Viral Single-Strand DNA Induces p53-Dependent Apoptosis in Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication

Human Embryonic Stem Cells Have Constitutively Active Bax at the Golgi and Are Primed to Undergo Rapid Apoptosis

Human embryonic stem (hES) cells activate a rapid apoptotic response after DNA damage but the underlying mechanisms are unknown. A critical mediator of apoptosis is Bax, which is reported to become active and translocate to the mitochondria only after apoptotic stimuli. Here we show that undifferentiated hES cells constitutively maintain Bax in its active conformation. Surprisingly, active Bax was maintained at the Golgi rather than at the mitochondria, thus allowing hES cells to effectively minimize the risks associated with having pre-activated Bax. After DNA damage, active Bax rapidly translocated to the mitochondria by a p53-dependent mechanism. Interestingly, upon differentiation, Bax was no longer active and cells were not acutely sensitive to DNA damage. Thus, maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death, likely to prevent the propagation of mutations during the early critical stages of embryonic development

The p53 tumour suppressor inhibits glucocorticoid‐induced proliferation of erythroid progenitors

Hypoxia encountered at high altitude, blood loss and erythroleukemia instigate stress erythropoiesis, which involves glucocorticoid-induced proliferation of erythroid progenitors (ebls). The tumour suppressor p53 stimulates hematopoietic cell maturation and antagonizes glucocorticoid receptor (GR) activity in hypoxia, suggesting that it may inhibit stress erythropoiesis. We report that mouse fetal liver ebls that lack p53 proliferate better than wild-type cells in the presence of the GR agonist dexamethasone. An important mediator of GR-induced ebl self-renewal, the c-myb gene, is induced to higher levels in p53(–/–) ebls by dexamethasone. The stress response to anemia is faster in the spleens of p53(–/–) mice, as shown by the higher levels of colony forming units erythroids and the increase in the CD34/c-kit double positive population. Our results show that p53 antagonizes GR-mediated ebl expansion and demonstrate for the first time that p53–GR cross-talk is important in a physiological process in vivo: stress erythropoiesis

Uncommon cause for anterior knee pain - Aggressive aneurysmal bone cyst of the patella

A 56-year-old man presented with a two month history of increasing anterior knee pain without previous trauma. As usual we recommended physiotherapy with stretching exercises of the quadriceps muscle. Since symptoms did not improve after 6 weeks MRI was performed. Surprisingly a hyperintense lobulated mass of the patella with small fluid-filled cavities at the inferior pole was revealed. We performed an open biopsy to exclude any malignancy and diagnosed an aneurysmal bone cyst. Further examination with CT scans showed an aggressive behaviour with cortical breakthrough

Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm

Sox2 is expressed in developing foregut endoderm, with highest levels in the future esophagus and anterior stomach. By contrast, Nkx2.1 (Titf1) is expressed ventrally, in the future trachea. In humans, heterozygosity for SOX2 is associated with anopthalmiaesophageal-genital syndrome (OMIM 600992), a condition including esophageal atresia (EA) and tracheoesophageal fistula (TEF), in which the trachea and esophagus fail to separate. Mouse embryos heterozygous for the null allele, Sox2EGFP, appear normal. However, further reductions in Sox2, using Sox2LP and Sox2COND hypomorphic alleles, result in multiple abnormalities. Approximately 60% of Sox2EGFP/COND embryos have EA with distal TEF in which Sox2 is undetectable by immunohistochemistry or western blot. The mutant esophagus morphologically resembles the trachea, with ectopic expression of Nkx2.1, a columnar, ciliated epithelium, and very few p63+ basal cells. By contrast, the abnormal foregut of Nkx2.1-null embryos expresses elevated Sox2 and p63, suggesting reciprocal regulation of Sox2 and Nkx2.1 during early dorsal/ventral foregut patterning. Organ culture experiments further suggest that FGF signaling from the ventral mesenchyme regulates Sox2 expression in the endoderm. In the 40% Sox2EGFP/COND embryos in which Sox2 levels are ~18% of wild type there is no TEF. However, the esophagus is still abnormal, with luminal mucus-producing cells, fewer p63+ cells, and ectopic expression of genes normally expressed in glandular stomach and intestine. In all hypomorphic embryos the forestomach has an abnormal phenotype, with reduced keratinization, ectopic mucus cells and columnar epithelium. These findings suggest that Sox2 plays a second role in establishing the boundary between the keratinized, squamous esophagus/forestomach and glandular hindstomach

The Prosensory Function of Sox2 in the Chicken Inner Ear Relies on the Direct Regulation of Atoh1

The proneural gene Atoh1 is crucial for the development of inner ear hair cells and it requires the function of the transcription factor Sox2 through yet unknown mechanisms. In the present work, we used the chicken embryo and HEK293T cells to explore the regulation of Atoh1 by Sox2. The results show that hair cells derive from Sox2-positive otic progenitors and that Sox2 directly activates Atoh1 through a transcriptional activator function that requires the integrity of Sox2 DNA binding domain. Atoh1 activation depends on Sox transcription factor binding sites (SoxTFBS) present in the Atoh1 3′ enhancer where Sox2 directly binds, as shown by site directed mutagenesis and chromatin immunoprecipitation (ChIP). In the inner ear, Atoh1 enhancer activity is detected in the neurosensory domain and it depends on Sox2. Dominant negative competition (Sox2HMG-Engrailed) and mutation of the SoxTFBS abolish the reporter activity in vivo. Moreover, ChIP assay in isolated otic vesicles shows that Sox2 is bound to the Atoh1 enhancer in vivo. However, besides activating Atoh1, Sox2 also promotes the expression of Atoh1 negative regulators and the temporal profile of Atoh1 activation by Sox2 is transient suggesting that Sox2 triggers an incoherent feed-forward loop. These results provide a mechanism for the prosensory function of Sox2 in the inner ear. We suggest that sensory competence is established early in otic development through the activation of Atoh1 by Sox2, however, hair cell differentiation is prevented until later stages by the parallel activation of negative regulators of Atoh1 function

The role of neuregulin-ErbB4 interactions on the proliferation and organization of cells in the subventricular zone

Coordinated regulation of neuronal progenitor differentiation in the subventricular zone (SVZ) is a fundamental feature of adult neurogenesis. However, the molecular control of this process remains mostly undeciphered. Here, we investigate the role of neuregulins (NRGs) in this process and show that a NRG receptor, ErbB4, is primarily expressed by polysialylated neural cell adhesion molecule immature neuroblasts but is also detected in a subset of GFAP+ astroglial cells, ependymal cells, and Dlx2+ precursors in the SVZ. Of the NRG ligands, both NRG1 and -2 are expressed by immature polysialylated neural cell adhesion molecule neuroblasts in the SVZ. NRG2 is also expressed by some of the GFAP+ putative stem cells lining the ventricles. Infusion of exogenous NRG1 leads to rapid aggregation of Dlx2+ cells in the SVZ and affects the initiation and maintenance of organized neuroblast migration from the SVZ toward the olfactory bulb. In contrast, the infusion of NRG2 increased the number of Sox2 and GFAP+ precursors in the SVZ. An outcome of this NRG2 effect is an increase in the number of newly generated migrating neuroblasts in the rostral migratory stream and GABAergic interneurons in the olfactory bulb. The analysis of conditional null mice that lack NRG receptor, ErbB4, in the nervous system revealed that the observed activities of NRG2 require ErbB4 activation. These results indicate that different NRG ligands affect distinct populations of differentiating neural precursors in the neurogenic regions of the mature forebrain. Furthermore, these studies identify NRG2 as a factor capable of promoting SVZ proliferation, leading to the formation of new neurons in vivo

The role of neuregulin-ErbB4 interactions on the proliferation and organization of cells in the subventricular zone

Coordinated regulation of neuronal progenitor differentiation in the subventricular zone (SVZ) is a fundamental feature of adult neurogenesis. However, the molecular control of this process remains mostly undeciphered. Here, we investigate the role of neuregulins (NRGs) in this process and show that a NRG receptor, ErbB4, is primarily expressed by polysialylated neural cell adhesion molecule immature neuroblasts but is also detected in a subset of GFAP+ astroglial cells, ependymal cells, and Dlx2+ precursors in the SVZ. Of the NRG ligands, both NRG1 and -2 are expressed by immature polysialylated neural cell adhesion molecule neuroblasts in the SVZ. NRG2 is also expressed by some of the GFAP+ putative stem cells lining the ventricles. Infusion of exogenous NRG1 leads to rapid aggregation of Dlx2+ cells in the SVZ and affects the initiation and maintenance of organized neuroblast migration from the SVZ toward the olfactory bulb. In contrast, the infusion of NRG2 increased the number of Sox2 and GFAP+ precursors in the SVZ. An outcome of this NRG2 effect is an increase in the number of newly generated migrating neuroblasts in the rostral migratory stream and GABAergic interneurons in the olfactory bulb. The analysis of conditional null mice that lack NRG receptor, ErbB4, in the nervous system revealed that the observed activities of NRG2 require ErbB4 activation. These results indicate that different NRG ligands affect distinct populations of differentiating neural precursors in the neurogenic regions of the mature forebrain. Furthermore, these studies identify NRG2 as a factor capable of promoting SVZ proliferation, leading to the formation of new neurons in vivo

Syntheses, structures and redox properties of tris(pyrazolyl)borate-capped ruthenium vinyl complexes.

Reaction of RuHCl(CO)(PPh3)3 with aryl alkynes HCCC6H4R-4 [1: R = N(C6H4Me-4)2 (a), OMe (b), Me (c), CO2Me (d), NO2 (e)] gives the five-coordinate vinyl complexes Ru(CHCHC6H4R-4)Cl(CO)(PPh3)2 (2a–e). Reaction of 2a with excess PMe3 gives crystallographically characterised Ru{CHCHC6H4N(C6H4Me-4)2-4}Cl(CO)(PMe3)3 (3a), whilst reaction of 2a–e with KTp affords Ru(CHCHC6H4R-4)(CO)(PPh3)Tp (4a–e) bearing the facially capping Tp− ligand. Electrochemical and spectroelectochemical properties of 4a–e are consistent with substantial redox activity associated with the vinyl ligand, and these properties have been satisfactorily modelled by DFT based calculations of electronic structure