EMBO/sd-character-level-ner huggingface dataset:
(C) Experimental outline: 15 days-old (P15) female and male wt and Pidd1-/- mice were injected with DEN (25 mg/kg) or PBS at t(0). The livers were analyzed after t(8h) and t(48h), or the mice were weaned at P19 to induce polyploidization and harvested 5 days later - t(weaning+5d). t(8h) PBS: wt n=5, Pidd1-/- n=5; DEN: wt n=3, Pidd1-/- n=3. t(48h) PBS: wt n=3, Pidd1-/- n=3; DEN: wt n=5, Pidd1-/- n=3. t(weaning+5d) PBS: wt n=3, Pidd1-/- n=3; DEN: wt n=3, Pidd1-/- n=3.
(a) Paradigms of early life trauma in mice and humans. In mice, early life trauma consists in an exposure to unpredictable maternal separation combined with unpredictable maternal stress (MSUS). In humans, exposure involves paternal loss and maternal separation (PLMS) in early childhood (age 5-11).
The Arabidopsis pi4kβ1 pi4kβ2 double mutant displays a growth defect and cytokinetic defects. The growth phenotype of the Arabidopsis pi4kβ1 pi4kβ2 double mutant can be complemented by ectopic expression of PI4Kβ1 or PI4Kβ2, or by mCherry-tagged PI4Kβ1 expressed from a genomic fragment. Plants shown are one month old. Scale bar, 10 cm.
(e) Sections of the liver and muscle tissues from fasted 12-week-old GFP-LC3+-Atg7+/+ and GFP-LC3+-Atg7+/− mice were subjected to fluorescent microscopy to examine GFP-LC3 puncta (left). The number of GFP-LC3 puncta was counted (right). Scale bars, 10 μm. *P0.05; Student's t-test, n=5.
Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Hot-plate test (H).
C, D Velocity analysis based on differences between spliced and unspliced transcripts for all cell clusters (C) and astrocyte clusters (D).
(A, B) The nucleolin GAR domain binds a kinesin light chain, directly linking nucleolin-mRNA complexes to kinesin motors for axonal transport (A). The GAR domain further mediates membrane association of nucleolin. GAR-mediated subcellular targeting of nucleolin complexes enables export of key mRNAs to the axon, and the local translation of their encoded proteins for local functions in the axon, or for retrograde transport to the cell body. This reciprocal transport mechanism provides intrinsic regulation of axon length and growth, and indeed deletion or mutation of the GAR domain (B) perturbs mRNA localization to axons and increases axonal elongation.
d. Representative example of the frequency of IFN+Foxp3+ Tregs isolated ex vivo from RRMS patients (lower row) and healthy controls (HC, upper row) and stimulated in the presence of vehicle (left histogram), LY294002 (middle histogram) or MK2206 (right histogram) for 3 days. Staining was performed after a 4-hour stimulation with PMA and ionomycin. e. Statistical analysis of the frequency of IFN+Foxp3+ Tregs in HC (white bars) and RRMS patients (black bars) stimulated in the presence of either vehicle, LY294002 or MK2206 (n=10).
F Regression analysis of the correlation between TERT expression and percentage of CD4+ cells in colon tumours. n = 36.
(A) The first instar TBPH-/- larvae were treated with Rapamycin for 72 h. Then the third instar larvae of W1118 and TBPH-/- were subjected to larval locomotion assays. Data from three independent experiments represented as means ± S.E.M., *, p<0.05; **, p<0.01, one-way ANOVA.
BJcl2 cells with or without hTRF2 overexpression were inoculated into the backs of immunodeficient Nude mice and treated with anti-GR1 antibody or isotypic control (200µg/IP) as indicated in (J)
(B) Endogenous DOR leaves the nucleus under starvation. C2C12 myoblasts were incubated for 30 min at 37°C with DMEM (basal) or HBSS (starvation). DOR is shown in red and nuclei are in blue (DAPI staining). Scale bars, 5 μm.
(C) Screening of Phm8's phosphatase activity against 90 phosphorylated compounds. Phosphatase activity was measured in the presence of 0.5 mM substrate and 5 mM Mg2+ (pH=7.0, 30°C). Compounds with specific activity higher than 0.1 μmol/mg/min are shown. The x axis represents specific phosphatase activity (μmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates).
B. Transmission EM demonstrates absence of synaptic ribbons in presynaptic terminals of bipolar cellribbon synapses in RBEKO/KOmice, while synaptic ribbons are readily visible in the terminals of RBEWT/WTmice. Except for the absence of synaptic ribbons, the ultrastructural appearance of the presynaptic terminals is comparable to RBEWT/WTmice. Abbreviations are the same as in A. Scale bars: 1 µm (B1, B2, B4, B5); 300 nm (B3, B6).
(G) Fifty indicated cells were transplanted with 2 ×105 competitor cells into irradiated recipients. After 12 weeks, the chimerism of peripheral blood was analyzed. White or gray circles represent donor cell chimerism in multilineage-reconstructed recipient mice (> 0.5% for myeloid and B- and T-lymphoid lineages) or unreconstructed mice after 20 weeks, respectively.
Unsupervised hierarchical clustering based on the average degree of methylation of the 500 most variable promoters (red - low/violet -high methylation levels); * - relapse (REL) sample of patient P4 with blast content of 8%
B) Electrophoretic mobility shift assay (EMSA) examination of the binding affinity of CENPC-CT and CM peptide to a CENP-A nucleosome. Binding to a canonical H3 nucleosome was also examined using EMSA.
V. cholerae ΔvrrA ΔmicV strains carrying pBAD-micV, pBAD-vrrA or an empty vector control (pCtr) were cultivated to early stationary phase (OD600 of 1.5) in LB. Cells were treated with L-arabinose (0.2% final conc.) and RNA samples were collected at the indicated time points after induction. Northern blot analysis was performed to determine VrrA, MicV and ompT levels. 5S rRNA served as loading control. For comparison, RNA samples of a wild-type strain carrying pCtr were collected during various growth phases, which indicated ~18-fold and ~7-fold higher levels of VrrA and MicV expressed from the pBAD plasmids, respectively (see Source data for quantifications).
E The mitochondrial fission complex is required for cyclin C-dependent Bax activation. Extracts were prepared MEF cultures with the indicated genotypes treated with H2O2 (0.4 mM, 4 h). The extracts were treated as in A. Molecular weight markers (kDa) are indicated on the left.
(C HeLa cells were co-transfected with the indicated TDP-NLS reporters as well as GFP or GA175-GFP, and treated with MG132 (10µM) or vehicle for 16h.
(a) Quantification of mitochondria numbers from Atg18 control (Atg18KG03090/wild type) and mutant (Atg18KG03090/Df(3L)6112) or Atg7 control (Atg7d30/d14) and mutant (Atg7d77/d14) TEM images. Mitochondria were quantified from 2 distinct 25 μm2 regions per cell from 2 cells per animal from at least 3 different animals per genotype.
(A) ADP-Glo ATPase assay showing stimulation of S. cerevisiae Hrr251-394 (blue circles), Hrr251-394 K38R (red diamonds), Hrr25 full-length:Mam187-191 (black triangles), Hrr251-394:Mam187-191 (orange squares), and Hrr251-394:Mam187-191 R131A (green triangles) by bovine casein. See Figure EV6A for SDS-PAGE analysis of purified proteins.
(C) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds in (B). The lid-1(xd288) and atgl-1(xd314) mutations significantly increase the number of surviving neurons. Neuron-specific expression of atgl-1 or lid-1 reverses the protective effect of atgl-1 or lid-1 mutation. The data were analyzed using one-way ANOVA with Turkey's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n = 5, with at least 50 animals per strain analyzed in each experiment.
The tubular stress marker Lipocalin-2 (LCN2) is not expressed in early time points but increases in time course in late stages when fibrosis is more prominent and mRNA level
Flowcytometry analysis of YTHDF1 protein expression in GFPhigh/low/neg populations from Lgr5-GFP-IRES-creERT2 mice.
Flow cytometric analysis showing the histograms and MFI of O-propagyl-puromysin (OPP) incorporation in LT-HSC in: IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,43), AML patients plasma (Orange, n=6), or human CD34+ cells (blue, n=3) normalized to vehicle-injected contralateral femurs after subtracting the background fluorescence.
C Principal component analysis of scaled element counts (sum of barcodes tagging same genomic construct in mRNA) in resting and stimulated CD4 T cells from 12 donors. Dotted lines indicate samples from the same donor.
SENP3 knockdown abolishes the DFP-induced increase in LC3-II in HeLa cells. Nsi or SENP3i (I) was transfected into HeLa cells. Two days post-transfection the cells were treated with DFP for a further 24 h. Whole cell lysate samples were blotted as indicated (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; siRNA concentration, 20nM; DFP, 1mM for 24 h; values normalized to the control value; n=3, biological replicates; ***, p<0.001; N.S., not significant, p>0.05; paired t-test).
(A) The number of HeLa cells with N-terminal- or C-terminal-tagged PINK1 localized to the mitochondria was counted in >100 cells.
(E) Quantification of the cumulated period of calcium signals in individual tumor cells expressed as the percentage of total time. Each dot represents one tumor cell. Bars represent mean values. Data information: Data were pooled of four independent experiments. (*p<0.05, ***p<0.001, unpaired t‑test).
(A) Root meristem zones of wild type (Col), kup9 mutants (kup9-1 and kup9-2) and the kup9-1/ProKUP9:KUP9 complementation lines (COM1 and COM2). Seeds were germinated and grown on LK or HK medium for 7 d. The meristem zone lengths are marked with red lines. Scale bars, 50 μm.
Inhibition of TNF resistance of S mice by RU486. B and S mice (all groups n = 5) were injected with 5 μg TNF (= LD100 in RU486-pretreated mice from preliminary experiments), 30 min after injection of RU486 or DMSO control. Both B (black dashed line) and S mice (grey dashed line) pretreated with RU were significantly sensitized for TNF lethality compared to DMSO/TNF controls (full lines) (top left panel) and for TNF-induced hypothermia measured 6 h after injection of 5 μg TNF. IL-6 levels 6 h after injection of TNF were equally high in RU486-sensitized B and S mice. RU486 injection in S mice (n = 4) led to significantly increased TNFR1 protein expression in liver and spleen 6 h later compared to DMSO vehicle-treated S mice (n = 4).
E qRT-PCR for TET family members normalized to the average of RPS16, Bact, and Ubch5b transcripts in naïve and activated CD4+ T cell cDNA.
Inhibition of miR-130a by its sponge enhances IFNγ-inducible PD-L1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected to immunoblot
A, Schematic representation of the dsRNA sensing mechanism, downstream signalling events and expected phenotypes.
A, PTPN3 wildtype, phosphatase-dead D811A and ICC-derived mutant L232R exhibit similar patterns of subcellular localization and co-localization with TβRI. HaCaT cells were transfected with HA-TβRI and Flag-PTPN3 (wildtype or its mutants). 24 h later, cells were harvested for immunofluorescence with the indicated antibodies. Scale bar = 10 μm.
293T cells were transfected with a plasmid encoding FLAG-Stx17 wild type (WT) or the indicated constructs. At 24 h after transfection, cell lysates were immunoprecipitated (IP) with anti-FLAG M2 beads, and analyzed by IB using antibodies against PGAM5 and FLAG. Five percent of lysates was analyzed as input.
E - LC/MS-MS traces showing acetylcholine (Ach) levels in SVF isolated from ChATfl/fl and ChATfl/fl;Vav-iCre IWAT compared to an internal control (d4-Ach).
(B) Mavs+/- and Mavs-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs. The level of d2GFP in Cy5+ cells was measured at various timepoints post-transfection as indicated. Histograms plots show d2GFP level 48 hrs post-transfection. Bar graphs show 3 timepoints as indicated.
B VP1388 and VgrG1 co-precipitate with VP1390. Immunoprecipitation using α-FLAG antibodies from V. parahaemolyticus ∆hns/∆hcp1/∆vp1390 derivatives harboring plasmids for the arabinose-inducible expression of FLAG-tagged sfGFP or VP1390. Cells were grown in MLB media supplemented with chloramphenicol to maintain the plasmids, and 0.1% arabinose. Endogenous VP1388 and VgrG1 were detected using α-VP1388 and α-VgrG1 antibodies, respectively.
ceramide measurement in HeLa cells infected with Shigella-WT or mock-treated, analyzed at 3 hpi Ceramide is shown normalized to mock-treated cells
(D) PBMCs were transduced with increasing doses of GFP reporter vectors (vectors were normalized to the same number of infectious units after titering them on permissive cells), left plot. Vectors were integrated at similar levels as measured by GFP Taqman qPCR, right plot. A 293T cell line with a single vector copy integrant (PGK-GFP) was used as a control to estimate the absolute copy numbers. A representative experiment of 3 experiments is shown here. Two-tailed unpaired t test p-value 0.0288 (dose 3). Error bars show SD.
D, Heatmap illustrating the steady-state levels of significantly regulated lipids in THEM6 OE CWR22res cells when compared to EV (p ≤ 0.05, FC = 1.2). Values are expressed as log2(FC). Data information: Data reproducibility: n = 3 independent biological experiments. EV: Empty Vector; DAG: diacylglycerol; TG: triglyceride; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; LysoPC: lysophosphatidylcholine; LysoPE: lysophosphatidylethanolamine; Cer: Ceramide; SM: sphingomyelin; CE: Cholesteryl ester.
Real-time PCR analysis of Oct4 and several lineage differentiation markers expression during EB formation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).
Illustrations show the two major SVs associated with filament/s in the wild-type RA-SV pool at rest; synaptic ribbon (R, red), SVs (dark green) with filament/s (blue). Not drawn to scale. (A,B) Ri-attached SVs (Ri.) and (C,D) interconnected SVs (In.) harbor single (A,C) or multiple (B,D) filaments. Example virtual section from resting wild-type; single Ri. (A',A'') and In. (C',C''), multiple Ri. (B',B'') and In. (D',D''). Magnification 12,000x; scale bar, 40 nm. Structures are color-coded similarly to the illustrations, the number of ribbon-attached filaments per SV are indicated with red arrowhead/s and interconnectors per SV with purple arrowhead/s in A'', B'', C'' and D''. Magnification 12,000x; scale bar, 40 nm.
D. Exocytic ΔCm (top) and corresponding Ca2+ current integrals, QCa (bottom) for various depolarization durations. Data are grand averages of the cells' means. Exocytic ΔCm was reduced in the knockout from 20 ms onwards, indicating reduced sustained exocytosis. Data from OtofPga/Pga animals were replotted for direct comparison from (Pangrsic et al, 2010).
F Overall survival of gastric cancer patients (all stages) analyzed depending on TRF2, HSPG genes (HS3ST4, GPC6 and VCAN) and MDSC level (Lo or Hi). Data information: For all panels, the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.
F, immunoblotting of NPC1L1 in MCs, MPCs and RCs of Du145TXR or MCF-7ADR. G, immunoblotting of NPC1L1 in Du145TXR or MCF-7ADR cells transfected with siNPC1L1 or siScramble.
Therapeutic effects of GSK-650394 in SNCA-PD model mice. mDA neuron degeneration in the SN (O-U) of the SNCA-PD mice were assessed 12 weeks post- SNCA injection after sacrifice SNCA pathology assessed by p129-α-syn immunoreactivity in TH+ mDA neurons in the SN. n= 5 GSK-650394-treated SNCA-PD mice, 5 vehicle-treated SNCA-PD mice, and 8 SNCA-untreated non-PD mice.
(f) BJAB and Jurkat cells expressing control or Atg5 shRNA were treated with Fas ligand (15 ng ml−1) or TRAIL (12.5 ng ml−1) for 24 h (same technical replicate as d). Cells were then re-plated at low density and allowed to recover for 5-6 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0089). Atg7 knockdown data were not included for long-term viability owing to the growth suppressive effect of Atg7 knockdown in the absence of drug treatment. Uncropped images of blots are shown in Supplementary Fig. 6.
Representative H&E images of human lung tissues from normal background and bronchiolitis obliterans (BO) with progressive fibrosis.
(D,E) Colony-forming units (CFUs) were analyzed from (D) abdominal lavage and (E) liver homogenates. Significance maps depict p-values from a Kruskal-Wallis ANOVA with controlled false-discovery rates applying the Benjamini-Hochberg procedure.
Quantification of stochastic gene expression for the indicated p53 target genes before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). The fraction (f) of active promoters (proxy for burst frequency) increases, while the transcription rate (µ) (proxy for burst size) at active TSS remains similar upon DNA damage for all time points. Left panel: The percentage of cells with active TSS is shown as stacked bar graphs. We subdivided the population in cells with strong TSS activity (> 75% of TSS active, solid colors) and those with partial TSS activity (at least one, but less than 75% of TSS active, shaded colors). The mean fraction of active promoters (ratio of all active TSS to the total number of genomic loci analyzed) is indicated above each bar. Right panel: Distributions of calculated transcription rates µ [RNAs/h] at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The number of TSS analyzed is indicated in each plot (compare Fig EV2C).
A-E) Overall survival (OS) of patients with tumors containing mutations in the genes A) TP53, B) PIK3CA, C) ERBB2, and D) PTEN. E) OS by PTEN-MutExp genotype ("low" defined as PTEN mutation or PTEN expression in the lower quartile across the cohort, "normal" otherwise) stratified by groups receiving no systemic treatment (n=336), endocrine therapy only (Endo only; n=1,579), endocrine and chemotherapy (Endo + Chemo ± any; n=914), as well as HER2 treatment with any other treatment or none (HER2 ± any; n=348). Specific treatments in these groups are detailed in Table EV5. In each Kaplan-Meier plot, wildtype (wt) and normal cases are plotted in blue, mutated (mut) and low cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for mutation/low is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG. Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade.
A) H3K9me3 ChIP-sequencing tracks from the q21.12 arm of chromosome 7 in RPE-1 cells (Parental and Taxol-resistant clones). The ABCB1 gene region is shown in pink. Each colored track shows the H3k9me3 profile of a different Taxol-resistant clone.
(A) Differential interference contrast micrograph of live T. agilis cell. The arrow points to the cell anterior, where the rostrum is located; arrowheads point to some of the flagella.
Cell viability assays for MEFs with indicated genotypes treated with Mdm2 inhibitors ± 0.4 µg/ml Doxo. n=3.
Western blot detection of the presence of GST-cGAS and His-STING in the immunoprecipitates of cGAMP agarose. Purified GST-cGAS and His-STING were incubated separately or together with cGAMP agarose. The input and immunoprecipitates were immunoblotted.
(B) Mitochondrial morphology quantification. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). *P0.05 versus Scr group.
(f) Effects of C18PC on caspase-dependent or caspase-independent cell death were determined in MEFs isolated from Bax−/− Bak−/− or Casp3−/− Casp7−/− mice and from wild-type (Bax+/+ Bak+/+) or Casp3+/− Casp7+/− mice, used as controls. Data shown are an average of at least three experiments ± s.d. (*P 0.05).
The R23A/R109A HMGB1 double mutant has chemotactic activity but is not inhibited by DFL (the experiment was carried out as described in panel B). The data points represent three biological replicates in one experiment (of 2 performed in different days); the avg ± sd is shown.
The three PCA analyses are based on the 43 genes selected in Appendix Figure S2 and highlight the transcriptional differences between wt (in blue), ko (in red) and ko CX546 treated (light and dark green for early and late treatment respectively) samples.
F) Scatterplot comparing inter-REC8 distances along on X-chromosomes with the distances between flanking REC8 foci at sites of LSAEs in the Stag3 mutant. 211 inter-REC8 distances measured along X-chromosomes and 10 distances measured between flanking REC8 foci in Stag3 mutant. Grey area represents 15% of the chromosome axis length. Horizontal lines indicate the median. p < 0.0001, obtained with two-tailed Mann-Whitney test.
(D) Quantification of the absolute number of B1, C and A cells (identified as in B) in the SVZ of 4D- (white) and 4D+ (black) mice regardless of RFP expression (RFP+/-).
(D) Representative images of insulin granule fusion events (yellow circles) in mice of indicated genotypes in response to administration of 15mM glucose (scale bar = 10μm). Quantification of the proportion of responding cells and the density of granule fusion events are presented in graphs in the lower panel (mean±SEM, 8-10 islets/mouse, >4 mice/genotype, **p=<0.01, ***p=<0.001 of cre relative to tm1d, Students t-test).
A Representative western blot analysis of recognized autophagy signaling molecules (pJNK, JNK, pAKT, and AKT, pmTOR, and mTOR) in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis of autophagy upstream signaling molecules on HC (#1-5) and DM (#1-18) individuals. (pAKT/AKT; p=0.0747, pJNK/JNK; *p=0.001, pmTOR/mTOR; p=0.3356 vs. HC). GAPDH was used as the loading control.
D, E Quantification of total PCNA‐positive cells per crypt revealed a significant increase when Snai1 levels were elevated (n = 4, P = 0.009). This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of (E) PCNA‐positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue (n = 4, P = 0.0005). Bars represent mean ± SD.
(A) ATZ glycosylation mutants and their electrophoretic mobility. N is the acceptor site asparagine; Q is for the mutation to glutamine that prevents glycosylation. The E342K mutation of ATZNNN is in blue.
E, Knockdown of PTPN3 abolishes TGF-β-induced p21 transcription. HaCaT cells were transfected with siRNA and then treated with TGF-β (2 ng/mL) for 0, 4, 8 or 24 h as indicated. qRT-PCR was carried out Data are shown as mean ± SEM; n = 3.
At day 13, tumor infiltrated immune cells were isolated and stained for CD206 and CD11b.
Venn diagram of LPS- and DIM-upregulated genes (in basal condition) in ileum, as identified by RNA-seq (n=3).
C U937 cells were co-transfected with the IFNβ luciferase reporter plasmid and the PB1-F2 or NS1 expressing plasmid and treated with polyI:C for 12 h before harvesting. IFNβ promoter activity was determined by luciferase reporter assay. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).
A. Genomic views of Cpt1a mRNA and genome binding of Tfcp2l1 in WT and Tfcp2l1_KO 2i-ESCs.
Overview of TiLO using a subregion of HUVEC chr5 as an example. TADs along each chromosome are treated as nodes in an interaction network and inter-TAD Hi-C signal is used to infer network connections. Inferred connections are then sliced and network robustness is assessed iteratively to obtain the final clustering. TAD clusters are visualized in the space of the nucleus using Chrom3D.
A-B Surface representations of the macrodomain structures for the human macroH2A isoforms macroH2A1.1 (PDB ID 1ZR3) and macroH2A2 (PDB ID 6FY5), respectively, with electrostatic potential included. A yellow-framed box indicates the location of the major surface pocket accommodating ADP ribose in macroH2A1.1.