Superoxide Accumulation Research Articles

Cell wall and reactive oxygen metabolism responses of strawberry fruit during storage to low voltage electrostatic field treatment

Low voltage electrostatic field (LVEF) is a new, non-thermal and low-cost fresh-keeping technology. However, there are few studies to evaluate the effects of LVEF on cell wall metabolism and reactive oxygen metabolism of strawberries. In this investigation, strawberries were stored in LVEF at 4 ± 1 °C for 10 d to explore the mechanism of cell wall metabolism and the effect of reactive oxygen on fruit quality. After 10 d of storage, strawberries were of better qualities compared to control group, with fruit firmness increased by 17 %, decay incidence decreased by 38%, weight loss reduced by 25 % and relative conductivity reduced by 38 %. Meanwhile, LVEF treatment significantly inhibited the accumulation of malondialdehyde, hydrogen peroxide and superoxide anion, and induced an increase of superoxide dismutase, catalase and ascorbate peroxidase activities. LVEF treatment also retained antioxidant components such as ascorbic acid and reduced glutathione. In addition, LVEF treatment slowed down the degradation of protopectin to water-soluble pectin and inhibited the activities of cellular degradation enzymes, including pectin methylesterase, polygalacturonase, cellulase, β-xylosidase and Xyloglucan endotransglycosylase/hydrolase. These results demonstrated that LVEF treatment might be an effective method for extending strawberries shelf life.

Co-Application of 1-MCP and Laser Microporous Plastic Bag Packaging Maintains Postharvest Quality and Extends the Shelf-Life of Honey Peach Fruit.

Honey peach (Prunus persica L.) is highly nutritious; it is an excellent source of sugars, proteins, amino acids, vitamins, and mineral elements. However, it is a perishable climacteric fruit that is difficult to preserve. In this study, “Feicheng” honey peach fruit was used as a test material to investigate the synergistic preservation effect of 1-methylcyclopropene (1-MCP) and laser microporous film (LMF). The peach fruits were fumigated for 24 h with 2 μL L−1 1-MCP, then packed in LMF. In comparison with the control treatment, 1-MCP + LMF treatment markedly decreased the respiration rate, weight loss, and rot rate of peach fruits. Moreover, the combination of 1-MCP and LMF suppressed the increase in soluble solids (SS) and reducing sugars (RS), as well as the decrease in titratable acid (TA) and ascorbic acid (AsA). The combined application also maintained a high protopectin content and low soluble pectin content; it reduced the accumulation of superoxide anions (O2−) and hydrogen peroxide (H2O2). Except in a few samples, the catalase (CAT) and ascorbate peroxidase (APX) activities were higher when treated by 1-MCP + LMF. Conversely, the phenylalanine deaminase (PAL), peroxidase (POD), lipase, lipoxygenase (LOX), polygalacturonase (PG), β-glucosidase, and cellulase (Cx) activities were lower than in the control. Furthermore, 1-MCP + LMF treatment reduced the relative abundances of dominant pathogenic fungi (e.g., Streptomyces, Stachybotrys, and Issa sp.). The combined treatment improved the relative abundances of antagonistic fungi (e.g., Aureobasidium and Holtermanniella). The results indicated that the co-application of 1-MCP and LMF markedly reduced weight loss and spoilage, delayed the decline of nutritional quality, and inhibited the physiological and biochemical metabolic activities of peach during storage. These changes extended its shelf-life to 28 days at 5 °C. The results provide a reference for the commercial application of this technology.

Neuroprotective Function of Fucoxanthin in Oxidative Stress-Mediated Mitochondrial Dysfunction

ObjectivesOxidative stress plays an important role in neuronal loss associated with mitochondrial dysfunction. Excess reactive oxygen species (ROS) production damages mitochondria, impairing neuronal energy metabolism. The damaged mitochondria promote aberrant ROS production leading to neuronal death. Fucoxanthin, a carotenoid with antioxidant properties primarily found in brown seaweeds, has been shown to protect mitochondria in various disease models. However, limited studies have demonstrated the mechanisms of fucoxanthin-mediated neuroprotection. In this study, we hypothesize that fucoxanthin regulates DJ-1, an oxidative stress sensing protein, and protects neurons against ROS-mediated mitochondrial dysfunction.MethodsRat primary hippocampal neurons were treated with fucoxanthin, hydrogen peroxide, or a combination of both. After 6 h of incubation, mitochondrial superoxide and the mitochondrial membrane potential were measured using mitoSOX and TMRM, respectively. Middle-aged male Sprague Dawley rats were supplemented with or without fucoxanthin (1 mg/kg, 5 d/w for 4 wk). After supplementation was completed, brain tissues were harvested, and DJ-1 protein levels were quantified using immunoblotting.ResultsTreatment with fucoxanthin decreased mitochondrial superoxide accumulation and prevented loss of mitochondrial membrane potential against ROS challenge in rat primary hippocampal neurons. Oral supplementation of fucoxanthin increased DJ-1 protein levels in the hippocampal tissues isolated from middle-aged rats.ConclusionsWe found that fucoxanthin treatment upregulates DJ-1 expression in the hippocampus in vivo and protects mitochondria during ROS challenges in primary hippocampal neurons in vitro. Our data suggest fucoxanthin has neuroprotective potential against ROS-associated mitochondrial dysfunction.Funding SourcesRGC Program (University of Alabama); Crenshaw Research Fund (University of Alabama).

Simulated gastrointestinal digestion and colonic fermentation of blue honeysuckle: Phenolic profile and protectivity on ethyl carbamate-induced oxidative damage

Blue honeysuckle (Lonicera caerulea L.) is well-known fruit that belongs to the berries family, and is widely cultivated in China, Russia, and Japan. This study aimed to evaluate the impact of in vitro gastrointestinal digestion followed by gut microbiota fermentation on the phenolic profile of blue honeysuckle as well as the protective effect of digested-blue honeysuckle against ethyl carbamate (EC)-induced oxidative damage. Results of phenolic profile showed that cyanidin-3-O-glucoside was identified as the predominant anthocyanin in blue honeysuckle and was significantly decreased after in vitro digestion. The generation of some metabolites (gallic acid, ferulic acid, and 3,4-dihydroxybenzoic acid) could be attributed to the degradation of chlorogenic acid and anthocyanins. Cellular studies indicated that digested-blue honeysuckle (0.5 and 1 mg/mL) exhibited significant protection against EC-induced cytotoxicity. Digested-blue honeysuckle at a concentration 1.0 mg/mL exhibited significant (p < 0.05) protective effect against EC-induced cytotoxicity in Caco-2 cells via inhibiting ROS and superoxide anion accumulation, attenuating GSH depletion, and inhibiting mitochondrial membrane potential collapse compared to EC-treated group. Blue honeysuckle could be a promising resource for ameliorating EC-induced cellular oxidative damage, which could be an ingredient for functional foods. However, further in vivo studies are recommended to prove the functionality of blue honeysuckle.

Patient-derived microphysiological model identifies the therapeutic potential of metformin for thoracic aortic aneurysm.

SummaryBackgroundThoracic aortic aneurysm (TAA) is the permanent dilation of the thoracic aortic wall that predisposes patients to lethal events such as aortic dissection or rupture, for which effective medical therapy remains scarce. Human-relevant microphysiological models serve as a promising tool in drug screening and discovery.MethodsWe developed a dynamic, rhythmically stretching, three-dimensional microphysiological model. Using patient-derived human aortic smooth muscle cells (HAoSMCs), we tested the biological features of the model and compared them with native aortic tissues. Drug testing was performed on the individualized TAA models, and the potentially effective drug was further tested using β-aminopropionitrile-treated mice and retrospective clinical data.FindingsThe HAoSMCs on the model recapitulated the expressions of many TAA-related genes in tissue. Phenotypic switching and mitochondrial dysfunction, two disease hallmarks of TAA, were highlighted on the microphysiological model: the TAA-derived HAoSMCs exhibited lower alpha-smooth muscle actin expression, lower mitochondrial membrane potential, lower oxygen consumption rate and higher superoxide accumulation than control cells, while these differences were not evidently reflected in two-dimensional culture flasks. Model-based drug testing demonstrated that metformin partially recovered contractile phenotype and mitochondrial function in TAA patients’ cells. Mouse experiment and clinical investigations also demonstrated better preserved aortic microstructure, higher nicotinamide adenine dinucleotide level and lower aortic diameter with metformin treatment.InterpretationThese findings support the application of this human-relevant microphysiological model in studying personalized disease characteristics and facilitating drug discovery for TAA. Metformin may regulate contractile phenotypes and metabolic dysfunctions in diseased HAoSMCs and limit aortic dilation.FundingThis work was supported by grants from National Key R&D Program of China (2018YFC1005002), National Natural Science Foundation of China (82070482, 81771971, 81772007, 51927805, and 21734003), the Science and Technology Commission of Shanghai Municipality (20ZR1411700, 18ZR1407000, 17JC1400200, and 20YF1406900), Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), and Shanghai Municipal Education Commission (Innovation Program 2017-01-07-00-07-E00027). Y.S.Z. was not supported by any of these funds; instead, the Brigham Research Institute is acknowledged.

Tetramethylpyrazine nitrone TBN extends the lifespan of C. elegans by activating the Nrf2/SKN-1 signaling pathway

SKN-1, the ortholog of mammalian Nrf2 proteins, is a transcription factor that plays an important role in oxidative stress resistance and longevity. Similar to other defense systems, the Nrf2-mediated stress response is compromised in aging and neurodegenerative diseases. Our previous studies demonstrated that tetramethylpyrazine nitrone (TBN), a derivative of tetramethylpyrazine armed with a potent free radical-scavenging nitrone moiety, exerted multifunctional neuroprotection in neurological and other diseases. However, the ability of TBN to extend a healthy lifespan and its underlying mechanisms of action are not yet clear. C. elegans have become a popular animal model in aging research. Herein, we demonstrate that TBN can extend the lifespan, promote age-associated health indicators, and restore mitochondrial function in C. elegans. TBN also significantly reduced ROS levels and superoxide accumulation in C. elegans. We show that TBN-mediated lifespan extension is SKN-1dependent. The present study provides valuable insights into the mechanisms by which TBN inhibits aging via the Nrf2/SKN-1 pathway in C. elegans.

The Root Hair Development of Pectin Polygalacturonase PGX2 Activation Tagging Line in Response to Phosphate Deficiency.

Pectin, cellulose, and hemicellulose constitute the primary cell wall in eudicots and function in multiple developmental processes in plants. Root hairs are outgrowths of specialized epidermal cells that absorb water and nutrients from the soil. Cell wall architecture influences root hair development, but how cell wall remodeling might enable enhanced root hair formation in response to phosphate (P) deficiency remains relatively unclear. Here, we found that POLYGALACTURONASE INVOLVED IN EXPANSION 2 (PGX2) functions in conditional root hair development. Under low P conditions, a PGX2 activation tagged line (PGX2AT) displays bubble-like root hairs and abnormal callose deposition and superoxide accumulation in roots. We found that the polar localization and trafficking of PIN2 are altered in PGX2AT roots in response to P deficiency. We also found that actin filaments were less compact but more stable in PGX2AT root hair cells and that actin filament skewness in PGX2AT root hairs was recovered by treatment with 1-N-naphthylphthalamic acid (NPA), an auxin transport inhibitor. These results demonstrate that activation tagging of PGX2 affects cell wall remodeling, auxin signaling, and actin microfilament orientation, which may cooperatively regulate root hair development in response to P starvation.

Cell Localization of DPI-Dependent Production of Superoxide in Reproductive Tissues of the Olive Tree (Olea europaea L.)

Reactive Oxygen Species (ROS) are compounds derived from oxygen with important implications in biological processes in plants, some of them related to reproduction. Among ROS, superoxide is the primary oxidant, since an array of other ROS are eventually derived from this anion. Therefore, analysis of the molecular systems able to generate this molecule and the cellular compartmentalization of these events is of paramount importance. We have used the fluorochrome DCFH2-DA and the chromogenic substrate NBT in association with DPI (a specific inhibitor of Rboh enzymes generating superoxide in plants) in combination with confocal microscopy and stereomicroscopy, respectively to identify cell localization of ROS in general, and superoxide accumulation in olive reproductive tissues. A significant production of both ROS and superoxide has been described, showing a fairly precise spatial and temporal location throughout olive flower development. The reduction of the NBT signal after the addition of DPI suggests that the generation of superoxide is largely due to Rboh or other flavin oxidase activity. At the subcellular level, accumulation of O2●− has been located in the plasma membrane of mature pollen and germinated pollen, as well as in the rough endoplasmic reticulum and in mitochondria.

PgLEA, a gene for late embryogenesis abundant proteinfrom Panax ginseng, enhances drought and salt tolerancein transgenic Arabidopsis thaliana

Late embryogenesis abundant (LEA) proteins are important for promoting the growth and stress tolerance of plants. They are widely involved in plant growth regulation and responses to hormones and environmental factors. However, knowledge of the functions of the LEA gene in ginseng species remains limited. In this study, a Panax ginseng LEA gene (PgLEA) expression vector was constructed, and stable transgenic Arabidopsis lines were established. The PgLEA protein was classified in the LEA-2 subgroup. Reverse-transcription quantitative PCR analysis showed that the expression of PgLEA increased under 300 mM NaCl or 10 % (m/v) polyethylene glycol treatments. Under salt and osmotic stresses, overexpression of PgLEA in transgenic Arabidopsis plants improved germination rate, root length, and survival rate compared to wild-type plants. In response to drought or salt stress, transgenic plants increased proline accumulation, decreased malonaldehyde content and ion leakage. Furthermore, the transgenic plants exhibited significantly increased activity of superoxide dismutase, peroxidase, and catalase, and reduced accumulation of hydrogen peroxide and superoxide. Moreover, overexpression of PgLEA affected the expression of genes related to salt/drought stress. Taken together, PgLEA is a positive regulator of drought and salinity stress, and positively functioned in pleiotropic effects through regulating osmotic balance, reactive oxygen species scavenging and inducing transcription of stress-related genes. PgLEA may enable ginseng plants to adapt to adverse environments. The data presented herein imply that PgLEA may be useful for breeding new stress-tolerant ginseng cultivars.

Protection against glutathione depletion-associated oxidative neuronal death by neurotransmitters norepinephrine and dopamine: Protein disulfide isomerase as a mechanistic target for neuroprotection.

Oxidative stress is extensively involved in neurodegeneration. Clinical evidence shows that keeping the mind active through mentally-stimulating physical activities can effectively slow down the progression of neurodegeneration. With increased physical activities, more neurotransmitters would be released in the brain. In the present study, we investigated whether some of the released neurotransmitters might have a beneficial effect against oxidative neurodegeneration in vitro. Glutamate-induced, glutathione depletion-associated oxidative cytotoxicity in HT22 mouse hippocampal neuronal cells was usedas an experimental model. We showed that norepinephrine (NE, 50 µM) or dopamine (DA, 50 µM) exerted potent protective effect against glutamate-induced cytotoxicity, but this effect was not observed when other neurotransmitters such as histamine, γ-aminobutyric acid, serotonin, glycine and acetylcholine were tested. In glutamate-treated HT22 cells, both NE and DA significantly suppressed glutathione depletion-associated mitochondrial dysfunction including mitochondrial superoxide accumulation, ATP depletion and mitochondrial AIF release. Moreover, both NE and DA inhibited glutathione depletion-associated MAPKs activation, p53 phosphorylation and GADD45α activation. Molecular docking analysis revealed that NE and DA could bind to protein disulfide isomerase (PDI). In biochemical enzymatic assay in vitro, NE and DA dose-dependently inhibited the reductive activity of PDI. We further revealed that the protective effect of NE and DA against glutamate-induced oxidative cytotoxicity was mediated through inhibition of PDI-catalyzed dimerization of the neuronal nitric oxide synthase. Collectively, the results of this study suggest that NE and DA may have a protective effect against oxidative neurodegeneration through inhibition of protein disulfide isomerase and the subsequent activation of the MAPKs‒p53‒GADD45α oxidative cascade.

Melatonin alleviates copper stress to promote rice seed germination and seedling growth via crosstalk among various defensive response pathways

Copper (Cu) contamination dramatically affects crop growth and thus threatens crop production; while applications of melatonin (MT) serve as an effective way to tolerate Cu stress for plant development, the underlying mechanism remains largely unknown in rice. Here, we found that Cu toxicity remarkably decreased germination rates and seedling growth compared to the untreated control (CK), while seed priming with a solution of 100 μM MT significantly alleviated the adverse effects on Cu-stressed seeds. In addition, the MT treatment decreased the accumulation of Cu in seedlings at 7 days after imbibition (DAI), possibly through enhanced Cu sequestration, and improved reserve mobilization through the promoted activity of α-amylase and protease in seeds under Cu stress. Interestingly, gibberellin (GA) synthesis was restored to or even exceeded the CK levels in the MT presoaking treatment, while the abscisic acid (ABA) content decreased compared to those of the Cu-stressed seeds, suggesting crosstalk between MT and other phytohormones, e.g., GA and ABA. More importantly, MT pretreatment also significantly promoted the growth of postgermination seedlings. This was largely ascribed to the MT-ameliorated antioxidant system, which consequently reduced the accumulation of Cu stress-induced oxidative products, e.g., hydrogen peroxide (H2O2), malondialdehyde (MDA), and superoxide (O2·_). Collectively, these results demonstrate that seed priming with MT could greatly mitigate the adverse effects of Cu stress on seed germination and subsequent postgermination growth through crosstalk among various defensive response pathways. This study provides vital guidance for applications of MT in agronomic production.

RIP3-Dependent Accumulation of Mitochondrial Superoxide Anions in TNF-α-Induced Necroptosis.

Excessive production of reactive oxygen species (ROS) is a key phenomenon in tumor necrosis factor (TNF)-α-induced cell death. However, the role of ROS in necroptosis remains mostly elusive. In this study, we show that TNF-α induces the mitochondrial accumulation of superoxide anions, not H2O2, in cancer cells undergoing necroptosis. TNF-α-induced mitochondrial superoxide anions production is strictly RIP3 expression-dependent. Unexpectedly, TNF-α stimulates NADPH oxidase (NOX), not mitochondrial energy metabolism, to activate superoxide production in the RIP3-positive cancer cells. In parallel, mitochondrial superoxide-metabolizing enzymes, such as manganese-superoxide dismutase (SOD2) and peroxiredoxin III, are not involved in the superoxide accumulation. Mitochondrial-targeted superoxide scavengers and a NOX inhibitor eliminate the accumulated superoxide without affecting TNF-α-induced necroptosis. Therefore, our study provides the first evidence that mitochondrial superoxide accumulation is a consequence of necroptosis.

Effect of pH on aluminum uptake and differential aluminum tolerance in cyanobacterial strains: A bioresource for agricultural and environmental sustainability

The nitrogen-fixing cyanobacterial strains Scytonema sp., Nostoc muscorum, Cylindrospermum sp., and Anabaena sp., were subjected to different concentrations of aluminum at different pH 4.0, 6.0, and 7.0 along with control. Al metal uptake was highest at pH 4. Enhanced peroxidase, superoxide dismutase activity and the accelerated lipid peroxidation depicted a significant accumulation of superoxide and peroxide radicals in Nostoc muscorum exposed to higher concentrations of Al (8 and 16 μM). The non-enzymatic antioxidants, proline and total phenols also increased in a dose-dependent manner Overall, acid Al stress led to differential tolerance and bioavailability of Al metal in various cyanobacterial strains. Among all the strains, Nostoc muscorum tolerated acid/Al stress exceedingly well. The results of the present study suggest plausible implication of Al tolerant cyanobacterial strain as bio-fertilizers in acidic soils. The intrinsic abilities of cyanobacterial strain to adsorb Al from soil may reduce its bioavailability there by increasing crop productivity.

MpSnRK2.10 confers salt stress tolerance in apple via the ABA signaling pathway

Sucrose non-fermentation-1 related protein kinase 2 (SnRK2) family members play an important role in the abscisic acid (ABA) signaling pathway and adaptation to various environments in plants. Here, we found that salt stress and ABA treatment induced the expression of MpSnRK2.10, and overexpression of this gene alleviated growth restriction caused by salt stress in apple. After treated with salt stress for 15 d, the relative electrolyte leakage, malondialdehyde content, and reactive oxygen species production were lower in overexpression (OE) lines, and the chlorophyll content, superoxide dismutase activity, photosynthetic capacity, and accumulation of several amino acids were higher in OE lines than in wild-type (WT) plants. OE plants also accumulated less Na+ in response to salt stress, and several genes involved in Na+ balance under salt stress were up-regulated in OE plants compared with WT plants. Overexpression of MpSnRK2.10 altered the role of the ABA pathway in the response to salt stress. We found that the ABA content and stomatal aperture were greater in OE plants than in WT plants. Exogenous ABA and the ABA inhibitor nordihydroguaiaretic acid (NDGA) altered the morphology and physiological status of plants under salt stress, especially OE plants, which became more sensitive to salt stress treatment. The ABA content and stomatal aperture were also altered in response to ABA and NDGA. In sum, our results showed that MpSnRK2.10 alleviated the salt stress inhibition of apple plants by altering the Na+ balance and ABA pathway.

Mitochondrial Toxicity Associated with Imatinib and Sorafenib in Isolated Rat Heart Fibers and the Cardiomyoblast H9c2 Cell Line.

Tyrosine kinase inhibitors (TKIs) are associated with cardiac toxicity, which may be caused by mitochondrial toxicity. The underlying mechanisms are currently unclear and require further investigation. In the present study, we aimed to investigate in more detail the role of the enzyme complexes of the electron transfer system (ETS), mitochondrial oxidative stress, and mechanisms of cell death in cardiac toxicity associated with imatinib and sorafenib. Cardiac myoblast H9c2 cells were exposed to imatinib and sorafenib (1 to 100 µM) for 24 h. Permeabilized rat cardiac fibers were treated with both drugs for 15 min. H9c2 cells exposed to sorafenib for 24 h showed a higher membrane toxicity and ATP depletion in the presence of galactose (favoring mitochondrial metabolism) compared to glucose (favoring glycolysis) but not when exposed to imatinib. Both TKIs resulted in a higher dissipation of the mitochondrial membrane potential in galactose compared to glucose media. Imatinib inhibited Complex I (CI)- and CIII- linked respiration under both conditions. Sorafenib impaired CI-, CII-, and CIII-linked respiration in H9c2 cells cultured with glucose, whereas it inhibited all ETS complexes with galactose. In permeabilized rat cardiac myofibers, acute exposure to imatinib and sorafenib decreased CI- and CIV-linked respiration in the presence of the drugs. Electron microscopy showed enlarged mitochondria with disorganized cristae. In addition, both TKIs caused mitochondrial superoxide accumulation and decreased the cellular GSH pool. Both TKIs induced caspase 3/7 activation, suggesting apoptosis as a mechanism of cell death. Imatinib and sorafenib impaired the function of cardiac mitochondria in isolated rat cardiac fibers and in H9c2 cells at plasma concentrations reached in humans. Both imatinib and sorafenib impaired the function of enzyme complexes of the ETS, which was associated with mitochondrial ROS accumulation and cell death by apoptosis.

Expressing the sunflower transcription factor HaHB11 in maize improves waterlogging and defoliation tolerance.

The sunflower (Helianthus annuus) transcription factor HaHB11 (H. annuus Homeobox 11) belongs to the homeodomain-leucine zipper family and confers improved yield to maize (Zea mays) hybrids (HiII × B73) and lines. Here we report that transgenic maize lines expressing HaHB11 exhibited better performance under waterlogging, both in greenhouse and field trials carried out during three growth cycles. Transgenic plants had increased chlorophyll content, wider stems, more nodal roots, greater total aerial biomass, a higher harvest index, and increased plant grain yield. Under severe defoliation caused by a windstorm during flowering, transgenic genotypes were able to set more grains than controls. This response was confirmed in controlled defoliation assays. Hybrids generated by crossing B73 HaHB11 lines with the contrasting Mo17 lines were also tested in the field and exhibited the same beneficial traits as the parental lines, compared with their respective controls. Moreover, they were less penalized by stress than commercial hybrids. Waterlogging tolerance increased via improvement of the root system, including more xylem vessels, reduced tissue damage, less superoxide accumulation, and altered carbohydrate metabolism. Multivariate analyses corroborated the robustness of the differential traits observed. Furthermore, canopy spectral reflectance data, computing 29 vegetation indices associated with biomass, chlorophyll, and abiotic stress, helped to distinguish genotypes as well as their growing conditions. Altogether the results reported here indicate that this sunflower gene constitutes a suitable tool to improve maize plants for environments prone to waterlogging and/or wind defoliation.

In Vivo Cardiac-specific Expression of Adenylyl Cyclase 4 Gene Protects against Klotho Deficiency-induced Heart Failure

Klotho is an aging-suppressor gene. Klotho gene deficiency causes heart failure in Klotho-hypomorphic mutant (KL (-/-)) mice. RNA-seq and western blot analysis showed that adenylyl cyclase type IV (AC4) mRNA and protein expression was largely decreased in cardiomyocytes of KL (-/-) mice. The objective of this study was to investigate whether in vivo cardiac-specific expression of AC4 gene protects against Klotho deficiency-induced heart failure. Interestingly, in vivo AAV-based cardiac-specific AC4 gene expression increased left ventricular fractional shortening, ejection fraction, stroke volume, and left ventricular end-diastolic volume in KL (-/-) mice, suggesting that cardiac-specific AC4 gene expression improves Klotho deficiency-induced heart dysfunction. Cardiac-specific AC4 gene expression also decreased Klotho deficiency-induced cardiac hypertrophy. Cardiac-specific AC4 gene expression alleviated Klotho deficiency-induced cardiac fibrosis and calcification. Furthermore, cardiac-specific AC4 gene expression attenuated mitochondrial dysfunction, superoxide accumulation and cardiomyocyte apoptotic cell death. Thus, downregulation of AC4 may contribute to Klotho deficiency-induced heart failure. Mechanistically, AAV2/9-αMHC-AC4 increased cardiomyocytic cAMP levels and thus regulated the PKA-PLN-SERCA2 signal pathway, which is critical in modulating calcium flux and mitochondrial function. In conclusion, cardiac-specific AC4 gene expression protects against Klotho deficiency-induced heart failure through increasing cardiomyocytic cAMP levels, which alleviates cAMP-dependent mitochondrial dysfunction, superoxide accumulation and apoptotic cell death. AC4 regulates superoxide levels via the cAMP-PKA pathway. AC4 could be a potential therapeutic target for heart failure associated with Klotho deficiency. Heart failure is the major cause of mortality in patients with chronic kidney disease (CKD). A decrease in Klotho levels is linked to CKD.

Season-long exposure of bilberry plants to realistic and future ozone pollution improves the nutraceutical quality of fruits

Ozone (O3) is a phytotoxic air pollutant capable of limiting plant yield and growth, and altering the quality of edible plant products. This study aimed to investigate the effects of long-term O3 exposure at realistic and future concentrations (applied during fruit development) not only on morphological, physiological, and biochemical plant/leaf traits of Vaccinium myrtillus but also on its fruit yield and quality. Three-year-old saplings were grown from May to July under three levels of O3 concentration [1.0, 1.5 and 2.0 times the ambient air concentrations, denoted as AA, 1.5_AA and 2.0_AA], using a new-generation O3 Free Air Controlled Exposure system. Ozone induced oxidative pressure and membrane denaturation as confirmed by the accumulation of anion superoxide, hydrogen peroxide (•O2−: +39 and + 29%; H2O2: +55 and + 59% in 1.5_AA and 2.0_AA, respectively, compared with AA), and malondialdehyde by-product (1.4- and 2.5-fold higher than AA, in 1.5_AA and 2.0_AA, respectively). The observed oxidative burst likely affected several cellular structures interested by photosynthetic processes (e.g., decrease of the maximum rate of carboxylation: −30%). This constraint likely induced a decline in plant vitality and a different partitioning of biomass allocation between above and below organs. An accelerated maturation of bilberries due to O3 was reported, suggesting that plants grown under harsher environmental conditions suffered from metabolic changes associated with early ripening. Increasing O3 concentrations might be responsible for an alteration of the ratio between oxidation and reduction processes mechanisms that was followed by a loss of integrity of membranes, so limiting the availability of energy/resources, triggering enzymatic oxidation of phenols to red/purple pigments, and promoting fruit maturation. To the best of our knowledge, this is the first research showing that long-term O3 exposure during bilberry fruit development influenced not only several plant/leaf traits, but also fruit nutraceutical quality at the time of harvest.

Four-Octyl Itaconate Protects Chondrocytes against H2O2-Induced Oxidative Injury and Attenuates Osteoarthritis Progression by Activating Nrf2 Signaling.

Nrf2 is a critical regulator of the antioxidant defense systems in cellular protection. Emerging evidence has shown that four-octyl itaconate (OI) activates Nrf2 cascade. In this study, the chondroprotective effects of OI on H2O2-stimulated chondrocytes and DMM-induced osteoarthritis (OA) progression were investigated. In primary murine chondrocytes, OI interrupted the binding of Keap1 and Nrf2, leading to accumulation and nuclear translocation of Nrf2 protein, as well as transcription and expression of Nrf2-dependent genes, such as HO-1, NQO1, and GCLC. Furthermore, OI inhibited cell death and apoptosis, as well as H2O2-stimulated ROS generation, lipid peroxidation, superoxide accumulation, and mitochondrial depolarization in chondrocytes, which were abolished by the silence or depletion of Nrf2. In addition, in vivo experiments revealed the therapeutic effects of OI on OA progression in a DMM mouse model. Collectively, these results suggested that OI might serve as a potential treatment for OA progression.

Interaction of Periplasmic Fab Production and Intracellular Redox Balance in Escherichia coli Affects Product Yield

Antibody fragments such as Fab’s require the formation of disulfide bonds to achieve a proper folding state. During their recombinant, periplasmic expression in Escherichia coli, oxidative folding is mediated by the DsbA/DsbB system in concert with ubiquinone. Thereby, overexpression of Fab’s is linked to the respiratory chain, which is not only immensely important for the cell’s energy household but also known as a major source of reactive oxygen species. However, the effects of an increased oxidative folding demand and the consequently required electron flux via ubiquinone on the host cell have not been characterized so far. Here, we show that Fab expression in E. coli BL21(DE3) interfered with the intracellular redox balance, thereby negatively impacting host cell performance. Production of four different model Fab’s in lab-scale fed-batch cultivations led to increased oxygen consumption rates and strong cell lysis. An RNA sequencing analysis revealed transcription activation of the oxidative stress-responsive soxS gene in the Fab-producing strains. We attributed this to the accumulation of intracellular superoxide, which was measured using flow cytometry. An exogenously supplemented ubiquinone analogue improved Fab yields up to 82%, indicating that partitioning of the quinone pool between aerobic respiration and oxidative folding limited ubiquinone availability and hence disulfide bond formation capacity. Combined, our results provide a more in-depth understanding of the profound effects that periplasmic Fab expression and in particular disulfide bond formation has on the host cell. Thereby, we show new possibilities to elaborate cell engineering and process strategies for improved host cell fitness and process outcome.