Journal: bioRxiv

Article Title: Cell-free, methylated DNA in blood samples reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1101/2022.04.12.487966

Figure Lengend Snippet: (A) Tree dendrogram depicting the relationship between human reference Whole Genome Bisulfite Sequencing (WGBS) datasets included in the analysis. Methylation status of the top 30,000 variable blocks was used as input for the unsupervised hierarchical clustering. Samples from cell types with greater than n=3 replicates were merged. (B) UMAP projection of human WGBS reference datasets, colored by tissue and cell-type. (C) UMAP projection of mouse WGBS reference datasets, colored by tissue and cell type. *Acronyms: CAEC = coronary artery endothelial cell, CMEC = cardiac microvascular endothelial cell, CPEC = joint cardio-pulmonary endothelial cell, HUVEV = human umbilical vein endothelial cell, LSEC = liver sinusoidal endothelial cell, MK = megakaryocyte, NK = natural killer cell, PAEC = pulmonary artery endothelial cell, PMEC = pulmonary microvascular endothelial cell.

Article Snippet: Cryopreserved passage 1 human liver sinusoidal endothelial cells (LSEC) were purchased from ScienCell research laboratories (SKU#5000).

Techniques: Methylation Sequencing, Methylation

Journal: bioRxiv

Article Title: Cell-free, methylated DNA in blood samples reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1101/2022.04.12.487966

Figure Lengend Snippet: (A-B) Heatmaps of differentially methylated cell-type specific blocks identified from reference WGBS data compiled from healthy cell types and tissues in human (A) and mouse (B). Each cell in the plot marks the methylation score of one genomic region (rows) at each of the 20 cell types in human and 9 in mouse (columns). Up to 100 blocks with the highest methylation score are shown per cell type. The methylation score represents the number of fully unmethylated or methylated read-pairs / total coverage for hypo- and hyper-methylated blocks, respectively. (C) Heatmap of distance scores between gene-set pathways identified from GeneSetCluster. Genes adjacent to human cell-type specific methylation blocks were identified using HOMER and pathway analysis was performed using both Ingenuity Pathway Analysis (IPA) and GREAT. Significantly enriched gene-set pathways (p<0.05) from differentially methylated blocks identified in immune, cardiomyocyte, hepatocyte, and lung epithelial cell types were analyzed using GeneSetCluster. Cluster analysis was performed to determine the distance between all identified gene-set pathways based on the degree of overlapping genes from each individual gene-set compared to all others. Over-representation analysis was implemented in the WebgestaltR (ORAperGeneSet) plugin to interpret and functionally label identified gene-set clusters. *Acronyms: CPEC = cardio-pulmonary endothelial cell, HUVEV = human umbilical vein endothelial cell, LSEC = liver sinusoidal endothelial cell, NK = natural killer cell.

Article Snippet: Cryopreserved passage 1 human liver sinusoidal endothelial cells (LSEC) were purchased from ScienCell research laboratories (SKU#5000).

Techniques: Methylation

Journal: bioRxiv

Article Title: Cell-free, methylated DNA in blood samples reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1101/2022.04.12.487966

Figure Lengend Snippet: (A) Example of the NOS3 locus specifically unmethylated in endothelial cells. This endothelial-specific, differentially methylated block (DMB; highlighted in light blue) is 157bp long (7 CpGs), and is located within the NOS3 gene, an endothelial-specific gene (upregulated in paired RNA-sequencing data as well as in vascular endothelial cells, GTEx inset). The alignment from the UCSC genome browser (top) provides the genomic locus organization and is aligned with the average methylation (purple tracks) across cardiomyocyte, lung epithelial, liver sinusoidal endothelial (LSEC), cardiopulmonary endothelial (CPEC), hepatocyte, and immune (PBMC) samples (n=3 / cell-type group). Results from RNA-sequencing generated from paired cell-types are depicted (green tracks) as well as peak intensity from H3K27ac and H3K4me3 published ChIP-seq data generated in endothelial cells (blue tracks). (B) Significant functions of genes adjacent to endothelial-specific methylation blocks (all p<0.05). Blue color indicates nearby hypomethylated regulatory blocks. Yellow color indicates nearby hypermethylated regulatory blocks. (C) Pathways supporting the biological significance of endothelial-specific methylation blocks (all p<0.05). Unique pathways for distinct tissue-specific endothelial cells are highlighted in distinct colors. (D) Expression levels of genes adjacent to tissue-specific endothelial methylation blocks. Expression data were generated from paired RNA-sequencing of the same cardiopulmonary endothelial (CPEC) and liver sinusoidal endothelial (LSEC) cell populations used to generate methylation reference data. Pan-endothelial genes upregulated in both populations (ALL) are identified as common endothelial-specific methylation blocks to both LSEC and CPEC tissue-specific endothelial populations. (E) Top 5 transcription factor binding sites enriched among endothelial-specific hypomethylated blocks, using HOMER de novo and known motif analysis. The background for the HOMER analysis was composed of 3,574 non-endothelial cell-type specific hypomethylated blocks. *Acronyms: CPEC = cardio-pulmonary endothelial cell, HUVEV = human umbilical vein endothelial cell, LSEC = liver sinusoidal endothelial cell.

Article Snippet: Cryopreserved passage 1 human liver sinusoidal endothelial cells (LSEC) were purchased from ScienCell research laboratories (SKU#5000).

Techniques: Methylation, Blocking Assay, RNA Sequencing Assay, Generated, ChIP-sequencing, Expressing, Binding Assay

Journal: bioRxiv

Article Title: Cell-free, methylated DNA in blood samples reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1101/2022.04.12.487966

Figure Lengend Snippet: (A) Representative 3D-CRT treatment planning for patients with right-sided (i and ii) and left-sided (iii and iv) breast cancer, respectively. Computed tomography simulation of coronal and sagittal images depicting the anatomic position of the target volume in relation to nearby organs. The color map represents different radiation dose levels or isodose lines (Green: 95% of prescription dose, yellow: 90% isodose line, cyan: 80% isodose line, orange: 70% isodose line, brown: 50% isodose line). (B) Heatmaps of differentially methylated cell-type specific blocks identified from all reference WGBS data compiled from healthy human cell-types and tissues. Each cell in the plot marks the methylation score of one genomic region (rows) at each of the 20 human cell types (columns). Up to 100 blocks with the highest methylation score are shown per cell type. Differential blocks identified from cell types comprising the target organs-at-risk from radiation (lungs, heart, and liver) were selected for generation of a radiation-specific methylation atlas, separating these solid organ cell types from all other immune cell types. *Acronyms: CAEC = coronary artery endothelial cell, CMEC = cardiac microvascular endothelial cell, CPEC = joint cardio-pulmonary endothelial cell, HUVEC = human umbilical vein endothelial cell, LSEC = liver sinusoidal endothelial cell, MK = megakaryocyte, NK = natural killer cell, PAEC = pulmonary artery endothelial cell, PMEC = pulmonary microvascular endothelial cell.

Article Snippet: Cryopreserved passage 1 human liver sinusoidal endothelial cells (LSEC) were purchased from ScienCell research laboratories (SKU#5000).

Techniques: Computed Tomography, Methylation

Journal: bioRxiv

Article Title: Cell-free, methylated DNA in blood samples reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1101/2022.04.12.487966

Figure Lengend Snippet: (A-C Human) (A) Predicted human immune-derived cfDNA in Geq. Human Geq are calculated by multiplying the relative fraction of cell-type specific cfDNA x initial concentration cfDNA ng/mL x the weight of the haploid human genome. Immune cfDNA was assessed at n = 222 methylation blocks found to separate immune cell types from solid organ cell types. (g1 = Bcell, CD4Tcell, CD8Tcell, NK, MK, Erythroblast, Monocyte, Macrophage, Neutrophil; g2 = breast basal/luminal epi, lung epi, hepatocyte, kidney podocyte, pancreas islet, colon epi, cardiomyocyte, LSEC, CPEC, HUVEC, neuron, and skeletal muscle). (B) Predicted human solid organ-derived cfDNA in Geq where %solid organ is defined as 100-%immune using these same n=222 methylation blocks. (C) Fold change in human immune versus solid organ Geq at EOT and recovery relative to baseline. Friedman test was performed comparing paired results at baseline, EOT, and recovery timepoints. The results were considered significant when *P < 0.05. (D-E Mouse) (D) Predicted mouse immune-derived cfDNA in Geq. Mouse Geq are calculated by multiplying the relative fraction of cell-type specific cfDNA x initial concentration cfDNA ng/mL x the weight of the haploid mouse genome. Immune cfDNA was assessed at n = 148 methylation blocks found to separate immune cell types from solid organ cell types. (g1 = Bcell, CD4Tcell, CD8Tcell, Neutrophil; g2 = mammary epi, cardiomyocyte, hepatocyte, lung endothelial, cerebellum, hypothalamus, colon, intestine, kidney). (E) Predicted mouse solid organ-derived cfDNA in Geq. Kruskal-Wallis test was used for comparisons amongst groups. ns, P ≥ 0.05; *, P < 0.05.

Article Snippet: Cryopreserved passage 1 human liver sinusoidal endothelial cells (LSEC) were purchased from ScienCell research laboratories (SKU#5000).

Techniques: Derivative Assay, Concentration Assay, Methylation

Journal: bioRxiv

Article Title: Cell-free, methylated DNA in blood samples reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1101/2022.04.12.487966

Figure Lengend Snippet: (A, B) Hepatocyte cfDNA (in Geq/mL) in serum samples collected at different times. Fragment-level deconvolution used hepatocyte specific methylation blocks (top n=200). (C) Fold change in hepatocyte cfDNA after treatment (EOT) and at recovery relative to baseline. (D, E) Liver sinusoidal endothelial (LSEC) cfDNA (in Geq/mL) in the same serum samples. Fragment-level deconvolution used LSEC specific methylation blocks (n=89). (F) Fold change in LSEC cfDNA Geq at EOT and recovery relative to baseline levels. * Geq were calculated by multiplying the relative fraction of cell-type specific cfDNA x initial concentration cfDNA ng/mL x 3.3 x 10 -12 grams per haploid genome equivalent. Wilcoxon matched pairs signed rank test was performed between groups and results were considered significant when *P < 0.05; ns, P ≥ 0.05.

Article Snippet: Cryopreserved passage 1 human liver sinusoidal endothelial cells (LSEC) were purchased from ScienCell research laboratories (SKU#5000).

Techniques: Methylation, Concentration Assay

Journal: Frontiers in Genetics

Article Title: The role of microRNAs in defining LSECs cellular identity and in regulating F8 gene expression

doi: 10.3389/fgene.2024.1302685

Figure Lengend Snippet: microRNAs targeting F8 gene. (A) Relative expression of F8 gene (Probe ID: ILMN_1675083) in the used ECs and LSECs samples. (B) Left panel: Unsupervised, 3D-PCA displaying different endothelial cells for 25 expressed microRNAs that bind to F8 gene according to IPA. Middle panel: heat map showing the expression of these 25 microRNAs in three LSEC samples. Right panel: Correlation between F8 expression in different endothelial cells and PCA-1, X-axis represents F8 expression and y-axis represents PCA-1. (C) A depiction of the 1808-base pair long 3′ mRNA sequence of the F8 gene along with the 25 expressed microRNAs known to bind to this sequence. (D) Box plots illustrating the expression levels of four microRNAs in different endothelial cells, potentially binding to the F8 gene, as well as the transcription factors binding to the F8 gene promoter. (E) Top Panel: An IPA-generated figure showing the relationships between potentially F8 -binding microRNAs and transcription factors identified using TRANSFAC, which bind to the F8 promoter. Sequence logos of individual transcription factors are displayed on the right. Bottom Panel: Representation of the F8 promoter, covering 1,200 base pairs with 1 kb before the transcription start site and 200 base pairs after the transcription start site. The numbers represent the transcription factors binding sites identified using TRANSFAC (shown and numbered in top panel above).

Article Snippet: In this study, Primary adult Human Liver Sinusoidal Endothelial Cells (LSECs) were procured from Biozol Diagnostica Vertrieb GmbH (product lot No. HEC03020491) and cultured in a specialized endothelial cell culture medium, denoted as ENDO-Growth Medium MED001, supplemented with growth factors and antibiotics.

Techniques: Expressing, Sequencing, Binding Assay, Generated