EMBO/soda-nel huggingface dataset:
SRD-1 in AWA neurons is the receptor for female volatile sex pheromones in C. elegans males (2019)
Figure 6

Figure 6. SRD-1 C-terminal (CT) region contributes to species-specific activities of SRD-1 homologs in sex pheromone perception.

A) Predicted topological analysis revealing that SRD-1 from 4 closely related Caenorhabditis species feature similar structures, which contain 7 transmembrane helices and show considerable species-to-species variation in the CT region. Sequences of the 4 SRD-1 proteins are highly conserved. For the 4 sequences, the alignment identity score was analyzed by using Benchling with Clustal Omega.

B) Wild-type C. elegans males are less attracted to sex pheromone than wild-type C. remanei males.

C) Expression level of srd-1 cDNA in srd-1 transgenic lines. srd-1 mutant males were rescued using, separately, wild-type C. elegans and wild-type C. remanei srd-1 cDNAs. Transgenic lines expressing the molecule at a similar level were selected for use in chemoattraction assays. Four independent transgenic lines were selected, and the expression-level data were combined into a single bar. The expression levels were measured using qPCR.

D) srd-1 mutant males rescued with Ce-srd-1 cDNA showed lower responsiveness than those rescued with Cre-srd-1 cDNA, which indicates that sex pheromone perception ability depends primarily on the protein coding region and not the regulatory sequences in the promoter. The sex pheromone perception defect of srd-1 mutants was not rescued in males expressing CT-truncated SRD-1 proteins. The findings show that the CT region is essential for sex pheromone perception.

E) srd-1 mutant males rescued by using Ce-srd-1 cDNA together with Cre-srd-1 CT cDNA were more responsive to sex pheromone than those rescued using Cre-srd-1 cDNA with Ce-srd-1 CT cDNA. The results of the CT-swapping experiment demonstrated that sex pheromone perception ability is determined by the CT region and not the rest of the SRD-1 protein.

Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. For transgenic-line studies, each bar represents a total of 1200 males from 4 independent transgenic lines; 20 assays were performed for 2 independent transgenic lines and 10 assays were performed for the other 2 independent transgenic lines, and the tests were conducted over 2-4 separate days. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.
Myeloid-resident Neuropilin-1 promotes Choroidal Neovascularization while Mitigating Inflammation (2021)
Figure 1

Figure 1. NRP1 ligands are elevated in patients with NV AMD and in a mouse model of CNV.

A. Optical coherence tomography (OCT) horizontal B-scan and thickness map of neovascular age related macular degeneration (NV AMD) patient with active lesions (1), cystoid macular edema (2), subretinal fibrin deposits (3) and serous retinal detachment (4).

B. Optical coherence tomography (OCT) horizontal B-scan and thickness map of control patient with a medium sized, stage 3, full thickness macular hole (5).

C-G. Vitreous humor analyzed by ELISA for VEGF-A (C); n = 7 (Ctrl), 9 (NV AMD), SEMA3A (D); n = 10 (Ctrl), 10 (NV AMD), TGFβ (E); n = 8 (Ctrl), 7 (NV AMD), PDGF-BB (F); n = 5 (Ctrl),6 (NV AMD), PGF (G): n = 5 (Ctrl), 5 (NV AMD). Dots represent concentrations of individual patient samples.

H. Micron IV infrared and fluorescein in vivo imaging of naïve mouse fundus and following laser-induced CNV at D0, D3, D7, D14.

I-M. Time course of mRNA expression of NRP1 ligands in mouse RPE-choroid-sclera complexes relative to naïve (no burn), 3 (D3), 7 (D7), 10 (D10), and 14 (D14) days after burn for Vegfa (I); n = 11 (No burn), 6 (D3) , 7 (D7) , 4 (D10 and D14), Sema3a (J); n = 17 (No burn), 6 (D3) , 4 (D7) , 3 (D10 and D14), Tgfb1 (K); n = 14 (No burn), 3 (D3) , 7 (D7) , 3 (D10), 6 (D14), Pdgfb (L); n = 9 (No burn), 5 (D3 and D7), 4 (D10 and D14), Pgf (M); n = 6 (No burn), 4 (D3) , 3 (D7) , 6 (D10), 3 (D14).

Data information: All comparisons between groups were analyzed using a Student's unpaired t-test (C-G) or a one-way analysis of variance (ANOVA) and Dunnett's multiple comparisons test (I-M); *P < 0.05, **P < 0.01, ***P < 0.001; error bars represent mean ± SEM; exact P values listed in table 2.
Myeloid-resident Neuropilin-1 promotes Choroidal Neovascularization while Mitigating Inflammation (2021)
Figure 2

Figure 2. NRP1-expressing mononuclear phagocytes increase in the retina upon injury and promote CNV

A. Representative FACS plots of retinas and sclera-choroid-RPE cell complexes from naïve and burned mice 3 days (D3) after laser-burn.

B. Quantification of mononuclear phagocytes (Ly6G-, F4/80+, CD11b+) in retinas and sclera-choroid-RPE cell complexes at D3 relative to Naïve; n = 5 (Naïve), 4 (CNV).

C. Quantification of NRP1+ mononuclear phagocytes (Ly6G-, F4/80+, CD11b+, NRP1+) in retinas and sclera-choroid-RPE cell complexes at D3 relative to Naïve; n = 5 (Naïve), 4 (CNV).

D. mRNA expression of Nrp1 in BMDM relative to LysM-Cre/Nrp1+/+; n = 3 (LysM-Cre/Nrp1+/+), n = 4 (LysM-Cre/Nrp1fl/fl).

E. Quantification of NRP1 protein expression in LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl BMDM; n= 6.

F. Representative Western Blot showing NRP1 expression in LysM-Cre/Nrp1+/+ (Ctrl) and LysM-Cre/Nrp1fl/fl (Nrp1fl/fl).

G. Representative confocal images of NRP1 and IBA1-stained mononuclear phagocytes on choroidal flat mounts from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D3. Arrow heads indicate NRP1-positive mononuclear phagocytes. Scale bar: 20μm.

H. Representative confocal images of IBA-1-stained mononuclear phagocytes on choroidal flat mounts from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D7 and D14. Examples of macrophage quantification (yellow stars) are presented in side panels. Scale bar: 20μm.

I, J. Total number of IBA-1-positive mononuclear phagocytes counted around laser impact area on confocal images of choroidal flat mounts at D7 (I) and D14 (J); n = 19 burns (D7 LysM-Cre/Nrp1+/+), n = 25 burns (D7 LysM-Cre/Nrp1fl/fl) n = 37 burns (D7 LysM-Cre/Nrp1+/+), n = 23 burns (D7 LysM-Cre/Nrp1fl/fl), 3-5 mice with ~4 burns per eye.

K. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and IB4-stained laser impact area from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14. Scale bar: 20μm.

L-N. Quantification of area of FITC-dextran-labeled CNV (L), isolectin B4 (IB4)-stained laser impact area (M) and the ratio of FITC/IB4 per laser-burn (N) relative to LysM-Cre/Nrp1+/+ at D14; n = 23 burns (LysM-Cre/Nrp1+/+), n = 27 burns ( LysM-Cre/Nrp1fl/fl).

Data information: All comparisons between groups were analyzed using a Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P< 0.0001; error bars represent mean ± SEM; exact P values listed in table 2.
Myeloid-resident Neuropilin-1 promotes Choroidal Neovascularization while Mitigating Inflammation (2021)
Figure 3

Figure 3. NRP1-expressing mononuclear phagocytes display a pro-angiogenic alternatively activated phenotype.

A-D. mRNA expression of inflammation markers relative to LysM-Cre/Nrp1+/+ in mouse RPE-choroid-sclera complexes at D3 for Il1b (A); n = 10 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Il6 (B); n = 7 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Vegfa (C); n = 10 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Tnf (D); n = 5 (LysM-Cre/Nrp1+/+), n = 10 (LysM-Cre/Nrp1fl/fl).

E-H. mRNA expression of inflammation markers relative to LysM-Cre/Nrp1+/+ in mouse RPE-choroid-sclera complexes at D7 for Il1b (E); n = 6 (LysM-Cre/Nrp1+/+), n = 10 (LysM-Cre/Nrp1fl/fl), Il6 (F); n = 3 (LysM-Cre/Nrp1+/+), n = 4 (LysM-Cre/Nrp1fl/fl), Vegfa (G); n = 6 (LysM-Cre/Nrp1+/+), n = 10 (LysM-Cre/Nrp1fl/fl), Tnf (H); n = 8 (LysM-Cre/Nrp1+/+), n = 12 (LysM-Cre/Nrp1fl/fl).

I. Representative Western Blot showing pNF-κB expression in LysM-Cre/Nrp1+/+ (Ctrl) and LysM-Cre/Nrp1fl/fl (Nrp1fl/fl).

J. Quantification of pNF-κB expression in LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl BMDM; n= 6.

K, L. mRNA expression relative to LysM-Cre/Nrp1+/+ of inflammation markers in mouse BMDMs for Il1b (K); n = 4 (LysM-Cre/Nrp1+/+), n = 5 (LysM-Cre/Nrp1fl/fl) and Il6 (L); n = 3 (LysM-Cre/Nrp1+/+), n = 2 (LysM-Cre/Nrp1fl/fl)

M. Representative FACS plots of M1 and M2-Like macrophages in LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl BMDMs.

N, O. Quantification of M1-Like macrophages (F4/80+, CD11b+, CD11c+, CD206-)(N), M2-Like macrophages (F4/80+, CD11b+, CD11c-, CD206+) (O) in LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl BMDMs relative to LysM-Cre/Nrp1+/+; n= 5.

P. Heatmap (left) and enrichment plot (right) of GO Angiogenesis gene set enrichment analysis (GSEA) of wildtype and LysM-Cre/Nrp1fl/fl peritoneal macrophages; n = 2. NES, normalized enrichment score; FDR, false discovery rate.

Data information: All comparisons between groups were analyzed using a Student's unpaired t-test; *P < 0.05, **P < 0.01; error bars represent mean ± SEM; exact P values listed in table 2.
Myeloid-resident Neuropilin-1 promotes Choroidal Neovascularization while Mitigating Inflammation (2021)
Figure 4

Figure 4. Therapeutic intravitreal administration of soluble NRP1 reduces CNV in mice

A. Schematic representation of a soluble receptor Neuropilin-1, consisting of five domains; two CUB motifs (A1, A2), two coagulation factor domains (B1, B2) and the MAM domain (C).

B. Rate constant and binding affinities of Sema3A and VEGF to immobilized trap obtained using a one-site Langmuir binding model.

C, D. Representative SPR sensorgrams for various concentrations of Sema3A (C) and VEGF (D) binding to immobilized Trap.

E. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and isolectin B4 (IB4)-stained laser impact area from Vehicle and NRP1-derived trap treated wildtype mice. Scale bar: 20μm.

F-H. Quantification of area of FITC-dextran-labeled CNV (F), IB4-stained laser impact area (G) and the ratio of FITC/IB4 per laser-burn (H) relative to Vehicle at D14; n = 24 burns (Vehicle), n = 26 burns (NRP1-derived trap).

I. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and IB4-stained laser impact area from Vehicle and NRP1-derived trap treated LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14. Scale bar: 20μm.

J-L. Quantification of area of FITC-dextran-labeled CNV (J), isolectin B4 (IB4)-stained laser impact area (K) and the ratio of FITC/IB4 per laser-burn (L) relative to LysM-Cre/Nrp1+/+ + Vehicle in Vehicle and NRP1-derived trap treated LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14; n = 16 burns (LysM-Cre/Nrp1+/+ + Vehicle), n = 16 burns (LysM-Cre/Nrp1+/+ + Trap), n = 13 burns (LysM-Cre/Nrp1fl/fl + Vehicle) , n = 19 burns (LysM-Cre/Nrp1fl/fl + Trap).

M. Proposed therapeutic paradigm. From a therapeutic perspective, intravitreal injection of NRP1-derived traps reduces pathological angiogenesis associated with CNV.

Data information: Comparisons between groups were analyzed using a Student's unpaired t-test; (E-G) or one-way ANOVA with Tukey's multiple comparisons test (I-K); *P < 0.05, **P < 0.01, ***P < 0.001, ****P< 0.0001; error bars represent mean ± SEM; exact P values listed in table 2.

Expanded View Figure legends
Conformational dynamics of the ABC transporter McjD seen by single-molecule FRET (2018)
Figure 1

Figure 1. Biochemical characterization of McjD and smFRET assay design.

A) Schematic representation of the smFRET assay using crystal-structure snapshots of McjD. On the right side McjD is also shown as a cartoon. In the crystal structure nucleotides and ATP-Mg in orange sticks, the peptide MccJ25 in pink. Each half transporter is colored in dark and light grey. McjD is shown in a view along the plane of the membrane which is depicted in light grey. Colored balls show the position of the cysteines used for probing conformational changes. PBD codes for McjD in inward-occluded conformation are 5OFP (left) (Bountra et al, 2017) and in the outward-occluded conformation 4PL0 (right) (Choudhury et al, 2014).

B) Coomassie stained SDS-PAGE of McjD-C547 reconstituted in liposomes. The amount reconstituted was evaluated against detergent purified protein.

C) Chromatogram of McjD C547 (black) labelled with Alexa 555 (blue) and Alexa 647 (red) maleimide fluorophore using a Superdex 200 size-exclusion column. The fraction containing the highest degree of labeling is marked by a square.

D) The ATPase activity of all labelled variants C547, Y64C, L67C was determined in liposomes and compared to wild-type, wt. All labelled McjD variants show basal- and ligand-induced ATPase activities in liposomes comparable to that of wild type protein, indicating that labelling does not interfere with their activity. Error bars were calculated from two replicates from two independent reconstitutions (mean ± standard deviation, n=2). The ruler character of FRET using the fluorophore pair Alexa555/Cy5 was supported by data of dsDNA in Figure EV1B, where different base-pair distances between both dyes correlate directly with the apparent FRET efficiency.
Conformational dynamics of the ABC transporter McjD seen by single-molecule FRET (2018)
Figure 2

Figure 2. Freely diffusing McjD in detergent studied by smFRET.

A, B) Schematic overview of McjD labelling schemes (A) and diffusion-based experiments (B) using confocal microscopy. Stochastic labeling is performed on the NBD (native cysteine C547) and TMD mutants (Y64C; L67C).

C) Confocal single molecule analysis with ALEX of labeled McjD in detergent under different conditions as indicated.

D) Apparent ligand binding affinity values of McjD for AMPPNP (top), ATP (middle), and ADP-vanadate (bottom). Data points were obtained from the areas of fits from the E* histograms considering the ratio ligand-free/(ligand-free+ligand-bound) at the indicated substrate concentrations. Extraction of Kd values from a fit to the data is described in the Material and Methods section. Data for detergent-solubilized McjD for additional biochemical conditions is shown in Figure EV2.
Conformational dynamics of the ABC transporter McjD seen by single-molecule FRET (2018)
Figure 3

Figure 3. Conformational states of surface-immobilized McjD in proteoliposomes.

A) schematic showing the immobilization of McjD to the surface: labeled McjD reconstituted in biotinylated DOPE lipid was immobilized to a PEG-biotin-coated surface in a flow cell via a neutravidin-tag on the proteoliposome. A typical surface scan is shown in false-color representation on the right (orange, double-labeled McjD; green, McjD with donor fluorophore only; red, McjD with acceptor fluorophore only).

B) Representative fluorescence time traces (blue, donor signal; red, acceptor signal; black, FRET signal; yellow, fit) of McjD labeled with Alexa555 and Alexa647-maleimide with indicated concentrations of corresponding substrate: (B) McjD L67C with 10 mM ATP and 200 µM MccJ25, McjD Y64C with 10 mM ATP and 200 µM MccJ25, and McjD C547 with 10 mM ATP. The respective right panels are the projections of accumulated time traces in the absence and presence of substrate for each variant. We note that differences between the setup dependent apparent FRET values are not influencing our interpretations since only relative changes of FRET efficiency are interpreted. Experimental support for proper reconstitution is provided in Figure EV3A showing longer bursts and distinct TEV cleavage for liposomes compared to detergent.
Conformational dynamics of the ABC transporter McjD seen by single-molecule FRET (2018)
Figure 4

Figure 4. Intrinsic conformational dynamics of the NBDs in surface-immobilized McjD in proteoliposomes.

A) Representative FRET time traces (black, FRET signal; yellow, fit) of McjD NBD C547 labeled with Alexa 555 and Alexa 647-maleimide under apo conditions at 10 ms time resolution. Approximately ~85% of all apo-McjD traces show no significant fluctuations beyond shot-noise. Apo-McjD is predominantly in the low FRET and thus open state with E* values <0.3.

B, C) Within the complete data set of apo-McjD (N = 80 traces) comprising donor and acceptor, ~15%, show infrequent fluctuations to a higher FRET efficiency state with a lifetime of (C) 82 ± 25 ms where E* values > 0.5 are observed during short dwells. The data set represents a total recording time of 4.6 mins with a temporal resolution of 10 ms. To exclude unwanted influence on McjD due to surface-immobilization, experimental verification is provided that ATP-induced NBD switching occurs similarly with detergent solubilized McjD on the surface in Figure EV4.
Conformational dynamics of the ABC transporter McjD seen by single-molecule FRET (2018)
Figure 5

Figure 5. ATP-induced conformational dynamics of the NBDs in surface-immobilized McjD in proteoliposomes.

A) Representative FRET time traces (black, FRET signal; yellow, fit) of McjD NBD C547 labeled with Alexa 555 and Alexa 647-maleimide in apo conditions at 10 ms time resolution. This panel shows stable ATP-loaded (left) and ATP-free (right) McjD.

B) Representative FRET time traces that show switching between ATP free McjD (E* ~ 0.3) and ATP-loaded (high FRET >0.5) at ~4s s (left) and ~1.4 s (right). The traces show photobleaching events of the acceptor after ~10 s (left) and ~3.3 s (right).

C) Representative FRET time traces that show switching between ATP-loaded (high FRET >0.5) and ATP free McjD (E* ~ 0.3) at 2 s (left) and 5 s (right). The traces show photobleaching events after ca. 40 s (left, donor-bleaching) and 70 s (right, acceptor-bleaching).
MiR-29 coordinates age-dependent plasticity brakes in the adult visual cortex (2020)
Figure 1

Figure 1: Developmental increase of miR-29a regulates the expression of extracellular and epigenetic remodeling factors.

a) Venn diagram showing the intersection between putative miR-29a target genes (grey) and age-upregulated (green, bottom) or age-downregulated (red, top) genes. Ellipse area is proportional to the size of the sets. The numbers indicate the numerosity of the sets. 1051 miR-29a predicted targets by Targetscan, 5429 genes downregulated with age; 554 genes in the intersection, odds ratio 4.40; Fisher exact test, p< 0.0001; 4339 upregulated genes with age; 164 genes in the intersection, odds ratio 0.93; Fisher exact test p=0.427.

b) Box plot showing the distribution of log2(FC) of all or selected miR-29a targets in the Data ref: Mazziotti et al, 2017 dataset of age-regulated genes in the visual cortex. Notches represent confidence intervals for the median. Whiskers indicate 95th and 5th percentile, the box marks the 25th and 75th percentile. Significance vs. all detected genes was assessed by Wilcoxon's test (miR-29a targets p=4.9x10-14, top 200 miR-29a targets p=3.7x10-38).

c) DAVID functional annotation clustering analysis showing the most significantly overrepresented GO terms in age-downregulated miR-29 targets.

(d,e) Mature miR-29a (n=3/4 mice for each age; one-way ANOVA, p<0.0001; post hoc Tukey's test ***p<0.001, ****p<0.0001, ns not significant) and Dnmt3a expression level (normalized to P10) at different ages measured by qPCR (n=3/4 mice for each age; one-way ANOVA, p<0.0001; post hoc Tukey's test: *p<0.05, **p<0.01, ****p<0.0001, ns not significant). One way ANOVA p-values are reported. Error bars represent ±SEM.

f) Scatter plot showing correlation of miR-29a and Dnmt3a levels in the visual cortex display a strong negative correlation (Pearson correlation: r=-0.817, p<0.001). Each circle represents data from a single animal.

g) Representative examples of in situ hybridization on visual cortex (left P25, right P120) using a LNA probe against miR-29a. Scale bar 180 µm. Cortical layers are delimited by roman numerals and brackets

h) Example of co-staining for PV (green), miR-29a (red) and nuclear marker (blue) in a P120 mouse. White triangles represent PV+/miR-29a+ cells. Scale bar 20 µm.
MiR-29 coordinates age-dependent plasticity brakes in the adult visual cortex (2020)
Figure 2

Figure 2: miR-29a expression is not experience-dependent.

(a-d) Mature miR-29a expression (miR29) in visually deprived mice: a) normal rearing (NR, N=5) vs dark rearing (DRb, N=5); t-test, p=0.19, b) NR (N=4) vs dark rearing from P10 (DRp10, N=5; t-test, p=0.70. In both experiments, analysis was performed at P25, c) one week period of dark rearing beginning at P30 (DRw) followed by a 4 hour exposure to light (DRw+4h light) vs control animals (ctr, N=4 for each group; one-way ANOVA, p=0.68), d) days of MD beginning at P25, contralateral cortex vs ipsilateral cortex to deprived eye, N=5; paired t test, p=0.52). Error bars represent ±SEM, symbols represent individual cases.
MiR-29 coordinates age-dependent plasticity brakes in the adult visual cortex (2020)
Figure 3

Figure 3: miR-29a upregulation blocks OD plasticity of young mice.

a) Experimental design for miR-29a mimic treatment in young mice. Primary visual cortex (V1).

b) MiR-29a and Dnmt3a expression in the cortex treated with scrambled or miR-29a mimic (N=3 mice). Data were normalized to the untreated cortex of the same animal. Mir-29a: scr vs mim29a, t-test, p=0.005; Dnmt3a scr vs mim29a t-test, p<0.0001.

c) IOS analysis. Ocular dominance index (ODI) of deprived animals treated with scrambled (scr, N=6 black lines) or miR-29a mimic (mimic29, N=8 orange lines), or left untreated (MD no treat, N=5 grey lines) before and after 3 days of MD. Dashed lines represent single animals, continuous lines the group average. two-way ANOVA RM, treatment X time, p= 0.028; post hoc Sidak's comparison, post-MD mim29a vs scr p<0.05, mim29a vs MD no treat p<0.01; pre-MD scr vs post-MD scr p<0.001; pre-MD MD no treat vs post-MD MD no treat p<0.01; other comparisons not significant).

d) The effects of MD on expression of Npas IV and Bdnf exIV is reported as ratio between expression level of deprived and nondeprived cortex for animals treated with scrambled (scr+MD) and miR-29a mimic (mim29a+MD). Each symbol represents the result of a single mouse. (MD+scr vs MD+mim29a; N=4; t-test, Npas4: p=0.038; Bdnf exIV: p=0.049).

Data information: Error bars represent ±SEM, asterisks statistical significance, *p<.05, **p<0.001, ***p<0.001.