EMBO/sd-character-level-ner huggingface dataset:
Nhp2 bridges components of the SSU and H2AQ105me.Figure 4A) Peptide pulldowns from indicated TAP-tagged strains. Pulldown efficiency was analyzed by western blotting. B) Spot test of the indicated strains on YPD or YPD supplemented with the given antibiotics. Overnight yeast cultures were adjusted to OD600=1 and then serially diluted 5-fold before spotting on indicated media. C) Ribosome slippage assay using a tandem luciferase reporter separated by a sequence allowing for slippage. The sequences allowing for slippage are given below the graph. n=8, biological replicates, error bars are standard error of the mean. Significance was tested using 2-tailed, unpaired t-test. D) Proteomic analysis of Nhp2 interactors. Highlighted in red are the known members of the H/ACA pseudouridylation complex. The red dashed line indicates significance cutoff E) Intersection of Nhp2 and H2AQ105me pulldown. 17 proteins were enriched in both experiments and the majority are members of the small subunit processome (highlighted in red). F) GO enrichment for these 17 commonly identified proteins shown in Figure 4E. G) Peptide pulldowns from human cells (HEK293T) to test for conservation of the reported interactions between Nhp2 and members of the SSU processome complex on H2AQ105me. Dyskering (Cbf5 homolog) was probed to test for the presence of members of the H/ACA complex. Pulldown efficiency was analyzed by western blotting using the indicated antibodies. H) Simplified schematic overview over some processing steps of the rRNA. Cut sites are indicated as A0-A3. Blue bars indicate Northern probes to detect 18S and 25S mature rRNA, respectively (please also see Figure EV4). Labelled bars on top of the scheme indicate used probes. I) Quantification of Northern Blot results. Normalised to GAPDH, n=3, error bars are standard error of the mean. Asterix indicates p-value (*, p<0.05, based on unpaired, 2-tailed t-test).
Figure 6(A) 12-day-old WT or 35S::GLV2 8-5 seedlings were treated with mock or 1 μM GLV2 for 24h before protein extraction. Western blots were probed with α-FLS2 or α-BAK1 antibodies. CBB: Coomassie brilliant blue. Data information: Similar results were obtained in 3 independent experiments. (B) Quantitative real-time PCR of FLS2 transcripts from 12-day-old seedlings treated with mock or 1 μM GLV2 for the indicated time, n=3 biological replicates. UBQ was used as a house keeping gene. Students T-test revealed no statistical difference for each of the different timepoints. Data information: Similar results were obtained in 3 independent experiments. (C) Protein extraction was performed from 12-day-old seedlings of the indicated genotypes. Western blot was probed with α-FLS2 antibodies. (D) Protein extraction was performed from 12-day-old seedlings after 1 hour treatment with mock or 50 μM MG132. Western blots were probed with α-FLS2 antibodies. Data information: Similar results were obtained in 3 independent experiments.
Atg1 binds Atg8 in a LIR‐dependent manner.Figure 2(A) Schematic representation of budding yeastAtg1 and sequence alignment of its putative LIR motif with Atg1 homologues from various species. Conserved residues of the predicted LIR motif are indicated with grey boxes, residues targeted by mutagenesis are marked with asterisks (*). The Atg1 kinase domain is underlined and the Atg13‐binding region is shaded in black. Circled 'P' mark known phosphorylation sites on Atg1. N: amino‐terminus; C: carboxy‐terminus. (B) GST‐Atg8 or GST‐Ubiquitin (Ub) was purified from E. coli and bound to GSH beads. Wild‐type Atg1‐TAP or the VE and EYE mutants were purified from yeast, incubated with the immobilized GST proteins and bound proteins analysed by western blotting. (C) (C) GST‐Atg8 and GST‐Ubiquitin were immobilized as described in (B), and probed for their ability to bind Atg1‐TAP isolated from wild‐type or atg13Δ cells. (D) atg1Δatg13Δ cells expressing as indicated wild‐type Atg1‐TAP or the VE or EYE mutants, and wild‐type Atg13 or the Atg13‐FV mutant were grown to mid log phase. Atg1 was immunoprecipitated and its association with Atg13 was determined by western blotting. (E) GST, GST‐LC3B, GST‐GATE16 and GST‐GABARAP were purified from E. coli and bound to beads. HEK293 cell lysates prepared from cells expressing wild‐type (wt) HA‐ULK1 or the DFP mutant were incubated with the beads, and bound proteins analysed by western blotting. (F) HEK293 cells were co‐transfected with GFP or GFP‐GABARAP along with either wild‐type HA‐ULK1 or the DFP mutant. GFP and GFP‐GABARAP were immobilized on GFP‐Trap resin and HA‐ULK1 binding was analysed by western blotting. Figure source data can be found with the Supplementary data.
Figure 7A UMAP plots of sorted cell subsets from Dataset 3 colored according to cell cycle phases. B Bar plot showing the percentage of cells in the G0/G1 (red bars), S (blue bars) and G2/M (green bars) phases of the cell cycle among each of the 7 sorted cell subsets of Dataset 3. C Heatmap showing scaled expression of up to 25 DEG from each of the seven sorted cell subsets of Dataset 3. In contrast to Figure 5C, the analyzed cells were further subdivided according to cell cycle phases (G0/G1, S, and G2/M) and 100 cells were randomly selected and analyzed per sample and per phase. The origin of each cell sample is indicated above columns and the color code corresponding to each seven sorted cell subsets is indicated at the bottom of the heatmap. A green star indicates that in the case of DN3a cells, only 32 cells were in G2/M phase and analyzed.
Ras, MEK and ERK control bronchial epithelial gene expression.Figure 3(a) Acute versus chronic MEK inhibition. Acute (panels 1 and 2): Cells were seeded sparsely and incubated for 4 days in normal media, then subjected to a calcium switch and recovery, in the presence of DMSO (panel 1) or 500nM GSK1120212 (panel 2). Chronic (panels 3 and 4): Cells were seeded sparsely and incubated for 4 days in DMSO (panel 3) or 500nM GSK1120212 (panel 4). Cells were subjected to a calcium switch and recovery, in the presence of DMSO (panel 3) or 500nM GSK1120212 (panel 4). Cells were fixed and stained for ZO-1 and DNA. Scale bar represents 20m. (b-d) Microarray analysis. 16HBE cells were stably infected with pQCXIP (control) or pQCXIP expressing DN HRas. Control cells were treated with DMSO, GSK1120212 (500nM), or SCH772984 (1µM) for 4 days. RNA was isolated and analysed using an Illumina gene array; n=3 independent samples were prepared for each condition. (b) Venn diagram representing genes downregulated by >1.6-fold versus control, with an unadjusted p-value <0.05. (c) Relative expression levels of 33 genes downregulated by DN HRas, MEK and ERK inhibition, expressed as % of DMSO control. (d) Relative expression levels of EMP1. Error bars denote mean ± SEM, dots indicate individual data points. **, p < 0.002 (DN Ras = 0.0019, ERKi = 0.0016); ****, p <0.0001.
Blood vessels intrinsically control radial glia expansion.Figure 2A,B, Staining for Isolectin B4+ blood vessels in control (A) and Gpr124ECcKO (B) forebrains. Asterisks mark the lateral ventricles. C,D, Staining for pimonidazole (PIMO, green) and Isolectin B4+ blood vessels (red) in ctrl (C) and Gpr124ECcKO (D) cortices. E,F, Immunostaining for Pax6 in control (E) and Gpr124ECcKO (F) forebrain sections to reveal the lateral extension of the neocortex (dashed line) between the cortical hem (arrowhead) and the lateral ganglionic eminence (arrow). G, Quantification the lateral extension of the cortex in control and Gpr124ECcKObrains (mean±SEM; N=6 for ctrl and N=8 for Gpr124ECcKO; * p<0.05). H,I, Staining for EdU (red) and Ki67 (green) in control (H) and Gpr124ECcKO (I) cortices at E13.5, 24 hours after EdU injection. J, Quantification of Ki67+ NPCs in the in VZ (blue), SVZ/CP (grey) and of Ki67- neurons (cyan) generated from EdU-labeled NPCs in control and Gpr124ECcKO embryos (mean±SEM; N=4; * p<0.05, ** P<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm K,L, Immunostaining for Tbr2 (red) and Ngn1 (green) in control (K) and Gpr124KO (L) cortices. M-O, Quantification of neurogenic (Ngn1+) RGs and BPs (M), newborn BPs (N) or expanding (Tbr2- Ngn1-) RGs (O) in control and Gpr124ECcKO cortices (mean±SEM; N=4; * p<0.05, ** p<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm
Figure 2Schematic diagram of experimental pipeline for preparation and optimization of cell-free expression systems. Optimization of transcriptional output using different concentrations of Mg-glutamate and K-glutamate in 10 bacterial cell-free expression systems with Broccoli as a reporter (solid lines, optimal buffer composition shown in red). No DNA template controls are shown as grey dashed lines. 12.5 nM of DNA template was used in all systems, except L. lactis (50 nM used).
Figure 1A H&E staining of sections of intestinal tissues obtained from endoscopic biopsy of COVID-19 patients who presented with gastrointestinal (GI) symptoms. The inflammatory infiltrates were indicated by a yellow arrow. The edema area was indicated by a red star. Scale bars, 100 µm. B The bar chart shows the degrees of intestinal inflammation in patients with COVID-19. Number of samples for each group as indicated. Data information: All data are shown as mean ± SD. C Cytokine levels in the plasma of COVID-19 patients with (n=12) or without (n=6) GI symptoms by cytokine assay. Data information: All data are shown as mean ± SD. P values are determined by Student's t-test. D Levels of plasma VEGF in COVID-19 patients with (n=8) or without (n=10) disease progression by cytokine assay. Data information: All data are shown as mean ± SD. P values are determined by Student's t-test. E Temporal course of plasma VEGF at the early and late stage of COVID-19 infection by cytokine assay. Data shown are the levels of plasma VEGF in patients with (n=8) and without (n=10) disease progression at the early stage (one to three days after laboratory-confirmed for COVID-19) and late stage (more than three days after laboratory-confirmed for COVID-19). F Levels of plasma VEGF in COVID-19 patients with (n=7) or without (n=6) interstitial vasodilation by cytokine assay. G Levels of plasma VEGF in COVID-19 patients with (n=8) or without (n=5) interstitial edema by cytokine assay. Data information: All data are shown as mean ± SD. P values are determined by Student's t-test.
Homozygosity and compound heterozygosity for null mutations in GRK2 in ATD.Figure 2(A) Sanger sequence trace showing homozygosity for the c.469C>T mutation, predicting a stop codon in exon 6 of GRK2 (arrow). (B) Exome analysis showing the biallelic changes c. 1348_1349del and c.555 +1G>A. Red arrow points to the place of mutation. Orange and green boxes indicate the reference nucleotide and its substitution, respectively. (C) Domain composition of GRK2 and the corresponding location of the mutations (RGS, regulator of G protein signaling domain; PH, pleckstrin homology domain). (D) Expression of GRK2 as measured by RT-PCR in three replicates each of control and R05-365A fibroblasts, demonstrating absence of GRK2 transcript in ATD cells. GAPDH served as a positive control. (E) Two control and ATD fibroblast samples were immunoblotted for GRK2. Note the absence of GRK2 protein in the ATD cells. Actin served as a loading control. The data shown are representative of three independent experiments.
Figure 6(A) Sall4, Plzf or Crbn mRNA expression in E4 chicken embryos was analysed by whole-mount in situ hybridisation. Upper panel shows whole chicken embryo and lower panel shows right forelimb bud. (B) Sall4, Plzf, or Crbn mRNA expression in E4 chicken limb bud was analysed by section in situ hybridisation. (C) Immunoblot analysis of AGIA-GgPLZF in AGIA-GgPLZF expressing DF-1 cells transfected with shContol (shGFP) or shPLZF expression vector. (D) Immunoblot analysis of PLZF from tissue of chicken forelimb or hindlimb bud. Endogenous PLZF protein expression was detected by immunoblot using E4.5 chicken embryos infected with RCAN virus packaging shControl or shPlzf (forelimb shControl (n = 4), forelimb shPLZF (n = 4), hindlimb shControl (n = 2) or hindlimb shPlzf (n = 3)). (E) Skeletal patterning of forelimb and hindlimb in E6 chicken embryos infected RCAN virus packaging shControl (n = 10) or shPLZF (n = 9) were analysed by Victoria blue staining. h; humerus, r; radius, u; ulna, fe; femur, fi; fibula, t; tibia. Data in bar graphs were calculated as relative bone length with bone length of shControl as 100. Error bars denote ± standard deviation (forelimb and hindlimb shControl (n = 3), forelimb shPLZF (n = 5) or hindlimb shPLZF (n = 6)). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01 and ****P < 0.0001). (F) Teratogenic phenotypes of chicken embryos
KSHV dysregulates a novel ncRNA network during lytic replication.Figure 8Schematic of proposed ncRNA network manipulated by KSHV. circHIPK3 is upregulated by KSHV, partly mediated through ORF57, this leads to increased sponging of miR-30c and increased DLL4 levels. DLL4 has downstream effects on cyclins leading to changes in the cell cycle aiding KSHV replication. Created with BioRender.com.
Detection of oscillations in metabolite concentrations throughout the cell cycle in HeLa cells revealed by LC-MS based metabolomics of synchronized HeLa cells and computational deconvolution.Figure 1(a) Synchronization dynamics of a population of HeLa cells within almost 3 complete cell cycles measured via PI staining followed by FACS analysis. The increase in cell number following each mitosis is shown by an overlaid curve (in orange; mean and s.d. of n=3). (b) Computational modelling of the synchronization loss, considering 11% cell-cell variation in doubling time, shows that the simulated fraction of the cells in G1, S, and G2/M in the synchronized cells throughout the cell cycle (straight lines) match experimental measurements (marked with asterisk). (c) The measured average cell volume in the synchronized cell population (red; mean and s.d. of n=3; in Equation 6), the deconvoluted signal (in case of no synchronization loss; green; in Equation 6), and the simulated average cell volume considering the loss in synchronization (black; matching the measured concentration data; Equation 6). (d) The measured concentration of CTP in synchronized cells shown in red (mean and s.d. of n=5; ) in Equation 7; the deconvoluted concentration dynamics, in case of no synchronization loss (green; in Equation 7); and the expected concentration dynamics based on the deconvoluted concentrations and considering the loss in synchronization, matching the measured concentrations (black; Equation 7). The measured concentrations converge towards the steady-state concentrations measured in non-synchronized cells (horizontal blue line).
Spectral features of oCI coding.Figure 3(A) Exemplar STCs in response to SGN stimulation with four blocks of four µLEDs inserted via the round window, from apical to basal direction (A-D). White markers define the best electrode (BE), and measurement of the spread of excitation (SoE) is indicated in the left panel. In STCs where more than one peak was detected, the dorsal- and ventral-most electrodes have been considered as the boundaries of the STC in order to avoid underestimation of the SoE. (B) Relative BE as a function of stimulus location, referring to the apical-most μLED evoking a response. The black line indicates the tonotopic axis as expected based on literature values, dashed lines indicate the regression line of individual (blue; correlation coefficient r = 0.33, p = 0.00066) and blockwise (purple; correlation coefficient r = 0.58, p = 1.96*10-7) oCI stimulation. Black markers indicate the data shown in panel A. Negative values can appear if the STC in response to a more basal emitter as compared to the apical-most emitter have a more dorsal BE. (C) Spectral spread of excitation upon SGN stimulation with µLED-based oCIs (blocks of 4 µLEDs, solid lines; mean ± standard error of the mean), acoustic (black dashed line), laser-coupled optical fibers (blue dashed line), as well as mono- and bipolar electrical stimulation with a clinical-style eCI (dashed and dotted red lines, respectively). Average data from stimulation via optical fibers and eCIs are replotted from Dieter et al (Dieter et al, 2019).
Figure 1(a) Representation of a zebrafishembryo at 70 % epiboly. Animal (A) and vegetal (V) poles and φ (embryo surface - red) and φ (internal to external axis - green) are indicated. The enveloping layer (EVL) is a single-cell layer (dark grey) that stands at the animal cap surface surrounding the inner deep cells (DCs) (blue). It is attached to the yolk cell membrane (purple). The YSL and Yolk Cytoplasmic Layer (YCL) (yellow) at the periphery and yolk granules fill the yolk cell. Progression of epiboly is represented by a curved black arrow. Geometrical axes are shown in grey. (b) Kinematics of the embryo surface. A kymograph of the embryo surface (φ axis) from a Tg (β-actin:m-GFP) embryo shows the continuous displacement of the EVL / yolk margin, which accelerates after 50% epiboly, while the yolk cell membrane does not show any shift (see Movie EV1). (c) Theoretical kymograph from 50 % to 70 % epiboly superimposed, between the red lines, on the experimental kymograph(b). Membrane signal intensity is color-coded (blue to red). White arrowheads point to flow lines (black). (d) AV velocities, normalized to EVL / yolk margin velocity, were measured by PIV of time-lapse recordings of the surface of lectin-soaked embryos. The plots correspond to three different epiboly stages (30 %, blue; 60 %, red; and 90 %, green). Blue and red arrows point to the width of the E-YSL at 30 and 60 % epiboly respectively (from Appendix Figure S1). (e) The yolk cell membrane is highly convoluted (white brackets) ahead of the EVL leading cells and condenses over time. Tg (β-actin:m-GFP) embryo at 30 % (left) and 60 % (right) epiboly. Scale bar 25 μm. Inset shows a high magnification of the wrinkled area (arrowhead). Scale bar 10 μm. (f) Actin accumulates within wrinkles at the E-YSL. The yolk cell membrane of LifeAct-GFP injected embryos (actin - green) was stained with lectin-TRITC (red) (top). Orthogonal projection of the wrinkled area (bottom). Wrinkles are outlined with a white dotted line. Scale bar 2 μm.
Figure 2Yeast cells that express galactose-inducible GFP-tagged hTDP-43 only or co-expressing mCherry-tagged WT or catalytic-inactive mutant of OGT, were spotted onto plates containing glucose or galactose. Cell growth was assessed after 2 days. Yeast cells expressing the indicated constructs were induced in galactose-containing medium, and representative images were shown using fluorescence microscopy to visualize TDP-43 aggregates. DIC: Differential interference contrast. TDP-43 aggregate foci numbers from individual cell of (B) were quantified from over 50 cells each of the indicated yeast strains. The foci numbers in individual cell > 3 were labelled with yellow, and the foci numbers in individual cell < 3 are labelled with blue. Expression levels of TDP-43 in strains were examined by immunoblotting.
Hippocampal synaptic transmission and plasticity in Grin2aS/S and Grin2a+/S mice with the GluN2A(N615S) mutation.Fig. 2a (left) In the absence of extracellular Mg2+ the synaptic AMPA/NMDA ratio of CA1 pyramidal cells in acute hippocampal slices is not altered in Grin2a+/S and Grin2aS/S mice compared to control littermates. (right) In the presence of extracellular Mg2+ the strong reduction of the AMPA/NMDA ratio in Grin2aS/S mice relates to the increased NMDA currents at CA1 synapses (Tukey's test). Example traces are depicted to the right of each bar graph. Data from the same experimental group were pooled across animals and are presented as mean ± SEM (see also ref. 35) with p < 0.05 being designated as statistically significant. Numbers in bar graphs indicate the number of slices. b Paired-pulse facilitation at CA3-to-CA1 synapses excluded strong alterations of presynaptic function in mutant mice. The stimulation strengths (in nC) necessary to elicit a pre-volley of 1.0 and 1.5 mV and the resulting fEPSP amplitudes were comparable in all genotypes but showed only a trend towards lower fEPSP amplitudes recorded at 1.5 mV pre-volley amplitudes in Grin2aS/S mice. The fEPSP amplitudes necessary to elicit a just detectable population spike (1) and a population spike of 2 mV amplitude (2) and the paired-pulse facilitation ratio (PPF) at an interstimulus interval of 50 ms did not indicate any synaptic impairments in Grin2aS/S and Grin2a+/S mice compared to WT littermates. The number of slices is indicated in the bar graphs or in brackets. c Field LTP (fLTP) at CA3-to-CA1 synapses, induced by tetanic stimulation in slices (1 s; 100 Hz; arrow), was comparable in all three genotypes. The inset in c gives a schematic view of the stimulating (white arrow) and the recording (green arrow) electrode positions in str. radiatum (r) and str. oriens (o). d Similarly, CA1-to-CA3 fLTP (induction: 2 × 1 s; 100 Hz; arrow) in freely moving mice was comparable between Grin2aS/S and Grin2a+/+ mice. The inset shows a Nissl-stained slice of one recorded mouse post mortem. e In mutant mice, but not in control littermates, the GluN2B-containing NMDAR contributes significantly to the magnitude of LTP, since LTP was significantly reduced by the GluN2B-specific antagonist CP101,106 (CP). Recordings of the non-tetanized control pathway in c and e are given as dashed lines. Error bars represent mean ± SEM (for statistics: Supplementary Statistics to Fig. 2).
Figure 6In the ventral pancreatic program, the ventral foregut endoderm cells first differentiate into Pdx1low intermediate progenitors, which can generate hepatoblasts, extrahepatobiliary cells, and Pdx1high pancreatic progenitors. In the dorsal pancreatic program, the dorsal foregut endoderm cells directly differentiate into Pdx1highpancreatic progenitors, a fraction of which further differentiates into Pdx1lowprecocious pancreatic endocrine lineages. In both programs, Pdx1high cells are identical at the transcriptomic level and generate the majority of pancreatic lineages.