Journal: eLife

Article Title: Activation of targetable inflammatory immune signaling is seen in myelodysplastic syndromes with SF3B1 mutations

doi: 10.7554/eLife.78136

Figure Lengend Snippet: ( A ) CDK2 structure (PDB:1GY3) bound to ATP is shown in cartoon representation. The putative TRAF6 binding peptide (magenta) is located at a loop between αG and αH in the C-lobe of the kinase. Sequence alignment of CDK2 peptide with common binding sequences with TRAF6. The consensus pattern with conserved P-2, P0, and P3 positions is shown at the bottom. ( B ) MM-GBSA docking of CDK2 peptide to substrate binding domain of TRAF6 (PDB:1LB5). The top 1 populated cluster is shown. TRAF6 and CDK2 peptide are shown in ribbon and surface representation, respectively. Positions P-2, P0, and P3 correspond to three distinct sub-pockets in the binding site, formed largely by residues Y473, M450, G472 (pink), A458, G470, K469 (yellow), and H376, V374, R392 (white). Capital lettering of conserved residues P, E, and W within the shown peptides depicts successful docking in corresponding sub-pockets. ( C ) Lys63-linked proteins were immunoprecipitated in K562 cells with wild type (WT) and K700E SF3B1 mutation and CDK2 ubiquitination was determined by CDK2 specific antibody. ( D ) Ubiquitination of CDK2 was determined in HEK-293T cells transfected with indicated plasmids by immunoprecipitating with HA specific antibody and immunoblotting with either Lys48 or Lys63 antibody. ( E ) Lys63-linked proteins were immunoprecipitated in HEK-293T cells transfected with indicated plasmids and CDK2 ubiquitination was determined by Myc-tag specific antibody. Sequence alignment of optimum amino-acids sequence predicted for TRAF6 with WT and mutant CDK2 (CDK2-122 and CDK2-123) ( F–G ) Myelodysplastic syndromes (MDS) patient derived samples (bone marrow/peripheral blood) were treated with DMSO or Dinaciclib (10 nM) or AT7519 (10 nM) for 14 days on methylcellulose clonogenic assays. The samples were evaluated for colony formation CFU-GM (colony forming unit - granulocyte macrophage) and for myeloid differentiation on colonies and were subjected to flow cytometry analysis. Figure 3—source data 1. Ubiquitination of CDK2 in HEK293T cells. Figure 3—source data 2. Ubiquitination of CDK2 mutants in HEK293T cells. Figure 3—source data 3. Ubiquitination of CDK2 in K562 CTL and K700E cells.

Article Snippet: The antibodies used for flow cytometric analysis of human MDS and AML cells included Mouse CD45 FITC; Human CD45 PeCy7 (e-Bioscience); Human CD4-Pacific Orange; Human CD8-Pacific Orange; Human CD19-Pacific Blue; Human CD20-Pacific Blue; Human CD11b-APC (Thermo Fisher); Human CD34-PE (Miltenyi Biotec);and Human CD33-APC-Cy7 (Abcam).

Techniques: Binding Assay, Sequencing, Immunoprecipitation, Mutagenesis, Ubiquitin Proteomics, Transfection, Western Blot, Derivative Assay, Flow Cytometry

Journal: eLife

Article Title: Activation of targetable inflammatory immune signaling is seen in myelodysplastic syndromes with SF3B1 mutations

doi: 10.7554/eLife.78136

Figure Lengend Snippet: ( A ) MMGBSA docking (see Materials and methods) of CDK2 peptide to substrate binding domain of TRAF6 (PDB:1LB5). Various potential clusters are shown. TRAF6 and CDK2 peptide are shown in ribbon and surface representation, respectively. High-lettering of conserved residues P, E, W within the shown peptides depicts successful docking in corresponding sub-pockets. ( B ) Lys63 ubiquitination of CDK2 was determined in myelodysplastic syndromes (MDS)-L cells treated with IRAK4 inhibitors. Immunoprecipitation with CDK2 and immunoblotting with anti-Lys63 ubiquitin antibody shows decreased overall smear after IRAK4 inhibition. Figure 3—figure supplement 1—source data 1. Ubiquitination of CDK2 in MDS-L cells with IRAK4 inhibitors CA-4948 (Emavusertib) and PF06650833.

Article Snippet: The antibodies used for flow cytometric analysis of human MDS and AML cells included Mouse CD45 FITC; Human CD45 PeCy7 (e-Bioscience); Human CD4-Pacific Orange; Human CD8-Pacific Orange; Human CD19-Pacific Blue; Human CD20-Pacific Blue; Human CD11b-APC (Thermo Fisher); Human CD34-PE (Miltenyi Biotec);and Human CD33-APC-Cy7 (Abcam).

Techniques: Binding Assay, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Inhibition

Journal: eLife

Article Title: Activation of targetable inflammatory immune signaling is seen in myelodysplastic syndromes with SF3B1 mutations

doi: 10.7554/eLife.78136

Figure Lengend Snippet: ( A ) IRAK4 expression in 183 samples from MDS patient bone marrow CD34+ cells was correlated with platelet counts, RBC transfusion dependency, and blast counts. Cohort with high IRAK4 expression (>median) were associated with lower platelet counts, higher transfusion dependence, and higher blast counts (t-test, p<0.05). ( B ) NSG mice were xenografted with MDS cells with SF3B1 mutation. After engraftment, mice were treated with either vehicle or IRAK4 inhibitor (CA-4849, 12.5 mg/kg) and bone marrow aspirates were used to evaluate human cells engraftment by flow cytometry. ( C–D ) Summary of human cell engraftment for xenografted mice treated with either IRAK4 inhibitor (CA-4948) or vehicle. Representative MDS sample xenograft with SF3B1 K700E mutation is shown. ( E ) IRAK4 expression in sorted HSCs (Lineage –ve, CD34+, CD38−) cells from MDS/acute myeloid leukemia (AML) cases (N=9) and controls (N=5). Significantly increased IRAK4 expression is seen in samples with complex karyotype (CK) when compared to HC (t-test, p<0.05). ( F ) Gene expression signature associated with high IRAK4 expression (>median, from set of 183 MDS CD34+ cells) was compared to know leukemic stem cell signature and showed significant similarity by GSEA analysis. ( G–H ) MDS bone marrow cells were isolated and purified from mice either treated with vehicle or CA-4948 in and then xenografted in secondary recipient NSG mice. Human cell engraftment was evaluated after 4 weeks by flow cytometry on bone marrow aspirates.

Article Snippet: The antibodies used for flow cytometric analysis of human MDS and AML cells included Mouse CD45 FITC; Human CD45 PeCy7 (e-Bioscience); Human CD4-Pacific Orange; Human CD8-Pacific Orange; Human CD19-Pacific Blue; Human CD20-Pacific Blue; Human CD11b-APC (Thermo Fisher); Human CD34-PE (Miltenyi Biotec);and Human CD33-APC-Cy7 (Abcam).

Techniques: Expressing, Mutagenesis, Flow Cytometry, Gene Expression, Isolation, Purification

Journal: eLife

Article Title: Activation of targetable inflammatory immune signaling is seen in myelodysplastic syndromes with SF3B1 mutations

doi: 10.7554/eLife.78136

Figure Lengend Snippet: Proposed schematic of myelodysplastic syndromes (MDS) pathogenesis due to mis-splicing of IRAK4 because of SF3B1 mutations.

Article Snippet: The antibodies used for flow cytometric analysis of human MDS and AML cells included Mouse CD45 FITC; Human CD45 PeCy7 (e-Bioscience); Human CD4-Pacific Orange; Human CD8-Pacific Orange; Human CD19-Pacific Blue; Human CD20-Pacific Blue; Human CD11b-APC (Thermo Fisher); Human CD34-PE (Miltenyi Biotec);and Human CD33-APC-Cy7 (Abcam).

Techniques:

Journal: Oncology Letters

Article Title: Integrated bioinformatic analysis of microarray data reveals shared gene signature between MDS and AML

doi: 10.3892/ol.2018.9237

Figure Lengend Snippet: Summary of individual studies included in the meta-analysis.

Article Snippet: Sternberg et al , 2005 , GSE2779 , MDS , Bone marrow CD34+ cells , Affymetrix human genome U133A array , ( ) .

Techniques: Microarray, Expressing

Journal: Oncology Letters

Article Title: Integrated bioinformatic analysis of microarray data reveals shared gene signature between MDS and AML

doi: 10.3892/ol.2018.9237

Figure Lengend Snippet: Summary of individual studies included in the meta-analysis.

Article Snippet: Pellagatti et al , 2006 , GSE4619 , MDS , Bone marrow CD34+ cells , Affymetrix human genome U133 plus 2.0 array , ( ) .

Techniques: Microarray, Expressing

Journal: Metabolites

Article Title: Analysis of Different Methods of Extracting NSAIDs in Biological Fluid Samples for LC-MS/MS Assays: Scoping Review

doi: 10.3390/metabo12080751

Figure Lengend Snippet: Systematic investigation and analysis of different drug extraction methods.

Article Snippet: Banda, 2016 India, [ ] , Bioanalytical Assay , Olsalazine Sodium , UHPLC (Shimadzu, Kyoto, Japan) MS/MS Triple quadrupole API-6500 (MDS Sciex, Ontário, Canada) , Turbo Ion spray , LLE , Human Plasma.

Techniques: Liquid Chromatography, High Performance Liquid Chromatography, Blocking Assay, Solid-phase Microextraction

Journal: Cell stem cell

Article Title: Human iPSC-derived cerebral organoids model cellular features of lissencephaly and reveal prolonged mitosis of outer radial glia

doi: 10.1016/j.stem.2016.12.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human MDS iPSC lines “MDS3-3” and “MDS3-4” from 18 fetal weeks male , Coriell Institute for Medical Research (GM09208-fibroblasts); Bershteyn et al., 2014 (iPSC lines) , MDS3-3/MDS3-4.

Techniques: Recombinant, cDNA Synthesis, Sample Prep, RNA Sequencing, Derivative Assay, Plasmid Preparation, Software

Journal: Bioinformatics

Article Title: DECO: decompose heterogeneous population cohorts for patient stratification and discovery of sample biomarkers using omic data profiling

doi: 10.1093/bioinformatics/btz148

Figure Lengend Snippet: Datasets for assessing DECO in comparison with other methods

Article Snippet: Myelodysplastic syndrome MDS-1 , Bone marrow CD34+ Cells , Affymetrix Human GenomeU133 Plus2.0 , 41 , RMA , RAEB1-MDS, RAEB2-MDS , Supervised (two classes) , RAEB1 , RAEB2 , GSE19429 (GEO).

Techniques: