Journal: Nature

Article Title: Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs

doi: 10.1038/s41586-024-07472-3

Figure Lengend Snippet: a , Experimental design. CD, chow diet. b , Glucose tolerance of unexposed male offspring (F 1 ) of HFD-exposed bucks. Top: AUC ipGTT . Bottom: frequency distribution analysis to identify tolerant and intolerant animals. n = 60 male mice across 4 cohorts with 5 litters each and 3 males per litter (eLFD and eHFD bucks); n = 10–15 (sLFD and sHFD bucks) including 1 cohort with 5 litters and 3 males per litter. c , d , Glucose tolerance ( c ; mean ± s.e.m.) and insulin sensitivity ( d ; mean ± s.e.m.) of male offspring (F 1 ) of HFD-exposed bucks. Data represent a re-phenotyping of the animals in c carried out 8 weeks after the first phenotyping. Significance calculated by a two-way ( c ; n in graph) or one-way ( d ; n as in c ) analysis of variance (ANOVA; *** P < 10 −4 ). ITT, insulin tolerance test. e , f , PCA plot ( e ) and functional enrichment analysis (KEGG (Kyoto Encyclopedia of Genes and Genomes); f ) of peripheral tissue RNA-seq data from HFDt and HFDi F 1 animals. eWAT, epididymal white adipose tissue; metab., metabolism; AA, amino acid; TH, thyroid hormone; OXPHOS, oxidative phosphorylation; FA, fatty acid; TCA, tricarboxylic acid. g , Left: overlap between genes differentially expressed in tissues from HFDi mice and their human orthologues associated with childhood obesity. Right: functional enrichment analysis (KEGG) of the overlapping genes ( n = 693) pre-classified as protective and risk genes for childhood obesity on the basis of the β -score for BMI-SDS. DEGs, differentially expressed genes; Hs , Homo sapiens ; FDR, false discovery rate; NSCLC, non-small cell lung cancer; CML, chronic myeloid leukaemia; NAFLD, non-alcoholic fatty liver disease. h , Scatter plot of children’s body weight trajectories as a function of paternal BMI at conception in families with mothers who were lean (red line; r = 0.2611; P value < 10 −4 ) or overweight (blue line; r = 0.3467; P value < 10 −4 ) at conception. Significant association calculated by linear regression analysis. i , Insulin sensitivity, measured as ISI Matsuda (top) or homeostatic model assessment for insulin resistance (HOMA-IR; bottom) indices in children as a function of parental weight status at conception. n lean–lean = 106; overweight–lean = 184; lean–overweight = 114; overweight–overweight = 415. Data represented as mean ± s.e.m. Significance calculated by two-way ANOVA (details in the graph).

Article Snippet: Reads mapping and differential expression analysis were carried out using the A.I.R. (Artificial Intelligence RNA-Seq) software from Sequentia Biotech with the following pipeline: BBDuk (reads trimming; BBDUkguide ), STAR (reads mapping to the mouse genome GRCm38 (ENSEMBL); https://github.com/alexdobin/STAR ), featureCounts (gene expression quantification; https://subread.sourceforge.net/featureCounts.html ) and NOISeq (statistical analysis of differentially expressed genes; http://bioinfo.cipf.es/noiseq/doku.php ).

Techniques: Functional Assay, RNA Sequencing Assay

Journal: Nature

Article Title: Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs

doi: 10.1038/s41586-024-07472-3

Figure Lengend Snippet: ( a ) Representative H&E (top) and TRA98 (bottom) staining in testes from LFD (left) or HFD (right) fed mice. N = 6-7 mice/group independently stained and analysed ( b ) Quantification of the testis tubule diameter (average of the horizontal and vertical diameters) from H&E-stained testes (N = 60–70 seminiferous tubules – 10/mouse/group – significance calculated with Mann-Whitney test to compare ranks). ( c ) Motility of cauda spermatozoa from LFD and HFD-fed mice (N = 7 – significance calculated by two-tailed t-test). ( d - e ) First cleavage (d) and positive IVF (% of fertilised oocytes that complete pre-implantation development - e) rates in embryos generated via IVF with sperm from LFD or HFD-fed mice (N = 4 IVF each done with a pool of spermatozoa from 10 mice – significance calculated by two-tailed t-test). ( f ) PCA representation of the RNA-seq analysis of Round Spermatids (RS) isolated from testes of mice fed with LFD or HFD (f). ( g - h ) Scatter plot (g) and heatmap (h) representation of differentially expressed genes in round spermatids. ( i - j ) UMAP representation (i) and cluster annotation (j - based on publicly available datasets - GSE112393 ) of single-cell RNA-seq analysis of testes from LFD and HFD-fed mice (n = 3). ( k ) Representative marker genes for the different germ cell populations.

Article Snippet: Reads mapping and differential expression analysis were carried out using the A.I.R. (Artificial Intelligence RNA-Seq) software from Sequentia Biotech with the following pipeline: BBDuk (reads trimming; BBDUkguide ), STAR (reads mapping to the mouse genome GRCm38 (ENSEMBL); https://github.com/alexdobin/STAR ), featureCounts (gene expression quantification; https://subread.sourceforge.net/featureCounts.html ) and NOISeq (statistical analysis of differentially expressed genes; http://bioinfo.cipf.es/noiseq/doku.php ).

Techniques: Staining, MANN-WHITNEY, Two Tailed Test, Generated, RNA Sequencing Assay, Isolation, Marker

Journal: Nature

Article Title: Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs

doi: 10.1038/s41586-024-07472-3

Figure Lengend Snippet: a , Distribution of sncRNA biotypes in cauda spermatozoa from LFD- and HFD-fed mice. n = 3 samples per diet. lincRNA, long intergenic non-coding RNA; miRNA, microRNA. b , Biotype-specific differential expression analysis ( P adj < 0.05; log 2 [fold change (FC)] > |1|) of cauda spermatozoa sncRNAs. c , Histogram (bars at a bin centre of 0.5 with the overlapping non-linear fit curve) showing the distribution of the log 2 [FC(HFD versus LFD)] in nuclear- (n) and mitochondrial- (mt) derived tsRNAs (left) and rsRNAs (right). d , Volcano plot representation of differentially expressed mt-tsRNAs (differential expression calculated by EdgeR; significance defined as P adj < 0.05). e , Fragmentation pattern of mt-tRNAs in cauda spermatozoa from LFD- and HFD-fed mice. Significance tested by a two-tailed t -test, HFD versus LFD (mean ± s.e.m.; n = 3; * P < 0.05). f , Pearson-based correlation analysis of individual sncRNA biotype expression in human sperm with BMI (two-tailed P value with 95% confidence interval). g , Differentially expressed tsRNAs in human sperm from lean and overweight donors calculated by DESeq2-based continuous differential expression analysis ( n = 18 donors). VST, variance-stabilizing transformation. h , Heat map representation of mature mt-tRNA levels in cauda spermatozoa from LFD- and HFD-fed mice. i , Relative abundance of the indicated sncRNA biotypes in epididymosomes (Epi) or spermatozoa (Sp) isolated from the caput, corpus and cauda epididymis. Data reanalysed from refs. , . j , Uniform manifold approximation and projection (UMAP) representation of mtDNA transcription during spermatogenesis from testis single-cell RNA-seq data.

Article Snippet: Reads mapping and differential expression analysis were carried out using the A.I.R. (Artificial Intelligence RNA-Seq) software from Sequentia Biotech with the following pipeline: BBDuk (reads trimming; BBDUkguide ), STAR (reads mapping to the mouse genome GRCm38 (ENSEMBL); https://github.com/alexdobin/STAR ), featureCounts (gene expression quantification; https://subread.sourceforge.net/featureCounts.html ) and NOISeq (statistical analysis of differentially expressed genes; http://bioinfo.cipf.es/noiseq/doku.php ).

Techniques: Expressing, Derivative Assay, Two Tailed Test, Transformation Assay, Isolation, RNA Sequencing Assay

Journal: Nature

Article Title: Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs

doi: 10.1038/s41586-024-07472-3

Figure Lengend Snippet: a , MA plot representation of differentially expressed genes in HFD_A versus LFD embryos. b , Gene Ontology-based functional enrichment analysis of differentially expressed genes in HFD_A versus LFD embryos (see for details). c , d , Heat map representation of the relative expression (HFD versus LFD) of OXPHOS genes in early two-cell embryos ( c ) and in a publicly available dataset of single-embryo RNA-seq analysis of mouse pre-implantation development ( d ). TE_blast, trophectoderm blastocyst; ICM_blast, inner cell mass blastocyst. e , PCA-based representation of the developmental timing in HFD and LFD embryos laid over the analysis of mouse pre-implantation development of ref. . f , RNA-seq-based quantification of the expression of genes important for mitochondrial function across adult tissues and germ cells of male mice exposed to 2 weeks of HFD as well as early embryos and adult tissues from unexposed male offspring of HFD-fed fathers. 2CEs, two-cell embryos; TF, transcription factor; ROS, reactive oxygen species; gastro, gastrocnemius. g , Pearson-based correlation matrix of IMPC-derived metabolic phenotypes in WT offspring of parents heterozygous (het.) for IMPC-selected genes (see for details). h , Bar plot representation of the relative (WT versus control) glucose intolerance (measured as AUC ipGTT ) in WT offspring of fathers heterozygous for genes important for mitochondrial structure and function. Black arrows indicate genes for which cryopreserved heterozygous sperm samples were analysed. i , Distribution of sncRNA biotypes in cauda spermatozoa from the indicated mutant mice ( n = 10 mice per gene). j , Heat map representation of the relative abundance of 5′ n-tsRNAs and 5′ mt-tsRNAs in mutant spermatozoa. Control, LFD and HFD samples are cryopreserved and resequenced to serve as reference and technical controls.

Article Snippet: Reads mapping and differential expression analysis were carried out using the A.I.R. (Artificial Intelligence RNA-Seq) software from Sequentia Biotech with the following pipeline: BBDuk (reads trimming; BBDUkguide ), STAR (reads mapping to the mouse genome GRCm38 (ENSEMBL); https://github.com/alexdobin/STAR ), featureCounts (gene expression quantification; https://subread.sourceforge.net/featureCounts.html ) and NOISeq (statistical analysis of differentially expressed genes; http://bioinfo.cipf.es/noiseq/doku.php ).

Techniques: Functional Assay, Expressing, RNA Sequencing Assay, Derivative Assay, Control, Mutagenesis

Journal: Iubmb Life

Article Title: Tyrosine and Phenylalanine Activate Neuronal DNA Repair but Exhibit Opposing Effects on Global Transcription and Adult Female Mice Are Resilient to TyrRS / YARS1 Depletion

doi: 10.1002/iub.70030

Figure Lengend Snippet: cis ‐RSV and trans ‐RSV have opposite effects on Aβ‐induced neurite degeneration and DNA damage response. (A) cis‐ RSV and trans ‐RSV have opposite effects on Aβ‐induced TyrRS depletion. Representative spectral images (scale bar, 20 μm) and quantification of TyrRS protein in rat cortical neurons (DIV 10) treated with cis ‐RSV or trans ‐RSV, alone or in combination with nMAβ 42 (50 nM) for 16 h. (B) cis ‐RSV prevents, while trans‐RSV exacerbates nMAβ42‐induced neurite degeneration. Representative images (scale bar, 20 μm) of cortical neurons pre‐treated with cis ‐RSV and trans ‐RSV (50 μM) for 16 h, followed by nMAβ 42 (50 nM) exposure for 24 h. Neurons were stained with MAP2 (neurite marker, magenta) and DAPI (nuclear marker, blue). Neurons were immunoassayed with anti‐MAP2 antibody and quantified for neurite degeneration ( n = 15 images per condition with N = 3 independent experiments). (C) cis ‐RSV and trans ‐RSV have opposite effects on nMAβ42‐mediated DNA damage accumulation. Immunostaining images (scale bar, 10 μm) for DNA damage marker, pSer139‐H2AX foci (γ‐H2AX, green; DAPI—nuclear marker, blue) in cortical neurons (DIV 10) treated with cis ‐RSV and trans ‐RSV (50 μM) alone or in combination with nMAβ 42 (50 nM) for 24 h. The graph represents the average number of γ‐H2AX foci per nuclei for n = 30 neurons per treatment condition for n = 3 independent experiments. (D) cis‐ RSV and trans ‐RSV have opposite effects on nMAβ42‐induced histone H3 phosphorylation. Immunostaining images (scale bar, 10 μm) for pSer‐10‐H3 (pSer‐10‐H3, green; DAPI—nuclear marker, blue) in cortical neurons (DIV 10) treated with cis ‐RSV and trans ‐RSV (50 μM) alone or in combination with nMAβ 42 (50 nM) for 24 h. The graph represents pSer‐10‐H3 for n = 30 neurons per condition for N = 3 independent experiments with p values represented in the figure (* ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, and **** ≤ 0.0001). Statistical analysis was done using 2 way ANOVA with Tukey's multiple comparisons test.

Article Snippet: MAP2 , Aves labs , SKU: MAP , 1:500.

Techniques: Staining, Marker, Immunostaining, Phospho-proteomics