Alignment of Synchronized Time-Series Data Using the Characterizing Loss of Cell Cycle Synchrony Model for Cross-Experiment Comparisons.

Investigating the cell cycle often depends on synchronizing cell populations to measure various parameters in a time series as the cells traverse the cell cycle. However, even under similar conditions, replicate experiments display differences in the time required to recover from synchrony and to traverse the cell cycle, thus preventing direct comparisons at each time point. The problem of comparing dynamic measurements across experiments is exacerbated in mutant populations or in alternative growth conditions that affect the synchrony recovery time and/or the cell-cycle period. We have previously published a parametric mathematical model named Characterizing Loss of Cell Cycle Synchrony (CLOCCS) that monitors how synchronous populations of cells release from synchrony and progress through the cell cycle. The learned parameters from the model can then be used to convert experimental time points from synchronized time-series experiments into a normalized time scale (lifeline points). Rather than representing the elapsed time in minutes from the start of the experiment, the lifeline scale represents the progression from synchrony to cell-cycle entry and then through the phases of the cell cycle. Since lifeline points correspond to the phase of the average cell within the synchronized population, this normalized time scale allows for direct comparisons between experiments, including those with varying periods and recovery times. Furthermore, the model has been used to align cell-cycle experiments between different species (e.g., Saccharomyces cerevisiae and Schizosaccharomyces pombe), thus enabling direct comparison of cell-cycle measurements, which may reveal evolutionary similarities and differences.

From the factory to the field: considerations of product characteristics for insecticide-treated net (ITN) bioefficacy testing.

BACKGROUND: Insecticide-treated nets (ITNs) undergo a series of tests to obtain listing by World Health Organization (WHO) Prequalification. These tests characterize the bioefficacy, physical and chemical properties of the ITN. ITN procurers assume that product specifications relate to product performance. Here, ITN test methods and their underlying assumptions are discussed from the perspective of the ITN manufacturing process and product characteristics. METHODS: Data were extracted from WHO Pesticide Evaluation Scheme (WHOPES) meeting reports from 2003 to 2017, supplemented with additional chemical analysis to critically evaluate ITNs bioassays with a focus on sampling, washing and wash resistance, and bioefficacy testing. Production methods for ITNs and their impact on testing outcomes are described. RESULTS AND RECOMMENDATIONS: ITNs are not homogenous products. They vary within panels and between the sides and the roof. Running tests of wash resistance using a before/after tests on the same sample or band within a net reduces test variability. As mosquitoes frequently interact with ITN roofs, additional sampling of the roof when evaluating ITNs is advisable because in nets where roof and sides are of the same material, the contribution of roof sample (20-25%) to the average is less than the tolerance for the specification (25%). Mosquito mortality data cannot be reliably used to evaluate net surface concentration to determine regeneration time (RT) and resistance to washing as nets may regenerate beyond the insecticide concentrations needed to kill 100% of susceptible mosquitoes. Chemical assays to quantify surface concentration are needed. The Wash Resistance Index (WRI) averaged over the first four washes is only informative if the product has a log linear loss rate of insecticide. Using a WRI that excludes the first wash off gives more reliable results. Storage conditions used for product specifications are lower than those encountered under product shipping and storage that may exceed 50 °C, and should be reconsidered. Operational monitoring of new ITNs and linking observed product performance, such as bioefficacy after 2 or 3 years of use, with product characteristics, such as WRI, will aid the development of more robust test methods and product specifications for new products coming to market.

RoboCOP: jointly computing chromatin occupancy profiles for numerous factors from chromatin accessibility data.

Chromatin is a tightly packaged structure of DNA and protein within the nucleus of a cell. The arrangement of different protein complexes along the DNA modulates and is modulated by gene expression. Measuring the binding locations and occupancy levels of different transcription factors (TFs) and nucleosomes is therefore crucial to understanding gene regulation. Antibody-based methods for assaying chromatin occupancy are capable of identifying the binding sites of specific DNA binding factors, but only one factor at a time. In contrast, epigenomic accessibility data like MNase-seq, DNase-seq, and ATAC-seq provide insight into the chromatin landscape of all factors bound along the genome, but with little insight into the identities of those factors. Here, we present RoboCOP, a multivariate state space model that integrates chromatin accessibility data with nucleotide sequence to jointly compute genome-wide probabilistic scores of nucleosome and TF occupancy, for hundreds of different factors. We apply RoboCOP to MNase-seq and ATAC-seq data to elucidate the protein-binding landscape of nucleosomes and 150 TFs across the yeast genome, and show that our model makes better predictions than existing methods. We also compute a chromatin occupancy profile of the yeast genome under cadmium stress, revealing chromatin dynamics associated with transcriptional regulation.

Linking the dynamics of chromatin occupancy and transcription with predictive models.

Though the sequence of the genome within each eukaryotic cell is essentially fixed, it exists within a complex and changing chromatin state. This state is determined, in part, by the dynamic binding of proteins to the DNA. These proteins-including histones, transcription factors (TFs), and polymerases-interact with one another, the genome, and other molecules to allow the chromatin to adopt one of exceedingly many possible configurations. Understanding how changing chromatin configurations associate with transcription remains a fundamental research problem. We sought to characterize at high spatiotemporal resolution the dynamic interplay between transcription and chromatin in response to cadmium stress. Whereas gene regulatory responses to environmental stress in yeast have been studied, how the chromatin state changes and how those changes connect to gene regulation remain unexplored. By combining MNase-seq and RNA-seq data, we found chromatin signatures of transcriptional activation and repression involving both nucleosomal and TF-sized DNA-binding factors. Using these signatures, we identified associations between chromatin dynamics and transcriptional regulation, not only for known cadmium response genes, but across the entire genome, including antisense transcripts. Those associations allowed us to develop generalizable models that predict dynamic transcriptional responses on the basis of dynamic chromatin signatures.

Local nucleosome dynamics and eviction following a double-strand break are reversible by NHEJ-mediated repair in the absence of DNA replication.

We interrogated at nucleotide resolution the spatiotemporal order of chromatin changes that occur immediately following a site-specific double-strand break (DSB) upstream of the PHO5 locus and its subsequent repair by nonhomologous end joining (NHEJ). We observed the immediate eviction of a nucleosome flanking the break and the repositioning of adjacent nucleosomes away from the break. These early chromatin events were independent of the end-processing Mre11-Rad50-Xrs2 (MRX) complex and preceded the MRX-dependent broad eviction of histones and DNA end-resectioning that extends up to ∼8 kb away from the break. We also examined the temporal dynamics of NHEJ-mediated repair in a G1-arrested population. Concomitant with DSB repair by NHEJ, we observed the redeposition and precise repositioning of nucleosomes at their originally occupied positions. This re-establishment of the prelesion chromatin landscape suggests that a DNA replication-independent mechanism exists to preserve epigenome organization following DSB repair.

Teacher-made models: the answer for medical skills training in developing countries?

BACKGROUND: The advantages of using simulators in skills training are generally recognized, but simulators are often too expensive for medical schools in developing countries. Cheaper locally-made models (or part-task trainers) could be the answer, especially when teachers are involved in design and production (teacher-made models, TM). METHODS: We evaluated the effectiveness of a TM in training and assessing intravenous injection skills in comparison to an available commercial model (CM) in a randomized, blind, pretest-posttest study with 144 undergraduate nursing students. All students were assessed on both the TM and the CM in the pre-test and post-test. After the post-test the students were also assessed while performing the skill on real patients. RESULTS: Differences in the mean scores pre- and post-test were marked in all groups. Training with TM or CM improved student scores substantially but there was no significant difference in mean scores whether students had practiced on TM or CM. Students who practiced on TM performed better on communication with the patient than did students who practiced on CM. Decreasing the ratio of students per TM model helped to increase practice opportunities but did not improve student's mean scores. The result of the assessment on both the TM and the CM had a low correlation with the results of the assessment on real persons. CONCLUSIONS: The TM appears to be an effective alternative to CM for training students on basic IV skills, as students showed similar increases in performance scores after training on models that cost considerably less than commercially available models. These models could be produced using locally available materials in most countries, including those with limited resources to invest in medical education and skills laboratories.