Mitochondrial Oxidase Research Articles

Combined FRET-FLIM and NAD(P)H FLIM to Analyze B Cell Receptor Signaling Induced Metabolic Activity of Germinal Center B Cells In Vivo.

Affinity maturation of B cell clones within germinal centers constitutes an important mechanism for immune memory. During this process, B cell receptor signaling capacity is tested in multiple rounds of positive selection. Antigen stimulation and co-stimulatory signals mobilize calcium to switch on gene expression leading to proliferation and survival and to differentiation into memory B cells and plasma cells. Additionally, all these processes require adaption of B cell metabolism, and calcium signaling and metabolic pathways are closely interlinked. Mitochondrial adaption, ROS production, and NADPH oxidase activation are involved in cell fate decisions, but it remains elusive to what extent, especially because the analysis of these dynamic processes in germinal centers has to take place in vivo. Here, we introduce a quantitative intravital imaging method for combined measurement of cytoplasmic calcium concentration and enzymatic fingerprinting in germinal center B cells as a possible tool in order to further examine the relationship of calcium signaling and immunometabolism.

Inhibition of mitosomal alternative oxidase causes lifecycle arrest of early-stage Trachipleistophora hominis meronts during intracellular infection of mammalian cells.

Mitosomes are highly reduced forms of mitochondria which have lost two of the 'defining' features of the canonical organelle, the mitochondrial genome, and the capacity to generate energy in the form of ATP. Mitosomes are found in anaerobic protists and obligate parasites and, in most of the studied organisms, have a conserved function in the biosynthesis of iron-sulfur clusters (ISC) that are indispensable cofactors of many essential proteins. The genomes of some mitosome-bearing human pathogenic Microsporidia encode homologues of an alternative oxidase (AOX). This mitochondrial terminal respiratory oxidase is absent from the human host, and hence is a potential target for the development of new antimicrobial agents. Here we present experimental evidence for the mitosomal localization of AOX in the microsporidian Trachipleistophora hominis and demonstrate that it has an important role during the parasite's life cycle progression. Using a recently published methodology for synchronising T. hominis infection of mammalian cell lines, we demonstrated specific inhibition of T. hominis early meront growth and replication by an AOX inhibitor colletochlorin B. Treatment of T. hominis-infected host cells with the drug also inhibited re-infection by newly formed dispersive spores. Addition of the drug during the later stages of the parasite life cycle, when our methods suggest that AOX is not actively produced and T. hominis mitosomes are mainly active in Fe/S cluster biosynthesis, had no inhibitory effects on the parasites. Control experiments with the AOX-deficient microsporidian species Encephalitozoon cuniculi, further demonstrated the specificity of inhibition by the drug. Using the same methodology, we demonstrate effects of two clinically used anti-microsporidian drugs albendazole and fumagillin on the cell biology and life cycle progression of T. hominis infecting mammalian host cells. In summary, our results reveal that T. hominis mitosomes have an active role to play in the progression of the parasite life cycle as well as an important role in the biosynthesis of essential Fe/S clusters. Our work also demonstrates that T. hominis is a useful model for testing the efficacy of therapeutic agents and for studying the physiology and cell biology of microsporidian parasites growing inside infected mammalian cells.

Near‐Infrared Windows I and II Phosphors for Theranostic Applications: Spectroscopy, Bioimaging, and Light‐Emitting Diode Photobiomodulation

AbstractThe importance and practicality of near‐infrared (NIR) light for the biomedical field has attached importance to the academe and industry with the development of technology. Luminescent materials with different doping strategies are promising candidates for covering near‐infrared biological windows from 700 nm to 1 700 nm. The relationship between NIR light, nanophosphors, and biological tissue towards the function of diagnostic and therapy in different NIR windows is explored clearly, which corresponds to diffuse reflection spectroscopy with bioimaging and light‐emitting diode (LED) photobiomodulation. The diseases can be monitored with specific chromophore absorption under the NIR light source, which also can be traced to the dynamic change of the biological environment by luminescent materials. Especially in brain diseases, cytochrome c oxidase in mitochondria as the crucial photoreceptor can be activated under NIR irradiation to achieve the non‐invasive treatment of neuron protection and recovery. NIR light attributed to LED or nanophosphors show the potential of theranostic approaches in biomedical technology. Cr3+‐ and Ni2+‐activated phosphors covering NIR windows I and II are presented with optical properties to explore the feasibility and practicability of non‐invasive precise medical treatment in the future.

Identifying antibiotics based on structural differences in the conserved allostery from mitochondrial heme-copper oxidases

Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.

Treponema pallidum membrane protein Tp47 promotes angiogenesis through ROS-induced autophagy.

Pathological angiogenesis is an important manifestation of syphilis, but the underlying mechanism of Treponema pallidum subspecies pallidum (T.pallidum)-induced angiogenesis is poorly understood. The objective of this study is to investigate the role and related mechanism of the T.pallidum membrane protein Tp47 in angiogenesis. The proangiogenic activity of recombinant T.pallidum membrane protein Tp47 in human umbilical vein endothelial cells (HUVECs) was assessed by tube formation assay, three-dimensional angiogenesis analysis and experiments with a zebrafish embryo model. The effects of mitochondrial ROS and NADPH oxidase on intracellular ROS induced by Tp47 were further investigated. Furthermore, the levels of autophagy-related proteins and autophagic flux were measured. Finally, the role of ROS-induced autophagy in angiogenesis was studied. Tp47 promoted tubule formation and the formation of angiogenic sprouts in vitro. In addition, a significant increase in the number of subintestinal vessel branch points in zebrafish injected with Tp47 was observed using a zebrafish embryo model. Tp47 also significantly increased intracellular ROS levels in a dose-dependent manner. Tp47-induced tube formation and angiogenic sprout formation were effectively prevented by the ROS inhibitor NAC. In addition, Tp47 enhanced the production of mitochondrial ROS and expression of the NADPH oxidase-related proteins Nox2 and Nox4. The production of mitochondrial ROS and intracellular ROS was reduced by the NADPH oxidase inhibitors DPI and apocynin. Furthermore, Tp47 significantly increased expression of the autophagy-related proteins P62 and Beclin 1 and the LC3-II/LC3-I ratio and promoted an increase in autophagic flux, which could be effectively rescued by coincubation with the ROS inhibitor NAC. Further intervention with the autophagy inhibitor BafA1 significantly inhibited tube formation and angiogenic sprout formation. Tp47-induced NADPH oxidase enhanced intracellular ROS production via mitochondrial ROS and promoted angiogenesis through autophagy mediated by ROS. These findings may contribute to our understanding of pathological angiogenesis in syphilis.

The Mitochondrial Alternative Oxidase in Ustilago maydis Is Not Involved in Response to Oxidative Stress Induced by Paraquat.

It has been shown that the alternative oxidase in mitochondria of fungi and plants has important functions in the response against stress conditions, although their role in some organisms is still unknown. This is the case of Ustilago maydis. There is no evidence of the participation of the U. maydis Aox1 in stressful conditions such as desiccation, high or low temperature, and low pH, among others. Therefore, in this work, we studied the role of the U. maydis Aox1 in cells exposed to oxidative stress induced by methyl viologen (paraquat). To gain insights into the role of this enzyme, we took advantage of four strains: the FB2 wild-type, a strain without the alternative oxidase (FB2aox1Δ), other with the Aox1 fused to the Gfp under the control of the original promoter (FB2aox1-Gfp), and one expressing constitutively de Aox1-Gfp (FB2Potef:aox1-Gfp). Cells were incubated for various times in the presence of 1 mM paraquat and growth, replicative capacities, mitochondrial respiratory activity, Aox1 capacity, and the activities of several antioxidant enzymes (catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase) were assayed. The results show that (1) the response of U. maydis against oxidative stress was the same in the presence or absence of the Aox1; (2) the activities of the antioxidant enzymes remained constant despite the oxidative stress; and (3) there was a decrease in the GSH/GSSG ratio in U. maydis cells incubated with paraquat.

Empagliflozin suppresses urinary mitochondrial DNA copy numbers and interleukin-1β in type 2 diabetes patients

Sodium-glucose co-transporter 2 (SGLT2) inhibitors improve cardiovascular and renal outcomes in type 2 diabetes mellitus (T2DM) patients. However, the mechanisms by which SGLT2 inhibitors improve the clinical outcomes remain elusive. We evaluated whether empagliflozin, an SGLT2 inhibitor, ameliorates mitochondrial dysfunction and inflammatory milieu of the kidneys in T2DM patients. We prospectively measured copy numbers of urinary and serum mitochondrial DNA (mtDNA) nicotinamide adenine dinucleotide dehydrogenase subunit-1 (mtND-1) and cytochrome-c oxidase 3 (mtCOX-3) and urinary interleukin-1β (IL-1β) in healthy volunteers (n = 22), in SGLT2 inhibitor-naïve T2DM patients (n = 21) at baseline, and in T2DM patients after 3 months of treatment with empagliflozin (10 mg, n = 17 or 25 mg, n = 4). Both urinary mtDNA copy numbers and IL-1β levels were higher in the T2DM group than in healthy volunteers. Baseline copy numbers of serum mtCOX-3 in the T2DM group were lower than those in healthy volunteers. Empagliflozin induced marked reduction in both urinary and serum mtND-1 and mtCOX-3 copy numbers, as well as in urinary IL-1β. Empagliflozin could attenuate mitochondrial damage and inhibit inflammatory response in T2DM patients. This would explain the beneficial effects of SGLT2 inhibitors on cardiovascular and renal outcomes.

Abstract 12842: Adipose Tissue Derived Ceramides Regulate the Myocardial Redox State and Predict Cardiovascular Outcomes

Introduction: Ceramides biosynthesis is dysregulated in obesity, yet their role as mediators of obesity-induced myocardial dysfunction is unclear. Objective: We investigate the causal role of AT-derived ceramides in regulating the myocardial redox state and explore their prognostic value in patients with CVD. Methods: In 880 cardiac surgery patients, 20 sphingolipids were measured in plasma, subcutaneous-, thoracic- and epicardial-AT biopsies and their secretomes. Myocardial redox state was measured and the contribution of NOXs, uncoupled eNOS and mitochondrial oxidases to O 2 .- production quantified. Median follow-up was 8.3 years. Genetic variants were identified to predict CVD outcomes using cis-Mendelian Randomisation. The underlying mechanisms were explored using differentiated H9c2 cardiomyocytes in vitro and human myocardial tissue ex vivo . Results: C16:0-ceramide (CerC16) was the most abundant ceramide secreted from AT (p<0.0001). Patients with high plasma CerC16 and C16:0-glucosylceramide (GlcC16) had significantly increased myocardial O 2 .- production (vs low/int.) (A) . Patients with rs112572487 (B) , an intronic SNP in UGCG , had significantly increased plasma GlcC16 (C) and myocardial NOX-derived O 2 .- (D) vs those without. Exogenous CerC16 (20nM) induced O 2 .- production in H9c2 cardiomyocytes and human myocardial tissue (E) , an effect prevented by the UGCG inhibitor D-PDMP. Importantly, high plasma GlcC16 led to a higher risk of cardiac death and/or heart failure (HR=2.128 [1.101, 4.115], p=0.025) vs low/int. levels, a relationship also seen with rs112572487 (F) . rs112572487 also led to an increased risk of new, post-operative atrial fibrillation in hospital (HR=1.805 [1.188, 2.742], p=0.006). Conclusions: We demonstrate for the first time, that AT-derived ceramides are causally related with dysregulated myocardial redox signalling and adverse cardiovascular disease outcomes in patients with advanced atherosclerosis.

Экспериментальная биология растений: физиолого-биохимические исследования на Европейском Северо-Востоке (обзор)

The stages of formation and the main results of the phytophysiology development in the Komi Republic were considered. The contribution of the Institute of Biology to the study of the fundamental processes of plant life was noted. Achievements in the study of the physiological and biochemical bases of the production process of agricultural crops, functional plasticity, and stress resistance of wild-growing plants were analyzed. Results that significantly deepen the understanding of the respiration and respiratory pathways involvement in the plant cells metabolism and bioenergetics have been obtained. The participation of alternative mitochondrial oxidase in the complex protective system of the cell, signaling, and the redox balance maintenance under stress has been proven. Current research and priority directions for the long-term period were outlined.

Correction of the Mitochondrial NADH Oxidase Activity, Peroxidation and Phospholipid Metabolism by Haplogenin-7-Glucoside in Hypoxia and Ischemia

In this study, it was used haplogenin-7-glucoside flavonol (C16H12O8) isolated from the plant Haplophyllum perforatum. It was studied the effect of haplogenin-7-glycoside on NADH oxidase activity, peroxidation, and phospholipid metabolism in rat liver mitochondria under in vitro conditions. Incubation of mitochondria at 37 ℃ leads to disruption of the integrity of mitochondrial membranes, as a result, in cytochrome с desorption from the inner membrane into the intermembrane space, which leads to a decrease in the activity of rotenone-sensitive NADH oxidase and an increase in the activity of rotenone-insensitive NADH oxidase. Haplogenin-7-glucoside exhibits membrane stabilization by reducing these alterations. Under ischemia conditions, the effect of haplogenin-7-glucoside on the LPO process in mitochondria decreases LPO in mitochondria. Quantitative changes in phosphatidylserine in mitochondrial membranes under LPO conditions during incubation of mitochondria at 37 ℃ were observed to accelerate controlled (auto-oxidation) LPO over time (0 - 90 min). Peroxidation of phosphatidylserine in mitochondria when the addition of haplogenin-7-glucoside to the incubation medium at 30, 60 and 90 min, LPO was found to be reduced. The increasing amount of phosphatidylinositol has not happened depend on time. However, a slight increase was found under the influence of haplogenin-7-glucoside.
 HIGHLIGHTS
 
 Cytochrome c desorption from the inner membrane into the interstitial space leads to a decrease in the activity of rotenone-sensitive NADH-oxidase and an increase in the activity of non-rotenone-sensitive NADH-oxidase
 Haplogenin-7-glycoside showed membrane-stabilizing properties by significantly reducing these changes; the addition of haplogenin-7-glucoside to mitochondria leads to a decrease for MDA, a decrease in the LPO process
 At incubation of mitochondria at 37℃, the amount of phosphatidic acid and lysophosphatidic acid in mitochondria under LPO conditions was also found to be less than control under the influence of haplogenin-7-glucoside. Thus, haplogenin-7-glucoside reduces the rate of peroxidation of phospholipids in liver mitochondria and has a corrective effect
 
 GRAPHICAL ABSTRACT

Mitochondrial VDAC1: A Potential Therapeutic Target of Inflammation-Related Diseases and Clinical Opportunities.

The multifunctional protein, voltage-dependent anion channel 1 (VDAC1), is located on the mitochondrial outer membrane. It is a pivotal protein that maintains mitochondrial function to power cellular bioactivities via energy generation. VDAC1 is involved in regulating energy production, mitochondrial oxidase stress, Ca2+ transportation, substance metabolism, apoptosis, mitochondrial autophagy (mitophagy), and many other functions. VDAC1 malfunction is associated with mitochondrial disorders that affect inflammatory responses, resulting in an up-regulation of the body’s defensive response to stress stimulation. Overresponses to inflammation may cause chronic diseases. Mitochondrial DNA (mtDNA) acts as a danger signal that can further trigger native immune system activities after its secretion. VDAC1 mediates the release of mtDNA into the cytoplasm to enhance cytokine levels by activating immune responses. VDAC1 regulates mitochondrial Ca2+ transportation, lipid metabolism and mitophagy, which are involved in inflammation-related disease pathogenesis. Many scientists have suggested approaches to deal with inflammation overresponse issues via specific targeting therapies. Due to the broad functionality of VDAC1, it may become a useful target for therapy in inflammation-related diseases. The mechanisms of VDAC1 and its role in inflammation require further exploration. We comprehensively and systematically summarized the role of VDAC1 in the inflammatory response, and hope that our research will lead to novel therapeutic strategies that target VDAC1 in order to treat inflammation-related disorders.

Adipose tissue derived ceramides regulate myocardial redox state and predict cardiovascular outcomes

Abstract Background Obesity is linked to both dysfunctional adipose tissue (AT) and heart failure, but the exact mechanisms mediating these associations are unknown. Although ceramides biosynthesis is dysregulated in obesity, their role as mediators of obesity-induced myocardial dysfunction is unclear. Purpose We investigate the causal role of AT-derived ceramides in the regulation of myocardial redox state and explore their role in predicting cardiovascular outcomes. Methods The study population included a total of 880 patients undergoing cardiac surgery. A panel of 20 sphingolipids was measured in plasma as well as in biopsies of subcutaneous AT (ScAT), thoracic AT (ThAT) and epicardial AT (EpAT) and their secretome, obtained from a subgroup of n=48. Myocardial redox state was measured using lucigenin chemiluminescence and the contribution of NOXs, uncoupled nitric oxide synthases and mitochondrial oxidases in O2•– production was quantified. The cohort was followed up for a median of 8.3 years. Genome-wide genetic analysis was done using the UK Biobank array. A total of 99,524 SNPs within 50kb of 110 genes involved in sphingolipid biosynthesis were analysed to identify genetic variants that could predict CVD outcomes using cis-Mendelian Randomisation. The underlying mechanisms were then explored further, using differentiated H9c2 cardiomyocytes in vitro and human right atrial tissue ex vivo. Results The production and secretion of C16:0-ceramide (CerC16) was higher in visceral AT (EpAT and ThAT) compared to ScAT (p<0.0001). Patients with high plasma levels of CerC16 and its derivative C16:0-glucosylceramide (GlcC16) had higher myocardial O2•– production vs those with low/int. levels (p<0.05 for both) (A). To test the causality of this association, we performed a targeted single-SNP analysis for the genetic prediction of GlcC16 levels demonstrating that rs112572487, an intronic variant in UGCG (an enzyme that catalyses glucosylceramide formation from ceramides), was the top hit (B). Indeed, those with the rs112572487 minor allele (G) displayed significantly increased myocardial NOX-derived O2•– (C) and plasma GlcC16 levels (D) vs those without. Exogenous CerC16 (20nM) induced NOX-derived O2•– production in H9c2 cardiomyocytes, an effect prevented by the UGCG inhibitor D-PDMP (E), suggesting that GlcC16 is a modifiable regulator of myocardial NOX-O2•–. Importantly, high plasma GlcC16 levels were associated with a higher risk of cardiac death and/or heart failure (adj. HR=2.128 [95% CI: 1.101, 4.115], p=0.025, for high vs low/int. levels), a relationship also seen with rs112572487 (F). Conclusions We demonstrate for the first time, that AT-derived ceramides are causally related with dysregulated myocardial redox signalling and adverse cardiovascular disease outcomes in patients with advanced atherosclerosis. As such, GlcC16 may be an important therapeutic target for the prevention and treatment of cardiovascular complications in obesity and diabetes. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): The British Heart FoundationBritish Heart Foundation Chair Award

KCNH6 Enhanced Hepatic Glucose Metabolism through Mitochondrial Ca2+ Regulation and Oxidative Stress Inhibition.

KCNH6 has been proven to affect glucose metabolism and insulin secretion both in humans and mice. Further study revealed that Kcnh6 knockout (KO) mice showed impaired glucose tolerance. However, the precise function of KCNH6 in the liver remains unknown. Mitochondria have been suggested to maintain intracellular Ca2+ homeostasis; ROS generation and defective mitochondria can cause glucose metabolism disorders, including type 2 diabetes (T2D). Here, we found that Kcnh6 attenuated glucose metabolism disorders by decreasing PEPCK and G6pase abundance and induced Glut2 and IRS2 expression. Overexpression of Kcnh6 increased hepatic glucose uptake and glycogen synthesis. Kcnh6 attenuated intracellular and mitochondrial calcium levels in primary hepatocytes and reduced intracellular ROS and mitochondrial superoxide production. Kcnh6 suppressed oxidative stress by inhibiting mitochondrial pathway activation and NADPH oxidase expression. Experiments demonstrated that Kcnh6 expression improved hepatic glucose metabolism disorder through the c-Jun N-terminal kinase and p38MAPK signaling pathways. These results were confirmed by experiments evaluating the extent to which forced Kcnh6 expression rescued metabolic disorder in KO mice. In conclusion, KCNH6 enhanced hepatic glucose metabolism by regulating mitochondrial Ca2+ levels and inhibiting oxidative stress. As liver glucose metabolism is key to T2D, understanding KCNH6 functions may provide new insights into the causes of diabetes.

Coupling of phagocytic NADPH oxidase activity and mitochondrial superoxide production.

Superoxide radical plays an important role in redox cell signaling and physiological processes; however, overproduction of superoxide or insufficient activity of antioxidants leads to oxidative stress and contributes to the development of pathological conditions such as endothelial dysfunction and hypertension. Meanwhile, the studies of superoxide in biological systems represent unique challenges associated with short lifetime of superoxide, insufficient reactivity of the superoxide probes, and lack of site-specific detection of superoxide. In this work we have developed 15N-and deuterium-enriched spin probe 15N-CAT1H for high sensitivity and site-specific detection of extracellular superoxide. We have tested simultaneous tracking of extracellular superoxide by 15N-CAT1H and intramitochondrial superoxide by conventional 14N-containing spin probe mitoTEMPO-H in immune cells isolated from spleen, splenocytes, under basal conditions or stimulated with inflammatory cytokines IL-17A and TNFα, NADPH oxidase activator PMA, or treated with inhibitors of mitochondrial complex I rotenone or complex III antimycin A. 15N-CAT1H provides two-fold increase in sensitivity and improves detection since EPR spectrum of 15N-CAT1 nitroxide does not overlap with biological radicals. Furthermore, concurrent use of cell impermeable 15N-CAT1H and mitochondria-targeted 14N-mitoTEMPO-H allows simultaneous detection of extracellular and mitochondrial superoxide. Analysis of IL-17A- and TNFα-induced superoxide showed parallel increase in 15N-CAT1 and 14N-mitoTEMPO signals suggesting coupling between phagocytic NADPH oxidase and mitochondria. The interplay between mitochondrial superoxide production and activity of phagocytic NADPH oxidase was further investigated in splenocytes isolated from Sham and angiotensin II infused C57Bl/6J and Nox2KO mice. Angiotensin II infusion in wild-type mice increased the extracellular basal splenocyte superoxide which was further enhanced by complex III inhibitor antimycin A, mitochondrial uncoupling agent CCCP and NADPH oxidase activator PMA. Nox2 depletion attenuated angiotensin II mediated stimulation and inhibited both extracellular and mitochondrial PMA-induced superoxide production. These data indicate that splenocytes isolated from hypertensive angiotensin II-infused mice are “primed” for enhanced superoxide production from both phagocytic NADPH oxidase and mitochondria. Our data demonstrate that novel 15N-CAT1H provides high sensitivity superoxide measurements and combination with mitoTEMPO-H allows independent and simultaneous detection of extracellular and mitochondrial superoxide. We suggest that this new approach can be used to study the site-specific superoxide production and analysis of important sources of oxidative stress in cardiovascular conditions.

REDD1 interacts with AIF and regulates mitochondrial reactive oxygen species generation in the keratinocyte response to UVB

Non-melanoma skin cancer (NMSC) incidence is rising, especially in high-risk, immunocompromised groups such as organ transplant patients, who often develop numerous, aggressive cutaneous squamous cell carcinomas. Identifying the pathways that support NMSC development will result in new approaches for prevention and therapy. Our goal is to define the function of REDD1 (Regulated in DNA Damage and Development 1) in the UVB stress response. REDD1 is rapidly induced by a variety of stressors to repress mechanistic target of rapamycin complex I (mTORC1), and it has been reported that REDD1 loss causes dysfunctional mitochondria with increased reactive oxygen species (ROS) and impaired oxidative phosphorylation (OXPHOS). We now show that knockout of REDD1 in human keratinocytes sensitizes them to UVB-induced apoptosis in an mTORC1-independent manner and intensifies mitochondrial ROS generation. Upon REDD1 knockout, we observe reduced levels of apoptosis inducing factor (AIF), a mitochondrial intermembrane space NADH oxidase that is required for electron transport chain Complex I biogenesis. Further, we show that keratinocyte REDD1 interacts with both AIF and the mitochondrial import protein CHCHD4, a direct binding partner of AIF that ensures functional OXPHOS. Our results support the hypothesis that REDD1 is part of a mitochondrial complex that protects cells from UVB-induced ROS toxicity and suggest novel therapeutic targets for prevention and therapy of NMSC.

Nucleolar function is regulated by the mitochondrial protein sulfite oxidase (SUOX)

Ribosome biogenesis occurs in the nucleolus and relies upon RNA Polymerase I (Pol I) transcription of the rDNA into rRNA. This process is critical for normal cells but is dysregulated in diseases such as Alzheimer’s disease and cancer. To date, however, we have not yet explored the potential relationship between nucleoli and mitochondria. Mitochondria are critical for cellular energy and mitochondrial disorders exhibit dysregulation of nutrient and energy homeostasis. Like nucleoli, mitochondria are also dysregulated in Alzheimer’s disease and cancer, suggesting we may have overlooked a regulatory relationship between these two organelles.In pursuing novel regulators of nucleoli number and function, we have identified a mitochondrial protein that when individually depleted by siRNA in breast epithelial cells (MCF10As) causes a change in nucleolar number – sulfite oxidase (SUOX). Upon SUOX depletion, the nucleolar number decreases from the typical 2‐3 nucleoli to 1 nucleolus. SiRNA‐mediated SUOX depletion reduces nucleolar rRNA levels in a high‐throughput 5‐EU assay (Bryant et al. 2021) and reduces global protein translation by a puromycin incorporation assay, indicating defective nucleolar function. Furthermore, combined metabolomic and transcriptomic analyses following siRNA‐mediated SUOX depletion reveal disruption in the activated methyl cycle, consistent with aberrant ribosome biogenesis. Experiments are underway to validate defective methylation experimentally at specific steps in ribosome biogenesis. The glutamate cycle is also disrupted, congruent with the presentation of the recessive nervous system disorder Isolated Sulfite Oxidase Deficiency (ISOD). Thus, we have defined a novel mitochondrial regulator of nucleolar number and function, nucleolar rRNA biogenesis, and protein translation with implications in human disease. Our work will open up a new avenue of nucleolus‐mitochondria relationships and regulation, and potentially connect mitochondrial disorders with ribosome biogenesis as well as identify how mitochondrial homeostasis is critical for nucleolar function.

Mitochondrial Dysfunction Contributes To Zinc-induced Neurodegeneration: a Link with NADPH Oxidase.

Mitochondrial dysfunction and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) are the major sources of augmentation in free radical generation leading to neurodegeneration. Although NADPH oxidase involvement is reported in zinc (Zn)-induced neurodegeneration, contribution of the mitochondrial dysfunction and its association with NADPH oxidase are not known. Therefore, the study was aimed to decipher the role of mitochondrial dysfunction and its link with NADPH oxidase in Zn-induced Parkinsonism. Zn reduced the motor activities, the number of tyrosine hydroxylase (TH)-positive neurons, and level of TH protein. Conversely, Zn increased the mitochondrial reactive oxygen species (ROS) production, lipid peroxidation (LPO), and superoxide dismutase (SOD) activity and reduced the mitochondrial membrane potential and catalytic activities of complex I and III. Zn also attenuated B-cell lymphoma-2 (Bcl-2) and pro-caspase 9/3 levels and augmented the translocation of cytosolic Bcl-2 associated X (Bax) protein to the mitochondria and cytochrome c release into cytosol from the mitochondria. Cyclosporine A, a mitochondrial outer membrane transition pore inhibitor and apocynin, a NADPH oxidase inhibitor, independently, ameliorated the Zn-induced changes. Similarly, Zn reduced cell viability through mitochondrial dysfunction and apoptosis in human neuroblastomaSH-SY5Y cells, which were notably normalized in the presence of cyclosporine or apocynin. The results demonstrate that mitochondrial dysfunction contributes to Zn-induced neurodegeneration, which could be partially aided by the NADPH oxidase.

Crosstalk Between the Oxidative Stress and Glia Cells After Stroke: From Mechanism to Therapies.

Stroke is the second leading cause of global death and is characterized by high rates of mortality and disability. Oxidative stress is accompanied by other pathological processes that together lead to secondary brain damage in stroke. As the major component of the brain, glial cells play an important role in normal brain development and pathological injury processes. Multiple connections exist in the pathophysiological changes of reactive oxygen species (ROS) metabolism and glia cell activation. Astrocytes and microglia are rapidly activated after stroke, generating large amounts of ROS via mitochondrial and NADPH oxidase pathways, causing oxidative damage to the glial cells themselves and neurons. Meanwhile, ROS cause alterations in glial cell morphology and function, and mediate their role in pathological processes, such as neuroinflammation, excitotoxicity, and blood-brain barrier damage. In contrast, glial cells protect the Central Nervous System (CNS) from oxidative damage by synthesizing antioxidants and regulating the Nuclear factor E2-related factor 2 (Nrf2) pathway, among others. Although numerous previous studies have focused on the immune function of glial cells, little attention has been paid to the role of glial cells in oxidative stress. In this paper, we discuss the adverse consequences of ROS production and oxidative-antioxidant imbalance after stroke. In addition, we further describe the biological role of glial cells in oxidative stress after stroke, and we describe potential therapeutic tools based on glia cells.

Identification of a biological form in the Anopheles stephensi laboratory colony using the odorant-binding protein 1 intron I sequence.

Anopheles stephensi Listen (1901) is a major vector of malaria in Asia and has recently been found in some regions of Africa. The An. stepehnsi species complex is suspected to have three sibling species: type, intermediate, and mysorensis, each with its own vector competence to the malaria parasite and ecology. To identify the members of the species complex in our An. stephensi insectary colony, we used the morphological features of eggs and genetic markers such as AnsteObp1 (Anopheles stephensi odorant binding protein 1), mitochondrial oxidases subunit 1 and 2 (COI and COII), and nuclear internal transcribed spacer 2 locus (ITS2). Eggs were collected from individual mosquitoes (n = 50) and counted for the number of ridges under stereomicroscope. Genomic DNA was extracted from female mosquitoes. After the amplification of partial fragments of AnsteObp1, COI, COII and ITS2 genes, the PCR products were purified and sequenced. Phylogenetic analysis was performed after aligning query sequences against the submitted sequences in GenBank using MEGA 7. The range of ridges number on each egg float was 12-13 that corresponds to the mysorensis form of An. stephensi. The generated COI, COII and ITS2 sequences showed 100%, 99.46% and 99.29% similarity with the sequences deposited for Chinese, Indian and Iranian strains of An. stephensi, respectively. All the generated AnsteObp1 intron I region sequences matched 100% with the sequences deposited for An. stephensi sibling species C (mysorensis form) from Iran and Afghanistan. This manuscript precisely describes the morphological and molecular details of the 'var mysorensis' form of An. stephensi that could be exploited in elucidating its classification as well as in differentiation from other biotypes of the same or other anopheline species. Based on our findings, we recommend AnsteObp1 as a robust genetic marker for rapid and accurate discrimination (taxonomic identification) of the An. stephensi species complex, rather than the COI, COII, and ITS2 marker, which could only be utilized for interspecies (Anopheles) differentiation.

Infection of Endothelial Cells by Dengue Virus Induces ROS Production by Different Sources Affecting Virus Replication, Cellular Activation, Death and Vascular Permeability.

Exacerbated inflammatory response and altered vascular function are hallmarks of dengue disease. Reactive oxygen species (ROS) production has been associated to endothelial barrier disturbance and microvascular alteration in distinct pathological conditions. Increased ROS has been reported in in vitro models of dengue virus (DENV) infection, but its impact for endothelial cell physiology had not been fully investigated. Our group had previously demonstrated that infection of human brain microvascular endothelial cells (HBMEC) with DENV results in the activation of RNA sensors and production of proinflammatory cytokines, which culminate in cell death and endothelial permeability. Here, we evaluated the role of mitochondrial function and NADPH oxidase (NOX) activation for ROS generation in HBMEC infected by DENV and investigated whether altered cellular physiology could be a consequence of virus-induced oxidative stress. DENV-infected HBMECs showed a decrease in the maximal respiratory capacity and altered membrane potential, indicating functional mitochondrial alteration, what might be related to mtROS production. Indeed, mtROS was detected at later time points after infection. Specific inhibition of mtROS diminished virus replication, cell death, and endothelial permeability, but did not affect cytokine production. On the other hand, inhibition of NOX-associated ROS production decreased virus replication and cell death, as well as the secretion of inflammatory cytokines, including IL-6, IL-8, and CCL5. These results demonstrated that DENV replication in endothelial cells induces ROS production by different pathways, which impacts biological functions that might be relevant for dengue pathogenesis. Those data also indicate oxidative stress events as relevant therapeutical targets to avoid vascular permeability, inflammation, and neuroinvasion during DENV infection.