Phosphorylation of the Leukemic Oncoprotein EVI1 on Serine 196 Modulates DNA Binding, Transcriptional Repression and Transforming Ability

The EVI1 (ecotropic viral integration site 1) gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML) is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1 mediates transcriptional regulation, signalling, and epigenetic modifications by interacting with DNA, proteins and protein complexes. To explore to what extent protein phosphorylation impacts on EVI1 functions, we analysed endogenous EVI1 protein from a high EVI1 expressing Fanconi anaemia (FA) derived AML cell line. Mass spectrometric analysis of immunoprecipitated EVI1 revealed phosphorylation at serine 196 (S196) in the sixth zinc finger of the N-terminal zinc finger domain. Mutated EVI1 with an aspartate substitution at serine 196 (S196D), which mimics serine phosphorylation of this site, exhibited reduced DNA-binding and transcriptional repression from a gene promotor selectively targeted by the N-terminal zinc finger domain. Forced expression of the S196D mutant significantly reduced EVI1 mediated transformation of Rat1 fibroblasts. While EVI1-mediated serial replating of murine haematopoietic progenitors was maintained by EVI1-S196D, this was associated with significantly higher Evi1-trancript levels compared with WT-EVI1 or EVI1-S196A, mimicking S196 non-phosphorylated EVI1. These data suggest that EVI1 function is modulated by phosphorylation of the first zinc finger domain

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Stakeholders' inclusion for translating healt systems research into policy and action: expereinces in Africa, China and Latin Amrerica.

info:eu-repo/semantics/nonPublishe

Long-term results of total elbow arthroplasty in patients with hemophilia

HYPOTHESIS: It was hypothesized that the long-term survivorship and clinical outcome are reasonable, justifying total elbow arthroplasty (TEA) in patients with end-stage hemophilic arthropathy. METHODS: From 2002 to 2012, 13 primary TEAs (Coonrad-Morrey design) were implanted in 9 consecutive patients with an average age of 55 (range, 39-76) years. Type A hemophilia was diagnosed in 7 patients and type B hemophilia in 2 patients. Clinical and radiographic results of all (11 TEAs) but 1 patient were retrospectively analyzed. RESULTS: After a mean of 9.1 (range, 5-14) years, the mean visual analog scale score for pain, total Mayo Elbow Performance Score, and subjective elbow value were significantly improved from 5 (standard deviation, ±3) to 2 (±2; P = .007) points, from 64 (±16) to 89 (±11; P = .008) points, and from 47% (±15%) to 81% (±11%; P < .001), respectively. Whereas the flexion arc remained unchanged (P = .279), mean active pronation improved significantly (P = .024). Postoperative complications were recorded in 8 TEAs (62%), whereas 5 TEAs (38%) underwent partial component exchange after a mean of 7.2 (range, 3-10) years: 2 for periprosthetic infection, 2 for polyethylene wear, and 1 for humeral component loosening. Of the living patients after partial component exchange (n = 3), the mean final total Mayo Elbow Performance Score, flexion and rotation arc, visual analog scale score for pain, and subjective elbow value were comparable with the results of the living patients without revision surgery (n = 8). CONCLUSIONS: TEA for patients with advanced hemophilic arthropathy is associated with a substantial complication and revision rate. However, even after revision without implant removal, it provides good functional and subjective long-term results

Serial replating of murine hematopoietic progenitor cells transduced with wtEvi1 and Evi1 phosphorylation site mutants.

<p><b>(A)</b> Western blot analysis of Evi1 expression in PLAT-E cells two days after transfection with retroviral vectors encoding wtEVI1, Evi1-S196A, Evi1-S196D or empty vector control. <b>(B)</b> Total colony numbers (mean±s.d, n = 5) formed in serial replating assays by c-kit positive cells transduced with wtEVI1, S196A, S196D or control retroviral expression vectors. <b>(C)</b> Percentage of cell types (blast, macrophage or other) enumerated by morphological analysis of May-Grünwald Giemsa stained cytospins after the third round of replating. Mean, n = 4. Photomicrographs of May-Grünwald-Giemsa-stained cytospins after the third round of replating of transduced cells expressing WT or mutant EVI1 as shown. Scale bar indicates 50 µM. <b>(D)</b> Photomicrographs of May-Grünwald-Giemsa-stained cytospins after the third round of replating of transduced cells expressing WT or mutant EVI1 as shown. Scale bar indicates 50 µM. <b>(E)</b> Relative <i>Evi1</i> expression in c-kit+ cells after the first round of plating determined by qRT-PCR analysis. Mean±s.d, n = 4. *P<0.01. <b>(G)</b> Sanger-Sequencing of <i>Evi1</i> transcripts from c-kit+ cells after the first round of plating. Electropherograms illustrating DNA-sequence around codon encoding S196. Nucleotide and amino acid sequence (single letter amino acid code) are shown.</p

Structural context of phosphorylation and substitutions at EVI1 S196

<p>. Main chain is shown in white and side chains in blue. Oxygen atoms on residue 196 are indicated by red spheres. (<b>A</b>) The zinc finger domain with unmodified serine, (<b>B</b>) phosphorylated serine, (<b>C</b>) aspartate substitution and (<b>D</b>) alanine substitution. All residues can adopt a similar conformation and extend into the solvent.</p

EVI1 S196 phosphorylation abrogates Rat1 transformation

<p>. (<b>A</b>) Representative photomicrographs of Rat1 fibroblast colonies transduced with wtEVI1 or S196 mutants (S196A and S196D) retroviral expression vectors. (<b>B</b>) Assays were scored after 24 days and colonies enumerated for number and size (mean±s.d., n = 3, *P< 0.05). <b>(C</b>) Detection of Evi1 protein in transduced Rat1 fibroblasts shown by western blot on whole cell lysates using the Evi1-SP137 antibody.</p