EMBO/sd-character-level-ner huggingface dataset:
Modulation of the UPR and down-stream responses in T.Figure 7(A) After incubation of BMDCs with HK parasites or STAg, TLRs are stimulated and induce transcriptional activation of the three pathways of the UPR. The IRE1α/XBP1s pathway stimulates the secretion of IL-6 and IL-12 by BMDCs. Blue arrows indicate down-regulated pathways and red arrows, up-regulated pathways. (B) In BMDCs infected with live Pru parasites for 16h, the IRE1α/XBP1s pathway is activated in a MyD88-dependent manner while the PERK pathway is not induced and the ATF6 pathway is repressed. XBP1s stimulates the secretion of IL-6 and IL-12 and IRE1α promotes MHC-I presentation of secreted parasite antigens. The secretion of IL-23 is independent of XBP1s and CHOP and only partially depends on MyD88. Our data also indicate that live parasites down-regulate the induction of XBP1s and CHOP induced by TLRs. Blue arrows indicate down-regulated pathways and red arrows, up-regulated pathways.
Figure 5A. A schematic depicts the procedure of the establishment of PDX and drug administration. B, C. SCID male mice with PDX tumors were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg), CPI-637 (30 mg/kg), combination of JQ1 (50 mg/kg) and CPI-637 (30 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of drug treatment were photographed (B) and tumor growth are shown in (C). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; *** P < 0.001. PDX tumors in each treatment group were harvested for protein For western blot analysis, three tumors each group (n = 3) were used for protein extraction and mixed with equal ration to detect the indicated antibodies in (D). PDX tumors in each treatment group were harvested for RNA extraction. For RT-qPCR, three tumors each group (n = 3) were extracted RNA for RT-qPCR in (E). Data are shown as means ± SEM. The P value comparing vehicle with each treatment group was calculated by unpaired two-tailed Student's t-test and indicated in the graph.
Pharmacological and genetic inhibition of Dyn2 function also reduces starvation-induced LD breakdown.Figure 2.HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: Dynasore (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM. (C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM Dyn2 for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. (G) Whole-cell lysates and LD fractions isolated from HuH-7 hepatocytes under resting or starved (2 h HBSS starvation) conditions. (H) Primary hepatocyte expressing Dyn2-GFP, showing an absence of colocalization with the LD surface (stained with Oil Red O). Bar, 20 µM. Inset shows magnification of boxed region (bar, 2 µM).
Human IFITMs inhibit infection by SARS-CoV-2.Figure 2A) Flow cytometry histograms showing stable expression of the indicated IFITMs in HEK293T cells. B) Flow cytometry plots of HEK293T cells transfected with ACE2-GFP and infected with SARS-CoV-2 (MOI 0.5) for 24 h showing SARS-CoV-2 infection occurs in ACE2-GFP positive cells. C) Stable HEK293T lines were infected with SARS-CoV-2 (MOI 1) for 24 h and analyzed for percent infection by flow cytometry. Representative example plots are shown for each cell line. D) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test. E) The indicated cell lines were infected with influenza A virus (IAV) for 24h and percent infection was determined by flow cytometry. Graph depicts percent infection of triplicate samples. Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05., NS, not significant, by ANOVA followed by Tukey's multiple comparisons test.
Fig.Figure 3A Experimental timelines for CSDS, ARL67156 administration and behavioral study. B The ATPase activity of mice exposure to ARL67156 (n = 7, 7, 5, 5 mice. Treatment F3, 20 = 16.81, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P < 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0405. One-way ANOVA with Fisher's LSD test). C The social avoidance behavior of susceptible mice with intracerebroventricular infusion of ARL67156 (n = 12, 10, 9, 9 mice. Interaction F3, 36 = 5.119, P = 0.0047; Target F1, 36 = 0.005, P = 0.9437; Drug F3, 36 = 3.626, P = 0.022. For Target, CSDS - ACSF vs Ctrl -ACSF, P = 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0054. Two-way ANOVA with Tukey's post-test). D The sucrose preference of susceptible mice with intracerebroventricular infusion of ARL67156 (n = 12, 10, 10, 10 mice. Interaction F1, 38 = 5.352, P = 0.0262; Group F1, 38 = 5.711, P = 0.0219; Drug F1, 38 = 9.703, P = 0.0035; CSDS - ACSF vs Ctrl - ACSF, P = 0.0083; Ctrl - ARL67156 vs CSDS - ACSF, P = 0.0027; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0031. Two-way ANOVA with Tukey's post-test). The social avoidance behavior (E) in mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 9, 9, 9, 8 mice. For social behavior, Interaction F3, 31 = 5.579, P = 0.0035; Target F1, 31 = 0.5121, P = 0.4796; Drug F3, 31 = 8.367, P = 0.0003; CSDS - ACSF vs Ctrl - ACSF, P < 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0131. sucrose preference (F) in mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 9, 9, 9, 8 mice. Sucrose, Interaction F1, 30 = 0.3342, P = 0.5675; Group F1, 30 = 17.22, P = 0.0003; Drug F1, 30 = 0.05903, P = 0.8097; CSDS- ACSF vs Ctrl - ACSF, P = 0.0761. Two-way ANOVA with Tukey's post-test). G The immobility time in the TST of mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 16, 12, 21, 19 mice. Treatment F 3, 64= 13.61, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P = 0.0059; Ctrl - ARL67156 vs Ctrl - ACSF, P = 0.0011; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0002. One-way ANOVA with Fisher's LSD test). H The immobility time in the FST with intra-hippocampal infusion of ARL67156 after CSDS (n = 21, 12, 23, 19 mice. Treatment F 3, 71 = 12.94, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P = 0.0036; Ctrl - ARL67156 vs Ctrl - ACSF, P = 0.0428; CSDS - ARL67156 vs CSDS - ACSF, P < 0.0001. One-way ANOVA with Fisher's LSD test).Data information: data are expressed as the means ± SEM. NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Ctrl, control.
Figure 5Panels from A to D show that the variability in foci formation contributes to variability in free protein abundance. A. Confocal microscopy images of URA3agg and URA3sol incubated for 18 hours at 30°C. B. Coefficient of variation and p-values (Wilcoxon test) of the total fluorescence measured from populations of URA3agg and URA3sol. C. Boxplot showing the distribution of foci fluorescence (left) or cytoplasmic fluorescence (right), from the foci containing subset of a population of URA3agg grown at 30ºC. Coefficient of Variation and p-values of each boxplot are shown below. D. Schematic showing how the formation of protein deposit can affect the abundance of free protein in a cell. Pie charts showing the average fraction of deposited and free protein of URA3agg in different media composition and 30ºC. E. Confocal microscopy image of URA3agg incubated in the presence of 5FOA after 18 hours at 30°C. F. Schematic showing that in our system, the phase separation-promoting region can modulate Ura3p activity and thus its phenotypic effect in a cell. G. Schematic highlighting that higher/lower level of free protein inside the cell results in higher/lower Ura3p activity leading to the manifestation of a stronger/weaker phenotype. H. Boxplots of the cytoplasmic fluorescence normalized by the cell area (FCYTOagg/cell area) for individual cells of the URA3agg population in 5FOA. Light green box, cells with foci containing at least 70% of the total fluorescence (big foci). Dark green box, cells with foci containing less than 70% of the total fluorescence (small foci). I-J. Cell cycle arrest phenotype. Density plots showing the fluorescence distribution of individual cells from the URA3agg population stained with propidium iodide to measure their DNA content. The cells were incubated for 18 hours at 30°C without (I) and with 5FOA (J).
Fig.Figure 2(A) Reverse transcription of RNA (500 ng) with oligo-dT in the presence of increasing doses of (PR)20. Data represent the fluorometric quantification of the resultant cDNA (n=3), mean values ± SD. (B) Percentage of RNA (1 μg) remaining after a 15´ digestion with increasing doses of RNase A in the presence or absence of (PR)20 (5 μM) (n=3). Data represent mean values ± SEM. (C, D) High-throughput microscopy (HTM)-mediated analysis of 5-ethynyl-uridine (EU) (C) and polyA+ mRNA (D) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 16 h. EU was added 30´ prior to fixation (n=3). Black lines indicate median values in both panels. In D, boxes define upper and lower quartiles and whiskers mark the highest and lowest observations. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm. (E) Effect of (PR)20 (10 μM, 16 h) in the number of Cajal Bodies (CB) identified by immunofluorescence with anti-COILIN and anti-SMN antibodies (n=3). Data represent mean values ± SD. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm. (F) Accumulation of Sindibis virus (SV) genomic RNA in BHK-21 cells in the absence or presence of increasing doses of (PR)20. The amount of viral and cellular RNA was quantified from total RNA isolated 4 h after infection by quantitative real-time PCR (qRT-PCR) with specific primers (n=3). Data represent mean values ± SEM.
Figure 4(A) EI24 knockout promotes ER stress-induced apoptosis. Control (sgCtrl) and EI24 knockout (sgEI24) HeLa cells were treated with the indicated concentrations of tunicamycin (TM) for 8 hr. Percentages of early apoptotic cells were measured by flow cytometry. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test (B) Knockdown of IP3R1 prevents ER stress-induced apoptosis in EI24 knockout cells. Cells were cultured with or without 4 μM TM for 8 hr. Percentages of early apoptotic cells were measured by flow cytometry. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test (C) Administration of calcium chelator BAPTA prevents ER stress-induced apoptosis in EI24 knockout cells. Cells were cultured in the presence of 50 μM BAPTA for 1 hr and then treated with or without 4 μM TM for 8 hr. Percentages of early apoptotic cells were measured by flow cytometry. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test (D) Knockdown of IP3R1 prevents ER stress-induced apoptosis. Cells were treated with 4 μM TM for the indicated times. Protein expressions were analyzed by immunoblotting. (E) EI24 knockout promotes ER stress-induced, but not death ligand-induced, apoptosis. Control and EI24 knockout HeLa cells were treated with 4 μM TM or with 10 ng/mL TNFα plus 4 μg/mL cycloheximide (TNFα/CHX) for the indicated times. Protein expressions were analyzed by immunoblotting. (F-H) Reintroduction of full-length EI24, but not the TM34- or C-terminus-deleted mutant (F, G) or the C-terminus alone (H), inhibited ER stress-induced apoptosis in EI24 knockout cells. Cells treated with or without TM were analyzed for apoptosis by flow cytometry (F, H) or by immunoblotting (G). Arrows in (H) denote decreased or increased apoptotic levels. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test for F), one-tailed Student's t-test for (H).
Figure 2(A) Representative western blot showing a mildly altered protein acetylation pattern (Anti AcK) between 1 week (young) and 4 week (midlife) old flies. Tubulin served as a loading control. N=4-5 per group. Tub, Tubulin. (B) Heat map showing altered protein acetylation sites between young and midlife flies, based on the acetyl-lysine enriched peptide fractions analyzed by MS and after normalization to protein input (Detailed statistical method in extended 'Methods'). N=5 per group. fc, fold change. (C) The lysine deacetylase inhibitor (KDACi) sodium butyrate (SB) induces a rapid increase in oxygen consumption in the heads of young flies, but this increase is milder in midlife flies. The dashed line indicates the addition of SB. Data was normalized to the measurement prior to addition of SB (100%) and collected after five cycles of measurements, corresponding to approximately 33 minutes. N=9 young vehicle, 8 young SB, 9 midlife vehicle and 7 midlife SB (**P<0.01, ***P<0.001). Error bars indicate the S.E.M. (D) High sodium butyrate (SB) treated flies reach the end of the pre-mortality plateau phase at earlier age of 11 days. Survival 90% control = 26 days, 15 mM SB = 24 days, 150 mM SB = 11 days. Median survival for control = 45 days, 15 mM SB = 41 days, 150 mM SB = 30. N=357 (vehicle), 294 (15 mM SB) and 320 (150 mM SB). SB 15mM Log Rank Test, χ2 =14.83, p=0.0001. SB 150mM Log Rank Test, χ2 =151.1, p<0.0001. Unpaired two tailed t-tests were used for calculating the p-values in C. All male flies were collected from a mixed male/female population.
Characterization of anti-BACE1 VHH.Figure 1(A) VHHs inhibiting BACE1 were identified using an in vitro APP cleavage assay. VHHs were recombinantly expressed in bacteria, purified and added to the assay at a final concentration of 5 μM. VHH-B9, VHH-10C4 and VHH-4A2 consistently inhibited BACE1 activity, compared to PBS or control VHH (Aβ3 and BCIILP, raised against Aβ peptide and β-lactamase BCII 659/H, respectively). Values are mean ± range, n=2, technical replicates. (B) The EC50 values (95% confidence interval) for VHH-B9, VHH-10C4 and VHH-4A2 are 99.2 nM (83.8-117.5 nM), 112.5 nM (101.1-125.2 nM) and 788.7 nM (682.7-911.2 nM), respectively. Values are mean ± S.D., n=3, technical replicates. (C, D) Primary cultured neurons were transduced by Semliki Forest virus (SFV) expressing wild type human APP695 and treated with 3 µM of the indicated VHHs. PBS and anti-GFP VHH were used as controls. (C) Western blot analysis of conditioned media for sAPP⍺ and sAPPβ, as well as cell extracts for full length APP, CTF⍺ and CTFβ. (D) ELISA measurements of Aβ1-40 and Aβ1-42 in conditioned media. Values are mean ± S.E.M., n=3 cultures for each analysis. One-way ANOVA, *p<0.5, **p<0.01, *** p<0.0001. (E, F) Dose-dependent inhibition of BACE1 in primary cultured neurons by VHH-B9. Cultured neurons were transduced by SFV expressing wild type human APP695 and treated with PBS (control: CT) or decreasing concentrations of VHH-B9, ranging from 10 µM to 0.7 nM. (E) Western blot analysis of conditioned media for sAPP⍺ and sAPPβ, as well as cell extracts for full length APP, CTF⍺ and CTFβ. (F) Conditioned media was analyzed by ELISA to assess levels of Aβ1-40. Values are mean ± S.D., n=3 cultures for each analysis. The EC50 value (95 % confidence interval) was estimated as 85.4 nM (52.5-125.6 nM).
Figure 1(A) BMDMs from WT (Pml+/+) and Pml-/- mice were pre-treated with zVAD (20 μM), Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 30 min followed by co-treatment with AT-406 (1 μM). Cell viability was evaluated by ATP release after 18 h. Data show mean ± SD of four biological replicates. (B) WT and Pml-/- BMDM were treated with TNF (100 ng/ml), zVAD (20 μM) and Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. (C) Normal expression of RIPK1, RIPK3 and MLKL in Pml-/- BMDMs. Lysates from WT and Pml-/- BMDMs were analysed for their levels of RIPK1, RIPK3 and MLKL. (D) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM), and GSK872 (10 μM), as indicated, for 8 h. Cell viability was determined by ATP assay. Data show mean ± SD of three biological replicates. (E) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml),IKK inhibitor BMS-345541 (10 mM) and Nec-1, as indicated, for 6 h and then cell death was assayed. Data show mean ± SD of five biological replicates, except the Nec-1-containing experiment (n=2).
Figure 1Dual-color SMLM shows co-localization of BAX and DRP1 at the nanoscale. A) Overview SMLM image of a HeLa cell transfected with GFP-BAX stained with anti-GFP-nanobody-AF647 and anti-DRP1 antibody probed with a CF680-labelled secondary antibody, 3 h after apoptosis induction. Scale bar 2 μm. Dual-color SMLM shows co-localization of BAX and DRP1 at the nanoscale. B) Magnified SMLM images of GFP-BAX and DRP1 assemblies co-localizing up to 30 nm during apoptosis. Scale bar 100 nm. C) Co-localization control of DRP1 immunostaining with the same primary and different secondary antibodies (labelled with AF647 and CF680) in HeLa cells. Scale bar 100 nm. D) Quantification of the distance from GFP-BAX to DRP1 structures (distance measured at the center of each structure) from dual-color SMLM images. Data are quantified from n=4 independent experiments with a total of 720 BAX structure. E,F) Representative confocal microscopy images of HeLa cells transfected with ddFP RA-BAX and GB-DRP1 (E) in untreated or apoptotic (STS) conditions, or with RA-BAX and GB-BAX during apoptosis (F). RA-BAX/GB-DRP1 and RA-BAX/GB-BAX complexes shown in green, mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm. Right panels are zoomed areas representing individual and merged channels. Scale bar 5 μm. G) BAX/DRP1 interaction compared to BAX/BAX and BCL‑xL/DRP1 by ddFP. % mito-BFP transfected cells that show RA/GB signal was quantified in HeLa cells untreated (untr.) and 3 h after apoptosis induction (STS). Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, *** p < 0.001) compared to BAX/DRP1 after apoptotic induction (STS).
Fig.Figure 6Volcano plot of the proteomic analysis of CSF from AD patients treated with acitretin or placebo for 30 days. Every circle represents one protein. The log10 transformed t-test p-values (again treatment/baseline for every patient) are plotted against the log2 transformed label-free quantification intensity ratios of acitretin and placebo CSF. ADAM10 substrates are indicated in bold writing. In total, more than 50 ADAM10 substrates were identified, but only the significantly altered substrates as well as NrCAM and APP are indicated.
Fig.Figure 7Schematic representation of the strategy for antibiotic treatment of Casp8WT and Casp8ECko mice (A) analysis of the most prominent bacterial phyla (B; n= 4 Control, 6-8 Antibiotics; multiple t-tests with Holm-Sidak method) by qPCR analysis, In B, the cycle number of 16S rDNA expression compared to the total number of cycles is shown. Data information: Data shown as mean ± SEM. ** P <0.01, *** P <0.001, **** P <0.0001; ns: not significant. Scale bars: 100μm. Graphs showing the relative weight (C; n= 11 WT, 7 ECko, 10 ECko+ antibiotics one-way ANOVA with tukey's multiple comparison) upon tamoxifen treatment in the presence of an antibiotic cocktail. Graphs showing the survival of mice (D; n= 6 WT, 7 ECko, 7 ECko+ antibiotics; Log-Rank test) upon tamoxifen treatment in the presence of an antibiotic cocktail. E. Representative images of the gut of Casp8WT and Casp8ECko mice with antibiotic treatment 30 days after tamoxifen. F-H. Representative images of H&E staining (F), quantification of intestinal pathology (G) and mucosal hemorrhages (H) in Casp8WT and Casp8ECko mice treated with antibiotics compared to untreated Casp8ECko mice (G; n= 6 WT, 4 ECko+ antibiotics, 5 ECko; one-way ANOVA with tukey's multiple comparison, H; n= 6 WT, 4 ECko+ antibiotics, 4 ECko; one-way ANOVA with tukey's multiple comparison). Data information: Data shown as mean ± SEM. ** P <0.01, *** P <0.001, **** P <0.0001; ns: not significant. Scale bars: 100μm.
Figure 6(a) DamID domainograms of NL interactions after activation of CCSER1, GRID2, or both. All data are from human RPE-1 cells. (b) DamID domainograms after activation of ADAM22, ABCB1, or the genes ABCB4, ABCB1, ADAM22 and RUNC3B simultaneously. All data are from human RPE-1 cells. DamID data of activation of ADAM22 and ABCB1 alone are same as in Figure 4a, b.
Structural analysis of TREM2 transmembrane helix (TMH) in dodecyl-phosphocholine (DPC) micelles.Figure 1(a) Proteolytic processing­­ of TREM2. First, ADAM10 or 17 cleave off the ectodomain followed by sequential cleavage of the CTF by γ-secreta­­­se creating a p3-like extracellular peptide and the intracellular domain (ICD). Only an intact CTF can bind to DAP12. CTF: C-terminal fragment. (b) 2D-[1H;15N]-TROSY spectrum of 400 µM 2H,13C,15N-labeled TREM2-TMH (residues R161-H206) in DPC micelles at 37 °C. Assigned resonances are labeled. (c) Secondary chemical shifts plotted against the residue number indicates an α-helical region between residues 173 and 198. The used TREM2-TMH construct along with the predicted transmembrane helix content (aa 175-195) is also indicated. (d) Circular dichroism (CD) spectrum of TREM2-TMH in DPC micelles indicates an α-helical secondary structure content of ~50%. (e) 3D-15N-edited NOESY spectra show that the transmembrane helix is interrupted at residues 190-192. Dashed red lines indicate sequential amide NOE contacts that are characteristic for an α-helical secondary structure. (f) Final NMR-derived structural bundle of the 20 lowest-energy structures of TREM2 shows that the C-terminal end of the transmembrane helix can adopt various positions relative to the well-defined TMH. r.m.s.d.: root mean squared deviation.