Coulomb Excitation of Proton-rich N = 80 Isotones at HIE-ISOLDE

A projectile Coulomb-excitation experiment was performed at the radioactive ion beam facility HIE-ISOLDE at CERN. The radioactive ¹⁴⁰Nd and ¹⁴²Sm ions were post accelerated to the energy of 4.62 MeV/A and impinged on a 1.45 mg/cm²-thin ²⁰⁸Pb target. The γ rays depopulating the Coulomb-excited states were recorded by the HPGe-array MINIBALL. The scattered charged particles were detected by a double-sided silicon strip detector in forward direction. Experimental γ-ray intensities were used for the determination of electromagnetic transition matrix elements. Preliminary results for the reduced transition strength of the B(M1;23+→21+)=0.35(19)μN2 of ¹⁴⁰Nd and a first estimation for ¹⁴²Sm have been deduced using the Coulomb-excitation calculation software GOSIA. The 2³₊ states of ¹⁴⁰Nd and ¹⁴²Sm show indications of being the main fragment of the proton-neutron mixed-symmetry 2⁺₁,ms state

IDH1R132H in Neural Stem Cells: Differentiation Impaired by Increased Apoptosis.

BACKGROUND:The high frequency of mutations in the isocitrate dehydrogenase 1 (IDH1) gene in diffuse gliomas indicates its importance in the process of gliomagenesis. These mutations result in loss of the normal function and acquisition of the neomorphic activity converting α-ketoglutarate to 2-hydroxyglutarate. This potential oncometabolite may induce the epigenetic changes, resulting in the deregulated expression of numerous genes, including those related to the differentiation process or cell survivability. METHODS:Neural stem cells were derived from human induced pluripotent stem cells following embryoid body formation. Neural stem cells transduced with mutant IDH1R132H, empty vector, non-transduced and overexpressing IDH1WT controls were differentiated into astrocytes and neurons in culture. The neuronal and astrocytic differentiation was determined by morphology and expression of lineage specific markers (MAP2, Synapsin I and GFAP) as determined by real-time PCR and immunocytochemical staining. Apoptosis was evaluated by real-time observation of Caspase-3 activation and measurement of PARP cleavage by Western Blot. RESULTS:Compared with control groups, cells expressing IDH1R132H retained an undifferentiated state and lacked morphological changes following stimulated differentiation. The significant inhibitory effect of IDH1R132H on neuronal and astrocytic differentiation was confirmed by immunocytochemical staining for markers of neural stem cells. Additionally, real-time PCR indicated suppressed expression of lineage markers. High percentage of apoptotic cells was detected within IDH1R132H-positive neural stem cells population and their derivatives, if compared to normal neural stem cells and their derivatives. The analysis of PARP and Caspase-3 activity confirmed apoptosis sensitivity in mutant protein-expressing neural cells. CONCLUSIONS:Our study demonstrates that expression of IDH1R132H increases apoptosis susceptibility of neural stem cells and their derivatives. Robust apoptosis causes differentiation deficiency of IDH1R132H-expressing cells

Expression of glial and neuronal markers by differentiated ebiNSc.

<p>Double staining with antibodies to GFAP (red) and MAP2 (green) after 14 days of differentiation of non-transduced ebiNSc showing populations of astrocytic and neuronal cells. Photomicrographs demonstrate <b>(A)</b> Merged image of GFAP (red), MAP2 (green) and DAPI (blue) showing populations of GFAP and MAP2 positive cells with some overlap between the two markers; DAPI was used to estimate the percentage of cells positive for GFAP and MAP2; <b>(B)</b> GFAP (red); <b>(C)</b> MAP2 (green). The images at 200x magnification, scale bars mark 100μm.</p

Primers sequences.

<p>Primers sequences.</p

IDH1 expression in ebiNSc.

<p><b>(A)</b> Endogenous wild type IDH1 expression in the non-transuded ebiNSc controls. Arrows mark the characteristic punctuate expression pattern. <b>(B)</b> Lack of the endogenous IDH1^R132H expression in the non-transuded ebiNSc controls. <b>(C)</b> Expression of wild type IDH1 in ebiNSc transduced with wild type gene, ebiNSc^IDH1wt. Arrows mark the punctuate pattern characteristic for the endogenous IDH1. Arrowheads mark the strong, diffuse pattern of the induced expression. <b>(D)</b> Expression of IDH1^R132H in ebiNSc transduced with the mutant gene, ebiNSc^IDH1R132H. Arrows mark cells lacking the induced expression. Arrowheads mark the strong, diffuse pattern of the induced expression. <b>(E)</b> Expression of endogenous wild type IDH1 in ebiNSc transduced with the empty vector, ebiNSc^empty. Arrows mark the characteristic punctuate expression pattern. Each image was taken at magnification 400x; scale bars mark 50μm. <b>(F)</b> <i>IDH1</i> (non-mutation-specific) expression at the mRNA level in the four cultures Error bars indicate SEM. Statistical significance calculated by One-way ANOVA with Tukey's post-comparisons test. ***, p<0.005; ns, not significant.</p

Expression of stem cell markers by undifferentiated ebiNSc.

<p>Double staining with antibodies against Nestin (red) and SOX2 (green) showing expression of neural stem cell markers in the same cells. Each image was taken at magnification 400x; scale bars mark 50μm.</p

IDH1^R132H increases apoptosis susceptibility of induced neural stem cells and their derivatives.

<p><b>(A)</b> Micrographs showing apoptotic cells in control ebiNSc and ebiNSc^IDH1R132H cultured in non-differentiating medium with synthetic reporter of Caspase 3/7 activity. Increased apoptosis visible in IDH1^R132H-expressing cells. <b>(B)</b> Apoptotic cells in control ebiNSc and ebiNSc^IDH1R132H after 7 days of differentiation. Increased number of apoptotic cells was observed in ebiNSc^IDH1R132H cells compared to differentiating control ebiNSc. Experiment was conducted in a manner similar to that described in A. Each image was taken at magnification 100x, scale bars mark 50μm. <b>(C)</b> Western Blot analysis for PARP in non-transduced ebiNSc and ebiNSc^IDH1R132H under differentiating and self-renewing conditions. In case of undifferentiated cells (0d), more cleaved form of PARP was observed in ebiNSc^IDH1R132H than in control. The decrease in PARP level during differentiation of ebiNSc^IDH1R132H may be correlated with reduced total protein amount as indicated by actin.</p

Expression of wild type IDH1 protein in different cell types.

<p>Endogenous wild type IDH1 expression in the ebiNSc, neurospheres derived from glioblastoma primary culture, neurons and astrocytes. Each image was taken at magnification 400x; scale bars mark 50μm.</p