μM Hydrogen Peroxide Research Articles

Reaction to Hydrogen-Peroxide Action in Nicotiana tabacum Plants Transformed by the Cholinoxidase Gene (codA)

Genetic engineering of glycine betaine (GB) synthesis offers a new approach to improvements in the productivity and stress resistance of cultivated plants. The possible effect of the bacterial heterologous sequence on the reaction of plants under oxidative stress (OS) induced by 3 mM of hydrogen peroxide (H2O2) was determined. The independent transgenic tobacco lines (Nicotiana tabacum L.) Cod 16 and Cod 45 with the choline oxidase (codA) gene from Arthrobacter gloiformis, which is responsible for GB synthesis, were used. The transgenic lines differed from wild-type plants with lower resistance to H2O2, which led to a more significant decrease in the pool of photosynthetic pigments and a greater intensity of lipid peroxidation and dynamics of superoxide dismutase (SOD) and peroxidase (PO) activity. In terms of OS manifestation, transgenic lines with initially similar values of GB accumulation (0.825–0.910 mmol/g) had significant differences among themselves. In the Cod 16 line, the reaction to OS induction was earlier (1 h after H2O2 treatment), and there was a more significant increase in PO activity and proline content than in the Cod 45 line and the original variety. In the Cod 45 line, there is a significant increase in SOD activity and proline content in response to OS induction 24 h after treatment, and, in contrast to the wild-type plants and the Cod 16 line, it is accompanied by a significantly higher content of carotenoids in the leaves. When genetic-engineering methods are used to increase the productivity and resistance of plants to stress, it is necessary to take into account the functional interaction of antioxidants and osmoprotectors.

Tamarix articulata Extracts Exhibit Antioxidant Activity and Offer Protection against Hydrogen Peroxide-Mediated Toxicity to Human Skin Fibroblasts.

Tamarix articulata (TA) is a wild halophytic plant growing in extremely harsh environmental conditions in the deserts of Saudi Arabia. Evaluating the protective effect of the methanolic extract of different parts (fresh and dry leaves, stem, and root) of TA was determined by MTT assay using Hs27 skin fibroblasts as the cellular model. The study was designed and conducted in two sets. The first set assesses the toxicity profile of TA extracts in both concentration- and time-dependent ways on Hs27 cells. Our MTT results showed that methanolic extracts from all four parts of TA at varying doses (27.5, 55, 110, and 220 μg/mL) display negligible toxicity when exposed for 4 h. However, exposure of Hs27 cells to varying doses of all four TA extracts for 24 and 48 h promotes significant 23%, 24%, 26%, and 25% (p < 0.05) and 35%, 36%, 39%, and 41% (p < 0.05) cell toxicity at 220 μg/mL of all four TA extracts compared to untreated control cells. To evaluate the protection offered by TA extracts against H₂O₂, we perform a second set of experiments to preincubate Hs27 cells with the TA extracts in both dose- and time-dependent way. This is followed by 300 μM hydrogen peroxide- (H₂O₂-) mediated oxidative insult for 1 h. Using MTT assay, we found that methanolic extracts of TA at different time points (4, 24, and 48 h) and higher doses (220 μg/mL) provide significant protection in cell viability when challenged with H2O2-induced oxidative stress in Hs27 cells. The protective effect was more pronounced at 48 h and 220 μg/mL and the amounts were 39%, 41%, 41%, and 44% for stem, root, fresh leaf, and dry leaf TA extracts (p < 0.05), respectively, compared to untreated cells (2–4%). Collectively, the current study demonstrates that methanolic extracts of TA contain potential bioactive compounds and offer significant protection against H2O2-mediated oxidative stress in Hs27 skin fibroblasts.

Antioxidant Effect of α-Lipoic Acid on the Oxidation of Phosphatidylcholine Initiated by the Fenton Reaction

Mass spectrometry with negative-ion electrospray ionization proves the existence of not only monomers of α-lipoic acid (LA) but also its dimers and trimers in the aqueous phase. Upon incubation with ferrous ions (0.1 mM), new mass ions at m/z 261, 467, and 727 appear in the mass chromatogram in the positive ionization mode, and a mass ion at m/z 671 appears in the negative ionization mode. These mass ions are complexes of LA with ferrous ions, formed due to coordination bonds. The presence of complexes depends on the LA concentration. At concentrations of 0.25 to 4.00 mM, four complexes are observed. LA at a concentration of 4 mM (the maximum concentration that can be achieved by dissolving the acid in water) in the presence of 1 mM of hydrogen peroxide and 0.1 mM of Fe2+ inhibits phospholipid oxidation by 25%. Such an effect was not observed at the LA concentrations below 4 mM. At a concentration of 0.06 mM, only one complex with iron ions is formed (m/z 261). We found by IR spectroscopy that in the presence of iron ions and hydrogen peroxide, one or both sulfur atoms in the LA molecule are oxidized by the hydroxyl radical. The results suggest two mechanisms for the implementation of the antioxidant properties of LA: oxidation by a hydroxyl radical and chelation of ferrous ions.

Mitochondrial DNA drives noncanonical inflammation activation via cGAS\u2013STING signaling pathway in retinal microvascular endothelial cells

BackgroundPathological stimuli cause mitochondrial damage and leakage of mitochondrial DNA (mtDNA) into the cytosol, as demonstrated in many cell types. The cytosolic mtDNA then drives the activation of noninfectious inflammation. Retinal microvascular endothelial cells (RMECs) play an important role in the inner endothelial blood–retinal barrier (BRB). RMEC dysfunction frequently occurs in posterior-segment eye diseases, causing loss of vision. In this study, we investigated the involvement of cytosolic mtDNA in noninfectious immune inflammation in RMECs under pathological stimuli.MethodsRMECs were stimulated with 100 ng/ml lipopolysaccharide (LPS), 200 μM hydrogen peroxide (H2O2), or 25 mM d-glucose. After 24 h, immunofluorescent staining was used to detect the opening of the mitochondrial permeability transition pore (MPTP). Cytosolic mtDNA was detected with immunofluorescent staining and PCR after stimulation. mtDNA was then isolated and used to transfect RMECs in vitro, and the protein levels of cGAS were evaluated with western blotting. Real-time PCR was used to examine cGAS mRNA expression levels at different time points after mtDNA stimulation. The activation of STING was detected with immunofluorescent staining 6 h after mtDNA stimulation. Western blotting was used to determine the expression of STING and IFNβ, the phosphorylation status of TBK1, IRF3, and nuclear factor-κB (NF-κB) P65, and the nuclear translocation of IRF3 and NF-κB P65 at 0, 3, 6, 12, and 24 h. The mRNA expression of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were determined with real-time PCR, together with the concentrations of intercellular adhesion molecule 1 (ICAM-1) mRNA.ResultsPathological stimuli caused mtDNA to leak into the cytosol by opening the MPTP in RMECs after 24 h. Cytosolic mtDNA regulated the expression of cGAS and the distribution of STING in RMECs. It promoted ICAM-1, STING and IFNβ expression, TBK1, IRF3, and NF-κB phosphorylation and the nuclear translocation in RMECs at 12 and 24 h after its transfection. The mRNAs of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were significantly elevated at 12 and 24 h after mtDNA stimulation.ConclusionsPathological stimulation induces mtDNA escape into the cytosol of RMECs. This cytoplasmic mtDNA is recognized by the DNA sensor cGAS, increasing the expression of inflammatory cytokines through the STING–TBK1 signaling pathway.Ba7zzwjjDYYV6NH7beoBySVideo . (MP4 37490 kb)

Enhanced sonophotocatalytic degradation of paracetamol in the presence of Fe-doped TiO2 nanoparticles and H2O2

The presence of pharmaceutical compounds, particularly, paracetamol in water is of significant environmental concern due to their undesirable effects on aquatic organisms and poor biodegradability. The present work investigated the efficient degradation of paracetamol in water using sonophotocatalytic process in the presence of iron (Fe)-doped titanium dioxide (TiO2) nanoparticles. The effects of Fe doping into TiO2 and calcination temperatures were investigated using field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction, nitrogen adsorption–desorption analysis and X-ray photoelectron spectroscopy. It was discovered that 3 wt% Fe dopants into TiO2 nanoparticles with suitable calcination temperature at 600 °C could improve the sonophotocatalytic activity remarkably toward the degradation of paracetamol. The degradation efficiency of 100% was achieved under optimal experimental conditions, namely 5 mg/L initial paracetamol, 1 g/L 3 wt% Fe-doped TiO2-600, solution pH of 5, 0.5 mM of hydrogen peroxide (H2O2) after 30-min ultrasonic power of 296 W, ultrasonic frequency of 50 kHz and ultraviolet wavelength of 302 nm. These findings are crucial for designing Fe-doped TiO2 nanoparticles with high catalytic performance for the degradation of paracetamol and appeared as an alternative solution to treat pharmaceutical compounds effectively.

Salicylic Acid and H2O2 Induce PPO Derived GUS Expression in Arabidopsis

Polyphenol oxidases (PPOs) are ubiquitous enzymes of plant defense which transform polyphenols into quinones that response to biotic and abiotic stresses. Transgenic Arabidopsis thaliana (L.) Heynh. lines harboring OsPPO promoter fused to β-glucoronidase (GUS) reporter gene, were treated exogenously with 0.1, 0.5 and 1 mM concentrations of salicylic acid (SA) and 5, 10 and 15 mM of hydrogen peroxide (H2O2). Plant samples were harvested after 1, 3 and 5 days of treatment. Transcriptional profiling of OsPPOGUS by qRT-PCR revealed the highest 12.96-fold induction with 0.5 mM SA after 3 days of treatment and the lowest fold induction (0.57) with 1 mM SA after one day. With H2O2, the OsPPOGUS mRNA transcript showed the highest accumulation of 21.08-fold change with 10 mM after 5 days of treatment and the lowest induction (3-fold) after 1 day of treatment by 15 mM H2O2. This specific OsPPO induction in response to SA and H2O2 clearly links OsPPO in potential responses to such stressors.

Electrocatalytic reduction of Cr(VI) over heterophase MoS2 film electrode

Electrochemical reduction and treatment of Cr(VI)-contaminated wastewater normally suffer from a high overpotential, due to the extravagant consumption of electrons by the adverse reactions of hydrogen evolution and oxygen reduction. In this work, the MoS2 film electrode prepared by electrochemical deposition-annealing method was applied into the electrochemical reduction of Cr(VI), with the aim to suppress the over-potential of the reduction and improve the utilization of electrons. Results demonstrated that the 2H/1T′ heterostructure of MoS2 enhanced the reduction efficiency of Cr(VI) up to 99% at a lower potential of −1.0 V vs. Ag/AgCl. The in-situ electrochemically generated hydrogen peroxide was found to play a critical role in accelerating the reduction of Cr(VI). Selective oxygen reduction reaction (ORR) via a two-electron pathway on the MoS2 cathode was responsible for the formation of hydrogen peroxide, where the MoS2 film electrode annealed at 700 °C can obtain a high productivity of 360 μM hydrogen peroxide within 60 min. The plenty of preserved interfaces between 2H and 1 T′ in the heterophase MoS2 were crucial to the selective electrocatalytic activity. In addition, this electrode also presented an excellent electrocatalytic activity in hydrogen evolution reaction (HER), which could form atomic hydrogen on the surface of cathode to facilitate the reduction of Cr(VI) under the anoxic condition. This study paves a novel pathway to in-situ deposit the MoS2 film electrode for the sustainable electrochemical reduction and treatment of Cr(VI)-containing wastewater.

Hemistepsin A protects human keratinocytes against hydrogen peroxide-induced oxidative stress through activation of the Nrf2/HO-1 signaling pathway

Hemistepsin A, a sesquiterpene lactone compound isolated from Hemistepta lyrata, has been identified a variety of pharmacological actions including anti-hepatotoxic, anti-inflammatory and anti-cancer activities. Nevertheless, the antioxidant effects of hemistepsin A and the underlying mechanisms have not been investigated properly. Therefore, in the present study, we investigated the protective effect of hemistepsin A against oxidative stress in HaCaT human keratinocytes. The results demonstrated that hemistepsin A suppressed 500 μM hydrogen peroxide (H2O2)-induced cytotoxicity and DNA damage by blocking ROS accumulation. 10 μM Hemistepsin A also prevented apoptosis by preventing the mitochondrial dysfunction and the cytosolic release of cytochrome c, reducing the rate of Bax/Bcl-2 expression, and decreasing the activation of caspase-9 and caspase-3, suggesting that hemistepsin A protected cells from H2O2-induced mitochondria-mediated apoptosis. In addition, hemistepsin A markedly promoted the activation of nuclear factor-erythroid-2-related factor 2 (Nrf2), which was associated with the enhanced expression and activity of heme oxygenase-1 (HO-1) in the presence of 500 μM H2O2. However, inhibiting the expression of HO-1 by artificially blocking the expression of Nrf2 or HO-1 using siRNA significantly eliminated the protective effect of hemistepsin A, indicating that hemistepsin A activates the Nrf2/HO-1 signaling pathway in HaCaT cells to protect against oxidative stress. Therefore, these results suggest that hemistepsin A may be useful as a potential therapeutic agent against various oxidative stress-related skin diseases.

Determination of digitoxin-induced elevated hydrogen peroxide levels in SK-Hep-1 cells using a chemiluminescent method

Drug-induced intracellular hydrogen peroxide overproduction is proposed as an upstream event in apoptosis signaling. It is therefore important to develop a sensitive method to quantify hydrogen peroxide in biological samples undergoing cell death. We report herein a luminol-based chemiluminescent assay with a suitable detection range of best linearity from 1 to 0.1 μM hydrogen peroxide with a CV range of 5.7% to 21.7% for intraday measurement at the detection range. We hypothesized that digitoxin-treated human hepatoma SK-Hep-1 cells produce hydrogen peroxide to cause cytotoxicity, which was verified by measuring the spent media by our chemiluminescent method in a dose-dependent manner. Taken together, our sensitive luminol-based chemiluminescent assay has potential use in the quantification of trace amounts of digitoxin overdose-induced hydrogen peroxide in biological samples.

Conflicting actions of 4-vinylcatechol in rat lymphocytes under oxidative stress induced by hydrogen peroxide

4-Vinylcatechol (4VC) has been identified as an aroma compound in roasted foods, especially coffee. It is also a component in traditional herbal medicines. This compound may be subconsciously ingested through foods and herbs. Recent experimental evidence has shown that 4VC possesses an antioxidative action. However, the antioxidative action of 4VC at cellular levels is not well characterized. The effects of 4VC (0.1–100 µM) were examined on rat thymic lymphocytes without and with oxidative stress induced by 300 µM hydrogen peroxide (H2O2). Cell treatment with 100 µM 4VC alone for 4 h significantly increased the population of dead cells. Thus, 4VC at 100 µM or above elicits cytotoxicity. However, 4VC at sublethal concentrations (1–10 µM) significantly attenuated the H2O2-induced increase in cell lethality in a concentration-dependent manner. While application of 10 µM 4VC slowed the process of cell death induced by H2O2, 4VC did not antagonize the H2O2-induced reduction of cellular nonprotein thiols. Although 4VC at 10 µM did not affect intracellular Ca2+ and Zn2+ levels, the agent potentiated the H2O2-induced increases in these levels. These actions of 10 µM 4VC are adverse to the cells under the oxidative stress. However, 10 µM 4VC partly attenuated the cell death induced by 100 nM A23187, a calcium ionophore. There are conflicting actions of 4VC at 1–100 µM on the cells under oxidative stress although the agent is used for an antioxidant. Thus, caution is required when using 4VC as a therapeutic agent.

Functional analysis of mesencephalic astrocyte-derived neurotrophic factor in retinal ganglion cells under oxidative stress.

Glaucoma is optic neuropathy that is characterized by progressive neurodegeneration of the retinal ganglion cells (RGCs) and axons. This condition will lead to visual impairment and bring glaucoma to become the second cause of blindness globally. Neuroprotection in glaucoma is needed to prevent the progression of optic neuropathy. In this study, we examined the effects of the superior colliculus (SC), and mesencephalic astrocyte-derived neurotrophic factor (MANF) secreted from the SC, on RGC survival after oxidative stress. SC slices and RGCs from rats (3-day old) were co-cultured using a 3D-transwell system. In addition, primary RGCs from 4 to 5-day-old rats were cultured and treated with 100 μM hydrogen peroxide (H2 O2 ), together with stimulation by MANF. Immunoblot and immunofluorescence analyses indicated down-regulated expression levels of several survival markers of RGCs. Extension of neurites was decreased in RGCs treated with 100 μM H2 O2 . Following co-culture with SC slices, or the addition of MANF, we found that both the down-regulated expression of neural markers and extension of neurites caused by oxidative stress in RGCs were blocked. Furthermore, we found a decrease in the expression of neural markers and extension of neurites after co-culture with MANF siRNA-treated SC slices compared with slices treated with mock siRNA, but, RGCs co-cultured with SC slices treated with MANF siRNA displayed no-changed about to apoptosis. These results suggest that MANF secreted from the SC may play an important role in maintenance of function and survival of RGCs. It is also possible that MANF is an important factor in neuroprotection of RGCs. SIGNIFICANCE OF THE STUDY: Glaucoma is a progressive neurodegenerative disease of the retinal ganglion cell (RGC) and their axons. Neuroprotection is aimed at protecting those neurons that are damaged glaucomatous optic neuropathy. We have now examined the effects of superior colliculus, or msencephalic astrocyte-derived neutrophic factor (MANF), secreted from superior colliculus, on RGC survival using co-culture system. Our results suggested that MANF may important key factor in neuroprotection of RGC.

The Potent Antioxidant MitoQ Protects Against Preeclampsia During Late Gestation but Increases the Risk of Preeclampsia When Administered in Early Pregnancy.

Aims: Although preeclampsia (PE) has been attributed to excessive oxidative stress (OS) in the placenta, mild antioxidants failed to prevent PE in clinical trials. As mitochondria are a major source of OS, this study assessed the potential of a potent mitochondria-targeting antioxidant MitoQ in the prevention of PE. Results: Placentas from women with PE and from reduced uterine perfusion pressure (RUPP) mice demonstrated significantly higher OS, along with increased mitochondrial damage and compromised glutathione peroxidase (GPx) activities. MitoQ administration during late gestation alleviated RUPP-induced PE; whereas early-pregnancy MitoQ treatment not only exacerbated blood pressure, fetal growth restriction, and proteinuria but also reduced the labyrinth/spongiotrophoblast ratio and blood sinuses in the labyrinth. Invasion (Matrigel transwell) and migration (wound healing assay) of trophoblasts were greatly improved by 1 μM hydrogen peroxide (H2O2), but this improvement was abolished by MitoQ or MitoTempo. Mild OS enhanced the expression of miR-29b-3p, which regulates five genes involved in viability and mobility, in HTR8-S/Vneo cells. Innovation and Conclusions: Although the potent mitochondrial-targeting antioxidant MitoQ protects against hypertension and kidney damage induced by RUPP in mice when administered in late gestation, it exacerbates the PE-like phenotype when given in early gestation by interfering with placenta formation because mild OS is required to stimulate trophoblast proliferation, invasion, and migration. Eliminating trophoblastic OS during early pregnancy may lead to compromised placentation and a risk of diseases of placental origin. Therefore, antioxidant therapy for pregnant women should be carefully considered.

Hematite/Graphitic Carbon Nitride Nanofilm for Fenton and Photocatalytic Oxidation of Methylene Blue

Hematite (α-Fe2O3)/graphitic carbon nitride (g-C3N4) nanofilm catalysts were synthesized on fluorine-doped tin oxide glass by hydrothermal and chemical vapor deposition. Scanning electron microscopy, energy-dispersive spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopy analyses of the synthesized catalyst showed that the nanoparticles of g-C3N4 were successfully deposited on α-Fe2O3 nanofilm. The methylene blue degradation efficiency of the α-Fe2O3/g-C3N4 composite catalyst was 2.6 times greater than that of the α-Fe2O3 single catalyst under ultraviolet (UV) irradiation. The methylene blue degradation rate by the α-Fe2O3/g-C3N4 catalyst increased by 6.5 times after 1 mM of hydrogen peroxide (H2O2) was added. The photo-Fenton reaction of the catalyst, UV, and H2O2 greatly increased the methylene blue degradation. The results from the scavenger experiment indicated that the main reactants in the methylene blue decomposition reaction are superoxide radicals photocatalytically generated by g-C3N4 and hydroxyl radicals generated by the photo-Fenton reaction. The α-Fe2O3/g-C3N4 nanofilm showed excellent reaction rate constants at pH 3 (Ka = 6.13 × 10−2 min−1), and still better efficiency at pH 7 (Ka = 3.67 × 10−2 min−1), compared to other methylene blue degradation catalysts. As an immobilized photo-Fenton catalyst without iron sludge formation, nanostructured α-Fe2O3/g-C3N4 are advantageous for process design compared to particle-type catalysts.

Structural and Functional Tailoring of Melanin-Like Polydopamine Radical Scavengers

Melanin is a ubiquitous but poorly understood polymeric, dark brown to black pigment biomaterial found both in nature and by organic synthesis. Its structural heterogeneity and disordered covalent/...

Integrative analysis of an lncRNA‑associated competing endogenous RNA network in human trabecular meshwork cells under oxidative stress.

Long non-coding RNAs (lncRNAs) are a group of non-coding transcripts of >200 nucleotides. They can act as competing endogenous RNAs (ceRNAs) and suppress microRNA (miRNA) function by preventing them from binding to and interacting with target mRNAs. However, the specific role of the lncRNA-associated ceRNA network in the pathogenesis of glaucoma has not yet been elucidated. To study this, data were downloaded from the Gene Expression Omnibus database (GSE126170), which contained three human trabecular meshwork cell (HTMC) samples treated with 300 µm hydrogen peroxide and three control samples treated with vehicle. Differentially expressed lncRNAs and mRNAs of HTMCs were obtained using the R package limma. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of differentially expressed mRNAs were performed using the R package clusterProfiler. Finally, the ceRNA network was constructed using the mircode, miRDB, miRTarBase and TargetScan databases, and visualized using Cytoscape v3.6.1. The results showed that 70 lncRNAs and 558 mRNAs were identified to be significantly dysregulated (|log2FoldChange| >1 and adjusted P<0.05) in HTMCs under oxidative stress compared to those in HTMCs under control conditions. Moreover, 24 lncRNAs, 24 miRNAs and 40 mRNAs were closely connected, and were part of the ceRNA network. Among these, the expression levels of 19 lncRNAs were upregulated, and those of 5 lncRNAs were downregulated. To conclude, using bioinformatics analysis, the differential expression profiles of lncRNAs were reported and a lncRNA-associated ceRNA network in HTMCs under oxidative stress was constructed. These results may bring to light a new pathological mechanism or a potential therapeutic target for glaucoma.

Teriflunomide preserves peripheral nerve mitochondria from oxidative stress-mediated alterations.

Mitochondrial dysfunction is a common pathological hallmark in various inflammatory and degenerative diseases of the central nervous system, including multiple sclerosis (MS). We previously showed that oxidative stress alters axonal mitochondria, limiting their transport and inducing conformational changes that lead to axonal damage. Teriflunomide (TFN), an oral immunomodulatory drug approved for the treatment of relapsing forms of MS, reversibly inhibits dihydroorotate dehydrogenase (DHODH). DHODH is crucial for de novo pyrimidine biosynthesis and is the only mitochondrial enzyme in this pathway, thus conferring a link between inflammation, mitochondrial activity and axonal integrity.Here, we investigated how DHODH inhibition may affect mitochondrial behavior in the context of oxidative stress. We employed a model of transected murine spinal roots, previously developed in our laboratory. Using confocal live imaging of axonal mitochondria, we showed that in unmanipulated axons, TFN increased significantly the mitochondria length without altering their transport features. In mitochondria challenged with 50 µM hydrogen peroxide (H2O2) to induce oxidative stress, the presence of TFN at 1 µM concentration was able to restore mitochondrial shape, motility, as well as mitochondrial oxidation potential to control levels. No effects were observed at 5 µM TFN, while some shape and motility parameters were restored to control levels at 50 µM TFN.Thus, our data demonstrate an undescribed link between DHODH and mitochondrial dynamics and point to a potential neuroprotective effect of DHODH inhibition in the context of oxidative stress-induced damage of axonal mitochondria.

Pseudoexfoliation glaucoma – Do genes or environment matter more?

AbstractPurposeLysyl oxidase like 1 (LOXL1) catalyzes collagen and elastin crosslinking. Single nucleotide polymorphisms (SNPs) within LOXL1 are associated with pseudoexfoliation (PXF) syndrome and PXF glaucoma (PXFG). These SNPs are present in 50%–80% of the general population, suggesting environmental factors also play a role in disease development. Our aim was to investigate LOXL1 levels in Human Tenons Fibroblasts (HTFs) and aqueous humour (AqH) from PXF, PXFG and cataract controls (CAT) and to establish if there was an association with genotype, epigenetic regulation (methylation) or environmental factors (hypoxia, oxidative stress, UV exposure and caffeine intake).MethodsPatients with PXF, PXFG and CAT were recruited and genotyped. LOXL1 expression was measured by qPCR and Western Blot and LOXL1 activity by ELISA. CAT HTFs were cultured in hypoxic (1% O2) and oxidative stress conditions (200 μm hydrogen peroxide). Epigenetic analysis was determined by global DNA methylation and qPCR of methylation‐associated enzymes. Environmental factors were recorded in a RedCap™ database.ResultsIncreased high‐risk LOXL1 gene haplotypes were observed in PXFG compared with CAT. LOXL1 expression and activity was decreased in PXFG HTFs vresus CAT (p &lt; 0.01). This was replicated in CAT HTFs cultured under hypoxic (p &lt; 0.05) and oxidative stress conditions (p &lt; 0.01). Global DNA methylation and DNMT1 and 3A expression was increased in PXFG compared to CAT (p &lt; 0.05). DNMT inhibition using 5‐azacytidine restored LOXL1 expression in PXFG (p &lt; 0.05). Outdoor occupation was associated with PXFG. PXFG patients with the high‐risk (G‐G) haplotype had higher rates of trabeculectomy and numbers of IOP lowering drugs.ConclusionsA combination of genetic, epigenetic and environmental factors contributes to LOXL1 expression in PXFG. LOXL1 genotyping may offer a means of screening for risk of disease progression and epigenetic modifiers such as 5‐azacytidine may represent a future therapeutic target for PXFG.

The Charcot-Marie Tooth Disease Mutation R94Q in MFN2 Decreases ATP Production but Increases Mitochondrial Respiration under Conditions of Mild Oxidative Stress.

Charcot–Marie tooth disease is a hereditary polyneuropathy caused by mutations in Mitofusin-2 (MFN2), a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. Autosomal-dominant inheritance of a R94Q mutation in MFN2 causes the axonal subtype 2A2A which is characterized by early onset and progressive atrophy of distal muscles caused by motoneuronal degeneration. Here, we studied mitochondrial shape, respiration, cytosolic, and mitochondrial ATP content as well as mitochondrial quality control in MFN2-deficient fibroblasts stably expressing wildtype or R94Q MFN2. Under normal culture conditions, R94Q cells had slightly more fragmented mitochondria but a similar mitochondrial oxygen consumption, membrane potential, and ATP production as wildtype cells. However, when inducing mild oxidative stress 24 h before analysis using 100 µM hydrogen peroxide, R94Q cells exhibited significantly increased respiration but decreased mitochondrial ATP production. This was accompanied by increased glucose uptake and an up-regulation of hexokinase 1 and pyruvate kinase M2, suggesting increased pyruvate shuttling into mitochondria. Interestingly, these changes coincided with decreased levels of PINK1/Parkin-mediated mitophagy in R94Q cells. We conclude that mitochondria harboring the disease-causing R94Q mutation in MFN2 are more susceptible to oxidative stress, which causes uncoupling of respiration and ATP production possibly by a less efficient mitochondrial quality control.

Cytoprotective Preconditioning of Osteoblast-Like Cells with N-Acetyl-L-Cysteine for Bone Regeneration in Cell Therapy.

Oxidative stress hinders tissue regeneration in cell therapy by inducing apoptosis and dysfunction in transplanted cells. N-acetyl-L-cysteine (NAC) reinforces cellular antioxidant capabilities by increasing a major cellular endogenous antioxidant molecule, glutathione, and promotes osteogenic differentiation. This study investigates the effects of pretreatment of osteoblast-like cells with NAC on oxidative stress-induced apoptosis and dysfunction and bone regeneration in local transplants. Rat femur bone marrow-derived osteoblast-like cells preincubated for 3 h with and without 5 mM NAC were cultured in a NAC-free osteogenic differentiation medium with continuous exposure to 50 μM hydrogen peroxide to induce oxidative stress. NAC preincubation prevented disruption of intracellular redox balance and alleviated apoptosis and negative impact on osteogenic differentiation, even under oxidative stress. Autologous osteoblast-like cells with and without NAC pretreatment in a collagen sponge vehicle were implanted in critical-size defects in rat femurs. In the third week, NAC-pretreated cells yielded complete defect closure with significantly matured lamellar bone tissue in contrast with poor bone healing by cells without pretreatment. Cell-tracking analysis demonstrated direct bone deposition by transplanted cells pretreated with NAC. Pretreatment of osteoblast-like cells with NAC enhances bone regeneration in local transplantation by preventing oxidative stress-induced apoptosis and dysfunction at the transplanted site.

Impact of Hydrogen Peroxide on the Secondary Metabolites, Enzyme Activities and Photosynthetic Pigment Accumulation of Vigna unguiculata L. (Walp)

The effects of exogenous application of hydrogen peroxide on the secondary metabolites, enzyme activities and photosynthetic pigment accumulation of Vigna unguiculata was investigated. The hereby study was carried out under a screen house to minimize extraneous factors. The experiment was laid down in completely randomized design (CRD) with eight replicates. After two weeks of sowing when the seedlings become fully established, the sample pots were divided into two regimes. The first regimes were supplied daily with 500 mL of 0.1 mM of hydrogen peroxide and the second regime was watered daily with tap water. After fifth week of sowing, the leaves of Vigna unguiculata from each regime were randomly selected and harvested. Secondary metabolites, enzyme assay and photosynthetic pigment screening was carried out on the harvested leaves. The impact of hydrogen peroxide on the parameters studied was done using one way analysis of variance. Mean values were separated using Fisher’s LSD at p≤0.05. The results showed that hydrogen peroxide strongly increased the accumulation of secondary metabolites such as flavonoids, glycosides, saponins, carbohydrates and alkaloids. It also induced α-amylase and lipase activities than the control plants and promotes the accumulation and biosynthesis of chlorophylls and carotenoids. In conclusion, hydrogen peroxide could be used as potential molecule to improve some secondary metabolites (flavonoids, glycosides, saponins, carbohydrates and alkaloids), α-amylase and lipase activities and photosynthetic pigments.