mRNA Fragmentation Pattern Detected by SHAPE.

The success of messenger RNA (mRNA) vaccines in controlling COVID-19 has warranted further developments in new technology. Currently, their quality control process largely relies on low-resolution electrophoresis for detecting chain breaks. Here, we present an approach using multi-primer reverse transcription sequencing (MPRT-seq) to identify degradation fragments in mRNA products. Using this in-house-made mRNA containing two antigens and untranslated regions (UTRs), we analyzed the mRNA completeness and degradation pattern at a nucleotide resolution. We then analyzed the sensitive base sequence and its correlation with the secondary structure. Our MPRT-seq mapping shows that certain sequences on the 5' of bulge-stem-loop structures can result in preferential chain breaks. Our results agree with commonly used capillary electrophoresis (CE) integrity analysis but at a much higher resolution, and can improve mRNA stability by providing information to remove sensitive structures or sequences in the mRNA sequence design.

Effects of freeze-thaw cycles on soil greenhouse gas emissions: A systematic review.

In the context of global warming, increasingly widespread and frequent freezing and thawing cycles (FTCs) will have profound effects on the biogeochemical cycling of soil carbon and nitrogen. FTCs can increase soil greenhouse gas (GHG) emissions by reducing the stability of soil aggregates, promoting the release of dissolved organic carbon, decreasing the number of microorganisms, inducing cell rupture, and releasing carbon and nitrogen nutrients for use by surviving microorganisms. However, the similarity and disparity of the mechanisms potentially contributing to changes in GHGs have not been systematically evaluated. The present study consolidates the most recent findings on the dynamics of soil carbon and nitrogen, as well as GHGs, in relation to FTCs. Additionally, it analyzes the impact of FTCs on soil GHGs in a systematic manner. In this study, particular emphasis is given to the following: (i) the reaction mechanism involved; (ii) variations in soil composition in different types of land (e.g., forest, peatland, farmland, and grassland); (iii) changes in soil structure in response to cycles of freezing temperatures; (iv) alterations in microbial biomass and community structure that may provide further insight into the fluctuations in GHGs after FTCs. The challenges identified included the extension of laboratory-scale research to ecosystem scales, the performance of in-depth investigation of the coupled effects of carbon, nitrogen, and water in the freeze-thaw process, and analysis of the effects of FTCs through the use of integrated research tools. The results of this study can provide a valuable point of reference for future experimental designs and scientific investigations and can also assist in the analysis of the attributes of GHG emissions from soil and the ecological consequences of the factors that influence these emissions in the context of global permafrost warming.

Low-carbon treatment and remediation of oil sludge in mid-to-high latitude regions: A coupled approach of freeze-thaw and supercritical CO2 extraction.

The oil sludge produced while extracting large oil and gas fields in the middle and high latitude regions has caused serious pollution to the surrounding soil. The key to solving this problem in the future is to unify the remediation of soil and the treatment of oil sludge. This study uses supercritical carbon dioxide(scCO2) technology to construct a low-carbon method, providing a new approach to achieve this goal. The study determines the optimal extraction conditions for black calcareous soil with 15% oil content to be 55 °C, 25 MPa, and 90 min through single factor and response surface experiments. Experiments on the scCO2 extraction coupled with freeze-thaw cycles show that oil sludge with a water content of 10% can improve the extraction efficiency of scCO2 by about 2.69% after less than five freeze-thaw cycles. The study also compares the extraction efficiency of the four soils, with a difference of 6.03% observed under the same conditions. Additionally, we analyze the impact of the extraction process on changes in the properties of the oil and soil in the oil sludge. Comprehensive tests, including scanning electron microscope (SEM), nutrient detection, X-ray powder diffractometer (XRD), fourier transform infrared spectroscopy (FTIR), and Gas Chromatography (GC), have been conducted. Results show that standalone scCO2 extraction can remove up to 98.2% of petroleum hydrocarbons from the oil sludge, while simultaneously causing small changes to the soil microstructure and the crystal structure of the oil sludge. Furthermore, this process does not lead to a significant depletion of key nutrients or the generation of new pollutants.

Varieties of P fractions in biochar-amended reconstructed soils as impacted by freeze-thaw interference.

With low cost and stable chemical properties, biochar has great potential in environmental pollution control and improving soil quality. Reusing tailings slag to reconstruct soil ecosystems and applying amendments such as biochar to enhance soil quality are significant for restoring waste mine lands. Phosphorus (P) as the restrictive nutrition element for plant growth is easily affected by freeze-thaw cycles (FTCs). However, effective information about FTCs on P dynamics in biochar-amended reconstructed soil is scanty. To further understand the effect of FTCs on P in reclaimed mine soils, three reconstructed soils composed of equal brown soil and tailings slag with the respective application of no amendment, 5% biochar and 5% powder both derived from Gleditsia japonica shells (GS), were prepared to evaluate P fraction changes after FTCs. The results indicated that GS biochar increased soil pH, total organic matter (TOM), and moisture content (MC). GS biomass had a similar impact on TOM and MC but decreased soil pH. The two agricultural amendments increased active P and microbial biomass P (MBP) by 46.13%-101.63% and 162.8%-185.7%, which might be largely contributed by soil organic matter and moisture. FTC numbers (0, 3, 6, 10, 15) significantly decreased MBP contents and slightly converted non-labile P into labile fractions while FTC temperature (-20∼5 °C and -10∼5 °C) hardly influenced soil P behavior. In addition, GS conditioners simultaneously enhanced available P content and P fixation potential by soil under FTCs.

Effects of freeze-thaw cycles on nutrient removal from bioretention cells.

There have been numerous summaries of the runoff purification characteristics of bioretention cells in warm climates. However, little has been done on the effects of freeze-thaw cycles (FTCs) that frequently occur in cold regions on bioretention cell performance. Three experimental columns were constructed to simulate the operation of the bioretention cell under the FTCs. The effects of FTCs on the nutrient removal efficiency of different filling bioretention cells were analyzed. The results showed that the ammonia nitrogen (NH4+-N) concentration in the effluent of the wood chip bioretention cell under the T3 conditions (WBCF) (2.35 mg/L) was significantly higher than that of the wood chip bioretention cell operating at room temperature (WBCR) (0.62 mg/L). The effluent NH4+-N concentration of aluminum sludge bioretention cell (ABCF) (0.096 mg/L) under the FTCs was lower than that of WBCF (0.91 mg/L). Under the T3 condition, the effluent nitrate nitrogen (NO3--N) and total nitrogen (TN) concentrations of WBCF (5.33 mg/L and 8.86 mg/L) were higher than those of WBCR (5 mg/L and 6.11 mg/L) at room temperature. Under FTCs conditions, both WBCF and ABCF had high NO3--N removal efficiency (up to 85.87% and 24.75%) at the initial stage of thawing of the filler, and the efficiency gradually decreased with the thawing of the filler. With the increase of FTCs, the NO3--N removal efficiency of WBCF gradually decreased (always higher than 13.6%), while the removal efficiency of ABCF fluctuated wildly (the removal efficiency was primarily negative). The total phosphorus (TP) concentration in the effluent of WBCF (0.11 mg/L) under the T3 conditions was lower than that of WBCR (0.02 mg/L) at room temperature, and the TP concentration of ABCF (0.021 mg/L) in the effluent under the FTCs was slightly lower than that of WBCF (0.031 mg/L). The FTCs have a more significant impact on removing nitrogen pollutants in runoff, but have little effect on phosphorus. Compared with aluminum sludge, wood chips are more suitable for efficient removal of nitrogen pollutants in runoff under the FTCs. The experimental conclusions can provide a reference for the construction of bioretention cells in cold regions.

Variability of green infrastructure performance due to climatic regimes across Sweden.

In the context of increasing urbanization and global warming, there is a growing interest in the implementation of green infrastructure (GI) across different climates and regions. Identifying an appropriate GI design criteria is essential to ensure that the design is tailored to satisfy local environmental requirements. This article aims to compare the hydrological performance of GI facilities in eleven Swedish cities by isolating the effect of climatic conditions using an identical GI design configuration. Long-term simulations based on 23-years of meteorological time-series were used as inputs for the Storm Water Management Model (SWMM) with Low Impact Development (LID) controls representing two types of facilities: a biofilter cell (BC) and a green roof. (GR). Large differences in potential annual and seasonal runoff retention were found between locations, driven mainly by the extent of winter/spring season, and the distribution of precipitation patterns (for BCs) and the sequence of rainy days-dry periods and evapotranspiration rates (for GRs). Winter/spring and summer demonstrated the highest/lowest differences between the seasons, results that suggest that implications for design might be aligned to the spatio-temporal distribution of precipitation patterns, and runoff regimes generated by snowmelt and rain-on-snow events, in locations where snowmelt represent high portion of runoff generation.

Effects of biochar and straw application on the soil structure and water-holding and gas transport capacities in seasonally frozen soil areas.

To explore the effects of different regulation modes on the soil structure and gas transport characteristics in seasonal permafrost regions, freeze-thaw cycles (FTCs) were used as boundary conditions and three typical soils on the Songnen Plain were used: black soil, baijiang soil and meadow soil. Four treatments were established: biochar addition (B1), straw addition (S1), biochar combined with straw addition (B1S1) and an untreated control (CK). The changes in the proportion of soil water-stable aggregates, total soil porosity (TP), soil water characteristic curves (SWRCs), soil dissolved organic carbon (DOC) and soil air permeability (PL) were analyzed. The results showed that biochar and straw influenced the structure of the three soil types. The proportions of large (2-0.5 mm) and medium (0.5-0.25 mm) aggregates increased significantly. The soil aggregate stability indexes of the treated soils were better than those of the CK, and the three-phase ratios of the treated soils were closer to ideal. The different treatments had particularly obvious effects on the black soil; the generalized soil structure index (GSSI) values reached 95.59, 94.36 and 98.74 in the B1, S1 and B1S1 treatments, respectively. An interaction effect was observed between biochar and straw. B1S1 had a stronger effect than the other treatments, and the soil water holding capacity was significantly improved (FC = 0.317 cm3 cm-3). Under the B1S1 treatment, the DOC contents in black soil, baijiang soil and meadow soil were 160.78 mg/kg, 272.828 mg/kg and 271.912 mg/kg, respectively. Moreover, biochar and straw combined effectively reduced PL fluctuations under FTCs and improved the long-term stability of the soil structure. These results can aid in rational straw and biochar use to achieve comprehensive agricultural waste utilization.

Pregnancy outcome of freeze thaw cycles of polycystic ovary syndrome patients regarding the anti-Müllerian hormone percentile.

Patients with polycystic ovary syndrome display increased levels of anti-Müllerian hormone. Frozen-thawed embryo transfer (FET) prevents ovarian hyperstimulation and results in better pregnancy outcome in PCOS patients. Therefore, we aimed to evaluate the effect of serum AMH levels on the pregnancy outcome of FET cycles in PCOS patients. 110 infertile women with PCOS who were recommended for embryo cryopreservation followed by FET. The patients' AMH levels were evaluated, and the age-related AMH percentiles were determined. The patients were then grouped according to AMH percentiles, namely, 75th-90th percentile (Group 1) and higher than the 90th percentile (Group 2).A total of 110 PCOS patients who conceived in Frozen Embryo Transfer (FET)-In-vitro Fertilisation (IVF) cycles were included in this study. The preterm delivery rates in FET cycles were higher in the group of PCOS patients with AMH levels greater than the 90th percentile than in patients in the 75th-90th percentile group (50% vs 28.8%, p = .024). In conclusion, PCOS patients with AMH levels higher than the 90th percentile had substantially higher preterm delivery rates than those with AMH levels at the 75th-90th percentile, suggesting the need for closer follow-up. Further studies are needed to elucidate the underlying mechanisms behind this correlation.IMPACT STATEMENTWhat is already known on this subject? The association of AMH levels with the risk of adverse pregnancy outcomes has been previously investigated. In women with PCOS, substantially elevated AMH levels were significantly associated with preterm birth.What do the results of this study add? Results showed that the PCOS patients with higher AMH levels and underwent assisted reproductive treatment demonstrated an increased risk of preterm labour than the PCOS patients with lower AMH levels.What are the implications of these findings for clinical practice and/or further research? In women with PCOS, substantially elevated AMH levels were significantly related to preterm birth, suggesting the need for closer follow-up in this population and the need for further studies to elucidate the underlying mechanisms behind this correlation.

Do freeze-thaw cycles affect the cadmium accumulation, subcellular distribution, and chemical forms in spinach (Spinacia oleracea L.)?

To date, although there are many studies investigating the toxicity of heavy metal to plant, little research exists in the seasonal freeze-thaw (FT) regions where FT cycles often happen during the plant growing process. To reveal the adaptive mechanisms of plants to the combination stresses of cadmium (Cd) and FT, the Cd accumulation, subcellular distribution, chemical forms, and antioxidant enzyme activity (peroxidase (POD)) were investigated in spinach (Spinacia oleracea L.) growing under different soil Cd levels (i.e., 0.10 mg Cd kg-1 soil (low), 1.21 mg Cd kg-1 soil (medium), and 2.57 mg Cd kg-1 soil (high)). Compared to the non-freeze-thaw (NFT) treatments, higher Cd concentrations in the root and lower translocation factors from root to leaf were found for the plants experiencing FT cycles. FT significantly decreased the Cd concentrations in the leaves under the low- and medium-Cd treatments, while similar values were found for the high-Cd treatments. Generally, FT could decrease the concentrations and proportions of Cd stored in the cell wall and soluble fractions and increase them in the organelle fractions for the medium- and high-Cd treatments, while opposite tendency was found for the low-Cd treatments. Moreover, larger Cd amounts in the inorganic and water-soluble forms were found for the low- and medium-Cd treated plants under FT, while lower values were found for the high-Cd treatments. Additionally, POD, which presented higher activities at the low- and medium-Cd treatments and lower activities at the high-Cd treatments under FT, were also significantly influenced by the Cd × FT interaction. This study indicated that FT could significantly change the accumulations of Cd in plant, and it provided a new insight into the Cd accumulation by plants in the seasonal FT region.

Temperatures and precipitation affect vegetation dynamics on Scandinavian extensive green roofs.

Standard succulent vegetation mixes developed mostly in temperate climates are being increasingly used on green roofs in different climate zones with uncertain outcome regarding vegetation survival and cover. We investigated vegetation on green roofs at nine temperate, cold, and/or wet locations in Norway and Sweden covering wide ranges of latitude, mean annual temperature, annual precipitation, frequencies of freeze-thaw cycles, and longest annual dry period. The vegetation on the roofs were surveyed in two consecutive years, and weather data were compiled from meteorological databases. At all sites we detected a significant decline in species compared to originally intended (planted/sown) species. Both the survival rate and cover of the intended vegetation were positively related to the mean annual temperature. Contrary to a hypothesis, we found that intended vegetation cover was negatively rather than positively related to mean annual precipitation. Conversely, the unintended (spontaneous) vegetation was favoured by high mean annual precipitation and low mean annual temperature, possibly by enabling it to colonize bare patches and outcompete the intended vegetation. When there is high mortality and variation in cover of the intended vegetation, predicting the strength of ecosystem services the vegetation provides on green roofs is difficult. The results highlight the needs for further investigation on species traits and the local factors driving extinction and colonizations in order to improve survivability and ensure a dense vegetation throughout the successional stages of a green roof.

Effect of Freeze-Thaw Cycles on the Oxidation of Protein and Fat and Its Relationship with the Formation of Heterocyclic Aromatic Amines and Advanced Glycation End Products in Raw Meat.

The aim of this research was to investigate the effect of the number of freeze-thaw cycles (0, 1, 3, 5, and 7) on porcine longissimus protein and lipid oxidation, as well as changes in heterocyclic aromatic amines (HAAs) and advanced glycation end products (AGEs) and their precursors. We analyzed the relationship among HAAs, AGEs, oxidation, and precursors and found the following results after seven freeze-thaw cycles. The HAAs, Norharman and Harman, were 20.33% and 16.67% higher, respectively. The AGEs, Nε-carboxyethyllysine (CEL) and Nε-carboxymethyllysine (CML), were 11.81% and 14.02% higher, respectively. Glucose, creatine, and creatinine were reduced by 33.92%, 5.93%, and 1.12%, respectively after seven freeze-thaw cycles. Norharman was significantly correlated with thiobarbituric acid reactive substances (TBARS; r2 = 0.910) and glucose (r2 = -0.914). Harman was significantly correlated to TBARS (r2 = 0.951), carbonyl (r2 = 0.990), and glucose (r2 = -0.920). CEL was correlated to TBARS (r2 = 0.992) and carbonyl (r2 = 0.933). These changes suggest that oxidation and the Maillard reaction during freeze-thaw cycles promote HAA and AGE production in raw pork.

Annual ecosystem respiration is resistant to changes in freeze-thaw periods in semi-arid permafrost.

Warming in cold regions alters freezing and thawing (F-T) of soil in winter, exposing soil organic carbon to decomposition. Carbon-rich permafrost is expected to release more CO2 to the atmosphere through ecosystem respiration (Re) under future climate scenarios. However, the mechanisms of the responses of freeze-thaw periods to climate change and their coupling with Re in situ are poorly understood. Here, using 2 years of continuous data, we test how changes in F-T events relate to annual Re under four warming levels and precipitation addition in a semi-arid grassland with discontinuous alpine permafrost. Warming shortened the entire F-T period because the frozen period shortened more than the extended freezing period. It decreased total Re during the F-T period mainly due to decrease in mean Re rate. However, warming did not alter annual Re because of reduced soil water content and the small contribution of total Re during the F-T period to annual Re. Although there were no effects of precipitation addition alone or interactions with warming on F-T events, precipitation addition increased total Re during the F-T period and the whole year. This decoupling between changes in soil freeze-thaw events and annual Re could result from their different driving factors. Our results suggest that annual Re could be mainly determined by soil water content rather than by change in freeze-thaw periods induced by warming in semi-arid alpine permafrost.

Lead immobilization processes in soils subjected to freeze-thaw cycles.

Soil freeze-thaw cycles (FTCs) change the physical and chemical properties of soils; however, information is limited about the consequences for heavy metal sorption and desorption. Lead (Pb) sorption isotherms and successive desorption tests were measured for three soils from North China (Chestnut, Lou and Black), following multiple freeze-thaw cycles (0, 1, 3, 6 and 9 FTCs) of -5 °C for 12 h and then +5 °C for 12 h. Lead adsorption dominated the sorption processes for all soils, and sorption capacity increased with additional FTCs. The Freundlich affinity parameter of soils for Pb sorption (i.e. A; Lß mmol1-ß kg-1), was linearly correlated with carbonate content for soils with multiple FTCs. The effects of FTCs on lead adsorption may be more dependent on carbonate and clay contents than organic matter (OM), cation exchange capacity (CEC) and amorphous iron content. Repeated FTCs increased the pH of soil solutions at applied Pb concentrations >1.4 mmol L-1, which could facilitate formation of inner-sphere complexes of Pb in studied soils. Cation exchange, a weak association, could occupy specific adsorption sites with increasing Pb doses in soils and it can also be facilitated by FTCs. Our results demonstrate the great potential for increasing Pb immobilization with repeated FTCs, by facilitating the formation of both inner-sphere and outer-sphere complexes. Hence, these findings provide useful information on Pb immobilization in contaminated soils that undergo frequent FTCs and offer an additional insight into predicting Pb behavior in cold and freezing environments like the polar regions.

Small-Baseline Approach for Monitoring the Freezing and Thawing Deformation of Permafrost on the Beiluhe Basin, Tibetan Plateau Using TerraSAR-X and Sentinel-1 Data.

The dynamic changes of the thawing and freezing processes of the active layer cause seasonal subsidence and uplift over a large area on the Qinghai-Tibet Plateau due to ongoing climate warming. To analyze and investigate the seasonal freeze-thaw process of the active layer, we employ the new small baseline subset (NSBAS) technique based on a piecewise displacement model, including seasonal deformation, as well as linear and residual deformation trends, to retrieve the surface deformation of the Beiluhe basin. We collect 35 Sentinel-1 images with a 12 days revisit time and 9 TerraSAR-X images with less-than two month revisit time from 2018 to 2019 to analyze the type of the amplitude of seasonal oscillation of different ground targets on the Beiluhe basin in detail. The Sentinel-1 results show that the amplitude of seasonal deformation is between -62.50 mm and 11.50 mm, and the linear deformation rate ranges from -24.50 mm/yr to 5.00 mm/yr (2018-2019) in the study area. The deformation trends in the Qinghai-Tibet Railway (QTR) and Qinghai-Tibet Highway (QTH) regions are stable, ranging from -18.00 mm to 6 mm. The InSAR results of Sentinel-1 and TerraSAR-X data show that seasonal deformation trends are consistent, exhibiting good correlations 0.78 and 0.84, and the seasonal and linear deformation rates of different ground targets are clearly different on the Beiluhe basin. Additionally, there are different time lags between the maximum freezing uplift or thawing subsidence and the maximum or minimum temperature for the different ground target areas. The deformation values of the alpine meadow and floodplain areas are higher compared with the alpine desert and barren areas, and the time lags of the freezing and thawing periods based on the Sentinel-1 results are longest in the alpine desert area, that is, 86 days and 65 days, respectively. Our research has important reference significance for the seasonal dynamic monitoring of different types of seasonal deformation and the extensive investigations of permafrost in Qinghai Tibet Plateau.

Effects of Cyclic Freeze-Thaw on the Steel Bar Reinforced New-To-Old Concrete Interface.

Frost damage of concrete has significant effects on the safety and durability of concrete structures in cold regions, and the concrete structures after repair and reinforcement are still threatened by cyclic freezing and thawing. In this study, the new-to-old concrete interface was reinforced by steel bar. The shear strength of the new-to-old concrete interface was tested after the new-to-old combination was subjected to cyclic freeze-thaw. The effects of the diameter of the steel bar, the compressive strength of new concrete, the number of freeze-thaw cycles and the freezing temperatures on the shear properties of new-to-old concrete interface were studied. The results showed that, in a certain range, the shear strength of the interface was proportional to the diameter of the steel bar and the strength of the new concrete. Meanwhile, the shear strength of the reinforced interface decreased with the decreasing of the freezing temperature and the increasing of the number of freeze-thaw cycles.

Fetal bovine serum-free cryopreservation methods for clinical banking of human adipose-derived stem cells.

The use of fetal bovine serum (FBS) as a cryopreservation supplement is not suitable for the banking of mesenchymal stem cells (MSCs) due to the risk of transmission of disease as well as xenogeneic immune reactions in the transplanted host. Here, we investigated if human serum albumin (HSA), human serum (HS), or knockout serum replacement (KSR) can replace FBS for the cryopreservation of MSCs. In addition, we examined the characteristics of MSCs after multiple rounds of cryopreservation. Human adipose-derived stem cells (ASCs) cryopreserved with three FBS replacements, 9% HSA, 90% HS, or 90% KSR, in combination with 10% dimethyl sulfoxide (Me2SO) maintained stem cell properties including growth, immunophenotypes, gene expression patterns, and the potential to differentiate into adipogenic, osteogenic, and chondrogenic lineages, similar to ASCs frozen with FBS. Moreover, the immunophenotype, gene expression, and differentiation capabilities of ASCs were not altered by up to four freeze-thaw cycles. However, the performance of three or four freeze-thaw cycles significantly reduced the proliferation ability of ASCs, as indicated by the longer population doubling time and reduced colony-forming unit-fibroblast frequency. Together, our results suggest that HSA, HS, or KSR can replace FBS for the cryopreservation of ASCs, without altering their stemness, and should be processed with no more than two freeze-thaw cycles for clinical approaches.

Prolonged culture of blastocysts after thawing as a tool for improving prediction of success.

PURPOSE: A few years ago, we started to use a new freeze-thaw protocol for the frozen embryo transfer cycles. Instead of thawing the embryos 2-4 h prior to the transfer, we started thawing the embryos 20-22 h prior to the transfer. The aim of this study was to compare the pregnancy rate in cases of embryos that continued to develop in the post-thawing culture to that of embryos that did not. METHODS: A retrospective cohort study of blastocyst freeze/thaw cycles vitrified on day 5, thawed and transferred after 20-22 h in the culture, between January 2012 and December 2016. RESULTS: A total of 375 patients were included in the analysis. Two hundred twenty-eight embryos graded as good, 87 graded as fair, and 60 graded as poor embryos were transferred. The clinical pregnancy rate (50% vs. 19.5% vs 3.3% p < 0.01) and the ongoing pregnancy rate (38.5% vs. 13.6% vs 1.7% p < 0.01) were higher in cases of good embryo quality compared with fair and poor-quality embryos, respectively. For good embryos, progressing to a better grade during the culture did not change the clinical pregnancy rate (51.3% vs. 46.2% p = NS) or the ongoing pregnancy rate (38.5% vs. 37.5% p = NS). For fair embryos, progressing to a better grade during the culture resulted in a higher clinical pregnancy rate (25.4% vs 9% p = 0.05). CONCLUSIONS: The development of the fair embryos in the culture has a highly positive impact on the pregnancy rate and this factor should be taken into consideration before deciding how many embryos to transfer.

Effect of multiple freeze-thaw cycles on selected biochemical serum components.

BACKGROUND: To maintain the best performance a frozen serum sample should be thawed once to prevent repeated freeze-thaw cycles. Archival biobanks often have one tube of a sample available, causing repeated freeze-thaw cycles when the sample is used in multiple research projects. In this study, we investigated potential effects of freeze-thaw cycles on several biochemical components in serum. METHODS: Serum from 40 fasting donors of both genders, aged 30-60 years, were frozen at -25 °C. Aliquots of the 40 different samples went through 1, 2, 3, 4, 5 and 10 thaws, respectively. They were analyzed after 3 month of storage for 15 serum components including electrolytes and metabolites, proteins and enzymes, lipids, hormones and vitamins. One-way analyses of variance (ANOVA) with repeated measurements and equivalence tests were used to examine differences in component levels. RESULTS: Albumin, aspartate-aminotransferase (ASAT), cholesterol, creatinine, C-reactive protein, glucose, immunoglobulin G, potassium, testosterone, triglycerides, urea and vitamin B12 levels did not show significant difference for pairwise comparisons after 10 repeated thaws. Although albumin, ASAT, bilirubin, potassium, sodium, testosterone and thyroid stimulating hormone (TSH) showed overall statistically significant changes in serum levels, only bilirubin, sodium and TSH were significant for the pairwise comparisons investigated. Clinical significance were shown for albumin, ASAT, bilirubin, sodium and testosterone. CONCLUSIONS: Twelve components (albumin, ASAT, cholesterol, creatinine, C-reactive protein, glucose, immunoglobulin G, potassium, testosterone, triglycerides, urea and vitamin B12) were robust to 10 repeated thaws compared to baseline level. Three components (bilirubin, sodium and TSH) showed statistical significant difference for pairwise comparisons, however, TSH was not clinically affected.

Growth of high-elevation Cryptococcus sp. during extreme freeze-thaw cycles.

Soils above 6000 m.a.s.l. are among the most extreme environments on Earth, especially on high, dry volcanoes where soil temperatures cycle between -10 and 30 °C on a typical summer day. Previous studies have shown that such sites are dominated by yeast in the cryophilic Cryptococcus group, but it is unclear if they can actually grow (or are just surviving) under extreme freeze-thaw conditions. We carried out a series of experiments to determine if Cryptococcus could grow during freeze-thaw cycles similar to those measured under field conditions. We found that Cryptococcus phylotypes increased in relative abundance in soils subjected to 48 days of freeze-thaw cycles, becoming the dominant organisms in the soil. In addition, pure cultures of Cryptococcus isolated from these same soils were able to grow in liquid cultures subjected to daily freeze-thaw cycles, despite the fact that the culture medium froze solid every night. Furthermore, we showed that this organism is metabolically versatile and phylogenetically almost identical to strains from Antarctic Dry Valley soils. Taken together these results indicate that this organism has unique metabolic and temperature adaptations that make it able to thrive in one of the harshest and climatically volatile places on Earth.

Influence of time, storage temperature and freeze/thaw cycles on the activity of digestive enzymes from gilthead sea bream (Sparus aurata).

In this study, we tested the effects of long-term storage (2 years) at -20 °C and short-term storage (several hours) in ice and freeze/thaw cycles on the activities of pancreatic, gastric and intestinal (brush border and cytosolic) digestive enzymes in a teleost fish species. The results revealed a significant lose in activity of pancreatic (trypsin, chymotrypsin, total alkaline proteases and α-amylase) and intestinal cytosolic (leucine-alanine peptidase) enzymes between 140 and 270 days of storage at -20 °C, whereas in contrast, the activity of all the assayed brush border enzymes remained constant during the first 2 years of storage at -20 °C. During short-term storage conditions, the most stable enzymes assayed were those of the enterocytes of the brush border, which did not show any change in activity after being held for 5 h in ice. Five freezing and thawing cycles did not affect the activity of the intestinal brush border enzymes and the cytosolic ones, whereas the activity of trypsin, α-amylase and bile-salt-activated lipase was significantly affected by the number of freezing and thawing cycles. No changes in pepsin activity were found in samples exposed to 1 and 2 freezing and thawing cycles.