No association between intravenous fluid volume and endothelial glycocalyx shedding in patients undergoing resuscitation for sepsis in the emergency department

Endothelial glycocalyx (EG) shedding is associated with septic shock and described following intravenous (IV) fluid administration. To investigate the possible impact of IV fluids on the pathobiology of septic shock we investigated associations between biomarkers of EG shedding and endothelial cell activation, and relationships with IV fluid volume. Serum samples were obtained on admission (T0) and at 24 h (T24) in patients undergoing haemodynamic resuscitation for suspected septic shock in the emergency department. Biomarkers of EG shedding—Syndecan-1 (Syn-1), Syndecan-4 (Syn-4), Hyaluronan, endothelial activation—Endothelin-1 (ET-1), Angiopoeitin-2 (Ang-2), Vascular Endothelial Growth Factor Receptor-1(VEGF-1) and leucocyte activation/inflammation—Resistin, Neutrophil Gelatinase Associated Lipocalin (NGAL) and a marker of cardiac stretch—Pro-Atrial Natriuretic Peptide (Pro-ANP) were compared to the total IV fluid volume administered using Tobit regression. Data on 86 patients (52 male) with a mean age of 60 (SD 18) years were included. The mean fluid volume administered to T24 was 4038 ml (SD 2507 ml). No significant association between fluid volume and Pro-ANP or any of the biomarkers were observed. Syn-1 and Syn-4 were significantly correlated with each other (Spearman Rho 0.43, p \u3c 0.001) but not with Hyaluronan. Syn-1 and Syn-4 both correlated with VEGFR-1 (Rho 0.56 and 0.57 respectively, p \u3c 0.001) whereas Hyaluronan correlated with ET-1 (Rho 0.43, p \u3c 0.001) and Ang-2 (Rho 0.43, p \u3c 0.001). There was no correlation between Pro-ANP and any of the EG biomarkers. Distinct patterns of association between biomarkers of EG shedding and endothelial cell activation were observed among patients undergoing resuscitation for sepsis. No relationship between IV fluid volume and Pro-ANP or any of the other biomarkers was observed

A MYC-ZNF148-ID1/3 regulatory axis modulating cancer stem cell traits in aggressive breast cancer

The MYC proto-oncogene (MYC) is one of the most frequently overexpressed genes in breast cancer that drives cancer stem cell-like traits, resulting in aggressive disease progression and poor prognosis. In this study, we identified zinc finger transcription factor 148 (ZNF148, also called Zfp148 and ZBP-89) as a direct target of MYC. ZNF148 suppressed cell proliferation and migration and was transcriptionally repressed by MYC in breast cancer. Depletion of ZNF148 by short hairpin RNA (shRNA) and CRISPR/Cas9 increased triple-negative breast cancer (TNBC) cell proliferation and migration. Global transcriptome and chromatin occupancy analyses of ZNF148 revealed a central role in inhibiting cancer cell de-differentiation and migration. Mechanistically, we identified the Inhibitor of DNA binding 1 and 3 (ID1, ID3), drivers of cancer stemness and plasticity, as previously uncharacterized targets of transcriptional repression by ZNF148. Silencing of ZNF148 increased the stemness and tumorigenicity in TNBC cells. These findings uncover a previously unknown tumor suppressor role for ZNF148, and a transcriptional regulatory circuitry encompassing MYC, ZNF148, and ID1/3 in driving cancer stem cell traits in aggressive breast cancer

Interspecies variation in the functional consequences of mutation of cytochrome c

The naturally occurring human cytochrome c variant (G41S) is associated with a mild autosomal dominant thrombocytopenia (Thrombocytopenia Cargeeg) caused by dysregulation of platelet production. The molecular basis of the platelet production defect is unknown. Despite high conservation of cytochrome c between human and mouse (91.4% identity), introducing the G41S mutation into mouse cytochrome c in a knockin mouse (Cycs (G41S/G41S)) did not recapitulate the low platelet phenotype of Thrombocytopenia Cargeeg. While investigating the cause of this disparity we found a lack of conservation of the functional impact of cytochrome c mutations on caspase activation across species. Mutation of cytochrome c at residue 41 has distinct effects on the ability of cytochrome c to activate caspases depending on the species of both the cytochrome c and its binding partner Apaf-1. In contrast to our previous results showing the G41S mutation increases the ability of human cytochrome c to activate caspases, here we find this activity is decreased in mouse G41S cytochrome c. Additionally unlike wildtype human cytochrome c, G41S cytochrome c is unable to activate caspases in Xenopus embryo extracts. Taken together these results demonstrate a previously unreported species-specific component to the interaction of cytochrome c with Apaf-1. This suggests that the electrostatic interaction between cytochrome c and Apaf-1 is not the sole determinant of binding, with additional factors controlling binding specificity and affinity. These results have important implications for studies of the effects of cytochrome c mutations on the intrinsic apoptosis pathway

Mutations at residue 41 have species-specific effects on caspase activation.

<p>A. and B. Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in A. mouse WEHI164 (100 μg) and B. human U937 (100 μg) cytosols with the addition of 0 nM, 25 nM, 50 nM or 100 nM mouse somatic WT (white bars) or G41S (black bars) cytochrome <i>c</i> at pH 7.25. All reactions contained 1 mM dATP. Data are presented as mean ± SD (n = 3). *<i>P</i> < 0.01, **<i>P</i> < 0.001 compared to WT. C. Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in Xenopus cytosolic extracts (2 μg) with the addition of 100 nM human cytochrome <i>c</i> without (white bars) or with (black bars) the addition of human Apaf-1. Data are presented as mean ± SD (n = 3). *p < 0.01, **p < 0.001 compared to without human Apaf-1. Progress curves are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130292#pone.0130292.s002" target="_blank">S2B–S2D Fig</a>).</p

Variation at residue 44 does not alter the functional impact of the G41S mutation in mouse somatic cytochrome <i>c</i>.

<p>A and B. Human cytochrome <i>c</i> and mouse somatic cytochrome <i>c</i> differ in sequence at surface exposed residues. A. Human ferricytochrome <i>c</i> with side chains of residues differing between human and mouse somatic shown as sticks with carbon in pale blue, oxygen in red and nitrogen in blue. The backbone of the 40-to-57 Ω loop is in cyan. Drawn from Protein Data Bank entry 3NWV. B. Clustal Omega sequence alignment of human (P99999) and mouse somatic (P62897) cytochromes <i>c</i> with the initiating Met removed. The residues comprising the 40-to-57 Ω loop are in cyan and Gly41 is in bold. C. Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in mouse WEHI164 (100 μg) cytosols with the addition of 50 nM, 100 nM or 150 nM of mouse somatic WT (white bars), G41S (light grey bars), A44P (dark grey bars) or G41S/A44P (black bars) cytochrome <i>c</i>. The rate in the absence of cytochrome <i>c</i> was 2.74 ± 0.24 pmol/min AMC. Data are presented as mean ± SD (n = 3). *<i>P</i> < 0.001 compared to WT. Progress curves are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130292#pone.0130292.s004" target="_blank">S4 Fig</a>.</p

Cytochromes c from different specifies have different abilities to activate caspases.

<p>Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in human U937 (100 μg, A) and mouse WEHI164 (100 μg, B) cytosols with the addition of 0 nM, 25 nM, 50 nM or 100 nM human (white bars), mouse somatic (grey bars) or horse (black bars) cytochrome <i>c</i>. Data are presented as mean ± SD (n = 3). *<i>P</i> < 0.05, **<i>P</i> < 0.001 compared to human (A) or mouse (B).</p

Platelet counts and formation are normal in <i>Cycs</i>^G41S/G41S mice.

<p>A. Platelet counts of 8 week old <i>Cycs</i>^<b>+/+</b>, <i>Cycs</i>^{<b>+/G41S</b>} and <i>Cycs</i>^{<b>G41S/G41S</b>} mice. B. Anti-platelet serum was injected into <i>Cycs</i>^<b>+/+</b> mice (□, n = 7–11) and <i>Cycs</i>^{<b>G41S/G41S</b>} mice (△, n = 8–13) to deplete platelets and platelet count was monitored over 10 days. Data are presented as mean ± SEM.</p

Interaction between cytochrome <i>c</i> and Apaf-1 in a human apoptosome model.

<p>Apaf-1 in grey (NBD, HD1, HD2 and WHD), pale blue (8 blade ß-propeller), lemon (7 blade ß-propeller) with the ß7 loop in orange. Cytochrome <i>c</i> in salmon-pink with the 40-to-57 Ω loop in magenta. Inset shows expansion of the cytochrome <i>c</i> 40-to-57 Ω loop with Gly-41 as a sphere and Apaf-1 as an electrostatic surface. Drawn from Protein Data Bank entry 3J2T.</p

Zfp281 (ZBP-99) plays a functionally redundant role with Zfp148 (ZBP-89) during erythroid development.

Erythroid maturation requires the concerted action of a core set of transcription factors. We previously identified the Krüppel-type zinc finger transcription factor Zfp148 (also called ZBP-89) as an interacting partner of the master erythroid transcription factor GATA1. Here we report the conditional knockout of Zfp148 in mice. Global loss of Zfp148 results in perinatal lethality from nonhematologic causes. Selective Zfp148 loss within the hematopoietic system results in a mild microcytic and hypochromic anemia, mildly impaired erythroid maturation, and delayed recovery from phenylhydrazine-induced hemolysis. Based on the mild erythroid phenotype of these mice compared with GATA1-deficient mice, we hypothesized that additional factor(s) may complement Zfp148 function during erythropoiesis. We show that Zfp281 (also called ZBP-99), another member of the Zfp148 transcription factor family, is highly expressed in murine and human erythroid cells. Zfp281 knockdown by itself results in partial erythroid defects. However, combined deficiency of Zfp148 and Zfp281 causes a marked erythroid maturation block. Zfp281 physically associates with GATA1, occupies many common chromatin sites with GATA1 and Zfp148, and regulates a common set of genes required for erythroid cell differentiation. These findings uncover a previously unknown role for Zfp281 in erythroid development and suggest that it functionally overlaps with that of Zfp148 during erythropoiesis

Zfp281 (ZBP-99) plays a functionally redundant role with Zfp148 (ZBP-89) during erythroid development

Erythroid maturation requires the concerted action of a core set of transcription factors. Wepreviously identified the Krüppel-type zinc finger transcription factor Zfp148 (also called ZBP-89) as an interacting partner of the master erythroid transcription factor GATA1. Here we report the conditional knockout of Zfp148 in mice. Global loss of Zfp148 results in perinatal lethality from nonhematologic causes. Selective Zfp148 loss within the hematopoietic system results in a mild microcytic and hypochromic anemia, mildly impaired erythroid maturation, and delayed recovery from phenylhydrazine-induced hemolysis. Based on the mild erythroid phenotype of these mice compared with GATA1- deficient mice, we hypothesized that additional factor(s) may complement Zfp148 function during erythropoiesis. We show that Zfp281 (also called ZBP-99), another member of the Zfp148 transcription factor family, is highly expressed in murine and human erythroid cells. Zfp281 knockdown by itself results in partial erythroid defects. However, combined deficiency of Zfp148 and Zfp281 causes a marked erythroid maturation block. Zfp281 physically associates with GATA1, occupies many common chromatin sites with GATA1 and Zfp148, and regulates a common set of genes required for erythroid cell differentiation. These findings uncover a previously unknown role for Zfp281 in erythroid development and suggest that it functionally overlaps with that of Zfp148 during erythropoiesis