EMBO/sd-character-level-ner huggingface dataset:
Figure 4A-B. Bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins or domains fused to the T18 or T25 domain of the Bordetella adenylate cyclase were spotted on X-Gal indicator plates. The blue color of the colony reflects the interaction between the two proteins. TolB and Pal are two proteins known to interact but unrelated to the T6SS or the MltE proteins. The experiment was performed in triplicate and a representative result is shown. C. Co-immunoprecipitation assay. The solubilized lysates from 2×1010 E. coli K-12 W3110 cells co-producing the indicated FLAG-tagged TssMP variants (exported in the periplasm) and VSV-G-tagged MltE protein (Total, T) were subjected to immune precipitation on anti-FLAG-coupled agarose beads. The immunoprecipitated material (IP) was subjected to 12.5%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (upper panel, TssM domains) and anti-VSV-G (lower panel, MltE) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.
Generation of viral cDNA clones and recovery of recombinant SARS-CoV-2 and SARS-CoV-2-GFP.Figure 3.Generation of viral cDNA clones and recovery of recombinant SARS-CoV-2 and SARS-CoV-2-GFP.a, Schematic representation of SARS-CoV-2 (upper panel) and SARS-CoV-2-GFP (lower panel) genome organisation with 12 and 14 viral subgenomic overlapping cDNA fragments, respectively, used for TAR cloning. pT7, T7 RNA polymerase promoter; UTR, untranslated region; An, poly (A) tail; J represents assembly junction between two overlapping DNA fragments. b, Representation of predicted RNA stem-loop (SL) secondary structures formed in the 5'-UTR of SARS-CoV-2. The SARS-CoV-2 RNA secondary structures were manually adjusted based on RNA structure predictions by Chan et al.22. Black letters and numbering represents the SARS-CoV-2 5'-terminal sequence. Red letters depict nucleotides that are different within the SARS-CoV 5'-terminal sequence ("-" indicates nucleotide deletion in SARS-CoV versus SARS-CoV-2). N20 indicates 20 nucleotides. SL structures 1, 2, and 4 are shown according to Chan et al.22. Note that recombinant virus construct 1 (rSARS-CoV-2 clone 1) and construct 4 (rSARS-CoV-GFP clone 4) contain the SARS-CoV-2 5'-terminus with nucleotides 3-5 (UAA) exchanged by nucleotides 3-5 (AUU) of SARS-CoV; recombinant virus construct 2 (rSARS-CoV-2 clone 2) and construct 5 (rSARS-CoV-2-GFP clone 5) contain a 5'-terminus with the first 124 nucleotides from SARS-CoV; and recombinant virus construct 3 (rSARS-CoV-2 clone 3) and construct 6 (rSARS-CoV-GFP clone 6) contain the authentic SARS-CoV-2 5'-terminus. Rescue of recombinant rSARS-CoV-2 and rSARS-CoV-2-GFP. The experimental setting is illustrated in the upper panel. Two clones of each construct 1-6 were used to prepare YAC DNAs and in-vitro transcribed viral genome RNAs were electroporated into BHK-21 cells and/or BHK-SARS-N together with an mRNA encoding the SARS-CoV-2 N protein. Electroporated cells were co-cultivated with VeroE6 cells in a T25 flask and cell culture supernatants were transferred to 12-well plates to perform plaque assay for rSARS-CoV-2 constructs 1-3 and fluorescence microscopy for rSARS-CoV-2-GFP constructs 4-6. Plaque assays are shown in the lower panel for SARS-CoV-2 clones 1.1, 2.2, and 3.1. VeroE6 cells were infected with 10−1, 10−2, and 10−3 dilutions of 1 ml supernatant from the individual rescue experiments and are compared to non-infected cells (Mock). The expression of GFP in SARS-CoV-2-GFP infected VeroE6 cells is shown for clones 4.1, 5.2, and 6.1. Mock represents non-infected VeroE6 cells. Summary of the timeline of events of the SARS-CoV-2 outbreak and the work leading to the reconstruction and recovery of rSARS-CoV-2.
Figure 2A. Single-letter amino acid (aa) sequence of FadA. The intact pre-FadA consists of 129 aa, with the first 18-aa (top row) constitute the signal peptide. The remaining 111 aa constitute mFadA. The residues that are replaced in the mutant proteins used in this study are shown in red. B. Analysis of detergent resistant recombinant FadA polymers expressed in E. coli. An aliquot of 50 μg detergent-resistant pellets prepared from E. coli BL21(DE3) carrying the cloning vector pET21(b), pYWH417-6 (p_FadAc), or pYWH418 (p_mFadA) were analyzed by SDD-AGE, followed by Western blot analysis. FadA polymers were only detected in E. coli expressing FadAc, but not in mFadA or the vector control. C. Reactivity of recombinant FadA and its variants with anti-amyloid fibril antibody OC. A total of 10 μg each of recombinant protein mFadA, FadAc, FadA-L-9A, FadA-L14A, FadA-S71A, and FadA-L76A purified from E.coli was slot-blotted onto nitrocellulose and incubated with polyclonal OC antibody at 1:5,000 dilution. Amyloid-like recombinant Rim4 (Berchowitz et al., 2015) was used as a positive control and histone H1 was used as a negative control. Ponceau stain (pink color) is shown as a loading control. D. Thioflavin-T binding assay. FadA proteins at indicated concentrations were incubated with 10 μM Thioflavin-T at room temperature for 10 min. The fluorescent intensity was measured at excitation wavelength of 440 nm and emission wavelength of 500 nm. The experiment was performed in duplicate and repeated three times. The error bars indicate SD. *p<0.05, **p< 0.01, ***p<0.001 (compared to BSA, t-test). E. Thioflavin-T binding assay in the presence of 0.1% SDS. FadA proteins (0.8 mg/ml) were incubated with 10 μM Thioflavin-T at room temperature for 10 min. The fluorescent intensity was measured at excitation wavelength of 440 nm and emission wavelength of 500 nm. The experiment was performed in duplicate and repeated three times. The error bars indicate SD. *p<0.05, **p< 0.01 (compared to BSA, t-test). F. Western blot analysis of Fn12230 (Fn), fadA-deletion mutant US1 (∆fadA) and spontaneous mutant lam following SDS-PAGE. A total of 400 μl of each culture grown to OD600 of 1.0 was pelleted and loaded onto 12% SDS-PAGE. FadA protein was detected using anti-FadA mAb 7H7 at 1:4,000 dilution. Compared to the wild type, lam produced significantly less pre-FadA, but not mFadA. G. Analysis of amyloid-like FadA produced by Fn 23726 and its ∆fap2 mutant. An aliquot of 50 μg detergent-resistant pellets was loaded onto each lane, followed by SDD-AGE and Western blot analysis as described above. The mutant produced significantly less amyloid-like polymers than the wild type.
Figure 3(A) Linear representation of HIV-1SF2 NEF. To identify a motif of NEF critical for suppression of the humoral immune response, highlighted regions were either deleted or mutated. (B) Production of anti-HEL IgG at the indicated day post immunization with HEL-OVA/CFA following adoptive transfer of BHEL and CD4OT-II cells expressing NEF or various NEF mutants. Shown are relative levels of HEL IgG production with the median of IgG levels in mice receiving control cells on day 14 post immunization arbitrarily set to 1. Each dot represents one animal. Shown are mean values with SD from 5-12 mice from 4 experiments. (C) HEL-OVA pulsed- BHEL cells (blue) and CD4OT-II cells (green) were placed on PLL-coated cover glasses and subjected to live-cell time-lapse imaging. Shown are representative still images. BHEL cells were loaded with SiRAct to visualize actin polymerization in B cells (F-actin displayed in red). Filled and empty arrowheads indicate presence or absence of F-actin accumulation at the IS, respectively. Scale bars, 5µm. (D) Quantification of B cell actin polymerization as shown in C with frequency of actin polymerization in B cells co-cultured with control T cells arbitrarily set to 100%. Shown are mean values with SD from three independent experiments with at least 30 conjugates evaluated per condition. (E-G) Effects of NEF expression in CD4OT-II cells on gene expression in BHEL cells. After 24h of B-T co-culture, BHEL cells were separated from CD4OT-II using magnetic beads before RNA isolation for microarray analysis. The top 235 deregulated genes (see Dataset EV2) were grouped into genes that were highly induced (E), moderately induced (F) or repressed (G) by Ag stimulation in control cells and the effects of NEF WT or NEF ∆12-39 displayed as heat maps. Heat map values are plotted as average Log2 FC (n=3 biological repeats) relative to unstimulated BHEL. Red and green represents higher and lower expression, respectively. (H) Relative surface expression of CD4, CD28, OX40, ICOS, CD45 and CD54 on T cells expressing NEF WT or the indicated NEF mutants. Analysis of surface expression was performed using flow cytometry 3-7 d post transduction of T cells. Depicted are mean values with SD from 3-6 independent experiments, with control T cells arbitrarily set to 100% as indicated by the dashed line. (I) Representative confocal images of the subcellular distribution of IL-4 in CD4OT-II cells expressing the indicated proteins engaged in an IS with BHEL cells. B cells and T cells were placed on PLL-coated cover glasses for 60-90min, fixed and stained for intracellular IL-4. Filled and empty arrowheads indicate presence or absence of IL-4 accumulation at the IS, respectively. Scale bars, 5µm. (J) Quantification of IL-4 accumulation at the IS as depicted in I. Shown are mean values with SD from 3 independent experiments relative to control cells set to 100%.
Figure 5(A) Representative fluorescence microscopy images of wild type (w1118), NimB4sk2, draper, eater1 and croquemort△ third instar larvae macrophages stained with LysoTracker Red (live imaging). Overlay of fluorescence and differential interference contrast microscopy (DIC). Scale bar = 10 μm. (B) Mean fluorescence intensity after staining with LysoTracker Red (live confocal imaging). Values from at least three independent experiments are represented as mean ± SD ( ** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests). (C) Representative transmission electron micrographs of macrophages from wild type (top, w1118) and NimB4sk2 (bottom) L3 wandering larvae. Scale bar: 2 μm (D) Quantification of the number of vesicles per macrophages in the electron micrographs. Values from at least three independent experiments are represented as mean ± SD (**** P<0.0001 by Mann-Whitney test). (E) Representative confocal imaging of Rab7EYFP immunostaining in wild type (w1118) and NimB4 sk2 macrophages. Tissues were stained with anti-GFP (red, Rab7), counterstained with phalloidin (gray) and DAPI (blue). The arrows indicate the enlarged vesicles are decorated with Rab7EYFP in the NimB4sk2 macrophages. Scale bar = 10 μm. (F) Quantification of the diameter of the vesicles. Values from at least three independent experiments are represented as mean ± SD (**** P<0.0001 by Mann-Whitney test).
Figure 2A. Schematic diagram showing the dual chamber assay. SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). *** P < 0.001 against PBS, one-way ANOVA. NT, c-src KD #1 and #2 SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells for 12 h, and then fixed and immunostained with anti-EGFP antibody. Values were derived from four independent experiments (n=4). *** P < 0.001 compared to NT cells, one-way ANOVA , D. primary cortical neurons were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). *** P < 0.001 against PBS, one-way ANOVA. , E. primary cortical neurons downregulating c-src were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells for 12 h, and then fixed and immunostained with anti-EGFP antibody. Values were derived from four independent experiments (n=4). *** P < 0.001 compared to NT cells one-way ANOVA unpaired t-test SH-SY5Y cells were transfected with plasmids of WT c-src, Y529F c-src (kinase active), or K297M c-src (kinase dead) tagged with EGFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 h and then fixed and immunostained with anti-α-syn antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). *** P < 0.001, * P < 0.05 compared to mock positive cells, one-way ANOVA. G. FcγRIIB KD SH-SY5Y cells were transfected with plasmids of WT c-src, Y529F c-src (kinase active), or K297M c-src (kinase dead) tagged with EGFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 h and then fixed and immunostained with anti-α-syn antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). *** P < 0.001, * P < 0.05 compared to mock positive cells, one-way ANOVA.
Figure 7(A) Specific GALC enzymatic activity in single brain slices of WT untreated (UT; white bars, physiological levels), WT LV.hGALC-treated (JV02) and Krabbe-affected LV.hGALC-treated (JV02) animals (filled grey bars, injected hemisphere; striped grey bars, contralateral hemisphere). Two-way ANOVA followed by Bonferroni's multiple comparison tests; *p<0.05, **p<0.01 vs matched slices of WT UT. (B-C) Summary of GALC activity in brain (B) samples from JT02 and JV02, as well as from WT and Krabbe UT controls. Data in A-C are represented as floating bars (min to max, line at mean; n=2-6 blocks/slice). Number of blocks analysed/animal: 16 (WT UT), 24 (JT02), 22 (JV02). GALC activity in Krabbe UT samples is < 0.001 nmol/h*mg; n=3 blocks. Kruskal-Wallis test and Dunn's multiple comparison test; *p<0.05, ***p<0.001 vs WT UT. (B-C) Summary of GALC activity in spinal cord (C; n=3 blocks/animal) samples from JT02 and JV02, as well as from WT and Krabbe UT controls. Data in A-C are represented as floating bars (min to max, line at mean; n=2-6 blocks/slice). Number of blocks analysed/animal: 16 (WT UT), 24 (JT02), 22 (JV02). GALC activity in Krabbe UT samples is < 0.001 nmol/h*mg; n=3 blocks. Kruskal-Wallis test and Dunn's multiple comparison test; *p<0.05, ***p<0.001 vs WT UT. (D) Chromatographic profile of GALC in selected brain slices and spinal cord (SC) tissue of untreated (UT) WT and LV.hGALC-treated Krabbe NHP (JT02). Extracts were run through a Sephadex S-300 gel filtration column. Fractions (0.2 ml) were assayed for enzyme activity using MUGAL substrate in the presence () or absence of 11µM AgNO3 (●). (E) GALC activity in the CSF of JT02 (pre- and post-treatment) and JV02 (post-treatment). Dots represent technical replicates. (F) Beginning at two months (m) of age, animals were evaluated monthly for motor performance using a modified Bayley's scale for infant development. For the study animals (JV02 and JT02), scores are reported as pre-surgery and post-surgery. Historical data for normal and affected animals are presented as means plus one standard deviation. Affected animals score significantly lower than normal animals at any age considered. JT02's motor scores increase over time post LV.GALC injection, being close to the mean observed in normal juveniles at 5 months. Historical data are analyzed by two-way ANOVA and Bonferroni posttests, p<0.05 at 6m, p<0.001 at all other ages. n=5-20 animals/group, except for normal animals at 6 months (n=1).
Molecular features of C3b-bound MCP and SPICE and comparison with C3b-FH.Figure 3A. Superposition of MCP and FH domains CCP3-4 (shown as blue and orange cartoons, resp.) on the surface of C3b. B. Cartoon representation of the superposition of domains CCP3-4 of MCP (blue), FH (orange) and SPICE (green). Despite the strong structural similarity and the conserved angle between domains CCP3 and CCP4, MCP and SPICE show a rotation of approximately 100° in the position of CCP4 compared to FH. C. The reported mutagenesis data on MCP CCP3 and CCP4 (data from Liszewki et al. (Liszewski et al, 2000)) are consistent with the crystal structure. Shown are two orientations of the surface of MCP CCP3-4 as in the crystal structure of the C3b-MCP complex, highlighting the contact sites with the C3b platform (purple), the MCP mutations identified as critical for C3b binding (red) and those not affecting the interaction (blue). The GB24 antibody epitope is shown as yellow contour on the MCP surface. D. Model of the putative positioning of the flexible CCP1-2 domains of MCP. The model has been generated by superposing the structure of CCP1-4 of MCP from PDB 3O8E (Persson et al, 2010) onto the structure of the C3b-MCP (CCP1-4) complex. CCP1-2 domains of MCP, not visible in the C3b-MCP (CCP1-4) structure, are shown in grey. E. Highlight of the primary sequence differences between SPICE and VCP. The 11 mutations distinguishing the two viral regulators are shown on the surface of SPICE in purple. The contact surface of C3b on the regulator in the C3b-SPICE (CCP1-4) complex is shown in green on the right panel.
Figure 1(A) Venn diagram depicting the total number of genes upregulated in MEFs (1504) or mHEPs (532) undergoing reprogramming and the 454 common genes upregulated in both MEFs and mHeps at day 6 of reprogramming. Of these, 30 genes, listed in the adjacent table, encode for Transcriptional Regulators (TRs). The top diagram indicates the experimental outline of the DNA Microarrays expression studies performed in MEFs and mHEPs undergoing reprogramming. (B) Bar graph summarizing the efficiency of single RNAi knockdowns for 28 out of the 30 TRs, and their effects in the reprogramming efficiency in comparison to control cells expressing scramble shRNA. The efficiency of each TR's knockdown (KD) does not correlate with its effect in reprogramming efficiency. The knockdown of 9 (Class III) out of the 28 TRs decreased the efficiency of the reprogramming in a statistically significant manner (unpaired two-tailed Student's t-test, p-value <0.05). Data are shown as mean ± SEM of at least two independent experiments. Class I corresponds to one TR inhibiting reprogramming, whereas Class II corresponds to 18 TRs having a weak or no effect in repogramming. Shown are the effects of 28 out of 30 TRs, because we did not succeed in knocking down Cphx and Phox2a. (C) Left: Shown is a line graph depicting normalized mRNA expression levels of the 9 TRs at the indicated time points during the reprogramming of MEFs. The data were plotted after normalization with the endogenous Gapdh using the "ΔCt method". The grey vertical dashed line depicts day 6 in which the expression levels of nearly all TRs peaks. Notice that the y-axis is broken to accommodate the large spread of expression levels. Right: Shown are western blots using antibodies specific for Irf6, Tead4, Taf1c, Rcan1 and Tfap4. Whole cell extracts were prepared from MEFs at day 6 of reprogramming and run side-by-side with extracts from control samples (day 0). Gapdh, mTOR and b-Tubulin were used as loading controls. (D) Same as in (B) except MEFs were co-transduced with lentiviruses expressing all possible pairwise combinations of shRNAs for the nine TRs. Left: Heat map depicting the effects of paired KD combinations in the Reprogramming Efficiency (RE%). Shown are the average REs values from at least two independent experiments. The REs of the double KDs were evaluated against the RE of the corresponding single KDs. Right: Alkaline Phosphatase (AP) stained cultures of terminal reprogrammed MEFs upon representative single and double KDs of our shRNA-based screens.
Dcp-1 alters ATP levels that in turn regulate autophagy.Figure 6.(A) Total cellular ATP levels were measured in l(2) mbn cells treated with control or Dcp-1 dsRNA. Data represent ± SEM of three independent experiments (n = 3). Statistical significance was determined using a two-tailed Students t test (*, P = 0.02). (B) Total cellular ATP levels were measured in ovaries from fed or starved w1118 and Dcp-1Prev1 flies. Data represent ± SEM of five independent experiments (n = 5). Statistical significance was determined using a two-tailed Student's t test (**, P = 0.014; ***, P < 0.001). (C) Fed or starved w1118 and Dcp-1Prev1 flies were treated with DMSO or 25 µg/ml oligomycin A, and Ref(2) P levels were assessed by immunoblot analysis. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graph represents ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P = 0.02). (D and E) Control UASp-GFP-mCherry-DrAtg8a/+;nosGAL4/+ flies (D) and Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies (E) were subjected to starvation conditions supplemented with DMSO or 25 µg/ml oligomycin A. Bars, 25 µm. Quantifications show percentage of autolysosomes (autolysosomes/total autophagic structures). At least eight egg chambers were manually quantitated per genotype per condition (n = 8). Statistical testing was determined using a two-tailed Student's t test. ***, P = 0.0002.
Figure 3(A) IFN‑α (left) and IFN‑β (right) production by WT and Irf3-/-Irf7-/- BMDCs plated at high density (2x105 cells/cm2) and stimulated with poly(I:C) (blue dots) or left unstimulated (grey dots). Dotted lines represent the ELISA threshold of detection. Data are representative of 2 independent experiments. (B) CD86 and CD40 expression was quantified on WT (blue dots) and Irf3-/-Irf7-/- (orange dots) BMDCs plated at high density (2x105 cells/cm2) and stimulated with poly(I:C) or left unstimulated. Data are pooled from 2 independent experiments. (C) CD86 and CD40 expression on Irf3-/-Irf7-/- BMDCs plated at 2x105 cells/cm2 and stimulated with poly(I:C) (dark blue dots), IFN-α (light blue dots) or a combination of both (green dots). Data are representative of 2 independent experiments. (D-E) WT and Irf3‑/‑Irf7‑/‑ BMDCs were mixed at different ratios for a total number of 2x105 cells/cm2 and stimulated with poly(I:C) or left unstimulated. (D) Representative dot plots showing CD86 and CD40 expression on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) BMDCs. (E) Flow cytometric quantification of CD86 and CD40 expression on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) BMDCs expressed as a fold change in gMFI over unstimulated controls. (F) CD86 and CD40 expression on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) BMDCs expressed as a fold change in the proportion of CD86+ CD40+ cells over unstimulated controls. Data are pooled from 2 independent experiments.
Figure 4(A) Assay of FoxA2 mRNA by RT-PCR in FoxA2-Tet ES cell lines 48 hr after treatment with the indicated dose of DOX. Average and SD of two independent experiments from 2 independent lines. B) FoxA2 immunostaining of FoxA2-Tet ES cells 48 hr after DOX treatment. Left, no DOX control. Middle and right, 10ng/ml DOX. DAPI staining shown in blue. Right panel shows middle image without DAPI signal. (C) Assay of FoxA2 and CXCR4 mRNA by RT-PCR on day 3 of differentiation with DOX at the indicated dose. Average and SD of two experiments from two independent cell lines. (D) Assay of Sox17 mRNA by RT-PCR on day 7 of differentiation with addition of 3 µM CH and/or 10 ng/ml DOX at the indicated time points (in days). Average and SD of two independent experiments from two independent cell lines. (E) Sox17 immunostaining of FoxA2-Tet ES cells at day 7 of differentiation in the presence or absence of 10 ng/ml DOX and the presence and absence of 3 µM CH as indicated. Far right panel shows image to its left without DAPI.