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Studies measuring the acetoacetate/beta-hydroxybutyrate in different brain regions with aging and DR are needed to determine the extent to which DR leads to a reduction of the brain mitochondrial [NAD + ]/[NADH], possibly driving neuroprotection and slowing the rate of brain aging

Properly formulated serums (pH 5.56.5) show no cytotoxicity

19 The primers used for RT-PCR is listed below: GAPDH F: 5-TGGCCTTCCGTGTTCCTAC-3 GAPDH R: 5-GAGTTGCTGTTGAAGTCGCA-3 IL1A F: 5-CGAAGACTACAGTTCTGCCATT-3 IL1A R: 5-GACGTTTCAGAGGTTCTCAGAG-3 IL1B F: 5-GCAACTGTTCCTGAACTCAACT-3 IL1B R: 5-ATCTTTTGGGGTCCGTCAACT-3 IL6 F: 5-TAGTCCTTCCTACCCCAATTTCC-3 IL6 R: 5-TTGGTCCTTAGCCACTCCTTC-3 MMP3 F: 5-ACATGGAGACTTTGTCCCTTTTG-3 MMP3 R: 5-TTGGCTGAGTGGTAGAGTCCC-3 MMP9 F: 5-CTGGACAGCCAGACACTAAAG-3 MMP9 R: 5-CTCGCGGCAAGTCTTCAGAG-3 CCL2 F: 5-TTAAAAACCTGGATCGGAACCAA-3 CCL2 R: 5-GCATTAGCTTCAGATTTACGGGT-3 CCL4 F: 5-TTCCTGCTGTTTCTCTTACACCT-3 CCL4 R: 5-CTGTCTGCCTCTTTTGGTCAG-3 BCL2 F: 5-GTCGCTACCGTCGTGACTTC-3 BCL2 R: 5-CAGACATGCACCTACCCAGC-3 BCL2A1A F: 5-GGCTGAGCACTACCTTCAGTA-3 BCL2A1A R: 5-TGGCGGTATCTATGGATTCCAC-3 CCND2 F: 5-GAGTGGGAACTGGTAGTGTTG-3 CCND2 R: 5-CGCACAGAGCGATGAAGGT-3 RNA-Sequencing (RNA-Seq) library preparation and data analysis For RNA-Seq library preparation, total RNA was first extracted using Trizol, as previously described, 21, 22 from control and ESCC tissues ( n =3 each)

The analysis was done after the fact rather than as a primary endpoint, and the clocks were measured in blood, so they track systemic aging rather than the brain in particular

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