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This work aimed to study the effect of trehalose in protecting cells against Sod1 proteinopathy associated with amyotrophic lateral sclerosis (ALS). Humanized yeast cells in which native Sod1 was replaced by wild-type human Sod1 or an ALS mutant (WT-A4V Sod1 heterodimer) were used as the experimental model. Cells were treated with 10% trehalose (p/v) before or after the appearance of hSod1 proteinopathy induced by oxidative stress. In both conditions, trehalose reduced the number of cells with Sod1 inclusions, increased Sod1 activity, and decreased the levels of intracellular oxidation, demonstrating that trehalose avoids Sod1 misfolding and loss of function in response to oxidative stress. The survival rates of ALS Sod1 cells stressed in the presence of trehalose were 60% higher than in their absence. Treatment with trehalose after the appearance of Sod1 inclusions in cells expressing WT Sod1 doubled longevity; after 5 days, non-treated cells did not survive, but 15% of cells treated with sugar were still alive. Altogether, our results emphasize the potential of trehalose as a novel therapy, which might be applied preventively in ALS patients with a family history of the disease or after diagnosis in ALS patients who discover the disease following the first symptoms.
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In Escherichia coli, the disaccharide trehalose can be metabolized as a carbon source or be accumulated as an osmoprotectant under osmotic stress. In hypertonic environments, E. coli accumulates trehalose in the cell by synthesis from glucose mediated by the cytosolic enzymes OtsA and OtsB. Trehalose in the periplasm can be hydrolyzed into glucose by the periplasmic trehalase TreA. We have previously shown that a treA mutant of extraintestinal E. coli strain BEN2908 displayed increased resistance to osmotic stress by 0.6 M urea, and reduced production of type 1 fimbriae, reduced invasion of avian fibroblasts, and decreased bladder colonization in a murine model of urinary tract infection. Since loss of TreA likely results in higher periplasmic trehalose concentrations, we wondered if deletion of otsA and otsB genes, which would lead to decreased internal trehalose concentrations, would reduce resistance to stress by 0.6 M urea and promote type 1 fimbriae production. The BEN2908ΔotsBA mutant was sensitive to osmotic stress by urea, but displayed an even more pronounced reduction in production of type 1 fimbriae, with the consequent reduction in adhesion/invasion of avian fibroblasts and reduced bladder colonization in the murine urinary tract. The BEN2908ΔtreAotsBA mutant also showed a reduction in production of type 1 fimbriae, but in contrast to the ΔotsBA mutant, resisted better than the wild type in the presence of urea. We hypothesize that, in BEN2908, resistance to stress by urea would depend on the levels of periplasmic trehalose, but type 1 fimbriae production would be influenced by the levels of cytosolic trehalose.
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Fímbrias Bacterianas , Osmorregulação , Trealose , Bexiga Urinária , Infecções Urinárias , Animais , Trealose/metabolismo , Camundongos , Bexiga Urinária/microbiologia , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/genética , Infecções Urinárias/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Modelos Animais de Doenças , Feminino , Pressão Osmótica , Escherichia coli Extraintestinal Patogênica/metabolismo , Escherichia coli Extraintestinal Patogênica/genética , Ureia/metabolismo , Trealase/metabolismo , Trealase/genética , Deleção de Genes , Glucose/metabolismoRESUMO
Introdução: A lipoenxertia é um enxerto autólogo de células do tecido celular subcutâneo, que pode ser utilizada como técnica complementar na reconstrução mamária. Diante disso, a criopreservação de células-tronco mesenquimais provenientes de tecido adiposo (CTDAs) poderia ser uma maneira de realizar a coleta em um tempo cirúrgico e após realizar a lipoenxertia de forma fracionada. O dimetilsulfóxido (DMSO) é um criopreservante utilizado em pesquisas com células, porém é potencialmente tóxico, o que impossibilitaria a utilização de CTDAs criopreservadas na prática clínica. Novos criopreservantes celulares, sem toxicidade, vêm sendo descritos na literatura científica experimental, como as substâncias L-prolina e trealose. Com isso, esse trabalho teve como objetivo avaliar a viabilidade de CTDAs criopreservadas com a combinação de L-prolina e trealose, em um período de até 90 dias. Método: Estudo experimental, no qual foram obtidas amostras de lipoaspirado provenientes de 9 pacientes. A fração celular foi processada e congelada com L-prolina (1,5M) + trealose (0,2M), ou com DMSO + soro fetal bovino (SFB), como controle. Após 30 e 90 dias, as amostras foram descongeladas e a viabilidade celular foi avaliada pela técnica de MTT. Resultados: A análise das CTDAs, após 1 e 3 meses de congelamento, indicou que as amostras tratadas com L-prolina + trealose apresentaram viabilidade semelhante àquelas preservadas com DMSO e SFB (p=0,444). Conclusão: A associação de L-prolina e trealose manteve CTDA viáveis por 30 e 90 dias de congelamento, podendo ser uma alternativa como criopreservante celular sem toxicidade e viabilizando o uso de lipoenxertia seriada.
Introduction: Fat grafting is an autologous graft of cells from subcutaneous tissue, which can be used as a complementary technique in breast reconstruction. Given this, the cryopreservation of adipose tissue-derived mesenchymal stem cells (ADMSCs) could be a way to collect them in one surgical procedure and after performing fractional fat grafting. Dimethyl sulfoxide (DMSO) is a cryopreservative used in cell research, but it is potentially toxic, which would make it impossible to use cryopreserved ADMSCs in clinical practice. New cellular cryopreservatives, without toxicity, have been described in the experimental scientific literature, such as the substances L-proline and trehalose. Therefore, this work aimed to evaluate the viability of ADMSCs cryopreserved with the combination of L-proline and trehalose over up to 90 days. Method: Experimental study in which lipoaspirate samples were obtained from 9 patients. The cellular fraction was processed and frozen with L-proline (1.5M) + trehalose (0.2M) or with DMSO + fetal bovine serum (FBS) as control. After 30 and 90 days, the samples were thawed, and cell viability was assessed using the MTT technique. Results: The analysis of ADMSCs, after 1 and 3 months of freezing, indicated that samples treated with L-proline + trehalose showed similar viability to those preserved with DMSO and SFB (p=0.444). Conclusion: The association of L-proline and trehalose kept ADMSC viable for 30 and 90 days of freezing, and could be an alternative as a cellular cryopreservative without toxicity and enabling the use of serial fat grafting.
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Yeasts from cold environments have a wide range of strategies to prevent the negative effects of extreme conditions, including the production of metabolites of biotechnological interest. We investigated the growth profile and production of metabolites in yeast species isolated from cold environments. Thirty-eight strains were tested for their ability to grow at different temperatures (5-30 °C) and solute concentrations (3-12.5% NaCl and 50% glucose). All strains tested were able to grow at 5 °C, and 77% were able to grow with 5% NaCl at 18 °C. We were able to group strains based on different physicochemical/lifestyle profiles such as polyextremotolerant, osmotolerant, psychrotolerant, or psychrophilic. Five strains were selected to study biomass and metabolite production (glycerol, trehalose, ergosterol, and mycosporines). These analyses revealed that the accumulation pattern of trehalose and ergosterol was related to each lifestyle profile. Also, our findings would suggest that mycosporines does not have a role as an osmolyte. Non-conventional fermentative yeasts such as Phaffia tasmanica and Saccharomyces eubayanus may be of interest for trehalose production. This work contributes to the knowledge of non-conventional yeasts with biotechnological application from cold environments, including their growth profile, metabolites, and biomass production under different conditions.
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Basidiomycota , Trealose , Trealose/metabolismo , Cloreto de Sódio/metabolismo , Leveduras , Ergosterol/metabolismo , Temperatura BaixaRESUMO
BACKGROUND: The lesser grain borer (Rhyzopertha dominica), a worldwide primary pest of stored grain, causes serious economic losses and threatens stored food safety. R. dominica can respond to changes in temperature, especially the adaptability to heat. In this study, transcriptome analysis of R. dominica exposed to different temperatures was performed to elucidate differences in gene expression and the underling molecular mechanism. RESULTS: Isoform-sequencing generated 17,721,200 raw reads and yielded 20,416 full-length transcripts. A total of 18,880 (92.48%) transcripts were annotated. We extracted RNA from R. dominica reared at 5 °C (cold stress), 15 °C (cold stress), 27 °C (ambient temperature) and 40 °C (heat stress) for RNA-seq. Compared to those of control insects reared at 27 °C, 119, 342, and 875 differentially expressed genes (DEGs) were identified at 5 °C, 15 °C, and 40 °C, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that pathways associated with "fatty acid metabolism", "fatty acid biosynthesis", "AMPK signaling pathway", "neuroactive ligand receptor interaction", and "longevity regulating pathway-multiple species" were significantly enriched. The functional annotation revealed that the genes encoding heat shock proteins (HSPs), fatty acid synthase (FAS), phospholipases (PLA), trehalose transporter (TPST), trehalose 6-phosphate synthase (TPS), and vitellogenin (Vg) were most likely involved in temperature regulation, which was also validated by RT-qPCR. Seven candidate genes (rdhsp1, rdfas1, rdpla1, rdtpst1, rdtps1, rdvg1, and rdP450) were silenced in the RNA interference (RNAi) assay. RNAi of each candidate gene suggested that inhibiting rdtps1 expression significantly decreased the trehalose level and survival rate of R. dominica at 40 °C. CONCLUSIONS: These results indicated that trehalose contributes to the high temperature resistance of R. dominica. Our study elucidates the molecular mechanisms underlying heat tolerance and provides a potential target for the pest management in R. dominica.
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Aclimatação , Besouros , Trealose , Aclimatação/genética , Ácidos Graxos , FosfatosRESUMO
The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) is a membrane protein that is destabilized during purification in the absence of calcium ions. The disaccharide trehalose is a protein stabilizer that accumulates in the yeast cytoplasm when under stress. In the present work, SERCA was purified by including trehalose in the purification protocol. The purified SERCA showed high protein purity (~95%) and ATPase activity. ATP hydrolysis was dependent on the presence of Ca2+ and the enzyme kinetics showed a hyperbolic dependence on ATP (Km = 12.16 ± 2.25 µM ATP). FITC labeling showed the integrity of the ATP-binding site and the identity of the isolated enzyme as a P-type ATPase. Circular dichroism (CD) spectral changes at a wavelength of 225 nm were observed upon titration with ATP, indicating α-helical rearrangements in the nucleotide-binding domain (N-domain), which correlated with ATP affinity (Km). The presence of Ca2+ did not affect FITC labeling or the ATP-mediated structural changes at the N-domain. The use of trehalose in the SERCA purification protocol stabilized the enzyme. The isolated SERCA appears to be suitable for structural and ligand binding studies, e.g., for testing newly designed or natural inhibitors. The use of trehalose is recommended for the isolation of unstable enzymes.
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Since the number of aged people will increase in the next years, neurodegenerative diseases, including Parkinson's Disease (PD), will also rise. Recently, we demonstrated that autophagy stimulation with rapamycin decreases dopaminergic neuronal death mediated by oxidative stress in the paraquat (PQ)-induced PD model. Assessing the neurotherapeutic efficacy of autophagy-inducing molecules is critical for preventing or delaying neurodegeneration. Therefore, we evaluated the autophagy inducers metformin and trehalose effect in a PD model. Autophagy induced by both molecules was confirmed in the SH-SY5Y dopaminergic cells by detecting increased LC3-II marker and autophagosome number compared to the control by western blot and transmission electron microscopy. Both autophagy inducers showed an antioxidant effect, improved mitochondrial activity, and decreased dopaminergic cell death induced by PQ. Next, we evaluated the effect of both inducers in vivo. C57BL6 mice were pretreated with metformin or trehalose before PQ administration. Cognitive and motor deteriorated functions in the PD model were evaluated through the nest building and the gait tests and were prevented by metformin and trehalose. Both autophagy inducers significantly reduced the dopaminergic neuronal loss, astrocytosis, and microgliosis induced by PQ. Also, cell death mediated by PQ was prevented by metformin and trehalose, assessed by TUNEL assay. Metformin and trehalose induced autophagy through AMPK phosphorylation and decreased α-synuclein accumulation. Therefore, metformin and trehalose are promising neurotherapeutic autophagy inducers with great potential for treating neurodegenerative diseases such as PD.
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Metformina , Neuroblastoma , Doença de Parkinson , Humanos , Animais , Camundongos , Idoso , Doença de Parkinson/tratamento farmacológico , Trealose/farmacologia , Trealose/uso terapêutico , Camundongos Endogâmicos C57BL , Autofagia , Dopamina , Metformina/farmacologia , Metformina/uso terapêuticoRESUMO
BACKGROUND: Trehalose is a non-reducing disaccharide synthesized by lower organisms. It has recently received special attention because of its neuroprotective properties by stimulating autophagy in Parkinson's disease (PD) models. Therefore, evaluating whether trehalose affects metabolic organs is vital to determine its neurotherapeutic safety. METHODS: We validated the trehalose neuroprotective dosage in a PD model induced with intraperitoneal paraquat administration twice weekly for 7 weeks. One week before paraquat administration, mice were treated with trehalose in the drinking water and continued along with paraquat treatment. Histological and morphometrical analyses were conducted on the organs involved in trehalose metabolism, including the liver, pancreas, and kidney. RESULTS: Paraquat-induced dopaminergic neuronal loss was significantly decreased by trehalose. After trehalose treatment, the liver morphology, the mononucleated/binucleated hepatocytes percentage, and sinusoidal diameter remained unchanged in each liver lobes. Endocrine and exocrine pancreas's histology was not affected, nor was any fibrotic process observed. The islet of Langerhans's structure was preserved when analyzing the area, the largest and smallest diameter, and circularity. Renal morphology remained undamaged, and no changes were identified within the glomerular basement membrane. The renal corpuscle structure did not suffer alterations in the Bowman's space, area, diameter, circularity, perimeter, and cellularity. Besides, the renal tubular structures's luminal area and internal and external diameter were preserved. CONCLUSION: Our study demonstrates that systemic trehalose administration preserved the typical histological architecture of the organs involved in its metabolism, supporting its safety as a potential neuroprotective agent.
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Paraquat , Trealose , Camundongos , Animais , Trealose/farmacologia , Trealose/uso terapêutico , Rim , Fígado , PâncreasRESUMO
This work proposes a combination of polyethylene glycol 400 (PEG) and trehalose as a surface modification approach to enhance PLGA-based nanoparticles as a drug carrier for neurons. PEG improves nanoparticles' hydrophilicity, and trehalose enhances the nanoparticle's cellular internalization by inducing a more auspicious microenvironment based on inhibiting cell surface receptor denaturation. To optimize the nanoprecipitation process, a central composite design was performed; nanoparticles were adsorbed with PEG and trehalose. PLGA nanoparticles with diameters smaller than 200 nm were produced, and the coating process did not considerably increase their size. Nanoparticles entrapped curcumin, and their release profile was determined. The nanoparticles presented a curcumin entrapment efficiency of over 40%, and coated nanoparticles reached 60% of curcumin release in two weeks. MTT tests and curcumin fluorescence, with confocal imaging, were used to assess nanoparticle cytotoxicity and cell internalization in SH-SY5Y cells. Free curcumin 80 µM depleted the cell survival to 13% at 72 h. Contrariwise, PEG:Trehalose-coated curcumin-loaded and non-loaded nanoparticles preserved cell survival at 76% and 79% under the same conditions, respectively. Cells incubated with 100 µM curcumin or curcumin nanoparticles for 1 h exhibited 13.4% and 14.84% of curcumin's fluorescence, respectively. Moreover, cells exposed to 100 µM curcumin in PEG:Trehalose-coated nanoparticles for 1 h presented 28% fluorescence. In conclusion, PEG:Trehalose-adsorbed nanoparticles smaller than 200 nm exhibited suitable neural cytotoxicity and increased cell internalization proficiency.
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Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is an essential signal metabolite in plants, linking growth and development to carbon status. Our current understanding of Tre6P metabolism and signaling pathways in plants is based almost entirely on studies performed with Arabidopsis thaliana, a model plant that performs C3 photosynthesis. Conversely, our knowledge on the molecular mechanisms involved in Tre6P regulation of carbon partitioning and metabolism in C4 plants is scarce. This topic is especially relevant due to the agronomic importance of crops performing C4 photosynthesis, such as maize, sorghum and sugarcane. In this review, we focused our attention on recent developments related to Tre6P metabolism in C4 species and raised some open questions that should be addressed in the near future to improve the yield of economically important crops.
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Arabidopsis , Trealose , Trealose/metabolismo , Plantas/metabolismo , Arabidopsis/metabolismo , Fotossíntese , Carbono/metabolismo , Fosfatos/metabolismoRESUMO
Trehalose is a type of carbohydrate that protects against different types of stress and is also used as a source of carbon storage in prokaryotes. There are four different ways of synthesizing trehalose in Acidithiobacillus ferrivorans and two in Acidithiobacillus ferrooxidans, but its purpose remains unknown. This study aimed to measure the production of trehalose under different conditions by quantifying it in three culture media at two different temperatures. The growth kinetics of both species were also assessed, and the trehalose concentration was analysed during the early stationary phase using an enzymatic method. The results showed that the modified 9K medium with ferrous iron at 28°C had the highest production of trehalose, with A. ferrivorans CF27 having a higher production of 0.34 µmol/mg protein compared to A. ferrooxidans ATCC 23270 at 0.31 µmol/mg protein. When using CuS, the production of trehalose was lower, with 0.02 and 0.03 µmol/mg protein for A. ferrivorans CF27 and A. ferrooxidans ATCC 23270, respectively, while no trehalose was detected in the presence of zinc. At 15°C, the enzymatic method did not detect any trehalose in all three culture media, this would indicate that this carbohydrate does not protect against low temperatures in either species.
La trehalosa es un tipo de carbohidrato, que en procariotas protege contra diferentes tipos de estrés y también se utiliza como fuente de almacenamiento de carbono. Hay cuatro formas diferentes de sintetizar trehalosa en Acidithiobacillus ferrivorans y dos en Acidithiobacillus ferrooxidans, pero su propósito sigue siendo desconocido. Este estudio tuvo como objetivo medir la producción de trehalosa en diferentes condiciones mediante su cuantificación en tres medios de cultivo a dos temperaturas diferentes. También se evaluó la cinética de crecimiento de ambas especies y se analizó la concentración de trehalosa durante la fase estacionaria temprana mediante un método enzimático. Los resultados mostraron que el medio 9K modificado con hierro ferroso a 28 °C tuvo la mayor producción de trehalosa, con A. ferrivorans CF27 con una mayor producción de 0.34 µmol/mg de proteína en comparación con A. ferrooxidans ATCC 23270 a 0.31 µmol/mg de proteína. Al utilizar CuS, la producción de trehalosa fue menor, con 0.02 y 0.03 µmol/mg de proteína para A. ferrivorans CF27 y A. ferrooxidans ATCC 23270, respectivamente, mientras que en presencia de zinc no se detectó trehalosa. A 15°C, el método enzimático no detectó trehalosa en los tres medios de cultivo, lo que indicaria que este carbohidrato no protege contra las bajas temperaturas en ninguna de las especies.
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The regenerative therapies with stem cells (SC) has been increased by the cryopreservation, permitting cell storage for extended periods. However, the permeating cryoprotectant agents (CPAs) such as dimethylsulfoxide (DMSO) can cause severe adverse effects. Therefore, this study evaluated equine mesenchymal stem cells derived from adipose tissue (eAT-MSCs) in fresh (Control) or after slow freezing (SF) in different freezing solutions (FS). The FS comprise DMSO and non-permeating CPAs [Trehalose (T) and the SuperCool X-1000 (X)] in association or not, totalizing seven different FS: (DMSO; T; X; DMSO+T; DMSO+X; T+X, and DMSO+T+X). Before and after cryopreservation were evaluated, viability, colony forming unit (CFU), and cellular differentiation capacity. After freezing-thawing, the viability of the eAT-MSCs reduced (P< 0.05) in all treatments compared to the control. However, the viability of frozen eAT-MSCs in DMSO (80.3 ± 0.6) was superior (P<0.05) to the other FS. Regarding CFU, no difference (P>0.05) was observed between fresh and frozen cells. After freezing-thawing, the eAT-MSCs showed osteogenic, chondrogenic, and adipogenic lineages differentiation potential. Nonetheless, despite the significative reduction in the osteogenic differentiation capacity between fresh and frozen cells, no differences (P > 0.05) were observed among FS. Furthermore, the number of chondrogenic differentiation cells frozen in DMSO+X solution reduced (P<0.05) comparing to the control, without differ (P>0.05) to the other FS. The adipogenic differentiation did not differ (P>0.05) among treatments. In conclusion, although these findings confirm the success of DMSO to cryopreserve eAT-MSCs, the Super Cool X-1000 could be a promise to reduce the DMSO concentration in a FS.
As terapias regenerativas com células-tronco (CT) têm sido incrementadas pela criopreservação, permitindo o armazenamento celular. No entanto, os agentes crioprotetores (ACPs) penetrantes, como DMSO, podem causar efeitos adversos graves. Portanto, este estudo avaliou células-tronco mesenquimais equinas derivadas de tecido adiposo (CTM-TAe) in natura (Controle) ou após congelamento lento (CL) em diferentes soluções de congelamento (SC). As SCs compreendem DMSO e ACPs não permeáveis [Trealose (T) e o SuperCool X-1000 (X)] associados ou não: (DMSO; T; X; DMSO+T; DMSO+X; T +X e DMSO+T+X). Antes e após a criopreservação foram avaliados, viabilidade, unidade formadora de colônia (UFC) e capacidade de diferenciação celular. Após o congelamento-descongelamento, a viabilidade das CTM-TAe reduziu (P< 0,05) em todos os tratamentos em relação ao controle. Entretanto, a viabilidade das CTM-TAe congeladas em DMSO (80,3 ± 0,6) foi superior (P<0,05) às demais SC. Em relação às UFC, não houve diferença (P>0,05) entre células frescas e congeladas. Após congelamento-descongelamento, as CTM-TAe apresentaram potencial de diferenciação de linhagens osteogênicas, condrogênicas e adipogênicas. No entanto, apesar da redução significativa na capacidade de diferenciação osteogênica entre células frescas e congeladas, não foram observadas diferenças (P > 0,05) entre SCs. Além disso, o número de células de diferenciação condrogênica congeladas em solução de DMSO+X reduziu (P<0,05) em relação ao controle, sem diferir (P>0,05) das demais SCs. A diferenciação adipogênica não diferiu (P>0,05) entre os tratamentos. Em conclusão, embora esses achados confirmem o sucesso do DMSO para criopreservar CTM-TAe, o Super Cool X-1000 pode ser uma promessa para reduzir a concentração de DMSO.
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This study aimed to detect intracellular trehalose in boar sperm that were cryopreserved with liposomes and conduct an analysis of its effects on some characteristics of thawed sperm, including rheological properties. First, soybean lecithin cholesterol-based liposomes were produced and characterized in the presence of 300 mM trehalose. Next, semen samples were frozen in two freezing media: a control medium with 300 mM trehalose and an experimental medium supplemented with 300 mM trehalose and 10% liposomes, both of which were thawed and then studied to ascertain their integrity, motility, rheological response, and trehalose quantities by testing two methods of spermatic lysis via high-performance liquid chromatography with an evaporative light-scattering detector (HPLC-ELSD). The results found spherical liposomes measuring 357 nm that were relatively stable in an aqueous medium and had an entrapment efficiency of 73%. An analysis of the cryopreserved ejaculates showed that their viability and motility did not significantly differ between groups (P > 0.05). The viscous response of the samples was influenced by the extracellular medium rather than by the freezing-thawing process, which resulted in a loss of interaction between the cells and cryoprotectants. Finally, intracellular trehalose levels were determined using HPLC-ELSD, with no differences observed (P > 0.05) when comparing both sperm lysis methods. The use of liposomes with trehalose appears to be a promising option for boar semen cryopreservation, with a marked effect on rheological properties. The proposed HPLC-ELSD method was effective for measuring trehalose in cryopreserved cell samples.
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Preservação do Sêmen , Sêmen , Masculino , Suínos , Animais , Sêmen/fisiologia , Trealose , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Lipossomos , Motilidade dos Espermatozoides/fisiologia , Dissacarídeos , Criopreservação/veterinária , Criopreservação/métodos , Crioprotetores/farmacologia , Espermatozoides/fisiologiaRESUMO
Cancer-related metabolic features are in part maintained by hexokinase 2 upregulation, which leads to high levels of glucose-6-phosphate (G6P) and is needed to provide energy and biomass to support rapid proliferation. Using a humanized model of the yeast Saccharomyces cerevisiae, we explored how human hexokinase 2 (HK2) behaves under different nutritional conditions. At high glucose levels, yeast presents aerobic glycolysis through a regulatory mechanism known as catabolic repression, which exerts a metabolic adaptation like the Warburg effect. At high glucose concentrations, HK2 did not translocate into the nucleus and was not able to shift the metabolism toward a highly glycolytic state, in contrast to the effect of yeast hexokinase 2 (Hxk2), which is a crucial protein for the control of aerobic glycolysis in S. cerevisiae. During the stationary phase, when glucose is exhausted, Hxk2 is shuttled out of the nucleus, ceasing catabolic repression. Cells harvested at this condition display low glucose consumption rates. However, glucose-starved cells expressing HK2 had an increased capacity to consume glucose. In those cells, HK2 localized to mitochondria, becoming insensitive to G6P inhibition. We also found that the sugar trehalose-6-phosphate (T6P) is a human HK2 inhibitor, like yeast Hxk2, but was not able to inhibit human HK1, the isoform that is ubiquitously expressed in almost all mammalian tissues. In contrast to G6P, T6P inhibited HK2 even when HK2 was associated with mitochondria. The binding of HK2 to mitochondria is crucial for cancer survival and proliferation. T6P was able to reduce the cell viability of tumor cells, although its toxicity was not impressive. This was expected as cell absorption of phosphorylated sugars is low, which might be counteracted using nanotechnology. Altogether, these data suggest that T6P may offer a new paradigm for cancer treatment based on specific inhibition of HK2.
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Hexoquinase , Fosfatos Açúcares , Animais , Humanos , Hexoquinase/metabolismo , Saccharomyces cerevisiae/metabolismo , Glicólise , Glucose/metabolismo , MamíferosRESUMO
Abstract Background: In artificial insemination, chicken egg yolk is added to bovine semen to protect it during the cryopreservation process, although it contains substances that can affect the microbiological quality and metabolism of sperm. Objective: To evaluate post-thaw quality of bovine cryopreserved semen added with centrifuged and non-centrifuged egg yolk, low-density lipoproteins (LDL), and trehalose (T). Methods: Ten ejaculates from five bulls were cryopreserved under the treatments T1: pure egg yolk (PEY) at 20% v/v, T2: centrifuged egg yolk (CEY) at 20% v/v, T3: LDL at 8% v/v, T4: T at 100 mM, and T5: T at 100 mM plus LDL at 8% v/v (TLDL). Spermatic motility and kinetics, functional membrane integrity (FMI), structural membrane integrity (SMI), sperm vitality (SV) and abnormal morphology (AM) were assessed using the Sperm Class Analyzer (SCA®) system, hypoosmotic test (HOST), SYBR/PI probes, and eosin-nigrosin staining, respectively. A completely randomized design was used. Normal distribution of the variables was validated through the Kolmogórov- Smirnov test. A generalized linear model was used to determine sources of variation. Means were compared using the Tukey test. Results: Inclusion of CEY or LDL had a similar effect on sperm protection, and were superior for motility, kinetics and membrane integrity compared to the other treatments (p<0.05). CEY was superior for progressive motility (p<0.05). The cryoprotective action of LDL was similar to TLDL for motility and kinetics, SMI, SV, and AM (p<0.05). Inclusion of PEY and T resulted in the lowest semen quality (p<0.05). The use of T resulted in a reduction in FMI and SMI (p<0.05). No differences in AM between treatments were found (p>0.05). Conclusions: Egg yolk can be replaced by centrifuged egg yolk or low-density lipoproteins in the freezing extender used for bovine semen used in artificial insemination.
Resumen Antecedentes: la yema de huevo de gallina se agrega al semen bovino usado en inseminación artificial para protegerlo durante el proceso de criopreservación, aunque ésta tiene sustancias que pueden afectar el metabolismo espermatico y la calidad microbiológica del semen. Objetivo: evaluar la calidad post-descongelación del semen bovino criopreservado agregado con yema de huevo centrifugada y no centrifugada, lipoproteínas de baja densidad (LDL) y trehalosa (T). Métodos: diez eyaculados de cinco toros se criopreservaron bajo los tratamientos, T1: yema de huevo pura (PEY) al 20% v/v, T2: yema de huevo centrifugada (CEY) al 20% v/v, T3: LDL al 8% v/v, T4: T a 100 mM, y T5: T a 100 mM más LDL al 8% v/v (TLDL). La movilidad y la cinética espermática, la integridad funcional de la membrana (FMI), la integridad estructural de la membrana (SMI), la vitalidad espermática (SV) y la morfología anormal (AM), se determinaron mediante el sistema Sperm Class Analyzer (SCA®), la prueba hipoosmótica (HOST), las sondas SYBR/PI y la tinción con eosina-nigrosina, respectivamente. Se utilizó un diseño completamente al azar. La normalidad de las variables se validó mediante la prueba de Kolmogórov-Smirnov. Se utilizó un modelo lineal generalizado para determinar las fuentes de variación. Las medias de los tratamientos se compararon mediante la prueba de Tukey. Resultados: CEY y LDL tuvieron un efecto similar en la protección de los espermatozoides, siendo superiores a los demás tratamientos respecto a movilidad, cinética e integridad de la membrana (p<0,05). CEY fue superior para la movilidad progresiva (p<0,05). La acción crioprotectora de LDL fue similar a TLDL para movilidad y cinética, SMI, AM y SV (p<0,05). PEY y T resultaron en la más baja calidad seminal (p<0,05). El uso de T redujo la FMI y la SMI (p<0,05). No se encontraron diferencias en AM entre los tratamientos (p>0,05). Conclusiones: la yema de huevo puede reemplazarse por yema de huevo centrifugada o por lipoproteinas de baja densidad en el diluyente de congelación usado para semen bovino destinado a inseminacion artificial.
Resumo Antecedentes: a gema de ovo de galinha tem sido utilizada com a finalidade de proteger o sêmen bovino durante o processo de criopreservação, embora tenha substâncias que possam afetar o metabolismo dos espermatozóides e a qualidade microbiológica do sêmen utilizado para a inseminação artificial. Objetivo: avaliar a qualidade pós-descongelamento do sêmen bovino criopreservado com gema de ovo centrifugada e não centrifugada, lipoproteínas de baixa densidade (LDL) e trealose (T). Métodos: dez ejaculados de cinco touros foram criopreservados sob os tratamentos, T1: gema de ovo pura (PEY) 20% v/v, T2: gema de ovo centrifugada (CEY) 20% v/v, T3: LDL 8% v/v, T4: T 100 mM e T5: T 100 mM mais LDL 8% v/v (TLDL). Mobilidade e cinética espermática, integridade funcional da membrana (FMI), integridade estrutural da membrana (SMI), vitalidade espermática (SV) e morfologia anormal (AM) foram determinadas por o sistema Sperm Class Analyzer (SCA®), teste hiposmótico (HOST), coloração com SYBR/PI e eosina-nigrosina, respectivamente. Um design completamente aleatoriedade foi usado. A normalidade das variáveis foi validada pelo teste de Kolmogorov-Smirnov. Um modelo linear generalizado foi utilizado para determinar as fontes de variação. As médias dos tratamentos foram comparadas pelo teste de Tukey. Resultados: T2 (CEY) e T3 (LDL) tiveram efeito similar na proteção espermática, sendo superior aos demais tratamentos para mobilidade, cinética e integridade da membrana (p<0,05). T2 (CEY) foi superior para mobilidade progressiva (p<0,05). A ação crioprotetora de T3 (LDL) foi semelhante à T5 (TLDL) para motilidade e cinética, SMI, SV e AM (p<0,05). T1 (PEY) e T4 (T) tiveram a menor qualidade seminal (p<0,05). O uso de T4 (T) produziu uma redução na SMI e FMI (p<0,05). Não foram encontradas diferenças na AM entre os tratamentos (p>0,05). Conclusões: a gema de ovo pode ser substituída por gema de ovo centrifugada ou lipoproteínas de baixa densidade no diluente de congelamento de sêmen bovino.
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Defoliation tolerance (DT) in Amaranthus cruentus is known to reach its apex at the panicle emergence (PE) phase and to decline to minimal levels at flowering (FL). In this study, defoliation-induced changes were recorded in the content of non-structural carbohydrates and raffinose family oligosaccharides (RFOs), and in the expression and/or activity of sugar starvation response-associated genes in plants defoliated at different vegetative and reproductive stages. This strategy identified sugar-starvation-related factors that explained the opposite DT observed at these key developmental stages. Peak DT at PE was associated with increased cytosolic invertase (CI) activity in all organs and with the extensive induction of various class II trehalose-phosphate synthase (TPS) genes. Contrariwise, least DT at FL coincided with a sharp depletion of starch reserves and with sucrose (Suc) accumulation, in leaves and stems, the latter of which was consistent with very low levels of CI and vacuolar invertase activities that were not further modified by defoliation. Increased Suc suggested growth-inhibiting conditions associated with altered cytosolic Suc-to-hexose ratios in plants defoliated at FL. Augmented cell wall invertase activity in leaves and roots, probably acting in a regulatory rather than hydrolytic role, was also associated with minimal DT observed at FL. The widespread contrast in gene expression patterns in panicles also matched the opposite DT observed at PE and FL. These results reinforce the concept that a localized sugar starvation response caused by C partitioning is crucial for DT in grain amaranth.
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This review commemorates the 50th anniversary of the Nobel Prize in Chemistry awarded to Luis F. Leloir 'for his discovery of sugar-nucleotides and their role in the biosynthesis of carbohydrates'. He and his co-workers discovered that activated forms of simple sugars, such as UDP-glucose and UDP-galactose, are essential intermediates in the interconversion of sugars. They elucidated the biosynthetic pathways for sucrose and starch, which are the major end-products of photosynthesis, and for trehalose. Trehalose 6-phosphate, the intermediate of trehalose biosynthesis that they discovered, is now a molecule of great interest due to its function as a sugar signalling metabolite that regulates many aspects of plant metabolism and development. The work of the Leloir group also opened the doors to an understanding of the biosynthesis of cellulose and other structural cell wall polysaccharides (hemicelluloses and pectins), and ascorbic acid (vitamin C). Nucleotide-sugars also serve as sugar donors for a myriad of glycosyltransferases that conjugate sugars to other molecules, including lipids, phytohormones, secondary metabolites, and proteins, thereby modifying their biological activity. In this review, we highlight the diversity of nucleotide-sugars and their functions in plants, in recognition of Leloir's rich and enduring legacy to plant science.
Assuntos
Parede Celular , Plantas , Metabolismo dos Carboidratos , Parede Celular/metabolismo , Glicosiltransferases/metabolismo , Uridina Difosfato Glucose/metabolismoRESUMO
Saccharides have bioprotective properties, with a high capacity to preserve biological proteins and membranes during sperm cryopreservation. The aim of this study was to evaluate how replacing the lactose of cryopreservation media by sucrose (SUC) or trehalose (TRE) at concentrations of 0.2 M (SUC-1 and TRE-1) and 0.25 M (SUC-2 and TRE-2) affects frozen/thawed pig spermatozoa. The media used were composed of medium A (saccharide/egg yolk) and B (saccharide/egg yolk/glycerol), their osmolality being determined prior to freezing. Cell viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), lipid peroxidation, thiol group oxidation, total reactive oxygen species (ROS), peroxynitrite and superoxide anion (O2â-) were determined through flow cytometry; total motility (TM), progressive motility (PM) and kinetic parameters motility were determined immediately after thawing (T0) and again 30 (T30) and 60 (T60) minutes later. The SUC-2 and TRE-2 groups maintained viability significantly and presented fewer lipid membrane disorders, respectively, both with a significant increase in MMP. The production of O2â- and peroxynitrite was lower in the TRE-2 groups compared to the control (P < 0.05). Total motility at T0 was greater in the TRE-2 group (P < 0.05). Sperm kinetics was not affected by the treatment. The use of saccharides SUC and TRE at a concentration of 0.25 M improves sperm quality, so that both non-penetrating cryoprotectants can be utilized in pig sperm freezing media.
Assuntos
Preservação do Sêmen , Animais , Criopreservação/métodos , Crioprotetores/farmacologia , Congelamento , Masculino , Estresse Oxidativo , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides , Espermatozoides , SuínosRESUMO
Yeasts play an important role in the cocoa fermentation process. Although the most relevant function is the degradation of sugars and the production of ethanol, there is little understanding of the enzyme activities and attributes that allow them to survive even after drying. The present study explored the functional biodiversity of yeasts associated with Criollo Colombian cocoa fermented beans, able to survive after drying. Twelve species belonging to 10 genera of osmo-, acid-, thermo-, and desiccation-tolerant yeasts were isolated and identified from fermented and dry cocoa beans, with Pichia kudriavzevii and Saccharomyces cerevisiae standing out as the most frequent. For the first time, we reported the presence of Zygosaccharomyces bisporus in cocoa fermented beans. It was found that resistance to desiccation is related to the different degradation capacities of fermentation substrates, which suggests that associative relationships may exist between the different yeast species and their degradation products. Besides, the increased thermotolerance of some species was related to the presence of polyphenols in the medium, which might play a fundamental role in shaping the microbial community composition.
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High-gravity (HG) brewing has broader application to succeed on beer differentiation and production optimization. However, such process imposes a handicap to yeasts, which must be able to deal with stressful conditions in fermentation. In this work, we assessed different physiological traits of 24 Saccharomyces cerevisiae strains isolated from Brazilian bioethanol distilleries for the selection of novel starters for HG brewing. Five yeast strains were selected with ability to overcome different stressors under HG beer fermentation, showing high fermentability rates, resilience to ethanol stress, low production of foam and hydrogen sulfide, as well as similar flocculation rates to brewer's yeasts. After five fermentation recycles, most strains sustained a viability rate higher than 90% and were able to efficiently accumulate trehalose and glycogen, besides presenting no detectable petite mutants at the final stage. In the sensory analysis, the beers obtained from the five selected strains showed greater aromatic complexity, with predominance of 'spicy', 'dried' and 'fresh fruits' descriptors. In conclusion, this study sheds light on the potential of yeast strains from Brazilian bioethanol process to produce distinctive specialty beers, aside from proposing an effective selection methodology based on relevant physiological attributes for HG brewing process.