Regulation of the dynamic RNA Pol II elongation rate in Drosophila embryos.

An increasing number of studies have shown the key role that RNA polymerase II (RNA Pol II) elongation plays in gene regulation. We systematically examine how various enhancers, promoters, and gene body composition influence the RNA Pol II elongation rate through a single-cell-resolution live imaging assay. By using reporter constructs containing 5' MS2 and 3' PP7 repeating stem loops, we quantify the rate of RNA Pol II elongation in live Drosophila embryos. We find that promoters and exonic gene lengths have no effect on elongation rate, while enhancers and the presence of long introns may significantly change how quickly RNA Pol II moves across a gene. Furthermore, we observe in multiple constructs that the RNA Pol II elongation rate accelerates after the transcriptional onset of nuclear cycle 14 in Drosophila embryos. Our study provides a single-cell view of various mechanisms that affect the dynamic RNA Pol II elongation rate, ultimately affecting the rate of mRNA production.

A Measurement-Based Protocol Improves Interrater Agreement and Accuracy of Right Ventricular Systolic Pressure Assessment by Echocardiography in Children: A Call for Quality Improvement.

BACKGROUND: Echocardiographic measurements carry the promise of improving interrater (IR) agreement over subjective assessment. The aim of this study was to assess the effect of implementing a measurement-based protocol on IR agreement and accuracy in reporting of right ventricular (RV) systolic pressure in children. The effect of this reporting protocol on IR agreement in reporting RV dilation, hypertrophy, and systolic function was also evaluated. METHODS: Five echocardiography readers reported their assessment of RV systolic pressure, dilation, hypertrophy, and systolic function on 40 deidentified echocardiograms using their individual accustomed methods and then using an agreed-upon protocol based solely on RV measurements. IR agreement was assessed using κ statistics. Accuracy of RV systolic pressure ratings was assessed using the McNemar test in comparison with hemodynamic data obtained by cardiac catheterization. The reliability of the RV measurements was assessed using intraclass correlation coefficient (ICC) and coefficient of variation. RESULTS: IR agreement and accuracy of RV systolic pressure assessment improved after using the measurement-based protocol (agreement from 0.39 [95% CI, 0.27-0.5] to 0.62 [95% CI, 0.48-0.76] and accuracy from 18 of 40 to 29 of 40 [P = .03]). IR agreement of RV dilation improved (from 0.36 [95% CI, 0.25-0.48] to 0.63 [95% CI, 0.48-0.79]), while IR agreement of RV hypertrophy (from 0.29 [95% CI, 0.17-0.42] to 0.35 [95% CI, 0.15-0.55]) and RV systolic function (from 0.57 [95% CI, 0.45-0.69] to 0.53 [95% CI, 0.41-0.66]) did not improve. The reliability of the measurements was good (ICC > 0.8), except for RV free wall thickness (ICC = 0.62, coefficient of variation = 24%) and RV fractional area change (ICC = 0.47, coefficient of variation = 22%), suggesting a possible reason for the lack of improvement in IR agreement of RV hypertrophy and RV systolic function. Heteroscedasticity was observed in the reliability of RV measurements, with the ICC being significantly lower at larger magnitudes for all RV measurements. CONCLUSIONS: Standardization of reporting protocols using RV measurements in place of subjective assessment improved IR agreement and accuracy of RV systolic pressure assessment. Reliable measurements (RV systolic pressure and dilation) resulted in improvement in IR agreement while unreliable measurements (RV hypertrophy and systolic function) did not. Special attention to measurements' reliability and heteroscedasticity of reliability is required when designing clinical protocols to decrease IR disagreement as a source of error.

Diverse heterochromatin-associated proteins repress distinct classes of genes and repetitive elements.

Heterochromatin, typically marked by histone H3 trimethylation at lysine 9 (H3K9me3) or lysine 27 (H3K27me3), represses different protein-coding genes in different cells, as well as repetitive elements. The basis for locus specificity is unclear. Previously, we identified 172 proteins that are embedded in sonication-resistant heterochromatin (srHC) harbouring H3K9me3. Here, we investigate in humans how 97 of the H3K9me3-srHC proteins repress heterochromatic genes. We reveal four groups of srHC proteins that each repress many common genes and repeat elements. Two groups repress H3K9me3-embedded genes with different extents of flanking srHC, one group is specific for srHC genes with H3K9me3 and H3K27me3, and one group is specific for genes with srHC as the primary feature. We find that the enhancer of rudimentary homologue (ERH) is conserved from Schizosaccharomyces pombe in repressing meiotic genes and, in humans, now represses other lineage-specific genes and repeat elements. The study greatly expands our understanding of H3K9me3-based gene repression in vertebrates.

Vulnerability and Well-Being Decades After Leaving Care.

One of the most important goals of out of home placements is to reduce vulnerability and to enable well-being in the long term. This article hermeneutically reconstructs biographies decades after leaving-care to understand the impact of residential care experiences on selected dimensions of care-leavers' well-being, that were discovered in the data material. For this article three analytic areas were selected from the core of the narratives of former care leavers: Social networks, parenthood and state interventions. The selected findings on long-term outcomes presented here are based on a qualitative research project funded by the Swiss National Science Foundation on life trajectories after residential care (1950-1990). The authors have conducted 37 biographical narrative interviews with former children placed in residential care between 1950 and 1990 in the Canton of Zurich, Switzerland. The analysis of these narrative interviews was structured by the inductive procedures of Grounded Theory. Its foundation is the conceptualisation and dimensionalisation of data through inductive coding within the narratives. Research question: We mainly were interested in aspects of transitions exclusively relevant from the actors' point of view. The objective of this paper is to learn for the future by taking biographical experiences and long-term outcome in account. As we know residential care facilities have changed in last decades, but structurally some key figures are still continuing. They still interrupt the life course two times: when you start to the live in the institution and when you leave. One main question is how young people manage to integrate residential experiences through their life course and where they keep on struggling until the end of their lives. From a life-course perspective, the impact of social service intention on individual life courses, behind sending the individuals to such facilities, are important to investigate. They implicate relevant information concerning current practice and impact of placing children in residential care. Social networks and experiences of parenthood show why we must frame and accompany transitions out of care.

Regulation of spatiotemporal limits of developmental gene expression via enhancer grammar.

The regulatory specificity of a gene is determined by the structure of its enhancers, which contain multiple transcription factor binding sites. A unique combination of transcription factor binding sites in an enhancer determines the boundary of target gene expression, and their disruption often leads to developmental defects. Despite extensive characterization of binding motifs in an enhancer, it is still unclear how each binding site contributes to overall transcriptional activity. Using live imaging, quantitative analysis, and mathematical modeling, we measured the contribution of individual binding sites in transcriptional regulation. We show that binding site arrangement within the Rho-GTPase component t48 enhancer mediates the expression boundary by mainly regulating the timing of transcriptional activation along the dorsoventral axis of Drosophila embryos. By tuning the binding affinity of the Dorsal (Dl) and Zelda (Zld) sites, we show that single site modulations are sufficient to induce significant changes in transcription. Yet, no one site seems to have a dominant role; rather, multiple sites synergistically drive increases in transcriptional activity. Interestingly, Dl and Zld demonstrate distinct roles in transcriptional regulation. Dl site modulations change spatial boundaries of t48, mostly by affecting the timing of activation and bursting frequency rather than transcriptional amplitude or bursting duration. However, modulating the binding site for the pioneer factor Zld affects both the timing of activation and amplitude, suggesting that Zld may potentiate higher Dl recruitment to target DNAs. We propose that such fine-tuning of dynamic gene control via enhancer structure may play an important role in ensuring normal development.

Pre-exposure prophylaxis accessibility research and evaluation (PrEPARE Study).

Tenofovir-emtricitabine (TDF-FTC) has demonstrated effectiveness as HIV preexposure prophylaxis (PrEP), but it is not commonly prescribed. Our study was designed to determine the barriers preventing utilization of PrEP among men who have sex with men (MSM), the group at greatest risk for HIV infection in the United States. A population-based sample of MSM presenting for HIV testing at 'Early Test' HIV testing and counseling sites in San Diego, California were offered PrEP and education about potential efficacy. Eligible individuals reported having unprotected sex within the past 12 months and who tested negative for HIV were offered study participation. Despite offering procedures for evaluation and prescription for PrEP to 416 eligible subjects, less than 0.5 % of participants received the drug. Surveys collected from 54 of those who declined study participation revealed multiple barriers to PrEP among MSM including cost, low perceived risk of infection and concerns about taking a daily medication and potential long-term side effects. Efforts should be made to address these barriers, especially lowering the cost of TDF-FTC, education about PrEP side effects and awareness of HIV risks.