Journal: Nature Metabolism

Article Title: NADH inhibition of SIRT1 links energy state to transcription during time-restricted feeding

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: a , ( left ) Representative wheel-running behaviour and ( middle ) quantification of average daily onset of activity for TRF-Reg (n = 5) and TRF-CR (n = 5) mice throughout the duration of the 4-wk intervention (double plotted for clarity). ( right ) Background-corrected PER2::LUCIFERASE readings from excised suprachiasmatic nucleus (SCN) of TRF-Reg (n = 6) and TRF-CR (n = 6) mice. The grey shading indicates mean values ± SEM. Experiments were performed in 4–6 mo old male C57BL6/J mice. b , Body weight of TRF-Reg (n = 15) and TRF-CR (n = 15) mice over the duration of the 4-wk study. c , Blood glucose and serum insulin during oral glucose tolerance testing performed during the daytime (at ZT4) in TRF-Reg (n = 6) and TRF-CR (n = 6) mice. d , GAP/DHAP mass isotopomer distribution determined by mass spectrometry of liver from TRF-Reg (n = 3) and TRF-CR (n = 2) mice 30 min following i.p. administration of a 10:1 mix of U 13 C-lactate:U 13 C-pyruvate (1 g/kg). e-f , ( left ) Relative concentration of (e) NADH and (f) NAD + by HPLC in liver of TRF-CR compared to TRF-Reg mice during the day (ZT4) (n = 12 for TRF-Reg, n = 11 for TRF-CR,) and at night (ZT16) (n = 9 for each diet) for each time point. ( right ) Concentration of (e) NADH and (f) NAD + in ad lib fed wild-type mouse liver by HPLC every 6 hrs for 24 hrs (n = 7). g-h , Log 2 -FC in daytime liver acyl-carnitine levels in (g) TRF-CR compared to TRF-Reg mice and (h) Lb NOX- compared to null-overexpressing TRF-CR mice (n = 3). Data are presented as mean values ± SEM. Statistics were performed with unpaired, two-tailed student’s t test except as otherwise noted in the figure. *p < 0.5, ***p < 0.01.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: Activity Assay, Luciferase, Mass Spectrometry, Concentration Assay, Two Tailed Test

Journal: Nature Metabolism

Article Title: NADH inhibition of SIRT1 links energy state to transcription during time-restricted feeding

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: a , Model for the TRF-CR diet using an automated feeder system in 4–6-month-old male C57BL/6J mice. Control mice received a 300 mg pellet of regular chow every 1.2 h throughout the dark period (TRF-Reg), whereas TRF-CR mice received a 300 mg pellet of a carbohydrate-depleted, nutrient-controlled chow every 2 h throughout the dark period, resulting in a 40% reduction in calories. b , Relative concentration of NADH and NAD + by HPLC in liver of TRF-Reg ( n = 12) and TRF-CR ( n = 11) mice during the daytime (ZT4). c , log 2 (fold change) in daytime serum acyl-carnitine levels during TRF-CR compared with TRF-Reg mice ( n = 3). d , Body temperature rhythms monitored non-invasively using subcutaneous probes in TRF-Reg ( n = 12) and TRF-CR ( n = 9) mice over 24 h (double plotted for clarity). e , Model depicting NADH-consuming reaction of Lb NOX. Representative tissue-specific expression profile of cytonuclear Lb NOX in null- and Lb NOX-transduced mice relative to Lb NOX-transduced liver. f , Relative concentration of NADH and NAD + in liver of null- and Lb NOX-transduced TRF-Reg ( n = 6) or TRF-CR ( n = 5 for NADH, n = 6 for NAD + ) mice during the daytime. g , log 2 (fold change) in daytime serum acyl-carnitine levels in TRF-CR mice transduced with cytonuclear Lb NOX compared with TRF-CR mice transduced with null virus ( n = 3). h , Body temperature rhythms in Lb NOX-expressing TRF-Reg ( n = 12) or TRF-CR ( n = 9) mice over 24 h (double plotted for clarity). Data are presented as mean ± s.e.m. Statistics were performed with unpaired, two-tailed Student’s t test unless otherwise noted in the figure. * P < 0.05, *** P < 0.001. ANOVA, analysis of variance; Cereb, cerebellum; DC, dicarboxylate; Gastroc, gastrocnemius muscle; gWAT, gonadal white adipose tissue; Hypoth, hypothalamus; i, isomer; iWAT, inguinal white adipose tissue; OH, hydroxy; Quad, quadriceps muscle.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: Control, Concentration Assay, Expressing, Transduction, Virus, Two Tailed Test

Journal: Nature Metabolism

Article Title: NADH inhibition of SIRT1 links energy state to transcription during time-restricted feeding

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: a , RNA-Seq in liver in the morning (ZT4) demonstrating the effect of TRF-CR in null-transduced (left) ( n = 6) and Lb NOX-transduced (middle) ( n = 6 for TRF-Reg, n = 5 for TRF-CR) 4–6-month-old male C57BL/6J mice for genes differentially expressed (DESeq2 FDR-adjusted P < 0.05) by TRF-CR in null-transduced mice (930 genes). Venn diagram (right) displays overlap in differentially expressed genes by TRF-CR in null- and Lb NOX-transduced mice. b , Quadrant plot comparing transcriptional responses between TRF-CR ( x axis) and Lb NOX in TRF-CR ( y axis). Each point indicates a gene that is differentially expressed by TRF-CR in null-transduced mice (930 genes). Colouring indicates genes within quadrant 2 (green) and quadrant 4 (red), and the percentages within each quadrant are shown. c , d , For the genes within quadrants 2 and 4 from b , the top 15 ( c ) Kyoto Encyclopedia of Genes and Genomes (KEGG) terms enriched ( P < 0.05) following gene ontology analysis and ( d ) JASPAR motifs enriched ( P < 0.05) following HOMER DNA motif enrichment analysis are shown. e , Quadrant plots comparing the transcriptional response to TRF-CR in null-transduced mice ( x axis) with that of genetic ablation of either Pparα (top) or Bmal1 (bottom) ( y axis ) in animals fed ad libitum. Each point indicates a gene that is differentially expressed by TRF-CR in null-transduced animals. Genes that have an absolute log (fold change) > 0.5 for both comparisons are coloured blue or black, and the percentage of genes was determined by quadrant ( n = 3). f , LC–MS metabolomics profiling of amino acids in liver during the daytime (ZT4). The log (fold change) from TRF-CR (blue) ( n = 5) and Lb NOX in TRF-CR (white) ( n = 6) is shown for select differential amino acids (two-tailed, unpaired Student’s t test with Benjamini and Hochberg adjustment for multiple measures FDR P < 0.05; see Supplementary Table for full list of amino acids). Box and whisker plots depict the following: line, median; box limits, first and third quartiles; whiskers, 10th and 90th percentiles. g , Model depicting the interrelationship of NADH during TRF-CR to the activity of PPARα and BMAL1 and the transcription of downstream oxidative gene networks.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: RNA Sequencing, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Whisker Assay, Activity Assay

Journal: Nature Metabolism

Article Title: NADH inhibition of SIRT1 links energy state to transcription during time-restricted feeding

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: a , ( left ) RNA-seq reads per 10 million sequenced reads (RPM) that align to Lb NOX in Lb NOX- vs null-transduced TRF-Reg (n = 6 per genotype) and TRF-CR (n = 6 for null; n = 5 for Lb NOX) liver. ( right ) Representative in situ immunohistochemistry against FLAG- Lb NOX in liver of null- and Lb NOX-transduced mice. Experiments were performed in 4–6 mo old male C57BL6/J mice unless otherwise noted. b , Heatmap depicting log 2 -FC in gene expression from TRF-CR and Lb NOX in TRF-CR for genes within gluconeogenic and glycolytic gene ontology groups. c , Unbiased principal components analysis during the day (ZT4) or night (ZT16) of null- and Lb NOX-expressing TRF-Reg or TRF-CR mice for genes that are differentially-expressed by TRF-CR in null mice at respective time points (DESeq2 p-adj<0.05) (n as in panel a). d , Log 2 -transformed FC in expression of select fatty acid metabolism genes during TRF-CR (blue) (n = 6), following Lb NOX-overexpression during TRF-CR (white) (n = 5), and in Bmal1 knockout liver (purple) (n = 3). e , 24-hour fasted body temperatures in 4–6 mo old female liver-specific Bmal1 fx/fx mice before and after retro-orbital administration of AAV8–TBG-iCre (n = 6) ( left : ANOVA, p < 0.01; right: *p < 0.05 in paired, two-way student’s t test). f , Log 2 -transformed FC in expression of genes within the methionine pathway for conditions (n as in panel d). g , ( top ) Summary of methionine pathway and ( bottom ) metabolomics of effect of TRF-CR and Lb NOX in TRF-CR on end-products of methionine metabolism in liver and serum performed during the daytime (ZT4) (n = 3). Data are presented as mean values ± SEM.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: RNA Sequencing, In Situ, Immunohistochemistry, Gene Expression, Expressing, Transformation Assay, Over Expression, Knock-Out

Journal: Nature Metabolism

Article Title: NADH inhibition of SIRT1 links energy state to transcription during time-restricted feeding

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: a , Lineweaver-Burk transformation of data from SAMDI-MS. b , Relative NADH quantified by HPLC following supplementation with pyruvate or lactate compared to controls (n = 3). c , Densitometric quantification of western blots from liver of 4–6 mo old male mice in indicated genotypes at ZT4 for p53-(K379)Ac ( left ) and FOXO1-(K242, K245, K262)Ac ( right ) relative to control TRF-Reg (from Fig. ) (n = 3). d , FOXO1 ChIP-seq from liver collected during the daytime comparing effect of FOXO1 binding in TRF-CR (x-axis) with the effect of hepatic SIRT1 ablation ( L-Sirt1 − / − ) (y-axis). Each point indicates a FOXO1 peak in control liver. Peaks that have absolute log 2 (fold change) >0.5 for both comparisons are colored blue or black and counted by quadrant (n = 2). e , Densitometric quantification of western blots as in (c) relative to null-transduced TRF-Reg mice (from Fig. ) (Ac-p53: n = 4 for TRF-CR, Lb NOX. n = 6 for all other conditions; Ac-FOXO1: n = 2 for TRF-CR, Lb NOX. n = 3 for all other conditions). f , FOXO1 ChIP-seq from liver during the daytime comparing effect of TRF-CR (x-axis) (n = 3) with effect of (f) Lb NOX in TRF-CR mice (y-axis) (n = 2) with coloring and counting as in panel d. (n = 2–3). g , Western blotting for Ac-FOXO1 during the nighttime (ZT16) in null- and Lb NOX-transduced mice on TRF-Reg and TRF-CR (n.s. non-specific) and densitometric quantification (n = 3). h , BMAL1 ChIP-seq from liver during the daytime comparing the effect of TRF-CR and L-Sirt1 − / − as in panel d (n = 3). Uncropped Western blot scans labelled with molecular weight markers are presented in the Source Data Files. i , RNA-seq reads mapping to the exon of Sirt1 that is flanked by LoxP sites (Ex4) relative to exon 9 (Ex9) of Sirt1 , and RNA-seq reads per 10 million sequenced reads (RPM) that align to iCre and Lb NOX in null- or Lb NOX-transduced TRF-CR mice co-transduced with iCre (n = 6). Null-expressing mice on TRF-CR (blue) (n = 5) are shown as reference. j , Quadrant plot comparing transcriptional responses to TRF-CR in null-transduced mice (x-axis) (n = 6) and Lb NOX-expression in TRF-CR, L-Sirt1 − / − mice (y-axis) (n = 6). Each point indicates a gene that is DE by TRF-CR in null-transduced mice (930 genes). Percentages of genes within each quadrant are shown. k , Average 48-hour fasting body temperature in 4–6 mo old female liver-specific Sirt1 − / − mice (n = 5). Data are presented as mean values ± SEM. Statistics were performed with unpaired, two-tailed student’s t test except as otherwise noted in the figure. *p < 0.05.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: Transformation Assay, Western Blot, Control, ChIP-sequencing, Binding Assay, Molecular Weight, RNA Sequencing, Transduction, Expressing, Two Tailed Test

Journal: Nature Metabolism

Article Title: NADH inhibition of SIRT1 links energy state to transcription during time-restricted feeding

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: a , Model to examine the role of SIRT1 in the NADH-dependent effects on RNAs, metabolites and body temperature during TRF-CR. b , Deacetylation rate for SIRT1 with increasing concentrations of NAD + and NADH assayed by SAMDI-MS ( n = 4). c , Model of pyruvate/lactate equilibrium shows that supplementation with pyruvate and lactate reduces and elevates NADH, respectively. Western blotting for SIRT1 targets, Ac-H3K9 and Ac-H4K16, in immortalized mouse embryonic fibroblasts treated with pyruvate or lactate to modulate NADH ( n = 3). d , e , Western blotting for SIRT1 targets, Ac-p53 and Ac-FOXO1, in TRF-Reg or TRF-CR liver of 4–6-month-old male ( d ) control or liver-specific Sirt1 − / − mice or ( e ) null- or Lb NOX-transduced mice. n.s., non-specific. Uncropped western blot scans labelled with molecular weight markers are presented in the Source Data files. f , BMAL1 ChIP-Seq in liver of TRF-Reg or TRF-CR mice. Peaks demonstrating an absolute log 2 (fold change) > 0.5 are coloured black. Box–whisker plots of BMAL1 ChIP-Seq demonstrating the effect of TRF-CR in null- ( n = 6) and Lb NOX-transduced ( n = 3) liver on BMAL1 peaks identified in controls and with an absolute log 2 (fold change) > 0.5 in controls. Box and whisker plots depict the following: line, median; box limits, first and third quartiles; whiskers, 10th and 90th percentiles. g , Heatmap depicting log 2 (fold change) in gene expression (left), liver metabolite concentrations (middle) and serum acyl-carnitine levels (right) in indicated conditions/genotypes at ZT4. Heatmaps are subdivided into the genes, metabolites and acyl-carnitines that are regulated by Lb NOX during TRF-CR through mechanisms requiring SIRT1 and sorted by effect of TRF-CR (RNA-Seq, n = 6; metabolomes, n = 5–6; acyl-carnitines n = 3). h , Body temperature rhythms over 24 h from subcutaneous probes implanted in null- ( n = 6 for control, n = 5 for L-Sirt1 − / − ) or Lb NOX-expressing ( n = 5) control and liver-specific Sirt1 − / − mice on TRF-CR. Data are presented as mean values ± s.e.m.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: Western Blot, Control, Molecular Weight, ChIP-sequencing, Whisker Assay, Gene Expression, RNA Sequencing, Expressing

Journal: Nature Metabolism

Article Title: NADH inhibition of SIRT1 links energy state to transcription during time-restricted feeding

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: TRF-CR generates rhythmic bouts of daytime torpor through increased levels of NADH in liver, inhibition of SIRT1 and downregulation of oxidative gene networks controlling acyl-carnitines and core body temperature. Reducing levels of NADH in the morning through the transduction of Lb NOX in TRF-CR mice increases lipid oxidation through the activation of SIRT1, resulting in elevated daytime body temperature rhythms. These findings identify NAD(H) redox state as a link between TRF-CR and whole-body metabolism.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: Inhibition, Transduction, Activation Assay

Journal: bioRxiv

Article Title: Native plasmid-encoded mercury resistance genes are functional and demonstrate natural transformation in environmental bacterial isolates

doi: 10.1101/764415

Figure Lengend Snippet: Plasmid map of p5343_UC57. The p5343 part (depicted in blue) encodes Mercuric ion reductase (MerA), Mercuric ion uptake protein (MerF), Hyp: Hypothetical protein (Hyp2), Mercuric transport protein (MerP), Mercuric transport protein (MerT), Regulator of Mercury resistance genes (MerR), Mobilization protein A (MobA), Mobilization protein C (MobC), Plasmid replication protein (RepA), Helix-turn-helix domain protein (HTH), RelE toxin (RelE), while the pUC57 part (depicted in grey) codes for promotor sequence for Ampicillin resistance gene (AmpR Promotor), Ampicillin resistance marker (AmpR), origin of replication, Lac promotor, LEU2 selection marker (Leu2) and elements ensuring plasmid maintenance (CEN/ARS).

Article Snippet: To propagate the synthesized DNA in the model bacterium E. coli , p5343 was cloned into the vector pUC57 resulting in plasmid p5343_UC57 (plasmid and sequence available via Addgene 126645).

Techniques: Plasmid Preparation, Sequencing, Marker, Selection

Journal: bioRxiv

Article Title: Native plasmid-encoded mercury resistance genes are functional and demonstrate natural transformation in environmental bacterial isolates

doi: 10.1101/764415

Figure Lengend Snippet: Improved mercury resistance in Escherichia coli DH10B containing p5343_UC57 (blue) versus only the empty vector pUC57 (green), grown in the presence of varying mercuric chloride concentrations.

Article Snippet: To propagate the synthesized DNA in the model bacterium E. coli , p5343 was cloned into the vector pUC57 resulting in plasmid p5343_UC57 (plasmid and sequence available via Addgene 126645).

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Native plasmid-encoded mercury resistance genes are functional and demonstrate natural transformation in environmental bacterial isolates

doi: 10.1101/764415

Figure Lengend Snippet: Results of the HGT assay used to screen environmental isolates for uptake of a plasmid-encoded trait via natural transformation. The 58 bacterial isolates are depicted on a 16S rRNA sequence-based phylogenetic tree (constructed using Maximum Likelihood). The colored bars indicate different bacterial classes, namely, orange is Actinobacteria, red is Cytophagia (Bacteriodetes phylum), yellow is Alphaproteobacteria, peach is Betaproteobacteria and pink is Gammaproteobacteria. The heat map plots optical density in the presence of p5343_UC57 compared to pUC57 in the presence of different mercury chloride concentrations normalized by the no mercury chloride control. Only the strains showing improved plasmid-based growth in the presence of mercury chloride were subjected to merA based colony PCR. The strains positive for colony PCR are indicated in black circles while those negative are depicted in grey circles. The genus of the strains positive for natural transformation as depicted on the figure are Arthrobacter (Ar), Dermacoccus (De), Acidovorax (Ax), Cupriavidus (Cu), Acinetobacter (Ac) and Pseudomonas (Ps). Figure constructed using ITOL 46 .

Article Snippet: To propagate the synthesized DNA in the model bacterium E. coli , p5343 was cloned into the vector pUC57 resulting in plasmid p5343_UC57 (plasmid and sequence available via Addgene 126645).

Techniques: Plasmid Preparation, Transformation Assay, Sequencing, Construct

Journal: bioRxiv

Article Title: Native plasmid-encoded mercury resistance genes are functional and demonstrate natural transformation in environmental bacterial isolates

doi: 10.1101/764415

Figure Lengend Snippet: Confirmation of HGT in strains 5, 12 and DH10B. Growth curves from second growth regime in the presence of varying mercury chloride concentrations are depicted when strains 5, 12 and DH10B were naturally transformed with plasmid p5343_UC57 (blue) and pUC57 (green). Assay design provided in Figure S2.

Article Snippet: To propagate the synthesized DNA in the model bacterium E. coli , p5343 was cloned into the vector pUC57 resulting in plasmid p5343_UC57 (plasmid and sequence available via Addgene 126645).

Techniques: Transformation Assay, Plasmid Preparation