EMBO/sd-character-level-ner huggingface dataset:
In contrast to the normal mouse which exhibits normal cognitive ability (left panel), developmental arsenic exposure induces Dnmt3a degradation through direct arsenic-Dnmt3a interaction, by which downregulates genome-wide DNA methylation and dysregulates ECM-related genes (middle panel). MET combination treatment normalizes global DNA methylation level and ECM-related gene expression in the mouse hippocampus, by which ameliorates the arsenic-induced cognitive impairment in mouse (right panel).
(B-C) Northern blot analysis and corresponding quantification of mitochondrial (B) rRNAs and mRNAs or (C) tRNAs. 18S rRNA is used as loading control. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S n=5 and the rest of samples n=4).
B Upper panel: Western blot analysis of mitochondrial and cytosolic aconitases. Lower panel: quantification of mAco and cAco by densitometry.
E. Raw 264.7 cells were co-transfected with eGFP(N-term)-Rab10 and mRuby(N-term)-Rab5 for 48 hours, with live-cell recordings for ~10 minutes. A representative vesicle is shown over the presented time-lapse. Scale bar represents 1 μm. F. Fluorescence intensities of individual vesicles are calculated over time. Dots show mean values calculated from n=8 recorded vesicles from cells from n=3 biologically independent experiments. Error bars show ± SEM.
Normal, si-control-transfected, or si-PTEN-transfected SGC7901 cells were treated with CDDP (1 µg/ml) and M2-si-control-exo or M2-si-CRNDE-exo. Si-control, the negative control of si-PTEN. The (C) cell proliferation, in SGC7901 cells.
(P) OVCAR-3 and MDA-MB-436 (WT and DR5-KO) cells were treated with KMTR2 followed by ubiquitin and S5a immunoblotting from total lysates. GAPDH is loading control.
(E) The half-life of SOD1G85R is reduced from 8.5 h to 5 h upon knockdown of UBE4B and LSD1. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
(E) Extract of H1299 expressing MDMX at indicated amounts were analyzed for β-catenin S45 kinase activity in a titration assay.
B Experimental scheme of tamoxifen injection paradigm: perinatal mice were injected twice (12 h apart) to label early perinatal rNSCs and killed at postnatal day (P) 3, 7, 10, or 14 to analyse the rNSC-generated progeny within the DG layers hilus, granular zone (GZ), and molecular layer (ML).
(I) P-Taf12, total Taf12, and Ser546-phosphorylated Gad8AKT were followed in WT cells, grown in rich conditions or over a time-course after a shift to starvation medium. Gad8AKT phosphorylation at Ser546 is a proxy of TORC2 activity in S. pombe.
Sample spectrogram of WWOX KO slice shows a gradual increase in activity upon addition of 4AP (marked by red arrow) for up to 2 min, and a decrease after 25 min. All traces were filtered with a 60 Hz notch filter and 0.5 Hz high-pass filter.
B, C, D, Catalytic activity of OTUB1 is not required to inhibit RAS ubiquitination. 6xHis-tagged ubiquitin and RAS expression constructs were introduced into HEK293T cells expressing wt HA-OTUB1, the catalytically dead mutant HA-OTUB1 C91S, or empty vector (V). Ubiquitinated RAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody.
(A) MEF cell lines derived from 3 p53-/-;MDMXSG/SG and 3 p53-/-;MDMXwt/wt embryos were analyzed for β-catenin pS45 level by western blot.
WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with epinephrine (3µg/10g). (K) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after epinephrine stimulation
A-C. Overexpressed Snq2-GFP accumulates in the ER and induces UPR in ypt1-1 mutant cells. Snq2-yEGFP was overexpressed in WT and ypt1-1 mutant cells and the following effects were analyzed as described for Fig 1A-1C, respectively: increase in the level of Snq2-yEGFP protein (A), accumulation of Snq2-yEGFP in aberrant ER structures (B),
H, OTUB1 mRNA overexpression is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients were stratified by tumor stages (T1-T4) and OTUB1 expression levels as described in Materials and Methods. Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.
A. I.29µ+ B lymphoma cells were treated with LPS to induce differentiation towards IgM secretion. At the indicated times, cells were lysed in TritonX114 to separate soluble and integral membrane proteins (aqueous and detergent phases, respectively). Aliquots from the two phases were resolved electrophoretically and the blot was decorated by anti-µ antibodies. The blue arrow points at a band likely corresponding to µs2L2C575OX, the intensity of which increases upon LPS stimulation.
The APC/C requires the Cdc20 coactivator subunit to ubiquitinate Nek2A. Nek2A ubiquitination reactions performed with the APC/C in absence and in presence of increasing concentrations of Cdc20 using the E2 UbcH10.
Principal-component analysis of SQCLC (n=44; blue) and HNSCC (n=30; red) protein expression. Shown are the first two principal components, accounting for 8.51% and 8.03% of the total variance in the data, respectively.
(f) Immunofluorescent analysis of mitochondrial and lysosomal proteins. Liver Ter119+ cells (E18.5) of the indicated mice were stained with anti-Tom20 (mitochondrial marker) and anti-Lamp2 (lysosomal marker) antibodies, and observed by fluorescent microscopy. Scale bar, 2 μm. Green, red, white, and blue indicate Tom20, Lamp2, Ter119 and DAPI (DNA), respectively. Mitochondrial and lysosomal markers have separate distributions in Ulk1gt/gt cells and DKO cells.
C Mitochondrial protein synthesis was monitored in hippocampal neurons, using ATTO 590-azide (magenta) labelling and cells immunostained for TOM20 (green). A representative deconvolved STED microscopy image is shown. Scale bars, 1 µm.
Figure 4. Restored p53 activity by nutlin3a impairs MGC formation and inos dysregulation A Representative images of unstimulated BMMΦ from wt and p53-/- mice at day 6 with hemacolor staining (left), LP stimulation for 6 days and quantification for MGC formation by hemacolor staining (right). Depicted are means ± SEM of 3 independent experiments; scale bar, 50µm.
(A) mRNA from TAΔDHPR and TACtrl tissues were extracted and nNOS expression was quantified by qRT-PCR. Results are expressed as mean±s.e.m., **P0.001, n=4.
(J) Representative images showing proliferation of respective shRNA resistant reconstitution of POLH in 293A and HMCES KO cells stably expressing POLH shRNA. Experiment was repeated two independent times in duplicates.
D. Distributions of OCT4 pulse frequencies in pro-pluripotent, pro-mixed, and pro-differentiated cell populations. To gain an unbiased look at preexisting determinants of cell fate in panels only cells who completed their entire cell cycle duration before BMP4 addition (t = 0) were included in the analysis.
(C) Grk5 mRNA levels in NRCMs depleted of and overexpressing Cand2 measured by RT-qPCR. t-test, n=6-9 biological replicates. Data information: Error bars indicate means ± SEM. * - P ≤ 0.05, *** - P ≤ 0.001.
Figure 4. Dynamic reorganization of chromosomes during mitosis. (H, I) Distributions of intra-chromosomal contacts made by a 20 kb cen-proximal rDNA flanking region (highlighted in grey) with the rest of chr12 in cdc20, cdc15, cdc15 released (+40 min, +60 min), nocodazole treated (G2/M noc, cdc15 noc) and asynchronous (asyn) cells. Schematic representations of chr12 are displayed atop of the graphes. Grey lines indicate centromere position. Pink arrowheads points at the right arm anaphase loop.
(E and F) Intact and lysed mitochondria (E) or intact mitochondria isolated from l(2) mbn cells (F) were treated with proteinase K (PK). The effects of proteinase K treatment were assessed by antibodies to VDAC, ATPsyn-α, Pink1, and Dcp-1.
(A) NF-κB activity in TLR2-transfected HEK293T cells (n=3) Data information I one representative of 'n' independent experiments is shown (mean+SD) Student's t-tes
(C) Scatter plots showing that the expression pattern in the hta9-1/11-1 mutant is correlated with that in the chr11/17, mbd9-1, and pie1 mutants. The differentially expressed genes identified in the hta9-1/11-1 mutant were subjected to the analysis.
J. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 3 h (indicated as F) or 100 μM DFO for 3 h after treatment with 10 μg/ml FAC for 12 h (indicated as D), and then cultured with DMSO or 50 μM biotin for 30 min. Triton-soluble cell lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed by immunoblotting with the indicated antibodies. The black and white arrowheads indicate hNCOA4-TurboID and endogenous NCOA4, respectively.
A, HBMEC were incubated with AG1478 (1 µM) 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h.
Tetrad dissection was used to show that the double mutant sen1∆ rpc37-V189D is dead.
(B) Distribution of nucleosomes over TSS of all protein-coding genes in the WT, chr11/17, pie1, and mbd9-1 mutants. The MNase-seq results were generated from two independent biological replicates.
E. Differentiated A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 5 μM saracatinib (sara) or 10 μM SKI-1 in the presence of DMSO or 50 μM bafilomycin A1 (BafA) for 12 h.
A. A schematic overview of in vivo experiments on orthotopic GBM8. The tumor growth was monitored by luciferase imaging (WBI), and expressed in photon flux per second. Mice assigned to the treatment and control groups were treated with miR-10b inhibitors or corresponding control oligonucleotides in different formulations.
, Gains of OTUB1 and KRAS mutation are mutually exclusive in lung adenocarcinomas. OncoPrint showing the distribution of KRAS somatic mutations and OTUB1 copy number alterations in TCGA lung adenocarcinomas and squamous cell carcinomas obtained from cBioPortal (Cerami et al, 2012; Gao et al, 2013). Co-occurrence analysis showing significant mutual exclusivity between KRAS mutation and OTUB1 gain.
B. 2'-O-MOE-POmiR-10b inhibitor (miR-10b-i) or non-targeting control (1 μg of each) formulated with InVivo-jet PEI were injected intratumorally at days 20 and 25 after cells implantation. The efficacy of miR-10b inhibition was assessed by qRT-PCR analysis of the resected tumors, with miR-10b expression levels normalized to miR-125b.
(B) Activity of mAb 2A10 on the proportion of motile sporozoites (left panel) and blood vessel entry (right panel). Four-minute videos of mice inoculated with mAb 2A10 and naïve controls were manually counted at 10 min post P. falciparum sporozoite inoculation. 5 videos for each condition, from 5 independent imaging sessions were scored for motility. 4 videos per condition from 4 independent imaging sessions were analyzed for blood vessel entry. Statistical analysis: Percent motile, Mann-Whitney, p=0.0317; Blood vessel entry, Fisher's exact test, p=0.0163
(d) Association of GAPDH (5 μg) to starved rat liver lysosomes in the presence of increasing concentrations of WT or G/S LRRK2. Inset: representative immunoblot n = 4-6.
(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of cohesin. P-values (Student's t test): **p < 0.01.
Number of Ki67-positive cells on day 4 in uterine epithelium of Rb1d/d mice was higher than that of Rb1f/f mice (mean ± SEM, Student's t test; n=5 mice for each group). Scale bar, 100μm; le, luminal epithelium; s, stroma.
(A) Epifluorescence microscopy analysis examining the subcellular localization of de novo made alcAp-AzgA-GFP and alcAp-FurA-GFP, after 6-8 hours transcriptional derepression in strains where the expression of Sec24, SedV, GeaA, HypB, RabE, AP-1σ and SsoA was repressed ab initio (o/n) by addition of thiamine. Images showing that sorting of AzgA or FurA to the PM does not require early (SedV, GeaA), late (HypB) or post-Golgi (RabE, AP-1σ ) key proteins. On the other hand, COPII coat protein Sec24 and PM t-SNARE SsoA are essential for proper localization of the two transporters to the PM. Scale bar: 5 μm.
(A) Heat maps representing enriched HCDR3 sequences across the different panning sets for the short- and long-HCDR3 (upper and lower panel, respectively) phagemid libraries. HCDR3 sequences were selected based on NGS counts in 100'000 analyzed sequences (Z-score values; Appendix Table S2) and clustered according to the NGS-binding profiles. Red and blue: high and low number of NGS counts of the HCDR3 sequence, respectively. Donut charts (the right side) of each heat map indicate the number of clones with NGS-identified HCDR3 for a predicted PrP epitope. (B) Bar graphs showing the number of rare (one count in mouse recPrP23‑231 panning) and predominant (count ≧ 20) clones binding to the various PrP regions for the short- and long-HCDR3 libraries.
B. Immunostaining of P5 cerebellum with indicated antibodies. Boxplots of the intensity of CCND1 immuno-signal (B'''', normalized to the mean intensity of AT+GCPs). We measured the intensities of CCND1 in ATOH1+/NEUROD1- cells and NEUROD1+/ ATOH1- cells. ATOH1+/ NEUROD1+ cells were not included in this quantification. These boxplots are showing the median (central band), 25% / 75% quartiles (boxes) and the ranges (lower whiskers represent the regions of lower box - 1.5 * IQR (inter-quartile range), upper whisker represent the regions of upper box - 1.5 * IQR). Wilcoxon test were performed between AT+GCPs and ND+GCPs. ***p < 0.001.
C Confocal images of rNSC-derived astrocytes expressing GFP (green) and astrocyte-specific markers (ACSBG1, GFAP, S100β; magenta) at different timepoints (P7, P10, P14); lower pictures depict single-channel images the astrocyte markers (grey) used above. Data information: ; scale bars (C = 10 µm,
(F) Failure of KLF4 PARylation leads to increased aneuploidy population in U2OS cells. n=3, p values were labeled in figure, one-way ANOVA assay.
(E) Percentage of mated with proven stud males and age at onset of puberty in the female WT and L-HRD KO mice. (n=10 for each group) Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.
Genomic region encompassing the E(spl)-C; green graphs indicate ChIP enrichment for Su(H) in Kc167 cells (Log2 scale is -0.5 to 2.9, data published previously in [4]); gene models are depicted in dark blue. Positions of primer pairs used in qPCR experiments are indicated with black arrows. Abbreviations are as follows: "igr" = intergenic region, "tr" = transcribed region and "enh" = enhancer.
a) Spontaneous neutrophil apoptosis. Apoptosis was associated with the appearance of a 24K anti-Atg5-reactive protein. 33K Atg5 disappeared during apoptosis in a time-dependent manner. HeLa cells overexpressing Atg5 served as positive control.
A: Impaired y-H2AX and 53BP1 accumulation in Ubc9 D100A cells. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed. Left panel: representative immunofluorescence images. Right panel: quantification of cells with γ-H2AX and 53BP1 foci. Error bars represent S.E.M, n=3 independent experiments * p < 0.05.
Fbp1 protein abundance in wildtype and Gid10 overexpressing yeast strains grown in ethanol medium (YPE) for 19 hours, and following 30 minutes and 2 hours glucose recovery. Bars represent mean, error bars represent standard deviation, significance was determined by one-way ANOVA (unpaired) followed by Tukey's multiple comparison test, * indicates p<0.05, ** indicates p<0.01, (n=3 biological replicates).
F. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon are shown. The body weight gain data are presented as a percentage of the initial body weight. The fat weight data are presented as a percentage of the body weight.
(H) WT mice were i.v. injected with αGalCer or PBS (control) and activation of splenicILC3s was assessed by flow-cytometry as CD69 up-regulation at 6 h or 16 h after injection. Grey filled profile= ILC3 from αGalCer-injected mice; Empty profile= ILC3 from PBS-injected mice; Dotted line= CD45-cells. Right graph, percentage of CD69+ILC3s 6 h after injection of αGalCer (grey) or PBS (white). (n=3) *p<0.05 two-tailed unpaired t-test.
(D, E) Cells treated as in C were immunostained with anti-α-tubulin (white), Hoechst (blue) (D Scale bars, 5 μm. The intensities of astral MTs relative to total MTs were determined from confocal microscopy images and the graph in (D) shows the average of four separate experiments (10 cells per condition/experiment) (±S.E). *p<0.05 and **p<0.01, Student's t-test
Time-lapse confocal imaging analysis of the co-localization of YFP-mTOR (red) with GFP-COPA (green) and lysosomes (blue) in live C2C12 cells under nutrient starvation (0 min), and in response to AA re-stimulation for indicated time (0.5 min, 2 min, 6 min and 8 min). Arrows highlight COPA puncta that were co-localized with mTOR or COPA puncta alone prior to their association with mTOR and lysosomes.. Scale bar, 5 μm.
(C) Subcellular localization of neosynthesized alcAp-GFP-ChsB in the absence (left panel) or presence (≈40 min) of benomyl (middle panel) or latrunculin B (right panel), after ≈2h of derepression. Notice the abolishment of proper polar localization of ChsB at the apical tip in both cases. Scale bars: 5 μm.
G Cell viability analyses of resistant SKMEL28 cells. Cells were transfected with the indicated siRNAs for 5 days. Relative cell viability, compared with that of control siRNA-transfected cells, was measured by CCK8 assay.
Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (J) The cAMP concentration after isoprenaline stimulation (10 μM) for 10 minutes was determined using the cAMP assay kit in the presence of 10 μM IBMX (n = 3/group).
A Cytokine release by PBMCs challenged with heat inactivated S. aureus (hiSa), and/or RNase A (RA) treated solution, or Sa19 (TLR13 activating ORN) upon TLR2 blockade (T2.5; n.p., not performed; **, p ≤ 0.01; n = 3).
(B) Expression levels of various BCL‐2 homologues in the cortices of animals of the indicated genotypes.
B- Western blot verifying the expression of the CTD variants RPB1-52R and RPB1-25R in U2OS cells in whole cell extracts. U1 snRNP was used as a loading control.
(c) Production of TNF was analyzed in mouse RAW 264.7 macrophages bEnd.3 microvascular endothelial cells as assessed in vitro by multi-channel flow cytometry upon exposure to recombinant murine uPA-PAI-1, TNF, or saline, quantitative data are shown (mean±SEM for n=4 experiments per group; #p<0.05 vs. saline; One-way ANOVA / t test).
In situ proximity ligation assay (PLA) shows endogenous parkin-Beclin-1 complexes in (G) WT C57BL/6mice (N = 5) and (H) parkin−/− as control.PLA in Tg-APPmice IP injected once daily for 3 weeks with (I) DMSO (J) 5 mg/kg Bosutinib and (K) 10 mg/kg Nilotinib (N = 5).
G. Kaplan-Meier survival curves of mice bearing T3359 GSC-derived xenografts treated with C646 or the vehicle control (Vehicle control: n=9 mice; C646: n= 8 mice). Median survival: Vehicle control, 27 days; C646, 33.5 days.
(D) Representative immunoblot of Cand2 protein from different cardiac cell types CM- cardiomyocytes, EC - endothelial cells, FB - fibroblast.
Quantification of IL-6 and IL-12 secretion by BMDC treated with 250 nM cEC or EC for 60 min before stimulation with R837 (5 μg/ml; 18 h). Bars represent mean ± SEM from one of three independent experiments. **P < 0.01; ***P < 0.001; one-way ANOVA adjusted by Dunnett's multiple comparisons test.
(D) V5-tagged Dcp-1C<A or V5 vector only control was expressed in l(2) mbn cells and subjected to nutrient-full or starvation conditions for 2 h. After IP with anti-V5 antibodies, lysates were separated by SDS-PAGE. Proteins were visualized with colloidal Coomassie stain.
(C) Iron-binding measured by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Xanthan and various glucans were mixed with 2 mM FeSO4, dialysed against deionized water before determination of Fe2+ content by ICP-OES. Data shown are mean ± SD (n≥3). ** indicates p-value < 0.01 and *** indicates p-value < 0.001 by paired student t-test.
The dynamic range (H), and glutamate release threshold (V10) (I) show differences for the three clusters. Cluster 1 is composed of linear synapses with wider dynamic range and lower threshold compared to the other clusters. Pillar synapses are depicted as white-filled circles, and modiolar ones are depicted as gray-filled circles. Box plots indicate first quartile (25th percentile), median and third quartile (75th percentile) with whiskers reaching from 10-90%. The clusters were compared by one-way ANOVA test, followed by a posthoc Tukey's test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001
(E, F) Quantitative assessment of cell viability changes due to the siRNA-mediated knock down of Dnmt1, Dnmt3a or Dnmt3b in NSC97Q cells with DHT (E) or without DHT (F) using the WST-8 assay (n = 3).
Human placental syncytiotrophoblast cells express markers of cellular senescence, p16 Sections of normal human post-partum, third trimester, placenta were evaluated by immunofluorescence co-staining for senescence regulators (A) p16 Green label: the syncytiotrophoblast marker βHCG, red label: p16 Blue label: DAPI nuclear stain. Arrowheads indicate positively stained syncytiotrophoblast cells βHCG+/p16+ (A) Scale bar, 50 µm.
Validation of inducible knockout of KLF4 in mouse intestine. Five weeks after injection of AAV7-Cre-mCherry, mouse intestine was removed and followed by the preparation of tissue section. The KLF4 expression in the intestine was then detected by immunohistochemistry
(D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib) (vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, von Willebrand Factor in green) and pulmonary vascular smooth muscle cells (SMC, aSMA in white); and quantification.
AGO2KO cells were transduced with a lentiviral vector coding for FLAG-HA-tagged AGO2 (AGO2KO_FH AGO2) or for GFP as a control (AGO2KO_GFP). Quantification of telomerase activity in AGO2KO_FH AGO2 and AGO2KO_GFP, as assessed by TRAP, was plotted (n=3 experimental replicates).
A, Serum estradiol-17β (E2) levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice (mean ± SEM, Student's t test; n=4 mice for each group).
(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.
Colony-forming activity of HeLaS3 and AGO2KO cells was determined by colony-formation assay (n=3 experimental replicates).
(H) BALF IL-6 and TNF from intratracheally treated WT or Tlr2 KO mice (n=4-5 mice each) one-way ANOVA with Dunnett's multiple compariso
(d) Replicative lifespan analysis of BY4741 wild-type yeast cells after separation into old (fraction V) and young (fraction II) cells by elutriation centrifugation. The remaining lifespan with or without (control) spermidine (1 mM, applied after elutriation) on 2% glucose synthetic complete medium is shown.
b, Quantification of PanIN from mice of the indicated genotype and age. Fold increase and male/female ratios are shown. Box plots express the first (Q1) and third (Q3) quartiles set by the upper and lower limits of the box, whereas the median is shown by the horizontal line inside the box. The whiskers extend upward and downward to the highest or lowest observation within the upper (Q3 + 1.5 × the interquartile range) and lower (Q1 − 1.5 × interquartile range) limits. Values outside the upper and lower limits are 'outliers' and are shown by individual symbols.