EMBO/soda-nel huggingface dataset:
A critical period of pre-hearing spontaneous Ca2+ spiking is required for hair-bundle maintenance in inner hair cells (2022)
Figure 7

Figure 7. Ca2+ spikes in IHCs regulate bundle morphology over a critical period during the second postnatal week of development.

A,B, Maximum intensity projections of confocal z-stacks showing the IHCs of the apical cochlear region from control (A) and Kir2.1-OE (B) pups with the females being in the continuous presence of DOX in the drinking water from conception up to when the pups were P5 (upper panels). Middle and bottom panels show IHCs at P7 and P14 following the removal of DOX at P5 for both control (A) and Kir2.1-OE mice (B). IHCs were stained with antibodies against the K+ channel Kir2.1 (green) and Myosin 7a (Myo7a, blue: cell marker). Note that after 2 days following the removal of DOX, Kir2.1overexpression was already largely downregulated. At least 3 mice for each genotype were used.

C, D, Calcium action potentials in IHCs from control (C) and Kir2.1-OE (D) mice during the second-postnatal week (P8-P9). IHC voltage responses were recorded during the application of 1.3 mM Ca2+ extracellular solution. The voltage-clamp data recorded from the same two IHCs displayed in panel C and D are shown in Appendix Fig S2; the IHCs from Kir2.1-OE mice show a strongly reduced Kir2.1 current. DOX was removed from the drinking water at P5

E, F, Spontaneous Ca2+ action potentials in IHCs from control (E, 60 sec out of 92 sec recording time) and Kir2.1-OE (F, 60 sec out of 103 sec recording time) mice during the second-postnatal week in the presence of the in vivo endolymph-like 0.3 mM Ca2+. Note that, different from when DOX was present throughout development (Fig. 2A-D), the removal of DOX at P5 restored the ability of IHCs from Kir2.1-OE mice to generate spontaneous intrinsic Ca2+ action potentials.

G, IHC resting membrane potentials from 2 control (4 IHCs) and 3 Kir2.1-OE (7 IHCs) mice in the presence of 1.3 mM Ca2+ (left) or 0.3 mM Ca2+ (right) in the extracellular solution. One-way ANOVA followed by Bonferroni's post-test: ns, P>0.9990 (1.3 mM Ca2+); ns, P=0.8864 (0.3 mM Ca2+); all other comparisons were *P<0.0001.

H,I, Maximum intensity projections of confocal z-stacks showing images of the hair bundles from apical-coil IHCs of P14 control (H) and Kir2.1-OE (I) mice stained with phalloidin. DOX was removed from the mother's drinking water when the pups were P5. At least 3 mice for each genotype were used.

J,K, SEM images showing the normal structure of the hair bundles of the IHCs in the apical coil of the cochlea of P14 control (J) and P14 Kir2.1-OE (K) mice. DOX was removed from the mother's drinking water when the pups were P5. Note that the morphological profile of the hair bundles in IHCs is comparable between control and Kir2.1-OE mice, indicating that the removal of the intrinsic Ca2+ action potentials prior to the second postnatal week has no effect on the mechanoelectrical transduction apparatus. At least 3 mice for each genotype were used.
Memory IgM protects endogenous insulin from autoimmune destruction (2021)
Figure 1

Figure 1. Polyvalent native self-molecules induce robust autoreactive IgG responses in vivo.

A: Schematic illustration of proinsulin-derived full-length C-peptide and Insulin.

B: Table comparing human to murine C-peptide amino acid sequences. Underlined: sequence used as peptide.

C: Schematic immunization schedule.

D-H: (D) Serum anti-C-peptide-immunoglobulins (coating: C-peptide) titers of mice immunized with cCP (n=9 for d14 and d28; n=5 for d49) and (G) anti-insulin-immunoglobulins (coating: Insulin) titers of mice immunized with complex native insulin (cInsulin, n=5) and control-immunized (CI: CpG only, n=3) measured by ELISA at indicated days. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Mann-Whitney-U test. (E, H) ELISpot assays showing CP-specific IgG (n=4 mice/group) (E) and insulin-specific IgG (n=3 mice/group) (H) producing splenocytes at day 14. Top lane showing representative images of wells. Mean ± SD, statistical significance was calculated by using Mann-Whitney-U test, *P < 0.05, **P < 0.01.

F: Schematic illustration of native insulin (cInsulin) and full length C-peptide (cCP) tetrameric polyvalent complexes.

I: Flow cytometric analysis of splenocytes of mice immunized with cInsulin or control immunization on day 26. Left and middle panel showing germinal center (GC) B cells (GL-7+) pre-gated on B cells (CD19+ B220+). Right panel showing insulin-reactive GC B cells pre-gated on GL-7+ cells in a histogram. Data are representative of two independent experiments with n=4/group.
Memory IgM protects endogenous insulin from autoimmune destruction (2021)
Figure 4

Figure 4. Soluble Insulin-A-peptides modulate autoimmune responses.

A: Serum anti-Insulin-immunoglobulin titers of mice immunized with InsA-peptide (red: 0:1, n=10 and green: 100:1, n=5) and control-immunization (CI: CpG only, grey, n=3) measured by ELISA at indicated days (coating: Insulin). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test, *P < 0.05, ***P < 0.001.

B: Ratios of IgG to IgM derived from ELISA values plotted against molar ratios of antigens for n=5/group. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test, *P < 0.05, ***P < 0.001.

C: Western blot analysis of insulin-specific serum IgG derived from InsA-peptide immunized mice. Top panel (green): 100:1 serum, lower panel (red): 0:1 serum (sInsA:cInsA). Serum of mice was used as primary antibody and detection was done with anti-mouse-IgG-HRP. Black filled arrow: Proinsulin (12 kD), Black non-filled arrow: Insulin (6 kD), β-actin (42 kD, loading control). Representative of two independent experiments.

D: ELISpot of InsA-peptide immunized (red, n=11) and CI mice (grey, n=8) showing insulin-specific IgG-producing splenocytes on day 14. Representative wells are shown (top lane). Mean ± SD, statistical significance was calculated by using Mann-Whitney-U test, ****P < 0.0001.

E, F: Blood glucose levels of InsA-peptide immunized (red: 0:1, n=5 for E and n=10 for F; green: 100:1, n=5 and n=4 for F, d30, d32) and CI (grey, n=5 for E and n=10 for F) mice were assessed at indicated days. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test (E) and repeated measure ANOVA test (F), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

G: Urine glucose levels of InsA peptide immunized (red: n=10 for d28, d32 and n=8 for d30; green: n=5 for d28, n=4 for d30, n=3 for d32) and CI (grey, n=3) mice were monitored at indicated days post immunization. Dots represent individual mice. For 100:1: representative data are shown for two independent experiments with total n=10. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, ***P < 0.001.

H: Table comparing human to murine Insulin-A-chain amino acid sequences. Underlined: sequence used as peptide.
Memory IgM protects endogenous insulin from autoimmune destruction (2021)
Figure 5

Figure 5. Monovalent Insulin-A-peptides induce immune tolerance via autoreactive memory IgM.

A: Immunization scheme. *monitoring: diabetes symptoms observed (red bar).

B: Blood glucose levels of InsA-peptide immunized mice (n=3) and CI (n=7) mice were assessed at indicated days post immunization. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, all comparisons were not significant.

C-F: (C, F) Urine glucose levels of InsA peptide immunized (n=3) and CI (n=3) mice were monitored at indicated days post immunization. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, *P < 0.05. (D, E) Serum anti-Insulin-immunoglobulin titers of mice immunized with InsA-peptide (day 59, day 78: n=3, day 49: n=7) and control-immunization (CI: CpG only, n=3) measured by ELISA at indicated days (coating: Insulin). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Mann-Whitney-U test (D) and repeated measure ANOVA test (E). *P < 0.05, **P < 0.01.
Memory IgM protects endogenous insulin from autoimmune destruction (2021)
Figure 6

Figure 6. High-affinity memory IgM counteracts IgG-mediated self-destructive autoimmune responses in vivo.

A: Ratios of IgG to IgM derived from ELISA values plotted on a two-dimensional graph against blood glucose levels (left panel) and urine glucose levels (right panel) for n=5/group. Central bands: median, boxes: interquartile range, whiskers: max./min., statistical significance was calculated by using Mann-Whitney-U test, **P < 0.01.

B: Serum anti-Insulin-IgM titers of mice immunized with InsA-peptide with a γ/µ ratio < 0.1 (black, n=5) and CI (grey, n=3) mice measured by ELISA at indicated days (coating: Insulin). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, *P < 0.05.

C: Blood glucose levels of InsA-peptide immunized mice (γ/µ < 0.1, black, n=5) and CI (grey, n=6) mice were assessed at indicated days post immunization with a commercial blood glucose monitor device. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test, all comparisons were not significant.

D: Insulin-specific IgM affinity-maturation of InsA-peptide immunized mice at indicated days was measured by ELISA. Ratios of IgM binding to InsA(1) and InsA(4) (referring to molar antigen density) were calculated and plotted as relative units (n=5 for d7, d28; n=3 for d78). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, **P < 0.01. Serum dilutions: 1:25.

E: Coomassie stained SDS-page showing purified serum IgM of complex InsA peptide (cInsA) immunized and CI mice under reducing (+ β-ME), and non-reducing conditions. Representative of two independent experiments.

F: Anti-Insulin affinity of PR-IgM (blue), primary IgM (red) and isotype control (black) measured by bio-layer interferometry. IgM binding to insulin was acquired in pm and used to calculate the dissociation constant shown in the graph (Kd = 1/Ka). Graph is showing antigen-antibody association phase. Data are representative for three independent experiments.

G: Blood glucose levels of intravenously injected mice with purified IgM from control immunization (CI, n=5), complex InsA peptide (cInsA) immunization day 7 (total IgM d7, n=4) and day 85 (total IgM d85, n=5). Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test showing comparison of red to black line, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Memory IgM protects endogenous insulin from autoimmune destruction (2021)
Figure 7

Figure 7. Differences in the affinity and specificity of primary versus memory IgM control autoimmune responses.

A: Schematic illustration of immunization schedule with complex InsA peptides (cInsA) intraperitoneally and insulin-specific protective IgM (PR-IgM) in 48 hours cycles intravenously (i.v.). *monitoring: diabetes symptoms were only observed within cInsA only group.

B: Blood and urine glucose levels of mice immunized with complex InsA-peptides (cInsA) (red, n=5) and cInsA plus intravenously injected (i.v.) pIgM (salmon, n=5) at day 7. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Mann-Whitney-U test, *P < 0.05.

C: Blood glucose levels of mice intravenously injected with total IgG pulldown of cInsA immunized mice (red, n=4), or total IgG (cInsA) together with anti-insulin PR-IgM (black, n=4). Measurements were done 5 hours post intravenous injection. Data were collected from two independent experiments. Mean ± SD, statistical significance was calculated by using Mann-Whitney-U test, **P < 0.01.

D, F: (D) Serum (dilution: 1:50) and (F) insulin-specific IgM (concentration: 500 ng/mL) tested for dsDNA (left panel, coating: calf thymus dsDNA) and insulin (right panel, coating: Insulin) reactivity measured by ELISA. Mice were immunized with cInsA and serum was collected on day 7 (D: n=8, left; n=5, right; F: n=4) and day 85 (D: n=4, left; n=5, right, F: n=4) post immunization. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Mann-Whitney-U test, **P < 0.01, ****P < 0.0001.

E, G: Anti-nuclear-IgM (ANA) of control-immunized (CI, n=3) and complex InsA peptide (cInsA) immunized mice on day 7 (n=3) and day 85 (n=3) analyzed by indirect immunofluorescence. We used (E) total serum (dilutions: 1:20) or (G) Insulin-specific IgM (concentration: 500 ng/mL) purified from serum at indicated days on commercial HEp-2 slides. Scale bar: 10 µm. Green fluorescence indicates IgM bound to nuclear structures. Data are representative for three independent experiments with isotype control: n=3, day 7: n=3, day 85: n=3.

H: Coomassie stained SDS-page showing primary (cInsA d7) and memory (cInsA d85) Insulin-specific purified IgM pre-incubated with Insulin and calf thymus dsDNA. Samples were loaded onto the gel after size exclusion with a cut-off at 10.000 kD (fractions referring to >/< 104 kD). IgM heavy chain (HC): 69 kD, IgM light chain (LC): 25 kD, J-chain: 15 kD. Data shown are representative of three independent experiments.

I: Blood glucose levels of mice intravenously injected with either IgM isotype ctrl (n=6), anti-insulin PR-IgM (d85, n=5), anti-insulin primary IgM (d7, n=4) after Insulin-specific pulldown. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test showing comparison of red and black line, ***P < 0.001, ****P 0.0001.
MicroRNA-202 safeguards meiotic progression by preventing premature SEPARASE-mediated REC8 cleavage (2022)
Figure 3

Figure 3. miR-202 knockout disrupts meiosis I prophase.

(A) Immunostaining of SYCP3 and γH2AX in sections from adult WT mice. Arrow and arrowhead indicate diffuse and dot-like γH2AX staining, respectively. White dotted line indicates the outline of testis tubules.

(B) Proportion of tubules of γH2AX expression patterns. At least 100 tubules of each mouse were counted. n=4 for WT and n=5 for KO.

(C) Average number of spermatocytes with γH2AX expression patterns per tubule. At least 50 tubules of each mouse were counted. n=3.

(D-H) Representative nuclear spreads of WT and KO spermatocytes at leptonema (D), zygonema (E), early pachynema (F), late pachynema (G) and diplonema (H). Spermatocytes were immunostained for SYCP3, γH2AX, and H1t. Magnified views are indicated by dashed areas. Schematic representation shows homolog characteristic revealed by SYCP3 signals at different backgrounds and stages. Arrowheads indicate bivalent stretches lacking SYCP3 signals in KO.

(I) Proportion statistics of spermatocytes with discontinuous SYCP3 signals. At least 200 cells of each mouse were counted. n=3 for WT and n=4 for KO.

(J) Meiotic stage frequencies in (D-H). At least 200 cells of each mouse were counted. n=3.

Data information: All values are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant. Statistical analyses were performed using two-tailed Student's t test. Scale bars: 50 μm (A) and 10 μm (D-H).
MicroRNA-202 safeguards meiotic progression by preventing premature SEPARASE-mediated REC8 cleavage (2022)
Figure 4

Figure 4. Loss of miR-202 causes inter-sister synapsis and impaired crossover formation.

(A) Nuclear-spread spermatocytes from WT and KO mice were immunostained for SYCP3 and SYCP1. Representative images of WT and KO pachynema are shown. Enlargements of boxes are shown below the respective full nucleus images, where "X" marks the XY body regions. Schematic representation shows inter-sister synapsis in bubble and fork regions. Arrows indicate the PAR. Arrowheads indicate inter-sister synapsis besides the boxed regions.

(B) Schematic representation shows the SYCP3 and SYCP1 patterns for XY body and autosomes in WT and KO pachytene spermatocytes in (A). In the lower panel, percentages of KO pachytene spermatocytes with and without inter-sister synapsis in XY body and autosomes are respectively shown. n=3 mice.

(C) Nuclear-spread spermatocytes from WT and KO mice were immunostained for SYCP3 and MLH1. Representative images of pachynema are shown. Magnified views are indicated by dashed areas.

(D) Number of bivalents without an MLH1 focus per pachytene spermatocyte in (C). n=3 mice. Nor, normal pachynema; L 1, pachynema-like 1; L 2, pachynema-like 2.

(E-H) Nuclear-spread spermatocytes from WT and KO mice were immunostained for SYCP3 together with RAD51 (E) or DMC1 (G) and quantification of RAD51 (F) or DMC1 (H) focus numbers at early zygonema, late zygonema, and pachynema. Representative images of early zygonema are shown. n=3 mice.

Data information: All values are shown as mean ± SEM. *P < 0.05, ***P < 0.001. NS, not significant. Statistical analyses were performed using one-tailed Mann-Whitney tests. Scale bars: 10 μm.
MicroRNA-202 safeguards meiotic progression by preventing premature SEPARASE-mediated REC8 cleavage (2022)
Figure 5

Figure 5. REC8 is progressively lost in miR-202 KO mice.

(A-D) Representative nuclear spreads of WT and KO spermatocytes at leptonema (A), zygonema (B), pachynema (C) and diplonema (D). Spermatocytes were immunostained for REC8 and SYCP3. REC8 foci distribute along SYCP3-labeled structures. Magnified views are indicated by dashed areas.

(E) Inter-REC8 distances along axial elements. Five stretches or homologs were measured and the average distance was calculated for each nucleus. Each point represents one nucleus. The upper panel is the schematic representation for distance measurement. n=3 mice.

(F) Western blot analyses for REC8 and SEPARASE in spermatocytes isolated by FACS after Hoechst staining. n=3 mice.

(G) Normalized density of REC8 in (F). n=3 mice.

Data information: All values are shown as mean ± SEM. **P < 0.01, ***P < 0.001. NS, not significant. Statistical analyses were performed using one-tailed Mann-Whitney tests (E) or two-tailed Student's t test (G). Scale bars: 10 μm.
MicroRNA-202 safeguards meiotic progression by preventing premature SEPARASE-mediated REC8 cleavage (2022)
Figure 6

Figure 6. SEPARASE-mediated cleavage of REC8 is aberrantly activated in miR-202 KO mice

(A-D) Representative nuclear spreads of WT and KO spermatocytes at lepnema (A), zygonema (B), pachynema (C) and diplonema (D). Spermatocytes were immunostained for SEPARASE and SYCP3. SEPARASE signals distribute along SYCP3-labeled structures from zygonema to diplonema in KO.

(E) Western blot analyses for SECURIN and CYCLIN B1 in spermatocytes isolated by FACS after Hoechst staining. n=3 mice.

(F) Normalized density of SECURIN and CYCLIN B1 in (E). n=3 mice.

Data information: All values are shown as mean ± SEM. NS, not significant. Statistical analyses were performed using two-tailed Student's t test. Scale bars: 10 μm.
MicroRNA-202 safeguards meiotic progression by preventing premature SEPARASE-mediated REC8 cleavage (2022)
Figure 7

Figure 7. miR-202-3p directly targets Separase mRNA.

(A) Validation of Separase mRNA as a direct target of miR-202-3p by dual luciferase assay. Data are normalized to the scrambled negative control (NC) mimic. Left panel: the sequence of miR-202-3p, and predicted miRNA regulatory elements at the 3' UTR of Separase mRNA and mutated 3' UTR sequence. n=5 biological replicates.

(B) Diagram for biotin-labeled mimic pull-down assays in cultured spermatogonial stem cells. Spermatogonial stem cells were transfected with biotin-labeled NC or miR-202-3p mimic and treated with RA. One day later, the cells were harvested and analyzed by qPCR.

(C) qPCR analyses of Separase mRNA enrichment in (B). Data are shown as as the percentage of input. n=4 biological replicates.

(D) qPCR analyses for Separase mRNA in spermatocytes isolated by FACS after Hoechst staining. Data are normalized to WT. n=3 mice.

(E) Diagram for verification of SEPARASE activation and REC8 cleavage in cultured spermatogonial stem cells. Spermatogonial stem cells were transfected with SEPARASE or NC siRNA. Ten hours later, the cells were treated with RA (retinoic acid) for 24 hours. Four days after induction, the cells were harvested and analyzed.

(F) Western blot analyses for REC8 and SEPARASE for cells in (E). n=3 biological replicates.

(G) Normalized density of REC8 in (F). n=3 biological replicates.

(H) Western blot analyses for SECURIN and CYCLIN B1 for cells in (E). n=3 biological replicates.

(I) Normalized density of SECURIN and CYCLIN B1 in (H). n=3 biological replicates.

Data information: All values are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant. Statistical analyses were performed using two-tailed Student's t test.

Expanded View Figure Legends
Helminthic dehydrogenase drives PGE2 and IL-10 production in monocytes to potentiate Treg induction (2022)
Figure 2

Figure 2. CLys- and CSN-induced Tregs with CNS and lymphoid tissues homing capacity.

A CD4+CD127-CD25hiFoxP3+CTLA4+ Treg induction from PBMCs incubated with CVF, CLys and CSN (10µg/ml) after 4h, 24h, 48h, 72h (plots and graphs shown), 120h and 168h.

B Trafficking receptors (CXCR3, CCR4, CCR6 and CCR7) evaluation on induced Treg (CD25hiCD127-) and non-Treg cells (CD127+CD25-).

C Induced Treg proliferation with Ki67 staining and Treg expression of IL-10 and IFNγ.

Data information: For (C), populations were pre-gated on CD4+CD127-CD25hi. Graphs are representative of 6-8 biological replicates (n=6-8) for (A-B) and 5-8 biological replicates (n=5-8) for (C). Results are expressed as means ± SEM. Asterisks show significant statistical differences analyzed using Kruskal-Wallis one-way ANOVA followed by a Dunn's multiple comparison test. * p<0.05; ** p< 0.01.
Helminthic dehydrogenase drives PGE2 and IL-10 production in monocytes to potentiate Treg induction (2022)
Figure 3

Figure 3. Treg induction by CLys and CSN is PGE2- and IL-10-dependent.

A Levels of pro- and anti-inflammatory cytokines in PBMC culture supernatants with CVF, CLys and CSN (10µg/ml) after 72h determined using a LUMINEX-based technology.

B Composition and levels of bioactive lipid mediators (LMs), including prostaglandins, leukotrienes, lipoxins, thromboxanes, and their precursors in culture with CVF, CLys and CSN quantified by LC-MS/MS-based profiling. Heat maps show relative amounts of LMs detected by LC-MS/MS in supernatants. Asterisks show statistical differences analyzed using two-way ANOVA followed by Turkey's multiple comparison test.

C Dynamic of PGE2 release correlated with Treg induction by CLys (upper panel) and CSN (lower panel) using a Pearson correlation.

D Treg induction by CLys or CSN following PBMC pre-incubation with 10µM mPGES1 inhibitor 934 (or DMSO as a control).

E Induction of Tregs with PGE2 analog (DINP) (5µM) and/or recombinant human IL-10 (rhIL-10) (10ng/ml) as compared to CLys.

Data information: Graphs are representative of 7-8 biological replicates (n=7-8). Results are expressed as means ± SEM. Asterisks show significant statistical differences analyzed using Kruskal-Wallis one-way ANOVA followed by a Dunn's multiple comparison test. * p<0.05; ** p< 0.01; *** p<0.001; **** p<0.0001.