HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway

Moderate environmental and physiological stressors are known to initiate protective heat shock response (HSR) leading to cell survival. HSR is largely mediated by the activation of heat shock factor (HSF), resulting in increased heat shock protein expression. Dysregulation of the HSR signaling has been associated with various diseases including cancer, inflammation and neurodegenerative disorders. Compounds that can modulate HSR have been pursued for the treatment of these diseases. To facilitate the discovery of HSR modulators, we developed a high-throughput amenable betalactamase transcriptional reporter gene assay for monitoring the function of HSF. HeLa cells were engineered to express the beta-lactamase reporter under the control of HSF response elements (HSE) present in the HSP70 gene promoter. The HSE-beta lactamase (HSE-bla) reporter gene assay was validated by using HSF-specific siRNAs and known small molecule modulators. Taking the advantage of fluorescence resonance energy transfer (FRET)-based cell permeable betalactamase substrate, this assay can be miniaturized into 1536-well format. Our results demonstrate that the assay is robust and can be applied to high-throughput screening (HTS) for modulators of HSR

Walking the Talk in Bullying Prevention: Teacher Implementation Variables Related to Initial Impact of the \u27Steps to Respect\u27 Program

This study examined relationships between teacher implementation of a comprehensive bullying prevention program and student outcomes. Implementation in third- through sixth-grade classrooms (N = 36) was measured by observation and teacher report. Student outcomes were measured by student surveys and teacher ratings of peer social skills (N = 549) and observations of playground behaviors (n = 298). Multilevel modeling showed that teacher coaching of students involved in bullying was associated with less observed victimization and destructive bystander behavior among students engaged in these problems at pretest, and less observed aggression among fifth- and sixth-grade students. Support for skill generalization related to reductions in observed aggression and victimization among older students. Adherence to lessons was associated with higher ratings of peer social skills. Quality of lesson instruction corresponded to greater self-reported victimization, as well as more perceived difficulty responding assertively to bullying. Implications for school-based practice and future research directions are discussed

Reducing Playground Bullying and Supporting Beliefs: An Experimental Trial of the \u27Steps to Respect\u27 Program

Six schools were randomly assigned to a multilevel bullying intervention or a control condition. Children in Grades 3–6 (N = 1,023) completed pre- and posttest surveys of behaviors and beliefs and were rated by teachers. Observers coded playground behavior of a random subsample (n = 544). Hierarchical analyses of changes in playground behavior revealed declines in bullying and argumentative behavior among intervention-group children relative to control-group children, increases in agreeable interactions, and a trend toward reduced destructive bystander behavior. Those in the intervention group reported enhanced bystander responsibility, greater perceived adult responsiveness, and less acceptance of bullying/aggression than those in the control group. Self-reported aggression did not differ between the groups. Implications for future research on the development and prevention of bullying are discussed

Setting Sail for Early Learning Success: Using a Data-based Decision Making Process to Measure and Monitor Outcomes in Early Childhood Programs

The use of data to inform decision-making and monitor individual student progress is recognized as an important, yet elusive practice in early childhood programs. In this article, Data-based Navigation is presented as a five step data-based decision making process designed to help early childhood professionals measure and monitor desired programmatic outcomes. A case study that focuses on the reduction of challenging behaviors is provided to illustrate the process

Zinc-Regulated DNA Binding of the Yeast Zap1 Zinc-Responsive Activator

The Zap1 transcription factor of Saccharomyces cerevisiae plays a central role in zinc homeostasis by controlling the expression of genes involved in zinc metabolism. Zap1 is active in zinc-limited cells and repressed in replete cells. At the transcriptional level, Zap1 controls its own expression via positive autoregulation. In addition, Zap1's two activation domains are regulated independently of each other by zinc binding directly to those regions and repressing activation function. In this report, we show that Zap1 DNA binding is also inhibited by zinc. DMS footprinting showed that Zap1 target gene promoter occupancy is regulated with or without transcriptional autoregulation. These results were confirmed using chromatin immunoprecipitation. Zinc regulation of DNA binding activity mapped to the DNA binding domain indicating other parts of Zap1 are unnecessary for this control. Overexpression of Zap1 overrode DNA binding regulation and resulted in constitutive promoter occupancy. Under these conditions of constitutive binding, both the zinc dose response of Zap1 activity and cellular zinc accumulation were altered suggesting the importance of DNA binding control to zinc homeostasis. Thus, our results indicated that zinc regulates Zap1 activity post-translationally via three independent mechanisms, all of which contribute to the overall zinc responsiveness of Zap1

The state of the Martian climate

60°N was +2.0°C, relative to the 1981–2010 average value (Fig. 5.1). This marks a new high for the record. The average annual surface air temperature (SAT) anomaly for 2016 for land stations north of starting in 1900, and is a significant increase over the previous highest value of +1.2°C, which was observed in 2007, 2011, and 2015. Average global annual temperatures also showed record values in 2015 and 2016. Currently, the Arctic is warming at more than twice the rate of lower latitudes

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD