E2F and p53 Induce Apoptosis Independently during Drosophila Development but Intersect in the Context of DNA Damage

In mammalian cells, RB/E2F and p53 are intimately connected, and crosstalk between these pathways is critical for the induction of cell cycle arrest or cell death in response to cellular stresses. Here we have investigated the genetic interactions between RBF/E2F and p53 pathways during Drosophila development. Unexpectedly, we find that the pro-apoptotic activities of E2F and p53 are independent of one another when examined in the context of Drosophila development: apoptosis induced by the deregulation of dE2F1, or by the overexpression of dE2F1, is unaffected by the elimination of dp53; conversely, dp53-induced phenotypes are unaffected by the elimination of dE2F activity. However, dE2F and dp53 converge in the context of a DNA damage response. Both dE2F1/dDP and dp53 are required for DNA damage-induced cell death, and the analysis of rbf1 mutant eye discs indicates that dE2F1/dDP and dp53 cooperatively promote cell death in irradiated discs. In this context, the further deregulation in the expression of pro-apoptotic genes generates an additional sensitivity to apoptosis that requires both dE2F/dDP and dp53 activity. This sensitivity differs from DNA damage-induced apoptosis in wild-type discs (and from dE2F/dDP-induced apoptosis in un-irradiated rbf1 mutant eye discs) by being dependent on both hid and reaper. These results show that pro-apoptotic activities of dE2F1 and dp53 are surprisingly separable: dp53 is required for dE2F-dependent apoptosis in the response to DNA damage, but it is not required for dE2F-dependent apoptosis caused simply by the inactivation of rbf1

Transcriptional and Post-Transcriptional Mechanisms for Oncogenic Overexpression of Ether À Go-Go K+ Channel

The human ether-à-go-go-1 (h-eag1) K+ channel is expressed in a variety of cell lines derived from human malignant tumors and in clinical samples of several different cancers, but is otherwise absent in normal tissues. It was found to be necessary for cell cycle progression and tumorigenesis. Specific inhibition of h-eag1 expression leads to inhibition of tumor cell proliferation. We report here that h-eag1 expression is controlled by the p53−miR-34−E2F1 pathway through a negative feed-forward mechanism. We first established E2F1 as a transactivator of h-eag1 gene through characterizing its promoter region. We then revealed that miR-34, a known transcriptional target of p53, is an important negative regulator of h-eag1 through dual mechanisms by directly repressing h-eag1 at the post-transcriptional level and indirectly silencing h-eag1 at the transcriptional level via repressing E2F1. There is a strong inverse relationship between the expression levels of miR-34 and h-eag1 protein. H-eag1antisense antagonized the growth-stimulating effects and the upregulation of h-eag1 expression in SHSY5Y cells, induced by knockdown of miR-34, E2F1 overexpression, or inhibition of p53 activity. Therefore, p53 negatively regulates h-eag1 expression by a negative feed-forward mechanism through the p53−miR-34−E2F1 pathway. Inactivation of p53 activity, as is the case in many cancers, can thus cause oncogenic overexpression of h-eag1 by relieving the negative feed-forward regulation. These findings not only help us understand the molecular mechanisms for oncogenic overexpression of h-eag1 in tumorigenesis but also uncover the cell-cycle regulation through the p53−miR-34−E2F1−h-eag1 pathway. Moreover, these findings place h-eag1 in the p53−miR-34−E2F1−h-eag1 pathway with h-eag as a terminal effecter component and with miR-34 (and E2F1) as a linker between p53 and h-eag1. Our study therefore fills the gap between p53 pathway and its cellular function mediated by h-eag1

An E2F1-Mediated DNA Damage Response Contributes to the Replication of Human Cytomegalovirus

DNA damage resulting from intrinsic or extrinsic sources activates DNA damage responses (DDRs) centered on protein kinase signaling cascades. The usual consequences of inducing DDRs include the activation of cell cycle checkpoints together with repair of the damaged DNA or induction of apoptosis. Many DNA viruses elicit host DDRs during infection and some viruses require the DDR for efficient replication. However, the mechanism by which DDRs are activated by viral infection is poorly understood. Human cytomegalovirus (HCMV) infection induces a DDR centered on the activation of ataxia telangiectasia mutated (ATM) protein kinase. Here we show that HCMV replication is compromised in cells with inactivated or depleted ATM and that ATM is essential for the host DDR early during infection. Likewise, a downstream target of ATM phosphorylation, H2AX, also contributes to viral replication. The ATM-dependent DDR is detected as discrete, nuclear γH2AX foci early in infection and can be activated by IE proteins. By 24 hpi, γH2AX is observed primarily in HCMV DNA replication compartments. We identified a role for the E2F1 transcription factor in mediating this DDR and viral replication. E2F1, but not E2F2 or E2F3, promotes the accumulation of γH2AX during HCMV infection or IE protein expression. Moreover, E2F1 expression, but not the expression of E2F2 or E2F3, is required for efficient HCMV replication. These results reveal a novel role for E2F1 in mediating an ATM-dependent DDR that contributes to viral replication. Given that E2F activity is often deregulated by infection with DNA viruses, these observations raise the possibility that an E2F1-mediated mechanism of DDR activation may be conserved among DNA viruses

Human Papillomaviruses Activate the ATM DNA Damage Pathway for Viral Genome Amplification upon Differentiation

Human papillomaviruses (HPV) are the causative agents of cervical cancers. The infectious HPV life cycle is closely linked to the differentiation state of the host epithelia, with viral genome amplification, late gene expression and virion production restricted to suprabasal cells. The E6 and E7 proteins provide an environment conducive to DNA synthesis upon differentiation, but little is known concerning the mechanisms that regulate productive viral genome amplification. Using keratinocytes that stably maintain HPV-31 episomes, and chemical inhibitors, we demonstrate that viral proteins activate the ATM DNA damage response in differentiating cells, as indicated by phosphorylation of CHK2, BRCA1 and NBS1. This activation is necessary for viral genome amplification, as well as for formation of viral replication foci. In contrast, inhibition of ATM kinase activity in undifferentiated keratinocytes had no effect on the stable maintenance of viral genomes. Previous studies have shown that HPVs induce low levels of caspase 3/7 activation upon differentiation and that this is important for cleavage of the E1 replication protein and genome amplification. Our studies demonstrate that caspase cleavage is induced upon differentiation of HPV positive cells through the action of the DNA damage protein kinase CHK2, which may be activated as a result of E7 binding to the ATM kinase. These findings identify a major regulatory mechanism responsible for productive HPV replication in differentiating cells. Our results have potential implications for the development of anti-viral therapies to treat HPV infections

Non-affirmative Theory of Education as a Foundation for Curriculum Studies, Didaktik and Educational Leadership

This chapter presents non-affirmative theory of education as the foundation for a new research program in education, allowing us to bridge educational leadership, curriculum studies and Didaktik. We demonstrate the strengths of this framework by analyzing literature from educational leadership and curriculum theory/didaktik. In contrast to both socialization-oriented explanations locating curriculum and leadership within existing society, and transformation-oriented models viewing education as revolutionary or super-ordinate to society, non-affirmative theory explains the relation between education and politics, economy and culture, respectively, as non-hierarchical. Here critical deliberation and discursive practices mediate between politics, culture, economy and education, driven by individual agency in historically developed cultural and societal institutions. While transformative and socialization models typically result in instrumental notions of leadership and teaching, non-affirmative education theory, previously developed within German and Nordic education, instead views leadership and teaching as relational and hermeneutic, drawing on ontological core concepts of modern education: recognition; summoning to self-activity and Bildsamkeit. Understanding educational leadership, school development and teaching then requires a comparative multi-level approach informed by discursive institutionalism and organization theory, in addition to theorizing leadership and teaching as cultural-historical and critical-hermeneutic activity. Globalisation and contemporary challenges to deliberative democracy also call for rethinking modern nation-state based theorizing of education in a cosmopolitan light. Non-affirmative education theory allows us to understand and promote recognition based democratic citizenship (political, economical and cultural) that respects cultural, ethical and epistemological variations in a globopolitan era. We hope an American-European-Asian comparative dialogue is enhanced by theorizing education with a non-affirmative approach

ASYMMETRIC SHORTER-DUPLEX SIRNA STRUCTURES TRIGGER EFFICIENT GENE SILENCING WITH REDUCED NONSPECIFIC EFFECTS

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that mediate efficient gene silencing in a sequence-specific manner by utilizing the endogenous RNA interference (RNAi) pathway. The current standard synthetic siRNA structure harbors a 19-base-pair duplex region with 3' overhangs of 2 nucleotides (the so-called 19+2 form). However, the synthetic 19+2 siRNA structure exhibits several sequence-independent, nonspecific effects, which has posed challenges to the development of RNAi therapeutics and specific silencing of genes in research. In this study, we report on the identification of truncated siRNA backbone structures with duplex regions shorter than 19 bp (referred to as asymmetric shorter-duplex siRNAs or asiRNAs) that can efficiently trigger gene silencing in human cell lines. Importantly, this asiRNA structure significantly reduces nonspecific effects triggered by conventional 19+2 siRNA scaffold, such as sense-strand-mediated off-target gene silencing and saturation of the cellular RNAi machinery. Our results suggest that this asiRNA structure is an important alternative to conventional siRNAs for both functional genomics studies and therapeutic applications.X116469sciescopu

What does distributed cognition tell us about student learning of science?

This paper reports multi-layered analyses of student learning in a science classroom using the theoretical lens of Distributed Cognition (Hollan et al. 1999; Hutchins 1995). Building on the insights generated from previous research employing Distributed Cognition, the particular focus of this study has been placed on the “public space of interaction” (Alac and Hutchins 2004, p. 639) that includes both participants’ interaction with each other and their interaction with artefacts in their environment. In this paper, a lesson from an Australian science classroom was examined in detail, in which a class of grade-seven students were investigating the scientific theme of gravity by designing pendulums. The video-stimulated post-lesson interviews with both the teacher and the student groups offered complementary accounts (Clarke 2001a) that assisted the interpretation of the classroom data. The findings of this study provide supporting evidence to demonstrate the capacity of Distributed Cognition for advancing our understanding of the nature of learning in science classrooms