Nonrigid registration of 3-d multichannel microscopy images of cell nuclei.

We present an intensity-based nonrigid registration approach for the normalization of 3-D multichannel microscopy images of cell nuclei. A main problem with cell nuclei images is that the intensity structure of different nuclei differs very much; thus, an intensity-based registration scheme cannot be used directly. Instead, we first perform a segmentation of the images from the cell nucleus channel, smooth the resulting images by a Gaussian filter, and then apply an intensity-based registration algorithm. The obtained transformation is applied to the images from the nucleus channel as well as to the images from the other channels. To improve the convergence rate of the algorithm, we propose an adaptive step length optimization scheme and also employ a multiresolution scheme. Our approach has been successfully applied using 2-D cell-like synthetic images, 3-D phantom images as well as 3-D multichannel microscopy images representing different chromosome territories and gene regions. We also describe an extension of our approach, which is applied for the registration of 3D + t (4-D) image series of moving cell nuclei.

Xist yeast artificial chromosome transgenes function as X-inactivation centers only in multicopy arrays and not as single copies.

X-chromosome inactivation in female mammals is controlled by the X-inactivation center (Xic). This locus is required for inactivation in cis and is thought to be involved in the counting process which ensures that only a single X chromosome remains active per diploid cell. The Xist gene maps to the Xic region and has been shown to be essential for inactivation in cis. Transgenesis represents a stringent test for defining the minimal region that can carry out the functions attributed to the Xic. Although YAC and cosmid Xist-containing transgenes have previously been reported to be capable of cis inactivation and counting, the transgenes were all present as multicopy arrays and it was unclear to what extent individual copies are functional. Using two different yeast artificial chromosomes (YACs), we have found that single-copy transgenes, unlike multicopy arrays, can induce neither inactivation in cis nor counting. These results demonstrate that despite their large size and the presence of Xist, the YACs that we have tested lack sequences critical for autonomous function with respect to X inactivation.

Functional analysis of the DXPas34 locus, a 3' regulator of Xist expression.

X inactivation in female mammals is controlled by a key locus on the X chromosome, the X-inactivation center (Xic). The Xic controls the initiation and propagation of inactivation in cis. It also ensures that the correct number of X chromosomes undergo inactivation (counting) and determines which X chromosome becomes inactivated (choice). The Xist gene maps to the Xic region and is essential for the initiation of X inactivation in cis. Regulatory elements of X inactivation have been proposed to lie 3' to Xist. One such element, lying 15 kb downstream of Xist, is the DXPas34 locus, which was first identified as a result of its hypermethylation on the active X chromosome and the correlation of its methylation level with allelism at the X-controlling element (Xce), a locus known to affect choice. In this study, we have tested the potential function of the DXPas34 locus in Xist regulation and X-inactivation initiation by deleting it in the context of large Xist-containing yeast artificial chromosome transgenes. Deletion of DXPas34 eliminates both Xist expression and antisense transcription present in this region in undifferentiated ES cells. It also leads to nonrandom inactivation of the deleted transgene upon differentiation. DXPas34 thus appears to be a critical regulator of Xist activity and X inactivation. The expression pattern of DXPas34 during early embryonic development, which we report here, further suggests that it could be implicated in the regulation of imprinted Xist expression.

The dynamics of imprinted X inactivation during preimplantation development in mice.

In the mouse, there are two forms of X chromosome inactivation (XCI), random XCI in the fetus and imprinted paternal XCI, which is limited to the extraembryonic tissues. While the mechanism of random XCI has been studied extensively using the in vitro XX ES cell differentiation system, imprinted XCI during early embryonic development has been less well characterized. Recent studies of early embryos have reported unexpected findings for the paternal X chromosome (Xp). Imprinted XCI may not be linked to meiotic silencing in the male germ line but rather to the imprinted status of the Xist gene. Furthermore, the Xp becomes inactivated in all cells of cleavage-stage embryos and then reactivated in the cells of the inner cell mass (ICM) that form the epiblast, where random XCI ensues.

Integrated kinetics of X chromosome inactivation in differentiating embryonic stem cells.

Inactivation of the X chromosome during early female development and the subsequent maintenance of this transcriptionally inert state through countless cell divisions remain a paradigm for epigenetic regulation in mammals. Nevertheless, the exact mechanisms underlying this chromosome-wide silencing process remain unclear. Using differentiating female embryonic stem (ES) cells as a model system, we recently found that histone H3 tail modifications are among the earliest known chromatin changes in the X inactivation process, appearing as soon as Xist RNA accumulates on the X chromosome, but prior to transcriptional silencing of X-linked genes (Heard et al., 2001). In this report we present an integrated analysis of the sequence of early events and chromatin modifications underlying X inactivation in differentiating female ES cells. We have extended our previous analysis concerning changes in histone tail modification states. We find that the hypomethylation of Arg-17 and that of Lys-36 on histone H3 also characterize the inactive X chromosome, and that these profiles show a similarly early onset during the initiation of X inactivation. In addition, we have investigated the kinetics of the shift in replication timing of the X chromosome undergoing inactivation. This event occurs slightly later than Xist RNA coating and the chromatin modifications. Finally, from an early stage in the X inactivation process, characteristic histone modification patterns can be found on the X chromosome at mitosis, suggesting that they represent true epigenetic marks of the inactive state.

Sentinel node localization in oral cavity and oropharynx squamous cell cancer.

OBJECTIVE: To evaluate the feasibility and predictive ability of the sentinel node localization technique for patients with squamous cell carcinoma of the oral cavity or oropharynx and clinically negative necks. DESIGN: Prospective, efficacy study comparing the histopathologic status of the sentinel node with that of the remaining neck dissection specimen. SETTING: Tertiary referral center. PATIENTS: Patients with T1 or T2 disease and clinically negative necks were eligible for the study. Nine previously untreated patients with oral cavity or oropharyngeal squamous cell carcinoma were enrolled in the study. INTERVENTIONS: Unfiltered technetium Tc 99m sulfur colloid injections of the primary tumor and lymphoscintigraphy were performed on the day before surgery. Intraoperatively, the sentinel node(s) was localized with a gamma probe and removed after tumor resection and before neck dissection. MAIN OUTCOME MEASURES: The primary outcome was the negative predictive value of the histopathologic status of the sentinel node for predicting cervical metastases. RESULTS: Sentinel nodes were identified in 9 previously untreated patients. In 5 patients, there were no positive nodes. In 4 patients, the sentinel nodes were the only histopathologically positive nodes. In previously untreated patients, the sentinel node technique had a negative predictive value of 100% for cervical metastasis. CONCLUSIONS: Our preliminary investigation shows that sentinel node localization is technically feasible in head and neck surgery and is predictive of cervical metastasis. The sentinel node technique has the potential to decrease the number of neck dissections performed in clinically negative necks, thus reducing the associated morbidity for patients in this group.

The dental patient with renal disease: precautions and guidelines.

Hemodialysis and related methods of treatment are keeping renal patients alive and able to carry on many of their daily activities. These patients, however, are limited in the scope within which they can function. Dietary requirements, fluid intake, and most medications must be rigidly controlled. Many commonly used drugs can damage the kidneys and must be avoided. Dentists who treat patients with renal insufficiency must avoid prescribing or administering nephrotoxic drugs or agents that become dangerous with loss of kidney function.

Cdyl, a new partner of the inactive X chromosome and potential reader of H3K27me3 and H3K9me2.

X chromosome inactivation is a remarkable example of chromosome-wide gene silencing and facultative heterochromatin formation. Numerous histone posttranslational modifications, including H3K9me2 and H3K27me3, accompany this process, although our understanding of the enzymes that lay down these marks and the factors that bind to them is still incomplete. Here we identify Cdyl, a chromodomain-containing transcriptional corepressor, as a new chromatin-associated protein partner of the inactive X chromosome (Xi). Using mouse embryonic stem cell lines with mutated histone methyltransferase activities, we show that Cdyl relies on H3K9me2 for its general association with chromatin in vivo. For its association with Xi, Cdyl requires the process of differentiation and the presence of H3K9me2 and H3K27me3, which both become chromosomally enriched following Xist RNA coating. We further show that the removal of the PRC2 component Eed and subsequent loss of H3K27me3 lead to a reduction of both Cdyl and H3K9me2 enrichment on inactive Xi. Finally, we show that Cdyl associates with the H3K9 histone methyltransferase G9a and the MGA protein, both of which are also found on Xi. We propose that the combination of H3K9me2 and H3K27me3 recruits Cdyl to Xi, and this, in turn, may facilitate propagation of the H3K9me2 mark by anchoring G9a.

Chromatin structure and nuclear organization dynamics during X-chromosome inactivation.

Early development of female mammals is accompanied by transcriptional inactivation of one of their two X chromosomes. This leads to monoallelic expression of most of the X chromosome and ensures dosage compensation with respect to males (XY). One of the most surprising aspects of this phenomenon is that the two X homologs are treated differently even though they are present within the same nucleus. In eutherian mammals, such as humans and mice, either the maternal or the paternal X is inactivated during early embryogenesis. Once set up, the silent state is epigenetically transmitted as cells divide, so that adult females are mosaics of clonal cell populations, which express either of their two X chromosomes. The past years have been marked by the discovery of several molecular events that accompany chromosome-wide silencing.

Sensing X chromosome pairs before X inactivation via a novel X-pairing region of the Xic.

Mammalian dosage compensation involves silencing of one of the two X chromosomes in females and is controlled by the X-inactivation center (Xic). The Xic, which includes Xist and its antisense transcription unit Tsix/Xite, somehow senses the number of X chromosomes and triggers Xist up-regulation from one of the two X chromosomes in females. We found that a segment of the mouse Xic lying several hundred kilobases upstream of Xist brings the two Xics together before the onset of X inactivation. This region can autonomously drive Xic trans-interactions even as an ectopic single-copy transgene. Its introduction into male embryonic stem cells is strongly selected against, consistent with a possible role in trans-activating Xist. We propose that homologous associations driven by this novel X-pairing region (Xpr) of the Xic enable a cell to sense that more than one X chromosome is present and coordinate reciprocal Xist/Tsix expression.

RNA and protein actors in X-chromosome inactivation.

In female mammals, one of the two X chromosomes is converted from the active euchromatic state into inactive heterochromatin during early embryonic development. This process, known as X-chromosome inactivation, results in the transcriptional silencing of over a thousand genes and ensures dosage compensation between the sexes. Here, we discuss the possible mechanisms of action of the Xist transcript, a remarkable noncoding RNA that triggers the X-inactivation process and also seems to participate in setting up the epigenetic marks that provide the cellular memory of the inactive state. So far, no functional protein partners have been identified for Xist RNA, but different lines of evidence suggest that it may act at multiple levels, including nuclear compartmentalization, chromatin modulation, and recruitment of Polycomb group proteins.

Rapid-fire improvement with short-cycle kaizen.

Continuous improvement is an attractive idea, but it is typically more myth than reality. SCK is no myth. It delivers dramatic improvements in traditional measures quickly. SCK accomplishes this via kaizens: rapid, repeated, time-compressed changes for the better in bite-sized chunks of the business.

Role play in X-inactivation.

Initiation of X-inactivation is known to depend on the presence of a unique region or locus known as the X-inactivation center, or Xic, defined by the study of chromosomal rearrangements. Its mode of action is currently unknown but is the scene of much research effort. This review explores the recent literature concerning the definition of Xic and its relationship both to Xist and to another genetic entity thought to play a role in the initiation of X-inactivation, the X-controlling element, or Xce.

X-chromosome inactivation: counting, choice and initiation.

In many sexually dimorphic species, a mechanism is required to ensure equivalent levels of gene expression from the sex chromosomes. In mammals, such dosage compensation is achieved by X-chromosome inactivation, a process that presents a unique medley of biological puzzles: how to silence one but not the other X chromosome in the same nucleus; how to count the number of X's and keep only one active; how to choose which X chromosome is inactivated; and how to establish this silent state rapidly and efficiently during early development. The key to most of these puzzles lies in a unique locus, the X-inactivation centre and a remarkable RNA--Xist--that it encodes.

A large inverted duplicated DNA region associated with an amplified oncogene is stably maintained in a YAC.

In the polyoma virus (Py) transformed 3B rat cell line the Py oncogene and adjacent cellular DNA are amplified in arrays of very large inverted duplications. A region of the 3B amplified DNA was cloned as a 550 kb insert in a Yeast Artificial Chromosome (YAC) vector, designated y3B01. Analysis of the y3B01 cloned insert revealed it contained a large inverted duplicated DNA region which was approximately 400 kb in size (two palindromic arms of about 200 kb). At least 420 kb of the 550 kb YAC insert has been identified as being derived from the 3B amplified DNA and the amplicon in 3B cells is at least 220 kb in size. No DNA instability of the y3B01 YAC clone was detected. The y3B01 DNA replicated as efficiently as yeast chromosomes and was structurally stable in yeast cells during more than 30 cell divisions. Comparison of the restriction endonuclease maps of the inverted duplicated region of the y3B01 DNA insert and the amplified 3B genomic DNA did not reveal any gross differences suggesting that no rearrangements had occurred during or after the cloning into the YAC vector. These results suggest that large inverted duplications, which can show instability in prokaryotic cloning systems, can be stably cloned and maintained in YAC vectors in yeast.

Creation of a deletion series of mouse YACs covering a 500 kb region around Xist.

Two mouse YACs, PA-2 and PA-3, contain the Xist gene and are 460 kb and 3.3 Mb long respectively. While PA-2 is non-chimeric, PA-3 contains a substantial proportion of non-contiguous DNA. As a prerequisite to functional studies of the role of this region in X inactivation, we have created a deletion series of YACs that are spaced at approximately 50 kb intervals and were able to eliminate the unwanted chimeric sequences in YAC PA-3. For this purpose, we have constructed mouse B1 fragmentation vectors based on those described for human Alu fragmentation. Having created this series of YAC deletion derivatives, we were able to eliminate efficiently the 10-15% aberrant YACs that arise during the course of a fragmentation experiment by assessing their marker content. The overlap and the opposite orientation of the two YAC inserts permitted the creation of deletions on both sides of the 500 kb region around Xist. The use of this series of YACs in a biological assay will help us define the extent of the sequences necessary to bring about X chromosome inactivation.

Xce haplotypes show modified methylation in a region of the active X chromosome lying 3' to Xist.

During early mammalian embryogenesis, one of the two X chromosomes in somatic cells of the female becomes inactivated through a process that is thought to depend on a unique initiator region, the X-chromosome inactivation center (Xic). The recently characterized Xist sequence (X-inactive-specific transcript) is thought to be a possible candidate for Xic. In mice a further genetic element, the X chromosome-controlling element (Xce), is also known to influence the choice of which of the two X chromosomes is inactivated. We report that a region of the mouse X chromosome lying 15 kb distal to Xist contains several sites that show hypermethylation specifically associated with the active X chromosome. Analysis of this region in various Xce strains has revealed a correlation between the strength of the Xce allele carried and the methylation status of this region. We propose that such a region could be involved in the initial stages of the inactivation process and in particular in the choice of which of the two X chromosomes present in a female cell will be inactivated.