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Leber’s hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mitochondrial DNA (mtDNA) mutations. LHON-linked ND6 14484T > C (p.M64V) mutation affected structural components of complex I but its pathophysiology is poorly understood. The structural analysis of complex I revealed that the M64 forms a nonpolar interaction Y59 in the ND6, Y59 in the ND6 interacts with E34 of ND4L, and L60 of ND6 interacts with the Y114 of ND1. These suggested that the m.14484T > C mutation may perturb the structure and function of complex I. Mutant cybrids constructed by transferring mitochondria from lymphoblastoid cell lines of one Chinese LHON family into mtDNA-less (ρo) cells revealed decreases in the levels of ND6, ND1 and ND4L. The m.14484T > C mutation may affect mitochondrial mRNA homeostasis, supported by reduced levels of SLIRP and SUPV3L1 involved in mRNA degradation and increasing expression of ND6, ND1 and ND4L genes. These alterations yielded decreased activity of complex I, respiratory deficiency, diminished mitochondrial ATP production and reduced membrane potential, and increased production of reactive oxygen species in the mutant cybrids. Furthermore, the m.14484T > C mutation promoted apoptosis, evidenced by elevating Annexin V-positive cells, release of cytochrome c into cytosol, levels in apoptotic proteins BAX, caspases 3, 7, 9 and decreasing levels in anti-apoptotic protein Bcl-xL in the mutant cybrids. Moreover, the cybrids bearing the m.14484T > C mutation exhibited the reduced levels of autophagy protein LC3, increased levels of substrate P62 and impaired PINK1/Parkin-dependent mitophagy. Our findings highlighted the critical role of m.14484T > C mutation in the pathogenesis of LHON.

Complex I deficiency, the most common respiratory chain defect, is genetically heterogeneous: mutations in 8 nuclear and 7 mitochondrial DNA genes encoding complex I subunits have been described. However, these genes account for disease in only a minority of complex I–deficient patients. We investigated whether there may be an unknown common gene by performing functional complementation analysis of cell lines from 10 unrelated patients. Two of the patients were found to have mitochondrial DNA mutations. The other 8 represented 7 different (nuclear) complementation groups, all but 1 of which showed abnormalities of complex I assembly. It is thus unlikely that any one unknown gene accounts for a large proportion of complex I cases. The 2 patients sharing a nuclear complementation group had a similar abnormal complex I assembly profile and were studied further by homozygosity mapping, chromosome transfers, and microarray expression analysis. NDUFS6, a complex I subunit gene not previously associated with complex I deficiency, was grossly underexpressed in the 2 patient cell lines. Both patients had homozygous mutations in this gene, one causing a splicing abnormality and the other a large deletion. This integrated approach to gene identification offers promise for identifying other unknown causes of respiratory chain disorders.

Methamphetamine (METH) is a highly abused psychostimulant that is neurotoxic to dopaminergic (DAergic) nerve terminals in the striatum and increases the risk of developing Parkinson’s disease (PD). In vivo, METH-mediated DA release, followed by DA-mediated oxidative stress and mitochondrial dysfunction in pre- and postsynaptic neurons, mediates METH neurotoxicity. METH-triggered oxidative stress damages parkin, a neuroprotective protein involved in PD etiology via its involvement in the maintenance of mitochondria. It is not known whether METH itself contributes to mitochondrial dysfunction and whether parkin regulates complex I, an enzymatic complex downregulated in PD. To determine this, we separately assessed the effects of METH or DA alone on electron transport chain (ETC) complexes and the protein parkin in isolated striatal mitochondria. We show that METH decreases the levels of selected complex I, II, and III subunits (NDUFS3, SDHA, and UQCRC2, respectively), whereas DA decreases the levels only of the NDUFS3 subunit in our preparations. We also show that the selected subunits are not decreased in synaptosomal mitochondria under similar experimental conditions. Finally, we found that parkin overexpression does not influence the levels of the NDUFS3 subunit in rat striatum. The presented results indicate that METH itself is a factor promoting dysfunction of striatal mitochondria; therefore, it is a potential drug target against METH neurotoxicity. The observed decreases in ETC complex subunits suggest that DA and METH decrease activities of the ETC complexes via oxidative damage to their subunits and that synaptosomal mitochondria may be somewhat “resistant” to DA- and METH-induced disruption in mitochondrial ETC complexes than perikaryal mitochondria. The results also suggest that parkin does not regulate NDUFS3 turnover in rat striatum.

Background: NADH dehydrogenase 5 (ND5) is one of 44 subunits forming Complex I in the mitochondrial respiratory chain. Therefore, a mitochondrially encoded ND5 (MT-ND5) gene mutation causes mitochondrial oxidative phosphorylation (OXPHOS) disorder, resulting in the development of mitochondrial diseases, such as mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. Although focal segmental glomerulosclerosis (FSGS) is induced by several mitochondrial diseases, no reports have yet confirmed that an MT-ND5 mutation causes FSGS. Case presentation: A Japanese woman at 29 years old was found to have proteinuria and renal dysfunction in an annual health check-up for the first time. Because her proteinuria and renal dysfunction were persistent, she was admitted for a kidney biopsy to investigate the reason at 33 years of age. Although there were no abnormal neurological findings, she was diagnosed with sensorineural hearing loss by an otolaryngologist. Renal histology showed perihilar variant-type FSGS with podocytes filled with abnormal mitochondria. In addition, the renal pathological findings showed granular swollen epithelial cells (GSECs) in tubular cells, age-inappropriately disarranged and irregularly sized vascular smooth muscle cells (AiDIVs), and red-colored podocytes (ReCPos) by acidic dye, which are assumed to be characteristic changes of mitochondrial nephropathy. Genetic analysis using peripheral mononuclear blood cells and urine sediment cells detected the m.13513 G>A variant, which has already been confirmed as a pathogenic variant of the MT-ND5 gene. The heteroplasmy rates of the blood cells and urine sediments were 10.3% and 62.2%, respectively. Therefore, this patient was diagnosed with FSGS due to an MT-ND5 mutation. Conclusion: This is the first case report to show that a gene mutation encoding subunits of Complex I causes FSGS with podocytes filled with abnormal mitochondria. The routine evaluation of GSECs, AiDIVs, and ReCPos is expected to be useful for detecting mitochondrial nephropathies, which could be latent in etiology-unknown FSGS or glomerulosclerosis cases.

Nad dehydrogenase complex in mtDNA has a significant role in cellular respiration. One of the largest subunits in the complex is subunit 5 (Nad5). Four cDNAs of the Hordeum vulgare subsp. spontaneum nad5 gene have been characterized and subjected to four phases of 0.5 M salinity, at 0 h (control, accession no. MT235236), after 2 h (acc. no. MT235237), after 12 h (acc. no. MT235238) and after 24 h (acc. no. MT235239). Utilizing raw data from RNA-seq, ten RNA editing sites were reported. Seven sites have common editing from C to U in positions (C1490, C1859, C1895, C1900, C1901, C1916, C1918). A rare editing event U to C was detected in two positions (U1650 and U1652) and a novel editing event U to G was for the first time in positions nad5-U231. The highest editing level was shown in 2 and 12 h after salinity exposure. After 24 h, these edits were disrupted, possibly due to the launch of the programed cell death mechanism. However, the RNA editing in positions U1650, U1652 and U231 was fixed at all exposure times. Although study clarified the role of salinity stress in nad5 RNA editing sites, the main achievements are first report of U to G RNA editing in plants at position U231 and first report of U to C editing in the nad5 gene at U1650 and U1652.

Leigh syndrome (LS) is an early-onset progressive neurodegenerative disorder associated with mitochondrial deficiency. m.14597A>G (p.Ile26Thr) in the MT-ND6 gene was reported to cause Leberʼs hereditary optic neuropathy (LHON) or dementia/dysarthria. In previous reports, less than 90% heteroplasmy was shown to result in adult-onset disease. Here, by whole mitochondrial sequencing, we identified m.14597A>G mutation of a patient with LS. PCR–RFLP analysis on fibroblasts from the patient revealed a high mutation load (> 90% heteroplasmy). We performed functional assays using cybrid cell models generated by fusing mtDNA-less rho0 HeLa cells with enucleated cells from patient fibroblasts carrying the m.14597A>G variant. Cybrid cell lines bearing the m.14597A>G variant exhibited severe effects on mitochondrial complex I activity. Additionally, impairment of cell proliferation, decreased ATP production and reduced oxygen consumption rate were observed in the cybrid cell lines bearing the m.14597A>G variant when the cells were metabolically stressed in medium containing galactose, indicating mitochondrial respiratory chain defects. These results suggest that a high mutation load of m.14597A>G leads to LS via a mitochondrial complex I defect, rather than LHON or dementia/dysarthria.

Currently, Tigers (the top predator of an ecosystem) are on the list of endangered species. Thus the need is to understand the tiger's population genomics to design their conservation strategies. We analyzed the molecular evolution of tiger diversity using NADH dehydrogenase subunit 4 (ND4), a significant electron transport chain component. We have analyzed nucleotide composition and distribution pattern of ND genes, molecular evolution, evolutionary conservation pattern and conserved blocks of NADH, phylogenomics of ND4, and estimating species divergence, etc., using different bioinformatics tools and software, and MATLAB programming and computing environment. The nucleotide composition and distribution pattern of ND genes in the tiger genome demonstrated an increase in the number of adenine (A) and a lower trend of A+T content in some place of the distribution analysis. However, the observed distributions were not significant (P > 0.05). Evolutionary conservation analysis showed three highly align blocks (186 to 198, 406 to 416, and 527 to 545). On mapping the molecular evolution of ND4 among model species (n = 30), we observed its presence in a broader range of species. ND4 based molecular evolution of tiger diversity and time divergence for a tiger (20 different other species) shows that genus Panthera originated more or less at a similar time. The nucleotide composition and nucleotide distribution pattern of tiger ND genes showed the evolutionary pattern and origin of tiger and Panthera lineage concerning the molecular clock, which will help to understand their adaptive evolution.

Background The transplantation of human umbilical cord blood (UCB) CD34+ cells has been successfully used to treat hematological disorders but one major limitation has been the low cell numbers available. Mesenchymal stem cells (MSCs) lying within the bone marrow in vivo behave like a scaffold on which CD34+ cells interact and proliferate. We therefore evaluated the use of allogeneic MSCs from the human UC Wharton’s jelly (hWJSCs) as stromal support for the ex vivo expansion of CD34+ cells. Methods We performed an in-depth evaluation of the primitiveness, migration, adhesion, maturation, mitochondrial behavior, and pathway mechanisms of this platform using conventional assays followed by the evaluation of engraftment potential of the expanded CD34+ cells in an in vivo murine model. Results We demonstrate that hWJSCs and its conditioned medium (hWJSC-CM) support the production of significantly high fold changes of CD34+, CD34+CD133+, CD34+CD90+, CD34+ALDH+, CD34+CD45+, and CD34+CD49f+ cells after 7 days of interaction when compared to controls. In the presence of hWJSCs or hWJSC-CM, the CD34+ cells produced significantly more primitive CFU-GEMM colonies, HoxB4, and HoxA9 gene expression and lower percentages of CD34+CXCR4+ cells. There were also significantly higher N-cadherin+ cell numbers and increased cell migration in transwell migration assays. The CD34+ cells expanded with hWJSCs had significantly lower mitochondrial mass, mitochondrial membrane potential, and oxidative stress. Green Mitotracker-tagged mitochondria from CD34+ cells were observed lying within red CellTracker-tagged hWJSCs under confocal microscopy indicating mitochondrial transfer via tunneling nanotubes. CD34+ cells expanded with hWJSCs and hWJSC-CM showed significantly reduced oxidative phosphorylation (ATP6VIH and NDUFA10) and increased glycolytic (HIF-1a and HK-1) pathway-related gene expression. CD34+ cells expanded with hWJSCs for 7 days showed significant greater CD45+ cell chimerism in the bone marrow of primary and secondary irradiated mice when transplanted intravenously. Conclusions In this report, we confirmed that allogeneic hWJSCs provide an attractive platform for the ex vivo expansion of high fold numbers of UCB CD34+ cells while keeping them primitive. Allogeneic hWJSCs are readily available in abundance from discarded UCs, can be easily frozen in cord blood banks, thawed, and then used as a platform for UCB-HSC expansion if numbers are inadequate.

Acute myeloid leukemia (AML) is a type of hematological malignancy caused by uncontrolled clonal proliferation of hematopoietic stem cells. The special energy metabolism mode of AML relying on oxidative phosphorylation is different from the traditional 'Warburg effect'. However, its mechanism is not clear. In the present study, it was demonstrated that the mRNA expression levels of NADH dehydrogenase subunit 1, 4 and 5 (ND1, ND4 and ND5) were upregulated in AML samples from The Cancer Genome Atlas database using the

Chemically modified forms of tetraiodothyroacetic acid (tetrac), an L-thyroxine derivative, have been shown to exert their anticancer activity at plasma membrane integrin αvβ3 of tumor cells. Via a specific hormone receptor on the integrin, tetrac-based therapeutic agents modulate expression of genes relevant to cancer cell proliferation, survival and energy metabolism. P-bi-TAT, a novel bivalent tetrac-containing synthetic compound has anticancer activity in vitro and in vivo against Glioblastoma Multiforme (GBM) and other types of human cancers. In the current study, microarray analysis was carried out on a primary culture of human GBM cells exposed to P-bi-TAT (10-6 tetrac equivalent) for 24 h. P-bi-TAT significantly affected expression of a large panel of genes implicated in cancer cell stemness, growth, survival, and angiogenesis. Recent interest elsewhere in ATP synthase as a target in GBM cells caused us to focus attention on expression of genes involved in energy metabolism. Significantly downregulated transcripts included multiple energy metabolism-related genes: electron transport chain genes ATP5A1 (ATP synthase 1), ATP51, ATP5G2, COX6B1 (cytochrome c oxidase subunit 6B1), NDUFA8 (NADH dehydrogenase [ubiquinone] FA8), NDUFV2I and other NDUF genes. The NDUF and ATP genes are also relevant to control of oxidative phosphorylation and transcription. Qualitatively similar actions of P-bi-TAT on expression of energy metabolism-linked genes were also detected in established human GBM and pancreatic cancer cell lines. In conclusion, acting at αvβ3 integrin, P-bi-TAT caused downregulation in human cancer cells of expression of a large number of genes involved in electron transport and oxidative phosphorylation. These observations suggest that cell surface thyroid hormone receptors on αvβ3 regulate expression of genes relevant to tumor cell stemness and energy metabolism.

Leber's hereditary optic neuropathy (LHON) is the maternal inheritance of eye disorder. LHON-linked mitochondrial DNA (mtDNA) mutations affect the ND1, ND4 or ND6 genes encoding essential subunits of complex I. However, the role of mitochondrial tRNA defects in the pathogenesis of LHON is poorly understood. In this report, Sanger sequence analysis of 22 mitochondrial tRNA genes identified 139 variants in a cohort of 811 Han Chinese probands and 485 control Chinese subjects. Among these, 32 (4 known and 28 novel/putative) tRNA variants in 71 probands may contribute to pathogenesis of LHON, as these exhibited (1) present in 1% of controls; (2) evolutionary conservation; (3) potential and significance of structural and functional modifications. Such variants may have potentially compromised structural and functional aspects in the processing of tRNAs, structure stability, tRNA charging, or codon-anticodon interactions during translation. These 32 variants presented either singly or with multiple mutations, with the primary LHON-linked ND1 3640G A, ND4 11778G A or ND6 14484 T C mutations in the probands. The thirty-eight pedigrees carrying only one of tRNA variants exhibited relatively low penetrances of LHON, ranging from 5.7% to 42.9%, with an average of 19%. Strikingly, the average penetrances of optic neuropathy among 33 Chinese families carrying both a known/putative tRNA variant and a primary LHON-associated mtDNA mutation were 40.1%. These findings suggested that mitochondrial tRNA variants represent a significant causative factor for LHON, accounting for 8.75% cases in this cohort. These new insights may lead to beneficial applications in the pathophysiology, disease management, and genetic counseling of LHON.

Atrial fibrillation (AF) is one of the most common types of arrhythmia worldwide; although a number of theories have been proposed to explain the mechanisms of AF, the treatment of AF is far from satisfactory. Energy metabolism is associated with the development of AF. Mitochondrial transcription factor A (TFAM) serves a role in the maintenance and transcription of mitochondrial DNA. The present study aimed to investigate the association between TFAM and AF and the effect of TFAM on ATP content in cardiomyocytes. Left atrial appendage tissues were collected from 20 patients with normal sinus rhythm (SR) and 20 patients with AF, and the expression levels of TFAM in SR and AF tissues were evaluated. In addition, a tachypacing model of primary cultured cardiomyocytes was constructed to assess ATP content, cell viability and expression levels of TFAM, mitochondrially encoded (MT)-NADH dehydrogenase 1 (ND1), MT-cytochrome c oxidase 1 (CO1), NADH ubiquinone oxidoreductase core subunit 1 (NDUFS1) and cytochrome c oxidase subunit 6C (COX6C). Finally, the effects of overexpression and inhibition of TFAM on ATP content, cell viability and the expression levels of MT-ND1 and MT-CO1 were investigated. The expression levels of TFAM were decreased in AF tissues compared with SR tissues (P0.05). Overexpression of TFAM increased ATP content, cell viability and expression levels of MT-ND1 and MT-CO1 (P

Tuberculosis caused by Mycobacterium tuberculosis is one of the main global health concerns. In this study, the entire mitochondrial genome from blood samples of tuberculosis patients was analyzed to understand the possible mtDNA variants. The potential impact of non-synonymous substitutions on protein functions were determined using prediction tools. 28 non- synonymous variants were found of which 2 variants (MT-ND2 g. A > G4824 p.T119A and MT-ND6 g. T > C14180 p.Y165C) were found to be deleterious among the cases only. Majority of the variants lie in the D-loop of the non-protein coding region of the mitochondrial DNA. We propose that mutations in the mitochondrial genome need to be validated further to understand their association with tuberculosis.

Leigh syndrome is a genetically heterogeneous disorder resulting from deficient oxidative energy biogenesis. The syndrome is characterized by subacute episodic decompensations, transiently elevated lactate, and necrotizing brain lesions most often in the striatum and brainstem. Acute decompensation is often triggered by viral infections. Sequalae from repeated episodes leads to progressive neurological deterioration and death. The severity of Leigh syndrome varies widely, from a rapid demise in childhood to rare adult presentations. Although the causes of Leigh syndrome include genes affecting a variety of different pathways, more than 75 of them are nuclear or mitochondrial encoded genes involved in the assembly and catalytic activity of mitochondrial respiratory complex I. Here we report the detailed clinical and molecular phenotype of two adults with mild presentations of NDUFS3 and NDUFAF6-related Leigh Syndrome. Mitochondrial assays revealed slightly reduced complex I activity in one proband and normal complex I activity in the other. The proband with NDUFS3-related Leigh syndrome was mildly affected and lived into adulthood with novel biallelic variants causing aberrant mRNA splicing (NM_004551.2:c.419G > A; p.Arg140Gln; NM_004551.2:c.381 + 6 T > C). The proband with NDUFAF6-related Leigh syndrome had biallelic variants that cause defects in mRNA splicing (NM_152416.3:c.371 T > C; p.Ile124Thr; NM_152416.3:c.420 + 2_420 + 3insTA). The mild phenotypes of these two individuals may be attributed to some residual production of normal NDUFS3 and NDUFAF6 proteins by NDUFS3 and NDUFAF6 mRNA isoforms alongside mutant transcripts. Taken together, these cases reported herein suggest that splice-regulatory variants to complex I proteins could result in milder phenotypes.

Leber hereditary optic neuropathy (LHON) is a neurodegenerative disorder characterised by bilateral, painless, subacute, central vision loss caused by pathogenic sequence variants in mitochondrial DNA (mtDNA). Over the course of 20 years, 734 people were systematically screened by our diagnostic laboratory for suspected LHON or for being at risk of LHON, with 98 found to harbour one of the three primary pathogenic mtDNA variants. Detection incidences were: 0.95% for NC_012920.1(MT-ND1):m.3460G>A; 9.4% for (MT-ND4):m.11778G>A; and 2.9% for (MT-ND6):m.14484T>C. The median age for symptomatic males was 27.3 years and for females 29.5 years, with a male to female ratio of 4.4:1 (62 males; 14 females). Most pathogenic variant carriers were propositi with the other individuals belonging to one of 14 pedigrees with noteworthy intra-family variability of clinical severity of the disease.

Reactive oxygen species (ROS) have already been demonstrated to impede the migratory ability in non-melanocytic cell lines by depleting mitochondrial ATP production. Therefore, understanding the mitochondrial metabolic response to migration in the presence of ROS should be a key to understanding repigmentation in vitiligo. This study aimed to investigate the energy mechanism associated with the ROS-mediated attenuation of melanocyte migration. After melanocytes were pretreated with H2 O2 , their ATP production, migratory ability, ultrastructural changes and Mitochondrial Permeability Potential were analysed. The results showed that, in parallel with the decreased ATP production, the migratory ability of melanocytes was significantly inhibited by oxidative stress. Supplementation with exogenous ATP reversed the suppressed ATP-dependent migration of melanocytes. Melanocytes were then stressed with H2 O2 and Agilent Whole Human Genome microarray analysis identified 763 up-regulated mRNAs and 1117 down-regulated mRNAs. Among them, 11 of the encoded proteins were involved in mitochondrial ATP production and their expression levels were verified. The decreased expression of NADH dehydrogenase 2(ND2) , cytochrome c oxidase 1(COX1) and cytochrome c oxidase 3(COX3) was shown to be involved in the depletion of mitochondrial ATP production, which was coupled with the impaired migratory potential. These results indicate that the migration of melanocytes relies heavily on an inexhaustible supply of ATP from mitochondria.

The enzyme 6-phosphogluconate dehydrogenase catalyzes the conversion of 6-phosphogluconate to ribulose-5-phosphate. It represents an important reaction in the oxidative pentose phosphate pathway, producing a ribose precursor essential for nucleotide and nucleic acid synthesis. We succeeded, for the first time, to determine the three-dimensional structure of this enzyme from an acetic acid bacterium, Gluconacetobacter diazotrophicus (Gd6PGD). Active Gd6PGD, a homodimer (70 kDa), was present in both the soluble and the membrane fractions of the nitrogen-fixing microorganism. The Gd6PGD belongs to the newly described subfamily of short-chain (333 AA) 6PGDs, compared to the long-chain subfamily (480 AA; e.g., Ovis aries, Homo sapiens). The shorter amino acid sequence in Gd6PGD induces the exposition of hydrophobic residues in the C-terminal domain. This distinct structural feature is key for the protein to associate with the membrane. Furthermore, in terms of function, the short-chain 6PGD seems to prefer NAD+ over NADP+ , delivering NADH to the membrane-bound NADH dehydrogenase of the microorganisms required by the terminal oxidases to reduce dioxygen to water for energy conservation. ENZYME: ECnonbreakingspace1.1.1.343. DATABASE: Structural data are available in PDB database under the accession number 6VPB.

Recent studies have demonstrated a significant role of mitochondrial DNA (mtDNA) defects in the pathogenesis of many human and some canine tumours. The aim of this study was to identify mutations in the ND2 and ND4 mitochondrial genes in canine mast cell tumours and determine their association with the process of neoplastic transformation and the phenotypic traits of dogs. In total, 136 gene sequences from 68 biological samples, including blood and neoplastic tissue samples from 34 dogs with diagnosed MCTs, were analysed. The study consisted in DNA sequencing of the ND2 and ND4 genes as well as bioinformatics and statistical analyses. For the first time, mutations in NADH-dehydrogenase genes were detected in dogs with MCTs. In total, 22 polymorphic loci and 19 mutations in the ND2 and ND4 genes were identified. The majority of the identified mutations were homoplasmic, and tumour heteroplasmy was detected in eight nucleotide positions in three dogs. Seven of the ND2 mutations and two of the ND4 mutations caused an amino acid change. The changes in non-synonymous protein-coding SNPs did not exert an adverse effect on proteins. A statistically significant correlation of the presence of mutations/polymorphisms with the sex, age, and size of the dogs and the tumour location was demonstrated. Polymorphisms and mutations in NADH-dehydrogenase genes, including mastocyte-specific changes, in canine mast cell tumours that had not been reported earlier in the literature were identified. Some of these changes may imply that these are the hotspot mutations in canine mast cell tumours. It cannot be excluded that the molecular changes are directly associated with the development of mast cell tumours, and further investigations are needed to verify whether they can become molecular markers of MCTs in the future.

Nad complex plays a very important role during cellular respiration. nad3 (nad dehydrogenase subunit 3) is one of the biggest subunits in this complex. Four cDNAs of nad3 gene were characterized in Hordeum vulgare subsp. spontaneum at exposed to four periods of 500 mM salinity, 0 h or control (accession no. MN066165), after 2 h (accession no. MN066166), after 12 h (accession no. MN066167) and after 24 h (accession no. MN066168) using RNA-seq raw data. Seventeen RNA editing sites were found in positions (or nucleotide nos. C5, C39, C44, C61, C62, C79, C80, C147, C185, C190, C191, C208, C209, C275, C317, C344, C349) within the nad3 coding region. These alterations represent differential editing at four exposure times. The maximum editing rate was revealed 2 and 12 h after salinity exposure. However, these edits were disrupted after 24 h probably due to the initiation of program cell death machinery. We found that RNA editing not only improved protein function but also may improve codon bias by altering the nucleotide without any change in amino acid. Characterization of pentatricopeptide repeat-containing protein At4g13650 (PPRSp1) in wild barley helped us to understand the behavior of editing sites C190 and C191 under salinity. Position - 6 in cis-element upstream editing sites of C155, C190 and C191 may be vital to the editing process in these sites by PPRSp1 protein. The differential editing of this gene under salinity led to a relationship between RNA editing and cellular respiration regulation.

The mutations in the genes encoding the subunits of complex I of the mitochondrial electron transport chain are the most common cause of Leber’s hereditary optic neuropathy (LHON), a maternal hereditary disease characterized by retinal ganglion cell (RGC) degeneration. The characteristics of incomplete penetrance indicate that nuclear genetic and environmental factors also determine phenotypic expression of LHON. Therefore, further understanding of the role of mutant mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit proteins and nuclear genetic factors/environmental effects in the etiology of LHON is needed. In this study, we generated human-induced pluripotent stem cells (hiPSCs) from healthy control, unaffected LHON mutation carrier, and affected LHON patient. hiPSC-derived RGCs were used to study the differences between affected and unaffected carriers of mitochondrial DNA point mutation m.11778G > A in the MT-ND4 gene. We found that both mutated cell lines were characterized by increase in reactive oxygen species production, however, only affected cell line had increased levels of apoptotic cells. We found a significant increase in retrograde mitochondria and a decrease in stationary mitochondria in the affected RGC axons. In addition, the messenger RNA and protein levels of KIF5A in the LHON-affected RGCs were significantly reduced. Antioxidant N-acetyl-L-cysteine could restore the expression of KIF5A and the normal pattern of mitochondrial movement in the affected RGCs. To conclude, we found essential differences in the mutually dependent processes of oxidative stress, mitochondrial transport and apoptosis between two LHON-specific mutation carrier RGC cell lines, asymptomatic carrier and disease-affected, and identified KIF5A as a central modulator of these differences.

Background: Mitochondrial dysfunction may involve in the progression of human non-small cell lung cancers (NSCLCs). We analyzed the mitochondrial DNA (mtDNA) copy number, the expression levels of mitochondrial biogenesis-related proteins including pyruvate dehydrogenase, mitochondrial transcription factor A (TFAM) and mtDNA-encoded peptide NADH dehydrogenase subunit 1 (ND1), and the expression level of hexokinase II (HK-II) in human NSCLCs both ex vivo and in vitro. Materials and Methods: Paired cancerous and non-cancerous pathological specimens from 20 resected NSCLCs and an NSCLC cell line, the H23, were used in this study. H23 was infected by lentiviral particles to knockdown (KD) the expression of TFAM. TFAM-Null and TFAM-KD represent the control and TFAM knocked-down H23 cells, respectively. The mtDNA copy number was measured by quantitative real-time polymerase chain reaction and the protein expression levels were measured by immunohistochemical staining and Western blotting, respectively. Results: Low TFAM expression (P = 0.066) and low mtDNA copy number of NSCLCs (P = 0.009) were poor prognostic variables in NSCLC patients. Advanced T4 NSCLCs had lower TFAM expression (P = 0.021), lower expression of mtDNA-encoded ND1 polypeptide (P = 0.049), and lower mtDNA copy number (P = 0.050) than did T1 or T2/T3 NSCLCs, respectively. TFAM-KD cells expressed lower levels of TFAM protein (P Conclusion: Mitochondrial dysfunction caused by lower levels of TFAM, mtDNA copy number, and mtDNA-encoded ND1 polypeptide may play an important role in the progression of NSCLCs.

Polymerase Chain Reaction (PCR) is an accurate, simple and fast analytical method. This technique is widely used in the identification of meat adulteration and meat-based processed food products. Three Mitochondrial DNA (mt-DNA) primers NADH Dehydrogenase sub unit 5 (ND5), D-Loop, and Cytochrome b (Cyt-b) were tested for their specificity in detecting of pig (Sus scrofa) DNA fragments. DNA genome from 6 meat samples (pork, beef, goat, lamb, and chicken) was amplified by PCR technique using three pairs of primers (ND5, D-Loop, and Cyt-b) and sequenced. The results of amplification using the three primers produced specific DNA bands with the lengths of 232 bp, 951 bp, and 404 bp, respectively. Comparison results with ND5, D-Loop, and Cyt-b gene sequences resulted in similarity values of 100%, 97%, and 99%, respectively. These showed that the mt-DNA primers of ND5, D-Loop, and Cyt-b genes can be recommended as specific primers in detecting pig (Sus scrofa) DNA fragments.

Cyclic electron transport/flow (CET/CEF) in chloroplasts is a regulatory process essential for the optimization of plant photosynthetic efficiency. A crucial CEF pathway is catalyzed by a membrane-embedded NADH dehydrogenase-like (NDH) complex that contains at least 29 protein subunits and associates with photosystem I (PSI) to form the NDH-PSI supercomplex. Here, we report the 3.9 Å resolution structure of the Arabidopsis thaliana NDH-PSI (AtNDH-PSI) supercomplex. We constructed structural models for 26 AtNDH subunits, among which 11 are unique to chloroplasts and stabilize the core part of the NDH complex. In the supercomplex, one NDH can bind up to two PSI-light-harvesting complex I (PSI-LHCI) complexes at both sides of its membrane arm. Two minor LHCIs, Lhca5 and Lhca6, each present in one PSI-LHCI, interact with NDH and contribute to supercomplex formation and stabilization. Collectively, our study reveals the structural details of the AtNDH-PSI supercomplex assembly and provides a molecular basis for further investigation of the regulatory mechanism of CEF in plants.

Myocardial infarction (MI) is the leading cause of premature death among adults. Cardiomyocyte death and dysfunction of the remaining viable cardiomyocytes are the main pathological factors of heart failure after MI. Mitochondrial complexes are emerging as critical mediators for the regulation of cardiomyocyte function. However, the precise roles of mitochondrial complex subunits in heart failure after MI remain unclear. Here, we show that NADH:ubiquinone oxidoreductase core subunit S1 (Ndufs1) expression is decreased in the hearts of heart failure patients and mice with myocardial infarction. Furthermore, we found that cardiac-specific Ndufs1 overexpression alleviates cardiac dysfunction and myocardial fibrosis in the healing phase of MI. Our results demonstrated that Ndufs1 overexpression alleviates MI/hypoxia-induced ROS production and ROS-related apoptosis. Moreover, upregulation of Ndufs1 expression improved the reduced activity of complex I and impaired mitochondrial respiratory function caused by MI/hypoxia. Given that mitochondrial function and cardiomyocyte apoptosis are closely related to heart failure after MI, the results of this study suggest that targeting Ndufs1 may be a potential therapeutic strategy to improve cardiac function in patients with heart failure.

Prenatal ambient particulate matter (PM2.5) exposure impacts infant development and alters placental mitochondrial DNA abundance. We investigated whether the timing of PM2.5 exposure predicts placental mitochondrial mutational load using NextGen sequencing in 283 multi-ethnic mother-infant dyads. We observed increased PM2.5 exposure, particularly during mid- to late- pregnancy and among genes coding for NADH dehydrogenase and subunits of ATP synthase, was associated with a greater amount of nonsynonymous mutations. The strongest associations were observed for participants of African ancestry. Further work is needed to tease out the role of mitochondrial genetics and its impact on offspring development and emerging disease disparities.

Fasciola gigantica is a major pathogen that causes fasciolosis in Africa. A recent study in Uganda demonstrated that Fasciola flukes were present in 65.7% of slaughtered cattle. However, molecular identification of Fasciola species has not yet been performed in the country. In the present study, 292 Fasciola flukes were collected from Kampala and Gulu, Uganda. The samples were identified as F. gigantica using a multiplex polymerase chain reaction (PCR) assay for phosphoenolpyruvate carboxykinase (pepck) and a PCR-restriction fragment length polymorphism (RFLP) assay for DNA polymerase delta (pold). A significant genetic difference between F. gigantica obtained from cattle slaughtered at Kampala and Gulu was observed by analyzing the mitochondrial markers NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1). Fasciola collected from Gulu had a more diversified population than that collected from Kampala, probably because of differences in livestock management systems. One of the possible reasons for this observation is that cattle slaughtered in Gulu were reared under an extensive communal grazing system, which is suitable for maintaining parasite diversity, whereas cattle slaughtered in Kampala mainly originated from fenced/closed farms, which limits parasite diversity. However, the cause of the difference between these two locations was not clearly defined by the results of this study. The F. gigantica population from Uganda was related to that obtained from Zambia. A star-like phylogeny was detected in a median-joining network analysis, which indicated rapid population expansion and suggested that the F. gigantica populations from both countries are maintained by domestic ruminants in eastern Africa. Interestingly, the F. gigantica population from Uganda was not related to those from Egypt and Nigeria. The results of the present study suggest that F. gigantica populations in African countries are indigenous to each country or region.

Brown adipose tissue (BAT) is functionally linked to skeletal muscle because both tissues originate from a common progenitor cell, but the precise mechanism controlling muscle-to-brown-fat communication is insufficiently understood. This report demonstrates that the immunoglobulin superfamily containing leucine-rich repeat (Islr), a marker of mesenchymal stromal/stem cells, is critical for the control of BAT mitochondrial function and whole-body energy homeostasis. The mice loss of Islr in BAT after cardiotoxin injury resulted in improved mitochondrial function, increased energy expenditure, and enhanced thermogenesis. Importantly, it was found that interleukin-6 (IL-6), as a myokine, participates in this process. Mechanistically, Islr interacts with NADH: Ubiquinone Oxidoreductase Core Subunit S2 (Ndufs2) to regulate IL-6 signaling; consequently, Islr functions as a brake that prevents IL-6 from promoting BAT activity. Together, these findings reveal a previously unrecognized mechanism for muscle-BAT cross talk driven by Islr, Ndufs2, and IL-6 to regulate energy homeostasis, which may be used as a potential therapeutic target in obesity.

Coenzyme Q (CoQ, ubiquinone) is the electron-carrying lipid in the mitochondrial electron transport system (ETS). In mammals, it serves as the electron acceptor for nine mitochondrial inner membrane dehydrogenases. These include the NADH dehydrogenase (complex I, CI) and succinate dehydrogenase (complex II, CII) but also several others that are often omitted in the context of respiratory enzymes: dihydroorotate dehydrogenase, choline dehydrogenase, electron-transferring flavoprotein dehydrogenase, mitochondrial glycerol-3-phosphate dehydrogenase, proline dehydrogenases 1 and 2, and sulfide:quinone oxidoreductase. The metabolic pathways these enzymes are involved in range from amino acid and fatty acid oxidation to nucleotide biosynthesis, methylation, and hydrogen sulfide detoxification, among many others. The CoQ-linked metabolism depends on CoQ reoxidation by the mitochondrial complex III (cytochrome bc1 complex, CIII). However, the literature is surprisingly limited as for the role of the CoQ-linked metabolism in the pathogenesis of human diseases of oxidative phosphorylation (OXPHOS), in which the CoQ homeostasis is directly or indirectly affected. In this review, we give an introduction to CIII function, and an overview of the pathological consequences of CIII dysfunction in humans and mice and of the CoQ-dependent metabolic processes potentially affected in these pathological states. Finally, we discuss some experimental tools to dissect the various aspects of compromised CoQ oxidation.

Leigh syndrome (LS) is one of the most common mitochondrial diseases in children, for which at least 90 causative genes have been identified. However, many LS patients have no genetic diagnosis, indicating that more disease-related genes remain to be identified. In this study, we identified a novel variant, m.3955G > A, in mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1) in two unrelated LS patients, manifesting as infancy-onset frequent seizures, neurodegeneration, elevated lactate levels, and bilateral symmetrical lesions in the brainstem, basal ganglia, and thalamus. Transfer of the mutant mtDNA with m.3955G > A into cybrids disturbed the MT-ND1 expression and CI assembly, followed by remarkable mitochondrial dysfunction, reactive oxygen species production, and mitochondrial membrane potential reduction. Our findings demonstrated the pathogenicity of the novel m.3955G > A variant, and extend the spectrum of pathogenic mtDNA variants.

NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is a nuclear-encoded core subunit of human mitochondrial complex I. Defects in NDUFS8 are associated with Leigh syndrome and encephalomyopathy. Cell-penetrating peptide derived from the HIV-1 transactivator of transcription protein (TAT) has been successfully applied as a carrier to bring fusion proteins into cells without compromising the biological function of the cargoes. In this study, we developed a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFS8 defects. Two fusion proteins (TAT-NDUFS8 and NDUFS8-TAT) were exogenously expressed and purified from Escherichia coli for transduction of human cells. In addition, similar constructs were generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFS8 and NDUFS8-TAT were delivered into mitochondria and correctly processed. Interestingly, the mitochondrial import of TAT-containing NDUFS8 was independent of mitochondrial membrane potential. Treatment with TAT-NDUFS8 not only significantly improved the assembly of complex I in an NDUFS8-deficient cell line, but also partially rescued complex I functions both in the in-gel activity assay and the oxygen consumption assay. Our current findings suggest the considerable potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency.

Pathogenic mitochondrial NADH dehydrogenase (ND) gene mutations enhance the invasion and metastasis of various cancer cells, and they are associated with metastasis in human non-small cell lung cancer (NSCLC). Moreover, monocarboxylate transporter 4 (MCT4) is overexpressed in solid cancers and plays a role in cancer cell proliferation and survival. Here, we report that MCT4 is exclusively expressed in mouse transmitochondrial cybrids with metastasis-enhancing pathogenic ND6 mutations. A high level of MCT4 is also detected in human NSCLC cell lines and tissues predicted to carry pathogenic ND mutations and is associated with poor prognosis in NSCLC patients. MCT4 expression in the cell lines is suppressed by N-acetyl-L-cysteine. Phosphatidylinositol-3 kinase (PI3K), AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) are involved in the regulation of MCT4 expression in the transmitochondrial cybrid cells. An MCT1/4 inhibitor effectively kills NSCLC cells with predicted pathogenic ND mutations, but an MCT1/2 inhibitor does not have the same effect. Thus, MCT4 expression is augmented by pathogenic ND mutations and could be a biomarker and a therapeutic target in pathogenic ND mutation-harbouring metastatic tumours.

Certain mutations in mitochondrial DNA (mtDNA) are associated with Leber's hereditary optic neuropathy (LHON). In particular, the well‑known NADH dehydrogenase 4 (ND4) m.11778G>A mutation is one of the most common LHON‑associated primary mutations worldwide. However, how specific mtDNA mutations, or variants, affect LHON penetrance is not fully understood. The aim of the current study was to explore the relationship between mtDNA mutations and LHON, and to provide useful information for early detection and prevention of this disease. Following the molecular characterization of a Han Chinese family with maternally inherited LHON, four out of eight matrilineal relatives demonstrated varying degrees of both visual impairment and age of onset. Through PCR amplification of mitochondrial genomes and direct Sanger sequencing analysis, a homoplasmic mitochondrial‑encoded ND4 m.11778G>A mutation, alongside a set of genetic variations belonging to human mtDNA haplogroup B5b1 were identified. Among these sequence variants, alanine transfer RNA (tRNA)Ala m.5601C>T was of particular interest. This variant occurred at position 59 in the TψC loop and altered the base pairing, which led to mitochondrial RNA (mt‑RNA) metabolism failure and defects in mitochondrial protein synthesis. Bioinformatics analysis suggested that the m.5601C>T variant altered tRNAAla structure. Therefore, impaired mitochondrial functions caused by the ND4 m.11778G>A mutation may be enhanced by the mt‑tRNAAla m.5601C>T variant. These findings suggested that the tRNAAla m.5601C>T variant might modulate the clinical manifestation of the LHON‑associated primary mutation.

Summary Pectobacterium atrosepticum SCRI1043 is a phytopathogenic Gram‐negative enterobacterium. Genomic analysis has identified that genes required for both respiration and fermentation are expressed under anaerobic conditions. One set of anaerobically expressed genes is predicted to encode an important but poorly understood membrane‐bound enzyme termed formate hydrogenlyase‐2 (FHL‐2), which has fascinating evolutionary links to the mitochondrial NADH dehydrogenase (Complex I). In this work, molecular genetic and biochemical approaches were taken to establish that FHL‐2 is fully functional in P. atrosepticum and is the major source of molecular hydrogen gas generated by this bacterium. The FHL‐2 complex was shown to comprise a rare example of an active [NiFe]‐hydrogenase‐4 (Hyd‐4) isoenzyme, itself linked to an unusual selenium‐free formate dehydrogenase in the final complex. In addition, further genetic dissection of the genes encoding the predicted membrane arm of FHL‐2 established surprisingly that the majority of genes encoding this domain are not required for physiological hydrogen production activity. Overall, this study presents P. atrosepticum as a new model bacterial system for understanding anaerobic formate and hydrogen metabolism in general, and FHL‐2 function and structure in particular., Pectobacterium atrospecticum contains the genes for formate hydrogenlyase‐2, considered the ancient progenitor of Complex I. Here, P. atrosepticum was harnessed as a new model system for advancing new knowledge in FHL‐2. The complex was found to contain an unusual selenium‐free formate dehydrogenase and a [NiFe]‐hydrogenase‐4 with a large membrane arm. FHL‐2 was established as the major source of hydrogen gas; however, some components of the membrane arm were surprisingly not essential for this activity.

Due to its pivotal role in NADH oxidation and ATP synthesis, mitochondrial complex I (CI) emerged as a crucial regulator of cellular metabolism. A functional CI relies on the sequential assembly of nuclear- and mtDNA-encoded subunits; however, whether CI assembly status is involved in the metabolic adaptations in CI deficiency still remains largely unknown. Here, we investigated the relationship between CI functions, its structure and the cellular metabolism in 29 patient fibroblasts representative of most CI mitochondrial diseases. Our results show that, contrary to the generally accepted view, a complex I deficiency does not necessarily lead to a glycolytic switch, i.e. the so-called Warburg effect, but that this particular metabolic adaptation is a feature of CI assembly defect. By contrast, a CI functional defect without disassembly induces a higher catabolism to sustain the oxidative metabolism. Mechanistically, we demonstrate that reactive oxygen species overproduction by CI assembly intermediates and subsequent AMPK-dependent Pyruvate Dehydrogenase inactivation are key players of this metabolic reprogramming. Thus, this study provides a two-way-model of metabolic responses to CI deficiencies that are central not only in defining therapeutic strategies for mitochondrial diseases, but also in all pathophysiological conditions involving a CI deficiency.

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.

The stimulation of adenosine monophosphate-activated protein kinase (AMPK) is a prime target to decrease the hyperglycemic condition, hence it is a lutein (L) and oxidised lutein (OXL) is a target molecule for the treatment of type II diabetes. In the current study, a plausible interaction of L and OXL with AMPK was investigated by molecular docking. In addition, the effect of L and OXL for the activation of AMPK that triggers the downstream regulator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), TFAM expression, mitochondrial DNA (mtDNA), mitochondrial biogenesis and superoxide dismutase 2 (SOD2) in high glucose treated HepG2 cells were investigated by quantitative polymerase chain reaction and Western blot analysis. Molecular docking reveals higher binding affinity of L (ΔG = -6.3 kcal/mol) and OXL (ΔG = -15.5 kcal/mol) with AMPK, compared with metformin (ΔG = -5.0 kcal/mol). The phosphorylation of AMPK increased by 1.3- and 1.5-fold with L and OXL treatment, respectively, in high glucose induced HepG2 cells. The activation of PGC-1α is significant (P < 0.05) in OXL group than L. Similarly, TFAM expression is increased with L and OXL compared with the high glucose group. Further increase in SOD2 and mtDNA, confirms the efficacy of L and OXL in restoring the mitochondrial biogenesis in high glucose induced cells through AMPK, PGC-1α, and TFAM.

Analysis of rat DNA contamination in meatball meat circulating in Makassar by PCR (Polymerase Chain Reaction) method has been carried out. In this study, a polymerase chain reaction method will be developed to analyze the presence of rat meat contamination in beef meatballs. There are three stages in the PCR amplification process carried out with 30 cycles, which are 95oC temperature denaturation, 51oC attachment, and 72oC extension. DNA analysis included agarose gel electrophoresis, measurement of concentration and purification, and analysis of rat DNA using PCR. The results of PCR amplification using mouse-specific primers namely primary ND1 (NADH dehydrogenase 1) showed no bands seen in UV light. So that it can be proven that beef meatball samples in the Makassar region did not contain rat DNA. t.

The electron transport systems in mitochondria of many organisms contain alternative respiratory enzymes distinct from those of the canonical respiratory system depicted in textbooks. Two of these enzymes, the alternative NADH dehydrogenase and the alternative oxidase, were of interest to a limited circle of researchers until they were envisioned as gene therapy tools for mitochondrial disease treatment. Recently, these enzymes were discovered in several animals. Here, we analyse the functioning of alternative NADH dehydrogenases and oxidases in different organisms. We propose that both enzymes ensure bioenergetic and metabolic flexibility during environmental transitions or other conditions which may compromise the operation of the canonical respiratory system.

Cysteamine bitartrate is a US Food and Drug Administration-approved therapy for nephropathic cystinosis also postulated to enhance glutathione biosynthesis. We hypothesized this antioxidant effect may reduce oxidative stress in primary mitochondrial respiratory chain (RC) disease, improving cellular viability and organismal health. Here, we systematically evaluated the therapeutic potential of cysteamine bitartrate in RC disease models spanning three evolutionarily distinct species. These pre-clinical studies demonstrated the narrow therapeutic window of cysteamine bitartrate, with toxicity at millimolar levels directly correlating with marked induction of hydrogen peroxide production. Micromolar range cysteamine bitartrate treatment in Caenorhabditis elegans gas-1(fc21) RC complex I (NDUFS2(−/−)) disease invertebrate worms significantly improved mitochondrial membrane potential and oxidative stress, with corresponding modest improvement in fecundity but not lifespan. At 10 to 100 μm concentrations, cysteamine bitartrate improved multiple RC complex disease FBXL4 human fibroblast survival, and protected both complex I (rotenone) and complex IV (azide) Danio rerio vertebrate zebrafish disease models from brain death. Mechanistic profiling of cysteamine bitartrate effects showed it increases aspartate levels and flux, without increasing total glutathione levels. Transcriptional normalization of broadly dysregulated intermediary metabolic, glutathione, cell defense, DNA, and immune pathways was greater in RC disease human cells than in C. elegans, with similar rescue in both models of downregulated ribosomal and proteasomal pathway expression. Overall, these data suggest cysteamine bitartrate may hold therapeutic potential in RC disease, although not through obvious modulation of total glutathione levels. Careful consideration is required to determine safe and effective cysteamine bitartrate concentrations to further evaluate in clinical trials of human subjects with primary mitochondrial RC disease.

Recently, medaka has been used as a model organism in various research fields. However, even though it possesses several advantages over zebrafish, fewer studies were done in medaka compared to zebrafish, especially with regard to its behavior. Thus, to provide more information regarding its behavior and to demonstrate the behavioral differences between several species of medaka, we compared the behavioral performance and biomarker expression in the brain between four medaka fishes, Oryzias latipes, Oryzias dancena, Oryzias woworae, and Oryzias sinensis. We found that each medaka species explicitly exhibited different behaviors to each other, which might be related to the different basal levels of several biomarkers. Furthermore, by phenomics and genomic-based clustering, the differences between these medaka fishes were further investigated. Here, the phenomic-based clustering was based on the behavior results, while the genomic-based clustering was based on the sequence of the nd2 gene. As we expected, both clusterings showed some resemblances to each other in terms of the interspecies relationship between medaka and zebrafish. However, this similarity was not displayed by both clusterings in the medaka interspecies comparisons. Therefore, these results suggest a re-interpretation of several prior studies in comparative biology. We hope that these results contribute to the growing database of medaka fish phenotypes and provide one of the foundations for future phenomics studies of medaka fish.

Complex I is the largest and most intricate of the protein complexes of mitochondrial electron transport chain (ETC). This L-shaped enzyme consists of a peripheral hydrophilic matrix domain and a membrane-bound orthogonal hydrophobic domain. The interfacial region between these two arms is known to be critical for binding of ubiquinone moieties and has also been shown to be the binding site of Complex I inhibitors. Knowledge on specific roles of the ETC interfacial region proteins is scarce due to lack of knockout cell lines and animal models. Here we mutated nuclear encoded NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2), one of three protein subunits of the interfacial region, in a human embryonic kidney cell line 293 using a CRISPR/Cas9 procedure. Disruption of NDUFS2 significantly decreased cell growth in medium, Complex I specific respiration, glycolytic capacity, ATP pool and cell-membrane integrity, but significantly increased Complex II respiration, ROS generation, apoptosis, and necrosis. Treatment with idebenone, a clinical benzoquinone currently being investigated in other indications, partially restored growth, ATP pool, and oxygen consumption of the mutant. Overall, our results suggest that NDUFS2 is vital for growth and metabolism of mammalian cells, and respiratory defects of NDUFS2 dysfunction can be partially corrected with treatment of an established mitochondrial therapeutic candidate. This is the first report to use CRISPR/Cas9 approach to construct a knockout NDUFS2 cell line and use the constructed mutant to evaluate the efficacy of a known mitochondrial therapeutic to enhance bioenergetic capacity.