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The formation of natural gas hydrates presents significant economic and safety challenges to the petroleum and gas industry, necessitating the development of effective prevention strategies. This study investigates an environmentally sustainable Tenebrio molitor antifreeze protein (TmAFP) modified to be a potential kinetic hydrate inhibitor. The aim of this study was to enhance the inhibitory activity of TmAFP by systematically substituting threonine (Thr) residues with glycine (Gly), alanine (Ala), or serine (Ser) at positions 29, 39, and 53. The Ala mutant demonstrated superior inhibition of hydrate formation, attributed to its optimized spatial conformation and enhanced hydrophobic interactions, followed by the Gly and Ser mutants. The wild-type TmAFP showed limited efficacy. The radial distribution function (RDF) analysis indicated that the mutations facilitated a better accommodation of adjacent residues within the hydrate crystal structure by adjusting the distance between Thri and Thri+2 to closely match the second peak in the RDF of methane molecules at 6.4 Å. The potential of mean force (PMF) calculations revealed that the Ala and Ser mutants exhibited enhanced interactions with hydrate cages, with PMF values of −0.73 and −0.71 kJ/mol, respectively, compared to the Gly mutant, which had a PMF value of 1.46 kJ/mol. By identifying the optimal mutation combination (T29 39 53A) to significantly increase the potency of TmAFP, this study provides a fundamental basis for the further development of hydrate inhibition strategies. [ABSTRACT FROM AUTHOR]

In 2012, the molecular structure of a new, broad class of ice-binding proteins, classified as "domain of unknown function" (DUF) 3494, was described for the first time. These proteins have a common tertiary structure and are characterized by a very wide spectrum of antifreeze activity (from weakly active to hyperactive). The ice-binding surface (IBS) region of these molecules differs significantly in its structure from the IBS of previously known antifreeze proteins (AFPs), showing a complete lack of regularity and high hydrophilicity. The presence of a regular, repeating structural motif in the IBS region of hitherto known AFP molecules, combined with the hydrophobic nature of this surface, promotes the formation of an ice-like ordering of the solvation water layer and, as a result, facilitates the process of transformation of this water layer into ice. It is, therefore, surprising that the newly discovered DUF3494 class of proteins clearly breaks out of this characteristic. In this paper, using molecular dynamics simulations, we analyze the solvation water structure of the IBS region of both DUF3494 family molecules and AFPs. As we show, although the IBS of DUF3494 molecules does not form an ice-like water structure in the solvation layer, this is compensated by the formation of the equivalent of "anchored clathrate water," in the form of a relatively large number of water molecules bound to the surface of the protein molecule and providing potential binding sites for it to the ice surface. [ABSTRACT FROM AUTHOR]

In this study, peptides designed using fragments of an antifreeze protein (AFP) from the freeze-tolerant insect Tenebrio molitor, TmAFP, were evaluated as inhibitors of clathrate hydrate formation. It was found that these peptides exhibit inhibitory effects by both direct and indirect mechanisms. The direct mechanism involves the displacement of methane molecules by hydrophobic methyl groups from threonine residues, preventing their diffusion to the hydrate surface. The indirect mechanism is characterized by the formation of cylindrical gas bubbles, the morphology of which reduces the pressure difference at the bubble interface, thereby slowing methane transport. The transfer of methane to the hydrate interface is primarily dominated by gas bubbles in the presence of antifreeze peptides. Spherical bubbles facilitate methane migration and potentially accelerate hydrate formation; conversely, the promotion of a cylindrical bubble morphology by two of the designed systems was found to mitigate this effect, leading to slower methane transport and reduced hydrate growth. These findings provide valuable guidance for the design of effective peptide-based inhibitors of natural-gas hydrate formation with potential applications in the energy and environmental sectors. [ABSTRACT FROM AUTHOR]

Using molecular dynamics simulations, we show that a molecule of moderately active antifreeze protein (type III AFP, QAE HPLC-12 isoform) is able to interact with ice in an indirect manner. This interaction occurs between the ice binding site (IBS) of the AFP III molecule and the surface of ice, and it is mediated by liquid water, which separates these surfaces. As a result, the AFP III molecule positions itself at a specific orientation and distance relative to the surface of ice, which enables the effective binding (via hydrogen bonds) of the molecule with the nascent ice surface. Our results show that the final adsorption of the AFP III molecule on the surface of ice is not achieved by chaotic diffusion movements, but it is preceded by a remote, water-mediated interaction between the IBS and the surface of ice. The key factor that determines the existence of this interaction is the ability of water molecules to spontaneously form large, high-volume aggregates that can be anchored to both the IBS of the AFP molecule and the surface of ice. The results presented in this work for AFP III are in full agreement with the ones obtained by us previously for hyperactive CfAFP, which indicates that the mechanism of the remote interaction of these molecules with ice remains unchanged despite significant differences in the molecular structure of their ice binding sites. For that reason, we can expect that also other types of AFPs interact with the ice surface according to an analogous mechanism. [ABSTRACT FROM AUTHOR]

Sperm cryopreservation is crucial in reproductive biotechnology; however, the longevity of frozen and thawed semen is limited by the deterioration of sperm cell integrity. This study aimed to examine the effects of adding antifreeze protein III (AFP III) to the diluent, using samples from eight healthy mature dogs. The ejaculates were divided into aliquots and diluted with a standard Tris-fructose-egg yolk extender containing AFP III at concentrations of 0, 0.75, 1.0, and 2.0 μg/ml. After thawing, the samples were analyzed for kinematic parameters, membrane Integrity, lipid peroxidation, viability, acrosome integrity, intracellular hydrogen peroxide, mitochondrial membrane potential and apoptotic metrics. The results show that while motility and velocity were not significantly different between the treated and control groups (p > 0.05), the treated groups generally performed better. Specifically, the 0.75 and 1.0 μg/ml groups exhibited better movement compared to the 2.0 μg/ml group. Additionally, there was a significant difference (p < 0.05) in membrane integrity between the control and treated groups, though no differences were observed among the treated groups. Significant differences (p < 0.05) were also observed in viability and acrosome integrity, with the 0.75 and 1.0 μg/ml groups outperforming the control and 2.0 μg/ml groups. There were no significant variations (p > 0.05) in phosphatidylserine translocation, lipid peroxidation, mitochondrial membrane potential, or hydrogen peroxide levels. However, the 0.75 and 1.0 μg/ml groups demonstrated superior effects compared to both the control and the 2.0 μg/ml groups. These results suggest that the addition of antifreeze proteins, specifically AFP III, markedly improves the protection of canine sperm during cryopreservation. This enhancement is evident in various parameters, underscoring the beneficial effects of AFP III in maintaining sperm quality. [ABSTRACT FROM AUTHOR]

Simple Summary: This study investigated the effects of MitoQ and antifreeze protein III (AFP III) on the frozen and thawed semen of dogs. The samples were mixed with solutions containing MitoQ (200 nM/mL) and AFP III (0.75, 1.0, and 2.0 µg/mL). After thawing, we observed significant improvements in sperm movement and structure with AFP III and MitoQ. AFP III also improved sperm viability and membrane integrity, especially at 0.75 and 1.0 µg/mL concentrations. There were no significant changes in ROS-H2O2 levels or mitochondrial function, except in the 1.0 µg/mL AFP III group. The combined treatment with MitoQ and AFP III significantly reduced dead sperm cells. In conclusion, AFP III and MitoQ can protect canine sperm from cryodamage. Cryopreservation can adversely affect sperm motility, structural integrity, and fertilization ability. This study investigated the effects of MitoQ and antifreeze protein III (AFP III) on frozen–thawed semen from eight adult dogs using a Tris–fructose extender. Ejaculates were divided and diluted with a standard Tris–fructose–egg yolk extender containing MitoQ (200 nM/mL) and AFP III (0.75, 1.0, 2.0 µg/mL), individually or combined. Post-thaw, samples were evaluated for motility, viability, membrane and acrosome integrity, lipid peroxidation, apoptosis indicators, mitochondrial function, and reactive oxygen species (ROS-H2O2). The results showed significant (p < 0.05) improvements in motility rate, progressive motility, VAP, VSL, VCL, ALH, and BCF with MitoQ or AFP alone. AFP III (0.75, 1.0 µg/mL) showed higher values than controls (p > 0.05), while MitoQ alone showed no significant effect. Viability and acrosome integrity improved with AFP III. Membrane integrity and lipid peroxidation were better in 0.75 and 1.0 µg/mL AFP III groups. ROS-H2O2 levels and mitochondrial membrane potential were unaffected except at 1.0 µg/mL AFP III. The phosphatidylserine translocation assay showed no significant differences in dead sperm between controls and individual treatments, but significant differences occurred with combined MitoQ/AFP III. In conclusion, AFP III and MitoQ in diluents protect canine sperm cells from cryodamage. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The effects of antifreeze proteins (AFP) on the quality changes of pork during freezing were investigated in comparison to those of the commercial antifreeze agent alginate (4 mg/mL). Samples with no added AFP were used as a control. The effects of adding different amounts of AFP (0.05, 0.1, 0.2, and 0.3 mg/mL) on the quality, physicochemical properties of myofibrillar proteins, microstructure, and water distribution of frozen pork were investigated. The results showed that AFP decreased the water loss of frozen pork, which was beneficial to maintaining pork color and microstructure. Addition of AFP at 0.3 mg/mL had the highest cryoprotective effect on the cooking loss, thawing loss and color characteristics of pork during frozen storage, followed by AFP at 0.2 mg/mL and alginate at 4 mg/mL. The shear force and total sulfhydryl content of myofibrillar proteins in the 0.2 and 0.3 mg/mL AFP groups were significantly higher than those of the control group (P < 0.05). nuclear magnetic resonance imaging (NMRI) discovered that the infiltration of AFP reduced the loss of bound and immobile water in pork. Scanning electron microscopy showed that in the presence of 0.2 mg/mL AFP, the arrangement of muscle fibers was orderly, appearing more compact and denser. Overall, AFP addition had a positive effect on improving the quality of frozen pork and might have the potential of being a safe and efficient cryoprotection method.

In liquid water, there is a natural tendency to form aggregates that consist of water molecules linked by hydrogen bonds. Such spontaneously formed aggregates are surrounded by a "sea" of disordered water molecules, with both forms remaining in equilibrium. The process of creating water aggregates also takes place in the solvation water of proteins, but in this case, the interactions of water molecules with the protein surface shift the equilibrium of the process. In this paper, we analyze the structural properties of the solvation water in antifreeze proteins (AFPs). The results of molecular dynamics analysis with the use of various parameters related to the structure of solvation water on the protein surface are presented. We found that in the vicinity of the active region responsible for the binding of AFPs to ice, the equilibrium is clearly shifted toward the formation of "ice-like aggregates," and the solvation water has a more ordered ice-like structure. We have demonstrated that a reduction in the tendency to create "ice-like aggregates" results in a significant reduction in the antifreeze activity of the protein. We conclude that shifting the equilibrium in favor of the formation of "ice-like aggregates" in the solvation water in the active region is a prerequisite for the biological functionality of AFPs, at least for AFPs having a well-defined ice binding area. In addition, our results fully confirm the validity of the "anchored clathrate water" concept, formulated by Garnham et al. [Proc. Natl. Acad. Sci. U. S. A. 108, 7363 (2011)]. [ABSTRACT FROM AUTHOR]

Alanine-rich, alpha-helical type I antifreeze proteins (AFPs) in fishes are thought to have arisen independently in the last 30 Ma on at least four occasions. This hypothesis has recently been proven for flounder and sculpin AFPs, which both originated by gene duplication and divergence followed by substantial gene copy number expansion. Here, we examined the origins of the cunner (wrasse) and snailfish (liparid) AFPs. The cunner AFP has arisen by a similar route from the duplication and divergence of a GIMAP gene. The coding region for this AFP stems from an alanine-rich region flanking the GTPase domain of GIMAPa. The AFP gene has remained in the GIMAP gene locus and has undergone amplification there along with the GIMAPa gene. The AFP gene originated after the cunner diverged from its common ancestor with the closely related spotty and ballan wrasses, which exhibit similar gene synteny but lack AFP genes. Snailfish AFPs have also recently evolved because they are confined to a single genus of this family. In these AFP-producing species, the AFP locus does not share any similarity to functional genes. Instead, it is replete with repetitive DNAs and transposons, several stretches of which could encode alanine tracts with a dominant codon (GCC) that matches the bias observed in the AFP genes. All four known instances of type I AFPs occurring in fishes are independent evolutionary events that occurred soon after the onset of Northern Hemisphere Cenozoic glaciation events. Collectively, these results provide a remarkable example of convergent evolution to one AFP type. [ABSTRACT FROM AUTHOR]

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

• Ice particles were generated in aqueous solution with various solute concentration. • Effects of AFP and PVA on the prevention of the ice particle growth were evaluated. • AFP was more effective to prevent the ice particle growth than PVA. • Effects of AFP and PVA became weak in thick solute concentration solution. Ice slurry is a promising functional fluid with high thermal energy density and high heat transfer rate. However, large ice particles in the ice slurry can cause blocking of tubes during ice slurry flow. Therefore, it is preferable to use fine ice particles to prepare ice slurry in many fields. In this study, ice slurries were generated from supercooled solutions containing additives, such as anti-freeze protein and polyvinyl alcohol, to prevent the increase in the ice particle size in the formation process of the ice slurry. The concentration of the solute and amount of the additive were varied as experimental parameters, and the size of the ice particles was evaluated. The average area of the ice particles decreased with the addition of the additives. In particular, anti-freeze protein was effective for generating fine ice particles in the ice slurry. However, the effects of the additives became weaker for higher concentration of the solute, and the size of the ice particles was almost the same regardless of the concentration of the solute and the amount of the additive. Moreover, it was found that particular properties, such as the freezing-point depression, kinematic viscosity of the solution, and effective latent heat of fusion, did not affect the size of the ice particles in the ice slurry generation process. [ABSTRACT FROM AUTHOR]

Simple Summary: Bees, while individually poikilothermic, can maintain group warmth, which is vital for biodiversity. Apis cerana outperforms Apis mellifera in cold tolerance, particularly as a pollinator in harsh conditions. Here, we utilized transcriptome data to identify alternative splicing events and alternative polyadenylation genes associated with cold stress in A. cerana. These genetic changes are linked to signal transduction, energy metabolism, and oxidative stress, with key genes like HSP70 and BAG increasing in expression and others showing a pattern of an initial decrease followed by an increase. This post-transcriptional regulation may help bees adapt to cold, providing insights for breeding cold-resistant bee strains and advancing cold resistance research in insects. Bees, as individual poikilotherms capable of group homeothermy, possess temperature tolerance and regulatory mechanisms crucial for biodiversity conservation. The Asian honeybee (Apis cerana), a key pollinator for alpine and winter-flowering plants, exhibits superior cold tolerance over Apis mellifera, making it a dominant model species for insect cold resistance studies. While extensive research has delved into the behavioral and physiological facets of honeybee cold resistance, the post-transcriptional regulation in response to cold stress remains understudied. In this study, full-length transcriptome data obtained from PacBio and Illumina sequencing identified 25,443 events of alternative splicing (AS) and 2821 genes of alternative polyadenylation (APA) involved in the cold stress response of honeybees. GO and KEGG functional annotations were performed on the differential AS and APA between the cold stress (T vs. CK) and cold tolerance (TR vs. CK) groups in A. cerana workers of different ages, (3 days (Ac3d), 10 days (Ac10d), and 21 days (Ac21d) old), and the results indicated that these differentially expressed AS (DASs) and differentially expressed APAs (DAPAs) implicate signal transduction, energy metabolism, and oxidative metabolism. Venn analysis, expression clustering, and trend analysis highlight the roles of DASs in regulating high-stress proteins like HSP70 and BAG, with increasing expression levels, while DAPAs are mainly associated with signal transduction and energy metabolism, including genes like CUBN and UGCG, with gene expression levels initially decreasing and then increasing. We verified the isoforms of three DASs and the polyadenylation sites of three DAPAs using RT-PCR and 3′ RACE. In conclusion, the post-transcriptional regulation of AS and APA in A. cerana may respond to chilling stress by participating in the regulation of different metabolic pathways and antifreeze proteins. The results of this study have reference value for the study of the molecular mechanisms of insect cold resistance and provide clues for the development of molecular breeding targets for cold-resistant strains of bees. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Oocyte cryopreservation is not yet considered a reliable technique since it can reduce the quality and survival of oocytes in several species. This study determined the effect of different concentrations of antifreeze protein I (AFP I) on the vitrification solution of immature cat oocytes. For this, oocytes were randomly distributed in three groups and vitrified with 0 μg/mL (G0, 0 μM); 0.5 μg/mL (G0.5, 0.15 μM), or 1 μg/mL (G1, 0.3 μM) of AFP I. After thawing, oocytes were evaluated for morphological quality, and compared to a fresh group (FG) regarding actin integrity, mitochondrial activity and mass, reactive oxygen species (ROS) and glutathione (GSH) levels, nuclear maturation, expression of GDF9 , BMP15 , ZAR-1 , PRDX1 , SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes), and ultrastructure. G0.5 and G1 presented a higher proportion of COCs graded as I and while G0 had a significantly lower quality. G1 had a higher percentage of intact actin in COCs than G0 and G0.5 (P < 0.05). There was no difference (P > 0.05) in the mitochondrial activity between FG and G1 and they were both higher (P < 0.05) than G0 and G0.5. G1 had a significantly lower (P < 0.05) mitochondrial mass than FG and G0, and there was no difference among FG, G0, and G0.5. G1 had higher ROS than all groups (P < 0.05), and there was no difference in GSH levels among the vitrified groups (P > 0.05). For nuclear maturation, there was no difference between G1 and G0.5 (P > 0.05), but these were both higher (P < 0.05) than G0 and lower (P < 0.05) compared to FG. Regarding gene expression, in G0 and G0.5, most genes were downregulated compared to FG, except for SIRT1 and SIRT3 in G0 and SIRT3 in G0.5. In addition, G1 kept the expression more similar to FG. Regardless of concentration, AFP I supplementation in vitrification solution of immature cat oocytes improved maturation rates, morphological quality, and actin integrity and did not impact GSH levels. In the highest concentration tested (1 μg/mL), AFP maintained the mitochondrial activity, reduced mitochondrial mass, increased ROS levels, and had the gene expression more similar to FG. Altogether these data show that AFP supplementation during vitrification seems to mitigate some of the negative impact of cryopreservation improving the integrity and cryosurvival of cat oocytes. [Display omitted] • The use of AFP I in the vitrification solution of immature cat oocytes improved their maturation rate. • AFP I also improved the morphological quality and actin integrity of immature vitrified cat oocytes. • In the highest concentration tested (1 μg/mL), AFP I kept the gene expression more similar to fresh oocytes. • 1 μg/mL of AFP I seems to be the suitable concentration for improving oocyte vitrification in this species. [ABSTRACT FROM AUTHOR]

Antifreeze proteins (AFPs) have unique features to sustain life in sub-zero environments due to ice recrystallization inhibition (IRI) and thermal hysteresis (TH). AFPs are in demand as agents in cryopreservation, but some antifreeze proteins have low levels of activity. This research aims to improve the cryopreservation activity of an AFPIV. In this in silico study, the helical peptide afp1m from an Antarctic yeast AFP was modeled into a sculpin AFPIV, to replace each of its four α-helices in turn, using various computational tools. Additionally, a new linker between the first two helices of AFPIV was designed, based on a flounder AFPI, to boost the ice interaction activity of the mutants. Bioinformatics tools such as ExPASy Prot-Param, Pep-Wheel, SOPMA, GOR IV, Swiss-Model, Phyre2, MODFOLD, MolPropity, and ProQ were used to validate and analyze the structural and functional properties of the model proteins. Furthermore, to evaluate the AFP/ice interaction, molecular dynamics (MD) simulations were executed for 20, 100, and 500 ns at various temperatures using GROMACS software. The primary, secondary, and 3D modeling analysis showed the best model for a redesigned antifreeze protein (AFP1mb, with afp1m in place of the fourth AFPIV helix) with a QMEAN (Swiss-Model) Z score value of 0.36, a confidence of 99.5%, a coverage score of 22%, and a p value of 0.01. The results of the MD simulations illustrated that AFP1mb had more rigidity and better ice interactions as a potential cryoprotectant than the other models; it also displayed enhanced activity in limiting ice growth at different temperatures. [ABSTRACT FROM AUTHOR]

Ice slurry is a promising functional fluid with high thermal energy density and high heat transfer rate. However, the agglomeration of the ice particles in flowing ice slurry can cause blocking of tubes. In this study, anti-freeze protein (AFP) and polyvinyl alcohol (PVA) were selected as additives to prevent the agglomeration of ice particles in ice slurry. Ice slurry was generated using the release of the supercooled state of 3 mass% NaCl solution containing the additives. The size of the ice particles in the ice slurry was observed, and the average area of the ice particles was evaluated to reveal the effects of the additives on the ice particle size. Furthermore, the flow behavior of the ice slurry in a tube was observed, and the effects of the additives on the flow behavior and prevention of the agglomeration of the ice particles were evaluated experimentally. It was found that the average area decreased with increasing concentration of the additives, and the effect of AFP on the average area was particularly significant. In the case of without the additives, the ice particles flowed in the tube while the ice particles agglomerated. On the other hand, the ice particles in the ice slurry with the additives flowed without agglomeration in the tube, especially in the case of AFP addition. It was found that AFP prevented the agglomeration of the ice particles in the ice slurry flow. Moreover, the ice slurries with AFP show the significant tendency for pseudo-plastic fluid. [ABSTRACT FROM AUTHOR]

Antifreeze agents play a critical role in various fields including tissue engineering, gene therapy, therapeutic protein production, and transplantation. Commonly used antifreeze agents such as DMSO and other organic substances are known to have cytotoxic effects. Antifreeze proteins sourced from cold-adapted organisms offer a promising solution by inhibiting ice crystal formation; however, their effectiveness is hindered by a dynamic ice-shaping (DIS) effect and thermal hysteresis (TH) properties. In response to these limitations, antifreeze peptides (AFPs) have been developed as alternatives to antifreeze proteins, providing similar antifreeze properties without the associated drawbacks. This review explores the methods for acquiring AFPs, with a particular emphasis on chemical synthesis. It aims to offer valuable insights and practical implications to drive the realm of sub-zero storage. [ABSTRACT FROM AUTHOR]

The development of new materials for antifreezing and anti‐icing applications is a big challenge in industry and academic area. Inspired by the antifreeze proteins, latex particles with superhydrophilic zwitterionic shells and superhydrophobic cores are synthesized by reversible addition‐fragmentation chain transfer emulsion polymerization, and the applications of the latex particles in antifreezing and anti‐icing applications are investigated. In antifreezing study, the critical aggregate temperature (CAT) of the latex particles decreases, and the separation of the melting and freezing temperature of ice increases with the particle concentration. Enzyme molecules can be cryopreserved in the particle solution, and their bioactivities are well maintained. Latex particles are casted into latex films with dynamic surfaces. Anti‐icing performances, including antifrosting properties, freezing delay time, and ice adhesion strengths, are studied; and the water‐treated latex films present stronger anti‐icing properties than other films, due to the synergistic effects of the superhydrophilic and superhydrophobic components. In addition, latex particles with zwitterionic shells and poly(n‐butyl methacrylate) cores, and latex particles with small molecular surfactant on the surfaces are synthesized. The antifreezing performances of the latex particles and anti‐icing properties of the latex films are compared. [ABSTRACT FROM AUTHOR]

A decline in diversity from the equator to the poles is a common feature of Earth's biodiversity. Here, we examine body shape diversity in marine fishes across latitudes and explore the role of time and evolutionary rate in explaining the diversity gradient. Marine fishes' occupation of upper latitude environments has increased substantially over the last 80 million years. Fishes in the highest latitudes exhibit twice the rate of body shape evolution and one and a third times the disparity compared to equatorial latitudes. The faster evolution of body shape may be a response to increased ecological opportunity in polar and subpolar oceans due to (1) the evolution of antifreeze proteins allowing certain lineages to invade regions of cold water, (2) environmental disturbances driven by cyclical warming and cooling in high latitudes, and (3) rapid transitions across depth gradients. Our results add to growing evidence that evolutionary rates are often faster at temperate, not tropical, latitudes. [ABSTRACT FROM AUTHOR]

The Chinese rare minnow (Gobiocypris rarus), an important model fish in China, faces endangerment in the wild. Sperm cryopreservation facilitates the development of new strains and germplasm conservation, but the quality of its cryopreserved sperm remains low. This study evaluates the protective effects of different concentrations of antifreeze proteins (AFP I and AFP III) on the cryopreservation of Chinese rare minnow sperm. Cryopreserved sperm showed significant declines in progressive motility, curvilinear velocity (VCL), average path velocity (VAP), and lifespan compared to fresh sperm, except for straight-line velocity (VSL). The cryomedium containing 10 μg/mL AFP I improved these parameters to their highest levels. However, no significant difference was found in progressive motility and kinetic parameters between cryopreserved sperm with and without AFPs. Cryopreserved sperm with 10 μg/mL AFP I showed the highest plasma membrane integrity, mitochondrial activity, and DNA integrity, significantly better than without AFPs; importantly, the fertilization rate of cryopreserved sperm with 10 μg/mL AFP I was not significantly different from that of fresh sperm. These results indicate that the addition of 10 μg/mL AFP I to the cryomedium for Chinese rare minnow sperm does not improve kinetic parameters but significantly enhances sperm quality, aiding in its new strain development and germplasm conservation. [ABSTRACT FROM AUTHOR]

Frozen dough technology is widely used and may guarantee bread's freshness and so prolong its shelf life. However, a variety of issues, including the restriction of yeast activity and structural damage to the dough, may arise while frozen dough is being made and store, ultimately resulting in a loss of quality. After fermentation, the bread's ability to retain CO2 and the yeast's ability to produce CO2 determine the frozen dough's quality. Ice crystals are thought to be the primary cause of both decreased viability of yeast and the breakdown of the dough network structure, which two significant elements are contributing to the decline in dough quality. Several factors affect yeast's resistance to freezing and thawing, such as its physiological state before freezing; for instance, yeast cells in growth standstill are more resistant to freezing than those in the exponential growth phase. Numerous strategies and methods have been created to raise the freezing baker's yeast resistance and, consequently, the quality of frozen dough. These strategies include the use of genetic engineering, the incorporation of chemicals like hydrocolloids and antifreeze proteins (AFP), the improvement of freezing times and circumstances of storage, and the creation of novel freezing techniques like ultrasonic freezing. These techniques for enhancing the freezing resistance of baker's yeast are outlined in the publication.

Anti-freeze proteins (AFPs) protect organisms at freezing conditions by attaching to the ice surface and arresting its growth. Each adsorbed AFP locally pins the ice surface, resulting in a metastable dimple for which the interfacial forces counteract the driving force for growth. As supercooling increases, these metastable dimples become deeper, until metastability is lost in an engulfment event where the ice irreversibly swallows the AFP. Engulfment resembles nucleation in some respects, and this paper develops a model for the "critical profile" and free energy barrier for the engulfment process. Specifically, we variationally optimize the ice–water interface and estimate the free energy barrier as a function of the supercooling, the AFP footprint size, and the distance to neighboring AFPs on the ice surface. Finally, we use symbolic regression to derive a simple closed-form expression for the free energy barrier as a function of two physically interpretable, dimensionless parameters. [ABSTRACT FROM AUTHOR]

Cryopreservation is a crucial technique for the long-term ex situ conservation of plant genetic resources, particularly in the context of global biodiversity decline. This process entails freezing biological material at ultra-low temperatures using liquid nitrogen, which effectively halts metabolic activities and preserves plant tissues over extended periods. Over the past seven decades, a plethora of techniques for cryopreserving plant materials have been developed. These include slow freezing, vitrification, encapsulation dehydration, encapsulation–vitrification, droplet vitrification, cryo-plates, and cryo-mesh techniques. A key challenge in the advancement of cryopreservation lies in our ability to understand the molecular processes underlying plant freezing tolerance. These mechanisms include cold acclimatization, the activation of cold-responsive genes through pathways such as the ICE–CBF–COR cascade, and the protective roles of transcription factors, non-coding RNAs, and epigenetic modifications. Furthermore, specialized proteins, such as antifreeze proteins (AFPs) and late embryogenesis abundant (LEA) proteins, play crucial roles in protecting plant cells during freezing and thawing. Despite its potential, cryopreservation faces significant challenges, particularly in standardizing protocols for a wide range of plant species, especially those from tropical and subtropical regions. This review highlights the importance of ongoing research and the integration of omics technologies to improve cryopreservation techniques, ensuring their effectiveness across diverse plant species and contributing to global efforts regarding biodiversity conservation. [ABSTRACT FROM AUTHOR]

Antifreeze proteins (AFPs) are found in a variety of marine cold‐water fishes where they prevent freezing by binding to nascent ice crystals. Their diversity (types I, II, III and antifreeze glycoproteins), as well as their scattered taxonomic distribution hint at their complex evolutionary history. In particular, type I AFPs appear to have arisen in response to the Late Cenozoic Ice Age that began ~ 34 million years ago via convergence in four different groups of fish that diverged from lineages lacking this AFP. The progenitor of the alanine‐rich α‐helical type I AFPs of sculpins has now been identified as lunapark, an integral membrane protein of the endoplasmic reticulum. Following gene duplication and loss of all but three of the 15 exons, the final exon, which encoded a glutamate‐ and glutamine‐rich segment, was converted to an alanine‐rich sequence by a combination of frameshifting and mutation. Subsequent gene duplications produced numerous isoforms falling into four distinct groups. The origin of the flounder type I AFP is quite different. Here, a small segment from the original antiviral protein gene was amplified and the rest of the coding sequence was lost, while the gene structure was largely retained. The independent origins of type I AFPs with up to 83% sequence identity in flounder and sculpin demonstrate strong convergent selection at the level of protein sequence for alanine‐rich single alpha helices that bind to ice. Recent acquisition of these AFPs has allowed sculpins to occupy icy seawater niches with reduced competition and predation from other teleost species. [ABSTRACT FROM AUTHOR]

Determining the origins of novel genes and the mechanisms driving the emergence of new functions is challenging yet crucial for understanding evolutionary innovations. Recently evolved fish antifreeze proteins (AFPs) offer a unique opportunity to explore these processes, particularly the near-identical type I AFP (AFPI) found in four phylogenetically divergent fish taxa. This study tested the hypothesis of protein sequence convergence beyond functional convergence in three unrelated AFPI-bearing fish lineages. Through comprehensive comparative analyses of newly sequenced genomes of winter flounder and grubby sculpin, along with available high-quality genomes of cunner and 14 other related species, the study revealed that near-identical AFPI proteins originated from distinct genetic precursors in each lineage. Each lineage independently evolved a de novo coding region for the novel ice-binding protein while repurposing fragments from their respective ancestors into potential regulatory regions, representing partial de novo origination—a process that bridges de novo gene formation and the neofunctionalization of duplicated genes. The study supports existing models of new gene origination and introduces new ones: the innovation–amplification–divergence model, where novel changes precede gene duplication; the newly proposed duplication–degeneration–divergence model, which describes new functions arising from degenerated pseudogenes; and the duplication–degeneration–divergence gene fission model, where each new sibling gene differentially degenerates and renovates distinct functional domains from their parental gene. These findings highlight the diverse evolutionary pathways through which a novel functional gene with convergent sequences at the protein level can evolve across divergent species, advancing our understanding of the mechanistic intricacies in new gene formation. [ABSTRACT FROM AUTHOR]

Frozen dough technology is widely used and may guarantee bread's freshness and so prolong its shelf life. However, a variety of issues, including the restriction of yeast activity and structural damage to the dough, may arise while frozen dough is being made and store, ultimately resulting in a loss of quality. After fermentation, the bread's ability to retain CO2 and the yeast's ability to produce CO2 determine the frozen dough's quality. Ice crystals are thought to be the primary cause of both decreased viability of yeast and the breakdown of the dough network structure, which two significant elements are contributing to the decline in dough quality. Several factors affect yeast's resistance to freezing and thawing, such as its physiological state before freezing; for instance, yeast cells in growth standstill are more resistant to freezing than those in the exponential growth phase. Numerous strategies and methods have been created to raise the freezing baker's yeast resistance and, consequently, the quality of frozen dough. These strategies include the use of genetic engineering, the incorporation of chemicals like hydrocolloids and antifreeze proteins (AFP), the improvement of freezing times and circumstances of storage, and the creation of novel freezing techniques like ultrasonic freezing. These techniques for enhancing the freezing resistance of baker's yeast are outlined in the publication. [ABSTRACT FROM AUTHOR]

The adsorption of large rod-like molecules or crystallites on a flat crystal face, similar to Buffon's needle, requires the rods to "land," with their binding sites in precise orientational alignment with matching sites on the surface. An example is provided by long, helical antifreeze proteins (AFPs), which bind at specific facets and orientations on the ice surface. The alignment constraint for adsorption, in combination with the loss in orientational freedom as the molecule diffuses toward the surface, results in an entropic barrier that hinders the adsorption. Prior kinetic models do not factor in the complete geometry of the molecule, nor explicitly enforce orientational constraints for adsorption. Here, we develop a diffusion-controlled adsorption theory for AFP molecules binding at specific orientations to flat ice surfaces. We formulate the diffusion equation with relevant boundary conditions and present analytical solutions to the attachment rate constant. The resulting rate constant is a function of the length and aspect ratio of the AFP, the distance threshold associated with binding, and solvent conditions such as temperature and viscosity. These results and methods of calculation may also be useful for predicting the kinetics of crystal growth through oriented attachment. [ABSTRACT FROM AUTHOR]

To realize the soluble expression of human-derived Tumstatin in Escherichia coli. Five recombinant E. coli (BL21(DE3)) expressed by fusion of different solubility-promoting fusion tags and tumstatin genes were constructed, and isopropyl-beta-D-thiogalactopyranoside (IPTG) was used to induce the recombinant bacteria to express the target protein. SDS-PAGE and Western-Blot were employed to analyze and screen for the recombinant bacteria that could solubility express Tumstatin. The results indicated that BL21/pGEX-6p-1-Tum (containing glutathione mercaptotransferase tag -GST tag), BL21/pET28a-MBP-Tum (containing maltose-binding protein tag-MBP tag), BL21/pET28a-Tum (no tag) all expressed the fusion protein Tumstatin in the form of inclusion bodies. Only the recombinant bacteria BL21/pET28a-XXA-Tum (containing the reverse protein of antifreeze protein-XXA tag) expressed fusion proteins in a soluble form, and the target protein accounts for more than 45% of the total protein of the bacteria. The target protein was purified by affinity chromatography and analyzed by Western-Blot. The band of interest with a molecular weight of 50.9 kDa was obtained, which was consistent with the theoretical molecular weight. Furthermore, the inducer concentration, addition time, induction temperature, and induction time of recombinant bacteria BL21/pET28a-XXA-Tum were optimized. Finally, the induction conditions of tumstatin fusion protein were selected as follows: Inducer concentration 0.1 mmol/L, inducer added at 2 h, induction temperature 16°C, induction time 36 h. The soluble expression of human Tumstatin in E. coli laid the foundation for the preparation of soluble human-derived Tumstatin in a large scale, which could provide a reference for the soluble expression of functional polypeptides in E. coli. [ABSTRACT FROM AUTHOR]

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, ΔT*, for which AFPs are able to resist engulfment is widely believed to scale as the inverse of the separation, L, 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, ΔT* scales as L-2 and not as L-1. We further show that ΔT* 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. [ABSTRACT FROM AUTHOR]

Polar environments pose extreme challenges for life due to low temperatures, limited water, high radiation, and frozen landscapes. Despite these harsh conditions, numerous macro and microorganisms have developed adaptive strategies to reduce the detrimental effects of extreme cold. A primary survival tactic involves avoiding or tolerating intra and extracellular freezing. Many organisms achieve this by maintaining a supercooled state by producing small organic compounds like sugars, glycerol, and amino acids, or through increasing solute concentration. Another approach is the synthesis of ice-binding proteins, specifically antifreeze proteins (AFPs), which hinder ice crystal growth below the melting point. This adaptation is crucial for preventing intracellular ice formation, which could be lethal, and ensuring the presence of liquid water around cells. AFPs have independently evolved in different species, exhibiting distinct thermal hysteresis and ice structuring properties. Beyond their ecological role, AFPs have garnered significant attention in biotechnology for potential applications in the food, agriculture, and pharmaceutical industries. This review aims to offer a thorough insight into the activity and impacts of AFPs on water, examining their significance in cold-adapted organisms, and exploring the diversity of microbial AFPs. Using a meta-analysis from cultivation-based and cultivation-independent data, we evaluate the correlation between AFP-producing microorganisms and cold environments. We also explore small and large-scale biotechnological applications of AFPs, providing a perspective for future research. [ABSTRACT FROM AUTHOR]

A class of proteins known as antifreeze proteins (AFPs) appear in some organisms, allowing them to survive in low-temperature environments. These AFPs irreversibly adsorb to the surfaces of ice crystals and reduce the freezing temperature without significantly affecting the equilibrium melting point. Ice crystal growth is inhibited in the temperature gap between the melting point and the non-equilibrium freezing point, referred to as thermal hysteresis (TH). The irreversible adsorption of AFPs on the surfaces of ice crystals has been questioned because it is not consistent with the concentration dependence of the TH activity obtained from experimental studies. In this study, based on adsorption–inhibition theory, a random sequential adsorption model of AFPs was used to investigate the irreversible adsorption of hyperactive AFPs that have been found in the yellow mealworm beetle Tenebrio molitor (TmAFPs). The occupied fractions covered by TmAFP on ice crystal surfaces were obtained. The time and concentration dependence of the TH activity of TmAFP was analyzed. The theoretical results obtained from this model were consistent with reported experimental data. This work provides ideas and a theoretical basis for understanding the TH activity during the irreversible adsorption of AFPs. [ABSTRACT FROM AUTHOR]

Cryopreservation is a valuable technique used to assist in the genetic improvement of cultured stocks and provide a continuous supply of good‐quality semen for artificial insemination. Conserving semen by cryopreservation serves several purposes (e.g. artificial reproductive technologies and species conservation) and is also used in the clinical treatment of human infertility. However, the lifespan of cryopreserved semen is influenced by a range of factors, including storage temperature, cooling rate, chemical composition of the extender, the concentration of cryoprotectant, reactive oxygen species, seminal plasma composition and hygienic control. The choice of cryoprotectant is a vital factor underlying the success of animal semen cryopreservation. In this regard, extensive research has been carried out on various cryoprotectants, such as egg yolk, dimethyl sulfoxide, methanol, ethylene glycol and dimethylacetamide. Recent studies have also described the use of a range of new cryoprotectants for cryopreservation, including compounds of plant origin (soy), amino acids, antifreeze proteins, carbohydrates and cyclodextrins. Moreover, semen cryopreservation and storage require the use of liquid nitrogen or ultralow refrigeration methods for both long‐ and short‐term storage. This review summarizes the general methods used for freezing semen and discusses the use of traditional and newly emerging cryoprotectants (permeable and non‐permeable) for the cryopreservation of semen in selected fish and mammalian species. [ABSTRACT FROM AUTHOR]

Background: Cold hardiness is fundamental for amphibians to survive during the extremely cold winter on the Qinghai-Tibet plateau. Exploring the gene regulation mechanism of freezing-tolerant Rana kukunoris could help us to understand how the frogs survive in winter. Results: Transcriptome of liver and muscle of R. kukunoris collected in hibernation and spring were assisted by single molecule real-time (SMRT) sequencing technology. A total of 10,062 unigenes of R. kukunoris were obtained, and 9,924 coding sequences (CDS) were successfully annotated. Our examination of the mRNA response to whole body freezing and recover in the frogs revealed key genes concerning underlying antifreeze proteins and cryoprotectants (glucose and urea). Functional pathway analyses revealed differential regulated pathways of ribosome, energy supply, and protein metabolism which displayed a freeze-induced response and damage recover. Genes related to energy supply in the muscle of winter frogs were up-regulated compared with the muscle of spring frogs. The liver of hibernating frogs maintained modest levels of protein synthesis in the winter. In contrast, the liver underwent intensive high levels of protein synthesis and lipid catabolism to produce substantial quantity of fresh proteins and energy in spring. Differences between hibernation and spring were smaller than that between tissues, yet the physiological traits of hibernation were nevertheless passed down to active state in spring. Conclusions: Based on our comparative transcriptomic analyses, we revealed the likely adaptive mechanisms of R. kukunoris. Ultimately, our study expands genetic resources for the freezing-tolerant frogs. [ABSTRACT FROM AUTHOR]

The development of nontoxic and efficient antifreeze agents for organ cryopreservation is crucial. However, the research remains highly challenging. In this study, we designed and synthesized a series of peptoid oligomers using the solid-phase submonomer synthesis method by mimicking the amphiphilic structures of antifreeze proteins (AFPs). The obtained peptoid oligomers showed excellent antifreeze properties, reducing the ice crystal growth rate and inhibiting ice recrystallization. The effects of the hydrophobicity and sequence of the peptoid side chains were also studied to reveal the structure–property relationship. The prepared peptoid oligomers were detected as non-cytotoxic and considered to be useful in the biological field. We hope that the peptoid oligomers presented in this study can provide effective strategies for the design of biological cryoprotectants for organ preservation in the future. [ABSTRACT FROM AUTHOR]

Freezing, a technique extensively employed within the food industry, is considered to be among the most prevalent preservation methods. Conventional techniques of freezing have the potential to cause certain forms of quality degradation, such as harm to cellular structure, and heightened loss of moisture. Therefore, innovative techniques for freezing have been devised to mitigate the drawbacks. Certain naturally occurring biomaterials that possess environmentally friendly, sustainable, non-harmful, remarkably efficient properties and the freezing point regulators (such as antifreeze proteins) have been scientifically proven to manage the freezing and thawing cycle, thus demonstrating promising prospects for utilization in the realm of food and food-related sectors. The purpose of this review is to thoroughly investigate the advanced freezing methods, and the cryoprotective impact of antifreeze proteins (AFPs), emphasizing their function in the food freeze–thaw process. Moreover, this review highlights the advantages and challenges of AFP employment in food preservation. The characteristics of AFPs are derived from their ability to exhibit thermal hysteresis, alter the crystal morphology, and prevent the process of ice recrystallization. Hence, AFPs have been effectively utilized to maintain the quality of a diverse range of refrigerated and frozen food products as a potential cryoprotectant agent in food industry. [ABSTRACT FROM AUTHOR]

Shifts in environmental conditions can impose strong selection for adaptive traits. During the Cenozoic era, as the oceans cooled, many marine teleost fish species were at risk of freezing. This led to the independent emergence of distinct ice‐binding antifreeze proteins (AFPs). The report in this issue by Graham and Davies reveals the development of AFP genes in shorthorn and longhorn sculpin from a copy of the lunapark gene. The predicted sculpin AFP sequences are unrelated to that of lunapark; the coding sequences for the AFPs appear to have arisen from small portions of the lunapark gene by codon frameshifting along with a series of mutations. [ABSTRACT FROM AUTHOR]

Summary: Freezing 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. [ABSTRACT FROM AUTHOR]

Many polar organisms produce antifreeze proteins (AFPs) and ice-binding proteins (IBPs) to protect themselves from ice formation. As IBPs protect cells and organisms, the potential of IBPs as natural or biological cryoprotective agents (CPAs) for the cryopreservation of animal cells, such as oocytes and sperm, has been explored to increase the recovery rate after freezing–thawing. However, only a few IBPs have shown success in cryopreservation, possibly because of the presence of protein denaturants, such as dimethyl sulfoxide, alcohols, or ethylene glycol, in freezing buffer conditions, rendering the IBPs inactive. Therefore, we investigated the thermal and chemical stability of FfIBP isolated from Antarctic bacteria to assess its suitability as a protein-based impermeable cryoprotectant. A molecular dynamics (MD) simulation identified and generated stability-enhanced mutants (FfIBP_CC1). The results indicated that FfIBP_CC1 displayed enhanced resistance to denaturation at elevated temperatures and chemical concentrations, compared to wildtype FfIBP, and was functional in known CPAs while retaining ice-binding properties. Given that FfIBP shares an overall structure similar to DUF3494 IBPs, which are recognized as the most widespread IBP family, these findings provide important structural information on thermal and chemical stability, which could potentially be applied to other DUF3494 IBPs for future protein engineering. [ABSTRACT FROM AUTHOR]

Highlights: Cryopreservation generates ice recrystallization. D. antarctica apoplastic proteins show antifreeze activity. PMI of S. salar sperm can be maintained with AFPs. High MMP of sperm increases with AFPs. D. antarctica apoplastic proteins act as nonpermeable cryoprotectants. Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media. [ABSTRACT FROM AUTHOR]

Antifreeze (glyco) proteins [AF(G)Ps], which are widely present in various extreme microorganisms, can control the formation and growth of ice crystals. Given the significance of cryogenic technology in biomedicine, climate science, electronic energy, and other fields of research, scientists are quite interested in the development and synthesis high-efficiency bionic antifreeze protein materials, particularly to reproduce their dynamic ice shaping (DIS) characteristics. Single ice crystal shaping materials, a promising class of ice-controlling materials, can alter the morphology and growth rate of ice crystals at low temperatures. This review aims to highlight the development of single ice crystal shaping materials and provide a brief comparison between a series of natural and bionic synthetic materials with DIS ability, which include AF(G)Ps, polymers, salts, and nanomaterials. Additionally, we summarize their applications in cryopreservation. Finally, this paper presents the current challenges and prospects encountered in developing high-efficiency and practical single ice crystal shaping materials. The formation and growth of ice crystals hold a significant importance to an incredibly broad range of fields. Therefore, the design and fabrication of the single ice crystal shaping materials have gained the increasing popularity due to its key role in dynamic ice shaping (DIS) characteristics. Especially, single ice crystal shaping materials are considered one of the most promising candidates as ice inhibitors, presenting tremendous prospects for enhancing cryopreservation. In this work, we focus on the molecular characteristics, structure–function relationships, and DIS mechanisms of typical natural and biomimetic synthetic materials. This review may provide inspiration for the design and preparation of single ice crystal shaping materials and give guidance for the development of effective cryopreservation agent. [Display omitted] [ABSTRACT FROM AUTHOR]

Ice-binding proteins are crucial for the adaptation of various organisms to low temperatures. Some of these, called antifreeze proteins, are usually thought to inhibit growth and/or recrystallization of ice crystals. However, prior to these events, ice must somehow appear in the organism, either coming from outside or forming inside it through the nucleation process. Unlike most other works, our paper is focused on ice nucleation and not on the behavior of the already-nucleated ice, its growth, etc. The nucleation kinetics is studied both theoretically and experimentally. In the theoretical section, special attention is paid to surfaces that bind ice stronger than water and thus can be "ice nucleators", potent or relatively weak; but without them, ice cannot be nucleated in any way in calm water at temperatures above −30 °C. For experimental studies, we used: (i) the ice-binding protein mIBP83, which is a previously constructed mutant of a spruce budworm Choristoneura fumiferana antifreeze protein, and (ii) a hyperactive ice-binding antifreeze protein, RmAFP1, from a longhorn beetle Rhagium mordax. We have shown that RmAFP1 (but not mIBP83) definitely decreased the ice nucleation temperature of water in test tubes (where ice originates at much higher temperatures than in bulk water and thus the process is affected by some ice-nucleating surfaces) and, most importantly, that both of the studied ice-binding proteins significantly decreased the ice nucleation temperature that had been significantly raised in the presence of potent ice nucleators (CuO powder and ice-nucleating bacteria Pseudomonas syringae). Additional experiments on human cells have shown that mIBP83 is concentrated in some cell regions of the cooled cells. Thus, the ice-binding protein interacts not only with ice, but also with other sites that act or potentially may act as ice nucleators. Such ice-preventing interaction may be the crucial biological task of ice-binding proteins. [ABSTRACT FROM AUTHOR]

Antifreeze proteins (AFPs) are biopolymers capable of interfering with ice growth. Their antifreeze action is commonly understood considering that the AFPs, by pinning the ice surface, force the crystal–liquid interface to bend forming an ice meniscus, causing an increase in the surface free energy and resulting in a decrease in the freezing point ΔTmax. Here, we present an extensive computational study for a model protein adsorbed on a TIP4P/Ice crystal, computing ΔTmax as a function of the average distance d between AFPs, with simulations spanning over 1 µs. First, we show that the lower the d, the larger the ΔTmax. Then, we find that the water–ice–protein contact angle along the line ΔTmax(d) is always larger than 0°, and we provide a theoretical interpretation. We compute the curvature radius of the stable solid–liquid interface at a given supercooling ΔT ≤ ΔTmax, connecting it with the critical ice nucleus at ΔT. Finally, we discuss the antifreeze capability of AFPs in terms of the protein–water and protein–ice interactions. Our findings establish a unified description of the AFPs in the contest of homogeneous ice nucleation, elucidating key aspects of the antifreeze mechanisms and paving the way for the design of novel ice-controlling materials. [ABSTRACT FROM AUTHOR]

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The integration of solar‐driven interfacial evaporation and electricity co‐generation is considered a promising approach to simultaneously alleviate freshwater scarcity and the energy crisis. However, affected by intermittent solar irradiation/uncontrollable weather, the overall performance of solar‐driven evaporation in the real world is greatly reduced. Herein, inspired by antifreeze proteins in beetles that survive in extreme climates, all‐weather solar‐driven interfacial evaporators with a sandwich structure are designed. The top and bottom layers composed of MnO2‐modified cotton cloth are used for photothermal conversion and water transport, meanwhile, the middle layer made of a phase change microcapsule/hydrogel composite serves for heat storage and release. Under 1 kW m−2 irradiation, the evaporator exhibits a high evaporation rate of 2.67 kg m−2 h−1 and an efficiency of 89.5%. In the dark, the heat released from the phase change layer supports an evaporation rate of 0.43 kg m−2 h−1, 3.6 times that of pure water. Additionally, assembled with a thermoelectric module, the hybrid device achieves a stable output electricity power of 0.42 W m−2 under 1‐sun illumination and a prolonged output for 30 min in the dark. This work provides a novel approach for full‐time solar‐powered steam‐electricity co‐generation and a proof of concept for biomimetic steam generation/heat management integration. [ABSTRACT FROM AUTHOR]

Antifreeze proteins (AFPs) are multifunctional polypeptides that adsorb onto ice crystals to inhibit their growth and onto cells to protect them from nonfreezing hypothermic damage. However, the mechanism by which AFP exerts its hypothermic cell protective (HCP) function remains uncertain. Here, we assessed the HCP function of three types of fish-derived AFPs (type I, II, and III AFPs) against human T-lymphoblastic lymphoma by measuring the survival rate (%) of the cells after preservation at 4 °C for 24 h. All AFPs improved the survival rate in a concentration-dependent manner, although the HCP efficiency was inferior for type III AFP compared to other AFPs. In addition, after point mutations were introduced into the ice-binding site (IBS) of a type III AFP, HCP activity was dramatically increased, suggesting that the IBS of AFP is involved in cell adsorption. Significantly, high HCP activity was observed for a mutant that exhibited poorer antifreeze activity, indicating that AFP exerts HCP- and ice-binding functions through a different mechanism. We next incubated the cells in an AFP-containing solution, replaced it with pure EC solution, and then preserved the cells, showing that no significant reduction in the cell survival rate occurred for type I and II AFPs even after replacement. Thus, these AFPs irreversibly bind to the cells at 4 °C, and only tightly adsorbed AFP molecules contribute towards the cell-protection function. • Antifreeze proteins bind to ice crystals and cells to perform different functions. • The correlation between antifreeze and cell protective functions of AFP was studied. • AFPs protect the white blood cell line in a concentration-dependent manner at 4 °C. • Irreversible binding at the ice-binding site of AFP leads to its protective function. [ABSTRACT FROM AUTHOR]

The possibility of the protein backbone adopting lasso-like entangled motifs has attracted increasing attention. After discovering the surprising abundance of natively entangled protein domain structures, it was shown that misfolded entangled subpopulations might become thermosensitive or escape the homeostasis network just after translation. To investigate the role of entanglement in shaping folding kinetics, we introduce a novel indicator and analyze simulations of a coarse-grained, structure-based model for two small single-domain proteins. The model recapitulates the well-known two-state folding mechanism of a non-entangled SH3 domain. However, despite its small size, a natively entangled antifreeze RD1 protein displays a rich refolding behavior, populating two distinct kinetic intermediates: a short-lived, entangled, near-unfolded state and a longer-lived, non-entangled, near-native state. The former directs refolding along a fast pathway, whereas the latter is a kinetic trap, consistently with known experimental evidence of two different characteristic times. Upon trapping, the natively entangled loop folds without being threaded by the N-terminal residues. After trapping, the native entangled structure emerges by either backtracking to the unfolded state or threading through the already formed but not yet entangled loop. Along the fast pathway, trapping does not occur because the native contacts at the closure of the lasso-like loop fold after those involved in the N-terminal thread, confirming previous predictions. Despite this, entanglement may appear already in unfolded configurations. Remarkably, a longer-lived, near-native intermediate, with non-native entanglement properties, recalls what was observed in cotranslational folding. Author summary: Recently, a surprisingly large fraction of protein structures was shown to host topologically entangled motifs, whereby one protein chain portion is lassoed by a second portion, that loops between two residues in non-covalent contact with each other. Moreover, there is growing evidence that failure in adopting the correct entangled motifs may produce misfolded structures with impaired biological functions. Such structures are otherwise similar to the correct ones and can escape the cell quality control system for protein expression, leading to soluble and less functional protein species. Here, we study in detail the folding kinetics of an entangled small anti-freeze protein, using a simplified representation of the protein chain. We find a very rich folding behavior, unusual for small proteins, with different folding pathways. A fast pathway is followed if a crucial set of contacts is formed before lassoing takes place. If not, a misfolded structure which acts as a kinetic trap is formed, slowing down folding; in such structure, most of the contacts are correctly in place yet the lasso is not formed. The detailed understanding that we provide for a small protein may pave the way for similar studies for larger entangled proteins. [ABSTRACT FROM AUTHOR]

Freshwater fungi play an important role in the decomposition of organic matter of leaf litter in rivers and streams. They also possess the necessary mechanisms to endure lower temperatures caused by habitat and weather variations. This includes the production of coldactive enzymes and antifreeze proteins. To better understand the physiological activities of freshwater fungi in their natural environment, different methods are being applied, and genome sequencing is one in the spotlight. In our study, we sequenced the first genome of the freshwater fungus Filosporella fistucella (45.7 Mb) and compared the genome with the evolutionary close-related species Tricladium varicosporioides (48.2 Mb). The genomes were annotated using the carbohydrate-active enzyme database where we then filtered for leaflitter degradation-related enzymes (cellulase, hemicellulase, laccase, pectinase, cutinase, amylase, xylanase, and xyloglucanase). Those enzymes were analyzed for antifreeze properties using a machine-learning approach. We discovered that F. fistucella has more enzymes to participate in the breakdown of sugar, leaf, and wood than T. varicosporioides (855 and 719, respectively). Filosporella fistucella shows a larger set of enzymes capable of resisting cold temperatures than T. varicosporioides (75 and 66, respectively). Our findings indicate that in comparison with T. varicosporioides, F. fistucella has a greater capacity for aquatic growth, adaptability to freshwater environments, and resistance to low temperatures. [ABSTRACT FROM AUTHOR]