EMBO/sd-character-level-ner huggingface dataset:
Figure 1(a) Lipid phosphatase activity of Paladin, wild-type and phosphatase-dead C/S mutant, towards PI(4,5) P2 and PI(3,4,5) P3 substrates. Positive control; wild-type phosphatase and tensin homolog (PTEN); negative control; lipid phosphatase-dead C124S PTEN. Mean±SEM, Paired t-test, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (b) Representative confocal image of HDMEC stained for Paladin (green), Vascular Endothelial Cadherin (VEC, red) and nuclei (DAPI, blue), scale bar: 10 μm. White boxes in the upper image are showed at higher magnification below, scale bar: 3 µm. (c) HDMEC stained for VEGFR2 (green), Paladin (red) and nuclei (DAPI, blue) and with or without stimulation of 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm. (d) Quantification of VEGFR2-Paladin co-staining before and after VEGF-A stimulation as shown in (C). Mean±SEM, One-way ANOVA. n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (e) HDMEC analysed using Proximity Ligand Assay (PLA) for Paladin and VEGFR2. Green dots indicate complex formation, VE-cadherin (red) and nuclei (DAPI, blue). Cells stimulated with 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm. (f) Quantification of (e), the number of PLA Paldin-VEGFR2 complexes per cell at 0, 2 and 10 minutes after VEGF-A stimulation (top). Quantification of (e), average distance for the PLA complexes to the nearest VE-cadherin positive junction at 0, 2 and 10 minutes after VEGF-A stimulation (bottom). Mean±SEM, One-way ANOVA. n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (g) HDMEC stained for Paladin (green) and EEA1 (red), colocalization in yellow, and nuclei (DAPI, blue) after VEGF-A stimulation for 0, 2 or 10 minutes, a representative image from a single confocal plane is shown, scale bar: 10 µm. White boxes in the upper image are showed at higher magnification below, scale bar 3 µm. (h) Quantification of (g), EEA1/Paladin double positive particles per field of view at 0, 2 and 10 minutes after VEGF-A stimulation. Mean±SEM, One-way ANOVA. n= 3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 1(A) Akt is deubiquitinated during fasting. Left: Soluble fractions of TA muscles from fed and fasted mice were analyzed by SDS-PAGE and immunoblot using Akt antibody. Right: densitometric measurement of presented blots (n=3). * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM. (B) Akt was immunoprecipitated from the soluble fraction of muscles from fed or fasted (2d) mice. Percipitated were analyzed by immunoblotting using an anti-ubiquitin conjugates. Mouse IgG was used as a control for non-specific binding. (C) Scheme for DUBs screening experiment to identify the enzyme that directly deubiquitinates Akt in vitro. )D( Ubiquitinated Akt is a substrate for USP1 in vitro. Top: Akt was immunoprecipitated from the soluble fraction of TA muscle from fed mice, and was subjected to an in vitro deubiquitination by a panel of purified DUBs arrayed in a multi-well plate. The dividing line indicates the removal of an intervening lane for presentation purposes. Bottom: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each well. (E) shRNA-mediated knockdown of USP1 in Hela cells. Soluble extracts were analyzed by immunoblotting. (F) USP1 deubiquitinates Akt during fasting in vivo. Left: Soluble fractions of TA muscles transfected with shLacz control or shUSP1 from fed and fasted mice were analyzed by SDS-PAGE and immunoblotting using anti-Akt. The actin blot serves as a loading control. Right: densitometric measurements of presented blots (n=3). Data is presented as ratio of ubiquitinated Akt to total Akt normalized to actin. * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM. (G) USP1 removes K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing USP1(C90S) or control plasmids. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates. (H) USP1 removes polyubiquitin chains linked to K8 on Akt. Akt was immunoprecipitated from the soluble fraction of TA muscles transfected with USP1(C90S) , HA-Akt(K8R) or control from fasted mice, and protein precipitates were analyzed by immunoblotting with anti-Akt antibody. Mouse IgG was used as a control for non-specific binding. Right: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane (n=2).
a) HeLa cells were transiently transfected with pcDNA (vector), Ptch1 or Ptch2 constructs for 48 h.f2(a) HeLa cells were transiently transfected with pcDNA (vector), Ptch1 or Ptch2 constructs for 48 h. Where indicated, cells were treated with bafA1 for the last 4 h. LC3-II levels were detected by western blotting and levels were quantified by densitometric analysis relative to tubulin and shown in graph. (b) HeLa were transfected with Ptch1 or Ptch2 together with HA-HttQ74 and the percentage of transfected cells with aggregates was assessed by HA immunofluorescence. P-values were calculated by odds ratio. (c) Atg5+/+ or Atg5−/− MEFs were transfected with GFP-HttQ74 for 48 h, cells were fixed and the percentage of transfected cells with aggregates was scored by direct fluorescence. (d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with 100 mM trehalose in the last 24 h (d) Quantification and statistical analysis is shown in graphs. (d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with400 nM bafA1 for 4 h (e). Quantification and statistical analysis is shown in graphs (f) siRNA-targeting Ptch2 was transfected into HeLa cells stably expressing mRFP-GFP-LC3 and representative confocal microscopy images are shown. Scale bars represent 26 μm. Percentage of GFP-positive vesicles (autophagosomes), RFP-positive vesicles (total number of vesicles) or percentage of autolysosomes (calculated by subtracting numbers of GFP from RFP vesicles), quantified using a Cellomics array scan, was calculated relative to control siRNA-transfected cells. Although an increase in autophagosome biogenesis generally increases both the number of GFP+ vesicles and the number of GFP-/RFP+ vesicles as a result of an enhanced autophagy flux, defects in autophagosome degradation, such as in bafA1-treated cells, increase GFP+ vesicles but not GFP−/RFP+ structures, as GFP remains intact. Graph shows the mean value obtained from four independent experiments in triplicate and with control conditions set to 100. In all panels, unless indicated, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test: **P0.01; *P0.05; NS, not significant.
The two CoV2-miR-O7a isoforms are produced in COVID-19 patients.Figure 7(A) Expression levels of CoV2-miR-O7a.1, CoV2-miR-O7a.2 and a 22 nt region from the ORF6 of the viral genome that does not produce detectable levels of small RNAs (Control ORF6) analyzed by stem-loop RT-qPCR from nasopharyngeal swabs of patients tested positive for COVID-19 or another seasonal human coronavirus (HCoV). Relative expression to hsa-miR-let-7a is shown. Ct values in parenthesis refer to the Ct value for the detection of viral genome in patient swab samples. (B) SARS-CoV-2 genomic view showing the distribution of normalized 22nt small RNA reads from nasopharyngeal swabs of three patients tested positive for COVID-19. The most abundant small RNAs are marked by the red boxes and correspond to the CoV2-miR-O7a. (C) Model for the production and function of Cov2-miR-O7a in COVID-19 patients.
Figure 2A. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG) or for 30 min with DHPG and pretreated with Xestospungin C and S107 (XeC/S107 + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 or 20 cells each, p < 0.0001, Student's t test. B. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG), or for 30 min with DHPG and pretreated with S107 (S107 + DHPG), or for 30 min with DHPG and pretreated with Xestospungin C (XeC + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, ANOVA. C. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under control or 10 min Thapsigargin (TG) treatment. Bottom. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. D. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with treatment with either no DHPG or with DHPG for 30 min, after 15 min of Thapsigargin. Bottom. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, Student's t test. E. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min, after 5 min of BAPTA. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, p < 0.001, Student's t test. F. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min after 5 min of BAPTA-AM. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. All error bars represent s.e.m.
Tamoxifen decreases hypoxia and increases vascularisation: (A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control of tamoxifen, scale bar 100 μm.Figure 1(A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control of tamoxifen, scale bar 100 μm (B Quantification of GLUT1 (hypoxia marker Control (n=5), 2mg (n=5), and 5mg (n=4). In all cases, bars represent mean ± s.e.m (C) Relative values of protein levels for Glut1 in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates) ,D) Quantification o CD31 (endothelial cell marker). Control (n=5), 2mg (n=5), and 5mg (n=4). In all cases, bars represent mean ± s.e.m. (C (E,F) Expression levels of DEGs relevant to response to hypoxia (left) and blood vessel morphogenesis (right). The values were normalized by tubulin family genes (G) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control and 5mg of tamoxifen, scale bar 100 μm (H) Quantification of HIF-1A in PDAC tissues Control (n=5), and 5mg (n=4). In the box-and-whisker plot, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value (I) qPCR levels of HIF-1A in PSCs, normalized to RPLP0 and relative to control (J) Western blot bands for protein expression in PSCs (p-Tmod is post-translational modification). The plot shows the quantification of the sum of band intensities corresponding to isoform 1, isoform 2, isoform 3, and post-transcriptionally modified HIF-1A (n=8 control and n=8 tam)
KCC4 protein levels in the kidneys and metabolic acidosis status of SIRT7-deficient mice and renal tubule-specific SIRT7-deficient mice.Figure 5A KCC4 protein levels were determined in the kidneys of wild-type (SIRT7+/+) and SIRT7-deficient mice (SIRT7-/-). SIRT7 protein expression are absent in the kidneys of SIRT7-deficient mice. B KCC4 protein levels, acetylation status of KCC4, and interaction with SIRT7 in kidneys of SIRT7+/+ and SIRT7-/-mice under normal conditions (Control) or with metabolic acidosis. C Immunolocalization of KCC4 and H-ATPase in the kidneys of SIRT7+/+ and SIRT7-/- mice under normal conditions (Control) or with metabolic acidosis (NH4Cl). (KCC4, red; H-ATPase, green). Scale bar: 20 μm. Cell count was performed automatically using the Gen5 software to report the percentage of KCC4+ cells among H-ATPase+ cells (lower panel). D NCC and NKCC2 protein levels in the kidney of SIRT7+/+ and SIRT7-/- mice under basal conditions. E Scheme illustrating the proposed mechanism by which SIRT7 regulates KCC4 in the kidney. Briefly, KCC4 maintains a low intracellular Cl- concentration to ensure that basolateral Cl-/HCO3- exchange remains active to allow extrusion of HCO3- from the cell, specifically in α-intercalated cells of the collecting duct, and therefore warranting a constant production of H+ that are secreted through the apical membrane to be eliminated in the urine. During stress, such as metabolic acidosis, SIRT7 levels increase in the cytoplasm, promoting the interaction and stability of KCC4. This interaction induces an increase in KCC4 protein levels and activity and therefore contributes to the increase of basolateral Cl- efflux, which decreases the intracellular Cl- concentration, thus maintaining the gradient for the Cl-/HCO3- exchanger to eliminate bicarbonate from the cells and increase the secretion of H+ to reestablish acid-base homeostasis.
Coq9Q95X mice exhibited higher CoQ levels compared with Coq9R239X mice.Figure 3A-F Residual CoQ9 levels in tissue homogenates from brain (A), cerebellum (B), heart (C), kidney (D), liver (E) and skeletal muscle (F) of Coq9+/+, Coq9Q95X and Coq9R239X mice at 1, 3 and 5 months of age. Data are expressed as mean ± SD. **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; ##P < 0.01; ###P < 0.001; Coq9Q95X versus Coq9R239Xmice (one-way ANOVA with a Tukey's post hoc test; n = 8 for each group; numbers above columns indicate P-values of the one-way ANOVA test).
SCOC interacts with UVRAG and the interaction is reduced in starvation.Figure 7(A) SCOC interacts with UVRAG. HEK293 cells were transfected with FLAG-SCOC and Myc-UVRAG, lysed and incubated with anti‐FLAG (left) or anti‐Myc (right) antibodies. Co‐immunoprecipitation of Myc-UVRAG (left) and FLAG-SCOC (right) was confirmed using anti‐Myc and anti‐FLAG antibodies for western blotting. Asterisk in (A-C) and (D) indicates non‐specific bands. (B) Interaction of SCOC with UVRAG is unaffected by FEZ1-GFP. As in (A), co‐immunoprecipitation was preformed in HEK293 cells transfected with FLAG-SCOC, Myc-UVRAG and FEZ1-GFP. Myc-UVRAG was detected as in (A), FEZ1-GFP was detected with anti‐GFP antibody. (C) The SCOC and UVRAG interaction is reduced by starvation, and this reduction is inhibited by FEZ1-GFP. HEK293 cells were transfected as in (B) and 24 h later either treated with FM or ES for 2 h. FLAG-SCOC or Myc-UVRAG was immunoprecipitated with anti‐FLAG or anti‐Myc antibodies and the bound proteins were detected with anti‐Myc, ‐GFP or ‐FLAG antibodies by western blotting. Quantification of the amount of FLAG-SCOC co‐immunoprecipitated with Myc-UVRAG in FM or ES, without overexpressed FEZ1-GFP (left) or with overexpressed FEZ1-GFP (right). Significance was determined using a two‐tailed paired t‐test (n=3). (D) FEZ1 interacts directly with endogenous UVRAG and is required for SCOC interaction. HEK293 cells were transfected with FEZ1-GFP, GFP or FLAG-SCOC alone or in combination. Top panel input, bottom panel immunoprecipitation with anti‐GFP or anti‐FLAG. Western blot for endogenous UVRAG, GFP-FEZ1 GFP or FLAG-SCOC as indicated. Arrowhead: endogenous UVRAG. (E) UVRAG can interact with both FEZ1 and SCOC in vitro. GST, GST-FEZ1 or GST-SCOC were immobilised and incubated with 35S‐labelled Myc-UVRAG as indicated. Arrowheads indicate GST-FEZ1 (left) and GST-SCOC (right). (F) Endogenous UVRAG partially colocalises with endogenous FEZ1 in HEK293 cells. Arrows show colocalisation. Scale bar=10 μm.
Figure 1A Mass spectrometric analysis of LSD1 methylation. Immunoprecipitation (IP) was performed using anti-Flag antibody in HEK-293T cells overexpressing Flag-tagged LSD1, followed by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analysis. The fragmentation of the LSD1 peptide IADQFLGAMYTLPR identified a dimethylated residue at R838. B Interactions between LSD1 and CARM1 were detected by co-immunoprecipitation (Co-IP) assay with anti-Flag in HEK-293T cells transfected with Flag-LSD1 or Flag-CARM1, respectively. C Interaction of endogenous LSD1 with CARM1 was analyzed by Co-IP assay using anti-CARM1 antibody in MDA-MB-231 cells. Purified GST-tagged CARM1 was pulled down with the Flag-LSD1 purified from HEK-293T cells. The amounts of GST and GST-tagged CARM1 were visualized by Coomassie Blue staining (asterisk: GST-CARM1). E In vitro methylation assays. Purified fusion protein of GST-tagged LSD1 WT or the four methylated arginine mutants (R-A), or four methylated arginine mutants in combination (4RA), were incubated with GST-CARM1 in the presence of 3H-SAM. Methylated proteins were detected via autoradiography and total amounts of proteins were visualized by Coomassie Blue staining (arrows: the position of LSD1; arrowheads: the position of CARM1). F Increasing amounts of GST-CARM1 incubated with GST-LSD1 in presence of SAM at 30°C for 1 h. And methylation of LSD1 was analyzed by immunoblotting with anti-LSD1 R838me2a antibody. G GST-CARM1 incubated with GST-LSD1 WT or GST-LSD1 R838A/R838K in presence of SAM at 30°C for 1 h, followed by immunoblotting with anti-LSD1 R838me2a to detect LSD1 methylation. H Sequence alignments of LSD1 among mammals. LSD1 residue at R838 is denoted in the protein sequences.
Genetic and biochemical interactions between Atg1 and spaghetti‐squash activator (Sqa).Figure 2(A-K) Genetic interactions between Atg1, Sqa, and spaghetti‐squash (Sqh). Compared with the ptc‐GAL4 controls (A), expression of Atg1 or Sqa by ptc‐GAL4 resulted in missing anterior cross‐vein phenotypes (B, C). However, RNAi‐mediated downregulation of Atg1 (Atg1RNAi) or Sqa (SqaRNAi) did not cause wing vein defects (D, E). Depletion of Atg1 and Sqa suppressed Atg1 and Sqa‐induced wing vein defects, respectively (F, I). Atg1‐induced wing defects were modulated by depletion of Sqa (G) or by co‐expression of SqhA20A21 (H). Nevertheless, Sqa‐induced wing vein defects were suppressed by co‐expression of SqhA20A21 (J) but not Atg1RNAi (K). (L) (L) Schematic presentation of the domain structures of Sqa and deletion mutants. (M-N) Atg1 physically interacted with Sqa. (M) 293T cells were transfected with HA‐tagged Sqa WT (wild‐type) or KA, together with Flag‐tagged Atg1 WT or KR. The cells were lysed 48 h after transfection and immunoprecipitated (IP) with anti‐Flag antibodies. The immunoprecipitated proteins and the total cell lysates (TCL) were analysed by immunoblotting (IB) with antibodies as indicated. (N) 293T cells transfected with Flag-Atg1‐KR and various HA‐tagged Sqa contracts were subjected to immunoprecipitations with anti‐HA antibody. The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated. (O) Atg1 directly phosphorylated Sqa in vitro. Flag‐tagged Atg1 WT or KR immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant Sqa‐K1, Sqa‐K2, and Sqa‐C in an in vitro kinase assay. The lower panels represent equal input of His‐fusion proteins and Atg1 immunoprecipitates.
Figure 6(A Measurement of serum indices including liver enzymes (AST, ALT and GGT) from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) to reflect liver and kidney functions (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test). B) Measurement of kidney markers (CREA and UA) from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) to reflect liver and kidney functions (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test). (C) Morphological examination of liver and kidney from wild-type C57BL/6 (C57), untreated mdx controls (mdx) and treated mdx mice (scale bar: 100 μm). (D Immunohistochemistry of macrophages, T cells and monocytes in quadriceps and diaphragm from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO (n=3) (scale bar: 100 μm). The arrowheads point to CD68+ macrophages, CD3+ T cells or CD11b+ monocytes (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test). E) quantification of macrophages, T cells and monocytes in quadriceps and diaphragm from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO (n=3) (scale bar: 100 μm). The arrowheads point to CD68+ macrophages, CD3+ T cells or CD11b+ monocytes (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).
Figure 2A Dissociation constants for ERAD E2-SS-Ub/E3 pairs. KD values were determined by ITC titration of Hrd1 or Doa10 to Ubc6-SS-Ub and U7BR/Ubc7-SS-Ub, respectively. Due to the weak binding, errors are high, so the constants are reported to just a single significant figure. B In vitro substrate ubiquitylation assay for Hrd1 with Ubc7 (left) and Ubc6 (right). Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; "no E3" reactions do not contain S-Hrd1, "no ATP" reactions do not contain ATP, but S-Hrd1. The RNase-Ub2 band co-migrates with a nonspecific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity, only significances related to the "no E3" control of a given E2 are shown. C In vitro Ub nucleophile discharge assays for Hrd1 with U7BR/Ubc7 (top - yellow) and Ubc6 (bottom - green) with ethanolamine as nucleophile. Representative Coomassie gels are shown. D Quantification of Ub nucleophile discharge assays for Hrd1 with U7BR/Ubc7 (left) and Ubc6 (right). Plots of E2~Ub discharge (dots) as a function of time with first-order reaction models fitted to the discharge data (lines) are shown. Values for each time point are reported as means ± standard deviation (n = 3). Insets show reaction rates derived from these fits. Significances were determined by student T-test; * p < 0.05.
Figure 3A) Preservation analysis between BrainSpan dorsolateral prefrontal cortexgene expression data and the ASD + control blood data. Zsummary statistic (e.g., Zsummary > 10 means highly preserved, Zsummary in between 2 and 10 is weak to moderate preservation, Zsummary<2 is little to no preservation and median rank (modules with lowest rank are highly preserved). Median Rank and Zsummary values indicate high module preservation between the two datasets. B) Boxplot showing module eigengene (ME) values the BrainSpan cell cycle module for fetal versus postnatal time-points. C) Scatterplot indicating the BrainSpan cell cycle module trajectory across development (vertical line indicates birth; time-points to the left of the line are fetal time-points, while time-points to the right of the line are postnatal time points; best-fit curve indicates a 4th order polynomial fit).