Respiratory Metabolism Research Articles

Acidic electrolyzed water treatment delays the senescence of ‘Lingwu long’ jujube fruit during cold storage by regulating energy and respiratory metabolism

Jujube fruit may rapidly undergo maturity and senescence during storage, seriously affecting its commercialization. The study aimed to evaluate the role of acidic electrolyzed water (AEW) on energy and respiratory metabolism, and senescence in jujube fruit during cold storage. The results indicated that AEW improved ATP content and energy charge via increasing succinate dehydrogenase, cytochrome C oxidase, H+-ATPase, and Ca2+-ATPase activities and delayed jujube fruit senescence. The treatment was found to improve the activities of nicotinamide adenine dinucleotide kinase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase, and the content of NADPH, and reduce oxidative stress. Transcriptome analysis suggested that AEW regulated the expression of the genes associated with glycolysis, citrate cycle, pentose phosphate pathway, oxidative phosphorylation, ROS metabolism, transcription factors, phenylpropane biosynthesis, cell wall metabolism, and plant hormone signal transduction. Therefore, we concluded that AEW may delay the senescence of postharvest jujube fruit by regulating expression of the relevant genes and metabolic pathways.

Confining Prepared Ultra-Small Nanozymes Loading Ato for Lung Cancer Catalytic Therapy/Immunotherapy.

Nanozymes with inherent enzyme-mimic catalytic properties combat malignant tumor progression via catalytic therapy, while the therapeutic efficacy still needs to be improved. In this work, we innovatively synthesized ultra-small platinum nanozymes (nPt) in confined domain of wormlike pore channel in gold nanobipyramidal-mesoporous silica dioxide nanocomposites, producing nanozyme carriers AP-mSi with photo-enhanced peroxidase ability. Afterwards, based on the prepared AP-mSi, the lung cancer nanozymes probe (AP-HAI) was ingeniously produced by removing SiO2 template, modifying HSA, and loading ATO as well as IR780. Under NIR light irradiation, inner AuP and IR780 collaborated for photothermal process, thus facilitating the peroxidase-like catalytic process of H2 O2 . Additionally, loaded atovaquone molecules (ATO), a cell respiration inhibitor, could impair tumor respiration metabolism and cause oxygen retention, hence enhancing IR780's PDT effectiveness. In a result, IR780's PDT property and nPt nanozymes' photo-enhanced peroxidase-like ability endowed probes a high ROS productivity, eliciting anti-tumor immune responses to destroy tumor tissue. Systematic studies revealed that the obvious ROS generation was obtained by the strategy of using nPt nanozymes and reducing oxygen consumption by ATO, which in turn enabled lung cancer synergetic catalytic therapy/ICD-based immunotherapy. The results of this work would provide theoretical justification for the practical use of photo-enhanced nanozyme probes. This article is protected by copyright. All rights reserved.

Priming methods affected deterioration speed of primed rice seeds by regulating reactive oxygen species accumulation, seed respiration and starch degradation.

Seed priming is a pre-sowing seed treatment that is beneficial for rice seed germination and seedling growth, but the reduced seed longevity after seed priming greatly limited its adoption. The deterioration of primed seeds showed large differences among different studies, and the priming method might play an important role in regulating the deterioration speed of primed seeds. However, whether and how the priming method affected the deterioration of primed rice seeds during storage remains unknown. In this study, two typical seed priming methods, namely hydropriming (HP) and osmopriming (PEG) were compared under artificially accelerated aging conditions, the changes in germination performance, starch metabolism, seed respiration and reactive oxygen species accumulation before and after accelerated aging were determined. Hydroprimed rice seeds exhibited significantly faster deterioration speed than that of PEG-primed seeds in terms of germination speed and percentage. Meanwhile, α-amylase activity and total soluble sugar content in hydroprimed seeds were reduced by 19.3% and 10.0% respectively after aging, as compared with PEG-primed seeds. Such effects were strongly associated with the increased reactive oxygen generation and lipid peroxidation, as the content of superoxide anion, hydrogen peroxide and malondialdehyde in hydroprimed seeds were 4.4%, 12.3% and 13.7% higher than those in PEG-primed seeds after aging, such effect could be attributed to the increased respiratory metabolism in hydroprimed seeds. In addition, the simultaneous use of N-acetylcysteine with HP and PEG priming greatly inhibited the deterioration of primed rice seeds, suggesting that the ability to scavenge reactive oxygen species may be the key factor affecting the speed of deterioration in primed rice seeds during storage.

Mitochondrial structure and respiratory metabolism in cold resistance of alfalfa seedling root

Mitochondrial structure and respiratory metabolism in cold resistance of alfalfa seedling root

Enhanced methane oxidation efficiency by digestate biochar in landfill cover soil: Microbial shifts and carbon metabolites insights

The ability of biochar to enhance the oxidation of methane (CH4) in landfill cover soil by promoting the growth and activity of methane-oxidizing bacteria (MOB) has attracted significant attention. However, the optimal characteristics of digestate-derived biochar (DBC) for promoting the MOB community and CH4 removal performance remain unclear. This study examined how the CH4 oxidation capacity and respiratory metabolism of MOB life process are affected by the application of DBC compared with the most commonly used woody-derived biochar (WBC). The addition of both WBC and DBC enhanced CH4 oxidation, with DBC exhibiting a nearly twofold increase in cumulative CH4 oxidation mass (7.14 mg CH4 g−1) compared to WBC. The high ion-exchange capacity of DBC was found to be more favorable for the growth of Type I MOB, which have more efficient metabolic pathways for CH4 oxidation. Type I MOB which are abundant in DBC may prefer monovalent positive ions, while the charge-rich nature of DBC may also have hindered extracellular protein aggregation. The superiority of DBC in terms of CH4 oxidation thus highlights the underlying mechanisms of biochar-MOB interactions, offering potential biochar options for landfill cover soil.

Transcriptome analysis reveals the mechanism of polyethylene atmosphere packaging delaying browning of postharvest Pleurotus eryngii

To elucidate the mechanism of low-density polyethylene(LDPE) atmosphere packaging on delaying browning of Pleurotus eryngii under a postharvest low-temperature storage, this study focused on exploring the effects of two thicknesses (0.02 and 0.05 mm) LDPE packaging on some antioxidant enzymes, and stress markers in P. eryngii. The results showed that LDPE atmosphere packaging maintained the phenolic content and higher superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) levels, reduced malondialdehyde (MDA) content, electrolyte leakage rate and polyphenol oxidase (PPO) levels in the mushroom. Also, transcriptome analyses showed that LDPE atmosphere packaging regulated the expression of enzymes PPO, POD, CAT and APX-related genes, inhibited the expression of lipoxygenases (LOX) and phospholipase D (PLD)-related genes and regulated the l-glutathione (GSH) antioxidant system. Overall, LDPE atmosphere packaging delayed the reduction of unsaturated fatty acids and energy loss by maintaining the integrity of cell membranes and regulating important genes involved in membrane lipid metabolism, energy metabolism, and respiratory metabolism of P. eryngii. In addition, it activated the expression of genes related to GSH synthesis and metabolism, thus enhancing the antioxidant capacity of mushroom body, reducing the accumulation of ROS and delaying browning.

Starch/carboxymethyl cellulose-based photodynamic packaging material delays the quality deterioration of snap beans via ROS metabolism regulation

Snap beans (Phaseolus vulgaris L.) are prone to rapid senescence and quality deterioration during postharvest storage. In this study, the effect of a novel carbon dot/g-C3N4-mediated starch/carboxymethyl cellulose-based photodynamic film (CCPF) on the postharvest quality deterioration and reactive oxygen species (ROS) metabolism of snap bean pods was investigated. CCPF packaging influenced gas composition regulation, ethylene degradation and microbial infection, which resulted in snap bean pods with lower respiratory metabolism and less quality loss. In addition, the snap bean pods in the CCPF group maintained the content of nonenzymatic antioxidants, low activities of pro-oxidative enzymes and high activities of antioxidant enzymes compared to other groups, thus decreasing electrolyte leakage, malondialdehyde accumulation, H2O2 accumulation and the O2·- generation rate. Overall, CCPF packaging effectively delayed postharvest senescence and extended the shelf life of snap beans by reducing respiration and regulating ROS metabolism. Pearson’s correlation analysis confirmed that postharvest senescence correlated with ROS metabolism in snap bean pods. This study offers theoretical evidence for a method to preserve snap bean pods and can be used as a valuable reference for the practical application of novel CCPF packaging.

In-vitro antibacterial activity and mechanism of Monarda didyma essential oils against Carbapenem-resistant Klebsiella pneumoniae

To fight the global epidemic of drug-resistant bacteria, essential oils have gained increasing attention as a new source of antibiotics. The antimicrobial activity of Monarda didyma essential oils (MDEO) for the Carbapenem-resistant Klebsiella pneumoniae (CRKP) strains were determined by agar disc diffusion assay and broth microdilution assay. To further understand MDEO efficacy, a time-growth curve was performed. The biofilm formation of CRKP were determined by crystalline violet staining method, additionally, changes in intracellular Adenosine triphosphate (ATP), protein, Alkaline phosphatase (AKP) activities, and membrane integrity were investigated to assess the influence of MDEO on cell membrane damage. Finally, the activities of key enzymes in the tricarboxylic acid (TCA) pathways and pentose phosphate (PPP) pathways were examined to determine the effect of MDEO on the respiratory metabolism of CRKP. This study presents the antibacterial mechanism of MDEO against CRKP with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 1.25 mg/ml. To understand MDEO efficacy, a time-kill kinetics approach was performed. The bactericidal effect of MDEO was evident at 2 h compared to the control at its MIC and 2MIC. Surface electron microscopic and ATP assay studies provided evidence for the multi-target action of MDEO against CRKP. MDEO could inhibit CRKP biofilm formation. MDEO could also cause irreversible damage to the CRKP cell membrane, resulting in the leakage of biological macromolecules (protein, ATP) and the reduction of intracellular enzymes (AKP) activities. Finally, MDEO affected the pathways of respiratory metabolism, such as PPP and TCA pathways. MDEO could reduce the activity of key enzymes (Glucose-6-phosphate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase) in the PPP and TCA pathways to exert its biological effects against CRKP. These results suggest MDEO can exert inhibitory effects on CRKP, and potential mechanisms of action including inhibition of biofilm formation, damage of cell membrane structure and inhibition of energy metabolism.

Regulation of Embden-Meyerhof-Parnas (EMP) Pathway and Tricarboxylic Acid (TCA) Cycle Concerning Aberrant Chilling Injury Behavior in Postharvest Papaya (Carica papaya L.).

Postharvest abnormal chilling injury (CI) behavior in papaya (Carica papaya L.) fruit is a rare phenomenon that may be associated with respiratory metabolism. This study thus aimed to investigate the impacts of storage temperatures (1 and 6 °C) on the respiratory metabolism of postharvest papaya and its impact on CI development. Results demonstrated that 1 °C storage reduced the activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), citrate synthase (CS), and α-ketoglutarate dehydrogenase (α-KGDH) and regulated the expression of corresponding enzymes in the Embden-Meyerhof-Parnas (EMP) pathway and tricarboxylic acid (TCA) cycle compared with 6 °C storage, resulting in a lower respiration rate of the EMP-TCA pathway and mitigating the development of CI. Meanwhile, lower contents of nicotinamide adenine dinucleotide (hydrogen) (NAD(H)) were observed in papaya fruit stored at 1 °C. Notably, papaya fruit stored at 1 °C maintained higher activity and transcriptional levels of SDH and IDH during the whole storage period. These findings suggest that 1 °C storage reduced the respiration rate of the EMP-TCA pathway by reducing the expression level and activity of related enzymes, which is conducive to the reduction of respiration substrate consumption and finally alleviating the occurrence of CI.

Silicon improves the growth of Glycyrrhiza uralensis Fisch. under salt and/or drought stress by regulating respiration metabolism

Drought and salt stress severely threaten the growth and productivity of Glycyrrhiza uralensis Fisch. (G.uralensis), and usually, they occur concomitantly. Silicon (Si) addition is well known to enhance the tolerance of plants. Here, we analyzed the changes in parameters related to growth and respiration, including glycolysis pathway (EMP), tricarboxylic acid (TCA) cycle, and pentose phosphate pathway (PPP) by Si in G. uralensis seedlings under salt and/or drought stresses. Results showed that Si effectively alleviated the effect of salt, drought and combined stress on the growth and respiration metabolism of G. uralensis seedlings. Under salt stress, Si increased leaf number by 15.13%, upregulated the TCA pathway at the transcriptional level, and increased PFK, PK, and content of acetyl CoA in both aerial and underground, ICDHm, α-KGDH in aerial part and NAD-MDH in underground part, thus alleviating the inhibitory effect of salt stress on the EMP–TCA process, but weakening the PPP by declining G6PDH and 6-PGDH in aerial part by 34.10% and 55.54%, and G6PDH in underground part by 83.86%. Under drought stress, Si increased underground biomass by 11.40%, slowed the EMP by decreasing PFK in aerial part by 21.76%, PK and PFK in underground part by 38.11% and 64.71%, weakened PPP by reducing 6-PGDH in aerial part by 72.9%, G6PDH and 6-PGDH in underground part by 77.00% and 64.71%. Si increased SDH and phosphoenolpyruvate carboxylase (PEPC) by 28.66% and 145.48% with the TCA cycle and GABA shunt as the main respiratory pathways in the underground part of drought-stressed seedlings. Under combined stresses, Si addition increased leaf number and aboveground biomass by 11.96% and 23.88%, upregulated the EMP and TCA pathways at the transcriptional level, and alleviated the effects of combined stress on EMP–TCA metabolic processes at the physiological and biochemical levels by increasing PDH, α-KGDH and NAD-MDH in aerial part by 27.22%, 18.85% and 140.37%, HK, PK and PEPC in underground part by 93.97%, 25.81% and 31.19%, initiated the replenishment mechanism and inhibited GABA shunt in underground part of G. uralensis to provide energy for vital activities. In conclusion, Si can effectively regulate respiration metabolism process to provide more substrates for subsequent physiological processes, thus improving tolerance to salt and drought stress, and thereby promote the growth of G. uralensis seedlings under stress conditions.

Effect of an environmental microplastic mixture from the Seine River and one of the main associated plasticizers, dibutylphthalate, on the sentinel species Hediste diversicolor

The aim of this study was to explore the adverse effects of a microplastic (MP) mixture obtained from litter accumulated in the Seine River (France) compared to those of their major co-plasticizer, dibutylphthalate (DBP), on the sentinel species Hediste diversicolor. A suite of biomarkers has been investigated to study the impacts of MPs (100 mg kg−1 sediment), DBP (38 μg kg−1 sediment) on worms compared to non-exposed individuals after 4 and 21 days. The antioxidant response, immunity, neurotoxicity and energy and respiratory metabolism were investigated using biomarkers. After 21 days, worms exposed to MPs showed an increasing aerobic metabolism, an enhancement of both antioxidant and neuroimmune responses. Energy-related biomarkers demonstrated that the energy reallocated to the defence system may come from proteins. A similar impact was depicted after DBP exposure, except for neurotoxicity. Our results provide a better understanding of the ecotoxicological effects of environmental MPs and their associated-contaminants on H. diversicolor.

Carbon Fluxes in Potato (Solanum tuberosum) Remain Stable in Cell Cultures Exposed to Nutritional Phosphate Deficiency.

Nutritional phosphate deficiency is a major limitation to plant growth. Here, we monitored fluxes in pathways supporting respiratory metabolism in potato (Solanum tuberosum) cell cultures growing in control or limiting phosphate conditions. Sugar uptake was quantified using [U-14C]sucrose as precursor. Carbohydrate degradation through glycolysis and respiratory pathways was estimated using the catabolism of [U-14C]sucrose to 14CO2. Anaplerotic carbon flux was assessed by labeling with NaH14CO3. The data showed that these metabolic fluxes displayed distinct patterns over culture time. However, phosphate depletion had relatively little impact on the various fluxes. Sucrose uptake was higher during the first six days of culture, followed by a decline, which was steeper in Pi-sufficient cells. Anaplerotic pathway flux was more important at day three and decreased thereafter. In contrast, the flux between sucrose and CO2 was at a maximum in the mid-log phase of the culture, with a peak at Day 6. Metabolization of [U-14C]sucrose into neutral, basic and acidic fractions was also unaffected by phosphate nutrition. Hence, the well-documented changes in central metabolism enzymes activities in response to Pi deficiency do not drastically modify metabolic fluxes, but rather result in the maintenance of the carbon fluxes that support respiration.

Effects of cadmium exposure on metabolism, antioxidant defense, immune function, and the hepatopancreas transcriptome of Cipangopaludina cathayensis

Cadmium (Cd) is a common contaminant in aquatic environments. However, little is known about the mechanisms underlying Cd toxicity in the freshwater snail Cipangopaludina cathayensis (Heude, 1890). This study to investigate the toxic effects of Cd on the standard metabolism, antioxidant activities, immune function, and hepatopancreas transcriptome profiles of C. cathayensis. C. cathayensis was exposed to 0.25, 0.5, 1.0, or 1.5 mg/L Cd for 3 h, with results showing that Cd significantly inhibited oxygen consumption and ammonia excretion and disrupted the respiratory metabolism of C. cathayensis. In addition, the O:N ratio dropped below 7, indicating that C. cathayensis may rely exclusively on proteins as an energy source under Cd stress. To understand how Cd impacts the antioxidant activities, immune function, and transcriptional profiles, C. cathayensis were exposed to 0.5 (low exposure, L14) or 1.5 (high exposure, H14) mg/L Cd for 14 days. Our results indicate that Cd exposure leads to oxidative stress and immunosuppression, with the latter effect being larger for exposure to higher Cd concentrations. A total of 2172 differentially expressed genes (DEGs) were identified by transcriptome analysis of the hepatopancreas, of which 885 were upregulated and 1287 were downregulated. Gene ontology and KEGG analyses revealed that the DEGs in the H14 group are enriched for energy generation terms and the “oxidative phosphorylation” pathway, respectively. Therefore, up-regulation of energy metabolism may be an adaptive strategy under Cd stress. Moreover, several genes involved in antioxidant activity were downregulated, whereas genes related to reactive oxygen species generation were upregulated. In addition, many immunity-related genes were identified within the DEGs, indicating that Cd toxicity may affect immune defense. Further, DEGs in the H14 group were enriched for disease-associated pathways. Taken together, our results indicate that Cd exposure leads to metabolic disorders, oxidative stress, and immunosuppression and thus may potentially contribute to disease outbreaks.

Elevated nocturnal respiratory rates in the mitochondria of CAM plants: current knowledge and unanswered questions.

Crassulacean acid metabolism (CAM) is a metabolic adaptation that has evolved convergently in 38 plant families to aid survival in water-limited niches. Whilst primarily considered a photosynthetic adaptation, CAM also has substantial consequences for nocturnal respiratory metabolism. Here, we outline the history, current state and future of nocturnal respiration research in CAM plants, with a particular focus on the energetics of nocturnal respiratory oxygen consumption. Throughout the 20th century, research interest in nocturnal respiration occurred alongside initial discoveries of CAM, although the energetic and mechanistic implications of nocturnal oxygen consumption and links to the operation of the CAM cycle were not fully understood. Recent flux balance analysis (FBA) models have provided new insights into the role that mitochondria play in the CAM cycle. Several FBA models have predicted that CAM requires elevated nocturnal respiratory rates, compared to C3 species, to power vacuolar malic acid accumulation. We provide physiological data, from the genus Clusia, to corroborate these modelling predictions, thereby reinforcing the importance of elevated nocturnal respiratory rates for CAM. Finally, we outline five unanswered questions pertaining to nocturnal respiration which must be addressed if we are to fully understand and utilize CAM plants in a hotter, drier world.

Fiber supplementation protects from antibiotic-induced gut microbiome dysbiosis by modulating gut redox potential

Antibiotic-induced gut dysbiosis (AID) is a frequent and serious side effect of antibiotic use and mitigating this dysbiosis is a critical therapeutic target. We propose that the host diet can modulate the chemical environment of the gut resulting in changes to the structure and function of the microbiome during antibiotic treatment. Gut dysbiosis is typically characterized by increases in aerobic respiratory bacterial metabolism, redox potential, and abundance of Proteobacteria. In this study, we explore dietary fiber supplements as potential modulators of the chemical environment in the gut to reduce this pattern of dysbiosis. Using defined-diets and whole-genome sequencing of female murine microbiomes during diet modulation and antibiotic treatment, we find that fiber prebiotics significantly reduced the impact of antibiotic treatment on microbiome composition and function. We observe reduced abundance of aerobic bacteria as well as metabolic pathways associated with oxidative metabolism. These metatranscriptomic results are corroborated by chemical measurements of eH and pH suggesting that fiber dampens the dysbiotic effects of antibiotics. This work indicates that fiber may act as a potential therapeutic for AID by modulating bacterial metabolism in the gut to prevent an increase in redox potential and protect commensal microbes during antibiotic treatment.

Discovery of Novel Salicylaldehyde Derivatives Incorporating an α-Methylene-γ-Butyrolactone Moiety as Antifungal Agents.

Plant diseases caused by phytopathogenic fungi and oomycetes pose a serious threat to ensuring crop yield and quality. Finding novel antifungal candidates based on natural products is one of the critical methods for developing effective and environmentally friendly pesticides. In this study, a series of salicylaldehyde derivatives containing an α-methylene-γ-butyrolactone moiety were designed, synthesized, and their fungicidal activities were evaluated. The bioassay studies indicated that compound C3 displayed an excellent in vitro activity against Rhizoctonia solani with an EC50 value of 0.65 μg/mL, higher than that of pyraclostrobin (EC50 = 1.44 μg/mL) and comparable to that of carbendazim (EC50 = 0.33 μg/mL). For Valsa mali and Phytophthora capsici, compound C3 also showed good fungicidal activities with EC50 values of 0.91 μg/mL and 1.33 μg/mL, respectively. In addition, compound C3 exhibited promising protective in vivo activity against R. solani (84.1%) at 100 μg/mL, which was better than that of pyraclostrobin (78.4%). The pot experiment displayed that compound C3 had 74.8% protective efficacy against R. solani at 200 μg/mL, which was comparable to that of validamycin (78.2%). The antifungal mode of action research indicated that compound C3 could change the mycelial morphology and ultrastructure, increase cell membrane permeability, affect respiratory metabolism by binding to complex III, and inhibit the germination and formation of sclerotia, thereby effectively controlling the disease. The present study provides support for the application of these salicylaldehyde derivatives as promising potential pesticides with remarkable and broad-spectrum fungicidal activities against phytopathogenic fungi and oomycetes in crop protection. This article is protected by copyright. All rights reserved.

ATP yield of plant respiration: potential, actual and unknown.

The ATP yield of plant respiration (ATP/hexose unit respired) quantitatively links active heterotrophic processes with substrate consumption. Despite its importance, plant respiratory ATP yield is uncertain. The aim here was to integrate current knowledge of cellular mechanisms with inferences required to fill knowledge gaps to generate a contemporary estimate of respiratory ATP yield and identify important unknowns. A numerical balance sheet model combining respiratory carbon metabolism and electron transport pathways with uses of the resulting transmembrane electrochemical proton gradient was created and parameterized for healthy, non-photosynthesizing plant cells catabolizing sucrose or starch to produce cytosolic ATP. Mechanistically, the number of c subunits in the mitochondrial ATP synthase Fo sector c-ring, which is unquantified in plants, affects ATP yield. A value of 10 was (justifiably) used in the model, in which case respiration of sucrose potentially yields about 27.5 ATP/hexose (0.5 ATP/hexose more from starch). Actual ATP yield often will be smaller than its potential due to bypasses of energy-conserving reactions in the respiratory chain, even in unstressed plants. Notably, all else being optimal, if 25 % of respiratory O2 uptake is via the alternative oxidase - a typically observed fraction - ATP yield falls 15 % below its potential. Plant respiratory ATP yield is smaller than often assumed (certainly less than older textbook values of 36-38 ATP/hexose) leading to underestimation of active-process substrate requirements. This hinders understanding of ecological/evolutionary trade-offs between competing active processes and assessments of crop growth gains possible through bioengineering of processes that consume ATP. Determining the plant mitochondrial ATP synthase c-ring size, the degree of any minimally required (useful) bypasses of energy-conserving reactions in the respiratory chain, and the magnitude of any 'leaks' in the inner mitochondrial membrane are key research needs.

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures.

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

Acidic electrolyzed oxidizing water delayed the breakdown occurrence in pulp of fresh longan by regulating the metabolisms of respiratory and energy

The efficacy of acidic electrolyzed oxidizing water (AEOW) treatment on the respiratory and energy metabolism during storage of harvested longan and its relationship with pulp breakdown were investigated. Results confirmed that the AEOW treatment exerted a significant inhibitory effect on longan pulp breakdown, with 15.5 % lower pulp breakdown index than the control at 6 d. Compared to the control fruit, the AEOW treatment displayed lower activities of polyphenol oxidase (PPO), ascorbic acid oxidase (AAO), alternate oxidase (AOX), cytochrome C oxidase (CCO), succinate dehydrogenase (SDH), pulp phosphohexose isomerase (PGI), and higher levels of nicotinamide adenine dinucleotide kinase (NADK), glucose-6-phosphate dehydrogenase (G-6-PDH) and glucose-6-phosphate dehydrogenase (6-PGDH). Furthermore, the AEOW treatment increased the amount of NADPH and NADP while decreasing that of NADH and NAD. In terms of energy metabolism, the AEOW treatment maintained higher levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), energy charge, H+-ATPase, Ca2+-ATPase, and Mg2+-ATPase, but lower content of adenosine monophosphate (AMP). Therefore, the AEOW treatment inhibited respiratory metabolism by decreasing the terminal oxidase activities, tricarboxylic acid cycle (TCA cycle) and glycolytic pathway (EMP), and increasing the pentose phosphate pathway (PPP), which helped to maintain a higher energy status and further led to attenuation of longan pulp breakdown.

Antitumor effect of trimetazidine in a model of solid Ehrlich carcinoma is mediated by inhibition of glycolytic pathway and AKT signaling

Disturbance in glucose metabolism was proposed to be a pathogenetic mechanism of breast cancer. Trimetazidine (TMZ) inhibits β-oxidation of fatty acids through blocking the activity of 3-ketoacylCoA thiolase enzyme, leading to enhancement of glucose oxidation and metabolic respiration. The present study aimed to examine the cytotoxic effect of TMZ in both in vivo and in vitro models of breast cancer, focusing on its impact on the expression of some glycolytic enzymes and AKT signaling. The cytotoxic effect of TMZ was screened against breast (MCF-7) cancer cell line at different concentrations [0.01–100 μM]. In vivo, graded doses (10, 20, 30 mg/kg) of TMZ were tested against solid Ehrlich carcinoma (SEC) in mice. Tumor tissues were isolated for assessment of the expression of glucose transporter-1 (GLUT-1) and glycolytic enzymes by quantitative PCR. The protein expression of AKT and cellular myelocytomatosis (c-Myc) was determined by western blotting, while p53 expression was evaluated by immunohistochemistry. Molecular docking study of TMZ effect on AKT and c-Myc was performed using Auto-Dock Vina docking program. TMZ showed a cytotoxic action against MCF-7 cells, having IC50 value of 2.95 μM. In vivo, TMZ reduced tumor weight, downregulated the expression of glycolytic enzymes, suppressed AKT signaling, but increased p53 expression. Molecular docking and in silico studies proposed that TMZ is an AKT and c-Myc selective inhibitor. In conclusion, TMZ demonstrated a viable approach to suppress tumor proliferation in biological models of breast cancer.