Antifreeze Mechanism Research Articles

Functional and evolutionary analysis of key enzymes triacylglycerol lipase, glycogen hydrolases in the glycerol and glucose biosynthesis pathway and cellular chaperones for freeze-tolerance of the Rice stem borer, Chilo suppressalis

Freeze-tolerance is an important physiological trait for terrestrial environmental adaptation and intraspecific geographic-lineage diversification in ectothermic animals, yet there remains a lack of systematic studies on its underlying genetic mechanisms and evolution. To address this problem, we employed the widely distributed rice pest, the Chilo suppressalis, as a model to explore the genetic mechanisms and evolutionary history of freeze-tolerance. First, we systematically characterized its antifreeze mechanisms by performing functional validation of potential key genes in laboratory-reared lines. This revealed the functional roles of glycerol biosynthesis in freeze-tolerance, including the triacylglycerol-originated pathway via triacylglycerol lipase (Tgl) hydrolysis and the glycogen-originated pathway via α-amylase (Aa) and maltase (Ma) hydrolysis, as well as the roles of the cellular chaperones Hsc70 and Hsf1. Then, we investigated the evolution of freeze-tolerance by collecting representative geographical samples and performing population genetic analyses, which suggested differentiated strategies of cold adaptation in different geographic populations. Taken together, our findings demonstrate the functional basis of cold resistance in Chilo suppressalis and reveal the evolutionary history of freeze-tolerance in natural populations, providing insights into organismal freeze-tolerance and clues for pest control.

Molecular mechanisms of natural antifreeze phenomena and their application in cryopreservation.

Cryopreservation presents a critical challenge due to cryo-damage, such as crystallization and osmotic imbalances that compromise the integrity of biological tissues and cells. In contrast, various organisms in nature exhibit remarkable freezing tolerance, leveraging complex molecular mechanisms to survive extreme cold. This review explores the adaptive strategies of freeze-tolerant species, including the regulation of specific genes, proteins, and metabolic pathways, to enhance survival in low-temperature environments. We then discuss recent advancements in cryopreservation technologies that aim to mimic these natural phenomena to preserve cellular and tissue integrity. Special focus is given to the roles of glucose metabolism, microRNA expression, and cryoprotective protein modulation in improving cryopreservation outcomes. The insights gained from studying natural antifreeze mechanisms offer promising directions for advancing cryopreservation techniques, with potential applications in medical, agricultural, and conservation fields. Future research should aim to further elucidate these molecular mechanisms to develop more effective and reliable cryopreservation methods.

Nondestructive frozen protein ink: Antifreeze mechanism, processability, and application in 3D printing

Antifreeze peptide (AFP) including in frozen protein ink is an inevitable trend because AFP can make protein ink suitable for 3D printing after freezing. AFP-based surimi ink (ASI) was firstly investigated, and the AFP significantly enhanced 3D printability of frozen surimi ink. The rheological and textural results of ASI show that the τ0, K, and n values are 321.14 Pa, 2.2259 × 105 Pa·sn, and 0.19, respectively, and the rupture strength of the 3D structure is up to 217.67 g. Circular dichroism, intermolecular force, and differential scanning calorimeter show ASI has more undenatured protein after freezing when compared that surimi ink (SI), which was denatured, and the α-helix changed to a β-sheet due to the destruction of hydrogen bonds and the exposure of hydrophobic groups. The water distribution, water holding capacity, and microstructure indicate that ASI effectively binds free water after freezing, while SI has weak water binding capacity and a large amount of free water is formed. ASI is suitable for 3D printing, and can print up to 40.0 mm hollow isolation column and 50.0 mm high Wuba which is not possible with SI. The application of AFP provides guidance for 3D printing frozen protein ink in food industry.

Composition-antifreeze property relationships of gelatin and the corresponding mechanisms

The inherent functional fractions (gelation and ice-affinitive fractions) of gelatin enable it as a promising cryoprotectant alternative. However, the composition-antifreeze property relationships of gelatin remain to be investigated. In this study, the HW-PSG and LW-PSG fractions of gelatin from fish scales were obtained, according to the critical gelation conditions and ice-binding measurements, respectively. Thermal hysteresis (THA) value, associated with ice nucleation, of LW-PSG was higher than that of HW-PSG. Besides, the relatively low-sized ice crystals (210–550 μm2) indicated that HW-PSG showed strong ice recrystallization inhibition (IRI) ability, compared to other groups. These results suggested that LW-PSG inhibited ice nucleation, while HW-PSG displayed the strong IRI ability. Furthermore, the antifreeze mechanisms were clarified through IRI measurements and molecular dynamics simulation. The minimum size of ice crystals was found for HW-PSG gels with dense microstructure, suggesting the HW-PSG retarded the growth of ice crystals by restricting the migration and phase transformation of water molecules. The hydrogen bond interactions between the ice crystal surface and ASN1294 and PRO1433 residues of LW-PSG, and hydrophobic interactions contributed to inhibiting the nucleation of ice crystals. This study provided some references to further enhance antifreeze performance of gelatin by modulating fragment composition.

Ice growth inhibition by poly(vinyl alcohol): Insights from near-infrared spectroscopy and molecular dynamics simulation

Poly(vinyl alcohol) (PVA) is a well-known ice recrystallization inhibitor, yet its antifreeze mechanism remains debated. Here, the mechanism of PVA is elucidated by means of near-infrared spectroscopy and molecular dynamics (MD) simulations. Analysis of the spectra of PVA solutions during the ice growth process reveals a dynamic interaction conversion from PVA-water to PVA-ice, indicating that PVA inhibits ice growth by directly binding to ice. Moreover, we observed OH binding to the ice surface through one or two hydrogen bonds but did not witness the previously reported formation of three hydrogen bonds when the OH group perfectly embeds into the ice crystal lattice. In addition, the spectral feature of CH2 groups of PVA indirectly indicates their significant contribution to inhibiting ice growth through hydrophobic interactions. The MD simulations further confirm the effective roles of both OH and CH2 groups for inhibiting ice growth.

Identification and isolation of a novel antifreeze peptide from crayfish shells

In this study, a novel antifreeze peptide was isolated from crayfish shells and its antifreeze mechanism and activity were investigated. Firstly, the crayfish shells peptides (CSPs) were prepared by Enzymatic Hydrolysis Technology (alkaline protease and trypsin) and the CSPs could significantly enhance the survival rate of Saccharomyces cerevisiae from 12.5% to 88.5% after 24h freeze-thaw cycle. Then, a total 1004 peptides were identified from CSPs by liquid chromatography/tandem mass spectrometry (LC-MS/MS), among those peptides, two peptide sequences YWDHPIRDGFAPH (AFP1) and GPPGKPGIPDIVDW (AFP1) were selected as potential antifreeze polypeptides (AFPs) according the physicochemical properties: instability index, hydrophilicity, and confidence level. Furthermore, the antifreeze activity and mechanism of AFP1 and AFP2 were studied through molecular dynamics simulation, revealing that the AFP1 and AFP2 could adsorb to ice surface with 7 and 14 hydrogen bonds, respectively. Moreover, AFP2 has a more stable binding capacity and could interact with 33 water molecules. At last, the results of differential scanning calorimetry (DSC) and cold-stage polarization microscope indicated that the thermal hysteresis (TH) values of AFP2 were 1.62–2.09 °C and could inhibit ice recrystallization. Therefore, this study provides a theoretical basis for developing safe peptide-based cryoprotectants which will improve the quality of frozen foods.

Key anti-freeze genes and pathways of Lanzhou lily (Lilium davidii, var. unicolor) during the seedling stage.

Temperature is one of the most important environmental factors for plant growth, as low-temperature freezing damage seriously affects the yield and distribution of plants. The Lanzhou lily (Lilium davidii, var. unicolor) is a famous ornamental plant with high ornamental value. Using an Illumina HiSeq transcriptome sequencing platform, sequencing was conducted on Lanzhou lilies exposed to two different temperature conditions: a normal temperature treatment at 20°C (A) and a cold treatment at -4°C (C). After being treated for 24 hours, a total of 5848 differentially expressed genes (DEGs) were identified, including 3478 significantly up regulated genes and 2370 significantly down regulated genes, accounting for 10.27% of the total number of DEGs. Quantitative real-time PCR (QRT-PCR) analysis showed that the expression trends of 10 randomly selected DEGs coincided with the results of high-throughput sequencing. In addition, genes responding to low-temperature stress were analyzed using the interaction regulatory network method. The anti-freeze pathway of Lanzhou lily was found to involve the photosynthetic and metabolic pathways, and the key freezing resistance genes were the OLEO3 gene, 9 CBF family genes, and C2H2 transcription factor c117817_g1 (ZFP). This lays the foundation for revealing the underlying mechanism of the molecular anti-freeze mechanism in Lanzhou lily.

Antifreeze Polysaccharides from Wheat Bran: The Structural Characterization and Antifreeze Mechanism.

Exploring a novel natural cryoprotectant and understanding its antifreeze mechanism allows the rational design of future sustainable antifreeze analogues. In this study, various antifreeze polysaccharides were isolated from wheat bran, and the antifreeze activity was comparatively studied in relation to the molecular structure. The antifreeze mechanism was further revealed based on the interactions of polysaccharides and water molecules through dynamic simulation analysis. The antifreeze polysaccharides showed distinct ice recrystallization inhibition activity, and structural analysis suggested that the polysaccharides were arabinoxylan, featuring a xylan backbone with a majority of Araf and minor fractions of Manp, Galp, and Glcp involved in the side chain. The antifreeze arabinoxylan, characterized by lower molecular weight, less branching, and more flexible conformation, could weaken the hydrogen bonding of the surrounding water molecules more evidently, thus retarding the transformation of water molecules into the ordered ice structure.

Recent advances, challenges and functional applications of antifreeze protein in food industry

SummaryFreezing is a common food preservation method that can extend the shelf life of food, but the ice crystals and recrystallisation during freezing, storage and transportation cause significant damage to frozen food. Antifreeze proteins are a class of proteins that widely exist in organisms living in cold conditions, such as fish, insects, plants and microorganisms in Antarctica and other cold regions. Antifreeze proteins can reduce the freezing point of water, inhibit recrystallisation effects, modify ice crystal morphology and effectively suppress the quality deterioration caused by ice crystals during food freezing. This review comprehensively introduces the source, antifreeze mechanism, application in food, influencing factors and regulating methods of antifreeze activity of antifreeze proteins, as well as the limitations of antifreeze proteins. It also looks forward to the future application of antifreeze proteins in food.

Development of a radical polymerization algorithm for molecular dynamics simulations of antifreezing hydrogels with double-network structures

The development and understanding of antifreezing hydrogels are crucial both in principle and practice for the design and delivery of new materials. The current antifreezing mechanisms in hydrogels are almost exclusively derived from their incorporation of antifreezing additives, rather than from the inherent properties of the polymers themselves. Moreover, developing a computational model for the independent yet interconnected double-network (DN) structures in hydrogels has proven to be an exceptionally difficult task. Here, we develop a multiscale simulation platform, integrating ‘random walk reactive polymerization’ (RWRP) with molecular dynamics (MD) simulations, to computationally construct a physically-chemically linked PVA/PHEAA DN hydrogels from monomers that mimic a radical polymerization and to investigate water structures, dynamics, and interactions confined in PVA/PHEAA hydrogels with various water contents and temperatures, aiming to uncover antifreezing mechanism at atomic levels. Collective simulation results indicate that the antifreezing property of PVA/PHEAA hydrogels arises from a combination of intrinsic, strong water-binding networks and crosslinkers and tightly crosslinked and interpenetrating double-network structures, both of which enhance polymer-water interactions for competitively inhibiting ice nucleation and growth. These computational findings provide atomic-level insights into the interplay between polymers and water molecules in hydrogels, which may determine their resistance to freezing.

Characterization and the mechanism underlying the cryoprotective activity of a peptide from large yellow croaker (Pseudosciaena crocea)

Ice crystal-induced protein denaturation is the main cause of the deterioration of fish during frozen storage and transportation. In this study, the ultra-performance liquid chromatography − quadrupole − time of flight (UPLC-Q-TOF) technique was used to identify and screen tryptic peptides Ile-Glu-Glu-Leu-Glu-Glu-Leu-Glu-Ala-Glu-Arg (IEELEEELEAER) from large yellow croaker (Pseudosciaena crocea). The results were used study their cryoprotective effects on turbot fish meat during freeze–thaw cycles at different concentrations, and to investigate their anti-freezing mechanism. The results showed that the I-2.0 group effectively inhibiting the degeneration and structure changes of myofibrillar proteins after three freeze–thaw cycles, and the Ca2+-ATPase activity (1.65 μmolPi/mg/h), increased by 55.86% compared with that of the control group. Additionally, peptide IEELEEELEAER could provide antifreeze protection by binding to the surface of ice crystals and inhibiting their transformation. This peptide acts as a natural cryoprotectant and might be used for the cryogenic storage and transportation of fish products.

Living in the mountains: Thermal ecology and freezing tolerance of the lizard Abronia taeniata (Squamata: Anguidae)

The impact of daily and seasonal variation in environmental temperature on lizards is important, since their physiological processes are body temperature dependent. Lizards that occupy mountainous areas must have been favoured to colonize such habitats through selection on thermal biology traits to thermoregulate effectively. Moreover, mountain lizards may be able to maintain their activity near their minimum critical temperature and even have antifreeze mechanisms. Tolerance of freezing is related to the biosynthesis of cryoprotective molecules, such as glucose, whose concentration may increase after freezing. The aims of the present work were: (1) study the thermoregulation of the viviparous lizard Abronia taeniata, and (2) determine its survival and/or tolerance to freezing. This species occurs in pine forests, pine-oak forests, and mountain mesophilic forests in areas that reach freezing temperatures. In the field, we recorded air, substrate, and body temperatures at capture time of the lizards, and registered operative temperatures at the study area. In the laboratory, we determined thermal preferences, crystallization point, and blood glucose levels of individuals before and after freezing. We found out that A. taeniata sustains activity in a wide range of temperatures, actively avoids thermally favourable microhabitats in spring, and is a moderate thermoregulator during autumn and winter. In A. taeniata, the body temperatures are tightly linked to air and substrate temperatures. Seasonality had an effect over body temperature, preferred temperatures and thermoregulatory effectiveness indices. When exposed to temperatures below zero, A. taeniata showed an increase in blood glucose levels, which aided them in surviving freezing. Taken together, our results suggest that A. taeniata may sustain activity at low environmental temperatures, due to an effective behavioural thermoregulation, and in case temperatures of its habitat go below zero, is also capable of tolerate freezing.

Rational Design of and Mechanism Insight into an Efficient Antifreeze Peptide for Cryopreservation

The development of effective antifreeze peptides to control ice growth has attracted a significant amount of attention yet still remains a great challenge. Here, we propose a novel design method based on in-depth investigation of repetitive motifs in various ice-binding proteins (IBPs) with evolution analysis. In this way, several peptides with notable antifreeze activity were developed. In particular, a designed antifreeze peptide named AVD exhibits ideal ice recrystallization inhibition (IRI), solubility, and biocompatibility, making it suitable for use as a cryoprotective agent (CPA). A mutation analysis and molecular dynamics (MD) simulations indicated that the Thr6 and Asn8 residues of the AVD peptide are fundamental to its ice-binding capacity, while the Ser18 residue can synergistically enhance their interaction with ice, revealing the antifreeze mechanism of AVD. Furthermore, to evaluate the cryoprotection potential of AVD, the peptide was successfully employed for the cryopreservation of various cells, which demonstrated significant post-freezing cell recovery. This work opens up a new avenue for designing antifreeze materials and provides peptide-based functional modules for synthetic biology.

Integrated Full-Length Transcriptome and Metabolome Profiling Reveals Flavonoid Regulation in Response to Freezing Stress in Potato.

Cold stress impairs plant growth and development, resulting in crop failure. Cultivated potato (Solanum tuberosum L.) is sensitive to freezing, while its wild relative, S. commersonii, has a strong freezing tolerance. To decipher the anti-freezing mechanism of CM, we carried out a transcriptomic and metabolomic analysis of an anti-freezing variety of CM (a type of S. commersonii) and a freeze-sensitive variety of DM (a type of Solanum tuberosum L.). A total of 49,232 high-quality transcripts from 12,811 gene loci, including 46,772 coding sequences and 2018 non-coding RNAs, were identified. KEEG enrichment analysis of differentially expressed genes (DEGs) between the two varieties showed that the flavonoid biosynthesis pathway was strongly induced by freezing stress, which was proven by flavonoid metabolome analysis. Consistent with the accumulation of more flavonoids, nearly all the pathway genes were significantly upregulated in CM than those in DM. The transcript levels of two chalcone synthase (CHS-1) isoforms and four isoforms of flavonoid 3'-hydroxylase (F3'H-1) were confirmed by qRT-PCR. Co-expression analysis identified one Myb-related and three UGTs (UDP-glycosyltransferase) that were significantly upregulated in CM during freezing stress. Our findings support that the flavonoid pathway was significantly enhanced by freezing stress and the greater accumulation ofglycosylatedflavonoids in resistant types than that of sensitive types, maybe accounting for the increased freezing tolerance of freeze-resistant potato varieties.

Advances in Antifreeze Molecules: From Design and Mechanisms to Applications

Freezing can cause serious hazards in various fields, such as cryoinjury in cell and organ cryopreservation, food quality deterioration, and ice accretion on surfaces including energy facilities (wind turbines, overhead transmission lines, and towers), aircraft, etc., causing serious equipment damages and economic losses. To alleviate these problems, antifreeze molecules have attracted significant attention and have been applied extensively in cryopreservation and anti-icing coatings. In this review, the progress on antifreeze molecules is summarized and discussed, including their classifications, mechanisms, and applications. The antifreeze molecules are divided into natural and artificial molecules, and their characteristics are introduced. Moreover, their antifreeze mechanisms are comprehensively outlined involving depression of freezing points, dynamic ice shaping, and ice recrystallization inhibition. Then, the representative applications in cryobiology, anti-icing surfaces, food technology, and agriculture are also summarized and discussed. Finally, the challenges and development prospects of antifreeze molecules are further provided.

Perturbation of Hydrogen Bonding Network Plays an Important Role in Determining Antifreeze Activity

In order to develop new antifreeze agents, it is crucial to understand the antifreeze activity. The adsorption-inhibition mechanism has been widely accepted, and most previous studies focus on ice-binding affinity of additive molecules but rarely discuss their influence on the hydrogen bonding network of ice. Herein, we investigate the ice recrystallization inhibition (IRI) activity of two structurally similar molecules, histidine (His) and phenylalanine (Phe). It is verified by experiment that Phe has excellent IRI activity, while His does not. Molecular dynamics simulation is conducted to study the process of ice growth with amino acids and to explore the origin of the difference between His and Phe. We find that His is prone to be swallowed up by ice and thus no longer hinders its growth, whereas Phe can remain unswallowed on the ice surface for a long period, which is attributed to their different abilities to perturb the hydrogen bonding network. Based on a statistical analysis of oxygen–hydrogen bond orientations, we propose a method to evaluate the order of the hydrogen bonding network. With this method, the IRI activity of His and Phe can be well interpreted, and the influence of different groups on the hydrogen bonding network can be quantified. This study takes the structural integrity of ice into consideration and provides a possibility to investigate the influence of different additive molecules on hydrogen bonding network, which is expected to improve the understanding of antifreeze mechanism.

Freeze-thaw resistance of concrete containing azodicarbonamide expansive agent

Azodicarbonamide expansive agent (ADC) and air-entraining agent (AEA) have similarities in introducing pores into concrete and can both be applied in constructions served in severe cold environments. This paper investigates the effect of ADC on the freeze-thaw resistance of concrete and compares the differences between ADC and AEA in the anti-freezing mechanism. The results show that ADC improves the freeze-thaw resistance of concrete, while its effect is weaker than that of AEA. The non-hydrophobic bubble shell formed by ADC cannot reduce the freezing temperature and delay freezing time as well as the shell consisted of hydrophobic calcium salt formed by AEA. A rosette-shaped pore structure and non-dense shell of air voids formed by ADC have both positive and negative effects on freeze-thaw resistance: they release hydraulic pressure to improve freezing resistance, while they reduce the suction force resulting in weakened freezing resistance. The air-void spacing factor instead of the air content at fresh stage is more suitable for assessing the freeze-thaw resistance of concrete with ADC.

Revealing the Mechanism of Irreversible Binding of Antifreeze Glycoproteins to Ice.

Antifreeze glycoproteins (AFGPs) are a special kind of antifreeze proteins with strong flexibility. Whether their antifreeze activity is achieved by reversibly or irreversibly binding to ice is widely debated, and the molecular mechanism of irreversible binding remains unclear. In this work, the antifreeze mechanism of the smallest AFGP isoform, AFGP8, is investigated at the atomic level. The results indicate that AFGP8 can bind to ice both reversibly through its hydrophobic methyl groups (peptide binding) and irreversibly through its hydrophilic disaccharide moieties (saccharide binding). Although peptide binding occurs faster than saccharide binding, free-energy calculations indicate that the latter is energetically more favorable. In saccharide binding, at least one disaccharide moiety is frozen in the grown ice, resulting in irreversible binding, while the other moieties significantly perturb the water hydrogen-bonding network, thus inhibiting ice growth more effectively. The present study reveals the coexistence of reversible and irreversible bindings of AFGP8, both contributing to the inhibition of ice growth and further provides molecular mechanism of irreversible binding.

Inhibition of ice recrystallization by tamarind (Tamarindus indica L.) seed polysaccharide and molecular weight effects

This study aimed to investigate the inhibition effects of tamarind seed polysaccharide (TSP) on ice recrystallization and to figure out its possible molecular weight-dependent effects. TSP fractions (2412.38–20.75 kDa) were prepared while preserving the natural structure. Ice recrystallization was effectively inhibited by TSP. Decreasing the molecular weight to a certain range, such as 224.04 kDa and 90.41 kDa, could enhance the activity of TSP due to the reduction of self- and intermolecular aggregation. Adding TSP into water decreased the melting temperature of bulk ice. Raman spectra showed that partial group vibrations or deformations of TSP molecules were restricted upon solution freezing and also revealed a destructuring effect of TSP on the H-bond network of water. These findings suggested the potential of TSP as a novel food cryoprotectant and help produce TSP fractions with enhanced activity, and shed new light on understanding the antifreeze mechanism of natural polysaccharides.

Spatially confined building of environmental-adaptive hydrogel electrolyte for supercapacitors

Developing hydrogel electrolytes combining enhanced ionic conductivity, great electrolyte/electrode interfacial contact and high electrochemical stability represents the basic demand of flexible electronic devices for cryogenic applications, but is still a formidable challenge at present. Herein, inspired by the antifreeze mechanism of antifreeze proteins (AFP), we developed a facile spatially confined strategy to stabilize water in molecular level so as to prevent freezing meanwhile maintain other critical features of hydrogels at subzero temperature. Meriting from unique AFP-mimetic structure, the polyacrylic acid-DMSO hydrogel prepared by rapid one-pot photo-polymerization embodies merits of excellent flexibility, large ionic conductivity, and strong interfacial adhesiveness with almost all types of surfaces at temperature down to −40 °C. As-assembled supercapacitor delivers remarkable specific capacitance of 73 F g−1 at −40 °C (∼70% of the capacitance at 20 °C), 84.5% of capacitance retention after 5000 charge-discharge cycles at −40 °C, and 85.8% of capacitance retention after 1000 cycles at an extreme bending angle of 180° and −40 °C, evidently manifesting high efficiency/stability toward cyclic charge-discharge operation and structure deformation in cold climates. It is believed that this work will play an exemplary role in designing anti-freezing hydrogel electrolytes for reliable, flexible electronic devices working at extremely cold environments.