EMBO/sd-character-level-ner huggingface dataset:
A. The RT-PCR product generated from cells treated with oligo#5 was directly sequenced using Sanger sequencing method. The fluorescent peaks after capillary electrophoresis are shown. Letters in gray boxes indicate the editing sites (also shown in Figure 1C).
(A) Representative GFP-labeled blastomeres that contributed to the chimeric porcine RPE cells are shown with immunofluorescence staining. Scale bars, 25 μm
K: Relative mtDNA copy number analyzed by RT-qPCR for regions of ND1 and nuclear gene APP in all iPSC-derived DA neurons (n=3, technical replicates per clone).
Relative tumor volume (RTV) of Panc1-derived tumors in NMRI nu/nu mice. Eleven days after implantation, mice were randomized according to tumor volume. Treatment with mocetinostat (60 or 90 mg/kg/day) and gemcitabine (25 mg/kg/day) was implemented (day 0) as depicted in the scheme. Shown are the group medians of the RTVs over time (left) and the individual RTVs on day 32 (right). n = 5 for each treatment group; nonparametric Mann-Whitney U-test.
F) Immunoblot showing the levels of phosphorylated ATM (Ser1987) and ATM in the Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment (10µM CPT for 2 hours). Bar graph (lower panel) represents average band density of P-ATM in Mybl2Δ/Δ ESCs treated with CPT relative to ATM and relative to wild type CPT treated cells from three independent repeats. Error bars represent SD. Statistical analysis was carried out using a two-tailed unpaired t-test.
RKO, but not HEK293A cells, expressed both full-length TAZ and cTAZ transcripts. RT PCR primers targeting different regions
A Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was analyzed by SDS-PAGE and silver staining. The input sample and mock precipitation using total mouse IgG are also shown. The gel slices indicated by boxes were excised and subjected to LC-MS/MS analysis.
F Heat map of same cells as (D) showing expression of the top 20 marker genes in each cluster. Color bars at the top of the plot show cluster membership (colors as in (D)) and pre- or post-menopausal status (blue and yellow, respectively).
D. DNA sequence confirming the mutation from K64K65 to AA in PbkKI/KI mice from the tail genomic DNA compared with PbkWT/WT mice.
(C) Chs5-GFP displays reduced membrane association in the absence of ChAPs. Differential centrifugation of cell lysates obtained from Chs5 GFP WT and ΔΔΔΔ strains. TCL, total cell lysate; S10, 10,000 g supernatant; P10, 10,000 g pellet; S100, 100,000 g supernatant; P100, 100,000 g pellet. Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker. A representative immunoblot of three independent biological experiments is shown.
B) TDP-43 condensates without RNA control or in the presence of A(GU)6 (8 μM), A(GU)18 (2.6 μM), or A(CA)6 (8 μM) (Supplementary Table 1) at 150 mM NaCl (4 μM TDP-43).
(a) Association of MOFA factors to time to next treatment using a univariate Cox models. Error bars denote 95% confidence intervals. Numbers on the right denote p-values for each predictor
Oxygen consumption rate (OCR) (H) were measured in BAT from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucpcre+ (Cre+) mice kept at 30℃ or at 4℃ for 72 h.
D. 293T cells were transiently co-transfected with empty vector and NDUFA4-GFP, or OMA1-E324Q-Flag and NDUFA4-GFP. Cell lysates were used for coimmunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with antibodies against Flag or GFP (n = 3 independent experiments).
(F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.
(D) Constructs for expressing full-length HA-SPOP or HA-SPOP mutants capable of oligomerization through only one (or neither) of the self-association domains were transfected into NIH 3T3 cells. DAPI was used to stain the nucleus and SPOP localization was identified using an anti-HA antibody. Experiments were performed at least twice on four biological samples. Multiple cells were examined and representative cells are shown. For additional images see Appendix Fig S3. Scale bar represents 5 µm.
B. ENZA acutely decreases AR levels (up to 18 h) while SDHB silencing rescued the early ENZA-mediated AR downregulation in LNCaP cells.
(G,H) Representative immunofluorescence images (G) and quantification (H) of PML NBs (green, maximal projection) and SUMO 2/3 (red, maximal projection) during mock and productive HIV-1 infection of primary CD4+ T-cells at 7dpi. Scale bar 10μm.
A, B Effects of Noc overproduction on cell division and nucleoid morphology in E. coli. Cells of strain DWA261 carrying pDWA37 (PA1/04/03‐noc) were examined after growth in LB with either no additions (A) or after induction for 1 h with 1 mM IPTG (B).
K - Schematic depicting the experimental strategy for transcriptomic profiling of ChAT-eGFP+ and ChAT-eGFP- MΦ from IWAT of mice housed at 4oC for 4 h.
(G-H) Stable expression of CSAG2 in HCT116 (G) and CSAG2-negative H460 (H) cells increases anchorage-independent growth in soft agar colony formation assays, n=3 biological replicates. Data are represented as the mean ± SD.
Representative images (F) and cfu quantification (G) of the re-infection assays with Salmonella ΔfliC, ΔfliC/pFliC or ΔflhC mutant strains in HeLa cells primarily infected with Shigella WT or mock-treated
(j-k) Scattered labeling of Atoh1+ cells in control conditions, while DSS injury lead to Atoh1 lineage labeled crypts lacking Lgr5+ stem cells. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm Data are represented as mean ± SD analyzed using Student's t-test with Welch's correction *P≤0.05, ***P≤0.001.
Visual representation of (D) iWAT and (E) pWAT sizes in sham, sham starved and CLP mice 24h post-surgery. Data are representative of 3 independent experiments.
(F) MT intensity at MTOCs at the time of GVBD quantified from SiR-tubulin signal that was normalized to mean cytoplasmic SiR-tubulin signal. Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group (means ± SDs, Mann-Whitney-test). Total of 184 MTOC in 17 control oocytes and a total of 170 MTOCs in 17 IPZ-treated oocytes were analyzed. (G) Time course of the MT intensity in the area of spindle after GVBD. Mean of MT intensity was arbitrarily centered to 1 in the control group (means ± SDs). Oocyte numbers are indicated in brackets.
(D) Amino acid sequence alignments of the STI-II regions of UBQLN1 and UBQLN4. UBQLN4-specific residues are shown as red characters. HS: Homo sapiens (Mammalia), MM: Mus musculus (Mammalia), GG: Gallus gallus (Aves), PS: Pelodiscus sinensis (Reptilia), and XL: Xenopus laevis (Amphibia).
D ChIP-qPCR analysis of the relative binding of CDF4 to the promoter of CAT2. An anti-HA monoclonal antibody was used for DNA immunoprecipitation from 32-d-old pER8::CDF4-HA transgenic plants. Black columns indicate the enrichment fold-changes normalized to that of ACT2. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.
(A) Overlay of the proton NMR spectrum fragments of the following suspensions (from top to down): untreated first (lysosomal) cell fraction after ultracentrifgation and one washing (F1*), iodixanole (Optiprep) suspension, lower (F3), middle (F2) and upper fraction (F1) of the ultracentrifugates after two washings. The chemical shifts of the spectra were calibrated to the Choline/Phosphatidylcholine (Cho/PC) signal. Apart from iodixanol, we could identify the PUFA groups (methylene groups situated between two CH = CH groups), Cho/PC and glycerophosphocholine (GPC) in F1* and F1-3. Furthermore we observed lactate and the methylene groups of fatty acids (at 1.3 ppm), citrate (at 2.55 and 2.7 ppm) and phosphoethanolamine (at 3.1 ppm) in the F1* but not in the F1-F3 groups..
Representative immunohistochemistry staining for (a) ubiquitinated protein (brown); (b) p62 (brown) and (c) GFP-LC3 (brown) in paraffin sections of heart revealing increased accumulation of p62, ubiquitinated protein and LC3 in Hace1−/− sTAChearts. Hearts from three mice in each group were analysed (scale bars, 10 μm).
C, D Levels of p-Lyn, Lyn, E-cadherin and SNX10 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24h were determined by immunoblots (C) and quantified by ImageJ software (D) (n = 3 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.
D. GFP-positive colonies of mouse iPSCs generated by Oct4 mutants in combination with Sox2, Klf4, and c-Myc. Colonies were imaged 16 days after second viral infection, using a fluorescence microscope. Scale bars: 250 μm; objective 10x.
B The mRNA levels of CK14, Trp63, Sox2 and Mash1 in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; CK14: p=0.2135, Trp63: p=0.1930, Sox2: p=0.5190, *p<0.05; Student's t-test).
B. Models of intertwined loop "spike" interacting with the cellular membrane. The disordered "spike" regions are indicated by dash lines in DENV2 and WNV NS1 structures, which consist of hydrophobic or positively charged amino acids.
SB stability in dark state rhodopsin: I) WT, J) T94I and K) G90D rhodopsin. L) OD440 is plotted as a function of time and fitted to a single exponential decay. All decay rates were measured at 55 C.
Cytoplasmic localization of WT LIN28A and LIN28A(R192G) proteins. HeLa cells transfected with pFLAG-WT-LIN28 and pFLAG-LIN28A(R192G) were stained with FLAG antibody and counterstained with DAPI.
(C) Representative Western blots of LAMP1, EZRIN, EZRIN-pT567, CLN5, TRPML1, SQSTM1/p62, CTSD and LC3 proteins from DMSO or NSC668394 treated cells. Note an increase of both proCTSD and its maturation CTSD heavy chain (hc). The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control. Bar graphs represent mean values ± s.e.m. of at least n = 6 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05, ***p ≤ 0.005.
(Top) Number of K-Orange expressing cells observed in chimaeric embryos obtained using control ES cells, Mta123∆ ES cells, or Mta123∆ ES cells in which Mta2 was reintroduced on a constitutively expressed transgene (Mta123∆+Mta2TG). P-values calculated using a two-tailed t-test. (Middle) Mean number of K-Orange cells per embryo separated by Sox2 expression. P-values calculated using a two-tailed t-test. (Bottom) Number of K-Orange and Caspase-3 positive cells per embryo. P-values calculated using a one-tailed t-test: *P < 0.05, ****P < 0.0001, "ns" = not significant.
A Western blot analysis of B16F1 WT and two independent XIAPKO cell lines. Actin was used as a loading control.
A) GC cells (SGC7901 and MGC803) were transfected with (A) shUHMK1-#1,-#2, -#3, or control shRNA lentiviral vector, or (B) with a shRNA-resistant expression construct, UHMK1Δ. Western blotting was used to measure the levels of UHMK1. NIH ImageJ software was used to quantify the band intensity. Western blotting assay were conducted for three replicates. SGC7901 cells were transfected with or without shUHMK1-#1 or the shRNA-resistant expression construct UHMK1Δ. CCK-8 assays were conducted. Scale bars=5mm. Data information: **P< 0.01, ***P< 0.001, # marked no significance.
F. The endogenous MERVL was up-regulated significantly in ICSI embryos compared with SCNT embryos at 2-cell stages, as determined by RT-qPCR. Error bars, s.e.m., n ≥ 3
D 2D [1H,15N] HSQC spectra of EDC3104-197 with (red; 1:2 molar ratio) and without (blue) Pim1. We titrated Pim1 into 15N-labeled EDC3, and the 2D [1H,15N] HSQC spectrum of EDC3 (residues 104-197) when bound to Pim1 was measured. Direct comparison with the unbound 2D [1H,15N] HSQC spectrum of EDC3 showed that while the majority of EDC3 peaks are unchanged, ~30 peaks become either broadened beyond detectability demonstrating they directly bind to Pim1 or show small chemical shift perturbations. Upon completion of the sequence-specific backbone assignment of the EDC3 IDR, we identified that EDC3 residues 137 to 167 are necessary for the interaction with Pim1.
(e) Illustration of experimental protocol outlining DT treatment of Atoh1CreERT2;ROSA26tdTomato;Lgr5DTR-GFP mice.
F In heavily infected cells, addition of Shld-1 at 32 hours post-inoculation for 4 hours leads to premature egress of DDmyc-PKArG321E-Ty parasites but not of the DDmyc-PKAr-Ty control line as assessed by IFA.Data information: In C and E-H, data are presented as mean ± SEM. Scale bars = 5 µm (F)
(B) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with FAPs isolated from mdx mice treated either with vehicle (+FAPs CTR) or TSA (0.6 mg/kg/day for 15 days by i.p.) (+FAPs TSA), and transfected with scramble (Scr siRNA) or Drosha siRNA (Drosha siRNA) prior to co-culture with MuSCs. Scale bar = 50 μm.
(C) ts85 cells were treated as in (B) and cell extracts applied to size exclusion chromatography. Ferritin content in selected fractions was measured by ELISA. Black bars represent assembled ferritin (>400 kDa), gray bars represent monomeric (50 kDa).
Subcellular localization analysis of CALIC. RNAs were isolated from the nuclear and cytoplasmic fractions of HCT116 cells and quantified by qRT-PCR (n = 3). Nuclear controls: U6, MALAT1; Cytoplasmic control: GAPDH.
(A) Schematic of the synthetic lethal screen used in this study. EMS treated vac17∆ cells which have a plasmid expressing yEmRFP and VAC17, exhibit a red color colony due to over-expression of the yEmRFP protein. Colonies that retain the red color are likely those that cannot lose the plasmid on non-selective medium, and therefore indicate candidate mutations which are synthetically lethal with the vac17∆ mutant.
H1-T6SS dynamics in tagged parental strain (∆retS TssB1-mCherry2, referred to as WT H1) and ΔtssA1PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. Scale bars: 2 µm.
F Representative images of MuERV-L::tdTomato relative to GFP signal in the same embryo when WT embryos reached the corresponding stages. Zygotes were injected with the MuERV-L::tdTomato reporter plasmid and polyadenylated Gfp mRNA (as a positive control of microinjection), then allowed to develop in vitro. Cultured embryos were imaged at 24 h and 40 h after hCG injection. DIC, differential interference contrast. Scale bar = 100 μm.
E. Ear skin sections from 10 week-old mice stained with anti-CD68 (red) or anti-osteopontin antibody (green); counterstained with Hoechst (blue). Osteopontin-positive macrophages in wt/Act and HPV8/Act mice are indicated with arrows. Representative of N=2 wt/wt and N=3 HPV8/wt, wt/Act and HPV8/Act mice. Boxed areas in top panel are shown at higher magnification in bottom panel. Scale bar top: 100μm, bottom: 20μm.
The assembly of V-ATPase V1-V0 holoenzymes in WT RPE1 cells over-expressing the α-tubulin deacetylase HDAC6 or EGFP as a control. Representative immunoblots for ATP6V0D, ATP6V1A, ATP6V1C1, LAMP1 and GAPDH are shown in (H).
(B) Levels of COUP-TFII, LDHA, and phosphorylated S6K (T389) in COUP-TFII-silenced HCT116 and DLD-1 cells.
(d-m) Immunofluorescence of the SVZ (i-m) of P28mice (d,e,i,j). Boxed areas are shown in more detail in insets (e,j) and/or panels below (i,j). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (d,e), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. Mean ± s.e.m. of the number of GFAP+nestin+ and GFAP+SOX2+ radial glia (f,h), and NSCs (k,m), and GFAP+nestin− astrocytes (g,l) per section are shown. Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001
Pa's population size at the onset of its diauxic shift correlates to the supply concentration of whichever resource is supplied in a more limiting amount, indicating coutilization. Data Information: Data were collected at 600nm.
(C) Quantification of fibronectin intensity density o FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by PSCs
(F) 293T cells were co‐transfected with expression vectors for Beclin‐1-MYC, BI‐1-HA, and BCL‐XL-FLAG. Cell extracts were prepared in CHAPS buffer and Beclin‐1-MYC immunoprecipitated, and the possible co‐precipitation of BI‐1-HA, and BCL‐XL-FLAG was assessed by western blot analysis (N=3). Asterisks indicate BI‐1 oligomers.
B. Amino acid conservation of Mis18 proteins among eukaryotes. The alignment includes orthologues from S. pombe (sp), G. gallus (gg), M. musculus (mm), H. sapiens (hs) and B. Taurus (bt). Predicted (light orange; using PsiPred, http://bioinf.cs.ucl.ac.uk/psipred) and observed (bright orange; from the crystal structure shown in Fig 1D) secondary structure elements are shown below the aligned sequences. Amino acid residues mutated in this study are highlighted with circles (dimer interface residues) and asterisks (putative substrate-binding pocket residues).
Percentage of cells showing γH2AX foci in carcinomas of the indicated genotypes (left panel). Representative images of γH2AX immunohistochemistry (right panel).
IF staining of γH2AX in the frontal cortices and quantification of the immunoreactive cells from 8-mon-old of WT+Vehicle, Tg+Vehicle, Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm. * p = 0.0421 or **** p < 0.0001 by multiple comparison. Data information: number of cells in each view is presented.
(F) The startle responses were significantly lower in Cox6a2-/- mice when the loud stimulus (120 dB) was presented alone the first 5 times. Two-way ANOVA followed by Tukey's multiple comparisons tests.
(I) IHC of p65 on skin sections from HS and rosacea. Scale bar, 50 μm. (J) Percentage of nuclear p65 positive cells in the epidermis from HS (n = 8) and rosacea (n = 13). All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test (F and G) or two-tailed unpaired Student's t-test (J) was used.
A. LRP6 level is reduced during serum starvation. HEK293 cells were incubated in serum-free medium for the indicated hours. Cells were lysed and the cell lysates were analyzed by immunoblotting.
(D) HT29 cells were infected with Shigella WT or ∆ospC1 mutant and incubated for 8 h. Infected cells were then fixed and stained with cleaved caspase-8 (green), rhodamine-phalloidin (red), and DAPI (blue). Percentages of positive cells are shown in the graph at right (*P<0.05; unpaired two-tailed Student's t-test). Scale bar: 100 μm.
A 3D-PCR amplicon was digested in vitro with recombinant ISG20 and increasing concentration of manganese chloride.
B) Graphs showing RNA levels for the representative genes shown in (A) measured by qPCR.
(C) Graph represents the mean percentage of males born at 25°C either expressing eyGal4-driven UASPoloT182D together with UASlacZ or in combination with UASRodRNAi (n≥3 independent crosses for each condition). The number of males with the genotype of interest born in each cross was normalized to the mean total number of males that are born in a control cross (eyGal4>UASlacZ, n=9 independent crosses). Data information: Data are shown as mean ± SD. Scale bar: 5 μm.
E. Overlay of the N-terminal tail of CENP-A (red) and H3 (green; PDB 6ESF), illustrating shorter αN helix of CENP-A (obstructed by the presence of bulky CENP-AW47) and DNA moved by 4 Å. F. The N-terminal tail of CENP-A (red) makes contacts with the DNA (cyan) at the entry/exit sites.
C) HR modeled Pressure field (positive pressure - red, negative pressure - blue). D) HR modeled Power density (positive power / active contribution - red, negative power / resisting tissue - blue). Data information: Scale bar = 40 µm.
E- G. Recombinant protein interaction analysis using the indicated epitope tags and purification methods. Domain architecture of bait and prey are indicated.
[B] (Adapted from Hiruma et al., 2016) Pi-deficient plants allow symbiosis with certain fungi because endophytic fungi can transfer phosphate to plants, whereas Pi-sufficient plants need no fungi-assisted Pi uptake and so they deploys Trp-derived secondary metabolites to defend against fungi invasion.
Figure 6. Experimental control of microbial swarmbot safeguard by other circuits.Demonstration of modular safeguard performance of swarmbots with different collective survival systems. For the swarmbots containing cells carrying the QS-CAT circuit, pulsing flow of medium containing 100µg/ml chloramphenicol established safeguard control but not for the static condition. For the swarmbots with the QS-BlaM circuit, the static condition of 100 µg/ml carbenicillin was sufficient for safeguard control. For all the conditions, safeguard did not occur in the absence of antibiotic (top panels from each condition). All the images were from the swarmbots cultured at 37˚C for 16 hours. The scale bar is 250 µm.
(B) Schematic representation of the GltPh transport cycle showing one protomer. Comparatively rapid and slow steps of the cycle are shaded pink and blue, respectively.
(B) Representative images of acceptor neurons that were either (i) co-cultured with donor neurons (upper panel, Co-culture), (ii) physically separated from donor cells (middle panel, No contact) or (iii) cultured with the conditioned medium of donor neurons (bottom panel, CM). In red: α-synucleinfibrils, in white: acceptor neurons and in blue: nuclei. Scale bar represents 10 μm.
Curvature time-traces (smoothed running averages over 11 ns widows) from individual replicates (n=20) quantify the bicelle shape transformation process during simulations. Black lines (mean value) and shaded region (s.e.m) denote the average behaviour of the system. From the 20 replicates, data on waiting times (t) were collected to compute rates and acceleration factors (Table EV3). Vesicle formation is marked by bilayer-disc curvature, |H| = 0.14 nm−1.
B,C Quantified fluorescence of UbG76V-GFP normalized to (C) mCherry in the intestine from L4+48 hour animals exposed to feeding RNAi for either an empty vector control or the indicated xenobiotic stress gene. ****P<0.0001, ***P<0.001, **P<0.01, ANOVA, Tukey's multiple comparison test compared to control. N=20 animals per genotype and timepoint. Error bars indicate SEM.
B, Multiple sequence alignment of the AGO1 putative TR region across vertebrates.
(F) Centrosomal distance over time in RPE1 cells stably expressing CETN1-GFP and treated with DHCB. Time zero corresponds to the frame preceding anaphase onset. Coloured dots (lower panel) summarize the clustering time for each cell, mean ± standard deviation in black. Data calculated from four-dimensional imaging as in (E). N = 10 cells.
(R) Wound closure in FSP1-TK and WT littermate mice. N=10 biological replicates per group, data are from two independent experiments. Two-way ANOVA performed comparing WT to TK mice.
(A) Determining the number and density of CD22, CD19, IgM and IgD on the surface of wild-type primary B cells. Cells were stained with PE-conjugated antibody against CD22 (blue profile), CD19 (red profile), IgM (red profile) or IgD (red profile) and their fluorescence profiles compared to those of standard beads (grey profiles), which each possess a well-defined number of binding sites (st0=blank, st1-st4=increasing number of binding sites) against the primary antibody. For IgM, where the range of expression is large, the low and high 25 percentiles were calculated as in Mattila et al. 2013 and shown in brackets.Data are representative of three independent experiments.
(D) Boxplot/Jitterplot representing the percentage of GC nucleotide contents in the peaks from DPPA2, DPPA4 and DUX ChIP, and in a random shuffle of peaks.