Journal: Chemical Science

Article Title: Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives

doi: 10.1039/d2sc06665c

Figure Lengend Snippet: Examples of commercial molecular diagnostic assays for the detection of SARS-CoV-2 a

Article Snippet: Agena Bioscience, Inc. , MassARRAY SARS-CoV-2 Panel , rRT-PCR, MALDI-TOF , N gene (N1, N2, and N3 regions), ORF1, and ORF1ab , Nasopharyngeal swabs, oropharyngeal swabs, nasal and mid-turbinate swabs , 310–2500 copies per mL , 98.9% , 100% , H , .

Techniques: Diagnostic Assay, Amplification, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Microarray, Hybridization, Kinetic Assay, Helicase-dependent Amplification

Journal: Chemical Science

Article Title: Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives

doi: 10.1039/d2sc06665c

Figure Lengend Snippet: Comparison of the three types of SARS-CoV-2 antibody assay diagnostic kits

Article Snippet: Agena Bioscience, Inc. , MassARRAY SARS-CoV-2 Panel , rRT-PCR, MALDI-TOF , N gene (N1, N2, and N3 regions), ORF1, and ORF1ab , Nasopharyngeal swabs, oropharyngeal swabs, nasal and mid-turbinate swabs , 310–2500 copies per mL , 98.9% , 100% , H , .

Techniques: Comparison, Diagnostic Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, High Throughput Screening Assay

Journal: Chemical Science

Article Title: Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives

doi: 10.1039/d2sc06665c

Figure Lengend Snippet: Types of SARS-CoV-2 pseudovirus neutralizing tests

Article Snippet: Agena Bioscience, Inc. , MassARRAY SARS-CoV-2 Panel , rRT-PCR, MALDI-TOF , N gene (N1, N2, and N3 regions), ORF1, and ORF1ab , Nasopharyngeal swabs, oropharyngeal swabs, nasal and mid-turbinate swabs , 310–2500 copies per mL , 98.9% , 100% , H , .

Techniques: Plasmid Preparation, Expressing, Virus, Luciferase, Mutagenesis, Clinical Proteomics, Bioprocessing

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Analysis of the correlation between FUS protein and myocardial infarction. (a) Enrichment Analysis Bar Plot based on differential gene expression profiles in lncRNA microarray analysis.(b) Detection information about lncRNA LOC101928697 binding to FUS proteins in AnnoLnc2 database. (c) Detection information about lncRNA LOC101928697 binding to FUS protein in RBPDP database. (d) Scores in the RPISeq database on the model of lncRNA LOC101928697 binding to FUS protein. (e-g) Prediction information about lncRNA LOC101928697 binding to FUS protein in catRAPID website, (e) Statistical map information about protein and RNA binding sites, (f) Total scoring information, and (g) Interaction map showing the interaction region between protein and RNA. (h-i) Analyses about bioinformatics techniques based on GSE163772 in the GEO database, where (h) is a statistical map of FUS gene expression in endothelial cells of a mouse model of myocardial infarction, and (i) A scatter plot about the correlation between the level of FUS gene expression and the disease state (control vs. myocardial infarction).

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Gene Expression, Microarray, Binding Assay, RNA Binding Assay, Control

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Interaction of exosomal lncRNA LOC101928697 with FUS proteins. (a and b) The western blot detection of FUS protein expression in each group of cells and the statistical graph. (c) Statistical graph of RT-qPCR to detect the expression of FUS at the mRNA level in each group of cells. (d) The fluorescence graph of fluorescence in situ hybridization (FISH) experiment. In which FUS was labeled with green fluorescence, lncRNA LOC101928697 was labeled with red fluorescence, and the nucleus was labeled with blue fluorescence (20×). (e) Western blot detection of FUS protein following RNA pull-down using sense or antisense LOC101928697 transcripts. (f) Quantification of FUS protein enrichment in sense RNA pull-down versus antisense control, based on densitometric analysis. (g-h) Western blot detection of FUS protein expression in each group of cells after knockdown or overexpression of lncRNA LOC101928697 and the statistical graphs. (i) Statistical graph of mRNA level expression of FUS in each group of cells after knockdown or overexpression of lncRNA LOC101928697 by RT-qPCR assay. a p < 0.05 compared to control group. b p < 0.05 compared to exosome group. c p < 0.05 compared to siRNA + exosome group.

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Labeling, Protein Enrichment, Control, Knockdown, Over Expression

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a) Distribution of annotated single hits over MEG3 gene, with statistically filtered EZH2-FLASH reads from two biological replicates in HUVECs. b) The occupancy of EZH2 hits over MEG3 features. Total reads per feature are given with exons being mostly occupies vs introns. c) Proportion of overlapping features over MEG3. The occupancy of EZH2 over each MEG3 exon is shown for two constitutively expressed transcripts. For both given transcripts there is high occupancy of exon 3. d) RNA immunoprecipitation (RIP) for EZH2 and H3K27me3 (repressive chromatin) followed by qPCR analysis. RIP-purified RNA from UV crosslinked HUVECs was used to prepare cDNA for qPCR analysis with primers against MEG3 (exon 3 region). Primers against U1snRNA gene serves as a negative control. Side diagram of EHZ2-MEG3 interacting region is charted as per FLASH hits and sequence. e) Distribution of EZH2 hybrids hits over MEG3 gene. Intermolecular MEG3-RNA interactions found in chimeras are captured by EZH2-FLASH-seq. Hits represent MEG3:MEG3 hybrids (black). IgG hybrids are plotted but are <1. f) Total MEG3:MEG3 hybrid count against predicted free energy of hybridization (dG) for MEG3 interactions ( red lncRNA:MEG3, blue mRNA:MEG3, green MEG3:antisense, purple snoRNA:MEG3) with free hybridization energy cutoff at dG<-10 kcal mol -1 , as captured by EZH2-FLASH-seq ( i ) vs. IgG control ( ii ) .

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Immunoprecipitation, Purification, Negative Control, Sequencing, Hybridization, Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Overview of the critical steps to obtain MEG3-bound genomic loci and intersections with EZH2 and H3K27me3 signals (obtained from GEO databases for HUVECs). In addition, enhancer regions were mapped within the genomic tracks. The intersection between GEO EZH2 ChIP data, GEO H3K27me3 ChIP data and statistically filtered MEG3-ChIRP data from two biological replicates was performed. The number of genes and degree of overlap is obtained between MEG3 and PRC2-dependent genes. The p-values are a result of hypergeometric test. b. Distribution of MEG3 peaks overlapping EZH2-ChIP peaks or H3K27me3-peaks with intersecting reads in relation to (i) gene regions and (ii) gene-type. c. Maximum peak score of ChIP signal for EZH2 and H3K27me3 intersecting the top enriched MEG3 peaks associated with nearest genes. Highest EZH2 peak score is over ITGA4, whereas H3K27me3 was detected in ITGA4, ITGA7, ITGA8 and ITGA9, members of ITGA family. d. Normalized reads from RNA-seq de novo analysis of GEO: GSE71164 dataset on Hg38, and expression of ITGA4 gene between Scr and siEZH2 depleted HUVECs, showing that ITGA4 is targeted by EZH2. Dataset in d and e is compared using Student’s t-test. e. ITGA4 expression from microarray analysis in C2C12 cells depleted of MEG3 (10nM, LNA GapMer) as per GEO dataset: GSE73524. The data shows that ITGA4 is a direct target of MEG3. f. (i) Total number of representable peaks (mRNA, antisense and lncRNA genes) from ChIP-seq analysis of Scr vs. MEG3 KD HUVECs. (ii ) Depletion of MEG3 gene in HUVECs (10nM LNA gapmers) was achieved with relative expression showing ∼70% reduction compared with Scr control. g. (i) Heat map showing distribution of reads and EZH2 densities at all unique RefSeq genes within TSSs ± 3 kb, sorted by EZH2 occupancy, in Control vs. MEG3 deficient (10nM) HUVECs. (ii) Overlap of ChIP-results between MEG3 and EZH2-dependent genes, with overlapped genes belonging to the biological pathway regulating cell adhesion. The common targets had lost or reduced EZH2 ChIP-signal.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Sequencing, Expressing, Microarray, ChIP-sequencing, Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a) Computational analysis pipeline used to obtain orthologous peaks in human and intersect regions and genes enriched in repressive chromatin (H3K27me3) from ChIP-seq public dataset GSE114283. Up- and down-regulated genes were obtained associated with the peak region within 2000bp, and relevant function and biological pathway were associated using GREAT and DAVID analysis b) Overlap of the GEO datasets from a (Microarray GSE73524 ) and b (RNA-seq GSE71164 ) and the GSE114283 ChIP-seq reads of H3K27me 3 distribution in mouse MN cells depleted of MEG3 vs. control. ChIP extracted peaks unique to Ctrl vs. MEG3 KD were obtained, and associated mouse gene list composed based on reduction in H3K27me 3 signal. Using gene orthologous analysis in gProfiler we obtained human orthologous targets that was used for data intersection. c) Maximum peak scores of the overlapping signal over ITGA4 promoter, obtained by intersection of EZH2 ChIP signal with MEG3-ChIRP signal at this region. Upon depletion of MEG3 the EZH2 signal is significantly reduced whereby no overlap with MEG3 ChIRP signal is seen. d) Relative expression of ITGA4 in HUVEC measuring the levels of ITGA4 following addition of siRNA (50nM).

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: ChIP-sequencing, Microarray, RNA Sequencing, Control, Expressing

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Venn diagram showing the intersection between statistically filtered FLASH data from two biological replicates of our MEG3-ChIRP-seq-data (green), de novo hg38 analysed GEO RNA-seq data from siEZH2 deficient HUVECs (GSE71164, blue), and EZH2 ChIP-seq following MEG3 KD (yellow) and FLASH-seq transcriptome following EZH2 IP (pink). b. Correlation between gene expression levels and FLASH signal. Gray, expressed RefSeq genes with reproducible FLASH signal consistently detected in RNA-seq. Blue, genes with the highest RNA-seq signals and no reproducible FLASH signal belonging to integrin cell surface interaction pathway. Red , expressed ITGA4 gene, and green, ITGB1 gene, without reproducible FLASH signals. Data are from two biological replicates of each EZH2 FLASH sample and three biological replicates of EZH2 RNA-seq samples (Scr vs. siEZH2, GSE71164). c. Genomic tracks showing ChIRP-seq signal (MEG3 Odd, Even and LacZ) in HUVECs over ITGA4 gene only. The MEG3 binding site is located upstream of the ITGA4 gene in the promoter region, and it overlaps with the H3K27me3 signal and EZH2; as well as downstream within the ITGA4 gene body, where it overlaps with within the EZH2 signal in the intronic region of the gene. d. MEG3-ChIRP followed by qPCR, analysis of MEG3 binding region on ITGA4 in HUVECs. The crosslinked cell lysates were incubated with combined biotinylated probes against MEG3 lncRNA and the binding complexes recovered by magnetic streptavidin-conjugated beads. The qPCR was performed to detect the enrichment of specific region that associated with MEG3, peaks were related to input control and compared vs. the non-biotynilated control. e. ChIP-QPCR enrichment for EZH2 and H3K27me3 over ITGA4 promoter region in HUVECs depleted of MEG3 vs. Control.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Sequencing, ChIP-sequencing, Gene Expression, Binding Assay, Incubation, Control, ChIP-qPCR

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. ChIP signal enrichment vs . 1% input for EZH2 and H3K27me3 mark over ITGA4 promoter regions in HUVECs treated with A-395 (5µM, 24h) inhibitor of PRC2 vs. Control (DMSO). The expression was measured using two sets of primers against the same promoter region of ITGA4. Representative graphs are average of three qPCR datasets ± SEM. b. ITGA4 expression in the presence of A-395 vs . DMSO control, N=6 independent experiments compared using t -test. c. Measuring the expression levels of ITGA4 upon depletion of MEG3 using LNA GapmeRs (10nM, 48h), data is mean of N=5 independent experiments (biological replicates). d. Representative image of immunofluorescence staining for ITGA4 protein levels in ECs treated with A-395 vs . DMSO, or upon MEG3 depletion like in b . e. Intra-cellular localisation of MEG3 (chromatin associated lncRNA) between different cellular compartments in HUVECs treated with A-395 vs. DMSO, whereby the distribution of MEG3 has shifted upon PRC2 inhibition with A-395; from the nucleus (where it was highly chromatin bound) into the cytoplasm. Representative bars were compared by t-test and on-way Anova. f. MEG3-ChIRP followed by qPCR, N =3, analysis of MEG3 binding over ITGA4 promoter region in HUVECs treated with A-395 (5µM, 24h) vs. DMSO. MEG3-ChIRP HUVEC lysates treated with A-395 resulted in reduced engagement of MEG3 with ITGA4 site compared with either DMSO control or ChIRP with non-biotinylated probes. The non-biotin probes served as a negative control, and we detected the background level <1.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: Control, Expressing, Immunofluorescence, Staining, Inhibition, Binding Assay, Negative Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Measure of cell migratory capacity using ECIS functional analysis in ECs treated with control or A-395 (5µM, 24h) inhibitor. Experiments were performed in duplicates (technical replicates) and four experiments were run for migration assay and six for adhesion (biological replicates). The data showing ECIS trace (left hand side) is mean ±SD as calculated by the ECIS. The graph on the right is mean±SEM with N =6, data was compared using ordinary one-way ANOVA with Dunnett’s multiple comparisons tests. b. Adhesion to Fibronectin, FN (20µg/ml) was used to coat the culture plates and assess adhesion of endothelial cells within 3h of ECIS assay, following cell pre-treatment with A-395, 24h. The difference in resistance change was calculated over 3h. c. Subcutaneous Matrigel plug injection (200µl) into mice ( N =5) treated with DMSO (control, left flange) and A-395 (1mg/ml, right flange) was done for 2 weeks. Matrigel plugs were collected and processed for histology. Staining for H3K27me3 was done, displaying nuclear positivity with strong intensity in control (<0.02% DMSO in water) and the A-395 treatment decreased total H3K27me3 staining, as compared by t-test. d. Staining for arterioles was performed to assess vessel growth as angiogenesis and data was compared using Student’s t-test. The data shows increased area of staining for Isolectin B4 (Iso-B4) dye in A-395 vs. DMSO treated Matrigel plugs with increased neovascularization, P<0.05. e. A-395 has increased the percentage of vessels positive for ITGA4 (red) within the Isolectin B4 positive cells, compared with the DMSO using t -test. f. Graphical abstract. 1 Maternally Expressed Gene–MEG3 is highly expressed with hypoxia and bound to EZH2 in endothelial cells (EC) affected by ischaemic insult. 2 Such MEG3:EZH2 complex assembles onto the target genes to 3 direct the EZH2 activity to “write” H3K27me3 trimethylation repressive mark and block expression of target gene i.e. integrin alpha 4 (ITGA4) and its ability to dimerise with integrin beta 1 (ITGB1), leading to 4 reduced EC function as measured by adhesion and migration. Hence 5 targeted disruptions of MEG3:EZH2 interaction, or inhibition of EZH2 activity could increase EC function under ischaemia.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: Functional Assay, Control, Migration, Injection, Staining, Activity Assay, Blocking Assay, Expressing, Inhibition