Differences of size and shape of active and inactive X-chromosome domains in human amniotic fluid cell nuclei

It is a widely held belief that the inactive X-chromosome (Xi) in female cell nuclei is strongly condensed as compared to the largely decondensed active X-chromosome (Xa). We have reconsidered this problem and painted X-chromosome domains in nuclei of subconfluent, female and male human amniotic fluid cell cultures (46, XX and 46, XY) by chromosomal in situ suppression (CISS) hybridization with biotinylated human X-chromosome specific library DNA. FITC-conjugated avidin was used for probe detection and nuclei were counterstained with propidium iodide (PI). The shape of these nuclei resembling flat ellipsoids or elliptical cylinders makes them suitable for both two-dimensional (2D) and three-dimensional (3D) analyses. 2D analyses of Xi- and Xa-domains were performed in 34 female cell nuclei by outlining of the painted domains using a camera lucida. Identification of the sex chromatin body in DAPI-stained nuclei prior to CISS-hybridization was confirmed by its colocalization with one of the two painted X-domains. In 31 of the 34 nuclei the area AXi for the inactive X-domain was smaller than the area AXa for the active domain (mean ratio AXa/AXi = 1.9 ± 0.8 SD, range 1.0-4.3). The signed rank test showed a highly significant (P r(Xi) demonstrating a generally more elongated structure of Xa. For 3D analysis a confocal scanning laser fluorescence microscope (CSLFM) was used. Ten to 20 light optical sections (PI-image, FITC-image) were registered with equal spacings (approx. 0.4 m). A thresholding procedure was applied to determine the PI-labeled nuclear and FITC-labeled X-domain areas in each section. Estimated slice volumes were used to compute total nuclear and X-domain volumes. In a series of 35 female nuclei most domains extended from the top to the bottom nuclear sections. The larger of the two X-chromosome domains comprised (3.7 ± 1.7 S.D.)% of the nuclear volume. A mean ratio of 1.2 ± 0.2 SD (range 1.1-2.3) was found for the volumes of the larger and the smaller X-domains in these female nuclei. In a series of 27 male amniotic fluid cell nuclei the relative X-chromosome domain volume comprised (4.0 ± 2.6 S.D.)%. These findings indicate that differences in the 3D expansion of active and inactive X-chromosome domains are less pronounced than previously thought. A current model suggests that chromosome domains consist of a compact core surrounded by loosely coiled outer chromatin fiber loops. The latter fraction may be considerably larger in Xa- as compared to Xi-domains. We suggest that the interactive outlining procedure used in the 2D analyses included the loosely structured domain periphery more accurately, while the threshold algorithm applied to light optical sections delineated the more compact core of the domains, leading to smaller and more similar volume estimates of Xa and Xi. Present limitations of nuclear and chromosome domain volume measurements using confocal laser scanning microscopy are discussed

Large-scale computations on histology images reveal grade-differentiating parameters for breast cancer

BACKGROUND: Tumor classification is inexact and largely dependent on the qualitative pathological examination of the images of the tumor tissue slides. In this study, our aim was to develop an automated computational method to classify Hematoxylin and Eosin (H&E) stained tissue sections based on cancer tissue texture features. METHODS: Image processing of histology slide images was used to detect and identify adipose tissue, extracellular matrix, morphologically distinct cell nuclei types, and the tubular architecture. The texture parameters derived from image analysis were then applied to classify images in a supervised classification scheme using histologic grade of a testing set as guidance. RESULTS: The histologic grade assigned by pathologists to invasive breast carcinoma images strongly correlated with both the presence and extent of cell nuclei with dispersed chromatin and the architecture, specifically the extent of presence of tubular cross sections. The two parameters that differentiated tumor grade found in this study were (1) the number density of cell nuclei with dispersed chromatin and (2) the number density of tubular cross sections identified through image processing as white blobs that were surrounded by a continuous string of cell nuclei. Classification based on subdivisions of a whole slide image containing a high concentration of cancer cell nuclei consistently agreed with the grade classification of the entire slide. CONCLUSION: The automated image analysis and classification presented in this study demonstrate the feasibility of developing clinically relevant classification of histology images based on micro- texture. This method provides pathologists an invaluable quantitative tool for evaluation of the components of the Nottingham system for breast tumor grading and avoid intra-observer variability thus increasing the consistency of the decision-making process

Stepped vitrification technique for human ovarian tissue cryopreservation

The advantage of stepped vitrification (SV) is avoiding ice crystal nucleation, while decreasing the toxic effects of high cryoprotectant concentrations. We aimed to test this method for human ovarian tissue cryopreservation. Ovarian cortex was taken from 7 fertile adult women. Samples were subjected to an SV protocol performed in an automatic freezer, which allowed sample transfer to ever higher concentrations of dimethyl sulfoxide (DMSO) as the temperature was reduced. Histological evaluation of the vitrified-warmed tissue showed large numbers of degenerated follicles after 24 hours of in vitro culture. We therefore evaluated DMSO perfusion rates by X-ray computed tomography, ice crystal formation by freeze-substitution, and cell toxicity by transmission electron microscopy, seeking possible reasons why follicles degenerated. Although cryoprotectant perfusion was considered normal and no ice crystals were formed in the tissue, ultrastructural analysis detected typical signs of DMSO toxicity, such as mitochondria degeneration, alterations in chromatin condensation, cell vacuolization and extracellular matrix swelling in both stromal and follicular cells. The findings indicated that the method failed to preserve follicles due to the high concentrations of DMSO used. However, adaptations can be made to avoid toxicity to follicles caused by elevated levels of cryoprotectants.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 2016/22947-

Chipping away at gamma-H2AX foci

The mammalian histone H2AX protein functions as a dosage-dependent genomic caretaker and tumor suppressor. Phosphorylation of H2AX to form gamma-H2AX in chromatin around DNA double strand breaks (DSBs) is an early event following induction of these hazardous lesions. For a decade, mechanisms that regulate H2AX phosphorylation have been investigated mainly through two-dimensional immunofluorescence (IF). We recently used chromatin immunoprecipitation (ChIP) to measure gamma-H2AX densities along chromosomal DNA strands broken in G(1) phase mouse lymphocytes. Our experiments revealed that (1) gamma-H2AX densities in nucleosomes form at high levels near DSBs and at diminishing levels farther and farther away from DNA ends, and (2) ATM regulates H2AX phosphorylation through both MDC1-dependent and MDC1-independent means. Neither of these mechanisms were discovered by previous if studies due to the inherent limitations of light microscopy. Here, we compare data obtained from parallel gamma-H2AX ChIP and three-dimensional IF analyses and discuss the impact of our findings upon molecular mechanisms that regulate H2AX phosphorylation in chromatin around DNA breakage sites

TFIIIC Localizes Budding Yeast ETC Sites to the Nuclear Periphery

Peer reviewedPublisher PD

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

The nuclear topography of splicing snRNPs, mRNA transcripts and chromosome domains in various mammalian cell types are described. The visualization of splicing snRNPs, defined by the Sm antigen, and coiled bodies, revealed distinctly different distribution patterns in these cell types. Heat shock experiments confirmed that the distribution patterns also depend on physiological parameters. Using a combination of fluorescencein situ hybridization and immunodetection protocols, individual chromosome domains were visualized simultaneously with the Sm antigen or the transcript of an integrated human papilloma virus genome. Three-dimensional analysis of fluorescence-stained target regions was performed by confocal laser scanning microscopy. RNA transcripts and components of the splicing machinery were found to be generally excluded from the interior of the territories occupied by the individual chromosomes. Based on these findings we present a model for the functional compartmentalization of the cell nucleus. According to this model the space between chromosome domains, including the surface areas of these domains, defines a three-dimensional network-like compartment, termed the interchromosome domain (ICD) compartment, in which transcription and splicing of mRNA occurs

Targeting EZH2-mediated methylation of H3K27 inhibits proliferation and migration of Synovial Sarcoma in vitro.

Synovial sarcoma is an aggressive soft tissue sarcoma genetically defined by the fusion oncogene SS18-SSX. It is hypothesized that either SS18-SSX disrupts SWI/SNF complex inhibition of the polycomb complex 2 (PRC2) methyltransferase Enhancer of Zeste Homologue 2 (EZH2), or that SS18-SSX is able to directly recruit PRC2 to aberrantly silence target genes. This is of potential therapeutic value as several EZH2 small molecule inhibitors are entering early phase clinical trials. In this study, we first confirmed EZH2 expression in the 76% of human synovial sarcoma samples. We subsequently investigated EZH2 as a therapeutic target in synovial sarcoma in vitro. Knockdown of EZH2 by shRNA or siRNA resulted in inhibition of cell growth and migration across a series of synovial sarcoma cell lines. The EZH2 selective small-molecule inhibitor EPZ005687 similarly suppressed cell proliferation and migration. These data support the hypothesis that targeting EZH2 may be a promising therapeutic strategy in the treatment of synovial sarcoma; clinical trials are initiating enrollment currently