Journal: Nature Communications

Article Title: Increased renal elimination of endogenous and synthetic pyrimidine nucleosides in concentrative nucleoside transporter 1 deficient mice

doi: 10.1038/s41467-023-38789-8

Figure Lengend Snippet: A Schematic workflow illustrating the Slc28a1 editing and genotyping strategy. B Representative DNA chromatogram illustrating the sequence alignment between the Slc28a1 reference and experimental sequence from Slc28a1 +/+ and Slc28a1 −/− mice with deleted nucleic acids indicated with a dash (-). Sequencing performed in two independent experiments. C Representative genotyping gel loaded with PCR-amplified Slc28a1 from Slc28a1 +/+ and Slc28a1 −/− with and without the Acul restriction digest. Genotyping performed in over ten independent experiments. D Relative Slc28a1 gene expression in Slc28a1 −/− mouse organs normalized to GAPDH and Slc28a1 +/+ control displayed as a fold change (2 −ΔΔCt ). Data represent mean ± SEM ( n = 3 mice/group mean ± SEM, * p < 0.05 by two-tailed t -test). Gene expression performed in two independent experiments. E Representative immunoblots of mCNT1 in Slc28a1 +/+ and Slc28a1 −/− mouse organs at 8 weeks of age with GAPDH as a loading control. Empty vector and mCNT1 overexpression (OE) controls were generated in HEK293 cells (#CRL 1573, ATCC). Immunoblotting performed in two independent experiments.

Article Snippet: Western blotting analysis was performed using an mCNT1 (Alomone Labs; ANT-061, 1:500) primary antibody with GAPDH (CST 97166, 1:5000) as a loading control.

Techniques: Sequencing, Amplification, Gene Expression, Control, Two Tailed Test, Western Blot, Plasmid Preparation, Over Expression, Generated

Journal: Nature Communications

Article Title: HIV-1 treatment timing shapes the human intestinal memory B-cell repertoire to commensal bacteria

doi: 10.1038/s41467-023-42027-6

Figure Lengend Snippet: a Dot plots comparing the serum anti-HIV-1 Env IgG (colored) and IgA (clear) antibody levels in eART (blue) and lART (red) individuals ( n = 3 per group). The y axis indicates the area under the curve (AUC) values of the ELISA binding curves shown in Supplementary Fig. . Bars correspond to the means. Samples were tested in two independent experiments. b t-SNE-based analysis comparing the subset distribution of single mucosal CD19 + cells between eART and lART donors ( n = 3; 2 × 10 5 cells per group) (top). Single-cell sorted B-cell sub-populations are shown in t-SNE plots (bottom). c Volcano plots comparing the immunoglobulin gene repertoires of intestinal antibody-secreting cells (ASC) and class-switched (CS) memory B cells ( n = 206 parameters) between e-ART (blue) and l-ART (red). Dashed lines indicate the statistically significant cut-off ( p < 0.05). d Plot showing the principal component analysis (PCA) of intestinal CS B cells in eART and lART (left). Contribution plot showing the fold changes of significantly diverging parameters between groups (right). Groups in ( c ) and ( d ) were compared using 2 × 2 Fisher’s Exact test. e Violin plots comparing the number of somatic mutations in the IgA/IgG V H ( n = 143 for eART and n = 138 for lART) and Vκ genes ( n = 96 for eART and n = 88 for lART) from single-sorted intestinal CS B cells between eART ( n = 3) and lART donors ( n = 3). f Violin plots comparing the number of somatic mutations in the IgA ( n = 12,265,476 and 12,779,331 sequences for eART and lART, respectively) and IgG V H ( n = 4,669,125 and 7,300,158 sequences for eART and lART, respectively) genes from mucosal and peripheral blood B cells analyzed by NGS between eART ( n = 7) and lART ( n = 7). The average number of mutations is indicated below each violin plot. Numbers of hypermutation in ( e ) and ( f ) were compared between groups using two-tailed unpaired Student’s t test with Welch’s correction. Bars in ( e ) and ( f ) represent the medians. g Divergence plots comparing the distribution of intestinal IgA + and IgG + B-cell sequences ( y -axis) between eART (dashed line) and lART (straight line) according to their V H -gene family and hypermutation frequencies ( x -axis). The average frequencies of mutations for eART (blue) and lART (red) are indicated in each plot. h Network visualization comparing the clonal expansion levels of intestinal of IgA + and IgG + B cells according to the somatic mutation loads between eART and lART HIV-1-infected individuals. Source data are provided as a Source Data file.

Article Snippet: Recombinant IgG antibodies (150 µg/ml final concentration) were assayed by indirect immunofluorescence assay (IFA) using the ANA HEp-2 AeskuSlides® kit (Aesku.Diagnostics) following the manufacturer’s instructions. mGO53, ED38 and kit’s control antibodies were included in each experiment.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Two Tailed Test, Mutagenesis, Infection

Journal: Nature Communications

Article Title: HIV-1 treatment timing shapes the human intestinal memory B-cell repertoire to commensal bacteria

doi: 10.1038/s41467-023-42027-6

Figure Lengend Snippet: a Plot comparing the ELISA reactivity of mucosal CS memory B-cell antibodies against HIV-1 Env gp140-F vs. p24 ( n = 200). Antibodies were tested in triplicate. b 3D dot plot comparing the polyreactive binding of mucosal CS memory B-cell antibodies from eART (blue) and lART (red) to HIV-1 proteins as measured by ELISA in Supplementary Fig. . c Violin plots comparing the ELISA polyreactivity of mucosal CS memory B-cell antibodies from eART (blue) and lART (red). The y -axes indicate the log 10 cumulative AUC (CAUC) values for polyreactivity as measured in Supplementary Fig. . Antibodies were tested in two independent experiments. Groups were compared using two-tailed Student’s t test with Welch’s correction. d Same as in ( c ) but for HEp-2-reactivitity with ELISA OD 405 nm values for the binding to HEp-2 cell antigens. Bars in ( c ) and ( d ) represent the medians. Antibodies were tested in triplicates. e Pie charts comparing the frequency of poly- and self-reactive of intestinal IgA + and IgG + CS B-cell antibodies between eART (blue) and lART (red) as measured in ( c ) and ( d ), respectively. The number of tested antibodies is indicated in the pie chart center, and the frequency of the reactive ones (colored) on the chart. Groups were compared using 2 × 2 Fisher’s Exact test. f Microscopic images showing representative antibody reactivities to HEp2-expressing self-antigens detected by indirect immunofluorescence assay (IFA) (representative from two independent experiments). Scale bars represent 15 µm. Pie charts summarizing the IFA data are shown. The number of tested antibodies is indicated in the pie chart center, and the frequency of the reactive ones (colored) on the chart. Groups were compared using 2 × 5 Fisher’s Exact test. g Microarray plots showing the reactivity profile of selected antibodies to human proteins. For each protein spot, Z -scores given by the reference (Ref: mGO53) and test antibody are depicted on the y - and x -axis, respectively. Immunoreactive proteins are indicated in each plot. Source data are provided as a Source Data file.

Article Snippet: Recombinant IgG antibodies (150 µg/ml final concentration) were assayed by indirect immunofluorescence assay (IFA) using the ANA HEp-2 AeskuSlides® kit (Aesku.Diagnostics) following the manufacturer’s instructions. mGO53, ED38 and kit’s control antibodies were included in each experiment.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Two Tailed Test, Expressing, Immunofluorescence, Microarray

Journal: Nature Communications

Article Title: HIV-1 treatment timing shapes the human intestinal memory B-cell repertoire to commensal bacteria

doi: 10.1038/s41467-023-42027-6

Figure Lengend Snippet: a Violin plots comparing the binding of purified serum IgA and IgG antibodies to gp140 between eART (blue, n = 38) and lART (red, n = 40). The mean AUC is indicated below each violin plot. Bars represent the medians. Groups were compared using two-tailed unpaired Student’s t test with Welch’s correction. Antibodies were tested in duplicate. b Graphs comparing the in vitro neutralization activity of purified serum IgGs against Bal.26 (Bal), YU2.DG (YU2) and PVO.4 (PVO) between eART ( n = 38) and lART ( n = 40). Means from triplicates from TZM-bl assay experiments are shown. Pie charts summarize the frequency of individuals with IgG-mediated neutralizing activities (>10%) against one (1), two (2) or three (3) viruses (colored). White color indicates that no neutralization was detected (0). Groups were compared using 2 × 5 Fisher’s Exact test. c Violin plots comparing the binding of serum IgA, IgA1 and IgA2 antibodies to LPS between eART (blue, n = 38) and lART (red, n = 40). Means of triplicate values are shown. d Same as in ( c ) but for purified serum IgG and IgA antibodies against selected commensal bacteria. Bars in ( c ) and ( d ) represent the medians. Groups were compared using two-tailed unpaired Student’s t test with Welch’s correction. Antibodies were tested in duplicate. e Graphs showing the reactivity profiles against commensal bacteria of purified serum antibodies (IgA, purple straight lines; IgG, purple dashed lines; n = 78 in total) and bacteria-reactive monoclonal antibodies (mucosal IgA, red line ( n = 10) and mucosal IgG, green line ( n = 5)). Data are presented as mean values ± SD. Cumulative distributions between groups were compared using the two-tailed Kolmogorov–Smirnov unpaired test. Source data are provided as a Source Data file.

Article Snippet: Recombinant IgG antibodies (150 µg/ml final concentration) were assayed by indirect immunofluorescence assay (IFA) using the ANA HEp-2 AeskuSlides® kit (Aesku.Diagnostics) following the manufacturer’s instructions. mGO53, ED38 and kit’s control antibodies were included in each experiment.

Techniques: Binding Assay, Purification, Two Tailed Test, In Vitro, Neutralization, Activity Assay, Bacteria

Journal: Nature Communications

Article Title: HIV-1 treatment timing shapes the human intestinal memory B-cell repertoire to commensal bacteria

doi: 10.1038/s41467-023-42027-6

Figure Lengend Snippet: a Density plots showing the t-SNE analysis of circulating blood IgA + and IgG + CD19 + cells from eART ( n = 5) and lART ( n = 4) (2 × 10 5 cells per group). b t-SNE plot showing the distribution of blood B-cell subsets (based on CD27 and β7 markers) in circulating blood IgA + and IgG + CD19 + cells from eART ( n = 5) and lART ( n = 4). Dot plots comparing the blood B-cell subset frequencies between eART (blue) and lART (red) are shown. The average frequency of positive cells is indicated below each dot plot. c t-SNE density plots comparing the distribution of HIV-1 gp140-specific cells (shown in red), with a phenotypic analysis of these cells based on CD27 and β7 surface expression at the bottom left-hand corner. d Dot plots comparing gp140 + B-cell frequencies between eART ( n = 5) and lART ( n = 4). The average frequency of reactive cells among total IgA + and IgG + B cells is indicated below each dot plot. e Same as in ( b ) but for polyreactive B cells (PolyR + ). Bars in ( b ), ( d ) and ( e ) represent the means. Groups were compared using two-tailed Mann–Whitney test. f Circos plots comparing the antibody sequence relationships between single-cell-sorted mucosal B cells and NGS libraries between cellular compartments. Interconnecting lines indicate sequences sharing identical V H and J H gene segments and at least 90% CDR H 3 amino acid sequence homology. Groups were compared using 2 × 2 Fisher’s Exact test. g Bar graph comparing the frequencies of polyreactive (gray and black for IgG and IgA, respectively) and non-polyreactive (white) intestinal memory B-cell antibody sequences found in the blood-derived NGS library between eART ( n = 3) and lART ( n = 3). Data are presented as mean values ± SD. Groups were compared using 2 × 2 Fisher’s Exact test. h Dot plot presenting the clustering of eART (blue) and lART (red) based on blood SIgA level and frequency of immunoglobulin sequences shared between the blood and the intestinal mucosa. Source data are provided as a Source Data file.

Article Snippet: Recombinant IgG antibodies (150 µg/ml final concentration) were assayed by indirect immunofluorescence assay (IFA) using the ANA HEp-2 AeskuSlides® kit (Aesku.Diagnostics) following the manufacturer’s instructions. mGO53, ED38 and kit’s control antibodies were included in each experiment.

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Sequencing, Derivative Assay