellates (43 taxa), diatoms (29 taxa) and cercozoans (12 taxa) accounted for ~80% of the sequences. The sympagic communities living within drift ice and land‐fast ice harbored taxonomically distinct communities and we highlight specific taxa of dinoflagellates and diatoms that may be indicators of land‐fast and drift ice.     

The Southern Ocean ecosystem under multiple climate change stresses ‐ an integrated circumpolar assessment

A quantitative assessment of observed and projected environmental changes in the Southern Ocean (SO) with a potential impact on the marine ecosystem shows: (i) large proportions of the SO are and will be affected by one or more climate change processes; areas projected to be affected in the future are larger than areas that are already under environmental stress, (ii) areas affected by changes in sea‐ice in the past and likely in the future are much larger than areas affected by ocean warming. The smallest areas (<1% area of the SO) are affected by glacier retreat and warming in the deeper euphotic layer. In the future, decrease in the sea‐ice is expected to be widespread. Changes in iceberg impact resulting from further collapse of ice‐shelves can potentially affect large parts of shelf and ephemerally in the off‐shore regions. However, aragonite undersaturation (acidification) might become one of the biggest problems for the Antarctic marine ecosystem by affecting almost the entire SO. Direct and indirect impacts of various environmental changes to the three major habitats, sea‐ice, pelagic and benthos and their biota are complex. The areas affected by environmental stressors range from 33% of the SO for a single stressor, 11% for two and 2% for three, to <1% for four and five overlapping factors. In the future, areas expected to be affected by 2 and 3 overlapping factors are equally large, including potential iceberg changes, and together cover almost 86% of the SO ecosystem.