Impact of structured reporting on developing head and neck ultrasound skills.

BACKGROUND: Reports of head and neck ultrasound examinations are frequently written by hand as free texts. This is a serious obstacle to the learning process of the modality due to a missing report structure and terminology. Therefore, there is a great inter-observer variability in overall report quality. Aim of the present study was to evaluate the impact of structured reporting on the learning process as indicated by the overall report quality of head and neck ultrasound examinations within medical school education. METHODS: Following an immersion course on head and neck ultrasound, previously documented images of three common pathologies were handed out to 58 medical students who asked to create both standard free text reports (FTR) and structured reports (SR). A template for structured reporting of head and neck ultrasound examinations was created using a web-based approach. FTRs and SRs were evaluated with regard to overall quality, completeness, required time to completion and readability by two independent raters (Paired Wilcoxon test, 95% CI). Ratings were assessed for inter-rater reliability (Fleiss' kappa). Additionally, a questionnaire was utilized to evaluate user satisfaction. RESULTS: SRs received significantly better ratings in terms of report completeness (97.7% vs. 53.5%, p < 0.001) regarding all items. In addition, pathologies were described in more detail using SRs (70% vs. 51.1%, p < 0.001). Readability was significantly higher in all SRs when compared to FTRs (100% vs. 54.4%, p < 0.001). Mean time to complete was significantly lower (79.6 vs. 205.4 s, p < 0.001) and user satisfaction was significantly higher when using SRs (8.5 vs. 4.1, p < 0.001). Also, inter-rater reliability was very high (Fleiss' kappa 0.93). CONCLUSIONS: SRs of head and neck ultrasound examinations provide more detailed information with a better readability in a time-saving manner within medical education. Also, medical students may benefit from SRs in their learning process due to the structured approach and standardized terminology.

Xist localization and function: new insights from multiple levels.

In female mammals, one of the two X chromosomes in each cell is transcriptionally silenced in order to achieve dosage compensation between the genders in a process called X chromosome inactivation. The master regulator of this process is the long non-coding RNA Xist. During X-inactivation, Xist accumulates in cis on the future inactive X chromosome, triggering a cascade of events that provoke the stable silencing of the entire chromosome, with relatively few genes remaining active. How Xist spreads, what are its binding sites, how it recruits silencing factors and how it induces a specific topological and nuclear organization of the chromatin all remain largely unanswered questions. Recent studies have improved our understanding of Xist localization and the proteins with which it interacts, allowing a reappraisal of ideas about Xist function. We discuss recent advances in our knowledge of Xist-mediated silencing, focusing on Xist spreading, the nuclear organization of the inactive X chromosome, recruitment of the polycomb complex and the role of the nuclear matrix in the process of X chromosome inactivation.

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci.

BACKGROUND: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super-resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). RESULTS: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an 'autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. CONCLUSIONS: 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi.

Spatial separation of Xist RNA and polycomb proteins revealed by superresolution microscopy.

In female mammals, one of the two X chromosomes is transcriptionally silenced to equalize X-linked gene dosage relative to XY males, a process termed X chromosome inactivation. Mechanistically, this is thought to occur via directed recruitment of chromatin modifying factors by the master regulator, X-inactive specific transcript (Xist) RNA, which localizes in cis along the entire length of the chromosome. A well-studied example is the recruitment of polycomb repressive complex 2 (PRC2), for which there is evidence of a direct interaction involving the PRC2 proteins Enhancer of zeste 2 (Ezh2) and Supressor of zeste 12 (Suz12) and the A-repeat region located at the 5' end of Xist RNA. In this study, we have analyzed Xist-mediated recruitment of PRC2 using two approaches, microarray-based epigenomic mapping and superresolution 3D structured illumination microscopy. Making use of an ES cell line carrying an inducible Xist transgene located on mouse chromosome 17, we show that 24 h after synchronous induction of Xist expression, acquired PRC2 binding sites map predominantly to gene-rich regions, notably within gene bodies. Paradoxically, these new sites of PRC2 deposition do not correlate with Xist-mediated gene silencing. The 3D structured illumination microscopy was performed to assess the relative localization of PRC2 proteins and Xist RNA. Unexpectedly, we observed significant spatial separation and absence of colocalization both in the inducible Xist transgene ES cell line and in normal XX somatic cells. Our observations argue against direct interaction between Xist RNA and PRC2 proteins and, as such, prompt a reappraisal of the mechanism for PRC2 recruitment in X chromosome inactivation.

Pluripotency factor binding and Tsix expression act synergistically to repress Xist in undifferentiated embryonic stem cells.

BACKGROUND: Expression of Xist, the master regulator of X chromosome inactivation, is extinguished in pluripotent cells, a process that has been linked to programmed X chromosome reactivation. The key pluripotency transcription factors Nanog, Oct4 and Sox2 are implicated in Xist gene extinction, at least in part through binding to an element located in Xist intron 1. Other pathways, notably repression by the antisense RNA Tsix, may also be involved. RESULTS: Here we employ a transgene strategy to test the role of the intron 1 element and Tsix in repressing Xist in ES cells. We find that deletion of the intron 1 element causes a small increase in Xist expression and that simultaneous deletion of the antisense regulator Tsix enhances this effect. CONCLUSION: We conclude that Tsix and pluripotency factors act synergistically to repress Xist in undifferentiated embryonic stem cells. Double mutants do not exhibit maximal levels of Xist expression, indicating that other pathways also play a role.

A scaffold for X chromosome inactivation.

X chromosome inactivation (XCI), the silencing of one of the two X chromosomes in XX female cells, equalises the dosage of X-linked genes relative to XY males. The process is mediated by the non-coding RNA X inactive specific transcript (Xist) that binds in cis and propagates along the inactive X chromosome elect, triggering chromosome-wide silencing. The mechanisms by which Xist RNA binds and spreads along the chromosome, and initiates Xist-mediated chromosome silencing remain poorly understood. Accumulating evidence suggests that chromosome and nuclear organisation are important in both processes. Notably, recent studies have identified specific factors, previously shown to be components of the nuclear matrix or scaffold, to play a role both in Xist RNA-binding and in Xist-mediated silencing. In this review we provide a perspective on these studies in the context of previous work on chromosome/nuclear architecture in XCI.

The ATRX-ADD domain binds to H3 tail peptides and reads the combined methylation state of K4 and K9.

Mutations in the ATRX protein are associated with the alpha-thalassemia and mental retardation X-linked syndrome (ATR-X). Almost half of the disease-causing mutations occur in its ATRX-Dnmt3-Dnmt3L (ADD) domain. By employing peptide arrays, chromatin pull-down and peptide binding assays, we show specific binding of the ADD domain to H3 histone tail peptides containing H3K9me3. Peptide binding was disrupted by the presence of the H3K4me3 and H3K4me2 modification marks indicating that the ATRX-ADD domain has a combined readout of these two important marks (absence of H3K4me2 and H3K4me3 and presence of H3K9me3). Disease-causing mutations reduced ATRX-ADD binding to H3 tail peptides. ATRX variants, which fail in the H3K9me3 interaction, show a loss of heterochromatic localization in cells, which indicates the chromatin targeting function of the ADD domain of ATRX. Disruption of H3K9me3 binding may be a general pathogenicity pathway of ATRX mutations in the ADD domain which may explain the clustering of disease mutations in this part of the ATRX protein.

Sensitive and viable quantification of inside-out signals for LFA-1 activation in human cytotoxic lymphocytes by flow cytometry.

An early step in immunosurveillance by cytotoxic lymphocytes is leukocyte functional antigen (LFA)-1-dependent adhesion to target cells, which is promoted by inside-out signals from receptors such as the T cell receptor and a variety of natural killer (NK) cell activating receptors. Inside-out signals induce a conformational change in LFA-1, resulting in an extension of the extracellular domain of the receptor. Here, we have evaluated several mAbs that specifically detect the extended conformation of LFA-1 and detail a protocol for flow cytometric quantification of ß2-integrin activation in human peripheral blood cytotoxic T cells and NK cells in response to target cell recognition. By comparison to the markers of degranulation and chemokine synthesis, e.g. surface CD107a expression and intracellular MIP-1ß expression, respectively, evaluation of LFA-1 conformational changes represent a sensitive and rapid parameter of primary cytotoxic lymphocyte activation that can facilitate isolation of viable cells. Potentially, combined with other read-outs of cytotoxic lymphocyte function, this technique is applicable to the assessment of various clinical conditions, including for the diagnosis of primary immunodeficiency syndromes and for evaluating the efficacy of tumor targeting by donor lymphocytes.