Journal: Scientific Reports

Article Title: T cell immunity to Zika virus targets immunodominant epitopes that show cross-reactivity with other Flaviviruses

doi: 10.1038/s41598-017-18781-1

Figure Lengend Snippet: CD4 T–cell epitopes to Zika envelope protein in HLA class II transgenic mice. Mice transgenic for HLA-DR1 (DRB1*0101) n = 6 ( A ); HLA-DR4 (DRB1*0401) n = 5 ( B ); HLA-DR15:01 (DRB1*1501) n = 4 ( C ) and HLA-DQ8 (DQB1*0302) n = 6 ( D ) were primed with 25 μg of recombinant zika envelope protein (Env). 10 days after immunisation, draining lymph node cells (DLNs) were assayed by IFNγ ELISpot for recall responses to the Env protein and to an overlapping panel of 50 Env peptides. Data are plotted as number of spot forming cells (SFC) per 10 6 cells for individual mice. Responses were considered positive (+) if the response was greater than 2 SD above the mean of the response in the absence of any antigen (shown as horizontal dotted line). Peptides that were defined as positive epitopes across 3 or more HLA class II transgenic mouse lines are highlighted.

Article Snippet: The frequency of cells producing IFNγ in response to antigen was quantified by ELISpot using a murine IFN gamma ELISpot set kit (862.031.020, Diaclone; 2B Scientific, Oxon, U.K) – .

Techniques: Transgenic Assay, Recombinant, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: T cell immunity to Zika virus targets immunodominant epitopes that show cross-reactivity with other Flaviviruses

doi: 10.1038/s41598-017-18781-1

Figure Lengend Snippet: T cells responding to ZIKV envelope protein peptide 1 are crossreactive with variant peptides from other flavivirus species. Mice transgenic for ( A ) HLA-DR1 (DRB1*0101) n = 6, ( B ) HLA-DR15:01 (DRB1*1501) n = 6 and ( C ) HLA-DQ8 (DQB1*0301) n = 6 were primed with 25 μg of recombinant ZIKV envelope protein (Env). 10 days after immunisation, DLNs were assayed by IFNγ ELISpot for recall responses to the Env protein, Env peptide 10, Env peptides as shown and the corresponding peptide variants of west nile virus (WNV), yellow fever virus (YFV), dengue virus 1 (D1), dengue virus 2 (D2), dengue virus 3 (D3) and dengue virus 4 (D4). Data are plotted as number of spot forming cells (SFC) per 10 6 cells for individual mice. Responses were considered positive (+) if the response was greater than 2 SD above the mean of the response in the absence of any antigen (shown as horizontal dotted line). HLA-DR1 ELISpot supernatants were collected prior to assay development and levels of ( D ) IL-17A and ( E ) IL-10 measured by ELISA. Data shown represent mean ± SEM. Statistical significance was determined using an unpaired t-test.

Article Snippet: The frequency of cells producing IFNγ in response to antigen was quantified by ELISpot using a murine IFN gamma ELISpot set kit (862.031.020, Diaclone; 2B Scientific, Oxon, U.K) – .

Techniques: Variant Assay, Transgenic Assay, Recombinant, Enzyme-linked Immunospot, Virus, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: T cell immunity to Zika virus targets immunodominant epitopes that show cross-reactivity with other Flaviviruses

doi: 10.1038/s41598-017-18781-1

Figure Lengend Snippet: ZIKV virus infection in AG129 mice is associated with a strong T-cell response to ZIKV Env peptide 1 which is crossreactive with variant peptides from other flavivirus species. AG129 mice were (i) mock infected (n = 4) or (ii) infected intraperitoneally with 10 5 FFU of ZIKV (PF13/251013-18) (n = 5). ( A ) Mock (grey circles) and ZIKV infected (black circles) mice were monitored daily for signs of weight loss and culled at 7 days post infection. ( B ) ZIKV RNA load was quantified by real time PCR. Splenocytes from both groups of mice were assayed by IFNγ ELISpot for recall responses to ( C ) ZIKV proteins Env, NS1, NS3 and NS5, ( D ) an overlapping panel of 50 Env peptides and flavivirus variant peptides from west nile virus (WNV), yellow fever virus (YFV), dengue virus 1 (D1), dengue virus 2 (D2), dengue virus 3 (D3) and dengue virus 4 (D4) for peptides 1 ( E ) and 31 ( H ). Data are plotted as number of spot forming cells (SFC) per 10 6 cells for individual mice. Responses were considered positive (+) if the response was greater than 2 SD above the mean of the response in the absence of any antigen (shown as horizontal dotted line). ELISpot supernatants from peptide 1 variants were also collected prior to assay development and levels of IL-17A ( F ) and IL-10 ( G ) measured by ELISA. Data shown represent mean ± SEM.

Article Snippet: The frequency of cells producing IFNγ in response to antigen was quantified by ELISpot using a murine IFN gamma ELISpot set kit (862.031.020, Diaclone; 2B Scientific, Oxon, U.K) – .

Techniques: Virus, Infection, Variant Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: Heterochromatin-dependent transcription links the PRC2 complex to small RNA-mediated DNA elimination

doi: 10.1038/s44319-024-00332-1

Figure Lengend Snippet: ( A ) IES retention score distribution after Fire1/2 or TFIIS4 silencing, for IESs with an IRS > 0.1. Histogram bars with more than 10,000 IESs have been truncated for clarity. Note that the cutoff is different from Fig. . ( B ) Venn diagram depicting shared IES retention between TFIIS4 and Fire1/2 silencing, for IESs with an IRS > 0.1. ( C ) Correlation plots calculated by hexagonal binning of IES retention scores generated using After_ParTIES (Swart et al, ) and the IES retention scores provided in Dataset EV . Pearson’s correlation coefficients are given above each subgraph. Red lines are for ordinary least-squares (OLS) regression, orange lines for LOWESS, and gray lines for orthogonal distance regression (ODR). From light green to dark blue, the correlation is stronger. ( D ) Localization of TFIIS4-GFP with and without Fire1/2, as well as Fire1-GFP with and without TFIIS4. Dotted white circles denote new MACs. Scale bar: 10 μm. ( E ) Northern blots using RNA extracted from the late stage of development in EV, TFIIS4 silencing and Fire1/2 silencing cultures, probed against TFIIS4, Fire1, or the 17S rRNA as a loading control. ( F ) Venn diagram depicting shared IES retention between Dcl2/3/5 and Fire1/2 silencing, for IESs with an IRS > 0.1. ( G ) Analysis of sRNA sizes and contents (color coded) of sRNAs extracted from EV control and Fire1/2-silenced samples at the Late stage of development, plotted as a proportion of the total sRNA population. IES internally eliminated sequence, OES other eliminated sequence, MDS macronuclear destined sequence (MAC matching), TE transposable element, Vector pGEM T and L4440 vectors, Klebsiella Klebsiella pneumoniae (food source for Paramecium ). See also Fig. and Appendix Fig. S . .

Article Snippet: Illumina TruSeq small RNA kit , Illumina , Cat# RS-200-0012.

Techniques: Generated, Northern Blot, Control, Sequencing, Plasmid Preparation

Journal: EMBO Reports

Article Title: Heterochromatin-dependent transcription links the PRC2 complex to small RNA-mediated DNA elimination

doi: 10.1038/s44319-024-00332-1

Figure Lengend Snippet: ( A ) Total iesRNA counts corresponding to Fire1/2-dependent or independent IESs, with a retention score cutoff of 0.1 (left) or 0.01 (right) to denote Fire1/2-dependent IESs. ( B ) iesRNA count per IES for Fire1/2-dependent or independent IESs, with a retention score cutoff of 0.1 (left) or 0.01 (right) to denote Fire1/2-dependent IESs. The bold line denotes the median, the lower and upper hinges the 25th and 75th percentiles and the whiskers extend to the largest and smallest values no larger than 1.5 x inter-quartile range (IQR). Outliers were omitted for better visualization. Number of IESs (n) from left to right: IRS > 0.1, 14684, 30243, 14684, 30243; IRS > 0.01, 26840, 18087, 26840, 18087. For all plots, 21 to 24 and 26 to 30 nt sRNA with perfect IES-matching sequences were selected as iesRNAs, and the reads were normalized to the 23 nt siRNAs mapped to the backbone of the L4440 vector.

Article Snippet: Illumina TruSeq small RNA kit , Illumina , Cat# RS-200-0012.

Techniques: Plasmid Preparation

Journal: EMBO Reports

Article Title: Heterochromatin-dependent transcription links the PRC2 complex to small RNA-mediated DNA elimination

doi: 10.1038/s44319-024-00332-1

Figure Lengend Snippet: The PRC2 complex sets H3K9me3 and H3K27me3 in the developing new MACs, one or both of which is read by the Pc-family proteins Fire1/2. Fire1/2 acts in tight association with the transcription elongation factor TFIIS4 to generate ncRNA transcripts that are used as scaffolds for targeting by sRNAs. Targeting by sRNAs recruits the nucleosome remodeler ISWI1, resulting in nucleosome depletion on IESs. Finally, nucleosome-poor IESs can be precisely removed by the excisase Pgm.

Article Snippet: Illumina TruSeq small RNA kit , Illumina , Cat# RS-200-0012.

Techniques:

Journal: EMBO Reports

Article Title: Heterochromatin-dependent transcription links the PRC2 complex to small RNA-mediated DNA elimination

doi: 10.1038/s44319-024-00332-1

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Illumina TruSeq small RNA kit , Illumina , Cat# RS-200-0012.

Techniques: Recombinant, Construct, Expressing, Sequencing, DNA Labeling, Software, Plasmid Preparation, Centrifugation