Journal: Folia microbiologica

Article Title: Unusual oral mucosal microbiota after hematopoietic cell transplantation with glycopeptide antibiotics: potential association with pathophysiology of oral mucositis.

doi: 10.1007/s12223-018-0596-1

Figure Lengend Snippet: Fig. 2 DGGE profiles of amplified 16S rDNA from mucosal bacterial samples of HCT patients. DGGE profiles of amplified 16S rDNA from mucosal bacterial samples of HCT patients are shown. a Quinolone prophylaxis and/or β- lactam monotherapy group. b β- Lactam-glycopeptide combination therapy group. bfr, before HCT sample; aft, after HCT sample. Intense bands (a a– ao, b a–z) were sequenced, and bacterial species identified from the sequence data are shown in Table 2. M, marker

Article Snippet: For DGGE analyses, the amplicons and DGGE marker I (Nippon Gene, Tokyo, Japan) were electrophoresed using a D-code DGGE complete system (Bio-Rad, Hercules, CA) operated at 60 °C for 12 h at 100 V in a linear 25–65% denaturing agent gradient (100% denaturing agent consisted of 7mol/L urea and 40% deionized formamide) with 8% polyacrylamide gels (polyacrylamide gel, ratio of acrylamide HG (Wako Pure Chemical Industries, Osaka, Japan) to bisacrylamide (Wako Pure Chemical Industries), 37.5:1).

Techniques: Amplification, Glycoproteomics, Sequencing, Marker

Journal: Communications Biology

Article Title: Large libraries of single-chain trimer peptide-MHCs enable antigen-specific CD8+ T cell discovery and analysis

doi: 10.1038/s42003-023-04899-8

Figure Lengend Snippet: a Workflow for SCT-facilitated capture of SARS-CoV-2 antigen-specific CD8 + T cells, single-cell TCR sequencing, and TCR cloning into autologous T cells (created with BioRender.com). b SCT tetramer-positive CD8 + T cells from HLA-matched COVID participants for SCT libraries ( n = 1). c Frequency of unique TCR clonotypes against peptides whose SCTs produced high % tetramer binding (red boxes of ( b )). d Representative flow cytometry plots of T cell cloning workflow. Autologous T cells ( d.i ), T cells after CRISPR-mediated TCR knockout ( d.ii ), tetramer+ T cells after lentiviral TCR cloning ( d.iii ), tetramer+ T cells after sort and REP ( d.iv ). Peptide sequences are found in Supplementary Tables – . A2 A*02:01, A24 A*24:02, B7 B*07:02, PLpro papain-like protease, P PLpro, S spike, PE phycoerythrin, ACN allophycocyanin, REP rapid expansion protocol.

Article Snippet: To probe the TCR repertoire of the SARS-CoV-2-specific T cells, selected expanded populations were stained with SCT-loaded DNA-barcoded dCODE dextramers (Immudex) to enable pairing of the SCT capture reagent with specific TCR clonotypes through 10× single-cell sequencing (Fig. ).

Techniques: Sequencing, Clone Assay, Produced, Binding Assay, Flow Cytometry, CRISPR, Knock-Out

Journal: medRxiv

Article Title: Human SARS-CoV-2 challenge resolves local and systemic response dynamics

doi: 10.1101/2023.04.13.23288227

Figure Lengend Snippet: ( a ) Gating strategy used to enrich SARS-CoV-2 antigen specific T cells via MACSQuant Tyto cell sorting. Cells were sequentially stained with a multi-allele panel of dCODE dextramer-PE complexes, with the addition of anti-human CD3-APC and CD14-FITC FACS antibodies as references to help us identify T cell specific binding. Debris and cell aggregates were gated out first using BSB-H (backscatter blue laser-height) SSC-H (side scatter-height). From the cells, DAPI+ dead cells were excluded. T cells (CD3+) and monocytes (CD14+) were then gated for (CD3+ and\or CD14+ population) and the sort gate defined from this population as all PE-dCODE Dextramer® positive cells. This lenient sorting strategy was decided upon in order to collect enough cells for 10X 5’ single cell analysis downstream and to ensure we were capturing all SARS-CoV-2 antigen specific cells. Any non-specific binding (e.g. to monocytes) and background noise could then be removed computationally. ( b ) Calculated quality control metrics for PE-dCODE Dextramer® positive sequenced cells showing gene reads per cell per sample. ( c ) Proportions of activated T cells bound to Dextramers loaded with selected SARS-CoV-2 antigens. The total amount of bound cells to each Dextramer is shown, color-coded by predicted cell state. If barcodes from several Dextramers were detected to be bound to the same cell, we only selected the Dextramer with the highest signal as bound. As a control to separate background and real binding, cells are separated based on the HLA haplotype compatibility with the tested Dextramer. Only Dextramers with at least 10 HLA matched bound cells are shown. FDR corrected p values were determined by a Fisher-exact test comparing the proportion of HLA matched activated T cells in the Dextramer bound cells to the proportion of unbound HLA matched activated T cells. N represents the number of cells in each bar. The right-most bar represents the overall distribution of cell types across all Dextramer experiments.

Article Snippet: In order to further validate and investigate the SARS-CoV-2 antigen-specific T-cell populations in our single cell dataset Day 10, 14 and 28 post-inoculation PBMCs samples, from all 16 participants, were further enriched and processed for single cell sequencing using a multi-allele panel of 44 SARS-CoV-2 antigen specific dCODETM Dextramer® (10x compatible) (Immudex, see Extended Data Table 1k for full panel).

Techniques: FACS, Staining, Binding Assay, Single-cell Analysis, Control