Analysis of the Barr body with super-resolution microscopy

X chromosome inactivation (XCI) in female mammalian cells is an ideal model system to study the relationship of epigenetic regulation and higher-order chromatin structure. However, light microscopic studies of chromosomal organization have long been limited by the diffraction barrier of optical resolution. Super-resolution 3D-structured illumination microscopy (3D-SIM) – one of several recent techniques that circumvent this limitation – enables multicolor optical sectioning of entire cells with eightfold-improved volumetric resolution compared to conventional fluorescence imaging methods. In the present work, 3D-SIM has been applied to analyze higher-order chromatin structure of the Barr body in mammalian nuclei, a characteristic hallmark of XCI, with yet unprecedented detail. First, the increased resolution prompted to reappraise the potential detrimental effect of the DNA-FISH procedure on chromatin structure. Comparative analyses revealed slight deteriorations at the resolution level of 3D-SIM, especially within more decondensed euchromatin sites within the nuclear interior. In contrast, overall nuclear morphology and the nuclear envelope as well as heterochromatic sites in general maintained well preserved. The results suggest that DNA-FISH studies can benefit from a combination with super-resolution microscopy. In particular, when keeping in mind the current developments of the FISH technique with increasingly small and higher-complexity probes. The compact shape of the Barr body led to the assumption of a contribution of this special higher-order chromatin structure to the establishment and maintenance of the silenced state in the inactive X chromosome (Xi). However, a confirmation of this view has always been hampered by the restrictions of conventional light microscopy. In this work, the 3D chromosomal organization of the Xi and autosomes has been investigated with 3D-SIM in various human and mouse somatic cells and in mouse embryonic stem cell (ESC) lines. The precise subchromosomal localization of a variety of factors involved in XCI in different developmental states was qualitatively and quantitatively assessed utilizing combined immunofluorescence, EdU- pulse and RNA-/DNA-FISH labeling protocols and novel data analysis tools customized for the special requirements of 3D-SIM. The results demonstrate that all autosomes are made of a three-dimensional interconnected network of chromatin domains (CDs, or topology associated domains, TADs) of highly-variable shape and dynamics. CDs/TADs are comprised of a compacted chromatin core enriched with repressive marks, which is collectively proposed to be the functionally passive chromatin compartment (PNC). This PNC is surrounded by a 50 – 150 nm locally defined, less compacted perichromatin region (PR) that is enriched with active histone modifications and pervaded by a three-dimensional interchromatin (IC) network. The PR and the IC are collectively referred to as being the functionally relevant active nuclear compartment (ANC) that harbors all major nuclear processes, including transcription and replication. 3D-SIM data revealed that the Barr body maintains this principle compartmentalization and that it is still pervaded by a narrow ANC network, which is able to fulfill its functional role as a hub for replication or rarely occurring expression of XCI-escape genes. Live-cell super-resolution imaging on HeLa H2B-GFP cells confirmed that the observed chromatin features do not reflect fixation artifacts. Xist RNA, the key factor of XCI, has been found to be preferentially located as distinct discernible foci within the ANC throughout the entire volume of the Barr body. Here, it is tightly associated with a Xi-specific form of the nuclear matrix protein SAF-A, which confirms a previously suggested role for this Xi-enriched protein in Xist RNA spreading. In contrast, Xist RNA shows no spatial correlation with repressive Xi-enriched histone marks that are found within compacted chromatin sites. This specific localization of Xist RNA reflects an intrinsic feature as it is already present during early spreading in differentiating female ESCs, where it precedes chromatin compaction concomitant with RNA Polymerase II exclusion. Its localization is further confirmed in a male ESC line carrying an inducible Xist transgene on an autosome, but where Xist RNA fails to form a true autosomal Barr body, which is less compacted and maintains transcriptional activity. Last, Xist RNA shows no direct association with PRC2, the mediator of H3K27me3, which is in contrast to the generally believed direct recruitment model of PRC2 to the Xi by Xist RNA. The data collected in this work reflects further support and a refinement of the not unequivocally accepted CT-IC (chromosome territory - interchromatin compartment) model of higher-order chromosome architecture. In addition, a first attempt has been made to integrate these findings with a recently growing number of studies using chromosome conformation capturing (3C)-based techniques and to complement them on the single-cell level. Finally, a novel model for Xist RNA function in XCI is presented, which proposes a sequence-independent structural role for gene silencing and the formation of a repressive chromatin compartment

Misleading graphs in context:Less misleading than expected

Misleading graphs are a source of misinformation that worry many experts. Especially people with a low graph literacy are thought to be persuaded by graphs that misrepresent the underlying data. But we know little about how people interpret misleading graphs and how these graphs influence their opinions. In this study we focus on the effect of truncating the y-axis for a line chart which exaggerates an upgoing trend. In a randomized controlled trial, we showed participants either a normal or a misleading chart, and we did so in two different contexts. After they had seen the graphs, we asked participants their opinion on the trend and to give an estimation of the increase. Finally we measured their graph literacy. Our results show that context is the only significant factor in opinion-forming; the misleading graph and graph literacy had no effect. None of these factors had a significant impact on estimations for the increase. These results show that people might be less susceptible to misleading graphs than we thought and that context has more impact than a misleading y-axis.Science Communication and Societ

Exploring Health Experts' and Creative Communicators' Focus in Pandemic Video Communication: A Qualitative Study

Pandemic video communication aimed at the general public often lacks creativity and fails to reach large audiences. Yet, the scientific content should not be compromised by attempts to improve the creativity or reach. This study explores the processes utilised by various health experts and professional communicators when creating communication, to identify similarities and differences, and how pandemic video communication thus can be improved through an interdisciplinary approach. We interviewed 12 individuals from 6 different professional domains: health, public health, film/science communication, video journalism, advertising, and social media/YouTube. Semi-structured individual interviews were conducted using the same interview guide. The interview data were subjected to thematic analysis with both deductive and inductive coding, and the results were visualised in a bubble chart. Our study has highlighted both similarities and differences between health professionals and creative communicators relating to their creative processes and their approaches to pandemic video communication. We found that participants from health domains assigned great importance to and efforts on the content, but were unsure or lacked experience in how content is translated through form and creativity. Creative communicators, on the other hand, emphasise and specialise in form, yet depend on health professionals, experts, and scientists to provide and validate content. The key to improving pandemic-related video communication appears to lie in striking the right balance between high-quality and evidence-based content and creativity. This study found that both health professionals and creative communicators play crucial roles in reaching a solid end result, and we suggest a fusion model approach to interdisciplinary collaboration.publishedVersio

Predicting 6-minute walking test outcomes in patients with chronic obstructive pulmonary disease without physical performance measures

Background and Objective: Chronic obstructive pulmonary disease (COPD) requires a multifactorial assessment, evaluating the airflow limitation and symptoms of the patients. The 6-min walk test (6MWT) is commonly used to evaluate the functional exercise capacity in these patients. This study aims to propose a novel predictive model of the major 6MWT outcomes for COPD assessment, without physical performance measurements. Methods: Cardiopulmonary and clinical parameters were obtained from fifty COPD patients. These parameters were used as inputs of a Bayesian network (BN), which integrated three multivariate models including the 6-min walking distance (6MWD), the maximum HR (HRmax ) after the walking, and the HR decay 3 min after (HRR3 ). The use of BN allows the assessment of the patients’ status by predicting the 6MWT outcomes, but also inferring disease severity parameters based on actual patient’s 6MWT outcomes. Results: Firstly, the correlation obtained between the estimated and actual 6MWT measures was strong (R = 0.84, MAPE = 8.10% for HRmax ) and moderate (R = 0.58, MAPE = 15.43% for 6MWD and R = 0.58, MAPE = 32.49% for HRR3 ), improving the classical methods to estimate 6MWD. Secondly, the classification of disease severity showed an accuracy of 78.3% using three severity groups, which increased up to 84.4% for two defined severity groups. Conclusions: We propose a powerful two-way assessment tool for COPD patients, capable of predicting 6MWT outcomes without the need for an actual walking exercise. This model-based tool opens the way to implement a continuous monitoring system for COPD patients at home and to provide more personalized care

Predicting 6-minute walking test outcomes in patients with chronic obstructive pulmonary disease without physical performance measures

Background and Objective Chronic obstructive pulmonary disease (COPD) requires a multifactorial assessment, evaluating the airflow limitation and symptoms of the patients. The 6-min walk test (6MWT) is commonly used to evaluate the functional exercise capacity in these patients. This study aims to propose a novel predictive model of the major 6MWT outcomes for COPD assessment, without physical performance measurements. Methods Cardiopulmonary and clinical parameters were obtained from fifty COPD patients. These parameters were used as inputs of a Bayesian network (BN), which integrated three multivariate models including the 6-min walking distance (6MWD), the maximum HR (HRmax) after the walking, and the HR decay 3 min after (HRR3). The use of BN allows the assessment of the patients’ status by predicting the 6MWT outcomes, but also inferring disease severity parameters based on actual patient's 6MWT outcomes. Results Firstly, the correlation obtained between the estimated and actual 6MWT measures was strong (R = 0.84, MAPE = 8.10% for HRmax) and moderate (R = 0.58, MAPE = 15.43% for 6MWD and R = 0.58, MAPE = 32.49% for HRR3), improving the classical methods to estimate 6MWD. Secondly, the classification of disease severity showed an accuracy of 78.3% using three severity groups, which increased up to 84.4% for two defined severity groups. Conclusions We propose a powerful two-way assessment tool for COPD patients, capable of predicting 6MWT outcomes without the need for an actual walking exercise. This model-based tool opens the way to implement a continuous monitoring system for COPD patients at home and to provide more personalized care.Peer ReviewedPostprint (published version

Wnt7a Decreases Brain Endothelial Barrier Function Via β-Catenin Activation

The blood-brain barrier consists of tightly connected endothelial cells protecting the brain’s microenvironment from the periphery. These endothelial cells are characterized by specific tight junction proteins such as Claudin-5 and Occludin, forming the endothelial barrier. Disrupting these cells might lead to blood-brain barrier dysfunction. The Wnt/β-catenin signaling pathway can regulate the expression of these tight junction proteins and subsequent barrier permeability. The aim of this study was to investigate the in vitro effects of Wnt7a mediated β-catenin signaling on endothelial barrier integrity. Mouse brain endothelial cells, bEnd.3, were treated with recombinant Wnt7a protein or XAV939, a selective inhibitor of Wnt/β-catenin mediated transcription to modulate the Wnt signaling pathway. The involvement of Wnt/HIF1α signaling was investigated by inhibiting Hif1α signaling with Hif1α siRNA. Wnt7a stimulation led to activation and nuclear translocation of β-catenin, which was inhibited by XAV939. Wnt7a stimulation decreased Claudin-5 expression mediated by β-catenin and decreased endothelial barrier formation. Wnt7a increased Hif1α and Vegfa expression mediated by β-catenin. However, Hif1α signaling pathway did not regulate tight junction proteins Claudin-5 and Occludin. Our data suggest that Wnt7a stimulation leads to a decrease in tight junction proteins mediated by the nuclear translocation of β-catenin, which hampers proper endothelial barrier formation. This process might be crucial in initiating endothelial cell proliferation and angiogenesis. Although HIF1α did not modulate the expression of tight junction proteins, it might play a role in brain angiogenesis and underlie pathogenic mechanisms in Wnt/HIF1α signaling in diseases such as cerebral small vessel disease

The effect of hunger state on hypothalamic functional connectivity in response to food cues

ACKNOWLEDGMENT The authors thank Lisette Charbonnier for her relentless efforts in setting up the study at all three sites and collecting the Dutch data. Open Access funding enabled and organized by Projekt DEAL. FUNDING INFORMATION This work was financially supported by the European Union Seventh Framework Programme (FP7/2007–2013) for research, technological development, and demonstration under grant agreement 266408 (Full4Health, www.full4health.eu). Furthermore, the study was supported in parts by a grant (01GI0925) from the Federal Ministry of Education and Research (BMBF) to the German Center for Diabetes Research (DZD e.V.).Peer reviewedPublisher PD

Observationally constrained surface mass balance of Larsen C IceShelf, Antarctica

Abstract. Combining several geophysical techniques, we reconstruct spatial and temporal patterns of surface mass balance (SMB) over Larsen C Ice Shelf (LCIS), Antarctic Peninsula. Continuous time series of snow height at five locations allow for multi-year estimates of seasonal and annual SMB over LCIS. There is high interannual variability, with an SMB of 395 ± 61 to 413 ± 42 mm w.e. y−1 in the north and a larger SMB of up to 496 ± 50 mm w.e. y−1 farther south. This difference between north and south is corroborated by winter snow accumulation derived from an airborne radar survey from 2009, which showed an average snow thickness of 0.95 m north of 76° S, and 1.12 m south of 78°. Analysis of ground-penetrating radar from several field campaigns allows for a longer-term perspective of spatial SMB: a particularly strong and coherent reflection horizon below 25–44 m w.e. of ice and firn is observed in radargrams collected across the shelf. We propose that this horizon was formed in a single melt season over the ice shelf. Combining ground and airborne radar with SMB output from a regional climate model confirms that SMB increases from north to south, overprinted by a gradient of increasing SMB to the west. Previous observations show a strong decrease in firn air content toward the west, which we attribute to spatial patterns of melt, refreezing, and densification, rather than SMB. </jats:p

3D-Image analysis platform monitoring relocation of pluripotency genes during reprogramming

Nuclear organization of chromatin is an important level of genome regulation with positional changes of genes occurring during reprogramming. Inherent variability of biological specimens, wide variety of sample preparation and imaging conditions, though pose significant challenges to data analysis and comparison. Here, we describe the development of a computational image analysis toolbox overcoming biological variability hurdles by a novel single cell randomizing normalization. We performed a comparative analysis of the relationship between spatial positioning of pluripotency genes with their genomic activity and determined the degree of similarity between fibroblasts, induced pluripotent stem cells and embryonic stem cells. Our analysis revealed a preferred positioning of actively transcribed Sox2, Oct4 and Nanog away from the nuclear periphery, but not from pericentric heterochromatin. Moreover, in the silent state, we found no common nuclear localization for any of the genes. Our results suggest that the surrounding gene density hinders relocation from an internal nuclear position. Altogether, our data do not support the hypothesis that the nuclear periphery acts as a general transcriptional silencer, rather suggesting that internal nuclear localization is compatible with expression in pluripotent cells but not sufficient for expression in mouse embryonic fibroblasts. Thus, our computational approach enables comparative analysis of topological relationships in spite of stark morphological variability typical of biological data sets