EMBO/sd-character-level-ner huggingface dataset:
(A) Left: differentially accessible regions (DARs) between WT Treg cells and Tet2/3 DKO Treg cells. 2,270 regions are more accessible in WT Treg cells (upper panel); 1,201 regions are less accessible in WT Treg cells (lower panel). Middle: 5hmC distribution assessed by CMS-IP in the DARs shown on the left. Right: DNA methylation status assessed by WGBS in DARs shown on the left: 11,303 CpGs in 2,270 regions more accessible in WT Treg cells (upper panel); 4,785 CpGs in 1,201 regions less accessible in WT Treg cells (lower panel). CD4+ naïve T cells were used as a reference. P-values are calculated using analysis of variance and boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.
(C) SCOC-GFP and SCOC-myc levels are reduced by SCOC siRNA duplex‐03. Anti‐GFP, ‐Myc and ‐Actin blots of HEK293 cells after treatment with ULK1, RISCfree or SCOC siRNA and overexpression of GFP, SCOC-myc or SCOC-GFP.
mRNA expression analysis from enriched cardiac endothelial cells (open columns, n=4 mice) or whole heart (closed columns, n= 3 mice). Data presented as mean ± StDev relative to Rpl19 expression. Enrichment factor is calculated as mean cardiac EC/mean whole heart and presented in the box.
DNA methylation status of a portion of the Rasgrf1 differentially methylated region (DMR) in 0−2-dpp Mili+/− and Mili−/− prospermatogonia. Regions corresponding to B, C, D, E, and F are demethylated in Mili−/− prospermatogonia. Vertical bars represent DNA methylation levels at CpG sites determined by whole genome bisulfite sequencing (see below). The top dark gray bar represents a portion of the Rasgrf1 gametic DMR (Tomizawa et al, 2011). The CpG cluster is shown. The deleted RMER4B sequence is indicated.
E Western blot of HA, Sec61 and Pgk1 from cell lysates and microsomes prepared from WT and ∆ice2 cells expressing Nem1-HA (SSY3140, 3141). Nem1 is undetectable in cell lysates due to its low abundance.
E Cells were distributed on YEPD plates with or without CPT at different concentrations to determine colony-forming units after 3 days at 25°C. Plotted values are the mean values with error bars denoting SD (n = 3)
Boxplot of GC contents of three groups of introns up-regulated upon YTHDC1 KD [1105 introns (∆logFC<0.5 in D), 659 HSATIII-sensitive introns (∆logFC≧0.5 in D), and 44 YTHDC1-mediated HSATIII-target introns]. The GC content of 54,222 whole annotated introns is shown as a reference. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above.
(A) Primary CD4 T cells were infected with WT or ΔVpu HIV-1 (NLAD8 strain). After 24h, productively infected cells were enriched by magnetic depletion of CD4high cells, and cultured with the indicated E430G-mutated antibodies and with NHS or HIHS. The % of Gag+ was measured in live cells by flow cytometry at days 1, 2, 3 and 6.
(A) Schematic of the p150 constructs used. The p135 isoform lacks the CAP-Gly domain, but retains part of the adjacent basic domain (red) and coiled-coil domains (blue). Right, Coomassie blue stained gel of the purified p150 and p135 proteins. Molecular weight markers are indicated.
CSF PGRN levels do not differ among MC participants carrying a PSEN1, PSEN2 or APP mutation.
H Representative time-course of histamine-induced changes in mitochondrial calcium for the experiments shown in (I).
Brightfield images and analysis of NPC markers by immunofluorescence staining (Sox1 and Nestin) or RT-qPCR (Nestin and Pax6) (n=3, mean ±SEM).
b, Kaplan-Meier overall survival after KRas reactivation in mice bearing regressed tumours treated for 2 weeks with oligomycin or vehicle.
G Quantification of the mean cell velocity of shIP3R(1,3) A (blue)‐, shIP3R(2,3) B (red)‐, and shIP3R(1,3) C (green)‐silenced immature DCs migrating in micro‐channels. shScramble‐infected DCs were used as a control (gray) (n > 100 cells from three independent experiments for shIP3R(1,3) A and shIP3R(1,3) C and two independent experiments for shIP3R(2,3) B). Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test.
(b) A53T α-synuclein (α-syn) expression was induced in stable PC12 cells for 48 h, then switched off for 24 h. Cells were treated with DMSO (control), or with 0.94 μM, 4.7 μM or 47 μM of SMER10; 0.86 μM, 4.3 μM or 43 μM of SMER18; or 0.9 μM, 4.7 μM or 47 μM of SMER28 added in the switch-off period. A53T α-syn levels were analyzed by immunoblotting with antibody against HA (left) and densitometry analysis relative to actin (right). Error bars denote s.e.m. P = 0.0917, P = 0.009, P = 0.0001 (for increasing concentrations of SMER10); P = 0.0068, P = 0.0023, P = 0.0002 (for increasing concentrations of SMER18); P = 0.0016, P 0.0001, P 0.0001 (for increasing concentrations of SMER28).
(a) Immunoblot for the indicated Cx of homogenates (Hom) and autophagic vacuoles (AV) isolated from fed or 6 h starved (Stv) mice. Quantification of enrichment is shown in Supplementary Fig. 1a.
C Binary combinations of directions in observed expression changes for perturbation 1, perturbation 2 and the interaction term grouped into four general classes. Based on classification implemented in (Dixit et al, 2016).
Treatment with systemic anti-TNF or local rmIL-10 led to impaired fracture repair at day 28, indicated by % callus mineralization. Data are presented as mean ± SEM. Treatment with PBS control versus anti-TNF, *P = 0.037, PBS control versus rmIL-10, *P = 0.037, by unpaired two-sided t-tests.
(H) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were detached or reseeded as in (C). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Arrowheads and arrows point to the mono- or di-ubiquitinated Merlin (see a longer exposed blotting results in Figure EV1D). The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.
C. Schematic showing early reprogramming processes of CMs and NCs isolated from triple transgenic mice.
serum FITC-dextran level of colon sections from the treated WT colitis mice on day 8 (n = 8).
Hydrolysis of fluorogenic substrates (Ac-PAL-AMC) for LMP2-activity of human (upper panel) or mouse (lower panel) 20S immunoproteasome at various concentrations of PRN1126 or ONX 0914. Data are presented as the means of fluorescence ± s.d. from quadruplicate assays. The experiments were repeated three times with similar results.
Sec61β wild-type (WT) and the 3R variant tagged at their N-terminus with the FLAG epitope were translated in RRL from RNAs lacking a stop codon. Ribosome-nascent chain complexes (RNCs) were recovered using anti-FLAG affinity resin, beads were washed, bound proteins eluted with 3xFLAG peptide and RNCs isolated by pelleting through a sucrose cushion Samples were resolved by SDS-PAGE and immunoblotted for endogenous rabbit SGTA, FLAG-tagged translation products, proteins of the 40S (Rps3) and 60S (Rpl17) ribosomal subunits or analysed by phosphorimaging (35S). The 3xFLAG peptide eluted material (lanes 4 and 5) corresponds to ~10% of the sedimented RNCs (lanes 7 and 8).
(a) Left, the structure of THPN. Right, THPN specifically induces the cell death in several melanoma cell lines but not in any other types of cancer cell lines. The cells were treated with THPN for 48 h, as indicated; cell survival was determined by flow cytometry. '-' represents vehicle treatment only.
C Usp27x interacts with endogenous BimEL independently of its catalytic activity293FT cells either carrying 3xFlag-Usp27x (293FT-TetR-3xFlag-usp27x) or the catalytic inactive mutant 3xFlag-Usp27xC87A under the control of the Tet-repressor were treated for 24h with dox to induce expression of Usp27x or Usp27xC87A. In all conditions PMA (to induce Bim-ubiquitination, 16.2 nM), and Q-VD-OPh (to inhibit apoptosis, 10 µM, see Fig. 3) were added at the time of Usp27x-induction. MG132 (to prevent Bim-degradation, 40 µM) was added in all conditions 4h prior to cell lysis. 3xFlag tagged Usp27x or Usp27xC87A was immunoprecipitated from whole cell lysates using anti-Flag resin. Interaction with BimEL or β-TrCP was detected by Western blotting using anti-Bim or anti-β-TrCP antibodies. Western blots are representative of n≥3 independent experiments.
(C) Immunoblot of cardiac GATA4 abundance in control (Con) and cardiomyocyte specific Gata4 knock-out mice (CM-G4-KO) at postnatal day (P) 1.(D) Quantification of the immunoblot shown in (C); *p=0.018.
Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 agomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96 (right) (n=3). Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96. Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 antagomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96 (right) (n=3). Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96.
UBE2D3˜ubiquitin-release assays analysed by non-reducing SDS-PAGE and immunodetection. His-tagged UBE2D3 was charged with ubiquitin by incubation with ATP and UAE, and charging was stopped by ATP depletion with apyrase. No CUL3 (negative control, lane 1), CUL3WT or CUL3Δ403-459 was then added to the charged E2 and the activity of the RING domain, RBX1, monitored by the hydrolysis of ubiquitin from UBE2D3, visualised by immunodetection with anti-His antibody (upper panel, "˜" denotes thioester bond). The lower panel is an immunoblot for CUL3, showing equivalent amount of both CUL3WT and CUL3Δ403-459 was added to the reaction mix; over time, the appearance of higher molecular weight bands represents the modification of CUL3 by covalent attachment of ubiquitin. The right two lanes are control lanes incubated for 10 min with E2˜Ubi but then treated with reducing agent prior to SDS-PAGE.
A Schematic representation of truncated mutants of the Smad6 MH2 domain and binding to Pellino-1N. HA-tagged Pellino-1N encodes amino acids 1-137 of Pellino-1.
entry.Pseudotypes harboring the indicated viral surface proteins were incubated with different dilutions of serum from a convalescent SARS patient and subsequently inoculated onto 293T cells that transiently express ACE2 in order to evaluate cross-neutralization. The results from a representative experiment with triplicate samples are shown and were confirmed in a separate experiment. Error bars indicate SD. Statistical significance was tested by two-way ANOVA with Dunnett posttest.
(c) HeLa cells stably expressing EGFP-LC3 were treated for 4 h with DMSO (control) or 200 nM bafilomycin A1 (Baf), or with 200 nM bafilomycin A1 and 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28. Cells were left untreated or pretreated with SMERs for 24 h before adding bafilomycin A1. Levels of EGFP-LC3-II were determined by immunoblotting with antibody against EGFP (above) and densitometry analysis relative to actin (below). Error bars denote s.e.m. P = 0.0259 (baf), P 0.0001 (SMER10), P = 0.0003 (SMER18 and SMER28) versus control; P = 0.0025 (SMER10), P = 0.0218 (SMER18), P = 0.0195 (SMER28) versus bafilomycin A1. ***P 0.001; **P 0.01; *P 0.05.
E: BT12 cells treated with 20nM MMA show a different cellular phenotype than with 100nM. Beyond 24h of exposure, there is evidence of mesenchymal differentiation and the appearance of maturing adipocytes. Scale bar (lower left): 150μm.
CLSMimages of UBFimmunofluorescence in mouseNIH 3T3cells treated for 5 h with α‐amanitin to inhibit Pol II transcription show the dispersion of nucleoli, as previously also imaged via RNAstaining (Caudron‐Herger et al, ).
Distinct co-elution of holo-CSN (observed at the expected fraction 35) and Mini-CSN CSN1/3/8 (observed eluting offset only one fraction late, F45, of the expected fraction, F44). Expected fractions are estimated from the cumulative sum of one copy per component and external size calibration. Coloring adapted to highlight subversion components and their partitioning across holo- and sub-complex. CSN1/3/8 interact and form a submodule within the CSN holo-complex structure (PDB accession 4D10). The observations are consistent between the two whole workflow replicates (see Appendix Figure S6A). Lower right panel, validation of distinct elution behavior of holo-CSN exclusive (CSN7A) and shared subunit (CSN8) by immunoblotting. For full immunoblotting data (CSN1, CSN3, CSN4, CSN5, CSN7A and CSN8) see Appendix Figure S7C).
Using the homodimeric kinesin OSM-3 as a marker for IFT, proper localisation is seen in the ts-che-3 mutant at the permissive temperature, with the strongest signal at the basal body (bb). At the restrictive temperature, most of the OSM-3 signal is found within the axoneme of the short, bulbous cilia. Scale bar is 4 µm
(B, C) The relative expressions of miR-342-3p and miR-342-5p were analyzed by northern blotting (B, representative blots from n = 3 biological replicates are shown) or quantitative real-time PCR (C, data are shown as the mean ± s.e.m. of n = 3 biological replicates) in Mtb-stimulated C3H and B6 BMDMs.
(A) SFA/UFA ratios of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. control (pEmpty). Data is presented as mean ± SD (n≥3). * p < 0.05, ** p < 0.01, **** p < 0.0001, n.s. not significant, unpaired two-tailed t-test of the indicated bar compared to WT pSCT1.
At homeostasis, RNAlow neoblasts are relatively slow-cycling and their size is restricted by Lrig-1. During regeneration, RNAlow neoblasts grow, extend projections, and enter the cell cycle by 48 hpa in a TORC1-dependent manner.
G, H IF of Lef1 at E60 and E90. (G, H') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.
Caco-2 cells incubated with P31-43 in the presence or absence of VX-770 (A) Immunoblot of PPARγ or phospho-ERK1/2 (phERK1/2) and densitometric analysis of protein levels relative to β-actin. Mean ± SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.
H ChIP-on break assay. (Top) Schematic of I-SceI substrate construct introduced into HT1080 cells, which contains a single I-SceI site located within a puromycin resistance cassette. Shown along the top are the coordinates for amplicons probed by Q-PCR. (Bottom) 53BP1 enrichment at the indicated loci in Scramble control or SirT7 knockdown cells. Q-PCR measurements were normalized to input DNA and non-I-SceI treated samples (No Cut) (mean ± SEM; one of two independent experiments shown). (Bottom Right) western blot demonstrating efficient knockdown of SirT7, with Histone H3 as a loading control.
(E) Bnip3‐mediated atrophy is not modified by mutating the BH3 domain (Bnip3BH3mut). Adult TA muscles were transfected with Bnip3 or Bnip3BH3mut expression plasmids and cross‐sectional area was quantified 7 days later and plotted as % of controls (*P0.001) n=440.
(N, O) Flow cytometry determination of p-Akt (N) and p-Erk1/2 (O) levels in CD4+ TFH-gated splenocytes from recipient mice transplanted with cells expressing the indicated proteins (bottom). In both panels, each point represents the values obtained with a single experimental mouse. n as in H. Data information: data represent the mean ± SEM. Statistical values obtained using the Student's t are given relative to control EGFP+ cells. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
D, E Co-IP assays showing that CBF1 interacted with phyA (D) and phyB (E) in vivo. (D) homozygous CBF1-myc seedlings were first grown at 22°C in continuous FR light for 4 d, then the total proteins were extracted and subsequently treated with 5-min R light, or 5-min R light followed by 5-min FR light (R+FR) before immunoprecipitation. (E) homozygous CBF1-myc seedlings were first grown at 22°C in continuous R light for 4 d, then the total proteins were extracted and subsequently treated with 5-min FR light, or 5-min FR light followed by 5-min R light (FR+R) before immunoprecipitation. After light treatments, total proteins were incubated with anti-c-myc Affinity Gel (Sigma-Aldrich). The total and precipitated proteins were examined by immunoblotting using antibodies against myc, phyA, phyB, and RPN6, respectively. The numbers below anti-phyA and anti-phyB blots indicate the relative band intensities of co-precipitated phyA or phyB normalized to those of precipitated CBF1-myc, respectively. The ratio of the first clear band was set to 100 for each blot.
Percentage of MEFs with primarily cytosolic or nuclear Lipin1mGFP. Chi-square test. **** p < 0.0001.
B. Carnitine-bound fatty acids commonly found in serum that were detected in mass spectrometry.
B,C Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at the indicated time point. *P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.
A A schematic representation of the p85 regulatory subunit SH2 consensus binding motif of PI3‐Kinase in KSHV‐TK.
A A549 and U937 cells were infected with each IAV at MOI 1 or 5 for 24 h, and lysates were analyzed by Western blotting using polyclonal anti-PB1-F2 antibody.
A Electron micrograph of wild-type yeast treated with DTT for 3 h. Inset 1 shows an ER whorl and inset 2 shows ER fragments in macroautophagic bodies (white arrows). Cy, cytosol; V, vacuole.
B Scheme for the in vivo mouse experiment involving the s.c. injection of CCR9+ (shControl) or CCR9− (shCCR9) M579-A2 tumor cells in the left and right flank, respectively, of the NSG mice. Following this, at d2 and d9, mice received i.v. injection of TIL 209 in PBS (n = 7) or PBS alone (control group for tumor growth; n = 3) and measured for tumor growth.
TIGAR expression modulates autophagy independently of p53. (A) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were transfected with scrambled or TIGAR siRNAs, and 48 h after transfection, cells were left untreated (t0) or treated with Bafilomycin A1 (100 nM) for 1 or 2 h (t1 and t2). The percentage of cells with GFP-LC3 puncta was calculated at the indicated time points. Data are shown as the mean and standard deviation from three independent experiments.
Schematic summary of the study design and patient cohort. Both blood and throat swabs were collected in different timepoint (1 to 5). Not all samples were analyzed in all omics approach due to different requirements of sample quality and amounts for different approach. The sample numbers for each omics data set were shown and specific sampling time for each sample in each data set was listed in Dataset EV1.
C A single variant (rs6927172) has the largest and most significant expression-modulating activity in resting (left panel) and stimulated CD4 T cells (right panel) with the risk allele reducing transcription. Plots represent median and IQR (box) and min-to-max (whiskers). FDR-corrected meta-analysis P value shown.
C Farnesylation of recombinant Ykt6 by FTase. Recombinant Ykt6 (5 μM) was incubated with FTase (0 or 50 nM) and 3H-FPP (1 μM; ~3,500 dpm/pmol) at 37ºC. Reactions were stopped at the indicated time points, and the amount of 3H-farnesyl transferred to Ykt6 was quantified by scintillation counting (mean ± SEM, n = 3).
(a) Size exclusion chromatogram of nanodiscs (black) and BakΔTM bound to nanodiscs (red). Inset: SDS-PAGE confirms the presence of Bak-loaded nanodiscs.
Cysteine conservation of AK2. AK2 contains four cysteines of which three (C40, C42 and C92) are highly conserved. AK2 exists in two isoforms that differ in the C-terminus with the "A" isoform containing C232 and the "B" isoform not. AK2 does not contain a mitochondrial targeting sequence. Logo plot was derived from alignments of AK2 from 163 species.
(D) Immunohistochemical staining of levels IL-1β expression in sections of pancreas from Abca12tm1d mice versus control animals at 24 weeks of age (bar = 25μm, islets outlined).
Viral titer for SARS-CoV-2 was quantitated by plaque assay on Vero E6 cells two days post inoculation.
(G) Dot plot showing the average pixel intensities at individual parent centrioles expressed as the PIDD1/CEP128 fluorescence ratio in RPE1 of the indicated genotypes. Mean values (red lines) ± s.e.m. are reported. N ≥ 50 centrosomes were assessed from as many individual cells for each condition, a.u. = arbitrary units. Statistical significance was assessed by Kruskal-Wallis test, comparing each sample to the wild type (****P < 0.0001).
C. Percentage of cell death of WT, Tfcp2l1_KO, and Tfcp2l1_KO 2i-ESCs with Cpt1a overexpression (OE) treated with 200 nM INK-128 for 24 hours. n=3 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.
(c) Confocal microscopy of differentiated THP-1 cells treated for 2 h with LPS (500 ng/ml) and ATP (3mM), immunostained for p62 (green) and ASC (red): left two images, merged channel volume and surface rendering of a three-dimensional reconstruction; right four images, enlargement of images at left (yellow arrow), with arrows indicating removal of part of the signal to reveal the red signal (ASC) inside the green-stained area (p62).
(F-H) The normalized fluorescence emission intensity profiles for F-actin (red line) and (F) EGFP, (G) EGFP-CA2, or (H) EGFP-CA7 (black line).
D. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre2 cleavage by western blot probed with anti-Sre2 serum or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown in the presence of oxygen.
A, A list of TSN-interacting proteins selected for the co-localization analysis.
(D) RT-qPCR-based analysis of expression changes of DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (E) RT-qPCR-based analysis of expression changes of KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (F) RT-qPCR-based analysis of expression changes of DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (G) RT-qPCR-based analysis of expression changes of KMT2D in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (H
(F) Visualization of pericyte coverage (NG2:green; IsoB4:red) in PBS- or COCO-injected retinas. Scale bar, 50 μm. (G) Quantification of percentage of IsoB4 vascular staining covered by NG2 staining (% overlay) (n=8).
Effects of U18666A, thioperamide and trimeprazine, compared with controls. Cells treated with the indicated compounds or with DMSO alone for 18h at 37°C, were processed for immunofluorescence microscopy. Low magnification views are shown after staining with anti-LBPA antibodies (red), filipin (green pseudo-color) and DAPI (blue). The scale bar is 20 µm.
B A recombinant polyhistidine‐ and SUMO‐tagged TFG fragment (Sumo‐TFGc, amino acids 194‐400; total molecular mass of 42 kDa) was expressed and purified from Escherichia coli extracts using nickel affinity resin. Coomassie‐stained gels of the peak elution fractions after separation of the recombinant protein on a S200 gel filtration column (top) or a 10-30% glycerol gradient (bottom) are shown. To determine the native molecular weight of the protein, the following equation was used: M = 6πηNas/(1 − υρ), where M is the native molecular weight, η is the viscosity of the medium, N is Avogadro's number, a is the Stokes radius, s is the sedimentation value, υ is the partial specific volume, and ρ is the density of the medium (Siegel Monty, ). These data suggest that this fragment of TFG is capable of forming dimers in solution.
Western blot analysis of c-PARP in GBM cells treated with DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol for 72 h.