Increased ATP Levels Research Articles

Concurrent Measurement of Mitochondrial DNA Copy Number and ATP Concentration in Single Bovine Oocytes.

To sustain energy-demanding developmental processes, oocytes must accumulate adequate stores of metabolic substrates and mitochondrial numbers prior to the initiation of maturation. In the past, researchers have utilized pooled samples to study oocyte metabolism, and studies that related multiple metabolic outcomes in single oocytes, such as ATP concentration and mitochondrial DNA copy number, were not possible. Such scenarios decreased sensitivity to intraoocyte metabolic relationships and made it difficult to obtain adequate sample numbers during studies with limited oocyte availability. Therefore, we developed and validated procedures to measure both mitochondrial DNA (mtDNA) copy number and ATP quantity in single oocytes. Validation of our procedures revealed that we could successfully divide oocyte lysates into quarters and measure consistent results from each of the aliquots for both ATP and mtDNA copy number. Coefficient of variation between the values retrieved for mtDNA copy number and ATP quantity quadruplicates were 4.72 ± 0.98 and 1.61 ± 1.19, respectively. We then utilized our methodology to concurrently measure mtDNA copy number and ATP quantity in germinal vesicle (GV) and metaphase two (MII) stage oocytes. Our methods revealed a significant increase in ATP levels (GV = 628.02 ± 199.53 pg, MII = 1326.24 ± 199.86 pg, p < 0.001) and mtDNA copy number (GV = 490,799.4 ± 544,745.9 copies, MII = 1,087,126.9 ± 902,202.8 copies, p = 0.035) in MII compared to GV stage oocytes. This finding is consistent with published literature and provides further validation of the accuracy of our methods. The ability to produce consistent readings and expected results from aliquots of the lysate from a single oocyte reveals the sensitivity and feasibility of using this method.

A pilot to assess target engagement of terazosin in Parkinson's disease

BackgroundImpaired brain energy metabolism is a key feature of Parkinson's disease (PD). Terazosin (TZ) binds phosphoglycerate kinase 1 and stimulates its activity, which enhances glycolysis and increases ATP levels. Preclinical and epidemiologic data suggest that TZ may be neuroprotective in PD. We aimed to assess target engagement and safety of TZ in people with PD. MethodsWe performed a 12-week pilot study in people with PD. Participants were randomized to receive 5 mg TZ or placebo. Participants and study personnel were blinded. We assessed TZ target engagement by measuring brain ATP with 31P-magnetic resonance spectroscopy (MRS) and whole blood ATP with a luminescence assay. Robust linear regression models compared changes between groups controlling for baseline brain and blood ATP levels, respectively. We also assessed clinical measures of PD and adverse events. ResultsThirteen participants were randomized. Mild dizziness/lightheadedness was more common in the TZ group, and three participants taking TZ dropped out because of dizziness and/or orthostatic hypotension. Compared to the placebo group, the TZ group had a significant increase in the ratio of βATP to inorganic phosphate in the brain. The TZ group also had a significant increase in blood ATP levels compared to the placebo group (p < 0.01). ConclusionsThis pilot study suggests that TZ may engage its target and change ATP levels in the brain and blood of people with PD. Further studies may be warranted to test the disease-modifying potential of TZ.

Mitochondriomics reveals the underlying neuroprotective mechanism of TrkB receptor agonist R13 in the 5×FAD mice

Decreased energy metabolism and mitochondrial biogenesis defects are implicated in the pathogenesis of Alzheimer's disease (AD). In present study, mitochondriomics analysis revealed significant effects of R13, a prodrug of 7,8-dihydroxyflavone, on mitochondrial protein expression profile, including the proteins related to the biological processes: fatty acid beta-oxidation, fatty acid metabolic process, mitochondrial electron transport, and mitochondrial respiratory chain. Cluster analysis demonstrated that R13 promoted mitochondrial oxidative phosphorylation (OXPHOS). The functional analysis showed that R13 increased ATP levels and enhanced OXPHOS including complex Ⅰ, Ⅱ, Ⅲ and Ⅳ. R13 treatment increased mitochondrial biogenesis by regulating the levels of p-AMPKα, p-CREB, PGC-1α, NRF1 and TFAM as a consequence of activation of TrkB receptor in the 5 × FAD mice. Finally, R13 significantly reduced the levels of tau phosphorylation and Aβ plaque. Our data suggest that R13 may be used for treating AD via enhancing mitochondrial biogenesis and metabolism.

Hyperbaric oxygen therapy does not alleviate tourniquet-induced acute ischemia-reperfusion injury in mouse skeletal muscles

During tourniquet application, blood flow is restricted to a limb to stop excessive limb hemorrhage in a trauma setting and to create a bloodless operating field in the surgical setting. During tourniquet-related ischemia, aerobic respiration stops, and ATP is depleted, and during subsequent reperfusion, there is an increase in reactive oxygen species (ROS) production and other endogenous substances, which leads to acute ischemia-reperfusion (IR) injuries, including tissue necrosis and skeletal muscle contractile dysfunction. Hyperbaric oxygen (HBO) therapy can increase the arterial oxygen tension in the tissues of patients with general hypoxia/anoxia, including carbon monoxide poisoning, circulatory arrest, and cerebral and myocardial ischemia. Here, we studied the protective effects of HBO pretreatment with 100% oxygen at 2.5 ATA against tourniquet/IR injury in mice. After one hour of HBO therapy with 100% oxygen at 2.5 ATA was administered to C57/BL6 mice, a rubber band was placed at the hip joint of the unilateral hindlimb to induce 3 h of ischemia and then released for 48 h of reperfusion. We analyzed gastrocnemius muscle morphology and contractile function and measured the levels of ATP and ROS accumulation in the muscles. HBO pretreatment did not improve tourniquet/IR-injured gastrocnemius muscle morphology and muscle contraction. Tourniquet/IR mice with HBO pretreatment showed no increase in ATP levels in IR tissues, but they did have a decreased amount of ROS accumulation in the muscles, compared to IR mice with no HBO pretreatment. These data suggest that one hour of HBO pretreatment with 100% oxygen at 2.5 ATA increases the antioxidant response to lower ROS accumulation but does not increase ATP levels in IR muscles and improve tourniquet/IR-injured muscle morphology and contractile function.

Mitochondrial bioenergetic, oxidative stress and burrowing responses in earthworm exposed to roxarsone in soil

The eco-risk of roxarsone (ROX) was evaluated using multiple responses of earthworm biomarkers under different ROX concentrations for 28 d. With the increasing total arsenic accumulation (TAs-E), biological responses in earthworm generally showed a two-stage changes of homeostasis dysregulation and dose-dependent alterations. At the early periods, ROX stress increased the reactive oxygen species (ROS) and lipid peroxidation (LPO) in a similar manner, and apparently disrupted mitochondrial calcium ([Ca2+]m). But earthworms regulated their mitochondrial and redox homeostasis through stable mitochondrial membrane potential (MMP) and increase of ATP level, superoxide dismutase (SOD) and catalase (CAT). After 14 d, the positively correlated mitochondrial effects of ROS, [Ca2+]m, MMP and ATP were related to the behavioral inhibition of burrow length, depth and reuse rate as well as antioxidant up-regulation of Nrf2, HO-1, sod1 and cat. These results contributed possible biomarkers from the dose-dependent relationship between mitochondrial, antioxidant and behavioral responses. Multiple biological detection in earthworms can better reflect the sub-chronic ecotoxicity of phenylarsenic pollutants in soil.

Glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase inhibition in the presence of pro-inflammatory cytokines contribute to the metabolic imbalance of diabetic retinopathy

Diabetic retinopathy (DR) is the leading cause of vision impairment in working age adults. In addition to hyperglycemia, retinal inflammation is an important driving factor for DR development. Although DR is clinically described as diabetes-induced damage to the retinal blood vessels, several studies have reported that metabolic dysregulation occurs in the retina prior to the development of microvascular damage. The two most commonly affected metabolic pathways in diabetic conditions are glycolysis and the glutamate pathway. We investigated the role of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutamine synthetase (GS) in an in-vitro model of DR incorporating high glucose and pro-inflammatory cytokines. We found that GAPDH and GS enzyme activity were not significantly affected in hyperglycemic conditions or after exposure to cytokines alone, but were significantly decreased in the DR model. This confirmed that pro-inflammatory cytokines IL-1β and TNFα enhance the hyperglycemic metabolic deficit. We further investigated metabolite and amino acid levels after specific pharmacological inhibition of GAPDH or GS in the absence/presence of pro-inflammatory cytokines. The results indicate that GAPDH inhibition increased glucose and addition of cytokines increased lactate and ATP levels and reduced glutamate levels. GS inhibition did not alter retinal metabolite levels but the addition of cytokines increased ATP levels and caused glutamate accumulation in Müller cells. We conclude that it is the action of pro-inflammatory cytokines concomitantly with the inhibition of the glycolytic or GS mediated glutamate recycling that contribute to metabolic dysregulation in DR. Therefore, in the absence of good glycemic control, therapeutic interventions aimed at regulating inflammation may prevent the onset of early metabolic imbalance in DR.

Cytochrome P450 3A5 polymorphism affects the metabolism of sorafenib and its toxicity for hepatocellular carcinoma cells in vitro.

Cytochrome P450 3A5 (CYP3A5) is a highly polymorphic gene and the encoded protein variants differ in catalytic activity, leading to inter-individual variation in metabolic ability. The aim of the current study was to investigate the effects of seven allelic variants on the ability of CYP3A5 to metabolize sorafenib in vitro and further explore the impacts of CYP3A5 polymorphism on the proliferation and apoptosis of hepatocellular carcinoma cell line (HepG2) induced by sorafenib. Wild-type and variant CYP3A5 enzymes were expressed in Spodoptera frugiperda insect cells using a baculovirus dual-expression system, and protein expression was checked by western blot. The enzymes were incubated with sorafenib at 37°C for 30 min, and formation of the major metabolite sorafenib N-oxide was assayed using ultra-performance liquid chromatography and tandem mass spectrometry. Intrinsic clearance values (Vmax/Km) were calculated for each enzyme. Additionally, recombinant HepG2 cells transfecting with CYP3A5 variants were used to investigate the effects of sorafenib on the proliferation of HepG2 cells. Intrinsic clearance of the six variants CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 was 26.41-71.04% of the wild-type (CYP3A5*1) value. In contrast, the clearance value of the variant CYP3A5*6 was significantly higher (174.74%). Additionally, the decreased ATP levels and cell viability and the increased cell apoptosis in HepG2 cells transfected with CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 were observed, whereas, the increased ATP levels and cell viability and the reduced cell apoptosis in HepG2 cells transfected with CYP3A5*6 were also investigated when compared to CYP3A5*1. Our results suggest that CYP3A5 polymorphism influences sorafenib metabolism and pharmacotherapeutic effect in hepatic carcinomas. These data may help explain differential response to drug therapy for hepatocellular carcinoma, and they support the need for individualized treatment.

Abstract 12755: Effects of Intravenously Delivered Extracellular Vesicles on Cardiac Function in Chemotherapy-Induced Cardiomyopathy

Introduction: Extracellular Vesicles (EV) are increasingly recognized as key mediators of the effects of cellular therapy but repeated dosing is likely required for optimizing their therapeutic benefits. This, in turn, requires a noninvasive delivery route. Hypothesis: Repeated intravenous (IV) EV infusions might improve cardiac function in chemotherapy-induced cardiomyopathy. Methods: All EV were collected from human iPSC-derived cardiovascular progenitor cells. In vitro experiments : iPSC-derived cardiomyocytes were stressed 3 times by doxorubicin (Dox, 0.2 μM), once every 48 hours (D0, D2, D4), and then exposed to EV once (D6) or twice (D6 and D8). Outcomes were assessed on D8 and D10 on mitochondrial function and intracellular ATP levels assessed by the Seahorse XF Cell Mito Stress Test and ATPlite Luminescence Assay System, respectively. In vivo experiments : Male BALB/c mice were subjected to 3 weekly injections of Dox (total cumulated dose: 12 mg/kg) followed by 3 IV infusions of EV (10 9 per dose, n=11) or an equivalent volume of saline (n=14) over 1-2 weeks. The main outcome measure was Global Longitudinal Strain (GLS), a robust and early marker of LV dysfunction, blindly assessed by MRI at baseline and 8-11 weeks following the onset of Dox treatment. Explanted hearts were then processed for histology and transcriptomics. Results: Compared with Dox-stressed controls, EV increased ATP levels by 30%±8% (n=7, each in triplicate, p=0.047; m±SEM) and enhanced both mitochondrial respiration and anaerobic glycolysis. In vivo , GLS at baseline averaged -14.98±0.24. It was well preserved after EV treatment with a decrease at end-study of only 2.96%±7.18% from baseline while it fell by 11.90%±3.51% in PBS-injected controls. Similar patterns of changes were seen for longitudinal endocardial strain which declined by 0.22%±7.29% and 8.39%±3.42% from baseline in EV- and PBS-injected hearts, respectively, while the corresponding declines for epicardial strains were 3.92%±7.20% and 13.44%±3.28%. Basal global circumferential strain was also better preserved in EV-treated hearts. Conclusions: Repeated IV infusions of EV decrease chemo-triggered cardiomyocyte toxicities and help preserving cardiac function in chemotherapy-induced cardiomyopathy.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Mitophagy mediates metabolic reprogramming of induced pluripotent stem cells undergoing endothelial differentiation

Pluripotent stem cells are known to shift their mitochondrial metabolism upon differentiation, but the mechanisms underlying such metabolic rewiring are not fully understood. We hypothesized that during differentiation of human induced pluripotent stem cells (hiPSCs), mitochondria undergo mitophagy and are then replenished by the biogenesis of new mitochondria adapted to the metabolic needs of the differentiated cell. To evaluate mitophagy during iPSC differentiation, we performed live cell imaging of mitochondria and lysosomes in hiPSCs differentiating into vascular endothelial cells using confocal microscopy. We observed a burst of mitophagy during the initial phases of hiPSC differentiation into the endothelial lineage, followed by subsequent mitochondrial biogenesis as assessed by the mitochondrial biogenesis biosensor MitoTimer. Furthermore, hiPSCs undergoing differentiation showed greater mitochondrial oxidation of fatty acids and an increase in ATP levels as assessed by an ATP biosensor. We also found that during mitophagy, the mitochondrial phosphatase PGAM5 is cleaved in hiPSC-derived endothelial progenitor cells and in turn activates β-catenin-mediated transcription of the transcriptional coactivator PGC-1α, which upregulates mitochondrial biogenesis. These data suggest that mitophagy itself initiates the increase in mitochondrial biogenesis and oxidative metabolism through transcriptional changes during endothelial cell differentiation. In summary, these findings reveal a mitophagy-mediated mechanism for metabolic rewiring and maturation of differentiating cells via the β-catenin signaling pathway. We propose that such mitochondrial-nuclear cross talk during hiPSC differentiation could be leveraged to enhance the metabolic maturation of differentiated cells.

Etavopivat, an Allosteric Activator of Pyruvate Kinase-R, Improves Sickle RBC Functional Health and Survival and Reduces Systemic Markers of Inflammation and Hypercoagulability in Patients with Sickle Cell Disease: An Analysis of Exploratory Studies in a Phase 1 Study

Etavopivat, an Allosteric Activator of Pyruvate Kinase-R, Improves Sickle RBC Functional Health and Survival and Reduces Systemic Markers of Inflammation and Hypercoagulability in Patients with Sickle Cell Disease: An Analysis of Exploratory Studies in a Phase 1 Study

Febuxostat treatment attenuates oxidative stress and inflammation due to ischemia-reperfusion injury through the necrotic pathway in skin flap of animal model

BackgroundIschemia-reperfusion (I/R) injury is a major contributor to skin flap necrosis, which is a serious complication of reconstructive surgery. The purpose of this study was to evaluate the protective effect of treatment with febuxostat, a selective xanthine oxidase inhibitor, on I/R injury in the skin flap of an animal (rat) model. MethodsSuperficial epigastric flaps were raised in Sprague-Dawley rats and subjected to ischemia for 3 h. Febuxostat at a dose of 10 mg/kg/day was administered to rats in drinking water from 1 week before the surgery (Feb group). Control animals received no drugs (Con group). The mean ratio of flap survival and contraction was evaluated and compared between animals with and without administration of febuxostat on day 5 after the surgery. In addition, infiltration by polymorphonuclear leukocytes and muscles of the panniculus carnosus in the flap were histologically evaluated using hematoxylin-eosin staining. Furthermore, xanthine oxidase activity, ATP levels, superoxide dismutase activity, and expression of 8-hydroxy-2′-deoxyguanosine (8-OHdG), tumor necrosis factor-α, and interleukin-1β were quantitatively assessed in the skin flap 24 h after the surgery. ResultsIn the Feb group, the survival and contraction rates at the 5 d timepoint post-surgery were significantly higher and lower than those in the Con group, respectively. Histological analysis showed significant reduction in polymorphonuclear leukocyte infiltration and muscle injury scores due to I/R injury in the Feb group. The expression of 8-OHdG was also significantly inhibited in animals administered febuxostat. Biochemical analysis showed a significant reduction in xanthine oxidase activity and significant increases in ATP levels and superoxide dismutase activity in the Feb group. Furthermore, the expression of interleukin-1β was significantly lower in the Feb group than in the Con group. ConclusionFebuxostat, which is clinically used for the treatment of hyperuricemia, was effective against necrosis of the skin flap via inhibition of oxidative stress and inflammation caused by I/R injury.

Neuroprotective effects of Withania somnifera in the SH-SY5Y Parkinson cell model

Parkinson's disease is the most frequent neurodegenerative motor disorder. The clinical syndrome and pathology involve motor disturbance and the degeneration of dopaminergic neurons in the substantia nigra. Root extracts of Withania. somnifera, commonly called Ashwagandha, contain several major chemical constituents known as withanolides. Studies have shown that W. somnifera extracts exhibit numerous therapeutic effects including inflammation and oxidative stress reduction, memory and cognitive function improvement. This study aimed to evaluate the protective effects of KSM-66, W. somnifera root extract, on 6-hydroxydopamine (6-OHDA)-induced toxicity in the human neuroblastoma SH-SY5Y cell line, as well as the associated oxidative response protein expression and redox regulation activity focused on S-glutathionylation. SH-SY5Y cells were treated with 6-OHDA preceded or followed by treatment with the KSM-66 extract. Using KSM-66 concentrations ranging from 0.25 to 1 mg/ml before and after treatment of the cells with 6-OHDA has resulted in an increased viability of SH-SY5Y cells. Interestingly, the extract significantly increased glutathione peroxidase activity and thioltransferase activity upon pre- or post- 6-OHDA treatment. KSM-66 also modulated oxidative response proteins: peroxiredoxin-I, VGF and vimentin proteins upon 6-OHDA pre/post treatments. In addition, the extract controlled redox regulation via S-glutathionylation. Pre-treatment of SH-SY5Y cells with KSM-66 decreased protein-glutathionylation levels in the cells treated with 6-OHDA. The rescue of mitochondria with 0.5 mg/ml KSM-66 extract showed an increase in ATP levels. These findings suggest that W. somnifera root extract acts as a neuroprotectant, thereby introducing a potential agent for the treatment or prevention of neurodegenerative diseases.

Asiatic acid alleviates ischemic myocardial injury in mice by modulating mitophagy- and glycophagy-based energy metabolism.

Myocardial infarction (MI) causes disturbances in myocardial energy metabolism, ultimately leading to a poor prognosis. Cytosolic glycogen autophagy (glycophagy) and mitochondrial autophagy (mitophagy) are upregulated in MI to optimize energy metabolism but to a limited extent. Asiatic acid (AA), a pentacyclic triterpene derived from the traditional Chinese herb Centella asiatica, displays anti-inflammatory, antioxidant, and antiapoptotic activities. AA has been found to alleviate focal cerebral and liver ischemic injury by reversing mitochondrial dysfunction. In this study, we investigated whether AA exerted cardioprotective effects against MI by activating glycophagy and mitophagy to improve the energy balance. In vitro cardioprotective effects were examined in neonatal mouse cardiomyocytes subjected to oxygen-glucose deprivation for 12 h. Treatment with AA (2-50 μM) significantly increased cell viability and improved the energy metabolism evidenced by increased ATP level and phosphocreatine/ATP ratio. In vivo cardioprotective effects were studied in a mouse model of MI. Administration of AA (5-125 mg·kg-1·d-1, ig) significantly reduced infarct size and ischemic myocardial injury, and improved cardiac function. AA treatment also promoted mitophagy and relieved mitochondrial edema evidenced by increased number of mitophagosomes in ischemic myocardium in vivo and increased mitochondria-light chain 3 (LC3)-II colocalization in ODG-treated cardiomyocytes in vitro. Mitophagy activation was accompanied by activation of the AMPK signaling pathway. Knockdown of AMPK abolished AA-activated mitophagy. Furthermore, we showed that glycophagy was upregulated in OGD cardiomyocytes evidenced by increased starch binding domain protein 1 (STBD1)-GABA type A receptor-associated protein-like 1(GABARAPL1) interaction and extracellular acidification rate, whereas AA treatment further promoted glycophagy accompanied by PI3K/Akt activation. PI3K inhibitor LY294002 or Akt inhibitor GSK690693 blocked the effects of AA on glycophagy and glycolysis. Finally, simultaneous inhibition of glycophagy and mitophagy abolished the cardioprotective effects and energy regulation of AA. These results demonstrate that AA protects ischemic cardiomyocytes by modulating glycophagy- and mitophagy-based energy metabolism through the PI3K/Akt and AMPK pathways.

Ent-kaurane diterpenes isolated from n-hexane extract of Baccharis sphenophylla by bioactivity-guided fractionation target the acidocalcisomes in Trypanosoma cruzi

BackgroundIn the present work the bioactivity-guided fractionation of n-hexane extract from aerial parts of Baccharis sphenophylla (Asteraceae) against trypomastigote forms of Trypanosoma cruzi was performed. PurposeTo evaluate the antitrypanosomal potential of diterpenes ent‑kaurenoic (1), grandifloric (2). and 15β-tiglinoyloxy‑ent-kaurenoic (3) acids, isolated from n-hexane extract from aerial parts of B. sphenophylla, and elucidate their mechanism of action against T. cruzi. Methods/Study designn-Hexane and MeOH extracts from aerial parts of B. sphenophylla were prepared and caused, respectively, 100% and 50% of death of trypomastigote forms of T. cruzi. Based on these results, the n-hexane extract was subjected to bioactivity-guided fractionation procedures to afford three related ent‑kaurane diterpenoids (1–3). Based on spectrofluorometric assays and flow cytometry analysis, the mechanism of action of compounds 1 and 3 was investigated. ResultsCompounds 1 and 3, isolated from n-hexane extract from aerial parts of B. sphenophylla, showed potent activity against parasites with EC50 values of 10.6 μM (SI > 18.8) and 2.4 μM (SI = 34.8), respectively. On the other hand, compound 2 was inactive against trypomastigotes. In mechanism of action studies using the fluorescent probe SYTOX Green, the plasma membrane permeability was unaltered after treatment with compounds 1 and 3, but compound 1 induced a depolarization of the plasma membrane electric potential (ΔΨp). No substantial alterations were observed in the mitochondria after treatment with compound 3, but a transient hyperpolarization of the mitochondrial membrane potential (ΔΨm) by compound 1. Despite the increased ATP levels induced by compounds 1 and 3, no alterations of ROS and Ca2+ levels were registered. However, both compounds promoted a time-dependent alkalinization of the acidocalcisomes, probably contributing to an osmotic imbalance of the cell. In silico physicochemical studies of compounds 1–3 suggested that lipophilicity and molecular complexity may play an important role in the antitrypanosomal activity. Moreover, no pan-assay interference compounds (PAINS) alerts were detected for compounds 1–3. ConclusionObtained data indicated that the isolated ent‑kaurane diterpenes from n-hexane extract from aerial parts of B. sphenophylla, especially compound 3, could be considered interesting prototypes for further modifications aiming the discovery of new hits against T. cruzi.

L-glycine and l-glutamic acid protect Pediococcus pentosaceus R1 against oxidative damage induced by hydrogen peroxide

The effects of l-glycine (Gly) and l-glutamic acid (Glu) on oxidative damage induced by hydrogen peroxide (H2O2) in Pediococcus pentosaceus R1 were investigated. Gly and Glu significantly reduce the production of intracellular reactive oxygen species and the levels of malondialdehyde and carbonylated proteins and concomitantly increase ATP levels in P. pentosaceus R1 under H2O2-induced stress (P < 0.05). Transmission electron microscopy and atomic force microscopy of bacteria under H2O2-induced stress revealed that Gly and Glu suppress bacterial membrane deformation and cell damage. Gly exhibited stronger ferrous ion-chelating ability, whereas Glu has higher radical scavenging activities and reducing power (P < 0.05). The abilities of Gly and Glu to inhibit lipid peroxidation are comparable. Gly and Glu significantly enhance the activities of superoxide dismutase and glutathione peroxidase, respectively, and increase the total antioxidant capacity of bacteria (P < 0.05). These findings indicate that Gly and Glu alleviate H2O2-induced oxidative stress via direct antioxidant effects and increase the activities of bacterial antioxidant enzyme.

TAZ as a novel regulator of oxidative damage in decidualization via Nrf2/ARE/Foxo1 pathway

TAZ, as a crucial effector of Hippo pathway, is required for spermatogenesis and fertilization, but little is known regarding its physiological function in uterine decidualization. In this study, we showed that TAZ was localized in the decidua, where it promoted stromal cell proliferation followed by accelerated G1/S phase transition via Ccnd3 and Cdk4 and induced the expression or activity of stromal differentiation markers Prl8a2, Prl3c1 and ALP, indicating the importance of TAZ in decidualization. Knockdown of TAZ impeded HB-EGF induction of stromal cell proliferation and differentiation. Under oxidative stress, TAZ protected stromal differentiation against oxidative damage by reducing intracellular ROS and enhancing cellular antioxidant capacity dependent on the Nrf2/ARE/Foxo1 pathway. TAZ strengthened the transcriptional activity of Nrf2 which directly bound to the antioxidant response element (ARE) of Foxo1 promoter region. Additionally, silencing TAZ caused accumulation of intracellular ROS through heightening NOX activity whose blockade by APO reversed the disruption in stromal differentiation. Further analysis revealed that TAZ might restore mitochondrial function, as indicated by the increase in ATP level, mtDNA copy number and mitochondrial membrane potential with the reduction in mitochondrial superoxide. Additionally, TAZ modulated the activities of mitochondrial respiratory chain complexes I and III whose suppression by ROT and AA resulted in the inability of TAZ to defend against oxidative damage to stromal differentiation. Moreover, TAZ prevented stromal cell apoptosis by upregulating Bcl2 expression and inhibiting Casp3 activity and Bax expression. In summary, TAZ might mediate HB-EGF function in uterine decidualization through Ccnd3 and ameliorate oxidative damage to stromal cell differentiation via Nrf2/ARE/Foxo1 pathway.

Comparative toxicities of BPA, BPS, BPF, and TMBPF in the nematode Caenorhabditis elegans and mammalian fibroblast cells

Bisphenol A (BPA) is a chemical compound commonly used in the production of plastics for daily lives and industry. As BPA is well known for its adverse health effects, several alternative materials have been developed. This study comprehensively analyzed the toxicity of BPA and its three substitutes including bisphenol S (BPS), bisphenol F (BPF), and tetramethyl bisphenol F (TMBPF) on aging, healthspan, and mitochondria using an in vivo Caenorhabditis elegans (C. elegans) model animal and cultured mammalian fibroblast cells. C. elegans treated with 1 mM BPA exhibited abnormalities in the four tested parameters related to development and growth, including delayed development, decreased body growth, reduced reproduction, and abnormal tissue morphology. Exposure to the same concentration of each alternative including TMBPF, which has been proposed as a relatively safe BPA alternative, detrimentally affected at least three of these events. Moreover, all bisphenols (except BPS) remarkably shortened the organismal lifespan and increased age-related changes in neurons. Exposure to BPA and BPF resulted in mitochondrial abnormalities, such as reduced oxygen consumption and mitochondrial membrane potential. In contrast, the ATP levels were noticeably higher after treatment with all bisphenols. In mammalian fibroblast cells, exposure to increasing concentrations of all bisphenols (ranging from 50 μM to 500 μM) caused a severe decrease in cell viability in a dose-dependent manner. BPA increased ATP levels and decreased ROS but did not affect mitochondrial permeability transition pores (mPTP). Notably, TMBPF was the only bisphenol that caused a significant increase in mitochondrial ROS and mPTP opening. These results suggest that the potentially harmful physiological effects of BPA alternatives should be considered.

Microvesicles transfer mitochondria and increase mitochondrial function in brain endothelial cells

We have demonstrated, for the first time that microvesicles, a sub-type of extracellular vesicles (EVs) derived from hCMEC/D3: a human brain endothelial cell (BEC) line transfer polarized mitochondria to recipient BECs in culture and to neurons in mice acute brain cortical and hippocampal slices. This mitochondrial transfer increased ATP levels by 100 to 200-fold (relative to untreated cells) in the recipient BECs exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. We have also demonstrated that transfer of microvesicles, the larger EV fraction, but not exosomes resulted in increased mitochondrial function in hypoxic endothelial cultures. Gene ontology and pathway enrichment analysis of EVs revealed a very high association to glycolysis-related processes. In comparison to heterotypic macrophage-derived EVs, BEC-derived EVs demonstrated a greater selectivity to transfer mitochondria and increase endothelial cell survival under ischemic conditions.

Effect of hydrogen sulfide on glycolysis-based energy production in mouse erythrocytes.

Hydrogen sulfide (H2 S) is a gasotransmitter that regulates both physiological and pathophysiological processes in mammalian cells. Recent studies have demonstrated that H2 S promotes aerobic energy production in the mitochondria in response to hypoxia, but its effect on anaerobic energy production has yet to be established. Glycolysis is the anaerobic process by which ATP is produced through the metabolism of glucose. Mammalian red blood cells (RBCs) extrude mitochondria and nucleus during erythropoiesis. These cells would serve as a unique model to observe the effect of H2 S on glycolysis-mediated energy production. The purpose of this study was to determine the effect of H2 S on glycolysis-mediated energy production in mitochondria-free mouse RBCs. Western blot analysis showed that the only H2 S-generating enzyme expressed in mouse RBCs is 3-mercaptopyruvate sulfurtransferase (MST). Supplement of the substrate for MST stimulated, but the inhibition of the same suppressed, the endogenous production of H2 S. Both exogenously administered H2 S salt and MST-derived endogenous H2 S stimulated glycolysis-mediated ATP production. The effect of NaHS on ATP levels was not affected by oxygenation status. On the contrary, hypoxia increased intracellular H2 S levels and MST activity in mouse RBCs. The mitochondria-targeted H2 S donor, AP39, did not affect ATP levels of mouse RBCs. NaHS at low concentrations (3-100 μM) increased ATP levels and decreased cell viability after 3 days of incubation in vitro. Higher NaHS concentrations (300-1000 μM) lowered ATP levels, but prolonged cell viability. H2 S may offer a cytoprotective effect in mammalian RBCs to maintain oxygen-independent energy production.