When is mitochondrial dna replicated

Cells expressing Twinkle mutants accumulated double-stranded mtDNA replication intermediates the main feature of the strand-coupled model with loss of RNA associated with the lagging strand the hallmark of the RITOLS model , compared to control cells. The single-strandness of the long-standing stranddisplacement model of mtDNA replication might, in the end, just be a technical artifact. The research has also gone beyond mammalian organisms, showing the possible conservation of mechanisms in birds Reyes et al.

Unfortunately for the field, researchers of the strand-displacement model have not maintained the same pace of investigations using in vivo data, which might compromise the credibility of the oldest model. All proteins involved in animal mtDNA maintenance are encoded by nuclear genes, and to date, only three of these proteins have been identified working at the mtDNA replication fork Figure 3. This ssDNA is also bound to the mitochondrial single-stranded DNA-binding protein mtSSB , which protects it from nucleolysis, without any associated catalytic activity.

Furthermore, replication does not appear to be primed by RNA derived from a dedicated primase see discussion below , but instead by extension of processed RNA transcripts laid down by the mitochondrial RNA polymerase mtRNApol Reyes et al. Interestingly, sequence and structural analyses indicate that the mtDNA replisome is a "chimeric machinery", consisting of proteins of different evolutionary origins. How these proteins of originally distinct functions have evolved to participate in mtDNA replication is still unclear, although the functional rewiring of ancestral proteins appears to have happened frequently in mitochondrial history Shutt and Gray, a; Camara et al.

The mutations found in POLG1 , are nearly uniformly distributed along the length of the gene Stumpf and Copeland, , but they do tend to cluster within distinct functional modules in the tridimensional structure of the enzyme, at least for the mutations associated with the severe Alpers' syndrome Euro et al. Interestingly, Alpers patients typically present compound heterozygosity, but there has not yet been a case in which a combination of two mutations from the same functional module was found.

Surprisingly, the most pronounced phenotypes of these mice were reduced longevity and premature aging, characterized by a loss of weight reduction in subcutaneous fat content and in whole-body bone mineral density and content , kyphosis, alopecia, anemia, infertility, hearing loss and reduced hair density, detected at a young age weeks.

Although it appears that the mtDNA mutator mouse does not have increased levels of reactive oxygen species ROS due to its mitochondrial dysfunctions, this animal model clearly establishes the role of mtDNA mutations as a cause of aging and age-related diseases. Mitochondrial ROS are generated when the transport of electrons in the respiratory chain is interrupted by chemical inhibition or mutations in subunits of the OXPHOS complexes , causing a leak of these electrons, which will consequently react directly with molecular oxygen, creating highly reactive molecules that have DNA, lipid and protein damaging properties.

This chain of events is part of the foundation of the mitochondrial theory of aging; data from the mutator mouse suggests that this theory needs some reevaluation to accommodate the idea that mtDNA mutations might be involved with other aging mechanisms, and not with elevated ROS production reviewed in Bratic and Larsson, Mutations in the human Twinkle gene C10orf2 were for the first time reported in as a cause of autosomal dominant PEO Table 1 , associated with multiple mtDNA deletions Spelbrink et al.

Combined with mutagenesis studies that demonstrated the roles of conserved residues in the Walker A and Walker B motifs and identified the arginine finger of Twinkle Matsushima and Kaguni, ; Matsushima et al. The sequence similarities between Twinkle and the bifunctional gp4 primase-helicase of bacteriophage T7, which suggest that these enzymes share ancestry, are most pronounced in the C-terminal helicase domain Spelbrink et al.

The N-terminal "primase"-like domain of the human Twinkle and all other vertebrates has diverged dramatically, losing nearly all conserved residues important for synthesis of RNA primers Shutt and Gray, b and, most importantly, showing no detectable primase activity in vitro Farge et al. Even the Drosophila melanogaster Twinkle, which unlike its vertebrate homologues, retained most of the conserved cysteines found in the primase domain of T7 gp4 Shutt and Gray, b , does not appear to need these residues during mtDNA replication in cultured S2 cells Matsushima and Kaguni, These observations give us the opportunity to speculate on the roles of the N-terminal "primase"-like domain of Twinkle and on the nature of the putative mtDNA primase.

Additionally, the N-terminus might be important for some of the putative roles of Twinkle as the initiator of mtDNA replication Jemt et al. Regardless by which mechanism mtDNA is replicated, the need for mtRNApol in this process is crucial for a more detailed review of the interface between transcription and replication in animal mitochondria, we recommend Kasiviswanathan et al.

In fact, disturbing some of the most conserved residues of the ssDNA-binding domain by alanine substitutions disrupted the ssDNA-binding properties of recombinant mtSSB, as expected Farr et al. The ability of mtSSB to stimulate in vitro the intrinsic activities of pol y and Twinkle has been shown in individual enzymatic assays Farr et al.

We can speculate that mtSSB coordinates the functions of these two enzymes, ensuring that the replication fork progresses smoothly during mtDNA duplication. The goal of the research efforts on mtDNA replication is to understand the basic cellular mechanism s that promote s duplication of this genome, ultimately providing insight into treatment options for human patients with mtDNA replication-related diseases.

However, as research in this area advances, a complex and diverse picture of these mechanisms emerges, giving the impression that treatment is far from being achieved.

Therefore, these findings need to be taken into careful consideration when developing treatments. We thank Crassos Caio de Oliveira for helping with the figures, and Dr. Mike Gerards for critical reading of the manuscript. License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abrir menu Brasil. Indeed, this outcome may merely reflect a difference between domestic and non-domestic mammals. A small-scale study comparing the effects of haplotype between two breeds of cattle demonstrated that one mtDNA lineage produced a greater number of blastocysts following SCNT Bruggerhoff et al.

In SCNT-bovine embryos, divergence up to 0. In SCNT-ovine embryos, divergence up to 0. A model approach using handmade cloning and phylogenetic analysis has gone some way to clarifying the relationship between donor cell and recipient oocyte mtDNA Bowles et al. Handmade clones can be generated by fusing a donor cell to one or more enucleated oocytes of varying genetic distance to determine whether the donor cell will preferentially replicate a population of mtDNA that is closer or genetically more divergent.

The outcomes of these studies demonstrated that, within limits of genetic distance, the donor cell favours a genetically more diverse mtDNA haplotype to that of its own with the most divergent genetic distance being at 0. Fusion with very close genetic partners appears to hinder development. Cytoplasmic hybrids, commonly known as cybrids, have been used to study nuclear—cytoplasmic interactions in a variety of species.

This technology requires either the karyoplast to be completely or partially depleted of its mtDNA and it is then fused to an enucleated cytoplast King and Attardi, The resultant fusions can then be used to understand either the proliferation of mtDNA mutations for specific cells in the disease state or the interactions between a nucleus from one species with a population of mtDNA from another. For example, the generation of mouse—rat cybrids, where rat mtDNA was efficiently replicated, transcribed and translated by the mouse nuclear transcription and replication factors, had decreased ATP output and oxygen consumption, and increased levels of cellular lactate and reduced activity in complexes I, III and IV of the ETC compared with the parental cell lines and mouse—mouse cybrids Barrientos et al.

Similar outcomes have been observed as primate mtDNA can only be replicated by a human nucleus when the human mtDNA has been eliminated Barrientos et al. In this instance, the activities of complexes II—V were unaltered, but there was a deficiency in complex I activity. Induced pluripotent cells iPSCs are somatic cells that have been induced to become ESC-like due to the introduction of defined factors Takahashi and Yamanaka, They then express key pluripotent markers such as Oct4 , Nanog and Sox-2 and have the ability to differentiate into multiple cell types.

It however remains to be determined whether such cells have the ability to regulate their mtDNA copy number in their undifferentiated state as ESCs do. It further needs to be demonstrated whether these cells can effectively regulate their patterns of mtDNA replication as the process of differentiation takes place in ESCs Facucho-Oliveira et al. It is unlikely that those cells generated through NT would be able to do so, due to the up-regulation of the mtDNA replication factors during early embryonic development Bowles et al.

Attempts at reprogramming cells using cell fusion Sumer et al. These may include heteroplasmy, because of two different sources of cells being used to generate the newly fused reprogrammed cell, and the failure to adopt appropriate numbers of mtDNA copies.

As reprogrammed cells have been proposed as models that can be used for the study of specific diseases and to screen drugs for their efficacy Bowles et al. Consequently, it would not be clear from the experimental outcome whether the cellular dysfunction arose through the genetic rearrangement or the cell's inability to generate sufficient levels of ATP through OXPHOS.

Equally so, the inability of the drug to induce, e. However, as such cells offer considerable hope as clinical entities, then it is worth investigating whether it is feasible to simply regulate the mtDNA content of these cells so that they are suitable for therapeutic and experimental use.

The segregation of mtDNA molecules tends to follow a pattern of random genetic drift reviewed in Shoubridge and Wai, Consequently, it is not possible to predict which cell types or tissues mutant molecules will migrate to during early development.

This is the case for both somatic and germ cells and specifically relevant to female primordial germ cells PGCs , which, when harbouring mutant molecules, would thus ensure their continued transmission to subsequent generations. PGCs originate from the extra-embryonic endoderm and migrate to the germinal ridge Surani et al. Consequently, those mature mouse oocytes that are competent for the process of fertilization or oocyte reconstruction will have between and mtDNA copies Cao et al.

Consequently, the combination of random genetic drift and clonal expansion of mtDNA would explain the large variation in mutant molecules found within the cohorts of oocytes from those women who are potential carriers of mtDNA mutations.

Following fertilization of the oocyte, mtDNA copy number remains constant during mouse preimplantation development up to the morula stage but is reduced in porcine Spikings et al.

However, this replication is specific to the trophectodermal cells, the cells that give rise to the placenta, whereas those cells giving rise to the embryo or ESCs, the ICM cells, have very few mitochondria present and thus copies of the mtDNA genome Spikings et al.

The ICM cells are likely to continue to reduce their mtDNA copy number as they continue to divide before they undergo gastrulation, as evidenced by human and mouse ESCs having as few as 10—20 mitochondria St John et al. This selective reduction in mtDNA copies in those cells giving rise to the fetus ensures that very few copies are available for clonal amplification in gametes and other tissues and that the maternal homoplasmic transmission of a single type of molecule is transmitted to the next generation.

This process, otherwise known as the mitochondrial bottleneck Hauswirth and Laipis, , also ensures that mutated molecules are either eliminated or fixed in the female germline according to the severity of the mutation within a generation so that either quality mtDNA is transmitted or the mutation is lethal and the oocytes do not mature Fan et al. The variable transmission of mtDNA has questioned the feasibility of using preimplantation genetic diagnosis PGD on embryos generated following either in vitro fertilization or intra-cytoplasmic sperm injection protocols to determine whether individual preimplantation blastomeres contain equal loading for mutant molecules.

Interestingly, although mutant load can be different in various mature cell types reviewed in McFarland et al. Although this is a reassuring outcome, it suggests that PGD is only useful as a predictor that mature cells might have high mutant mtDNA loading when a critical threshold of mutant loading is reached in preimplantation embryos.

For those preimplantation embryos with low levels of mutation, it can inform patients that their offspring would most unlikely be affected Poulten et al. However, the overriding question is: how sure can we be? It is likely that there is a refinement of mtDNA copy at various stages of development and these are likely to be linked to the expression of certain genes during the various different stages of development Facucho-Oliveira et al.

Early phases of replication are likely to be associated with replenishment of a genome and also to determine whether the replication machinery is capable of sustaining phasic changes, thus ensuring that, at various stages of differentiation, the cell is capable of adaptation.

Consequently, PGD may not reflect later stage outcomes. This is further likely as it appears that mtDNA rearrangements and wild-type molecules are able to intermix so that they do not remain as separate entities within distinct organelles or cells Nakada et al. Currently, there are no effective intervention strategies available to prevent the transmission of mtDNA disease from mother to offspring.

Experimental approaches concentrate on determining whether germinal vesicle or pro-NT might be appropriate approaches. In this case, the germinal vesicle or pronuclei from the affected oocyte would be transferred into an enucleated egg that was not carrying the mutant population Meirelles and Smith, The degree of donor cell transmission that arises or transfer of donor mitochondria following cytoplasmic transfer when at its higher levels could mimic conditions associated with mitochondrial disease.

Studies, for example, using mouse strains with variants in the coding sequences between the two strains would be similar to clinical-onset heteroplasmy and would produce similar outcomes to those observed following SCNT when different breeds of cattle and pig contributed by a donor cell and a recipient oocyte. As previously demonstrated, some replication of such mtDNA can take place during preimplantation development Meirelles and Smith, and persist at high levels in a few offspring Sato et al.

Consequently, it is worth questioning whether the degree of mtDNA heteroplasmy in cloned offspring would indeed account for some of the phenotypic diseases observed from NT. Although there is no direct evidence currently to support this, there is substantive evidence that is associated with those malformations in tissues from fetuses and offspring following SCNT Cibelli et al.

A recent study has also demonstrated that donor ooplasm transfer can affect growth in early life with interspecific germinal vesicle transfer causing growth abnormalities post-natally Cheng et al. Such tissues that might be affected are lung, heart and liver Bowles et al.

As the transmission of donor cell mtDNA in cloned offspring could also be explained by random genetic drift, as these molecules tend to segregate in a non-uniform manner, the up-regulation of mtDNA replication factors during preimplantation development Bowles et al. Consequently, any assisted reproductive technology that uses an oocyte cytoplasm to reprogramme the donor cell and allows mixing of mtDNA populations will affect any PGD outcomes as the population present in the preimplantation embryo will continue to be replicated unlike its fertilized counterpart and would be only predictive of that stage of development.

This outcome may be equally valid for the transfer of a germinal vesicle or a pronuclei which will bring some accompanying mtDNA as part of the transfer. Consequently, entire elimination of donor cell mtDNA would be perhaps an appropriate safeguard for such approaches Bowles et al. A recent attempt in M. The task for scientists at the moment is to elicit when these key stages are and how many of these key stages exist.

We have approaches that may be able to regulate mtDNA transmission in the new offspring through assisted reproductive technologies.

The task for scientists now is to develop approaches using stem cells that will allow dysfunctional tissues affected by mtDNA to be replaced with non-heteroplasmic populations which will alleviate the severe phenotype on the offspring.

Google Scholar. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Search methods. The mitochondrion. What is mtDNA? Mitochondrial DNA transmission, replication and inheritance: a journey from the gamete through the embryo and into offspring and embryonic stem cells.

John , Justin C. E-mail: justin. Oxford Academic. Joao Facucho-Oliveira. Yan Jiang. Richard Kelly. Rana Salah. Revision received:. Cite Cite Justin C. Select Format Select format. Permissions Icon Permissions. Figure 1. Open in new tab Download slide. Figure 2. The electrons then flow to the lipid-soluble carrier co-enzyme Q which in turn donates electrons to Complex III. Cytochrome c is the next electron acceptor and donates electrons to Complex IV.

The energy released during the transfer of electrons is used by Complexes I, III and IV to pump protons across the mitochondrial inner membrane and to generate an electrochemical gradient. Figure 3. ATAD3 are arranged peripherally. Nucleoid protein. Molecular weight kDa. Chromosomal location in human. TFAM Open in new tab. Figure 4. The main events of mitochondrial biogenesis and mtDNA proliferation.

However, the cells of the ICM continue to decrease their numbers with each cell division with undifferentiated and differentiating ESCs maintaining low copy number. Figure 5. Table II mtDNA content in cloned embryos, fetuses and offspring generated by intra- and interspecific nuclear transfer. Sample analysed. Degrees of heteroplamsy. Detection methods. Bovine Sampled blastomeres, blastocysts, tissues of a newborn clone NT-blastomere zygotes and blastocysts had similar levels of heteroplasmy donor cell— Heteroplasmic levels in NT-fibroblast zygotes—4.

High levels of heteroplasmy 6. Amount of donor cell mtDNA depended on donor cell type and tissue with significant differences between sertoli 0. Taurus nucleus and B. Donor cell. Recipient oocyte. Degree of heteroplasmy. Mixed-breed domestic cat fibroblasts Bovine 1-, 2-, 4- and 8-cell stage embryos Cat mtDNA stable from 1- to 8-cell. Real-time PCR Ma et al. Real-time PCR Hua et al. Rabbit mtDNA remained constant to the morula stage and increased sharply in both types of blastocyst. In late stage embryos post-vascularization only recipient oocyte mtDNA detected Species specific PCR on agarose gel electrophoresis with image analysis Sun et al.

Google Scholar Crossref. Search ADS. Almeida Santos. Mitochondrial content reflects oocyte variability and fertilization outcome. The expression of polymerase gamma and mitochondrial transcription factor A and the regulation of mitochondrial DNA content in mature human sperm.

Human xenomitochondrial cybrids. Cellular models of mitochondrial complex I deficiency. Patterns of organelle distribution in mouse embryos during preimplantation development.

Crystal structures of the Klenow fragment of DNA polymerase I complexed with deoxynucleoside triphosphate and pyrophosphate. Characterization of the helicase and primase activities of the kDa component of the bacteriophage T7 gene 4 protein. Google Scholar PubMed. Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolution.

The inheritance of genes in mitochondria and chloroplasts: laws, mechanisms, and models. Nuclear transfer: preservation of a nuclear genome at the expense of its associated mtDNA genome s. Contrasting effects of in vitro fertilization and nuclear transfer on the expression of mtDNA replication factors.

Mitochondrial DNA transmission and transcription after somatic cell fusion to one or more cytoplasts. Bovine somatic cell nuclear transfer using recipient oocytes recovered by ovum pick-up: effect of maternal lineage of oocyte donor. Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer.

The mitochondrial bottleneck occurs without reduction of mtDNA content in female mouse germ cells. Crystal structure and deletion analysis show that the accessory subunit of mammalian DNA polymerase gamma, Pol gamma B, functions as a homodimer.

Blastocyst formation, karyotype, and mitochondrial DNA of interspecies embryos derived from nuclear transfer of human cord fibroblasts into enucleated bovine oocytes. Interspecies implantation and mitochondria fate of panda-rabbit cloned embryos.

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes. Effects of ooplasm manipulation on DNA methylation and growth of progeny in mice. Leber hereditary optic neuropathy: does heteroplasmy influence the inheritance and expression of the GA mitochondrial DNA mutation? Dynamic changes in mitochondrial biogenesis and antioxidant enzymes during the spontaneous differentiation of human embryonic stem cells.

In vitro methylation of nuclear respiratory factor-1 binding site suppresses the promoter activity of mitochondrial transcription factor A. Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells. Birth of infant after transfer of anucleate donor oocyte cytoplasm into recipient eggs. A reduction of mitochondrial DNA molecules during embryogenesis explains the rapid segregation of genotypes.

Evaluation of parental mitochondrial inheritance in neonates born after intracytoplasmic sperm injection. Prospect of preimplantation genetic diagnosis for heritable mitochondrial DNA diseases. Functional constraints of nuclear-mitochondrial DNA interactions in xenomitochondrial rodent cell lines. Di Re. Fate of donor cell mitochondrial DNA in cloned bovine embryos produced by microinjection of cumulus cells.

Mitochondrial transcription factor A regulates mtDNA copy number in mammals. El Shourbagy. The relationship between pluripotency and mitochondrial DNA proliferation during early embryo development and embryonic stem cell differentiation. Mitochondrial DNA replication during differentiation of murine embryonic stem cells. A mouse model of mitochondrial disease reveals germline selection against severe mtDNA mutations.

The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos. DNA wrapping and bending by a mitochondrial high mobility group-like transcriptional activator protein. In vitro differentiation of embryonic stem cells into glial cells and functional neurons. DNA polymerases of tumor virus: specific effect of ethidium bromide on the use of different synthetic templates.

Isolation and characterization of all-trans-retinoic acid-responsive genes in the rat testis. The mitochondrial RNA polymerase contributes critically to promoter specificity in mammalian cells.

Mitochondrial DNA heteroplasmy in calves cloned by using adult somatic cell. Mitochondrial DNA polymerase gamma is essential for mammalian embryogenesis. Cryopreservation of nuclear material as a potential method of fertility preservation.

According to the manifestations of the disease, MDS can be divided into three categories: myopathic, encephalomyopathic and hepatocerebral. To illustrate these effects, two inherited POLG -originated diseases are described. PEO patients also manifest skeletal muscle weakness and wasting accompanied by exercise intolerance. This disease is also associated with specific neurologic syndromes such as familial forms of spastic paraplegia, spinocerebellar disorders, and sensorimotor peripheral neuropathy.

The variants of this disorder involve both autosomal dominant adPEO and recessive arPEO forms, as both the nuclear and the mitochondrial genome are implicated in this pathogenesis. This is a rare but very severe and usually lethal autosomal recessive MDS disease that appears within the first few years of life. Patients exhibit progressive spastic quadri-paresis, progressive cerebral degeneration leading to mental deterioration, cortical blindness, deafness and liver failure. The most widely studied class of drugs that inhibit mtDNA replication, thus generating drug-related toxicities, are the nucleoside analog reverse transcriptase inhibitors NRTI Fig.

They are administered as prodrugs that must be transported into the cell and phopshorylated to the metabolically active triphosphate in order to exert their therapeutic effect. These drugs are pharmacological analogues of native nucleosides that can be. Reverse Transcriptase Inhibitors are a group of antiretroviral drugs which inhibit the viral reverse transcriptase, a crucial enzyme of the HIV life cycle. This enzyme reverse-transcribes the viral RNA genome into DNA, which is then integrated into the host genome and replicated along with it.

Additional effects on mtDNA synthesis have also been suggested. Regardless of the mechanism by which mtDNA replication is compromised, it ultimately interferes with the synthesis of essential proteins of the mitochondrial ETC Chiao et al. Clinical experience with NRTI-including therapy has revealed the appearance of several side effects ranging from hyperlactatemia and lactic acidosis to lipodystrophy, myopathy, peripheral neuropathy, bone marrow suppression, insulin resistance and diabetes, as well as hepatosteatosis and pancreatitis, some of which develop into life-threatening condition Kakuda, The first report of NRTI-induced mitochondrial effects, described in , was myopathy in patients treated with zidovudine, who exhibited ragged red muscle fibers and reduced mtDNA content Dalakas et al.

Cardiomyopathy and bone marrow suppression were also described. This, together with the lower degree of lamivudine incorporation in the mtDNA chain, predicts reduced toxicity for this analogue, a finding which is supported by in vivo observations.

For example, a. This undermines mtDNA synthesis with a consequent depletion of the mtDNA-encoded subunits of the mitochondrial electron transport chain. Such an effect leads to impairment of the mitochondrial function manifested as compromised oxidative phosphorylation, a reduction in mitochondrial membrane potential and induction of oxidative stress. Tyr and Glu seem to interact with the rigid sugar rings of stavudine and abacavir. In vivo millimolar accumulation has been shown for the phosphorylated form of zidovudine Frick et al.

Moreover, interactions with host proteins during the process of activation of NRTI inside the cell, allow additional mechanisms for mitochondrial toxicity of these drugs. However, the adverse reactions associated with the long-term use of this therapy rash and hypersensibility reactions, hepatotoxicity, metabolic disturbances including lipodystrophy, hyperlactatemia, and CNS toxic effects have become a major concern.

As a result, research efforts are now focused on understanding the cellular mechanisms underlying these effects. However, other mitochondrial mechanisms and targets responsible for NRTI-induced mitotoxicity have also been suggested. In vivo studies have demonstrated that treatment with this drug leads to a disrupted cardiac mitochondrial ultrastructure and a diminished expression of mitochondrial cytochrome b mRNA, as well as induction of oxidative stress in heart mtDNA Sardao et al.

In addition, in cultured rat hepatocytes, stavudine, but not zidovudine or zalcitabine, impairs fatty acid oxidation in the absence of mtDNA depletion Igoudjil et al. Moreover, mitochondrial import of nucleoside drugs may also be related to their toxicity in this organelle.

Some of the nucleoside channels have been shown to transport stavudine, zalcitabine, zidovudine and didanosine Yamamoto et al. Hepatotoxicity has emerged as one of the most common adverse events associated with HAART and constitutes a major problem in the management of HIV-patients. Preliminary studies were performed in which cells were treated with NRTI Sequoia Research Products at therapeutic concentrations during a short period of time in order to avoid any effects due to a decrease in mtDNA content.

Subsequently, several parameters of mitochondrial function including mitochondrial respiration, generation of ATP and mitochondrial ROS production were determined. Measurements were recorded using the Duo. For these measurements, cells were incubated for 1h with the NRTI under study.

Protein concentrations were determined with the BCA protein assay kit. ROS production was analyzed in cells seeded in a black well plate.