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Direct pulp tissue grafting has been proposed in the past to void the tedious task of laboratory culturing. Migration of cells from dental pulp tissue explantation have shown multi differentiation potential in vitro like DPSC. These cells were named MP-MSC (minced pulp derived mesenchymal stem cells) by our group. Establishing that intact pulp tissue can be a potential source of dental pulp derived stem cells, we further wanted to investigate the possibility of pulp tissue banking. Cord tissue banking has gained popularity and success in the past decade and has shown successful in medicine. Out study demonstrated that cryopreserved pulp tissue has a potential to sustain viable cells and thesecells additionally showed that they can maintain the multi potent differentiation in vitrolike unfrozen tissue as well as expression of odontogenic marker. Additionally,our data also suggests that the perivascular niche can be maintained intact in the minced pulp tissue as well as frozen minced pulp tissue, suggesting the mesenchymal origin of the cells. Ex vivo tissue engineering showed migration of cells and attachment to dentin surface in presence of scaffold from frozen tissue is similar to unfrozen tissue. Additionally, RT-PCR data from our study shows that frozen pulp tissue derived cells showed differentiation and expression of ALP, OCN, DSPP markers suggesting that they maintained the differentiation potential after cryopreservation.

Low temperature studies with winter-dormant buds are severely limited by the lack of a rapid,non-destructive assay for their viability. Investigations involving the winter harvest of ecodormant buds of woody subjects, including cryopreservation, are restricted if viability cannot be assessed until dormancy is broken. If post-treatment grafting indicates low survival of the harvested population then further collection and study has to be delayed until the next winter season. This study trials the use of a portable gas exchange system able to discriminate between live and dead buds rapidly, with the assay confirmed as non-destructive by subsequent micropropagation. Active respiration was recorded for 85% of a winter-dormant Malus domestica buds population that showed 91% viability when grafted (n = 45). Lethally stressed material gave no false positive results. When micropropagated after respiratory measurement, a population viability of 76% was recorded. There was a significant, positive correlation between respiration and fresh weight for buds of mass >10 mg, from a population with a mean fresh weight of 17 mg.

Objective The aim of this study was to identify genes related to a freeze-thaw tolerance and to elucidate the tolerance mechanism in yeast Saccharomyces cerevisiae as an appropriate eukaryote model. Results In this study, one tolerant strain under exposure to freeze-thaw stress was isolated by screening a transposon-mediated mutant library and the disrupted gene was identified to be YCP4. In addition, this phenotype related to freeze-thaw tolerance was comfirmed by deletion and overexpressing of this corresponding gene. This mutant strain showed a freeze-thaw tolerance by the reduction in the intracellular level of reactive oxygen species (ROS) and the activation of the MSN2/4 and STRE-mediated genes such as CTT1 and HSP12. Conclusions Disruption of YCP4 in S. cerevisiae results in increased tolerance to freeze-thaw stress.

To find the gold standard out of three pre-established routine slow freezing (SF) methods, ovarian cortex tissues of nine transsexual individuals were cryopreserved and compared to each other, as well as the control (fresh) samples. Histological, genomic, and endocrinological effects of the SFs were assessed post-thawing and after a seven-day culture period. SF1 included 10% dimethyl-sulfoxide (Me2SO) in the base medium (BM), SF2 had 1.5 M/L ethylene-glycol (EG) and 0.1 M/L sucrose in the BM, and SF3 consisted of 6% Me2SO, 6% EG and 0.15 M/L sucrose in the BM. The cortical tissue strips went under a programmed cooling process and were stored in liquid nitrogen. Histological criteria (tissue damage and follicular quality), as well as gene expression levels, were assessed in the thawed and control tissues. Half of the thawed and control tissues were cultured for seven days and their histology, genetic profile, and hormonal status were examined as the reflection of the avascular tension effect. Post-thawing tissue damage was similar between all groups but significantly increased post-culture (P 0.05). The percentages of high-quality follicles diminished in all SFs after thawing and culture (P 0.05) except for the similarity of post-thawing SF3, compared to control. The genetic profile of the tissue after thawing and culture suggested quiescence/activation balance in SF1 and 2 and significant down-regulation in SF3, compared to the control specimens (P 0.05). Post-thawing BAX:BCL2 was higher than control in SF1 and SF3 (P 0.05), while this ratio in SF2 was similar to the control. However, after culture this ratio was similar to that of control in SF3 and diminished in SF1 and 2 (P 0.05). The expression levels of gap-junction genes showed dramatic pre- and post-thawing fluctuations in all groups. After culture, estradiol in SF3 was significantly higher than SF1 and 2 (P 0.05). In addition, progesterone in SF3 was similar to control but significantly lower in SF1 and 2 (P 0.05). In conclusion, all SFs showed advantages and disadvantages, and the follicular quality and its function depend on the type of cryoprotectant and the speed of thawing. The effects of freezing/thawing continue to appear during the seven days of culture. According to the results of this study, SF3 seems to be more promising in keeping the follicles functional and safe from cell damage during culture.

Cryopreservation can help to meet the demand for biosamples of high medical value. However, it remains difficult to effectively cryopreserve some sensitive cells, tissues, and reproductive organs. A coordinated effort from the perspective of the whole frozen biological system is necessary to advance cryopreservation technology. Animals that survive in cold temperatures, such as hibernators and cold-tolerant insects, offer excellent natural models. Their anti-cold strategies, such as programmed suppression of metabolism and the synthesis of cryoprotectants (CPAs), warrant systematic study. Furthermore, the discovery and synthesis of metabolism-regulating and cryoprotective biomaterials, combined with biotechnological breakthroughs, can also promote the development of cryopreservation. Further advances in the quality and duration of biosample storage inspired by nature will promote the application of cryopreserved biosamples in clinical therapy.

Background: Sperm-associated antigens (SPAGs) are 18 types of proteins, some of which play important roles in various biological functions associated with assisted reproductive technology outcomes,...

As the cornerstone of normal operation for Sponge City, the service performance of permeable pavement is directly related to the utilization rate of precipitation resources. However, the pore filling state (air, water and ice) of permeable pavement in Sponge City is easily affected by the service environment, especially low temperature. Thus, in the study, the influence of pore filling states and phase change processes on the service performance for permeable pavement were studied based on the models established on specimen section. Details of pore water phase change were visually displayed and described, and the strength variation of Marshall specimens after freeze-thaw cycle experiment also proved the rationality of simulation. Results show that the pore filling states in permeable pavement have different effects on the temperature field, and the influence of pore air on the temperature distribution for permeable pavement is greater than that of pore water (ice). In particular, weak positions between different pores are more susceptible to freezing stress, leading to stress concentration. Simulation results were also verified by macro experiments. Furthermore, the sustainable service performance of permeable pavement in Sponge City was inevitably affected. Therefore, the study results have certain significance for the development of permeable pavement in freezing and thawing regions, and are conducive to the wider application of Sponge City. HIGHLIGHTS Influence of pore air on the temperature distribution for permeable pavement is greater than that of pore water (ice).; The encounter of low temperature and precipitation will lead to pore water phase change.; Pore water phase change directly causes the pore wall to be subjected to freezing stress.; The freezing stress is greater at the narrow pore, resulting in stress concentration.

Incorporation of Coenzyme Q10 (CoQ10) to the freezing medium provides advantageous effect for sperm cryopreservation in a variety of animal species, yet which has not been tested in giant grouper (Epinephelus lanceolatus). This research was designed to elucidate if CoQ10 could be used as a potential additive to improve giant grouper sperm quality after cryopreservation. After the process of freezing and thawing, various sperm quality parameters including motility, viability, apoptosis, mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS) generation, DNA fragmentation as well as fertilization rate were evaluated with CoQ10 added at concentrations of 0, 25, 50 and 100 μM. Compared to the control group (0 μm), addition of CoQ10 in the medium yielded significantly higher total motility and curvilinear velocity, whereas the progressive motility, straight-line velocity and average path velocity were not differ from each other. An obvious improvement in viability was observed in spermatozoa cryopreserved with 25 and 50 μM CoQ10, while the apoptosis rate in CoQ10 treated groups (25, 50 and 100 μM) exhibited significantly lower values than that of the control. Besides, the production of ROS was significantly decreased with CoQ10 addition groups when compared with the control. In consistent with the improvement in antioxidant defense, CoQ10 supplementation in the medium also enhanced mitochondrial activity and reduced DNA fragmentation. In addition, freezing medium supplemented with CoQ10 also improved the fertilization success, a significantly higher fertilization rate was recorded at the concentration of 50 μM, but this value was not differ from that of 25 μM. Overall, the antioxidant CoQ10 provided an obvious beneficial effect on post-thaw quality of giant grouper spermatozoa. It was concluded that the optimal concentration of CoQ10 is 50 μM in the freezing medium.

Background As a biennial plant, Secale cereale L is usually harvested in the autumn in the northern part of China where the temperature difference between day and night is of great disparity Through the pot experiment, the seedlings were cut to 2, 6 and 10 cm stubble height, and the simulated freeze-thaw (FT) stress (10/− 5 °C) was carried out after 6 days regrowth. The physiological effects of FT with different stubble height were revealed by analyzing the relative water content (RWC), osmotic adjustment substance concentration (soluble sugar and protein), membrane peroxidation (MDA) and catalase (CAT) activity. Results The results demonstrated that under freeze stress (− 5 °C), the content of soluble protein and MDA decreased and the seedlings of 2 cm treatment kept higher level of soluble protein and MDA, while the seedlings of 6 and 10 cm treatments kept higher level of the RWC, soluble sugar content, and CAT activity. After FT stress, the content of soluble sugar and protein, RWC in the 6 cm treatment were higher than those in 2 cm and 10 cm treatments, and the CAT activity in 10 cm treatment was the highest while the MDA content is lower. Conclusion These data suggest that keeping high stubble height is more adaptive for short-term FT stress.

Background Winter carbon loss in northern ecosystems is estimated to be greater than the average growing season carbon uptake and is primarily driven by microbial decomposers. Viruses modulate microbial carbon cycling via induced mortality and metabolic controls, but it is unknown whether viruses are active under winter conditions (anoxic and sub-freezing temperatures). Results We used stable isotope probing (SIP) targeted metagenomics to reveal the genomic potential of active soil microbial populations under simulated winter conditions, with an emphasis on viruses and virus-host dynamics. Arctic peat soils from the Bonanza Creek Long-Term Ecological Research site in Alaska were incubated under sub-freezing anoxic conditions with H218O or natural abundance water for 184 and 370 days. We sequenced 23 SIP-metagenomes and measured carbon dioxide (CO2) efflux throughout the experiment. We identified 46 bacterial populations (spanning 9 phyla) and 243 viral populations that actively took up 18O in soil and respired CO2 throughout the incubation. Active bacterial populations represented only a small portion of the detected microbial community and were capable of fermentation and organic matter degradation. In contrast, active viral populations represented a large portion of the detected viral community and one third were linked to active bacterial populations. We identified 86 auxiliary metabolic genes and other environmentally relevant genes. The majority of these genes were carried by active viral populations and had diverse functions such as carbon utilization and scavenging that could provide their host with a fitness advantage for utilizing much-needed carbon sources or acquiring essential nutrients. Conclusions Overall, there was a stark difference in the identity and function of the active bacterial and viral community compared to the unlabeled community that would have been overlooked with a non-targeted standard metagenomic analysis. Our results illustrate that substantial active virus-host interactions occur in sub-freezing anoxic conditions and highlight viruses as a major community-structuring agent that likely modulates carbon loss in peat soils during winter, which may be pivotal for understanding the future fate of arctic soils' vast carbon stocks.

Amazonian palm berries (açaí, Euterpe oleracea Mart.) are fruits with high nutritional value and antioxidant activity and have aroused the interest of consumers, popularizing fruit pulps enriched with probiotics. Amazonian palm berries (açaí, Euterpe oleracea Mart.) are fruits with high nutritional potential, providing a source of carbohydrates, fibers, proteins, lipids, vitamins, and minerals. Furthermore, açai provides several health benefits, including antioxidant activity. Nutritionally enhanced foods have aroused the interest of consumers, popularizing fruit pulps enriched with probiotics. Probiotics are dietary supplements consisting of live, beneficial microorganisms in the host which improve the intestinal microbiota. The objective of this study was to isolate, identify, and characterize the probiotic potential of an isolated Schleiferilactobacillus harbinensis strain (dubbed Ca12) and provide an optimized bioprocess for its production, using the complete factorial and central rotational compound design to supplement the frozen açai pulp. The isolated strain S. harbinensis Ca12 presented adequate resistance to gastric juice and bile salts, microbial activity against different Candida strains, self-aggregation and coaggregation properties, high adhesion in HT-29 cells, and 35% inhibition of Salmonella in HT-29 cells. When optimized, the cellular biomass production of the S. harbinensis Ca12 strain was approximately 600% higher than the unsupplemented whey, with a production of 3.6 × 1010 CFU mL(−1). The S. harbinensis Ca12 strain’s viability in the creamy and traditional frozen açai pulp was shown to be stable for up to 6 months at 20 °C. The impact of this study involved for the first time the S. harbinensis Ca12 described in the Brazilian cocoa pulp with activity against Candida albicans of clinical importance, creating the potential of a new functional food with important benefits to human health as prevention for candidiasis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00559-3.

Freezing tolerance of triticale is a major trait contributing to its winter hardiness. The identification of genomic regions – quantitative trait loci (QTLs) and molecular markers associated with freezing tolerance in winter hexaploid triticale was the aim of this study. For that purpose a new genetic linkage map was developed for the population of 92 doubled haploid lines derived from ‘Hewo’ × ‘Magnat’ F1 hybrid. Those lines, together with parents were subjected to freezing tolerance test three times during two winter seasons. Plants were grown and cold-hardened under natural fall/winter conditions and then subjected to freezing in controlled conditions. Freezing tolerance was assessed as the plants recovery (REC), the electrolyte leakage (EL) and chlorophyll fluorescence parameters (JIP) after freezing. Three consistent QTLs for several fluorescence parameters, electrolyte leakage and the percentage of the survived plants were identified with composite interval mapping (CIM) and single marker analysis (SMA). The first locus Qfr.hm-7A.1 explained 9 % of variation of both electrolyte leakage and plants recovery after freezing. Two QTLs explaining up to 12 % of variation in plants recovery and shared by selected chlorophyll fluorescence parameters were found on 4R and 5R chromosomes. Finally, main locus Qchl.hm-5A.1 was detected for chlorophyll fluorescence parameters that explained up to 19.6 % of phenotypic variation. The common QTLs located on chromosomes 7A.1, 4R and 5R, clearly indicated physiological and genetic relationship of the plant survival after freezing with the ability to maintain optimal photochemical activity of the photosystem II and preservation of the cell membranes integrity. The genes located in silico in the identified QTLs include those encoding transmembrane helix proteins like potassium channel and phosphoric ester hydrolase involved in response to osmotic stress as well as proteins involved in the regulation of the gene expression, chloroplast RNA processing and pyrimidine salvage pathway. Additionally, our results confirm that the JIP test is a valuable tool to evaluate freezing tolerance of triticale under unstable winter environments.

The aim of the study was to perform histological differentiation of dorsal and ventral musculature of fresh and frozen/thawed carps (Cyprinus carpio). Histological findings of muscle fibres (Myofibra striata) of fresh carps did not show any changes. Single freezing at –10 ºС resulted in extracellular gaps in the central part of some of fibres. After single freezing at –18 ºС, muscle fibres with cell destruction in the central part were identified while the periphery remained intact. Completely destructured and deformed areas of muscle fibres were demonstrated after single freezing at –27 ºС. Double freezing at –10 ºС resulted in shrinkage, extracellular gaps and fragmentation of fibres, while muscle fibres double-frozen at –18 ºС were impaired, degraded and with visible defects. The histological findings in carp muscle, double-frozen at –27 ºС comprised severely deformed muscle fibres with increased extracellular gaps from degraded muscle tissue. On the basis of findings, it could be concluded that double freezing of carps was not an appropriate method of storage and shelf-life extension.

Made available in DSpace on 2022-04-29T08:32:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 This study aimed to describe successful cryopreservation of sperm from maned wolves (Chrysocyon brachyurus). Three ejaculates from 2 maned wolves were collected by digital manipulation of the penis and evaluated subjectively, centrifuged and frozen in BotuCrio® (Botupharma, Botucatu, Brazil) or Tris–yolk egg extender. Spermatozoa were thawed at 37ºC/30s or 70ºC/4s and evaluated for kinetics, morphology, plasma and acrosome membrane integrity, mitochondrial potential, hydrogen peroxide, superoxide anion and lipid peroxidation. From 5 thawed samples, two had sperm total motility >55% (56.0% and 64.0%) and progressive motility ~35% (35% and 40%), both frozen with Tris–yolk egg. Plasma and acrosome membrane integrity decreased and percentage of sperm defects increased post-thawing. We concluded that is possible to freeze spermatozoa from maned wolves using semen collection and processing methods applied for domestic dogs. Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) University of Franca (UNIFRAN) Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University (UNESP)

BACKGROUND AND OBJECTIVES Frozen plasma (FP) is thawed prior to transfusion and stored for ≤5 days at 1-6°C. The effect of temperature excursions on the quality and safety of thawed plasma during 5-day storage was determined. MATERIALS AND METHODS Four plasma units were pooled, split and stored at ≤-18°C for ≤90 days. Test units T30 and T60 were exposed to 20-24°C (room temperature [RT]) for 30 or 60 min, respectively, on days 0 and 2 of storage. Negative and positive control units remained refrigerated or at RT for 5 days, respectively. On Day 5, test units were exposed once to RT for 5 h. Quality assays included stability of coagulation factors FV, FVII, FVIII, fibrinogen and prothrombin time. Bacterial growth was performed in units inoculated with ~1 CFU/ml or ~100 CFU/ml of Serratia liquefaciens, Pseudomonas putida, Pseudomonas aeruginosa or Staphylococcus epidermidis on Day 0. RESULTS Testing results of all quality parameters were comparable between T30 and T60 units (p

Background C. panzhihuaensis is more tolerant to freezing than C. bifida but the mechanisms underlying the different freezing tolerance are unclear. Photosynthesis is one of the most temperature-sensitive processes. Lipids play important roles in membrane structure, signal transduction and energy storage, which are closely related to the stress responses of plants. In this study, the chlorophyll fluorescence parameters and lipid profiles of the two species were characterized to explore the changes in photosynthetic activity and lipid metabolism following low-temperature exposure and subsequent recovery. Results Photosynthetic activity significantly decreased in C. bifida with the decrease of temperatures and reached zero after recovery. Photosynthetic activity, however, was little affected in C. panzhihuaensis. The lipid composition of C. bifida was more affected by cold and freezing treatments than C. panzhihuaensis. Compared with the control, the proportions of all the lipid categories recovered to the original level in C. panzhihuaensis, but the proportions of most lipid categories changed significantly in C. bifida after 3 d of recovery. In particular, the glycerophospholipids and prenol lipids degraded severely during the recovery period of C. bifida. Changes in acyl chain length and double bond index (DBI) occurred in more lipid classes immediately after low-temperature exposure in C. panzhihuaensis compare with those in C. bifida. DBI of the total main membrane lipids of C. panzhihuaensis was significantly higher than that of C. bifida following all temperature treatments. Conclusions The results of chlorophyll fluorescence parameters confirmed that the freezing tolerance of C. panzhihuaensis was greater than that of C. bifida. The lipid metabolism of the two species had differential responses to low temperatures. The homeostasis and plastic adjustment of lipid metabolism and the higher level of DBI of the main membrane lipids may contribute to the greater tolerance of C. panzhihuaensis to low temperatures.

Cryopreservation of spermatogonial stem cells (SSCs) is a useful method for fertility preservation in preadolescent children suffering from cancer. However, SSCs may become damaged during cryopreservation due to the generation of reactive oxygen species (ROS). For this reason, various antioxidant agents have been used to protect SSCs from cryopreservation-induced damages. Recently, it has been reported that miR-30a-5p has antiapoptotic and antioxidant activity. The aim of this study was to assess the antiapoptotic and antioxidant effects of miR-30a-5p mimics in frozen-thawed SSCs. To this end, SSCs were isolated from male BALB/C mice (3-6 days old) and cultivated for 14 days. After the detection of optimum concentration, a miR-30a-5p mimic or miR-30a-5p inhibitor with Lipofectamine was transfected into SSCs and, finally, the cell groups were frozen for 1 week. After thawing, different properties, including cell viability (using MTT), colonization of SSCs (number and diameter of colonies), ROS generation (using DCFH-DA assay), levels of malondialdehyde (MDA) and superoxide dismutase (SOD), and gene expression of Bcl-2 and BAXBax (using quantitative real-time PCR), were investigated. The transfection of SSCs with miR-30a-5p mimics before the freezing-thawing process significantly increased the viability, number, and diameter of SSCs colonies. Also, the miR-30a-5p mimic decreased the levels of ROS production and MDA, but it increased the SOD levels. Moreover, the miR-30a-5p mimic decreased BAX and increased Bcl-2 expression in frozen-thawed SSCs. The transfection of SSCs with the miR-30a-5p mimic can increase cell viability and antioxidant defense, and it can decrease apoptosis during the freezing-thawing process. If SSC is able to produce spermatozoa after the transfection of miR-30a-5p and the freezing-thawing process, it can be suggested as a promising strategy for the cryopreservation of SSCs in prepubertal boys suffering from cancer.

In high-latitude regions, the cold hardiness of buds and canes of grapevine is important for budburst time and yield in the next season. The freezing resistance of buds and canes sampled from six wine grapes currently cultivated in Hokkaido, Japan, all of them grown from autumn to winter, was investigated. A significant difference between the cultivars in their freezing resistance was detected in the buds harvested in winter. In addition, outstanding differences in the lower temperature exotherms (LTE) related to the supercooling ability of tissue cells happened in the winter buds, and there is a close relationship between freezing resistance and LTE detected in the winter buds. This suggests that the supercooling ability of tissue cells in winter buds is strongly related to the freezing resistance. However, detailed electron microscopy exposed that the differences in freezing resistance among cultivars appeared in freezing behavior of leaf primordium rather than apical meristem. This indicated that as the water mobility from the bud apical meristem to the spaces around the cane phloem progressed, the slightly dehydrated cells improved the supercooling ability and increased the freezing resistance.

Single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA-seq (snRNA-seq) allow transcriptomic profiling of thousands of cells from a renal biopsy specimen at a single-cell resolution. Both methods are promising tools to unravel the underlying pathophysiology of glomerular diseases. This review provides an overview of the technical challenges that should be addressed when designing single-cell transcriptomics experiments that focus on glomerulopathies. The isolation of glomerular cells from core needle biopsy specimens for single-cell transcriptomics remains difficult and depends upon five major factors. First, core needle biopsies generate little tissue material, and several samples are required to identify glomerular cells. Second, both fresh and frozen tissue samples may yield glomerular cells, although every experimental pipeline has different (dis)advantages. Third, enrichment for glomerular cells in human tissue before single-cell analysis is challenging because no effective standardized pipelines are available. Fourth, the current warm cell-dissociation protocols may damage glomerular cells and induce transcriptional artifacts, which can be minimized by using cold dissociation techniques at the cost of less efficient cell dissociation. Finally, snRNA-seq methods may be superior to scRNA-seq in isolating glomerular cells; however, the efficacy of snRNA-seq on core needle biopsy specimens remains to be proven. The field of single-cell omics is rapidly evolving, and the integration of these techniques in multiomics assays will undoubtedly create new insights in the complex pathophysiology of glomerular diseases. ispartof: JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY vol:32 issue:8 pages:1838-1852 ispartof: location:United States status: published

Germ cell cryopreservation has been used to preserve many fish species. However, this method has not been established for crustaceans; thus, we attempted to do this herein. The efficiency of slow freezing was compared to vitrification methods for germ cell cryopreservation in two types of marine shrimp, Fenneropenaeus merguiensis and Penaeus monodon. In situ hybridization with a vasa probe was used to identify germ cells. The effects of three cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (GLY), and magnesium chloride (MgCl2), on germ cell viability and recovery rate were compared at three concentrations (5%, 10%, and 15%). The effects of thawing temperature, including 10 and 27 °C, were also investigated. We discovered that 10% DMSO with the vitrification is suitable for preserving the germ cells of F. merguiensis for a long time, whereas 10% GLY with vitrification is suitable for P. monodon. Moreover, the most suitable thawing temperature was 10 °C for both species. This is the first report of germ cell cryopreservation in crustaceans. Thus, we provide evidence that crustacean germ cells can be preserved long-term in liquid nitrogen; this is the first step in the sustainable preservation of crustaceans, especially shrimp.

Our general understanding of plant responses to sub-zero temperatures focuses on mechanisms that mitigate stress to the plasma membrane. The plant cell wall receives comparatively less attention, and questions surrounding its role in mitigating freezing injury remain unresolved. Despite recent molecular discoveries that provide insight into acclimation responses, the goal of reducing freezing injury in herbaceous and woody crops remains elusive. This is likely due to the complexity associated with adaptations to low temperatures. Understanding how leaf cell walls of herbaceous annuals promote tissue tolerance to ice does not necessarily lead to understanding how meristematic tissues are protected from freezing by tissue-level barriers formed by cell walls in overwintering tree buds. In this mini-review, we provide an overview of biological ice nucleation and explain how plants control the spatiotemporal location of ice formation. We discuss how sugars and pectin side chains alleviate adhesive injury that develops at sub-zero temperatures between the matrix polysaccharides and ice. The importance of site-specific cell-wall elasticity to promote tissue expansion for ice accommodation and control of porosity to impede ice growth and promote supercooling will be presented. How specific cold-induced proteins modify plant cell walls to mitigate freezing injury will also be discussed. The opinions presented in this report emphasize the importance of a plant’s developmental physiology when characterizing mechanisms of freezing survival.

Freezing of cell culture supernatant (CCS) is a standard procedure in process development of monoclonal antibody (mAb) platform processes as up- and downstream development are usually separated. In the manufacturing process of mAb, however, freezing is avoided, which poses the question of comparability and transferability from process development to manufacturing. In this case study, mAb CCS from Chinese hamster ovary (CHO) cells is frozen and thawed in a novel active freezing device and subsequently captured by protein A chromatography. Critical quality attributes such as host cell protein (HCP) concentration and soluble mAb dimer shares have been monitored throughout the case study. Furthermore, cryo-concentration of individual proteins was investigated. The main factors that drive cryo-concentration are diffusion and natural convection. Natural convection in freezing processes was found to increase at warmer freezing temperatures and thus slower freezing, leading to higher concentration gradients from top to bottom of a freezing chamber. The freeze concentration was dependent on protein size and correlated to diffusivity, where smaller proteins are exposed to higher cryo-concentration. Our results suggest that as a result of freezing processes, large particles based on mAb and specific host cell proteins (HCPs) expressing a certain affinity to mAbs are formed that have to be removed before purification. This leads to a significant improvement in HCP reduction by the protein A step, when compared with reference samples, where twice as much HCP remained in the eluate. Furthermore, HCP and mAb dimer concentrations in protein A eluate were dependent on the freezing temperature. As a conclusion, CCS should be frozen as rapidly as possible during process development to minimize issues of transferability from process development to manufacturing.

Background Sufficient low temperature accumulation is the key strategy to break bud dormancy and promote subsequent flowering in tree peony anti-season culturing production. Exogenous gibberellins (GAs) could partially replace chilling to accelerate dormancy release, and different kinds of GAs showed inconsistent effects in various plants. To understand the effects of exogenous GA3 and GA4 on dormancy release and subsequent growth, the morphological changes were observed after exogenous GAs applications, the differentially expressed genes (DEGs) were identified, and the contents of endogenous phytohormones, starch and sugar were measured, respectively. Results Morphological observation and photosynthesis measurements indicated that both GA3 and GA4 applications accelerated bud dormancy release, but GA3 feeding induced faster bud burst, higher shoot and more flowers per plant. Full-length transcriptome of dormant bud was used as the reference genome. Totally 124 110 459, 124 015 148 and 126 239 836 reads by illumina transcriptome sequencing were obtained in mock, GA3 and GA4 groups, respectively. Compared with the mock, there were 879 DEGs and 2 595 DEGs in GA3 and GA4 group, 1 179 DEGs in GA3 vs GA4, and 849 DEGs were common in these comparison groups. The significant enrichment KEGG pathways of 849 DEGs highlighted plant hormone signal transduction, starch and sucrose metabolism, cell cycle, DNA replication, etc. Interestingly, the contents of endogenous GA1, GA3, GA4, GA7 and IAA significantly increased, ABA decreased after GA3 and GA4 treatments by LC–MS/MS. Additionally, the soluble glucose, fructose and trehalose increased after exogenous GAs applications. Compared to GA4 treatment, GA3 induced higher GA1, GA3 and IAA level, more starch degradation to generate more monosaccharide for use, and promoted cell cycle and photosynthesis. Higher expression levels of dormancy-related genes, TFL, FT, EBB1, EBB3 and CYCD, and lower of SVP by GA3 treatment implied more efficiency of GA3. Conclusions Exogenous GA3 and GA4 significantly accelerated bud dormancy release and subsequent growth by increasing the contents of endogenous bioactive GAs, IAA, and soluble glucose such as fructose and trehalose, and accelerated cell cycle process, accompanied by decreasing ABA contents. GA3 was superior to GA4 in tree peony forcing culture, which might because tree peony was more sensitive to GA3 than GA4, and GA3 had a more effective ability to induce cell division and starch hydrolysis. These results provided the value data for understanding the mechanism of dormancy release in tree peony.

Until now, very little is known about the ability of adult and deutonymph water mites (Acari, Hydrachnidia) to survive in sub-zero temperatures. Information concerns mainly water mites from vernal astatic waters, and the knowledge has never been experimentally verified. To determine the sensitivity of water mites to freezing, experiments were conducted on (1) the impact of acclimatization, (2) temperature, and (3) duration of freezing on survival, (4) the survival rate of water mites from various types of water bodies, and (5) the survival rate of water mites from different climatic zones. The experiments were carried out in a phytotron chamber, and water mites were placed in containers (10 × 10 × 5 cm) filled with 4/5 of water for 10 specimens each. Water mites were identified to the species level after finishing the experiments. The temperature was lowered 1 °C every hour until the target temperature was reached. After a certain period of freezing (depending on the treatment) the temperature was raised by 1 °C every hour until it reached 4 °C. The time of the experiment was measured from the moment the desired temperature was reached (below 0 °C) until the ice thawed and the temperature of 4 °C was reached again. The highest survival rates had Limnochares aquatica, Piona nodata, Sperchon clupeifer and Lebertia porosa, followed by L. insignis, Hygrobates longipalpis, H. setosus, Limnesia undulatoides, Piona pusilla, Arrenurus globator, Hydrodroma despiciens, Piona longipalpis, Sperchonopsis verrucosa, Unionicola crassipes and Mideopsis crassipes; no specimens of Torrenticola amplexa survived. The following conclusions were drawn: (1) water mites can survive freezing to −2 °C, lower temperatures are lethal for them; (2) they survived better the short period of freezing (24–48 h) than the long period (168 h); (3) resistance to freezing seems to be an evolutionary trait of individual species, only partly related to the living environment; and (4) freezing survival rates are linked to the region of Europe and are much lower in Southern than in Central Europe.

Cryopreservation of embryos is important for long-distance embryo transfer and conservation of genetic resources. Porcine research is important for animal husbandry and biomedical research. However, porcine embryos are difficult to cryopreserve because of their high cytoplasmic lipid content and sensitivity to chilling stress. Vitrification is more efficient than slow freezing, and vitrification is mostly used in embryo cryopreservation. So far, the vitrification process of porcine embryos has been continuously improved, resulting in improved survival rates of warmed embryos and farrowing rates after the transplant procedure. It is worth noting that automatic vitrification has made great progress, which is expected to promote the standardization and application of vitrification. In this article, the vitrification process of porcine embryos at the blastula stage and early development stages is reviewed in detail. In addition, the efficiency of different vitrification systems was compared. In addition, we summarize technology that can improve the survival rate of cryopreserved porcine embryos, such as delipidation methods (including physical delipidation and chemical delipidation) and medium improvements (including chemically defined media and adding antioxidants). Meanwhile, gene expression changes during cryopreservation are also elaborated.

Korpan N.N., Goltsev A.N., Dronov О.I., Bondarovych M.O. Cryoimmunology: Opportunities and challenges in biomedical science and practice. Cryobiology. 2021, Vol.100: 1-11. Autologous and allogeneic cryoimmunological medicine is a brand new branch of biomedical science and clinical practice that examines the features and formation of the immune response to immunogenic properties of normal and malignant biological structures altered by ultralow temperature, as well as specific changes in the structural and functional characteristics of immune cells and tissues after cryopreservation. Cryogenic protein denaturation phenomenon provides important insights into the mechanisms underlying the damage to cryogenic lesions immediately after freeze–thawing sessions in bioscience and medicine applications. The newly formed cryocoagulated protein components (cryomodified protein components) are crucial in cryoimmunology from the perspective of the formation of immunological substances at ultralow temperatures. Dendritic cells and cryocell detritus (cryocell debris) formed in living biological tissue after exposure to ultralow temperature in vivo may be an indication of one of the essential mechanisms involved in the cryoimmunological response of living structures to the impact of ultralow temperature exposure. Hence, the formation of new autologous and allogeneic cryoinduced immunogenic substances is a novel concept in biomedical research globally. Accordingly, this review focuses on issues concerning the peculiarities of the interaction of the immune system with a dominant malignant neoplasm tissue after exposure to subzero temperatures, considering the original cryogenic technical approaches. We present an overview of the state-of-the-art methods of cryoimmunology, and their major developments, past and present. The need for the delineation of structural and functional characteristics of the biological substrates of the immune system after cryopreservation that can be used in adoptive cell therapy, especially in cancer patients, is emphasized.

Reliable transportation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patient samples from a swabbing station to a diagnostics facility is essential for the generation of accurate results. Therefore, cooling or freezing the samples is recommended in case of longer transportation times. In this study, the impact on SARS-CoV-2 detectability by reverse transcriptase PCR was assessed after prolonged unfrozen storage or repetitive freeze-thawing of SARS-CoV-2 samples. SARS-CoV-2–positive patient swabs stored in viral transport medium were exposed to different temperatures (4°C, 25°C, and 35°C) and to repetitive freeze-thawing, to assess the effect of storage conditions on reverse transcriptase PCR detection. SARS-CoV-2 RNA was still reliably detected by reverse transcriptase PCR after 21 days of storage in viral transport medium, even when the samples had been stored at 35°C. The change of Ct value per day was 0.023 to 0.046 (±0.018 to 0.019). In addition, viral RNA was still detected after 15 freeze-thaw cycles, with a change of Ct value of 0.106 to 0.197 (±0.009 to 0.061) per freeze-thaw cycle. However, compared with storage at 4°C, RNA was significantly less detectable when stored at 25°C or 35°C, or after repeated freeze-thawing. The results of this study indicate that viral RNA levels, as measured by reverse transcriptase PCR assays, are significantly, but not substantially, altered by prolonged unrefrigerated storage of up to 21 days at temperatures ranging up to 35°C or after repeated freeze-thaw cycles.

Genetic transformation is a valuable and essential method that provides powerful insights into the gene function of microorganisms and contributes to the construction of engineered bacteria. Here, we developed a novel genetic transformation system to easily knock out a highly GC‐rich gene (74.71% GC) from Burkholderia pyrrocinia JK‐SH007, a biocontrol strain of poplar canker disease. This system revealed a reliable selectable marker (trimethoprim resistance gene, Tmp) and a simplified, efficient transformation method (6,363.64 CFU/μg, pHKT2) that was developed via freeze‐thawing. The knockout recombineering of B. pyrrocinia JK‐SH007 was achieved through a suicide plasmid with a three‐fragment mutagenesis construct. The three‐fragment cassette for mutagenesis was generated by overlap extension and touchdown PCRs and composed of Tmp flanked by GC‐rich upstream and downstream fragments from B. pyrrocinia JK‐SH007. The mutant strain (ΔBpEG), which was verified by PCR, lost 93.3% of its ability to degrade carboxymethyl cellulose over 40 days. Overall, this system may contribute to future research on B. pyrrocinia traits., A novel genetic transformation system was developed to easily knock out a highly GC‐rich gene (74.7% GC) from Burkholderia pyrrocinia JK‐SH007 by homologous recombination with freeze‐thawing transformation.

After a freezing event, it can be challenging to extrapolate levels of freezing damage to plant growth viability based on the presence or absence of symptoms in specific bud tissues. This study investigated the relationship between freezing damage in terminal buds during ecodormancy and their viability during the subsequent growing season. We identified the bud structure that best explained this relationship, and developed a model to explain the changes in bud cold hardiness. Vertical shoots (uprights) of Vaccinium macrocarpon Ait. were sampled in central Wisconsin during Spring of 2018 and 2019. Sets of uprights with terminal buds were subjected to controlled freezing tests, followed by either visual freeze damage evaluation or assessment of shoot viability by growth assays. We determined the Browning Lethal‐Temperature50 (BLT50), as temperature for 50% damage (tissue browning) at each bud structure, and Growth Lethal‐Temperature50 (GLT50) temperature where 50% reduction in growth viability occurred. Two models were constructed to explain: (1) bud structure damage and growth viability, and (2) GLT50's seasonal changes, representing the cold hardiness variations, and environmental factors. The correlation between the BLT50 and GLT50 values was closest for the bud scales and bud axis, indicating the better correspondence between levels of freezing damage with the impact on the growth potential. In addition, the latter was also the most suitable candidate for modeling due to easier damage evaluation. The freezing stress damage of the bud axis explained comparatively best the resulting growth viability. Seasonal changes in GLT50 were best explained by temperature indices based on daily minimum and on maximum temperatures over 10‐day periods. However, among the model components, daily maximum temperatures had the greatest influence on V. macrocarpon cold hardiness changes during ecodormancy.

While most ferns avoid freezing as they have a tropical distribution or shed their fronds, wintergreen species in temperate and boreoalpine ecosystems have to deal with sub-zero temperatures. Increasing evidence has revealed overlapping mechanisms of desiccation and freezing tolerance in angiosperms, but the physiological mechanisms behind freezing tolerance in ferns are far from clear. We evaluated photochemical and hydraulic parameters in five wintergreen fern species differing in their ability to tolerate desiccation. We assessed frond freezing tolerance, ice nucleation temperature and propagation pattern, and xylem anatomical traits. Dynamics of photochemical performance and xanthophyll cycle were evaluated during freeze-thaw events under controlled conditions and, in selected species, in the field. Only desiccation-tolerant species, which possessed a greater fraction of narrow tracheids (18 μm) than sensitive species, tolerated freezing. Frond freezing occurred in the field at -3.4 ± 0.9 °C (SD) irrespective of freezing tolerance, freezable water content, or tracheid properties. Even in complete darkness, maximal photochemical efficiency of photosystem II was down-regulated concomitantly with zeaxanthin accumulation in response to freezing. This was reversible upon re-warming only in tolerant species. Our results suggest that adaptation for freezing tolerance is associated with desiccation tolerance through complementary xylem properties (which may prevent risk of irreversible cavitation) and effective photoprotection mechanisms. The latter includes de-epoxidation of xanthophylls in darkness, a process evidenced for the first time directly in the field.

The effects of multiple cycles of freeze-thaw treatment, combined with pullulanase debranching, on the structural properties and digestibility of lotus seed starch-glycerin monostearin complexes were investigated. The formation and melting of ice crystals during freeze-thaw treatment disrupted the crystalline structure of the starch granules, creating pores which facilitated access of pullulanase to the interior of the granules. Pullulanase debranching increased the free amylose content of the starch, which promoted the formation of starch-lipid complexes, which, in turn, increased the proportion of resistant starch and the overall resistance of the starch to digestive enzyme action. These effects increased with the number of freeze-thaw cycles, because more cycles increased both the disruption of the granule structure and the extent of pullulanase debranching. These findings provide a basis for the preparation of functional foods with low glycemic indices, which have strong potential for management of type II diabetes.

Serological assays are valuable tools to study SARS‐CoV‐2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS‐CoV‐2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS‐CoV‐2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID‐19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293‐E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme‐linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in‐depth characterization of SARS‐CoV‐2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS‐CoV‐2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high‐quality SARS‐CoV‐2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in‐depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID‐19 serologic tests., Spike and RBD were produced in human derived cells hosts. Shake flasks, stirred tank and wave bioreactors were compared. The impact of these different scalable bioprocessing approaches on Spike and RBD production yields and quality attributes was evaluated. An in‐depth characterization of SARS‐CoV‐2 Spike and RBD proteins was performed, namely the antigen s oligomeric state, glycosylation profiles and thermal stability during storage. The correlation of these quality attributes with ELISA performance is discussed.

Candida parapsilosis is a leading cause of invasive mycoses and the major cause of nosocomial fungaemia amongst low and very low birth weight neonates. However, the molecular and physiological characteristics of this fungus remain understudied. To advance our knowledge about the pathobiology of this pathogen, we sought to develop and validate an effective method for chemical transformation of C. parapsilosis. Chemical transformation is the primary procedure for introducing foreign DNA into Candida yeast as it requires no special equipment, although its performance efficacy drops rapidly when the size of the transforming DNA increases. To define optimal conditions for chemical transformation in C. parapsilosis, we selected a leucine auxotroph laboratory strain. We identified optimal cell density for transformation, incubation times, inclusion of specific enhancing chemicals, and size and amounts of DNA fragments that resulted in maximized transformation efficiency. We determined that the inclusion of dimethyl sulfoxide was beneficial, but dithiothreitol pretreatment reduced colony recovery. As a result, the modified protocol led to a 20–55-fold increase in transformation efficiency, depending on the size of the transforming fragment. We validated the modified methodology with prototrophic isolates and demonstrated that the new approach resulted in the recovery of significantly more transformants in 5 of 6 isolates. Additionally, we identified a medium in which transformation competent yeast cells could safely be maintained at −80°C for up to 6 weeks that reduces laboratory work and shortens the overall procedure. These modifications will significantly aid further investigations into the genetic basis for virulence in C. parapsilosis.

Background: Freezing is a common and ancient method for preservation of foods which is applicable both under household and industrial conditions. The objective of the study was to establish histological and microbiological changes in dorsal and abdominal muscles of rainbow trout (Oncorhynchus mykiss) after freezing once and twice. Methods: Forty-five fresh rainbow trout specimens were distributed into three groups of 15 fish each. The first group was subjected to histological and microbiological analysis immediately after delivery at the laboratory. The second fish group was frozen at -18 °C for 15 days, while the third group of fish was frozen at -18 °C for 15 days, thawed and frozen again at -18 °C for 15 days. Data were analyzed using GraphPad InStat 3 software. Results: After freezing once, muscle fibers with intracellular void spaces were observed and retained stable peripheral boundary. In some muscle fibers, the endomysium boundaries were visible and with retained integrity. After freezing twice, damages and deformities were observed resulting in completely destructured muscle fibers. Large void spaces among the muscle fibers and bundles were greatly the result of shrinking and grouping of fibers and the laceration of endomysium and perimysium internum. Total microbial count and Enterobacteriaceae count had no significant differences (p>0.05) between fresh, frozen once, and frozen twice trout. Conclusion: Muscles of rainbow trout (O. mykiss) are histologically damaged to a greater extent after freezing twice and thawing. However, microbiological indicators had no change significantly after freezing once and twice.

The preservative effects of low temperature on biological materials have been long recognised, and cryopreservation is now widely used in biomedicine, including in organ transplantation, regenerative medicine and drug discovery. The lack of organs for transplantation constitutes a major medical challenge, stemming largely from the inability to preserve donated organs until a suitable recipient is found. Here, we review the latest cryopreservation methods and applications. We describe the main challenges—scaling up to large volumes and complex tissues, preventing ice formation and mitigating cryoprotectant toxicity—discuss advantages and disadvantages of current methods and outline prospects for the future of the field.