table_1.PDF

<p>Disorders of sex development (DSD) consist of a wide range of conditions involving numerous genes. Nevertheless, about half of 46,XY individuals remain genetically unsolved. GATA4 gene variants, mainly related to congenital heart defects (CHD), have also been recently associated with 46,XY DSD. In this study, we characterized three individuals presenting with 46,XY DSD with or without CHD and GATA4 variants in order to understand the phenotypical variability. We studied one patient presenting CHD and 46,XY gonadal dysgenesis, and two patients with a history of genetically unsolved 46,XY DSD, also known as male primary hypogonadism. Mutation analysis was carried out by candidate gene approach or targeted gene panel sequencing. Functional activity of GATA4 variants was tested in vitro on the CYP17 promoter involved in sex development using JEG3 cells. We found two novel and one previously described GATA4 variants located in the N-terminal zinc finger domain of the protein. Cys238Arg variant lost transcriptional activity on the CYP17 promoter reporter, while Trp228Cys and Pro226Leu behaved similar to wild type. These results were in line with bioinformatics simulation studies. Additional DSD variations, in the LRP4 and LHCGR genes, respectively, were identified in the two 46,XY individuals without CHD. Overall, our study shows that human GATA4 mutations identified in patients with 46,XY DSD may or may not be associated with CHD. Possible explanations for phenotypical variability may comprise incomplete penetrance, variable sensitivity of partner genes, and oligogenic mechanisms.</p

table_2.PDF

<p>Disorders of sex development (DSD) consist of a wide range of conditions involving numerous genes. Nevertheless, about half of 46,XY individuals remain genetically unsolved. GATA4 gene variants, mainly related to congenital heart defects (CHD), have also been recently associated with 46,XY DSD. In this study, we characterized three individuals presenting with 46,XY DSD with or without CHD and GATA4 variants in order to understand the phenotypical variability. We studied one patient presenting CHD and 46,XY gonadal dysgenesis, and two patients with a history of genetically unsolved 46,XY DSD, also known as male primary hypogonadism. Mutation analysis was carried out by candidate gene approach or targeted gene panel sequencing. Functional activity of GATA4 variants was tested in vitro on the CYP17 promoter involved in sex development using JEG3 cells. We found two novel and one previously described GATA4 variants located in the N-terminal zinc finger domain of the protein. Cys238Arg variant lost transcriptional activity on the CYP17 promoter reporter, while Trp228Cys and Pro226Leu behaved similar to wild type. These results were in line with bioinformatics simulation studies. Additional DSD variations, in the LRP4 and LHCGR genes, respectively, were identified in the two 46,XY individuals without CHD. Overall, our study shows that human GATA4 mutations identified in patients with 46,XY DSD may or may not be associated with CHD. Possible explanations for phenotypical variability may comprise incomplete penetrance, variable sensitivity of partner genes, and oligogenic mechanisms.</p

Characterization of Potent SMAC Mimetics that Sensitize Cancer Cells to TNF Family-Induced Apoptosis - Fig 4

<p>Condensed structures (A,B, and C) of the compounds series described in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#pone.0161952.t001" target="_blank">1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#pone.0161952.t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#pone.0161952.t003" target="_blank">3</a>. (D) Structure of P₂ substituent of compound <b>18</b>.</p

Binding affinities of naphthyl series SMAC mimetic compounds for BIR domains of cIAP1 and cIAP2^a.

<p>Binding affinities of naphthyl series SMAC mimetic compounds for BIR domains of cIAP1 and cIAP2<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#t002fn002" target="_blank">^a</a>.</p

SMAC mimetic 38 induces degradation of cIAP1.

<p>MDA-MB-231 cells were seeded at 40,000 per well of 12 well plates and cultured overnight. The next day, cultures were either left untreated (No trtm) or were treated for 6 h. with DMSO, 5 μM of SMAC mimetic <b>38</b> or 5 μM of inactive analogue compound <b>40</b>. Cells were lysed in SDS-sample buffer and lysates were analyzed by SDS-PAGE/immunoblotting using antibodies specific for cIAP1 and beta-actin. Molecular weight markers are indicated in kilo-Daltons (kDa).</p

SMAC mimetics inhibit cIAP1 and cIAP2 binding to RIP1.

<p>HEK293T cells were transfected with myc-RIP1 plasmid and 24h later, cell lysates were prepared and divided into equal aliquots to which 7 μg of either GST-cIAP1-BIR3 or GST-cIAP2-BIR3 was added. Then, aliquots of either DMSO control or various concentrations of SMAC mimetics <b>37</b>, <b>38</b> or inactive analogue <b>40</b> were added. After overnight incubation, GST-cIAP1-BIR3 or GST-cIAP2-BIR3 proteins were recovered using glutathione-sepharose beads and the bound myc-RIP1 protein was detected by SDS-PAGE/immunoblotting using anti-myc antibody for detection of myc-RIP1 and anti-GST antibody for detection of GST-fusion proteins.</p

Correlation between K_i of the cIAP BIR domains and their EC₅₀.

<p>Log of the K_i values for SMAC peptide displacement as measured by FPA was plotted against cell viability EC₅₀ values using the data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#pone.0161952.g005" target="_blank">Fig 5</a> for either TNF or LT-α. Correlations coefficient (r) and p-values are indicated.</p

Effect of buffer on K_D of SMAC peptide binding to BIR domains.

<p>All assays contained TCEP at 1 mM, 0.005% Tween 20 and SMAC-rhodamine at 20 nM. The buffers used were PBS @ pH 7.4, 25 mM HEPES @ pH 7.5, 25 mM HEPES @ pH 7.5 with 20 mM β-glycerol phosphate, 10 mM Potassium Phosphate @ pH 7.4, or 50 mM TRIS @ pH 7.5. Proteins were diluted into 25 mM HEPES @ pH 7.5 with 1 mM TCEP. FPA data were collected on the Analyst at 0, 30 and 60 min. Time overlays are plotted in the figure. K_Ds were determined in Prism.</p

K_D determination of SMAC-rhodamine binding to BIR2 and BIR3 domains of cIAP1 and cIAP2.

<p>Data were for assay conditions consisting of 25 mM Hepes @ 7.5, 1 mM TCEP, 20 nM SMAC-rhodamine with varying concentrations of various BIR domains. For cIAP1-BIR3, assays included 40 mM β-glycerol phosphate. Plates were read on the Analyst and observed mP were plotted against the log of protein concentration.</p

Binding affinities of hydrazine series SMAC mimetic compounds for BIR domains of cIAP1 and cIAP2^a.

<p>Binding affinities of hydrazine series SMAC mimetic compounds for BIR domains of cIAP1 and cIAP2<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#t003fn002" target="_blank">^a</a>.</p