Journal: bioRxiv

Article Title: A novel Smg6 mouse model reveals regulation of circadian period and daily CRY2 accumulation through the nonsense-mediated mRNA decay pathway

doi: 10.1101/2022.07.01.498406

Figure Lengend Snippet: A . Schematic of the genetic model. Smg6 flox expresses wild-type SMG6 protein encoded by the blue exons; after Cre-mediated recombination to Smg6 mut , point-mutated exons 18 and 19 (yellow) lead to expression of mutant SMG6 (D1352A, D1391A). B . The mutated aspartic acid residues are within the catalytic triade of the PIN nuclease domain, shown in the structure of the human protein (PDB accession 2HWW; ). C . For cellular studies, tail fibroblasts from adult male mice ( Smg6 flox and wild-type littermates) were cultured until spontaneous immortalization, and tamoxifen-activatable CreERT2 expression was achieved by a retrovirus. Upon 4-hydroxytamoxifen (4-OHT) treatment, NMD mutants (yellow) were compared to different control cells (shades of blue). D . PCR-based genotyping of genomic DNA extracted from cells depicted in C. indicates efficient recombination upon 4-OHT treatment. E . A luciferase reporter containing an intron in the 3’ UTR is upregulated in 4-OHT-treated Smg6 flox/flox cells, as expected under NMD-inactive conditions. N=2-6 plates/group, adjusted p=0.0038; multiple Student’s t-test. F . Density plot showing transcriptome-wide mRNA/pre-mRNA ratio distributions calculated from RNA-seq, in NMD-inactive (yellow) vs. control cells. G . The difference in mRNA/pre-mRNA ratios between NMD-inactivated ( Smg6 flox/flox + 4-OHT) and control cells ( Smg6 flox/flox + vehicle) is consistent with higher stability of annotated NMD substrates (purple, N=2681) and transcripts with retained introns (green, N=3369). Moreover, the broad distribution and shift to positive values for non-NMD-annotated protein coding transcripts (beige, N=3232) is indicative of transcriptome-wide NMD regulation. H . Read coverage on the Hnrnpl1 and I . Srsf11 loci indicates the specific upregulation of transcript isoforms that are NMD-annotated (purple) and that can be identified via specific exons (marked by arrows in insets). J . Differential expression analysis at the exon level, comparing Smg6 flox/flox + 4-OHT vs. Smg6 flox/flox + vehicle conditions, reveals significant upregulation of NMD-annotated exons (purple; N=492; 169 genes), retained introns (green; N=521; 122 genes), and a sizeable number of constitutive exons (beige; N=1787, 382 genes), suggestive of NMD regulating many protein coding genes. K . Correlation analysis between mRNA/pre-mRNA ratio change upon NMD activation and expression levels in wild-type cells shows significant anticorrelation. The lengths of L . the 5’ UTR, M . the CDS and N . the 3’ UTR are all positively correlated with mRNA/pre-mRNA ratio change upon NMD inactivation. Pearson correlation coefficient (r), slope and p-values were calculated by a linear model.

Article Snippet: Male adult Smg6 flox/flox mice and their control littermates ( Smg6 +/+ ) received bilateral stereotactic injections of CMV.HI-Cre::eGFP AAV5 particles (AddGene, 105545) into the SCN (400 nl per site).

Techniques: Expressing, Mutagenesis, Cell Culture, Control, Luciferase, RNA Sequencing, Quantitative Proteomics, Activation Assay

Journal: bioRxiv

Article Title: A novel Smg6 mouse model reveals regulation of circadian period and daily CRY2 accumulation through the nonsense-mediated mRNA decay pathway

doi: 10.1101/2022.07.01.498406

Figure Lengend Snippet: A . Real-time recording of bioluminescence rhythms in immortalized fibroblasts of genotypes Smg6 flox/flox and Smg6 +/+ (both transduced with CreERT2 retrovirus and circadian reporter Dbp::Luc ); after temperature-entrainment (24-h periodic square wave; 35°C-37°C), cells were released into free-running conditions (37°C) from day 4. Representative traces show longer free-running period in Smg6 mut cells (yellow) as compared to control cells (blue). B . Quantification of several experiments as in A. Smg6 mutants (yellow) showed significantly longer periods in comparison to controls ( Smg6 flox/flox treated with vehicle in blue, or Smg6 +/+ with/without 4-OHT in grey). N=5 for Smg6 flox/flox cells; N=3 for Smg6 wt/wt cells; period difference between 4-OHT and vehicle-treated Smg6 flox/flox cells is 1.6 hours; Bonferroni’s multiple comparisons test adjusted p=0.0228. C . Adult Smg6 flox/flox male mice and their Smg6 +/+ littermates (all carrying tamoxifen-activatable A lbCreERT2 and the circadian reporter Per2::Luc ) were sacrificed 4-5 weeks after tamoxifen injections. Liver and kidney explants were used for real-time recording of luciferase rhythms. Representative traces from livers show strong free-running period lengthening in the Smg6 mutant (yellow) vs. control (blue). D . Quantification of several experiments as in C. Robustly increased periods, by almost 3 hours, were observed in NMD-deficient liver explants (yellow; mean=25.36 ± 2.23 h) in comparison to control livers (blue; mean = 22.0 ± 0.90 h). No period length difference was observed for kidney explants from the same animals (grey; flox/flox mean±SD = 25.2 ± 1.19 h, control mean±SD = 24.4 ± 1.83 h). Liver tissue: N=16 slices Smg6 flox/flox ; N=17 slices for controls; Mann-Whitney test p<0.0001. Kidney tissue: N=16 for Smg6 flox/flox ; N=20 for controls, Mann-Whitney test p=0.0771. 1-4 tissue slices were used per mouse; analyses performed blindly. E . Efficient recombination was validated by genotyping of livers (PCR analysis of genomic DNA). F . Cartoon depicting the in vivo recording setup (RT-Biolumicorder); Smg6 flox/flox or Smg6 +/+ mice carrying AlbCreERT2 and mPer2::Luc alleles were implanted with a luciferin-loaded mini osmotic pump 4 weeks after tamoxifen injections before placing in the recording device. G . Left panel: After 2 days under LD12:12, bioluminescence rhythms (photons) and activity (infrared signal) were recorded for 5 days under photoskeleton photoperiod conditions (indicated by yellow vertical lines at ZT12 and before ZT0). The plot shows mean signal (solid trace) and SEM (shaded) over the whole course of the experiment in the left panel, and compiled data, averaged from all days and mice, in the right panel. H . Quantification of PER2::LUC bioluminescence signal showed no difference in phase between tamoxifen-injected Smg6 flox/flox (yellow) and Smg6 +/+ (blue) control animals. N=6 per group; Mann-Whitney test p=0.7251.

Article Snippet: Male adult Smg6 flox/flox mice and their control littermates ( Smg6 +/+ ) received bilateral stereotactic injections of CMV.HI-Cre::eGFP AAV5 particles (AddGene, 105545) into the SCN (400 nl per site).

Techniques: Transduction, Control, Comparison, Luciferase, Mutagenesis, MANN-WHITNEY, In Vivo, Activity Assay, Injection

Journal: bioRxiv

Article Title: A novel Smg6 mouse model reveals regulation of circadian period and daily CRY2 accumulation through the nonsense-mediated mRNA decay pathway

doi: 10.1101/2022.07.01.498406

Figure Lengend Snippet: A . Representative microphotographs of SCN sections to assess effective targeting of the SCN. Viral expression can be estimated from GFP signal, encoded with Cre on the same virus. B . Same as A, but image taken during bioluminescence recording of SCN slices. C . Upper diagram: Rhythms of voluntary locomotor activity were recorded prior to and after the SCN injection of the Cre-and GFP-expressing AAV. Lower: Representative actograms of a Smg6 flox/flox and Smg6 +/+ mouse. The day of Cre::eGFP AAV injection is marekd by an arrow and a dot. D . Period lengths of circadian locomotor activity rhythms of Smg6 flox/flox (in yellow) and Smg6 +/+ (in blue) mice before (DD1) and after (DD2) stereotaxic surgery. E . Averaged traces of mPer::Luc rhythms of AAV-injected Smg6 flox/flox (yellow) and Smg6 +/+ (blue) SCN explants. F . Period lengths of mPer::Luc expression in AAV-injected Smg6 flox/flox (yellow) and Smg6 +/+ (blue) SCN explants. G . Recombination efficiency following Cre induction was evaluated by genotyping of genomic DNA extracted from SCN slices (liver-specific mutants served as controls for the genotyping).

Article Snippet: Male adult Smg6 flox/flox mice and their control littermates ( Smg6 +/+ ) received bilateral stereotactic injections of CMV.HI-Cre::eGFP AAV5 particles (AddGene, 105545) into the SCN (400 nl per site).

Techniques: Expressing, Virus, Activity Assay, Injection

Journal: bioRxiv

Article Title: A novel Smg6 mouse model reveals regulation of circadian period and daily CRY2 accumulation through the nonsense-mediated mRNA decay pathway

doi: 10.1101/2022.07.01.498406

Figure Lengend Snippet: A . Schematic of the around-the-clock RNA-seq experiment, which was carried out on a time series of liver samples collected from LD12:12-entrained male Smg6 mutant ( Smg6 flox/flox ; AlbCreERT2 ; tamoxifen-treated) and control ( Smg6 +/+ ; AlbCreERT2 ; tamoxifen-treated) mice. B.-J . RNA-seq data is plotted for indicated core clock genes for mRNA (upper panels; exonic reads) and pre-mRNA (lower panels; intronic reads) for Smg6 mutants (yellow) and controls (blue). RPKM values (Reads Per Kilobase of transcript, per Million mapped reads) of individual mice are shown as dots with solid lines connecting the means for each timepoint. The dashed lines represent the rhythmic data fit using the parameters from Metacycle . K . Circular plot representing the phases of peak mRNA abundances according to the Metacycle fits for Smg6 mutants (dashed) and controls (solid) for indicated core clock genes. Cry2, Nr1d2 and Ror γ accumulated several hours later in Smg6 mutants, whereas minor effects were seen for the other genes. L . As in K. but for pre-mRNA rhythms. Several core clock pre-mRNAs showed later phases, indicative of transcriptional shifts; notable exceptions being Per2 (almost invariable) and Cry2 and Nr1d2 , which both showed a phase advance. M . Similar to , mRNA/pre-mRNA ratios were calculated for the liver RNA-seq data; briefly, average mRNA counts were first averaged over all samples per genotype, before dividing by average pre-mRNA counts. Three components of the negative limb, Cry2, Cry1 and Per2 , show higher mRNA/pre-mRNA ratios in Smg6 mutants.

Article Snippet: Male adult Smg6 flox/flox mice and their control littermates ( Smg6 +/+ ) received bilateral stereotactic injections of CMV.HI-Cre::eGFP AAV5 particles (AddGene, 105545) into the SCN (400 nl per site).

Techniques: RNA Sequencing, Mutagenesis, Control

Journal: bioRxiv

Article Title: A novel Smg6 mouse model reveals regulation of circadian period and daily CRY2 accumulation through the nonsense-mediated mRNA decay pathway

doi: 10.1101/2022.07.01.498406

Figure Lengend Snippet: A . Read coverage on the Hnrnpl1 locus indicates the specific upregulation of transcript isoforms that are NMD-annotated and that can be identified by specific exons (see arrows in insets) in liver tissue. B . RNA-seq data is plotted for indicated genes – that all encode components of the NMD machinery itself – for mRNA (upper panels; exonic reads) and pre-mRNA (lower panels; intronic reads) for Smg6 mutants (yellow) and controls (blue). RPKM values of individual animals are shown as dots with solid lines connecting the means for each timepoint. The dashed lines represent the rhythmic data fit using the parameters from Metacycle. C . RNA-seq data is plotted for Atf4 and Atf5 for mRNA (upper panels; exonic reads) and pre-mRNA (lower panels; intronic reads) for Smg6 mutants (yellow) and controls (blue). RPKM values of individual animals are shown as dots with solid lines connecting the means for each timepoint. The dashed lines represent the rhythmic data fit using the parameters from Metacycle. D . Western blot analysis of liver tissue (as in ) for ATF5, eIF2α and phospho-eIF2α in Smg6 mutant and control liver samples; HSP90 served as loading control. E . Quantification of ATF5 signal, normalized to HSP90 as loading control, from Western blot shown in D.

Article Snippet: Male adult Smg6 flox/flox mice and their control littermates ( Smg6 +/+ ) received bilateral stereotactic injections of CMV.HI-Cre::eGFP AAV5 particles (AddGene, 105545) into the SCN (400 nl per site).

Techniques: RNA Sequencing, Western Blot, Mutagenesis, Control

Journal: bioRxiv

Article Title: A novel Smg6 mouse model reveals regulation of circadian period and daily CRY2 accumulation through the nonsense-mediated mRNA decay pathway

doi: 10.1101/2022.07.01.498406

Figure Lengend Snippet: A . mRNA/pre-mRNA ratios across individual liver samples – grouped into light (ZT0, 4, 8) and dark phase (ZT12,16, 20) samples – indicate Cry2 mRNA stability increase in Smg6 mutants, which is visible in particular in the dark phase; p-value=0.06; ANOVA. B . Western blot analysis of total liver proteins, for CRY2 and HSP90 (loading control). Each sample is a pool of 3 individual mice. C . Quantification of Western blot shown in B; CRY2 intensity was normalized to the loading control, HSP90. D . Western blot as in panel B, but from individual animals at ZT8 and ZT20, indicating that CRY2 is reproducibly more abundant at ZT20 in Smg6 mutants. E . Quantification of Western blot in D; p=0.1; Mann-Whitney non-parametric test. F . Western blot analysis of total protein extract from fibroblasts (cells as shown in ) reveals CRY2 upregulation specifically in 4-OHT-treated Smg6 flox/flox cells. G . Quantification of Western blot shown in F; p=0.002; Mann-Whitney non-parametric test. H . RNA-seq read coverage on the Cry2 locus (fibroblasts). Only one transcript isoform - carrying the 2.2 kb 3’ UTR - is expressed; there is no evidence for expression of the short UTR isoform. I . Schematic of the lentiviral dual luciferase system used in assays to test 3’ UTRs of interest for NMD regulation. J . Dual-luciferase assays reveal that the Cry2 3’ UTR confers NMD regulation. Non-treated cells for each genotype/reporter were internally set to 1. Vector UTR alone shows ca. 5-fold upregulation under Smg6 flox/flox + 4-OHT conditions (an effect coming from both Firefly luciferase up- and Renilla luciferase downregulation). Against this background of the assay, the Cry2 3’ UTR confers an additional >5-fold increase. Cells of each genotype/reporter condition without 4-OHT treatment were internally set to 1, and the signal of 4-OHT-treated cells relative to these untreated cells is reported; N=5 from 3 different experiments; p=0.001; Mann-Whitney non-parametric test. K . Bioluminescence traces of NIH/3t3 cells carrying the DBP-Luciferase reporter, with (orange) and without (grey) 0.6 µ M SMG1i treatment. Traces show average (mean) signal and standard deviation from 3 independent experiments. L . Quantification of experiments shown in K, showing reproducible period lengthening by ca. 4h in the presence of 0.6 µ M SMG1i (N=11-12; p<0.001; Mann-Whitney test). M . Western blot analysis of total protein extract from NIH/3t3 cells treated with vehicle or 0.6 µ M SMG1i; values of CRY2 abundance normalized to HSP90 (loading control) below the lanes. N . Primary fibroblast (genotype Smg6 +/+ , no 4-OHT) were stably transduced with luciferase reporters carrying different 3’ UTRs (as in panel I). Real-time recording of Firefly luciferase signal was carried until the signal reached stable state, before addition of 1 µ M SMG1i (orange) or vehicle (grey). The reporter carrying the Cry2 3’ UTR was specifically upregulated, as compared to Per1, Per2 or vector 3’ UTRs. Traces show average (mean) signal and standard deviation; N=3. O . In assays as in N., only full-length Cry2 3’ UTR showed upregulation upon SMG1i treatment, but not individual fragments (N=2). P . Western blot showing absence of CRY2 in Crispr/Cas9-generated Cry2 knockout NIH/3t3 cells. Q . Period length of Dbp -luciferase reporter traces in NIH/3t3 cells - controls (grey) or Cry2 knockouts (green) - treated with 0.1 µM, 0.3 µM or 0.6 µM of SMG1i or with vehicle (DMSO) corresponding to the volume used in highest SMG1i treatment.

Article Snippet: Male adult Smg6 flox/flox mice and their control littermates ( Smg6 +/+ ) received bilateral stereotactic injections of CMV.HI-Cre::eGFP AAV5 particles (AddGene, 105545) into the SCN (400 nl per site).

Techniques: Western Blot, Control, MANN-WHITNEY, RNA Sequencing, Expressing, Luciferase, Plasmid Preparation, Standard Deviation, Stable Transfection, Transduction, CRISPR, Generated, Knock-Out

Journal: bioRxiv

Article Title: A novel Smg6 mouse model reveals regulation of circadian period and daily CRY2 accumulation through the nonsense-mediated mRNA decay pathway

doi: 10.1101/2022.07.01.498406

Figure Lengend Snippet: A . Heatmap of transcripts with significant rhythms at the mRNA level in both genotypes. Expression levels are represented as Z-scores for mRNA or pre-mRNA with color code for low (blue) to high (red) expression. Z-scores were calculated separately for mRNA and pre-mRNA data, but on a common scale for both genotypes. Transcripts are phase-order for the control genotype. B . Heatmap as in A., but for transcripts with significant rhythms only in control animals (N=223) and not in mutants. C . Heatmap as in A., but for transcripts with significant rhythms only in mutant animals (N=329) and not in controls. D . Radial diagrams showing peak phase of rhythmic mRNAs in control (blue) and Smg6 mutant (yellow) liver for the common transcripts shown in A. Dark shaded: high amplitude rhythmic transcripts; light shaded: low amplitude rhythmic transcripts; high/low cut-off on log2 peak-trough amplitude of 1. E . Peak phase of mRNA in Smg6 mutants (orange) relative to control phase (blue), ranked according to the phase in the control ( Smg6 wt) for the transcripts shown in A (N=1257). F . Peak phase difference between mutant and control mice for all common rhythmic mRNAs (N=1257). G . Peak phase difference of commonly rhythmic mRNAs as in F., but restricted to loci with ChIP-seq binding sites for BMAL1 and CLOCK (N=130), according to ; p=0.0065; permutation test, calculated by 1000x subsampling of N=130 transcripts from the “all rhythmic transcripts” (N=1257) of panel F, then comparing the means of these subsampling groups with the observed mean (t-test). H . Peak phase difference of commonly rhythmic mRNAs as in F., but restricted to loci with ChIP-seq binding sites for the ensemble of proteins PER1, PER2, CRY1 and CRY2 according to ; p=0.0005; permutation test as in panel G. I . Peak phase difference of commonly rhythmic mRNAs as in F., but restricted to loci with no ChIP-seq binding sites for any of the proteins BMAL1, CLOCK, PER1, PER2, CRY1 or CRY2 according to ; p=0.18; permutation test as in panel G. J . Z-score amplitudes – defined as the difference between the maximum and minimum Z-score values, calculated independently for mRNAs and pre-mRNAs of commonly rhythmic transcripts (N=1257) - show lower mean mRNA (p=0.236) and higher mean pre-mRNA amplitudes in mutants (p=2e-16); significance calculations from a linear model (equivalent to t-test). K . Transcript mRNA/pre-mRNA Z-score amplitude ratios (from the N=1257 common rhythmic transcripts) stratified by genotype show decrease in mutants; p=2e-16; Student’s t-test. L . RNA-seq data is plotted for indicated genes for mRNA (upper panels; exonic reads) and pre-mRNA (lower panels; intronic reads) for Smg6 mutants (yellow) and controls (blue). Rhythmicity of mRNA levels observed in control (blue) is dampened or lost in Smg6 mutant liver (yellow). RPKM values of individual mice are shown as dots with solid lines connecting the means for each timepoint. The dashed lines represent the rhythmic data fit using the parameters from Metacycle . M . RT-Biolumicorder traces of individual mice in food shifting experiment. After 2 days under ad libitum feeding, bioluminescence rhythms (photons) and activity (infrared signal) were recorded for 4 additional days under light-phase-restricted feeding conditions (ZT10-20; horizontal black bar at top); skeleton photoperiod entrainment indicated by yellow vertical lines at ZT12 and ZT0. Each line represents the signal from a control (blue) or a liver-specific Smg6 mutant (yellow) animal N . Compiled data, averaged over the last two days of the experiment. Mean signal (solid trace) and SEM (shaded). Indicated phase differences calculated from rhythmic fits to the data.

Article Snippet: Male adult Smg6 flox/flox mice and their control littermates ( Smg6 +/+ ) received bilateral stereotactic injections of CMV.HI-Cre::eGFP AAV5 particles (AddGene, 105545) into the SCN (400 nl per site).

Techniques: Expressing, Control, Mutagenesis, ChIP-sequencing, Binding Assay, RNA Sequencing, Activity Assay

Journal: Nature

Article Title: ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation

doi: 10.1038/nature11228

Figure Lengend Snippet: a , Colon histopathology, b , percentage weight loss, and c , diarrhoea scores in control and DSS-treated Ace2 +/y and Ace2 −/y littermates. In a , note crypt damage (arrowheads), ulcerations (arrow), and infiltration of inflammatory cells (asterisks) in DSS-treated Ace2 −/y mice. Haematoxylin and eosin staining on day 7 after DSS challenge. Scale bars, 100 μm. d , Colon histopathology (haematoxylin and eosin staining, day 4 after DSS challenge; scale bars, 100 μm), e , percentage weight loss, and f , diarrhoea scores of DSS-treated Ace2 +/y and Ace2 −/y littermates fed either normal chow (Control) or a protein free diet (PFD; <0.2% protein). All values are mean ± s.e.m. of 5–9 mice per group. * P < 0.05, ** P < 0.01 comparing DSS-treated Ace2 +/y with Ace2 −/y littermates, or Ace2 +/y mice on normal diet with those on PFD (paired- t -test).

Article Snippet: For microbiota transplantation, caecal and ileum extracts were pooled from Ace2 mutant mice and ACE2 expressing littermates, suspended in 3 ml PBS, and administered (0.1 ml per mouse) immediately to sterile-packed 4-week-old Swiss-Webster germ-free mice (Taconic).

Techniques: Histopathology, Control, Staining

Journal: Nature

Article Title: ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation

doi: 10.1038/nature11228

Figure Lengend Snippet: a , Colon histopathology (haematoxylin and eosin, day 10 after DSS challenge; scale bars, 100 μm), b , percentage weight loss, and c , diarrhoea scores of DSS-treated Ace2 +/y and Ace2 −/y littermates that received vehicle or nicotinamide (NAM) in their drinking water. Nicotinamide treatment was started 3 days before DSS challenge. d , Colon histopathology (haematoxylin and eosin, day 7; scale bars, 100 μm), e , percentage weight loss, and f , crypt injury scores of Ace2 +/y and Ace2 −/y mice fed a di-peptidic tryptophan diet (Trp+) or normal chow (Control). Values are mean ± s.e.m. of 3–10 mice per group. * P < 0.05, ** P < 0.01 comparing Ace2 −/y mice on a normal diet with those on Trp+ diet, or vehicle- versus nicotinamide-treated Ace2 −/y mice. ## P < 0.01 comparing Ace2 +/y versus Ace2 −/y mice (paired- t -test).

Article Snippet: For microbiota transplantation, caecal and ileum extracts were pooled from Ace2 mutant mice and ACE2 expressing littermates, suspended in 3 ml PBS, and administered (0.1 ml per mouse) immediately to sterile-packed 4-week-old Swiss-Webster germ-free mice (Taconic).

Techniques: Histopathology, Control

Journal: Nature

Article Title: ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation

doi: 10.1038/nature11228

Figure Lengend Snippet: a , b , mRNA expression levels of antimicrobial peptides in epithelial cells isolated from the small intestine of a , unchallenged Ace2 +/y and Ace2 −/y littermates, and b , Ace2 +/y mice fed a tryptophan-free diet (Trp−) or normal chow (Control). c , mRNA expression levels of antimicrobial peptide Defa1 in Ace2 +/y and Ace2 −/y littermates fed a Trp+ diet or normal chow (Control) for 10 days. d , e , Immunohistochemistry to detect levels of phosphorylated S6 (brown) in the small intestine of d , unchallenged Ace2 +/y and Ace2 −/y littermates or e , Ace2 −/y mice fed a Trp+ or normal chow diet (Control). Scale bars, 200 μm. f , Colon histopathology (haematoxylin and eosin, day 8; scale bars, 100 μm) of DSS treated wild-type mice receiving vehicle or rapamycin (RAPA) i.p., initiated 6 days before DSS challenge. Values are mean ± s.e.m. of 5–6 mice per group. * P < 0.05, ** P < 0.01 comparing Ace2 +/y with Ace2 −/y mice; # P < 0.05, ## P < 0.01 comparing Ace2 +/y mice on normal diet with those on Trp− diet (paired- t -test).

Article Snippet: For microbiota transplantation, caecal and ileum extracts were pooled from Ace2 mutant mice and ACE2 expressing littermates, suspended in 3 ml PBS, and administered (0.1 ml per mouse) immediately to sterile-packed 4-week-old Swiss-Webster germ-free mice (Taconic).

Techniques: Expressing, Isolation, Control, Immunohistochemistry, Histopathology

Journal: Nature

Article Title: ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation

doi: 10.1038/nature11228

Figure Lengend Snippet: a , b , Principal coordinate analysis plots; a , calculated by Bray–Curtis algorithm and b , based on unweighted UniFrac analysis. Plots show the similarity among ileocaecal bacterial communities in Ace2 +/y and Ace2 −/y mice fed a Trp+ diet or normal chow (Control) for 10 days. Only the two axes with high R 2 values are shown (axis 1, R 2 = 0.335; axis 2, R 2 = 0.8116). Each dot represents data from an individual animal. c , Comparison of microbial communities in Ace2 +/y and Ace2 −/y mice fed a Trp+ diet or normal chow (Control). The heat map depicts abundance of the top 25 species level OTUs contributing significantly to the axis shown in the weighted principal coordinate analysis plot ( a ). d , Diarrhoea scores and e , colon histopathology (haematoxylin and eosin, day 7; scale bars, 100 μm) of DSS challenged germ-free mice that received intestinal microbiota from Ace2 +/y or Ace2 −/y littermates. Values are mean ± s.e.m. of 4–6 mice per group. * P < 0.05 (paired- t -test).

Article Snippet: For microbiota transplantation, caecal and ileum extracts were pooled from Ace2 mutant mice and ACE2 expressing littermates, suspended in 3 ml PBS, and administered (0.1 ml per mouse) immediately to sterile-packed 4-week-old Swiss-Webster germ-free mice (Taconic).

Techniques: Control, Comparison, Histopathology