Journal: PloS one

Article Title: Cross-protective peptide vaccine against influenza A viruses developed in HLA-A*2402 human immunity model.

doi: 10.1371/journal.pone.0024626

Figure Lengend Snippet: Figure 4. An accumulation of murine CD3+ and CD8+ cells around the bronchioles in intranasally immunized mice. A24Tg mice were immunized three times at 7 to 9 days intervals i.n.(A,C,D,E) or s.c.(B) with PA130–138, PB1430–438 and PB2549–557 peptides in the presence of CpG-ODN (B,C,D), Tyrosinase206–214 plus CpG-ODN (E) or CpG-ODN plus empty-liposome solution (A). Lungs were harvested at day 7 after the final immunization, embedded in O.C.T. compound, frozen in dry ice-2-propanol. Ten mm thick frozen sections were prepared. The sections were post- fixed in acetone:ethanol (1:1) solution and blocked endogenous avidin and biotin activity, then stained with anti-mouse CD3 (A,B,C) or anti-mouse CD8a (D,E). doi:10.1371/journal.pone.0024626.g004

Article Snippet: The sections were stained with biotinylated hamster anti-mouse CD3 (eBioscience, San Diego, CA) or biotinylated rat anti-mouse CD8a (R&D Systems ) antibodies.

Techniques: Avidin-Biotin Assay, Activity Assay, Staining

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension

doi: 10.1164/rccm.201906-1141oc

Figure Lengend Snippet: Figure 1: Characterisation of Expressed BMP9 Mutant Proteins. (a) Schematic of

Article Snippet: After washing, 0.04 μg/well of anti-human BMP9 detection antibody (R&D Systems BAF3209) was added in Copyright © 2019 by the American Thoracic Society 6 PBS/1% BSA containing 0.2% GS.

Techniques: Mutagenesis

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension

doi: 10.1164/rccm.201906-1141oc

Figure Lengend Snippet: Figure 2: Loss of Activity in Pro:BMP9 Mutants Predicted to be Pathogenic. (a,b)

Article Snippet: After washing, 0.04 μg/well of anti-human BMP9 detection antibody (R&D Systems BAF3209) was added in Copyright © 2019 by the American Thoracic Society 6 PBS/1% BSA containing 0.2% GS.

Techniques: Activity Assay

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension

doi: 10.1164/rccm.201906-1141oc

Figure Lengend Snippet: Figure 3: Loss of active BMP9 in PAH patients carrying putatively pathogenic GDF2

Article Snippet: After washing, 0.04 μg/well of anti-human BMP9 detection antibody (R&D Systems BAF3209) was added in Copyright © 2019 by the American Thoracic Society 6 PBS/1% BSA containing 0.2% GS.

Techniques:

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension

doi: 10.1164/rccm.201906-1141oc

Figure Lengend Snippet: Figure 4: Plasma BMP9 and pBMP10 levels are not reduced in PAH, but a subset of

Article Snippet: After washing, 0.04 μg/well of anti-human BMP9 detection antibody (R&D Systems BAF3209) was added in Copyright © 2019 by the American Thoracic Society 6 PBS/1% BSA containing 0.2% GS.

Techniques: Clinical Proteomics

Journal: Virus Evolution

Article Title: Deep mutational scanning of SARS-CoV-2 Omicron BA.2.86 and epistatic emergence of the KP.3 variant

doi: 10.1093/ve/veae067

Figure Lengend Snippet: Deep mutational scanning of the SARS-CoV-2 Omicron BA.2.86 RBD. (a) Diagram of the RBD substitutions that distinguish Omicron BA.2 from Wuhan-Hu-1 (top), and BA.2.86 from BA.2 (bottom). Italicized mutations in BA.2.86 indicate secondarily mutated (D339H, A484K) or reverted (R493Q) substitutions that originally changed from Wuhan-Hu-1, and dashed lines show propagation of BA.2. changes to BA.2.86. Wuhan-Hu-1 reference spike numbering is used throughout the manuscript. (b–d) Quality control of the BA.2.86 RBD site-saturation mutagenesis library as assessed by PacBio sequencing, illustrating the distribution of number of amino acid mutations per barcoded variant (b), the average number of mutations of each type across library variants (c), and the distribution of mutations across sites in the RBD over all variants (d). (e, f) FACS gates used to sort RBD + singlet cells for ACE2 titration (e) and RBD expression (f) deep mutational scanning experiments from one representative replicate. (g, h) Correlation in per-mutant deep mutational scanning measurements between independently barcoded replicate libraries for ACE2-binding affinity (g) and RBD expression (h) experiments.

Article Snippet: Induced cells were washed with phosphate buffered saline supplemented with bovine serum albumin (PBS-BSA , BSA 0.2 mg/l), split into 16-OD*ml aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 −6 to 10 −13 m at 1−log intervals, plus a 0 m sample.

Techniques: Control, Mutagenesis, PacBio Sequencing, Variant Assay, Titration, Expressing, Binding Assay

Journal: Virus Evolution

Article Title: Deep mutational scanning of SARS-CoV-2 Omicron BA.2.86 and epistatic emergence of the KP.3 variant

doi: 10.1093/ve/veae067

Figure Lengend Snippet: Effects of mutations in the BA.2.86 receptor-binding domain on ACE2-binding and RBD expression. (a) Heatmap illustrating the impacts of all mutations in the BA.2.86 RBD on ACE2-binding affinity as determined from FACS-seq experiments with yeast-displayed RBD mutant libraries. ACE2 contact residues (top row of yellow squares, bottom heatmap) defined as RBD residues with non-hydrogen atoms <5 Å from ACE2 in the BA.2.86 RBD structure (PDB 8QSQ; Liu et al. 2024 ). Antibody escape residues (bottom row of orange squares, bottom heatmap) defined as those with average >0.125 relative antibody escape from aggregated deep mutational scanning data ( Greaney et al. 2022a ). (b) Deep mutational scanning data from (a) mapped to the ACE2-bound BA.2.86 RBD structure (PDB 8QSQ; Liu et al. 2024 ), illustrating the average effect of mutations at a site (left), the maximal effect of any mutation at a site (center), or the effect of the single-codon deletion (right). Sites of interest are labeled, and ACE2 (key motifs only) is shown as transparent gray cartoon. (c) Heatmap illustrating the impacts of all mutations in the BA.2.86 RBD on yeast-surface expression levels, a proxy for folding and expression efficiency. (d) Scatterplot illustrating the average effect of mutations at each site on ACE2-binding affinity ( y -axis) versus RBD expression ( x -axis). Yellow points indicate direct structural contacts as in (a). Individual measurements from (a) and (c) are reported in , and an interactive version of these heatmaps is available at https://tstarrlab.github.io/SARS-CoV-2-RBD_DMS_Omicron-EG5-FLip-BA286/RBD-heatmaps/ .

Article Snippet: Induced cells were washed with phosphate buffered saline supplemented with bovine serum albumin (PBS-BSA , BSA 0.2 mg/l), split into 16-OD*ml aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 −6 to 10 −13 m at 1−log intervals, plus a 0 m sample.

Techniques: Binding Assay, Expressing, Mutagenesis, Labeling

Journal: Virus Evolution

Article Title: Deep mutational scanning of SARS-CoV-2 Omicron BA.2.86 and epistatic emergence of the KP.3 variant

doi: 10.1093/ve/veae067

Figure Lengend Snippet: Epistatic shifts in mutational effects on ACE2 binding. (a) Epistatic shift in the effects of mutations on ACE2 binding at each RBD position as measured in the Wuhan-Hu-1 [previously reported in ( Starr et al. 2022a )] or BA.2.86 background compared to those previously measured in Omicron BA.2 ( Starr et al. 2022b ). Shaded gray bars indicate sites of strong antibody escape, as defined in Fig. 2A . (b) Mutation-level plots of epistatic shifts between BA.2 and BA.2.86 at sites of interest. Each scatterplot shows the measured ACE2-binding affinity of each amino acid (plotting character) in the BA.2.86 versus BA.2. backgrounds. Horizontal and vertical red dashed lines mark the wildtype RBD affinities on each axis, and the diagonal gray dashed line indicates the additive (non-epistatic) expectation. Interactive plots enabling the comparison of all SARS-CoV-2 variants and scatterplots for all RBD sites are available at https://tstarrlab.github.io/SARS-CoV-2-RBD_DMS_Omicron-EG5-FLip-BA286/epistatic-shifts/ .

Article Snippet: Induced cells were washed with phosphate buffered saline supplemented with bovine serum albumin (PBS-BSA , BSA 0.2 mg/l), split into 16-OD*ml aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 −6 to 10 −13 m at 1−log intervals, plus a 0 m sample.

Techniques: Binding Assay, Mutagenesis, Comparison

Journal: Virus Evolution

Article Title: Deep mutational scanning of SARS-CoV-2 Omicron BA.2.86 and epistatic emergence of the KP.3 variant

doi: 10.1093/ve/veae067

Figure Lengend Snippet: Epistatic emergence of the KP.3 variant . (a) Cladogram showing relationships among select SARS-CoV-2 Omicron variants, with amino acid substitutions at positions 455, 456, and 493 indicated (other mutations not shown). (b) Triple mutant cycle diagram illustrating epistatic interactions between L455S, F456L, and Q493E underlying KP.3 variant evolution. Transparent points indicate duplicate measurements of each variant’s binding strength for human ACE2 (determined as the EC50 from titrations of monomeric human ACE2 over yeast-displayed RBD variants), and solid points and lines connect the averaged binding values for each genotype. Red-orange lines highlight the impact of introducing the Q493E mutation in different sequence backgrounds. Asterisk indicates expected triple-mutant binding affinity assuming additivity of the single-mutant effects as measured in the BA.2.86 wildtype background. (c) Subset of the sarbecovirus RBD sequence alignment showing unique combinations of residues at positions 455, 456, and 493 that have evolved across different sarbecoviruses. Sequence names are colored according to RBD phylogenetic clade as in Starr et al. (2022c ).

Article Snippet: Induced cells were washed with phosphate buffered saline supplemented with bovine serum albumin (PBS-BSA , BSA 0.2 mg/l), split into 16-OD*ml aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 −6 to 10 −13 m at 1−log intervals, plus a 0 m sample.

Techniques: Variant Assay, Mutagenesis, Binding Assay, Sequencing

Journal: Nature Communications

Article Title: Histone H3K36me2 and H3K36me3 form a chromatin platform essential for DNMT3A-dependent DNA methylation in mouse oocytes

doi: 10.1038/s41467-022-32141-2

Figure Lengend Snippet: a Representative images of germinal vesicles of FGOs immunostained for H3K36me2 ( n = 14, from two independent experiments). Other representative images are also shown in Supplementary Fig. . An arrowhead indicates relatively strong H3K36me2 signals near the nuclear envelope. The maximum intensity projection images are shown. DAPI 4’,6-diamidino-2-phenylindol. Scale bar, 10 μm. b Genome browser snapshots showing H3K36me2 and H3K36me3 enrichment and CG methylation in FGOs. The upper and lower panels show the representative regions of chromosomes 15 and X, respectively. H3K36me2 and H3K36me3 enrichment are indicated by ChIP/input. c Violin plots showing H3K36me2 enrichment in 5 Mb bins in autosomes ( n = 502) and X chromosome ( n = 35). Boxplots show median value and 25–75th percentiles, and whiskers show 1.5× interquartile range from the box. Source data are provided as a Source Data file. d Violin plots showing CG methylation levels of 10 kb bins in autosomes ( n = 238,084) and the X chromosome ( n = 15,494). The methylation level was determined in 10 kb bins. Horizontal bars indicate mean values. Pie charts show percentages of 10 kb bins categorized as HMRs, MMRs, and LMRs. e Violin plots showing CG methylation levels of 10 kb bins across the genome ( n = 253,578), H3K36me2-enriched regions (ChIP/input ≥ 1.4, n = 30,234), and H3K36me3-enriched regions (ChIP/input ≥ 1.5, n = 28,655). Horizontal bars indicate mean values. Pie charts show percentages of 10 kb bins categorized as HMRs, MMRs, and LMRs.

Article Snippet: The total number of reads mapped to the spike-in chromatin (SNAP-ChIP-Kmet Stat Panel, EpiCypher) was used to normalize the read counts in samples of different genotypes (Supplementary Table ). (The number of reads from the spike-in was not sufficient for H3K36me2 ChIP-seq replicate 2; therefore, normalization was performed for replicate 1 only).

Techniques: Methylation

Journal: Nature Communications

Article Title: Histone H3K36me2 and H3K36me3 form a chromatin platform essential for DNMT3A-dependent DNA methylation in mouse oocytes

doi: 10.1038/s41467-022-32141-2

Figure Lengend Snippet: a Representative images of control and K36M FGOs immunostained for H3K36me2 (left) and plots showing their signal intensities (right). Expression of HA-tagged H3.3K36M (H3.3K36M-HA) was confirmed by HA immunostaining. Signal intensity was measured in the control ( n = 10) and K36M FGOs ( n = 10) from two independent experiments. Scale bar, 10 μm. P -values (two-tailed Mann–Whitney U tests) are indicated. Error bars indicate the mean ± SD. Source data are provided as a Source Data file. b Heatmaps showing genome-wide H3K36me2 (left) and H3K36me3 enrichment (right) in 10 kb bins in control and K36M FGOs. Enrichment values were normalized using the spike-in control. c Genome browser snapshots showing H3K36me2 and H3K36me3 enrichment and CG methylation in control and K36M FGOs. A representative region on the X chromosome. CG methylation is lost upon H3K36me2 depletion in the regions highlighted in violet. The enrichment values were ChIP/input. d Scatter plots showing CG methylation levels in control and K36M FGOs. Fifty thousand randomly selected 10 kb bins were plotted with a color gradient for H3K36me2 enrichment in control FGOs. e Violin plots showing CG methylation levels in control and K36M FGOs across the genome (left), autosomes (middle), and X chromosome (right). Horizontal bars indicate mean values. Pie charts show percentages of 10 kb bins categorized as HMRs, MMRs, and LMRs. f Boxplots showing CG methylation differences between K36M and control FGOs in individual chromosomes. CG methylation differences were determined in 10 kb bins ( n = 19,191, 17,638, 15,612, 15,010, 14,673, 14,592, 13,734, 12,541, 12,115, 12,685, 11,859, 11,646, 11,673, 11,848, 10,067, 9495, 9155, 8737, and 5813 bins for chromosome 1, 2, …, and 19, respectively, and n = 15,494 bins for chromosome X). The box shows the median value and 25–75th percentiles, and whiskers show 1.5× interquartile range from the box. g Genome browser snapshots showing H3K36me2 enrichment in control FGOs and changes in CG methylation in K36M FGOs. CG methylation differences between K36M and control FGOs (∆CGme) are indicated. The upper and lower panels indicate the representative regions of chromosomes 15 and X, respectively. H3K36me2 enrichment is indicated by ChIP/input.

Article Snippet: The total number of reads mapped to the spike-in chromatin (SNAP-ChIP-Kmet Stat Panel, EpiCypher) was used to normalize the read counts in samples of different genotypes (Supplementary Table ). (The number of reads from the spike-in was not sufficient for H3K36me2 ChIP-seq replicate 2; therefore, normalization was performed for replicate 1 only).

Techniques: Control, Expressing, Immunostaining, Two Tailed Test, MANN-WHITNEY, Genome Wide, Methylation

Journal: Nature Communications

Article Title: Histone H3K36me2 and H3K36me3 form a chromatin platform essential for DNMT3A-dependent DNA methylation in mouse oocytes

doi: 10.1038/s41467-022-32141-2

Figure Lengend Snippet: a Heatmaps showing H3K36me3, H3K36me2, and H3K27me3 enrichment and CG methylation levels in control and K36M FGOs. CG methylation differences between K36M and control FGOs (ΔCGme). 10 kb bins from the whole genome were grouped into five clusters based on H3K36me2 and H3K36me3 enrichment statuses in control FGOs. b Plots showing H3K36me2 enrichment around genic regions in control and K36M FGOs. The analysis was performed for all genes (left, n = 23,081) and those with H3K36me2 loss (right, n = 2407). c Plots showing H3K27me3 enrichment around genic regions in control and K36M FGOs. The analysis was performed for all genes (left, n = 23,081) and those with H3K36me2 loss (right, n = 2407). d MA plots showing changes in gene expression between K36M and control FGOs (left, n = 3) and between K36M oocyte-derived and control late two-cell embryos (right, n = 8). Differentially expressed genes with false discovery rate (FDR) < 0.05, are colored in red. CPM, counts per million. e Boxplots showing expression levels of all genes ( n = 23,081) and genes with H3K36me2 loss ( n = 2407) in control FGOs (left) and two-cell embryos (right). Genes with H3K36me2 loss included weakly expressed genes, such as olfactory receptor ( Olfr ) and vomeronasal receptor ( Vmnr ) genes (Supplementary Data ). The box shows the median value and 25–75th percentiles, and whiskers show 1.5× interquartile range from the box.

Article Snippet: The total number of reads mapped to the spike-in chromatin (SNAP-ChIP-Kmet Stat Panel, EpiCypher) was used to normalize the read counts in samples of different genotypes (Supplementary Table ). (The number of reads from the spike-in was not sufficient for H3K36me2 ChIP-seq replicate 2; therefore, normalization was performed for replicate 1 only).

Techniques: Methylation, Control, Gene Expression, Derivative Assay, Expressing

Journal: Nature Communications

Article Title: Histone H3K36me2 and H3K36me3 form a chromatin platform essential for DNMT3A-dependent DNA methylation in mouse oocytes

doi: 10.1038/s41467-022-32141-2

Figure Lengend Snippet: a Scatter plots showing CG methylation levels in control and Setd2 KO FGOs. Fifty thousand randomly selected 10 kb bins were plotted. b Heatmaps showing H3K36me3, H3K36me2, H3K27me3, and H3K4me3 enrichment and CG methylation levels in 10 kb bins in control and Setd2 KO FGOs , . CG methylation differences between Setd2 KO and control FGOs (ΔCGme). The data were sorted in the same order as in Fig. . c Heatmaps showing H3K4me3 enrichment in 10 kb bins in control and Mll2 KO FGOs . d Violin plots showing CG methylation differences in 10 kb bins of cluster 4 ( n = 45,161) between Setd2 KO and control FGOs (left) or between Mll2 KO and control FGOs (right) . Boxplots show median value and 25–75th percentiles, and whiskers show 1.5× interquartile range from the box. e Scatter plots showing CG methylation levels in control and Setd2 KO FGOs across the genome (left), autosomes (middle), and X chromosome (right). Fifty thousand (left) or ten thousand randomly selected 10 kb bins (middle and right) were plotted with a color gradient for H3K36me2 enrichment in Setd2 KO FGOs .

Article Snippet: The total number of reads mapped to the spike-in chromatin (SNAP-ChIP-Kmet Stat Panel, EpiCypher) was used to normalize the read counts in samples of different genotypes (Supplementary Table ). (The number of reads from the spike-in was not sufficient for H3K36me2 ChIP-seq replicate 2; therefore, normalization was performed for replicate 1 only).

Techniques: Methylation, Control

Journal: Nature Communications

Article Title: Histone H3K36me2 and H3K36me3 form a chromatin platform essential for DNMT3A-dependent DNA methylation in mouse oocytes

doi: 10.1038/s41467-022-32141-2

Figure Lengend Snippet: a Scatter plots showing CG methylation levels in the control and DM FGOs. Fifty thousand randomly selected 10 kb bins were plotted. b Violin plots showing CG methylation levels in 10 kb bins across the genome in control, Setd2 KO, DM , and Dnmt3a KO FGOs. Horizontal bars indicate mean values. c Scatter plot showing CG methylation levels in Dnmt3a KO and DM FGOs. Fifty thousand randomly selected 10 kb bins were plotted. d Genome browser snapshots showing CG methylation levels in control, Setd2 KO, DM , and Dnmt3a KO FGOs. The upper and lower panels show representative regions from chromosomes 13 and X, respectively. e Western blotting detecting DNMT3A and DNMT3L proteins in DM FGOs. Representative images from three biological replicates. Pan-H3 was used as the loading control. Source data are provided as a Source Data file. f Violin plots showing CG methylation differences between Dnmt3a KO and control FGOs in 10 kb bins of DM affected ( n = 59,487) and DM unaffected regions ( n = 450). Boxplots show median value and 25–75th percentiles, and whiskers show 1.5× interquartile range from the box. g Boxplots showing CG methylation levels in nongrowing oocytes in 10 kb bins of DM affected ( n = 59,487) and DM unaffected regions ( n = 450). The box shows the median value and 25–75th percentiles, and whiskers show 1.5× interquartile range from the box. h A model summarizing H3K36me2/3-dependent CG methylation in mouse oocytes. H3K36me2 and H3K36me3 facilitate CG methylation in MMRs and HMRs, respectively (top). Upon H3K36me2 depletion, CG methylation is lost in MMRs (second), but upon H3K36me3 depletion, CG methylation is lost in H3K36me3-enriched regions and gained in H3K36me2-enriched regions (third). When both H3K36me2 and H3K36me3 are lost, CG methylation is severely lost (bottom). Open and filled circles indicate unmethylated and methylated CG sites, respectively.

Article Snippet: The total number of reads mapped to the spike-in chromatin (SNAP-ChIP-Kmet Stat Panel, EpiCypher) was used to normalize the read counts in samples of different genotypes (Supplementary Table ). (The number of reads from the spike-in was not sufficient for H3K36me2 ChIP-seq replicate 2; therefore, normalization was performed for replicate 1 only).

Techniques: Methylation, Control, Western Blot