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Patel, Maulik R., Aimee R. Littleton, Ganesh Miriyala, and Harmit S. Malik. "Disease consequences of conflict between mitochondrial and nuclear genomes." Mitochondrion 24 (September 2015): S20—S21. http://dx.doi.org/10.1016/j.mito.2015.07.061.
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Wade, Michael J., and Devin M. Drown. "Nuclear–mitochondrial epistasis: a gene's eye view of genomic conflict." Ecology and Evolution 6, no. 18 (August 18, 2016): 6460–72. http://dx.doi.org/10.1002/ece3.2345.
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Rand, David M., and Jim A. Mossman. "Mitonuclear conflict and cooperation govern the integration of genotypes, phenotypes and environments." Philosophical Transactions of the Royal Society B: Biological Sciences 375, no. 1790 (December 2, 2019): 20190188. http://dx.doi.org/10.1098/rstb.2019.0188.
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The mitonuclear genome is the most successful co-evolved mutualism in the history of life on Earth. The cross-talk between the mitochondrial and nuclear genomes has been shaped by conflict and cooperation for more than 1.5 billion years, yet this system has adapted to countless genomic reorganizations by each partner, and done so under changing environments that have placed dramatic biochemical and physiological pressures on evolving lineages. From putative anaerobic origins, mitochondria emerged as the defining aerobic organelle. During this transition, the two genomes resolved rules for sex determination and transmission that made uniparental inheritance the dominant, but not a universal pattern. Mitochondria are much more than energy-producing organelles and play crucial roles in nutrient and stress signalling that can alter how nuclear genes are expressed as phenotypes. All of these interactions are examples of genotype-by-environment (GxE) interactions, gene-by-gene (GxG) interactions (epistasis) or more generally context-dependent effects on the link between genotype and phenotype. We provide evidence from our own studies in Drosophila , and from those of other systems, that mitonuclear interactions—either conflicting or cooperative—are common features of GxE and GxG. We argue that mitonuclear interactions are an important model for how to better understand the pervasive context-dependent effects underlying the architecture of complex phenotypes. Future research in this area should focus on the quantitative genetic concept of effect size to place mitochondrial links to phenotype in a proper context. This article is part of the theme issue ‘Linking the mitochondrial genotype to phenotype: a complex endeavour’.
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Havird, Justin C., Ryan J. Weaver, Liliana Milani, Fabrizio Ghiselli, Ryan Greenway, Adam J. Ramsey, Ana G. Jimenez, et al. "Beyond the Powerhouse: Integrating Mitonuclear Evolution, Physiology, and Theory in Comparative Biology." Integrative and Comparative Biology 59, no. 4 (August 24, 2019): 856–63. http://dx.doi.org/10.1093/icb/icz132.
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Abstract Eukaryotes are the outcome of an ancient symbiosis and as such, eukaryotic cells fundamentally possess two genomes. As a consequence, gene products encoded by both nuclear and mitochondrial genomes must interact in an intimate and precise fashion to enable aerobic respiration in eukaryotes. This genomic architecture of eukaryotes is proposed to necessitate perpetual coevolution between the nuclear and mitochondrial genomes to maintain coadaptation, but the presence of two genomes also creates the opportunity for intracellular conflict. In the collection of papers that constitute this symposium volume, scientists working in diverse organismal systems spanning vast biological scales address emerging topics in integrative, comparative biology in light of mitonuclear interactions.
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Wang, Baohua, Yan Zhang, Peipei Wei, Miao Sun, Xiaofei Ma, and Xinyu Zhu. "Identification of nuclear low-copy genes and their phylogenetic utility in rosids." Genome 57, no. 10 (October 2014): 547–54. http://dx.doi.org/10.1139/gen-2014-0138.
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By far, the interordinal relationships in rosids remain poorly resolved. Previous studies based on chloroplast, mitochondrial, and nuclear DNA has produced conflicting phylogenetic resolutions that has become a widely concerned problem in recent phylogenetic studies. Here, a total of 96 single-copy nuclear gene loci were identified from the KOG (eukaryotic orthologous groups) database, most of which were first used for phylogenetic analysis of angiosperms. The orthologous sequence datasets from completely sequenced genomes of rosids were assembled for the resolution of the position of the COM (Celastrales–Oxalidales–Malpighiales) clade in rosids. Our analysis revealed strong and consistent support for CM topology (the COM clade as sister to the malvids). Our results will contribute to further exploring the underlying cause of conflict between chloroplast, mitochondrial, and nuclear data. In addition, our study identified a few novel nuclear molecular markers with potential to investigate the deep phylogenetic relationship of plants or other eukaryotic taxonomical groups.
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Klucnika, Anna, and Hansong Ma. "A battle for transmission: the cooperative and selfish animal mitochondrial genomes." Open Biology 9, no. 3 (March 2019): 180267. http://dx.doi.org/10.1098/rsob.180267.
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The mitochondrial genome is an evolutionarily persistent and cooperative component of metazoan cells that contributes to energy production and many other cellular processes. Despite sharing the same host as the nuclear genome, the multi-copy mitochondrial DNA (mtDNA) follows very different rules of replication and transmission, which translate into differences in the patterns of selection. On one hand, mtDNA is dependent on the host for its transmission, so selections would favour genomes that boost organismal fitness. On the other hand, genetic heterogeneity within an individual allows different mitochondrial genomes to compete for transmission. This intra-organismal competition could select for the best replicator, which does not necessarily give the fittest organisms, resulting in mito-nuclear conflict. In this review, we discuss the recent advances in our understanding of the mechanisms and opposing forces governing mtDNA transmission and selection in bilaterians, and what the implications of these are for mtDNA evolution and mitochondrial replacement therapy.
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GRZYWACZ-GIBAŁA, BEATA, DRAGAN P. CHOBANOV, and ELŻBIETA WARCHAŁOWSKA-ŚLIWA. "Preliminary phylogenetic analysis of the genus Isophya (Orthoptera: Phaneropteridae) based on molecular data." Zootaxa 2621, no. 1 (September 22, 2010): 27. http://dx.doi.org/10.11646/zootaxa.2621.1.2.
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A preliminary phylogenetic analysis involving 22 taxa of the genus Isophya was conducted using four molecular markers (sequences of mitochondrial cytochrome b (cyt b), fragments of mitochondrial cytochrome oxidase subunit II (COII) and the internal transcribed spacers I and II (ITS1 and ITS2)). Our results indicate that Isophya is a monophyletic group with the exception of one species, I. bivittata. The analysis revealed a high level of polymorphism in the mitochondrial and nuclear genes of all species. MtDNA performs better in the phylogenetic reconstruction of Isophya than the ITS markers. Although the results show some conflict with the systematics of the group known from morphological and bioacoustic data, the study can be the basis for future reconstructions of the phylogeny of Isophya.
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Wahlberg, Niklas, Elisabet Weingartner, Andrew D. Warren, and Sören Nylin. "Timing major conflict between mitochondrial and nuclear genes in species relationships of Polygonia butterflies (Nymphalidae: Nymphalini)." BMC Evolutionary Biology 9, no. 1 (2009): 92. http://dx.doi.org/10.1186/1471-2148-9-92.
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Johnson, Kevin P., and Dale H. Clayton. "A Molecular Phylogeny of the Dove Genus Zenaida: Mitochondrial and Nuclear DNA Sequences." Condor 102, no. 4 (November 1, 2000): 864–70. http://dx.doi.org/10.1093/condor/102.4.864.
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AbstractWe reconstructed a phylogeny for the seven species of doves in the genus Zenaida on the basis of a combined analysis of mitochondrial (ND2 and cytochrome b) and nuclear (fibrinogen intron 7) DNA sequences. This phylogeny, which is completely resolved, is well supported with all nodes showing greater than 50% bootstrap support. There was no significant conflict between trees based on each gene independently, although trees produced from fibrinogen intron 7 did not resolve relationships among five of the Zenaida species. The species status of Z. graysoni, as well as that of Z. meloda, is suggested based on their divergence from sister taxa (about 1% and 4%, respectively) and other differences. Zenaida can be divided into two major groups: Zenaida asiatica and Z. meloda versus Z. aurita, Z. galapagoensis, Z. auriculata, Z. graysoni, and Z. macroura.
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Sahoo, Ranjit Kumar, Andrew D. Warren, Niklas Wahlberg, Andrew V. Z. Brower, Vladimir A. Lukhtanov, and Ullasa Kodandaramaiah. "Ten genes and two topologies: an exploration of higher relationships in skipper butterflies (Hesperiidae)." PeerJ 4 (December 6, 2016): e2653. http://dx.doi.org/10.7717/peerj.2653.
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Despite multiple attempts to infer the higher-level phylogenetic relationships of skipper butterflies (Family Hesperiidae), uncertainties in the deep clade relationships persist. The most recent phylogenetic analysis included fewer than 30% of known genera and data from three gene markers. Here we reconstruct the higher-level relationships with a rich sampling of ten nuclear and mitochondrial markers (7,726 bp) from 270 genera and find two distinct but equally plausible topologies among subfamilies at the base of the tree. In one set of analyses, the nuclear markers suggest two contrasting topologies, one of which is supported by the mitochondrial dataset. However, another set of analyses suggests mito-nuclear conflict as the reason for topological incongruence. Neither topology is strongly supported, and we conclude that there is insufficient phylogenetic evidence in the molecular dataset to resolve these relationships. Nevertheless, taking morphological characters into consideration, we suggest that one of the topologies is more likely.
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Krajewski, Carey, Gregory R. Moyer, Justin T. Sipiorski, Matthew G. Fain, and Michael Westerman. "Molecular systematics of the enigmatic 'phascolosoricine' marsupials of New Guinea." Australian Journal of Zoology 52, no. 4 (2004): 389. http://dx.doi.org/10.1071/zo04020.
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Previous analyses of DNA sequences for mitochondrial cytochrome-b and 12S rRNA, along with the nuclear protamine P1 genes, suggested that the New Guinean dasyurid genera Phascolosorex and Neophascogale (phascolosoricines) form the sister groups of quolls (Dasyurus) and Tasmanian devils (Sarcophilus). This runs counter to a common perception that phascolosoricines are anatomically primitive and only distantly related to other dasyurids. We report the DNA sequences of two additional mitochondrial loci (tRNAVal and 16S rRNA) and nuclear loci (interphotoreceptor binding protein exon 1, beta-fibrinogen intron 7) from Phascolosorex dorsalis, Neophascogale lorentzii, and all but three other dasyurid species. These sequences, along with those previously published, comprise a dataset of 7053 nucleotides. Phylogenetic analyses indicate that phascolosoricines form a clade that is highly resolved as sister to Dasyurus + Sarcophilus. This result is obtained independently by mitochondrial and nuclear genes, and cannot be attributed to taxon- or gene-sampling bias, compositional heterogeneity, or the use of overly simplistic phylogenetic estimation procedures. A re-evaluation of published morphological data bearing on the relationships of phascolosoricines demonstrates that, although the conflict with molecular results is significant, it is limited to a small number of correlated dental features that show considerable homoplasy in their evolution.
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Fujii, S., C. S. Bond, and I. D. Small. "Selection patterns on restorer-like genes reveal a conflict between nuclear and mitochondrial genomes throughout angiosperm evolution." Proceedings of the National Academy of Sciences 108, no. 4 (January 10, 2011): 1723–28. http://dx.doi.org/10.1073/pnas.1007667108.
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Ogedengbe, Mosun E., Shiem El-Sherry, Joseph D. Ogedengbe, H. David Chapman, and John R. Barta. "Phylogenies based on combined mitochondrial and nuclear sequences conflict with morphologically defined genera in the eimeriid coccidia (Apicomplexa)." International Journal for Parasitology 48, no. 1 (January 2018): 59–69. http://dx.doi.org/10.1016/j.ijpara.2017.07.008.
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Leaché, Adam D. "Species trees for spiny lizards (Genus Sceloporus): Identifying points of concordance and conflict between nuclear and mitochondrial data." Molecular Phylogenetics and Evolution 54, no. 1 (January 2010): 162–71. http://dx.doi.org/10.1016/j.ympev.2009.09.006.
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BOCHKOV, ANDRE V., PAVEL B. KLIMOV, DA-HEE KIM, and MACIEJ SKORACKI. "Validation of the status of a species with high CO1 and low nuclear genetic divergences: the scab mite Caparinia ictonyctis stat. res. (Acariformes: Psoroptidae) parasitizing the African hedgehog Atelerix albiventris." Zootaxa 4544, no. 4 (January 14, 2019): 523. http://dx.doi.org/10.11646/zootaxa.4544.4.4.
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We report two host-specific lineages of scab mites of the genus Caparinia, parasitizing European and African hedgehogs. Based on morphology, these mite lineages are closely related sister groups. The morphological differences, however, are subtle and do not provide clear-cut evidence for the existence of separate species. CO1 divergence between these lineages was 7.4–7.8%, well above the CO1 barcoding gaps or thresholds commonly used to separate species, whereas divergence of five nuclear genes was very low, 0.06–0.53%, suggesting that these lineages could belong to a single species with gene flow between them. Thus, there is a conflict between the mitochondrial (CO1) gene and nuclear genes (i.e mito-nuclear discordance). We attribute this conflict to the ‘gray zone’ where species delimitation is ambiguous due to substantial gene flow. We also report another ‘gray zone’ species, Psoroptes ovis (a species of veterinary importance), whose within-species CO1 distances reached 6.0%. We provide a detailed morphological description and figures of C. ictonyctis stat. res. from the African hedgehog, using light and SEM microscopy and give morphometric data for this species and its sister species, Caparinia tripilis from Europe. For all known species of Caparinia, we document their host associations and give a key to species of the world based on results of our morphological and molecular analyses and a nearly exhaustive study of museum specimens.
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PETERS, JEFFREY L., KIMBERLY A. BOLENDER, and JOHN M. PEARCE. "Behavioural vs. molecular sources of conflict between nuclear and mitochondrial DNA: the role of male-biased dispersal in a Holarctic sea duck." Molecular Ecology 21, no. 14 (May 14, 2012): 3562–75. http://dx.doi.org/10.1111/j.1365-294x.2012.05612.x.
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Small, David M., Washington Y. Sanchez, Sandrine F. Roy, Christudas Morais, Heddwen L. Brooks, Jeff S. Coombes, David W. Johnson, and Glenda C. Gobe. "N-acetyl-cysteine increases cellular dysfunction in progressive chronic kidney damage after acute kidney injury by dampening endogenous antioxidant responses." American Journal of Physiology-Renal Physiology 314, no. 5 (May 1, 2018): F956—F968. http://dx.doi.org/10.1152/ajprenal.00057.2017.
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Oxidative stress and mitochondrial dysfunction exacerbate acute kidney injury (AKI), but their role in any associated progress to chronic kidney disease (CKD) remains unclear. Antioxidant therapies often benefit AKI, but their benefits in CKD are controversial since clinical and preclinical investigations often conflict. Here we examined the influence of the antioxidant N-acetyl-cysteine (NAC) on oxidative stress and mitochondrial function during AKI (20-min bilateral renal ischemia plus reperfusion/IR) and progression to chronic kidney pathologies in mice. NAC (5% in diet) was given to mice 7 days prior and up to 21 days post-IR (21d-IR). NAC treatment resulted in the following: prevented proximal tubular epithelial cell apoptosis at early IR (40-min postischemia), yet enhanced interstitial cell proliferation at 21d-IR; increased transforming growth factor-β1 expression independent of IR time; and significantly dampened nuclear factor-like 2-initiated cytoprotective signaling at early IR. In the long term, NAC enhanced cellular metabolic impairment demonstrated by increased peroxisome proliferator activator-γ serine-112 phosphorylation at 21d-IR. Intravital multiphoton microscopy revealed increased endogenous fluorescence of nicotinamide adenine dinucleotide (NADH) in cortical tubular epithelial cells during ischemia, and at 21d-IR that was not attenuated with NAC. Fluorescence lifetime imaging microscopy demonstrated persistent metabolic impairment by increased free/bound NADH in the cortex at 21d-IR that was enhanced by NAC. Increased mitochondrial dysfunction in remnant tubular cells was demonstrated at 21d-IR by tetramethylrhodamine methyl ester fluorimetry. In summary, NAC enhanced progression to CKD following AKI not only by dampening endogenous cellular antioxidant responses at time of injury but also by enhancing persistent kidney mitochondrial and metabolic dysfunction.
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ACEVEDO, ALDEMAR A., ORLANDO ARMESTO, and R. EDUARDO PALMA. "Two new species of Pristimantis (Anura: Craugastoridae) with notes on the distribution of the genus in northeastern Colombia." Zootaxa 4750, no. 4 (March 13, 2020): 499–523. http://dx.doi.org/10.11646/zootaxa.4750.4.3.
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We describe two new species of the genus Pristimantis from the western margin of the Eastern Cordillera of Colombia. The species have a sympatric distribution and are found in the humid tropical forests of the lower part of the Tamá National Natural Park (PNN Tamá) and its buffer zone. The new species were described from morphological comparisons and phylogenetic reconstruction from the sequencing of three mitochondrial and two nuclear genes. With these two new species, the number of Pristimantis species known in the department of Norte de Santander has increased to 10. In addition, we included new sequences for the species P. anolirex, P. nicefori, P. mondolfii, and P. yukpa. Our explorations in the last decade indicate that there are probably between five and 10 species that have not been described in the northeastern part of Colombia, especially in areas where the armed conflict has predominated in the last five decades.
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Shaw, K. L. "Conflict between nuclear and mitochondrial DNA phylogenies of a recent species radiation: What mtDNA reveals and conceals about modes of speciation in Hawaiian crickets." Proceedings of the National Academy of Sciences 99, no. 25 (November 25, 2002): 16122–27. http://dx.doi.org/10.1073/pnas.242585899.
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Mengual, Ximo, Gunilla Ståhls, and Santos Rojo. "First phylogeny of predatory flower flies (Diptera, Syrphidae, Syrphinae) using mitochondrial COI and nuclear 28S rRNA genes: conflict and congruence with the current tribal classification." Cladistics 24, no. 4 (August 2008): 543–62. http://dx.doi.org/10.1111/j.1096-0031.2008.00200.x.
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Tello, Jose G., and John M. Bates. "Molecular Phylogenetics of The Tody-Tyrant and Flatbill Assemblage of Tyrant Flycatchers (Tyrannidae)." Auk 124, no. 1 (January 1, 2007): 134–54. http://dx.doi.org/10.1093/auk/124.1.134.
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Abstract The tody-tyrant and flatbill assemblage, sensuLanyon (1988a), includes 12 genera of tyrant flycatchers known variously as “tody-tyrants” and “flatbills.” Lanyon supported the monophyly of the group based on similar skull morphology and nest form, and built intergeneric relationships based on syringeal characters. However, these comparisons were made without a phylogenetic framework. A more recent study assessing relationships in the tyrant flycatchers using published morphological and behavioral data failed to recover monophyly of this assemblage (Birdsley 2002). Using DNA sequence data, we test for the monophyly of the tody-tyrant and flatbill assemblage and compare and contrast phylogenetic signals from mitochondrial and nuclear DNA character systems. We discuss our results in light of results from previous studies. We include representatives of the other major tyrant flycatcher assemblages for a total of 42 individuals (representing 27 tyrannid genera and 36 species). We sequenced 3,022 base pairs (bp) of three mitochondrial genes (ND2, ND3, and cytochrome b) and one nuclear intron (FIB5). Our results resolve many of the basal relationships of the tody-tyrant and flatbill phylogeny, but separate and combined analyses of data partitions are necessary to understand the nature of conflict among data sets. The tody-tyrants constitute a monophyletic clade, but the genera Hemitriccus and Lophotriccus are not monophyletic, and the limits of Oncostoma-Lophotriccus need to be revised with more complete sampling at the species level. The flatbills as defined by Lanyon are not monophyletic (Onychorhynchus and Platyrinchus are not true flatbills). Pseudotriccus and Corythopis are sister taxa and, together with Leptopogon and Mionectes, are allied to the tody-tyrant and flatbill clade. Filogenia Molecular del Grupo de los Picochatos y Mosquiteros de la Familia Tyrannidae
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Shepherd, Kelly A., Brendan J. Lepschi, Eden A. Johnson, Andrew G. Gardner, Emily B. Sessa, and Rachel S. Jabaily. "The concluding chapter: recircumscription of Goodenia (Goodeniaceae) to include four allied genera with an updated infrageneric classification." PhytoKeys 152 (July 7, 2020): 27–104. http://dx.doi.org/10.3897/phytokeys.152.49604.
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Close scrutiny of Goodenia (Goodeniaceae) and allied genera in the ‘Core Goodeniaceae’ over recent years has clarified our understanding of this captivating group. While expanded sampling, sequencing of multiple regions, and a genome skimming reinforced backbone clearly supported Goodenias.l. as monophyletic and distinct from Scaevola and Coopernookia, there appears to be no synapomorphic characters that uniquely characterise this morphologically diverse clade. Within Goodenias.l., there is strong support from nuclear, chloroplast and mitochondrial data for three major clades (Goodenia Clades A, B and C) and various subclades, which lead to earlier suggestions for the possible recognition of these as distinct genera. Through ongoing work, it has become evident that this is impractical, as conflict remains within the most recently diverged Clade C, likely due to recent radiation and incomplete lineage sorting. In light of this, it is proposed that a combination of morphological characters is used to circumscribe an expanded Goodenia that now includes Velleia, Verreauxia, Selliera and Pentaptilon, and an updated infrageneric classification is proposed to accommodate monophyletic subclades. A total of twenty-five new combinations, three reinstatements, and seven new names are published herein including Goodenia subg. Monochila sect. Monochila subsect. Infracta K.A.Sheph. subsect. nov. Also, a type is designated for Goodenia subg. Porphyranthus sect. Ebracteolatae (K.Krause) K.A.Sheph. comb. et stat. nov., and lectotypes or secondstep lectotypes are designated for a further three names.
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Li, Jiahe, Jiacheng Ma, Michael J. Lacagnina, Sabina Lorca, Max A. Odem, Edgar T. Walters, Annemieke Kavelaars, and Peter M. Grace. "Oral Dimethyl Fumarate Reduces Peripheral Neuropathic Pain in Rodents via NFE2L2 Antioxidant Signaling." Anesthesiology 132, no. 2 (February 1, 2020): 343–56. http://dx.doi.org/10.1097/aln.0000000000003077.
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Abstract Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New Background Available treatments for neuropathic pain have modest efficacy and significant adverse effects, including abuse potential. Because oxidative stress is a key mechanistic node for neuropathic pain, the authors focused on the master regulator of the antioxidant response—nuclear factor erythroid 2-related factor 2 (NFE2L2; Nrf2)—as an alternative target for neuropathic pain. The authors tested whether dimethyl fumarate (U.S. Food and Drug Administration-approved treatment for multiple sclerosis) would activate NFE2L2 and promote antioxidant activity to reverse neuropathic pain behaviors and oxidative stress-dependent mechanisms. Methods Male Sprague Dawley rats, and male and female wild type and Nfe2l2-/- mice were treated with oral dimethyl fumarate/vehicle for 5 days (300 mg/kg; daily) after spared nerve injury/sham surgery (n = 5 to 8 per group). Allodynia was measured in von Frey reflex tests and hyperalgesia in operant conflict-avoidance tests. Ipsilateral L4/5 dorsal root ganglia were assayed for antioxidant and cytokine/chemokine levels, and mitochondrial bioenergetic capacity. Results Dimethyl fumarate treatment reversed mechanical allodynia (injury-vehicle, 0.45 ± 0.06 g [mean ± SD]; injury-dimethyl fumarate, 8.2 ± 0.16 g; P < 0.001) and hyperalgesia induced by nerve injury (injury-vehicle, 2 of 6 crossed noxious probes; injury-dimethyl fumarate, 6 of 6 crossed; P = 0.013). The antiallodynic effect of dimethyl fumarate was lost in nerve-injured Nfe2l2-/- mice, but retained in nerve-injured male and female wild type mice (wild type, 0.94 ± 0.25 g; Nfe2l2-/-, 0.02 ± 0.01 g; P < 0.001). Superoxide dismutase activity was increased by dimethyl fumarate after nerve injury (injury-vehicle, 3.96 ± 1.28 mU/mg; injury-dimethyl fumarate, 7.97 ± 0.47 mU/mg; P < 0.001). Treatment reduced the injury-dependent increases in cytokines and chemokines, including interleukin-1β (injury-vehicle, 13.30 ± 2.95 pg/mg; injury-dimethyl fumarate, 6.33 ± 1.97 pg/mg; P = 0.022). Injury-impaired mitochondrial bioenergetics, including basal respiratory capacity, were restored by dimethyl fumarate treatment (P = 0.025). Conclusions Dimethyl fumarate, a nonopioid and orally-bioavailable drug, alleviated nociceptive hypersensitivity induced by peripheral nerve injury via activation of NFE2L2 antioxidant signaling. Dimethyl fumarate also resolved neuroinflammation and mitochondrial dysfunction—oxidative stress-dependent mechanisms that drive nociceptive hypersensitivity after nerve injury.
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Giaglis, S., D. Daoudlarian, D. Kyburz, N. Venhoff, and U. Walker. "POS0108 PLASMA MITOCHONDRIAL DNA AS A BIOMARKER IN DIAGNOSIS AND FOLLOW-UP OF SYSTEMIC LUPUS ERYTHEMATOSUS." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 265.1–265. http://dx.doi.org/10.1136/annrheumdis-2021-eular.1833.
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Background:A fundamental role of mitochondria in systemic lupus erythematosus (SLE) was recently demonstrated (1). In brief, mitochondrial ROS participate in the formation of neutrophil extracellular traps (NETs) (2), while extrusion of cell-free mitochondria and highly oxidized interferogenic mtDNA causes disease in an animal model of SLE (3-5).Objectives:The diagnostic and prognostic value of cell-free DNA in SLE is still unknown. The aim of the present study was therefore to examine the clinical utility of cell-free DNA quantification as a non-invasive biomarker in SLE.Methods:Total DNA was isolated from platelet-free plasma samples of healthy individuals (HC) and consecutive SLE patients. Plasma and clinical data were collected at baseline and follow-up. Copy numbers were quantified by qPCR for mitochondrial (mt) DNA (ATP-6 gene) and nuclear (n) DNA (GAPDH gene).Results:Fifty-six HC (median age 48.3 ± 13.5, 64% female) and 103 SLE patients (median age 46.8 ± 15.8, 99% female, mean SLEDAI: 3 ± 4) were available for analysis.mtDNA levels were significantly elevated in SLE plasma (1.3x108 copies/ml plasma, 95% CI: 7.3x107 to 1.7 x108)), compared to HC plasma (8.6x106 copies/ml plasma, 95% CI: 6.9x106 to 1.0x107, p<0.0001). nDNA levels in contrast did not differ between SLE (8.3x106 copies/ml plasma, 95%CI: 5.9x106 to 1.4 x107) and HC (1.0x107 copies/ml plasma, 95%CI: 2.0x106 to 1.5 x107, p=0.61). Receiver operating characteristic curve analysis showed that a cut-off value of 1.9x107 mtDNA copy numbers differentiated between SLE and HC with 87.4% sensitivity, 94.6% specificity and an AUC of 0.95 (Figure 1a).mtDNA levels correlated with the SLE Disease Activity Index 2000 (SLEDAI-2K) (r=0.29, p=0.0026), less so also nDNA copy numbers (r=0.24, p=0.014). There was no association of mtDNA elevation with any particular type of SLE organ involvement and no correlation between mtDNA copy numbers in SLE plasma and dsDNA antibody levels.Follow-up data were available for 32 SLE patients (median follow-up 4.0 months, IQR: 4.0). delta mtDNA-levels robustly correlated with changes in SLEDAI-2K (r=0.51, p=0.0012, Figure 1b).Conclusion:The quantification of cell free mtDNA, but not nDNA copy numbers allows a sensitive and specific distinction between healthy individuals and patients with SLE. mtDNA levels correlate cross sectionally with disease activity in SLE patients and within individual SLE patients longitudinally with the SLEDAI. Plasma mtDNA quantification may therefore aid in the diagnosis of SLE and in monitoring SLE activity.References:[1]Riley JS, Tait SW. Mitochondrial DNA in inflammation and immunity. EMBO Rep. 2020;21(4):e49799.[2]West AP, Shadel GS. Mitochondrial DNA in innate immune responses and inflammatory pathology. Nat Rev Immunol. 2017;17(6):363-75.[3]Crow MK, Olferiev M, Kirou KA. Type I Interferons in Autoimmune Disease. Annu Rev Pathol. 2019;14:369-93.[4]Kim J, Gupta R, Blanco LP, Yang S, Shteinfer-Kuzmine A, Wang K, et al. VDAC oligomers form mitochondrial pores to release mtDNA fragments and promote lupus-like disease. Science. 2019;366(6472):1531-6.[5]Lood C, Blanco LP, Purmalek MM, Carmona-Rivera C, De Ravin SS, Smith CK, et al. Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease. Nat Med. 2016;22(2):146-53.Declaration of conflict of interest:UW is coinventor of patents owned by Freiburg University; NV is coinventor of patents owned by Freiburg University.Figure 1.(a) Receiver operating characteristic curve for mtDNA plasma concentrations to discriminate between HC and SLE patients. AUC: area under the curve. (b) Changes in plasma mtDNA levels in SLE patients correlate with the evolution of disease activity at follow-up.Disclosure of Interests:None declared
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Ishida, Yasuko, Taras K. Oleksyk, Nicholas J. Georgiadis, Victor A. David, Kai Zhao, Robert M. Stephens, Sergios-Orestis Kolokotronis, and Alfred L. Roca. "Reconciling Apparent Conflicts between Mitochondrial and Nuclear Phylogenies in African Elephants." PLoS ONE 6, no. 6 (June 8, 2011): e20642. http://dx.doi.org/10.1371/journal.pone.0020642.
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Olofsen, Patricia, Paulette van Strien, Onno Roovers, Hans De Looper, Remco Hoogenboezem, Dennis Bosch, Mehrnaz Ghazvini, Eric Bindels, Emma De Pater, and Ivo Touw. "PML Plays a Key Role in Severe Congenital Neutropenia with Mutant ELANE Causing Neutrophil Elastase Protein Misfolding." Blood 134, Supplement_1 (November 13, 2019): 213. http://dx.doi.org/10.1182/blood-2019-122423.
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Introduction: Severe congenital neutropenia (SCN) is a genetically heterogeneous disease characterized by recurrent infections and a predisposition for malignant transformation. A wide variety of autosomal dominant or sporadic mutations in ELANE encoding neutrophil elastase (NE) are the most frequent cause of SCN, whereas recessive mutations in HAX1 are responsible for the autosomal recessive form of SCN known as Kostmann syndrome. How ELANE and HAX1 mutations cause SCN is still unclear. A prevailing hypothesis is that cellular stresses either caused by protein misfolding or malfunction in the case of ELANE-SCN, or by mitochondrial dysfunction in the case of HAX1-SCN, are drivers of the neutropenia. We focused on the role of the promyelocytic leukemia protein (PML) because PML is implicated in controlling cellular stress responses caused by reactive oxygen species (ROS) and protein misfolding and may exert both oncogenic and tumor-suppressive functions. Aims: (1) To elucidate which cellular stress mechanisms are involved in different genetic subtypes of SCN. (2) To assess the role of PML in SCN with a predicted ELANE misfolding mutation. Methods: We generated induced pluripotent stem cells (iPSCs) from healthy control and SCN patients with non-overlapping mutations: ELANE-I60F, ELANE-R103L and HAX1-W44X. CD34+CD45+ Hematopoietic Stem and Progenitor cells (HSPCs) were derived from iPSCs using the STEMdiff™ Hematopoietic Kit (STEMCELL Technologies). PML-/- iPSCs were created by introducing a stop codon in exon 3, shared by all PML isoforms, using CRISPR/Cas9 mediated genome editing. Results: HSPCs derived from the SCN-iPSCs showed increased ROS levels as measured with CellROX Deep Red. Consequently, nuclear translocation of the antioxidant regulatory factor NRF2 was significantly elevated in both ELANE- and HAX1-mutant SCN HSPCs relative to controls. Mutation prediction analysis (Venselaar, BMC Bioinformatics 2010) showed that ELANE-I60F likely causes NE protein misfolding, whereas the ELANE-R103L mutation predictably causes NE malfunction by disrupting interactions with other proteins. The mutation in HAX1 was predicted to result in nonsense-mediated mRNA decay. Transcriptome analysis using Gene Set Enrichment Analysis (GSEA) confirmed upregulation of the nonsense mediated decay pathway in HAX1 mutant HSPCs and in line with previous studies (Klein et al, 2008), FACS analysis using TMRM and Mitotracker Red showed that loss of HAX1 protein reduced mitochondrial membrane integrity. Surprisingly, and in apparent conflict with the mutation prediction analysis, GSEA on ELANE-I60F HSPCs did not show increased expression of the classical unfolded protein response (UPR) pathway. Because PML has been implicated as an alternative player involved in degrading misfolded proteins (Guo, Mol Cell 2014), we investigated a possible link between ELANE-I60F and PML. Immunofluorescent staining showed increased numbers of PML nuclear bodies (PML-NBs) in ELANE-I60F derived HPSCs, but not in ELANE-R103L or HAX1-W44X HSPCs. Furthermore, GSEA showed upregulation of transcripts associated with PML chromatin binding in ELANE-I60F, but not in ELANE-R103L or HAX1-W44X cells. Deletion of PML by CRISPR-Cas9 revealed that PML enhanced MYC and mTORC1-induced transcription and cell cycle signatures in HSPCs from ELANE-I60F, suggestive of an oncogenic role of PML by inducing proliferation and metabolism in ELANE-I60F. In contrast, PML inhibited these pathways in HSPCs derived from healthy control iPSCs, indicative of its tumor-suppressive function in normal HSPCs. Finally, and perhaps most intriguingly, transcriptome analysis revealed that ELANE-I60F HSPCs expressed 5-fold higher levels of (mutant) ELANE transcripts than control HSPCs, which were reduced to basal levels after deletion of PML. Conclusion: HAX1 and ELANE mutations cause oxidative stress in SCN-HSPCs by distinct mechanisms. We provide evidence for a dual role of PML in the pathogenesis of SCN caused by an ELANE mutation (I60F) associated with NE misfolding: (1) NE misfolding and increased oxidative stress cause elevated formation of PML-NBs, leading to increased expression of proliferation, cell cycle and metabolism associated transcripts, (2) PML strongly enhances the levels of ELANE transcripts, thus driving the expression of the disease causative ELANE mutant through a feed-forward mechanism. Disclosures No relevant conflicts of interest to declare.
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Liang, Raymond, Vijay Menon, Miao Lin, Roberta Nowak, Velia M. Fowler, Geoffrey Girnun, and Saghi Ghaffari. "Mitochondrial Regulation Is Essential for Erythroid Nuclear Clearance." Blood 132, Supplement 1 (November 29, 2018): 843. http://dx.doi.org/10.1182/blood-2018-99-120063.
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Abstract The terminal steps of red blood cell (RBC) generation involve an extensive cellular remodeling. This encompasses alterations of cellular content through five erythroblast stages that result in the expulsion of the nucleus (enucleation) followed by loss of mitochondria and all other organelles, and a transition to anaerobic glycolysis. Whether there is any link between the erythroid removal of the nucleus and the function of any other organelle including mitochondria remains unknown. Here we show that mitochondria are essential for nuclear clearance. We first demonstrate through high-throughput single-cell imaging and confocal microscopy that as mouse bone marrow erythroblasts mature (Gate 3: TER119+,CD44low, FSClow), mitochondria migrate to one end of the cell, aggregate and trail behind the nucleus as it extrudes from the cell, a prerequisite for enucleation to complete. We further show that mitochondrial localization behind the nucleus has similar kinetics as nuclear polarization. This process is also conserved in mouse fetal liver erythroid cells as well as in primary human CD34+-derived erythroblasts in culture. Notably, kinesin inhibition disrupts mitochondrial motility and localization and reduces significantly erythroblast enucleation rate in the absence of any impact on dynein or tubulin. These results suggest that mitochondria function as necessary chaperones during erythroblast enucleation. Furthermore, mitochondrial activity distinguishes erythroblasts on the verge of enucleation from others at the same erythroblast stage (Gate 3). We show that active mitochondrial respiration facilitates nuclear condensation and is required for nuclear extrusion. To our surprise however, metabolite profiling revealed that late-stage erythroblasts sustain mitochondrial metabolism and subsequent enucleation primarily through extracellular pyruvate but independently of glucose oxidation or anapleorotic reactions of amino acids. 13C-labeled metabolite tracing also confirmed pyruvate incorporation into mitochondria while glycolysis was minimal in orthochromatic erythroblasts. Thus, we provide evidence for the first time of a link between erythroid enucleation and mitochondrial metabolism. The process described establishes a model of mitochondrial compartmentalization within the cell for providing essential metabolites in a precise spatial and temporal manner. These findings are likely to improve the in vitro production of RBC and might be relevant to anemias of congenital mitochondrial disorders and aging. Disclosures No relevant conflicts of interest to declare.
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Chou, Yin-Chen, Chia-Wei Chen, Yuan-Yeh Kuo, Liang-In Lin, and Chung-Yi Hu. "Botanical Alkyl Hydroquinone Derivative HQ17(3) Induces Calcium-Associated Mitochondrial Damage and Mitophagy to Exert Ctotoxicity to Philadelphila Chromosome(+) ALL Cells." Blood 132, Supplement 1 (November 29, 2018): 5800. http://dx.doi.org/10.1182/blood-2018-99-120307.
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Abstract Introduction: Acute lymphoblastic leukemias (ALLs) harboring t(9;22)(Ph+-ALL) are very high risk (VHR) ALL displaying poor clinical outcome irrespective of intensive chemotherapies plus tyrosine kinase inhibitor (TKI) treatment. HQ17(3)[10'(Z),13'(E),15'(E)-heptadecatrienyl hydroquinone] isolated from sap of the lacquer tree showed rapid (within 24hrs) and potent cytotoxic effect at micromolar concentration on several ALL cell lines, including Imatinib-refractory Ph+-ALL SUP-B15 cells, but spared normal PB leukocytes, and showed nontoxic in experimental rats after 28-day injection. Therefore HQ17(3) presents as a potential anti-leukemic agents and provide a platform for exploring anti-leukemic adjuvants. Our previous study showed HQ17(3)-induced rapid cell demise, characterized by oxidative stress, mitochondrial membrane potential disturbance, loss of membrane integrity, and nuclear DNA fragmentation. HQ17(3)-induced cell death is a caspase-independent program, and is different from the RIP1-mediated controlled necroptosis since both pan-caspase inhibitor and RIP-1 inhihitor failed to protect SUP-B15 cells from death. The ER stress markers (chaperon Grp78 and phosphorylated-eIF2α) were up-regulated as early as 5hrs after HQ17(3) treatment. Here we aim to illustrate the characters of the HQ17(3)-induced non-classical death on Ph+-SUP-B15 cells, focus on ER stress-associated mitochondrial Ca2+ homeostasis. Methods: Cell death and changes of mitochondria in response to HQ17(3) w/wo inhibitors were analyzed. Cells were stained by Annexin V/PI and analyzed by flow cytometry for cell death. Mitochondria mass, mitochondrial Ca2+ accumulation was detected by fluorescent Mitotracker Green and Rhod-2 probes, respectively. Mitochondrial superoxide was measured by Mitosox stain. Western blot analysis was used to analyze MFN1/2, OPA1 (mitochondrial markers). Nuclear accumulation of apoptosis inducing factor (AIF), co-localization of mitochondrial COX-IV and LC3-II (mitophagy) were revealed by immunofluorescence stain and confocal microscopy. Results: We showed mitochondrial Ca2+ accumulation at the early time when ER stress occurred (Fig 1), accompanied by mitochondrial superoxide elevation, followed by loss of mitochondrial membrane potential (MMP) and nuclear translocation of apoptosis-inducing factor (AIF). HQ17(3) treatment lead to decreased mitochondrial proteins MFN1/2 and OPA1, while Mitotracker Green stain showed significant loss of mitochondrial mass preceded cell death, indicating damaged mitochondria underwent fission followed by mitophagy. Immunofluorescence stain showed evidence of mitophagy (COX IV and LC3B co-localization). Calpain-1 inhibitor PD150606 blocked AIF nuclear translocation but only slightly reduced the HQ17(3)-induced cell death (Fig 2). Further, Ca2+ chelator Bapta-AM prevented mitochondrial superoxide production, MMP loss, mitophagy (Fig 3), and rescued cell death (Fig 1) more effectively. Conclusion: In Ph+-ALL SUP-B15 cells, HQ17(3) induce ER stress by yet-defined mechanism, this mobilizes Ca2+ to mitochondria and acts in multi-facet: a) results in AIF cleavage and translocation to mediate nuclear chromatin fragmentation, b) Ca2+-overload leads to oxidative stress and perturbs mitochondria integrity, c) damaged mitochondria trigger extensive mitophagy and cell death ensues. Therefore, agents that help elicit similar intricate effector network associated with ER/mitochondria stress will have potential to be adjuvants in aiding control of the Ph+ VHR-ALL cells refractory to conventional chemotherapies and TKI regime. Disclosures No relevant conflicts of interest to declare.
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Xu, Xiuling, and Jeff S. Friedman. "A Role for the Transcriptional Coactivator PRC1 in Mitochondrial Biogenesis During Erythroid Development." Blood 114, no. 22 (November 20, 2009): 3642. http://dx.doi.org/10.1182/blood.v114.22.3642.3642.
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Abstract Abstract 3642 Poster Board III-578 Red cell development requires unparalleled mitochondrial metabolic and biosynthetic capacities to support hemoglobin formation. In addition to meeting the energetic requirements of the erythroblast and reticulocyte, mitochondria are the site of multiple steps in heme biosynthesis and are essential for iron utilization. As such, perturbation of mitochondrial function in hematopoietic cells appears to differentially affect red cell development. For instance, we show that the drug chloramphenicol (an inhibitor of mitochondrial protein synthesis) preferentially inhibits erythroid colony formation with relatively little effect on other types of myeloid progenitors. Similarly, we show that knockout of the mitochondrial superoxide dismutase (Sod2) preferentially inhibits BFU-E, CFU-E and GEMM colonies. Clinically, several mutations in mitochondrial DNA or in nuclear encoded mitochondria-localized proteins preferentially disrupt erythroid development resulting in sideroblastic anemia. In order to better understand regulation of mitochondrial activity during red cell development/differentiation, we have investigated expression of transcription factors and coactivators implicated as regulators of mitochondrial biogenesis in other tissues including PGC-1 α and β, ERR proteins, NRF 1 and NRF 2 in normal and Sod2 deficient murine erythroblasts. Microarray and qPCR data suggest that neither of the classic transcriptional coactivators PGC-1 α or β are expressed at appreciable levels in erythroblasts. However, a PGC-1 related coactivator, PRC1 is detectable at both the transcript and protein level in developing erythroid cells. In the Sod2 deficiency model, one of the most prominent transcriptional changes we have observed in erythroblasts is a broad down regulation of expression of nuclear encoded mitochondria localized protein transcripts including multiple components of the electron transport chain, TCA cycle enzymes and mitochondrial ribosomal proteins. We show that PRC1 expression is down regulated in Sod2 deficient cells—suggesting that this coactivator plays a key role in mitochondrial biogenesis during erythroid development. Further, using the MEL cell line, we show that PRC1 expression is turned on following induction of differentiation with DMSO. These results identify PRC1 as a novel, and perhaps exclusive regulator of mitochondrial biogenesis during erythroid development. A better understanding of regulation of PRC1 expression and identification of transcriptional targets of PRC1 during erythroid development will improve our understanding of how perturbations in mitochondrial physiology affect the red cell compartment. Disclosures: No relevant conflicts of interest to declare.
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Ghaffari, Saghi. "Regulation of Hematopoietic Stem Cell Mitochondrial Metabolism." Blood 128, no. 22 (December 2, 2016): SCI—33—SCI—33. http://dx.doi.org/10.1182/blood.v128.22.sci-33.sci-33.
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Abstract Hematopoietic stem cells (HSCs) like most, if not all, adult stem cells are primarily quiescent but have the potential to become highly active on demand. HSC quiescence is maintained by glycolytic metabolism and low levels of reactive oxygen species (ROS), which indicate that mitochondria are relatively inactive in quiescent HSC. However, HSC cycling - and exit of quiescence state - require a swift metabolic switch from glycolysis to mitochondrial oxidative phosphorylation. To improve our understanding of mechanisms that integrate energy metabolism with HSC homeostasis, my laboratory has been focused on the transcription factor FOXO3, which is critical for the maintenance of HSC quiescence and redox state and is implicated in HSC aging. We showed recently that FOXO3 is key to HSC mitochondrial metabolism, independent of its inhibition of ROS or mTOR signaling. Mitochondria divide and fuse constantly in part to segregate and dispose of their damaged counterparts. These processes are influenced by and highly linked to mitochondrial metabolism. We have recently developed imaging approaches to study HSC mitochondrial divisions. Mechanisms by which FOXO3 regulates HSC mitochondria and the impact of impaired FOXO3 on the HSC health and activity, and mitochondrial network will be discussed. Detailed understanding of the mitochondrial metabolism and divisions in HSC and their relationship to nuclear transcription are likely to have broad implications for the state of HSC fitness, regenerative capacity and aging. Disclosures No relevant conflicts of interest to declare.
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Hurley, Imogen A., Rachel Lockridge Mueller, Katherine A. Dunn, Eric J. Schmidt, Matt Friedman, Robert K. Ho, Victoria E. Prince, Ziheng Yang, Mark G. Thomas, and Michael I. Coates. "A new time-scale for ray-finned fish evolution." Proceedings of the Royal Society B: Biological Sciences 274, no. 1609 (November 21, 2006): 489–98. http://dx.doi.org/10.1098/rspb.2006.3749.
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The Actinopterygii (ray-finned fishes) is the largest and most diverse vertebrate group, but little is agreed about the timing of its early evolution. Estimates using mitochondrial genomic data suggest that the major actinopterygian clades are much older than divergence dates implied by fossils. Here, the timing of the evolutionary origins of these clades is reinvestigated using morphological, and nuclear and mitochondrial genetic data. Results indicate that existing fossil-based estimates of the age of the crown-group Neopterygii, including the teleosts, Lepisosteus (gar) and Amia (bowfin), are at least 40 Myr too young. We present new palaeontological evidence that the neopterygian crown radiation is a Palaeozoic event, and demonstrate that conflicts between molecular and morphological data for the age of the Neopterygii result, in part, from missing fossil data. Although our molecular data also provide an older age estimate for the teleost crown, this range extension remains unsupported by the fossil evidence. Nuclear data from all relevant clades are used to demonstrate that the actinopterygian whole-genome duplication event is teleost-specific. While the date estimate of this event overlaps the probable range of the teleost stem group, a correlation between the genome duplication and the large-scale pattern of actinopterygian phylogeny remains elusive.
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Wallace, Douglas C. "Mitochondrial Physiology in Health and Disease: Changes with Aging." Blood 114, no. 22 (November 20, 2009): SCI—1—SCI—1. http://dx.doi.org/10.1182/blood.v114.22.sci-1.sci-1.
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Abstract Abstract SCI-1 The incidence and cost of the age-related metabolic and degenerative diseases is rising in the face of ever increasing investment. Therefore, something must be missing in our current hypotheses about these diseases. Until now, the prevailing paradigms in Western medicine have been the anatomical paradigm of disease which posits that tissue-specific symptoms are due to tissue-specific structural defects and the Mendelian paradigm of genetics which posits that if a disease is inherited according to the Laws of Mendel it is genetic and if not it is environmental. The epigenome is invoked to explain the environmental modulation of the nuclear gene expression. However, life requires both structure and energy and the over 1500 mitochondrial energy genes are dispersed across the chromosomes plus the maternally inherited mitochondrial DNA. Moreover, the cells and tissues most affected by aging and the age-related disease are those most reliant on mitochondrial energy and the mitochondrial lies at the interface between environmental calories and human physiology, diabetes and obesity. The epigenome and the signal transduction pathways are regulated by protein phosphorylation by ATP, acetylation via acetyl-CoA, and methylation by S-adenosylmethionine, all driven by mitochondrial high energy substrates modulated by available calories. Furthermore, mitochondrial redox chemistry regulates reactive oxygen production and thiol/disulfide chemistry and these also regulate cellular signaling and function. Therefore, bioenergetics and mitochondrial genetics are the missing factors which have inhibited our capacity of address the biology and genetics of the age-related metabolic and degenerative diseases, cancer and aging. Disclosures No relevant conflicts of interest to declare.
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MARK, Kristiina, Lauri SAAG, Andres SAAG, Arne THELL, and Tiina RANDLANE. "Testing morphology-based delimitation of Vulpicida juniperinus and V. tubulosus (Parmeliaceae) using three molecular markers." Lichenologist 44, no. 6 (October 8, 2012): 757–72. http://dx.doi.org/10.1017/s0024282912000448.
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AbstractThe delimitation of two morphologically similar and not easily separable Vulpicida species, V. juniperinus and V. tubulosus, is analyzed using nuclear ITS and Mcm7, and mitochondrial SSU DNA sequences. Seventy-nine Vulpicida specimens, most from the two focal taxa, are included in the three-locus gene tree. The results from Bayesian and parsimony analyses are presented. There are strong conflicts between the single locus gene trees. Vulpicida juniperinus and V. tubulosus are divided into two clearly distinguished groups in the ITS and concatenated B/MCMC tree. However, these species are mixed in both clades, appearing polyphyletic. Currently accepted V. juniperinus and V. tubulosus are not distinct according to the loci studied. Vulpicida pinastri appears monophyletic based on the available sequences.
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Pang, Qingsong, Ningbo Liu, Fengting Liu, Samir Agrawal, and Ping Wang. "Drp1 Activation Overcomes Diffuse Large B-Cell Lymphoma Cells Radioresistance." Blood 120, no. 21 (November 16, 2012): 5121. http://dx.doi.org/10.1182/blood.v120.21.5121.5121.
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Abstract Abstract 5121 Mitochondria undergo fusion and fission in response to physiological or pathological changes. Mitochondrial fusion is regulated by mitofusin-1 and 2 (MFN-1/2) and optic atrophy 1(OPA-1), whereas, mitochondrial fission (or fragmentation) is controlled by a Dynamin-related protein 1 (Drp1). Drp1 activation is triggered by dephosphorylation of Drp1 on its serine 637 site. Recent studies demonstrated that Drp1 activation plays an important role in mitochondrial fragmentation-induced cell death. The phosphorylation of Drp1 by serine kinase inhibits its GTPase activity and prevents mitochondrial fragmentation. Inhibition of Drp1 activation or loss of Drp1 function leads to slow down apoptosis and necrosis. Recent reports showed that patients who received consolidative radiotherapy (RT) combine R-CHOP had significant better 5-year event-free and overall survival rates than patients with diffuse large B-cell lymphoma who did not receive RT combination therapy. Although RT combination therapy significant increased survival of DLBCL, the mechanisms of RT induced cell death are reminded unclear. In this study, we investigated the roles of Drp1 activation in the sensitivity of DLBCL cells to radiotherapy. Radiation causes damage in both nuclear and mitochondrial DNAs and generation of reactive oxygen species (ROS) followed by caspase activation. In the radio-resistant DLBCL cells, radiation induced activation of Drp1, and led to mitochondrial fragmentation, caspase-3 activation and cell death. However, the sensitive DLBCL cells underwent cytochrome c release, caspase-3 activation and PARP cleavage but not the activation of Drip1. To confirm the role of Drp1 activation in radiation-induced cell death on radio-resistant cells, Drp1 was dephosphorylated by a serine kinase inhibitor STS. Co-treatment of radio-resistant DLBCL cells with STS and radiation greatly enhanced mitochondrial fragmentation and significantly increased the sensitivity of radio-resistant DLBCL cells to radiation-induced caspase-3 activation, PARP cleavage and apoptotic cell death. However, co-treatment with STS did not further increase the sensitivity of radio-sensitive cells to caspsase-3 activation and cell death. Our data indicate that radiation-induced cell death in radio-resistant DLBCL cells is regulated by Drp1. We therefore propose that Drp1 could be a potential target for overcoming radio-resistance. Disclosures: No relevant conflicts of interest to declare.
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Cooney, Jeffrey D., Gordon J. Hildick-Smith, Caterina Garone, Jonathan N. Thon, Orhan H. Akman, Caiyong Chen, Dhvanit I. Shah, et al. "Megaloblastic Anemia and Mitochondriopathy Caused by a Homozygous Mutation in Sideroflexin-4." Blood 120, no. 21 (November 16, 2012): 79. http://dx.doi.org/10.1182/blood.v120.21.79.79.
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Abstract Abstract 79 Megaloblastic anemias are characterized by impaired DNA metabolism, often due to deficiencies in vitamin B12 or folate. Genes underlying hereditary forms of megaloblastic anemia not caused by vitamin B12 or folate deficiencies, however, remain largely unknown. Here we characterize a genetic deficiency in a patient with infantile-onset megaloblastic anemia, developmental delays, and a mitochondrial disorder of unknown etiology. Analysis of peripheral blood smears from the patient revealed hypersegmented neutrophils and erythroid macrocytes, classic features of megaloblastic anemias. The patient's vitamin B12 and folate levels are normal, eliminating their deficiency as potential causes of the disease. Whole-exome sequencing of the proband cDNA identified a homozygous, single nucleotide deletion (c.231delC) in Sideroflexin-4 (SFXN4), a predicted mitochondrial multi-spanning transmembrane protein. We experimentally verified the mitochondrial localization of SFXN4 using a combination of western analyses on mitochondrial lysates and confocal fluorescence immunohistochemistry. Using trypsin-sensitivity assays on isolated mitoplasts, we further determined the submitochondrial localization of SFXN4 to the inner mitochondrial membrane. Bioinformatic analyses predict that the mutation introduces a frame shift and a premature stop codon (p.Pro78Leufs*25), resulting in a severely truncated polypeptide. To determine whether the mutant mRNA were expressed in vivo, we used qRT-PCR to assess the steady state level of SFXN4 mRNA in cultured fibroblasts from the proband. qRT-PCR revealed a 92% reduction in SFXN4 expression, consistent with nonsense-mediated decay of the mutant transcript. Genotyping of the index patient and 3 generations of her nuclear family using both Sanger sequencing and allele-specific oligonucleotide hybridization showed that the mutant allele is inherited in an autosomal recessive manner (Fig. A), the result of a presumed founder effect. We used complementary zebrafish and human fibroblast systems to model the megaloblastic anemia and mitochondrial disease in the patient, respectively. Using splice-blocking antisense morpholino oligomers (MO) targeting sfxn4, we induced a loss-of-function phenotype in zebrafish embryos (hereafter, referred to as “morphants”). qRT-PCR confirmed the efficient knockdown of sfxn4, as morphants have <10% sfxn4 mRNA. Knockdown of sfxn4 in transgenic, Tg(globin LCR:eGFP) zebrafish showed a gross reduction in GFP+ erythrocytes and hemoglobinized cells stained by o-dianisidine (Fig. B, top), while quantification of the red cell population by flow cytometry showed a 60% reduction in the red cell mass. To characterize the anemia, we performed cytospins of flow-sorted erythroid cells from sfxn4 morphants and analyzed their morphology. Wright staining revealed that sfxn4 morphants have red cells with large nuclei containing non-condensed chromatin (Fig. B, bottom), consistent with the features of megaloblastic anemia observed in the index patient. Enumeration of the nuclear: cytoplasmic area ratios showed that red cells from sfxn4 morphants have a nearly 3-fold increase in the ratio of nuclear to cytoplasmic size. We also investigated the mitochondrial disorder using patient fibroblasts, which showed a severe reduction in complex I (37%) and complex I+III (7%) activity. The over-expression of wild-type human SFXN4 in proband fibroblasts completely rescued the respiratory defect of complex I+III, while transfection of the mutant c.231delC SFXN4 construct failed to increase complex I+III activity. In a complementary strategy, the over-expression of wild-type SFXN4 cRNA from either zebrafish or human partially rescues the anemia in morphant embryos, validating their functional orthologous relationship. In summary, a recessive loss-of-function mutation in SFXN4, a previously uncharacterized gene, causes the megaloblastic anemia and mitochondrial disorder described in the index patient. Genetic complementation studies in patient fibroblasts and sfxn4-silenced zebrafish morphants validate the pathogenicity of the mutation. Our findings: (1) demonstrate the requirement of SFXN4 for mitochondrial homeostasis and erythropoiesis, and (2) establish SFXN4 as a new candidate gene for mitochondriopathies and megaloblastic anemias. Disclosures: No relevant conflicts of interest to declare.
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Sakhuja, Kiran B., Lina A. Gugliotti, Yutaka Suzuki, Alexander L. Kovalchuk, Hongsheng Wang, Dong-Mi Shin, Susana M. Cerritelli, Herbert C. Morse, and Robert J. Crouch. "Constitutive Lymphoid Expression of the Nuclear Form of RNase H1 Is Associated with Development of Immature B Cell Lymphomas." Blood 114, no. 22 (November 20, 2009): 1613. http://dx.doi.org/10.1182/blood.v114.22.1613.1613.
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Abstract Abstract 1613 Poster Board I-639 Ribonuclease H1, which cleaves the RNA of RNA/DNA hybrids, plays a vital role in the replication of mitochondrial DNA during mouse embryogenesis, but its contributions to later mammalian development and adult biology are not known. In higher eukaryotes two isoforms of RNase H1, a nuclear form and mitochondrial form, are produced from a single transcript. To investigate the effects of constitutive high level RNase H1 expression, we generated transgenic (TG) mice, strain M27F7, with lymphocyte-specific over-expression of nuclear RNase H1. Transcripts in B cells from TG mice were ∼100-fold higher than in wild type B cells. Although there was no obvious phenotype in young mice, some of the older mice, especially females, developed lymphadenopathy, splenomegaly and mediastinal masses and were diagnosed histologically as having lymphoblastic lymphomas. Cell lines established from the tumors were examined by FACS, gene expression profiling and SKY as well as by FISH for Igh and Myc. By FACS criteria, the tumors were B cell lineage and were arrested at the pre-B to immature stage of differentiation. Expression profiles showed the lines to have increased expression of anti-apoptotic genes and genes promoting cell cycle progression. Chromosomal studies revealed no signs of genomic instability or translocations, although there were trisomies of chromosomes 3 and 15. Myc was structurally intact in all lines. These results suggest that RNase H1 plays a previously unappreciated role in early B cell development and that constitutive overexpression contributes to lymphomagenesis. This work was supported by the Intramural Research Program of the National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development and National Institute of Allergy and Infectious Diseases. Disclosures No relevant conflicts of interest to declare.
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Friedman, Jeff S. "Mitochondria and Hematologic Disorders." Blood 114, no. 22 (November 20, 2009): SCI—3—SCI—3. http://dx.doi.org/10.1182/blood.v114.22.sci-3.sci-3.
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Abstract Abstract SCI-3 Mitochondria have a special relationship with the erythroid lineage. Although RBC are devoid of mitochondria, during RBC development the mitochondria is the site of multiple steps in heme biosynthesis, and is essential for proper utilization of iron. As evidence of this special relationship, multiple mutations in both mitochondrial DNA (hereditary and acquired) and in nuclear genes encoding mitochondrial localized proteins (hereditary) result in sideroblastic anemia—where the hallmark pathologic lesion is intramitochondrial iron accumulation in erythroid progenitors. The erythroid-lineage specific readout of these mitochondrial genetic lesions raises the possibility that mitochondrial dysfunction is a contributor to anemia in other contexts as well. In this view, red cell development can be considered an early warning system for mitochondrial dysfunction in hematopoiesis. A focus of our laboratory is to investigate how increased mitochondrial-derived reactive oxygen species affect hematopoietic development. Gene expression and proteomic analyses of erythroblasts demonstrate that mitochondrial biogenesis during erythroid development is inhibited by oxidant stress. Transcriptional control of mitochondrial biogenesis in erythroid cells involves induction of the distinct transcriptional coactivator PRC1—perhaps helping to explain the erythroid specificity of phenotypes noted above. As has been elegantly demonstrated by Wallace and others, mitochondrial dysfunction is an important determinant of age-related decline in functional capacity of many tissues. This decline in function is accompanied by an increase in mitochondrial DNA mutations—both point mutations and deletions found primarily in post-mitotic cells. Modeling of this process through creation of mice with an error prone mtDNA polymerase accelerates the appearance of age-related tissue changes—including the development of anemia. Transplantation of murine hematopoietic stem cells harboring a large deletion of mtDNA also leads to anemia in reconstituted animals. Are these findings relevant for age-related hematologic abnormalities in people—and if so, for what disorders? There is considerable epidemiologic evidence indicating an increase in the frequency of anemia in the elderly, peaking at a prevalence of greater than 20% for individuals in their 80's. Approximately 1/3 of these elderly anemic cases are idiopathic—that is, no underlying disease process is identified. In studying this group with idiopathic anemia, we have investigated a number of hypotheses including the possibility of mitochondrial dysfunction. To date we have found altered mitochondrial DNA content and a higher mutation frequency in mtDNA isolated from peripheral blood cells when comparing anemic versus age/sex matched controls. However, these studies are correlative, and do not prove causality. Proving a direct role for specific acquired mitochondrial DNA lesions in development of anemia, myelodysplasia or hematologic malignancy remains a technical challenge because of the difficulty in introducing specific mutant mtDNA's into relevant cells or tissues. The development of more facile methods for evaluation of mitochondria in few or even single cells promises to expand our understanding of how mitochondrial functional changes impact diverse hematopoietic cells, in addition to the erythroid lineage effects highlighted above. Disclosures No relevant conflicts of interest to declare.
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Zhao, Yan, Zhenzhen Yi, Eleni Gentekaki, Aibin Zhan, Saleh A. Al-Farraj, and Weibo Song. "Utility of combining morphological characters, nuclear and mitochondrial genes: An attempt to resolve the conflicts of species identification for ciliated protists." Molecular Phylogenetics and Evolution 94 (January 2016): 718–29. http://dx.doi.org/10.1016/j.ympev.2015.10.017.
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Sajdak, S. L., and R. B. Phillips. "Phylogenetic relationships among Coregonus species inferred from the DNA sequence of the first internal transcribed spacer (ITS1) of ribosomal DNA." Canadian Journal of Fisheries and Aquatic Sciences 54, no. 7 (July 1, 1997): 1494–503. http://dx.doi.org/10.1139/f97-057.
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Phylogenetic analyses based on morphometrics, data on protein electrophoresis, and mitochondrial DNA have given conflicting phylogenetic hypotheses for the subfamily Coregoninae (Prosopium, Coregonus, and Stenodus). To resolve these conflicts, phylogenetic analysis of 13 taxa of Coregoninae was done using a nuclear gene, the first internal transcribed spacer (ITS1) of the ribosomal DNA (rDNA). ITS1 sequences of all North American ciscoes were identical. In agreement with analyses based on other molecular data, the genus Stenodus was included in Coregonus, and there was no support for the monophyly of the ciscoes. The analysis based on rDNA also placed C. peled as the sister species to C. lavaretus, but this may represent a case of hybridization and introgression. A length polymorphism involving a repeating unit of 65-67 base pairs was found in the ITS1. All ciscoes except C. peled had one copy of the repeat. Coregonus peled, C. lavaretus, and Eurasian and Beringian C. clupeaformis had two copies of the repeat, while the other North American C. clupeaformis had three copies, suggesting that C. clupeaformis of northwestern North America is a form of C. lavaretus.
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Dupin, Julia, Pauline Raimondeau, Cynthia Hong-Wa, Sophie Manzi, Myriam Gaudeul, and Guillaume Besnard. "Resolving the Phylogeny of the Olive Family (Oleaceae): Confronting Information from Organellar and Nuclear Genomes." Genes 11, no. 12 (December 16, 2020): 1508. http://dx.doi.org/10.3390/genes11121508.
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The olive family, Oleaceae, is a group of woody plants comprising 28 genera and ca. 700 species, distributed on all continents (except Antarctica) in both temperate and tropical environments. It includes several genera of major economic and ecological importance such as olives, ash trees, jasmines, forsythias, osmanthuses, privets and lilacs. The natural history of the group is not completely understood yet, but its diversification seems to be associated with polyploidisation events and the evolution of various reproductive and dispersal strategies. In addition, some taxonomical issues still need to be resolved, particularly in the paleopolyploid tribe Oleeae. Reconstructing a robust phylogenetic hypothesis is thus an important step toward a better comprehension of Oleaceae’s diversity. Here, we reconstructed phylogenies of the olive family using 80 plastid coding sequences, 37 mitochondrial genes, the complete nuclear ribosomal cluster and a small multigene family encoding phytochromes (phyB and phyE) of 61 representative species. Tribes and subtribes were strongly supported by all phylogenetic reconstructions, while a few Oleeae genera are still polyphyletic (Chionanthus, Olea, Osmanthus, Nestegis) or paraphyletic (Schrebera, Syringa). Some phylogenetic relationships among tribes remain poorly resolved with conflicts between topologies reconstructed from different genomic regions. The use of nuclear data remains an important challenge especially in a group with ploidy changes (both paleo- and neo-polyploids). This work provides new genomic datasets that will assist the study of the biogeography and taxonomy of the whole Oleaceae.
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Xiong, Jie, Xingyi Kuang, Tingting Lu, Bingqing Cheng, Danna Wei, Xinyao Li, Weili Wang, Zhaoyuan Zhang, and Jishi Wang. "The Crucial Role of NR4A1 Dependent Apoptosis Induced By Fenretinide in Acute Myeloid Leukemia." Blood 132, Supplement 1 (November 29, 2018): 5266. http://dx.doi.org/10.1182/blood-2018-99-117776.
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Abstract OBJECTIVE: NR4A1 is a member of the orphan nuclear receptor family, which is involved in biological processes such as cell proliferation, apoptosis, metabolism, and differentiation. The expression of NR4A1 is increased in a variety of solid tumors, which promotes oncogenesis and enhances the viability of tumor cells. However, in hematological malignancies, its expression is significantly lower than normal. Studies have shown that clearance of NR4A1 can lead to the progression of mice to acute myeloid leukemia, reducing the amount of NR4A gene expression can make mice progress to MDS / MPN, and even acute myeloid leukemia. This indicates that NR4A1 is an important tumor suppressor in acute myeloid leukemia. The present study was to investigate the effect of drug-induced NR4A1 expression on apoptosis in acute myeloid leukemia and its related mechanisms. The results suggested that NR4A1 may become a new target for the treatment of acute myeloid leukemia. METHODS: In vitro, on the one hand, bone marrow mononuclear cells from patients with newly diagnosed or relapsed acute myeloid leukemia were extracted as samples, and the expression of NR4A1 mRNA and protein was detected by Real-time PCR and Western blot. Acute myeloid leukemia cell lines HL60 and Kasumi-1 were tested for fenretinide activity and apoptosis, as well as NR4A1 and mitochondrial apoptosis pathway-related protein expression. We used siNR4A1 to knockdown NR4A1 expression and LMB to inhibit nuclear export, the apoptosis rate and apoptosis protein were detected. The mechanism of anti-apoptotic effect of NR4A1 was verified by western blot, immunofluorescence and co-immunoprecipitation. AML mice model were established and fenretinide was injected into the tail veins to observe the effects of NR4A1 expression on survival time, blood routine, bone marrow and important organs. Results: 1. NR4A1 expression in patients with acute myeloid leukemia was significantly higher than normal. 2. The expression of NR4A1 increased in a time- and concentration-dependent manner under the action of fenretinide. 3. After siNR4A1, the apoptosis of acute myeloid leukemia cells was significantly decreased, and the expression of apoptotic proteins was decreased. 4. The expression of apoptotic proteins was decreased after fenretinide combined with leptomycin treatment. After fenretinide treatment, the expression of NR4A1 was decreased in nuclear extracts, and the expression of mitochondrial extract was increased. After drug treatment, NR4A1 interacted with Bcl-2, and the bcl-2 BH3 domain in cytoplasm was exposed to play an anti-apoptotic effect. After successful modeling of AML mice, the survival rate of the fenretinide treatment group was significantly prolonged compared with the control group. The peripheral blood leukocytes, hemoglobin and platelet values were significantly different. The liver and spleen volume of the treatment group were significantly smaller than that of the control group. The mRNA and protein levels of NR4A1 in the spleen were significantly higher than those in the control group. Conclusion: The effect of fenretinide on acute myeloid leukemia cells induces NR4A1 expression and plays a pro-apoptotic effect. This apoptosis effect depends on the nuclear export of NR4A1. NR4A1 is located from the nucleus to the mitochondria, and binds to the Bcl-2 BH3 domain. Key words: NR4A1, fenretinide, acute myeloid leukemia, apoptosis, nuclear export Disclosures No relevant conflicts of interest to declare.
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Ma, Yanping, and Zhenhua Qiao. "Apoptosis on U266 Cells Induced by Brucine by Death-Receptor Pathway." Blood 116, no. 21 (November 19, 2010): 5022. http://dx.doi.org/10.1182/blood.v116.21.5022.5022.
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Abstract Abstract 5022 Objective Multiple myeloma (MM) is an incurable hematological malignancy with high incidence in the elderly. The currently chemotherapeutic drugs show severe side effects, dose-limiting toxicity and development of resistance. The study in order to observe the Anti-proliferative of brucine which is a novel plant anti-cancer agents on multiple myeloma cell line-U266 and find the mechanism of apoptosis. Method U266 cells was cultured in 90% RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin, and maintained in a humidified atmosphere with 5% CO2 at 37°C. Cells (5×104) were plated in the presence or absence of the brucine (0, 0.05, 0.1, 0.2, 0.4 mg/ml) in 96 well culture plates for 24–72 h. The anti-proliferative response of brucine was assessed by MTT assay. Nuclear morphology of the treated cells was observed using Fluorescence microscope. The mitochondrial membrane potential of treated cell were measured by flow cytometry. The expression change of caspase-3 was detected using RT-PCR. Results The apoptotic effect of brucine show a dose and time dependent manner (p<0.05). The 50% inhibiting concentration of brucine was 0.16 mg/ml. Brucine treated cells show significant feature associated with apoptosis by Fluorescence microscope. The mitochondrial membrane potential using flow cytometry revealed no significant decline at different concentration. In addition, Brucine induced U266 cell apoptosis was caspase dependent, with caspase-3 activated by caspase-9 (p<0.05). Conclusions At 0.4mg/ml concentration range brucine can induce apoptosis in U266 cells. Brucine induces the apoptosis of U266 cell via death-receptor pathway. Disclosures: No relevant conflicts of interest to declare.
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Gomez, Esteban, Cassandra Calloway, Sang Hoon Lee, Jay Kim, Navpreet Dhillon, Ginny Gildengorin, and Ashutosh Lal. "Mitochondrial Genome Changes As a Measure Of Iron-Induced Mitochondrial Stress In Transfusion-Dependent Thalassemia." Blood 122, no. 21 (November 15, 2013): 2256. http://dx.doi.org/10.1182/blood.v122.21.2256.2256.
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Abstract Introduction Iron overload is a consequence of chronic blood transfusions to treat severe forms of thalassemia. While patient outcomes have improved with advances in iron overload assessment and chelation therapy, iron toxicity continues to be the primary determinant of morbidity. Mitochondrial dysfunction is of central importance in iron overload. However, clinical assessment of mitochondrial damage has not been feasible because current methods require invasive tissue sampling. Methods Novel bioassays were used to assess changes in the mitochondrial genome from a small amount of peripheral blood. We studied the relationship between mitochondrial DNA (mtDNA), mitochondrial respiratory complexes, and clinical measures of iron overload. Real-time PCR-based assays were used to measure mtDNA copy number per nuclear DNA copy (mtDNA:nDNA) and the frequency of the mtDNA 4977-bp deletion (ΔmtDNA4977). The quantity of mitochondrial respiratory complex I and IV in blood was measured using ELISA and expressed as a percentage of quantity in HepG2 cells. Results 44 individuals with thalassemia (median age 34.9 years, range 14 to 52.4 years) and 27 healthy controls (median age 22 years, range 19 to 39 years) were studied in this analysis. The median liver iron concentration (LIC) in thalassemia was 11.0 mg/g dry-weight (2.3-59.4 mg/g dry-weight), while the median ferritin was 2220 mcg/L (308-9470 mcg/L). The median cardiac T2* was 28.7 ms (6.5-45.8 ms). The mtDNA:nDNA was estimated and the median mtDNA copy number was 249.8 (87.6-738.3) in thalassemia, compared with 225.4 (101.9-348.7) in controls. The mtDNA copy number was significantly elevated in thalassemia (p=0.033, Mann-Whitney test). The mtDNA copy number increased with age in thalassemia (r=0.45, p=0.005), but no correlation with age was observed in the control group (r=0.06, p=n.s.). Myocardial iron deposition influenced the mtDNA copy number as evidenced by an inverse relationship with T2* (r= -0.45, p=0.006). The frequency of the ΔmtDNA4977deletion was analyzed in a subset of subjects and was found to be 8-fold higher in thalassemia versus controls (p=0.012). The median quantity of complex IV in thalassemia was 3.4% (0 to 104.2) compared with 2.5% (range 0 to 18.67) in controls. The median complex I activity in thalassemia was 6.3% (range 0 to 161.4) compared with 17.86% (range 0 to 55.0) in controls. These differences were not statistically significantly between the two groups. Higher values of mtDNA copy number were associated with increased quantity of complex IV (r=0.515, p=0.10) in thalassemia, but not in controls. No correlation of mtDNA copy number was observed with complex I activity. LIC, ferritin, and platelet count did not influence the mtDNA:nDNA ratios. Interpretation Alterations in mtDNA can be measured in blood samples of patients with thalassemia and iron overload. Increases in mitochondrial genome copy number and ΔmtDNA4977 deletion frequency may be a reflection of iron-induced mitochondrial stress. This hypothesis is supported by an increase in mtDNA copy number with age in thalassemia, but not in controls. The relation between mtDNA changes and myocardial iron suggests that this assay may possess physiological relevance. These preliminary results support the potential application of these non-invasive assays for the assessment of iron-induced organ damage. Disclosures: No relevant conflicts of interest to declare.
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Huang, Qin, Haihong Zhang, Wei Chen, Serhan Alkan, and Chunyang Fan. "Increased Mitochondrial Gene Deletions and ROS Production in Transgenic Mice Overexpressing a Human 8-Oxoguanine DNA-Glycosylase Gene: A Causal Association with the Development of Hematopoietic Neoplasms." Blood 124, no. 21 (December 6, 2014): 866. http://dx.doi.org/10.1182/blood.v124.21.866.866.
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Abstract Alterations of nuclear genes in human diseases including neoplasms have been well investigated in past several decades and unequivocally established their predominant role in the pathogenesis. However, the relationship of mitochondrial genome alteration with human diseases remains largely unknown. Mitochondria are dynamic organelles involved in oxidative phosphorylation and production of reactive oxygen species (ROS). Accumulated evidence supports that mitochondrial DNA damage and dysfunction play vital roles in the development of a wide array of mitochondria-related human diseases, such as obesity, diabetes, infertility, neurodegenerative disorders and malignant tumors. We previously described the development of a transgenic (TG) mouse model for mitochondrial DNA damage by overexpressing human mitochondrial isoform of 8-oxoguanine DNA Glycosylase 1 (hOGG1) gene. Over-expression of this gene produced a wide range of adverse biological phenotypes, manifesting early-onset obesity, metabolic disturbance, female infertility, high frequency of B-cell lymphoma and human essential thrombocythemia like myeloproliferative disorder, involving the lymph node, bone marrow, spleen, liver and other extranodal sites. Development of these hematopoietic neoplasms appeared to be age-dependent. In the current study, we focused on the pathogenesis of the hematopoietic neoplasms by characterization of the neoplasms via pathologic, biochemical and molecular approaches. While expression of mOGG1 was very similar in parallel organs from transgenic and wild-type control mice, the hOGG1 TG mice expressed very high levels of human OGG1 transgene mRNA, being 6.8- and 112-fold as high as the endogenous mouse OGG, in the spleen and bone marrow. By contrast, hOGG1 transgene mRNA were not detected at all in the above two organs from control mice, indicating that the transgene is highly expressed in the hematopoietic organs in TG mice. We then measured mitochondrial NADH dehydrogenase 1 (ND1) gene expression as an indirect measure of mitochondrial respiratory function. ND1 mRNA levels in the spleen (4) and lymphoma (4) of TG mice were 83% and 58% higher, respectively, than those in the spleen (4) of wild-type control mice (P < 0.01), indicative of increased mitochondrial respiration in the lymphoma and spleen of hOGG1 TG mice. We next measured the levels of intracellular H2O2 production in the lymphoma and spleen of hOGG1 transgenic (4) and the spleen from wild-type control (4) mice. The amount of H2O2 produced in the lymphoma and the spleen of hOGG1 transgenic mice was ~166% and ~66% higher, respectively, than that in the spleen from wild-type control mice (P < 0.001). The amount of H2O2 produced in the lymphoma was ~60% higher than that in the spleen from hOGG1 transgenic mice (P < 0.05). Finally, we examined mitochondrial DNA alterations in TG mice. Mitochondrial DNA samples were extracted from various organs and lymphoma tissues from hOGG1 TG and age-matched non-TG control animals and subjected to PCR analysis using specific primer sets franking the breakpoints of 7 major mitochondrial DNA deletions. Six deletions (3.7, 3.82, 3.86, 4.2, 4.9 and 5.2 kilobase in length) have been previously reported in the literatures. One novel deletion of 15.kilobase was identified in hOGG1 TG mouse in our laboratory. Among 7 major mitochondrial DNA deletion analyzed, Five (3.7, 3.86, 4.2, 5.2 and 15 kilobase in length) deletions were detected in higher frequency in various organs of hOGG1 TG but not in non-TG control mice, suggesting that those deletions might be resulted from overexpression of the transgene hOGG1. Notably, 3 deletions (del3729, del3868, and del15139) were present in significantly higher frequencies in spleen with myeloproliferative disorders or lymphoma from TG mice in comparison to the spleen of the age-matched wild type animals (P<0.01). While the precise mechanisms leading to the development of hematopoietic neoplasms remain elusive, we hypothesized that major mitochondrial gene deletions and increased mitochondrial respiration, as a result of overexpressed hOGG1 gene in the mitochondria, may contribute significantly to the increased intracellular ROS in hematopoietic progenitor cell populations, which, in turn, causes further genetic mutation and the development of lymphoma and myeloproliferative disorder seen in these TG mice. Disclosures No relevant conflicts of interest to declare.
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Demetrius, Lloyd A., and Jane A. Driver. "Preventing Alzheimer's disease by means of natural selection." Journal of The Royal Society Interface 12, no. 102 (January 2015): 20140919. http://dx.doi.org/10.1098/rsif.2014.0919.
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The amyloid cascade model for the origin of sporadic forms of Alzheimer's disease (AD) posits that the imbalance in the production and clearance of beta-amyloid is a necessary condition for the disease. A competing theory called the entropic selection hypothesis asserts that the primary cause of sporadic AD is age-induced mitochondrial dysregulation and the following cascade of events: (i) metabolic reprogramming—the upregulation of oxidative phosphorylation in compensation for insufficient energy production in neurons, (ii) natural selection—competition between intact and reprogrammed neurons for energy substrates and (iii) propagation—the spread of the disease due to the selective advantage of neurons with upregulated metabolism. Experimental studies to evaluate the predictions of the amyloid cascade model are being continually retuned to accommodate conflicts of the predictions with empirical data. Clinical trials of treatments for AD based on anti-amyloid therapy have been unsuccessful. We contend that these anomalies and failures stem from a fundamental deficit of the amyloid hypothesis: the model derives from a nuclear-genomic perspective of sporadic AD and discounts the bioenergetic processes that characterize the progression of most age-related disorders. In this article, we review the anomalies of the amyloid model and the theoretical and empirical support for the entropic selection theory. We also discuss the new therapeutic strategies based on natural selection which the model proposes.
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Seneviratne, Ayesh K., Juan J. Aristizabal Henao, G. Wei Xu, Rose Hurren, Sejin Kim, Neil MacLean, Xioaming Wang, et al. "Tafazzin (TAZ) Regulates the Differentiation of AML Cells By Reducing Levels of the Phospholipid Phosphatidylethanolamine." Blood 130, Suppl_1 (December 7, 2017): 788. http://dx.doi.org/10.1182/blood.v130.suppl_1.788.788.
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Abstract AML cells have unique mitochondrial characteristics with an increased reliance on mitochondrial metabolism and oxidative phosphorylation. To identify new biological vulnerabilities in the mitochondria of AML, we conducted a CRISPR knockout screen. CAS9-overexpressing human OCI-AML2 leukemia cells were transduced with a library of 91,320 sgRNAs in barcoded lentiviral vectors targeting 17,237 nuclear-encoded genes. Cells were harvested, genomic DNA was isolated, and the relative abundance of sgRNAs were determined by sequencing barcodes 14 days after puromycin selection. We focused on the sgRNAs targeting the 1050 mitochondrial proteins to identify targets in the mitochondrial proteome whose knockout reduced AML growth and viability. The cardiolipin remodeling enzyme tafazzin (TAZ) was among the top 1% of mitochondrial hits. Using individual sgRNA, we confirmed that knockout of TAZ reduced the growth of CAS9-OCI-AML2 cells by >70%, thus validating the findings from our screen. We also knocked down TAZ with 2 independent shRNA and demonstrated reductions in growth and viability of a panel of AML cells: OCI-AML2 (>80%), TEX (>50%), K562 (>50%), and U937 (>40%). Moreover, TAZ knockdown significantly reduced the engraftment of TEX leukemia cells in vivo by 80%, indicating that TAZ-knockdown reduces AML growth in vivo and can target leukemia initiating cells. In contrast, knockdown of TAZ in mouse models did not impair normal hematopoiesis nor reduced the abundance of hematopoietic stem cells, although more subtle defects in the hematopoietic stem cells might explain transient episodes of neutropenia seen in Barth's syndrome, a congenital condition associated with X-linked TAZ mutations. TAZ is responsible for the majority of Cardiolipin (CL) remodeling under physiological conditions. As expected the knockdown of TAZ in both AML and normal mouse hematopoietic cells increased the substrate (monlysocardiolipin) to product (CL) ratio of TAZ. CL is required for the proper localization, and efficient function of, respiratory chain enzymes. However, in AML cells, knockdown of TAZ did not alter respiratory chain complex activity, basal oxygen consumption, or respiratory chain reserve capacity. Recent studies have shown that mitochondrial pathways can regulate cell fate and differentiation independent of their effects on oxidative phosphorylation. Therefore, we examined changes in AML cell differentiation after TAZ knockdown. Knockdown of TAZ promoted the differentiation of AML cells as evidenced by increased non-specific esterase staining and increased CD11b expression on the cell surface. In breast cancer cells decreasing phosphatidylethanolamine (PE) levels, induced the differentiation of these cells. As TAZ regulates phospholipid remodeling, therefore we measured levels of PE and phosphatidylserine (PS) after TAZ-knockdown by spot densitometry. Interestingly, knockdown of TAZ in OCI-AML2 cells decreased PE and increased PS lipid levels. To determine whether alterations in PE and PS phospholipids are functionally important for differentiation of AML cells, we treated AML cells with MMV007285, an inhibitor of the phosphatidylserine decarboxylase (PISD), an enzyme that converts PS to PE. MMV007285 mimicked the effects of TAZ-knockdown and increased differentiation of OCI-AML2 and 8227 AML cells. In summary, the cardiolipin remodeling enzyme TAZ regulates the differentiation of AML cells by controlling levels of PS and PE, thereby highlighting a new mechanism by which phospholipids and mitochondrial enzymes regulate AML cell fate and differentiation. Moreover, PISD inhibition may be a novel therapeutic strategy to selectively promote the differentiation of AML. Disclosures No relevant conflicts of interest to declare.
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Ferdous, Maheen, Miguel Ganuza, Per Holmfeldt, Trent Hall, Megan Walker, and Shannon McKinney-Freeman. "The G Protein-Coupled Receptor Associated Sorting Proteins, Gprasp2 and Armcx1 Are Putative Negative Regulators of HSC Engraftment and Repopulation." Blood 126, no. 23 (December 3, 2015): 2386. http://dx.doi.org/10.1182/blood.v126.23.2386.2386.
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Abstract Although hematopoietic stem cell transplantation (HSCT) is used routinely to cure hematologic disease, the efficacy of transplantation is limited by the paucity of HSC. One way to overcome this is to increase the efficiency of HSC engraftment. Thus, we executed a functional screen for novel regulators of HSCT. Murine HSC were lentivirally transduced with shRNAs targeting prioritized gene candidates prior to transplantation into cohorts of lethally irradiated recipient mice. In total, around 1300 mice were transplanted to assess a putative role for about 50 genes in HSCT. We thereby identified Gprasp2 and Armcx1 as putative negative regulators of HSCT. When transplanted at a 1:4 disadvantage relative to control, recipients of either Gprasp2 or Armcx1 shRNA-treated CD45.2 Lineage- Sca-1+ c-Kit+ (LSK) cells displayed 3.12 (p=0.024) and 2.8 (p=0.04) fold enhanced CD45.2 chimerism in peripheral blood (PB) at 16 weeks post-transplant, respectively, relative to mice transplanted with CD45.2 LSK cells treated with control shRNAs. Although loss of each gene did not favor a particular PB lineage, CD45.2 chimerism was enhanced in all bone marrow (BM) HSC and progenitor (HSPC) compartments in these recipients, correlating with their enhanced PB chimerism. qRT-PCR reveals that both murine Armcx1 and Gprasp2 are highly enriched for expression in LSK CD150+CD48- cells relative to all downstream hematopoietic progeny. Further, HemaExplorer, a bioinformatics database of human hematopoietic gene expression, suggests that GPRASP2 and ARMCX1 are also highly expressed in human HSC. This prediction is currently being validated by qRT-PCR. Interestingly, Gprasp2 and Armcx1 both belong to the G protein-coupled receptor associated sorting protein (GASP) gene family, which has never before been implicated in HSC function. The closely related GASP family member, Gprasp1, sorts G protein-coupled receptors (GPCR) to lysosomes for degradation. As Gprasp1 and Gprasp2 both contain GPCR-binding domains and ~70% amino acid sequence conservation in their C-termini, Gprasp2 may also regulate GPCR trafficking and degradation in HSC. Although Gprasp1 was not tested in our screen, qRT-PCR analysis reveals that it is also highly expressed by murine HSC relative to downstream progeny, suggesting that it too may play a role in HSC function. We are currently assessing this using Gprasp1-shRNAs and competitive transplantation. In contrast, Armcx1 lacks the GPCR binding domain and contains both a nuclear and mitochondrial localization signal and has been shown to localize to mitochondrial networks when expressed in HEK-293 cells, suggesting a role in mitochondrial/nuclear communication. To determine how loss of Gprasp2 and Armcx1 promotes HSC engraftment, we are currently employing transplantation and ex vivo culture assays to analyze the effect of their loss on cell cycle, apoptosis, migration, and adhesion of HSPC post-transplant. Our work may help elucidate the mechanisms underlying efficient engraftment, adhesion, and retention of HSPC in the BM niche, which in turn may shed light on novel pathways that could be targeted to promote the efficiency of HSCT in the clinic. Disclosures No relevant conflicts of interest to declare.
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Cole, Alicia, Zezhou Wang, Rachel Mattson, Rose Hurren, Fengshu Lin, Marcela Gronda, Neil Maclean, et al. "Targeting the Mammalian Mitochondrial Clpp (mClpP) As a Novel Therapeutic Strategy for Acute Myeloid Leukemia." Blood 120, no. 21 (November 16, 2012): 3603. http://dx.doi.org/10.1182/blood.v120.21.3603.3603.
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Abstract Abstract 3603 The mammalian mitochondrial caseinolytic protease (mClpP) is a nuclear-encoded enzyme complex responsible for degrading excess proteins in the mitochondria. It is functionally similar to the proteasome complex in the cytoplasm and structurally homologous to the bacterial ClpP enzyme complex. While the proteasome has been well characterized as a therapeutic target for the treatment of hematologic malignancy, little is known regarding mClpP as a potential therapeutic target in malignant cells. Therefore, we evaluated the expression of mClpP in acute myeloid leukemia (AML) and normal hematopoietic cells by immunoblotting. Strikingly, mClpP was robustly expressed in AML cell lines (n=3/4) and primary AML patients (n=4/4), but was undetectable in normal bone marrow samples (n = 3) and G-CSF-mobilized peripheral blood mononuclear cells from consenting volunteers donating stem cells for allotransplant (n = 5). We also demonstrated over-expression of mClpP mRNA in these AML samples compared to normal by Q-RT-PCR. Next, we knocked down mClpP and its regulatory chaperone protein mClpX in OCI-AML2 and TEX human leukemia cells using 3 independent shRNA in lentiviral vectors. Target knockdown was confirmed by Q-RT-PCR and immunoblotting. Compared to cells infected with control sequences, knockdown of mClpP reduced the growth and viability of these leukemia cells by > 90%. Knockdown of the chaperone protein mClpX also reduced the growth and viability of these cells, but with less potency than mClpP knockdown. Rho-zero cells have been depleted of mitochondrial DNA by treatment with ethidium bromide and therefore lack mitochondrial protein synthesis. Through feedback mechanisms, expression of nuclear encoded proteins that contribute to oxidative metabolism are also reduced. Thus, these cells have lower rates of mitochondrial protein accumulation and turnover. We demonstrated that wild type and rho-zero 143B rhabdomyosarcoma cells both express mClpP protein, but levels were slightly lower in the rho-zero cells. We then tested the effects of mClpP knockdown in rho-zero cells and demonstrated that knockdown of mClpP reduced the growth and viability of wild type 143B cells, but had little effect on their rho-zero counterparts. As a chemical approach to evaluate the effects of mClpP inhibition on AML and normal hematopoietic cells, we synthesized a derivative of recently reported beta-lactone bacterial ClpP inhibitor and generated (3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one that we termed A2–32–01. We confirmed that A2–32–01 inhibited the enzymatic activity of recombinant bacterial ClpP similar to the activity of the reported beta-lactone inhibitors. A2–32–01 induced cell death in TEX, OCI-AML2, and K562 leukemia cells that express mClpP as measured by trypan blue staining. We also isolated mitochondria from these cells after treatment with A2–32–01 and demonstrated that the compound reduced the enzymatic activity of the mClpP protease as measured by cleavage of the fluorogenic substance N-succinyl-Leu-Tyr-7-amidomethylcoumarin (Suc-LY-AMC). In contrast, A2–32–01 was not cytotoxic to HL60 cells that had undetectable mClpP. Likewise, A2–32–01 induced death in 143B wild type cells, but not the rho-zero counterparts. Finally, we evaluated the effects of mClpP inhibition on primary AML and normal hematopoietic cells. Primary AML and normal hematopoietic cells were treated with increasing concentrations of A2–32–01 and cell viability was measured after 48 hours incubation by Annexin V/PI staining followed by flow cytometry. A2–32–01 did not kill normal hematopoietic cells (n = 3 samples), but was cytotoxic to 5/6 tested primary AML cells. In summary, we have used genetic and chemical approaches to highlight the mitochondrial protease, mClpP, as a novel therapeutic target for AML. Disclosures: No relevant conflicts of interest to declare.
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Kim, Hye Ran, Eun-Jeong Won, Hyun-Jung Choi, Hwan-Young Kim, James Moon, Jong-Hee Shin, Soon-Pal Suh, Dong-Wook Ryang, and Myung-Geun Shin. "Mitochondrial DNA Minisatellites Showed Higher Informativeness and Sensitivity Than the Nuclear DNA Markers for the Quantitative Determination of Chimerism After Allogeneic Stem Cell Transplantation,." Blood 118, no. 21 (November 18, 2011): 4004. http://dx.doi.org/10.1182/blood.v118.21.4004.4004.
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Abstract Abstract 4004 Background: Mitochondrial DNA (mtDNA) is widely used in forensic identification and anthropologic studies on account of its abundance resulting in preferential amplification, sequencing and inherent variability. We developed mtDNA markers to monitor donor cell engraftment after allogeneic stem cell transplantation(SCT), then compared with nuclear short tandem repeat (STR) markers. Patients and methods: The mtDNA control regions and six mtDNA minisatellites (mtMS) (303 poly C, 16184 poly C, 514 (CA) repeat, 3566 poly C, 12385 poly C and 12418 poly A) from the total DNA samples of 215 cases (donor, recipient and after allogeneic SCT) were amplified using the designated specific primers and PCR. The results were compared with those from the six short tandem repeat (STR) markers (D12S391, D18S51, F13A1, HUM RENA-4, HUM FABP2 and Amelogenin). Results: Polymorphisms in the mtDNA control region identify an informative marker in 88% (189 cases) of all cases. Among the six mtMS markers, the informativeness of 303 poly C and 16184 poly C mtMS was 63% and 67% respectively. A combination of direct sequencing through the mtDNA control region, 303poly C and 16184 poly C mtMS could completely distinguish the donor cells from the recipient cells. The results from a typical mixing experiment to determine the sensitivity revealed a detection limit (DL) of the gene scan analysis in a mtDNA mixture to be visible at 1% heteroplasmy in 303 poly C mtMS marker. However, the DL from D12S391 in the same mixing experiment was 5–10% heteroplasmy. Conclusions: mtMS markers, especially 303 poly C and 16184 poly C markers, can provide a sensitive, accurate and quantitative determination of mixed chimerism after a SCT. Disclosures: No relevant conflicts of interest to declare.
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Agarwal, Suneet, Anne B. C. Cherry, Erin M. McLoughlin, Paul H. Lerou, Justine D. Miller, Hongguang Huo, Odelya Hartung, et al. "Derivation of Disease-Free Induced Pluripotent Stem Cells From Patients with Pearson Marrow Pancreas Syndrome." Blood 116, no. 21 (November 19, 2010): 3. http://dx.doi.org/10.1182/blood.v116.21.3.3.
Full textAbstract:
Abstract Abstract 3 Mutations in mitochondrial DNA (mtDNA) cause several incurable diseases. In congenital mtDNA disorders, a mixture of normal and mutated mtDNA termed heteroplasmy exists at varying levels in different tissues, which determines the severity and phenotypic expression of the disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The clinical hallmarks of PS include sideroblastic anemia and other cytopenias, pancreatic insufficiency, metabolic acidosis, and other systemic organ dysfunction. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Somatic cells can be directly reprogrammed using defined genetic and chemical factors to yield “induced pluripotent stem” (iPS) cells, which have the capacity to differentiate into any tissue. iPS cells are particularly amenable for modeling mtDNA disorders, as cytoplasmic genetic material is retained during reprogramming. We sought to generate iPS cells from patients with PS and related mtDNA disorders to investigate the effects of mitochondrial dysfunction on stem cells and hematopoiesis. From a patient with PS, we generated bone marrow-derived fibroblasts carrying a high heteroplasmic burden of mutant mtDNA, and reprogrammed them into iPS cells. Although reprogramming efficiency was very low and kinetics of iPS colony emergence delayed, PS-iPS cells carrying the pathogenic mutation could be generated and displayed all hallmarks of pluripotency. We observed that PS-iPS cells initially demonstrated slow growth and a propensity for differentiation, but with ongoing passage in tissue culture, these characteristics improved. Unexpectedly, we found that the proportion of mutant mtDNA decreased rapidly in the PS-iPS lines as a function of passage. By subcloning, we were able to generate iPS cell lines with virtually undetectable amounts of mutant mtDNA, but which retained a viral integration pattern confirming their nuclear genetic identity to the original, highly heteroplasmic iPS clone. From “purged” PS-iPS cells, we generated hematopoietic progenitors free of detectable mutant mtDNA, thus yielding genetically identical, disease-free iPS cells and blood cells from a patient with Pearson syndrome. Disease-free iPS cells were readily obtained from the skin-derived fibroblasts of two other patients that carried a lower burden of mutant mtDNA. Our results suggest that mitochondrial dysfunction drives the segregation or elimination of mutant mtDNA in iPS cells as a function of passage, implying that maintenance of self-renewal and pluripotency are highly dependent on intact mitochondrial function. Importantly, a similar depletion of mutant mtDNA can be observed over time in vivo in certain tissues, such as hematopoietic cells, of patients with PS and other mtDNA disorders. This work provides a unique set of in vitro cellular models carrying varying degrees of mtDNA heteroplasmy to interrogate the effects of mitochondrial dysfunction on hematopoiesis. PS-iPS cells also provide a valuable opportunity to determine the factors driving changes in mtDNA heteroplasmy in stem cells, which holds important therapeutic implications for PS and a variety of congenital and acquired mtDNA disorders. Disclosures: No relevant conflicts of interest to declare.