Designed Azolopyridinium Salts Block Protective Antigen Pores In Vitro and Protect Cells from Anthrax Toxin

Background:Several intracellular acting bacterial protein toxins of the AB-type, which are known to enter cells by endocytosis, are shown to produce channels. This holds true for protective antigen (PA), the binding component of the tripartite anthrax-toxin of Bacillus anthracis. Evidence has been presented that translocation of the enzymatic components of anthrax-toxin across the endosomal membrane of target cells and channel formation by the heptameric/octameric PA63 binding/translocation component are related phenomena. Chloroquine and some 4-aminoquinolones, known as potent drugs against Plasmodium falciparium infection of humans, block efficiently the PA63-channel in a dose dependent way.Methodology/Principal Findings:Here we demonstrate that related positively charged heterocyclic azolopyridinium salts block the PA63-channel in the μM range, when both, inhibitor and PA63 are added to the same side of the membrane, the cis-side, which corresponds to the lumen of acidified endosomal vesicles of target cells. Noise-analysis allowed the study of the kinetics of the plug formation by the heterocycles. In vivo experiments using J774A.1 macrophages demonstrated that the inhibitors of PA63-channel function also efficiently block intoxication of the cells by the combination lethal factor and PA63 in the same concentration range as they block the channels in vitro.Conclusions/Significance:These results strongly argue in favor of a transport of lethal factor through the PA63-channel and suggest that the heterocycles used in this study could represent attractive candidates for development of novel therapeutic strategies against anthrax. © 2013 Beitzinger et al

The use of rapid review methods in health technology assessments: 3 case studies.

BACKGROUND: Rapid reviews are of increasing importance within health technology assessment due to time and resource constraints. There are many rapid review methods available although there is little guidance as to the most suitable methods. We present three case studies employing differing methods to suit the evidence base for each review and outline some issues to consider when selecting an appropriate method. METHODS: Three recently completed systematic review short reports produced for the UK National Institute for Health Research were examined. Different approaches to rapid review methods were used in the three reports which were undertaken to inform the commissioning of services within the NHS and to inform future trial design. We describe the methods used, the reasoning behind the choice of methods and explore the strengths and weaknesses of each method. RESULTS: Rapid review methods were chosen to meet the needs of the review and each review had distinctly different challenges such as heterogeneity in terms of populations, interventions, comparators and outcome measures (PICO) and/or large numbers of relevant trials. All reviews included at least 10 randomised controlled trials (RCTs), each with numerous included outcomes. For the first case study (sexual health interventions), very diverse studies in terms of PICO were included. P-values and summary information only were presented due to substantial heterogeneity between studies and outcomes measured. For the second case study (premature ejaculation treatments), there were over 100 RCTs but also several existing systematic reviews. Data for meta-analyses were extracted directly from existing systematic reviews with new RCT data added where available. For the final case study (cannabis cessation therapies), studies included a wide range of interventions and considerable variation in study populations and outcomes. A brief summary of the key findings for each study was presented and narrative synthesis used to summarise results for each pair of interventions compared. CONCLUSIONS: Rapid review methods need to be chosen to meet both the nature of the evidence base of a review and the challenges presented by the included studies. Appropriate methods should be chosen after an assessment of the evidence base

Do critical thinkers drink too much alcohol, forget to do class assignments, or cheat on exams? Using a critical thinking measure to predict college students’ real-world outcomes

Critical thinking is a higher-order way of reasoning composed of the skill and will to use cognitive abilities and knowledge on a daily basis. It is identified as essential by higher education institutions, corporations, and society in general. To analyze whether college students are critical thinkers in their daily lives, the Halpern Critical Thinking Assessment (HCTA; Halpern in Halpern Critical Thinking Assessment (Measurement instrument), Schuhfried, Mödling, 2012) and the real-world outcomes inventory (RWO; Butler in Appl Cogn Psychol 26(5):721–729, 2012) were administered to 238 students. We performed a cluster analysis (K-means-constrained clustering method), and ANOVAs for each cluster solution tested to identify the most suitable clustering solution, taking the RWO inventory dimensions as dependent variables and cluster membership as an independent variable. Four separate clusters emerged, each representing a different profile related to students’ everyday negative outcomes resulting from a lack of critical thinking. We performed multinomial logistic regression to examine which dimensions of the HCTA test, as well as gender, age, and disciplinary area, predicted the four singular groups of students that emerged: “Mature,” “Risk-taking,” “Lost in translation,” and “Reflective.” Results indicate that: (1) age is a relevant predictor of slackness, rashness, and health neglect, all characteristics of “Mature” students; (2) students who are particularly skilled in hypothesis testing tend to be “Risk-taking,” while it is less likely that students who are specifically competent in argument analysis will be in this group; (3) gender is relevant to predict “Lost in translation” students, while argument analysis is negatively related to the chances of being in this group. Our study supports the relevance of critical thinking in daily decisions and everyday outcomes.FCT -Fundação para a Ciência e a Tecnologia(Advanced Training)info:eu-repo/semantics/publishedVersio

Approaching Proof in the Classroom Through the Logic of Inquiry

The paper analyses a basic gap, highlighted by most of the literature concerning the teaching of proofs, namely, the distance between students' argumentative and proving processes. The analysis is developed from both epistemological and cognitive standpoints: it critiques the Toulmin model of reasoning and introduces a new model, the Logic of Inquiry of Hintikka, more suitable for bridging this gap. An example of didactical activity within Dynamic Geometry Environments is sketched in order to present a concrete illustration of this approach and to show the pedagogical effectiveness of the model

Tumor Endothelial Marker 8 Amplifies Canonical Wnt Signaling in Blood Vessels

Tumor Endothelial Marker 8/Anthrax Toxin Receptor 1 (TEM8/ANTXR1) expression is induced in the vascular compartment of multiple tumors and therefore, is a candidate molecule to target tumor therapies. This cell surface molecule mediates anthrax toxin internalization, however, its physiological function in blood vessels remains largely unknown. We identified the chicken chorioallantoic membrane (CAM) as a model system to study the endogenous function of TEM8 in blood vessels as we found that TEM8 expression was induced transiently between day 10 and 12 of embryonic development, when the vascular tree is undergoing final development and growth. We used the cell-binding component of anthrax toxin, Protective Antigen (PA), to engage endogenous TEM8 receptors and evaluate the effects of PA-TEM8 complexes on vascular development. PA applied at the time of highest TEM8 expression reduced vascular density and disrupted hierarchical branching as revealed by quantitative morphometric analysis of the vascular tree after 48h. PA-dependent reduced branching phenotype was partially mimicked by Wnt3a application and ameliorated by the Wnt antagonist, Dikkopf-1. These results implicate TEM8 expression in endothelial cells in regulating the canonical Wnt signaling pathway at this day of CAM development. Consistent with this model, PA increased beta catenin levels acutely in CAM blood vessels in vivo and in TEM8 transfected primary human endothelial cells in vitro. TEM8 expression in Hek293 cells, which neither express endogenous PA-binding receptors nor Wnt ligands, stabilized beta catenin levels and amplified beta catenin-dependent transcriptional activity induced by Wnt3a. This agonistic function is supported by findings in the CAM, where the increase in TEM8 expression from day 10 to day 12 and PA application correlated with Axin 2 induction, a universal reporter gene for canonical Wnt signaling. We postulate that the developmentally controlled expression of TEM8 modulates endothelial cell response to canonical Wnt signaling to regulate vessel patterning and density

Anthrax Edema Toxin Modulates PKA- and CREB-Dependent Signaling in Two Phases

Background: Anthrax edema toxin (EdTx) is an adenylate cyclase which operates in the perinuclear region of host cells. However, the action of EdTx is poorly understood, especially at molecular level. The ability of EdTx to modulate cAMPdependent signaling was studied in Jurkat T cells and was compared with that of other cAMP-rising agents: Bordetella pertussis adenylate cyclase toxin, cholera toxin and forskolin. Methodology/Principal Findings: EdTx caused a prolonged increase of the intracellular cAMP concentration. This led to nuclear translocation of the cAMP-dependent protein kinase (PKA) catalytic subunit, phosphorylation of cAMP response element binding protein (CREB) and expression of a reporter gene under control of the cAMP response element. Neither p90 ribosomal S6 kinase nor mitogen- and stress-activated kinase, which mediate CREB phosphorylation during T cell activation, were involved. The duration of phospho-CREB binding to chromatin correlated with the spatio-temporal rise of cAMP levels. Strikingly, EdTx pre-treated T cells were unresponsive to other stimuli involving CREB phosphorylation such as addition of forskolin or T cell receptor cross-linking. Conclusions/Significance: We concluded that, in a first intoxication phase, EdTx induces PKA-dependent signaling, which culminates in CREB phosphorylation and activation of gene transcription. Subsequently CREB phosphorylation is impaired and therefore T cells are not able to respond to cues involving CREB. The present data functionally link the perinuclea

Critical thinking for 21st-century education: A cyber-tooth curriculum?

It is often assumed that the advent of digital technologies requires fundamental change to the curriculum and to the teaching and learning approaches used in schools around the world to educate this generation of “digital natives” or the “net generation”. This article analyses the concepts of 21st-century skills and critical thinking, to understand how these aspects of learning might contribute to a 21st-century education. The author argues that, although both critical thinking and 21st-century skills are indeed necessary in a curriculum for a 21st-century education, they are not sufficient, even in combination. The role of knowledge and an understanding of differing cultural perspectives and values indicate that education should also fit local contexts in a global world and meet the specific needs of students in diverse cultures. It should also fit the particular technical and historical demands of the 21st century in relation to digital skills

Gender Differences in Early Reading Strategies: a Comparison of Synthetic Phonics Only with a Mixed Approach to Teaching Reading to 4-5 Year-Old Children

A survey of primary schools in England found that girls outperform boys in English across all phases (Ofsted in Moving English forward. Ofsted, Manchester, 2012). The gender gap remains an on-going issue in England, especially for reading attainment. This paper presents evidence of gender differences in learning to read that emerged during the development of a reading scheme for 4- and 5-year-old children in which 372 children from Reception classes in sixteen schools participated in 12-month trials. There were three arms per trial: Intervention non-PD (non-phonically decodable text with mixed methods teaching); Intervention PD (phonically decodable text with mixed methods teaching); and a ‘business as usual’ control condition SP (synthetic phonics and decodable text). Assignment to Intervention condition was randomised. Standardised measures of word reading and comprehension were used. The research provides statistically significant evidence suggesting that boys learn more easily using a mix of whole-word and synthetic phonics approaches. In addition, the evidence indicates that boys learn to read more easily using the natural-style language of ‘real’ books including vocabulary which goes beyond their assumed decoding ability. At post-test, boys using the nonphonically decodable text with mixed methods (Intervention A) were 8 months ahead in reading comprehension compared to boys using a wholly synthetic phonics approach