EMBO/soda-nel huggingface dataset:
Pool size estimations for dense-core vesicles in mammalian CNS neurons (2018)
Figure 5

Figure 5 - DCV release probability upon different stimulations

A Schematic representation of NPY-pHluorin as optical reporter for DCV fusion. Inset below shows F/F0 of single DCV fusion event. Scale bar: 1 µm.

B Zoomed confocal image of NPY-pHluorin expressing (green) hippocampal neuron (DIV14) immunostained for ChgB (red) and corresponding fluorescence intensity plot showing overlap between NPY-pHluorin and ChgB. Scale bar: 2 µm.

C Manders' coefficients of colocalization between NPY-pHluorin and ChgB per neuron.

D Total ChgB puncta per neuron in uninfected (control) and NPY-pHluorin infected neurons (NPY-pH). Student's t-test: n.s. = not significant, p = 0.90.

E Mean ChgB fluorescence intensity per puncta per neuron in uninfected (control) and NPY-pHluorin infected neurons (NPY-pH). Student's t-test: n.s. = not significant, p = 0.71.

F Correlation between ChgB puncta per neuron and total neurite length (mm) for uninfected control (grey) and NPY-pHluorin infected (green) neurons. Linear regression, inset shows goodness of fit (r2) and significance (p-value) per group. Slopes are not significantly different, ANCOVA: p = 0.19.

G-J Single isolated hippocampal neurons (DIV14) were labeled with NPY-pHluorin. DCV fusion events were quantified per neuron upon different stimulation paradigms. (G) Histogram of DCV fusion events per stimulation per time point. Stimulations include 1 action potential (AP) (grey), 16 bursts of 50 AP at 50 Hz (light-green), superfusion with 60 mM KCl Tyrode solution for 30 s. (dark-green) and superfusion with 5 μM Ionomycin for 30 s. (blue). Inset: Typical ∆F/F0 trace of rise in intracellular calcium (Fluo5-AM) upon different stimulations. Traces are corrected for baseline (first 10 frames) and normalized. (H) Cumulative plot of fusion events per neuron for different stimulations. (I) DCV fusion events per neuron per stimulation. Kruskal-Wallis with Dunn's correction: *p < 0.05, **p < 0.01, ***p < 0.001. 1AP vs. 5 μM Ionomycin, 16x50 AP,50Hz vs. 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non-significant, p > 0.05. (J) DCV release probability per neuron per stimulation. Kruskal-Wallis with Dunn's correction: **p < 0.01, ***p < 0.001. 1AP vs. 5 μM Ionomycin, 16x50 AP,50Hz vs. 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non-significant, p>0.05.

K DCV release probability per neuron in CaMKII-positive and negative hippocampus neurons (DIV14) upon repetitive electrical stimulation (16x50AP at 50 Hz). Mann-Whitney U test: n.s. = not significant, p = 0.12.

Data information: Stimulation period is represented by colored bars above graphs. Bars show mean ± SEM. N numbers represent number of experiments and number of (single) neuron observations in brackets are represented as dots.
Alternative lengthening of human telomeres is a conservative DNA replication process with features of break-induced replication (2016)
Figure 2

Figure 2. Conservative DNA replication at telomeres of human ALT cells

(A) Examples of chromosome arms exhibiting features of telomeric semiconservative (Semi) replication, conservative (Consrv) replication of the entire telomere (E) and conservative replication of part of the telomere (P). Idealized chromosome diagrams (left) and actual microscopy images (right) are shown. D-FISH, denaturing FISH; ND-CO-FISH (non-denaturing CO-FISH).

(B) Percentages of chromosome arms exhibiting conservative (Consrv) replication of the entire telomere (E), conservative replication of part of the telomere (P) and conservative replication of either the entire telomere of part of it (E+P). NE, U2OS cells expressing normal levels of cyclin E; OE, U2OS cells overexpressing cyclin E for four days. Bars represent means and standard errors of the mean from three independent experiments. Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres: P<0.025 for Consrv (P) and P<0.05 for Consrv (E+P), as calculated using the paired t test.
Alternative lengthening of human telomeres is a conservative DNA replication process with features of break-induced replication (2016)
Figure 3

Figure 3. Conservative DNA replication at telomeres and telomere function in ALT cells are dependent on PolD3 and PolD4

(A) Decrease in the fraction of chromosome arms exhibiting conservative (Consrv) DNA replication of the entire telomere length or part of the telomere (E+P) in U2OS cells after depletion of POLD3 or POLD4 by siRNA. si Ctrl, control siRNA; NE, cells expressing normal levels of cyclin E; OE, cells overexpressing cyclin E for four days. Bars represent means and standard errors of the mean from two independent experiments. For statistical comparisons the effects of PolD3 and Pold4 depletion were examined after grouping together the data from the NE and OE cells. PolD3 depletion reduced the fraction of conservatively replicated telomeres at a level of P=0.072; whereas for PolD4 depletion the decrease was significant at a level of P<0.025, as calculated using the paired t test.

(B) Decreased telomere length following PolD3 or PolD4 depletion in U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E (OE). The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,600 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6) for both NE and OE cells, as determined by unpaired t tests.

(C) Decreased telomere length following PolD3 or PolD4 depletion in parental U2OS cells. The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,800 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6), as determined by unpaired t tests.

(D) Increased frequency of chromosome end-to-end fusions following POLD3 or POLD4 depletion. U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E for four days (OE) were transfected with control siRNA (siCtrl) os siRNAs targeting POLD3 or POLD4. For each condition, 25 metaphases were examined (representing about 1,900 high quality chromosome arms per condition); the percentage of chromosome fusions was determined for each metaphase and used to calculate means and standard errors of the mean. For statistical analysis, the number of chromosome end-to-end fusions were compared by chi square tests among the various conditions. Cyclin E overexpression led to a higher number of fusions (P<0.0005); PolD3 and PolD4 depletion also led to a higher number of fusions (P<0.001 and P<0.025, respectively).
iPSC-derived neurons from GBA1-associated Parkinson’s disease patients show autophagic defects and impaired calcium homeostasis (2014)
f1

(a) Structure of the GBA1 gene on chromosome 1 and schematic overview of gene correction using ZFNs. Black boxes represent exons. (b) Mutated and gene-corrected (GC) iPSCs after sequencing (upper lanes: iPSCs with heterozygous mutations; lower lanes: iPSCs with homozygous wt-GBA1 sequences as indicated by arrows).
iPSC-derived neurons from GBA1-associated Parkinson’s disease patients show autophagic defects and impaired calcium homeostasis (2014)
f2

Immunostaining of indicated differentiated iPSC cultures at DIV65. Cells were stained for TH (red) and β-TubIII, FOXA2, NURR1, GIRK2 and VMAT2 (all green). Cell nuclei were counterstained with Hoechst (blue). (Bars, 20 μm).
iPSC-derived neurons from GBA1-associated Parkinson’s disease patients show autophagic defects and impaired calcium homeostasis (2014)
f3

(a) Gating strategy used for neuronal cell sorting. Live cells as determined by FSC/SSC gating were selected for CD184-APC-negative/low surface expression and subsequently separated for CD24-PE versus CD15/CD29/CD44 expression, detected in the FITC channel as indicated in the dot plots. Post-sort reanalysis shows representative samples of both fractions immediately after FACS enrichment, which were then plated for comparative in vitro analysis (phase contrast images. Bars, 50 μm). (b,c) Immunocytochemistry at DIV2 post-sort for TH (green) and the neuronal marker MAP2 (red) performed on the neuronal (b) and mixed (c) populations of a representative control and GBA-PD line. Nuclei were counterstained with Hoechst (blue). FACS enriches for both TH and neuronal marker positivity in the neuronal population compared with the non-neuronal population. (Bars, 50 μm.)
iPSC-derived neurons from GBA1-associated Parkinson’s disease patients show autophagic defects and impaired calcium homeostasis (2014)
f4

(a) Ganglioside composition of human fibroblasts, iPSCs, differentiated iPSCs and enriched iPSC-derived neurons (after FACS) from control (line C1-1), GBA-PD (line PD1-1) and GD (line GD1-1) subjects. Cell lipids were metabolically labelled with [1-3H]sphingosine and visualized by digital autoradiography. (b) Activities of glycohydrolases (GCase, GBA2, β-galactosidase, β-hexosaminidase) in control iPSCs and iPSC-derived enriched neurons at DIV65. Enzyme activities are expressed as pmoles mg−1 h−1. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.01; Student’s t-test. (c) GBA1, GBA2, β-galactosidase and HEXA and HEXB gene expression levels in control iPSCs and iPSC-derived enriched neurons at DIV65. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.05; **P0.01, Student’s t-test. (d) Representative western blots showing GCase, GBA2, β-galactosidase and HEXA and HEXB protein levels in control iPSCs and corresponding iPSC-derived neurons (DIV65).
iPSC-derived neurons from GBA1-associated Parkinson’s disease patients show autophagic defects and impaired calcium homeostasis (2014)
f5

(a) LC-MS/MS analysis showing the level of Glc-Cer in control, GBA-PD, isogenic controls and GD iPSC-derived neurons. Data are represented as mean+s.d.; n=3, *P0.01, One-way ANOVA. (b) Control iPSCs (line C1-1) were differentiated for 65 days and exposed to the GCase inhibitor CBE (25 μM) for 48 h. Representative western blot showing increased levels of α-syn in CBE-treated iPSC-derived neurons compared to untreated neurons. (c,d) Representative western blots showing the protein levels of α-syn in control, GBA-PD, gene-corrected isogenic controls and GD iPSC-derived neurons. (e) Optical densities of α-syn bands normalized by the averaged value of GAPDH and expressed as a percentage of the control (CTRL) line value. Data are represented as mean+s.d.; experiments were independently repeated five times. *P0.01, one-way ANOVA. (f) Average α-syn protein levels normalized by the averaged value of GAPDH and expressed as a percentage of the respective isogenic mutant lines. Data are represented as mean+s.d.; experiments were independently repeated five times. *P0.01, Student’s t-test.
iPSC-derived neurons from GBA1-associated Parkinson’s disease patients show autophagic defects and impaired calcium homeostasis (2014)
f6

(a) Glycohydrolase enzyme activities (GCase, GBA2, β-galactosidase, β-hexosaminidase) were measured in enriched neurons from control, GBA-PD, isogenic gene-corrected control and GD iPSC-derived neurons at DIV65. Enzyme activities are expressed as pmoles mg−1 h−1. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.05; **P0.01; ***P0.001 compared with controls, one-way ANOVA. (b,c) Representative western blots of GCase protein levels in control, GBA-PD, corresponding gene-corrected and GD iPSC-derived neurons. Data are represented as mean+s.d.; experiments were independently repeated three times. *P0.05; **P0.01, one-way ANOVA and Student’s t-test.
iPSC-derived neurons from GBA1-associated Parkinson’s disease patients show autophagic defects and impaired calcium homeostasis (2014)
f7

CSF glycohydrolase activities were measured in patients with idiopathic PD (iPD), GBA-PD patients and age-matched unaffected individuals (CTRL). Enzyme activities were normalized to total protein concentrations and expressed as pmoles mg−1 h−1. Data are represented as mean+s.e.m.; CTRL, n=14; iPD, n=15; GBA-PD, n=15. Experiments were performed in triplicate. *P0.05; **P0.01, Mann–Whitney U test.
iPSC-derived neurons from GBA1-associated Parkinson’s disease patients show autophagic defects and impaired calcium homeostasis (2014)
f8

(a) Immunostaining of differentiated iPSC cultures at DIV65 under basal conditions. Cells were stained for TH (green), β-TubIII (magenta) and LAMP1 (red). Cell nuclei were counterstained with Hoechst (blue). High magnification insets are shown in the right lane. (Bars, 20 μm and 10 μm in the insets.) (b) Average size and number of LAMP1+ particles in TH+ neurons were quantified by ImageJ. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01, **P0.001, one-way ANOVA. (c) Western blot analysis for LC3 in iPSC-derived neuronal cultures at DIV65, untreated (−) or treated with 200 μM leupeptin and 20 mM NH4Cl for 4 h (+). (d) Quantification of the basal levels of LC3-II relative to β-actin. Data are represented as mean+s.e.m.; experiments were independently repeated three times. *P0.01; **P0.001, one-way ANOVA. (e) Quantification of LC3 flux normalized to actin. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; **P0.001, one-way ANOVA.
iPSC-derived neurons from GBA1-associated Parkinson’s disease patients show autophagic defects and impaired calcium homeostasis (2014)
f9

(a) Cytosolic calcium levels were evaluated in iPSC-derived neurons at basal conditions by measuring the 340/380 nm ratio of Fura-2 acetoxymethyl ester. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; Student’s t-test. (Number of cells analysed: CTRL, n=58; RecNcil/wt, n=36; RecNcil/wt GC, n=107; L444P/wt, n=82; L444P/wt GC, n=33; L444P/L444P, n=62). (b) Caffeine (10 mM) was applied to iPSC-derived neurons, and the peak cytosolic calcium concentration was measured as a change in Fura-2 fluorescence. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; **P0.001, Student’s t-test. (Number of cells analysed: CTRL, n=58; RecNcil/wt, n=36; RecNcil/wt GC, n=107; L444P/wt, n=82; L444P/wt GC, n=33; L444P/L444P, n=62). (c) iPSC-derived neurons were treated with A23187 (0–5 μM) for 4 h and cytotoxicity was evaluated by released LDH activity. LDH release values were calculated as the percentage of untreated cells lysed by incubation with Triton X-100. Data are represented as mean+s.e.m.; experiments were independently repeated four times in triplicate. *P0.05, one-way ANOVA, control lines versus mutated lines; δP0.05, one-way ANOVA, mutated lines versus isogenic gene corrected controls. (d) Quantification of TH+ cells treated with A23187 (1 μM) for 4 h expressed as a percentage of DMSO-treated controls. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.01, one-way ANOVA. (e) Knockdown efficiency of NECAB2 determined by qRT-PCR and normalized to scrambled non-targeting shRNA. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. ***P0.0001, Student’s t-test. (f) Representative western blot for NECAB2 showing knockdown efficiency. 1, scrambled non-targeting shRNA; 2, NECAB2 shRNA. (g) Quantification of TH+ cells in GBA-PD and GD iPSC-derived neuronal cultures infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 μM for 4 h). Values were calculated as a percentage of scrambled shRNA-treated cells. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01, Student’s t-test. (h) Representative images showing GBA-PD iPSC-derived DA neurons infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 μM for 4 h). Cells were stained for β-TubIII (green) and TH (red). Nuclei were counterstained with Hoechst (blue). (Bar, 20 μm.)
Bacterial expression of a designed single-chain IL-10 prevents severe lung inflammation (2022)
Figure 1

Fig. 1. Effects of human IL-10 recombinant protein (hIL-10r) or IL-10 secreted by M. pneumoniae on macrophage primary cells on anti-inflammatory (A) or pro-inflammatory biomarkers (B).

A, B Data were normalised using the control group and are represented as fold-change (FC) of the mean fluorescence intensity (MFI). Macrophage cells were obtained from human donors (n = 4 biological replicas) and incubated with medium (CON) or recombinant human IL-10 (hIL-10r), MPN wild-type (WT) or MPN expressing IL-10 ORF (WT_IL10). Data are represented as mean ± Standard Error of the Mean (SEM). Statistical analysis was performed using one-way ANOVA + post-hoc Tukey multiple comparison test (*P < 0.05; **P < 0.001, ***P < 0.0001).
Bacterial expression of a designed single-chain IL-10 prevents severe lung inflammation (2022)
Figure 4

Fig. 4. Expression levels and apparent dissociation constant of selected IL-10 variants expressed by M. pneumoniae.

Each point in the figure is a biological replica.

(A) EC-50 (molar, M) for human IL-10 recombinant protein (hIL-10r) and IL-10 WT (IL-10 ORF) and different variants (MutM, hIL-10r, Mut1, Mut2, Mut3, MutSC1, and MutSC2) expressed by M. pneumoniae. Data are represented as mean ± SD. Statistical comparison was done by One-way ANOVA + post-hoc Bonferroni multiple comparison test, using the IL-10 ORF condition as a reference (*P < 0.05).

(B) Fold-change (FC) in expression levels (fg/CFU) of IL-10 variants Mut3 and MutSC1 secreted to the medium normalised by expression level of IL-10 ORF (ORF). Statistical comparison of mean ± SD was performed by One-way ANOVA + Tukey post-hoc test (*P < 0.05).

(C, D) Average and SD values for the FC in the relative EC-50 values determined by flow cytometry analysis of phosphorylated STAT3 after a 20-min exposure of the BlaER1 (C) or HAFTL (D) cell lines to IL-10 ORF (reference) or the mutant MutSC1 or MutSC2 (see Materials and Methods). Numbers indicate the average FC ± SD.
Bacterial expression of a designed single-chain IL-10 prevents severe lung inflammation (2022)
Figure 5

Fig. 5. Characterization of CV8 chassis in vivo.

C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post-infection (dpi) or 4-dpi (n ≥ 3 biological replicas/group).

(A) Average ± SD bacterial loads recovered of the WT (circle) and CV8 (square) strains in mouse lungs at 2-dpi or 4-dpi. Data are shown as log10 CFU/ lung homogenate.

(B) Inflammatory profile of mouse lungs inoculated with WT (dark blue), CV8 (light blue), or PBS (grey). Gene expression was analysed by RT-qPCR using gapdh as an endogenous control (see Materials and Methods). Data are shown as average ± SD of fold-change (FC) in mRNA expression (One-way ANOVA + Tukey post-hoc test; *P < 0.05).

(C) Histological findings of mouse lung samples at the analysed time points. Left, plots representing the quantitative evaluation (score: 0-5) of alveolar infiltrate (top panel) and peribronquial/olar infiltrate (FC) (bottom panel) (see Materials and Methods). Parameters were normalised using the average of the PBS group (FC = sample value/average PBS; FC control group, ~1). Data are shown as average ± SD of FC (one-way ANOVA + Tukey post-hoc test) (*P < 0.05). Right, representative images of lungs stained with hematoxylin-eosin (the line represents 250 μm).

(D) IL-10 secretion by the MPN WT or CV8 strain coding for hIL-10 WT (ORF), MutSC1, or MutSC2. Data are expressed as the mean ± SD of IL-10 supernatant concentration (μg/mL) by biomass (protein content in μg/mL) (n=3 biological replicas).
Bacterial expression of a designed single-chain IL-10 prevents severe lung inflammation (2022)
Figure 6

Fig. 6. Immunomodulatory effect of IL-10 variants in vivo.

Each point is a biological replica.

(A) Schematic representation of the experimental design performed

(B) CFUs of PAO1 (left) or Mycoplasma strains (right) recovered from lung samples. Data are shown as mean ± SD of Log10 CFU/lung homogenate. Two replicates were performed and individual symbols are shown (experiment 1, circle; experiment 2, square).

(C) Fold-change (FC) of mRNA gene expression of inflammatory markers determined by qPCR. The 2-ΔΔCt method was used to normalise the values, using gapdh as endogenous control (see Materials and Methods). Data are shown as average ± SD of FC. Statistical analyses were performed using unpaired t-test (* P < 0.05; ** P <0.01***; P <0.001, **** P <0.0001). Two replicates were performed and individual symbols are shown (Experiment 1, circle; Experiment 2, square).

(D) Quantitative analysis of immunochemistry (IHC) against neutrophil elastase (NE) of lung samples. Data are represented as the mean ± SD of percentage of positive cells, calculated as follows: % = 100 × (positive cells/positive cells + negative cells) (One-Way ANOVA + Tukey post-hoc test, * P <0.05).

(E) Representative images of IHC staining of lung samples against NE are shown. Arrows indicate NE-positive cells. Scale bar size: 50 µm.
A high-affinity, bivalent PDZ domain inhibitor complexes PICK1 to alleviate neuropathic pain (2020)
Figure 1

Figure 1. Development of a high affinity bivalent inhibitor of PICK1s.

Primary sequence of 5-mer peptide (C5) derived from the dopamine transporter C-terminus and the TAT conjugated variant, Tat-C5.

Fluorescence polarization (FP) competition derived affinity assessment of consecutive truncations of the DAT C13 C-terminus as indicated by the sequences. Bars indicate the mean fold change relative to C13 (Ki = 1.69 µM). Data is shown as mean with error-bars as SEM with n = 6. Representative binding curves are shown in EV1A.

FP competition binding of C5 and Tat-C5 to PICK1 with 5FAM-Tat-C5 (20 nM) as tracer. Saturation binding curve is shown in EV1B.

Backbone amide chemical shift changes (ΔδHN) of PICK1-PDZ-C10 after addition of Tat-C5 and addition of protease to cleave C10 from the PDZ domain and allow exchange. Numbers refer to residue number in PICK1 (Uniprot: Q9EP80). Red bars indicate a chemical shift larger than mean+SD.

1H-15N-HSQC 2D spectra of PICK1-PDZ-C10 (orange) following addition of Tat-C5 (green) and subsequent addition of protease (red) to allow for exchange between C10 and Tat-C5.

Docking model, for visual purposes, of PICK1-PDZ (PDB: 2LUI) in complex with Tat-C5 (blue), with perturbed residues indicated from D. in red.

Selectivity screen for Tat-C5 against a selection of 42 purified PDZ domains, performed with a fixed concentration of Tat-C5 (10 µM) in competition with PDZ domains and their respective fluorescent ligands. Data are normalized to binding in absence of peptide (full line). Dashed line represents the level of competition obtained for PICK1. Screen for C5 can be seen in EV1C.

Affinity gain of bivalent C5 peptides (Pn-(C5)2) N-terminally conjugated with different length PEG linkers as indicated. Bars indicate the mean fold change relative to C5 (Ki = 1.69 µM). Data is shown as mean with error-bars as SEM with n = 6. Representative binding curves are shown in EV1C.

FP competition binding of C5 (also used in C) and Tat-P4-(C5)2 to PICK1 with 5FAM-Tat-C5 (20 nM) as tracer.

Structure and highest obtained affinity of C5, TAT11-C5 and TAT11-P4-(C5)2 towards PICK1.

Data information: Data points and bars are shown as mean with error-bars as SEM of n ≥ 3. Docking in (F) was done using HADDOCK (van Zundert et al., 2016), with residues indicated from (D) as essential residues for interaction, but no further restraints, therefore being only for visual purposes.