Discussion

The aging population continues to grow at an unprecedented rate worldwide. Aging is associated with a   decline in cellular functions, damage accumulation,   and an increasing probability of chronic diseases that lead to systems collapse and eventual death [3–7].  The prevalence of age-related pathologies late in life has a significant impact on life quality and healthcare costs. Thus, there is an urgent need for interventions with an effective anti-aging activity that can be exploited as a geroprotector to delay aging and prolong healthspan.

Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a strong scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and has the ability to scavenge reactive oxygen species and prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.

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Cellular aging is controlled by a network of interconnected complex biological processes [8, 9].  Recent studies have shown that intervening in the aging biological processes can substantially increase the healthy lifespan of model organisms, including mammals [10, 14]. Chronological aging in budding yeast Saccharomyces cerevisiae is the well-established model system for determining the interventions of human post-mitotic cell aging [16–19]. 

We developed a new, fast, and cheap quantitative method called PICLS for measuring CLS using (1)   yeast cells incubated with media and test compounds on clear 96-well plates; (2) the fluorescent dye propidium iodide (PI), a cell impermeant that only enters dead cells [23–25]; and (3) a microplate reader for quantitative cell survival readout. To note, measuring cell density (and cell growth) at OD600nm can be performed from the same plate. For dead yeast cells on 96-well plates, we found a high linear correlation between PI fluorescence and OD600nm absorbance within an optimal cell density range corresponding to  0.05–12 ODs. This methodical approach can be utilized for quantifying cell viability in high-throughput assays.

There are several important innovations associated with PICLS: (i) PICLS is the currently only outgrowth-free method for CLS quantification. A critical time- and resource-demanding step is clipped from the protocol and, thus, PICLS is the first true high throughput screening (HTS) for chemical agents and culturing conditions for CLS measurement. (ii) The methodical idea behind PICLS can be generally formulated as a plate-based method where (a) the fraction of dead/surviving cells and (b) the total amount of cells is directly determined simultaneously from the same plate with an optical intensity measurement device (the commonly available plate reader). In this work, we used 96-well plates but 384-well plates appear also applicable (with possibly some lowered sensitivity due to the smaller amount of culture). Any coloring agent that distinguishes dead and surviving cells and that has fluorescence or absorption in a   channel that does not interfere with general cell density measurement (e.g., via absorption at OD600nm)   can be utilized. We applied propidium iodide that selectively marks dead cells but this is not critical for  PICLS.

Fig. 7 Testing the effect of 2,5-anhydro-D-mannitol analogs on the chronological lifespan of the yeast. The prototrophic yeast strain (CEN.PK113-7D) was grown in the synthetic defined medium with different concentrations of 2,5-anhydrous-Dmannitol (2,5-AM), D-fructose, D-mannitol, D-maltose, and  D-sorbitol in 96-well plates at 30 °C. A Cell growth OD600nm was measured at different time points 24  h, 48  h, and 72  h   using a microplate reader and graph plotted against different concentrations of 2,5-AM, fructose,  mannitol, maltose, and sorbitol. B The chronological lifespan (CLS) of different concentrations of 2,5-AM, fructose, mannitol, maltose, and sorbitol incubated cells was determined using the propidium iodide fluorescence–based method. Cell survival at different chronological age points was quantified and the growth time point  72 h was considered as day 1. C The CLS of the aged cells was determined by the outgrowth method in the YPD liquid medium.  The growth time point 72 h was considered as day 1. At various chronological age points, a 3-μL culture was transferred to a second 96-well plate containing 200μL YPD medium.  Outgrowth OD600nm in YPD liquid medium was measured after incubation for 24  h at 30  °C using a microplate reader.  The graph is plotted relative to day 1. D Outgrowth in YPD   liquid medium of a 96-well plate was photographed after incubation for 24 h at 30 °C. E At various chronological age points,  3-μL cultures were spotted on the YPD agar plate. Outgrowth was photographed after incubation for 48  h at 30  °C.  All data represent as means±SD.; *P<0.05, **P<0.01, and  ****P<0.0001 based on two-way ANOVA followed by Dunnett’s multiple comparisons tests (B and C). n.s, non-significant 

We note that black microplates are typically recommended for fluorescence assays because this material partially quenches autofluorescence. However, we evaluated the method in clear microplates and found similar effectiveness as with black microplates. As black microplates are associated with high costs, this is a significant advantage for large-scale high-throughput screening projects.

We validated our developed method with known anti-aging interventions such as rapamycin administration and calorie restriction [10–16] by reproducing their effect on the CLS of the yeast. These results suggest that our method is effective for identifying new anti-aging interventions. We have exploited our newly developed protocol for screening chemical agents to identify new anti-aging compounds (Fig. 8). We identified 2,5-anhydro-D-mannitol (2,5-AM) extending the CLS of the yeast. Based on our results, we suggest the usage of 2,5-AM individually or in combination with other anti-aging interventions.

2,5-AM is an analog of fructose, and both sugars can enter into the glycolytic pathway. Glycolysis is a   metabolic process in which glucose is first phosphorylated by hexokinase to form glucose-6-phosphate  (G6P). Phosphoglucoisomerase interconverts G6P to fructose-6-phosphate (F6P) that is further metabolized into different downstream glycolytic intermediates, including pyruvate that enters the mitochondrial  TCA cycle. Like glucose, fructose is also phosphorylated by hexokinase to form F6P. Phosphofructokinase converts F6P to fructose-1,6-bisphosphate (FBP),   which is further metabolized into different downstream glycolytic intermediates. 2,5-AM can also be phosphorylated by hexokinase to form 2,5-AM- 6-phosphate (2,5-AM6P) [39–41]. Furthermore, phosphofructokinase converts 2,5-AM6P to 2,5-AM- 1,6-bisphosphate (2,5-AMBP). However, 2,5-AMBP cannot be further metabolized into downstream glycolytic intermediates [39–41].

Since 2,5-AM is a fructose analog, we investigated whether fructose can also extend the lifespan of yeast.  However, we did not observe the anti-aging activity of fructose in extending the CLS of yeast. Mannitol and maltose can also enter into glycolysis and metabolize into downstream glycolytic intermediates.  We also examined the effect of mannitol and maltose on the lifespan of yeast. Fructose, mannitol, and maltose are also unable to extend the CLS of yeast.

As sorbitol, another yet non-metabolized sugar was previously reported to affect the CLS at very high concentrations (18% equivalent to 1 M) [20, 45],   we wished to clarify whether the anti-aging activity of 2,5-AM might be due to the increased osmolarity of the culture medium. Our experiments showed that sorbitol fails to increase the CLS of yeast at the lower concentrations that are effective in the case of 2,5- AM. Since sorbitol requires higher concentrations to increase the CLS of yeast, the anti-aging mechanism of 2,5-AM is independent of osmolarity effects.  These findings revealed that the anti-aging activity of  2,5-AM is specific and not paralleled by several other analogs with similar chemical structures.

However, how 2,5-AM extends the lifespan remains to be investigated. It will be interesting to examine whether 2,5-AM directly modulates the aging hallmarks [8]. TORC1 and AMPK are the glucose-sensing complexes involved in the aging process  [32, 43]. However, the chronological lifespan extended by the inhibition of TORC1 is reduced when AMPK is inhibited. Glucose and glycolytic intermediates regulate the activity of TORC1 and AMPK [34, 46, 47].  2,5-AMBP has been shown to inhibit the activity of aldolase that catalyzes the conversion of FBP into   glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) [39, 40]. DHAP is interconverted into G3P by triosephosphate isomerase. Recent evidence suggests that DHAP is the important glucose signal molecule that activates TORC1 [48]. 

Glycolytic fux is compromised when G3P and  DHAP synthesis is affected, leading to less ATP   production. Interestingly, AMPK is activated when the energy status of the cells is compromised and inhibits cell growth by inhibiting the TORC1 [46,  47]. Thus, AMPK’s and TORC1’s antagonistic relation is an efficient metabolic adaptation to ensure cellular homeostasis balance for healthy cells. Although,   we did not observe an effect of 2,5-AM on cell growth  (Figs. 6 and 7). These findings ruled out any glucose restriction effect that compromised the cell growth  (Fig. 5), however, extended the lifespan with a slow growth phenotype [49]. However, the extension of lifespan by rapamycin does not require a higher dose than compromises cell growth (Fig. 4). Therefore, we cannot exclude the possibility of anti-aging activity of 2,5-AM via TORC1/AMPK or independently of  TORC1/AMPK.

Unlike the inhibitory effect of 2,5-AMBP on aldolase, it is reported to activate the pyruvate kinase, the last glycolysis enzyme [39–41]. Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate to pyruvate, one of the sources for generating NAD+ (nicotinamide adenine dinucleotide). NAD+ is an important metabolite that regulates several cellular processes and functions critical for maintaining healthy cells and extending lifespan [50, 51]. Pyruvate is also the major substrate for mitochondrial reactions to generate various building block metabolites to synthesize vital elements, including amino acids, nucleic acids,   and ATP, essentially for cell survival and healthy aging [52–55]. Indeed, the decline in mitochondrial activity is associated with aging and age-related diseases [53–56]. Recently, 2,5-AM has been proposed as a potential therapeutic to treat acute myeloid leukemia (AML) cancer [57]. 

As a side note, we named our new method PICLS  (PI-based CLS method) with the wordplay “pickles” in mind. Whereas all elements of our new protocol have already been described independently elsewhere, their “fermented” combination makes the measurement simple and cheap, and finally high-throughput-ready.

In conclusion, we have developed a quantitative method for measuring the chronological lifespan of the yeast and identified 2,5-AM as a novel antiaging compound. A study is currently underway to investigate the mechanisms of anti-aging activity of 2,5-AM.

Materials and methods

Yeast strain, media, and chemicals

The prototrophic CEN.PK113-7D strain genetic background was used in this study [35, 36]. Standard-rich medium YPD (1% Bacto yeast extract, 2%  Bacto peptone, and 2% glucose), YPD agar (2.5%  Bacto agar), and synthetic defined (SD) medium containing 6.7  g/L yeast nitrogen base with ammonium sulfate without amino acids (DIFCO) and 2%   glucose. Rapamycin (Enzo) stock solution was prepared in dimethyl sulfoxide (Sigma). The final concentration of DMSO did not exceed 1% in any assay.  2,5-Anhydro-D-mannitol (Santa Cruz), D-fructose  (Sigma), D-mannitol (Sigma), D-maltose (Sigma),   and D-sorbitol (Sigma) working concentrations prepared fresh by directly dissolving the powder in medium and filter sterilized.

Yeast growth conditions

Yeast strain was recovered from frozen glycerol stock on YPD agar medium at 30  °C. Yeast was grown in SD medium overnight at 30 °C with shaking at 220  rpm. Cells grown overnight were diluted to OD600nm~0.2 in fresh SD medium to initiate the chronological lifespan experiments.

Chronological lifespan analysis

Chronological lifespan (CLS) experiments were performed in 96-well plates with a total of 200 µL yeast culture as described previously [58]. The cellular inoculum was transferred into the 96-well plate containing serially double-diluted concentrations  (0–10  nM) of rapamycin. For the caloric restriction assay, cellular inoculums were prepared in SD   medium containing 2%, 0.5%, and 0.25% glucose and transferred to 96-well plates. Likewise, the cellular inoculum was transferred into the 96-well plates containing serially double-diluted concentrations  (0–8  mM) of 2,5-anhydro-D-mannitol, D-fructose,  D-mannitol, D-maltose, and D-sorbitol. Cells were incubated at 30  °C, and the growth was measured at different time points. The growth time point 72 h   was considered as day 1 for the CLS assay. Cell survival was quantified at various age time points by three different approaches: (i) propidium iodide fluorescence–based method, (ii) outgrowth in YPD   liquid medium, and (iii) spotting assay.

(i) Propidium iodide fluorescence–based method: The detailed procedures for developing the propidium iodide (PI) fluorescence–based approach are mentioned in the result section. For the CLS  assay, 40-µl yeast cells on different age time points were transferred into a second 96-well plate. Cells were washed and incubated in 100 µl 1×PBS with  PI (5  µg/ml) for 15  min in the dark. Positive and negative samples control were included for quantitative analysis. Positive control (cells boiled at  100 °C for 15 min) was PI-stained and processed in the same 96-well plate. Samples without PI-stained cells served as the negative control. After incubation, cells were washed and resuspended in 100 µl  PBS. The sample's fluorescence reading (excitation at 535 nm, emission at 617 nm) and OD600nm were measured by the microplate reader (BioTek). The fluorescence intensity of each sample was normalized with OD600nm. The normalized fluorescence intensity of each sample was subtracted from the background signal of the unstained negative sample.  The obtained fluorescence intensity of the positive control sample (boiled dead cells) was considered  0% cell survival. We confirmed the cell's death b  allowing it to grow in the medium. Cell survival o different age time points samples was calculated b  normalizing the fluorescence intensity with a positive control sample (boiled cells).

where Iij(535nm∕617nm) is the fluorescent intensity measured at the well with plate coordinates i and j with excitation at 535 nm and emission at 617 nm,  ID(535nm∕617nm) is the fluorescent intensity measured for a cell with 100% dead cells stained with PI,  IC(535nm∕617nm) is the fluorescent intensity measured for cells without PI, and the respective OD values are the absorption measured for the respective cells at 600 nm normalizing for the number of cells.

(ii) Outgrowth in YPD liquid medium: Yeast stationary culture (3-μL) of different age time points was transferred to a second 96-well plate containing  200μL YPD medium and incubated for 24 h at 30 °C.  Outgrowth (OD600nm) of aged cells was measured by the microplate reader. Quantification of cell survival for each age point was determined relative to day 1 (considered 100% cell survival) as described previously [19, 20, 58]. 

(iii) Spotting assay: Yeast stationary culture (3  μL) of different age time points was spotted onto the YPD agar plate and incubated for 48 ho at 30 °C.  The outgrowth of aged cells on the YPD agar plate was photographed using the BioRad GelDoc imaging system.

Data analysis

Statistical analysis of all the results such as mean value, standard deviations, correlation (R2), significance, and graphing was performed using GraphPad  Prism v.9.3.1 software. The results were statistically compared using the ordinary one-way ANOVA and two-way ANOVA followed by multiples comparison by Dunnett’s post hoc test or Sidak’s post hoc test. In all the graph plots, P values are shown as *P<0.05,  **P<0.01, ***P<0.001, and ****P<0.0001 were considered significant. n.s, non-significant.

Acknowledgments The author's MA and FE are the inventors of two patent applications (10202201534P and 10202201536U) that includes part of the work described in this paper.

Author contribution MA conceived the project, performed the experiments, and analyzed the data. FE co-conceived the project, evaluated data, and devised a strategy. MA and FE wrote the paper, and all authors reviewed the paper and agreed to its final version.

Funding This work was supported by Bioinformatics Institute (BII), A*STAR Career Development Fund (C210112008),   and the Global Healthy Longevity Catalyst Awards grant  (MOH-000758–00).

Declarations

Competing interests The authors declare no competing interests.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution, and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

References

1. das nações Unidas, O. United Nations, Department of Economic and Social Afairs, Population Division (2019). World Population Prospects,  Highlights (ST/ESA/ SER.A/423). 2019. 

2. Robine J. Ageing populations: we are living longer lives, but are we healthier? United Nations, Department of Economics and Social Affairs, Population Division, UN DESA/ POP/2021/TP/NO. 2. (2021). 

3. Niccoli T, Partridge L. Ageing as a risk factor for disease. Curr Biol. 2012.

4. Hou Y, et al. Aging as a risk factor for neurodegenerative disease. Nat Rev Neurol. 2019. 

5. Harman D. The aging process: Major risk factor for disease and death. Proc Natl Acad Sci U S A. 1991. 

6. Partridge L, Deelen J, Slagboom PE. Facing up to the global challenges of aging. Nature. 2018. 

7. Fontana L, Kennedy BK, Longo VD. Medical research: treat aging. Nature. 2014. 

8. López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell. 2013. 

9. Kennedy BK, et al. Geroscience: Linking aging to chronic disease. Cell. 2014. 

10. Partridge L, Fuentealba M, Kennedy BK. The quest to slow aging through drug discovery. Nat Rev Drug Discovery. 2020.

11. Mattison JA, et al. Caloric restriction improves the health and survival of rhesus monkeys. Nat Commun. 

12. Bitto A, et al. Transient rapamycin treatment can increase lifespan and health span in middle-aged mice. Elife. 2016. 

13. Fontana L, Partridge L. Promoting health and longevity through diet: from model organisms to humans. Cell. 2015. 

14. Zhang Y, Zhang J, Wang S. The role of rapamycin in healthspan extension via the delay of organ aging. Ageing Res Rev. 2021. 

15. Selvarani R, Mohammed S, Richardson A. Effect of rapamycin on aging and age-related diseases—past and future. GeroScience. 2021. 

16. Fontana L, Partridge L, Longo VD. Extending healthy life span from yeast to humans. Science. 2010. 

17. Zimmermann A, et al. Yeast as a tool to identify anti-aging compounds. FEMS Yeast Res. 2018. 

18. Longo VD, Shadel GS, Kaeberlein M, Kennedy B. Replicative and chronological aging in Saccharomyces cerevisiae. Cell Metab. 2012. 

19. Powers RW, Kaeberlein M, Caldwell SD, Kennedy BK, Fields S. Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev. 2006. 

20. Murakami CJ, Burtner CR, Kennedy BK, Kaeberlein M. A method for high-throughput quantitative analysis of yeast chronological life span. J Gerontol. Ser. A Biol. Sci. Med. Sci. (2008).

21. Teng X, Hardwick JM. Quantification of genetically controlled cell death in budding yeast. Methods Mol Biol. 2013.

22. Garay E, et al. High-resolution profiling of stationary-phase survival reveals yeast longevity factors and their genetic interactions. PLoS Genet. 2014. 

23. Alfatah M, et al. Hypoculoside, a sphingoid base-like compound from Acremonium disrupts the membrane integrity of yeast cells. Sci Rep. 2019. 

24. Alfatah M, et al. Chemical-genetic interaction landscape of mono-(2-ethylhexyl)-phthalate using chemogenomic profiling in yeast. Chemosphere. 2019. 

25. Kwolek-Mirek M, Zadrag-Tecza R. Comparison of methods used for assessing the viability and vitality of yeast cells. FEMS Yeast Res. 2014.

26. Chadwick SR, et al. A Toolbox for rapid quantitative assessment of chronological lifespan and survival in Saccharomyces cerevisiae. Trafc. 2016. 

27. Pereira C, Saraiva L. Interference of aging media on the assessment of yeast chronological life span by propidium iodide staining. Folia Microbiol. (Praha). (2013). 

28. Lamming DW, Ye L, Sabatini DM, Baur JA. Rapalogs and mTOR inhibitors as anti-aging therapeutics. J Clin Investig. 2013. 

29. Arriola Apelo SI, Lamming DW. Rapamycin: an InhibiTOR of aging emerges from the soil of Easter Island. Journals of Gerontology - Series A Biological Sciences and Medical Sciences. 2016.

30. Wilkinson JE, et al. Rapamycin slows aging in mice. Aging Cell. 2012. 

31. Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. Cell. 2017;168:960–76. 

32. Johnson SC, Rabinovitch PS, Kaeberlein M. MTOR is a key modulator of aging and age-related disease. Nature. 2013. 

33. González A, Hall MN. Nutrient sensing and TOR signaling in yeast and mammals. EMBO J. 2017;36:397–408. 

34. Alfatah M, et al. TORC1 regulates the transcriptional response to glucose and the developmental cycle via the Tap42- Sit4-Rrd1/2 pathway in Saccharomyces cerevisiae. BMC Biol. 2021. 

35. Van Dijken JP, et al. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb Technol. 2000. 

36. Mülleder M, et al. A prototrophic deletion mutant collection for yeast metabolomics and systems biology. Nat Biotechnol. 2012. 

37. Alvers AL, et al. Autophagy is required for the extension of yeast's chronological life span by rapamycin. Autophagy. 2009. 

38. Zhang JH, Chung TDY, Oldenburg KR. A simple statistical parameter for use in the evaluation and validation of high throughput screening assays. J Biomol Screen. 1999. 

39. Riquelme PT, Wernette Hammond ME, Kneer NM, Lardy HA. Regulation of carbohydrate metabolism by 2,5-anhydro-D-mannitol. Proc Natl Acad Sci U S A. 1983. 

40. Riquelme PT, Wernette-Hammond ME, Kneer NM, Lardy HA. Mechanism of action of 2,5-anhydro-D-mannitol in hepatocytes. Effects of phosphorylated metabolites on enzymes of carbohydrate metabolism. J. Biol. Chem. (1984). 

41. Riquelme PT, Kneer NM, Wernette-Hammond ME, Lardy HA. Inhibition by 2,5-anhydromannitol of glycolysis in isolated rat hepatocytes and Ehrlich ascites cells. Proc Natl Acad Sci U S A. 

42. Hardie DG. AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nat Rev Mol Cell Biol. 2007. 

43. Burkewitz K, Zhang Y, Mair WB. AMPK at the nexus of energetics and aging. Cell Metab. 2014.

44. McCartney RR, Chandrashekarappa DG, Zhang BB, Schmidt MC. Genetic analysis of resistance and sensitivity to 2-deoxyglucose in Saccharomyces cerevisiae. Genetics. 2014. 

45. Burtner CR, Murakami CJ, Kennedy BK, Kaeberlein M. A molecular mechanism of chronological aging in yeast. Cell Cycle. 2009. 

46. Lin SC, Hardie DG. AMPK: sensing glucose as well as cellular energy status. Cell Metab. 2018. 

47. González A, Hall MN, Lin SC, Hardie DG. AMPK and TOR: the yin and yang of cellular nutrient sensing and growth control. Cell Metab. 2020. 

48. Orozco JM, et al. Dihydroxyacetone phosphate signals glucose availability to mTORC1. Nat Metab. 2020. 

49. Aguiar-Oliveira MH, Bartke A. Growth hormone deficiency: health and longevity. Endocr Rev. 2019. 

50. Orlandi I, Alberghina L, Vai M. Nicotinamide, nicotinamide riboside, and nicotinic acid—emerging roles in replicative and chronological aging in yeast. Biomolecules. 2020. 

51. Covarrubias AJ, Perrone R, Grozio A, Verdin E. NAD+ metabolism and its roles in cellular processes during aging. Nat Rev Mol Cell Biol. 2021. 

52. Spinelli JB, Haigis MC. The multifaceted contributions of mitochondria to cellular metabolism. Nat Cell Biol. 2018. 

53. Berry BJ, Kaeberlein M. An energetics perspective on geroscience: mitochondrial protonmotive force and aging. Gero-Science. 2021. 

54. Sun N, Youle RJ, Finkel T. The mitochondrial basis of aging. Mol Cell. 2016. 

55. Kauppila TES, Kauppila JHK, Larsson NG. Mammalian mitochondria and aging: an update. Cell Metab. 2017. 

56. Bratic A, Larsson NG. The role of mitochondria in aging. J Clin Investig. 2013. 

57. Chen WL, et al. Enhanced fructose utilization mediated by SLC2A5 is a unique metabolic feature of acute myeloid leukemia with therapeutic potential. Cancer Cell. 2016. 

58. Jing JL, Ning TCY, Natali F, Eisenhaber F, Alfatah M. Iron supplementation delays aging and extends cellular lifespan through potentiation of mitochondrial function. Cells 11, (2022).

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