The study by Skirmantas Kriaucionis and Nathaniel Heintz investigates the presence of 5-hydroxymethylcytosine (hmC) in Purkinje neurons and the brain. Using thin layer chromatography (TLC), high-pressure liquid chromatography (HPLC), and mass spectrometry (MS), they identified an unusual DNA nucleotide, 5-hydroxymethyl-2'-deoxycytidine (hmdC), in Purkinje cell nuclei. hmdC comprises 0.6% of total nucleotides in Purkinje cells and 0.2% in granule cells, but is not present in cancer cell lines. The presence of hmdC is enriched in the brain, suggesting a role in epigenetic control of neuronal function. The authors used transgenic mice to obtain pure preparations of Purkinje and granule cell nuclei, and observed a significantly lower amount of 5-methylcytosine (mC) in Purkinje DNA. They confirmed the identity of the unidentified spot "x" on TLC plates as hmdC through HPLC and MS analysis. The distribution of hmdC is enriched in the brain, particularly in the cortex and brainstem, and is not correlated with the age of adult mice. The findings suggest that hmC may have an important biological role in neuronal function, potentially as an intermediate in oxidative demethylation or a stable end-product that modulates protein affinity for mC signals.The study by Skirmantas Kriaucionis and Nathaniel Heintz investigates the presence of 5-hydroxymethylcytosine (hmC) in Purkinje neurons and the brain. Using thin layer chromatography (TLC), high-pressure liquid chromatography (HPLC), and mass spectrometry (MS), they identified an unusual DNA nucleotide, 5-hydroxymethyl-2'-deoxycytidine (hmdC), in Purkinje cell nuclei. hmdC comprises 0.6% of total nucleotides in Purkinje cells and 0.2% in granule cells, but is not present in cancer cell lines. The presence of hmdC is enriched in the brain, suggesting a role in epigenetic control of neuronal function. The authors used transgenic mice to obtain pure preparations of Purkinje and granule cell nuclei, and observed a significantly lower amount of 5-methylcytosine (mC) in Purkinje DNA. They confirmed the identity of the unidentified spot "x" on TLC plates as hmdC through HPLC and MS analysis. The distribution of hmdC is enriched in the brain, particularly in the cortex and brainstem, and is not correlated with the age of adult mice. The findings suggest that hmC may have an important biological role in neuronal function, potentially as an intermediate in oxidative demethylation or a stable end-product that modulates protein affinity for mC signals.