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ABSTRACTTemperate regions experiencing sub-zero temperatures negatively impact grapevine yield. Potassium has been claimed as a cryoprotectant to improve cold hardiness in grapevine. This study investigated the effect of foliar application of liquid potassium-based fertilizer, ReaXTM, on cold-hardiness of grapevine Vitis spp. “Chambourcin” along with its effect on yield and fruit quality. The vines were sprayed four to five times between the fruit set and veraison stage at a concentration of 1.5% (v/v) for two consecutive seasons. Petioles were analyzed for nutrients, clusters for yield and fruit quality, and cold hardiness was determined by differential thermal analysis and bud injury assessment. Potassium deficiency was observed in all treatments, and its content did not increase significantly in treated vines. Berry potassium levels and total soluble solids were generally higher in K-treated vines, however, there was no significant effect on yield and other fruit quality traits. Significant differences in cold hardiness levels were observed in both dormant seasons. Foliar application of potassium is a promising cultural practice to increase cold hardiness, but further studies are needed to understand the limits of its effectiveness.

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]

Abstract Background This study explores the biosynthesis, characteristics, and functional properties of exopolysaccharide produced by the strain Liquorilactobacillus mali T6-52. The strain demonstrated significant EPS production with a non-ropy phenotype. Results The genomic analysis unveiled genes associated with EPS biosynthesis, shedding light on the mechanism behind EPS production. These genes suggest a robust EPS production mechanism, providing insights into the strain’s adaptability and ecological niche. Chemical composition analysis identified the EPS as a homopolysaccharide primarily composed of glucose, confirming its dextran nature. Furthermore, it demonstrated notable functional properties, including antioxidant activity, fat absorption capacity, and emulsifying activity. Moreover, the EPS displayed promising cryoprotective activities, showing notable performance comparable to standard cryoprotective agents. The EPS concentration also demonstrated significant freeze-drying protective effects, presenting it as a potential alternative cryoprotectant for bacterial storage. Conclusions The functional properties of L. mali T6-52 EPS reveal promising opportunities across various industrial domains. The strain’s safety profile, antioxidant prowess, and exceptional cryoprotective and freeze-drying characteristics position it as an asset in food processing and pharmaceuticals.

Background: This study explores the biosynthesis, characteristics, and functional properties of exopolysaccharide produced by the strain Liquorilactobacillus mali T6-52. The strain demonstrated significant EPS production with a non-ropy phenotype. Results: The genomic analysis unveiled genes associated with EPS biosynthesis, shedding light on the mechanism behind EPS production. These genes suggest a robust EPS production mechanism, providing insights into the strain's adaptability and ecological niche. Chemical composition analysis identified the EPS as a homopolysaccharide primarily composed of glucose, confirming its dextran nature. Furthermore, it demonstrated notable functional properties, including antioxidant activity, fat absorption capacity, and emulsifying activity. Moreover, the EPS displayed promising cryoprotective activities, showing notable performance comparable to standard cryoprotective agents. The EPS concentration also demonstrated significant freeze-drying protective effects, presenting it as a potential alternative cryoprotectant for bacterial storage. Conclusions: The functional properties of L. mali T6-52 EPS reveal promising opportunities across various industrial domains. The strain's safety profile, antioxidant prowess, and exceptional cryoprotective and freeze-drying characteristics position it as an asset in food processing and pharmaceuticals. [ABSTRACT FROM AUTHOR]

Immunoglobulin, which is widely used in the formulation of protein drugs, is prone to aggregation due to freezing. The aggregated immunoglobulin exhibits decreased immune-reactivity and increasing immunogenicity. Accordingly, large amounts of excipients are added to immunoglobulin drugs to prevent aggregation. In the present study, we found that an Arabidopsis dehydrin (AtHIRD11), which is a stress-related intrinsically disordered protein, could efficiently inhibit the cryoaggregation of bovine gamma globulin (BGG). AtHIRD11 was 3 to 4 orders of magnitude more efficient than general protectants such as sugars and amino acids at the molar levels. The K-segment, which is a conserved sequence of dehydrin, was one of the protective sites of AtHIRD11. Amino acid substitution analysis indicated that the hydrophobic amino acids contributed to the cryoprotective activity of the K-segment. Moreover, the activity was roughly correlated with the hydropathy scores of hydrophobic amino acids. BGG and the K-segment individually migrated in size exclusion chromatography, showing that the K-segment did not bind to BGG in solution. This suggests that dehydrin may prevent the cryoaggregation of BGG via the K-segment through a transient hydrophobic interaction. Dehydrin may be utilized as an effective stabilizer of immunoglobulin to minimize aggregation under freezing conditions.

BACKGROUND: Dimethyl sulphoxide (DMSO, a small strongly polarized amphiphilic molecule) is used as a cryoprotector due to its possibility to easily penetrate cell membranes and to form strong H-bonds with water molecules, preventing by that arising of aqueous crystals. AIMS: We have investigated the vitality of hematopoietic stem cells after incubation at +4°C, +20°C and +37°C for 30 min, 60 min and 24 h in cryoprotective solution with 5% DMSO, and also after deep-freezing at –80°C and subsequent storage for 24 hours at –20°C and +4°C. MATERIALS AND METHODS: The vitality was evaluated counting the number of alive (uncoloured) and dead stem cells (blue coloured) using a staining test with Trypan blue dye. RESULTS: The obtained results disclose that DMSO has a cytotoxic effect which depends on the temperature and the time of cultivation: the cytotoxicity is insignificant at +4°C even for 24 hours; at +37°C about 90% cells remain vital for 1 hour, however after 24 hours they completely die out. CONCLUSION: Comparing the non-frozen cells with the deep-frozen at –80°C and subsequently defrozen cells in identical conditions (24 hours storage at +4°C) reveals that the freezing process causes an additional delayed damaging effect, which leads to death of half of the stem cells.. [ABSTRACT FROM AUTHOR]

The health effects of probiotics depend on the concentration of living cells of these beneficial bacteria (more than 106 to 107 log CFU/g) in the foodstuffs at the time of consumption, as well as after the passage of the digestive system. Therefore, applying various protective compounds has been studied to protect probiotic microorganisms using the encapsulation method. Optimal formulation (Whey protein isolate (WPI): 57.22%, bio-cellulose (BC): 25%, inulin: 17.78%) was obtained based on particle size, zeta potential, microcapsule resistance to acidic conditions, and encapsulation efficiency analyses. In the second stage, probiotic ice cream samples with encapsulated and free probiotic and control sample (ice cream without probiotic) were produced. During storage for 50 days, the physical, chemical, sensory, and microbial characteristics of ice cream were investigated. The results indicated that the free probiotic decreased 0.97 logs from day 1 to day 50, while the encapsulated probiotic bacteria decreased 0.61 logs. Encapsulation significantly preserved the probiotic in the food substrate during storage. The present study indicated that the living cells of free probiotic were decreased (2 log CFU/g), although that of microencapsulated probiotic merely decreased by less than 1 log CFU/g cycle. This shows the cryoprotectant potential of BC used as a microencapsulating agent against damage at freezing temperature. [ABSTRACT FROM AUTHOR]

Standard freezing protocols of clinically relevant cell lines commonly employ agents such as fetal bovine serum and dimethyl sulfoxide, which are a potential concern from both a regulatory and a patient safety perspective. The aim of this work was to develop formulations with safe and well tolerated excipients for the (cryo-) preservation of cell therapy products. We evaluated the cryoprotective capabilities of urea and glucose through measurements of cell metabolic activity. Freezing of clinically relevant human mesenchymal stromal/stem cells and human dermal fibroblasts at ≤ - 65°C at equimolar ratios of urea and glucose resulted in comparable viabilities to established dimethyl sulfoxide. Pre-incubation of human mesenchymal stromal/stem cells in trehalose and addition of mannitol and sucrose to the formulation further enhanced cell viability after freeze-thaw stress. Other cell types assessed (A549 and SK-N-AS) could not satisfactorily be preserved with urea and glucose, highlighting the need for tailored formulations to sustain acceptable cryopreservation. [ABSTRACT FROM AUTHOR]

Abstract Background A medium containing dimethyl sulfoxide (DMSO) (10% v/v) is most widely used for cell cryopreservation at –196 °C. However, residual DMSO consistently raises concerns because of its toxicity; thus, its complete removal process is required. Method As biocompatible polymers approved by the Food and Drug Administration for various biomedical applications for humans, poly(ethylene glycol)s (PEGs) with various molecular weights (400, 600, 1 K, 1.5 K, 5 K, 10 K, and 20 K Da) were studied as a cryoprotectant of mesenchymal stem cells (MSCs). Considering the cell permeability difference of PEGs depending on their molecular weight, the cells were preincubated for 0 h (no incubation), 2 h, and 4 h at 37 °C in the presence of PEGs at 10 wt.% before cryopreservation at –196 °C for 7 days. Then, cell recovery was assayed. Results We found that low molecular weight PEGs (400 and 600 Da) exhibit excellent cryoprotecting properties by 2 h preincubation, whereas intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) exhibit their cryoprotecting properties without preincubation. High molecular weight PEGs (10 K and 20 K Da) were ineffective as cryoprotectants for MSCs. Studies on ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), membrane stabilization, and intracellular transport of PEGs suggest that low molecular weight PEGs (400 and 600 Da) exhibit excellent intracellular transport properties, and thus the internalized PEGs during preincubation contribute to the cryoprotection. Intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) worked by extracellular PEGs through IRI, INI, as well as partly internalized PEGs. High molecular weight PEGs (10 K and 20 K Da) killed the cells during preincubation and were ineffective as cryoprotectants. Conclusions PEGs can be used as cryoprotectants. However, the detailed procedures, including preincubation, should consider the effect of the molecular weight of PEGs. The recovered cells well proliferated and underwent osteo/chondro/adipogenic differentiation similar to the MSCs recovered from the traditional DMSO 10% system. Graphical Abstract

BACKGROUND: Dimethyl sulphoxide (DMSO, a small strongly polarized amphiphilic molecule) is used as a cryoprotector due to its possibility to easily penetrate cell membranes and to form strong H-bonds with water molecules, preventing by that arising of aqueous crystals. AIMS: We have investigated the vitality of hematopoietic stem cells after incubation at +4°C, +20°C and +37°C for 30 min, 60 min and 24 h in cryoprotective solution with 5% DMSO, and also after deep-freezing at –80°C and subsequent storage for 24 hours at –20°C and +4°C. MATERIALS AND METHODS: The vitality was evaluated counting the number of alive (uncoloured) and dead stem cells (blue coloured) using a staining test with Trypan blue dye. RESULTS: The obtained results disclose that DMSO has a cytotoxic effect which depends on the temperature and the time of cultivation: the cytotoxicity is insignificant at +4°C even for 24 hours; at +37°C about 90% cells remain vital for 1 hour, however after 24 hours they completely die out. CONCLUSION: Comparing the non-frozen cells with the deep-frozen at –80°C and subsequently defrozen cells in identical conditions (24 hours storage at +4°C) reveals that the freezing process causes an additional delayed damaging effect, which leads to death of half of the stem cells..

Polymeric micelles are promising carriers for the delivery of poorly water-soluble drugs, providing enhanced drug solubility, blood circulation times, and bioavailability. Nevertheless, the storage and long-term stability of micelles in solution present challenges requiring the lyophilization and storage of formulations in the solid state, with reconstitution immediately prior to application. Therefore, it is important to understand the effects of lyophilization/reconstitution on micelles, particularly their drug-loaded counterparts. Herein, we investigated the use of β-cyclodextrin (β-CD) as a cryoprotectant for the lyophilization/reconstitution of a library of poly(ethylene glycol-b-ε-caprolactone) (PEG-b-PCL) copolymer micelles and their drug-loaded counterparts, as well as the effect of the physiochemical properties of different drugs (phloretin and gossypol). The critical aggregation concentration (CAC) of the copolymers decreased with increasing weight fraction of the PCL block (fPCL), plateauing at ~1 mg/L when the fPCL was >0.45. The blank (empty) and drug-loaded micelles were lyophilized/reconstituted in the absence and presence of β-CD (9% w/w) and analyzed via dynamic light scattering (DLS) and synchrotron small-angle X-ray scattering (SAXS) to assess for changes in aggregate size (hydrodynamic diameter, Dh) and morphology, respectively. Regardless of the PEG-b-PCL copolymer or the use of β-CD, the blank micelles displayed poor redispersibility (<10% relative to the initial concentration), while the fraction that redispersed displayed similar Dh to the as-prepared micelles, increasing in Dh as the fPCL of the PEG-b-PCL copolymer increased. While most blank micelles displayed discrete morphologies, the addition of β-CD or lyophilization/reconstitution generally resulted in the formation of poorly defined aggregates. Similar results were also obtained for drug-loaded micelles, with the exception of several that retained their primary morphology following lyophilization/reconstitution, although no obvious trends were noted between the microstructure of the copolymers or the physicochemical properties of the drugs and their successful redispersion. [ABSTRACT FROM AUTHOR]

The wood frog, Rana sylvatica (aka Lithobates sylvaticus) is the main model for studies of natural freeze tolerance among amphibians living in seasonally cold climates. During freezing, ∼65% of total body water can be converted to extracellular ice and this imposes both dehydration and hypoxia/anoxia stresses on cells. The current study analyzed the responses of the alpha subunit of the hypoxia-inducible transcription factor (HIF-1), a crucial oxygen-sensitive regulator of gene expression, to freezing, anoxia or dehydration stresses, examining six tissues of wood frogs (liver, skeletal muscle, brain, heart, kidney, skin). RT-PCR revealed a rapid elevation hif-1α transcript levels within 2 h of freeze initiation in both liver and brain and elevated levels of both mRNA and protein in liver and muscle after 24 h frozen. However, both transcript and protein levels reverted to control values after thawing except for HIF-1 protein in liver that dropped to ∼60% of control. Independent exposures of wood frogs to anoxia or dehydration stresses (two components of freezing) also triggered upregulation of hif-1α transcripts and/or HIF-1α protein in liver and kidney with variable responses in other tissues. The results show active modulation of HIF-1 in response to freezing, anoxia and dehydration stresses and implicate this transcription factor as a contributor to the regulation of metabolic adaptations needed for long term survival of wood frogs in the ischemic frozen state. [ABSTRACT FROM AUTHOR]

Cryosurgery is a minimally invasive approach to the treatment of focal prostate cancer (PCa). A major complication is the cryoinjury to the cavernous nerve in the neurovascular bundle (NVB). This nerve cryoinjury halts conduction of action potentials (APs) and can eventually result in erectile dysfunction and therefore diminished quality of life for the patient. Here, we propose the application of cryoprotective agents (CPA) to the regions of the nerves in the NVB, prior to prostate cryosurgery, to minimize non-recoverable loss of AP conduction. We modeled a cryosurgical procedure based on data taken during a clinical case and applied ex-vivo porcine phrenic nerves and rat sciatic nerve with temperature profile of NVB. The APs were measured before and after the CPA exposures and during 3 h of recovery. Comparisons of AP amplitude recovery with various CPA compositions reveal that certain CPAs (e.g., 5% DMSO + 7.5% Trehalose and 5% M22 for porcine and rat nerves, respectively) showed little or no toxicity and effective cryoprotection from freezing (on average 48% and 30% of recovered AP, respectively). In summary, we demonstrate that neural conduction can be preserved after exposure to freezing conditions if CPAs are properly selected and deployed onto the nerve. [ABSTRACT FROM AUTHOR]

Oncolytic viruses are highly promising for cancer treatment because they target and lyse tumor cells. These genetically engineered vectors introduce therapeutic or immunostimulatory genes into the tumor. However, viral therapy is not always safe and effective. Several problems are related to oncolytic viruses' targeted delivery to the tumor and immune system neutralization in the bloodstream. Cryoprotection and preventing viral particles from aggregating during storage are other critical issues. Aptamers, short RNA, or DNA oligonucleotides may help to crawl through this bottleneck. They are not immunogenic, are easily synthesized, can be chemically modified, and are not very demanding in storage conditions. It is possible to select an aptamer that specifically binds to any target cell, oncolytic virus, or molecule using the SELEX technology. This review comprehensively highlights the most important research and methodological approaches related to oncolytic viruses and nucleic acid aptamers. Here, we also analyze possible future research directions for combining these two methodologies to improve the effectiveness of cancer virotherapy. [ABSTRACT FROM AUTHOR]

Dehydrins are hydrophilic stress-induced proteins that are thought to protect cellular machinery from the adverse effect of dehydration caused by low temperature, drought, or salinity. In the previous study, acidic FSK 3 dehydrin DHN24 from Solanum sogarandinum was found to accumulate at multiple sites in phloem cells in response to cold treatment. This study investigated the biochemical and structural properties of recombinant DHN24. It was shown that the overexpression of DHN24 in Escherichia coli led to the inhibition of bacterial growth. The purified DHN24 was found to protect lactate dehydrogenase from freeze-induced denaturation. Circular dichroism (CD) analysis showed that DHN24 was disordered in aqueous solutions, but adopted α-helical conformation in a membrane-mimetic environment using sodium dodecyl sulfate micelles. DHN24 also interacted with anionic phosphatidic acid (PA). DHN24 contains four lysine-rich regions including three K-segments and a region upstream of the S-segment. The role of their local cationic nature is unknown. These segments are predicted to form helical structures. The CD analysis of mutant proteins in the membrane-mimetic environment matched these predictions most closely, revealing that the positively charged lysine residues in these regions promoted disorder-to-order transitions. Moreover, the inhibition of bacterial growth and interactions with PA were regulated by the local cationic nature of DHN24, while no such regulation was observed for its cryoprotective activity. The importance of the positive charge of the lysine-rich segments and disordered structure for DHN24 activity is discussed in relation to its possible biological function. • Cryoprotective activity of disordered DHN24 is independent of local positive charges. • Helicity gain of DHN24 is promoted by the cationic nature of lysine-rich segments. • Interactions with phosphatidic acid and bacterial growth inhibition are modulated by the local cationic nature of DHN24. [ABSTRACT FROM AUTHOR]

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A synergistic approach using cultivation methods, chemical, and bioinformatic analyses was applied to explore the potential of Pseudoalteromonas sp. S8-8 in the production of extracellular polymeric substances (EPSs) and the possible physiological traits related to heavy metal and/or antibiotic resistance. The effects of different parameters (carbon source, carbon source concentration, temperature, pH and NaCl supplement) were tested to ensure the optimization of growth conditions for EPS production by the strain S8-8. The highest yield of EPS was obtained during growth in culture medium supplemented with glucose (final concentration 2%) and NaCl (final concentration 3%), at 15 °C and pH 7. The EPS was mainly composed of carbohydrates (35%), followed by proteins and uronic acids (2.5 and 2.77%, respectively) and showed a monosaccharidic composition of glucose: mannose: galactosamine: galactose in the relative molar proportions of 1:0.7:0.5:0.4, as showed by the HPAE-PAD analysis. The detection of specific molecular groups (sulfates and uronic acid content) supported the interesting properties of EPSs, i.e. the emulsifying and cryoprotective action, heavy metal chelation, with interesting implication in bioremediation and biomedical fields. The analysis of the genome allowed to identify a cluster of genes involved in cellulose biosynthesis, and two additional gene clusters putatively involved in EPS biosynthesis. Key points: • A cold-adapted Pseudoalteromonas strain was investigated for EPS production. • The EPS showed emulsifying, cryoprotective, and heavy metal chelation functions. • Three gene clusters putatively involved in EPS biosynthesis were evidenced by genomic insights. [ABSTRACT FROM AUTHOR]

Dehydrins are intrinsically disordered proteins expressed ubiquitously throughout the plant kingdom in response to desiccation. Dehydrins have been found to have a cryoprotective effect on lactate dehydrogenase (LDH) in vitro, which is in large part influenced by their hydrodynamic radius rather than the order of the amino acids within the sequence (alternatively, this may be a sequence specific effect). However, it seems that a different mechanism may underpin the cryoprotection that they confer to the cold-labile yeast frataxin homolog-1 (Yfh1). Circular dichroism spectroscopy (CD) was used to assess the degree of helicity of Yfh1 at 1 °C, both alone and in the presence of several dehydrin constructs. Three constructs were compared to the wild type: YSK2-K→R (lysine residues substituted with arginine), YSK2-Neutral (locally neutralized charge), and YSK2-SpaceK (evenly distributed positive charge). The results show that sequence rearrangements and minor substitutions have little impact on the ability of the dehydrin to preserve LDH activity. However, when the positive charge of the dehydrin is locally neutralized or evenly distributed, the dehydrin becomes less efficient at promoting structure in Yfh1 at low temperatures. This suggests that a stabilizing, charge-based interaction occurs between dehydrins and Yfh1. Dehydrins are intrinsically disordered proteins, expressed by certain organisms to improve desiccation tolerance. These proteins are thought to serve many cellular roles, such as the stabilization of membranes, DNA, and proteins. However, the molecular mechanisms underlying the function of dehydrins are not well understood. Here, we examine the importance of positive charges in dehydrin sequences by making substitutions and comparing their effects in the cryoprotection of two different proteins. [ABSTRACT FROM AUTHOR]

Late embryogenesis abundant (LEA) proteins are hydrophilic proteins that lack a well-ordered tertiary structure and accumulate to high levels in response to water deficit, in organisms such as plants, fungi, and bacteria. The mechanisms proposed to protect cellular structures and enzymes are water replacement, ion sequestering, and membrane stabilization. The activity of some proteins has a limited shelf-life due to instability that can be caused by their structure or the presence of a stress condition that limits their activity; several LEA proteins have been shown to behave as cryoprotectants in vitro. Here, we report a group1 LEA from Azotobacter vinelandii AvLEA1, capable of conferring protection to lactate dehydrogenase, catechol dioxygenase, and Baylase peroxidase against freeze-thaw treatments, desiccation, and oxidative damage, making AvLEA a promising biological stabilizer reagent. This is the first evidence of protection provided by this LEA on enzymes with biotechnological potential, such as dioxygenase and peroxidase under in vitro stress conditions. Our results suggest that AvLEA could act as a molecular chaperone, or a "molecular shield," preventing either dissociation or antiaggregation, or as a radical scavenger, thus preventing damage to these target enzymes during induced stress. Key points: • This work expands the basic knowledge of the less-known bacterial LEA proteins and their in vitro protection potential. • AvLEA is a bacterial protein that confers in vitro protection to three enzymes with different characteristics and oligomeric arrangement. • The use of AvLEA as a stabilizer agent could be further explored using dioxygenase and peroxidase in bioremediation treatments. AvLEA1 protects against freeze-thaw treatments, desiccation, and oxidative damage on three different enzymes with biotechnological potential. [ABSTRACT FROM AUTHOR]

The development of serum-/xeno-free media may help avoid the drawbacks of using serum and its components, such as probable contamination, instability of composition, or difficulty in sterilization. The objectives of this research were to investigate the use of combinations of a permeating cryoprotective agent (Me 2 SO) and non-permeating polymers (polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, hydroxyethyl starch, dextran) for cryopreservation of interstitial cells (ICs) of rat testis, and to propose the mechanism of cryoprotection of such compositions. In the course of this study, the best combination was 100 mg/ml dextran (M.m. 40 kDa) (Dex40) with 0.7 M Me 2 SO in Ham's F12. The ICs were additionally cooled and warmed to different end temperatures (−30, −50, −50 and −196 °C) to determine which temperature intervals contributed most to the IC loss. Then, the cryoprotective action of this serum-/xeno-free medium was investigated in comparison with serum or albumin-containing media by differential scanning calorimetry (DSC) and thermomechanical analysis (TMA). The results showed that the medium based on Dex40 did not decrease the amount of ice formed. However, it could undergo other phase separation and phase transformation to form glassy states. Potential cell-damaging physical processes such as eutectic crystallization/melting, recrystallization of NaCl and/or Me 2 SO derivatives, found in serum-containing media and taking place in specific temperature intervals, were not observed in the Dex40 based media. This was in good correlation with indicators of cell survival. Additionally, the application of Dex40 allowed using Me 2 SO in lower concentrations (0.7 M) than required for serum-containing media (1.4 M), which may decrease the toxicity of serum-/xeno-free media. • This study examined the effect of combinations of Me 2 SO and polymers on testis interstitial cell cryopreservation. • The best combination was 100 mg/ml dextran 40 with 0.7 M Me 2 SO in Ham's F12. • Physical processes in the medium were investigated by differential scanning calorimetry and thermomechanical analysis. • The mechanisms of cryoprotection of this serum-/xeno-free medium based on dextran 40 were proposed. [ABSTRACT FROM AUTHOR]

Polymeric micelles are promising carriers for the delivery of poorly water-soluble drugs, providing enhanced drug solubility, blood circulation times, and bioavailability. Nevertheless, the storage and long-term stability of micelles in solution present challenges requiring the lyophilization and storage of formulations in the solid state, with reconstitution immediately prior to application. Therefore, it is important to understand the effects of lyophilization/reconstitution on micelles, particularly their drug-loaded counterparts. Herein, we investigated the use of β-cyclodextrin (β-CD) as a cryoprotectant for the lyophilization/reconstitution of a library of poly(ethylene glycol-b-ε-caprolactone) (PEG-b-PCL) copolymer micelles and their drug-loaded counterparts, as well as the effect of the physiochemical properties of different drugs (phloretin and gossypol). The critical aggregation concentration (CAC) of the copolymers decreased with increasing weight fraction of the PCL block (fPCL), plateauing at ~1 mg/L when the fPCL was >0.45. The blank (empty) and drug-loaded micelles were lyophilized/reconstituted in the absence and presence of β-CD (9% w/w) and analyzed via dynamic light scattering (DLS) and synchrotron small-angle X-ray scattering (SAXS) to assess for changes in aggregate size (hydrodynamic diameter, Dh) and morphology, respectively. Regardless of the PEG-b-PCL copolymer or the use of β-CD, the blank micelles displayed poor redispersibility (Dh to the as-prepared micelles, increasing in Dh as the fPCL of the PEG-b-PCL copolymer increased. While most blank micelles displayed discrete morphologies, the addition of β-CD or lyophilization/reconstitution generally resulted in the formation of poorly defined aggregates. Similar results were also obtained for drug-loaded micelles, with the exception of several that retained their primary morphology following lyophilization/reconstitution, although no obvious trends were noted between the microstructure of the copolymers or the physicochemical properties of the drugs and their successful redispersion.

In X‐ray macromolecular crystallography, cryoprotection of crystals mounted on harvesting loops is achieved when the water in the sample solvent transitions to vitreous ice before crystalline ice forms. This is achieved by rapid cooling in liquid nitrogen or propane. Protocols for protein crystal cryoprotection are based on either increasing the environmental pressure or reducing the water fraction in the solvent. This study presents a new protocol for cryoprotecting crystals. It is based on vapour diffusion dehydration of the crystal drop to reduce the water fraction in the solvent by adding a highly concentrated salt solution, 13 M potassium formate (KF13), directly to the reservoir. Several salt solutions were screened to identify KF13 as optimal. Cryoprotection using the KF13 protocol is non‐invasive to the crystal, high throughput and easy to implement, can benefit diffraction resolution and ligand binding, and is very useful in cases with high redundancy such as drug‐discovery projects which use very large compound or fragment libraries. An application of KF13 to discover new crystal hits from clear drops of equilibrated crystallization screening plates is also shown. [ABSTRACT FROM AUTHOR]

The goal of this study was to determine effects of trehalose (T) and Trolox (TX) additives on post-thaw viability of canine sperm after freezing. The sperm-rich portions of ejaculates obtained from 15 dogs were divided into three aliquots, which were diluted with Tris-based extender (TBE) (control), TBE + T (25 mM), and TBE + TX (1 mM) and put into straws. The samples were equilibrated at 4 °C for 1.5 hours, then frozen in liquid nitrogen vapour for 15 minutes and plunged into liquid nitrogen. Plasma membrane and acrosome integrity (PMAI) and high mitochondrial membrane potential (HMMP) were evaluated after thawing the straws in a water bath at 37 °C for 30 seconds. Motility, percentage of live spermatozoa, and hypo-osmotic swelling (HOS) were determined using phase-contrast microscopy. Motility (53.00 ± 6.46%), percentage of live spermatozoa (62.97% ± 6.84%), and HOS (40.40 ± 7.11%) of T were significantly higher (P <0.05) than the control (40.50 ± 4.76%, 50.63% ± 4.78%, and 30.47% ± 5.59%) and TX (40.50 ± 4.76%, 50.96 ± 5.84%, and 33.02% ± 8.77%) respectively. The PMAI and HMMP levels were higher (P <0.05) in T (35.51±8.72 and 24.36±7.15%) than in the control (19.18 ± 3.49 and 12.04 ± 3.83%). The TX group (35.62 ± 8.21%) had significantly higher PMAI (P <0.05) than the control (12.04 ± 3.83%). Thus, addition of T protected canine spermatozoa against cryogenic injury. Nonetheless, additional dose-response trials are recommended to determine the effects of Trolox on the quality of canine semen more accurately. [ABSTRACT FROM AUTHOR]

The survival of two strains of probiotic lactic acid bacteria Lactobacillus plantarum subsp. plantarum NBIMCC 3447 and Lactobacillus gasseri NBIMCC 2450 included in functional products based on hydrolyzed cow's milk and whey has been studied. Two types of cryoprotective medium were used − a first option combination of 3% sucrose and 2% pectin and a second option 0.2% locust bean gum solution. A comparative analysis of the number of viable cells (cfu/ml) after freezing and freeze-drying was performed. The results show that the import of cryoprotective medium to microorganisms in the fermentation medium has a positive effect and preserves cell survival after freezing and lyophilisation up to 95%, compared to control samples. It has been found that the solution of sucrose and pectin exhibits a better cryoprotective effect in milk environment, while in an environment with hydrolysed milk proteins Locust bean gum contributes to the preservation of a higher number of viable cells both after freezing and after lyophilisation of products. [ABSTRACT FROM AUTHOR]

The survival of the microflora of kefir from goat's milk was studied after freezing and freeze-drying with included natural biologically active components - fructooligosaccharides and gum locus bean, in their capacity of cryoprotective media. The effect of each cryoprotectant has been studied independently and in combination between them. To achieve the maximum number of vital and active cells after freeze-drying, the optimal process parameters were established by applying differential thermal analysis (DTA). The results show that the inclusion of kefir microorganisms in a protective medium leads to a higher survival after freezing and lyophilization, compared to the sample without cryprotector. Yeasts, followed by streptococci, have the highest resistance, and lactobacilli are the most sensitive to the process. The combination of 5% fructooligosaccharides with 1% gum locus bean can be considered optimal in terms of the cryoprotective effect on the main groups of microorganisms in kefir. Achieved high levels of survival allow the creation of lyophilized foods rich in viable synbiotic microflora. [ABSTRACT FROM AUTHOR]

Background Intrinsically disordered proteins (IDPs) have been shown to exhibit cryoprotective activity toward other cellular enzymes without any obvious conserved sequence motifs. This study investigated relationships between the physical properties of several human genome-derived IDPs and their cryoprotective activities. Methods Cryoprotective activity of three human-genome derived IDPs and their truncated peptides toward lactate dehydrogenase (LDH) and glutathione S-transferase (GST) was examined. After the shortest cryoprotective peptide was defined (named FK20), cryoprotective activity of all-D-enantiomeric isoform of FK20 (FK20-D) as well as a racemic mixture of FK20 and FK20-D was examined. In order to examine the lack of increase of thermal stability of the target enzyme, the CD spectra of GST and LDH in the presence of a racemic mixture of FK20 and FK20-D at varying temperatures were measured and used to estimate Tm. Results Cryoprotective activity of IDPs longer than 20 amino acids was nearly independent of the amino acid length. The shortest IDP-derived 20 amino acid length peptide with sufficient cryoprotective activity was developed from a series of TNFRSF11B fragments (named FK20). FK20, FK20-D, and an equimolar mixture of FK20 and FK20-D also showed similar cryoprotective activity toward LDH and GST. Tm of GST in the presence and absence of an equimolar mixture of FK20 and FK20-D are similar, suggesting that IDPs’ cryoprotection mechanism seems partly from a molecular shielding effect rather than a direct interaction with the target enzymes.

Oncolytic viruses are highly promising for cancer treatment because they target and lyse tumor cells. These genetically engineered vectors introduce therapeutic or immunostimulatory genes into the tumor. However, viral therapy is not always safe and effective. Several problems are related to oncolytic viruses’ targeted delivery to the tumor and immune system neutralization in the bloodstream. Cryoprotection and preventing viral particles from aggregating during storage are other critical issues. Aptamers, short RNA, or DNA oligonucleotides may help to crawl through this bottleneck. They are not immunogenic, are easily synthesized, can be chemically modified, and are not very demanding in storage conditions. It is possible to select an aptamer that specifically binds to any target cell, oncolytic virus, or molecule using the SELEX technology. This review comprehensively highlights the most important research and methodological approaches related to oncolytic viruses and nucleic acid aptamers. Here, we also analyze possible future research directions for combining these two methodologies to improve the effectiveness of cancer virotherapy.

Dehydrins are intrinsically disordered proteins expressed ubiquitously throughout the plant kingdom in response to desiccation. Dehydrins have been found to have a cryoprotective effect on lactate dehydrogenase (LDH) in vitro, which is in large part influenced by their hydrodynamic radius rather than the order of the amino acids within the sequence (alternatively, this may be a sequence specific effect). However, it seems that a different mechanism may underpin the cryoprotection that they confer to the cold-labile yeast frataxin homolog-1 (Yfh1). Circular dichroism spectroscopy (CD) was used to assess the degree of helicity of Yfh1 at 1 °C, both alone and in the presence of several dehydrin constructs. Three constructs were compared to the wild type: YSK2-K→R (lysine residues substituted with arginine), YSK2-Neutral (locally neutralized charge), and YSK2-SpaceK (evenly distributed positive charge). The results show that sequence rearrangements and minor substitutions have little impact on the ability of the dehydrin to preserve LDH activity. However, when the positive charge of the dehydrin is locally neutralized or evenly distributed, the dehydrin becomes less efficient at promoting structure in Yfh1 at low temperatures. This suggests that a stabilizing, charge-based interaction occurs between dehydrins and Yfh1. Dehydrins are intrinsically disordered proteins, expressed by certain organisms to improve desiccation tolerance. These proteins are thought to serve many cellular roles, such as the stabilization of membranes, DNA, and proteins. However, the molecular mechanisms underlying the function of dehydrins are not well understood. Here, we examine the importance of positive charges in dehydrin sequences by making substitutions and comparing their effects in the cryoprotection of two different proteins.

A soluble protein fraction from radish (Raphanus sativus L.) taproot had cryoprotective activity for lactate dehydrogenase (LDH). The activity was found mainly in the heat-stable fractions of soluble proteins. The cryoprotective protein, whose molecular mass was 43 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis, was obtained by successive chromatographies on TOYOPEARL SuperQ and TOYOPEARL DEAE. MALDI-TOF MS/MS analysis indicated that the purified protein was a radish vacuolar calcium-binding protein (RVCaB), which is reportedly related to calcium storage in the vacuoles of radish taproot. The purified RVCaB inhibited the cryoinactivation, cryodenaturation, and cryoaggregation of LDH. RVCaB had greater cryoprotective activity than general cryoprotectants. When RVCaB was divided into 15 segments (Seg01 to Seg15, 15 amino acids each), Seg03, which had a high hydrophobicity scale, showed remarkable cryoprotective activity. This indicated that RVCaB protected LDH from freezing and thawing damage presumably through a specific hydrophobic area (i.e., Seg03). • A protein with cryoprotective activity was purified from radish. • The cryoprotective protein was identified as a vacuolar calcium-binding protein. • The protein's hydrophobic area was involved in its cryoprotective activity. [ABSTRACT FROM AUTHOR]

Biological cryopreservation often involves using a cryoprotective agent (CPA) to mitigate lethal physical stressors cells endure during freezing and thawing, but effective CPA concentrations are cytotoxic. Hence, natural polysaccharides have been studied as biocompatible alternatives. Here, a subset of 26 natural polysaccharides of various chemical composition was probed for their potential in enhancing the metabolic post-thaw viability (PTV) of cryopreserved Vero cells. The best performing cryoprotective polysaccharides contained significant fucose amounts, resulting in average PTV 2.8-fold (up to 3.1-fold) compared to 0.8-fold and 2.2-fold for all non-cryoprotective and cryoprotective polysaccharides, respectively, outperforming the optimized commercial CryoStor™ CS5 formulation (2.6-fold). Stoichiometrically, a balance between fucose (18–35.7 mol%), uronic acids (UA) (13.5–26 mol%) and high molecular weight (MW > 1 MDa) generated optimal PTV. Principal component analysis (PCA) revealed that fucose enhances cell survival by a charge-independent, MW-scaling mechanism (PC1), drastically different from the charge-dominated ice growth disruption of UA (PC2). Its neutral nature and unique properties distinguishable from other neutral monomers suggest fucose may play a passive role in conformational adaptability of polysaccharide to ice growth inhibition, or an active role in cell membrane stabilization through binding. Ultimately, fucose-rich anionic polysaccharides may indulge in polymer-ice and polymer-cell interactions that actively disrupt ice and minimize lethal volumetric fluctuations due to a balanced hydrophobic-hydrophilic character. Our research showed the critical role neutral fucose plays in enhancing cellular cryopreservation outcomes, disputing previous assumptions of polyanionicity being the sole governing predictor of cryoprotection. [ABSTRACT FROM AUTHOR]

The eastern spruce budworm, Choristoneura fumiferana, is one of North America's most destructive forest insects. It survives the harsh winters by deploying both a sophisticated diapause program and a complex suite of cryoprotective molecules. The spruce budworm's cryoprotective biochemistry could revolutionize organ storage and transplants. Here we review the latest in C. fumiferana overwintering physiology and identify emerging theoretical and practical questions that are open for exploration. [ABSTRACT FROM AUTHOR]

Ice recrystallization inhibitors have emerged as novel cryoprotectants to improve cell viability for cryopreservation. Nanocelluloses were identified as new materials for ice recrystallization inhibition (IRI); however, conventional nanocelluloses aggregate and lose IRI activity at high ionic strengths, which limit their application as cryoprotectants. In this study, we synthesized a novel group of nanocelluloses - electrosterically stabilized cellulose nanocrystals (ECNCs), which remained dispersed and IRI-active at high ionic strengths. ECNCs improved the post-thaw viability of HCT-116 colorectal cancer cells in slow/fast freezing-slow thawing protocols in the presence of 1–20% v/v dimethyl sulfoxide (DMSO), as well as in slow/fast freezing-fast thawing protocols at reduced DMSO concentrations. The effectiveness in cryoprotection did not match the IRI activity in ECNCs, polyethylene glycol (PEG), and polyvinyl alcohol (PVA); and in ECNCs with different surface charge densities. Overall, ECNCs demonstrated IRI and cryoprotection activities, but the mechanism of cryoprotection remains unknown. • Electrosterically stabilized cellulose nanocrystals (ECNCs) were synthesized. • ECNCs remained dispersed at high ionic strengths. • ECNCs had ice recrystallization inhibition (IRI) activity at high ionic strengths. • ECNCs improved the post-thaw viability of HCT-116 cells in the presence of DMSO. • The cryoprotection effectiveness of ECNCs did not match with their IRI activity. [ABSTRACT FROM AUTHOR]

Several countries have established self-help cryonics groups whose mission is to cryopreserve human bodies or brains after legal death and ship them to cryonics organizations. The objective of this study was to report the first case of human brain cryopreservation in Argentina and complementary experiments in rats. After legal death, the body of a 78-year-old Caucasian woman was transported to a funeral home where her head was submitted to intracarotid perfusion with 5 L cold physiologic saline followed by the same volume of cold saline containing 13% dimethyl sulfoxide and 13% glycerol. The brain was removed, temporarily frozen at -80°C, and shipped to a U.S. cryostasis facility. Three groups of rats were intracardially perfused with fixative but not frozen (Reference group), vitrification solution VM1 (Control group), or the cryoprotection solution used in the patient (Experimental group). Control and Experimental brains were stored at -80°C and subsequently assessed by immunohistochemistry for the adult neuron marker (NeuN), the immature neuron marker doublecortin (DCX), the dopaminergic neuron marker tyrosine hydroxylase, and the presynaptic marker synaptophysin (SYN). The number of NeuN-positive neurons remained unchanged in the experimental brain cortex, whereas the number of immature DCX neurons in the hippocampus fell markedly in the cryoprotected brains. The results were highly variable for hypothalamic dopaminergic neurons. Confocal microscopy for SYN revealed that cryopreservation did not affect the synaptic network in the hippocampus. To our knowledge, this is the first report correlating a human cryoprotection procedure with results in complementary experiments in laboratory animals. [ABSTRACT FROM AUTHOR]

Sea-ice algae are an important source of primary production in polar regions, yet we have limited understanding of their responses to the seasonal cycling of temperature and salinity. Using a targeted liquid chromatography-mass spectrometry-based metabolomics approach, we found that axenic cultures of the Antarctic sea-ice diatom, 'Nitzschia lecointei', displayed large differences in their metabolomes when grown in a matrix of conditions that included temperatures of –1 and 4°C, and salinities of 32 and 41, despite relatively small changes in growth rate. Temperature exerted a greater effect than salinity on cellular metabolite pool sizes, though the N- or S-containing compatible solutes, 2, 3-dihydroxypropane-1-sulfonate (DHPS), glycine betaine (GBT), dimethylsulfoniopropionate (DMSP), and proline responded strongly to both temperature and salinity, suggesting complexity in their control. We saw the largest (> 4-fold) response to salinity for proline. DHPS, a rarely studied but potential compatible solute, had the highest intracellular concentrations among all compatible solutes of ~85 mM. When comparing the culture findings to natural Arctic sea-ice diatom communities, we found extensive overlap in metabolite profiles, highlighting the relevance of culture-based studies to probe environmental questions. Large changes in sea-ice diatom metabolomes and compatible solutes over a seasonal cycle could be significant components of biogeochemical cycling within sea ice.

Dehydrins are intrinsically disordered proteins composed of several well conserved sequence motifs known as the Y-, S-, F-, and K-segments, the latter of which is a defining feature of all dehydrins. These segments are interspersed by regions of low sequence conservation and are organized modularly, which results in seven different architectures: Kn, SKn, YnSKn, YnKn, KnS, FnK and FnSKn. Dehydrins are expressed ubiquitously throughout the plant kingdom during periods of low intracellular water content, and are capable of improving desiccation tolerance in plants. In vitro evidence of dehydrins shows that they are involved in the protection of membranes, proteins and DNA from abiotic stresses. However, the molecular mechanisms by which these actions are achieved are as of yet somewhat unclear. With regards to macromolecule cryoprotection, there is evidence to suggest that a molecular shield-like protective effect is primarily influenced by the hydrodynamic radius of the dehydrin and to a lesser extent by the charge and hydrophobicity. The interaction between dehydrins and membranes is thought to be a surface-level, charge-based interaction that may help to lower the transition temperature, allowing membranes to maintain fluidity at low temperatures and preventing membrane fusion. In addition, dehydrins are able to protect DNA from damage, showing that these abiotic stress protection proteins have multiple roles.

ABSTRACT: This study evaluated the effect of vitrification solutions and exposure time on the cryopreservation of Brazilian green dwarf coconut plumules (BGD) using the droplet vitrification technique. Explants were excised from BGD mature fruits from the Active Germplasm Bank of Embrapa Tabuleiros Costeiros, Sergipe, Brazil. Firstly, embryos were disinfected, and after excision, plumules were pre-cultivated for 72 hours in Y3 + 0.6 M sucrose + 2.2 g L-1 Gelrite® culture medium. Plumules were exposed to PVS2 and PVS3 solutions for 15 and 30 minutes and rapidly immersed in liquid nitrogen (-196 ºC). After cryopreservation, they were thawed in culture medium solution (Y3 + 1.2 M sucrose) and cultured in regeneration medium. The experimental design was completely randomized in a 2x2 factorial scheme (vitrification solutions per exposure times), with five replicates per treatment. Data were compared by the Tukey’s test at 5% probability. Significant differences were observed in the callogenesis percentage for the solutions x exposure time interaction for non-cryopreserved cultures (-NL) and for exposure time after cryopreservation (+NL). PVS2 and PVS3 combined with 15 minutes of exposure promoted the highest callus formation (70 and 100%, respectively) in control cultures. The exposure time of 30 min, regardless of vitrification solution, resulted in 30% embryogenic callus formation after cryopreservation. These results contributed to the long-term conservation of coconut palm.

This study evaluated the effect of vitrification solutions and exposure time on the cryopreservation of Brazilian green dwarf coconut plumules (BGD) using the droplet vitrification technique. Explants were excised from BGD mature fruits from the Active Germplasm Bank of Embrapa Tabuleiros Costeiros, Sergipe, Brazil. Firstly, embryos were disinfected, and after excision, plumules were pre-cultivated for 72 hours in Y3 + 0.6 M sucrose + 2.2 g L-1 Gelrite® culture medium. Plumules were exposed to PVS2 and PVS3 solutions for 15 and 30 minutes and rapidly immersed in liquid nitrogen (-196 °C). After cryopreservation, they were thawed in culture medium solution (Y3 + 1.2 Msucrose) and cultured in regeneration medium. The experimental design was completely randomized in a 2x2 factorial scheme (vitrification solutions per exposure times), with five replicates per treatment. Data were compared by the Tukey 's test at 5% probability. Significant differences were observed in the callogenesis percentage for the solutions x exposure time interaction for non-cryopreserved cultures (-NL) and for exposure time after cryopreservation (+NL). PVS2 and PVS3 combined with 15 minutes of exposure promoted the highest callus formation (70 and 100%, respectively) in control cultures. The exposure time of 30 min, regardless of vitrification solution, resulted in 30% embryogenic callus formation after cryopreservation. These results contributed to the long-term conservation of coconut palm. [ABSTRACT FROM AUTHOR]

Carbohydrates are a class of macromolecules that has significant potential across several domains, including the organisation of genetic material, provision of structural support, and facilitation of defence mechanisms against invasion. Their molecular diversity enables a vast array of essential functions, such as energy storage, immunological signalling, and the modification of food texture and consistency. Due to their rheological characteristics, solubility, sweetness, hygroscopicity, ability to prevent crystallization, flavour encapsulation, and coating capabilities, carbohydrates are useful in food products. Carbohydrates hold potential for the future of therapeutic development due to their important role in sustained drug release, drug targeting, immune antigens, and adjuvants. Bio-based packaging provides an emerging phase of materials that offer biodegradability and biocompatibility, serving as a substitute for traditional non-biodegradable polymers used as coatings on paper. Blending polyhydroxyalkanoates (PHA) with carbohydrate biopolymers, such as starch, cellulose, polylactic acid, etc., reduces the undesirable qualities of PHA, such as crystallinity and brittleness, and enhances the PHA's properties in addition to minimizing manufacturing costs. Carbohydrate-based biopolymeric nanoparticles are a viable and cost-effective way to boost agricultural yields, which is crucial for the increasing global population. The use of biopolymeric nanoparticles derived from carbohydrates is a potential and economically viable approach to enhance the quality and quantity of agricultural harvests, which is of utmost importance given the developing global population. The carbohydrate biopolymers may play in plant protection against pathogenic fungi by inhibiting spore germination and mycelial growth, may act as effective elicitors inducing the plant immune system to cope with pathogens. Furthermore, they can be utilised as carriers in controlled-release formulations of agrochemicals or other active ingredients, offering an alternative approach to conventional fungicides. It is expected that this review provides an extensive summary of the application of carbohydrates in the realms of food, pharmaceuticals, and environment. [ABSTRACT FROM AUTHOR]

Polysaccharides are becoming potential candidates for developing food-grade cryoprotectants due to their extensive accessibility and health-promoting effects. However, unremarkable ice recrystallization inhibition (IRI) activity and high viscosity limit their practical applications in some systems. Our previous study found a galactoxyloglucan polysaccharide from tamarind seed (TSP) showing moderate IRI activity. Herein, the enhancement of the IRI performance of TSP via enzymatic depolymerization and degalactosylation-induced self-assembly was reported. TSP was depolymerized and subsequently removed ∼40 % Gal, which induced the formation of supramolecular rod-like fiber self-assembles and exhibited a severalfold enhancement of IRI. Ice shaping assay did not show obvious faceting of ice crystals, indicating that both depolymerized and self-assembled TSP showed very weak binding to ice. Molecular dynamics simulation confirmed the absence of molecular complementarity with ice. Further, it highlighted that degalactosylation did not cause significant changes in local hydration properties of TSP from the view of a single oligomer. The inconsistency between molecular simulation and macroscopic IRI effect proposed that the formation of unique supramolecular self-assemblies may be a key requirement for enhancing IRI activity. The findings of this study provided a new opportunity to enhance the applied potential of natural polysaccharides in food cryoprotection. [Display omitted] • Depolymerization and degalactosylation of tamarind seed polysaccharide (TSP) • Supramolecular rod-like self-assemblies of modified TSP • A enhancement of ice recrystallization inhibition activity of self-assembled TSP • Inconsistency between molecular dynamics simulation and macroscopic observation • Size/or architecture-related effects on ice recrystallization inhibition activity [ABSTRACT FROM AUTHOR]

Previously we found that the soluble fraction of radish (Raphanus sativus L.) taproots contained a large amount of an intrinsically disordered protein, which we identified as radish vacuolar calcium binding protein (RVCaB). Although RVCaB is known to inhibit cryo-damage to freeze-sensitive enzyme lactate dehydrogenase, there is no information on its cryoprotection of other types of macromolecules, such as biomembranes. In this study we found that RVCaB inhibited the cryoaggregation of phospholipid liposomes to a greater extent than common cryoprotectants, such as trehalose, glycine betaine, proline, bovine serum albumin (BSA), and polyethylene glycol (PEG). RVCaB inhibited the freeze/thaw-induced membrane fusion of liposomes. It became structurally disordered in solution, and this disorder was maintained in the presence of liposomes. Finally, the mechanism underlying RVCaB's inhibition of liposome cryoaggregation was discussed. • RVCaB is a major soluble protein in radish taproots. • RVCaB inhibited liposome cryoaggregation more efficiently than general protectants. • RVCaB prevented cryo-induced membrane fusion of liposomes. • The structural disorder of RVCaB may enhance its cryoprotection of liposomes. [ABSTRACT FROM AUTHOR]

Trehalose is a natural cell preservation agent with the inability to cross the mammalian cellular membrane which limits its preservative action to the extracellular compartment. In order to address this, endocytosis was identified as the cellular mechanism that allowed trehalose entry. The cellular machinery behind endocytosis is complex and required inhibitors to reveal a specific mechanism that facilitates the entry of trehalose. This study, therefore, used human dermal fibroblast cultured and incubated in different trehalose extracellular concentrations and incubation times. The trehalase-based assay was used to calculate the intracellular trehalose and FITC was used to indicate the trehalose entry in a PKH26 membrane-stained fibroblast. Pitstop-2, dynasore and cytochalasin-D were used to inhibit trehalose entry. Cryopreservation used the slow-freezing method using the trehalose as cryomedium. Intracellular trehalose was accumulated in a time and concentration-dependent manner. Pitstop-2, dynasore, and the combination of both inhibitors reduced as much as 60% of the trehalose entry. Cytochalasin-D also reduced the trehalose entry in a dose-dependent manner. Additional pre-treatment incubation time and extracellular trehalose in cryomedium provided increased protection of the cryopreserved fibroblast in the trehalose. The presence of trehalose on both sides of the membrane also improved cellular adhesion. In conclusion, pitstop-2 and dynasore reduced the trehalose entry, indicating a clathrin-mediated mechanism. However, clathrin-independent, dynamin-independent, and actin engagement were also involved in facilitating trehalose entry. [ABSTRACT FROM AUTHOR]

Intrinsically disordered stress proteins have been shown to act as chaperones, protecting proteins from damage caused by stresses such as freezing and thawing. Dehydration proteins (dehydrins) are intrinsically disordered stress proteins that are found in almost all land plants. They consist of a variable number of the short, semi‐conserved, Y‐, S‐, and K‐segments, with longer stretches of poorly conserved sequences in between. Previous studies have provided conflicting views on the details of the dehydrin cryoprotective mechanism of enzymes. Experiments with polyethylene glycol (PEG) have shown that PEG cryoprotective efficiency is the same as dehydrins of the same hydrodynamic radius, suggesting that the protein's disordered and polar nature is important, rather than the specific order of the residues. To further elucidate the mechanism, we created scrambled variants of the wild grape dehydrins K2 and YSK2 and tested their ability to protect lactate dehydrogenase and yeast frataxin homolog‐1 from freeze/thaw damage. The results show that for preventing aggregation, it is the sequence composition and the size of the dehydrin that is the most important factor in protection, while for freeze/thaw damage causing loss of secondary structure, it is the sequence composition that is most significant. [ABSTRACT FROM AUTHOR]

Dryophytes chrysoscelis (formerly Hyla chrysoscelis, Cope's gray treefrog) is a freeze‐tolerant anuran that accumulates glycerol and urea during cold acclimation and freezing. It is hypothesized that glycerol and urea function as cryoprotectants by minimizing osmotically induced cell damage during freezing and thawing, thereby improving the postfreeze viability of red blood cells (RBCs) when frozen in medium containing those solutes. To test this, erythrocytes were obtained from warm (22°C) and cold‐acclimated (4°C) frogs and suspended in 280 mOsM phosphate‐buffered saline (PBS). RBCs were frozen in 280 mOsM, isosmotic/isotonic, PBS, or in PBS made hyperosmotic by addition of 150 mM solutes. Postfreeze viability was determined with a hemolysis assay. Postfreeze viability of cells from warm‐acclimated frogs improved from 18.9 ± 1.3% in PBS to 47.4 ± 5.2% in PBS with urea (p < 0.01). The addition of other solutes (glycerol, glucose, NaCl, or sorbitol) had no effect. RBCs from cold‐acclimated frogs had 45.8 ± 3.4% viability when frozen in 280 mOsM PBS, and this improved to 71.6 ± 8.9% or 71.9 ± 1.6%, respectively, when frozen with glycerol (p < 0.01) or urea (p < 0.001). The viability of RBCs from cold‐acclimated frogs was not different between unfrozen cells 86.7–88.4%) and those frozen with glycerol (71.6 ± 8.9%, p > 0.05) or with urea (71.9 ± 1.6%, p > 0.05). These data suggest that (a) cold acclimation induces cellular changes in RBCs that result in improved postfreeze viability, and (b) glycerol and urea are part of a complex cryoprotectant system in D. chrysoscelis. Cold acclimation of the freeze‐tolerant anuran Dryophytes (Hyla) chrysoscelis induces cellular changes in RBCs that result in improved postfreeze viability that is further enhanced by glycerol and urea as part of a complex cryoprotectant system. [ABSTRACT FROM AUTHOR]

Cold-adapted microorganisms inhabiting permanently low-temperature environments were initially just a biological curiosity but have emerged as rich sources of numerous valuable tools for application in a broad spectrum of innovative technologies. To overcome the multiple challenges inherent to life in their cold habitats, these microorganisms have developed a diverse array of highly sophisticated synergistic adaptations at all levels within their cells: from cell envelope and enzyme adaptation, to cryoprotectant and chaperone production, and novel metabolic capabilities. Basic research has provided valuable insights into how these microorganisms can thrive in their challenging habitat conditions and into the mechanisms of action of the various adaptive features employed, and such insights have served as a foundation for the knowledge-based development of numerous novel biotechnological tools. In this review, we describe the current knowledge of the adaptation strategies of cold-adapted microorganisms and the biotechnological perspectives and commercial tools emerging from this knowledge. Adaptive features and, where possible, applications, in relation to membrane fatty acids, membrane pigments, the cell wall peptidoglycan layer, the lipopolysaccharide component of the outer cell membrane, compatible solutes, antifreeze and ice-nucleating proteins, extracellular polymeric substances, biosurfactants, chaperones, storage materials such as polyhydroxyalkanoates and cyanophycins and metabolic adjustments are presented and discussed. [ABSTRACT FROM AUTHOR]

Marinobacter sp. W1-16 from Antarctic surface seawater was analysed for the production of extracellular polymeric substances (EPSs). Enhancement of the EPS biosynthesis was carried out by evaluating the influences of the carbon source (type and concentration), temperature, pH and salinity. EPS yields varied strongly depending on sugar substrate and temperature, while pH and salinity did not strongly affect levels of EPS production. Marinobacter sp. W1-16 produced the highest quantity of EPSs when growing at 15°C and pH 8, in the presence of 2% glucose and 3% NaCl. The EPS chemical characterization revealed a molecular weight of about 260 kDa. Colorimetric assays determined a higher quantity of carbohydrate than of proteins and uronic acids, as well as the presence of sulphate, in the extracted EPSs. The monosaccharidic composition resulted in Glc:Man:Gal:GalN:GalA:GlcA in relative molar proportions of 1:0.9:0.2:0.1:0.1:0.01. Some biotechnological potentialities (i.e. emulsifying and cryoprotective actions, and heavy metal binding properties) of the EPSs were proved, suggesting possible industrial and bioremediation applications. [ABSTRACT FROM AUTHOR]

The terrestrially hibernating wood frog (Rana sylvatica) is well-known for its iconic freeze tolerance, an overwintering adaptation that has received considerable investigation over the past 35 years. Virtually, all of this research has concerned frogs indigenous to the temperate regions of its broad range within North America. However, recent investigations have shown that frogs of subarctic populations are extremely cold hardy, being capable of surviving freezing for longer periods and at much lower temperatures as compared to conspecifics from temperate regions. Their exceptional freeze tolerance is partly supported by an enhanced cryoprotectant system that uses very high levels of urea and glucose to limit ice formation, regulate metabolism, and protect macromolecules and cellular structures from freezing/thawing stresses. In the weeks before they begin hibernating, northern frogs undertake radical physiological transitions, such as depletion of fat stores and catabolism of muscle protein, that prime the cryoprotectant system by accruing urea and stockpiling glycogen from which glucose is mobilized during freezing. Concentrations of cryoprotectants ultimately achieved in Alaskan frogs when freezing occurs vary among tissues but generally are higher than those of frogs inhabiting milder climates. This review summarizes the molecular, biochemical, and physiological adaptations permitting this northern phenotype to survive the long and harsh winters of the region. [ABSTRACT FROM AUTHOR]

The cryoprotective effects of different amounts of protein hydrolysates prepared from by-products of silver carp using Protamex and Alcalase on surimi that were subjected to six freeze-thaw cycles were investigated. Commercial cryoprotectant (8% w/w 1:1 sucrose-sorbitol blend, SuSo) and control (without cryoprotectant) groups were used for comparison. After six freeze-thaw cycles, the lowest actomyosin extractability, Ca2+-ATPase activity and total sulfhydryl content, along with the highest surface hydrophobicity of actomyosin, were observed in the control group (P < 0.05). On the contrary, the group with addition of 2 g of hydrolysate prepared by Protamex hydrolysis (PH-2) displayed the highest actomyosin extractability, Ca2+-ATPase activity and correspondingly, lowest surface hydrophobicity of actomyosin (P < 0.05). Total sulfhydryl content of actomyosin and textural properties of heat-set surimi gels were similar between samples with PH-2 and those with SuSo (P > 0.05). Differences in molecular weight distribution, total and free amino acid compositions between the hydrolysates prepared by Protamex and Alcalase hydrolysis were possible reasons attributing to their variable cryoprotective effects on freeze-thawed surimi. Results from this study clearly support that hydrolysate prepared by Protamex hydrolysis at an appropriate amount could serve as an effective cryoprotectant without increasing the sweetness of surimi products. Furthermore, our findings suggest that the hydrolysates follow a different cryoprotection mechanism compared to SuSo (sucrose-sorbitol blend). [ABSTRACT FROM AUTHOR]