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Objectives: We evaluated the effects of metformin (Met, 1,1‑dimethylbiguanide hydrochloride) combined or not with sodium selenite (Ss, Na 2 SeO 3 ) on the functional activities of LPS-activated GM-CSF monocyte-derived macrophages (GM-MDM)., Materials and Methods: Human GM-MDMs from three healthy donors were treated with Met or Ss alone, or with the combination of Met and Ss, and assayed for various biological activities and cytokines expression., Results: Met alone and Ss alone had significantly different effects on phagocytosis and killing capacities and IL-β production, but had similar effects on the downregulation of inducible nitric oxide synthase (iNOS) activity, relative nicotinamide adenine dinucleotide reduced (NADH) dehydrogenase (Complex I), intracellular free calcium ions ( if Ca 2+ ), and on the upregulation of arginase activity. Additionally, iNOS activity-to-arginase activity ratio was downregulated in Met or Ss treated-GM-MDMs, and, conversely, upregulated in GM-MDMs treated with Met + Ss in combination, indicating that arginase activity dominates that of iNOS when the two treatments are associated. Moreover, combination of Met with Ss significantly upregulated hydrogen peroxide (H 2 O 2 ) production and phagocytic capacity, but significantly downregulated the production of IL-1β, iNOS activity and killing capacity. On the contrary, we show that Met alone induced significant downregulation of phagocytic capacity and slight upregulation of killing capacity. Nevertheless, Ss seems to accentuate the effect of Met on the downregulation of NO production, as well as to reverse its effect on both phagocytic and killing capacities. On the other hand, all treatments induced a sharp decrease in relative levels of NADH dehydrogenase, and a marked decrease in the levels of if Ca 2+ . Finally, we found that GM-MDMs treated with Met or Ss, or Met combined with Ss exhibited different functional activation phenotypes, as indicated by the surface expression of co-stimulatory and cell activation and presentation molecules CD14, CD80, CD86 and HLA-DR., Conclusions: Our results demonstrated that Met/Ss combination can play an important role in the modulation of functional activities of human LPS-activated GM-MDMs. Additionally, the overall effects of Met and the induction of "M2" GM-MDMs-associated arginase could be influenced by its combination with Ss., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ustilago maydis is an aerobic basidiomycete that depends on oxidative phosphorylation for its ATP supply, pointing to the mitochondrion as a key player in its energy metabolism. Mitochondrial respiratory complexes I, III 2 , and IV occur in supramolecular structures named respirasome. In this work, we characterized the subunit composition and the kinetics of NADH:Q oxidoreductase activity of the digitonine-solubilized respirasome (1600 kDa) and the free-complex I (990 kDa). In the presence of 2,6-dimethoxy-1,4-benzoquinone (DBQ) and cytochrome c, both the respirasome NADH:O 2 and the NADH:DBQ oxidoreductase activities were inhibited by rotenone, antimycin A or cyanide. A value of 2.4 for the NADH oxidized/oxygen reduced ratio was determined for the respirasome activity, while ROS production was less than 0.001% of the oxygen consumption rate. Analysis of the NADH:DBQ oxidoreductase activity showed that respirasome was 3-times more active and showed higher affinity than free-complex I. The results suggest that the contacts between complexes I, III 2 and IV in the respirasome increase the catalytic efficiency of complex I and regulate its activity to prevent ROS production., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The novel arylamidine T-2307 exhibits broad-spectrum in vitro and in vivo antifungal activities against clinically significant pathogens. Previous studies have shown that T-2307 accumulates in yeast cells via a specific polyamine transporter and disrupts yeast mitochondrial membrane potential. Further, it has little effect on rat liver mitochondrial function. The mechanism by which T-2307 disrupts yeast mitochondrial function is poorly understood, and its elucidation may provide important information for developing novel antifungal agents. This study aimed to determine how T-2307 promotes yeast mitochondrial dysfunction and to investigate the selectivity of this mechanism between fungi and mammals. T-2307 inhibited the respiration of yeast whole cells and isolated yeast mitochondria in a dose-dependent manner. The similarity of the effects of T-2307 and respiratory chain inhibitors on mitochondrial respiration prompted us to investigate the effect of T-2307 on mitochondrial respiratory chain complexes. T-2307 particularly inhibited respiratory chain complexes III and IV not only in Saccharomyces cerevisiae but also in Candida albicans , indicating that T-2307 acts against pathogenic fungi in a manner similar to that of yeast. Conversely, T-2307 showed little effect on bovine respiratory chain complexes. Additionally, we demonstrated that the inhibition of respiratory chain complexes by T-2307 resulted in a decrease in the intracellular ATP levels in yeast cells. These results indicate that inhibition of respiratory chain complexes III and IV is a key factor for selective disruption of yeast mitochondrial function and antifungal activity., (Copyright © 2019 Yamashita et al.)

Mitochondrial complex I (CI) is the largest multi-subunit oxidative phosphorylation (OXPHOS) protein complex. Recent availability of a high-resolution human CI structure, and from two non-human mammals, enabled predicting the impact of mutations on interactions involving each of the 44 CI subunits. However, experimentally assessing the impact of the predicted interactions requires an easy and high-throughput method. Here, we created such a platform by cloning all 37 nuclear DNA (nDNA) and 7 mitochondrial DNA (mtDNA)-encoded human CI subunits into yeast expression vectors to serve as both 'prey' and 'bait' in the split murine dihydrofolate reductase (mDHFR) protein complementation assay (PCA). We first demonstrated the capacity of this approach and then used it to examine reported pathological OXPHOS CI mutations that occur at subunit interaction interfaces. Our results indicate that a pathological frame-shift mutation in the MT-ND2 gene, causing the replacement of 126 C-terminal residues by a stretch of only 30 amino acids, resulted in loss of specificity in ND2-based interactions involving these residues. Hence, the split mDHFR PCA is a powerful assay for assessing the impact of disease-causing mutations on pairwise protein-protein interactions in the context of a large protein complex, thus offering a possible mechanistic explanation for the underlying pathogenicity.

Changes in the gut microbiota and the mitochondrial genome are both linked with the development of disease. To investigate why, we examined the gut microbiota of mice harboring various mutations in genes that alter mitochondrial function. These studies revealed that mitochondrial genetic variations altered the composition of the gut microbiota community. In cross-fostering studies, we found that although the initial microbiota community of newborn mice was that obtained from the nursing mother, the microbiota community progressed toward that characteristic of the microbiome of unfostered pups of the same genotype within 2 months. Analysis of the mitochondrial DNA variants associated with altered gut microbiota suggested that microbiome species diversity correlated with host reactive oxygen species (ROS) production. To determine whether the abundance of ROS could alter the gut microbiota, mice were aged, treated with N -acetylcysteine, or engineered to express the ROS scavenger catalase specifically within the mitochondria. All three conditions altered the microbiota from that initially established. Thus, these data suggest that the mitochondrial genotype modulates both ROS production and the species diversity of the gut microbiome, implying that the connection between the gut microbiome and common disease phenotypes might be due to underlying changes in mitochondrial function., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The genetic causes of Leigh syndrome are heterogeneous, with a poor genotype-phenotype correlation. To date, more than 50 nuclear genes cause nuclear gene-encoded Leigh syndrome. NDUFS6 encodes a 13 kiloDaltons subunit, which is part of the peripheral arm of complex I and is localized in the iron-sulfur fraction. Only a few patients were reported with proven NDUFS6 pathogenic variants and all presented with severe neonatal lactic acidemia and complex I deficiency, leading to death in the first days of life. Here, we present a patient harboring two NDUFS6 variants with a phenotype compatible with Leigh syndrome. Although most of previous reports suggested that NDUFS6 pathogenic variants invariably lead to early neonatal death, this report shows that the clinical spectrum could be larger. We found a severe decrease of NDUFS6 protein level in patient's fibroblasts associated with a complex I assembly defect in patient's muscle and fibroblasts. These data confirm the importance of NDUFS6 and the Zn-finger domain for a correct assembly of complex I.

Dysregulation of miRNA expression is associated with multiple diseases, including cancers, in which small RNAs can have either oncogenic or tumor suppressive functions. Here we investigated the potential tumor suppressive function of miR-450a, one of the most significantly downregulated miRNAs in ovarian cancer. RNA-seq analysis of the ovarian cancer cell line A2780 revealed that overexpression of miR-450a suppressed multiple genes involved in the epithelial-to-mesenchymal transition (EMT). Overexpression of miR-450a reduced tumor migration and invasion and increased anoikis in A2780 and SKOV-3 cell lines and reduced tumor growth in an ovarian tumor xenographic model. Combined AGO-PAR-CLIP and RNA-seq analysis identified a panel of potential miR-450a targets, of which many, including TIMMDC1, MT-ND2, ACO2, and ATP5B, regulate energetic metabolism. Following glutamine withdrawal, miR-450a overexpression decreased mitochondrial membrane potential but increased glucose uptake and viability, characteristics of less invasive ovarian cancer cell lines. In summary, we propose that miR-450a acts as a tumor suppressor in ovarian cancer cells by modulating targets associated with glutaminolysis, which leads to decreased production of lipids, amino acids, and nucleic acids, as well as inhibition of signaling pathways associated with EMT. SIGNIFICANCE: miR-450a limits the metastatic potential of ovarian cancer cells by targeting a set of mitochondrial mRNAs to reduce glycolysis and glutaminolysis. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3294/F1.large.jpg., (©2019 American Association for Cancer Research.)

Mitochondrial dysfunction plays a major role in the pathogenesis of cardiovascular diseases. MicroRNAs (miRNAs) are small RNAs that act as negative regulators of gene expression, but how miRNAs affect mitochondrial function in the heart is unclear. Using a miRNA microarray assay, we found that miR-762 predominantly translocated in the mitochondria and was significantly upregulated upon anoxia/reoxygenation (A/R) treatment. Knockdown of endogenous miR-762 significantly attenuated the decrease in intracellular ATP levels, the increase in ROS levels, the decrease in mitochondrial complex I enzyme activity and the increase in apoptotic cell death in cardiomyocytes, which was induced by A/R treatment. In addition, knockdown of miR-762 ameliorated myocardial ischemia/reperfusion (I/R) injury in mice. Mechanistically, we showed that enforced expression of miR-762 dramatically decreased the protein levels of endogenous NADH dehydrogenase subunit 2 (ND2) but had no effect on the transcript levels of ND2. The luciferase reporter assay showed that miR-762 bound to the coding sequence of ND2. In addition, knockdown of endogenous ND2 significantly decreased intracellular ATP levels, increased ROS levels, reduced mitochondrial complex I enzyme activity and increased apoptotic cell death in cardiomyocytes, which was induced by A/R treatment. Furthermore, we found that the inhibitory effect of miR-762 downregulation was attenuated by ND2 knockdown. Thus, our findings suggest that miR-762 participates in the regulation of mitochondrial function and cardiomyocyte apoptosis by ND2, a core assembly subunit of mitochondrial complex I. Our results revealed that mitochondrial miR-762, as a new player in mitochondrial dysfunction, may provide a new therapeutic target for myocardial infarction.

Rationale: Hypoxic pulmonary vasoconstriction (HPV) optimizes systemic oxygen delivery by matching ventilation to perfusion. HPV is intrinsic to pulmonary artery smooth muscle cells (PASMCs). Hypoxia dilates systemic arteries, including renal arteries. Hypoxia is sensed by changes in mitochondrial-derived reactive oxygen species, notably hydrogen peroxide (H 2 O 2 ) ([H 2 O 2 ] mito ). Decreases in [H 2 O 2 ] mito elevate pulmonary vascular tone by increasing intracellular calcium ([Ca 2+ ] i ) through reduction-oxidation regulation of ion channels. Although HPV is mimicked by the Complex I inhibitor, rotenone, the molecular identity of the O 2 sensor is unknown., Objective: To determine the role of Ndufs2 (NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 2), Complex I's rotenone binding site, in pulmonary vascular oxygen-sensing., Methods and Results: Mitochondria-conditioned media from pulmonary and renal mitochondria isolated from normoxic and chronically hypoxic rats were infused into an isolated lung bioassay. Mitochondria-conditioned media from normoxic lungs contained more H 2 O 2 than mitochondria-conditioned media from chronic hypoxic lungs or kidneys and uniquely attenuated HPV via a catalase-dependent mechanism. In PASMC, acute hypoxia decreased H 2 O 2 within 112±7 seconds, followed, within 205±34 seconds, by increased intracellular calcium concentration, [Ca 2+ ] i . Hypoxia had no effects on [Ca 2+ ] i in renal artery SMC. Hypoxia decreases both cytosolic and mitochondrial H 2 O 2 in PASMC while increasing cytosolic H 2 O 2 in renal artery SMC. Ndufs2 expression was greater in PASMC versus renal artery SMC. Lung Ndufs2 cysteine residues became reduced during acute hypoxia and both hypoxia and reducing agents caused functional inhibition of Complex I. In PASMC, siNdufs2 (cells/tissue treated with Ndufs2 siRNA) decreased normoxic H 2 O 2 , prevented hypoxic increases in [Ca 2+ ] i , and mimicked aspects of chronic hypoxia, including decreasing Complex I activity, elevating the nicotinamide adenine dinucleotide (NADH/NAD + ) ratio and decreasing expression of the O 2 -sensitive ion channel, Kv1.5. Knocking down another Fe-S center within Complex I (Ndufs1, NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 1) or other mitochondrial subunits proposed as putative oxygen sensors (Complex III's Rieske Fe-S center and COX4i2 [cytochrome c oxidase subunit 4 isoform 2] in Complex IV) had no effect on hypoxic increases in [Ca 2+ ] i . In vivo, siNdufs2 significantly decreased hypoxia- and rotenone-induced constriction while enhancing phenylephrine-induced constriction., Conclusions: Ndufs2 is essential for oxygen-sensing and HPV.

It is generally accepted that alterations in metabolism are critical for the metastatic process; however, the mechanisms by which these metabolic changes are controlled by the major drivers of the metastatic process remain elusive. Here, we found that S100 calcium-binding protein A4 (S100A4), a major metastasis-promoting protein, confers metabolic plasticity to drive tumor invasion and metastasis of non-small cell lung cancer cells. Investigating how S100A4 regulates metabolism, we found that S100A4 depletion decreases oxygen consumption rates, mitochondrial activity, and ATP production and also shifts cell metabolism to higher glycolytic activity. We further identified that the 49-kDa mitochondrial complex I subunit NADH dehydrogenase (ubiquinone) Fe-S protein 2 (NDUFS2) is regulated in an S100A4-dependent manner and that S100A4 and NDUFS2 exhibit co-occurrence at significant levels in various cancer types as determined by database-driven analysis of genomes in clinical samples using cBioPortal for Cancer Genomics. Importantly, we noted that S100A4 or NDUFS2 silencing inhibits mitochondrial complex I activity, reduces cellular ATP level, decreases invasive capacity in three-dimensional growth, and dramatically decreases metastasis rates as well as tumor growth in vivo Finally, we provide evidence that cells depleted in S100A4 or NDUFS2 shift their metabolism toward glycolysis by up-regulating hexokinase expression and that suppressing S100A4 signaling sensitizes lung cancer cells to glycolysis inhibition. Our findings uncover a novel S100A4 function and highlight its importance in controlling NDUFS2 expression to regulate the plasticity of mitochondrial metabolism and thereby promote the invasive and metastatic capacity in lung cancer., (© 2019 Liu et al.)

Pentatricopeptide repeat (PPR) proteins are important RNA regulators in chloroplasts and mitochondria, aiding in RNA editing, maturation, stabilisation or intron splicing, and in transcription and translation of organellar genes. In this review, we summarise all PPR proteins documented so far in plants and the green alga Chlamydomonas. By further analysis of the known target RNAs from Arabidopsis thaliana PPR proteins, we find that all organellar-encoded complexes are regulated by these proteins, although to differing extents. In particular, the orthologous complexes of NADH dehydrogenase (Complex I) in the mitochondria and NADH dehydrogenase-like (NDH) complex in the chloroplast were the most regulated, with respectively 60 and 28% of all characterised A. thaliana PPR proteins targeting their genes., (© 2019 Scandinavian Plant Physiology Society.)

Glycerol is an attractive substrate for microbial fermentations due to its higher degree of reduction compared to glucose. The replacement of the native FAD-dependent glycerol catabolic pathway in Saccharomyces cerevisiae by an artificial NADH-delivering dihydroxyacetone (DHA) pathway is supposed to facilitate the capturing of electrons in fermentation products. This requires that the electrons from the cytosolic NADH are not exclusively transferred to oxygen. However, the external NADH dehydrogenases (Nde1/2) and the L-glycerol 3-phosphate shuttle (composed of Gpd1/2 and Gut2), both coupled to the respiratory chain, are known to contribute to cytosolic NAD+ regeneration during growth on non-fermentable carbon sources. In order to evaluate the role of these mechanisms during growth on glycerol, we deleted GPD1/2, GUT2 as well as NDE1/2, separately and in combinations in both the glycerol-utilizing wild-type strain CBS 6412-13A and the corresponding engineered strain CBS DHA in which glycerol is catabolized by the DHA pathway. Particularly, the nde1Δ mutants showed a significant reduction in growth rate and the nde1∆ nde2∆ double deletion mutants did not grow at all in synthetic glycerol medium. The current work also demonstrates a positive impact of deleting NDE1 on the production of the fermentation product 1,2-propanediol in an accordingly engineered S. cerevisiae strain., (© FEMS 2019.)

In angiosperms, cyclic electron transport (CET) around photosystem I (PSI) consists of two pathways, depending on PGR5/PGRL1 proteins and the chloroplast NDH complex. In single mutants defective in chloroplast NDH, photosynthetic electron transport is only slightly affected at low light intensity, but in double mutants impaired in both CET pathways photosynthesis and plant growth are severely affected. The question is whether this strong mutant phenotype observed in double mutants can be simply explained by the additive effect of defects in both CET pathways. In this study, we used the weak mutant allele of pgr5-2 for the background of double mutants to avoid possible problems caused by the secondary effects due to the strong mutant phenotype. In two double mutants, crr2-2 pgr5-2 and ndhs-1 pgr5-2, the plant growth was unaffected and linear electron transport was only slightly affected. However, NPQ induction was more severely impaired in the double mutants than in the pgr5-2 single mutant. A similar trend was observed in the size of the proton motive force. Despite the slight reduction in photosystem II parameters, PSI parameters were severely affected in the pgr5-2 single mutant, the phenotype that was further enhanced by adding the NDH defects. Despite the lack of ∆pH-dependent regulation at the cytochrome b 6 f complex (donor-side regulation of PSI), the plastoquinone pool was more reduced in the double mutants than in the pgr5-2 single mutants. This phenotype suggests that both PGR5/PGRL1- and NDH-dependent CET contribute to supply sufficient acceptors from PSI by balancing the ATP/NADPH production ratio., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum, the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase (NDH2) of P. falciparum, PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasite's susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc1 complex. Our results suggest that PfNDH2 is not likely a good antimalarial drug target., Competing Interests: The authors have declared that no competing interests exist.

The alternative respiratory chain (aRC), comprising the alternative NADH dehydrogenases (NDX) and quinone oxidases (AOX), is found in microbes, fungi and plants, where it buffers stresses arising from restrictions on electron flow in the oxidative phosphorylation system. The aRC enzymes are also found in species belonging to most metazoan phyla, including some chordates and arthropods species, although not in vertebrates or in Drosophila. We postulated that the aRC enzymes might be deployed to alleviate pathological stresses arising from mitochondrial dysfunction in a wide variety of disease states. However, before such therapies can be contemplated, it is essential to understand the effects of aRC enzymes on cell metabolism and organismal physiology. Here we report and discuss new findings that shed light on the functions of the aRC enzymes in animals, and the unexpected benefits and detriments that they confer on model organisms. In Ciona intestinalis, the aRC is induced by hypoxia and by sulfide, but is unresponsive to other environmental stressors. When expressed in Drosophila, AOX results in impaired survival under restricted nutrition, in addition to the previously reported male reproductive anomalies. In contrast, it confers cold resistance to developing and adult flies, and counteracts cell signaling defects that underlie developmental dysmorphologies. The aRC enzymes may also influence lifespan and stress resistance more generally, by eliciting or interfering with hormetic mechanisms. In sum, their judicious use may lead to major benefits in medicine, but this will require a thorough characterization of their properties and physiological effects., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

NADH dehydrogenase plays an important role in the central metabolism of almost all organisms, including acetic acid bacteria (AAB). In this study, the gene diversity of the NADH dehydrogenases in AAB was investigated. The distribution of the genes of the type I and type II NADH dehydrogenases in AAB was mostly congruent with their phylogenetic relationships. There are two phylogenetically distinct type I NADH dehydrogenase complexes, complex IA and complex IE. Complex IA', which lacks the nuoM gene from complex IA, was only conserved in the genera Acetobacter, Gluconacetobacter and Komagataeibacter, which all have the ability to perform acetic acid fermentation, whereas the complex IE gene cluster was found randomly in several species of AAB. Almost all AAB, excluding the early-diverged species, had the type II NADH dehydrogenase, while some of the species also had the homologue with an amino acid replacement at the residue responsible for NADPH oxidation ability. Thus, the gene repertoire of NADH dehydrogenases shows a history of adaptation towards their habitats through gene expansions and losses and neo-functionalization in AAB.

Increasing the availability of NADPH is commonly used to improve lysine production by Corynebacterium glutamicum since 4 mol of NADPH are required for the synthesis of 1 mol of lysine. Alternatively, engineering of enzymes in lysine synthesis pathway to utilize NADH directly can also be explored for cofactor balance during lysine overproduction. To achieve such a goal, enzyme mining was used in this study to quickly identify a full set of NADH-utilizing dehydrogenases, namely aspartate dehydrogenase from Pseudomonas aeruginosa (PaASPDH), aspartate-semialdehyde dehydrogenase from Tistrella mobilis (TmASADH), dihydrodipicolinate reductase from Escherichia coli (EcDHDPR), and diaminopimelate dehydrogenase from Pseudothermotoga thermarum (PtDAPDH). This allowed us to systematically perturb cofactor utilization of lysine synthesis pathway of C. glutamicum for the first time. Individual overexpression of PaASPDH, TmASADH, EcDHDPR, and PtDAPDH in C. glutamicum LC298, a basic lysine producer, increased the production of lysine by 30.7%, 32.4%, 17.4%, and 36.8%, respectively. Combinatorial replacement of NADPH-dependent dehydrogenases in C. glutamicum ATCC 21543, a lysine hyperproducer, also resulted in significantly improved lysine production. The highest increase of lysine production (30.7%) was observed for a triple-mutant strain (27.7 g/L, 0.35 g/g glucose) expressing PaASPDH, TmASADH, and EcDHDPR. A quadruple-mutant strain expressing all of the four NADH-utilizing enzymes allowed high lysine production (24.1 g/L, 0.30 g/g glucose) almost independent of the oxidative pentose phosphate pathway. Collectively, our results demonstrated that a combination of enzyme mining and cofactor engineering was a highly efficient approach to improve lysine production. Similar strategies can be applied for the production of other amino acids or their derivatives., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

2-(2-nitrovinyl) furan is a broad-spectrum antibacterial agent with activity against Gram-positive and Gram-negative bacteria. In this study, the contributions of reactive oxygen species, oxidative DNA damage and glutathione depletion to its activity against Acinetobacter baumannii was investigated. Inactivation of sodB, katG and recA lowered the minimum inhibitory concentration of 2-(2-nitrovinyl) furan. Furthermore, the inactivation increased the superoxide anion radical and hydrogen peroxide contents of 2-(2-nitrovinyl) furan-treated A. baumannii. Antioxidant (thiourea) reversed the elevated levels of superoxide anion radical and hydrogen peroxide. In addition, thiourea lowered the susceptibility of A. baumannii to 2-(2-nitrovinyl) furan. 2-(2-nitrovinyl) furan depleted reduced glutathione (GSH) contents of parental, sodB, katG and recA strains of A. baumannii. NAD+/NADH ratio parental, sodB, katG and recA strains of A. baumannii exposed to 2-(2-nitrovinyl) furan increased significantly. Inactivation of type-I NADH dehydrogenase lowered the reactive oxygen species generation in 2-(2-nitrovinyl) furan-treated A. baumannii. It is evident from this study that 2-(2-nitrovinyl) furan stimulates respiratory chain activity of A. baumannii leading to enhanced ROS generation, which depletes GSH and reacts with Fe 2+ to produce hydroxyl radical that damage DNA., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Colon cancer is one of the most frequently occurring types of cancers in the world. Primary tumours are treated very efficiently, but the metastatic cases are known to have severe outcomes. Therefore, the aim of the present study was to obtain a greater understanding of the transformation of primary colon cancer cells into metastatic phenotypes. Small changes in protein expression provoke the metastatic phenotype transformation. More sensitive methods to detect small variations are required. A murine colon cancer cell line with metastatic characteristics in a very early phase was created in order to investigate the first steps of transformation using a murine liver metastasis model. The protein expression patterns of metastatic and non‑metastatic cells were compared using the stable isotope labelling by amino acids in cell culture method in combination with mass spectrometry. Quantitative proteomics data indicated that nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase complex I was overexpressed in metastatic cells with respect to non‑metastatic cells. Since the NADH dehydrogenase complex catalyses the oxidation of NADH to NAD+, the functionality of the complex was studied by measuring the amount of NADH. The results revealed that metastatic cells accumulate more NADH and reactive oxygen species. In addition, the mitochondrial membrane potential of metastatic cells was lower than that of non‑metastatic cells, indicating that the activity of NADH dehydrogenase and the mitochondrial oxidative chain were decreased in metastatic cells. During the incipient transformation of primary cancer cells, NADH dehydrogenase complex I was overexpressed but then became inactive due to the Warburg effect, which inhibits mitochondrial activity. In the first step of transformation, the high energy demand required in an adverse environment is fulfilled by overexpressing components of the respiratory chain, a fact that should be considered for future anti‑metastatic therapies.

Endurance exercise has been shown to be a positive regulator of skeletal muscle metabolic function. Changes in mitochondrial dynamics (fusion and fission) have been shown to influence mitochondrial oxidative capacity. We therefore tested whether genetic disruption of mitofusins (Mfns) affected exercise performance in adult skeletal muscle. We generated adult-inducible skeletal muscle-specific Mfn1 (iMS-Mfn1KO), Mfn2 (iMS-Mfn2KO), and Mfn1/2 (iMS-MfnDKO) knockout mice. We assessed exercise capacity by performing a treadmill time to exhaustion stress test before deletion and up to 8 wk after deletion. Analysis of either the iMS-Mfn1KO or the iMS-Mfn2KO did not reveal an effect on exercise capacity. However, analysis of iMS-MfnDKO animals revealed a progressive reduction in exercise performance. We measured individual electron transport chain (ETC) complex activity and observed a reduction in ETC activity in both the subsarcolemmal and intermyofibrillar mitochondrial fractions specifically for NADH dehydrogenase (complex I) and cytochrome- c oxidase (complex IV), which was associated with a decrease in ETC subunit expression for these complexes. We also tested whether voluntary exercise training would prevent the decrease in exercise capacity observed in iMS-MfnDKO animals ( n = 10/group). However, after 8 wk of training we did not observe any improvement in exercise capacity or ETC subunit parameters in iMS-MfnDKO animals. These data suggest that the decrease in exercise capacity observed in the iMS-MfnDKO animals is in part the result of impaired ETC subunit expression and ETC complex activity. Taken together, these results provide strong evidence that mitochondrial fusion in adult skeletal muscle is important for exercise performance. NEW & NOTEWORTHY This study is the first to utilize an adult-inducible skeletal muscle-specific knockout model for Mitofusin (Mfn)1 and Mfn2 to assess exercise capacity. Our findings reveal a progressive decrease in exercise performance with Mfn1 and Mfn2 deletion. The decrease in exercise capacity was accompanied by impaired oxidative phosphorylation specifically for complex I and complex IV. Furthermore, voluntary exercise training was unable to rescue the impairment, suggesting that normal fusion is essential for exercise-induced mitochondrial adaptations.

Background: Araucaria angustifolia extract (AAE) is a polyphenol-rich extract that has gained interest as a natural anticancer agent. Recent work suggests that AAE induces oxidative damage and apoptosis through its action on decreasing complex I activity of the mitochondrial Electron Transport Chain (ETC)., Aims and Methods: In the present study, we aimed to further examine the specific targets by which AAE exerts proapoptotic effects in HEp-2 cancer cells. Specifically, the effect of AAE on the: 1) levels of pyruvate dehydrogenase was assessed by ELISA assay; 2) levels of mitochondrial ETC complexes, focusing on complex I at the gene transcript and protein level relevant to ROS generation was evaluated by multiplex ELISA followed by qRT-PCR and immunoblotting; 3) mitochondrial network distribution analysis was assessed by MitoTracker Red CMXRos; and 4) chemical variations on DNA was evaluated by dot-blotting in HEp-2 cells., Results: Results demonstrated that AAE increased protein levels of PDH, switching energy metabolism to oxidative metabolism. Protein expression levels of complex I and III were found decreased in AAE-treated HEp-2 cells. Analyzing the subunits of complex I, changes in protein and gene transcript levels of NDUFS7 and NDUFV2 were found. Mitochondria staining after AAE incubation revealed changes in the mitochondrial network distribution. AAE was able to induce DNA hypomethylation and decreased DNA (cytosine-5)-methyltransferase 1 activity., Conclusion: Our data demonstrate for the first time that AAE alters expression of NDUFS7 and NDUFV2 mitochondrial subunits and induce epigenetic changes in HEp-2 cancer cells leading to a possible suppression of oncogenes., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Hypoxia resulting from reduced oxygen (O 2 ) levels in the arterial blood is sensed by the carotid body (CB) and triggers reflex stimulation of breathing and blood pressure to maintain homeostasis. Studies in the past five years provided novel insights into the roles of heme oxygenase-2 (HO-2), a carbon monoxide (CO)-producing enzyme, and NADH dehydrogenase Fe-S protein 2, a subunit of the mitochondrial complex I, in hypoxic sensing by the CB. HO-2 is expressed in type I cells, the primary O2-sensing cells of the CB, and binds to O 2 with low affinity. O 2 -dependent CO production from HO-2 mediates hypoxic response of the CB by regulating H 2 S generation. Mice lacking NDUFS2 show that complex I-generated reactive oxygen species acting on K + channels confer type I cell response to hypoxia. Whether these signaling pathways operate synergistically or independently remains to be studied., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

In angiosperms, such as Arabidopsis and barley, the chloroplast NADH dehydrogenase-like (NDH) complex associates with two copies of photosystem I (PSI) supercomplex to form an NDH-PSI supercomplex for the stabilization of the NDH complex. Two linker proteins, Lhca5 and Lhca6, are members of the light-harvesting complex I (LHCI) family and mediate this supercomplex formation. The liverwort Marchantia polymorpha has branched from the basal land plant lineage and has neither Lhca5 nor Lhca6. Consequently, the NDH complex does not form a supercomplex with PSI in this plant. The Lhca6 gene does not seem to exist also in the moss Physcomitrella patens (Physcomitrella). Conversely, the Lhca5 gene has been found in Physcomitrella, although experimental evidence is still lacking for its contribution to NDH-PSI supercomplex formation as a linker. Here, we biochemically characterized the Lhca5 knock-out mutant (lhca5) in Physcomitrella. The NDH-PSI supercomplex observed in wild-type Physcomitrella was absent in the lhca5 mutant. Lhca5 protein was detected in this NDH-PSI supercomplex. Some PSI and NDH subunits were co-immunoprecipitated with Lhca5-HA. These results indicate that the Physcomitrella gene is the functional ortholog of Lhca5 reported in Arabidopsis. Between Physcomitrella and Arabidopsis, the stromal loop region is highly conserved in Lhca5 proteins but not in other LHCI members. We found that Lhca5 contributed to the stable accumulation of the NDH complex, but part of the NDH complex was still sensitive to high light intensity, even in the wild-type. We considered that angiosperms acquired another linker protein, Lhca6, to further stabilize the NDH complex., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Cell models of mitochondrial complex I (CI) deficiency display activation of glycolysis to compensate for the loss in mitochondrial ATP production. This adaptation can mask other relevant deficiency-induced aberrations in cell physiology. Here we investigated the viability, mitochondrial morphofunction, ROS levels and ATP homeostasis of primary skin fibroblasts from Leigh Syndrome (LS) patients with isolated CI deficiency. These cell lines harbored mutations in nuclear DNA (nDNA)-encoded CI genes (NDUFS7, NDUFS8, NDUFV1) and, to prevent glycolysis upregulation, were cultured in a pyruvate-free medium in which glucose was replaced by galactose. Following optimization of the cell culture protocol, LS fibroblasts died in the galactose medium, whereas control cells did not. LS cell death was dose-dependently inhibited by pyruvate, malate, oxaloacetate, α-ketoglutarate, aspartate, and exogenous NAD + (eNAD), but not by lactate, succinate, α-ketobutyrate, and uridine. Pyruvate and eNAD increased the cellular NAD + content in galactose-treated LS cells to a different extent and co-incubation studies revealed that pyruvate-induced rescue was not primarily mediated by NAD + . Functionally, in LS cells glucose-by-galactose replacement increased mitochondrial fragmentation and mass, depolarized the mitochondrial membrane potential (Δψ), increased H 2 DCFDA-oxidizing ROS levels, increased mitochondrial ATP generation, and reduced the total cellular ATP content. These aberrations were differentially rescued by pyruvate and eNAD, supporting the conclusion that these compounds rescue galactose-induced LS cell death via different mechanisms. These findings establish a cell-based strategy for intervention testing and enhance our understanding of CI deficiency pathophysiology.

Background: Trauma causes inflammation by releasing mitochondria that act as Danger-Associated Molecular Patterns (DAMPs). Trauma also increases susceptibility to infection. Human mitochondria contain 13 N-formyl peptides (mtFPs). We studied whether mtFPs released into plasma by clinical injury induce neutrophil (PMN) inflammatory responses, whether their potency reflects their similarity to bacterial FPs and how their presence at clinically relevant concentration affects PMN function., Methods: N-terminal sequences of the 13 mtFPs were synthesized. Changes in human PMN cytosolic Ca concentration ([Ca]i) and chemotactic responses to mtFPs were studied. Sequence similarity of mtFPs to the canonical bacterial peptide f-Met-Leu-Phe (fMLF/fMLP) was studied using the BLOcks SUbstitution Matrix 62 (BLOSUM 62) system. The presence of mtFPs in plasma of trauma patients was assayed by Enzyme-linked immunosorbent assay (ELISA). The effects of the most potent mtFP (ND6) on PMN signaling and function were then studied at ambient clinical concentrations by serial exposure of native PMN to ND6, chemokines and leukotrienes., Results: Five mtFPs (ND6, ND3, ND4, ND5, and Cox 1) induced [Ca]i flux and chemotaxis in descending order of potency. Evolutionary similarity to fMLF predicted [Ca]i flux and chemotactic potency linearly (R = 0.97, R = 0.95). Chemoattractant potency was also linearly related to [Ca]i flux induction (R = 0.92). Active mtFPs appear to circulate in significant amounts immediately after trauma and persist through the first week. The most active mtFP, ND6, suppresses responses to physiologic alveolar chemoattractants (CXCL-1, leukotriene B4) as well as to fMLF where CXCL-1 and leukotriene B4 do not suppress N-formyl peptide receptor (FPR)-1 responses to mtFPs. Prior FPR-1 inhibition rescues PMN from heterologous suppression of CXCR-1 and BLT-1 by mtFPs., Conclusion: The data suggest mtFPs released by injured tissue may attract PMN to trauma sites while suppressing PMN responses to other chemoattractants. Inhibition of mtFP-FPR1 interactions might increase PMN recruitment to lung bacterial inoculation after trauma. These findings suggest new paradigms for preventing infections after trauma., Level of Evidence: Therapeutic, Level IV.

Mitochondrial genes of Euglenozoa (Kinetoplastida, Diplonemea, and Euglenida) are notorious for being barely recognizable, raising the question of whether such divergent genes actually code for functional proteins. Here we demonstrate the translation and identify the function of five previously unassigned y genes encoded by mitochondrial DNA (mtDNA) of diplonemids. As is the rule in diplonemid mitochondria, y genes are fragmented, with gene pieces transcribed separately and then trans-spliced to form contiguous mRNAs. Further, y transcripts undergo massive RNA editing, including uridine insertions that generate up to 16-residue-long phenylalanine tracts, a feature otherwise absent from conserved mitochondrial proteins. By protein sequence analyses, MS, and enzymatic assays in Diplonema papillatum , we show that these y genes encode the subunits Nad2, -3, -4L, -6, and -9 of the respiratory chain Complex I (CI; NADH:ubiquinone oxidoreductase). The few conserved residues of these proteins are essentially those involved in proton pumping across the inner mitochondrial membrane and in coupling ubiquinone reduction to proton pumping (Nad2, -3, -4L, and -6) and in interactions with subunits containing electron-transporting Fe-S clusters (Nad9). Thus, in diplonemids, 10 CI subunits are mtDNA-encoded. Further, MS of D. papillatum CI allowed identification of 26 conventional and 15 putative diplonemid-specific nucleus-encoded components. Most conventional accessory subunits are well-conserved but unusually long, possibly compensating for the streamlined mtDNA-encoded components and for missing, otherwise widely distributed, conventional subunits. Finally, D. papillatum CI predominantly exists as a supercomplex I:III:IV that is exceptionally stable, making this protist an organism of choice for structural studies., (© 2018 Valach et al.)

5-Hydroxymethylcytosine (5hmC) exists in DNA, RNA, and mitochondrial DNA (mtDNA) and plays an important role in many diseases. Specifically, 5hmC is involved in promoting gene expression, and this process is regulated by Tet enzymes. In this study, we identified that there is no difference in male mice and female mice at first; then we examined the levels of 5hmC in mtDNA and explored the relationship among 5hmC, mitochondrial gene expression and ATP production after acute brain ischemia. The abundance of mtDNA 5hmC was increased at 1 d and peaked at 2 d after ischemic injury, whereas that of mtDNA 5mC was unchanged. Furthermore, increased mitochondrial Tet2 expression was found to be responsible for the increase in mtDNA 5hmC. Tet2 inhibition decreased the mtDNA 5hmC abundance and increased the ATP levels in mitochondria, suggesting an association between the cellular ATP levels and mtDNA 5hmC abundance. We also demonstrated that mtDNA 5hmC increased the mRNA levels of mitochondrial genes after ischemia/reperfusion (I/R) injury., (© 2018 Wiley Periodicals, Inc.)

Extracellular electron transfer (EET) describes microbial bioelectrochemical processes in which electrons are transferred from the cytosol to the exterior of the cell 1 . Mineral-respiring bacteria use elaborate haem-based electron transfer mechanisms 2-4 but the existence and mechanistic basis of other EETs remain largely unknown. Here we show that the food-borne pathogen Listeria monocytogenes uses a distinctive flavin-based EET mechanism to deliver electrons to iron or an electrode. By performing a forward genetic screen to identify L. monocytogenes mutants with diminished extracellular ferric iron reductase activity, we identified an eight-gene locus that is responsible for EET. This locus encodes a specialized NADH dehydrogenase that segregates EET from aerobic respiration by channelling electrons to a discrete membrane-localized quinone pool. Other proteins facilitate the assembly of an abundant extracellular flavoprotein that, in conjunction with free-molecule flavin shuttles, mediates electron transfer to extracellular acceptors. This system thus establishes a simple electron conduit that is compatible with the single-membrane structure of the Gram-positive cell. Activation of EET supports growth on non-fermentable carbon sources, and an EET mutant exhibited a competitive defect within the mouse gastrointestinal tract. Orthologues of the genes responsible for EET are present in hundreds of species across the Firmicutes phylum, including multiple pathogens and commensal members of the intestinal microbiota, and correlate with EET activity in assayed strains. These findings suggest a greater prevalence of EET-based growth capabilities and establish a previously underappreciated relevance for electrogenic bacteria across diverse environments, including host-associated microbial communities and infectious disease.

Breast cancer is the second cause of death among women all around the world. One out of every eight women is diagnosed with breast cancer in Iran. There are many reasons for cancer, one of which is the mutations in the mitochondrial genome observed in most breast cancer studies. However, the aim of this study is to evaluate the genetic region of NADH dehydrogenase subunit 4 in patients with breast cancer. First, the genomic DNA was extracted from a tissue. The NADH dehydrogenase subunit 4 coding region was amplified by PCR, and then the SSCP was sequenced. After that, the molecular dynamics were employed. The association between the mutations and the prognostic factors such as ER, PR, HER-2, and age were statistically examined. The sequence of the ND4 area was determined in 24 suspected patients, and 15 nucleotide changes were reported. The role of this variations was investigated by in-silico. The harmful mutations were predicted based on some servers. The molecular dynamics results showed that there is a significant relationship between the mutant protein and the changes in the structural conformation. Our results showed that the mutation in the ND4 area plays an important role in developing breast cancer. So, it can be concluded that the mitochondrial NADH dehydrogenase analysis may help to detect breast cancer in the early stages., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Groundwater arsenic contamination has become a serious global concern due to its adverse effects on human health. Arsenic-induced reactive oxygen species trigger oxidative stress inside mitochondria, which initiate a cascade of events including altered mitochondrial (mt) membrane potential, uncoupling of electron transport chain, and mtDNA damage. A case-control study was conducted to examine the association between arsenic exposure and differences in mtDNA methylation and to assess the downstream consequences. We recruited 221 arsenic-exposed individuals, including 106 individuals with skin lesions (WSL) and 115 subjects without any skin lesions (WOSL) from the Murshidabad district, West Bengal, India. The unexposed group included 101 individuals from the arsenic unexposed area in East Midnapore. We analyzed the status of mtDNA methylation in D-loop region and ND6 gene by methylation-specific PCR. Gene expression was studied by quantitative real-time PCR. Significant hypomethylation in both D-loop and ND6 was observed with a consequent increase in their target gene expression and higher mtDNA copy number in arsenic-exposed populations compared to controls. Further mechanistic insights regarding mitochondrial epigenetic alteration in arsenic exposure will be of critical importance for the prevention of adverse health effects., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The effects of triterpenic acids-enriched fraction from Cyclocarya paliurus (CPT) on nonalcoholic fatty liver disease (NAFLD) were investigated using in vivo and in vitro models. In high fat diet-induced Wister rats, CPT significantly increased superoxide dismutase (SOD) activity and glutathione/oxidized glutathione (GSH/GSSG) ratio, reduced malondialdehyde (MDA) and protein carbonyl (PCO) levels. Moreover, CPT restored mitochondrial membrane potential dysfunction, decreased cytochrome P450 enzyme 2E1 (CYP2E1) activity, improved nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2-mediated antioxidant enzyme heme oxygenase1 (HO-1) expression. In free fatty acids-induced HepG2 cells, CPT dramatically decreased ROS content, increased mitochondrial NADH dehydrogenase (Complex I) and mitochondrial cytochrome C oxidase (Complex IV) levels. Furthermore, CPT could upregulate HO-1, quinine oxidoreductase 1 (NQO1) expression, and increase Nrf2 translocation from cytoplasm-to-nucleus. The results indicated CPT could protect mitochondria function and improve oxidative stress by activating Nrf2. Therefore, it can be inferred that CPT may be a potential agent against NAFLD., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Rapid protein degradation and replacement is an important response to photodamage and a means of photoprotection by recovering proteostasis. Protein turnover and translation efficiency studies have discovered fast turnover subunits in cytochrome b 6 f and the NAD(P)H dehydrogenase (NDH) complex, in addition to PSII subunit D1. Mutations of these complexes have been linked to enhanced photodamage at least partially via cyclic electron flow. Photodamage and photoprotection involving cytochrome b 6 f, NDH complex, cyclic electron flow, PSI, and nonphotochemical quenching proteins have been reported. Here, we propose that the rapid turnover of specific proteins in cytochrome b 6 f and the NDH complex need to be characterised and compared with the inhibition of PSII by excess excitation energy and PSI by excess electron flux to expand our understanding of photoinhibition mechanisms., (Copyright © 2018. Published by Elsevier Ltd.)

For optimal plant growth, carbon and nitrogen availability needs to be tightly coordinated. Mitochondrial perturbations related to a defect in complex I in the Arabidopsis thaliana frostbite1 ( fro1 ) mutant, carrying a point mutation in the 8-kD Fe-S subunit of NDUFS4 protein, alter aspects of fundamental carbon metabolism, which is manifested as stunted growth. During nitrate nutrition, fro1 plants showed a dominant sugar flux toward nitrogen assimilation and energy production, whereas cellulose integration in the cell wall was restricted. However, when cultured on NH₄⁺ as the sole nitrogen source, which typically induces developmental disorders in plants (i.e., the ammonium toxicity syndrome), fro1 showed improved growth as compared to NO₃ - nourishing. Higher energy availability in fro1 plants was correlated with restored cell wall assembly during NH₄⁺ growth. To determine the relationship between mitochondrial complex I disassembly and cell wall-related processes, aspects of cell wall integrity and sugar and reactive oxygen species signaling were analyzed in fro1 plants. The responses of fro1 plants to NH₄⁺ treatment were consistent with the inhibition of a form of programmed cell death. Resistance of fro1 plants to NH₄⁺ toxicity coincided with an absence of necrotic lesion in plant leaves.

Energy generation is a promising area of drug discovery for both bacterial pathogens and parasites. Type II NADH dehydrogenase (NDH-2), a vital respiratory membrane protein, has attracted attention as a target for the development of new antitubercular and antimalarial agents. To date, however, no potent, specific inhibitors have been identified. Here, we performed a site-directed screening technique, tethering-fragment based drug discovery, against wild-type and mutant forms of NDH-2 containing engineered active-site cysteines. Inhibitory fragments displayed IC 50 values between 3 and 110 μM against NDH-2 mutants. Possible binding poses were investigated by in silico modelling, providing a basis for optimisation of fragment binding and improved potency against NDH-2., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The extraordinary physicochemical properties of graphene-based nanomaterials (GBNs) make them promising tools in nanotechnology and biomedicine. Considering the skin contact as one of the most feasible exposure routes to GBNs, the mechanism of toxicity of two GBNs (few-layer-graphene, FLG, and graphene oxide, GO) towards human HaCaT skin keratinocytes was investigated. Both materials induced a significant mitochondrial membrane depolarization: 72 h cell exposure to 100 μg mL-1 FLG or GO increased mitochondrial depolarization by 44% and 56%, respectively, while the positive control valinomycin (0.1 μg mL-1) increased mitochondrial depolarization by 48%. Since the effect was not prevented by cyclosporine-A, it appears to be unrelated to mitochondrial transition pore opening. By contrast, it seems to be mediated by reactive oxygen species (ROS) production: FLG and GO induced time- and concentration-dependent cellular ROS production, significant already at the concentration of 0.4 μg mL-1 after 24 h exposure. Among a panel of specific inhibitors of the major ROS-producing enzymes, diphenyliodonium, rotenone and allopurinol significantly reverted or even abolished FLG- or GO-induced ROS production. Intriguingly, the same inhibitors also significantly reduced FLG- or GO-induced mitochondrial depolarization and cytotoxicity. This study shows that FLG and GO induce a cytotoxic effect due to a sustained mitochondrial depolarization. This seems to be mediated by a significant cellular ROS production, caused by the activation of flavoprotein-based oxidative enzymes, such as NADH dehydrogenase and xanthine oxidase.

The opportunistic pathogen Streptococcus agalactiae is the major cause of meningitis and sepsis in a newborn's first week, as well as a considerable cause of pneumonia, urinary tract infections, and sepsis in immunocompromised adults. This pathogen respires aerobically if heme and quinone are available in the environment, and a functional respiratory chain is required for full virulence. Remarkably, it is shown here that the entire respiratory chain of S. agalactiae consists of only two enzymes, a type 2 NADH dehydrogenase (NDH-2) and a cytochrome bd oxygen reductase. There are no respiratory dehydrogenases other than NDH-2 to feed electrons into the respiratory chain, and there is only one respiratory oxygen reductase to reduce oxygen to water. Although S. agalactiae grows well in vitro by fermentative metabolism, it is shown here that the absence of NDH-2 results in attenuated virulence, as observed by reduced colonization in heart and kidney in a mouse model of systemic infection. The lack of NDH-2 in mammalian mitochondria and its important role for virulence suggest this enzyme may be a potential drug target. For this reason, in this study, S. agalactiae NDH-2 was purified and biochemically characterized, and the isolated enzyme was used to screen for inhibitors from libraries of FDA-approved drugs. Zafirlukast was identified to successfully inhibit both NDH-2 activity and aerobic respiration in intact cells. This compound may be useful as a laboratory tool to inhibit respiration in S. agalactiae and, since it has few side effects, it might be considered a lead compound for therapeutics development. IMPORTANCE S. agalactiae is part of the human intestinal microbiota and is present in the vagina of ~30% of healthy women. Although a commensal, it is also the leading cause of septicemia and meningitis in neonates and immunocompromised adults. This organism can aerobically respire, but only using external sources of heme and quinone, required to have a functional electron transport chain. Although bacteria usually have a branched respiratory chain with multiple dehydrogenases and terminal oxygen reductases, here we establish that S. agalactiae utilizes only one type 2 NADH dehydrogenase (NDH-2) and one cytochrome bd oxygen reductase to perform respiration. NADH-dependent respiration plays a critical role in the pathogen in maintaining NADH/NAD + redox balance in the cell, optimizing ATP production, and tolerating oxygen. In summary, we demonstrate the essential role of NDH-2 in respiration and its contribution to S. agalactiae virulence and propose it as a potential drug target., (Copyright © 2018 Lencina et al.)

Cells respond to iron deficiency by activating iron-regulatory proteins to increase cellular iron uptake and availability. However, it is not clear how cells adapt to conditions when cellular iron uptake does not fully match iron demand. Here, we show that the mRNA-binding protein tristetraprolin (TTP) is induced by iron deficiency and degrades mRNAs of mitochondrial Fe/S-cluster-containing proteins, specifically Ndufs1 in complex I and Uqcrfs1 in complex III, to match the decrease in Fe/S-cluster availability. In the absence of TTP, Uqcrfs1 levels are not decreased in iron deficiency, resulting in nonfunctional complex III, electron leakage, and oxidative damage. Mice with deletion of Ttp display cardiac dysfunction with iron deficiency, demonstrating that TTP is necessary for maintaining cardiac function in the setting of low cellular iron. Altogether, our results describe a pathway that is activated in iron deficiency to regulate mitochondrial function to match the availability of Fe/S clusters., Competing Interests: The authors declare no conflict of interest.

Acute O 2 sensing by peripheral chemoreceptors is essential for mammalian homeostasis. Carotid body glomus cells contain O 2 -sensitive ion channels, which trigger fast adaptive cardiorespiratory reflexes in response to hypoxia. O 2 -sensitive cells have unique metabolic characteristics that favor the hypoxic generation of mitochondrial complex I (MCI) signaling molecules, NADH and reactive oxygen species (ROS), which modulate membrane ion channels. We show that responsiveness to hypoxia progressively disappears after inducible deletion of the Ndufs2 gene, which encodes the 49 kDa subunit forming the coenzyme Q binding site in MCI, even in the presence of MCII substrates and chemical NAD + regeneration. We also show contrasting effects of physiological hypoxia on mitochondrial ROS production (increased in the intermembrane space and decreased in the matrix) and a marked effect of succinate dehydrogenase activity on acute O 2 sensing. Our results suggest that acute responsiveness to hypoxia depends on coenzyme QH 2 /Q ratio-controlled ROS production in MCI., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Resurrection plants, which are the "gifts" of natural evolution, are ideal models for studying the genetic basis of plant desiccation tolerance. Here, we report a high-quality genome assembly of 301 Mb for the diploid spike moss Selaginella tamariscina, a primitive vascular resurrection plant. We predicated 27 761 protein-coding genes from the assembled S. tamariscina genome, 11.38% (2363) of which showed significant expression changes in response to desiccation. Approximately 60.58% of the S. tamariscina genome was annotated as repetitive DNA, which is an almost 2-fold increase of that in the genome of desiccation-sensitive Selaginella moellendorffii. Genomic and transcriptomic analyses highlight the unique evolution and complex regulations of the desiccation response in S. tamariscina, including species-specific expansion of the oleosin and pentatricopeptide repeat gene families, unique genes and pathways for reactive oxygen species generation and scavenging, and enhanced abscisic acid (ABA) biosynthesis and potentially distinct regulation of ABA signaling and response. Comparative analysis of chloroplast genomes of several Selaginella species revealed a unique structural rearrangement and the complete loss of chloroplast NAD(P)H dehydrogenase (NDH) genes in S. tamariscina, suggesting a link between the absence of the NDH complex and desiccation tolerance. Taken together, our comparative genomic and transcriptomic analyses reveal common and species-specific desiccation tolerance strategies in S. tamariscina, providing significant insights into the desiccation tolerance mechanism and the evolution of resurrection plants., (Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.)

Complex I (NADH:ubiquinone oxidoreductase) uses the reducing potential of NADH to drive protons across the energy-transducing inner membrane and power oxidative phosphorylation in mammalian mitochondria. Recent cryo-EM analyses have produced near-complete models of all 45 subunits in the bovine, ovine and porcine complexes and have identified two states relevant to complex I in ischemia-reperfusion injury. Here, we describe the 3.3-Å structure of complex I from mouse heart mitochondria, a biomedically relevant model system, in the 'active' state. We reveal a nucleotide bound in subunit NDUFA10, a nucleoside kinase homolog, and define mechanistically critical elements in the mammalian enzyme. By comparisons with a 3.9-Å structure of the 'deactive' state and with known bacterial structures, we identify differences in helical geometry in the membrane domain that occur upon activation or that alter the positions of catalytically important charged residues. Our results demonstrate the capability of cryo-EM analyses to challenge and develop mechanistic models for mammalian complex I.

Although mitochondriotoxicity plays a major role in drug-induced hepatotoxicity, alteration of mitochondrial DNA (mtDNA) homeostasis has been described only with a few drugs. Because it requires long drug exposure, this mechanism of toxicity cannot be detected with investigations performed in isolated liver mitochondria or cultured cells exposed to drugs for several hours or a few days. Thus, a first aim of this study was to determine whether a 2-week treatment with nine hepatotoxic drugs could affect mtDNA homeostasis in HepaRG cells. Previous investigations with these drugs showed rapid toxicity on oxidative phosphorylation but did not address the possibility of delayed toxicity secondary to mtDNA homeostasis impairment. The maximal concentration used for each drug induced about 10% cytotoxicity. Two other drugs, zalcitabine and linezolid, were used as positive controls for their respective effects on mtDNA replication and translation. Another goal was to determine whether drug-induced mitochondriotoxicity could be modulated by lipid overload mimicking nonalcoholic fatty liver. Among the nine drugs, imipramine and ritonavir induced mitochondrial effects suggesting alteration of mtDNA translation. Ritonavir toxicity was stronger in nonsteatotic cells. None of the nine drugs decreased mtDNA levels. However, increased mtDNA was observed with five drugs, especially in nonsteatotic cells. The mtDNA levels could not be correlated with the expression of key factors involved in mitochondrial biogenesis, such as peroxisome proliferator-activated receptor- γ coactivator 1 α (PGC1 α ), PGC1 β , and AMP-activated protein kinase α -subunit. Hence, drug-induced impairment of mtDNA translation might not be rare, and increased mtDNA levels could be a frequent adaptive response to slight energy shortage. Nevertheless, this adaptation could be impaired by lipid overload., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

The mitochondrial respiratory chain in vertebrates and arthropods is different from that of most other eukaryotes because they lack alternative enzymes that provide electron transfer pathways additional to the oxidative phosphorylation (OXPHOS) system. However, the use of diverse experimental models, such as human cells in culture, Drosophila melanogaster and the mouse, has demonstrated that the transgenic expression of these alternative enzymes can impact positively many phenotypes associated with human mitochondrial and other cellular dysfunction, including those typically presented in complex IV deficiencies, Parkinson's, and Alzheimer's. In addition, these enzymes have recently provided extremely valuable data on how, when, and where reactive oxygen species, considered by many as "by-products" of OXPHOS, can contribute to animal longevity. It has also been shown that the expression of the alternative enzymes is thermogenic in cultured cells, causes reproductive defects in flies, and enhances the deleterious phenotype of some mitochondrial disease models. Therefore, all the reported beneficial effects must be considered with caution, as these enzymes have been proposed to be deployed in putative gene therapies to treat human diseases. Here, we present a brief review of the scientific data accumulated over the past decade that show the benefits and the risks of introducing alternative branches of the electron transport into mammalian and insect mitochondria, and we provide a perspective on the future of this research field., (© 2018 International Federation for Cell Biology.)

Shewanella oneidensis MR-1 is a metal-reducing bacterium with the ability to utilize many different terminal electron acceptors, including oxygen and solid-metal oxides. Both metal oxide reduction and aerobic respiration have been studied extensively in this organism. However, electron transport chain processes upstream of the terminal oxidoreductases have been relatively understudied in this organism, especially electron transfer from NADH to respiratory quinones. Genome annotation indicates that S. oneidensis MR-1 encodes four NADH dehydrogenases, a proton-translocating dehydrogenase (Nuo), two sodium ion-translocating dehydrogenases (Nqr1 and Nqr2), and an "uncoupling" dehydrogenase (Ndh), but none of these complexes have been studied. Therefore, we conducted a study specifically focused on the effects of individual NADH dehydrogenase knockouts in S. oneidensis MR-1. We observed that two of the single-mutant strains, the Δ nuoN and Δ nqrF1 mutants, exhibited significant growth defects compared with the wild type. However, the defects were minor and only apparent under certain growth conditions. Further testing of the Δ nuoN Δ nqrF1 double-mutant strain yielded no growth in minimal medium under oxic conditions, indicating that Nuo and Nqr1 have overlapping functions, but at least one is necessary for aerobic growth. Coutilization of proton- and sodium ion-dependent energetics has important implications for the growth of this organism in environments with varied pH and salinity, including microbial electrochemical systems. IMPORTANCE Bacteria utilize a wide variety of metabolic pathways that allow them to take advantage of different energy sources, and to do so with varied efficiency. The efficiency of a metabolic process determines the growth yield of an organism, or the amount of biomass it produces per amount of substrate consumed. This parameter has important implications in biotechnology and wastewater treatment, where low growth yields are often preferred to minimize the production of microbial biomass. In this study, we investigated respiratory pathways containing NADH dehydrogenases with varied efficiency (i.e., the number of ions translocated per NADH oxidized) in the metal-reducing bacterium Shewanella oneidensis MR-1. We observed that two different respiratory pathways are used concurrently, and at least one pathway must be functional for growth under oxic conditions., (Copyright © 2018 Duhl et al.)

A decrease in the specific activity of an enzyme is commonly observed when the enzyme is inappropriately handled or is stored over an extended period. Here, we reported a functional transition of an FMN-bound diaphorase (FMN-DI) that happened during the long-term storage process. It was found that FMN-DI did not simply lose its β-nicotinamide adenine diphosphate (NADH) dehydrogenase activity after a long-time storage, but obtained a new enzyme activity of NADH oxidase. Further mechanistic studies suggested that the alteration of the binding strength of an FMN cofactor with a DI protein could be responsible for this functional switch of the enzyme., (© 2017 International Union of Biochemistry and Molecular Biology, Inc.)

The coordination of protein synthesis and degradation regulating protein abundance is a fundamental process in cellular homeostasis. Today, mass spectrometry-based technologies allow determination of endogenous protein turnover on a proteome-wide scale. However, standard dynamic SILAC (Stable Isotope Labeling in Cell Culture) approaches can suffer from missing data across pulse time-points limiting the accuracy of such analysis. This issue is of particular relevance when studying protein stability at the level of proteoforms because often only single peptides distinguish between different protein products of the same gene. To address this shortcoming, we evaluated the merits of combining dynamic SILAC and tandem mass tag (TMT)-labeling of ten pulse time-points in a single experiment. Although the comparison to the standard dynamic SILAC method showed a high concordance of protein turnover rates, the pulsed SILAC-TMT approach yielded more comprehensive data (6000 proteins on average) without missing values. Replicate analysis further established that the same reproducibility of turnover rate determination can be obtained for peptides and proteins facilitating proteoform resolved investigation of protein stability. We provide several examples of differentially turned over splice variants and show that post-translational modifications can affect cellular protein half-lives. For example, N-terminally processed peptides exhibited both faster and slower turnover behavior compared with other peptides of the same protein. In addition, the suspected proteolytic processing of the fusion protein FAU was substantiated by measuring vastly different stabilities of the cleavage products. Furthermore, differential peptide turnover suggested a previously unknown mechanism of activity regulation by post-translational destabilization of cathepsin D as well as the DNA helicase BLM. Finally, our comprehensive data set facilitated a detailed evaluation of the impact of protein properties and functions on protein stability in steady-state cells and uncovered that the high turnover of respiratory chain complex I proteins might be explained by oxidative stress., (© 2018 Zecha et al.)

Reactive oxygen species (ROS) produced in vivo during various electron transfer reactions are generally kept at a certain level since they are harmful to cells. However, it can sensitize hematopoietic progenitors to differentiation, and plays a signaling role in the regulation of hematopoietic cell fate. In the present study, the transcriptomes of crab HPT and hemocytes were sequenced using the Ion Torrent Proton sequencing platform. A total of 51,229,690 single end reads were obtained from six single-end libraries, which were assembled into 31346 unireads as reference. After mapping and transcript assembling, 362 differently expressed genes were identified and 301 of them were deemed to be more abundant in HPT. GO annotation revealed that they were mostly implicated in DNA, RNA and protein synthesis, cell division, mitochondria activities and energy metabolism. The expression level of mitochondrial complexes I (mitochondrial NADH-ubiquinone oxidoreductase) which was the main natural producers of mitochondrial ROS was found to be 8.6-fold (p < 0.01) higher in HPT than that in hemocytes. In hemocytes, the proteinase genes associated with proPO activation from the 61 up-regulated genes in hemocytes were the main up-regulated genes which might be the potential markers for mature hemocytes. ROS level in HPT cells was relatively higher which was confirmed with the high expression level of mitochondria related genes identified by transcriptome sequencing. After the ROS level was depressed by N-acetyl-l-cysteine (NAC), the production of hemocytes from HPT was inhibited, and the recovery of the total hemocytes counts was delayed. These results collectively indicated that the genes in redox system were more active in HPT, and ROS could function as an important modulator in the hematopoiesis of crab and promote the production of hemocytes from HPT., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Evidence has demonstrated iron accumulation in specific brain regions of patients suffering from neurodegenerative disorders, and this metal has been recognized as a contributing factor for neurodegeneration. Using an experimental model of brain iron accumulation, we have shown that iron induces severe memory deficits that are accompanied by oxidative stress, increased apoptotic markers, and decreased synaptophysin in the hippocampus of rats. The present study aims to characterize iron loading effects as well as to determine the molecular targets of cannabidiol (CBD), the main non-psychomimetic compound of Cannabis sativa, on mitochondria. Rats received iron in the neonatal period and CBD for 14 days in adulthood. Iron induced mitochondrial DNA (mtDNA) deletions, decreased epigenetic modulation of mtDNA, mitochondrial ferritin levels, and succinate dehydrogenase activity. CBD rescued mitochondrial ferritin and epigenetic modulation of mtDNA, and restored succinate dehydrogenase activity in iron-treated rats. These findings provide new insights into molecular targets of iron neurotoxicity and give support for the use of CBD as a disease modifying agent in the treatment of neurodegenerative diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Asthenozoospermia (AS), an important cause of male infertility, is characterized by reduced sperm motility. Among the aetiologies of AS, inflammation seems to be the main cause. DJ-1, a conserved protein product of the PARK7 gene, is associated with male infertility and plays a role in oxidative stress and inflammation. Although our previous studies showed that a reduction in DJ-1 was accompanied by mitochondrial dysfunction in the sperm of patients with AS, the specific mechanism underlying this association remained unclear. In this study, we found that compared to the patients without AS, the expression of mitochondrial protein nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3) was also significantly decreased in the sperm of patients with AS. Similarly, decreased expression of DJ-1 and NDUFS3 and reduced mitochondria complex I activity were evident in a rat model of AS. Moreover, we showed that the interaction between DJ-1 and NDUFS3 in rat testes was weakened by ORN treatment. These results suggest that the impaired mitochondrial activity could be due to the broken interaction between DJ-1 and NDUFS3 and that downregulation of DJ-1 in sperm and testes contributes to AS pathogenesis.

Proper mitochondrial activity depends upon proteins encoded by genes in the nuclear and mitochondrial genomes that must interact functionally and physically in a precisely coordinated manner. Consequently, mito-nuclear allelic interactions are thought to be of crucial importance on an evolutionary scale, as well as for manifestation of essential biological phenotypes, including those directly relevant to human disease. Nonetheless, detailed molecular understanding of mito-nuclear interactions is still lacking, and definitive examples of such interactions in vivo are sparse. Here we describe the characterization of a mutation in Drosophila ND23 , a nuclear gene encoding a highly conserved subunit of mitochondrial complex 1. This characterization led to the discovery of a mito-nuclear interaction that affects the ND23 mutant phenotype. ND23 mutants exhibit reduced lifespan, neurodegeneration, abnormal mitochondrial morphology, and decreased ATP levels. These phenotypes are similar to those observed in patients with Leigh syndrome, which is caused by mutations in a number of nuclear genes that encode mitochondrial proteins, including the human ortholog of ND23 A key feature of Leigh syndrome, and other mitochondrial disorders, is unexpected and unexplained phenotypic variability. We discovered that the phenotypic severity of ND23 mutations varies depending on the maternally inherited mitochondrial background. Sequence analysis of the relevant mitochondrial genomes identified several variants that are likely candidates for the phenotypic interaction with mutant ND23 , including a variant affecting a mitochondrially encoded component of complex I. Thus, our work provides an in vivo demonstration of the phenotypic importance of mito-nuclear interactions in the context of mitochondrial disease., (Copyright © 2018 by the Genetics Society of America.)