Epigenomic Convergence of Neural-Immune Risk Factors in Neurodevelopmental Disorder Cortex.

Neurodevelopmental disorders (NDDs) affect 7-14% of all children in developed countries and are one of the leading causes of lifelong disability. Epigenetic modifications are poised at the interface between genes and environment and are predicted to reveal insight into NDD etiology. Whole-genome bisulfite sequencing was used to examine DNA cytosine methylation in 49 human cortex samples from 3 different NDDs (autism spectrum disorder, Rett syndrome, and Dup15q syndrome) and matched controls. Integration of methylation changes across NDDs with relevant genomic and genetic datasets revealed differentially methylated regions (DMRs) unique to each type of NDD but with shared regulatory functions in neurons and microglia. NDD DMRs were enriched within promoter regions and for transcription factor binding sites with identified methylation sensitivity. DMRs from all 3 disorders were enriched for ontologies related to nervous system development and genes with disrupted expression in brain from neurodevelopmental or neuropsychiatric disorders. Genes associated with NDD DMRs showed expression patterns indicating an important role for altered microglial function during brain development. These findings demonstrate an NDD epigenomic signature in human cortex that will aid in defining therapeutic targets and early biomarkers at the interface of genetic and environmental NDD risk factors.

Equine meniscal degeneration is associated with medial femorotibial osteoarthritis.

BACKGROUND: There is limited information available concerning normal equine meniscal morphology, its degeneration and role in osteoarthritis (OA). OBJECTIVES: To characterise normal equine meniscal morphology and lesions and to explore the relationship between equine meniscal degeneration and femorotibial OA. STUDY DESIGN: Ex vivo cadaveric study. METHODS: Menisci were harvested from 7 normal joints (n = 14 menisci) and 15 joints with OA (n = 30 menisci). A macroscopic femorotibial OA score (cartilage degeneration and osteophytosis) was employed to measure disease severity in each compartment. The femoral and tibial meniscal surfaces were scored for macroscopic fibrillation and tears (1-4). Histological sections (regions: cranial and caudal horn; body) were also scored for microscopic fibrillation and tears (0-3) and inner border degeneration (0-3). RESULTS: Partial meniscal tears were present on both femoral and tibial surfaces in all 3 regions and most frequently identified on the femoral surface of the cranial horn of the medial meniscus and body of the lateral meniscus. There was a significantly positive correlation between the global medial meniscal macroscopic scores and osteophyte (r = 0.7, P = 0.002) or cartilage degeneration (r = 0.5, P = 0.03) scores within the medial femorotibial joint. The global medial meniscal macroscopic score was greater (P = 0.004) in the advanced OA joints compared with control joints. MAIN LIMITATIONS: The menisci were principally from abattoir specimens without a known clinical history because of the challenge in obtaining a large number of specimens with a clinical diagnosis of femorotibial OA. CONCLUSIONS: This study is the first to describe normal equine meniscal morphology and lesions. Meniscal lesions were identified in all segments and on both articular surfaces. Meniscal degeneration significantly correlated with OA severity in the equine medial femorotibial joint. The relationship between OA and meniscal pathology remains to be elucidated.

X chromosome gene methylation in peripheral lymphocytes from monozygotic twins discordant for scleroderma.

Scleroderma (SSc) is a rare connective tissue disease characterized by fibrosis, microvasculopathy and autoimmune features. The role of genetics is limited in SSc, as suggested by similar concordance rates in monozygotic and dizygotic twin pairs, while environmental factors may act through epigenetic changes, as demonstrated for specific genes. Further, sex chromosome changes have been reported in SSc and may explain the female preponderance. In the present study we compared the methylation profile of all X chromosome genes in peripheral blood mononuclear cells from monozygotic twins discordant (n=7) and concordant (n=1) for SSc. Methylated DNA immunoprecipitations from each discordant twin pair were hybridized to a custom-designed array included 998 sites encompassing promoters of all X chromosome genes and randomly chosen autosomal genes. Biostatistical tools identified sites with an elevated probability to be consistently hypermethylated (n=18) or hypomethylated (n=25) in affected twins. Identified genes include transcription factors (ARX, HSFX1, ZBED1, ZNF41) and surface antigens (IL1RAPL2, PGRMC1), and pathway analysis suggests their involvement in cell proliferation (PGK1, SMS, UTP14A, SSR4), apoptosis (MTM1), inflammation (ARAF) and oxidative stress (ENOX2). In conclusion, we propose that X chromosome genes with different methylation profiles in monozygotic twin pairs may constitute candidates for SSc susceptibility.

Tailoring treatment to the individual in type 2 diabetes practical guidance from the Global Partnership for Effective Diabetes Management.

Good glycaemic control continues to be the most effective therapeutic manoeuvre to reduce the risk of development and/or progression of microvascular disease, and therefore remains the cornerstone of diabetes management despite recent scepticism about tight glucose control strategies. The impact on macrovascular complications is still a matter of debate, and so glycaemic control strategies should be placed in the context of multifactorial intervention to address all cardiovascular risk factors. Approaches to achieve glycaemic targets should always ensure patient safety, and results from recent landmark outcome studies support the need for appropriate individualisation of glycaemic targets and of the means to achieve these targets, with the ultimate aim to optimise outcomes and minimise adverse events, such as hypoglycaemia and marked weight gain. The primary goal of the Global Partnership for Effective Diabetes Management is the provision of practical guidance to improve patient outcomes and, in this article, we aim to support healthcare professionals in appropriately tailoring type 2 diabetes treatment to the individual. Patient groups requiring special consideration are identified, including newly diagnosed individuals with type 2 diabetes but no complications, individuals with a history of inadequate glycaemic control, those with a history of cardiovascular disease, children and individuals at risk of hypoglycaemia. Practical guidance specific to each group is provided.

Chromosome 15q11-13 duplication syndrome brain reveals epigenetic alterations in gene expression not predicted from copy number.

BACKGROUND: Chromosome 15q11-13 contains a cluster of imprinted genes essential for normal mammalian neurodevelopment. Deficiencies in paternal or maternal 15q11-13 alleles result in Prader-Willi or Angelman syndromes, respectively, and maternal duplications lead to a distinct condition that often includes autism. Overexpression of maternally expressed imprinted genes is predicted to cause 15q11-13-associated autism, but a link between gene dosage and expression has not been experimentally determined in brain. METHODS: Postmortem brain tissue was obtained from a male with 15q11-13 hexasomy and a female with 15q11-13 tetrasomy. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure 10 15q11-13 transcripts in maternal 15q11-13 duplication, Prader-Willi syndrome, and control brain samples. Southern blot, bisulfite sequencing and fluorescence in situ hybridisation were used to investigate epigenetic mechanisms of gene regulation. RESULTS: Gene expression and DNA methylation correlated with parental gene dosage in the male 15q11-13 duplication sample with severe cognitive impairment and seizures. Strikingly, the female with autism and milder Prader-Willi-like characteristics demonstrated unexpected deficiencies in the paternally expressed transcripts SNRPN, NDN, HBII85, and HBII52 and unchanged levels of maternally expressed UBE3A compared to controls. Paternal expression abnormalities in the female duplication sample were consistent with elevated DNA methylation of the 15q11-13 imprinting control region (ICR). Expression of non-imprinted 15q11-13 GABA receptor subunit genes was significantly reduced specifically in the female 15q11-13 duplication brain without detectable GABRB3 methylation differences. CONCLUSION: Our findings suggest that genetic copy number changes combined with additional genetic or environmental influences on epigenetic mechanisms impact outcome and clinical heterogeneity of 15q11-13 duplication syndromes.

The team approach to diabetes management: partnering with patients.

The recent United Nations (UN) Resolution on diabetes calls for action to curb the severe risks posed by diabetes and its complications, and encourages member states to improve awareness, treatment and care of diabetes worldwide. Overcoming barriers to good glycaemic control is a pressing need as we work towards fulfilling the UN resolution. In this article, the Global Partnership for Effective Diabetes Management highlights diabetes care strategies worldwide which employ a patient-centered approach that has improved patient care and health outcomes. Examples include implementation of multidisciplinary teams and forging of effective patient partnerships to motivate and empower individuals with type 2 diabetes to take control of their condition. These real-world case studies provide practical ways to facilitate effective diabetes care across the spectrum of resource settings worldwide.

Particle pinch with fully noninductive lower hybrid current drive in Tore Supra.

Recently, plasmas exceeding 4 min have been obtained with lower hybrid current drive (LHCD) in Tore Supra. These LHCD plasmas extend for over 80 times the resistive current diffusion time with zero loop voltage. Under such unique conditions the neoclassical particle pinch driven by the toroidal electric field vanishes. Nevertheless, the density profile remains peaked for more than 4 min. For the first time, the existence of an inward particle pinch in steady-state plasma without toroidal electric field, much larger than the value predicted by the collisional neoclassical theory, is experimentally demonstrated.

Detection of low level HER-2/neu gene amplification in prostate cancer by fluorescence in situ hybridization.

PURPOSE: Although expression of the HER-2/neu oncogene has been correlated with tumor progression in prostate cancer, the biologic significance of detecting HER-2/neu gene amplification by fluorescence in situ hybridization (FISH) or evidence for protein overexpression by immunohistochemistry (IHC) remains unclear. In this study, we directly compared HER-2/neu FISH and IHC to determine which may be more predictive of the response to trastuzumab. PATIENTS AND METHODS: Forty patients with prostate cancer were analyzed for gene amplification by FISH performed with HER-2/neu and chromosome 17 (CEP 17) DNA probes (Vysis). Protein expression was examined by immunofluorescence and by IHC using the DAKO HercepTest antibody protocol and a monoclonal antibody to Her-2/neu on archival paraffin sections. The patients included 30 men with primary tumors that were treated with radical prostatectomy. Of these, 15 demonstrated subsequent disease progression within 3 years. Five patients with prostatic intraepithelial neoplasia were tested, as were five with metastatic disease whose samples were obtained before androgen ablation therapy. RESULTS: None of the 30 primary prostate cancer specimens showed overexpression for HER-2/neu by immunofluorescence or by IHC with the DAKO protocol. One sample showed 3+ membrane expression with the monoclonal antibody. In contrast, low copy number gene amplifications (3-8 HER-2/neu signals/nucleus) were detected in 16 of 30 samples (53%) by FISH. Most amplified cells were diploid for CEP 17, demonstrating that amplification was not due to total cell aneuploidy. FISH and IHC determined that prostatic intraepithelial neoplasia samples were normal. Four of five (80%) metastatic samples were amplified for HER-2/neu by FISH. Nearly 70% of metastatic cancer cells among all five specimens demonstrated aneuploidy. A single lymph node metastasis showed 3+ membrane staining by IHC (DAKO). CONCLUSIONS: In contrast to breast cancer, FISH detects HER-2/neu amplification in a substantial proportion of prostate cancers that do not overexpress HER-2/neuby IHC. Although the biologic significance of this finding is uncertain, it has implications for the direction of current and planned clinical trials of trastuzumab in advanced prostate cancer, including determination of patient eligibility.

Quantitative localization of heterogeneous methyl-CpG-binding protein 2 (MeCP2) expression phenotypes in normal and Rett syndrome brain by laser scanning cytometry.

Rett syndrome (RTT) is an X-linked, dominant neurodevelopmental disorder caused by mutations in MECP2, encoding the methyl-CpG-binding protein 2 (MeCP2). A major paradox in the pathogenesis of RTT is how mutations in ubiquitously transcribed MECP2 result in a phenotype specific to the central nervous system (CNS) during postnatal development. To address this question, we have used a novel approach for quantitating the level and distribution of wild-type and mutant MeCP2 in situ by immunofluorescence and laser scanning cytometry. Surprisingly, cellular heterogeneity in MeCP2 expression level was observed in normal brain with a subpopulation of cells exhibiting high expression (MeCP2(hi)) and the remainder exhibiting low expression (MeCP2(lo)). MeCP2 expression was significantly higher in CNS compared with non-CNS tissues of human and mouse by automated quantitation of MeCP2 on multiple tissue arrays. Quantitative localization of MeCP2 expression phenotypes in normal human brain showed a mosaic, but distinct, distribution pattern, with MeCP2(hi) neurons highest in layer IV of the cerebrum and MeCP2(lo )neurons highest in the granular layer of the cerebellum. In female RTT brains, MECP2 mutant-expressing cells were identified as cells negative for the MeCP2 C-terminal epitope. MECP2 mutant-expressing cells were randomly localized in Rett cerebrum and cerebellum and showed normal MeCP2 expression with N-terminal-specific anti-MeCP2. These results demonstrate a CNS-specific cellular phenotype of MeCP2 high expression and suggest that MECP2 mutations in RTT are only manifested in MeCP2(hi) cells. In addition, our results demonstrate the power of laser scanning cytometry in examining complex cellular phenotypes in disease pathogenesis.

Automated quantitation of cell-mediated HIV type 1 infection of human syncytiotrophoblast cells by fluorescence in situ hybridization and laser scanning cytometry.

Infection of human placental syncytiotrophoblast cells with HIV requires direct contact with infected leukocytes. In vitro investigations into mechanisms regulating placental HIV transmission and into the development of therapeutic interventions have been hampered by difficulties inherent in quantitating HIV levels in cocultures of infected lymphocytes and adherent multinucleated syncytiotrophoblast cells. Here, we have used fluorescence in situ hybridization (FISH) for the direct detection of HIV-1 RNA within syncytiotrophoblast cells combined with laser scanning cytometry (LSC) to quantitate HIV levels exclusively in the syncytiotrophoblast cells. HIV-1-infected lymphocytic MOLT-4 cells were cocultured with primary human syncytiotrophoblast cells. Lymphocytic cells were identified with an anti-vimentin antibody and Cy5. HIV RNA was localized by in situ hybridization, using a digoxigenin-labeled riboprobe detected by Oregon Green, and nuclei were stained with 7-aminoactinomycin D. The three-color cocultures were analyzed by LSC to remove unwanted cell populations and quantitate HIV expression levels. The total HIV RNA level (green fluorescence integral) in each colony was normalized for cell size by dividing by the total DNA content (red fluorescence integral). The nuclear-normalized fluorescence integral was 2.3 times higher in infected cocultures than in uninfected cultures. When cocultures were incubated with 10 microM AZT, the green/red fluorescence integral value was significantly lower than that of cocultures incubated in the absence of AZT, corresponding to a 78% reduction in fluorescence. Laser scanning cytometry can be used to quantitate cell-mediated HIV infection in syncytiotrophoblast cells and should allow drug assessment studies and studies aimed at understanding the mechanism of virus entry into trophoblast cells to be carried out.

Clonal maintenance of imprinted expression of SNRPN and IPW in normal lymphocytes: correlation with allele-specific methylation of SNRPN intron 1 but not intron 7.

DNA methylation is a heritable and reversible modification to CpG sites in the mammalian genome. Parental allele-specific methylation is hypothesized to be important in the establishment and maintenance of imprinted gene expression; however, dynamic changes in allele-specific patterns have been observed. The upstream regulatory region of the small nuclear riboprotein N gene (SNRPN) is an important imprinting control region (ICR) for establishing and maintaining the methylation imprint in the locus on 15q11-13 associated with Prader-Willi and Angelman syndromes (PWS). To compare directly the role of allele-specific methylation patterns and the maintenance of imprinted expression in the PWS region, clonal populations of normal T lymphocytes were cultured for 22-25 generations. A novel long-range semi-nested polymerase chain reaction (PCR) strategy was utilized in order to span two different methylation sites, and a polymorphism within SNRPN was used so that allele-specific methylation of both sites could be determined. Reverse transcription/PCR followed by polymorphism analysis was also performed in order to determine parental allele-specific transcription. Exclusive paternal expression at both SNRPN and IPW was maintained in all T cell clones and correlated with maternal methylation of the intron 1 NotI site. In contrast, biallelic methylation was observed in all clones at the previously described paternally methylated HpaII site in intron 7. These results demonstrate that the maintenance of paternal expression of SNRPN and IPW correlates with a strict clonal maintenance of allele-specific methylation at the CpG-dense 5' end of SNRPN. Differential maintenance of methylation sites within imprinted genes may depend on the density and chromatin organization of surrounding CpG sites.

Flow cytometry and FISH to investigate allele-specific replication timing and homologous association of imprinted chromosomes.

Chromosome replication banding studies show that homologous regions on a pair of autosomes generally replicate at the same time in S phase (1). Izumikawa et al. first observed that this was not the case for the imprinted chromosomal region 15q11-q13 (2). This observation has been confirmed in other replication banding studies (3) as well by the fluorescence in situ hybridization (FISH) replication assay (4-9). The latter technique has also been used to observe DNA replication asynchrony in association with allelic inactivation of genes such as those encoding olfactory receptors and the cytokine, interleukin 2 (10,11). The latter genes are not imprinted but display random silencing of an allele in individual cells. In imprinted regions, DNA replication was generally observed to occur earlier on the paternal homologue (5,6,9,12,13). The patterns of allele-specific replication in the cells of Prader- Willi (PWS) and Angelman syndrome (AS) patients, however, have generally been synchronous (5,6,14). Furthermore, an investigation of the kinetics of allele-specific replication timing in the GABRB3/A5 cluster on 15q11-13 revealed that cells from PWS and AS have lost the strict replication timing observed on the parental chromosomes of normal cells (12). These results suggested the requirement of a biparental contribution for the regulation of replication asynchrony and lead to the hypothesis that allelic cross-talk, perhaps via pairing of homologous chromosomes, might play a role in the imprinting process.

Internal transport barrier with ion-cyclotron-resonance minority heating on tore supra

Recently, reversed magnetic shear operation was performed using only ion-cyclotron-resonance frequency minority heating (ICRH) during current ramp-up. A wide region of reversed magnetic shear has been obtained. For the first time, an electron internal transport barrier sustained by ICRH is observed, with a dramatical drop of density fluctuations. This barrier was maintained, on the current flat top, for about 2 s.

Parents' perceptions of chronic illness trajectories.

The notion of a small, generic set of chronic illness trajectories that can be independent of specific medical diagnoses, though controversial, has some theoretical, clinical, and qualitative research support. The purpose of this study was to quantitatively describe trajectories among parents of children with a chronic condition. It was hypothesized that factor analysis would confirm 3 trajectories similar to those in the qualitative literature and that parents' perceptions of their child's trajectory would differ significantly from medically based perceptions. A total of 140 parents provided data on their perceptions of the past, present, and future course of the condition of their repeatedly hospitalized child. Fourteen time-related items from the Coping Health Inventory for Parents Questionnaire on Resources and Stress and the Parenting Stress Index were analyzed. Pre- and post-hospitalization factor analyses extracted the same 8 items to construct 3 trajectories: Life Threatening; Declining; and Stable, Optimistic. The views of approximately one third of the parents differed from medically based classifications. Type of nursing care had no bearing on the perceptions of the parents.

Clonal heterogeneity at allelic methylation sites diagnostic for Prader-Willi and Angelman syndromes.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are developmental disorders resulting from the absence of the paternal or maternal contribution to the 15q11-13 region, respectively. Allele-specific methylation at D15S63 (PW71) has routinely been used as a diagnostic indicator of PWS and AS in DNA samples derived from peripheral blood. Extensive variation in allele-specific methylation patterns, however, has been observed at this site in different tissues, but the frequency or mechanism of this variation has remained uncharacterized. Herein, we have investigated the cellular basis of variation in methylation patterns at four sites of allelic methylation near SNRPN by using DNA samples derived from a panel of primary T lymphocyte clones. Interclonal variability was observed at three of these sites, including the diagnostic PW71 site. Changes in allele-specific methylation patterns occurred at a frequency of about one change in 50% of the cells every 22-25 doublings. In contrast, stable allele-specific methylation was observed in these clonal populations at exon 1 of SNRPN and the androgen receptor locus on the inactive X chromosome, suggesting that methylation at some CpG sites is more faithfully maintained than others. Clonal heterogeneity at PW71 was not an artifact of cell culture because the absence of allelic methylation was also observed in about 20% of the alleles in unstimulated peripheral blood. These results demonstrate that variation in allele-specific methylation at PW71 and other sites in the PWS/AS region appear to depend on the clonal complexity of the particular tissue and on the lack of strict maintenance of methylation within clones.

High-resolution analysis of DNA replication domain organization across an R/G-band boundary.

Establishing how mammalian chromosome replication is regulated and how groups of replication origins are organized into replication bands will significantly increase our understanding of chromosome organization. Replication time bands in mammalian chromosomes show overall congruency with structural R- and G-banding patterns as revealed by different chromosome banding techniques. Thus, chromosome bands reflect variations in the longitudinal structure and function of the chromosome, but little is known about the structural basis of the metaphase chromosome banding pattern. At the microscopic level, both structural R and G bands and replication bands occupy discrete domains along chromosomes, suggesting separation by distinct boundaries. The purpose of this study was to determine replication timing differences encompassing a boundary between differentially replicating chromosomal bands. Using competitive PCR on replicated DNA from flow-sorted cell cycle fractions, we have analyzed the replication timing of markers spanning roughly 5 Mb of human chromosome 13q14.3/q21.1. This is only the second report of high-resolution analysis of replication timing differences across an R/G-band boundary. In contrast to previous work, however, we find that band boundaries are defined by a gradient in replication timing rather than by a sharp boundary separating R and G bands into functionally distinct chromatin compartments. These findings indicate that topographical band boundaries are not defined by specific sequences or structures.

RAB22 and RAB163/mouse BRCA2: proteins that specifically interact with the RAD51 protein.

The human RAD51 protein is a homologue of the bacteria RecA and yeast RAD51 proteins that are involved in homologous recombination and DNA repair. RAD51 interacts with proteins involved in recombination and also with tumor suppressor proteins p53 and breast cancer susceptibility gene 1 (BRCA1). We have used the yeast two-hybrid system to clone murine cDNA sequences that encode two RAD51-associated molecules, RAB22 and RAB163. RAB163 encodes the C-terminal portion of mouse BRCA2, the homologue of the second breast cancer susceptibility gene protein in humans, demonstrating an in vitro association between RAD51 and BRCA2. RAB22 is a novel gene product that also interacts with RAD51 in vitro. To detect RAD51 interactions in vivo, we developed a transient nuclear focus assay that was used to demonstrate a complete colocalization of RAB22 with RAD51 in large nuclear foci.

Homologous association of oppositely imprinted chromosomal domains.

Human chromosome 15q11-q13 encompasses the Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) loci, which are subject to parental imprinting, a process that marks the parental origin of certain chromosomal subregions. A temporal and spatial association between maternal and paternal chromosomes 15 was observed in human T lymphocytes by three-dimensional fluorescence in situ hybridization. This association occurred specifically at the imprinted 15q11-q13 regions only during the late S phase of the cell cycle. Cells from PWS and AS patients were deficient in association, which suggests that normal imprinting involves mutual recognition and preferential association of maternal and paternal chromosomes 15.

Domain organization of allele-specific replication within the GABRB3 gene cluster requires a biparental 15q11-13 contribution.

Imprinting marks the parental origin of chromosomes, resulting in allele-specific changes in chromatin organization, transcription and replication. We report a 50-60 kb domain of allele-specific replication between the gamma-aminobutyric acid receptor subunit beta 3 (GABRB3) and alpha 5 (GABRA5) genes. Replication of this domain occurs in early S phase on the maternal chromosome 15 but is delayed until the end of S phase on the paternal homologue. In contrast, the genomic regions flanking this domain exhibit paternal earlier replication in mid to late S phase. Uniparental disomy or hemizygous deletion of chromosome 15 results in altered allele-specific replication kinetics compared with normals, suggesting that allele-specific replication within the GABRB3/A5 region may be regulated by reciprocal imprints on the maternal and paternal chromosomes.

T cell anergy.

T cell clonal anergy is a proposed mechanism of immunologic self tolerance in which T cells become functionally inactivated after previous stimulation. MHC class II-restricted antigen presentation by different cell types has been speculated to have a role in determining activation vs. anergy in responding T cells. Human T cells express MHC class II after activation and have been shown to present high concentrations of degraded peptide antigen to autologous T cells resulting in clonal anergy. In contrast to low antigen dose T cell clonal anergy, which occurs in the absence of costimulation, T cell anergy induced by human T cell presentation of antigen results in both primary proliferation and secondary unresponsiveness to high-dose antigenic stimulation. Although clonal anergy was previously thought to prevent autoreactive T cells from ever responding to self antigen presented without costimulation, we postulate that T cell presentation of antigen represents a "fail-safe" mechanism of immunologic self tolerance that would anergize clonally expanded autoreactive T cells when they are surrounded by a high extracellular concentration of degraded self antigen.