Synergistic effect of residual sugar on freeze-thaw stability of high internal phase emulsions using glycosylated cod protein as interface stabilizer.

Nowadays, glycosylated protein seems to be one of the most effective stabilizers for preparing freeze-thaw stabile emulsion; nevertheless, few papers mentioned the relationship between the residual free sugars after the glycosylation reaction and the freeze-thaw stability of high internal phase emulsions (HIPEs). Herein, glucose was used to prepare glycosylated cod proteins (GCPs). The synergistic effect was related to the grafting degree of GCP, and the amount of glucose added to prepare freeze-thaw stable HIPEs was reduced from 20% to 4% when the grafting degree of GCP increased from 0% to 31.58% (i.e. 12% GCP). This might be due to fewer ice crystals forming in water phase or less destruction of emulsion droplets by ice crystals. The obtained results in this study will allow developing freeze-thaw stable HIPEs or new frozen ingredients.

Texture maintenance and degradation mechanism of ice-stored grass carp (Ctenopharyngodon idella): A scope of intramuscular connective tissue.

Although intramuscular connective tissue (IMCT) is low in fish, its impact on texture cannot be ignored due to its special location. Therefore, this study was aimed to investigate the contribution of IMCT degradation to fish softening and its mechanism induced by endogenous proteases. Results showed that IMCT honeycomb-like structure collapsed entirely on the 10th day of ice storage, along with a decrease of shear force by 36.5%. Meanwhile, IMCT and myofibrils (MF) degradation accelerated softening by 25.1% and 15.3% during 10 days of ice storage, respectively. Next, IMCT deterioration was indicated to be highly correlated with decorin degradation (0.956**), followed by elastin (0.928**) and collagen (0.904**). Ulteriorly, endogenous collagenase was shown to degrade IMCT crucial components, while endogenous cathepsins had little effect. In conclusion, this study confirmed that IMCT played an essential role in maintaining fish texture and was mainly degraded by endogenous collagenase.

Programmable scaffolds with aligned porous structures for cell cultured meat.

Porous scaffolds for cell cultured meat are currently limited in the food-grade material requirements, the cell adhesion, proliferation, and differentiation capacities, and the ignored appearance design. We proposed programmable scaffolds specially tailored for cell cultured meat. The scaffold with aligned porous structures was fabricated with the ice-templated directional freeze-drying of the food-grade collagen hydrogel. Due to the abundant tripeptide presence and well-aligned porous structures, the scaffold could not only provide sites for cell adhesion and proliferation, but also promote the oriented growth and differentiation of cells. The up-regulation of myogenic related genes, synthesis of myogenic related proteins and formation of matured myotubes furtherly proved the differentiation of cells on aligned scaffold. These characteristics would facilitate the traditional meat characteristics simulation of cell cultured meat in term of texture and microstructure. Meanwhile, patterned scaffolds were achievable as well with the help of mold-assisted ice templating, which would improve the people's interest, recognition, and acceptance of the tailored cell cultured meat. These characteristics indicate great application prospects of the proposed programmable scaffolds in cell cultured meat.

Fall and collision related injuries among pedestrians, sickness absence and associations with accident type and occupation.

OBJECTIVES: This study explores pedestrian fall accidents and collisions with other road users in the Swedish road transport system, and sickness absence (SA) in relation to accident type, injury, and occupation. Further, it studies the associations between accident type, occupation, and duration of SA. METHODS: Data from several national registers were used that included 15,359 working age pedestrians (20-64 years) receiving healthcare after a fall or collision throughout 2014-2016. Individual characteristics, accident type, injury, and occupation were presented and related to SA. Logistic regression was used to estimate odds ratios (OR), with 95% confidence intervals, for associations between accident type, occupation, and SA duration. RESULTS: About 11,000 pedestrians (72%) were involved in fall accidents in the road traffic environment and well over 4,000 in collisions with another road user; 22% of all injured pedestrians had a new SA. The population had a higher proportion of women and individuals in older age groups (≥45). Of the falls, 31% were due to snow or ice, and these were associated with a higher OR for both short SA (<90 days) 1.76 (95% CI 1.56-1.98) and long SA (≥90 days) 1.81 (95% CI 1.51-2.18), compared to the group slipping, tripping, and stumbling. The working sectors health & social care, and construction had the highest ORs for SA. A higher OR was found for health & social care, short SA 1.58 (95% CI 1.38-1.81), long SA 1.79 (95% CI 1.45-2.20) and for construction, short SA 1.56 (95% CI 1.24-1.96), long SA 1.75 (95% CI 1.26-2.44), compared to the sector finance, communication, & cultural service. CONCLUSIONS: The OR for having short and long SA was higher in falls due to snow or ice and differed between occupational sectors. PRACTICAL IMPLICATIONS: This information contributes to the knowledge base for planning a safe road transport system for pedestrians.

Ice Recrystallization Inhibition Activity of Silk Proteins.

The cryopreservation of cells, tissue, and organs is essential in both fundamental research and practical applications, such as modern regenerative medicine and technological applications. However, the formation of ice crystals during ice recrystallization can have harmful or even fatal effects on biological systems. To address this challenge, we explore the ice recrystallization inhibition (IRI) activity of two natural silk proteins of Bombyx mori, fibroin and sericin. We found that silk fibroin (SF) had higher ice recrystallization inhibition activity than silk sericin (SS). Moreover, SF aqueous solutions perform better in inhibiting ice recrystallization than SF phosphate-buffered saline solutions. Sum-frequency generation spectroscopy shows that stronger electrostatic interactions are responsible for the higher IRI ability of SF. This work is significant for broadening the applications of silk proteins in biomedical fields.

Anomalous Dynamics of Labile Metabolites in Cold Human Blood Detected Using 1H NMR Spectroscopy.

Recent efforts in our laboratory have enabled access to an unprecedented number (∼90) of quantifiable metabolites in human blood by a simple nuclear magnetic resonance (NMR) spectroscopy method, which includes energy coenzymes, redox coenzymes, and antioxidants that are fundamental to cellular functions [ J. Magn. Reson. Open 2022, 12-13, 100082]. The coenzymes and antioxidants, however, are notoriously labile and are extremely sensitive to specimen harvesting, extraction, and measurement conditions. This problem is largely underappreciated and carries the risk of grossly inaccurate measurements and incorrect study outcomes. As a part of addressing this challenge, in this study, human blood specimens were comprehensively and quantitatively investigated using 1H NMR spectroscopy. Freshly drawn human blood specimens were treated or not treated with methanol, ethanol, or a mixture of methanol and chloroform, and stored on ice or on bench, at room temperature for different time periods from 0 to 24 h, prior to storing at -80 °C. Interestingly, the labile metabolite levels were stable in blood treated with an organic solvent. However, their levels in blood in untreated samples increased or decreased by factors of up to 5 or more within 3 h. Further, surprisingly, and contrary to the current knowledge about metabolite stability, the variation of coenzyme levels was more dramatic in blood stored on ice than on bench, at room temperature. In addition, unlike the generally observed phenomenon of oxidation of redox coenzymes, reduction was observed in untreated blood. Such preanalytical dynamics of the labile metabolites potentially arises from the active cellular metabolism. From the metabolomics perspective, the massive variation of the labile metabolite levels even in blood stored on ice is alarming and stresses the critical need to immediately quench the cellular metabolism for reliable analyses. Overall, the results provide compelling evidence that warrants a paradigm shift in the sample collection protocol for blood metabolomics involving labile metabolites.

Detoxification of arsenite by iodide in frozen solution.

The oxidation of arsenite (As(III)) to arsenate (As(V)) has received significant attention because it helps mitigate the hazardous and adverse effects of As(III) and subsequently improves the effectiveness of arsenic removal. This study developed an efficient freezing technology for the oxidative transformation of As(III) based on iodide (I-). For a sample containing a very low concentration of 20 µM As(III) and 200 µM I- frozen at -20 °C, approximately 19 µM As(V) was formed after reaction for 0.5 h at pH 3. This rapid conversion has never been achieved in previous studies. However, As(V) was not generated in water at 25 °C. The acceleration of the oxidation of As(III) by I- in ice may be attributed to the freeze-concentration effect. During freezing, all components (i.e., As(III), I-, and protons) are highly concentrated in the ice grain boundary regions, resulting in thermodynamically and kinetically favorable conditions for the redox reaction between As(III) and I-. The efficiency of the oxidation of As(III) using I- increased at high I- concentrations and low pH values. The low freezing temperature (below -20 °C) hindered the oxidative transformation of As(III) by I-. The efficiency of the oxidation of As(III) significantly increased using a fixed initial concentration of I- by subjecting the system to six freezing-melting cycles. The outcomes of this study suggest the possibility of the self-detoxification of As(III) in the natural environment, indicating the potential for developing an eco-friendly method for the treatment of As(III)-contaminated areas in regions with a cold climate. It also demonstrates radical remediation to almost completely remove a very small amount of As(III) that was input in As(III)-contaminated wastewater detoxification, a benchmark that existing methods have been unable to achieve.

Role of Poloxamer 188 in Preventing Ice-Surface-Induced Protein Destabilization during Freeze-Thawing.

The phase behavior of poloxamer 188 (P188) in aqueous solutions, characterized by differential scanning calorimetry (DSC) and synchrotron X-ray diffractometry, revealed solute crystallization during both freezing and thawing. Sucrose and trehalose inhibited P188 crystallization during freeze-thawing (FT). While trehalose inhibited P188 crystallization only during cooling, sucrose completely suppressed P188 crystallization during both cooling and heating. Lactate dehydrogenase (LDH) served as a model protein to evaluate the stabilizing effect of P188. The ability of P188, over a concentration range of 0.003-0.800% w/v, to prevent LDH (10 µg/mL) destabilization was evaluated. After five FT cycles, the aggregation behavior (by dynamic light scattering) and activity recovery were evaluated. While LDH alone was sensitive to interfacial stress, P188 at concentrations of ≥0.100% w/v stabilized the protein. However, as the surfactant concentration decreased, protein aggregation after FT increased. The addition of sugar (1.0% w/v; sucrose or trehalose) improved the stabilizing function of P188 at lower concentrations (≤0.010% w/v), possibly due to the inhibition of surfactant crystallization. Based on a comparison with the stabilization effect of polysorbate (both 20 and 80), it was evident that P188 could be a promising alternative surfactant in frozen protein formulations. However, when the surfactant concentration is low, the potential for P188 crystallization and the consequent compromise in its functionality warrant careful consideration.

Electrochemical cells from water ice? Preliminary methods and results.

Electrochemical cells from ice will be an important seasonal addition to power generation in cold regions. We demonstrate power generation on the order of 0.1 mW at 0.3 V and 0.13 m2 surface area using an electrochemical cell with 2% HCl providing a pH gradient in ice, and suggest a solar add-on effect due to temperature changes under direct sunlight. Different models are discussed, and data are presented related to different additives: (1) solutes such as NaCl and monopotassium phosphate; (2) pH modifying agents such as acids and bases; (3) particulate suspensions with kaolinite and other substances. The results are positive and suggest viable use of electrochemical cells from ice with low fabrication costs and safe environmental impact for ephemeral power generation, especially with future material improvements and refinement of technique. Current research in this nascent field is also briefly introduced. The model presented has implications both for power systems and for biology: an icy-worlds hypothesis for the origin of life suggests a protometabolism with an ice-based pH gradient.

Rhizoglyphus robini, a pest mite of saffron, is unable to resist extracellular ice formation.

The saffron mite, Rhizoglyphus robini Claparède (Acari, Astigmata: Acaridae), is one of the most important pests of saffron-producing regions in Iran. It causes yellowing and decreases saffron growth, and finally it destroys the bulbs. In this research, the cold tolerance and supercooling point (SCP) of the saffron mite were measured in three populations and two temperature regimes. Our results showed that the mean SCP of the saffron mite was approximately -14.6 °C without significant difference among the populations. On the contrary, acclimation of the mites significantly decreased their SCP to a mean of approximately -16.5 °C. Exposure of the mites for 24 h to 0 and -2.5 °C had no significant effect on the survival of the mites but when the mites were exposed to -5.0 °C for 24 h, survival of the three populations reached the lowest level of roughly 60%. By 24-h exposure to -7.5 °C, survival of the mites was almost negligible. As a large proportion of mortality was observed above the SCP, and LT50 > SCP, it can be inferred that the saffron mite is likely a chill-susceptible species. This suggests that the saffron mite lacks the ability to withstand extracellular ice formation. Overall, the results of the current study suggest no significant physiological differences between populations of the saffron mite.

Copper Oxide Spike Grids for Enhanced Solution Transfer in Cryogenic Electron Microscopy.

The formation of uniform vitreous ice is a crucial step in the preparation of samples for cryogenic electron microscopy (cryo-EM). Despite the rapid technological progress in EM, controlling the thickness of vitreous ice on sample grids with reproducibility remains a major obstacle to obtaining high-quality data in cryo-EM imaging. The commonly employed classical blotting process faces the problem of excess water that cannot be absorbed by the filter paper, resulting in the formation of thick and heterogeneous ice. In this study, we propose a novel approach that combines the recently developed nanowire self-wicking technique with the classical blotting method to effectively control the thickness and homogeneity of vitrified ice. With simple procedures, we generated a copper oxide spike (COS) grid by inducing COSs on commercially available copper grids, which can effectively remove excess water during the blotting procedure without damaging the holey carbon membrane. The ice thickness could be controlled with good reproducibility compared to non-oxidized grids. Incorporated into other EM techniques, our new modification method is an effective option for obtaining high-quality data during cryo-EM imaging.

Strong Inhibition of Ice Growth by Biomimetic Crowding Coacervates.

The freezing of biological fluids is intensively studied but remains elusive as it is affected not only by the various components but also by the crowding nature of the biological fluids. Herein, we constructed spherical crowders, fibrous crowders, and coacervates by various components ranging from surfactants to polymers and proteins, to mimic three typical crowders in biological fluids, i.e., globular proteins, fibrous networks, and condensates of biomolecules. It is elucidated that the three crowders exhibit low, moderate, and strong ice growth inhibition activity, respectively, resulting from their different abilities in slowing down water dynamics. Intriguingly, the coacervate consisting of molecules without obvious ice growth inhibition activity strongly inhibits ice growth, which is firstly employed as a highly-potent cryoprotectant. This work provides new insights into the survival of freezing-tolerant organisms and opens an avenue for the design of ice-controlling materials.

Aerosolization flux, bio-products, and dispersal capacities in the freshwater microalga Limnomonas gaiensis (Chlorophyceae).

Little is known on the spreading capacities of Limnomonas gaiensis across freshwater lakes in Northern Europe. In this study, we show that the species could successfully be aerosolized from water sources by bubble bursting (2-40 particles.cm-3), irrespectively of its density in the water source or of the jet velocity used to simulate wave breaking. The species viability was impacted by both water turbulences and aerosolization. The survival rate of emitted cells was low, strain-specific, and differently impacted by bubble busting processes. The entity "microalga and bionts" could produce ethanol, and actively nucleate ice (principally ≤-18 °C) mediated soluble ice nucleation active proteins, thereby potentially impacting smog and cloud formation. Moreover, smallest strains could better cope with applied stressors. Survival to short-term exposure to temperatures down to -21 °C and freezing events further suggest that L. gaiensis could be air dispersed and contribute to their deposition.

Ice holes microrefugia harbor genetically and functionally distinct populations of Vaccinium vitis-idaea (Ericaceae).

In the mountain terrain, ice holes are little depressions between rock boulders that are characterized by the exit of cold air able to cool down the rock surface even in summer. This cold air creates cold microrefugia in warmer surroundings that preserve plant species probably over thousands of years under extra-zonal climatic conditions. We hypothesized that ice hole populations of the model species Vaccinium vitis-idaea (Ericaceae) show genetic differentiation from nearby zonal subalpine populations, and high functional trait distinctiveness, in agreement with genetic patterns. We genotyped almost 30,000 single nucleotide polymorphisms using restriction site-associated DNA sequencing and measured eight functional traits indicative of individual performance and ecological strategies. Genetic results showed high differentiation among the six populations suggesting isolation. On siliceous bedrock, ice hole individuals exhibited higher levels of admixture than those from subalpine populations which could have experienced more bottlenecks during demographic fluctuations related to glacial cycles. Ice hole and subalpine calcareous populations clearly separated from siliceous populations, indicating a possible effect of bedrock in shaping genetic patterns. Trait analysis reflected the bedrock effect on populations' differentiation. The significant correlation between trait and genetic distances suggests the genetic contribution in shaping intraspecific functional differentiation. In conclusion, extra-zonal populations reveal a prominent genetic and phenotypic differentiation determined by history and ecological contingency. Therefore, microrefugia populations can contribute to the overall variability of the species and lead to intraspecific-driven responses to upcoming environmental changes.

Comparison of the effects PRICE and POLICE treatment protocols on ankle function in patients with ankle sprain.

BACKGROUND: Ankle sprain is a frequent reason for presentation to the emergency department. Current treatment modalities include Protection, Optimal Loading, Ice, Compression, and Elevation (POLICE) and Protection, Rest, Ice, Compression, and Elevation (PRICE). This study aimed to compare the effects of PRICE and POLICE treatment protocols. METHODS: This randomized controlled study was conducted between October 15, 2020, and October 15, 2021, at Ankara University's Department of Emergency Medicine. Double-blind randomization was used to assign patients to either the POLICE or PRICE treatment groups. RESULTS: In total, 109 patients were included. In the POLICE group, the median difference between the American Orthopedic Foot and Ankle Scores on admission and the 14th day following the injury was 34.5 (IQR: 27.25-41.75), while that of the PRICE group was 24 (IQR: 15.5-35). In the POLICE group, the median value of the difference in the Foot and Ankle Disability Index scores on admission and the 14th day following the injury was 42 (IQR: 35.25-50), while that of the PRICE group was 31 (IQR: 22-41.5). CONCLUSION: The POLICE treatment protocol provided more effective and faster recovery than the PRICE treatment protocol.

Accumulation of Antifreeze Proteins on Ice Is Determined by Adsorption.

Antifreeze proteins (AFPs) facilitate the survival of diverse organisms in frigid environments by adsorbing to ice crystals and suppressing their growth. The rate of AFP accumulation on ice is determined by an interplay between AFP diffusion from the bulk solution to the ice-water interface and the subsequent adsorption of AFPs to the interface. To interrogate the relative importance of these two processes, here, we combine nonequilibrium fluorescence experiments with a reaction-diffusion model. We find that as diverse AFPs accumulate on ice, their concentration in the aqueous solution does not develop a gradient but remains equal to its bulk concentration throughout our experiments. These findings lead us to conclude that AFP accumulation on ice crystals, which are smaller than 100 µm in radius, is not limited by the diffusion of AFPs, but by the kinetics of AFP adsorption. Our results imply that mass transport limitations do not hinder AFPs from performing their biological function.

Mechanism of antifreeze protein functioning and the "anchored clathrate water" concept.

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)].

Preferential Ordering and Organization of Hydration Water Favor Nucleation of Ice by Ice-Nucleating Proteins over Antifreeze Proteins.

Bacteria containing ice-nucleating proteins (INPs) evolved in nature to nucleate ice at the high sub-zero ambiance. The ability of the INPs to induce order in the hydration layer and their aggregation propensity appear to be key factors of their ice nucleation abilities. However, the mechanism of the process of ice nucleation by INPs is yet to be understood clearly. Here, we have performed all-atom molecular dynamics simulations and analyzed the structure and dynamics of the hydration layer around the proposed ice-nucleating surface of a model INP. Results are compared with the hydration of a topologically similar non-ice-binding protein (non-IBP) and another ice-growth inhibitory antifreeze protein (sbwAFP). We observed that the hydration structure around the ice-nucleating surface of INP is highly ordered and the dynamics of the hydration water are slower, compared to the non-IBP. Even the ordering of the hydration layer is more evident around the ice-binding surface of INP, compared to the antifreeze protein sbwAFP. Particularly with increasing repeat units of INP, we observe an increased population of ice-like water. Interestingly, the distances between the hydroxyl groups of the threonine ladder and its associated channel water of the ice-binding surface (IBS) of INP in the X and Y direction mimic the oxygen atom distances of the basal plane of hexagonal ice. However, the structural synergies between the hydroxyl group distances of the threonine ladder and its associated channel water of the IBS of sbwAFP and oxygen atom distances of the basal plane are less evident. This difference makes the IBS of the INP a better template for ice nucleation than AFP, although both of them bind to the ice surface efficiently.

Phthalate esters delivery to the largest European lagoon: Sources, partitioning and seasonal variations.

Phthalate esters (PAEs) due to their ability to leach from plastics, widely used in our daily life, are intensely accumulating in wastewater water treatment plants (WWTP) and rivers, before being exported to downstream situated estuarine systems. This study aimed to investigate the external sources of eight plasticizers to the largest European lagoon (the Curonian Lagoon, south-east Baltic Sea), focusing on their seasonal variation and transport behaviour through the partitioning between dissolved and particulate phases. The obtained results were later combined with hydrological inputs at the inlet and outlet of the lagoon to estimate system role in regulating the transport of pollutants to the sea. Plasticizers were detected during all sampling events with a total concentration ranging from 0.01 to 6.17 µg L-1. Di(2-ethylhexyl) phthalate (DEHP) was the most abundant PAEs and was mainly found attached to particulate matter, highlighting the importance of this matrix in the transport of such contaminant. Dibutyl phthalate (DnBP) and diisobutyl phthalate (DiBP) were the other two dominant PAEs found in the area, mainly detected in dissolved phase. Meteorological conditions appeared to be an important factor regulating the distribution of PAEs in environment. During the river ice-covered season, PAEs concentration showed the highest value suggesting the importance of ice in the retention of PAEs. While heavy rainfall impacts the amount of water delivered to WWTP, there is an increase of PAEs concentration supporting the hypothesis of their transport via soil leaching and infiltration into wastewater networks. Rainfall could also be a direct source of PAEs to the lagoon resulting in net surplus export of PAEs to the Baltic Sea.