Ice-Binding Proteins and Their Function.

Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities.

On the engulfment of antifreeze proteins by ice.

Antifreeze proteins (AFPs) are remarkable biomolecules that suppress ice formation at trace concentrations. To inhibit ice growth, AFPs must not only bind to ice crystals, but also resist engulfment by ice. The highest supercooling, [Formula: see text], for which AFPs are able to resist engulfment is widely believed to scale as the inverse of the separation, [Formula: see text], between bound AFPs, whereas its dependence on the molecular characteristics of the AFP remains poorly understood. By using specialized molecular simulations and interfacial thermodynamics, here, we show that in contrast with conventional wisdom, [Formula: see text] scales as [Formula: see text] and not as [Formula: see text]. We further show that [Formula: see text] is proportional to AFP size and that diverse naturally occurring AFPs are optimal at resisting engulfment by ice. By facilitating the development of AFP structure-function relationships, we hope that our findings will pave the way for the rational design of AFPs.

Engineered Compounds to Control Ice Nucleation and Recrystallization.

One of the greatest concerns in the subzero storage of cells, tissues, and organs is the ability to control the nucleation or recrystallization of ice. In nature, evidence of these processes, which aid in sustaining internal temperatures below the physiologic freezing point for extended periods of time, is apparent in freeze-avoidant and freeze-tolerant organisms. After decades of studying these proteins, we now have easily accessible compounds and materials capable of recapitulating the mechanisms seen in nature for biopreser-vation applications. The output from this burgeoning area of research can interact synergistically with other novel developments in the field of cryobiology, making it an opportune time for a review on this topic.

Temperature-Modulated Reversible Clustering of Gold Nanorods Driven by Small Surface Ligands.

Temperature-modulated colloidal phase of plasmonic nanoparticles is a convenient playground for resettable soft-actuators or colorimetric sensors. To render reversible clustering under temperature change, bulky ligands are required, especially if anisotropic morphologies are of interest. This study showcases thermoresponsive gold nanorods by employing small surface ligands, bis (p-sulfonatophenyl) phenyl-phosphine dihydrate dipotassium salt (BSPP) and native cationic surfactant. Temperature-dependent analysis in real-time allowed to describe the structural features (interparticle distance and cluster size) as well as thermal parameters, melting and freezing temperatures. These findings suggest that neither covalent Au-S bonds nor bulky ligands are required to obtain a robust thermoresponsive system based on anisotropic gold nanoparticles, paving the way to stimuli-responsive nanoparticles with a wide range of sizes and geometries.

Room-temperature magnetocapacitance spanning 97 K hysteresis in molecular material.

Magnetic capacitor, as a new type of device, has broad application prospects in fields such as magnetic field sensing, magnetic storage, magnetic field control, power electronics and so on. Traditional magnetic capacitors are mostly assembled by magnetic and capacitive materials. Magnetic capacitor made of a single material with intrinsic properties is very rare. This intrinsic property is magnetocapacitance (MC). The studies on MC effect have mainly focused on metal oxides so far. No study was reported in molecular materials. Herein, two complexes: (CETAB)2[CuCl4] (1) and (CETAB)2[CuBr4] (2) (CETAB = (2-chloroethyl)trimethylammonium) are reported. There exist strong H-Br and Br-Br interactions and other weak interactions in complex 2, so the phase transition energy barrier is high, resulting in the widest thermal hysteresis loop on a molecular level to date. Furthermore, complexes 1 and 2 show large MC parameters of 0.247 and 1.614, respectively, which is the first time to observe MC effect in molecular material.

Brassica juncea leaf cuticle contains xylose and mannose (xylomannan) which inhibit ice recrystallization on the leaf surface.

MAIN CONCLUSION: Conjugated sugars showed antifreeze activity in the cuticle by ice recrystallization inhibition rather than thermal hysteresis, enhancing freezing capacity at the surface of B. juncea leaves. Antifreeze biomolecules play a crucial role in mitigating the physical damage from frost by controlling extracellular ice crystal growth in plants. Antifreeze proteins (AFPs) are reported from the apoplast of different plants. Interestingly, there is no report about antifreeze properties of the cuticle. Here, we report the potential antifreeze activity in the Brassica juncea (BJ) leaf cuticle. Nano LC-MS/MS analysis of a cuticle protein enriched fraction (CPE) predicted over 30 putative AFPs using CryoProtect server and literature survey. Ice crystal morphology (ICM) and ice recrystallization inhibition (IRI) analysis of ABC supernatant showed heat and pronase-resistant, non-protein antifreeze activities as well as hexagonal ice crystals with TH of 0.17 °C and IRI 46%. The ZipTip processed ABC supernatant (without peptides) had no effect on TH activity, confirming a non-protein antifreeze molecule contributing to activity. To understand the origin and to confirm the source of antifreeze activity, cuticular membranes were isolated by pectinase and cellulase hydrolysis. FTIR analysis of the intact cuticle showed xylose, mannose, cellulose, and glucose. Xylanase and cellulase treatments of the ZipTip processed ABC supernatant led to an increase in sugar content and 50% loss in antifreeze activity. UV spectroscopy and NMR analysis supported the finding of FTIR and enzyme hydrolysis suggesting the contribution of xylose and mannose to antifreeze activity. By TLC analysis, conjugated sugars were found in the cuticle. This work has opened up a new research area where the antifreeze capacity needs to be established with regard to complete characterization and mechanism of action of the antifreeze carbohydrates (conjugated sugars) on the leaf surface.

Thermal Hysteresis and Ordering Behavior of Magnetic Skyrmion Lattices.

The physics of phase transitions in two-dimensional (2D) systems underpins research in diverse fields including statistical mechanics, nanomagnetism, and soft condensed matter. However, many aspects of 2D phase transitions are still not well understood, including the effects of interparticle potential, polydispersity, and particle shape. Magnetic skyrmions are chiral spin-structure quasi-particles that form two-dimensional lattices. Here, we show, by real-space imaging using in situ cryo-Lorentz transmission electron microscopy coupled with machine learning image analysis, the ordering behavior of Néel skyrmion lattices in van der Waals Fe3GeTe2. We demonstrate a distinct change in the skyrmion size distribution during field-cooling, which leads to a loss of lattice order and an evolution of the skyrmion liquid phase. Remarkably, the lattice order is restored during field heating and demonstrates a thermal hysteresis. This behavior is explained by the skyrmion energy landscape and demonstrates the potential to control the lattice order in 2D phase transitions.

Structural Insights into Hysteretic Spin-Crossover in a Set of Iron(II)-2,6-bis(1H-Pyrazol-1-yl)Pyridine) Complexes.

Bistable spin-crossover (SCO) complexes that undergo abrupt and hysteretic (ΔT1/2 ) spin-state switching are desirable for molecule-based switching and memory applications. In this study, we report on structural facets governing hysteretic SCO in a set of iron(II)-2,6-bis(1H-pyrazol-1-yl)pyridine) (bpp) complexes - [Fe(bpp-COOEt)2 ](X)2 ⋅CH3 NO2 (X=ClO4 , 1; X=BF4 , 2). Stable spin-state switching - T1/2 =288 K; ΔT1/2 =62 K - is observed for 1, whereas 2 undergoes above-room-temperature lattice-solvent content-dependent SCO - T1/2 =331 K; ΔT1/2 =43 K. Variable-temperature single-crystal X-ray diffraction studies of the complexes revealed pronounced molecular reorganizations - from the Jahn-Teller-distorted HS state to the less distorted LS state - and conformation switching of the ethyl group of the COOEt substituent upon SCO. Consequently, we propose that the large structural reorganizations rendered SCO hysteretic in 1 and 2. Such insights shedding light on the molecular origin of thermal hysteresis might enable the design of technologically relevant molecule-based switching and memory elements.

Electro-Elastic Modeling of Thermal Spin Transition in Diluted Spin-Crossover Single Crystals.

Spin-crossover solids have been studied for many years for their promising applications as optical switches and reversible high-density memories for information storage. This study reports the effect of random metal dilution on the thermal and structural properties of a spin-crossover single crystal. The analysis is performed on a 2D rectangular lattice using an electro-elastic model. The lattice is made of sites that can switch thermally between the low-spin and high-spin states, accompanied by local volume changes. The model is solved by Monte Carlo simulations, running on the spin states and atomic positions of this compressible 2D lattice. A detailed analysis of metal dilution on the magneto-structural properties allows us to address the following issues: (i) at low dilution rates, the transition is of the first order; (ii) increasing the concentration of dopant results in a decrease in cooperativity and leads to gradual transformations above a threshold concentration, while incomplete spin transitions are obtained for big dopant sizes. The effects of the metal dilution on the spatiotemporal aspects of the spin transition along the thermal transition and on the low-temperature relaxation of the photo-induced metastable high-spin states are also studied. Significant changes in the organization of the spin states are observed for the thermal transition, where the single-domain nucleation caused by the long-range elastic interactions is replaced by a multi-droplet nucleation. As to the issue of the relaxation curves: their shape transforms from a sigmoidal shape, characteristic of strong cooperative systems, into stretched exponentials for high dilution rates, which is the signature of a disordered system.

Antifreeze Proteins: Novel Applications and Navigation towards Their Clinical Application in Cryobanking.

Antifreeze proteins (AFPs) or thermal hysteresis (TH) proteins are biomolecular gifts of nature to sustain life in extremely cold environments. This family of peptides, glycopeptides and proteins produced by diverse organisms including bacteria, yeast, insects and fish act by non-colligatively depressing the freezing temperature of the water below its melting point in a process termed thermal hysteresis which is then responsible for ice crystal equilibrium and inhibition of ice recrystallisation; the major cause of cell dehydration, membrane rupture and subsequent cryodamage. Scientists on the other hand have been exploring various substances as cryoprotectants. Some of the cryoprotectants in use include trehalose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), sucrose, propylene glycol (PG) and glycerol but their extensive application is limited mostly by toxicity, thus fueling the quest for better cryoprotectants. Hence, extracting or synthesizing antifreeze protein and testing their cryoprotective activity has become a popular topic among researchers. Research concerning AFPs encompasses lots of effort ranging from understanding their sources and mechanism of action, extraction and purification/synthesis to structural elucidation with the aim of achieving better outcomes in cryopreservation. This review explores the potential clinical application of AFPs in the cryopreservation of different cells, tissues and organs. Here, we discuss novel approaches, identify research gaps and propose future research directions in the application of AFPs based on recent studies with the aim of achieving successful clinical and commercial use of AFPs in the future.

Synthesis and Characterization of [Fe(Htrz)2(trz)](BF4)] Nanocubes.

Compounds that exhibit spin-crossover (SCO) type behavior have been extensively investigated due to their ability to act as molecular switches. Depending on the coordinating ligand, in this case 1H-1,2,4-triazole, and the crystallite size of the SCO compound produced, the energy requirement for the spin state transition can vary. Here, SCO [Fe(Htrz)2(trz)](BF4)] nanoparticles were synthesized using modified reverse micelle methods. Reaction conditions and reagent ratios are strictly controlled to produce nanocubes of 40-50 nm in size. Decreases in energy requirements are seen in both thermal and magnetic transitions for the smaller sized crystallites, where, compared to bulk materials, a decrease of as much as 20 °C can be seen in low to high spin state transitions.

Discovery of Hyperactive Antifreeze Protein from Phylogenetically Distant Beetles Questions Its Evolutionary Origin.

Beetle hyperactive antifreeze protein (AFP) has a unique ability to maintain a supercooling state of its body fluids, however, less is known about its origination. Here, we found that a popular stag beetle Dorcus hopei binodulosus (Dhb) synthesizes at least 6 isoforms of hyperactive AFP (DhbAFP). Cold-acclimated Dhb larvae tolerated -5 °C chilled storage for 24 h and fully recovered after warming, suggesting that DhbAFP facilitates overwintering of this beetle. A DhbAFP isoform (~10 kDa) appeared to consist of 6-8 tandem repeats of a 12-residue consensus sequence (TCTxSxNCxxAx), which exhibited 3 °C of high freezing point depression and the ability of binding to an entire surface of a single ice crystal. Significantly, these properties as well as DNA sequences including the untranslated region, signal peptide region, and an AFP-encoding region of Dhb are highly similar to those identified for a known hyperactive AFP (TmAFP) from the beetle Tenebrio molitor (Tm). Progenitor of Dhb and Tm was branched off approximately 300 million years ago, so no known evolution mechanism hardly explains the retainment of the DNA sequence for such a lo-ng divergence period. Existence of unrevealed gene transfer mechanism will be hypothesized between these two phylogenetically distant beetles to acquire this type of hyperactive AFP.

Magnetic Phase Diagram of the MnxFe2-xP1-ySiy System.

The phase diagram of the magnetocaloric MnxFe2-xP1-ySiy quaternary compounds was established by characterising the structure, thermal and magnetic properties in a wide range of compositions (for a Mn fraction of 0.3 ≤ x < 2.0 and a Si fraction of 0.33 ≤ y ≤ 0.60). The highest ferromagnetic transition temperature (Mn0.3Fe1.7P0.6Si0.4, TC = 470 K) is found for low Mn and high Si contents, while the lowest is found for low Fe and Si contents (Mn1.7Fe0.3P0.6Si0.4, TC = 65 K) in the MnxFe2-xP1-ySiy phase diagram. The largest hysteresis (91 K) was observed for a metal ratio close to Fe:Mn = 1:1 (corresponding to x = 0.9, y = 0.33). Both Mn-rich with high Si and Fe-rich samples with low Si concentration were found to show low hysteresis (≤2 K). These compositions with a low hysteresis form promising candidate materials for thermomagnetic applications.

Effect of in vitro cold acclimation of Deschampsia antarctica on the accumulation of proteins with antifreeze activity.

Deschampsia antarctica has managed to colonize the maritime Antarctic. One of the main factors associated with its tolerance to low temperatures is the presence of apoplastic proteins with antifreeze activity. This work focuses on the effect of cold acclimation of D. antarctica on the accumulation of apoplastic proteins with antifreeze activity. Antifreeze proteins present in apoplastic extracts were purified by ice affinity purification, and their identity was determined by protein sequencing. D. antarctica plants were subjected to 22 days of cold acclimation at 4 °C. The highest content of apoplastic proteins with antifreeze activity was obtained at between 12 and 16 days of acclimation. Protein sequencing allowed their identification with >95% probability. Percentage coverage was 74% with D. antarctica ice recrystallization inhibition protein 1 (DaIRIP1) and 55% with DaIRIP3. Cold acclimation of D. antarctica improved the yield of apoplastic proteins, and resulted in an increase in the antifreeze activity of apoplastic extracts. An in silico analysis suggested that the fluctuations presented by the three-dimensional structures of DaIRIPs help to explain the presence of certain DaIRIPs in apoplastic extracts under the cold acclimation conditions evaluated.

Giant Single-Crystal Shape Transformation with Wide Thermal Hysteresis Actuated by Synergistic Motions of Molecular Cations and Anions.

Manipulating the collective molecular movements to implement macroscopic mechanical response of bulk material is attractive and challenging. Here, an organic-inorganic hybrid single crystal is synthesized, which exhibits a giant macroscopic shape transformation with a remarkable thermal hysteretic feature. The colossal anisotropic shape change, which manifests as an abrupt elongation of ca. 9 % along the crystallographic c-axis and a concomitant contraction of ca. 9 % in a perpendicular direction, is induced by a significant reorientation of imidazolium, accompanied with a substantial configurational variation in CuBr4 2- complex anions. The synergistic motions of both the molecular cations and anions engender a remarkable large thermal hysteresis (>30 K) in the shape transformation of the single crystal, implying that this material may play a role in alternating memory media. Furthermore, due to the stable crystal lattice, a single crystal that demonstrates naked-eye detectable large shape transformation was used as a thermal actuator to spontaneously control an electric circuit by temperature variation.

A Method to Increase the Frequency Stability of a TCXO by Compensating Thermal Hysteresis.

Due to the rapid growth of electronic information technology, the need for the higher stability of crystal oscillators has increased. The temperature-compensated X'tal (crystal) oscillator (TCXO), a type of crystal oscillator with high frequency stability, has been widely used in communications, sensor networks, automotive electronics, industrial control, measuring devices, and other equipment. The traditional TCXO only performs frequency compensation based on the current temperature, without considering the error caused by thermal hysteresis. As the frequency stability of the TCXO improves, the thermal hysteresis of the crystal oscillator has a negligible influence on the frequency stability of the crystal oscillator. This study measured different compensation tables for hysteresis curves at different temperatures and used a microprocessor to store the historical information of crystal temperature changes. Furthermore, corresponding algorithms were designed to select the correct values, according to the temperature change history, to compensate for the thermal hysteresis of the crystal oscillator error. Experiments show that this method can reduce the hysteresis error of the crystal oscillator from 700 to 150 ppb (-40 to 80 °C).

Relative Contributions of Packaging Elements to the Thermal Hysteresis of a MEMS Pressure Sensor.

Piezoresistive silicon pressure sensor samples were thermally cycled after being consecutively packaged to three different levels. These started with the absolute minimum to allow measurement of the output and with each subsequent level incorporating additional packaging elements within the build. Fitting the data to a mathematical function was necessary both to correct for any testing uncertainties within the pressure and temperature controllers, and to enable the identification and quantification of any hysteresis. Without being subjected to any previous thermal preconditioning, the sensors were characterized over three different temperature ranges and for multiple cycles, in order to determine the relative contributions of each packaging level toward thermal hysteresis. After reaching a stabilised hysteretic behaviour, 88.5% of the thermal hysteresis was determined to be related to the bond pads and wire bonds, which is likely to be due to the large thermal mismatch between the silicon and bond pad metallisation. The fluid-fill and isolation membrane contributed just 7.2% of the total hysteresis and the remaining 4.3% was related to the adhesive used for attachment of the sensing element to the housing. This novel sequential packaging evaluation methodology is independent of sensor design and is useful in identifying those packaging elements contributing the most to hysteresis.

Ice-binding proteins and the applicability and limitations of the kinetic pinning model.

Ice-binding proteins (IBPs) are unique molecules that bind to and are active on the interface between two phases of water: ice and liquid water. This property allows them to affect ice growth in multiple ways: shaping ice crystals, suppressing the freezing point, inhibiting recrystallization and promoting nucleation. Advances in the protein's production technologies make these proteins promising agents for medical applications among others. Here, we focus on a special class of IBPs that suppress freezing by causing thermal hysteresis (TH): antifreeze proteins (AFPs). The kinetic pinning model describes the dynamics of a growing ice face with proteins binding to it, which eventually slow it down to a halt. We use the kinetic pinning model, with some adjustments made, to study the TH dependence on the solution's concentration of AFPs by fitting the model to published experimental data. We find this model describes the activity of (moderate) type III AFPs well, but is inadequate for the (hyperactive) Tenebrio molitor AFPs. We also find the engulfment resistance to be a key parameter, which depends on the protein's size. Finally, we explain intuitively how TH depends on the seeding time of the ice crystal in the protein solution. Using this insight, we explain the discrepancy in TH measurements between different assays. This article is part of the theme issue 'The physics and chemistry of ice: scaffolding across scales, from the viability of life to the formation of planets'.

Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins.

Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFPs as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFPs using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFPs: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application.

The First Observation of Hidden Hysteresis in an Iron(III) Spin-Crossover Complex.

Molecular magnetic switches are expected to form the functional components of future nanodevices. Herein we combine detailed (photo-) crystallography and magnetic studies to reveal the unusual switching properties of an iron(III) complex, between low (LS) and high (HS) spin states. On cooling, it exhibits a partial thermal conversion associated with a reconstructive phase transition from a [HS-HS] to a [LS-HS] phase with a hysteresis of 25 K. Photoexcitation at low temperature allows access to a [LS-LS] phase, never observed at thermal equilibrium. As well as reporting the first iron(III) spin crossover complex to exhibit reverse-LIESST (light-induced excited spin state trapping), we also reveal a hidden hysteresis of 30 K between the hidden [LS-LS] and [HS-LS] phases. Moreover, we demonstrate that FeIII spin-crossover (SCO) complexes can be just as effective as FeII systems, and with the advantage of being air-stable, they are ideally suited for use in molecular electronics.