EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A-E) GFP-pho8Δ60-expressing wild-type (WT), atg5Δ, atg7Δ, and atg5Δpep4Δ cells were starved (A) or treated with AmphoB (B-E) and subjected to western blotting. Cells were treated with 2.5 µg/ml AmphoB for the indicated time (B) or with the indicated doses for 24 hr (C-E). Generation of free GFP was a marker of proteolysis. (F) GFP-pho8Δ60- and Pep4-mRFP-expressing atg5Δ cells were cultured with (AmphoB) or without (NT) AmphoB (2.5 µg/ml, 24 hr), and localization of GFP-pho8Δ60 and Pep4-mRFP was observed by confocal microscopy. Arrowheads indicate the cells in which GFP-pho8Δ60 was delivered to the vacuoles. Scale bars = 2 µm. (G, H) Accumulation of autophagic body (AB)-like structures by AmphoB in atg5Δpep4Δ cells. pep4Δ and atg5Δpep4Δ cells were starved for 3 hr or treated with AmphoB (2.5 µg/ml, 24 hr). Scale bars = 2 µm. Cells containing AB-like structures were counted under phase-contrast microscopy. Representative images (G) and the percentage of cells with AB-like structures (H) are shown. *p < 0.01 vs the value of pep4Δ cells. (I-K) Induction of AB-like structures in atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr). Representative images of quick-frozen replicas (I) and thin section of the frozen-substituted material (J) are shown. (G, H) Accumulation of autophagic body (AB)-like structures by AmphoB in atg5Δpep4Δ cells. pep4Δ and atg5Δpep4Δ cells were starved for 3 hr or treated with AmphoB (2.5 µg/ml, 24 hr). Scale bars = 2 µm. Cells containing AB-like structures were counted under phase-contrast microscopy. Representative images (G) and the percentage of cells with AB-like structures (H) are shown. *p < 0.01 vs the value of pep4Δ cells. (I-K) Induction of AB-like structures in atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr). Representative images of quick-frozen replicas (I) and thin section of the frozen-substituted material (J) are shown. (I) AB-like structures accumulated in the vacuole. Scale bar = 0.5 µm. (J) AB-like structures containing lipid particles and mitochondria accumulated in the cross-section of the vacuole. An autophagosome (AP)-like double-membrane compartment containing a ribosome and Golgigranule is present in the cytosol. Scale bar = 0.5 µm. A magnified photo is available in Appendix Fig. S4B. (K) The cells containing AB-like structures were counted under electron microscopy [no treatment (NT): n = 20, AmphoB: n = 192]. * p < 0.01 vs the value of NT.
Identification of anti-PrP autoantibodies in an unselected hospital patient cohort.Figure 5(A) Schematic workflow for the HTS of patient plasma samples for anti-PrP autoantibodies. (B) Age pyramid, separated for females and males, of the 37,894 unselected hospital patients tested in the HTS. (C) Boxplot with half-violin plot displaying the distribution of autoantibody reactivity in 48,718 plasma samples. Twenty-seven samples over the reactivity threshold of -log(EC50) ≥ 2 and fitting error < 20% were considered as hits in the primary screen. Black dots correspond to single samples. The boxplot divides the data set into three quartiles. The box extends from the first to the third quartile values of the data set, with a line at the median. Whiskers show the range of the data (minimum and maximum). (D) Frequency distribution of hits based on age. (E) Heat map representation for the validation of the hits from the primary screen (21 out of 27 hits). Confirmed hits displayed selective reactivity against human recPrP23‑230. Controls: Patients #11-13, ApoE4 and BSA. Red: high reactivity; blue: low reactivity. (F) Heat maps representing autoantibody clonality of hits analyzed by ELISA. Autoantibodies were mostly constituted by lambda light chains (λ), while the kappa light chains (κ) showed prevalence in the total IgG fraction. (G) Determination of antibody reactivity (-log(EC50)) for three individuals at two different time points. Individuals were selected by displaying strong anti-PrP IgG reactivity in the first test (Time 1). Reactivity was maintained for even more than one year (Time 2). (H) Purified IgGs from patients with strong anti-PrP reactivity were tested against PrP variants and BSA (negative control) in dose-dependent ELISAs. Patient #5 and #6 showed binding mostly to the human recPrP121‑230. Patient #1 displayed a polyreactive pattern with binding to the human recPrP121‑230 and mouse recPrP23-110.
Figure 1(A) Immunoblotting for Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of AR-97Q, AR-24Q, and wild-type mice. (B) Quantitative densitometry analysis indicated that Dnmt1 spinal cord level was up-regulated in AR-97Q mice (n = 3). Dnmt3a and Dnmt3b spinal cord level was not significantly different among wild-type, AR-24Q, and AR-97Q mice (n = 3). (C) Quantification of the Dnmts mRNA levels of the spinal cord in wild-type, AR-24Q, and AR-97Q mice using RT-qPCR (n = 3). (D and E) Representative immunohistochemical images depicting the expression of Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of male AR-97Q, AR-24Q, and wild-type mice and quantification of the immunoreactivity for Dnmts (n = 25 neurons from three mice of each group). (F) Double-immunofluorescence staining for Dnmt1 and 1C2 in the spinal motor neurons of an AR-97Q mouse. (G) Immunoblotting for Dnmt1 of the skeletal muscle of wild-type and AR-97Q mice. (H) Quantification of the signal intensity of Dnmt1 bands (n = 3).
Figure 6Representative photomicrographs of confocal microscopy of sections from differently injected day 1 wounds stained for Ly6G+ neutrophils (green) and F4/80+ macrophages (red). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 1 wounds injected with PBS (control), non-primed MSCs, LPS primed MSCs and LPS primed TLR4-silenced MSCs Double stained cells indicate phagocytic engulfment of neutrophils by macrophages. To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Scale bar: 100µm. Quantification of Ly6G and F4/80 double positive cells on sections of differently injected day 1 wounds. Wounds were injected as described in (a). Double positive cells were counted and statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates. Quantification of Ly6G+ neutrophils and F4/80+ macrophages on sections of differently injected day 1 wounds. Wounds were injected as described in (a). Single positive cells were counted and statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates. Representative photomicrographs of confocal microscopy of sections from differently injected day 3 wounds double-stained for TGFβ-1 (red) and for F4/80+ macrophages (green). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 5 wounds injected with PBS (control), non-primed MSCs, LPS primed MSCs and LPS primed TLR4-silenced MSCs. Scale bar: 50µm. Representative photomicrographs of sections of day 5 wounds immunostained for CD31 (indicative of endothelial cells and newly formed vessels) and for α-SMA (indicative of myofibroblasts differentiation) after injection of LPS primed MSCs, non-primed MSCs, LPS primed TLR4-silenced MSCs or PBS (middle and lower panel). To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Scale bars: 50µm. Double positive macrophages stained for TGFβ-1 and F4/80 were counted and statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates. Quantitative analysis of CD31 positive endothelial cells in sections of wounds injected with PBS, non-primed MSCs, LPS primed MSCs and LPS primed TLR4 silenced LPS Cell counting was performed on immunostained wound sections and statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates. Western blot analysis of lysates from day 5 wounds (left panel) and the corresponding densitometric analysis (right panel) depicts enhanced α-SMA protein expression in wounds injected with LPS primed MSCs as opposed to the respective control groups. Actin served as loading control. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, three biological replicates.
Prom1 deficiency defects glucagon-induced gluconeogenesis A.Figure 2A. Levels of G6pc and Pck1 mRNAs in the liver of mice after 18 hours fasting were determined by qRT-PCR. The level of each mRNA was normalized to the level of 18s rRNA (n = 3/fed, 7 or 8/fasted). B. Levels of phospho-PKA substrates (p-PKA substrates), phospho-CREB (p-CREB), CREB, G6pc Pck, Prom1 and GAPDH were determined after 18 hours fasting. Band intensity for p-CREB was quantified using Photoshop software. C. Blood glucose levels (mg/dL) of mice after fasted for 24 hours (n = 12 or 14/group). D. Serum glucagon level in the fed or fasted mice (n = 3/fed, 7 or 8/fasted). E. Glycogen contents in the liver of mouse in each condition was shown by PAS (periodic acid schiff) staining. Scale bar, 500 μm. Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. F. Levels of the G6pc and Pck1 mRNAs 2 hours after glucagon stimulation (10 nM) were determined by qRT-PCR. The level of each mRNA was normalized to the level of the 18S rRNA. For these experiments, primary hepatocytes were cultured under 3D conditions using a Matrigel matrix (n = 3 mice/group). Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. G. Levels of phospho-PKA substrates (p-PKA substrates), phospho-CREB (p-CREB), CREB, phospho-Inositol trisphosphate receptor (p-IP3R), IP3R, phospho-Hormone-Sensitive Lipase (p-HSL), HSL, Prom1 and Gapdh 10 minutes after glucagon stimulation (10 nM) were determined by immunoblotting. Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. H. The nuclear localization of p-CREB 10 minutes after glucagon stimulation (10 nM) was determined by immunofluorescence staining. Scale bar, 20 μm. Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. I. Relative PKA activities 10 minutes after glucagon stimulation (10 nM) were determined using the PKA assay kit in the absence or presence of 10 μM IBMX (n = 3 mice/group). Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. J. The cAMP concentration 10 minutes after glucagon stimulation (10 nM) was determined using the cAMP assay kit in the absence or presence of 10 μM IBMX (n = 3 mice/group).
Figure 2A. Validation of the top candidate genes in the C11 cell line. C11 cells were infected by lentiCRISPR v2.0 packaged lentiviruses with sgRNA following by screening for 14 days with 2 µg/ml puromycin. The percentage of GFP-positive cells was measured by flow cytometry to determine the level of HIV-1 reactivation. The effect of candidate genes on HIV latency was further verified in J-Lat 10.6 (B) models of HIV latency. GFP expression in J-Lat 10.6 cells was analyzed by flow cytometry The effect of candidate genes on HIV latency was further verified in ACH2 (C) models of HIV latency. p24 in ACH2 cells was analyzed by ELISA, respectively. D. PEBP1 was deleted after CRISPR/Cas9 knockout. PCR products related to PEBP1 from control or PEBP1 knockout cells were cloned, and then sequenced. PEBP1-sg1 target sites are shown in red letters. Dashes indicate the deleted bases relative to the wild-type PEBP1 gene sequence. E. PEBP1 protein levels were measured by Western blot after knock out in C11 cells by LvPEBP1-sg1. Mock C11 cells served as control.
Figure 2(a) Species relative abundance as a function of time for all pairwise communities. Experimental measurements and model fits based on T3 are represented as data points and lines, respectively. In each subplot, time and species relative abundance are displayed on the x and y-axis, respectively. Stars denote datasets with a sum of mean squared errors greater than 0.15. Error bars represent 1 s.d. from the mean of at least three biological replicates. (b) Temporal changes in species relative abundance of a selected set of pairwise assemblages inoculated at 5% species A, 95% species B or 95% species A, 5% species B based on OD600 values. Time and relative abundance are represented on the x and y-axis, respectively. Data points and lines represent experimental measurements and model fits to T3, respectively. Error bars represent 1 s.d. from the mean of at least three biological replicates. Stars denote datasets with a sum of mean squared errors greater than 0.15. (c) Inferred inter-species interaction coefficients for the gLV model trained on T3. Gray and green edges denote negative (αij < 0) and positive (αij > 0) interaction coefficients. The edge width and node size represent the magnitude of the inter-species interaction coefficient and steady-state monospecies abundance (xe = -μiαii-1), respectively. To highlight significant interactions, inter-species interaction coefficients with a magnitude less than 1e-5 were not displayed.
Figure 3The following properties of Schaffer collateral-CA1 synaptic responses were recorded from wt and NPC1nmf164 mice. A. Basal synaptic transmission expressed as mean ± SEM EPSP slope (n= 9 slices from 5 WT mice and n=8 slices from 5 NPC1nmf164 mice, all mice 10 week-old, unpaired Student t test, * p150μA = 0.0368, p200μA = 0.0138); B. Mean ± SEM fibre volley amplitude (n=10 slices from 5 WT mice and n= 7 slices from 5 NPC1nmf164 mice, all mice 10 week-old); C. Mean ± SEM paired pulse facilitation (n=11 slices from 5 WT mice and n=10 slices from 5 NPC1nmf164 mice, all mice 10 week-old, unpaired Student t test, **p = 0.0011); D. LTP expressed as mean ± SEM percentage of EPSP slope over baseline (n=7 slices form 5 WT mice for WT and n=10 slices from 5 NPC1nmf164 mice, all mice 10 week-old, unpaired Student t test, ***p < 0.0001 in the last 10 min recording) The following properties of Schaffer collateral-CA1 synaptic responses were recorded from wt and NPC1nmf164 mice. E. LTD expressed as mean ± SEM percentage of EPSP slope over baseline (n=7 slices from 5 WT mice and n=12 slices from 5 NPC1nmf164 mice, all mice 10 week-old). The behavioural tests were recorded from wt and NPC1nmf164 mice. F. Mean ± SEM exploration time of objects in novel and familiar location in the Object Placement Recognition test (n=10 mice, 10 week-old, 2-way ANOVA, *p = 0.0167). The behavioural tests were recorded from wt and NPC1nmf164 mice. G. Mean ± SEM percentage of entry to the novel arm of the Y maze test (n=10 mice, 10 week-old; unpaired Student t test, **p = 0.0015). H. Mean ± SEM percentage of freezing time in the Contextual Fear Conditioning test (n=10 mice, 10 week-old; unpaired Student t test, ***p = 0.0002). I. Mean ± SEM distance covered during a 5 min period in the Open Field test (n=10 mice, 10 week-old).
Figure 2(A) Heatmap depicting the relative expression level measurements of promoter constructs with different pairs of the nucleosomal sequences block 1 and block 7 compared to B1.11 + B7.11 expressions (log2 scale). Results were pooled for all tested native core promoters to calculate the average deviation to B1.11 + B7.11 expressions. (B) Heatmap depicting the relative expression level measurements of promoter constructs with different free combinations of block 1 and block 7 compared to B1.11 + B7.11 expressions (marked with a red rectangle). Results were pooled for all tested native core promoters to calculate the average deviation to B1.11 + B7.11 expressions. Bar plots on the top and the left represent the GC content of each block 1 and block 7 sequence. Block 7 with column "w/o B7" represents the results obtained from promoters without block 7 sequence. (C) Boxplots depicting block 1 effects for tested core promoters. Effects of different block 7s were merged in each column (within the same block 1): the median SD is 0.66 for developmental promoters compared to 1.23 for constitutive promoters (lower right corner); Wilcoxon rank-sum test ***p = 3.1×10-4, significant. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements. (D) Boxplots depicting block 7 effects for tested core promoters. Effects of different block 1s were merged in each column (within the same block 7): the median SD is 0.54 for developmental promoters compared to 0.64 for constitutive promoters (lower right corner); Wilcoxon rank-sum test p = 0.3, not significant. Block 7 with column "w/o B7" represents the results obtained from promoters without block 7 sequence. Developmental and constitutive promoters are highlighted in green and red, respectively. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements.
Figure 4(A) Schematic representation of the experimental data sets and the integration procedure used to reconstruct the 9TR GRN. DEGs: Differentially Expressed Genes; TRs: Transcriptional Regulators; KDs: Knockdowns; RE: Reprogramming Efficiency; GRN: Gene Regulatory Network (B) The sequential assembly of a novel gene regulatory network required for reprogramming. Each connection (line) represents the regulatory interaction between the indicated nine TRs during the reprogramming course. The red connections indicate functional synergy between the TRs. The light grey circles shown at 18 hrs timepoint represent the TR genes that have not yet been expressed. The O/S/K/M labeled beads placed on the gene circles denote the direct O/S/K/M binding at putative gene regulatory regions as deciphered from our ChIP-seq binding profiles. O: Oct4, S: Sox2, K: Klf4 and M: c-Myc. (C) Heatmap summarizing the effects of each of the nine TRs single knockdowns on the expression of the other TRs on day 6; The white color denotes no change in expression levels. (D) Scatter plots depicting the extent of co-expression of either the broadly- or early-iPSCs-specifically expressed TRs in individual cells obtained from the early-iPSCs colonies or from the rest of the population (left and right panel respectively). Each dot depicts the percentage of cells co-expressing a unique combination of two or three TRs. Data are shown as mean ± SEM of at least two independent experiments. (E) Heatmap summarizing the most striking effects of double nine TRs KDs on the expression of the other TRs and on the expression of the Epcam and Lin28a pluripotency markers; the white color indicates no change in expression levels. The genes examined have been ranked in the heatmap according to their order of upregulation during reprogramming; Lin28a is the latest upregulated gene.
a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.figf5(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and ER and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm. (b) Quantification of LC3-positive spots as seen in a using ImageJ software. Depicted are mean ±  s.e.m. of the number of LC3-positive spots divided by the number of DAPI-positive cells (n=4). *P0.05. (c) MSCs expressing FOXO3(A3)-ER were treated with or without 4-OHT and BafA1 (100 nM). At the indicated time points, cell lysates were analysed for protein levels of glutamine synthetase (GS), LC3, pS6 (Ser 235/236) and actin. Shown are representative blots (n=2). (d) IL-3 was withdrawn from Ba/F3 cells in the presence or absence of BafA1 (100 nM) as indicated. Cell lysates were analysed for protein levels of glutamine synthetase, LC3 and hsp90. Shown are representative blots (n=2). (e) DLD1 cells were stimulated with l-glutamine (l-Gln) in glutamine-free DMEM containing 0.1% FBS. After 24 h, cell lysates were analysed for protein levels of LC3, p62, p-S6K (Thr 389) and actin. Shown are representative blots (n=3). (f) DLD1 cells were starved and stimulated with glutamine (5 and 10 mM). Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy, and the co-localization between mTOR and LAMP2 was quantified by ImageJ. Depicted are the means of the percentage of co-localized pixels (n=2). (g) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-WIPI-1 and subsequently stimulated with 4-OHT in the presence or absence of MSO in glutamine-free DMEM containing 0.1% FCS or medium without amino acids for 24 h. GFP-WIPI-1 puncta formation analysis was performed by confocal microscopy. Depicted are the percentages of cells positive for GFP-WIPI-1 puncta. Shown are the mean ±  s.e.m. (n=4). *P0.05 and **P0.01. Uncropped images of blots are shown in Supplementary Fig. S8.
Figure 2A-D. Analysis of IL-1R1 expression by different populations of murine (A) CD4 T cells and (B) Th17 cells isolated from the dLN 9 days after immunization; and (C) CD4T cells and (D) Th17 cells isolated from the CNS at the peak of EAE.Cells (A-D) were restimulated in the presence of MOG for 6 h. Data consist of n=4 wild type mice immunized with MOG/CFA/PTx.Data (A, C) are representative FACS histogram overlay of VD-TCRβ+CD4+ cells gated on IL-17A+, IL-17A-IFNγ+ (IFNγ+), IL-17A-GM-CSF+ (GM-CSF+) or IL-17A-FoxP3+ (FoxP3+) cells (also see Appendix Fig S1) and quantifications shown as bar diagram (mean +SEM). Data (B, D, F) are FACS plots of Th17 cells and histograms of IL-1R1 staining of indicated cell populations with mean fluorescent intensity (MFI) ±SEM or mean frequencies per group. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t-test. Experiments were performed twice with similar results. E-F. Analysis of IL-1R1 expression by different populations of human (E) CD4 T cells and (F) Th17 cells isolated from the peripheral blood.Cells (E-F) were restimulated with PMA/Ionomycin for 4 h. Data consist of PBMC isolated from n=4 healthy individuals.Data (E) are FACS plots of cells gated on CD14-CD3+CD4+cells with mean frequencies per group and quantifications shown as bar diagram (mean +SEM). Data (B, D, F) are FACS plots of Th17 cells and histograms of IL-1R1 staining of indicated cell populations with mean fluorescent intensity (MFI) ±SEM or mean frequencies per group. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t-test. Experiments were performed twice with similar results.
Vasculotide delays progression of experimental lung metastases but not lymphatic metastases from a human breast cancer cell line (LM2-4luc).Figure 2A-H LM2-4luctumor cells were implanted intravenously into SCIDmice, generating predominantly lung-specific metastases in one experiment (n = 5-6; A-D) and predominantly lymphatic-specific metastases in another experiment (n = 12; E-H). (C and G) Photographs of typical metastases in the lungs and caudal lymphatics, respectively. Kaplan-Meier survival analysis showed that Vasculotide (VT) significantly delayed mortality due to lungmetastases (A) but not lymphaticmetastases (E). Quantitative analysis of in vivo bioluminescent images taken at 24DPI (B) showed that concurrent VT treatment effectively reversed the sunitinib (SU)-induced acceleration of lungmetastases (D). With regard to lymphaticmetastases (F, H), SU treatment did not accelerate their progression. Geometric means ± 95%CI are depicted in (D and H); P values were derived by one-way ANOVA (D) and Kruskal-Wallis test (H). The same trends were reproduced in confirmatory experiments summarized in Supplementary Fig S5.