Changes in cell morphology due to plasma membrane wounding by acoustic cavitation.

Acoustic cavitation-mediated wounding (i.e., sonoporation) has great potential to improve medical and laboratory applications requiring intracellular uptake of exogenous molecules; however, the field lacks detailed understanding of cavitation-induced morphologic changes in cells and their relative importance. Here, we present an in-depth study of the effects of acoustic cavitation on cells using electron and confocal microscopy coupled with quantitative flow cytometry. High resolution images of treated cells show that morphologically different types of blebs can occur after wounding conditions caused by ultrasound exposure as well as by mechanical shear and strong laser ablation. In addition, these treatments caused wound-induced nonlytic necrotic death resulting in cell bodies we call wound-derived perikarya (WD-P). However, only cells exposed to acoustic cavitation experienced ejection of intact nuclei and nearly instant lytic necrosis. Quantitative analysis by flow cytometry indicates that wound-derived perikarya are the dominant morphology of nonviable cells, except at the strongest wounding conditions, where nuclear ejection accounts for a significant portion of cell death after ultrasound exposure.

Outer membrane-associated serine protease involved in adhesion of Shewanella oneidensis to Fe(III) oxides.

The facultative anaerobe Shewanella oneidensis MR-1 respires a variety of anaerobic electron acceptors, including insoluble Fe(III) oxides. S. oneidensis employs a number of novel strategies for respiration of insoluble Fe(III) oxides, including localization of respiratory proteins to the cell outer membrane (OM). The molecular mechanism by which S. oneidensis adheres to and respires Fe(III) oxides, however, remains poorly understood. In the present study, whole cell fractionation and MALDI-TOF-MS/MS techniques were combined to identify a serine protease (SO3800) associated with the S. oneidensis OM. SO3800 contained predicted structural motifs similar to cell surface-associated serine proteases that function as bacterial adhesins in other gram-negative bacteria. The gene encoding SO3800 was deleted from the S. oneidensis genome, and the resulting mutant strain (DeltaSO3800) was tested for its ability to adhere to and respire Fe(III) oxides. DeltaSO3800 was severely impaired in its ability to adhere to Fe(III) oxides, yet retained wild-type Fe(III) respiratory capability. Laser Doppler velocimetry and cryoetch high-resolution SEM experiments indicated that DeltaSO3800 displayed a lower cell surface charge and higher amount of surface-associated exopolysaccharides. Results of this study indicate that S. oneidensis may respire insoluble Fe(III) oxides at a distance, negating the requirement for attachment prior to electron transfer.

A permanent change in protein mechanical responses can be produced by thermally-induced microdomain mixing.

Electrospinning was employed to fabricate 3-D fiber networks from a recombinant amphiphilic elastin-mimetic tri-block protein polymer and the effects of moderate thermal conditioning (60 degrees C, 4 h) on network mechanical responses investigated. Significantly, while cryo-high resolution scanning electron microscopy (cryo-HRSEM) revealed that the macroscopic and microscopic morphology of the network structure was unchanged, solid-state (1)H-NMR spectroscopy demonstrated enhanced interphase mixing of hydrophobic and hydrophilic blocks. Significantly, thermal annealing triggered permanent changes in network swelling behavior (28.75 +/- 2.80 non-annealed vs. 13.55 +/- 1.39 annealed; P < 0.05) and uniaxial mechanical responses, including Young's modulus (0.170 +/- 0.010 MPa non-annealed vs. 0.366 +/- 0.05 MPa annealed; P < 0.05) and ultimate tensile strength (0.079 +/- 0.008 MPa vs. 0.119 +/- 0.015 MPa; P < 0.05). To our knowledge, these investigations are the first to note that mechanical responses of protein polymers can be permanently altered through a temperature-induced change in microphase mixing.

D-periodic collagen-mimetic microfibers.

Self-assembling peptides have been previously designed that assemble into macroscopic membranes, nanotapes, and filaments through electrostatic interactions. However, the formation of highly ordered collagen-like fibrils, which display D-periodic features, has yet to be achieved. In this report, we describe for the first time a synthetic peptide system that self-assembles into a fibrous structure with well-defined periodicity that can be visualized by transmission electron microscopy (TEM). Specifically, we designed and synthesized a peptide that utilizes charged amino acids within the ubiquitous Xaa-Yaa-Gly triad sequence to bias the self-assembly into collagen-like homotrimeric helices that are capable of fibrillogenesis with the production of D-periodic microfibers. Potential molecular mechanisms for peptide assembly into triple-helical protomers and their subsequent organization into structurally defined, linear assemblies were explored through molecular dynamics (MD) simulations. The formation of thermodynamically stable complexes was attributed to the presence of strong electrostatic and hydrogen bond interactions at staggered positions along the linear assembly. This unexpected mimicry of native collagen structure using a relatively simple oligopeptide sequence establishes new opportunities for engineering linear assemblies with highly ordered nano- and microscale periodic features. In turn, the capacity to precisely design periodic elements into an assembly that faithfully reproduces these features over large length scales may facilitate the fabrication of ordered two- and three-dimensional fiber networks containing oriented biologically, chemically, or optically active elements.

Macroscale assembly of peptide nanotubes.

Simple oligopeptides that self-assemble into homogeneous nanotubes can be directed to further assemble into macroscale parallel arrays through protein "salting out" strategies.

Semagenesis and the parasitic angiosperm Striga asiatica.

Over the last several years, intermediates in the reduction of dioxygen have been attributed diverse functional roles ranging from protection against pathogen attack to the regulation of cellular development. Evidence now suggests that parasitic angiosperms, which naturally commit to virulence through the growth of new organs, depend on reduced oxygen intermediates, or reactive oxygen species (ROS), for signal generation. Clearly, the role of ROS in both plant defense and other physiological responses complicates any models that employ these intermediates in host plant recognition. Here we exploit the transparent young Striga asiatica seedling to (i) localize the site of H(2)O(2) accumulation to the surface cells of the primary root meristem, (ii) demonstrate the accumulation of H(2)O(2) within cytoplasmic and apoplastic compartments, and (iii) document precise regulation of H(2)O(2) accumulation during development of the host attachment organ, the haustorium. These studies reveal a new active process for signal generation, host detection and commitment that is capable of ensuring the correct spatial and temporal positioning for attachment.

Scanning electron microscopic analysis of defects in polymer coatings of three commercially available stents: comparison of BiodivYsio, Taxus and Cypher stents.

BACKGROUND: Although the use of polymer-based drug-eluting stents appears to markedly reduce the risk of in-stent restenosis, there are concerns about their safety including polymer layer integrity. OBJECTIVES: The objective of this study was to investigate the morphology of the polymer layer of 3 commercially available polymercoated stents, including the effects of balloon catheter expansion, by scanning electron microscopy (SEM). METHODS: We assessed discontinuities and other irregularities in the polymer layer of BiodivYsio, Taxus and Cypher stents by SEM after balloon expansion in saline solution at 37 degrees C. RESULTS: Distinctive polymer layer morphologies were found among the 3 stent types, including responses to balloon expansion and withdrawal. The BiodivYsio stent showed no waving or other irregularities on the outer surface, but excess polymer was present on stent edges and polymer was peeled off from the inner surface. The Taxus stent showed no irregularities on the outer surface, but there were polymer bridgings across strut loops and linear cracking of the bridges, as well as inner surface polymer defects with bare-metal exposure. The Cypher stent showed a rough surface with irregularities and waving on the outer surface. There also appeared to be polymer defects with bare-metal exposure in the loop region and peeling of the top-coated polymer layer in the loop. CONCLUSIONS: We found several types of defects in the polymer layers on commercially available polymer-coated stents. Some of these indicate potential risks of thrombosis, coronary microembolism of polymer layer pieces and late inflammatory or neointimal reactions.

Polymeric sulfated amino acid surfactants: a class of versatile chiral selectors for micellar electrokinetic chromatography (MEKC) and MEKC-MS.

In this work, three amino acid-derived (l-leucinol, l-isoleucinol, l-valinol) sulfated chiral surfactants are synthesized and polymerized. These chiral sulfated surfactants are thoroughly characterized to determine critical micelle concentration, aggregation number, polarity, optical rotation, and partial specific volume. For the first time the morphological behavior of polymeric sulfated surfactants is revealed using cryogenic high-resolution electron microscopy. The polysodium N-undecenoyl-l-leucine sulfate shows distinct tubular structure, while polysodium N-undecenoyl-l-valine sulfate also shows tubular morphology but without any distinct order of the tubes. On the other hand, polysodium N-undecenoyl-l-isoleucine sulfate (poly-l-SUCILS) displays random distribution of coiled/curved filaments with heavy association of tightly and loosely bound water. All three polymeric sulfated surfactants are compared for enantioseparation of a broad range of structurally diverse racemic compounds at very acidic, neutral, and basic pH conditions in micellar electrokinetic chromatography (MEKC). A small combinatorial library of 10 structurally related phenylethylamines (PEAs) is investigated for chiral separation under acidic and moderately acidic to neutral pH conditions using an experimental design. In contrast to neutral pH conditions, at acidic pH, significantly enhanced chiral resolution is obtained for class I and class II PEAs due to the compact structure of polymeric sulfated surfactants. It is observed that the presence of a hydroxy group on the benzene ring of PEAs resulted in deterioration of enantioseparation. A sensitive MEKC-mass spectrometry (MS) method is developed for one of the PEAs (e.g., (+/-)-pseudoephedrine) in human urine. Very low limit of detection (LOD) is obtained at pH 2.0 (LOD 325 ng/mL), which is approximately 16 times better compared to pH 8.0 (LOD 5.2 microg/mL). Another broad range of chiral analytes (beta-blockers, phenoxypropionic acid, benzoin derivatives, PTH-amino acids, benzodiazepinones) studied also provided improved chiral separation at low pH compared to high-pH conditions. Among the three polymeric sulfated surfactants, poly-l-SUCILS with two chiral centers on the polymer head group provided overall higher enantioresolution for the investigated acidic, basic, and neutral compounds. This work clearly demonstrates for the first time the superiority of chiral separation and sensitive MS detection at low pH over conventional high-pH chiral separation and detection employing anionic chiral polymeric surfactants in MEKC and MEKC-MS.

The effect of a recombinant elastin-mimetic coating of an ePTFE prosthesis on acute thrombogenicity in a baboon arteriovenous shunt.

A recombinant elastin-mimetic triblock protein polymer with an inverse transition temperature (approximately 20 degrees C) was used to impregnate small-diameter (4 mm i.d.) expanded polytetrafluoroethylene (ePTFE) vascular grafts. Scanning electron microscopy confirmed that initial elastin impregnation of the graft followed by further multilayer coating with elastin films filled in the fibril and node structure of the luminal surface of the ePTFE graft and was macroscopically smooth. Elastin protein polymer impregnation reduced the advancing contact angle of the luminal surface to 43 degrees, which was comparable to the advancing contact angle of 47 degrees for a cast elastin film. Attenuated total reflection infrared spectroscopy and Coomassie blue staining revealed little discernable change in the protein surface film after 24 h of shear at 500 s(-1) and 37 degrees C. Excellent short-term blood-contacting properties as determined by minimal fibrin and platelet deposition were demonstrated using a baboon extracorporeal femoral arteriovenous shunt model. The results of this study demonstrate the applicability of an elastin-mimetic triblock protein polymer as a non-thrombogenic coating or as a component of a tissue-engineered composite.

Mechanism of intracellular delivery by acoustic cavitation.

Using conditions different from conventional medical imaging or laboratory cell lysis, ultrasound has recently been shown to reversibly increase plasma membrane permeability to drugs, proteins and DNA in living cells and animals independently of cell or drug type, suggesting a ubiquitous mechanism of action. To determine the mechanism of these effects, we examined cells exposed to ultrasound by flow cytometry coupled with electron and fluorescence microscopies. The results show that cavitation generated by ultrasound facilitates cellular incorporation of macromolecules up to 28 nm in radius through repairable micron-scale disruptions in the plasma membrane with lifetimes >1 min, which is a period similar to the kinetics of membrane repair after mechanical wounding. Further data suggest that cells actively reseal these holes using a native healing response involving endogenous vesicle-based membrane resealing. In this way, noninvasively focused ultrasound could deliver drugs and genes to targeted tissues, thereby minimizing side effects, lowering drug dosages, and improving efficacy.

Rational design of a reversible pH-responsive switch for peptide self-assembly.

Peptide TZ1H, based on the heptad sequence of a coiled-coil trimer, undergoes fully reversible, pH-dependent self-assembly into long-aspect-ratio helical fibers. Substitution of isoleucine residues with histidine at the core d-positions of alternate heptads introduces a mechanism by which self-assembly is coupled to the protonation state of the imidazole side chain. Circular dichroism spectroscopy, transmission electron microscopy, and microrheology techniques revealed that the self-assembly of TZ1H coincides with a distinct coil-helix conformational transition that occurs within a narrow pH range near the pKa of the imidazole side chains of the core histidine residues.

Geobacter lovleyi sp. nov. strain SZ, a novel metal-reducing and tetrachloroethene-dechlorinating bacterium.

A bacterial isolate, designated strain SZ, was obtained from noncontaminated creek sediment microcosms based on its ability to derive energy from acetate oxidation coupled to tetrachloroethene (PCE)-to-cis-1,2-dichloroethene (cis-DCE) dechlorination (i.e., chlororespiration). Hydrogen and pyruvate served as alternate electron donors for strain SZ, and the range of electron acceptors included (reduced products are given in brackets) PCE and trichloroethene [cis-DCE], nitrate [ammonium], fumarate [succinate], Fe(III) [Fe(II)], malate [succinate], Mn(IV) [Mn(II)], U(VI) [U(IV)], and elemental sulfur [sulfide]. PCE and soluble Fe(III) (as ferric citrate) were reduced at rates of 56.5 and 164 nmol min(-1) mg of protein(-1), respectively, with acetate as the electron donor. Alternate electron acceptors, such as U(VI) and nitrate, did not inhibit PCE dechlorination and were consumed concomitantly. With PCE, Fe(III) (as ferric citrate), and nitrate as electron acceptors, H(2) was consumed to threshold concentrations of 0.08 +/- 0.03 nM, 0.16 +/- 0.07 nM, and 0.5 +/- 0.06 nM, respectively, and acetate was consumed to 3.0 +/- 2.1 nM, 1.2 +/- 0.5 nM, and 3.6 +/- 0.25 nM, respectively. Apparently, electron acceptor-specific acetate consumption threshold concentrations exist, suggesting that similar to the hydrogen threshold model, the measurement of acetate threshold concentrations offers an additional diagnostic tool to delineate terminal electron-accepting processes in anaerobic subsurface environments. Genetic and phenotypic analyses classify strain SZ as the type strain of the new species, Geobacter lovleyi sp. nov., with Geobacter (formerly Trichlorobacter) thiogenes as the closest relative. Furthermore, the analysis of 16S rRNA gene sequences recovered from PCE-dechlorinating consortia and chloroethene-contaminated subsurface environments suggests that Geobacter lovleyi belongs to a distinct, dechlorinating clade within the metal-reducing Geobacter group. Substrate versatility, consumption of electron donors to low threshold concentrations, and simultaneous reduction of electron acceptors suggest that strain SZ-type organisms have desirable characteristics for bioremediation applications.

Fabrication of a phospholipid membrane-mimetic film on the luminal surface of an ePTFE vascular graft.

A stabilized, membrane-mimetic film was produced on the luminal surface of an ePTFE vascular graft by in situ photopolymerization of an acrylate functonalized phospholipid using a fiber optic diffusing probe. The phospholipid monomer was synthesized, prepared as unilamellar vesicles, and fused onto close-packed octadecyl chains that were components of an amphiphilic terpolymer anchored onto the polyelectrolyte multilayer (PEM) by electrostatic interactions. Scanning electron microscopy (SEM) confirmed that gelatin impregnation of the graft followed by the subsequent biomimetic film coating filled in the fibril and node structure of the luminal surface of the ePTFE graft and was smooth. The lipid film displayed an initial advancing contact angle of 44 degrees , which increased to 55 degrees after being subjected to a wall shear rate of 500s(-1) for 24h at 37 degrees C in phosphate buffered saline (PBS). Fourier transform (FT-IR) spectroscopy was used to characterize the stages of biomimetic film assembly and confirmed the stability of the film under shear flow conditions. In vivo assessment using a baboon femoral arteriovenous shunt model demonstrated minimal platelet and fibrinogen deposition over a 1-h blood-contacting period. The results of this study confirm the versatility of a biomimetic film coating system by successfully transferring the methodology previously developed for planar substrates to the luminal surface of an ePTFE vascular graft.

Quantitative PCR confirms purity of strain GT, a novel trichloroethene-to-ethene-respiring Dehalococcoides isolate.

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 micromol liter-1 day-1, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76+/-0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.

Shamrock surfactants with terminal carboxylate headgroups and a central phosphorodithioate or quaternary ammonium headgroup.

Surfactants 3 (tripotassium O,O'-di-[11-(carboxylato)undecyl]phosphorodithioate) and 4 (sodium 12-[dimethyl-(11-carboxylatoundecyl)ammonio]dodecanoate), which are new shamrock surfactants, were prepared and characterized. Shamrock surfactants represent a novel class of surfactants that contain a central headgroup connected to two flanking headgroups by hydrocarbon chains; they do not contain long-chain alkyl groups. Surfactants 3 and 4 were characterized in water by measurement of their Krafft temperatures and critical aggregation concentrations, and their aggregates were studied by 1H and 31P NMR spectroscopy, dynamic laser light scattering, and phase-contrast optical microscopy. Aqueous 3 and 4 were also studied by cryoetch high-resolution scanning electron microscopy, which revealed fences with interposed lacelike patterns for the former and compartments formed by irregular fences for the latter. Coacervates were likely formed upon the undisturbed hydration of 3 and 4, as determined by phase-contrast optical microscopy.

Surfactant quaternary ammonium hydrotris(1-pyrazolyl)borates.

Quaternary ammonium surfactants with the hydrotris(1-pyrazolyl)borate anion as an unusual counterion were prepared and characterized. Dodecyl- (1a), tetradecyl- (1b), and hexadecyltrimethylammonium hydrotris(1-pyrazolyl)borate (1c), as well as didodecyldimethylammonium hydrotris(1-pyrazolyl)borate (2), were prepared by metathesis reactions with the corresponding quaternary ammonium bromides and potassium hydrotris(1-pyrazolyl)borate (4). Although the surfactants have limited stabilities in water at 23 degrees C, they were characterized by measurement of their Krafft temperatures and critical aggregation concentrations. (1)H NMR spectroscopy suggested that the single-chain surfactants 1 form small aggregates in water and revealed an association of the quaternary ammonium and hydrotris(1-pyrazolyl)borate ions above their critical aggregation concentrations. Cryo-etch high-resolution scanning electron microscopy indicated that 1a and 1b most likely form segregation patterns upon plunge-freezing and cryo-etching of their aqueous solutions, and a single-crystal X-ray diffraction study of 1a was performed.

Imaging amyloid beta peptide oligomeric particles in solution.

While all protein misfolding diseases are characterized by fibrous amyloid deposits, the favorable free energy and strongly cooperative nature of the self-assembly have complicated the development of therapeutic strategies aimed at preventing their formation. As structural models for the amyloid fibrils approach atomic resolution, increasing evidence suggests that early folding intermediates, rather than the final structure, are more strongly associated with the loss of neuronal function. For that reason we now demonstrate the use of cryo-etch high-resolution scanning electron microscopy (cryo-HRSEM) for the direct observation of pathway intermediates in amyloid assembly. A congener of the Abeta peptide of Alzheimer's disease, Abeta(13-21), samples a variety of time-dependent self-assembles in a manner similar to those seen for larger proteins. A morphological description of these intermediates is the first step towards their structural characterization and the definition of their role in both amyloid assembly and neurotoxicity.

Design and synthesis of asymmetric acyclic phospholipid bolaamphiphiles.

[structure: see text] A synthetic route was devised for the generation of asymmetric lipid bolaamphiphiles through the sequential esterification of an alkyldioic acid, bearing distinct terminal protecting groups, with propanylamine and lyso-phosphatidylcholine headgroups. Bolaamphiphile self-assembly was investigated in solvent mixes of varying polarity by nuclear magnetic resonance (NMR) and Fourier transform-infrared (FT-IR) spectroscopy, as well as in water by cryo-high-resolution scanning electron microscopy (cryo-HRSEM). We anticipate that asymmetric lipid bolaamphiphiles will provide facile building blocks for engineering a variety of unique membrane-mimetic structures.

Shamrock surfactants: synthesis and characterization.

Two types of a new class of surfactants with three headgroups, which possess the general structure 1, have been prepared. Within structure 1, a central headgroup is connected to two flanking headgroups by hydrocarbon chains. The term "shamrock" is used to describe surfactants of structure 1, denoting their triple-headed character and reflecting the fact that shamrocks have leaflets in groups of three. The major lipophilic character of shamrock surfactants is provided by the two hydrocarbon chains linking the three headgroups and not by long-chain alkyl groups appended to the linking hydrocarbon chains or the headgroups. The new surfactants are 2a (2,2,15,15,28,28-hexamethyl-2,15,28-triazonianonacosane triiodide), 2b (2,2,15,15,28,28-hexamethyl-2,15,28-triazonianonacosane trichloride), 3a (O,O'-di-[10-(N,N,N-tripropylammonio)decyl]phosphorodithioate bromide), and 3b (O,O'-di-[10-(N,N,N-tributylammonio)decyl]phosphorodithioate bromide). Compound 14 (2,2,9,9,16,16-hexamethyl-2,9,16-triazoniaheptadecane triiodide) was prepared for comparison with 2a. Surfactants 2 and 3 were characterized in water by measurement of their Krafft temperatures and critical aggregation concentrations, and their aggregates were studied by 1H NMR spectroscopy, dynamic laser light scattering, and phase-contrast optical microscopy. Aqueous 2b was also studied by cryo-etch high-resolution scanning electron microscopy, which revealed irregularly shaped cells containing a complex matrix of surfactant. Coacervates were observed by optical microscopy upon the hydration of 2 and 3.

Ultrastructure in frozen/etched saline solutions: on the internal cleansing of ice.

Seawater, with its 3.5% salt content, freezes into hexagonal ice (Ih) that encloses concentrated brine within its matrix. When unsubmerged sea ice reaches a certain height and temperature, the brine drains downward through narrow channels. This mechanism was now modeled by frozen 2-3.5% saline as investigated by cryo-etch high-resolution secondary electron microscopy. Thus, saline was either plunge-frozen in liquid ethane at -183 degrees C or else high-pressure frozen to -105 degrees C in 5-6 ms. Ice from a freshly exposed surface was then subjected to a high-vacuum sublimation ("etching"), a procedure that removes pure bulk ice in preference to ice from frozen hydrated salt. After chromium-coating the etched surface with a 2-nm film, the sample was examined by cryo-HRSEM. Granular icy "fences" were seen surrounding empty areas where amorphous ice had originally resided. Since the fences, about 1-2 mum high, survived the etching, it is likely that they consist of frozen brine. The presence of such fences suggests that, during freezing, saline can purge itself of salt with remarkable speed (5-6 ms). Alternatively, channels (perhaps routed around submicroscopic crystallites of cubic ice (Ic) embedded in the amorphous ice at -105 degrees C) can guide the migration of salt to the periphery of ice patches. Macromolecules fail to form fences because they diffuse too slowly or because they are too large to pass through the channels.