Effect of storage temperature on gentamicin release from antibiotic-coated bone chips

Freezing is the most common method for storing bones until use in skeletal reconstruction. However, the effect of freezing on antibiotic delivery from antibiotic-coated bone has not been evaluated. In this study, we compared antibiotic delivery in vitro from gentamicin-coated human bone stored at different temperatures. Bone chips obtained from human femur heads were chemically cleaned and mixed with gentamicin sulfate. Samples were stored for 4 months at ?20 °C, 4 months at ?80 °C, or evaluated immediately without freezing. Antibiotic release from the bone chips was measured using Bacillus subtilis as an indicator strain. Zones of inhibition and rates of gentamicin release were similar in all three groups. Storage at ?20 and ?80 °C for bone allografts has no effect on gentamicin release from chemically cleaned bone chips.     

Modification of the intracellular sugar content alters the incidence of freeze-induced membrane lesions of protoplasts isolated from Arabidopsis thaliana leaves

Sugar content and freezing tolerance of protoplasts of Arabidopsis thaliana leaves were manipulated by incubating seedlings in a sucrose solution before protoplast isolation. Incubation in a 400 mM sucrose solution at 2 °C in the dark increased their freezing tolerance equivalent to that achieved after a conventional cold acclimation at 2 °C. The increased freezing tolerance was due to a decrease in the incidence of freeze‐induced lesions: expansion‐induced lysis (EIL) between ?2 and ?4 °C and loss of osmotic responsiveness (LOR) between ?5 and ?12 °C. The concentration of sucrose in the incubation medium required to minimize the incidence of the lesions was substantially different: 10–35 mM for EIL and 30–400 mM for LOR. Incubation in the sucrose solution at 23 °C decreased LOR only at ?5 and ?6 °C but less than that incubated at 2 °C, and there was no effect on EIL. Incubation in sorbitol solutions at 2 °C also decreased LOR at ?5 and ?6 °C but much less than in the sucrose solution. These results suggest that low concentrations of sucrose act as a metabolic substrate for the low‐temperature‐induced alterations required for the amelioration of EIL and, at higher concentrations, sucrose has a direct cryoprotective effect to minimize LOR.     

Rate and extent of enzymatic lipolysis at subfreezing temperatures

Little attention has been given to the effects that various freezing treatments have on rates of enzyme-catalyzed reactions in frozen systems and to the relationship between subfreezing temperatures and the ultimate extent to which a given reaction proceeds. Both of these aspects were explored using a model system consisting of lipase in an emulsion of tributyrin in water. The ultimate extent to which tributyrin was hydrolyzed decreased from 5.4% at ?2 °C to 4.0% at ?12 °C. Hydrolysis proceeded almost to completion at temperatures above 0 °C. Rapid freezing to ?80 °C produced a substantially slower initial reaction rate at ?8 °C than rapid freezing to ?20 °C, or than slow freezing, regardless of the temperature nadir.     

Comparison of the cold hardiness of two larval Lepidoptera (Noctuidae)

Abstract Diapause larvae of the European corn borer (Ostrinia nubilalis (Hubn.)) and the related Mediterranean noctuid Sesamia cretica Led. possess sufficient supercooling ability to avoid freezing over their normal environmental temperature ranges. In progressive chilling experiments (10 days acclimation at each 5° step in the temperature range from 15 to ?5°C), mean supercooling points (measured at a cooling rate of 0.1°C min^?1) were lowered from ?20.4°C at 15°C to ?24.0°C at 5°C (lower lethal temperatures: c.?28°C) in O.nubilalis, compared with ?15.0 to ?17.2°C (lower lethal temperatures: ?15 to ?17°C respectively) in S.cretica. Concentrations of glycerol and trehalose determined by gas chromatography of whole body extracts were consistently higher in the former than in the latter species at both 15 and 5°C, and may be responsible for the deeper supercooling in O.nubilalis larvae. Acclimation to 5°C increased glycerol levels in O. nubilalis extracts compared with 15°C, and this was enhanced in larvae exposed for a further 10 days at each of 0 and ?5°C (glycerol being 438μmol ml^?1 body water). Haemolymph glycerol concentrations showed a similar pattern to whole body extracts in this species. Fat body glycogen was reduced during low temperature acclimation in both species. Body water contents did not change with acclimation in O.nubilalis, whilst S.cretica, containing significantly more water, lost c.7% during acclimation from 15 to 5°C. Haemolymph osmolalities increased during acclimation, especially in Ostrinia larvae, probably as a result of the accumulation of cryoprotectants. The majority of O.nubilalis larvae survived freezing under the conditions of the cooling experiments, whilst larvae of S.cretica did not, thereby confirming an element of freezing tolerance in the former.     

Freezing mammalian embryos: A review of the techniques

This article reviews the literature on freezing mammalian oocytes and embryos, with emphasis on embryos of domestic animals. Mammalian embryos must be stored in a quiescent state to retain viability for long periods. This has been accomplished by freezing and storing the embryos at ?196°C. To freeze embryos, a cryoprotectant like dimethyl sulfoxide (DMSO) or glycerol was required, slow cooling (0.1 to 2.0°C/min) and warming (1 to 50°C/min) rates were used, enucleation or seeding the freezing medium was a necessity, and stepwise addition and removal of the cryoprotectant at room temperature seemed to be beneficial. Using the above parameters embryos have been frozen and stored at ?196°C for several years and upon thawing and transfer to a suitable recipient, viable offspring have developed. Initially embryo viability was low after freezing-thawing, but with refinement of freezing-thawing techniques has increased sufficiently so that freezing embryos is no longer a laboratory technique, but is applicable to field use.     

Sensitivity of pigmented mucosa and skin to freezing injury.

Pigmented areas of canine skin and oral mucosa were subjected to freezing in situ at various temperatures for the purpose of investigating possible differences in the sensitivity of epidermal cells to cold injury. Destruction of melanocytes occurred in the range of ?4 to ?7 °C, while squamous cells resisted injury even at ?20 °C. Replacement of the lost pigmented cells occurred from the normal tissue at the periphery of the injured area. The experiments suggest that selective destruction of pigmented lesions in clinical conditions may be achieved by freezing tissue to ?4 to ?7 °C or colder, but not to exceed ?20 °C in order to avoid destruction of squamous cells. The experiments also support wider use of cryosurgery for pigmented lesions of the skin and oral cavity.     

Effects of freeze-thaw on the biomechanical and structural properties of the rat Achilles tendon

Rodent models are commonly used to investigate tendon healing, with the biomechanical and structural properties of the healed tendons being important outcome measures. Tendon storage for later testing becomes necessary when performing large experiments with multiple time-points. However, it is unclear whether freezing rodent tendons affects their material properties. Thus the aim of this study was to determine whether freezing rat Achilles tendons affects their biomechanical or structural properties. Tendons were frozen at either −20 °C or −80 °C directly after harvesting, or tested when freshly harvested. Groups of tendons were subjected to several freeze-thaw cycles (1, 2, and 5) within 3 months, or frozen for 9 months, after which the tendons were subjected to biomechanical testing. Additionally, fresh and thawed tendons were compared morphologically, histologically and by transmission electron microscopy. No major differences in biomechanical properties were found between fresh tendons and those frozen once or twice at −20 °C or −80 °C. However, deterioration of tendon properties was found for 5-cycle groups and both long-term freezing groups; after 9 months of freezing at −80 °C the tear resistance of the tendon was reduced from 125.4 ± 16.4N to 74.3 ± 18.4N (p = 0.0132). Moreover, tendons stored under these conditions showed major disruption of collagen fibrils when examined by transmission electron microscopy. When examined histologically, fresh samples exhibited the best cellularity and proteoglycan content of the enthesis. These properties were preserved better after freezing at −80 °C than after freezing at −20 °C, which resulted in markedly smaller chondrocytes and less proteoglycan content. Overall, the best preservation of histological integrity was seen with tendons frozen once at −80 °C. In conclusion, rat Achilles tendons can be frozen once or twice for short periods of time (up to 3 months) at −20 °C or −80 °C for later testing. However, freezing for 9 months at either −20 °C or −80 °C leads to deterioration of certain parameters.     

False codling moth Thaumatotibia leucotreta (Lepidoptera,Tortricidae) larvae are chill‐susceptible

Abstract This study reports on the low temperature tolerance and cold hardiness of larvae of false codling moth, Thaumatotibia leucotreta. We found that larvae have mean critical thermal minima (lower limits of activity) of 6.7°C which was influenced by feeding status. The effects of low temperature exposure and duration of exposure on larval survival were assessed and showed that the temperature at which 50% of the population survives is ?11.5 ± 0.3°C after 2 h exposure. The supercooling point (SCP, i.e., freezing temperature) was investigated using a range of cooling rates and under different conditions (feeding and hydration status) and using inoculative freezing treatments (in contact with water or orange juice). The SCP decreased significantly from ?15.6°C to ?17.4°C after larvae were fasted for 24 h. Twenty‐four hour treatments at either high or low relative humidity (95.9% or 2.4%) also significantly decreased SCP to ?17.2°C and ?18.2°C respectively. Inoculative freezing (by water contact) raised SCP from ?15.6°C to ?6.8°C which could have important implications for post‐harvest sterilization. Cooling rates did not affect SCP which suggests that there is limited phenotypic plasticity of SCP during the larval life‐stage, at least over the short time‐scales investigated here. In conclusion, larvae of T. leucotreta are chill‐susceptible and die upon freezing. These results are important in understanding this pest's response to temperature variation, understanding pest risk status and improving post‐harvest sterilization efficacy.     

Cold,freezing, and desiccation tolerance of the limpet Acmaea digitalis (Eschscholtz)

Cold, freezing, and desiccation tolerance were examined in the limpet, Acmaea digitalis (Eschscholtz). Fifty percent of the experimental population survived freezing at temperatures between ?10 ° and ?12 °C for at least 24 hr. In this temperature range, 60–80 % of the body water was frozen. The LD₅₀ for water loss by desiccation was 76.5% ± 1.3_δ. At 70–80% body water loss, solutes were concentrated between 350 and 500%. Some limpets failed to survive immersion in 450% seawater for 6 hr (100% scawater = salinity of 31‰), suggesting that much of the damage from freezing and desiccation was from an increased solute concentration. No glycerol could be detected in extracts of the foot and the visceral mass of A. digitalis during winter, confirming similar results in studies carried out on other intertidal molluscs.     

Denaturation of Soybean Protein by Freezing

When a solution of soybean acid-precipitated or 11S protein was frozen and stored at ?1 to ?5°C, the protein became partially insoluble after thawing. Ultracentrifugation and disc-electrophoresis of freeze-stored 11S protein solution after removing insoluble components revealed that new components which may be aggregates or associates of the 11S component were formed. When concentrated and stored at 5°C, disc-electrophoresis of 11S component showed that associates were formed. Mercaptoethanol could dissolve the insoluble protein and also convert the associates to the original 11S component. NEM–11S was not insolubilized by frozen storage at ?5°C or storage at 5°C after being concentrated. From these facts it can be concluded that denaturation of soybean protein by freezing may be caused by intermolecular reactions through S-S bonds as a result of concentration by freezing. This may suggest a mechanism of the formation of sponge-like texture in kori-tofu which is made by frozen storage of soybean curd for 15 to 20 days at ?1 to ?3°C.     

Altering flux through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana modifies photosynthetic acclimation at low temperatures and the development of freezing tolerance

To test the hypothesis that the up‐regulation of sucrose biosynthesis during cold acclimation is essential for the development of freezing tolerance, the acclimation responses of wild‐type (WT) Arabidopsis thaliana (Heynh.) were compared with transgenic plants over‐expressing sucrose phosphate synthase (over‐sps) or with antisense repression of either cytosolic fructose‐1,6‐bisphosphatase (antifbp) or sucrose phosphate synthase (antisps). Plants were grown at 23 °C and then shifted to 5 °C. The leaves shifted to 5 °C for 10 d and the new leaves that developed at 5 °C were compared with control leaves on plants at 23 °C. Plants over‐expressing sucrose phosphate synthase showed improved photosynthesis and increased flux of fixed carbon into sucrose when shifted to 5 °C, whereas both antisense lines showed reduced flux into soluble sugars relative to WT. The improved photosynthetic performance by the over‐sps plants shifted to 5 °C was associated with an increase in freezing tolerance relative to WT (?9.1 and ?7.2 °C, respectively). In contrast, both antisense lines showed impaired development of freezing tolerance (? 5.2 and ?5.8 °C for antifbp and antisps, respectively) when shifted to 5 °C. In the new leaves developed at 5 °C the recovery of photosynthesis as typically seen in WT was strongly inhibited in both antisense lines and this inhibition was associated with a further failure of both antisense lines to cold acclimate. Thus, functional sucrose biosynthesis at low temperature in the over‐sps plants reduced the inhibition of photosynthesis, maintained the mobilization of carbohydrates from source leaves to sinks and increased the rate at which freezing tolerance developed. Modification of sucrose metabolism therefore represents an additional approach that will have benefits both for the development of freezing tolerance and over‐wintering, and for the supply of exportable carbohydrate to support growth at low temperatures.     

Preservation of Phakopsora pachyrhizi Uredospores

This study compared different temperatures and dormancy‐reversion procedures for preservation of Phakopsora pachyrhizi uredospores. The storage temperatures tested were room temperature, 5°C, ?20°C and ?80°C. Dehydrated and non‐dehydrated uredospores were used, and evaluations for germination (%) and infectivity (no. of lesions/cm²) were made with fresh harvested spores and after 15, 29, 76, 154 and 231 days of storage. The dormancy‐reversion procedures evaluated were thermal shock (40°C/5 min) followed or not by hydration (moist chamber/24 h). Uredospores stored at room temperature were viable only up to a month of storage, regardless of their hydration condition. Survival of uredospores increased with storage at lower temperatures. Dehydration of uredospores prior to storage increased their viability, mainly for uredospores stored at 5°C, ?20°C and ?80°C. At 5°C and ?20°C, dehydrated uredospores showed increases in viability of at least 47 and 127 days, respectively, compared to non‐dehydrated spores. Uredospore germination and infectivity after storage for 231 days (7.7 months), could only be observed at ?80°C, for both hydration conditions. At this storage temperature, dehydrated and non‐dehydrated uredospores exhibited 56 and 28% of germination at the end of the experiment, respectively. Storage at ?80°C also maintained uredospore infectivity, based upon levels of infection frequency, for both hydration conditions. Among the dormancy‐reversion treatments applied to spores stored at ?80°C, those involving hydration allowed recoveries of 85 to 92% of the initial germination.     

Freeze avoidance: a dehydrating moss gathers no ice

Using cryo‐SEM with EDX fundamental structural and mechanical properties of the moss Ceratodon purpureus (Hedw.) Brid. were studied in relation to tolerance of freezing temperatures. In contrast to more complex plants, no ice accumulated within the moss during the freezing event. External ice induced desiccation with the response being a function of cell type; water‐filled hydroid cells cavitated and were embolized at ?4 °C while parenchyma cells of the inner cortex exhibited cytorrhysis, decreasing to ～20% of their original volume at a nadir temperature of ?20 °C. Chlorophyll fluorescence showed that these winter acclimated mosses displayed no evidence of damage after thawing from ?20 °C while GCMS showed that sugar concentrations were not sufficient to confer this level of freezing tolerance. In addition, differential scanning calorimetry showed internal ice nucleation occurred in hydrated moss at ～?12 °C while desiccated moss showed no evidence of freezing with lowering of nadir temperature to ?20 °C. Therefore the rapid dehydration of the moss provides an elegantly simple solution to the problem of freezing; remove that which freezes.     

Improvement of Freezing Tolerance in Transgenic Tobacco Leaves by Expressing the hiC6 Gene

A cryoprotective protein, HIC6, was expressed transgenically in tobacco, a cold-sensitive plant, and the localization of the protein within the cell as well as freezing tolerance of the transgenic tobacco was investigated. For constitutive expression of HIC6 in tobacco, its corresponding gene was subcloned into pBI121. Through the transformation with pBI121/hiC6, fifteen transgenic tobacco lines were acquired, out of which twelve lines expressed the HIC6 protein. None of the transgenic tobacco lines, however, showed significant differences in freezing tolerance from the control plants (wild-type and transformed with pBI121) at ?1, ?3, and ?4°C, with the exception that their freezing temperature was ?2°C. In order to increase the accumulation level of HIC6, pBE2113 with a stronger promoter was used. Eight lines expressed the protein out of thirteen lines transformed with pBE2113/hiC6. The accumulation levels of the protein were clearly higher in the tobacco plants transformed with pBE2113/hiC6 than in those with pBI121/hiC6. The HIC6 protein seemed to be localized in mitochondria of the transgenic tobacco plants. Freezing-tolerance test at ?1 - ?4°C showed that the degree of electrolyte leakage was significantly lower in the plants with pBE2113/hiC6 than in the control plants. A leaf browning observation also showed that high accumulation of HIC6 significantly suppressed injury caused by freezing to the transgenic tobacco at ?3°C.     

Acclimatisation in the green spruce aphid, Elatobium abietinum

Thin layer chromatography separation of 80% ethanol extracts of adult Elatobium abietinum revealed the presence of the polyhydric alcohol mannitol in aphids overwintering outdoors but not in aphids kept permanently indoors at 15°C. After 3 days at 15°C no traces of mannitol were left in overwintering aphids. Mean freezing temperatures of outdoor, unfed instar I nymphs were about 4°C lower than those of unfed instar I nymphs produced at 15°C. Mean freezing temperatures of overwintering adults were considerably higher than those of unfed instar I nymphs and showed no changes associated with time at 15°C following transference indoors. Similarly, mean freezing temperatures of Sitka spruce needles transferred to 15°C did not change. It was concluded that, although freezing was mainly avoided by supercooling, the presence of mannitol lowered the true freezing temperature of aphid haemolymph and, consequently, the actual freezing temperatures of nymphs produced under cold conditions. However, the considerable increase in freezing point temperatures caused by imbibition of plant sap masked these acclimatisation changes in feeding aphids.     

野生鸡枞菌种长期保存方法比较

野生鸡枞菌种质资源的有效保存是对野生鸡枞加以保护和利用的前提。以自行分离的5个野生鸡枞菌株作为研究对象,采用蒸馏水保藏法和-80°C冻结保藏法对野生鸡枞菌种长期保存的方法进行了实验研究,蒸馏水法分别保存于室温和4°C,-80°C冻结保藏同时采用程控降温法和泡沫盒降温法,保存20个月后对4种不同方法保存的5个菌株的保存效果进行比较。实验结果表明:蒸馏水室温保存法菌种存活率为100%,萌发期较短,为4-10 d,是一种简便、实用、有效而成本低廉的长期保存方法;-80°C冻结保藏法的存活率为56%-76%,萌发期7-16 d,泡沫盒降温法可以很好地控制降温速度,是一种简便有效的控温方法。     

Magnetic resonance imaging (MRI) as a method for determination of freezing injury in strawberry crowns

In an experiment with controlled freezing, strawberry plants were exposed to 0, ?8, ?12, ?16 and ?20°C at a freeze and thaw rate of 2 °C/hour in March/April 1996. Crowns from the cultivar ‘Korona’ were examined using magnetic resonance imaging (MRI), showing a gradual increase of signal intensity from the centre of the crowns, as a result of the temperature drop, which might be caused by lipids. The increase in signal intensity was in accordance with the tissue browning of crowns, which increased substantially when the temperature dropped below ?12 °C. A similar reaction was shown in a field experiment comparing wintercovered and not wintercovered strawberry plants. The plants which had been exposed to temperatures between ?10 and ?16 °C were severely injured. This demonstrates that MRI has a potential as an objective method to determine freeze injury in the field, by «calibrating» the MRI instrument to freezing profiles in controlled experiments.     

Reversible phosphorylation regulation of NADPH-linked polyol dehydrogenase in the freeze-avoiding gall moth, Epiblema scudderiana: role in glycerol metabolism

Larvae of the goldenrod gall moth, Epiblema scudderiana, use a freeze avoidance strategy of cold hardiness to survive the winter. A key metabolic adaption that supports subzero survival is the accumulation of large amounts of glycerol as a colligative antifreeze. Production of glycerol relies on polyol dehydrogenase (PDH) which catalyzes the NADPH‐dependent conversion of glyceraldehyde into glycerol. Kinetic analysis of PDH from E. scudderiana revealed significant changes in properties as a result of subzero temperature acclimation; the K_m for glyceraldehyde in 5°C‐acclimated larvae was 7.0 mM and doubled in ? 15°C‐exposed larvae. This change suggested that PDH is regulated by a state‐dependent covalent modification. Indeed, high and low K_m forms could be interconverted by incubating larval extracts in vitro under conditions that stimulated either endogenous protein kinases or protein phosphatases. Protein kinase incubations doubled the K_m glyceraldehyde of the 5°C enzyme, whereas protein phosphatase incubations decreased the K_m of the ? 15°C enzyme by about 50%. PDH was purified by ion exchange and affinity chromatography steps and then subjected to electrophoresis. Staining with ProQ Diamond phosphoprotein stain showed a much higher phosphate content of PDH from ? 15°C‐acclimated larvae, a result that was further confirmed by immunoblotting that showed a much greater phosphoserine content on the ? 15°C enzyme. These experiments established that PDH is regulated by state‐dependent reversible phosphorylation in E. scudderiana and suggest that this regulatory mechanism makes a significant contribution to controlling the synthesis, maintenance, and degradation of glycerol pools over the winter months. © 2011 Wiley Periodicals, Inc.     

Design of experiments reveals critical parameters for pilot‐scale freeze‐and‐thaw processing of L‐lactic dehydrogenase

Freezing constitutes an important unit operation of biotechnological protein production. Effects of freeze‐and‐thaw (F/T) process parameters on stability and other quality attributes of the protein product are usually not well understood. Here a design of experiments (DoE) approach was used to characterize the F/T behavior of L‐lactic dehydrogenase (LDH) in a 700‐mL pilot‐scale freeze container equipped with internal temperature and pH probes. In 24‐hour experiments, target temperature between –10 and –38°C most strongly affected LDH stability whereby enzyme activity was retained best at the highest temperature of –10°C. Cooling profile and liquid fill volume also had significant effects on LDH stability and affected the protein aggregation significantly. Parameters of the thawing phase had a comparably small effect on LDH stability. Experiments in which the standard sodium phosphate buffer was exchanged by Tris‐HCl and the non‐ionic surfactant Tween 80 was added to the protein solution showed that pH shift during freezing and protein surface exposure were the main factors responsible for LDH instability at the lower freeze temperatures. Collectively, evidence is presented that supports the use of DoE‐based systematic analysis at pilot scale in the identification of F/T process parameters critical for protein stability and in the development of suitable process control strategies.