The Influence of Various Freezing-thawing Methods of Skin on Drug Permeation and Skin Barrier Function.

The selection of skin is crucial for the in vitro permeation test (IVPT). The purpose of this study was to investigate the influence of different freezing-thawing processes on the barrier function of skin and the transdermal permeability of granisetron and lidocaine. Rat and hairless mouse skins were thawed at three different conditions after being frozen at -20℃ for 9 days: thawed at 4℃, room temperature (RT), and 32℃. There were no significant differences in the steady-state fluxes of drugs between fresh and thawed samples, but compared with fresh skin there were significant differences in lag time for the permeation of granisetron in rat skins thawed at RT and 32℃. Histological research and scanning electron microscopy images showed no obvious structural damage on frozen/thawed skin, while immunohistochemical staining and enzyme-linked immunosorbent assay for the tight junction (TJ) protein Cldn-1 showed significantly impaired epidermal barrier. It was concluded that the freezing-thawing process increases the diffusion rate of hydrophilic drugs partly due to the functional degradation of TJs. It's recommended that hairless, inbred strains and identical animal donors should be used, and the selected thawing method of skin should be validated prior to IVPT, especially for hydrophilic drugs.

Evaluation of the effectiveness of the use of exosomes in the regulation of the mitochondrial membrane potential of frozen/thawed spermatozoa.

Numerous studies confirm the involvement of extracellular vesicles (EVs) in the regulation of physiological processes of mammalian sperm cells. It has been proven that they take part in the processes of capacitation, acrosonmal reaction, and anti-oxidation. Despite growing interest in the biomedical potential (including the search for new reproductive biomarkers) of EVs, the role of extracellular seminal vesicles in maintaining semen quality during cryopreservation has not yet been established. Therefore, the objective of this experiment was to evaluate the effectiveness of the use in the regulation of the mitochondrial membrane potential of bovine sperm and to explain the mechanisms of EV action during cell cryopreservation. Exosomes were isolated from bull semen plasma, measured, and used for extender supplementation. Semen samples were collected from Simmental bulls, diluted, and pre-evaluated. Then they were divided into equal fractions that did not contain EVs or were supplemented with 0.75; 1.5 and 2.25 mg/ml of EVs. The test samples were frozen/thawed and the mitochondrial membrane potential, DNA integrity, and viability were evaluated. EVs have been established to have a positive effect on cryopreserved sperm structures. The most favourable level of EVs was 1.5 mg / ml, which can be successfully to improve cell cryostability during freezing/thawing. In this study, exosomes isolated from the sperm plasma and supplemented with a concentrated dose in the extender for sperm freezing were shown to significantly improve cryostability of cells by supporting the potentials of the mitochondrial membrane and protecting the cytoplasmic membrane of spermatozoa.

High-speed cryo-microscopy reveals that ice-nucleating proteins of Pseudomonas syringae trigger freezing at hydrophobic interfaces.

Ice-nucleating proteins (INpro) trigger the freezing of supercooled water droplets relevant to atmospheric, biological, and technological applications. The high ice nucleation activity of INpro isolated from the bacteria Pseudomonas syringae could be linked to the aggregation of proteins at the bacterial membrane or at the air-water interface (AWI) of droplets. Here, we imaged freezing onsets, providing direct evidence of these proposed mechanisms. High-speed cryo-microscopy identified the onset location of freezing in droplets between two protein-repellent glass slides. INpro from sterilized P. syringae (Snomax) statistically favored nucleation at the AWI of the droplets. Removing cellular fragments by filtration or adding surfactants increased the frequency of nucleation events at the AWI. On the other hand, cultivated intact bacteria cells or lipid-free droplets nucleated ice without an affinity to the AWI. Overall, we provide visual evidence that INpro from P. syringae trigger freezing at hydrophobic interfaces, such as the AWI or the bacterial membrane, with important mechanistic implications for applications of INpro.

Proteomic analysis of Rana sylvatica reveals differentially expressed proteins in liver in response to anoxia, dehydration or freezing stress.

Ectothermic animals that live in seasonally cold regions must adapt to seasonal variation and specific environmental conditions. During the winter, some amphibians hibernate on land and encounter limited environmental water, deficient oxygen, and extremely low temperatures that can cause the whole body freezing. These stresses trigger physiological and biochemical adaptations in amphibians that allow them to survive. Rana sylvatica, commonly known as the wood frog, shows excellent freeze tolerance. They can slow their metabolic activity to a near halt and endure freezing of 65-70% of their total body water as extracellular ice during hibernation, returning to normal when the temperatures rise again. To investigate the molecular adaptations of freeze-tolerant wood frogs, a comprehensive proteomic analysis was performed on frog liver tissue after anoxia, dehydration, or freezing exposures using a label-free LC-MS/MS proteomic approach. Quantitative proteomic analysis revealed that 87, 118, and 86 proteins were significantly upregulated in dehydrated, anoxic, and frozen groups, suggesting potential protective functions. The presence of three upregulated enzymes, glutathione S-transferase (GST), aldolase (ALDOA), and sorbitol dehydrogenase (SORD), was also validated. For all enzymes, the specific enzymatic activity was significantly higher in the livers of frozen and anoxic groups than in the controls. This study reveals that GST, ALDOA, and SORD might participate in the freeze tolerance mechanism by contributing to regulating cellular detoxification and energy metabolism.

Predicting soil thermal properties in freeze-thaw cycles using EFAttNet: A comparative analysis.

This study investigates the thermal conductivity (λ) and volumetric heat capacity (C) of sandy soil samples under a variety of conditions, including freeze-thaw cycles at temperatures both above and below zero and differing moisture levels. To estimate these thermal properties, a novel predictive model, EFAttNet, was developed, which utilizes custom-designed embedding and attention-based fusion networks. When compared to traditional de Vries empirical models and other baseline algorithms, EFAttNet demonstrated superior accuracy. Preliminary measurements showed that λ values increased linearly with moisture content but decreased with temperature, whereas C values exhibited a rising trend with both moisture content and freezing temperature. Following freeze-thaw cycles, both λ and C were positively influenced by moisture content and freezing temperature. The EFAttNet-based model proved highly accurate in predicting thermal properties, particularly effective at capturing nonlinear relationships among the influencing factors. Among these factors, the degree of saturation had the most significant impact, followed by the number of freeze-thaw cycles, subzero temperatures, porosity, and moisture content. Notably, dry density exerted minimal influence on thermal properties, likely due to the overriding effects of other factors or specific soil characteristics, such as particle size distribution or mineralogical composition. These findings have significant implications for construction and engineering projects, especially in terms of sustainability and energy efficiency. The demonstrated accuracy of the EFAttNet-based model in estimating thermal properties under various conditions holds promise for practical applications. Although focused on specific soil types and conditions, the insights gained can guide further research and development in managing soil thermal properties across diverse environments, thereby enhancing our understanding and application in this field.

Imaging Bunyavirus Infections by Transmission Electron Microscopy: Conventional Sample Preparation vs High-Pressure Freezing and Freeze-Substitution.

Transmission electron microscopy significantly contributed to unveil the course of virus entry, replication, morphogenesis, and egress. For these studies, the most widely used approach is imaging ultrathin sections of virus-infected cells embedded in a plastic resin that is transparent to electrons. Before infiltration in a resin, cells must be processed to stabilize their components under the observation conditions in an electron microscope, such as high vacuum and irradiation with electrons. For conventional sample preparation, chemical fixation and dehydration are followed by infiltration in the resin and polymerization to produce a hard block that can be sectioned with an ultramicrotome. Another method that provides a superior preservation of cell components is high-pressure freezing (HPF) followed by freeze substitution (FS) before resin infiltration and polymerization. This chapter describes both procedures with cells infected with Bunyamwera virus (BUNV), a well characterized member of the Bunyavirales, and compares the morphological details of different viral structures imaged in the two types of samples. Advantages, disadvantages, and applications of conventional processing and HPF/FS are also presented and discussed.

Cryopreservation of human kidney organoids.

Recent advances in stem cell research have led to the creation of organoids, miniature replicas of human organs, offering innovative avenues for studying diseases. Kidney organoids, with their ability to replicate complex renal structures, provide a novel platform for investigating kidney diseases and assessing drug efficacy, albeit hindered by labor-intensive generation and batch variations, highlighting the need for tailored cryopreservation methods to enable widespread utilization. Here, we evaluated cryopreservation strategies for kidney organoids by contrasting slow-freezing and vitrification methods. 118 kidney organoids were categorized into five conditions. Control organoids followed standard culture, while two slow-freezing groups used 10% DMSO (SF1) or commercial freezing media (SF2). Vitrification involved V1 (20% DMSO, 20% Ethylene Glycol with sucrose) and V2 (15% DMSO, 15% Ethylene Glycol). Assessment of viability, functionality, and structural integrity post-thawing revealed notable differences. Vitrification, particularly V1, exhibited superior viability (91% for V1, 26% for V2, 79% for SF1, and 83% for SF2 compared to 99.4% in controls). 3D imaging highlighted distinct nephron segments among groups, emphasizing V1's efficacy in preserving both podocytes and tubules in kidney organoids. Cisplatin-induced injury revealed a significant reduction in regenerative capacities in organoids cryopreserved by flow-freezing methods, while the V1 method did not show statistical significance compared to the unfrozen controls. This study underscores vitrification, especially with high concentrations of cryoprotectants, as an effective approach for maintaining kidney organoid viability and structure during cryopreservation, offering practical approaches for kidney organoid research.

Effects of storage conditions on the stability of qPCR reagents: implications for environmental DNA detection.

OBJECTIVE: Environmental DNA (eDNA) detection is a transformative tool for ecological surveys which in many cases offers greater accuracy and cost-effectiveness for tracking low-density, cryptic species compared to conventional methods. For the use of targeted quantitative PCR (qPCR)-based eDNA detection, protocols typically require freshly prepared reagents for each sample, necessitating systematic evaluation of reagent stability within the functional context of eDNA standard curve preparation and environmental sample evaluation. Herein, we assessed the effects of long-term storage and freeze-thaw cycles on qPCR reagents for eDNA analysis across six assays. RESULTS: Results demonstrate qPCR plates (containing pre-made PCR mix, primer-probe, and DNA template) remain stable at 4 °C for three days before thermocycling without fidelity loss irrespective of qPCR assay used. Primer-probe mixes remain stable for five months of - 20 °C storage with monthly freeze-thaw cycles also irrespective of qPCR assay used. Synthetic DNA stocks maintain consistency in standard curves and sensitivity for three months under the same conditions. These findings enhance our comprehension of qPCR reagent stability, facilitating streamlined eDNA workflows by minimizing repetitive reagent preparations.

Autophagy is suppressed by low temperatures and is dispensable for cold acclimation in Arabidopsis.

Plants have evolved various mechanisms to adapt to the ever-changing external environment. Autophagy is one such mechanism and has been suggested to play a key role in responding to and adapting to abiotic stresses in plants. However, the role of autophagy in adaptation to cold and freezing stresses remains to be characterized in detail. Here, we investigated the role of autophagy in the low-temperature response of Arabidopsis using atg mutants. Both the atg5-1 and atg10-1 mutants exhibited normal freezing tolerance, regardless of cold acclimation. A comparison of fresh weights indicated that the difference in growth between the wild-type and atg plants under cold conditions was rather small compared with that under normal conditions. Analysis of COLD-REGULATED gene expression showed no significant differences between the atg mutants and wild type. Treatment with 3-methyladenine, an inhibitor of autophagy, did not impair the induction of COR15Apro::LUC expression upon exposure to low temperature. Evaluation of autophagic activity using transgenic plants expressing RBCS-mRFP demonstrated that autophagy was rarely induced by cold exposure, even in the dark. Taken together, these data suggest that autophagy is suppressed by low temperatures and is dispensable for cold acclimation and freezing tolerance in Arabidopsis.

Assessment of fecal bacterial viability and diversity in fresh and frozen fecal microbiota transplant (FMT) product in horses.

BACKGROUND: Currently, lack of standardization for fecal microbiota transplantation (FMT) in equine practice has resulted in highly variable techniques, and there is no data on the bacterial metabolic activity or viability of the administered product. The objectives of this study were to compare the total and potentially metabolically active bacterial populations in equine FMT, and assess the effect of different frozen storage times, buffers, and temperatures on an equine FMT product. Fresh feces collected from three healthy adult horses was subjected to different storage methods. This included different preservation solutions (saline plus glycerol or saline only), temperature (-20 °C or -80 °C), and time (fresh, 30, 60, or 90 days). Samples underwent DNA extraction to assess total bacterial populations (both live and dead combined) and RNA extraction followed by reverse transcription to cDNA as a proxy to assess viable bacteria, then 16s rRNA gene amplicon sequencing using the V1-V2 region. RESULTS: The largest difference in population indices and taxonomic composition at the genus level was seen when evaluating the results of DNA-based (total) and cDNA-based (potentially metabolically active) extraction method. At the community level, alpha diversity (observed species, Shannon diversity) was significantly decreased in frozen samples for DNA-based analysis (P < 0.05), with less difference seen for cDNA-based sequencing. Using DNA-based analysis, length of storage had a significant impact (P < 0.05) on the bacterial community profiles. For potentially metabolically active populations, storage overall had less of an effect on the bacterial community composition, with a significant effect of buffer (P < 0.05). Individual horse had the most significant effect within both DNA and cDNA bacterial communities. CONCLUSIONS: Frozen storage of equine FMT material can preserve potentially metabolically active bacteria of the equine fecal microbiome, with saline plus glycerol preservation more effective than saline alone. Larger studies are needed to determine if these findings apply to other individual horses. The ability to freeze FMT material for use in equine patients could allow for easier clinical use of fecal transplant in horses with disturbances in their intestinal microbiome.

Electromagnetic Field-Assisted Frozen Tissue Planarization Enhances MALDI-MSI in Plant Spatial Omics.

Plant samples with irregular morphology are challenging for longitudinal tissue sectioning. This has restricted the ability to gain insight into some plants using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Herein, we develop a novel technique termed electromagnetic field-assisted frozen tissue planarization (EMFAFTP). This technique involves using a pair of adjustable electromagnets on both sides of a plant tissue. Under an optimized electromagnetic field strength, nondestructive planarization and regularization of the frozen tissue is induced, allowing the longitudinal tissue sectioning that favors subsequent molecular profiling by MALDI-MSI. As a proof of concept, flowers, leaves and roots with irregular morphology from six plant species are chosen to evaluate the performance of EMFAFTP for MALDI-MSI of secondary metabolites, amino acids, lipids, and proteins among others in the plant samples. The significantly enhanced MALDI-MSI capabilities of these endogenous molecules demonstrate the robustness of EMFAFTP and suggest it has the potential to become a standard technique for advancing MALDI-MSI into a new era of plant spatial omics.

Nanoscale intracellular ultrastructures affected by osmotic pressure using small-angle X-ray scattering.

Although intracellular ultrastructures have typically been studied using microscopic techniques, it is difficult to observe ultrastructures at the submicron scale of living cells due to spatial resolution (fluorescence microscopy) or high vacuum environment (electron microscopy). We investigate the nanometer scale intracellular ultrastructures of living CHO cells in various osmolality using small-angle X-ray scattering (SAXS), and especially the structures of ribosomes, DNA double helix, and plasma membranes in-cell environment are observed. Ribosomes expand and contract in response to osmotic pressure, and the inter-ribosomal correlation occurs under isotonic and hyperosmolality. The DNA double helix is not dependent on the osmotic pressure. Under high osmotic pressure, the plasma membrane folds into form a multilamellar structure with a periodic length of about 6 nm. We also study the ultrastructural changes caused by formaldehyde fixation, freezing and heating.

Evaluation of the regeneration process of the sciatic nerve of a mouse animal model after application of freezing, crushing or electrocoagulation damage / Ocena procesu regeneracji nerwu kulszowego mysiego modelu zwierzecego po zastosowaniu uszkodzenia metoda mrozenia, zmiazdzenia i elektrokoagulacji.

The problem of regeneration of damaged peripheral nerves is an ongoing topic and has long been the subject of intensive research worldwide. This study examined the morphological and functional evaluation of the regeneration process within the damaged sciatic nerve, a mouse animal model. The effect of impaired expression of the TSC-1 gene on the process of nerve regeneration was evaluated, depending on the mode of damage. The research object consisted of 48, 2-month-old male TSC lines. The test group consisted of animals that underwent damage to the sciatic nerve by crushing, freezing and electrocoagulation, while the control group includes mice whose sciatic nerve was not damaged. Behavioral tests were conducted to evaluate the functional return of the limb, after 3,5,7 and 14 days. The first changes in the regeneration process of the damaged neurite are observed as early as day 3 after the injury, while on day 14 after the injury the functional return of the damaged limb was noted.

Repeated freezing to very low temperatures does not impact the amount ejected from EpiPen® and Jext® adrenaline autoinjectors.

It has previously been shown that EpiPen® autoinjectors are likely to activate normally following up to five excursions to -25°C but data about the post-freezing performance of other brands of adrenaline autoinjectors has not previously been published. Additionally, conditions experienced by polar medics may be substantially colder than this and the performance of adrenaline autoinjectors following more extreme freeze-thaw cycles remains uncharacterised. Investigators in Antarctica and the United Kingdom performed laboratory testing on two brands of adrenaline autoinjector, EpiPen® and Jext® (12 devices of each type). A single freeze-thaw cycle involved freezing the device to -80°C then allowing it to come to room temperature. Devices were exposed to 0, 1, 5 or 15 freeze-thaw cycles. The mass of liquid ejected from each device, when activated, was then measured. No significant differences in the mass of the liquid ejected was found between the test groups. Multiple freeze-thaw cycles to -80°C are unlikely to significantly impact the amount of adrenaline solution expelled from EpiPen® and EpiPen® autoinjectors. This preliminary finding encourages further work investigating the safety and effectiveness of adrenaline autoinjectors after exposure to very low temperatures. This information would be valuable for future polar medics planning and delivering medical provision in extreme environments.

[Effect of freeze-thaw cycles on aggregate breakdown of typical black soil during transportation]. / å†»èžå¯¹å ¸åž‹é»‘åœŸå›¢èšä½“è¾“ç§»ç ´ç¢Žç‰¹å¾çš„å½±å“.

During the snowmelt period, the external erosive forces are dominated by freeze-thaw cycles and snowmelt runoff. These forces may affect soil structure and aggregate stability, thereby influencing snowmelt erosion. The process of snowmelt runoff can lead to the breakdown of aggregates during their transportation. However, few studies examined the effects of freeze-thaw cycles on the breakdown of aggregates during transportation. Focusing on 5-7 and 3-5 mm soil aggregates of typical black soil region in Northeast China, we analyzed the composition of water-stable aggregates, mean weight diameter (MWD), normalized mean weight diameter (NMWD), as well as breakdown rate of soil aggregates (BR) under different freeze-thaw cycles (0, 1, 5, 10, 15 and 20 times) and different transport distances (5, 10, 15, 20, 25 and 30 m). We further investigated the contribution (CT) of both freeze-thaw cycles and transport distances to BR. The results showed that: 1) After freeze-thaw cycles, the 5-7 and 3-5 mm aggregates were mainly composed of particles with a diameter of 0.5-1 mm. With increasing frequency of freeze-thaw cycles, the MWD generally showed a downward trend. Moreover, under the same number of freeze-thaw cycles, the NMWD of 3-5 mm aggregates was higher than that of 5-7 mm aggregates. 2) As the transport distance increased, the BR of 5-7and 3-5 mm aggregates gradually increased. Compared that under control group, the BR under one freeze-thaw cycle increased by 59.7%, 32.2%, 13.7%, 6.2%, 13.4%, 7.5%, and 60.0%, 39.0%, 18.4%, 13.0%, 6.3%, 6.1% at the condition of 5, 10, 15, 20, 25 and 30 m transport distances, respectively. However, with increasing frequency of freeze-thaw cycles, the BR increased slowly. 3) The breakdown of soil aggregates was mainly influenced by the transport distance (CT=54.6%) and freeze-thaw cycles (CT=26.2%). Freeze-thaw cycles primarily altered the stability of soil aggregates, which in turn affected the BR. Therefore, during the snowmelt period, freeze-thaw cycles reduced the stability of soil aggregates, leading to severe breakdown of soil aggregates during snowmelt runoff process. This made the soil more susceptible to migration with snowmelt runoff, which triggered soil erosion. Therefore, more attention should be paid on the prevention of soil erosion during snowmelt period.

Maize residue retention shapes soil microbial communities and co-occurrence networks upon freeze-thawing cycles.

Maize residue retention is an effective agricultural practice for improving soil fertility in black soil region, where suffered from long freezing-thawing periods and intense freeze-thawing (FT) cycles. However, very few studies have examined the influence of maize residue retention on soil microbial communities under FT cycles. We investigated the response of soil microbial communities and co-occurrence networks to maize residue retention at different FT intensities over 12 cycles using a microcosm experiment conditioned in a temperature incubator. Our results indicated that maize residue retention induced dramatic shifts in soil archaeal, bacterial and fungal communities towards copiotroph-dominated communities. Maize residue retention consistently reduced soil fungal richness across all cycles, but this effect was weaker for archaea and bacteria. Normalized stochastic ratio analysis revealed that maize residue retention significantly enhanced the deterministic process of archaeal, bacterial and fungal communities. Although FT intensity significantly impacted soil respiration, it did not induce profound changes in soil microbial diversity and community composition. Co-occurrence network analysis revealed that maize residue retention simplified prokaryotic network, while did not impact fungal network complexity. The network robustness index suggested that maize residue retention enhanced the fungal network stability, but reduced prokaryotic network stability. Moreover, the fungal network in severe FT treatment harbored the most abundant keystone taxa, mainly being cold-adapted fungi. By identifying modules in networks, we observed that prokaryotic Module #1 and fungal Module #3 were enhanced by maize residue retention and contributed greatly to soil quality. Together, our results showed that maize residue retention exerted stronger influence on soil microbial communities and co-occurrence network patterns than FT intensity and highlighted the potential of microbial interactions in improving soil functionality.

Near-Infrared Spectroscopy for Rapid Differentiation of Fresh and Frozen-Thawed Common Carp (Cyprinus carpio).

This study aimed to investigate near-infrared spectroscopy (NIRS) in combination with classification methods for the discrimination of fresh and once- or twice-freeze-thawed fish. An experiment was carried out with common carp (Cyprinus carpio). From each fish, test pieces were cut from the dorsal and ventral regions and measured from the skin side as fresh, after single freezing at minus 18 °C for 15 ÷ 28 days and 15 ÷ 21 days for the second freezing after the freeze-thawing cycle. NIRS measurements were performed via a NIRQuest 512 spectrometer at the region of 900-1700 nm in Reflection mode. The Pirouette 4.5 software was used for data processing. SIMCA and PLS-DA models were developed for classification, and their performance was estimated using the F1 score and total accuracy. The predictive power of each model was evaluated for fish samples in the fresh, single-freezing, and second-freezing classes. Additionally, aquagrams were calculated. Differences in the spectra between fresh and frozen samples were observed. They might be assigned mainly to the O-H and N-H bands. The aquagrams confirmed changes in water organization in the fish samples due to freezing-thawing. The total accuracy of the SIMCA models for the dorsal samples was 98.23% for the calibration set and 90.55% for the validation set. For the ventral samples, respective values were 99.28 and 79.70%. Similar accuracy was found for the PLS-PA models. The NIR spectroscopy and tested classification methods have a potential for nondestructively discriminating fresh from frozen-thawed fish in as methods to protect against fish meat food fraud.

Optical Sensing of Tissue Freezing Depth by Sapphire Cryo-Applicator and Steady-State Diffuse Reflectance Analysis.

This work describes a sapphire cryo-applicator with the ability to sense tissue freezing depth during cryosurgery by illumination of tissue and analyzing diffuse optical signals in a steady-state regime. The applicator was manufactured by the crystal growth technique and has several spatially resolved internal channels for accommodating optical fibers. The method of reconstructing freezing depth proposed in this work requires one illumination and two detection channels. The analysis of the detected intensities yields the estimation of the time evolution of the effective attenuation coefficient, which is compared with the theoretically calculated values obtained for a number of combinations of tissue parameters. The experimental test of the proposed applicator and approach for freezing depth reconstruction was performed using gelatin-based tissue phantom and rat liver tissue in vivo. It revealed the ability to estimate depth up to 8 mm. The in vivo study confirmed the feasibility of the applicator to sense the freezing depth of living tissues despite the possible diversity of their optical parameters. The results justify the potential of the described design of a sapphire instrument for cryosurgery.

Quality analysis of dough and steamed bread under various freezing conditions.

Freezing is a crucial step in the process of frozen foods. In this study, the effects of different freezing methods, including liquid nitrogen immersion freezing (LF), quick-freezing machine freezing (QF), packaging immersion freezing (PF), and ultralow temperature refrigerator freezing (UF), and freezing time (0, 15, 30, and 60 days) on the textural properties, dynamic rheological properties, water distribution, and structure of dough and the quality of end steamed bread were evaluated. Freezing resulted in a decline in the physicochemical properties of dough. UF- and QF-doughs had higher storage modulus and loss modulus, compared with PF- and LF-doughs. LF enhanced the textural attributes of the dough, resulting in reduced hardness and increased springiness. At 15 days of freezing, QF- and LF-doughs exhibited a compact and continuous structure with a smooth surface. Additionally, the correlation analysis elucidated that the weight loss rate and the bound water content of the dough had discernible impacts on the texture of both the dough and the resulting steamed bread. Overall, LF demonstrated a relatively high freezing efficiency and effectively maintained the quality of the dough for up to 15 days of freezing. These results offer valuable insights for the applications of freezing methods and time in frozen foods.

A method to image brain tissue frozen at autopsy.

Magnetic Resonance Imaging (MRI) can provide the location and signal characteristics of pathological regions within a postmortem tissue block, thereby improving the efficiency of histopathological studies. However, such postmortem-MRI guided histopathological studies have so far only been performed on fixed samples as imaging tissue frozen at the time of extraction, while preserving its integrity, is significantly more challenging. Here we describe the development of cold-postmortem-MRI, which can preserve tissue integrity and help target techniques such as transcriptomics. As a first step, RNA integrity number (RIN) was used to determine the rate of tissue biomolecular degradation in mouse brains placed at various temperatures between -20 °C and +20 °C for up to 24 h. Then, human tissue frozen at the time of autopsy was immersed in 2-methylbutane, sealed in a bio-safe tissue chamber, and cooled in the MRI using a recirculating chiller to determine MRI signal characteristics. The optimal imaging temperature, which did not show significant RIN deterioration for over 12 h, at the same time giving robust MRI signal and contrast between brain tissue types was deemed to be -7 °C. Finally, MRI was performed on human tissue blocks at this optimal imaging temperatures using a magnetization-prepared rapid gradient echo (MPRAGE, isotropic resolution between 0.3-0.4 mm) revealing good gray-white matter contrast and revealing subpial, subcortical, and deep white matter lesions. RINs measured before and after imaging revealed no significant changes (n = 3, p = 0.18, paired t-test). In addition to improving efficiency of downstream processes, imaging tissue at sub-zero temperatures may also improve our understanding of compartment specificity of MRI signal.