Subunit Of NADH-ubiquinone Oxidoreductase Research Articles

Mitochondrially Targeted Gene Therapy Rescues Visual Loss in a Mouse Model of Leber's Hereditary Optic Neuropathy.

Leber's hereditary optic neuropathy (LHON) is a common mitochondrial genetic disease, causing irreversible blindness in young individuals. Current treatments are inadequate, and there is no definitive cure. This study evaluates the effectiveness of delivering wildtype human NADH ubiquinone oxidoreductase subunit 4 (hND4) gene using mito-targeted AAV(MTSAAV) to rescue LHOH mice. We observed a declining pattern in electroretinograms amplitudes as mice aged across all groups (p < 0.001), with significant differences among groups (p = 0.023; Control vs. LHON, p = 0.008; Control vs. Rescue, p = 0.228). Inner retinal thickness and intraocular pressure did not change significantly with age or groups. Compared to LHON mice, those rescued with wildtype hND4 exhibited improved retinal visual acuity (0.29 ± 0.1 cy/deg vs. 0.15 ± 0.1 cy/deg) and increased functional hyperemia response (effect of flicker, p < 0.001, effect of Group, p = 0.004; Interaction Flicker × Group, p < 0.001). Postmortem analysis shows a marked reduction in retinal ganglion cell density in the LHON group compared to the other groups (Effect of Group, p < 0.001, Control vs. LHON, p < 0.001, Control vs. Rescue, p = 0.106). These results suggest that MTSAAV-delivered wildtype hND4 gene rescues, at least in part, visual impairment in an LHON mouse model and has the therapeutic potential to treat this disease.

High expression of NADH Ubiquinone Oxidoreductase Subunit B11 induces catheter-associated venous thrombosis on continuous blood purification.

Venous thromboembolism (VTE) is a common vascular disease of venous return disorders, including deep venous thrombosis and pulmonary embolism (PE), with high morbidity and high mortality. However, the relationship between oxidative phosphorylation and NDUFB11 and venous thromboembolism is still unclear. The venous thromboembolism datasets GSE48000 and GSE19151 were downloaded, and the differentially expressed Genes (DEGs) were screened. The protein-protein interaction (PPI) network was constructed. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for functional enrichment analysis. The comparative toxicogenomics database (CTD) was used to identify the diseases most associated with the core genes. TargetScan was used to screen miRNA regulating central DEGs. Western blotting (WB) experiment and real-time quantitative PCR (RT-qPCR) experiment were performed. A total of 500 DEGs were identified. GO analysis showed that the DEGs were mainly enriched in ATP synthesis coupled electron transport, respiratory electron transport chain, cytoplasm, enzyme binding, nonalcoholic fatty liver disease, oxidative phosphorylation, and Alzheimer disease. Enrichment items were similar to GO and KEGG enrichment items of DEGs. The result of CTD showed that 12 genes (RPS24, FAU, RPLP0, RPS15A, RPS29, RPL9, RPL31, RPL27, NDUFB11, RPL34, COX7B, RPS27L) were associated with chemical and drug-induced liver injury, inflammation, kidney disease, and congenital pure red cell aplasia. WB and RT-qPCR results showed that the expression levels of 12 genes in venous thromboembolism were higher than normal whole blood tissue samples. NDUFB11 is highly expressed in catheter-related venous thromboembolism during continuous blood purification, which may lead to the formation of venous thrombosis through oxidative phosphorylation pathway.

Age-related flexibility of energetic metabolism in the honey bee Apis mellifera.

The mechanisms that underpin aging are still elusive. In this study, we suggest that the ability of mitochondria to oxidize different substrates, which is known as metabolic flexibility, is involved in this process. To verify our hypothesis, we used honey bees (Apis mellifera carnica) at different ages, to assess mitochondrial oxygen consumption and enzymatic activities of key enzymes of the energetic metabolism as well as ATP5A1 content (subunit of ATP synthase) and adenylic energy charge (AEC). We also measured mRNA abundance of genes involved in mitochondrial functions and the antioxidant system. Our results demonstrated that mitochondrial respiration increased with age and favored respiration through complexes I and II of the electron transport system (ETS) while glycerol-3-phosphate (G3P) oxidation was relatively decreased. In addition, glycolytic, tricarboxylic acid cycle and ETS enzymatic activities increased, which was associated with higher ATP5A1 content and AEC. Furthermore, we detected an early decrease in the mRNA abundance of subunits of NADH ubiquinone oxidoreductase subunit B2 (NDUFB2, complex I), mitochondrial cytochrome b (CYTB, complex III) of the ETS as well as superoxide dismutase 1 and a later decrease for vitellogenin, catalase and mitochondrial cytochrome c oxidase subunit 1 (COX1, complex IV). Thus, our study suggests that the energetic metabolism is optimized with aging in honey bees, mainly through quantitative and qualitative mitochondrial changes, rather than showing signs of senescence. Moreover, aging modulated metabolic flexibility, which might reflect an underpinning mechanism that explains lifespan disparities between the different castes of worker bees.

Breeding effects on durum wheat traits detected using GWAS and haplotype block analysis.

The recent boosting of genomic data in durum wheat (Triticum turgidum subsp. durum) offers the opportunity to better understand the effects of breeding on the genetic structures that regulate the expression of traits of agronomic interest. Furthermore, the identification of DNA markers useful for marker-assisted selection could also improve the reliability of technical protocols used for variety protection and registration. Within this motivation context, 123 durum wheat accessions, classified into three groups: landraces (LR), ancient (OC) and modern cultivars (MC), were evaluated in two locations, for 34 agronomic traits, including UPOV descriptors, to assess the impact of changes that occurred during modern breeding. The association mapping analysis, performed with 4,241 SNP markers and six multi-locus-GWAS models, revealed 28 reliable Quantitative Trait Nucleotides (QTNs) related to plant morphology and kernel-related traits. Some important genes controlling flowering time and plant height were in linkage disequilibrium (LD) decay with QTNs identified in this study. A strong association for yellow berry was found on chromosome 6A (Q.Yb-6A) in a region containing the nadh-ubiquinone oxidoreductase subunit, a gene involved in starch metabolism. The Q.Kcp-2A harbored the PPO locus, with the associated marker (Ku_c13700_1196) in LD decay with Ppo-A1 and Ppo-A2. Interestingly, the Q.FGSGls-2B.1, identified by RAC875_c34512_685 for flag leaf glaucosity, mapped less than 1 Mb from the Epistatic inhibitors of glaucousness (Iw1), thus representing a good candidate for supporting the morphological DUS traits also with molecular markers. LD haplotype block approach revealed a higher diversity, richness and length of haploblocks in MC than OC and LR (580 in LR, 585 in OC and 612 in MC), suggesting a possible effect exerted by breeding programs on genomic regions associated with the agronomic traits. Our findings pave new ways to support the phenotypic characterization necessary for variety registration by using a panel of cost-effectiveness SNP markers associated also to the UPOV descriptors. Moreover, the panel of associated SNPs might represent a reservoir of favourable alleles to use in durum wheat breeding and genetics.

Gene therapy restores mitochondrial function and protects retinal ganglion cells in optic neuropathy induced by a mito-targeted mutant ND1 gene

Therapies for genetic disorders caused by mutated mitochondrial DNA are an unmet need, in large part due barriers in delivering DNA to the organelle and the absence of relevant animal models. We injected into mouse eyes a mitochondrially targeted Adeno-Associated-Virus (MTS-AAV) to deliver the mutant human NADH ubiquinone oxidoreductase subunit I (hND1/m.3460G>A) responsible for Leber’s hereditary optic neuropathy, the most common primary mitochondrial genetic disease. We show that the expression of the mutant hND1 delivered to retinal ganglion cells (RGC) layer colocalizes with the mitochondrial marker PORIN and the assembly of the expressed hND1 protein into host respiration complex I. The hND1 injected eyes exhibit hallmarks of the human disease with progressive loss of RGC function and number, as well as optic nerve degeneration. We also show that gene therapy in the hND1 eyes by means of an injection of a second MTS-AAV vector carrying wild type human ND1 restores mitochondrial respiratory complex I activity, the rate of ATP synthesis and protects RGCs and their axons from dysfunction and degeneration. These results prove that MTS-AAV is a highly efficient gene delivery approach with the ability to create mito-animal models and has the therapeutic potential to treat mitochondrial genetic diseases.

Gene biomarker prediction in glioma by integrating scRNA-seq data and gene regulatory network

BackgroundAlthough great efforts have been made to study the occurrence and development of glioma, the molecular mechanisms of glioma are still unclear. Single-cell sequencing technology provides a new perspective for researchers to explore the pathogens of tumors to further help make treatment and prognosis decisions for patients with tumors.MethodsIn this study, we proposed an algorithm framework to explore the molecular mechanisms of glioma by integrating single-cell gene expression profiles and gene regulatory relations. First, since there were great differences among malignant cells from different glioma samples, we analyzed the expression status of malignant cells for each sample, and then tumor consensus genes were identified by constructing and analyzing cell-specific networks. Second, to comprehensively analyze the characteristics of glioma, we integrated transcriptional regulatory relationships and consensus genes to construct a tumor-specific regulatory network. Third, we performed a hybrid clustering analysis to identify glioma cell types. Finally, candidate tumor gene biomarkers were identified based on cell types and known glioma-related genes.ResultsWe got six identified cell types using the method we proposed and for these cell types, we performed functional and biological pathway enrichment analyses. The candidate tumor gene biomarkers were analyzed through survival analysis and verified using literature from PubMed.ConclusionsThe results showed that these candidate tumor gene biomarkers were closely related to glioma and could provide clues for the diagnosis and prognosis of patients with glioma. In addition, we found that four of the candidate tumor gene biomarkers (NDUFS5, NDUFA1, NDUFA13, and NDUFB8) belong to the NADH ubiquinone oxidoreductase subunit gene family, so we inferred that this gene family may be strongly related to glioma.

Cloperastine inhibits esophageal squamous cell carcinoma proliferation in vivo and in vitro by suppressing mitochondrial oxidative phosphorylation

Esophageal squamous cell carcinoma (ESCC) is a major type of esophageal cancer. The prognosis of patients with ESCC remains poor because of the high morbidity and mortality of the disease. One strategy for drug discovery for ESCC treatment or prevention is screening FDA-approved drugs. In the present study, we found that the antitussive agent cloperastine can inhibit the proliferation of ESCC cells. However, the underlying mechanism was unclear. To determine the mechanism of this inhibitory effect, we performed proteomic analysis using KYSE150 cells treated with cloperastine and DMSO. The results identified several down-regulated signaling pathways included those of three key proteins (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex 1, NADH ubiquinone oxidoreductase subunit S5, and cytochrome C oxidase subunit 6B1) involved in oxidative phosphorylation. Meanwhile, we observed that oxidative phosphorylation in mitochondria was inhibited by the drug. Importantly, cloperastine suppressed ESCC growth in a xenograft mouse model in vivo. Our findings revealed that cloperastine inhibits the proliferation of ESCC in vivo and in vitro by suppressing mitochondrial oxidative phosphorylation.

Changes in Metabolite and Protein Expression Profiles of Atypical Dark-cutting and Normal-pH Beef

Abstract The color of meat is an important deciding factor in consumers’ assessment of meat quality. To meat buyers, the bright cherry-red color of meat indicates freshness and wholesomeness. However, atypical dark-cutting beef represents dark-colored meat with a muscle pH between 5.6 and 5.8. Although previous studies have indicated that the ultimate pH of atypical dark-cutting beef is greater than normal, the mechanistic basis for the occurrence is not clear. Therefore, the objective of this study was to identify protein and metabolite profiles of normal and atypical dark-cutting beef. Longissimus thoracic (LT) muscles from 12 different animals (6 atypical dark-cutters and 6 normal-pH beef) were analyzed by comparing changes in metabolites and protein profiles using GC-MS non-targeted metabolomics and LC-MS/MS-based proteomics, respectively. Metabolomics analysis showed down-regulation of glycolytic metabolites such as glucose-6-phosphate, lactose, and glucose, indicating a reduced capacity to accumulate lactate postmortem in atypical dark-cutters compared with normal-pH beef. Mass spectrometry analysis identified 66 proteins with significant changes in protein expression profiles (P &amp;lt; 0.05) between atypical and normal-pH beef. Of these, 22 proteins were up-regulated while 14 were down-regulated in atypical dark-cutting beef. Functional annotation and protein-protein interaction network analyses revealed that up-regulated proteins such as NADH ubiquinone oxidoreductase subunit A7, ATPase Na+/K+ transporting subunit alpha 2, 3-oxoacid CoA-transferase 1, cytochrome b5 reductase 3, actinin alpha 4, tropomyosin 2, heat shock protein beta-7, and DnaJ heat shock protein family (Hsp40) member B4 are involved in energy synthesis, muscle contraction and stress responses. By contrast, down-regulated proteins, including phosphofructokinase, glycogen phosphorylase, amylo-alpha-1,6-glucosidase, phosphorylase kinase regulatory subunit alpha and gamma are involved in glycogen metabolism. In summary, lower glycolytic metabolites and glycogen degradation enzymes are associated with differences in muscle pH and color of atypical dark-cutting beef.

Reduced electron transport chain complex I protein abundance and function in Mfn2-deficient myogenic progenitors lead to oxidative stress and mitochondria swelling.

Mitochondrial remodeling through fusion and fission is crucial for progenitor cell differentiation but its role in myogenesis is poorly understood. Here, we characterized the function of mitofusin 2 (Mfn2), a mitochondrial outer membrane protein critical for mitochondrial fusion, in muscle progenitor cells (myoblasts). Mfn2 expression is upregulated during myoblast differentiation in vitro and muscle regeneration in vivo. Targeted deletion of Mfn2 gene in myoblasts (Mfn2MKO ) increases oxygen-consumption rates (OCR) associated with the maximal respiration and spare respiratory capacity, and increased levels of reactive oxygen species (ROS). Skeletal muscles of Mfn2MKO mice exhibit robust mitochondrial swelling with normal mitochondrial DNA content. Additionally, mitochondria isolated from Mfn2MKO muscles have reduced OCR at basal state and for complex I respiration, associated with decreased levels of complex I proteins NDUFB8 (NADH ubiquinone oxidoreductase subunit B8) and NDUFS3 (NADH ubiquinone oxidoreductase subunit S3). However, Mfn2MKO has no obvious effects on myoblast differentiation, muscle development and function, and muscle regeneration. These results demonstrate a novel role of Mfn2 in regulating mitochondrial complex I protein abundance and respiratory functions in myogenic progenitors and myofibers.

CoGRIM19 is required for invasive hyphal growth of Colletotrichum orbiculare inside epidermal cells of cucumber cotyledons

Colletotrichum orbiculare, an anthracnose disease fungus of cucurbit plants, extends penetration hyphae inside the epidermal cells of host plants. Unlike vegetative hyphae formed on a nutrient rich medium, this pathogen initially develops biotrophic penetration hyphae, which acquire nutrient resources from living host cells and secret effector proteins to suppress host defense responses. Subsequently, the nature of penetration hyphae changes from biotrophy to necrotrophy in response to the interaction with a host plant. Hence, controlling the extension of penetration hyphae is crucial for C. orbiculare infection. Here, we identified CoGRIM19 encoding Nadh-ubiquinone oxidoreductase subunit as a pathogenicity gene. Pathogenicity assays showed that the cogrim19 mutant caused no visible symptoms on cucumber cotyledons. Microscopic observations revealed that the cogrim19 mutant developed an appressorium and penetration hyphae under artificial conditions such as on coverslips or cellulose membranes, but the penetration hyphae of the mutant were retarded in the cucumber cotyledons. Microscopic observations of biotrophy-specific expression fluorescent signals revealed that the biotrophic stage was maintained in the retarded penetration hyphae of the cogrim19 mutant as the penetration of the wild type. In addition to cytological observations, pathogenicity assays using wounded leaves showed that the cogrim19 mutant had an attenuated pathogenesis. Taking our results together, CoGRIM19 is required for invasive hyphal growth inside the epidermal cells of cucumber cotyledons in C. orbiculare.

Mitochondrial Transfer of the Mutant Human ND6T14484C Gene Causes Visual Loss and Optic Neuropathy.

PurposeTo evaluate the long-term effects of mitochondrial gene transfer of mutant human NADH ubiquinone oxidoreductase subunit VI (hND6T14484C) in the mouse eye.MethodsAdult mice were injected intravitreally with mitochondrial-targeted adeno-associated virus carrying either hND6T14484C or mitochondrial encoded mCherry. The delivery and expression of the interest gene were detected by polymerase chain reaction (PCR), quantitative PCR (qPCR), and immunostaining. The pathologic effects of the mutant gene in live mice were assessed with RNA-seq, serial spectral domain optical coherence tomography (SD-OCT), and pattern electroretinogram (PERG).ResultsDelivered hND6 was found 30-fold greater than endogenous mouse ND6 in microdissected retinal ganglion cells of hND6-injected mice. Compared to controls injected with mCherry, PERG amplitude of hND6 mice dropped significantly at 3 (P = 0.0023), 6 (P = 0.0058), and 15 (P = 0.031) months after injection. SD-OCT revealed swelling of the optic nerve head followed by the progressive retinal and optic nerve atrophy in hND6 mice. Furthermore, RNA-seq data showed a change in 381 transcripts’ expression in these mice compared to mCherry mice. Postmortem analysis showed hND6 mice had marked atrophy of the entire optic nerve, from the globe to the optic chiasm, and a significant loss of retinal ganglion cells compared to age-matched control mice (P = 1.7E-9).ConclusionsDelivered hND6T14484C induces visual loss and optic neuropathy in mice, the hallmarks of human Leber's hereditary optic neuropathy (LHON).Translational RelevanceResults from this study will help establish a novel strategy not only to generate an LHON animal model but also to provide a potential to treat this or any other mitochondrial diseases.

Resistance to pyridaben in Canadian greenhouse populations of two-spotted spider mites, Tetranychus urticae (Koch)

Two-spotted spider mite (TSSM) Tetranychus urticae (Koch) is an important agricultural pest that causes considerable yield losses to over 150 field and greenhouse crops. Mitochondrial electron transport inhibitors (METI) acaricides are commonly used to control mite species in commercial Canadian greenhouses. Development of resistance to METIs in TSSM populations have been reported worldwide, but not until recently in Canada. The objectives of this study were to: 1) monitor the acaricide-susceptibility in greenhouse TSSM populations, and 2) investigate the resistance to pyridaben, a METI acaricide, in greenhouse resistant and pyridaben-selected (SRS) mite strains. The increased mortality to the pyridaben sub-lethal concentration (LC30) when SRS mites were exposed to piperonyl butoxide (PBO), a general cytochrome P450 monooxygenase inhibitor, and higher P450 activity compared to the greenhouse strain (RS) mites, indicated that P450s may be at least partially responsible for the resistance. The molecular mechanisms of target site insensitivity-mediated resistance in the pyridaben resistant strain of TSSM were investigated by comparing the DNA sequence of NADH dehydrogenase subunits TYKY and PSST, NADH-ubiquinone oxidoreductase chain 1 and 5 (ND1, ND5) and the NADH-ubiquinone oxidoreductase subunit 49 kDa from SRS to the reference strain (SS) and RS. Despite a number of nucleotide substitutions, none correlated with the pyridaben resistance. Understanding the underlying mechanisms of TSSM adaptation to acaricides is an essential part of resistance management strategy in any IPM program. The findings of this study will encourage growers to apply acaricides with different modes of action to reduce the rate at which acaricide resistance will occur in greenhouse TSSM populations.

Anti-stress phytohormones impact on proteome profile of green gram (Vigna radiata) under salt toxicity

Anti-stress phytohormones impact on proteome profile of green gram (Vigna radiata) under salt toxicity

Iron-induced energy supply deficiency and mitochondrial fragmentation in neurons.

Iron dyshomeostasis and mitochondrial impairments are both vitally important for the progression of many neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Nevertheless, how these two pathological phenomena are linked with one another remains unclear, especially in neurons. To address the question, a model of iron overload was established with exposure of rat primary cortical neurons to excessive iron. We first verified that iron overload resulted in a decrease in adenosine triphosphate (ATP) production in neurons. Meanwhile, the release of mitochondrial cytochrome c was significantly increased after iron overload and consequently triggered an apoptosis signal, as revealed by Caspase 3 cleavage. To explore the potential underlying molecular mechanisms, an unlabeled quantitative proteomics approach was applied to primary neurons. Gene Ontology enrichment analysis revealed that 58 mitochondria-associated proteins were significantly altered, including three subunits of mitochondrial complex I and optic atrophy 1(OPA1). Increased NADH-ubiquinone oxidoreductase 75kDa subunit and decreased NADH-ubiquinone oxidoreductase subunit A10 levels were further validated by a western blot, and more importantly, complex I activity markedly declined. Iron-induced down-regulation on the OPA1 level was also validated by a western blot, which was not reversed by the anti-oxidant but was reversed by the iron chelator. Moreover, an OPA1-associated key downstream effect, mitochondrial fragmentation, was found to be aggravated in neurons exposed to excessive iron, which is consistent with the down-regulation of OPA1. Furthermore, the protein level of PTEN-induced putative kinase 1, an important protein closely related to complex I activity and mitochondrial fragmentation, also significantly declined in neurons by iron overload. Thus, our findings may shed new light on the linkage between iron toxicity and mitochondrial impairments, such as energy supply deficiency and mitochondrial fragmentation, and further expand the toxic repertoire of iron in the central nerve system. Cover Image for this issue: doi: 10.1111/jnc.14205.

Role of mitochondrial function in the invasiveness of human colon cancer cells.

We investigated the role of mitochondrial function in the invasiveness of human colorectal cancer (CRC) cell lines, using paired primary SW480 and metastatic SW620 cells, and appraised the clinical relevance of the alteration of mtDNA copy number in 33pairs of CRC specimens after surgical resection. Suppression of mitochondrial function was achieved by the exposure of cells to oligomycinA (OA) or by knockdown of mitochondrial transcriptional factorA (TFAM) to evaluate their effects on energy metabolism, reactive oxygen species, protein expression levels of epithelial-mesenchymal transition (EMT) markers and invasive activity of CRC cells. We found that SW620 cells expressed higher levels of TFAM and mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit6 (ND6) and cytochromec oxidase subunitII (COX-II) and nuclear DNA-encoded NADH ubiquinone oxidoreductase subunitA9 (NDUFA9), iron-sulfur protein subunitB of succinate dehydrogenase (SDHB), ubiquinol‑cytochromec reductase core proteinI/II (UQCRC1/2) and cytochromec oxidase subunitIV (COX-IV) when compared with the SW480 cells. The mtDNA copy number, ADP-triggered oxygen consumption rate (OCR) and respiratory control ratio (RCR) of succinate-supported respiration in the SW620 cells were higher than those noted in the SW480 cells. The intracellular levels of H2O2 and O2-• in the SW620 cells were lower than levels noted in the SW480 cells. Moreover, SW620 cells displayed lower protein levels of hexokinaseII (HK-II), glucose 6-phosphate isomerase (GPI) and lactate dehydrogenase (LDH), and lower lactate production rate, and expressed higher levels of EMT markers N-cadherin, vimentin and Snail, and showed higher Transwell migration and invasion activities as compared with the SW480 cells. After OA treatment, SW620 cells exhibited a decrease in OCR and RCR of succinate-supported respiration, an increase in lactate production rate and intracellular levels of H2O2 and O2-•. Moreover, the level of vimentin and Transwell migration activity of the SW620 cells were decreased. After TFAM knockdown, the protein levels of TFAM, ND6 and COX-II, and mtDNA copy number, OCR and RCR of succinate-supported respiration in the SW620-KD#4 and SW620-KD#5 cells were all lower than those noted in the SW620‑Control cells. By contrast, the protein level of HK-II, lactate production rate, the intracellular levels of H2O2 and O2-• in the SW620-KD#4 and SW620-KD#5 cells were all higher than those noted in the SW620-Control cells. Subsequently, both SW620-KD#4 and SW620-KD#5 cells had lower Transwell invasion activity than did the SW620-Control cells. Furthermore, we found that deeper invasion (P=0.025) and longer tumor length (P=0.069) were associated with higher mtDNA copy ratios in the 33pairs of CRC specimens obtained from surgical resection. Taken together, we conclude that higher mtDNA copy number and mitochondrial function may confer an invasive advantage to CRCs.

Investigating the antifungal activity and mechanism of a microbial pesticide Shenqinmycin against Phoma sp.

Tea white scab (TWS) is a major disease affecting tea trees in mid-elevation regions and often occurs during rainy seasons with low temperatures. This disease is caused by the fungal pathogen Phoma sp. TWS can infect young stems, tender leaves, and tender shoots and lead to the production of low-quality tea. Owing to the absence of an effective control, TWS can result in substantial loss in tea production. In this study, we isolated and identified the pathogen from tea leaves infected by TWS and then evaluated in vitro the antifungal activity of Shenqinmycin, polyoxin, azoxystrobin, oligosaccharins, and tebuconazole against Phoma sp. Our results indicated that Shenqinmycin can inhibit the growth of Phoma sp. mycelia, with the EC50 value of 0.74μg/mL. After Phoma sp. being incubated in PDB liquid medium with Shenqinmycin, its mycelia were distorted and distended at 1.56μg/mL of minimum inhibitory concentration for 6h. Crucial genes associated with cell redox homeostasis, proteins synthesis, energy metabolism, and cytoskeleton were studied at mRNA and protein levels through RT-qPCR and Nano-LC-MS/MS. The results showed that the genes of 3-phosphate-glyceraldehyde dehydrogenase, citrate synthase, NADH-ubiquinone oxidoreductase subunit (NADH-subunit), ribosomal protein, eukaryotic initiation factor 4A–I, β-tubulin, and α-tubulin were up-regulated. Meanwhile, the genes of formate dehydrogenase (FDH), malate dehydrogenase, mitochondrial heat shock protein, and protein disulfide-isomerase (PDI) were up-regulated at mRNA level but down-regulated at protein level. These results indicated that Shenqinmycin contribute to cell redox homeostasis by up- or down-regulating NADH-subunit, FDH, and PDI.

Consequences of zygote injection and germline transfer of mutant human mitochondrial DNA in mice

Considerable evidence supports mutations in mitochondrial genes as the cause of maternally inherited diseases affecting tissues that rely primarily on oxidative energy metabolism, usually the nervous system, the heart, and skeletal muscles. Mitochondrial diseases are diverse, and animal models currently are limited. Here we introduced a mutant human mitochondrial gene responsible for Leber hereditary optic neuropathy (LHON) into the mouse germ line using fluorescence imaging for tissue-specific enrichment in the target retinal ganglion cells. A mitochondria-targeted adeno-associated virus (MTS-AAV) containing the mutant human NADH ubiquinone oxidoreductase subunit 4 (ND4) gene followed by mitochondrial-encoded mCherry was microinjected into zygotes. Female founders with mCherry fluorescence on ophthalmoscopy were backcrossed with normal males for eight generations. Mutant human ND4 DNA was 20% of mouse ND4 and did not integrate into the host genome. Translated human ND4 protein assembled into host respiratory complexes, decreasing respiratory chain function and increasing oxidative stress. Swelling of the optic nerve head was followed by progressive demise of ganglion cells and their axons, the hallmarks of human LHON. Early visual loss that began at 3 mo and progressed to blindness 8 mo after birth was reversed by intraocular injection of MTS-AAV expressing wild-type human ND4. The technology of introducing human mitochondrial genes into the mouse germ line has never been described, to our knowledge, and has implications not only for creating animal models recapitulating the counterpart human disorder but more importantly for reversing the adverse effects of the mutant gene using gene therapy to deliver the wild-type allele.

Heterologous expression of proteorhodopsin enhances H2 production in Escherichia coli when endogenous Hyd-4 is overexpressed

Proteorhodopsin (PR) is a light harvesting protein widely distributed among bacterioplankton that plays an integral energetic role in a new pathway of marine light capture. The conversion of light into chemical energy in non-chlorophyll-based bacterial systems could contribute to overcoming thermodynamic and metabolic constraints in biofuels production. In an attempt to improve biohydrogen production yields, H2 evolution catalyzed by endogenous hydrogenases, Hyd-3 and/or Hyd-4, was measured when recombinant proteorhodopsin (PR) was concomitantly expressed in Escherichia coli cells. Higher amounts of H2 were obtained with recombinant cells in a light and chromophore dependent manner. This effect was only observed when HyfR, the specific transcriptional activator of the hyf operon encoding Hyd-4 was overexpressed in E. coli, suggesting that an excess of protons generated by PR activity could increase hydrogen production by Hyd-4 but not by Hyd-3. Although many of the subunits of Hyd-3 and Hyd-4 are very similar, Hyd-4 possesses three additional proton-translocating NADH-ubiquinone oxidoreductase subunits, suggesting that it is dependent upon ΔμH+. Altogether, these results suggest that protons generated by proteorhodopsin in the periplasm can only enhance hydrogen production by hydrogenases with associated proton translocating subunits.

Gene delivery to mitochondria by targeting modified adenoassociated virus suppresses Leber’s hereditary optic neuropathy in a mouse model

To introduce DNA into mitochondria efficiently, we fused adenoassociated virus capsid VP2 with a mitochondrial targeting sequence to carry the mitochondrial gene encoding the human NADH ubiquinone oxidoreductase subunit 4 (ND4). Expression of WT ND4 in cells with the G11778A mutation in ND4 led to restoration of defective ATP synthesis. Furthermore, with injection into the rodent eye, human ND4 DNA levels in mitochondria reached 80% of its mouse homolog. The construct expressed in most inner retinal neurons, and it also suppressed visual loss and optic atrophy induced by a mutant ND4 homolog. The adenoassociated virus cassette accommodates genes of up to ∼5 kb in length, thus providing a platform for introduction of almost any mitochondrial gene and perhaps even allowing insertion of DNA encompassing large deletions of mtDNA, some associated with aging, into the organelle of adults.

Novel O-palmitolylated beta-E1 subunit of pyruvate dehydrogenase is phosphorylated during ischemia/reperfusion injury

BackgroundDuring and following myocardial ischemia, glucose oxidation rates are low and fatty acids dominate as a source of oxidative metabolism. This metabolic phenotype is associated with contractile dysfunction during reperfusion. To determine the mechanism of this reliance on fatty acid oxidation as a source of ATP generation, a functional proteomics approach was utilized.Results2-D gel electrophoresis of mitochondria from working rat hearts subjected to 25 minutes of global no flow ischemia followed by 40 minutes of aerobic reperfusion identified 32 changes in protein abundance compared to aerobic controls. Of the five proteins with the greatest change in abundance, two were increased (long chain acyl-coenzyme A dehydrogenase (48 ± 1 versus 39 ± 3 arbitrary units, n = 3, P < 0.05) and α subunit of ATP synthase (189 ± 15 versus 113 ± 23 arbitrary units, n = 3, P < 0.05)), while two were decreased (24 kDa subunit of NADH-ubiquinone oxidoreductase (94 ± 7 versus 127 ± 9 arbitrary units, n = 3, P < 0.05) and D subunit of ATP synthase (230 ± 11 versus 368 ± 47 arbitrary units, n = 3, P < 05)). Two forms of pyruvate dehydrogenase βE1 subunit, the rate-limiting enzyme for glucose oxidation, were also identified. The protein level of the more acidic form of pyruvate dehydrogenase was reduced during reperfusion (37 ± 4 versus 56 ± 7 arbitrary units, n = 3, P < 05), while the more basic form remained unchanged. The more acidic isoform was found to be O-palmitoylated, while both isoforms exhibited ischemia/reperfusion-induced phosphorylation. In silico analysis identified the putative kinases as the insulin receptor kinase for the more basic form and protein kinase Cζ or protein kinase A for the more acidic form. These modifications of pyruvate dehydrogenase are associated with a 35% decrease in glucose oxidation during reperfusion.ConclusionsCardiac ischemia/reperfusion induces significant changes to a number of metabolic proteins of the mitochondrial proteome. In particular, ischemia/reperfusion induced the post-translational modification of pyruvate dehydrogenase, the rate-limiting step of glucose oxidation, which is associated with a 35% decrease in glucose oxidation during reperfusion. Therefore these post-translational modifications may have important implications in the regulation of myocardial energy metabolism.