EMBO/sd-character-level-ner huggingface dataset:
(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B) or ELISA analysis (C
C. Two possible models for UPF3A/3B-dependent and -independent NMD. Model 1 predicts that mRNAs (represented by wavy lines) from one set of genes (mRNA 1) undergoes UPF3A/3B-dependent NMD and those from another set of genes (mRNA 2) are targeted by UPF3A/3B-independent NMD. Model 2 posits that same mRNAs are targeted to both UPF3A/3B-dependent and -independent NMD. The number of arrows signify the probability/rate of NMD commitment via each branch. In Model 2, commitment to NMD is higher in the presence of UPF3 paralogs and lower in their absence.
(L) Cellular insulin content in MIN6 cells after silencing of ABCA12 (mean±SEM, n=4 replicates from assays in quadruplicate).
B. NIH3T3 cells transfected with KV10.1 (red bars) showed markedly less cilia than control cells (empty vector, white bars). Subconfluent cultures grown in the presence of FCS were serum starved for 24 hours to induce ciliogenesis. Cilia were stained with anti acetylated α-tubulin antibody. To determine ciliary disassembly, cells were starved for 24 hours and then incubated for 4h in FCS to induce cell cycle re-entry and ciliary resorption.
Retrotransposon transcription in WT (Hu303), fft2Δ (Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). B) Northern blot for the Tf2 ORF; rRNA staining of the same membrane by methylene blue is shown below. Two different exposures of the molecular size marker are shown (left).
H: PAI-1 immunohistochemistry (left panels) and quantification of PAI-1-positive glomeruli (right panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. n = 4 for RT+ and RT- mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.
(B) Improvement of motor performance by the knockdown of Sk6II in Drosophila SCA1 model expressing ATXN1(82Q) in the central nervous system. Mean ± SEM, *P < 0.05, Linear mixed-effect model ANOVA, n = 10/genotype, four times of measurements.
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.
(D) The graphically presented amount of p65 of NC and CDC20si HEK293T cells was normalized with α-tublin and H3 expression. The percent of p65 protein was calculated accordingly. (n=6)
(C) Individual nuclear speckles were photobleached and FRAP was monitored for 90 sec. Data from 45 individual cells and FRAP events were corrected for background, normalized to the minimum and maximum intensity, and the mean is shown with error bars representing the standard error of the mean. The mean characteristic recovery time is indicated ± SEM.
Schematic illustration of proinsulin-derived full-length C-peptide and Insulin. Table comparing human to murine C-peptide amino acid sequences. Underlined: sequence used as peptide. Schematic immunization schedule.
B) Western blot detection (left panel) and quantification (right panel) of LC3 level in control and TMED2 overexpressing HEK293T cells treated or not with 50 µM chloroquine for 2 hours. Actin (ACT) served as a loading control. (*): p=0.0498.
G Treatment with GPN (200 µM), BAPTA-AM (20 µM), or ML-SI1 (20 µM) blocked ML-SA1-induced TRPML1-GCaMP6m responses in neurons. Data information: traces represent the mean values of each group.
(K) Immunoblots for FTMT, HIF1α and SP1 using the mitochondrial fraction lysate and whole cell lysate of Huh7 cells before and after DFP (1.0 mM) treatment with or without HIF1α or SP1 knockdown by siRNA. HSP60 and β-actin were used as loading controls for the mitochondrial fraction and whole cell lysates, respectively.
(C) Venn diagram of the overlap between the flg22-induced genes (fold change ≥2 and p-value ≤ 0.05) in wild-type and map4k4-1.
B. Co-binding analysis in functional KDM6A-bound enhancer and promoter regions revealed that HNF1A was the most enriched co-bound TF amongst three other acinar cell TFs. Binding regions of TAL1 in a non-pancreatic cell type and random binding sites were used as negative controls. P-values was determined by Fisher's exact test for peak comparisons using all enhancer and promoter regions as background.
Time-lapse confocal images of tPH-CynA-KikGR in giant Dictyostelium AX3 cells. Arrows point to vesicles.
(D) HeLa cells were lipofected with a cDNA encoding for an empty vector or GFP-DYRK3. Where indicated, cells were treated for 2 hrs with GSK (5 µM). Cell lysates were subjected to immunoprecipitation of GFP-DYRK3. Levels of GFP-DYRK3 and endogenous Hsp90 are shown in the input and bead fractions.
Upper panel, in vitro HDAC1 inhibition assay using bendamustine and CY. Lower panel, summary of CY IC50 against other HDACs.
D. As in (C), except cells were fixed with paraformaldehyde 20 min after DNA damage infliction and immunostained with PCNA antibody.
D. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP and 5 μM flag-TBK1 at 30 °C for 30 min and then followed by immunoblotting with the indicated Antibody.
(B) Representative metaphase spreads (Scale bar: 5µm) from HeLa cells transfected with indicated shRNAs or EV control and quantification of telomere fusions. Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>2,500 telomeres counted/condition/experiment). (***) P < 0.001; (**) P < 0.01 unpaired two-tailed Student's t-test.
(B) Left, representative images of cells in the transwell invasion assay. Right, quantification of cells that invaded through the matrigel-coated membrane following treatment with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody. Scale bar: 100 μm.
Representative maximum intensity z-stack-projected images of DAPI-stained (blue) U2OS cells (D), exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for REV-ERBα
Effect of Ld infection on the HuR expression in mouse liver. BALB/c mice of 6 weeks of age were infected with Ld via cardiac puncture. Animals were sacrificed after subsequent days; protein was extracted from liver tissue followed by western blot analysis for HuR. β- Actin was used as loading control (D, left panel). Each lane have designated individual animal. Band intensities were determined by ImageJ. In each case, HuR level was normalized by its respective β- Actin bands (D, right panel mean +/- s.e.m., n=5).
B) Venn diagram showing the overlap of the differentially expressed genes (FDR≤5%, absolute fold ≥2) between proliferating and senescent cells, and between senescent and MD senescent cells. Direction of change is indicated;
Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express SFTPC (SPC), green, markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm.
C. Quantification of hypocotyl length data Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median.
Effect of TMyc on neuronal survival. Primary cultures were incubated with TMyc (5, 15 or 25 μM, 30 min) and subjected to treatment with NMDA (100 µM) and glycine (10 µM) for 4 h. Specific neuronal viability was established and expressed relative to values in cultures with no treatment. Means ± SEM are represented (n = 8) and statistical analysis was performed by ANOVA test followed by post-hoc Tukey HSD test.
Images of mESCs treated with control (Ctrl, dimethyl sulfoxide (DMSO)), INK (INK128, 200 nM) for three days and released for three days in 2iL. Scale bar, 400 µm.
G. CDK-dependent binding of Plx1-PBD to the Apc1-loop500. MBP-fused Apc1-loop500 was incubated in the presence or absence of purified Cdk2/cyclinA at 30˚C for 2 h. MBP-fused Apc1-loop500 (CDK+/-) was isolated by amylose beads and further incubated with indicated amounts of WT Plx1-PBD on ice for 10 min. The bound proteins were recovered by amylose beads and analysed as described in (D).
F Comparison of differentially processed transcripts (RNAprof), differentially AS genes (AtRTD2-SUPPA) and differentially expressed genes (DEG) with genes significantly upregulated in the upf1upf3 mutant [56], indicating genes potentially regulated by NMD.
B. Male meiocytes immunostained for MSH2 (red) and ASY1 (green), and stained for DAPI (blue) in wild type (Col) or msh2.
A, Spontaneous rates of SGNs from OtofI515T/I515T (red, n=35) and Otof+/+ littermates (black, n=39) were not significantly different (p=0.83, Kolmogorov-Smirnov test).
Western blot analysis of (B) p845GluA1 and GluA1 expression (S n=9, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.
B Fluorescent microscopy of ISP and its N-terminal 20 residues (N20) fused with a BFP::3V5 peptide episomally expressed in zygotes. Scale bar = 5 μm.
E. Sporozoite ejection of immobilized concavin(-) infected mosquitoes on glass slides and quantification of ejected sporozoites from 20 mosquitoes. * indicates individual sporozoites in the ejected saliva. Scale bar: 10 µm.
A. Experimental approach used for the identification of pseudouridine sites in mitochondrial RNA. A combination of the CMC modification and the next-generation sequencing on pure mitochondrial RNA from control and siRNA treated cells was used. CMC-ψ modification causes skips in reverse transcription rather than stops, resulting in 1bp deletions in sequence alignments.
24OHC levels in spent media from LN229 (left) or GBM#P3 (right) cells treated with DMSO or 20 μM EFV for 72 h quantified using targeted LC-MS/MS.
TSC1 mRNA turnover determined by pulse and chase (Click-It, Invitrogen) over 8 h in P493-6 cells, either treated with tetracycline to repress MYC or left untreated (mean ± st.dev., n=3 technical replicates).
(D) Representative images of immunofluorescence assay for mDia2 and phalloidin staining for HCC cells with treatment as indicated (n = 3 independent experiments). WT-mDia2: expression vector encoding wild type mDia2. mDia2-NLS del.: expression vector encoding truncated mDia2 lacking NLS peptide. Scale bars, 20μm.
(g) Knockdown of EndoB1 or Cdk5 expression abolishes α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=4.
(c,d) Quantification of LC3 recruitment (c), and cell fate (d), of mixed cell-in-cell structures for which host or internalized cells were treated with ATG5 siRNA. Data represent mean±s.e.m.from three separate experiments; n, total number of structures analysed; *P0.01, **P0.004.
B Western blot analysis of p97 levels in HT1080 (B) cells treated with additional NaCl (+17, 34 and 51 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.
e, NCOA4 interaction network from cells expressing NCOA4-HA-Flag or FTH1-HA-Flag (Supplementary Table 9). Black lines (this study) depict directionality of interaction observed with line thickness weighted by weighted and normalized D score (WDN) score (293T data set). Dotted lines indicate data derived from the STRING database. Numbers in parentheses indicate log2(H:L) ratio of NCOA4, FTH1 and FTL from MCF7 Ex. 1 data set (Supplementary Table 4).
F Resting mitochondrial Ca2+ levels in mt-EMRE and scramble peptides treated cells, evaluated via ratiometric imaging of the mitochondrial targeted mt-riG6m. Mean ± s.d., student t-test, **p < 0.01, n = 3. Data information: Error bars for some points are too small to be visible.
(B Top 20 GO terms enriched by ATAC-DEGs. Rich factor represents the ratio of the number of mapped genes to the number of all the annotated genes located in the pathway.
(C) WGA staining of hearts from WT and miR-574-/- mice 4 weeks after TAC surgery (n≥500 cells). Scale bar: 20 μm. In the violin plot, black line shows median value for the group and peach dashed lines represent two quartile lines in each group.
TLN accumulation occurs in a compartment distinct from classic biosynthetic and endosomal pathways. (A-F) Early compartments. No colocalization was observed between TLN (green in all panels) and nuclei (A, TOPRO-3), ER-markers BIP (B) and calnexin (C), ERGIC-53 (D), β-COP (E) or GM130 (F), and labeling cis-Golgi. (G-L) Late compartments. TLN accumulations are not accessible for exogenous biotin (G). Early and recycling endosomes (H, EEA1) and transferrin receptor (I, TFR), late endosomes (J, LBPA), and lysosomes (K, Lamp-2; L, catD) also stained negative for TLN. Vertical sections (x-z, arrowheads in I and J) clearly distinguish TLN accumulations from recycling (I, TFR) and late endosomes (J, LBPA). Bars, 10 μm.
(L) weight as a function of time after treating Gbeys/ys mice with vehicle (n=8) or 144DG11 (n=9) at 6 months of age (at onset). Data information: Two way ANOVA with repeated measures show that, throughout the period, treatment values were higher than vehicle (p<0.15).
(B) Electron microscopy of ultrathin cryosections of HeLa cells co-transfected with EGFP-ORP5 and PTPIP51-HA and immunogold stained with anti-GFP (15 nm gold) and anti-HA (10 nm gold) showing EGFP-ORP5 localization at ER-mitochondria contacts (arrows). Arrowheads indicate the highly expanded ER membrane juxtaposed to mitochondria. Scale bar 200 nm. (C) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 and GFP-Sec61β in HeLa cells co-transfected with PTPIP51-HA. Results are presented as the percentage of the total number of gold particles (800 per condition, n=30-35 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (ER, reticular ER; ER-mito MCS, ER-mitochondria membrane contact sites; ER-PM MCS, ER-PM membrane contact sites). % gold particles ±SEM. Data are representative of three independent replicates. **P<0,001.
A-C Male cynomolgus monkeys were treated with vehicle control (n=2), AT845 (n=3) at 6×1013, 2×1014, or 5×1014 vg/kg, or Cyno-AT845 (n=3) at 2×1014 vg/kg and followed for approximately 12 weeks. (A) Anti-GAA antibody titer, (B) cardiac troponin I (cTnI), and (C) N-terminal pro b-type natriuretic peptide (BNP). Bars show mean and SEM.
A) Levels of murine TIMP1, MMP7 and TSP2 in plasma of mice bearing PDAC-PDX (HuPa4, HuPa8 and HuPa11) growing orthotopically in the pancreas (mean ± SEM; n≥3 for each group), *p<0.05 (Mann-Whitney). The exact n and p-values are indicated in Appendix Table S4A
(F) Immunofluorescence image and quantification of the expression of SOX2 in tumor tissues subjected to in vivo limiting dilution assay. Scale bar = 50 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0024, **P = 0.0088, and *P = 0.0342; two-tailed unpaired Student's t-test).
(a,b) Dynamics of HSFA2 and HSP101 transcript levels in control (NHS, NHS+187), 44°C heat-stressed non-colonized and SA187-colonized (HS, HS+187), thermoprimed (LAT) as well as thermoprimed and SA187-colonized plants (LAT+187) at 1 h, 24 h, 48 h, 72 h and 96 h of recovery at 22°C. SA187-colonized (HS+187) and thermoprimed (LAT) plants showed higher transcript levels in comparison to plants exposed at 44°C HS for 30 min (HS) after 1 h of recovery at 22°C. Data information: All plots represent the means of 3 biological replicates. Error bars represent SE. Asterisks indicate a statistical difference based on Student's t-test (*P ≤ 0.05
Figure 5 Comparison of immunostaining NUP160 after fixation with either PFA or glyoxal. HeLa cells were stained for the nucleoporin complex protein NUP160 after fixation with either PFA, glyoxal pH 4 or glyoxal pH 5. Fluorescence intensities (fold over background) were compared and are shown in the graph. The quantification of fluorescence signals shows that glyoxal pH 4 fixation allows for significantly brighter stainings. N = 73 - 156 cells per condition analyzed. Scale bar = 10 µm. *** p < 0.001
(D and E) Quantification of UBE2D1~UbAtto discharge assays. The loss of E2~Ub is plotted as the average of experimental duplicates (± s.d.).
(B) Signed test statistics from UK Biobank association data for 1463 disease- or medication-related traits for rs9387181. Black crosses denote the mean ± SEM for each group. Dotted horizontal line indicates the null.
(D) Representative immunofluorescence images showing Ki67 and p-cadherin (Pcad, labeling HG cells) expression in Sirt7−/− and Sirt7+/+ HFs on P74 and P90. The HF boundary and dermal papilla (DP) is denoted by the dotted and dashed lines, respectively.
A. Real-time fatty acid oxidation (FAO) profile of WT and Tfcp2l1_KO 2i-ESCs using the FAO stress test kit in Seahorse XF. Oxygen consumption rate (OCR) were measured. Error bars indicate SEM; n = 5 biological repeats. Palm-BSA, palmitate conjugated BSA, a substrate of FAO; Oligomycin, an inhibitor of ATP synthase (complex V); FCCP, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, a proton gradient uncoupler; Rotenone and Antimycin, a complex I inhibitor and a complex III inhibitor, respectively.
B. Purified Elongator was incubated with the primary amine crosslinker disuccinimidyl suberate (DSS) and analyzed by mass spectrometry. Schematic representation of the major crosslinking connectivity of Elp123 is shown.
(b) The proportions of EGFP-positive COS-7 cells with >5 EGFP-LC3 vesicles, transfected with EGFP-LC3 and pcDNA3.1 (empty vector) or rheb (1:3 ratio) for 4 h and treated with or without 10 μM calpastatin or 50 μM calpeptin for 24 h post-transfection. Error bars show 95% confidence interval.
Quantification of the % of WT load in cybrid sorted populations after transfection
D. Analysis of OXPHOS supercomplexes in control and patient fibroblasts. BN-PAGE from isolated mitochondria permeabilized with 6g/g (w/v) of digitonin immunoblotted on PVDF membrane and incubated with the indicated antibodies. SC, supercomplexes I+III2+IVn. The dividing lines correspond to gels sections visible in the raw data file.
(A) Huh7.5 cells were transfected with the negative control siRNA (NC) or the Rubicon (Rb) siRNA for 48 hours and then infected with 1 m.o.i. of HCV. Western-blot analysis of cell lysates at different time points after HCV infection. Actin served as the loading control.
Tubulogenesis of control and CEP41-knockdown cells for 18 h was compared via an in vitro angiogenesis assay. Scale bars, 600 μm. Quantification of tube node numbers in (F) and tube length in (G) from each field of view using the ImageJ angiogenesis analyzer at the indicated time points. The graph compares the relative length of control cells and CEP41-knockdown cells. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.
Blood glucose levels of InsA-peptide immunized mice (γ/µ < 0.1, black, n=5) and CI (grey, n=6) mice were assessed at indicated days post immunization with a commercial blood glucose monitor device. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test, all comparisons were not significant.
C ChIP-PCR analysis of H3K9Ac, H3K9me2, H3K9me3, H3K27Ac, H3K27me2, or H3K27me3 modification levels in Oct4 binding sites of MEFs infected with SKO plus Flag or Gadd45a on day 3. (**p≤0.01; n=3)
B Localisation of transiently expressed GFP-RAB35 in hTERT-RPE1 cells after 24 h serum starvation and staining for polyglutamylated tubulin (polyglu. tub.) and DNA. Insets show higher magnification images of the cilia region. Scale bars, 10 µm.
E MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h in presence of MG132 (10 μM) or DMSO, before LSD1 level was analyzed by immunoblotting.
Microglial volume was not affected by the Stat3 deletion (Mann-Whitney test), but peri-plaque microglia had significantly more microglial branches and junctions per plaque, and the total process length of peri-plaque microglia was increased (Mann-Whitney test; same mice as in C-D). Scale bar, 20 µm.
CXCR2 expression in human neutrophils, measured by FACS, was decreased by LPS treatment and restored by Smaducin-6 treatment.
One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (5-50 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-6). FGF3-4-8 expression was determined by sqRT-PCR. ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).
(C) The 400th residue plays a determinant role in the S. cerevisiae-S. bayanus incompatibility. Both Sb-CCM1E375Q and Sb-CCM1D400N mutants relieved the incompatibility in S. cerevisiae, but only Sb-CCM1D400N caused respiration defects in S. bayanus. The Sb-CCM1E375Q mutant was selected as a control in this experiment since it was a charged-to-polar substitution similar to Sb-CCM1D400N and also appeared three times in our screen. Sb, wild-type S. bayanus.
(B) High confidence protein-protein complexes identified by MIST (Molecular Interaction Search Tool) within the 105 genes identified in (A). Interactions between proteins can be direct or indirect within the complexes.
D Expression of genes on 6q23 in EU-containing mRNA (EU added at time of stimulation) showing that deletion of the NF-κB binding site specifically reduces transcription of TNFAIP3, but not other genes at this locus (n=6; one sample t-test). Representative data from the DH gRNA combination.
Defect in intra-Golgi trafficking in PTAR1 KO cells. (C) VSVG-GFP expressing WT and PTAR1 KO HeLa cells were cultured at 40ºC and then shifted to 32ºC. VSVG-GFP fluorescence images taken at the indicated times after the temperature shift are shown. The right panel shows quantification of VSVG-GFP fluorescence in the Golgi region after the temperature shift (mean ± SEM, n = 6).
(F) Straight immunoblotting of HA- and GFP-Parkin in the absence or presence of CCCP. Note the slower migrating ladders derived from ubiquitylation (Ub) in only the GFP-Parkin with CCCP lane.
D A list of novel CDKL5 substrate targets and phosphorylation sites identified. All phosphorylation sites are conserved in human. # MAP1S pS812 is identified based on sequence homology. Red = phosphorylated serine, bold = RPXS consensus sequence
(d) Atg5 knockout enhanced Wnt signalling. TopFlash reporter activity induced by Wnt3a CM in Atg5−/− or wild-type MEFs. Results are means ± s.d. after normalization to Renilla luciferase activity. (n = 3; asterisk, P 0.05).
B Imaging of GFP expression in the hippocampus 3 weeks after virus vector injection. Scale bar: 250 μm.
A. Sufu protein levels in mouse embryonic fibroblasts (MEFs) Ptch1+/+, Ptch1+/- and Ptch1-/- transfected with a non-targeting siRNA (Control) or two siRNAs targeting mouse Fbxl17 (1) and (2).
Western blot detection of FLAG-tagged ARS proteins in mascRNA anticodon stem-loop mutant Anti-Mut1 expressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. β-Tubulin was used as a loading control.
(A) Flow cytometry analysis of tail blood from Ch10/+ (control) at 30 days post tamoxifen administration, Ch10/+; Id1 Cre/+ at 30 days post tamoxifen administration, and Ch10/+; Id1Cre/+ at 80 days post tamoxifen administration.
c, d, Atg5-/- and ATG16L1HM MEFs were grown in the presence of the chemical inducer of autophagy rapamycin (Rap, 50 µg ml-1) and cyclohexamide (CHX, 5 µg ml-1) for 0, 4, 8 and 12 h. Cell lysates were analysed by western blot for loss of P62 expression (c). P62 levels were quantified by densitometry normalized to actin (n = 3; d). *P 0.04; **P 0.008; ***P = 0.0003