EMBO/soda-nel huggingface dataset:
TRIM16 controls assembly and degradation of protein aggregates by modulating the p62-NRF2 axis and autophagy (2018)
Figure 8

Figure 8. TRIM16 reduces cytotoxicity mediated by oxidative and proteotoxic stresses.

(A) MTT assays performed at different time points with HeLa and TRIM16KO cells, untreated or treated with H2O2 (400 µM, 2h). Data, mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test).

(B) Images of HeLa and TRIM16KO cells, untreated or treated with As2O3 (2.5 µM, 4h). Scale bar: 400 µm.

(C-F) Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (C) As2O3 (2.5 µM, 4h), (D) H2O2 (400 µM, 2h), (E) puromycin (5 µg/ml, 6h) and (F) MG132 (20 µM, 8h).

(G) Immunoblot blot analysis of HeLa and TRIM16KO lysates of cells treated with 5 µM of As2O3 for different durations as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure. Arrowheads indicate the cleaved form of PARP-1 and Casapse-3.
Adeno-associated virus-vectored influenza vaccine elicits neutralizing and Fc-gamma-receptor activating antibodies (2020)
Figure 1

Fig. 1: (H1N1)pdm-based AAV-vectored antigens are strongly expressed in vitro.

A 3D structure of a HA trimer (PDB 3UBE generated with PyMol). Each monomer consists of a HA1 (dark grey) and HA2 (light grey) subunit. The trimer can be divided into a membrane distal head which contains the RBS (yellow) and a proximal stalk region.

B HA and NP represent the Cal/7/9 wildtype proteins. Chimeric HA (cHA) 1 contains the head regions of H2 HA, cHA2 of H10 HA and cHA3 of H13 HA, while they all contain the Cal/7/9 HA-stalk region. Headless HA (HL) contains a deletion in the HA-head region (dashed line). Modified headless HA (mHL1 and mHL2) contain stabilizing mutations (black boxes) and lack additional internal parts. All constructs were codon-optimized and carry a V5-tag at their C-terminus.

C Frequency of C179+ or V5-tag+ 293T cells 24h after transfection of AAV-vector plasmids as measured by flow cytometry. Symbols represent single experiments and bars the mean ± SE (n = 3).

D Immunoblot of 293T cells 72 h after transduction with AAV-vectors at a MOI of 106. Antigen expression was detected with an anti-V5-tag antibody. Equal loading was controlled with a GAPDH antibody (n = 3).
Adeno-associated virus-vectored influenza vaccine elicits neutralizing and Fc-gamma-receptor activating antibodies (2020)
Figure 2

Fig. 2: AAV-vectored vaccines and WIV induce broadly reactive antibodies.

A Mice were intranasally given 1011 vg of AAV-vector in 50 µl volume three times in three-week intervals. 20 µg of Cal/7/9 WIV per 50 µl was given i.n. two times. Blood samples were taken at indicated time points (red drops). After influenza challenge, mice were monitored for survival and weight loss for two weeks before necropsy.

B Total IgG ELISA titers expressed as area under the curve (AUC) against homologous Cal/7/9 virus of pre-immune and pre-challenge sera of individual animals of the indicated vaccine groups (AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11). Background reactivity of pooled AAV-GFP pre-challenge serum is shown as dashed line. ELISAs were performed in technical duplicates.

C IgA ELISA titers in post-challenge lung homogenates against Cal/7/9 of individual mice of the indicated vaccine groups (AAV-HA, -cHA, -GFP, -NP, WIV n = 11). of the Cal/7/9 (blue symbols) and PR8 low dose (red symbols) challenge groups. ELISAs were performed in technical duplicates.

D to G Total IgG ELISA titers expressed as log10 of the mean AUC against indicated viruses in pre-challenge sera in AAV-HA (D, n = 18), AAV-cHA (E, n = 18), AAV-NP (F, n = 11) or WIV (G, n = 18) immunized animals (grey area). Reactivity of sera of AAV-GFP immunized animals is indicated as white area in the center of each web diagram. ELISAs were performed in technical duplicates.

Data information: Statistical significance between pre-immune and pre-challenge serum was determined using Wilcoxon matched pairs test (###p<0.001). Statistical significance between vaccine groups was determined using Kruskal-Wallis test with Dunn's multiple comparison testing (*p<0.05, **p<0.01, ***p<0.001). Lines indicate mean.
Adeno-associated virus-vectored influenza vaccine elicits neutralizing and Fc-gamma-receptor activating antibodies (2020)
Figure 3

Fig. 3: AAV-HA, AAV-cHA and WIV induce distinct HA-specific antibody profiles.

A Immunoblot analysis of purified H1N1 viruses A/California/7/2009 (Cal/7/9), A/Brevig Mission/1/1918 (BM/1/1918), A/Puerto Rico/8/1934 (PR8) or A/Brisbane/59/2007 (Bris/59/7) separated under reducing and denaturing conditions. Uncleaved HA0 and cleavage products HA1 and HA2 were detected with pooled pre-challenge sera (AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11) as indicated (n = 3).

B Significant peptides identified in the epitope screen with AAV-HA pooled pre-challenge serum ((AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11; data shown in Fig.EV2) were mapped onto HA1 (red) and HA2 (blue) of the trimeric HA (PDB 3UBE generated with PyMol) (n = 3, technical duplicates). The position of the RBS (yellow) and the C179 epitope (dashed line) is marked.

C to F Binding of C179 (C), or antibodies present in AAV-HA (D), AAV-cHA (E) or WIV (F) pooled pre-challenge sera (n = 18 mice per group) to Cal/7/9 or PR8 virus after incubation of the virions at pH = 7.2, 5.8, 5.4, 5.0, 4.4 or 4.4+DTT to induce conformational changes in HA or remove the HA1 subdomain. Mean ± SD (n = 3, in technical triplicates). Statistical significance between pH = 7.2 and other conditions was determined using Kruskal-Wallis test with Dunn's multiple comparison testing (*p<0.05, **p<0.01).
Adeno-associated virus-vectored influenza vaccine elicits neutralizing and Fc-gamma-receptor activating antibodies (2020)
Figure 4

Fig. 4: AAV-vectored vaccines but not WIV induce broadly reactive FcγR-activating antibodies.

A and B Influenza virus specific activation of the murine FcγRI, FcγRII, FcγRIII and FcγRIV by antibodies in pooled pre-challenge sera (AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11 mice per group). MDCKII cells were infected with Cal/7/9 (A) or PR8 (B) before mouse serum and FcγR-expressing reporter cells were added. Influenza specific Fc-FcγR interaction induces the production of IL-2 in the FcγR-expressing reporter cell, which was measured by anti-IL2 ELISA (n = 3, technical duplicates). Mean ± SE.

C HA-specific activation of FcγR by AAV-HA, AAV-cHA or WIV pooled pre-challenge sera (n = 18 mice per group). MDCKII cells were transfected with wildtype HA (pAAV-HA) or a stalk-only HA (pAAV-mHL1) before mouse serum and FcγR-I-expressing reporter cells were added (n = 2, technical duplicates). Mean ± SE.
Adeno-associated virus-vectored influenza vaccine elicits neutralizing and Fc-gamma-receptor activating antibodies (2020)
Figure 6

Fig. 6: AAV-HA and AAV-cHA have a protective effect in ferrets

A Ferrets (n = 4 per group) were immunized with AAV-vectors or quadrivalent inactivated vaccine (QIV). Serum was obtained at indicated time points (red drops). After challenge with a Cal/7/9-like (H1N1)pdm strain clinical signs, temperature and weight loss were monitored. At 3 dpi, animals were sacrificed and necropsied.

B Cal/7/9-specific serum antibody titers in individual sera at indicated time points. Black lines: individual animals, red lines: mean titer. Statistical significance between pre-immune and immune sera was determined using Friedman test with Dunn's multiple comparison testing (*p<0.05, **p<0.01). The lower table indicates Cal/7/9-specific MN50 and HAI mean titers. Assays were performed in technical duplicates.

C to E Clinical score (C), body temperature (D), and relative weight (E) during challenge period. Statistical significance between pre-immune and immune sera was determined using Friedman test with Dunn's multiple comparison testing (AAV-HA ##p<0.01; AAV-cHA §§p<0.01; AAV-GFP $p<0.05; $$p<0.01; QIV *p<0.05;). Mean ± SD

F Virus titers in nasal turbinates (NT), trachea or lung homogenates. Statistical significance between AAV-GFP and vaccine groups was determined using Kruskal-Wallis test with Dunn's multiple comparison testing (*p<0.05). Lines indicate the GMT. Assays were performed in technical duplicates.

G and H IHC staining of submucosal glands (SMG) (G) or bronchi (H) with an anti-H1N1 polyclonal antibody (bar: 100 µm; red: influenza antigen (neufuchsin); blue: nuclei (haematoxylin)).

I and J H&E stained histological sections of bronchi (I) and lung parenchyma with alveolar epithelial cells (AEC, J). Arrowheads: areas of suppurative bronchitis/bronchiolitis; arrows: interstitial pneumonia, necrosis of AEC (bar: (I) 100 µm; (J) 50 µm).

Expanded view figure legends:
Autologous, Lentivirus-Modified, T-Rapa Cell "Micropharmacies" for Lysosomal Storage Disorders (2022)
Figure 1

Figure 1: Transduced healthy (HDo) and Fabry donor (FDo)-derived T-Rapa "micropharmacies" (TRaMs) secrete functional lysosomal enzymes.

A. Schematic of TRaM production. CD4+ T cells were enriched by positive selection from leukaphereses of multiple HDos and 3 FDos - details are provided in Materials and Methods.

B. Enzyme compartments that are measured in C-I and Fig EV4G; intracellular, total secreted and colloidal particles (enclosed in red and yellow circles).

C. HDo-TRaMs were engineered with vectors relevant to treatment of Fabry, Gaucher, Farber and Pompe diseases. Intracellular lysosomal enzyme specific activities were measured in transduced T-Rapa and controls. Transductions varied across vectors/donors, as indicated.

D, E. Enzyme activity was assessed in transduced T-Rapa supernatants compared to controls in activated/dividing (D) and quiescent/resting (E) states.

F. Cells from all FDo were stably transduced, as indicated. Intracellular α-galactosidase A (α-gal A) specific activities were determined.

G, H. Secreted α-gal A activity was measured from FDo-derived TRaMs in activated (G), and quiescent/resting (H) states.

I. α-Gal A specific activity in Fabry patient-derived skin fibroblasts after 6 hours of exposure to conditioned media from TRaMs in the presence or absence of 1mM M6P.

Data information: Activities in (C-H) are reported as a mean of n=3 seeded wells per donor, error bars are standard deviation. Data for (C-E) are representative of at least 2 donors. Activities were compared between matched transduced and control cells using pairwise two-tailed Student's t-tests, *** p<0.001 and ** p<0.01. Activities in (I) are reported as a mean of n=3 independent treatments per condition, error bars are standard deviation. Two-way analysis of variance with Tukey's multiple comparison test was used to compare activities, ** p<0.01 and * p<0.05. Abbreviations - VCN/g: vector copy number per genome; GLA: α-gal A; GBA: β-glucocerebrosidase (GCase); ASAH1: acid ceramidase (ACDase); GAA: acid α-glucosidase; NT: non-transduced; LV: lentiviral transduced; CTRL: vehicle control.
Autologous, Lentivirus-Modified, T-Rapa Cell "Micropharmacies" for Lysosomal Storage Disorders (2022)
Figure 2

Figure 2: TRaMs result in elevated enzyme expression following xenotransplantation.

A. Schematic of the transplant protocol optimized for T-Rapa cells. NOD/SCID/Fabry (NSF) mice were conditioned and TRaMs were administered intravenously - details are indicated in Materials and Methods. Mice were euthanized 4 weeks post-infusion.

B. Engraftment was evaluated by flow cytometry for human CD3 expression.

C, D. α-Gal A activity in plasma, and specific activities in liver, spleen, heart and kidneys were measured after transplant of transduced healthy donor (HDo)-derived (C) or Fabry donor (FDo)-derived (D) TRaMs, non-transduced (NT) cells, or in sham-treated NOD/SCID (NS) and NSF mice.

Data information: %CD3 of lymphocyte-gated parent population in (B) and enzyme activities in (C, D) are reported as values for individual mice (n=4) with a line indicating the mean, and error bars showing standard error of the mean. The y-axis is logarithmic to base 10 in (c-d) to highlight activities in controls. One-way analyses of variance with Tukey's multiple comparison tests were used to compare activities between groups, *** p<0.001, ** p<0.01, * p<0.05 and ns p>0.05. Abbreviations - TRaMs: T-Rapa micropharmacies; LV/GLA: lentivirus transduced to express α-gal A; VCN/g: vector copy number per genome
Chromatin remodeler CHD7 is required for GABAergic neuron development by promoting PAQR3 expression (2021)
Figure 1

Figure 1 - Generation of a zebrafish chd7 mutant using CRISPR/Cas9.

A. Chromatograms showing the confirmation a 1-nucleotide insertion mutation by Sanger sequencing.

B. Gross morphological analyses of control (chd7+/+; top left image), heterozygous (chd7+/-; bottom left image) and knockout mutants (chd7-/-; images in right panel).

C. Kaplan-Meier Survival plot showing low survival of chd7 mutants after 12 dpf (N=5).

D. Measurement of head size of control (n=12) and mutants (n=14) showing significantly smaller head size in chd7-/- fish (****p<0.0001, Student's t-test).

E. Acetylated tubulin staining in 28 hpf controls (left) and mutants (right) showing severely affected outbranching of the trigeminal nerve (Vth cranial nerve). Notably, chd7-/- display reduced branching of the Vth cranial nerve (arrows) and axonal arborisation in the tectal area. Graphs showing quantitative analyses of percentage (n=5) and mean total length of peripheral projections (n=6) per zebrafish in controls and mutants (***p<0.001; **p<0.005, Student's t-test).

F. Locomotor activity of control (black), heterozygous (blue) and mutants (red) showing significant hyperactivity of mutants in dark and light cycles (N=3, n=48).

G. Average activity per second during dark cycle (left) is significantly increased in chd7-/- mutants compared to chd7+/+ (n=32; ****p<0.0001, Student's t-test). Representative swimming tracks during dark cycle of control and mutant fish (right). Mutant chd7-/- larvae displayed hyperactive swimming.

Data information: **** denotes p<0.0001; ***p<0.001; **p<0.005, Student's t-test. Data are presented as mean±SEM. Scale bar = 50 μm. n represents number of fish, N represents number of experimental repeats.
Chromatin remodeler CHD7 is required for GABAergic neuron development by promoting PAQR3 expression (2021)
Figure 2

Figure 2 - GABAergic neuron defects in zebrafish chd7 mutant brain.

A. Structural illustration of 5 dpf zebrafish brain from dorsal (top) and lateral (bottom) view (OB: Olfactory bulb, Tel: Telencephalon, OT: Optic tectum, CB: Cerebellum, HB: Hindbrain).

B. 5dpf dlx5a/6a transgenic line showing GFP+ GABAergic neurons are reduced in chd7-/- mutants (bottom) in comparison to controls (top) in both dorsal (left) and ventral (right) view.

C-H Total number of GABAergic neurons (GFP+ cells) in (C, D) the optic tectum (OT) and cerebellum (CB) regions of 5 dpf wild-type and chd7 mutant fish (n=16; ****p<0.0001; Student's unpaired t-test), (E, F) the hypothalamus (hyp) region (n=10; *p=0.0182; Student's t-test) and (G, H) the telencephalon (tel) (n=7; *p=0.0347; Student's t-test).

I. Treatment of control (dark grey) and mutants (light grey) with GABA agonists Baclofen (N=3, n=24; ns, p=0.1427; Student's t-test) and Muscimol (N=3, n=24; ns, p=0.3987; Student's t-test) showing recovery of hyperactive locomotor activity in chd7-/- mutants (vehicle: N=3, n=24, **** denotes p<0.0001; Student's t-test).

J, Functional analysis of GABAergic signalling shows increased responsiveness to GABA antagonist PTZ in both onset and overall locomotor activity (n=24; ****p<0.0001; one way ANOVA).

K. Average locomotor activity between 2 dpf controls (black) and chd7-/- mutants (red) shows increased activity after 3 mM PTZ exposure (n=24; ***p<0.001; two-way ANOVA).

Data information: Data are presented as mean±SEM. Scale bar = 50 μm. n represents number of fish used, N represents number of experimental repeats.
Chromatin remodeler CHD7 is required for GABAergic neuron development by promoting PAQR3 expression (2021)
Figure 3

Figure 3 - chd7 regulates GABAergic neuron development via MAPK/ERK signalling. Transcriptomic analysis was performed on dissected 5 dpf brains.

A. Volcano plot showing each gene plotted according to its log2 fold change. All highly differentially expressed genes with p<0.05 are in orange with fold-change>1.5.

B, C. Biological processes (B) and Molecular function (C) that are enriched in the differentially expressed genes.

D. ERK activation by phosphorylation is increased in mutants as shown by Western blot quantification (N=4; ***p<0.005; Student's t-test).

E. pERK immunohistochemistry showing increased ERK activation in the mutant brain (N=3, n=8).

F-G. Treatment with the ERK signalling inhibitor U0126 ameliorated the number of GABAergic neuron (n=10-15; **p<0.05; ****p<0.0001, one-way ANOVA).

H. Treatment with the ERK signalling inhibitor U0126 rescues locomotor hyperactivity phenotype in chd7 mutant fish (n=8 for chd7+/+ and chd7-/-; n=16 for U0126 treated fish; ****p<0.0001 ; ns, not significant, one-way ANOVA).

Data information: Data are presented as mean±SEM. Scale bar = 50 μm. n represents number of fish used, N represents number of experimental repeats.
Chromatin remodeler CHD7 is required for GABAergic neuron development by promoting PAQR3 expression (2021)
Figure 4

Figure 4 - CHD7 regulates PAQR3 expression in human cells.

A. qPCR analysis of PAQR3 expression in a CHD7-mutation positive patient compared to parental controls set (N=4; ****p<0.0001; Student's t-test). Fold change was calculated according to the 2(−ΔΔCt) method, using HPRT1 and RPS1 as housekeeping genes for normalization. All data were expressed as mean fold change ± SD across replicates, relative to control parents set to 1 (dotted line), N is the number of experimental repeats.

B. Schematic view of PAQR3 exon 1 and proximal promoter on chromosome 4 (hg19 assembly), obtained with the UCSC genome browser (https://genome.ucsc.edu/) and showing the sequence amplified in ChIP-qPCR assays in LCLs (thick black line) along with a previously described CHD7 ChIP-seq peak (thick grey line) and signal (green) in H1-hESC (ENCODE3).

C. ChIP-qPCR assays in LCLs showing decreased occupation of CHD7 on the PAQR3 proximal promoter in a CHD7-mutation positive patient (N=3) compared to parental controls (N=6); *p< 0.05; Student's t-test, All data were expressed as mean fold change ± SD, N is the number of experimental repeats.
Chromatin remodeler CHD7 is required for GABAergic neuron development by promoting PAQR3 expression (2021)
Figure 5

Figure 5 - Zebrafish paqr3b regulates GABAergic neuron development

A-B. GABAergic neuron defects in wild-type fish treated with ER/Golgi traffic inhibitor BFA (n=9; ****p<0.0001; ns, not significant; One-way ANOVA).

C-D. Overexpression of paqr3b mRNA improve the number of GABAergic neurons in 3dpf chd7 mutant fish (n=7 for chd7-/- and n=11 for chd7-/- + paqr3b mRNA; ****p<0.0001; **p<0.05; One-way ANOVA). Of note, the rescue experiment was performed at 3 dpf given the transient nature of mRNA.

E. ERK activation by phosphorylation in chd7-/- mutants was restored to normal levels upon overexpression of paqr3b mRNA shown by Western blot quantification (N=4; **p<0.05; One-way ANOVA, N is the number of experimental repeats.

Data information: Data are presented as mean±SEM. Scale bar = 50 μm.
Chromatin remodeler CHD7 is required for GABAergic neuron development by promoting PAQR3 expression (2021)
Figure 6

Figure 6 - GABAergic defects and behavioural anomalies are suppressed by ephedrine treatment in mut-chd7 C. elegans.

A. Movement scores of chd-7(gk290) mutants (red) compared to N2 wildtype (WT; black). Two-tailed T-test was performed for statistical analyses and movement score was considered different to WT (N=3, n=30; ****p < 0.0001, Student's t-test).

B. Lifespan analyses of chd-7(gk290) mutants (n=339; red) compared to WT (n=444; black). Log-rank test was performed for statistical analyses. ***p < 0.001.

C. WT and chd-7(gk290) animals with defects in the GABAergic (black) and cholinergic nervous system (grey) at the L4 stage (N=4, n=100; ****p<0.0001; Student's t-test).

D-F. Example pictures of the GABAergic nervous system cell bodies, commissures, axonal gaps and axonal breaks larger than 50 µm per worm in chd-7(gk290) mutants expressing unc-47p::mCherry.

G-I. GABAergic neurodevelopmental defects of WT (black) and chd-7(gk290) mutants (grey) at the L4 stage classified in axonal gaps per worm (G), number of GABAergic cell bodies (H) and number of commissures (I). N=4, n=100; ****p<0.0001, Student's t-test.

J. Chemical libraries (3850 compounds) were first screen in chd-7(gk290) C. elegans mutants and positive hits were tested on chd7 zebrafish mutants.

K. 49 compounds that improved motility phenotypes in chd-7(gk290) mutants were identified. These compounds were clustered in 6 main functional categories.

L. Movement scores of chd-7(gk290) mutants treated with ephedrine (blue) compared to the solvent DMSO (black). N=3, n>100; ****p<0.0001, Student's t-test.

M. Defects of the GABAergic nervous system at the L4 stage in chd-7(gk290) mutants treated with ephedrine (grey) compared to DMSO (black) (N=3, n>100). ****p<0.0001, Student's t-test.

N. Average activity per second is wild-type and mutants treated with ephedrine compared to non-treated mutant (wild-type treated with ephedrine is referred to as Control). Ephedrine suppresses hyperactivity in chd7 mutant fish. n=24; ****p<0.0001, one-way ANOVA.

O. Representative images of GABAergic neurons and total number of GABAergic neurons (GFP+ cells) in the optic tectum (OT) and cerebellum (CB) regions of wild-type, chd7 mutant and ephedrine-treated fish (n=9; ****p<0.0001; One-way ANOVA). Treatment with ephedrine ameliorated the number of GABAergic neurons.

P-Q. p-ERK is reduced in mutants following treatment with ephedrine as shown by Western blot quantification (N=4). ****p<0.0001, one-way ANOVA.

R. qPCR analysis of paqr3b expression in chd7+/+ , chd7-/- and chd7-/- treated with ephedrine (N=4; ****p<0.0001; ns, not significant, one-way ANOVA.

Data information: Data are presented as mean±SEM. Scale bar = 50 μm. n is the number of fish or worms used, N is the number of experimental repeats.

Expanded View Figure Legends
A synonymous variant in IRGM alters a binding site for miR-196 and causes deregulation of IRGM-dependent xenophagy in Crohn's disease (2011)
figf1

(a) In silico prediction of miR-196 and IRGM mRNA interactions showed differences in binding within the seed region. (b) IRGMC mRNA was significantly enriched in miR-196B complexes. Extracts of cells expressing FLAG-tagged-AGO1 and transfected with biotinylated miR-196B or control miR-20 as indicated and then with IRGMC or IRGMT plasmids were submitted to tandem affinity purification (immunoprecipitation with FLAG antibodies followed by affinity purification on streptavidin beads). IRGM mRNA variants were quantified using quantitative RT-PCR; results are presented as the ratio between miR-196B and miR-20 (non-relevant miRNA) pull-downs and the mean of three independent experiments ± standard deviation (s.d.). IP strep, immunoprecipitation straptavidine. (c) HEK293 cells (IRGMC/C) were transfected with either FLAG-tagged IRGMC or FLAG-tagged IRGMT plasmids and co-transfected with miR-196B. Immunoblotting with an IRGM antibody revealed the specificity of the downregulation effect mediated by the miRNA IRGM mRNA interaction. Quantification of the immunoblot signals are presented as IRGM expression relative to actin (mean of at least three independent experiments ± s.d.).