EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) Endogenous FAM134B forms oligomers. FAM134B knockout (KO) or wild type (WT) 293T cells were treated with 1μM of Thapsigargin (Tg) for different time and the cell lysates were prepared and analyzed by Western-blot. (B) Analysis the oligomer size of recombinant FAM134B WT using Native PAGE. (C) In vitro liposome fragmentation assay. After the injection of recombinant proteins (100μg/100μl) into the chamber (500μl), the morphological changes of liposomes were monitored by live imaging for 20 min. The images at different time points as indicated are presented. Scale bars, 10 μm. "ns" means no significance, one-way ANOVA, error bars indicate SEM (n=3). (D) Quantification of the time between protein addition and liposome fragmentation. ***P<0.001, one-way ANOVA, error bars indicate SEM (n=3). (E, F) Measurement of the intracellular ER-scission activity. U2OS cells transiently expressing EGFP-FAM134B (WT), EGFP-FAM134B (84-233) or EGFP-FAM134B (Δ84-233) at same levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1) or DMSO. GFP-positive puncta were quantified for each cell in (F). For Each group, at least 30 cells were counted. Scale bars, 10 μm. ***P<0.001, "ns" means no significance, one-way ANOVA, error bars indicate SEM. (G, H) Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (84-233) or mCherry-EGFP-FAM134B (Δ84-233) at same levels. ER-phagy is indicated by mCherry-positive but EGFP-negative puncta were quantified for each cell in (H). For mCherry-EGFP-FAM134B (WT), 50 cells were counted (n=50); for mCherry-EGFP-FAM134B (84-233), n=56; for mCherry-EGFP-FAM134B (Δ84-233), n=66. Scale bars, 10 μm. The scale bars in the magnification boxes are 2μm. ***P<0.001, one-way ANOVA, error bars indicate SEM. (I) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B WT-Flag, FAM134B (84-233)-Flag or FAM134B (Δ84-233)-Flag.
leukocytes and macrophages, and reduces tumor cell death and tumor growth (7 weeks model)(A) Growth of TNC-high (WT/shC) tumors upon cell engraftment and 5 weeks AMD treatment.Figure 5(A) Growth of TNC-high (WT/shC) tumors upon cell engraftment and 5 weeks AMD treatment. N = 20 tumors per condition, *** p = 0.0007, unpaired t-test.The central band represents the median, the ends of the box the first and the third quartiles, and the whiskers reach from each quartile to the minimum or maximum. (B, C) Imaging and signal quantification upon staining for cl. casp3 and DAPI in PBS and AMD treated tumors, N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. Scale bar, 50µm, arrows point at cl. Casp3. (D, E) CD8 TIL abundance and localization in PBS and AMD treated tumors, arrow points at CD8 TIL, scale bar, 50µm (D). (E) Quantification of CD8 T cells per randomly chosen fields. N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. (F) Gzmb levels in PBS and AMD treated tumors. N = 10 tumors, **** p < 0.0001, unpaired t-test. Mean ± SEM. ( (G, H) Heatmap of tumor cell apoptosis (G) and lymphocyte apoptosis (H) associated genes differentially expressed in PBS and AMD treated TNC-high (WT/shC) tumors, N = 2. (I, J) Abundance and quantification of F4/80+ macrophages in PBS and AMD treated tumors, (I) staining for the indicated molecules, (arrow, macrophages in the stroma; asterisk, macrophages in the tumor nest), (J) quantification of F4/80+ cells inside the tumor nest. N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. Scale bar, 50 µm.
Figure 9(A) MITOL was ubiquitylated only in the presence of Parkin when the mitochondrial membrane was decreased. After 15 µM CCCP treatment for 3 hours, HeLa cells stably expressing HA-Parkin and 3Flag-MITOL were immunoprecipitated with anti-Flag magnetic beads, and then immunoblotted with the indicated antibodies. Red bars indicate ubiquitylation; the black arrowhead indicates 3Flag-MITOL. (B) The characteristic ubiquitylation ladder was observed for wild-type MITOL, but was absent in the K268A and K54A/K268A mutants. Black arrowhead indicates 3Flag-MITOL. (C) The MITOL K268A and K268R mutants were targeted to mitochondria under steady-state conditions, whereas peroxisomal localization following CCCP treatment was considerably disrupted. HeLa cells stably expressing HA-Parkin were transfected with 3Flag-MITOL wild-type, K268A, or K268R mutants, treated with 15 µM CCCP for 3 hours, and then subjected to immuno-staining with anti-Flag and anti-catalase antibodies. Scale bars, 10 µm. (D) The double K54/K268 mutation did not change the MITOL localization pattern. The subcellular localization of the MITOL K268A or K268R mutants was not drastically changed by inclusion of the K54A or K54R mutations. Scale bars, 10 µm. (E) Correlation statistics for the localization of 3Flag-MITOL wild-type, K268A, or K268R mutants with catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. (F) The fold change in ubiquitylation of MITOL K268, K40, and K54 in valinomycin-treated samples versus untreated samples. After 3 hours of valinomycin treatment, PEX19 -/- HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were immunoprecipitated with anti-Flag magnetic beads, and then subjected to LC-MS/MS analysis for label-free quantification of ubiquitylated peptides. Error bars represent the mean ± s.e.m in three biological replicates.
Figure 5Fis1 is a bona fide target for SUMOylation. Fis1 is a SUMO target in mammalian cells. Flag-Fis1 together with pcDNA3 or His-SUMO-2 were transfected into HEK293 cells expressing Ubc9 for 48 h. His-pulldown (His-PD) and lysate samples were detected by immunoblotting for Flag or β-actin. Endogenous Fis1 is modified by His-SUMO-2 stably expressed in HEK293 cells. His-pulldown (His-PD) and lysate samples were blotted as indicated. Flag-Fis1 is deSUMOylated by SENP3. SENP3 knockdown enhances Fis1 SUMOylation. Nsi or SENP3i was transfected into HEK293 cells expressing Flag-Fis1, His-SUMO-2 and Ubc9 for 48 h. His-pulldown and lysate samples were detected by immunoblotting for Flag, SENP3 or β-actin. Values are presented as mean ± SEM (n=3, biological replicates; * p<0.05; paired-t test). SENP3 knockdown increases endogenous Fis1 SUMO-2/3-ylation in HeLa cells. SUMO-2/3 conjugates were immunoprecipitated (IP) from whole cell lysates prepared from HeLa cells using SUMO-2/3 affinity beads. IP and lysate samples were blotted as indicated. Amino acids including lysine 149 at the C-terminus of Fis1 are required for Fis1 SUMOylation. Flag-Fis1, Flag-Fis1 K149R mutant or Flag-Fis1 Δ149-152 mutant together with His-SUMO-2 were transfected into HEK293 cells expressing Ubc9 for 48 h. His-pulldown and lysate samples were blotted as indicated. Alignment (the right panel) shows amino acid sequences of the C-terminal tail of Fis1 from the indicated organisms: human (h), mouse (m), rat (r) and S. cerevisiae (y); red letters mark the lysine residue required for Fis1 SUMOylation. Schematic (the left panel) illustrates SUMO conjugation at the C-terminal tail of Fis1 (N, N-terminus; TM, Transmembrane domain; C, C-terminus).
Overview of non-synonymous mutations in terms of base substitution signatures, molecular subtype, and protein impactA) Contribution of base change types to the overall SNV composition in the ABiM cohort for captured DNA regions and mRNA in the captured DNA regions, as well as SCAN-B whole mRNA.Figure 2A) Contribution of base change types to the overall SNV composition in the ABiM cohort for captured DNA regions and mRNA in the captured DNA regions, as well as SCAN-B whole mRNA. B) Number of non-synonymous mutations per sample. Bars are colored by PAM50 subtypes Luminal A (dark blue), Luminal B (light blue), HER2-enriched (pink), basal-like (red), Normal-like (green) and Unclassified (gray). C-F) Lollipop plots showing the location, abundance, and impact of SNVs in C) TP53, D) PIK3CA, E) PTEN, and F) ERBB2 on the respective encoded protein. Protein change labels are shown for the most mutated amino acid positions, with residues ordered left to right by mutation frequency within each label.
a) Cells expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 as well as a plasmid-borne (pDJB12), mitochondrially targeted RFP (mtRFP) were incubated in SL-leucine medium for 3 days and imaged daily.f6(a) Cells expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 as well as a plasmid-borne (pDJB12), mitochondrially targeted RFP (mtRFP) were incubated in SL-leucine medium for 3 days and imaged daily. Aco2 and Idp1 show clear mitophagic targeting (white arrows point at specific examples) by day 3, whereas Idh2 and Hsp78 show weak or no mitophagy. In addition, Idh2 and Hsp78 show different degrees of segregation relative to the mtRFP marker, whereas Idp1-GFP and Aco2-GFP show a near-complete co-localization with mtRFP. Scale bar, 5 μm. (b) Quantification of the frequency of mitophagy from a; the percentage of cells showing mitophagic profiles in the red (magenta bars; plasmid-borne mtRFP) or green (turquoise bars; integrated C-terminal GFP chimerae) was calculated for each GFP chimera. Idp-GFP, red N=76, green N=82; Aco2-GFP, red N=79, green N=82; Idh2-GFP, red N=72, green N=72; Hsp78-GFP, red N=70, green N=70.
Figure 7The low bi-allelic expression of Tsix in mESCs allows the association of RIF1 with P2 on both Xist alleles. However, the presence of pluripotency-dependent inhibitors will not allow Xist up-regulation, despite the presence of RIF1. (1) Upon differentiation, the increase of Tsix levels weakens the association of RIF1 with P2. This opens the opportunity for a stochastic KAP1 binding to P2 of one of the two alleles (2). KAP1 is required for sustained high levels of Tsix, further reinforcing RIF1 exclusion from the KAP1-bound/Tsix high allele and establishing the asymmetry. It is not known whether KAP1 gains access to P2 to promote the increase of Tsix levels first, or whether the increase of Tsix levels is initially triggered by a differentiation-dependent factor. (3). The pluripotency Xist inhibitors having been silenced, RIF1 promotes Xist expression on the future Xi. A self-sustainable binary switch is thus created and it consolidates the choice of the future Xi and Xa.
Figure 7(A) Western blot analysis of ERK phosphorylation (p-ERK) in recipient serum-deprived HCT116 cells pre-treated with EVs isolated by SEC from glutamine-replete or -depleted HCT116 cells or vehicle (PBS). Bar chart shows ratio of p-ERK to ERK levels from triplicate independent experiments. (B) Bar chart shows HCT116 recipient cell growth over 120 h, after pre-treatment with EV preparations isolated as in (A) or with PBS, and then incubation for the first 24 h of culture in the presence or absence of the ERK inhibitor SCH772984 (1.00 µM). (C) Western blot analysis showing levels of the EGFR ligand, Amphiregulin (AREG) in EVs isolated by SEC of medium from HCT116 cells cultured in glutamine-replete (2.00 mM) or -depleted (0.15 mM) conditions for 24 h. Gel loading was normalised to cell lysate protein levels. AREG's molecular weight (approximately 26-28 kDa) suggests it is in its membrane-associated form (see Appendix Fig S8D). In the bar chart, the levels of putative exosome proteins were normalised to cell lysate protein levels. Significantly decreased levels are in blue and increased levels are in red.(C') Growth curves in low (1%) serum are for HCT116 recipient cells pre-treated with the EV preparations [isolated as in (C)], which had themselves been pre-treated with and without anti-AREG neutralising antibody or with a control IgG. Solid red line shows growth-promoting effect of EVs isolated under glutamine depletion, which is blocked by anti-AREG antibody (red dashed line). ***P colour denotes significant increase relative to EVs from glutamine-replete cells (black) and anti-AREG-treated cells (red). (C'') Western blot analysis of ERK phosphorylation (p-ERK) in recipient serum-deprived HCT116 cells pre-treated with EVs isolated by SEC from glutamine-replete (2.00 mM) or -depleted (0.15 mM) HCT116 cells or vehicle (PBS), which were pre-treated with anti-AREG (AREGab) or a control immunoglobulin (Ig). Note increase in ERK phosphorylation using EVs from glutamine-depleted cells, which is blunted by the addition of the anti-AREG antibody.
a, AMPK&agr1/&agr2 double knockout (DKO) and wild-type (WT) MEFs were deprived of glucose for 1 h and re-stimulated for 10 min.f4a) AMPKa1/a2 double knockout (DKO) and wild-type (WT) MEFs were deprived of glucose for 1 h and re-stimulated for 10 min. Wholecell extracts were immunoblotted for the indicated proteins. b, Immortalized MEFs of the indicated genotypes were deprived of growth factors, glucose, amino acids or glucose and amino acids for 1 h and re-stimulated with glucose and/or amino acids for 10 min. Whole-cell lysates were immunoblotted for the indicated proteins. c, RagA+/+ and RagAGTP/GTP immortalized MEFs were deprived of glucose or amino acids and surviving cells quantified in triplicate after 48 h. Cell number is indicated relative to cell number at the start of the treatment; mean ± s.d.; ***P  0.005 d, mTOR localization as detected by immunofluorescence. In HEK-293T cells, glucose deprivation causes mTOR to localize to diffuse small puncta throughout the cytoplasm. Re-addition of glucose leads to mTOR shuttling to the lysosomal surface, co-localizing with the lysosomal protein Lamp2. HEK-293T-RagBGTP cells show mTOR localized at the lysosomal surface, regardless of glucose concentrations. Scale bars, 10 μm. e, Glucose and amino acids affect the binding of the v-ATPase to the Ragulator complex. HEK-293T expressing Flag-p14 was deprived of glucose or amino acids for 90 min and re-stimulated for 20 min. Protein extracts and immunoprecipitates were immunoblotted for the indicated proteins. f, RagA+/+ and RagAGTP/GTP primary MEFs were cultured for 1 h in media with the glucose and amino-acid concentrations measured in neonates at birth (0 h) or after fasting for 1 h (1 h) and whole-cell protein extracts were analysed by immunoblotting.
Figure 1(A) Layout of the field trial. A total of 560 Zea mays B104 plants were grown in a grid of 10 rows by 56 columns. Border rows 0 and 9 are not shown on the plot, and the dimensions on the figure are not to scale. Cell colors indicate which data types are available for the plants, with grey indicating phenotype data only (p), orange transcriptome and phenotype data (t+p), and blue transcriptome, metabolome and phenotype data (t+m+p). Harvesting dates (DOH) are indicated by the cell border color. The RNA sequencing batch is indicated by cell border thickness. Plants belonging to different subgroups based on SNP analysis are indicated by the coloring of the plant ID numbers inside the cells. The designations 0 and 1 for the DOH, BATCH and SNP effect groups are used for the largest and smallest group, respectively. (B) Plot showing the first two principal components (PCs) in a PCA of the 60 single-plant transcriptomes. (C) Plot showing the first two PCs in a PCA of the 50 single-plant metabolomes. (D) Plot showing the first two PCs in a PCA of the plant phenomes for the 60 plants that were RNA-sequenced. Data information: The plants in panels (B-D) are numbered according to the numbering in panel (A). Plants belonging to different SNP and DOH subgroups are indicated by different markers and marker colors, respectively, and plants sequenced in the second, smallest batch are circled in panel (B).
Figure 6D-loop stimulation by Rad54 or Rdh54NRad54 in reactions with Rad51 (300 nM), tailed duplex substrate (15 nM) and a pUC19 plasmid dsDNA (0.3 nM).   Quantification of D-loop formation for Rad54 and Rdh54NRad54. Error bars represent s.d. of three independent experiments.   D-loop stimulation by Rdh54 and Rad54NRdh54.   Quantification of D-loop formation for Rad54 and Rdh54NRad54. Error bars represent s.d. of three independent experiments.   ATPase stimulation for GFP-tagged Rad54, Rdh54NRad54, Rdh54 or Rad54NRdh54 in the presence of Rad51-ssDNA. Error bars represent s.d. of three independent experiments. Quantification of Rad51 Atto565-tailed duplex PSCs bound per dsDNA molecule in dsDNA curtain homology search assays with Rad54 (N = 117), Rdh54NRad54 (N = 150) or Rad54NRdh54 (N=175). The error bars represent the s.d. for three independent experiments. Fraction translocating Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 117), Rdh54NRad54 (N = 150), Rad54NRdh54 (N = 0) and Rdh54 (N = 0). Translocation velocities of Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 86) and Rdh54NRad54 (N = 18). The error bars represent the s.d. of the data.
a–f) Immunofluorescence for PSA-NCAM (a–c) or NeuN (d–f) in the dentate gyrus (DG), SVZ, RMS and olfactory bulb (OB) of P28 mice.figf6(a-f) Immunofluorescence for PSA-NCAM (a-c) or NeuN (d-f) in the dentate gyrus (DG), SVZ, RMS and olfactory bulb (OB) of P28mice. Lines indicate boundaries of the DG (a,d), SVZ (a), and layers of the OB (d). Boxed areas in a indicate the RMS. Arrows mark PSA-NCAM+ cells. Mean ± s.e.m. of the number of PSA-NCAM+ cells (b,c) or NeuN+ cells (e,f) per section are shown. LV, lateral ventricle; ML, molecular layer; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. NS, not significant; *P 0.05. (g-i) Immunofluorescence of BrdU retention cells in the DG and OB of P31 mice. Boxed areas in left column of g are shown in more detail in right column. Dotted lines indicate the boundaries of the DG (left) and GZ (right). Arrows mark NeuN+BrdU+ cells and arrowheads mark NeuN−BrdU+ cells in the DG. Mean ± s.e.m. of the percentage of NeuN+BrdU+ cells of total BrdU retained cells in the DG (h) and OB (i) are shown. (j,k) Mean ± s.e.m. of percentage of GFAP+nestin−BrdU+ cells among total BrdU+ retained cells in the SVZ (j) and SGZ (k). (l,m) DHE and DAPI fluorescence at P28 in the DG (l) and SVZ (m) from FIP200GFAP cKO mice with or without NAC treatment, 2cKO mice and Trp53GFAP cKO mice. Dotted lines indicate the boundaries of the granular zone (GZ) (l) and SVZ (m). Arrows mark cells in the SGZ (l) and SVZ (m) and arrowheads mark cells in surrounding regions (the GZ in l and the striatum (ST) in m). n = 5 mice, ≥4 sections per mouse, >20 (h,j,k) or >200 (i) BrdU+ cells counted per mouse. cKO(n), NAC non-responder cKO mice; LV, lateral ventricle; ML, molecular layer; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. NS, not significant; *P 0.05.
Figure 2A. H&amp;amp;E stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 µm. Invasion index of KPC cells in the presence or absence of 10 µM H1152 ROCK inhibitor. Means ± SEM (n=9 for untreated, n=8 for H1152), p value by unpaired t test. B. Schematic representation of ROCK domains (RBD, Rho binding domain; PH, pleckstrin homology domain; CR, cysteine-rich). Conditionally-activated human ROCK1, human ROCK2 and GFP control fusion proteins (EGFP, enhanced green fluorescent protein; hbER, estrogen receptor hormone binding domain) were expressed in KPflC mouse PDAC cells and blotted with anti-GFP antibody. C. KPflC cells expressing GFP:ER, ROCK1:ER or ROCK2:ER fusion proteins were treated for 24 h with EtOH vehicle or 1 µM 4HT in the presence or absence of 1 µM or 10 µM H1152. Immunoblotting shows endogenous ROCK1 and ROCK2, ER-fusions, and phosphorylation of MLC2 (T18S19). Total MLC (MRCL3/MRLC2/MYL9) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were blotted as loading controls. D. H&amp;amp;E stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 µm. Invasion index of KPflC cells treated with 1 µM 4HT. Means ± SEM (n=6), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test. E. Cell proliferation determined by Ki67 immunofluorescence. Scale bar: 20 µm.F. Quantification of cell number at the collagen matrix surface per 0.046 mm2 field. Means ± SEM (n=30), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.G. Ki67 positive cell percentages at the surface and within the collagen matrix. Means ± SEM (n=30; n=12 for GFP:ER/Matrix), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test. H. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on uncoated plastic surfaces. Means ± SEM (n=3).I. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on collagen1-coated surfaces. Means ± SEM (n=3).
CUL3WT/Δ403–459 mice recapitulate PHAII electrolyte imbalances due to over-activation of the renal WNK4-SPAK pathway.Figure 4Western blot of whole kidney lysates from mice culled after a minimum 4-h fast. Following exsanguination after surgery, mouse tissues were rapidly harvested and stored, samples were then homogenised, clarified and quantified prior to SDS-PAGE. Immunodetection with the antibodies shown highlights elevated signalling through the WNK kinase pathway in CUL3WT/Δ403-459 versus CUL3WT mice. The lowest panel is an anti-CUL3 immunoprecipitation of the kidney lysate samples, whereby the CUL3 antibody was cross-linked to Protein G sepharose and used to affinity purify CUL3WT and CUL3Δ403-459 from kidney lysates. The samples were incubated together overnight to allow deneddylation of CUL3 proteins to occur and maximise CUL3 binding to the anti-CUL3 resin. Samples were thoroughly washed and eluted from the resin prior to SDS-PAGE and immunodetection for CUL3. The IP highlights that CUL3Δ403-459 is indeed present within the kidney lysate. Fresh frozen kidney tissues from transplant patient and donor cadavers were homogenised, clarified and quantified prior to SDS-PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human kidneys. Plasma aldosterone after a minimum 4-h fast was calculated by HTRF (homogeneous time-resolved fluorescence) aldosterone assay. The average aldosterone level per mouse was calculated from duplicate samples run in parallel on the assay. Blood was rapidly harvested in heparin-coated plasma extraction tubes following exsanguination after surgery, and samples were snap-frozen for storage. A 58% increase in aldosterone was detected in CUL3WT/Δ403-459 versus CUL3WT mice (*P = 0.0245). Two-tailed unpaired Student's t-test; data are mean ± SEM. Arterial blood biochemistries after a minimum 4-h fast. Under anaesthesia, the right carotid artery was cannulated to minimise atmospheric exposure of samples collected for iSTATbloodgas and electrolyte measurements. CUL3WT/Δ403-459mice present with abnormal electrolyte homoeostasis compared to CUL3WTmice, exhibiting hyperkalaemia (***P = 0.0004) and hyperchloraemia (***P = 9.5 × 10−5) with a compensated metabolic acidosis (P = 0.7766), marked by a decrease in bicarbonate () (***P = 3.4 × 10−5), base excess (BE) (***P = 9.1 × 10−5) and partial pressure of carbon dioxide (pCO2) (***P = 0.0038). Two-tailed unpaired Student's t-test; data are mean ± SEM.