Improvement of tolerance to freeze–thaw stress of baker’s yeast by cultivation with soy peptides.
Izawa, S., Ikeda, K., Takahashi, N. & Inoue, Y. (2007). Applied Microbiology and Biotechnology, 75(3), 533-537.
The tolerance to freeze–thaw stress of yeast cells is critical for frozen-dough technology in the baking industry. In this study, we examined the effects of soy peptides on the freeze–thaw stress tolerance of yeast cells. We found that the cells cultured with soy peptides acquired improved tolerance to freeze–thaw stress and retained high leavening ability in dough after frozen storage for 7 days. The final quality of bread regarding its volume and texture was also improved by using yeast cells cultured with soy peptides. These findings promote the utilization of soy peptides as ingredients of culture media to improve the quality of baker’s yeast.
Freezing-induced uptake of disaccharides for preservation of chromatin in freeze-dried stallion sperm during accelerated aging.
Oldenhof, H., Zhang, M., Narten, K., Bigalk, J., Sydykov, B., Wolkers, W. F. & Sieme, H. (2017). Biology of Reproduction, 7(6), 892-901.
Non-viable freeze-dried sperm have intact chromatin and can be used for fertilization via intracytoplasmic sperm injection. Freeze-dried sperm preferably should be stored at 4°C or lower, because DNA damage accumulates during storage at room temperature. Disaccharides are known to protect biomolecules both during freezing and drying, by forming a highly viscous glassy state. Their use for intracellular protection is challenging because cellular membranes are normally impermeable for disaccharides. In the current study, we demonstrate that membrane impermeable compounds, including lucifer yellow and trehalose, are taken up by stallion sperm when exposed to freezing. Trehalose uptake likely occurs during freezing-induced membrane phase transitions, but does not allow sperm to survive drying. Stallion sperm was freeze-dried in various formulations consisting of reducing or non-reducing sugars combined with albumin as bulking agent. Chromatin stability was studied during storage at 37°C, using the flow cytometric sperm chromatin structure assay and microscopic assessment of chromatin dispersion and DNA fragmentation after electrophoresis. Freeze-drying did not affect sperm chromatin, irrespective of the formulation that was used. DNA fragmentation index (DFI) values ranged from 5 - 8%. If sperm was freeze-dried without protectants or in a combination of glucose and proteins, DNA damage rapidly accumulated during storage at 37°C, reaching DFI values of respectively 95 ± 4 and 64 ± 42% after 1 month. DFI values of sperm freeze-dried with sucrose or trehalose ranged between 9 − 11% and 33 - 52% after 1 and 3 months storage, respectively. In conclusion, freeze-drying sperm with disaccharides results in uptake during freezing, which greatly reduces chromatin degradation during dried storage.
Starvation resistance and effects of diet on energy reserves in a predatory ground beetle (Merizodus soledadinus; Carabidae) invading the Kerguelen Islands.
Laparie, M., Larvor, V., Frenot, Y. & Renault, D. (2012). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 161(2), 122-129.
The relationship between nutritional requirements and the availability or quality of food is a prime parameter in determining the geographical expansion of invasive insects. At the sub-Antarctic Kerguelen Islands, the invasive ground beetle Merizodus soledadinus becomes the main invertebrate predator when it colonizes new habitats, leading to the local extinction of native fly species. Such changes in the structure of prey communities may alter the energy management (storage and expenditure) of this predator. In this species, we monitored survival and body mass during food deprivation, in addition to evaluating the effects of two distinct diets (maggots versus enchytraeids) on the consumption and restoration of body reserves (sugars and triglycerides). We found that adults can starve for more than 60 days, and feed every 3.76 days on average when food is available. We recorded higher predation rates on maggots, associated with steeper body mass variations, compared to enchytraeids. Sugars and triglycerides were significantly consumed during food deprivation and restored after refeeding, but varied similarly among individuals supplied on the distinct diets. Other parameters may determine the food preferences observed, such as salt content in prey tissues, because M. soledadinus mainly feeds in hypersaline foreshore habitats, and may limit the consumption of osmotic conformers.
Freezing tolerance and low molecular weight cryoprotectants in an invasive parasitic fly, the deer ked (Lipoptena cervi).
Nieminen, P., Paakkonen, T., Eerilä, H., Puukka, K., Riikonen, J., Lehto, V. P. & Mustonen, A. M. (2012). Journal of Experimental Zoology Part A: Ecological Genetics and Physiology, 317(1), 1-8.
Insect cold hardiness is often mediated by low molecular weight cryoprotectants, such as sugars, polyols, and amino acids (AA). While many free-living northern insects must cope with extended periods of freezing ambient temperatures (Ta), the ectoparasitic deer ked Lipoptena cervi imago can encounter subfreezing Ta only during a short autumnal period between hatching and host location. Subsequently, it benefits from the body temperature of the cervid host for survival in winter. This study investigated the cold tolerance of the species by determining its lower lethal temperature (100% mortality, LLT100) during faster and slower cold acclimation, by determining the supercooling point (SCP) and by measuring the concentrations of potential low molecular weight cryoprotectants. The LLT100 of the deer ked was approximately −16°C, which would enable it to survive freezing nighttime Ta not only in its current area of distribution but also further north. The SCP was −7.8°C, clearly higher than the LLT100, indicating that the deer ked displays freezing tolerance. The concentrations of free AA, especially nonessential AA, were higher in the cold-acclimated deer keds similar to several other insects. The concentrations of proline increased together with γ-aminobutyrate, arginine, asparagine, cystine, glutamate, glutamine, hydroxylysine, sarcosine, serine, and taurine. AA could be hypothesized to act as cryoprotectants by, e.g., protecting enzymes and lipid membranes from damage caused by cold.
A novel steamed bread making process using salt‐stressed baker’s yeast.
Yeh, L. T., Wu, M. L., Charles, A. L. & Huang, T. C. (2009). International Journal of Food Science & Technology, 44(12), 2637-2643.
The process of applying salt-stressed baker’s yeast during southern style Chinese steamed bread dough preparation was examined. Baker’s yeast was stressed in 7% salt solution then mixed into dough, which was then evaluated for dough fermentation producing gas, dough expansion, texture profile analysis (TPA), colour, specific volume, spread ratio and sensory analysis. The results of this study pointed out salt-stressed baker’s yeast produced significant amount of gas and dough expansion, particularly after 40 min of salt stressing. The texture of steamed bread was softer (463.08 g) than control (541.35 g) (P < 0.05), greater in specific volume (3.15 cm3 g-1) than control (2.89 cm3 g−1) (P < 0.05), had a lower spread ratio (1.45) than control (1.74) (P < 0.05) and a significantly improved sensory properties for taste (90.6) than control (81.6) (P < 0.05) were obtained.
Differences in cold and drought tolerance of high arctic and sub-arctic populations of Megaphorura arctica Tullberg 1876 (Onychiuridae: Collembola).
Bahrndorff, S., Petersen, S. O., Loeschcke, V., Overgaard, J. & Holmstrup, M. (2007). Cryobiology, 55(3), 315-323.
The springtail Megaphorura arctica (Onychiuridae: Collembola) inhabits the arctic and sub-arctic parts of the northern hemisphere where it on a seasonal basis will be exposed to severe cold and desiccating conditions. In the present study we compared how traits of stress resistance differed between two populations of M. arctica that were collected at a high arctic site (Spitsbergen) and a sub-arctic site (Akureyri, Iceland) with contrasting thermal environments. In addition we investigated how cold and desiccation affected the phospholipid fatty acid composition of M. arctica from Spitsbergen. The springtails from Spitsbergen were the most cold tolerant and this was linked to an almost three times higher level of trehalose accumulation during cryoprotective dehydration (15% and 5% of tissue dry weight in the Spitsbergen and Iceland populations, respectively). Although cryoprotective dehydration is intimately related to desiccation stress it was shown that M. arctica had a higher mortality when dehydrated over ice (−10 or −20°C) than when dehydrated at temperatures above 1°C. Thus, survival was lower after exposure to −10°C than after exposure to a relative humidity of 91.2% RH at +1°C although both treatments led to the same level of dehydration. Exposure to both cold (−10 and −20°C) and desiccation at +1°C caused significant changes in the phospholipid fatty acid composition with some similarities. These changes included a decrease in average chain length of the fatty acids due primarily to an increase in the phospholipid fatty acids 16:0 and a decrease in 18:3 and 20:4ω6.
Trehalose promotes the survival of Saccharomyces cerevisiae during lethal ethanol stress, but does not influence growth under sublethal ethanol stress.
Bandara, A., Fraser, S., Chambers, P. J. & Stanley, G. A. (2009). FEMS Yeast Research, 9(8), 1208-1216.
Trehalose is known to protect cells from various environmental assaults; however, its role in the ethanol tolerance of Saccharomyces cerevisiae remains controversial. Many previous studies report correlations between trehalose levels and ethanol tolerance across a variety of strains, yet variations in genetic background make it difficult to separate the impact of trehalose from other stress response factors. In the current study, investigations were conducted on the ethanol tolerance of S. cerevisiae BY4742 and BY4742 deletion strains, tsl1Δ and nth1Δ, across a range of ethanol concentrations. It was found that trehalose does play a role in ethanol tolerance at lethal ethanol concentrations, but not at sublethal ethanol concentrations; differences of 20–40% in the intracellular trehalose concentration did not provide any growth advantage for cells incubated in the presence of sublethal ethanol concentrations. It was speculated that the ethanol concentration-dependent nature of the trehalose effect supports a mechanism for trehalose in protecting cellular proteins from the damaging effects of ethanol.
Divergent strategies for adaptation to desiccation stress in two Drosophila species of immigrans group.
Parkash, R., Aggarwal, D. D., Ranga, P. & Singh, D. (2012). Journal of Comparative Physiology B, 182(6), 751-769.
Water balance mechanisms have been investigated in desert Drosophila species of the subgenus Drosophila from North America, but changes in mesic species of subgenus Drosophila from other continents have received lesser attention. We found divergent strategies for coping with desiccation stress in two species of immigrans group—D. immigrans and D. nasuta. In contrast to clinal variation for body melanization in D. immigrans, cuticular lipid mass showed a positive cline in D. nasuta across a latitudinal transect (10°46′–31°43′N). Based on isofemale lines variability, body melanization showed positive correlation with desiccation resistance in D. immigrans but not in D. nasuta. The use of organic solvents has supported water proofing role of cuticular lipids in D. nasuta but not in D. immigrans. A comparative analysis of water budget of these two species showed that higher water content, reduced rate of water loss and greater dehydration tolerance confer higher desiccation resistance in D. immigrans while the reduced rate of water loss is the only possible mechanism to enhance desiccation tolerance in D. nasuta. We found that carbohydrates act as metabolic fuel during desiccation stress in both the species, whereas their rates of utilization differ significantly between these two species. Further, acclimation to dehydration stress improved desiccation resistance due to increase in the level of carbohydrates in D. immigrans but not in D. nasuta. Thus, populations of D. immigrans and D. nasuta have evolved different water balance mechanisms under shared environmental conditions. Multiple measures of desiccation resistance in D. immigrans but reduction in water loss in D. nasuta are consistent with their different levels of adaptive responses to wet and dry conditions on the Indian subcontinent.
Sex-specific differences in the physiological basis of water conservation of Drosophila hydei from the western Himalayas.
Parkash, R., Singh, D. & Lambhod, C. (2014). Canadian Journal of Zoology, 92(6), 545-555.
In the cosmopolitan fruit fly Drosophila hydei – Sturtevant 1921 (Diptera: Drosophilidae), the relative abundance of males is significantly higher than females, but the physiological basis of such sex-specific differences are largely unknown. For wild populations of D. hydei, we found seasonal changes (summer versus autumn) in desiccation related traits but in all seasons the desiccation tolerance of males was higher than that of females. For desiccation related traits, we tested whether thermal developmental acclimation at three temperatures (17, 21 and 28°C) matched seasonal changes observed under wild conditions. Male flies showed significantly higher trait values for desiccation resistance, cuticular lipid mass, hemolymph, carbohydrate content and dehydration tolerance as compared with females when reared at lower or higher temperatures despite lack of significant sex-specific differences in the total body water content of flies reared at a particular growth temperature. We observed plastic changes in the amount of cuticular lipids consistent with corresponding differences in the rate of water loss. Treatment of cuticular surface with organic solvent (hexane) supported the role of cuticular lipids in affecting transcuticular water loss. We found significant thermal plastic effects for desiccation related traits of D. hydei but the sexual dimorphism was in the opposite direction i.e. males were more desiccation resistant than females in D. hydei while the reverse is true for many other Drosophila species (Diptera, Drosophilidae). Our results suggest that sex-specific differences in desiccation resistance level of D. hydei are good predictors of relative abundance levels of male and female flies under wild conditions.
Divergent strategy for adaptation to drought stress in two sibling species of montium species subgroup: Drosophila kikkawai and Drosophila leontia.
Ramniwas, S. & Kajla, B. (2012). Journal of Insect Physiology, 58(12), 1525-1533.
Drosophila leontia (warm adapted) has been considered as a sister species of Drosophila kikkawai (sub-cosmopolitan) with a very similar morphology. We found divergent strategies for coping with desiccation stress in these two species of montium subgroup. Interestingly, in contrast to clinal variation for body melanization in D. kikkawai, cuticular lipid mass showed a positive cline in D. leontia across a latitudinal transect. On the basis of isofemale line analysis, within population trait variability in cuticular lipid mass per fly is positively correlated with desiccation resistance and negatively correlated with cuticular water loss in D. leontia. A comparative analysis of water budget of these two species showed that higher abdominal melanization, reduced rate of water loss and greater dehydration tolerance confer higher desiccation resistance in D. kikkawai while the reduced rate of water loss is the only possible mechanism to enhance desiccation tolerance in D. leontia. The use of organic solvents has supported water proofing role of cuticular lipids in D. leontia but not in D. kikkawai. Thus, we may suggest that body melanization and cuticular lipids may represent alternative strategies for coping with dehydration stress in melanic versus non-melanic drosophilids. In both these species, carbohydrates were utilized under desiccation stress but a higher level of stored carbohydrates was evident in D. kikkawai. Further, we found increase desiccation resistance in D. kikkawai through acclimation while D. leontia lacks such a response. Thus, species specific divergence in water balance related traits in these species are consistent with their adaptations to wet and dry habitats.
Rapid effects of humidity acclimation on stress resistance in Drosophila melanogaster.
Aggarwal, D. D., Ranga, P., Kalra, B., Parkash, R., Rashkovetsky, E. & Bantis, L. E. (2013). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 166(1), 81-90.
We tested the hypothesis whether developmental acclimation at ecologically relevant humidity regimes (40% and 75% RH) affects desiccation resistance of pre-adults (3rd instar larvae) and adults of Drosophila melanogaster Meigen (Diptera: Drosophilidae). Additionally, we untangled whether drought (40% RH) acclimation affects cold-tolerance in the adults of D. melanogaster. We observed that low humidity (40% RH) acclimated individuals survived significantly longer (1.6-fold) under lethal levels of desiccation stress (0–5% RH) than their counter-replicates acclimated at 75% RH. In contrast to a faster duration of development of 1st and 2nd instar larvae, 3rd instar larvae showed a delayed development at 40% RH as compared to their counterparts grown at 75% RH. Rearing to low humidity conferred an increase in bulk water, hemolymph content and dehydration tolerance, consistent with increase in desiccation resistance for replicates grown at 40% as compared to their counterparts at 75% RH. Further, we found a trade-off between the levels of carbohydrates and body lipid reserves at 40% and 75% RH. Higher levels of carbohydrates sustained longer survival under desiccation stress for individuals developed at 40% RH than their congeners at 75% RH. However, the rate of carbohydrate utilization did not differ between the individuals reared at these contrasting humidity regimes. Interestingly, our results of accelerated failure time (AFT) models showed substantial decreased death rates at a series of low temperatures (0, −2, or −4°C) for replicates acclimated at 40% RH as compared to their counter-parts at 75% RH. Therefore, our findings indicate that development to low humidity conditions constrained on multiple physiological mechanisms of water-balance, and conferred cross-tolerance towards desiccation and cold stress in D. melanogaster. Finally, we suggest that the ability of generalist Drosophila species to tolerate fluctuations in humidity might aid in their existence and abundance under expected changes in moisture level in course of global climate change.
Laser induced injury caused hyperglycemia-like effect in Drosophila larva: a possible insect model for posttraumatic diabetes.
Okabe, F., Nakagiri, Y. & Yamada, T. (2015). Journal of Veterinary Medical Science, 77(5), 601-604.
Diabetic patients need particular care in case of infection, digestive disorder or external injury, because external stress often exasperates the glucose metabolism, which is known as “sick day management”. In addition, severe trauma can be a cause of hyperglycemia with insulin resistance. In spite of critical component of the treatment, the precise mechanisms of how trauma develops posttraumatic diabetes remain unknown. Here, we ablated body wall muscles in Drosophila larvae by laser beam and found that the level of trehalose, the principal sugar circulating in the hemolymph or in the tissues of most insects, was increased. The model may provide a helpful tool to understand the relationship between trauma and sugar metabolism.
Targeted mutagenesis and functional analysis of adipokinetic hormone-encoding gene in Drosophila.
Sajwan, S., Sidorov, R., Stašková, T., Žaloudíková, A., Takasu, Y., Kodrík, D. & Zurovec, M. (2015). Insect Biochemistry and Molecular Biology, 61, 79-86.
Adipokinetic hormones (Akhs) are small peptides (8-10 amino acid [aa] residues long) found in insects that regulate metabolic responses to stress by stimulating catabolic reactions and mobilizing energy stores. We employed Transcription activator-like effector nuclease (TALEN) mutagenesis and isolated an Akh1 mutant carrying a small deletion in the gene that resulted in a truncated peptide; the second aa (Leu) was missing from the functional octapeptide. This null Dmel/Akh mutant is suitable to study Akh function without any effect on the C-terminal associated peptide encoded by the same gene. The mutant flies were fully viable and compared to the control flies, had significantly low levels of hemolymph saccharides including trehalose and were resistant to starvation. These characteristics are similar to those obtained from the flies carrying targeted ablation of Akh-expressing neurons (reported earlier). We also found that the Akh1 mutants are slightly heavy and had a slow metabolic rate. Furthermore, we showed that the ectopic expression of Dmel/Akh reverses the Akh1 phenotype and restores the wild-type characteristics. Our results confirmed that Akh is an important regulator of metabolic homeostasis in Drosophila.
Use of Drosophila as an Evaluation Method Reveals imp as a Candidate Gene for Type 2 Diabetes in Rat Locus Niddm22.
Kawasaki, K., Yamada, S., Ogata, K., Saito, Y., Takahama, A., Yamada, T., Matsumoto, K. & Kose, H. (2015). Journal of Diabetes Research, 2015, Article ID 758564.
Type 2 diabetes (T2D) is one of the most common human diseases. QTL analysis of the diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats has identified numerous hyperglycemic loci. However, molecular characterization and/or gene identification largely remains to be elucidated due mostly to the weak genetic variances contributed by each locus. Here we utilized Drosophila melanogaster as a secondary model organism for functional evaluation of the candidate gene. We demonstrate that the tissue specific knockdown of a homologue of igf2bp2 RNA binding protein leads to increased sugar levels similar to that found in the OLETF rat. In the mutant, the expression of two of the insulin-like peptides encoded in the fly genome, dilp2 and dilp3, were found to be downregulated. Consistent with previous reports of dilp mutants, the imp mutant flies exhibited an extension of life span; in contrast, starvation tolerance was reduced. These results further reinforce the possibility that imp is involved in sugar metabolism by modulating insulin expression.
Short-term adaptation during propagation improves the performance of xylose-fermenting Saccharomyces cerevisiae in simultaneous saccharification and co-fermentation.
Nielsen, F., Tomás-Pejó, E., Olsson, L. & Wallberg, O. (2015). Biotechnology for Biofuels, 8(1), 219.
Background: Inhibitors that are generated during thermochemical pretreatment and hydrolysis impair the performance of microorganisms during fermentation of lignocellulosic hydrolysates. In omitting costly detoxification steps, the fermentation process relies extensively on the performance of the fermenting microorganism. One attractive option of improving its performance and tolerance to microbial inhibitors is short-term adaptation during propagation. This study determined the influence of short-term adaptation on the performance of recombinant Saccharomyces cerevisiae in simultaneous saccharification and co-fermentation (SSCF). The aim was to understand how short-term adaptation with lignocellulosic hydrolysate affects the cell mass yield of propagated yeast and performance in subsequent fermentation steps. The physiology of propagated yeast was examined with regard to viability, vitality, stress responses, and upregulation of relevant genes to identify any links between the beneficial traits that are promoted during adaptation and overall ethanol yields in co-fermentation. Results: The presence of inhibitors during propagation significantly improved fermentation but lowered cell mass yield during propagation. Xylose utilization of adapted cultures was enhanced by increasing amounts of hydrolysate in the propagation. Ethanol yields improved by over 30 % with inhibitor concentrations that corresponded to ≥2.5 % water-insoluble solids (WIS) load during the propagation compared with the unadapted culture. Adaptation improved cell viability by >10 % and increased vitality by >20 %. Genes that conferred resistance against inhibitors were upregulated with increasing amounts of inhibitors during the propagation, but the adaptive response was not associated with improved ethanol yields in SSCF. The positive effects in SSCF were observed even with adaptation at inhibitor concentrations that corresponded to 2.5 % WIS. Higher amounts of hydrolysate in the propagation feed further improved the fermentation but increased the variability in fermentation outcomes and resulted in up to 20 % loss of cell mass yield. Conclusions: Short-term adaptation during propagation improves the tolerance of inhibitor-resistant yeast strains to inhibitors in lignocellulosic hydrolysates and improves their ethanol yield in fermentation and xylose-fermenting capacity. A low amount of hydrolysate (corresponding to 2.5 % WIS) is optimal, whereas higher amounts decrease cell mass yield during propagation.
Effect of C-terminal domain truncation of Thermus thermophilus trehalose synthase on its substrate specificity.
Cho, C. B., Park, D. Y. & Lee, S. B. (2017). Enzyme and Microbial Technology, 96, 121-126.
The C-terminal domain of the three-domain-comprising trehalose synthase from Thermus thermophilus was truncated in order to study the effect on the enzyme’s activity and substrate specificity. Compared with the wild-type (WT) enzyme, the two truncated enzymes (DM1 and DM2) showed lower maltose- and trehalose-converting activities and a different transglycosylation reaction mechanism. In the mutants, the glucose moiety cleaved from the maltose substrate was released from the enzyme and intercepted by external glucose oxidase, preventing the production of trehalose. The WT enzyme, however, retained the glucose in the active site to effectively produce trehalose. In addition, DM1 synthesized much higher amounts of mannose-containing disaccharide trehalose analog (Man-TA) than did the WT and DM2. The results suggest that the C-terminal domain in the WT enzyme is important for retaining the glucose moiety within the active site. The mutant enzymes could be used to produce Man-TA, a postulated inhibitor of gut disaccharidases.
Cold and desiccation stress induced changes in the accumulation and utilization of proline and trehalose in seasonal populations of Drosophila immigrans.
Tamang, A. M., Kalra, B. & Parkash, R. (2017). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 203, 304-313.
Changes in the levels of energy metabolites can limit survival ability of Drosophila species under stressful conditions but this aspect has received less attention in wild populations collected in different seasons. We tested cold or desiccation triggered changes in the accumulation or utilization of two energy metabolites (trehalose and proline) in Drosophila immigrans flies reared under season specific environmental conditions. Such D.immigrans populations were subjected to different durations of cold (0°C) or desiccation stress (5% RH) or dual stress. We found stress induced effects of cold vs desiccation on the levels of trehalose as well as for proline. Different durations of cold stress led to accumulation of trehalose while desiccation stress durations revealed utilization of trehalose. In contrast, there was accumulation of proline under desiccation and utilization of proline with cold stress. Since accumulation levels were higher than utilization of each energy metabolite, the effects of dual stress showed additive effect. However, there was no utilization of total body lipids under cold or desiccation stress. We observed significant season specific differences in the amount of energy metabolites but the rate of metabolism did not vary across seasons. Stress triggered changes in trehalose and proline suggest possible link between desiccation and cold tolerance. Finally, stress specific (cold or desiccation) compensatory changes in the levels of trehalose and proline suggest possible energetic homeostasis in D.immigrans living under harsh climatic conditions of montane localities.
Thermoresponsive microgels containing trehalose as soft matrices for 3D cell culture.
Burek, M., Waśkiewicz, S., Lalik, A., Student, S., Bieg, T. & Wandzik, I. (2017). Biomaterials Science, 5(2), 234-246.
A series of thermoresponsive glycomicrogels with trehalose in the cross-links or with trehalose in the cross-links and as pending moieties was synthesized. These materials were obtained by surfactant-free precipitation copolymerization of N-isopropylacrylamide and various amounts of trehalose monomers. The resultant particles showed a spherical shape and a submicrometer hydrodynamic size with a narrow size distribution. At 25°C, glycomicrogels in solutions with physiological ionic strength formed stable colloids, which further gelled upon heating to physiological temperature forming a macroscopic hydrogel with an interconnected porous structure. These extremely soft matrices with dynamic storage modulus in the range of 9-70 Pa were examined in 3D culture systems for HeLa cell culture in comparison to traditional 2D mode. They showed relatively low syneresis over time, especially when glycomicrogels with a high content of hydrophilic trehalose were used as building blocks. An incorporated pending trehalose composed of two α, α′-1,1′-linked D-glucose moieties was used with the intention of providing multivalent interactions with glucose transporters (GLUTs) expressed on the cell surface. A better cell viability was observed when a soft hydrogel with the highest content of trehalose and the lowest syneresis was used as a matrix compared to a 2D control assay.
Adipokinetic hormone activities in insect body infected by entomopathogenic nematode.
Ibrahim, E., Hejníková, M., Shaik, H. A., Doležel, D. & Kodrík, D. (2017). Journal of Insect Physiology, 98, 347-355.
The role of adipokinetic hormone (AKH) in the firebug Pyrrhocoris apterus adults infected by the entomopathogenic nematode (EPN) Steinernema carpocapsae was examined in this study. It was found that co-application of EPN and AKH enhanced firebug mortality about 2.5 times within 24 h (from 20 to 51% in EPN vs. EPN + AKH treatments), and resulted in metabolism intensification, as carbon dioxide production in firebugs increased about 2.1 and 1.6 times compared to control- and EPN-treated insects, respectively. Accordingly, firebugs with reduced expression of AKH receptors showed a significantly lower mortality (by 1.6 to 2.9-folds), and lower general metabolism after EPN + AKH treatments. In addition, EPN application increased Akh gene expression in the corpora cardiaca (1.6 times), AKH level in the corpora cardiaca (1.3 times) and haemolymph (1.7 times), and lipid and carbohydrate amounts in the haemolymph. Thus, the outcomes of the present study demonstrate involvement of AKH into the anti-stress reaction elicited by the nematobacterial infection. The exact mechanism by which AKH acts is unknown, but results suggested that the increase of metabolism and nutrient amounts in haemolymph might play a role.
Freezing-induced uptake of trehalose into mammalian cells facilitates cryopreservation.
Zhang, M., Oldenhof, H., Sieme, H. & Wolkers, W. F. (2016). Biochimica et Biophysica Acta (BBA)-Biomembranes, 1858(6), 1400-1409.
The aim of this study was to investigate if membrane-impermeable molecules are taken up by fibroblasts when exposing the cells to membrane phase transitions and/or freezing-induced osmotic forces. The membrane-impermeable fluorescent dye lucifer yellow (LY) was used to visualize and quantify uptake during endocytosis, and after freezing-thawing. In addition, trehalose uptake after freezing and thawing was studied. Fourier transform infrared spectroscopic studies showed that fibroblasts display a minor non-cooperative phase transition during cooling at suprazero temperatures, whereas cells display strong highly cooperative fluid-to-gel membrane phase transitions during freezing, both in the absence and presence of protectants. Cells do not show uptake of LY upon passing the suprazero membrane phase transition at 30-10°C, whereas after freezing and thawing cells show intracellular LY equally distributed within the cell. Both, LY and trehalose are taken up by fibroblasts after freezing and thawing with loading efficiencies approaching 50%. When using 250 mM extracellular trehalose during cryopreservation, intracellular concentrations greater than 100 mM were determined after thawing. A plot of cryosurvival versus the cooling rate showed a narrow inverted-‘U’-shaped curve with an optimal cooling rate of 40°C min-1. Diluting cells cryopreserved with trehalose in isotonic cell culture medium resulted in a loss of cell viability, which was attributed to intracellular trehalose causing an osmotic imbalance. Taken together, mammalian cells can be loaded with membrane-impermeable compounds, including the protective agent trehalose, by subjecting the cells to freezing-induced osmotic stress.