Trehalose Assay Kit

Kveik is a term used to describe a group of traditional Saccharomyces cerevisiae strains originally employed by regional Norwegian brewers for the production of malt-based beverages and bread. A key feature of kveik strains is that they perform well at elevated temperatures and under high gravity conditions. These traits are believed to be indicative of general stress tolerance, however, empirical data on this subject does not currently exist. In this study, the tolerance of kveik strains to industrial challenges including heat, ethanol, oxidative and osmotic stress was quantitatively determined. The data obtained indicated that kveik yeast showed a remarkable tolerance to stress in general, and high temperature in particular, based on combined analyses of growth kinetics and metabolic activity tests (MTT cytotoxicity assay). Further analysis indicated that trehalose was observed to be elevated in kveik yeasts under both stressed- and non-stressed conditions, suggesting that this stress protectant is a principal factor contributing to tolerance. This data has direct implications for brewing, and broad commercial relevance for the global fermentation industry; the potential for using highly stress-tolerant strains forms a key strategy for sustainable practices through intensification of processes and maximizing efficiencies, leading to reductions in processing times and associated costs.

Trehalose has sparked considerable interest in a variety of pharmaceutical applications as well as in cryopreservation. Recently, there have been growing efforts in the development of trehalose delivery nanocarriers to address the issue of the poor bioavailability of trehalose. The majority of the strategies comprise physical entrapment of trehalose, since its covalent, yet biolabile, conjugation is challenging. Here, we present research on trehalose-releasing nanogels, in which covalent, yet biolabile, conjugation of trehalose was achieved through the co-incorporation of trehalose (meth)acrylate(s) together with hydrophilic primary/secondary acrylamides in one polymeric network. In this case, the primary and secondary amide groups participated in ester hydrolysis in the (meth)acrylate units, making the hydrolysis feasible under physiologically relevant conditions. A set of nanogels with precisely selected compositions were synthesized, characterized, and then studied to evaluate the influence of various structural and environmental factors on the release rate of trehalose. The study also provides insights into some other aspects that are important in view of potential biomedical applications, including specific interactions of nanogels through their terminal α-d-glucopyranosyl moieties from pendant trehalose, protein corona formation, and cellular uptake.

The long-term resistance to desiccation on abiotic surfaces is a key determinant of the adaptive success of Acinetobacter baumannii as a healthcare-associated bacterial pathogen. Here, the cellular and molecular mechanisms enabling A. baumannii to resist desiccation and persist on abiotic surfaces were investigated. Experiments were set up to mimic the A. baumannii response to air-drying that would occur when bacterial cells contaminate fomites in hospitals. Resistance to desiccation and transition to the “viable but nonculturable” (VBNC) state were determined in the laboratory-adapted strain ATCC 19606^T and the epidemic strain ACICU. Culturability, membrane integrity, metabolic activity, virulence, and gene expression profile were compared between the two strains at different stages of desiccation. Upon desiccation, ATCC 19606^T and ACICU cells lose culturability and membrane integrity, lower their metabolism, and enter the VBNC state. However, desiccated A. baumannii cells fully recover culturability and virulence in an insect infection model following rehydration in physiological buffers or human biological fluids. Transcriptome and chemical analyses of A. baumannii cells during desiccation unveiled the production of protective metabolites (L-cysteine and L-glutamate) and decreased energetic metabolism consequent to activation of the glyoxylate shunt (GS) pathway, as confirmed by reduced resuscitation efficiency of aceA mutants, lacking the key enzyme of the GS pathway. VBNC cell formation and extensive metabolic reprogramming provide a biological basis for the response of A. baumannii to desiccation, with implications on environmental control measures aimed at preventing the transmission of A. baumannii infection in hospitals.

The cabbage stem flea beetle (Psylliodes chrysocephala, CSFB) is a significant pest of winter oilseed rape crops in northern Europe. CSFB adults aestivate during the summer to protect themselves from heat and desiccation stress. Trehalose, the primary hemolymph sugar, has been linked to energy homeostasis and stress resilience, but its regulation and function during aestivation remain poorly understood. Here, we investigated the roles of two trehalose transporters, Tret-1 and Tret-2, in modulating trehalose dynamics across different adult stages in CSFB. Through spatiotemporal transcript profiling, we found that Tret-1 was predominantly expressed in the fat body, where it facilitates trehalose export to the hemolymph, whereas Tret-2 expression was higher in the Malpighian tubules, mediating trehalose uptake from the hemolymph. RNA interference experiments revealed that Tret-1 is involved in transporting trehalose from the fat body into the hemolymph, while Tret-2 works reciprocally to transport trehalose from the hemolymph into the Malpighian tubules. The disruption of trehalose transportation resulted in excess glucose, glycogen, and triglyceride levels, mainly in pre-aestivation beetles. Furthermore, the knockdown of either trehalose transporter caused a compensatory increase in feeding activity in pre-aestivation beetles, while the knockdown of Tret-2 compromised resilience to heat stress. Our findings uncover the reciprocal functions of Tret-1 and Tret-2 in regulating trehalose distribution and maintaining metabolic stability during aestivation, offering insights into the physiological strategies underpinning insect survival during aestivation.

Aestivation is a dormant state that allows animals to withstand hot and dry summer conditions and requires complex gene regulation. Nevertheless, the mechanisms involved in the regulation of genes necessary for aestivation remain unclear. MicroRNA (miRNA) are known to fine-tune gene expression at the post-transcriptional level and are important for various biological processes. In this study, we investigated the role of the miRNA pathway in the regulation of the obligatory aestivation stage in the cabbage stem flea beetle, a major pest of oilseed rape. Small RNA sequencing showed that ~25% of miRNAs were differentially abundant during aestivation. The inhibition of the miRNA pathway deregulated 116 proteins in aestivation, which were mainly associated with metabolism and catabolism, including peroxisome activity. Most proteins regulated by miRNA exhibited lower transcript levels during aestivation. RNA degradome sequencing confirmed the miRNA-mediated exonucleolytic decay of several transcripts. Furthermore, inhibiting the miRNA pathway resulted in altered body composition, compromised metabolic suppression, and lower resilience to high temperature during aestivation. Also, beetles could not suppress their feeding activity during the transition into aestivation. Our findings highlight the critical role of miRNA in regulating aestivation in the cabbage stem flea beetle, with important implications for climate change.

Trehalose production via a one-step enzymatic route using trehalose synthase (TreS) holds significant promise for industrial-scale applications due to its simplicity and utilization of low-cost substrates. However, the development of a robust whole-cell biocatalyst expressing TreS remains crucial for enabling practical and economically viable production. In this study, a high-sugar tolerant strain of S. cerevisiae was screened and employed as a host cell for the cell surface display of TreS from Acidiplasma aeolicum. The resultant strain, S. cerevisiae I3A, exhibited remarkable surface displayed TreS activity of 3358 U/g CDW and achieved approximately 64% trehalose yield (10.8 g/L/h productivity) from maltose. Interestingly, no glucose by-product was observed during trehalose production. The S. cerevisiae I3A cells exhibited reusability for up to 12 cycles leading to potential cost reduction of trehalose products. Therefore, our study demonstrated the development of a high-sugar tolerant S. cerevisiae strain expressing TreS on its surface as a whole-cell biocatalyst for efficient and economical trehalose production with potential applications in the food and pharmaceutical industries.

Yeast plays a crucial role in the fermentation industry, particularly in alcoholic beverage production, where robustness and metabolic flexibility are essential. This study aimed to investigate the stress tolerance and metabolic capabilities of seven commercial ale yeast strains under various stress conditions, including temperature, pH, osmotic pressure, glucose starvation, and ethanol concentration. Detailed growth assays and stress tolerance tests were utilized to evaluate fermentation efficiency, carbon source utilization, and stress adaptation. Significant variability was observed among the strains. ACY169 and ACY150 demonstrated high overall stress tolerance, making them suitable for high-gravity brewing and processes involving extreme temperature fluctuations. ACY10 showed robust performance under acid stress, making it ideal for sour beer production. In contrast, ACY5 exhibited limited adaptability under stress, with longer doubling times and reduced metabolic activity. The study also revealed differences in carbon source utilization, with ACY169 displaying exceptional metabolic versatility by efficiently fermenting various sugars, including glucose, fructose, maltose, and raffinose. ACY10 and ACY150 exhibited balanced fermentation profiles with high ethanol production rates, while ACY9 demonstrated the highest glucose consumption rate but lower ethanol yields and significant acidification.

Yeast populations can undergo diversification during their growth and ageing, leading to the formation of different cell-types. Differentiation into two major subpopulations, differing in cell size and density and exhibiting distinct physiological and metabolic properties, was described in planktonic liquid cultures and in populations of colonies growing on semisolid surfaces. Here, we compare stress resistance, metabolism and expression of marker genes in seven differentiated cell subpopulations emerging during cultivation in liquid fermentative or respiratory media and during colony development on the same type of solid media. The results show that the more-dense cell subpopulations are more stress resistant than the less-dense subpopulations under all cultivation conditions tested. On the other hand, respiratory capacity, enzymatic activities and marker gene expression differed more between subpopulations. These characteristics are more influenced by the lifestyle of the population (colony vs. planktonic cultivation) and the medium composition. Only in the population growing in liquid respiratory medium, two subpopulations do not form as in the other conditions tested, but all cells exhibit a range of characteristics of the more-dense subpopulations. This suggests that signals for cell differentiation may be triggered by prior metabolic reprogramming or by an unknown signal from the structured environment in the colony.

As traditionally cold areas become warmer due to climate change, temperature could no longer be a barrier to the establishment of non-native insects. This is particularly relevant for pest insects from warm and tropical areas, mainly those with some tolerance to moderately low temperatures, which could expand their range into these new locations. From this perspective, in this work we studied the morphological and biochemical responses of the Neotropical pest Paysandisia archon to low temperatures, as part of a possible strategy to colonize new areas. To that end, wild larvae were exposed for 7 days to either low (1 and 5°C) or ambient (23°C) temperatures. We then quantified the inner and outer morphological changes, by X-Ray Computer Tomography and Digital Holographic Microscopy, as well as the accumulation of metabolites acting as potential endogenous cryoprotectants, by Spectrophotometry. We found that Paysandisia archon developed a cold-induced response based on different aspects. On the one hand, morphological changes occurred with a significant reduction both in fluids susceptible to freezing and fat body, together with the thickening, hardening and increased roughness of the integument. On the other hand, we found an increase in the hemolymph concentration of cryoprotective substances such as glucose (6-fold) and glycerol (2-fold), while trehalose remained unchanged. Surprisingly, this species did not show any evidence of cold-induced response unless the environmental temperature was remarkably low (1°C). These results could be useful to improve models predicting the possible spread of such a pest, which should incorporate parameters related to its resistance to low temperatures.

Background: The intracellular molecule trehalose in Saccharomyces cerevisiae may have a major protective function under extreme environmental conditions. NTH1 is one gene which expresses trehalase to degrade trehalose. Small heat shock protein 12 (HSP12 expressed) plays a role in protecting membranes and enhancing freezing stress tolerance. Results: An optimized S. cerevisiae CRISPR-Cpf1 genome-editing system was constructed. Multiplex genome editing using a single crRNA array was shown to be functional. NTH1 or/and HSP12 knockout in S. cerevisiae enhanced the freezing stress tolerance and improved the leavening ability after freezing and thawing. Conclusions: Deleting NTH1 in the combination with deleting HSP12 would strengthen the freezing tolerance and protect the cell viability from high rates of death in longer-term freezing. It provides valuable insights for breeding novel S. cerevisiae strains for the baking industry through a more precise, speedy, and economic genome-editing system.

Honeybees, Apis mellifera, usually live in large colonies consisting of thousands of individuals. Within the colony, workers interact with their social environment frequently. The large workforce, division of labour, and other features may promote the ecological success of honeybees. For decades, artificial mini colonies in cages within the laboratory have become the gold standard, especially in experiments related to toxicology, effects of pesticides and pathogens. Experiments using caged bees and full-sized colonies yielded contradictory results. Here, the effect of cage experiments on the stress level of individual bees is analysed. Two different stress response were targeted, the heat shock response and the mobilization of energetic resources. While no differences were found for varying group sizes of bees, very strong effects emerged by comparing caged workers with bees from natural colonies. Caged workers showed increased levels of hsp expression and reduced haemolymph titres for trehalose, the energy storage sugar. These results reveal that the lack of the social environment (e.g., lack of queen, lack of sufficient group size) induce stress in caged bees, which might act synergistically when bees are challenged by additional stressors (e.g., pesticides, pathogens) resulting in higher mortality than observed under field conditions.

There have been many investigations on the negative effects of imidacloprid (IMD) on honey bees. IMD is known to disrupt honey bee physiology and colony health at a relatively low concentration compared to other pesticides. In this study, honey bee colonies were chronically exposed to field-realistic concentrations (5, 20, and 100 ppb) of IMD, and the body weight, flight performance, carbohydrate reserve, and lipid contents of forager bees analyzed. Transcriptome analyses followed by quantitative PCR were also conducted for both nurse and forager bees to elucidate any changes in energy metabolism related to phenotypic disorders. The body weights of newly emerged and nurse bees showed decreasing tendencies as the IMD concentration increased. In forager bees, however, IMD induced a biphasic change in body weight: body weight was decreased at the lower concentrations (5 and 20 ppb) but increased at the higher concentration (100 ppb). Nevertheless, the flight capability of forager bees significantly decreased in a concentration-dependent manner. The effects of IMD on target gene transcription in forager bees showed biphasic patterns between low (5 and 20 ppb) and high (100 ppb) concentrations. Nurse bees showed typical features of premature transition to foragers in a concentration-dependent manner. When exposed to low concentrations, forager bees exhibited downregulation of genes involved in carbohydrate and lipid metabolism and in the insulin/insulin-like growth factor signaling pathway, upregulation of transporter activity, and a dose-dependent body weight reduction, which were similar to insulin resistance and diabetic symptoms. However, increased lipid metabolism and decreased energy metabolism with body weight gain were observed at high IMD concentration. Considered together, these results suggest that field-realistic doses of IMD alter honey bee energy metabolism in distinctly different ways at low and high concentrations, both of which negatively affect honey bee colony health.

Sugar plays an important role in apple fruit development, appearance and quality as well as contributing to a plant's water stress response. Trehalose and the trehalose biosynthetic metabolic pathways are part of the sugar signaling system in plants, which are important regulator of water stress response in apple. The effect of water stress treatments applied to apple trees and the corresponding effects of ABA on developmental fruit quality were examined for indicators of fruit quality during fruit development. The results indicated that the severe water stress treatment (W2) occurring after the last stage of fruit cell division caused a decrease in the color and size of fruit. The moderate water stress (W1) occurring after the last stage of fruit cell enlargement (S2) caused an increase in the content of fructose and sorbitol while the apple fruit shape was not affected. These changes in sugar are related to the activity of sugar metabolic enzymes. While the enzymatic activity of vacuolar acid invertase (vAINV) was higher, that of sucrose-phosphate synthase (SPS) was lower in water stress treated fruit throughout the developmental period. This indicates that enhanced sucrose degradation and reduced sucrose synthesis leads to an overall reduced sucrose content during times of drought. Thus, water stress reduced sucrose content. Whereas the content of endogenous trehalose and ABA were the highest in water stress treated fruit. A moderate water stress (W1) imposed on apple trees via water restriction (60%–65% of field capacity) after the fruit cell enlargement phase of fruit development yielded sweeter fruit of higher economic value.

Environmental fluctuations often select for adaptations such as diapause states, allowing species to outlive harsh conditions. The natural sugar trehalose which provides both cryo- and desiccation-protection, has been found in diapause stages of diverse taxa. Here, we hypothesize that trehalose deposition in resting stages is a locally adapted trait, with higher concentrations produced in harsher habitats. We used resting stages, produced under standardized conditions, by 37 genotypes of Daphnia magna collected from Western Palaearctic habitats varying in their propensity to dry in summer and freeze in winter. Resting eggs produced by D. magna from populations from summer-dry habitats showed significantly higher trehalose than those from summer-wet habitats, suggesting that trehalose has a protective function during desiccation. By contrast, winter-freezing did not explain variation in trehalose content. Adaptations to droughts are important, as summer dryness of water bodies is foreseen to increase with ongoing climate change.

Vegetable-type soybean, also known as edamame, was recently introduced to South Africa. However, there is lack of information on its responses to drought. The aim of this study was to investigate the photosynthetic efficiency and carbohydrates responses of six edamame cultivars under drought stress. Photosynthetic efficiency parameters, including chlorophyll fluorescence and stomatal conductance, were determined using non-invasive methods, while pigments were quantified spectrophotometrically. Non-structural carbohydrates were quantified using Megazyme kits. Structural carbohydrates were determined using Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). Drought stress significantly increased the Fv/Fm and PIabs of AGS429 and UVE17 at pod filling stage. Chlorophyll-a, which was most sensitive to drought, was significantly reduced in AGS429 and UVE17, but chlorophyll-b was relatively stable in all cultivars, except UVE17, which showed a significant decline at flowering stage. AGS354 and AGS429 also showed reduced chlorophyll-b at pod filling. UVE17 showed a significant reduction in carotenoid content and a substantial reduction in stomatal conductance during pod filling. Drought stress during pod filling resulted in a significant increase in the contents of trehalose, sucrose and starch, but glucose was decreased. Chlorophyll-a positively correlated with starch. The FTIR and XRD results suggest that the cell wall of UVE14, followed by UVE8 and AGS429, was the most intact during drought stress. It was concluded that carotenoids, stomatal conductance, starch and hemicellulose could be used as physiological/biochemical indicators of drought tolerance in edamame. This information expands our knowledge of the drought defense responses in edamame, and it is essential for the physiological and biochemical screening of drought tolerance.