Volatiles found in microbes can inhibit HDACs in vitro

(A) Antennal and whole mount brain staining of Gr63a-Gal4 flies. Flies were exposed to diacetyl (headspace from 10-2 soln) or air for 2 to 5 days. Brains and antenna were dissected on the indicated days, fixed, and then stained for neuropil marker nc82 (red) and anti-GFP (green). (B) Mean of ab1C neurons expressing GFP after indicated days of odor exposure. d4on=2,3-butanedione. n=6, error bars=s.e.m. Schematic chemical structures of diacetyl, β-hydroxybutyrate and Sodium butyrate. (C) Dose-activity curves of class I HDACs: HDAC1, HDAC2, HDAC3, HDAC8 and class II HDAC6 treated with various concentrations of diacetyl. Percentage of HDAC activity is relative to the activity of each enzyme without diacetyl. IC50s are indicated in the chart areas. Error bars, S.E.M., n = 4-5. (E) Representative structures of odorants that inhibit HDACs, and (F) Average percentage inhibition of class I HDACs: HDAC1, HDAC3 and class II HDAC4, HDAC6 treated with 15mM of indicated volatiles. Error bar= S.D., each tested in duplicate.

Diacetyl increases level of histone acetylation in HEK293 cells

(A and B) Representative images from Western blots showing acetylation levels of H3K9 (left), H3K14 (middle) and H4K5 (right) in HEK293 cells after 2 hours (A) and 6 hours (B) of diacetyl exposure. PCNA (Proliferating cell nuclear antigen) is a 29 kDa nuclear protein used as a loading control for nuclear protein extracts. (C) Western blots showing acetylation levels of H3K9 in HEK293 cells treated with 100 μM diacetyl for 72, 96, and120 hours. PCNA is used for a loading control. (D) Bar graph showing the relative intensities of acetylated H3K9 in HEK293 cells treated with 100 μM diacetyl for 72 -120 hours. n = 4 samples, * p < 0.05, and **** p < 0.0001.

Remote exposure to HDACi volatile alters gene expression in an invertebrate, and vertebrate.

(A) Schematic of odor exposure protocol for transcriptome analysis from the antennae. (B) Plot highlighting up- and down-regulated genes in the diacetyl-exposed group. Red and blue dots represent up-regulated genes (false discovery rate (FDR) < 0.05, log2 fold change (LFC) > 1) and down-regulated genes (FDR < 0.05, LFC < 1), respectively. (C) Bar graphs denoting the protein classification of the genes up- and down-regulated after odor exposure. (D) Schematic of diacetyl exposure protocol for transcriptome analysis of mouse lung tissue. (E) Plot highlighting up- and down-regulated genes in the diacetyl-exposed groups. Red and blue dots represent up-regulated genes (FDR < 0.05, LFC > 1) and down-regulated genes (FDR < 0.05, LFC < 1), respectively in lungs. (F) Table showing pairwise tests of significance of overlap between gene sets. P-values from Fisher’s exact test, colored with associated odds ratio (strength of association). (G and H) Bar graphs denoting the protein classification of the genes up- and down-regulated in the lung after exposure to headspace above 0.1% (v/v) (G) and 1% (v/v) (H) of diacetyl solutions.

Gene expression changes are partly reversible, and overlaps with known HDACi drugs

(A) Schematic of odor exposure and recovery protocol for transcriptome analysis from the antennae. (B) Plot highlighting up- and down-regulated genes in the recovery from diacetyl exposure group. Red and blue dots represent up-regulated genes (FDR < 0.05, LFC > 1) and down-regulated genes (FDR < 0.05, LFC < 1), respectively. (C) Table showing pairwise tests of significance of overlap between gene sets. P-values from Fisher’s exact test, colored with associated odds ratio (strength of association). (D and E) Plots showing enrichment of up- and down-regulated genes in sodium butyrate-(D) and valproic acid-treated (E) groups. Red and blue dots represent up-regulated genes (FDR < 0.05, LFC > 1) and down-regulated genes (FDR < 0.05, LFC < 1), respectively. (F) Left: Venn diagrams showing the overlaps of up-regulated genes among diacetyl-, sodium butyrate- and valproic acid-treated groups. Right: Table showing pairwise tests of significance of overlap between gene sets. P-values from Fisher’s exact test. (G and H) GO-enrichment analysis of the common Up-regulated and down-regulated genes across all 3 treatments in (F).

Exposure to diacetyl vapor alters gene expression in the mouse brain

(A) Schematic of diacetyl exposure protocol for transcriptome analysis of mouse brain tissues. (B and C) Plot showing up- and down-regulated genes in the diacetyl-exposed groups. Red and blue dots represent up-regulated genes (FDR < 0.05, LFC > 1) and down-regulated genes (FDR < 0.05, LFC < 1), respectively in the brain. (D) Left: Table showing pairwise tests of significance of overlap between gene sets. P-values from Fisher’s exact test. Right: Venn diagrams showing the overlaps of DEGs between lung and brain groups. (E) Bar graphs denoting the protein classification of the brain genes up- and down-regulated after 1% diacetyl exposure. (F) Mean fold change of select neuroblastoma related genes in the brain of mice exposed to diacetyl vapors. (G) Cell counts of indicated cancer cell lines in tissue culture treated with solvent control or indicated concentration of diacetyl. N=3-6, p<0.001.

Odor exposure slows Huntington’s neurodegeneration model in fly eye

(A) Schematic diagram showing temperature of experimental condition and timing of the eye examination in pGMR-HTTQ120 flies. (B) Bar graph showing mean number of rhabdomeres in each ommatidium in solvent paraffin oil (PO, blue) and diacetyl-exposed (red) pGMR-HTTQ120 flies at 1, 5, and 10 days after eclosion (AE). n = 600 ommatidia from 15 flies, **** p < 0.0001. (C) A representative image of ommatidia of pGMR-HTTQ120 flies at 1 day AE. (D and E) Representative images of ommatidia of pGMR-HTTQ120 flies exposed to paraffin oil (PO) or diacetyl at 5 (D) and 10 (E) days AE. (F and G) Histogram showing the percent of the ommatidium with a given number of rhabdomeres indicated on the x axis at 5 (F) and 10 (G) days AE.