Journal: Nucleic Acids Research

Article Title: U6 snRNA m6A modification is required for accurate and efficient splicing of C. elegans and human pre-mRNAs

doi: 10.1093/nar/gkae447

Figure Lengend Snippet: The absence of mett-10 causes cis - and trans -splicing defects. ( A ) Overview of pre-mRNA cis -splicing and cis -splicing defects detectable by RNA sequencing. The grey bars represent exons adjacent to alternative splicing events. Orange and blue bars represent sequences that are excluded or included depending on the alternative splicing event type. ( B ) Overview of pre-mRNA trans-splicing and trans-splicing defects detectable by RNA sequencing. ( C ) Volcano plot of all transcripts tested for cis -splicing changes between mett-10 −/− and wild-type animals. The X-axis shows PSI differences between mett-10 and wild-type animals, and the y-axis shows the significance in P -values. Transcripts that show increased splicing in mett-10 −/− (p ≤ 0.05 and ΔPSI ≤ −0.1) at a given splice site are coloured green, and transcripts that show reduced splicing in mett-10 −/− ( P ≤ 0.05 and ΔPSI ≥ 0.1) at a given splice site are coloured purple. ( D ) Volcano plot of all transcripts tested for trans-splicing changes between mett-10 −/− and wild-type animals. Transcripts that show increased splicing in mett-10 −/− ( P ≤ 0.05 and ΔPSI ≤ −3) at a given splice site are coloured green, and transcripts that show reduced splicing in mett-10 −/− ( P ≤ 0.05 and ΔPSI ≥ 3) at a given splice site are coloured purple. −log 10 P -value of 1 gene has been lowered to fit into the graph. ( E ) Classification of all significant (FDR < 0.05) cis -splicing defects in mett-10 −/− animals. ( F ) Classification of all significant ( P -value < 0.05) trans-splicing defects in mett-10 −/− animals.

Article Snippet: 750 ng of recovered mRNA was used for library preparation using the direct RNA sequencing (DRS) kit following the manufacturer's instructions (Oxford Nanopore Technologies, SQK-RNA002).

Techniques: RNA Sequencing Assay, Alternative Splicing

Journal: Nucleic Acids Research

Article Title: U6 snRNA m6A modification is required for accurate and efficient splicing of C. elegans and human pre-mRNAs

doi: 10.1093/nar/gkae447

Figure Lengend Snippet: mett-10 sensitive 5′SSs predominantly have +4A. ( A ) The sequence motif and frequency analysis of mett-10 sensitive 5′SSs (−3 to + 6) and the alternative 5′SSs that are used more often in mett-10 −/− . The sequence motif shows the probability of bases at each position around the 5′SS. U5 and U6 snRNA binding sequences are shown under the sequence motif logo. The frequency table is coloured based on the U5 snRNA interacting sequence frequency on the y-axis (−2 and −1) and U6 snRNA interacting sequence frequency on the x-axis (+3, +4 and + 5). ( B ) Normalised coverage of RNA-Seq reads for the pch-2 intron 1 boundary. Alt. 5′SS 1 is used more often in wild-type animals and alt. 5′SS 2 is used more often in mett-10 −/− . Barplot shows the fraction of reads supporting splicing at the Alt. 5′SS 2 over total reads in mett-10 −/− and wild-type animals. ( C ) Normalised ONT-DRS alignments for the pch-2 intron 1 boundary in wild-type, mett-10 −/− , and mett-10 germline rescued animals. ( D ) Heatmap showing the correlation between splice site usage in mett-10 −/− (y-axis) and the specific base at position +4 of the 5′SS. ( E ) Effect size plot for the 5′SS positions −3 to + 6. Negative values indicate bases at the specific position are associated with significantly more alternative splicing, and positive values indicate bases at the specific position are associated with significantly less alternative splicing events. The circles' size corresponds to the base frequency at a given position across all 5′SSs in the genome (e.g. position 1 is predominantly G, and position 2 is predominantly U across all 5′SSs). ( F ) Histogram for the distance between alternative splice site pairs. The Y-axis shows the number of alternative splice site pairs, and the X-axis shows the distance between the pairs, with negative values indicating the alternative splice site moves upstream and positive values indicating the alternative splice site moves downstream of the original splice site.

Article Snippet: 750 ng of recovered mRNA was used for library preparation using the direct RNA sequencing (DRS) kit following the manufacturer's instructions (Oxford Nanopore Technologies, SQK-RNA002).

Techniques: Sequencing, Binding Assay, RNA Sequencing Assay, Alternative Splicing

Journal: Nucleic Acids Research

Article Title: U6 snRNA m6A modification is required for accurate and efficient splicing of C. elegans and human pre-mRNAs

doi: 10.1093/nar/gkae447

Figure Lengend Snippet: mett-10 −/- animals have increased intron retention and exon-skipping. ( A ) 5′SS motif analysis of introns with increased (left) and decreased (right) retention in mett-10 −/− . The frequency of sequences corresponding to U5 and U6 binding are shown in the heatmap. ( B ) Normalized RNA-Seq coverage of Y18H1A.11 intron 3 in mett-10 −/− and wild-type animals. The bar plot shows the fraction of intronic reads over the retained intron. ( C ) 5′SS motif analysis of exons with increased skipping (upper panel) and increased retention (bottom panel) in mett-10 −/− animals. Sequence motifs are shown for the upstream exon 5′SSs (grey) and the retained/skipped exon (orange). ( D ) Normalised ONT-DRS alignments for Y18H1A.11 intron 3 in wild-type, mett-10 −/− , and mett-10 germline rescued animals. ( E ) Normalized RNA-Seq coverage of aqp-2 exon 5 in mett-10 −/− and wild-type animals. Bar plots show the fraction of exonic reads over the skipped exon. ( F ) Normalized ONT-DRS alignments for aqp-2 exon 5 in wild-type, mett-10 −/− , and mett-10 germline rescued animals.

Article Snippet: 750 ng of recovered mRNA was used for library preparation using the direct RNA sequencing (DRS) kit following the manufacturer's instructions (Oxford Nanopore Technologies, SQK-RNA002).

Techniques: Binding Assay, RNA Sequencing Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: U6 snRNA m6A modification is required for accurate and efficient splicing of C. elegans and human pre-mRNAs

doi: 10.1093/nar/gkae447

Figure Lengend Snippet: The absence of mett-10 affects 3′SS usage. ( A ) 5′ and 3′SS motif analysis of transcripts that are mett-10 sensitive (upper panel) and the corresponding alternative 3′SSs whose usage increases in mett-10 −/− (bottom panel). ( B ) Histogram for the distance between alternative splice site pairs. The Y-axis shows the number of alternative splice site pairs, and the X-axis shows the distance between the pairs. Negative values indicate the alternative splice site moving upstream, and positive values indicate the alternative splice site moving downstream of the original splice site. ( C ) Normalized RNA-Seq coverage of B0001.7 intron 6. The canonical splice position is Alt. 3′SS 1. In wild-type animals, weak upstream splice site Alt. 3′SS 2 is also utilized. The bar plot shows the fraction of reads covering the exon sequence to the right of the canonical splice site. ( D ) Normalized ONT-DRS alignments for B0001.7 intron 6 in wild-type, mett-10 −/− and mett-10 germline rescued animals. ( E ) Heat map (left) and bar plots (right) showing the frequency of 5′SS usage at specific splice sites in wild-type and mett-10 −/− animals. ( F ) Heat map (left) and bar plots (right) showing the frequency of 3′SS usage at specific splice sites in wild-type and mett-10 −/− animals.

Article Snippet: 750 ng of recovered mRNA was used for library preparation using the direct RNA sequencing (DRS) kit following the manufacturer's instructions (Oxford Nanopore Technologies, SQK-RNA002).

Techniques: RNA Sequencing Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: U6 snRNA m6A modification is required for accurate and efficient splicing of C. elegans and human pre-mRNAs

doi: 10.1093/nar/gkae447

Figure Lengend Snippet: mett-10 is required for efficient trans-splicing. ( A ) The trans-splicing defects rescued by the germline expression of mett-10 are shown in orange over the total SL trans-splicing defects observed in mett-10 −/− animals as in Figure (blue). (B–D) Examples for the ( B ) outron retention, ( C ) alternative 3′ trans-splice site usage, ( D ) cis- spliced outron retention events showing the normalised coverage of ONT-DRS alignments (left) and the fraction of reads covering the outron sequence (right) in wild-type, mett-10 −/− and mett-10 germline rescued animals. ( E ) 3′ trans-splice site motif of background transcripts that do not show the trans-splicing defect (top panel), transcripts that show the weak trans-splicing defect (middle panel) and transcripts that show the strong trans-splicing defect (bottom panel).

Article Snippet: 750 ng of recovered mRNA was used for library preparation using the direct RNA sequencing (DRS) kit following the manufacturer's instructions (Oxford Nanopore Technologies, SQK-RNA002).

Techniques: Expressing, Sequencing

Journal: Nucleic Acids Research

Article Title: U6 snRNA m6A modification is required for accurate and efficient splicing of C. elegans and human pre-mRNAs

doi: 10.1093/nar/gkae447

Figure Lengend Snippet: Human METTL16 is required for efficient splicing of 5′SS with +4A, and in vivo , editing of 5′SS +4A to +4U can restore splicing in mett-10 −/− mutants. ( A ) Sequence motif analysis of METTL16 sensitive 5′SSs (left) and 5′SSs whose usage increases in METTL16 knock-down cells (right). The p-value for the comparison of motifs is 9.35 × 10 −156 . ( B ) Human U6 snRNA showing the positions of m6A and m2G methylations in red. The UACAGA box is highlighted with a red dashed line. Grey boxes indicate 5′ exons and depict interactions between m6A43 and m2G72 with the 5′SSs. M1 depicts the catalytic metal ion. ( C ) Sequence motif analysis of THUMPD2 sensitive 5′SSs (left) and 5′SSs whose usage increases in THUMPD2 knock-out cells (right). The p-value for the comparison of motifs is 0.98. ( D ) Normalized read coverage of pipp-4P intron 4 5′SSs. In wild-type animals, splicing was observed most frequently at Alt. 5′SS 1 position followed by Alt. 5′SS 2 position. In mett-10 mutants, splicing was observed most frequently at Alt. 5′SS 3 position followed by Alt. 5′SS 2 position. Adenosine position edited to uracil is shown in red. ( E ) cDNA visualisation of three splice isoforms for pipp-4P exon 4 and 5. Orange depicts the upstream exon, and green depicts the downstream exon. The hpy166I restriction enzyme recognition sequence is shown by the red bar. ( F ) Gel electrophoresis analysis of pipp-4P RT-PCR products followed by Hpy166I digestion. The orange arrow shows the undigested product size, and the red arrow shows the digested product size. The table shows the expected product sizes for RT-PCR and digestion. ( G ) Model for the function of U6 snRNA m6A modification in 5′SS recognition, alternative splicing of 3′SS and trans-splicing. m6A methylated U6 snRNA facilitates accurate recognition of 5′SSs with a //GURAG motif through non-canonical base pairing with +4A (Top panel). SNRNP27K likely functions together with m6A methylated U6 snRNA to recognize 5′SSs with +4A. Efficient recognition of 5′SSs can facilitate alternative splicing at weak 3′SSs. Similarly, efficient recognition of SL RNA 5′SSs by m6A methylated U6 snRNA can facilitate trans-splicing at weak 3′ trans -splice sites.

Article Snippet: 750 ng of recovered mRNA was used for library preparation using the direct RNA sequencing (DRS) kit following the manufacturer's instructions (Oxford Nanopore Technologies, SQK-RNA002).

Techniques: In Vivo, Sequencing, Knockdown, Comparison, Knock-Out, Nucleic Acid Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Modification, Alternative Splicing, Methylation

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet: B6 mice were transferred with PbT‐II cells, infected with Pcc, and treated with control (IgG; n = 1 biological replicate) or anti‐IL‐27 mAb (α‐IL‐27; n = 1 biological replicate) between −1 and 5 days of infection. PbT‐II cells were purified from these mice 7 days after infection and single‐cell RNA sequencing (scRNA‐seq) analysis was performed. Details of the experiments are shown in Fig . UMAP plots of PbT‐II cells from IgG control ( n = 4,030) and anti‐IL‐27 mAb‐treated mice ( n = 7,476) after unsupervised clustering of pooled single‐cell data from the two groups, with clusters colored by gene expression profiles. UMAP clustering of PbT‐II cells colored by cell cycle profiles. Summary graph of proportions of PbT‐II cells in each cluster for IgG and anti‐IL‐27 mAb‐treated mice in (A). Dot plots showing the expression of Th1‐, Tfh‐, Tcmp‐related genes (Ciucci et al , ), and other genes of interest in each UMAP cluster of PbT‐II cells from IgG and anti‐IL‐27 mAb‐treated mice. Dot colors represent the intensity of expression, while dot size represents the proportion of cells with the corresponding expression. Violin plots showing the expression of Th1‐, Tfh‐, Tcmp‐, and proliferation‐associated genes in PbT‐II cells from IgG (light blue) and anti‐IL‐27 mAb (blue) treated mice. Ridge plots showing the expression of published Th1, Tfh, Tmem, and Tcmp CD4 + T cell signatures in each of the UMAP clusters in (A) based on (Ciucci et al , ). Source data are available online for this figure.

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Infection, Control, Purification, RNA Sequencing, Gene Expression, Expressing

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet: B6 mice were transferred with PbT‐II cells, infected with Pcc, and were treated with either IgG or anti‐IL‐27 mAb between −1 and 7 days after infection ( n = 1 biological replicate per timepoint). PbT‐II cells were prepared from spleen at day 28 pi, stained for CD4/TCR/CD45.1 and for CD127, KLRG1, and CD49d with TotalSeq antibodies, sort purified, and processed for scRNA‐seq and CITE‐Seq analysis. Details of the experiment are found in Fig . A–G Comparative analysis of scRNA‐seq data from IgG and anti‐IL‐27 mAb‐treated PbT‐II cells. (A) UMAP plot colored of day 28 PbT‐II cells from IgG control ( n = 7,491) and anti‐IL‐27 mAb‐treated mice ( n = 4,944) after unsupervised clustering of pooled single cell data from the two groups, with clusters colored by gene expression profiles. Cluster labels were harmonized to reflect similar gene expression patterns in the clusters at day 7 pi (Fig ) and anti‐IL27 mAb day 7–28 PbT‐II analysis (Fig ). (B) UMAP clustering of PbT‐II cells colored by cell cycle profiles. (C) CITE‐seq analysis of PbT‐II cells for IgG2a (isotype control), CD127, KLRG1, and CD49d, shown in the same UMAP clustering as (A). (D) Proportions (%) of each cluster within PbT‐II cells, with bar graph sizes shown relative to the total number of PbT‐II cells in IgG (36.8 × 10 ) and anti‐IL‐27 mAb treated (265.7 × 10 ) mice. (E) Ridge plots of PbT‐II cells showing the expression of published CD4 + T cell signature genes (Ciucci et al , ). (F) Violin plots comparing the expression of the CD4 + T cell signature genes. (G) Dot plots showing the expression of Th1‐, Tfh‐, Tcmp‐, and proliferation‐associated genes in each cluster. Dot colors represent the intensity of expression, while dot size represents the proportion of cells with the corresponding expression. H Volcano plot of differentially expressed genes between major clusters 1* and 1** within PbT‐II cells from anti‐IL‐27‐treated mice and corresponding Gene Ontology enrichment analysis for the upregulated genes in each group using Metascape. Source data are available online for this figure.

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Infection, Staining, Purification, Control, Gene Expression, Expressing

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet: B6 mice were transferred with PbT‐II cells, treated with IgG or anti‐IL‐27 mAb on day −1, 2 and 5 for day 7 analysis, while mice were treated with anti‐IL‐27 mAb on day −1, 2, 5, and 7 for day 14 and 28 analysis ( n = 1 biological replicate per timepoint). PbT‐II cells were purified and subjected to single‐cell RNA sequencing (scRNA‐seq) and CITE‐seq analysis. The ProjecTILs algorithm (Andreatta et al , ) was used to analyze CD4 + T cell states of PbT‐II cells based on a published reference atlas (Andreatta et al , ). A Experimental scheme. B Gating strategy for the sorting of PbT‐II cells for the scRNA‐seq experiments: Spleen cells were stained for CD4, TCRβ, and CD45.1 to distinguish PbT‐II cells and for TotalSeq IgG2a, CD127, KLRG1, and CD49d for CITE‐seq analysis. C Flow cytometry profiles for each PbT‐II sample analyzed for single‐cell transcriptomics. D, E Predicted distribution of the projected PbT‐II cells in IgG and anti‐IL‐27 mAb‐treated mice on day 7 (D) and day 28 (E) after Pcc infection as density contours in a UMAP plot of a CD4 + T cell reference map (Andreatta et al , ). The bar graphs represent the proportions of the PbT‐II cells projected in the indicated reference subtype.

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Purification, RNA Sequencing, Staining, Flow Cytometry, Single-cell Transcriptomics, Infection

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet: scRNA‐seq data of PbT‐II cells from Pcc‐infected anti‐IL‐27 mAb‐treated mice (day7, 14, and 28) were pooled, and unsupervised clustering was performed. UMAP plot colored by gene expression clustering. Proportions (%) of each cluster for each time point. Feature plots of indicated genes across cell clusters as distributed in UMAP plots. Dot plots showing the expression of Th1‐, Tfh‐, Tmem‐, and proliferation‐associated genes in each cluster. Dot colors represent the intensity of expression, while dot size represents the proportion of cells with the corresponding expression. Ridge plots of PbT‐II cell clusters showing the expression of published CD4 + T cell signature genes (Ciucci et al , ).

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Infection, Gene Expression, Expressing

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet:

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Marker, Staining, FACS, Purification, Software, Cell Isolation

Journal: Nature Communications

Article Title: Spatially resolved multiomics on the neuronal effects induced by spaceflight in mice

doi: 10.1038/s41467-024-48916-8

Figure Lengend Snippet: Overview of the study workflow where brains from International Space Station (ISS; Flight mice) and ground control mouse groups (Ground control mice) were split into the two hemispheres for Spatial Gene Expression Analysis (Spatial Transcriptomics or ST) and Single Nuclei Multiomics analysis (snMultiomics).

Article Snippet: To identify specific cellular microenvironments affected by spaceflight, we combined the techniques of spatial transcriptomics (ST; 10X Genomics Visium) and single-nucleus multiomics (snMultiomics; gene expression and chromatin accessibility; 10× Genomics Single Cell Multiome ATAC + Gene Expression) on mouse brain.

Techniques: Control, Gene Expression

Journal: Nature Communications

Article Title: Spatially resolved multiomics on the neuronal effects induced by spaceflight in mice

doi: 10.1038/s41467-024-48916-8

Figure Lengend Snippet: A Distribution of UMIs per nucleus in the entire snRNA-seq dataset. nUMI/nuclei: number of UMIs detected in each nuclei. B Distribution of peaks per nucleus in the entire snATAC-seq dataset. nPeaks/nuclei: number of peaks detected per nuclei in the multiomics dataset. C Correlation between flight (y-axis) and ground control (x-axis) single nuclei multiomics samples (Pearson’s correlation coefficient, r = 0.95; p < 0.05) shown as a scatter plot. This is a two-sided Pearson correlation test with 95% confidence intervals performed on the average expression (log(1 + avgUMI)). avgUMI: average UMI counts per spot. D UMAP of single nuclei multiomics data and cluster annotations. E 11 functional multiomics clusters categories represented by their marker genes. F Distribution of UMIs per spot for the whole spatial transcriptomics (ST) dataset. nUMI/spot: number of UMIs detected per spot in the ST dataset. G Distribution of unique genes per spot for the whole spatial transcriptomics (ST) dataset. nGenes/spot: number of genes detected per spot in the ST dataset. H Correlation between flight (y-axis) and ground control (x-axis) ST samples (Pearson’s correlation coefficient, r = 0.99; p < 0.05) shown as a scatter plot. This is a two-sided Pearson correlation test with 95% confidence intervals performed on the average expression (log(1 + avgUMI)). avgUMI: average UMI counts per spot.

Article Snippet: To identify specific cellular microenvironments affected by spaceflight, we combined the techniques of spatial transcriptomics (ST; 10X Genomics Visium) and single-nucleus multiomics (snMultiomics; gene expression and chromatin accessibility; 10× Genomics Single Cell Multiome ATAC + Gene Expression) on mouse brain.

Techniques: Control, Expressing, Functional Assay, Marker

Journal: Nature Communications

Article Title: Spatially resolved multiomics on the neuronal effects induced by spaceflight in mice

doi: 10.1038/s41467-024-48916-8

Figure Lengend Snippet: A Clustering of spatial transcriptomics data, cluster annotations and spatial location of clusters visualized on flight and ground control mouse brain sections. B Marker genes for each ST cluster visualized as dotplot. C Spatial distribution of 3 genes (Wfs1 for CA1 region of hippocampus, Dkk3 for CA1 and CA3 hippocampal region and Prox1 for Dentate gyrus) in three flight (left column) and three ground control (right column) ST sections. D Significantly different pathways ( p < 0.05) between flight and ground control in ST cluster 9 (Cortical neurons, bottom layers). E Visualization of number of clusters identified by single-nuclei multiomics and their proportions in each ST cluster (x-axis; 0–17). Only multiomics clusters with higher proportions (>10%) are displayed in the barplot. F Cell type proportions mapped to spatial coordinates on three ground control (top row) and three flight (bottom row) mouse brain sections (Synaptic transmission I or multiomics cluster 1; Myelination or multiomics cluster 3; Neuronal activity, Synaptic transmission III or multiomics cluster 15).

Article Snippet: To identify specific cellular microenvironments affected by spaceflight, we combined the techniques of spatial transcriptomics (ST; 10X Genomics Visium) and single-nucleus multiomics (snMultiomics; gene expression and chromatin accessibility; 10× Genomics Single Cell Multiome ATAC + Gene Expression) on mouse brain.

Techniques: Control, Marker, Transmission Assay, Activity Assay

Journal: Nature Communications

Article Title: Spatially resolved multiomics on the neuronal effects induced by spaceflight in mice

doi: 10.1038/s41467-024-48916-8

Figure Lengend Snippet: A Dotplot showing the differentially expressed ligand receptor pairs found by CellPhoneDB between two interacting multiomics clusters (4 and 11) which are affected by spaceflight. These clusters showed the largest number of spaceflight DEGs, and four LR pairs were found significantly upregulated in these interactions. The null distribution of the mean expression of the LR pairs was estimated by employing a random permutation approach. The mean expression of the interacting LR molecule pairs are indicated by the dot colors and the dot sizes represent the p -values which refers to the enrichment of the LR pair in the interacting multiomics clusters. Scales for both dot size and color are presented below the plot. B Accessibility differences for motifs Atoh1, Zic1, and Zic2 in multiomics cluster 4 of flight mice and ground control mice. Spaceflight results in reduced accessibility of these motifs in flight samples. Two-sided Chi-square test statistic was used for differential testing with FDR correction (fdr <0.05). C Accessibility differences for motifs Pou5f1, and Sox2 in multiomics cluster 11 of flight and ground control mice. Spaceflight results in increased accessibility of these motifs in flight samples. Effects of spaceflight shown by increased accessibility of these motifs in flight samples. Two-sided Chi-square test statistic was used for differential testing with FDR correction (fdr <0.05). D (left) adjusted p -value of differential interactions found by MISTy in intraview (cell type and pathway activity colocalization) occuring only in flight (blue; n = 3 individual ST flight mouse samples) or in controls (red; n = 3 individual ST ground control mouse samples), tiles with black border identify statistically significant changes, (middle) correlation of MAPK pathway activity and Neurovasculature abundance, and mapped on Visium slide for two samples (right). Two-sided Student’s t tests with Benjamini–Hochberg multiple testing correction was used to determine the differential interactions. E adjusted p -value of differential interactions found by MISTy in paraview (cell type and pathway activity in local neighborhood) occuring only in flight (blue; n = 3 individual ST flight mouse samples) or in controls (red; n = 3 individual ST ground control mouse samples), tiles with black border identify statistically significant changes. Two-sided Student’s t tests with Benjamini–Hochberg multiple testing correction was used to determine the differential interactions. F Pearson correlation of Glis3 activity (left) containing vascular endothelial cells and MAPK activity ( n = 6 individual ST mouse samples, 3 flight, 3 ground controls), and their respective activities in Visium slides (4 plots on the right). Two-sided Student’s t - tests with Benjamini–Hochberg multiple testing correction was used to determine the changes in correlation. G Pearson correlation of Lef1 activity (left) within spots containing vascular endothelial cells and MAPK activity, and their respective activities in Visium slides (4 plots on the right). Two-sided Student’s t tests with Benjamini–Hochberg multiple testing correction was used to determine the changes in correlation. multiomics cl: multiomics cluster. The boxplots in D , F , and G show the median as a central line, the box boundaries denote the first and third quartiles and the whiskers extend to the most extreme point in the range within 1.5 times the interquartile range from the box.

Article Snippet: To identify specific cellular microenvironments affected by spaceflight, we combined the techniques of spatial transcriptomics (ST; 10X Genomics Visium) and single-nucleus multiomics (snMultiomics; gene expression and chromatin accessibility; 10× Genomics Single Cell Multiome ATAC + Gene Expression) on mouse brain.

Techniques: Expressing, Control, Activity Assay

Journal: Nature Communications

Article Title: Spatially resolved multiomics on the neuronal effects induced by spaceflight in mice

doi: 10.1038/s41467-024-48916-8

Figure Lengend Snippet: A Heatmap showing fold change differences (log2FC) between flight and ground control samples in oxidative phosphorylation pathway in both ST and multiomics datasets. There is a spaceflight-mediated inhibition seen for this pathway that is consistent across the two datasets. Two-sided Wilcoxon’s rank-sum test was done with FDR adjustment. B Heatmap showing fold change differences (log2FC) between flight and ground control samples in Arachidonic acid metabolism pathway in both ST and multiomics datasets. There is a deficit for this pathway seen in spaceflight samples in both the datasets. Two-sided Wilcoxon’s rank-sum test was done with FDR adjustment. C Heatmap showing fold change differences (log2FC) between flight and ground control samples in Fatty acid synthesis pathway in both ST and multiomics datasets. There is a spaceflight-mediated reduction observed for this pathway in both the modalities. Two-sided Wilcoxon’s rank-sum test was done with FDR adjustment. multiomics cl: multiomics cluster.

Article Snippet: To identify specific cellular microenvironments affected by spaceflight, we combined the techniques of spatial transcriptomics (ST; 10X Genomics Visium) and single-nucleus multiomics (snMultiomics; gene expression and chromatin accessibility; 10× Genomics Single Cell Multiome ATAC + Gene Expression) on mouse brain.

Techniques: Control, Phospho-proteomics, Inhibition