Single molecule MATAC-seq reveals key determinants for DNA replication efficiency

The stochastic nature of origin activation results in significant variability in the way genome replication is carried out from cell to cell. The reason for the diversity in efficiency and timing of individual origins has remained an unresolved issue for a long time. Cell-to-cell variability has been demonstrated to play a crucial role in cellular plasticity and cancer in mammalian cells. Although population-based methods have provided valuable insight into biological processes, it is necessary to use single molecule techniques to uncover events that are hidden by the population average. Many biological processes, such as DNA replication, transcription, and gene expression, are closely linked to the local chromatin structure. In yeast, although DNA replication origins have conserved DNA sequences, they display remarkable differences in timing and efficiency. Some origins initiate replication earlier during S-phase or more frequently than others, resulting in a high degree of heterogeneity among the cells in a population, with no two cells having the exact same replication profile. Our hypothesis is that the local nucleosomal structure may affect the DNA replication profile of individual origins. To explore this relationship, we have developed Methylation Accessibility of Targeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility maps of specific genomic locations after targeted purification in their native chromatin context. Our analysis of selected early-efficient (EE) and late-inefficient (LI) replication origins in Saccharomyces cerevisiae using MATAC-Seq revealed significant cell-to-cell heterogeneity in their chromatin states. Disrupting the INO80 or ISW2 chromatin remodeling complexes led to changes at individual nucleosomal positions that correspond to changes in replication efficiency. Our results show that a chromatin state with a narrow size of accessible origin DNA in combination with well-positioned surrounding nucleosomes and an open +2 linker region was a strong predictor for efficient origin activation. MATAC-Seq provides a single-molecule assay for chromatin accessibility that reveals the large spectrum of alternative chromatin states that coexist at a given locus, which was previously masked in population-based experiments. This provides a mechanistic basis for origin activation heterogeneity that occurs during DNA replication in eukaryotic cells. As a result, our single-molecule assay for chromatin accessibility will be ideal for defining single-molecule heterogeneity across many biological processes, such as transcription, replication, or DNA repair in vitro and ex vivo.Die stochastische Natur der Aktivierung von Replikationsursprüngen führt zu einer signifikanten Variabilität in der Art und Weise, wie die DNA Replikation von Zelle zu Zelle durchgeführt wird. Der Grund für die Diversität in Effizienz und Zeitpunkt der individuellen Aktivierung von Ursprüngen blieb lange ein ungelöstes Problem. Es wurde gezeigt, dass die Zell-zu-Zell-Variabilität eine entscheidende Rolle bei der zellulären Plastizität und Krebs in Säugetierzellen spielt. Obwohl populationsbasierte Methoden wertvolle Einblicke in biologische Prozesse geliefert haben, ist es notwendig, Einzelmolekültechniken zu verwenden, um Ereignisse aufzudecken, die durch das Durchschnittsverhalten aller Moleküle verborgen sind. Viele biologische Prozesse wie DNA-Replikation, Transkription und Genexpression sind eng mit der lokalen Chromatinstruktur verbunden. Obwohl die DNA-Replikationsursprünge in Hefe konservierte DNA-Sequenzen aufweisen, zeigen sie bemerkenswerte Unterschiede im Zeitpunkt und Effizienz der Replikation. Einige Ursprünge initiieren die Replikation früher während der S-Phase oder häufiger als andere, was zu einem hohen Grad an Heterogenität zwischen den Zellen in einer Population führt, wobei keine zwei Zellen das exakt gleiche Replikationsprofil aufweisen. Unsere Hypothese ist, dass die lokale nukleosomale Struktur das DNA-Replikationsprofil beeinflussen kann. Um diese Beziehung zu untersuchen, haben wir Methylation Accessibility of Targeted Chromatin Domain Sequencing (MATAC-Seq) entwickelt, um Einzelmolekül-Chromatin-Zugänglichkeitskarten spezifischer genomischer Orte nach gezielter Reinigung in ihrem nativen Chromatin-Kontext zu bestimmen. Unsere Analyse ausgewählter früh-effizient (EE) und spät-ineffizient (LI) feuernde Replikationsursprünge in Saccharomyces cerevisiae mit MATAC-Seq ergab eine signifikante Zell-zu-Zell-Heterogenität in ihren Chromatinzuständen. Die genetische Deletion der INO80- oder ISW2-Chromatin-Remodeling Komplexe führte zu Veränderungen an einzelnen nukleosomalen Positionen, die mit Veränderungen der Replikationseffizienz korrespondierten. Unsere Ergebnisse zeigten, dass ein Chromatinzustand mit einem engen Fenster an zugänglicher Replikationsursprungs-DNA in Kombination mit gut positionierten umgebenden Nukleosomen und einer offenen +2-Linkerregion ein starker Prädiktor für eine effiziente Ursprungsaktivierung war. MATAC-Seq bietet einen Einzelmolekül-Assay für die Zugänglichkeit von Chromatin, der das große Spektrum alternativer Chromatinzustände aufzeigt, die an einem bestimmten genomischen Lokus koexistieren, der zuvor in populationsbasierten Experimenten maskiert war. Dies liefert eine mechanistische Grundlage für die Heterogenität der Ursprungsaktivierung, die während der DNA-Replikation in eukaryotischen Zellen auftritt. Infolgedessen ist unser Einzelmolekül-Assay 5 für Chromatin-Zugänglichkeit ideal für die Definition der Einzelmolekül-Heterogenität über viele biologische Prozesse hinweg, wie z. B. Transkription, Replikation oder DNA-Reparatur in vitro und ex vivo

Hemostasis and complement in allogeneic hematopoietic stem cell transplantation: clinical significance of two interactive systems.

Hematopoietic stem cell transplantation (HCT) represents a curative treatment option for certain malignant and nonmalignant hematological diseases. Conditioning regimens before HCT, the development of graft-versus-host disease (GVHD) in the allogeneic setting, and delayed immune reconstitution contribute to early and late complications by inducing tissue damage or humoral alterations. Hemostasis and/or the complement system are biological regulatory defense systems involving humoral and cellular reactions and are variably involved in these complications after allogeneic HCT. The hemostasis and complement systems have multiple interactions, which have been described both under physiological and pathological conditions. They share common tissue targets, such as the endothelium, which suggests interactions in the pathogenesis of several serious complications in the early or late phase after HCT. Complications in which both systems interfere with each other and thus contribute to disease pathogenesis include transplant-associated thrombotic microangiopathy (HSCT-TMA), sinusoidal obstruction syndrome/veno-occlusive disease (SOS/VOD), and GVHD. Here, we review the current knowledge on changes in hemostasis and complement after allogeneic HCT and how these changes may define clinical impact

Leaving the Abstract for the Concrete in Written Expression in Elementary School in Benin

This article aims at analysing and influencing the pedagogical practices of six teachers involved in an action-research in written expression in the Elementary Course at the elementary school in Benin in order to optimize the learning capacities of the pupils. To this end, a research protocol was adopted that included a stage of identification of the problem, the establishment of an action plan, the implementation of the action, the evaluation of the effects of the action, the communication of the conclusions and the valorisation of the research. It was found that the teachers did not maximize the children's learning opportunities during the "chasse aux idées" phase; they did not get the children moving; they did not praise them properly; they remained in the abstract about the themes addressed. Several decisions were taken at each stage of the progression in the realization of the sequence II in written expression (first draft), the most important of which is the concretization based on clear and expressive images and reinforced by social writing

Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency

Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of four specific genomic loci. MATAC-Seq relies on preferential modification of accessible DNA by methyltransferases combined with Nanopore-Sequencing for direct readout of methylated DNA-bases. Applying MATAC-Seq to selected early-efficient and late-inefficient yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an accessible nucleosome-free region in combination with well-positioned +1 and +2 nucleosomes as a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during eukaryotic DNA replication. Consequently, our single-molecule chromatin accessibility assay will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo

Issues of geologically-focused situational awareness in robotic planetary missions: lessons from an analogue mission at Mistastin Lake impact structure, Labrador, Canada

Remote robotic data provides different information than that obtained from immersion in the field. This significantly affects the geological situational awareness experienced by members of a mission control science team. In order to optimize science return from planetary robotic missions, these limitations must be understood and their effects mitigated to fully leverage the field experience of scientists at mission control. Results from a 13-day analogue deployment at the Mistastin Lake impact structure in Labrador, Canada suggest that scale, relief, geological detail, and time are intertwined issues that impact the mission control science team‟s effectiveness in interpreting the geology of an area. These issues are evaluated and several mitigation options are suggested. Scale was found to be difficult to interpret without the reference of known objects, even when numerical scale data were available. For this reason, embedding intuitive scale-indicating features into image data is recommended. Since relief is not conveyed in 2D images, both 3D data and observations from multiple angles are required. Furthermore, the 3D data must be observed in animation or as anaglyphs, since without such assistance much of the relief information in 3D data is not communicated. Geological detail may also be missed due to the time required to collect, analyze, and request data. We also suggest that these issues can be addressed, in part, by an improved understanding of the operational time costs and benefits of scientific data collection. Robotic activities operate on inherently slow time-scales. This fact needs to be embraced and accommodated. Instead of focusing too quickly on the details of a target of interest, thereby potentially minimizing science return, time should be allocated at first to more broad data collection at that target, including preliminary surveys, multiple observations from various vantage points, and progressively smaller scale of focus. This operational model more closely follows techniques employed by field geologists and is fundamental to the geologic interpretation of an area. Even so, an operational time cost/benefit analyses should be carefully considered in each situation, to determine when such comprehensive data collection would maximize the science return. Finally, it should be recognized that analogue deployments cannot faithfully model the time scales of robotic planetary missions. Analogue missions are limited by the difficulty and expense of fieldwork. Thus, analogue deployments should focus on smaller aspects of robotic missions and test components in a modular way (e.g., dropping communications constraints, limiting mission scope, focusing on a specific problem, spreading the mission over several field seasons, etc.)

A three month controlled intervention of intermittent whole body vibration designed to improve functional ability and attenuate bone loss in patients with rheumatoid arthritis

Background: Rheumatoid arthritis (RA) is a chronic autoimmune condition that results in pain and disability. Patients with RA have a decreased functional ability and are forced into a sedentary lifestyle and as such, these patients often become predisposed to poor bone health. Patients with RA may also experience a decreased health related quality of life (HRQoL) due to their disease. Whole body vibration (WBV) is a form of exercise that stimulates bone loading through forced oscillation. WBV has also been shown to decrease pain and fatigue in other rheumatic diseases, as well as to increase muscle strength. This paper reports on the development of a semi randomised controlled clinical trial to assess the impact of a WBV intervention aiming to improve functional ability, attenuate bone loss, and improve habitual physical activity levels in patients with RA. Methods/Design: This study is a semi randomised, controlled trial consisting of a cohort of patients with established RA assigned to either a WBV group or a CON (control) group. Patients in the WBV group will undergo three months of twice weekly intermittent WBV sessions, while the CON group will receive standard care and continue with normal daily activities. All patients will be assessed at baseline, following the three month intervention, and six months post intervention. Main outcomes will be an improvement in functional ability as assessed by the HAQ. Secondary outcomes are attenuation of loss of bone mineral density (BMD) at the hip and changes in RA disease activity, HRQoL, habitual physical activity levels and body composition. Discussion: This study will provide important information regarding the effects of WBV on functional ability and BMD in patients with RA, as well as novel data regarding the potential changes in objective habitual physical activity patterns that may occur following the intervention. The sustainability of the intervention will also be assessed