BNIP3L/NIX and FUNDC1-mediated mitophagy is required for mitochondrial network remodeling during cardiac progenitor cell differentiation

Autophagy, Journal Year: 2019, Volume and Issue: 15(7), P. 1182 - 1198

Published: Feb. 11, 2019

Cell-based therapies represent a very promising strategy to repair and regenerate the injured heart prevent progression failure. To date, these have had limited success due lack of survival retention infused cells. Therefore, it is important increase our understanding biology cells utilize this information enhance their function in heart. Mitochondria are critical for progenitor cell survival. Here, we demonstrate importance mitochondrial autophagy, or mitophagy, differentiation process adult cardiac (CPCs). We found that mitophagy was rapidly induced upon initiation CPCs. also mediated by receptors, rather than PINK1-PRKN/PARKIN pathway. Mitophagy BNIP3L/NIX FUNDC1 not involved regulating fate determination, biogenesis, reprogramming. Instead, facilitated CPCs undergo proper network reorganization during differentiation. Abrogating BNIP3L- FUNDC1-mediated led sustained fission formation donut-shaped impaired mitochondria. It resulted increased susceptibility death failure survive infarcted Finally, aging associated with accumulation DNA (mtDNA) damage acquiring mtDNA mutations selectively disrupted differentiation-activated program These findings as regulator differentiation, well consequences accumulating mutations.Abbreviations: Baf: bafilomycin A1; BCL2L13: BCL2 like 13; BNIP3: interacting protein 3; BNIP3L: 3 like; CPCs: cells; DM: media; DNM1L: dynamin 1 EPCs: endothelial FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FUNDC1: FUN14 domain containing 1; HSCs: hematopoietic stem MAP1LC3B/LC3: microtubule-associated light chain beta; MFN1/2: mitofusin 1/2; MSCs: mesenchymal mtDNA: DNA; OXPHOS: oxidative phosphorylation; PPARGC1A: PPARG coactivator alpha; PHB2: prohibitin 2; POLG: polymerase gamma, catalytic subunit; SQSTM1: sequestosome TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester

Language: Английский

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The peroxisome: an update on mysteries 2.0

Histochemistry and Cell Biology, Journal Year: 2018, Volume and Issue: 150(5), P. 443 - 471

Published: Sept. 15, 2018

Peroxisomes are key metabolic organelles, which contribute to cellular lipid metabolism, e.g. the β-oxidation of fatty acids and synthesis myelin sheath lipids, as well redox balance. Peroxisomal dysfunction has been linked severe disorders in man, but peroxisomes now also recognized protective organelles with a wider significance human health potential impact on large number globally important diseases such neurodegeneration, obesity, cancer, age-related disorders. Therefore, interest their physiological functions significantly increased recent years. In this review, we intend highlight discoveries, advancements trends peroxisome research, present an update continuation two former review articles addressing unsolved mysteries astonishing organelle. We summarize novel findings biological peroxisomes, biogenesis, formation, membrane dynamics division, peroxisome–organelle contacts cooperation. Furthermore, peroxisomal proteins machineries at discussed. Finally, address role brain, neurological disorders, development cancer.

Language: Английский

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Mitochondria as central hub of the immune system

Redox Biology, Journal Year: 2019, Volume and Issue: 26, P. 101255 - 101255

Published: June 15, 2019

Nearly 130 years after the first insights into existence of mitochondria, new rolesassociated with these organelles continue to emerge. As essential hubs that dictate cell fate, mitochondria integrate physiology, signaling pathways and metabolism. Thus, recent research has focused on understanding how multifaceted functions can be used improve inflammatory responses prevent cellular dysfunction. Here, we describe role development function immune cells, highlighting metabolic aspects pointing out some metabolic- independent features sustain function.

Language: Английский

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Reactive oxygen species: a volatile driver of field cancerization and metastasis

Molecular Cancer, Journal Year: 2019, Volume and Issue: 18(1)

Published: March 30, 2019

Field cancerization and metastasis are the leading causes for cancer recurrence mortality in patients. The formation of primary, secondary tumors or is greatly influenced by multifaceted tumor-stroma interactions, which stromal components tumor microenvironment (TME) can affect behavior cells. Many studies have identified cytokines growth factors as cell signaling molecules that aid to communication. However, functional contribution reactive oxygen species (ROS), a family volatile chemicals, communication less understood. Cancer cells various tumor-associated produce secrete copious amount ROS into TME. Intracellular modulate cascades acquisition several hallmarks cancers. Extracellular help propagate, amplify, effectively create mutagenic oncogenic field facilitate multifoci act springboard metastatic In this review, we summarize our current knowledge atypical paracrine metastasis. often discussed separately; offer model placed these events with focal instigating agent broader "seed-soil" hypothesis.

Language: Английский

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Physiological Signaling Functions of Reactive Oxygen Species in Stem Cells: From Flies to Man

Frontiers in Cell and Developmental Biology, Journal Year: 2021, Volume and Issue: 9

Published: Aug. 6, 2021

Reactive oxygen species (ROS), superoxide anion and hydrogen peroxide, are generated as byproducts of oxidative phosphorylation in the mitochondria or via cell signaling-induced NADPH oxidases cytosol. In recent two decades, a plethora studies established that elevated ROS levels by eustress crucial physiological mediators many cellular developmental processes. this review, we discuss mechanisms generation regulation, current understanding functions maintenance adult embryonic stem cells, well process reprogramming to pluripotent state. Recently discovered cell-non-autonomous mediated growth factors for controlling differentiation immune response Drosophila . Importantly, may allow deciphering analogous processes human, which could potentially lead development novel therapeutic approaches ROS-associated diseases treatment.

Language: Английский

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Age-Associated Mitochondrial Dysfunction Accelerates Atherogenesis

Circulation Research, Journal Year: 2019, Volume and Issue: 126(3), P. 298 - 314

Published: Dec. 10, 2019

Rationale: Aging is one of the strongest risk factors for atherosclerosis. Yet whether aging increases atherosclerosis independently chronic hyperlipidemia not known. Objective: To determine if vascular before induction enhances atherogenesis. Methods and Results: We analyzed aortas young aged normolipidemic wild type, disease-free mice found that led to elevated IL (interleukin)-6 levels mitochondrial dysfunction, associated with increased mitophagy protein Parkin. In aortic tissue culture, we evidence dysfunction IL-6 exist in a positive feedback loop. triggered acute by inducing liver-specific degradation LDL (low-density lipoprotein) receptor combined 10-week western diet atherogenesis was enhanced wild-type mice. Hyperlipidemia further reduced function Parkin but Genetic disruption autophagy smooth muscle cells exposed levels, impaired function, Importantly, enhancing aged, hyperlipidemic via oral administration spermidine prevented increase Parkin, attenuated Conclusions: Before hyperlipidemia, elevates impairs within aorta, levels. These age-associated changes prime vasculature exacerbate upon hyperlipidemia. Our work implies novel therapeutics aimed at improving bioenergetics or reducing inflammation may reduce age-related

Language: Английский

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PPARγ/PGC1α signaling as a potential therapeutic target for mitochondrial biogenesis in neurodegenerative disorders

Pharmacology & Therapeutics, Journal Year: 2020, Volume and Issue: 219, P. 107705 - 107705

Published: Oct. 9, 2020

Language: Английский

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Icariin‐induced inhibition of SIRT6/NF‐κB triggers redox mediated apoptosis and enhances anti‐tumor immunity in triple‐negative breast cancer

Cancer Science, Journal Year: 2020, Volume and Issue: 111(11), P. 4242 - 4256

Published: Sept. 21, 2020

Abstract Abnormal activation of the nuclear factor‐kappa B (NF‐κB) signaling pathway is closely implicated in triple‐negative breast cancer growth, metastasis, and tumor immune escape. In this study, anti‐cancer effects icariin, a natural flavonol glycoside, toward cells underlying mechanisms were investigated. This investigation showed that icariin selectively inhibited proliferation triggered apoptosis concentration‐ time‐dependent manner, but exhibited little cytotoxicity normal cells. Moreover, induced cell via mitochondria‐mediated pathway, as indicated by upregulated ratio Bax/Bcl‐2 reactive oxygen species induction. Importantly, impaired NF‐κB/EMT evidenced upregulation SIRT6, resulting inhibition migration invasion Additionally, oss‐128167, an inhibitor dramatically attenuated anti‐migration anti‐invasion icariin. Transcriptomic analysis verified impairment NF‐κB led to selective function Notably, significant growth anti‐pulmonary metastasis effect mouse model MDA‐MB‐231 4T1 regulating immunosuppressive microenvironment. Together, these results could effectively trigger inhibit SIRT6/NF‐κB suggesting might serve potential candidate drug for treatment cancer.

Language: Английский

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From OCR and ECAR to energy: Perspectives on the design and interpretation of bioenergetics studies

Journal of Biological Chemistry, Journal Year: 2021, Volume and Issue: 297(4), P. 101140 - 101140

Published: Aug. 28, 2021

Biological energy transduction underlies all physiological phenomena in cells. The metabolic systems that support have been of great interest due to their association with numerous pathologies including diabetes, cancer, rare genetic diseases, and aberrant cell death. Commercially available bioenergetics technologies (e.g., extracellular flux analysis, high-resolution respirometry, fluorescent dye kits, etc.) made practical assessment parameters widely accessible. This has facilitated an explosion the number studies exploring, particular, biological implications oxygen consumption rate (OCR) substrate level phosphorylation via glycolysis (i.e., acidification (ECAR)). Though these demonstrated substantial utility broad applicability biology research, they are also susceptible historical assumptions, experimental limitations, other caveats led premature and/or erroneous interpretations. review enumerates various important considerations for designing interpreting cellular mitochondrial experiments, some common challenges pitfalls data interpretation, potential "next steps" be taken can address highlighted challenges. In recent decades, a multitude genetically modified cells organisms developed aim elucidating etiological pathophysiological mechanisms disease (1Jucker M. benefits limitations animal models translational research neurodegenerative diseases.Nat. Med. 2010; 16: 1210-1214Crossref PubMed Scopus (224) Google Scholar, 2Ruggeri B.A. Camp F. Miknyoczki S. Animal disease: Pre-clinical cancer applications drug discovery.Biochem. Pharmacol. 2014; 87: 150-161Crossref (199) 3King A.J.F. use diabetes research.Br. J. 2012; 166: 877-894Crossref (653) Scholar). These model often demonstrate features or compromised metabolism (4Cheng Z. Tseng Y. White M.F. Insulin signaling meets mitochondria metabolism.Trends Endocrinol. Metab. 21: 589-598Abstract Full Text PDF (292) 5Nagarajan A. Malvi P. Wajapeyee N. Oncogene-directed alterations Cancer. 2016; 2: 365-377Abstract (82) 6Gao C. Wang Q. Chung S.K. Shen Crosstalk factors neurogenic adult neurogenesis: Implication regulation mental neurological diseases.Neurochem. Int. 2017; 106: 24-36Crossref (14) Recent technological advances widespread commercial availability instrumentation reagents investigations into consequences gene variance systems, emphasis placed on processes related through oxidative (OxPhos) substrate-level (7Reily Mitchell T. Chacko B.K. Benavides G.A. Murphy M.P. Darley-Usmar V.M. Mitochondrially targeted compounds impact bioenergetics.Redox Biol. 2013; 1: 86-93Crossref (152) 8Gerencser A.A. Neilson Choi S.W. Edman U. Yadava Oh R.J. Ferrick D.A. Nicholls D.G. Brand M.D. Quantitative microplate-based respirometry correction diffusion.Anal. Chem. 2009; 81: 6868-6878Crossref (227) 9Mookerjee S.A. Goncalves R.L.S. Gerencser contributions respiration acid production.Biochim. Biophys. Acta. 2015; 1847: 171-181Crossref (170) 10Mookerjee Quantifying intracellular rates glycolytic ATP production using measurements.J. 292: 7189-7207Abstract (128) assays, exemplified by popular phosphorescent probe-based analysis (EFA) (8Gerencser Scholar), found areas biomedical research. For example, EFA contributed significant identification novel liabilities drugs (11Rana Aleo Gosink Will Evaluation vitro toxicity assays physicochemical properties prediction organ 228 pharmaceutical drugs.Chem. Res. Toxicol. 2019; 32: 156-167Crossref (0) 12Gohil Sheth Nilsson R. Wojtovich A.P. Lee J.H. Perocchi Chen W. Clish C.B. Ayata Brookes P.S. Mootha V.K. Nutrient-sensitized screening shift from glycolysis.Nat. Biotechnol. 28: 249-255Crossref (214) high-throughput comparison cancer-derived lines (13Wu Swift A.L. Moran Tamagnine Parslow D. Armistead Lemire K. Orrell Teich Chomicz Multiparameter reveals close link between attenuated bioenergetic function enhanced dependency human tumor cells.Am. Physiol. Cell 2007; 125-136Crossref (554) 14Martin S.D. McGee S.L. A systematic approach identify vulnerabilities breast lines.Cancer 7: 12Crossref 15Dier Shin D.H. Madhubhani L.P. Hemachandra Uusitalo L.M. Hempel Bioenergetic ovarian lines: Profiling histological subtypes mitochondria-defective line.PLoS One. 9e98479Crossref (42) elucidation links programmed death (16Robinson G.L. Dinsdale MacFarlane Cain Switching aerobic modulates sensitivity mantle lymphoma TRAIL.Oncogene. 31: 4996-5006Crossref (37) there application science, assumptions regarding organization persist may ultimately impede scientific progress. nuanced likely because observable conditions. As examples, "Warburg effect" cancerous noncancerous "dysfunction" diseases type II mellitus) both detected depending conditions, but remain disputed (17DeBerardinis Chandel N.S. We need talk about Warburg effect.Nat. 2020; 127-129Crossref (144) 18Muoio D.M. Neufer P.D. Lipid-induced stress insulin action muscle.Cell 15: 595-605Abstract (230) Additionally, measurements dramatically quality ECAR OCR ratios used "diagnose" effect subject several modifiers proportionality meant represent (9Mookerjee purpose this article is provide overview assay design, emphasizing key conceptual considerations, as well perspectives interpretation. Some flexible design covered first section, followed additional discussion three topic general importance: 1) ATP, 2) glycolysis, ECAR, partitioning, 3) dysfunction. Nearly take advantage principle steady-state mass balance determine fluxes. scheme, external internal fluxes measured, then net fit system(s) under study, unknown estimated. main discriminating factor among different approaches lies degree molecular "resolution," ability distinguish accurately pathways individual reactions. current modality highest resolution (MFA) (19Wiechert 13C analysis.Metab. Eng. 2001; 3: 195-206Crossref (587) method, measured fitting isotopomer labeling patterns simulation network interpolate reaction (20Antoniewicz M.R. guide biologist.Exp. Mol. 2018; 50: 1-13Crossref Two alternative methods include which involve time-resolved measurement only, paired inhibitor protocols reductively specific reactions/pathways. low compared MFA capable over short timescales, limitation typically long incubation times required achieve isotopic steady state Because technical challenges, tend performed researchers extensive experience biochemistry/metabolism. Respirometry will continued focus do not necessarily specialize Measuring respirometry) decades essential development modern understanding physiology (21Chance B. Williams G.R. simple rapid phosphorylation.Nature. 1955; 175: 1120-1121Crossref cells/tissues/organelles enclosed chamber exposed defined conditions tension media time. structure coupling stoichiometries electron transfer system (ETS) energetically coupled transport characterized variety types (22Hinkle P.C. P/O phosphorylation.Biochim. 2005; 1706: 1-11Crossref (197) 23Chance respiratory chain phosphorylation.Adv. Enzymol. Relat. Subj. Biochem. 1956; 17: 65-134PubMed allows interpolation OxPhos) data. There two currently employed measure OCR: potentiometric method uses platinum hydrogen (Clark) electrode, quenching fluorescence lifetime metal-porphyrin complexes (MPCs) presence elemental (24O'Donovan Hynes Yashunski Papkovsky D.B. Phosphorescent oxygen-sensitive materials applications.J. Mater. 2946-2951Crossref (57) 25Zhdanov A.V. Ogurtsov V.I. Taylor C.T. Monitoring oxygenation responses stimulation sensing technique.Integr. (Camb.). 443-451Crossref (51) Clark electrodes Oxygraph-2K; Oroboros) "homemade" apparatuses, principles described (26Haller Ortner Gnaiger E. respirometer investigating metabolism: Toward optimization studies.Anal. 1994; 218: 338-342Crossref 27Ripple M.O. Kim Springett Mammalian complex I pumps 4 protons per 2 electrons at high proton motive force living cells.J. 288: 5374-5380Abstract (41) Solid-state MPCs most technology extremely Seahorse Extracellular Flux Analyzer (Agilent technologies) although information explicitly community. prepared/purchased soluble form 28Zhdanov Favre O'Flaherty L. Adam O'Connor Pollard P.J. Comparative transformed budget platform.Integr. 2011; 1135-1142Crossref (23) 29O'Riordan T.C. Zhdanov Ponomarev G.V. Analysis mammalian fluorometry.Anal. 79: 9414-9419Crossref another parameter interpolated flux. Like lanthanide-based small-molecule probes (30Hynes O'Riordan Uray G. long-decay pH-sensitive lanthanide probe assay.Anal. 390: 21-28Crossref Several commonly conjunction electrochemical across inner membrane (IMM) proportional energetic ETS potentiometrically organic cations tetramethyl rhodamine methyl ester) (31Schmidt C.A. McLaughlin K.L. Boykov I.N. Mojalagbe Ranganathan Buddo K.A. Lin C.-T. Fisher-Wellman K.H. Aglycemic growth enhances carbohydrate induces menadione cultured tumor-derived cells.Cancer 2021; 9: 1-21Crossref 32Fisher-Wellman Te Ryan T.E. Reese L.R. Gilliam L.A.A. Cathey B.L. Lark D.S. Smith C.D. Muoio Pyruvate dehydrogenase nicotinamide nucleotide transhydrogenase constitute energy-consuming redox circuit.Biochem. 467: 271-280Crossref (81) reduced pyridine pool (NAD(P)H) within subcellular compartments monitored autofluorescence (ideally lifetime) (33Blacker T.S. Duchen Investigating NADH NADPH autofluorescence.Free Radic. 100: 53-65Crossref (169) 34Fisher-Wellman Davidson M.T. Narowski T.M. Koves T.R. Mitochondrial diagnostics: multiplexed platform comprehensive fluxes.Cell Rep. 24: 3593-3606.e10Abstract (33) enzyme activity cost-effective relatively perform reactions reveal allosteric interactions (34Fisher-Wellman Scholar) Many fluorometric measuring pH ion gradients (35Takahashi Zhang Centonze V.E. Herman Measurement situ.Biotechniques. 30: 804-815Crossref 36Roe M.W. Lemasters J.J. Assessment Fura-2 cytosolic free calcium.Cell Calcium. 1990; 11: 63-73Crossref (363) advancement encoded report changes pH, GSH/GSSG ratio, NAD(P)H state, etc. (37Shirmanova M.V. Druzhkova Lukina M.M. Matlashov M.E. Belousov V.V. Snopova L.B. Prodanetz N.N. Dudenkova Lukyanov Zagaynova E.V. Intracellular imaging tumors vivo new sensor SypHer2.Biochim. 1850: 1905-1911Crossref 38Fan Makar M.X. Ai H. thioredoxin red biosensor.Nat. 13: 1045-1052Crossref (39) 39Tao Zhao Chu Zhu X. Zou Shi Liu Su Du Zhou H.-M. Qian et al.Genetically sensors dynamic metabolism.Nat. Methods. 14: 720-728Crossref (119) Finally, platforms/instrumentation best suited preparations. Table 1.Table 1Considerations choosing studiesConsiderationsPotentiometric (electrode)Solid-state probeSoluble probeManufacturerOroboros O2KAgilent XF AnalyzerAgilent Mito-Xpress pH-XtraFormatSealed Chamber(s)Specialized MicroplateStandard (culture) microplateVariables measuredOCR, Membrane PotentialOCR, ECAROCR, ECARSuitable adherent cells?NoYesYesSuitable suspension cells/isolated mitochondria?YesNoYesSuitable permeabilized cells?YesYesYesMeasurement typeKineticKinetic (>4 Injections)Serial Open table tab major consideration buffer (or media) chemistry. media/buffer composition intact versus essentially opposite ionic sodium calcium/low potassium; permeabilized-high potassium/low calcium). Intact buffers designed lysed hypotonic nature (and vice versa). Both strength osmolarity should considered avoid effects kinetics energetics (40Bradshaw Pfeiffer D.R. Release Ca2+ Mg2+ yeast stimulated increased strength.BMC 2006; 1-12Crossref (110) 41Glancy Willis W.T. Chess D.J. Balaban R.S. Effect calcium cascade skeletal muscle mitochondria.Biochemistry. 52: 2793-2809Crossref (153) 42Golding E.M. Teague W.E. Dobson G.P. Adjustment K' varying pMg creatine kinase, adenylate kinase hydrolysis equilibria permitting quantitative assessment.J. Exp. 1995; 198: 1775-1782Crossref certain ions modulate own, could necessitate salts (inert) ions. alters isolated rates, circumvented tris-based To further emphasize tonicity osmolarity, consider many intermediates multivalent ions, contribute substantially parameters. 5 mmol/l addition citrate, imparts 20 mOsmol/l 30 buffer. citrate salt weak raise buffering capacity media, particularly unbuffered measurements. issue, reported buffer/assay descriptions course assay. controls included account relative changes, example: metabolically inert sugar alcohols mannitol) osmotic effects, counterions accordance preparation, acids pKa similar metabolite investigation effects. limited resolution, experiments compensate separating out components multiple reduce ambiguity 43Nicholls Ferguson Bioenergetics.4th Ed. Academic Press, London, UK2013Google 44Gnaiger Pathways Respiratory Control: An Introduction OXPHOS Analysis.5th Bioenergetics Communications, Innsbruck, Austria2020Google strategy known substrate-inhibitor titration (SUIT). strategy, while "input" (substrate) given pathway provided increasing concentration until saturating reached. obtain inhibitors steps titrated control exerted those set (45Kacser Burns J.A. Fell flux: 21 years on.Biochem. Soc. Trans. 23: 341-366Crossref implemented parallel more complete working picture 46Perry C.G.R. Kane Lanza I.R. Methods assessing diabetes.Diabetes. 62: 1041-1053Crossref (102) 47Brand Assessing dysfunction cells.Biochem. 435: 297-312Crossref (1433) Importantly, no single way SUIT protocol generalized few steps: define (metabolic model), experimentally measurable fluxes, candidate substrate/inhibitor combinations isolate components, 4) appropriate statistical comparisons interest. Notably, each must very carefully resulting articles books published serve excellent starting points mitochondria, cells/tissue, 48Wollenman L.C. Vander Ploeg Miller M.L. Bazil J.N. function.PLoS 12e0187523Crossref (10) 49Jaber S.M. Polster B.M. Mapping deficiencies respirometry: Beyond Mito Stress Test.Exp. Neurol. 328: 113282Crossref 50Makrecka-Kuka Krumschnabel High-resolution simultaneous peroxide cells, tissue homogenate mitochondria.Biomolecules. 5: 1319-1338Crossref Choice substrates catabolic flux) significantly influenced study. preparations, separated normal context. necessi

Language: Английский

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The Key Role of Mitochondrial Function in Health and Disease

Antioxidants, Journal Year: 2023, Volume and Issue: 12(4), P. 782 - 782

Published: March 23, 2023

The role of mitochondrial function in health and disease has become increasingly recognized, particularly the last two decades. Mitochondrial dysfunction as well disruptions cellular bioenergetics have been shown to be ubiquitous some most prevalent diseases our society, such type 2 diabetes, cardiovascular disease, metabolic syndrome, cancer, Alzheimer’s disease. However, etiology pathogenesis multiple yet elucidated, making it one significant medical challenges history. rapid advances knowledge metabolism coupled with novel understanding at molecular genetic levels show tremendous promise day elucidate mysteries this ancient organelle order treat therapeutically when needed. DNA mutations, infections, aging, a lack physical activity identified major players diseases. This review examines complexities function, whose incorporation into eukaryotic cells for energy purposes was key survival creation new species. Among these complexities, tightly intertwined derived from combustion alimentary substrates oxygen are necessary homeostasis, including production reactive discusses different etiological mechanisms by which mitochondria could dysregulated, determining fate tissues organs being protagonist many non–communicable Finally, is canonical evolutionary characteristic humans that remains embedded genes. normalization modern society led perception exercise an “intervention”. modus vivendi engrained genes sedentary real intervention collateral effect societies. It known leads and, hence, probably becomes factor affecting Since only stimulus we know can improve maintain emphasis on promotion should imperative prevent populations chronic where involved, individualized prescription crucial “metabolic rehabilitation” patients. From lessons learned elite athletes (the perfect human machines), possible translate apply concepts betterment

Language: Английский

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