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Successful oral delivery of liposomes requires formulations designed to withstand harsh gastrointestinal conditions, e.g., by converting to solid-state followed by loading into gastro-resistant delivery devices. The hypothesis was that the use of dextran-trehalose mixtures for spray drying would improve the rehydration kinetics of dried liposomes. The objectives were to determine the protective capacity of trehalose-dextran dehydration precursors and to increase the concentration of liposomes in the dry formulation volume. The study successfully demonstrated that 8.5% dextran combined with 76.5% trehalose protected CAF®04 liposomes during drying, with the liposome content maintained at 15% of the dry powder. Accordingly, the rehydration kinetics were slightly improved in formulations containing up to 8.5% dextran in the dry powder volume. Additionally, a 2.4-fold increase in lipid concentration (3 mM vs 7.245 mM) was achieved for spray dried CAF®04 liposomes. Ultimately, this study demonstrates the significance of trehalose as a primary carrier during spray drying of CAF®04 liposomes and highlights the advantage of incorporating small amounts of dextran to tune rehydration kinetics of spray-dried liposomes.

When dry soil is re-wet there is a rapid increase in CO2 efflux and rates can remain above those of well-watered controls for one or more days. These large pulses of CO2 efflux are known as the `Birch effect. To provide experimental evidence of different pools of C fuelling the Birch effect, we incubated a drying soil with 13C6glucose, re wet the soil and quantified 13C labelling of pools (microbial biomass, trehalose, extracellular, and old C) and soil CO2 efflux. We took advantage of trehalose being the most 13C-enriched pool (& delta;13C = +518%o) to obtain direct isotopic evidence of trehalose's contribution to respiration and microbial growth. For soil incubated with 13C6-glucose, the & delta;13C of soil respiration was +35%o in dry soil, increased to 100%o in the 10 min following rewetting, and subsequently decreased. During the first 5 h after re-wetting, trehalose must have been contributing to respiration given that & delta;13C of soil respiration was more 13C enriched than trehalose-free microbial biomass (& delta;13C = +30%o), extracellular C (& delta;13C = -17.7%o), and old C (& delta;13C = -22.9%o). A four-member isotopic mixing model suggested trehalose underpinned 16% of respiration in the 1st hour after rewetting, decreasing to 7% in the fifth hour. At times beyond 5 h after rewetting, trehalose underpinned 0-4% of respiration. In the seven days following rewetting, microbial biomass increased 2292 nmol C g-1. Isotopic mass balance indicated trehalose-C could account for no more than 5% of the gross influx of C into microbial biomass, instead the increase in microbial biomass was fuelled by unlabelled or weakly labelled pools such as old C and extracellular C. Collectively these data provide direct experimental evidence C from trehalose does not significantly contribute to microbial growth in re-wet soil, but instead contributes to respiration for the first 5 h after rewetting.

The HIV epidemic is still one of the world's most serious public health challenges, affecting about 38 million people worldwide, especially in sub-Saharan African and Southeast Asian countries. In recent years, tests have been developed to discriminate recent from long-term infection in HIV-infected populations, and these tools can help identify new outbreaks and networks of transmission and target prevention and treatment plans. New rapid tests for recent infection are being deployed in point-of-care settings; however, quality assurance programs need to be implemented to ensure consistency and reliability of the results. We have developed a dried tube specimen (DTS) stabilized with disaccharide trehalose as a quality control reagent for rapid recency testing that can be stored unrefrigerated prior to reconstitution at temperatures up to 37°C for up to 12 weeks. Analysis of 10 trehalose-stabilized DTSs showed that they maintained the same recency classification in all of the samples stored at 4°C and 37°C up to 12 weeks and at 56°C for 2 weeks, while the DTSs prepared without trehalose changed their classification from long-term to recent or recent to negative after storage at 37°C for 12 weeks. Development of DTS quality control reagents will facilitate proficiency and training programs, particularly in settings without cold chain capability in field environments. IMPORTANCE Implementation of stabilized dried tube specimens (DTSs) for quality control and training would facilitate HIV recency programs, especially in point-of-care settings without cold chain availability. This study shows that addition of the disaccharide trehalose to DTSs prior to drying the samples increased stability of the samples across a range of temperatures. This finding provides an affordable way to increase the availability of these key reagents for quality control in resource-constrained settings.

The mutualistic ectomycorrhizal (ECM) fungal genus Pisolithus comprises 19 species defined to date which colonize the roots of >50 hosts worldwide suggesting that substantial genomic and functional evolution occurred during speciation. To better understand this intra-genus variation, we undertook a comparative multi-omic study of nine Pisolithus species sampled from North America, South America, Asia, and Australasia. We found that there was a small core set of genes common to all species (13%), and that these genes were more likely to be significantly regulated during symbiosis with a host than accessory or species-specific genes. Thus, the genetic "toolbox" foundational to the symbiotic lifestyle in this genus is small. Transposable elements were located significantly closer to gene classes including effector-like small secreted proteins (SSPs). Poorly conserved SSPs were more likely to be induced by symbiosis, suggesting that they may be a class of protein that tune host specificity. The Pisolithus gene repertoire is characterized by divergent CAZyme profiles when compared with other fungi, both symbiotic and saprotrophic. This was driven by differences in enzymes associated with symbiotic sugar processing, although metabolomic analysis suggest that neither copy number nor expression of these genes is sufficient to predict sugar capture from a host plant or its metabolism in fungal hyphae. Our results demonstrate that intra-genus genomic and functional diversity within ECM fungi is greater than previously thought, underlining the importance of continued comparative studies within the fungal tree of life to refine our focus on pathways and evolutionary processes foundational to this symbiotic lifestyle.

Coronary endothelial dysfunction is a major cause of ischemia-reperfusion (I/R) injury. Trehalose, a natural disaccharide, has been reported to ameliorate endothelial dysfunction during aging by activating endothelial nitric oxide synthase (eNOS); however, its role in I/R injury is unknown. This study evaluated the effects of trehalose preconditioning on cardiac and coronary endothelial function after I/R. Langendorff-perfused rat hearts underwent 30 min of global ischemia followed by 80 min of reperfusion with or without trehalose preconditioning. Rate pressure product (RPP) and coronary flow (CF) were measured during reperfusion. Perivascular edema was assessed by hematoxylin and eosin staining. Myocardial oxidative stress and apoptosis were evaluated by immunohistochemistry and TUNEL staining, respectively. eNOS dimerization was determined by western blotting. An eNOS inhibitor was used to examine the role of eNOS. Trehalose preconditioning showed a higher recovery rate after I/R as indicated by high RPP (control vs. trehalose, 28 +/- 6% vs. 46 +/- 9%; P = 0.017, Cohen's d = 2.3) and CF values (35 +/- 10% vs. 55 +/- 9%; P = 0.025, d = 1.7). Furthermore, trehalose preconditioning reduced perivascular edema, myocardial oxidative stress, and apoptosis. The eNOS dimerization ratio was increased by trehalose (1.2 +/- 0.2 vs. 1.6 +/- 0.2; P = 0.023, d = 2.1), which was associated with the recovery of RPP and CF. These effects of trehalose were abolished by the eNOS inhibitor. Trehalose preconditioning showed protective effects on cardiac and coronary endothelial function after I/R through the eNOS signaling pathway.

This study evaluated the effectiveness of hyaluronic acid and trehalose (HA/trehalose) eyedrops in managing dry eye disease (DED) symptoms by measuring tear stability and administering a DED questionnaire. Sixty patients were treated with either HA/trehalose eyedrops (Tear A) or carmellose sodium eyedrops (Tear B) as controls. The tear breakup time (TBUT) and non-invasive breakup time (NIBUT) were monitored, and patients completed the standard patient evaluation of eye dryness (SPEED) questionnaire. After two months of twice-daily applications, patients treated with the HA/trehalose eyedrops demonstrated significant improvements in the NIBUT (12.98 ± 3.22 s) and TBUT (12.95 ± 2.98 s), indicating increased tear stability. Moreover, they reported lower dry eye sensation (6.70 ± 4.94 SPEED score points), suggesting a reduction in DED symptoms. These findings underscore the efficacy of HA/trehalose eyedrops in improving both the objective and subjective signs of DED, with twice-daily application enhancing ocular surface conditions and reducing patient-reported symptoms. © 2023 by the authors.

Sexual reproduction is prevalent in eukaryotic organisms and plays a critical role in the evolution of new traits and in the generation of genetic diversity. Environmental factors often have a direct impact on the occurrence and frequency of sexual reproduction in fungi. The regulatory effects of atmospheric relative humidity (RH) on sexual reproduction and pathogenesis in plant fungal pathogens and in soil fungi have been extensively investigated. However, the knowledge of how RH regulates the lifecycles of human fungal pathogens is limited. In this study, we report that low atmospheric RH promotes the development of mating projections and same-sex (homothallic) mating in the human fungal pathogen Candida albicans. Low RH causes water loss in C. albicans cells, which results in osmotic stress and the generation of intracellular reactive oxygen species (ROS) and trehalose. The water transporting aquaporin Aqy1, and the G-protein coupled receptor Gpr1 function as cell surface sensors of changes in atmospheric humidity. Perturbation of the trehalose metabolic pathway by inactivating trehalose synthase or trehalase promotes same-sex mating in C. albicans by increasing osmotic or ROS stresses, respectively. Intracellular trehalose and ROS signal the Hog1-osmotic and Hsf1-Hsp90 signaling pathways to regulate the mating response. We, therefore, propose that the cell surface sensors Aqy1 and Gpr1, intracellular trehalose and ROS, and the Hog1-osmotic and Hsf1-Hsp90 signaling pathways function coordinately to regulate sexual mating in response to low atmospheric RH conditions in C. albicans.

Background: Asexually developed fungal spores (conidia) are key for the massive proliferation and dispersal of filamentous fungi. Germination of conidia and subsequent formation of a mycelium network give rise to many societal problems related to human and animal fungal diseases, post-harvest food spoilage, loss of harvest caused by plant-pathogenic fungi and moulding of buildings. Conidia are highly stress resistant compared to the vegetative mycelium and therefore even more difficult to tackle. Results: In this study, complementary approaches are used to show that accumulation of mannitol and trehalose as the main compatible solutes during spore maturation is a key factor for heat resistance of conidia. Compatible solute concentrations increase during conidia maturation, correlating with increased heat resistance of mature conidia. This maturation only occurs when conidia are attached to the conidiophore. Moreover, conidia of a mutant Aspergillus niger strain, constructed by deleting genes involved in mannitol and trehalose synthesis and consequently containing low concentrations of these compatible solutes, exhibit a sixteen orders of magnitude more sensitive heat shock phenotype compared to wild-type conidia. Cultivation at elevated temperature results in adaptation of conidia with increased heat resistance. Transcriptomic and proteomic analyses revealed two putative heat shock proteins to be upregulated under these conditions. However, conidia of knock-out strains lacking these putative heat shock proteins did not show a reduced heat resistance. Conclusions: Heat stress resistance of fungal conidia is mainly determined by the compatible solute composition established during conidia maturation. To prevent heat resistant fungal spore contaminants, food processing protocols should consider environmental conditions stimulating compatible solute accumulation and potentially use compatible solute biosynthesis as a novel food preservation target.

Quercetin (QUER) is a natural polyphenolic compound endowed with beneficial properties for human health, with anti-aging effects. However, although this flavonoid is commercially available as a nutraceutical, target molecules/pathways underlying its pro-longevity potential have yet to be fully clarified. Here, we investigated QUER activity in yeast chronological aging, the established model for simulating the aging of postmitotic quiescent mammalian cells. We found that QUER supplementation at the onset of chronological aging, namely at the diauxic shift, significantly increases chronological lifespan (CLS). Consistent with the antioxidant properties of QUER, this extension takes place in concert with a decrease in oxidative stress. In addition, QUER triggers substantial changes in carbon metabolism. Specifically, it promotes an enhancement of a pro-longevity anabolic metabolism toward gluconeogenesis due to improved catabolism of C2 by-products of yeast fermentation and glycerol. The former is attributable to the Sir2-dependent activity of phosphoenolpyruvate carboxykinase and the latter to the L-glycerol 3-phosphate pathway. Such a combined increased supply of gluconeogenesis leads to an increase in the reserve carbohydrate trehalose, ensuring CLS extension. Moreover, QUER supplementation to chronologically aging cells in water alone amplifies their long-lived phenotype. This is associated with intracellular glycerol catabolism and trehalose increase, further indicating a QUER-specific influence on carbon metabolism that results in CLS extension.

Acidophiles maintain near-neutral intracellular pH using proton pumps. We have suggested the protective role of osmolytes and membrane lipids in the adaptation to an acidic environment. Previously we have observed, for the first time, high levels of trehalose in acidophilic basidiomycete Sistotrema brinkmannii. Here, we have studied the composition of both osmolytes and membrane lipids of two more acidophilic fungi. Trehalose and polyols were among the main osmolytes during growth under optimal conditions (pH 4.0) in basidiomycete Phlebiopsis gigantea and ascomycete Mollisia sp. Phosphatidic acids, phosphatidylethanolamines, phosphatidylcholines, and sterols, were predominant membrane lipids in both fungi. P. gigantea had a narrow optimum of growth at pH 4.0, resulting in a sharp decline of growth rate at pH 2.6 and 5.0, accompanied by a decrease in the number of osmolytes and significant changes in the composition of membrane lipids. In contrast, Mollisia sp. had a broad optimal growth range (pH 3.0–5.0), and the number of osmolytes either stayed the same (at pH 6.0) or increased (at pH 2.6), while membrane lipids composition remained unchanged. Thus, the data obtained indicate the participation of osmolytes and membrane lipids in the adaptation of acidophilic fungi.

The mutualistic ectomycorrhizal (ECM) fungal genus Pisolithus comprises 19 species defined to date which colonize the roots of >50 hosts worldwide suggesting that substantial genomic and functional evolution occurred during speciation. To better understand this intra-genus variation, we undertook a comparative multi-omic study of nine Pisolithus species sampled from North America, South America, Asia, and Australasia. We found that there was a small core set of genes common to all species (13%), and that these genes were more likely to be significantly regulated during symbiosis with a host than accessory or species-specific genes. Thus, the genetic "toolbox" foundational to the symbiotic lifestyle in this genus is small. Transposable elements were located significantly closer to gene classes including effector-like small secreted proteins (SSPs). Poorly conserved SSPs were more likely to be induced by symbiosis, suggesting that they may be a class of protein that tune host specificity. The Pisolithus gene repertoire is characterized by divergent CAZyme profiles when compared with other fungi, both symbiotic and saprotrophic. This was driven by differences in enzymes associated with symbiotic sugar processing, although metabolomic analysis suggest that neither copy number nor expression of these genes is sufficient to predict sugar capture from a host plant or its metabolism in fungal hyphae. Our results demonstrate that intra-genus genomic and functional diversity within ECM fungi is greater than previously thought, underlining the importance of continued comparative studies within the fungal tree of life to refine our focus on pathways and evolutionary processes foundational to this symbiotic lifestyle.

A fluorescence turn-on probe, an azide-masked and trehalosederivatized carbazole (Tre-Cz), was developed to image mycobacteria. The fluorescence turn-on is achieved by photoactivation of the azide, which generates a fluorescent product through an efficient intramolecular C-H insertion reaction. The probe is highly specific for mycobacteria and could image mycobacteria in the presence of other Gram-positive and Gram-negative bacteria. Both the photoactivation and detection can be accomplished using a handheld UV lamp, giving a limit of detection of 10(3) CFU/mL, which can be visualized by the naked eye. The probe was also able to image mycobacteria spiked in sputum samples, although the detection sensitivity was lower. Studies using heat-killed, stationary-phase, and isoniazid-treated mycobacteria showed that metabolically active bacteria are required for the uptake of Tre-Cz. The uptake decreased in the presence of trehalose in a concentration-dependent manner, indicating that Tre-Cz hijacked the trehalose uptake pathway. Mechanistic studies demonstrated that the trehalose transporter LpqY-SugABC was the primary pathway for the uptake of Tre-Cz. The uptake decreased in the LpqY-SugABC deletion mutants Delta lpqY, Delta sugA, Delta sugB, and Delta sugC and fully recovered in the complemented strain of Delta sugC. For the mycolyl transferase antigen 85 complex (Ag85), however, only a slight reduction of uptake was observed in the Ag85 deletion mutant Delta Ag85C, and no incorporation of Tre-Cz into the outer membrane was observed. The unique intracellular incorporation mechanism of Tre-Cz through the LpqY-SugABC transporter, which differs from other trehalose-based fluorescence probes, unlocks potential opportunities to bring molecular cargoes to mycobacteria for both fundamental studies and theranostic applications.

Lo sviluppo di “fabbriche cellulari” microbiche per la biosintesi di molecole di interesse è uno dei possibili approcci per affrontare l’aumento esponenziale della popolazione. Attualmente in letteratura si ritrovano innumerevoli esempi di questo approccio applicati a svariati prodotti. Trarre profitto dai percorsi metabolici noti è spesso possibile solo attraverso tecniche di ingegneria metabolica, per reindirizzare il metabolismo e forzarlo verso l’accumulo dei metaboliti di interesse. Tra tutti i microorganismi, il lievito Saccharomyces cerevisiae è una delle migliori fabbriche cellulari attualmente utilizzate in numerosi processi industriali, come ad esempio la produzione di biocarburanti. Il corpo principale di questo lavoro è incentrato sull'estensione della gamma di applicazioni per produrre molecole di forte interesse industriale sfruttando le capacità innate e non ancora esplorate di S. cerevisiae. Nella prima parte del lavoro abbiamo valutato la possibilità di produrre ammoniaca. Attualmente, la produzione avviene prevalentemente per sintesi chimica ma la necessità di ridurre l’inquinamento e trovare vie sintetiche rinnovabili ha condotto i ricercatori a testare le produzioni per via microbica. Ispirati dai precedenti studi con batteri, abbiamo indotto S. cerevisiae a sfruttare sostanze azotate naturali attraverso una strategia di evoluzione adattiva. I risultati hanno mostrato una marcata capacità di secernere ammoniaca utilizzando nutrienti non convenzionali. Mediante tecniche di coltivazione in bioreattore e sfruttando il metabolismo naturale del lievito, abbiamo raggiunto 2,03 grammi di ammoniaca totale prodotta che, ad oggi, è il più alto valore registrato con S. cerevisiae. Nella seconda parte di questo lavoro, lo scopo principale è stato quello di esaminare l'accumulo di un altro metabolita chiamato trealosio, esplorando le sue potenziali applicazioni. Abbiamo isolato un ceppo di lievito specifico in cui i geni responsabili della degradaz, The construction of microbial cell factories for the biosynthesis of molecules of interest is one of the essential prerogatives to face an exponential population increase. Taking full advantage of metabolism remains a challenge that has become extremely popular among researchers. Exploiting metabolic pathways using a rewarding approach is often only feasible through metabolic engineering techniques, which enable distinctive utilization of the route to produce compounds of interest. Among all, Saccharomyces cerevisiae is one of the best cell factories currently used in numerous industrial processes, such as the production of biofuels. The main body of this thesis focuses on extending the range of applications to produce molecules of strong industrial interest by exploiting the innate and hidden capabilities of S. cerevisiae. In the first part of this work, we explored the potential to produce ammonia. Currently, production is predominantly by chemical synthesis, and the need to move towards more sustainable society has led researchers to find microorganisms capable of producing chemicals. Intrigued by what had been done in previous studies with bacteria, we induce S. cerevisiae to take advantage of natural nitrogen substrates through an Adaptive Laboratory Evolution strategy. Results showed a marked ability to secrete ammonia revealing a pattern in the use of unconventional substrates. Using bioreactor cultivation techniques and exploiting the unknown natural metabolism of yeast, we achieved 2,03 grams of ammonia, which is the highest recorded with Saccharomyces cerevisiae to date. In the second part, the main purpose was to examine trehalose accumulation and explore its potential applicability. We isolated a specific yeast strain in which the genes responsible for the breakdown of trehalose were deleted (neutral and acid trehalose) and unexpectedly noticed an increased amount of this molecule being mobilized from the cytosol to the extracellular environment. The stu

The purpose of the research project was to extensively review the efficacy and safety of a trehalose tear-substitute treatment in cases of dry eye disease (DED). A systematic review that included only full-length randomized controlled studies (RCTs) reporting the effects of trehalose tear-substitute treatment in three databases, PubMed, Scopus andWeb of Science, was performed according to the PRISMA statement. The search period included papers published before 8 August 2023. The Cochrane risk-of-bias tool was used to analyze the quality of the studies selected. A total of 10 RCTs were included in this systematic review. Trehalose tear-substitute treatments achieved a higher improvement than did control group interventions in all reported variables. The mean differences between both groups were in favor of trehalose, and were as follows: ocular surface disease index (OSDI) questionnaire score of -8.5 +- 7 points, tear film breakup time (TBUT) of 1.9 +- 1 s, tear film thickness (TFT) of 0.25 +- 0.1 um, tear meniscus height (TMH) of 0.02 +- 0.02 mm, Schirmer test (ST) of 0.8 +- 1.4 mm, corneal fluorescein staining (CFS) of -0.7 +- 0.1 points and visual acuity (VA) of 0.3 +- 2.1 letters. No adverse events after trehalose tear-substitute treatments were reported. Trehalose tear substitutes are a safe and effective treatment for DED. Therefore, trehalose tear substitutes should be recommended for patients with dry eye disease. In addition, there is specific evidence to support its use in the preoperative cataract surgery period.

—: Investigation of the growth rate of Sistotrema brinkmannii at different pH values, temperature, and NaCl concentration showed that this fungus was a mesophile, preferred a salt-free medium, and was an obligate acidophile, since it had a pronounced growth optimum at pH 3.0–4.0 and did not grow at pH 7.0. To reveal the protective mechanisms allowing this fungus to develop under acidic conditions, the composition of its osmolytes and lipids was studied. This is the first report on occurrence of a large amount of trehalose (4.0‒6.6% of dry weight) in the mycelium of the fungus during its growth under optimal conditions, confirming the use of osmolytes by acidophiles for adaptation. At the same time, at the borders of the growth range (pH 2.6 and 6.0), the amount of trehalose in the mycelium of the fungus decreased by 2.5 times, which was in agreement with a narrow growth optimum of the fungus in its natural environments (pH 3.0–4.0). The composition of membrane lipids of the fungus was characterized by a high proportion of sphingolipids (up to 60% of the total), which decreased twofold during growth under optimal conditions. The main membrane lipids, apart from sphingolipids, were phosphatidic acids, phosphatidylethanolamines, and sterols; the proportion of these lipids increased with time. The composition of membrane lipids of the fungus at pH 2.6 did not differ much from the optimal conditions, while in the near-neutral region there was a twofold increase in the proportion of sphingolipids, indicating their adaptive value. The simultaneous decrease in the proportion of sphingolipids and the increase in the level of trehalose in the growth dynamics suggest association of these compounds in the protection of cell membranes.

The aim of this thesis was to increase our knowledge of the glassy state and the glass transition phenomenon and to evaluate the effect of water on the glassy state. To accomplish this, investigations were focused on the amorphous sucrose-water (paper I, II, III), trehalose-water (paper I), maltodextrin-water (paper I), and lysozyme-sucrose-water (paper IV) systems. We studied temperature-induced and isothermal glass transition (I, II, III, IV), as well as the impact of water on the activation energy of the relaxation process (II). It has been shown that water undergoes glass transition with disaccharides, but in polysaccharides water dynamics is uncoupled from the polymer matrix. This results in differences in the water diffusion coefficient: water moves several orders of magnitude faster in the polymers than in disaccharides (I). Water reduces the activation energy of the relaxation process in the sucrose-water system (II). Attenuated water diffusion at sub-zero temperatures leads to a delay in water crystallization/melting in the sucrose-water system, which does not happen in a polysaccharide - water system (III). The Tg of the lysozyme-sucrose system increases with increasing lysozyme concentration, i.e., the DCp of the mixtures does not follow the prediction based on the properties of the pure components. Consequently, lysozyme does not modulate the glass transition of the sucrose matrix and the increase of the Tg of the mixtures is a result of the confinement of amorphous sucrose in the space between lysozyme molecules. The amorphous structure and unfolding of lysozyme in the presence of sucrose was investigated by DSC and SAXS. These data revealed an increase of the protein-protein distance upon addition of sucrose and upon heating, as a result of lysozyme unfolding (IV).

A number of crop wild relatives can tolerate extreme stress to a degree outside the range observed in their domesticated relatives. However, it is unclear whether or how the molecular mechanisms employed by these species can be translated to domesticated crops. Paspalum (Paspalum vaginatum) is a self-incompatible and multiply stress-tolerant wild relative of maize and sorghum. Here, we describe the sequencing and pseudomolecule level assembly of a vegetatively propagated accession of P. vaginatum. Phylogenetic analysis based on 6,151 single-copy syntenic orthologues conserved in 6 related grass species places paspalum as an outgroup of the maize-sorghum clade. In parallel metabolic experiments, paspalum, but neither maize nor sorghum, exhibits a significant increase in trehalose when grown under nutrient-deficit conditions. Inducing trehalose accumulation in maize, imitating the metabolic phenotype of paspalum, results in autophagy dependent increases in biomass accumulation.

In this study, the protein stability of hen egg-white lysozymes (HEWL) by Fe3O4 and Fe3O4-coated trehalose (Fe3O4@Tre) magnetic nanoparticles (NPs) is investigated. For this purpose, the co-precipitation method was used to synthesize magnetic NPs. The synthesized NPs were characterized by XRD, FT-IR spectroscopy, FE-SEM, and VSM analysis. In addition, the stability of HEWLs exposed to different NP concentrations in the range of 0.001-0.1 mg mL(-1) was investigated by circular dichroism (CD) spectroscopy, fluorescence, and UV-Vis analysis. Based on the results, in the NP concentration range of 0.001-0.04 mg mL(-1) the protein structure is more stable, and this range was identified as the range of kosmotropic concentration. The helicity was measured at two concentration points of 0.02 and 0.1 mg mL(-1). According to the results, the alpha-helix at 0.02 mg mL(-1) of Fe3O4 and Fe3O4@Tre was increased from 35.5% for native protein to 37.7% and 38.7%, respectively. The helicity decreased to 36.1% and 37.4%, respectively, with increasing the concentration of Fe3O4 and Fe3O4@Tre to 0.1 mg mL(-1). The formation of hydrated water shells around protein molecules occurred by using Fe3O4@Tre NPs. Hence, it can be concluded that the trehalose as a functional group along with magnetic NPs can improve the stability of proteins in biological environments., QC 20220920

We evaluated a dimethyl sulfoxide (Me2SO)-free cryopreservation solution to freeze human adipose-derived mesenchymal stromal cells (hADSCs). In the first experiment, we compared the combined effects of 3% trehalose (3 T) and 5% dextran (5D) in lactated Ringer’s solution (LR) as a cryopreservation base solution containing 10% propylene glycol (PG). The cell viability of hADSCs immediately after thawing was significantly higher (p < 0.05) in LR supplemented with 3 T (LR-3 T) and with 3 T and 5D (LR-3 T-5D) than in LR. In the second experiment, we compared the cell characteristics of hADSCs freeze-thawed in LR-3 T-5D containing either 10% Me2SO or 10% PG. The cell viability, annexin V-positive ratio, colony-forming capacity, cell proliferation, cell surface antigen positivity, adipogenic differentiation, osteogenic differentiation, and genetic response to cytokine stimulation of hADSCs immediately after thawing were similar between the LR-3 T-5D containing 10% Me2SO and 10% PG. In the third experiment, we examined various concentrations of PG on the cell proliferative capacity of freeze-thawed hADSCs. The cell proliferative capacity of hADSCs frozen with LR-3 T-5D containing 2.5% to 5% PG was significantly higher (p < 0.05) than LR-3 T-5D containing 10% PG. Furthermore, the cell proliferative capacity of hADSCs frozen with LR-3 T-5D containing 4% PG was similar to that of fresh hADSCs. These results indicate that the combination of 3 T-5D in an LR solution as a basic solution is effective for post-thaw cell viability, and that the optimal concentration of PG to maintain the cell characteristics of hADSCs frozen with LR-3 T-5D is 2.5% to 5%, which is promising for cell therapy applications.

Recurrence of left ventricular (LV) remodeling after surgical ventricular reconstruction (SVR) for ischemic cardiomyopathy has been reported to be partially attributed to autophagy. We aimed to examine the effects of trehalose, an autophagy inducer, on the recurrence of LV remodeling after SVR. After SVR in rats with ICM, trehalose was orally administered. The changes in LV end-diastolic dimension (LVEDD) and fractional shortening (FS) were evaluated. The activation of myocardial autophagy was also estimated by autophagy markers: microtubule-associated light chain 3 II (LC3-II) and p62; the former usually increases and the latter decreases if autophagy is activated. Significant LV reverse remodeling was observed early after SVR. On the other hand, the 28th postoperative day SVR + trehalose was associated with smaller LVEDD and better FS than SVR alone (LVEDD, P = 0.043; FS, P < 0.01). LC3-II increased comparably in both groups, while p62 was significantly lower in the SVR + trehalose group than in the SVR alone group (P < 0.01). In conclusion, trehalose attenuated the recurrence of LV remodeling and changed autophagy markers after SVR in rats with ICM. Trehalose may be a candidate for adjuvant therapy to retain the effects of SVR.

Here, we present a two-step continuous flow enzymatic synthesis process in monolithic microreactors using basic sugars as substrates. In the first step UDP-glucose pyrophosphorylase (TaGalU) catalyses the synthesis of uridine-diphosphate-glucose (UDP-Glc) using uridine triphosphate (UTP) and glucose-1-phosphate (Glc-1-P). This is followed by the trehalose transferase (mCherry-TuTreT) catalysed reaction of UDP-Glc and Glc, to obtain trehalose. First, procedures for immobilisation of both enzymes on functionalised silica supports were studied and it was found that covalent bonding by amino groups using a glutaraldehyde linker gives highly active biocatalysts. Due to a drastic difference in temperature range of activity and stability of the immobilised enzymes a bi-reactor cascade was rationally the best solution. Depending on the applied flow rate and hence reaction (residence) time (1.5–10 min) the space-time-yield values varied, respectively, from 1.9 to 14.4 and 8.3 to 49.6 gproduct·L-1·h−1·mgprotein-1, for UDP-glucose pyrophosphorylase and trehalose transferase catalysed reactions. Prolonged (100 h) continuous flow operation showed that the system is operationally stable, but owing to neutral pH, it is prone to microbiological infections. They can be eliminated applying an antibacterial/antifungal therapy or preventive actions by storing and washing the reactors with a NaN3 solution. The presented process paves the way for the continuous in flow synthesis of natural and non-natural trehalose analogues and disaccharides., Green Open Access added to TU Delft Institutional Repository 'You share, we take care!' - Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public., BT/Biocatalysis

Background: Insulin resistance is a condition in which insulin cannot take up glucose, increasing blood glucose. Elderly people are more exposed to insulin resistance, requiring dietary interventions that extend longevity. Trehalose, a naturally occurring sugar, showed potentially reduce insulin resistance which can be measured using the HOMA-IR index. Objectives: This study aimed to assess HOMA-IR levels as a parameter of insulin resistance in old rats after giving trehalose sugar. Methods: Experimental research with 28 male Wistar rats (Rattus novergicus) was separated into 4 groups, the control group of young rats (Group A), the control group of old rats (Group B), a group of old rats that were given 2% Trehalose solution (Group C), and a group of old rats that given 2% sucrose solution (Group D) that observed for 8 weeks. Results: The results showed differences in HOMA-IR levels (p<0.001) between old and young subjects. The intervention in Group C was optimal in reducing levels of HOMA-IR (p<0.001) by 18.2% compared with the old control, while Group D increased levels of HOMA-IR by 14.3% (p<0.001) compared with the old control. The age of the subjects with HOMA-IR level is positively correlated (p<0.001; r=0.721) and the weight of subjects with the HOMA-IR level is also positively correlated (p<0.001; r=0.698), indicating that the older and the greater weight of subject resulting in the bigger of HOMA-IR value. Conclusion: Trehalose is effective in reducing HOMA-IR levels as a parameter of insulin resistance in old rats.

This paper describes the extension of a baker's yeast growth model to account for the intracellular trehalose storage and mobilization. Trehalose is a reserve carbohydrate that is accumulated and converted back to intracellular glucose when the yeast cells face certain stresses. This is modeled by a new macroscopic reaction, which is coupled to an existing macroscopic reaction scheme describing the coordinated uptake of glucose and ammonium by the yeast cells. The dynamics of the trehalose concentration is described by a delay differential equation as the available experimental data used to fit the model exhibits a time-delayed correlation between trehalose storage and glucose uptake as well as a time-delayed correlation between trehalose mobilization and ethanol respiration phases. The proposed extension contains 6 parameters of which 5 are estimated via nonlinear least squares identification. The proposed model predicts accurately the dynamics of trehalose storage and mobilization and can be used to optimize the intracellular trehalose accumulation in Saccharomyces cerevisiae, which is valuable for obtaining reinforced yeast cells, able to better withstand drying operations., SCOPUS: cp.p, info:eu-repo/semantics/published

Yeast metabolism depends on growing conditions, which include the chemical composition of the medium, temperature and growth time. Historically, fatty acid profiles have been used to differentiate yeasts growing in liquid media. The present study determined the fatty acids of Saccharomyces species in colonies. Using the same method, the effect of that the number of colonies and growth time had on solid media allowed us to determine the metabolomic profiles of the cells. Our results showed that the lipid and metabolomic profiles of the cells evolved as the colony grew. Interestingly, some strains of Saccharomyces cerevisiae have been were differentiated using the fatty acid profile of a colony; concretely indeed EC1118 and QA23 strains were separated from ICV-K1 and BM4x4. The synthesis of saturated fatty acids was greater than that of unsaturated fatty acids during the first two days of cell growth on a solid medium compared to a liquid medium. Unsaturated fatty acids subsequently became predominant. Finally, this methodology could be useful for carrying out physiological studies in a complete or defined solid growth medium allowing the supplementation of compounds, which inhibit or activate the growth of yeasts. © 2021

[ES] Helicobacter pylori es una bacteria patógena que causa graves problemas gástricos a gran parte de la población mundial, especialmente en países menos desarrollados. Los tratamientos tradicionales a base de antibióticos tienen efectos secundarios y no son efectivos al 100%. Estudios recientes demuestran que algunas cepas del género Lactobacillus son efectivas en el tratamiento contra Helicobacter pylori, reduciendo la colonización de este patógeno, lo que ha promovido su incorporación en la formulación de determinados alimentos. Sin embargo, la supervivencia de este microorganismo en alimentos es bastante limitada, especialmente si no son de origen lácteo. Este Trabajo de Final de Grado se plantea con el objeto de desarrollar un snack probiótico mediante impregnación a vacío con zumo de clementina inoculado con Lactobacillus salivarius spp. salivarius y posterior liofilización. Para aumentar la viabilidad del probiótico durante el procesado y almacenamiento del citado snack se propone la incorporación de trehalosa al líquido de impregnación antes de su inoculación con el probiótico y/o la homogenización a presión moderada tras su incubación. De acuerdo con los resultados obtenidos, la liofilización es una técnica adecuada para la estabilización de láminas de manzana impregnadas con el microorganismo probiótico ya que, a pesar de los daños estructurales ocasionados por la congelación y posterior sublimación del agua, tanto el recuento de viables como el contenido en compuestos con actividad antioxidante se vio incrementado tras esta operación de deshidratación. Sin embargo, pese a la baja actividad del agua alcanzada por las muestras al final del proceso, tanto la viabilidad del probiótico como el contenido en flavonoides disminuyeron tras 30 días de almacenamiento. En cuanto a la composición del líquido de impregnación, la adición de un 10% de trehalosa en peso y/o la homogeneización a 100 MPa aceleraron la pérdida de viabilidad del probiótico pero retardaron la, [EN] Helicobacter pylori is a pathogenic bacterium that causes severe gastric problems to a large part of the world's population, especially in less developed countries. Traditional treatments based on antibiotics have side effects and are not 100% effective. Recent studies show that some strains of the Lactobacillus genus are effective in the treatment against Helicobacter pylori, reducing the colonization of this pathogen, which has promoted the incorporation of Lactobacillus in the formulation of certain foods. However, the survival of this microorganism in food is rather limited, especially in not dairy products. The aim of this Final Degree Project is to develop a probiotic apple snack by vacuum impregnation with different clementine juices inoculated with Lactobacillus salivarius spp. salivarius and subsequent freeze drying. In order to increase the viability of the probiotic during the snack manufacture and storage, it is suggested to add trehalose to the juice formulation before its inoculation with the probiotic and-/-or to homogenizate the juice under moderate pressure after its incubation. According to the results obtained, freeze drying is a suitable technique for the stabilization of impregnated apple slices with the probiotic microorganism because, in spite of the structural damages caused by the freezing and following sublimation of the frozen water, both the count of viable cells and the content of compounds with antioxidant activity increased after this dehydration operation. However, despite the low water activity reached by the samples at the end of the process, both the viability of the probiotic and the flavonoid content decreased after 30 days of storage. As for the composition of the impregnation liquid, the addition of 10% of trehalose by weight and/or the homogenization at 100 MPa accelerated the loss of probiotic viability but delayed flavonoids degradation and promoted an increase in the amount of both phenols and total antioxidants. Final

[EN] To benefit the health of consumers, bioactive compounds must reach an adequate concentration at the end of the digestive process. This involves both an effective release from the food matrix where they are contained and a high resistance to exposure to gastrointestinal conditions. Accordingly, this study evaluates the impact of trehalose addition (10% w/w) and homogenization (100 MPa), together with the structural changes induced in vacuum impregnated apple slices (VI) by air-drying (AD) and freeze-drying (FD), on Lactobacillus salivarius spp. salivarius (CECT 4063) survival and the bioaccessibility of antioxidants during in vitro digestion. Vacuum impregnated apple slices conferred maximum protection to the lactobacillus strain during its passage through the gastrointestinal tract, whereas drying with air reduced the final content of the living cells to values below 10 cfu/g. The bioaccessibility of antioxidants also reached the highest values in the VI samples, in which the release of both the total phenols and total flavonoids to the liquid phase increased with in vitro digestion. The addition of trehalose and homogenization at 100 MPa increased the total bioaccessibility of antioxidants in FD and AD apples and the total bioaccessibility of flavonoids in the VI samples. Homogenizing at 100 MPa also increased the survival of L. salivarius during in vitro digestion in FD samples.

Autophagy is an intracellular pathway that degrades unnecessary proteins and organelles and provides energy substrates during cellular ischemic conditions. Although pharmacological myocardial pre -conditioning with an autophagy inducer has been reported to protect cells against ischemic reperfusion (I/R), the effects of preconditioning using naturally occurring substances are still unknown. We aimed to examine whether autophagic preconditioning with trehalose improves cardiac function after myocardial stunning by global ischemia in rats. Rat hearts were perfused by oxygenized Krebs Henseleit (KH) so-lution in Langendorff system. Ten rats were randomized into the following two groups according to the perfusates during the preconditioning: control (KH solution only, n = 5) and trehalose (KH & thorn; 2% trehalose, n = 5). After the 35-min preconditioning period and subsequent 20 min of global ischemia, the hearts were reperfused for 60 min. Cardiac function was assessed during the reperfusion. To evaluate autophagy, myocardial protein expression of microtubule-associated protein light chain 3 (LC3) II was evaluated by western blotting. During I/R, a systolic functional parameter, maximum dP/dt was signifi-cantly higher; meanwhile, coronary flow tended to be higher in the trehalose group than in the control group. Myocardial LC3-II expression after preconditioning was higher in the trehalose group than in the control group and decreased to the control level after I/R. In conclusion, in a rat model of global myocardial ischemia, trehalose preconditioning improved cardiac function during I/R. Further studies would be needed to identify the mechanism and effects of trehalose preconditioning. (c) 2021 Elsevier Inc. All rights reserved.

The research aim was to use orange essential oil and trehalose in a carrageenan matrix to form edible packaging. The edible packaging experimentally produced by casting from an aqueous solution were evaluated by the following analysis: UV-Vis spectrum, transparency value, transmittance, attenuated total reflectance Fourier-Transform spectroscopy (FTIR), scanning electron microscopy (SEM) and antimicrobial activity. The obtained results showed that the combination of orange essential oil with trehalose decreases the transmittance value in the UV and Vis regions (up to 0.14% +/- 0.02% at 356 nm), meaning that produced films can act as a UV protector. Most produced films in the research were resistant to Gram-positive bacteria (Staphylococcus aureus subsp. aureus), though most films did not show antibacterial properties against Gram-negative bacteria and yeasts. FTIR and SEM confirmed that both the amount of carrageenan used and the combination with orange essential oil influenced the compatibility of trehalose with the film matrix. The research showed how different combinations of trehalose, orange essential oils and carrageenan can affect edible film properties. These changes represent important information for further research and the possible practical application of these edible matrices.

Trehalose, a nonreducing disaccharide consisting of D-glucose with alpha,alpha-1,1 linkage, was evaluated as a functional material to improve the gut environment in preweaned calves. In experiment 1, 173 calves were divided into two groups; the trehalose group was fed trehalose at 30 g/animal/d with milk replacer during the suckling period, and the control group was fed nonsupplemented milk replacer. Medication frequency was lower in the trehalose group (P < 0.05). In experiment 2, calves (n = 20) were divided into two groups (control group [n = 10] and trehalose group [n =10] based on their body weight and reared under the same feeding regimens as in experiment 1. Fresh feces were collected from individual animals at the beginning of the trial (average age 11 d), 3 wk after trehalose feeding (experimental day 22), and 1 d before weaning, and the fecal score was recorded daily. Fecal samples were analyzed for fermentation parameters and microbiota. The fecal score was significantly lower in the trehalose group than in the control group in the early stage (at an age of 14 to 18 d; P < 0.05) of the suckling period. Calves fed trehalose tended to have a higher proportion of fecal butyrate on day 22 than calves in the control group (P = 0.08). Population sizes of Clostridium spp. were significantly lower (P = 0.036), whereas those of Dialister spp. and Eubacterium spp. tended to be higher in the feces of calves in the trehalose group on day 22 (P = 0.060 and P = 0.083). These observations indicate that trehalose feeding modulated the gut environment and partially contributed to the reduction in medication frequency observed in experiment 1.

The research aim was to use orange essential oil and trehalose in a carrageenan matrix to form edible packaging. The edible packaging experimentally produced by casting from an aqueous solution were evaluated by the following analysis: UV-Vis spectrum, transparency value, transmittance, attenuated total reflectance Fourier-Transform spectroscopy (FTIR), scanning electron microscopy (SEM) and antimicrobial activity. The obtained results showed that the combination of orange essential oil with trehalose decreases the transmittance value in the UV and Vis regions (up to 0.14% +/- 0.02% at 356 nm), meaning that produced films can act as a UV protector. Most produced films in the research were resistant to Gram-positive bacteria (Staphylococcus aureus subsp. aureus), though most films did not show antibacterial properties against Gram-negative bacteria and yeasts. FTIR and SEM confirmed that both the amount of carrageenan used and the combination with orange essential oil influenced the compatibility of trehalose with the film matrix. The research showed how different combinations of trehalose, orange essential oils and carrageenan can affect edible film properties. These changes represent important information for further research and the possible practical application of these edible matrices.

The research aim was to use orange essential oil and trehalose in a carrageenan matrix to form edible packaging. The edible packaging experimentally produced by casting from an aqueous solution were evaluated by the following analysis: UV-Vis spectrum, transparency value, transmittance, attenuated total reflectance Fourier-Transform spectroscopy (FTIR), scanning electron microscopy (SEM) and antimicrobial activity. The obtained results showed that the combination of orange essential oil with trehalose decreases the transmittance value in the UV and Vis regions (up to 0.14% +/- 0.02% at 356 nm), meaning that produced films can act as a UV protector. Most produced films in the research were resistant to Gram-positive bacteria (Staphylococcus aureus subsp. aureus), though most films did not show antibacterial properties against Gram-negative bacteria and yeasts. FTIR and SEM confirmed that both the amount of carrageenan used and the combination with orange essential oil influenced the compatibility of trehalose with the film matrix. The research showed how different combinations of trehalose, orange essential oils and carrageenan can affect edible film properties. These changes represent important information for further research and the possible practical application of these edible matrices.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) display the excellent oxygen-carrying properties of red blood cells, while overcoming some of the limitations of donor blood. Various encapsulation platforms have been explored to prepare HBOCs which aim to avoid or minimize the adverse effects caused by the administration of free Hb. Herein, we entrapped Hb within a poly(lac-tide-co-glycolide) (PLGA) core, prepared by the double emulsion solvent evaporation method. We study the effect of the concentrations of Hb, PLGA, and emulsifier on the size, polydispersity (PDI), loading capacity (LC), and entrapment efficiency (EE) of the resulting Hb-loaded PLGA nanoparti-cles (HbNPs). Next, the ability of the HbNPs to reversibly bind and release oxygen was thoroughly evaluated. When needed, trehalose, a well-known protein stabilizer that has never been explored for the fabrication of HBOCs, was incorporated to preserve Hb’s functionality. The optimized formulation had a size of 344 nm, a PDI of 0.172, a LC of 26.9%, and an EE of 40.7%. The HbNPs were imaged by microscopy and were further characterized by FTIR and CD spectroscopy to assess their chemical composition and structure. Finally, the ability of the encapsulated Hb to bind and release oxygen over several rounds was demonstrated, showing the preservation of its functionality.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) display the excellent oxygen-carrying properties of red blood cells, while overcoming some of the limitations of donor blood. Various encapsulation platforms have been explored to prepare HBOCs which aim to avoid or minimize the adverse effects caused by the administration of free Hb. Herein, we entrapped Hb within a poly(lactide-co-glycolide) (PLGA) core, prepared by the double emulsion solvent evaporation method.We study the effect of the concentrations of Hb, PLGA, and emulsifier on the size, polydispersity (PDI), loading capacity (LC), and entrapment efficiency (EE) of the resulting Hb-loaded PLGA nanoparticles (HbNPs). Next, the ability of the HbNPs to reversibly bind and release oxygen was thoroughly evaluated. When needed, trehalose, a well-known protein stabilizer that has never been explored for the fabrication of HBOCs, was incorporated to preserve Hb’s functionality. The optimized formulation had a size of 344 nm, a PDI of 0.172, a LC of 26.9%, and an EE of 40.7%. The HbNPs were imaged by microscopy and were further characterized by FTIR and CD spectroscopy to assess their chemical composition and structure. Finally, the ability of the encapsulated Hb to bind and release oxygen over several rounds was demonstrated, showing the preservation of its functionality.

[EN] Survival of probiotic microorganisms in dried foods is optimal for water activity (a(w)) values between 0.1 and 0.3. Encapsulating and adding low-molecular weight additives can enhance probiotic viability in intermediatea(w)food products, but the effectiveness of sub-lethal homogenization is still not proven. This study evaluates the effect of 10% (w/w) trehalose addition and/or 100 MPa homogenization onLactobacillussalivariusCECT 4063 counts and antioxidant properties of apple slices dried to different water activity values (freeze-drying to aa(w)of 0.25 and air-drying at 40 degrees C to aa(w)of 0.35 and 0.45) during four-week storage. Optical and mechanical properties of dried samples were also analyzed. Freeze-drying had the least effect on the microbial counts and air drying at 40 degrees C to aa(w)of 0.35 had the greatest effect. Antioxidant properties improved with drying, especially with convective drying. Decreases in both microbial and antioxidant content during storage were favored in samples with higher water activity values. Adding trehalose improved cell survival during storage in samples with a water activity of 0.35, but 100 MPa homogenization increased the loss of viability in all cases. Air-dried samples became more translucent and reddish, rather rubbery and less crispy than freeze-dried ones.

In a population, chemical communication determines the response of animals to changing environmental conditions, what leads to an enhanced resistance against stressors. In response to starvation, the nematode Caenorhabditis elegans arrest post-embryonic development at the first larval stage (L1) right after hatching. As arrested L1 larvae, C. elegans become more resistant to diverse stresses, allowing them to survive for several weeks expecting to encounter more favorable conditions. L1 arrested at high densities display an enhanced resistance to starvation, dependent on soluble compounds released beyond hatching and the first day of arrest. Here, we show that this chemical communication also influences recovery after prolonged periods in L1 arrest. Animals at high density recovered faster than animals at low density. We found that the density effect on survival depends on the final effector of the insulin signaling pathway, the transcription factor DAF-16. Moreover, DAF-16 activation was higher at high density, consistent with a lower expression of the insulin-like peptide DAF-28 in the neurons. The improved recovery of animals after arrest at high density depended on soluble compounds present in the media of arrested L1s. In an effort to find the nature of these compounds, we investigated the disaccharide trehalose as putative signaling molecule, since its production is enhanced during L1 arrest and it is able to activate DAF-16. We detected the presence of trehalose in the medium of arrested L1 larvae at a low concentration. The addition of this concentration of trehalose to animals arrested at low density was enough to rescue DAF-28 production and DAF-16 activation to the levels of animals arrested at high density. However, despite activating DAF-16, trehalose was not capable of reversing survival and recovery phenotypes, suggesting the participation of additional signaling molecules. With all, here we describe a molecular mechanism underlying social communicati

Mycobacterium avium is capable of an adaptive, reversible response to high-temperature survival depending on its growth temperature. Trehalose concentrations of M. avium cells grown at 42 °C were significantly higher compared to those of cells grown at 25 °C. Further, the survival of cells of M. avium grown at 42 °C and exposed to 65 °C were significantly higher than the survival of cells grown at 25 °C. This adaptive response to growth temperature may play a role in the persistence of M. avium in premise plumbing.

Abstract: Candidatus Accumulibacter phosphatis is an important microorganism for enhanced biological phosphorus removal (EBPR). In a previous study, we found a remarkable flexibility regarding salinity, since this same microorganism could thrive in both freshwater- and seawater-based environments, but the mechanism for the tolerance to saline conditions remained unknown. Here, we identified and described the role of trehalose as an osmolyte in Ca. Accumulibacter phosphatis. A freshwater-adapted culture was exposed to a single batch cycle of hyperosmotic and hypo-osmotic shock, which led to the release of trehalose up to 5.34 mg trehalose/g volatile suspended solids (VSS). Long-term adaptation to 30% seawater-based medium in a sequencing batch reactor (SBR) gave a stable operation with complete anaerobic uptake of acetate and propionate along with phosphate release of 0.73 Pmol/Cmol, and complete aerobic uptake of phosphate. Microbial analysis showed Ca. Accumulibacter phosphatis clade I as the dominant organism in both the freshwater- and seawater-adapted cultures (> 90% presence). Exposure of the seawater-adapted culture to a single batch cycle of hyperosmotic incubation and hypo-osmotic shock led to an increase in trehalose release upon hypo-osmotic shock when higher salinity is used for the hyperosmotic incubation. Maximum trehalose release upon hypo-osmotic shock was achieved after hyperosmotic incubation with 3× salinity increase relative to the salinity in the SBR adaptation reactor, resulting in the release of 11.9 mg trehalose/g VSS. Genome analysis shows the possibility of Ca. Accumulibacter phosphatis to convert glycogen into trehalose by the presence of treX, treY, and treZ genes. Addition of trehalose to the reactor led to its consumption, both during anaerobic and aerobic phases. These results indicate the flexibility of the metabolism of Ca. Accumulibacter phosphatis towards variations in salinity. Key points: • Trehalose is identified as an osmo, BT/Environmental Biotechnology

Autophagy is a highly conserved recycling pathway that promotes cell survival during periods of stress. We previously reported that induction of autophagy through the inhibition of the mechanistic target of rapamycin (MTOR) inhibits HIV replication in human macrophages and CD4+ T lymphocytes (T cells). However, the inhibition of MTOR has modulatory effects beyond autophagy that might affect viral replication. Here, we examined the effect on HIV replication of trehalose, a nontoxic, nonreducing disaccharide that induces autophagy through an MTOR-independent mechanism. Treatment of HIV-infected macrophages and T cells with trehalose inhibited infection in a dose-dependent manner. Uninfected and HIV-infected macrophages and T cells treated with trehalose exhibited increased markers of autophagy, including LC3B lipidation with further accumulation following bafilomycin A1 treatment, and increased levels of LAMP1, LAMP2, and RAB7 proteins required for lysosomal biogenesis and fusion. Moreover, the inhibition of HIV by trehalose was significantly reduced by knockdown of ATG5 Additionally, trehalose downregulated the expression of C-C motif chemokine receptor 5 (CCR5) in T cells and CD4 in both T cells and macrophages, which reduced HIV entry in these cells. Our data demonstrate that the naturally occurring sugar trehalose at doses safely achieved in humans inhibits HIV through two mechanisms: (i) decreased entry through the downregulation of CCR5 in T cells and decreased CD4 expression in both T cells and macrophages and (ii) degradation of intracellular HIV through the induction of MTOR-independent autophagy. These findings demonstrate that cellular mechanisms can be modulated to inhibit HIV entry and intracellular replication using a naturally occurring, nontoxic sugar.IMPORTANCE Induction of autophagy through inhibition of MTOR has been shown to inhibit HIV replication. However, inhibition of the mechanistic target of rapamycin (MTOR) has cellular effects that may alte

Water is critical for the survival of most cells and organisms. Remarkably, a small number of multicellular animals are able to survive nearly complete drying. The phenomenon of anhydrobiosis, or life without water, has been of interest to researchers for over 300 years. In this review we discuss advances in our understanding of protectants and mechanisms of desiccation tolerance that have emerged from research in three anhydrobiotic invertebrates: brine shrimp (Artemia), roundworms (nematodes), and tardigrades (water bears). Discovery of molecular protectants that allow each of these three animals to survive drying diversifies our understanding of desiccation tolerance, and convergent themes suggest mechanisms that may offer a general model for engineering desiccation tolerance in other contexts.

Mycobacterium avium is capable of an adaptive, reversible response to high-temperature survival depending on its growth temperature. Trehalose concentrations of M. avium cells grown at 42 °C were significantly higher compared to those of cells grown at 25 °C. Further, the survival of cells of M. avium grown at 42 °C and exposed to 65 °C were significantly higher than the survival of cells grown at 25 °C. This adaptive response to growth temperature may play a role in the persistence of M. avium in premise plumbing.

In a population, chemical communication determines the response of animals to changing environmental conditions, what leads to an enhanced resistance against stressors. In response to starvation, the nematode Caenorhabditis elegans arrest post-embryonic development at the first larval stage (L1) right after hatching. As arrested L1 larvae, C. elegans become more resistant to diverse stresses, allowing them to survive for several weeks expecting to encounter more favorable conditions. L1 arrested at high densities display an enhanced resistance to starvation, dependent on soluble compounds released beyond hatching and the first day of arrest. Here, we show that this chemical communication also influences recovery after prolonged periods in L1 arrest. Animals at high density recovered faster than animals at low density. We found that the density effect on survival depends on the final effector of the insulin signaling pathway, the transcription factor DAF-16. Moreover, DAF-16 activation was higher at high density, consistent with a lower expression of the insulin-like peptide DAF-28 in the neurons. The improved recovery of animals after arrest at high density depended on soluble compounds present in the media of arrested L1s. In an effort to find the nature of these compounds, we investigated the disaccharide trehalose as putative signaling molecule, since its production is enhanced during L1 arrest and it is able to activate DAF-16. We detected the presence of trehalose in the medium of arrested L1 larvae at a low concentration. The addition of this concentration of trehalose to animals arrested at low density was enough to rescue DAF-28 production and DAF-16 activation to the levels of animals arrested at high density. However, despite activating DAF-16, trehalose was not capable of reversing survival and recovery phenotypes, suggesting the participation of additional signaling molecules. With all, here we describe a molecular mechanism underlying social communicati

Survival of probiotic microorganisms in dried foods is optimal for water activity (aw) values between 0.1 and 0.3. Encapsulating and adding low-molecular weight additives can enhance probiotic viability in intermediate aw food products, but the effectiveness of sub-lethal homogenization is still not proven. This study evaluates the effect of 10% (w/w) trehalose addition and/or 100 MPa homogenization on Lactobacillus salivarius CECT 4063 counts and antioxidant properties of apple slices dried to different water activity values (freeze-drying to a aw of 0.25 and air-drying at 40 °C to a aw of 0.35 and 0.45) during four-week storage. Optical and mechanical properties of dried samples were also analyzed. Freeze-drying had the least effect on the microbial counts and air drying at 40 °C to a aw of 0.35 had the greatest effect. Antioxidant properties improved with drying, especially with convective drying. Decreases in both microbial and antioxidant content during storage were favored in samples with higher water activity values. Adding trehalose improved cell survival during storage in samples with a water activity of 0.35, but 100 MPa homogenization increased the loss of viability in all cases. Air-dried samples became more translucent and reddish, rather rubbery and less crispy than freeze-dried ones.

[EN] This study evaluates the effect of trehalose addition (10% or 20%, w/w) and/or sublethal homogenisation (25-150 MPa) on antioxidants content (vitamin C, total phenols and flavonoids) and activity (measured both by ABTS-TEAC and DPPH assays), as well as on microbial counts and survival to in vitro digestion of clementine juice inoculated with Lactobacillus salivarius spp. salivarius. Particle size, vacuum impregnation parameters and anti-Helicobacter pylori effect were also measured. Incubation with the probiotic improved the antioxidant properties of the juice. Homogenisation pressures below 100 MPa following incubation increased both the probiotic counts in the juice and its antioxidants bioaccesibility. Adding 10% (w/w) of trehalose to the juice was effective in preventing these bioactive compounds deterioration under adverse conditions. Once homogenised, liquids containing 10% (w/w) of trehalose became as able as those without trehalose to enter a food solid matrix. Inhibition of Helicobacter pylori growth was evident in all probiotic beverages.

Proliferation of microcystin (MC)-producing Microcystis aeruginosa in brackish waters has been described in several locations and represents a new concern for public and environmental health. While the impact of a sudden salinity increase on M. aeruginosa physiology has been studied, less is known about the mechanisms involved in salt tolerance after acclimation. This study aims to compare the physiological responses of two strains of M. aeruginosa (PCC 7820 and PCC 7806), which were isolated from contrasted environments, to increasing salinities. After acclimation, growth and MC production rates were determined and metabolomic analyses were conducted. For both strains, salinity decreased the biovolume, growth, and MC production rates and induced the accumulation of polyunsaturated lipids identified as monogalactosyldiacylglycerol. The distinct salt tolerances (7.5 and 16.9) obtained between the freshwater (PCC 7820) and the brackish-water (PCC 7806) strains suggested different strategies to cope with the osmotic pressure, as revealed by targeted and untargeted metabolomic analyses. An accumulation of trehalose as the main compatible solute was obtained in the freshwater strain, while sucrose was mainly accumulated in the brackish one. Moreover, distinct levels of glycine betaine and proline accumulation were noted. Altogether, metabolomic analysis illustrated a strain-specific response to salt tolerance, involving compatible solute production. IMPORTANCE Blooms of Microcystis aeruginosa and the production of microcystins are major issues in eutrophic freshwater bodies. Recently, an increasing number of proliferations of M. aeruginosa in brackish water has been documented. The occurrence of both M. aeruginosa and microcystins in coastal areas represents a new threat for human and environmental health. In order to better describe the mechanisms involved in Microcystis sp. proliferation in brackish water, this study used two M. aeruginosa strains isolated from fresh a