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Beautiful Nature View 2/2 at Oceanwalk! In New Smyrna Beach in New Smyrna Beach
- bedrooms 2
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This beautifully decorated 2/2 at Oceanwalk comes fully equipped
with a Queen in the Master along with twin bunks and a queen bed in the second
bedroom. There is also a Queen Sleeper sofa making this unit able to comfortably
sleep 8. This spacious unit also have a fully equipped kitchen and a DVD player
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Oceanwalk really does have it all!
2 exercise rooms and a dry sauna
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We have two lighted tennis courts, two shuffleboard courts, lighted basketball hoop, putting green, Bocce ball court, theatre room and much, much more including the Canaveral National Seashore Park just 3 miles to the south
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Copyright © 2013 The Authors All rights reserved.
Cell Reports, Volume 3, Issue 2, 291-300, 07 February 2013
Dynamics of 5-Hydroxymethylcytosine and Chromatin Marks in Mammalian Neurogenesis
Maria A. Hahn1, 5, Runxiang Qiu2, 5, Xiwei Wu3, Arthur X. Li4, Heying Zhang2, Jun Wang2, Jonathan Jui2, Seung-Gi Jin1, Yong Jiang1, Gerd P. Pfeifer1, 6, , and Qiang Lu2, 6, ,
1 Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
2 Department of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
3 Department of Molecular Medicine, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
4 Department of Information Sciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
Corresponding author
5 These authors contributed equally to this work
6 These authors contributed equally to this work
Graphical Abstract
► 5hmC levels increase in neuronal function-related genes during neurogenesis ► 5hmC gain is accompanied by H3K27me3 loss at promoters and gene bodies ► Gain of 5hmC is not associated with substantial DNA demethylation ► Polycomb and Tet protein together promote proper progression of neurogenesis
- DNA methylation in mammals is highly dynamic during germ cell and preimplantation development but is relatively static during the development of somatic tissues. 5-hydroxymethylcytosine (5hmC), created by oxidation of 5-methylcytosine (5mC) by Tet proteins and most abundant in the brain, is thought to be an intermediary toward 5mC demethylation. We investigated patterns of 5mC and 5hmC during neurogenesis in the embryonic mouse brain. 5hmC levels increase during neuronal differentiation. In neuronal cells, 5hmC is not enriched at enhancers but associates preferentially with gene bodies of activated neuronal function-related genes. Within these genes, gain of 5hmC is often accompanied by loss of H3K27me3. Enrichment of 5hmC is not associated with substantial DNA demethylation, suggesting that 5hmC is a stable epigenetic mark. Functional perturbation of the H3K27 methyltransferase Ezh2 or of Tet2 and Tet3 leads to defects in neuronal differentiation, suggesting that formation of 5hmC and loss of H3K27me3 cooperate to promote brain development.
Methylation of cytosine at CpG sequences is an epigenetic modification linked to gene regulation and developmental processes (Suzuki and Bird, 2008). It has been assumed that patterns of 5mC display a certain level of plasticity during development (Bernstein et al., 2007; Mohn and Schübeler, 2009). This model proposes that 5mC needs to be rapidly removed by DNA demethylation processes at certain developmental stages. Mechanisms of active DNA demethylation have remained controversial, however (Ooi and Bestor, 2008; Wu and Zhang, 2010). Tet protein-mediated oxidation of 5mC resulting in the formation of 5hmC is a plausible intermediate step in a replication-independent DNA demethylation pathway (Guo et al., 2011; Tahiliani et al., 2009; Wu and Zhang, 2010). To address the potential biological significance of a putative C → 5mC → 5hmC → C methylation-demethylation pathway, analysis of 5mC and 5hmC at two temporal stages in development or in two linearly related cell types becomes essential. 5hmC is particularly abundant in mammalian brain tissue (Jin et al., 2011b; Kriaucionis and Heintz, 2009; Münzel et al., 2010; Szulwach et al., 2011; Szwagierczak et al., 2010). In this study, we investigated the in vivo patterns of 5mC and 5hmC at the developmental transition of neural progenitor cells (NPCs) from self-renewal to differentiation, comparing global changes of the two modified cytosines between NPCs and daughter neurons. We used a dual reporter strategy by generating transgenic mice in which NPCs are labeled with GFP expressed from the nestin promoter and differentiated neurons are labeled with RFP expressed from the doublecortin promoter. The use of a differentiation reporter in conjunction with a progenitor cell-specific promoter helps alleviate the problem of "carryover" of GFP from a primitive cell to progeny, thus allowing effective copurification of NPCs and daughter neurons from the brain (Wang et al., 2011). We mapped the distribution of cytosine modifications and several key histone methylation marks during this important developmental step and studied the role of Tet proteins and the Polycomb complex in this in vivo system.
5hmC Increases during Neuronal Differentiation and Associates with Genes Important for Neuron Function
Our data revealed a dynamic change of 5hmC during neurogenesis. First, immunostaining showed a noticeable increase of 5hmC level in association with neuronal differentiation. NPCs in the ventricular zone (VZ) and young neurons in the intermediate zone (IZ) of the cortex contain lower levels of 5hmC, whereas maturing neurons in the cortical plate (CP) are enriched with 5hmC (Figure 1A). The higher cell density in the forming cortical plate gives an apparent minor increase for staining of 5mC as well, but in the case of 5hmC, the difference is much stronger (and consistent in multiple sections). 5mC levels did not show an obvious reduction as might be anticipated if 5hmC acts as an intermediate leading to substantial DNA demethylation (Figure 1A). In the CP, 5mC appeared to be present in smaller speckles, most likely indicating pericentromeric heterochromatin, whereas 5hmC was distributed throughout nuclei as a euchromatic mark (Figure 1B). Second, liquid chromatography tandem mass spectrometry (LC-MS/MS) quantification of cytosine modifications in NPCs and neurons isolated using the dual reporter approach indicated a doubling of 5hmC levels in neurons in comparison to NPCs (p < 0.0001, t test), whereas 5mC levels remain unchanged (p = 0.22, t test) (Figure 1C). Third, we performed antibody-based 5hmC immunoprecipitation (hMeDIP) combined with mouse whole-genome tiling array analysis to examine the gene-specific distribution of 5hmC during neurogenesis. We did not see a bias of hMeDIP toward CA-containing repeat sequences as was previously suggested (Matarese et al., 2011) (Figure S1A), and 5hmC profiling by hMeDIP or by T4 glycosyltransferase-based 5hmC enrichment (Song et al., 2011) gave similar data (Figures S1B and S1C). For several DNA regions, hMeDIP patterns were also confirmed by Tet-assisted bisulfite (TAB) sequencing (Figure 1F; see also Figure 3A), which is able to distinguish between 5hmC and 5mC (Yu et al., 2012).
In light of previous reports showing enrichment of 5hmC at enhancers in embryonic stem cells (ESCs) (Yu et al., 2012), we analyzed the 5hmC pattern at p300 binding sites previously mapped in mouse forebrain at embryonic day 11.5 (Visel et al., 2009). It was shown that p300 binding accurately predicts enhancers in vivo (Visel et al., 2009). In contrast to ESCs, cortical NPCs and neurons show an absence of 5hmC at p300 sites (Figures 1D–1F and S1D). However, sequences adjacent to p300 sites are characterized by 5hmC occupancy, which becomes enhanced during differentiation. The absence of 5hmC at p300 sites was confirmed by TAB sequencing for two p300 binding sites, an intragenic site in the Elk3 gene and a site located upstream of the Sox5 promoter (Figures 1F and S1D). As expected, p300 sites are associated with an increased CpG density in comparison to surrounding areas (Figure S1E).
The hMeDIP data revealed enrichment of 5hmC at promoters and gene bodies (Figure S1F). We observed an increase of 5hmC signal, mostly in intragenic regions, during neuronal differentiation (Figures 1G–1I) but little change of 5mC (Figure 1I). Levels of 5hmC are clearly dependent on 5mC because sequences that are lacking 5hmC also have very low levels of 5mC (dotted lines in Figure 1I). The data also indicate that the profiles of 5mC and 5hmC are similar toward the 3′ ends of genes but show distinctly opposite trends near promoters (Figure 1I).
We found that 5hmC levels at promoters or intragenic regions are differentially correlated with CpG density and gene activity. Genes with highest intragenic CpG density are enriched with 5hmC in the gene body (Figures S2A and S2B) whereas genes with moderate or low CpG density at the promoter and low transcriptional activity are characterized by frequent accumulation of 5hmC near the promoter (Figures S2C and S2D).
We inspected the genes strongly enriched with intragenic 5hmC. The intragenic 5hmC-rich genes included 2,782 genes and 3,879 genes in NPC and in neurons, respectively; 1,988 genes were common to both (Figure S2E; Table S1. Intragenic 5hmC Coverage, H3K27me3, and Transcriptional Changes during Neuronal Differentiation, Related to Figures 1 and 2). Intragenic 5hmC-enriched genes are associated with higher transcript levels than the rest of the genes, a trend that becomes more obvious in neurons (Figure S2F). This group of genes shows strong enrichment in genes expressing in brain (Figure S2G) and includes many genes critical for neuronal differentiation, migration, or axon guidance, for example, Nav2 (Figure 1G) and Robo1, Dab1, Dclk1, Tbr1, Sox5, Bcl11b, Satb2, Myt1l, Wnt7b, Cdk5r1, Rab3a, and Efna3 (Table S1. Intragenic 5hmC Coverage, H3K27me3, and Transcriptional Changes during Neuronal Differentiation, Related to Figures 1 and 2 and Table S2. Genes with Strong 5hmC Intragenic Coverage in Neurons Are Frequently Involved in Axonal Guidance, the Wnt/Beta-Catenin Pathway, and Ephrin Receptor Signaling, Related to Figure 1; Figure S2H).
Changes of 5hmC Are Negatively Correlated with Changes of H3K27me3
To evaluate whether and how 5hmC dynamics may work in concert with chromatin modifications during neurogenesis, we profiled H3K4me3, H3K36me3, and H3K27me3 histone marks. Recently, a negative crosstalk between H3K27me3 and 5mC was described where loss of 5mC caused by Dnmt3a knockout resulted in accumulation of H3K27me3 (Wu et al., 2010). Our heatmaps and composite profiles show that the 5mC mark did not change significantly with gain and loss of intragenic H3K27me3 during neuronal differentiation (Figure 2). We thus hypothesized that a negative association exists for H3K27me3 and 5hmC, and this was evident when sorting genes by changes of intragenic H3K27me3 during neuronal differentiation; loss of H3K27me3 is associated with gain of 5hmC in gene bodies (Figure 2A). Composite profiles of the 15% of genes with the most intensive loss of intragenic H3K27me3 during differentiation detected a strong increase of intragenic 5hmC (Figure 2B). Conversely, gain of intragenic H3K27me3 is linked to a loss of 5hmC in gene bodies.
Differential Expression of Many Genes during Neural Development Is Characterized by Epigenetic Switches Associated with H3K27me3, H3K4me3, H3K36me3, and 5hmC
Loss of H3K27me3 and gain of intragenic 5hmC appear to be features of many genes that become activated and are important for the progression of neuronal differentiation. Composite profiles for the most activated and repressed genes during neuronal differentiation showed that gene activation is often linked to decrease of H3K27me3 in gene bodies and promoters, gain of H3K4me3 at promoters, and gain of intragenic 5hmC (Figure 2C). Many neuronal differentiation regulators including Sox5, Bcl11b, Wnt7b, Ank3, Mir124a, and Myt1l undergo an epigenetic switch, which is characterized by a loss or reduction of promoter bivalent status due to decrease of H3K27me3 occupation at promoters (Table S1. Intragenic 5hmC Coverage, H3K27me3, and Transcriptional Changes during Neuronal Differentiation, Related to Figures 1 and 2). On the other hand, gene repression is associated with increase of H3K27me3 at the TSS, decrease of H3K4me3 levels at promoters and loss of H3K36me3 in gene bodies (Figure 2C). Accumulation of the H3K27me3 mark is mainly linked to silencing of genes responsible for maintaining an undifferentiated state such as Sox2, Pax6, Smad3, Rest, Jag1, Wwtr1, Prdm16, Nr2e1, Hes1, Notch1, Ccnd1, Rhoci, and Bmp7 (Table S3. Gene Ontology Analysis for Genes Undergoing Changes of H3K27me3 and 5hmC during Neuronal Differentiation, Related to Figure 2). Analysis of genes gaining intragenic 5hmC indicated a link to neuronal differentiation and axonogenesis (Table S3. Gene Ontology Analysis for Genes Undergoing Changes of H3K27me3 and 5hmC during Neuronal Differentiation, Related to Figure 2). In addition, transcriptional analysis revealed that accumulation of intragenic 5hmC together with loss of H3K27me3 is associated with the most significant gene activation during neuronal differentiation (Figure 2D).
Gene Activation Is Associated with an Increase of 5hmC in Gene Bodies but No Evidence for Substantial DNA Demethylation
To evaluate if 5hmC accumulation is associated with demethylation of cytosines, we performed bisulfite sequencing analysis on 11 regions that were abundant with 5hmC and apparently lost some 5mC during neuronal differentiation (Figures 3 and S3). Bisulfite sequencing cannot distinguish between 5mC and 5hmC but distinguishes both modified cytosines from cytosine (Huang et al., 2010; Jin et al., 2010). We did not see a substantial conversion of 5mC or 5hmC into C in 10 out 11 examined regions, where nine regions are located in genes (Nav2, Sox5, Bcl11b, Wnt7b, Ank3, Pde2a, Prex1, and Uchl1), which become activated during neuronal differentiation. Most (8/11) of the analyzed DNA regions indicated no change of unconverted cytosine or minimal loss, below 5% of the total analyzed CpGs, and two fragments were associated with increase of modified cytosine between 3%–4% (Figure 3C). In order to determine the exact frequency of 5hmC at selected loci, we performed TAB sequencing of five intragenic regions located in the Sox5, Nav2, Uchl1, Pde2a, and Elk3 genes (Figures 1E, N region, and 3A). This data indicated that 5hmC frequency is indeed doubling at some intragenic regions and reaches up to 20% of all CpGs in the analyzed regions and 25% of modified cytosines (5hmC level according to TAB sequencing divided by number of all modified cytosines detected by regular bisulfite sequencing). This data indicated that the rise of 5hmC can have a significant impact on sequence-specific 5mC levels during neuronal differentiation and also suggests substantial stability of this modification in the genome.
Polycomb and Tet Proteins Regulate the Normal Progression of Neuronal Differentiation in the Cortex
We next explored the functional significance of the dynamics of 5hmC and histone marks in relation to the developmental switch of neuronal differentiation. We targeted Tet proteins and the Ezh2 subunit of the Polycomb complex. In situ hybridization showed that Tet3 followed by Tet2 are the Tet genes most highly expressed in the cortex (Figure S4A). Their expression levels were upregulated during neuronal differentiation (Figures S4A and S4B) in parallel with the increase of 5hmC levels (Figure 1A). Expression of Ezh2 is reduced (RNA in situ based on http://genepaint.org and Figure S4B) during neurogenesis. We thus asked whether overexpression of Tet (Figure S4C) or inhibition of Ezh2 might be necessary for neuronal differentiation to proceed. RNA interference (RNAi) of Ezh2 in the cortex via in utero electroporation-mediated expression of an shRNA (Figures S4E and S4F) caused more cortical cells to translocate from the VZ into the IZ and CP (Figure 4A), an indication of differentiation of the affected NPCs (Murai et al., 2010; Qiu et al., 2008). This was supported by the observation that cells having moved into the CP were positive for neuronal marker β-III-tubulin but lacked dividing cell marker Ki67 (Figure 4B). Consistent with the observed cell distribution, quantification of acutely dissociated cells derived from electroporated cortices showed an increase of β-III-tubulin positive cells in the shEzh2-expressing population (Figure 4C). Overexpression of Tet3 and Tet2 caused a similar but less pronounced trend of early neuronal differentiation and induced a stronger effect when combined with knockdown of Ezh2 (Figures 4A and S4C). We next asked whether prolonged expression of Ezh2 (Figure S4D) or inhibition of Tet2 and Tet3 by shRNA (Figures S4E and S4F) might prevent differentiation. We found that, in contrast to the progression of neuronal differentiation in GFP control cells, for which differentiated progeny have progressively moved into the CP (Qiu et al., 2008), overexpression of Ezh2 caused many cells to remain in the VZ and IZ (Figure 4D). Ezh2-expressing cells were positive for nestin but negative for β-III-tubulin and some cells could incorporate BrdU (Figure 4E), suggesting that expression of Ezh2 could block differentiation. Reduction of Tet gene expression via coelectroporation of shRNAs directed against Tet3 and Tet2 (Ito et al., 2010) did not cause an overall shift of cell distribution as in the case of Ezh2 overexpression, but revealed a distinct effect (Figure 4F). Knockdown of Tet3 and Tet2 often (8/16 brains) led to abnormal accumulation of cell clusters along the radial axis in the IZ and VZ, whereas GFP control cells (Figure 4D) rarely (1/14 brains) produced such a phenomenon (p = 0.017; Fisher's exact test, two-tailed). Clustered cells did not express neuronal marker β-III-tubulin and some of the cells showed expression of nestin in their processes (Figure 4G), suggesting a defect in the progression of differentiation.
Taken together, our data suggest that formation of 5hmC along gene bodies is associated with gene activation during neuronal differentiation. The purpose of intragenic 5mC oxidation remains unclear. One possibility is that 5hmC is more strongly antagonizing Polycomb-mediated repression and H3K27me3 formation than 5mC, thus allowing efficient gene activation to occur and to be maintained. We find that many activated genes functioning in neuronal differentiation lose the Polycomb mark near promoters and in gene bodies concomitant with a gain of 5hmC. However, it is unlikely that the two processes are always directly linked, because loss of H3K27me3 is commonly observed at promoters and just downstream of the TSS whereas gain of 5hmC occurs along the entire gene body length. In agreement with this model, our functional data suggest that Polycomb and Tet proteins may act in sequence in regulation of neurogenesis. Loss of function of Ezh2 promoted neuronal differentiation, whereas gain of function of Ezh2 did the opposite, indicating a role of Ezh2 in guiding NPCs' decision to either self-renew or differentiate. Loss of function or gain of function of Tets did not swing the fate choice of NPCs, but appeared to affect the ability of NPCs to complete the differentiation process. Furthermore, in agreement with the functional data, we observed that prominent changes of H3K27me3 and 5hmC marks in relation to neuronal differentiation occurred in cell fate determinants or neuronal function-related genes, respectively. Thus collectively, our data suggest that Polycomb functions to regulate the switch of NPCs from expansion to differentiation, whereas Tet proteins are involved in maintaining the proper progression of the differentiation process after its initiation.
Our data indicate that 5hmC patterns in neuronal cells are dissimilar to those in ESCs that are associated with high levels of Tet1 and Tet2 and low levels of Tet3 (Koh et al., 2011). According to TAB sequencing data for ESCs, 5hmC is abundant at enhancers (p300 sites) and underrepresented in gene bodies (Yu et al., 2012). In contrast, cortical NPCs and neurons at E15.5 are characterized by low levels of 5hmC at p300 sites and by enrichment of 5hmC in gene bodies. These facts may suggest that Tet1 may play a prominent role in 5hmC formation at enhancers in ESCs, whereas Tet3 activity may be associated with intragenic 5hmC deposition during neuronal development. This information also suggests that cortical NPCs and neurons are able to maintain their enhancers unmethylated by mechanisms other than 5mC oxidation or that 5hmC turnover is more rapid at neuronal enhancers.
5hmC is thought to be an intermediate in enzyme-catalyzed active DNA demethylation. However, we could not substantiate a primary role of 5hmC in demethylation during neurogenesis. It is possible that 5hmC may slowly be converted to cytosine over time during the maturation of neurons; nevertheless, it appears that 5hmC is a rather stable epigenetic mark, as previously suggested from studies of early embryo development and tumorigenesis, in which passive, replication-dependent loss of 5hmC was observed (Gu et al., 2011; Inoue and Zhang, 2011; Iqbal et al., 2011; Jin et al., 2011a). 5hmC may be recognized by specific proteins, or, alternatively, it may represent a negative signal that interferes with protein complexes that bind to either 5mC, such as methyl-CpG binding proteins (Jin et al., 2010), or to CpG-rich DNA regions in general, such as the Polycomb complex. However, one publication reported that MBD3, one MBD family member, binds to 5hmC (Yildirim et al., 2011). Moreover, a very recent publication has suggested that the methyl-CpG binding protein MeCP2, an abundant protein in the brain, can in fact bind to 5hmC and accumulates in gene bodies (Mellén et al., 2012), where it may organize a nuclease-sensitive chromatin structure in active genes. Whatever the exact mechanism, formation of 5hmC along gene bodies appears to be an important signal that is linked to increased expression of genes critical for the neuronal differentiation process. Defects in this pathway or even subtle aberrations could manifest themselves in neurodevelopmental or neurological disorders.
Experimental Procedures
Purification of Neural Progenitors and Differentiating Neurons
Animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC). Purification of E15.5 cortical NPCs and neurons using a double reporter strategy and characterization of the purified cells were reported previously (Wang et al., 2011). Briefly, we bred heterozygous Nestin-GFP mice with homozygous DCX-RFP mice to yield both GFP/RFP double-positive and RFP single-positive littermate embryos, which were used for isolating GFP+RFP− cells (NPCs) and RFP+ cells (neurons), respectively.
The immunohistochemistry staining was done as described previously (Jin et al., 2011a; Qiu et al., 2008). Each antibody staining was performed on multiple brain sections and the experiment was repeated one or more times. We used the following primary antibodies: anti-5hmC (Active Motif, #39769, 1:1,000); anti-5mC (Diagenode, #BI-MECY-0100; 1:200); anti-nestin rat-401 (Developmental Studies Hybridoma Bank, 1:100); anti-βIII tubulin (Sigma-Aldrich, #T 8660, 1:400); anti-bromodeoxyuridine (BrdU) (Sigma-Aldrich, #B2531, 1:500); anti-Ki67 (BD PharMingen; #550609; 1:20).
RNA In Situ Hybridization and Western Blot
For RNA in situ hybridization, digoxigenin-labeled cDNAs were synthesized from linearized pBluescript constructs containing C-terminal fragments of Tet1, Tet2, and Tet3 cDNAs and hybridized to embryonic forebrain coronal sections with further detection by an anti-digoxigenin AP (alkaline phosphatase) approach. For western blot, transfected HEK cells or purified cortical cells were lysed in 2× SDS sample buffer, sonicated and boiled. Proteins were separated by SDS gel electrophoresis, transferred to PVDF membranes and incubated with Ezh2 (#39934, Active Motif), α-tubulin (T6199, Sigma-Aldrich), and rabbit anti-Tet3 antibody raised against an N-terminal 102 amino acid peptide of mouse CXXC-Tet3 fused to GST.
Electroporation and Phenotype Analysis
In utero electroporation-mediated functional assays and acutely dissociated cell assays were performed as previously described (Qiu et al., 2008). For overexpression, cDNAs encoding full-length Ezh2, Tet2, and CXXC-Tet3 were cloned into a CAG promoter plasmid.
shRNA Efficiency Screening
shRNAs were expressed under control of a mouse U6 promoter in pNUTS vector additionally containing EGFP expressed from the ubiquitin promoter. Candidate shRNAs were tested with targets cloned in psi-CHECK vector in transfected HEK293 cells, using a dual luciferase reporter assay (Promega, E1910). The sequences of shRNA for Tet transcripts were previously described by Ito et al., 2010. The shRNA for Ezh2 (Open Biosystems) had the following sequence: 5′-TTGAGTACTGTGGGCAATTTATTCAAGAGATAAATTGCCCACAGTACTCAA.
LC-MS/MS Quantification of 5hmdC and 5mdC in Genomic DNA
Global 5hmdC and 5mdC levels were measured by LC-MS/MS using stable isotope-labeled internal standards as described previously (Jin et al., 2011a).
Profiling of Methylated and Hydroxymethylated Cytosines
For analysis of 5mC, the methylated CpG island recovery assay (MIRA) was used as described previously (Rauch et al., 2006). Immunoprecipitated and input DNA were 5′-end- phosphorylated and blunt-ended with END-It DNA end-repair kit (Epicenter Biotechnologies) with further generation of overhanging A ends by Klenow Exo- (New England BioLabs) and DNA ligation with an overhanging "T" linker (5′-GCGGTGACCCGGGAGATCTGAATTCT, 5′-GAATTCAGATC) with T4 ligase (New England BioLabs). The genome amplification procedure was done as previously described (Hahn et al., 2008). For hMeDIP, linker ligated DNA was immunoprecipitated with 5hmC antibody (#39769, Active Motif; Carlsbad, CA).
After genome amplification, DNA obtained after MIRA or hMeDIP were hybridized versus input DNA on Mouse ChIP-chip 2.1M whole-genome tiling arrays and for additional biological replicates on tiling arrays of mouse chr7 (NimbleGen). For 5hmC profiling by a glycosylation-based method (Song et al., 2011), we used the Hydroxymethyl Collector Kit (Active Motif) according to the manufacturer's protocol. DNA was amplified and hybridized onto tiling arrays of mouse chr7 (NimbleGen).
Sequencing of Modified Cytosines
TAB conversion was performed by using the 5hmC TAB-Seq Kit (Wisegene) with two rounds of Tet1 oxidation. Bisulfite conversion of TAB-treated and untreated DNA was performed by using the EZ DNA methylation-Gold kit (Zymo Research) according to the manufacturer's instructions. PCR products were cloned by using CloneJet PCR cloning kit (ThermoScientific). The efficiency of Tet1 conversion was validated by TAB sequencing of DNA from neurons without glycosylation (Tet1-C in Figures 1F and 3A).
Chromatin Immunoprecipitation
The chromatin immunoprecipitation (ChIP) protocol was described previously (Hahn et al., 2008). The following antibodies were used: anti-H3K4me3 (39159, Active Motif), anti-H3K36me3 (ab9050, Abcam), and anti-H3K27me3 (07-449, Millipore). For obtaining the H3K4me3 profile, the H3K4me3 antibodies were preblocked with an H3K9me3 peptide (Abcam). After genome amplification, immunoprecipitated DNA was hybridized versus input DNA on Mouse ChIP-chip 2.1M whole genome tiling arrays (NimbleGen) and for additional biological replicates on chr7 tiling arrays.
Bioinformatics Analyses of Profiling Data
All analyses were performed using R statistical language, except gene ontology analysis, which was performed using DAVID annotation tools. The heatmaps were generated with Java Treeview v2.0. Refseq genes were downloaded from the UCSC mm9 annotation database (UCSC Genome Browser, Santa Cruz, CA). A detailed description of individual bioinformatics analyses can be found in the Extended Experimental Procedures.
Bioinformatics Analysis and Definitions
Generation of composite profiles and heatmaps has been described previously (Hahn et al., 2011). Promoter was defined as −1.5 kb to + 0.5 kb relative to the transcription start site (TSS). Gene body was defined as from 0.5 kb downstream of the TSS to the transcription end site (TES). CpG density was calculated as the number of CpG dinucleotide per 1000 bp in gene body or promoter regions.
Identification of Genes with 5hmC Enrichment in Promoter and Gene Body
5hmC enrichments were identified with a sliding window approach. Specifically, the probe log2 ratio within each 2 kb-window with 1 kb steps was examined. Windows with at least eight probes and > 50% of the probes with a log2 ratio ≥ 1 were denoted as 5hmC-positive (or 5hmC-enriched). The location of 5hmC enrichment in the genome was determined after joining adjacent windows. 5hmC enrichments were considered intergenic if they did not overlap with promoters or gene bodies. A Refseq gene (length > 2 kb) was designated as a gene with intragenic 5hmC enrichment if the length of all 5hmC enrichments within the gene body was > 20% of the gene's length. Genes with intensive 5hmC occupation were defined as genes with 5hmC enrichment and mean of 5hmC signal along the entire gene body of log2 ratio ≥ 0.4. Promoter regions with at least eight probes and > 50% of the probes with a log2 ratio ≥ 1 were denoted as promoters with 5hmC coverage. An equivalent approach was used to calculate 5mC coverage.
To identify genes gaining 5hmC within the gene body in neurons compared to NPCs, the average probe signals within each sliding window between neurons and NPCs were compared. Adjacent windows with a log2 ratio difference of > 0.5 were joined and considered as candidate regions. The cut-off was calculated with the following criteria: mean (diff) + 2 ∗ SD(diff). Genes with candidate region coverage > 20% were designated as genes gaining 5hmC in the gene body. In addition, genes with lower average probe log2 ratio within the entire gene body in neurons were excluded. A similar approach was used to identify genes losing 5hmC coverage within the gene body during neuronal differentiation.
Identification of Genes with H3K27me3 Changes
In order to identify genes with H3K27me3 changes, the mean probe log2 ratio of H3K27me3 signal at the promoter region and at the 5′ end of genes (−0.5 kb to +4.5 kb of the TSS) of each Refseq gene was calculated. Genes were considered losing H3K27me3 during differentiation if the mean signal in NPC was ≥ 0.1 and the difference between NPC and neurons was ≥ 0.38 (30% higher). A gene was considered gaining H3K27me3 during differentiation if the average signal in neurons was ≥ 0.1 and the difference between neurons and NPC was ≥ 0.38.
Gene Expression Data
The Affymetrix gene expression data of neurons and NPCs were generated previously (Wang et al., 2011). The differentially expressed genes were identified as genes with log2 fold change ≥ 1 and a false discovery rate (FDR) < 0.05. These genes were then sorted by their log2 fold changes and the top 25% most activated and 25% most repressed genes were selected for further analysis of association with epigenetic changes (Figure 2C). In gene ontology analysis, for genes which undergo epigenetic changes together with transcriptional changes, we used log2 fold changes ≥ 1.
This work was supported by NIH grants MH094599 from NIMH (Q.L. and G.P.P) and NS075393 from NINDS (Q.L.).
Accession Numbers
The Gene Expression Omnibus accession number for the 5mC, 5hmC, H3K27me3, H3K4me3, and H3K36me3 profiling data reported in this paper is GSE38118.
Supplemental Information
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Publication Information
Received: July 10, 2012
Revised: November 22, 2012
Accepted: January 14, 2013
Published: February 7, 2013


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The Pew Charitable Trusts is driven by the power of knowledge to solve today's most challenging problems. Pew applies a rigorous, analytical approach to improve public policy, inform the public and stimulate civic life.
Mar 17, 2010
As President Obama and Democratic leaders mounted what was characterized as the final push to pass health care reform legislation last week, the public followed the health care debate more closely than any other major story (33% say they followed this story most closely). The debate also topped media coverage.
The percentage of Americans who say they think Congress will pass health care legislation this year edged up to 43%, but about half (49%) still say lawmakers will not pass a bill in 2010. Among Democrats, 61% now say they think a health care reform bill will pass this year, up from 49% one week earlier. Only 27% of Republicans and 38% of independents say they think a bill will pass, unchanged from the previous week, according to the latest News Interest Index survey, conducted March 12-15 among 1,019 adults by the Pew Research Center for the People & the Press.
Read the full report Health Care Debate Dominates Interest and Coverage on the Pew Research Center for the People & the Press' Web site.


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NEW YORK NURSE: November 2009
Judy O'Donnell is a home care nurse with the Visiting Nurse Association of Staten Island.
She enters the homes of six or seven patients a day, driving her car from assignment to assignment. She has a caseload of 30 to 40 patients at any given time. For more than two decades, this has been her job for five days a week and one weekend a month.
"The acuity of the patients has changed drastically over that time," said O'Donnell. "When I started, I was working with 65- to 75-year-olds with adult onset illness. Now the patients are a generation older. People who used to be kept in the hospital for three weeks after heart bypass surgery are now released after five days. There's a greater risk for instability and the nurse really needs to be in touch with the physician."
"You see people in their environment and their whole family dynamic," O'Donnell continued. "You always have to keep your antenna up for elder abuse. Over the years I've had to call adult protective services and I've had to call child protective services. I called the ASPCA when a family took a duck out of Willowbrook Park and kept it in a crate under the kitchen table. I was afraid it was going to be dinner."
Home care nurses must constantly use their assessment skills with relatively little backup. This assessment goes beyond the client; it includes the family and the living environment.
"It's a very holistic form of nursing," O'Donnell explained. "Your judgment can be life-altering. You may detect underlying mental health issues or diabetes and refer clients to support groups or adult day care. You may see a grandchild with a developmental delay and call in an early intervention for evaluation and referral."
After more than 25 years in the community, O'Donnell has seen the fourth generation in some families. "I'll have taken care of the grandmother, and now the granddaughter has a premature baby that needs nursing care," she said.
O'Donnell has also witnessed changes in the homes she visits. "I've seen houses turn over and I notice how the new people redecorate," she said. "I'll tell them I like what they've done with the kitchen."
There is a team spirit among the 60 RNs working at the Staten Island VNA. "Everyone brings something different to the table," said O'Donnell. "Jen Masiello is younger. She's good with technology and administering IVs."
Jennifer Masiello has been a visiting nurse for six years. She worked previously in a hospital and then in a nursing home.
"The first six months were a huge adjustment, but Judy taught me the proper way to manage a home care patient and the other nurses offered a lot of support," she said. "The autonomy of the job can be scary at first. But now I like the autonomy. I like the freedom. There aren't ten people pulling at you at once. I feel like this is right for my personality. It's extremely interesting. It changes every single day."
O'Donnell agrees. "I've definitely found my niche. I'm not going anywhere. There's a challenge and a mystery behind every door."


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The study analyzed 72,000 California homes sold between 2000 and mid-2009, almost 2,000 of which had solar energy systems. It found that homes with solar sold for an average of $17,000 more than comparable non-solar homes.
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Search Tags: federal benefits
Press Roundtable: Inside perspectives, analysis, and predictions
Debra Roth hosts a press roundtable on the big issues affecting federal workers.
August 9, 2013
Tags: pay and benefits , workforce , acquisition , sequestration , furloughs , Congress , Debra Roth , Sean Reilly , Federal Times , Kellie Lunney , GovExec , Josh Hicks , Washington Post , Fed Talk
Minding the gap in federal benefits
Tony Vergnetti will hows a roundtable discussion of federal insurance benefits and what you can do to fill any holes in your coverage.
June 21, 2013
Tags: workforce , FEHBP , healthcare , life insurance , Tony Vergnetti , supplemental insurance , Mike Miles , Variplan , Jim De La Torre , Fed Advantage , Dennis Stephens , All Feds , Fed Talk
So, what's next?
Federal News Radio Senior Correspondent Mike Causey answers your calls and emails about a wide variety of issues affecting federal workers.
April 29, 2013
Tags: pay and benefits , budget 2014 , budget battle , sequestration , furloughs , federal pay raise , FERS supplement , FERS retirement program , budget cuts , retirement , retirement planning , Bob Leins , Tammy Flanagan , NITP , Mike Causey , Federal News Radio , For Your Benefit
FEHBP open season part 2
Insurance expert Walton Francis will answer your questions and emails about federal benefits.
December 5, 2012
Tags: pay and benefits , health care , Walton Francis , Checkbook Guide to Health Plans for Federal Employees , Open Season , Mike Causey , Your Turn
Open season, Medicare part B, Petraeus, & sequestration
Federal health benefits expert Walton Francis
talk about FEHBP open season, and Federal Times
reporter Sean Reilly will discuss sequestration
and other issues affecting federal workers.
November 14, 2012
Tags: Open Season , Medicare part B , Walton Francis , Checkbook Guide to Health Plans for Federal Employees , Sean Reilly , Petraeus , sequestration , USPS , Your Turn , Federal Times , Mike Causey , pay and benefits ,
Answers to your retirement questions
Co-hosts Bob Leins and John Elliott are joined by federal benefit expert David Redden.
February 27, 2012
Tags: pay and benefits , David Redden , Bob Leins , John Elliott , TSP , financial planning , For Your Benefit
Lawmakers try to water-down the pay freeze
Former Virginia Congressman Tom Davis said federal employees are on the chopping block because they are easy prey. Davis, now a director with Deloitte and Touche, said a letter from the Washington-D.C. area delegation could impact the potential two-year pay freeze the White House is proposing. The eight House members say feds are being targeted unfairly to help reduce the deficit.
Tags: pay and benefits , David Obey , Jack Lew , Steny Hoyer , Frank Wolf , House Appropriations Committee , OMB , Tom Davis , Deloitte and Touche , pay freeze , Jason Miller
Federal employee's guide to the known and unknown benefits universe
NITP presenters Bob Braunstein and James Marshall will answer your questions about Social Security, the TSP, and other federal benefits programs.
May 23, 2011
Tags: pay and benefits , TSP , FERS , CSRS , FERCCA , Social Security , LTC , Medicare , Medicare part B , NITP , Bob Braunstein , James Marshall , Bob Leins , For Your Benefit , insurance , retirement
Federal Benefit Roundup
Tags: P&B , FERS flu , sick leave , paid leave , equal benefits , John Palguta , Partnership for Public Service
Computers: Risky Business
Knowing how to use a computer is a must in most federal jobs, but Senior Correspondent Mike Causey says using it for the wrong reason can land you in a heap of trouble.
Tags: P&B , ethics , computer crimes , FEHP , sharks


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Article ID: 298074 - View products that this article applies to. This article was previously published under Q298074 This article has been archived. It is offered "as is" and will no longer be updated. SYMPTOMS
The French-language version of Internet Explorer 5.5 Service Pack 1 (SP1) incorrectly includes the debug version of Hlink.dll (packaged in Ie_1.cab):
Hlink.dll (French-language version of Internet Explorer 5.5 SP1)
This problem impacts the use of Microsoft Office programs such as Microsoft Excel, which may display the following assertion failure error message if you open an Excel spreadsheet by using Hypertext Transfer Protocol (HTTP):
File size: 192571 Version: 22.214.171.12413
Hlink Assert Assertion "0" failed MemLeak: Allocated at c:\binder\hlink\hlmem.cpp Line 86 Alloc #20 File c:\binder\hlink\hlmem.cpp, line 257 (A=exit; R=break; I=continue) Abort Retry Ignore RESOLUTIONTo resolve this problem, obtain the latest service pack for Internet Explorer 5.5. For additional information, click the following article number to view the article in the
Microsoft Knowledge Base:
The French-language version of this fix should have the following file attributes or later:
How to Obtain the Latest Service Pack for Internet Explorer 5.5
Date Time Version Size File name Platform --------------------------------------------------------------- 16/05/2001 15:10 188.8.131.5213 81 978 Hlink.dll Intel STATUSMicrosoft has confirmed that this is a problem in the Microsoft products that are listed at the beginning of this article. This problem was first corrected in Internet Explorer 5.5 Service Pack 2. Properties |


text:
Janice Morse reports:
Jeff Meadows, a West Chester-based attorney, earns nearly all his livelihood defending people who are charged with drunken driving.
Even Richard K. Jones, the sheriff of Butler County, where Meadows mostly practices, acknowledges: "Nobody deals in more DUIs than he does."
But Meadows has started selling a device that he hopes will put him out of business: a portable, disposable breath test.
Called "TESTorARREST," the device is a cigarette-sized clear plastic tube that sells for $9.99 for a two-pack. After a person breathes into the tube, a chemical inside interacts with breath alcohol. If the yellow crystals inside change to an aqua color, that indicates the person's alcohol level exceeds 0.08, Ohio's legal limit for drivers.
People who test over the limit may use their smart phones to access www.testorarrest.com to get "instant access to a local taxi," Meadows said.
The device's slogan: "If you don't pass, stay off the gas!"
"It's very inexpensive, costing the fraction of the price of a DUI attorney," said Meadows, who practices with the Lyons & Lyons law firm. "A few dollars now versus a few thousand later makes it a no-brainer."
The device is packaged with a disclaimer, telling consumers that abstaining from drinking alcohol is "the only way to be 100% certain that an individual is not impaired." (more…)


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Welcome to the General Government Service Center
The primary purpose of the General Government Service Center is to provide fiscal and human resource management services in support of Bureau and Departmental operations and programs. The Division furnishes all Bureaus with (a) consistent and uniform application of statewide budgetary policy; (b) application of generally accepted accounting and financial practices; (c) implementation and uniform application of collective baragaining agreements; (d) human resources and payroll administration; and (e) department billing services so that each line bureau within the Department may achieve its desired program goals and objectives, and remain in compliance with all laws and available budgetary resources.
The Division provides the same scope of services to the various departments and agencies throughout Maine State Government served by the following Service Centers; General Government, Natural Resources, Security and Employment, Corrections, Transportation and Department of Health and Human Services.
We look forward to providing a high level of service to all our customers.
Contact the Director:
Kimberly A. Smith
General Government Service Center
74 State House Station
Augusta, ME 04333-0074
(207) 624-7400


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We regret that currently, no events in English are on offer. For events in German please see the German website.
Between the Lines?
29 November 2013 to 6 April 2014
More Information
Guided tours for groups only by appointment. There is a special foreign language educational programme for school classes and adult groups.
To this day, in Nuremberg's southern districts, the remains of the buildings on the former Nazi Party Rally Grounds with their immense proportions are a vivid testimony to the megalomania of the National Socialist regime. This area of eleven square kilometres was intended as an impressive backdrop for the Nazi party rallies staged here to demonstrate their power.
The Documentation Centre Nazi Party Rally Grounds, often shortened to Dokuzentrum (Docu Centre), is located in the north wing of the Congress Hall, designed by the National Socialists to house 50,000 spectators, remained unfinished, though. With its "glass and steel arrow", piercing the north wing, the Documentation Centre is a widely visible architectural counterpoint.
On 1,300 square meters, the permanent exhibition "Fascination and Terror" looks at the causes, the context and the consequences of the National Socialist reign of terror.
It focuses on the topics which have a direct link to Nuremberg, and is centred on 19 exhibition areas which are structured in chronological order: the history of the party rallies, the buildings of the Nazi Party Rally Grounds, the "Nuremberg Racial Laws" of 1935, the "Nuremberg Trials" of the main perpetrators of Nazi crimes in 1945/46 and the twelve follow-up trials, as well as the difficult problem of dealing with the National Socialists' architectural heritage after 1945.
In addition to the permanent exhibition, the affiliated Education Forum is of essential importance for the Documentation Centre's educational work.
Educational Programme (foreign languages)
In special seminar rooms on the roof of the Congress Hall, Nuremberg Municipal Museums, in co-operation with various partners, offer in-depth educational programmes for school classes, groups of young people and adults. The portfolio of programmes ranges from 45 minute sessions after an exhibition visit to entire seminar days.
Since May 2006, a comprehensive information system (German/ English) located at various points throughout the Nazi Party Rally Grounds offers all visitors the opportunity of an in-depth look at the Nazi Party Rallies on the spot.
Information System


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- Northern New York Newspapers
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- Malone Telegram
Stormy N. Fields, 18, of 714 S. Massey St., was arrested by city police early Tuesday on multiple charges after she was stopped on the 300 block of Stone Street.
At 1:17 a.m., city police allegedly discovered heroin in two glassine envelopes in her pocket and inside her pants. She was charged with seventh-degree criminal possession of a controlled substance.
She also was charged with third-degree aggravated unlicensed operation and failure to notify the Department of Motor Vehicles of an address change.
She was taken to the Metro-Jefferson Public Safety Building and held on $1,000 bail pending arraignment in City Court.


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The criminal case against New York Jets running back Mike Goodson is headed to a grand jury.
He made a brief appearance in a New Jersey courtroom Wednesday.
Goodson pleaded not guilty to drugs and weapons charges after he was arrested last month following a traffic stop on Interstate 80.
Police say they found marijuana, a handgun and a hollow point bullet inside the vehicle.
Prosecutors say no plea deal is on the table.
Goodson played three seasons with the Carolina Panthers before he was signed by the Jets in March.


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2 hours), incontinence to apply lubricant to the penis and the cylinder cost, and confusion.
Missing 3) erection due to air leakage.
Missing 4) erection due to the pumping of high-speed or constant.
Create a bruise 5), the pain and inconvenience, pull the patient's pelvis and pubic organizations.
In order to maintain the erection patient, apply the constriction ring at the base of their penis. Ring contraction, inhibit the flow of blood from the penis to maintain the erection in this way. Must be the right diameter contractile ring to operate effectively. If it is too large, that the erection is maintained is not available. If it is too small, the patient will experience considerable pain of mild discomfort. You maybe interested in causes of loss of erection. Constriction ring patients to apply, you will need to install the ring of the first preliminary vacuum cylinder. To achieve this, VED some systems, the ring contraction special tool, the patient is characterized by using a ring applicator contractile ring constriction stretched out through the base of the cylinder. Other systems, you will need to lubricate the ring stretching for patients to be loaded, and to use brute force. Ring stretching into the cylinder, from the power cylinder, which is used to slip the ring on the base of the penis and to be loaded. There is constriction ring in position, and be sustained erection, the vacuum is released, the chamber is removed from the penis erect. Patients following sexual intercourse, you will need to remove the ring and more contraction. See penus enhancement. Ring should not be held for more than 30 minutes. To remove the ring, the patient, grasp the outer loop of the ring (ring all, note that you do not have the outer loop), pull on the outside, the penis loses its firmness of some while, you will need to hold for 15 seconds from 10. Check out also mbma erection tolerences. You can then ring to roll-off. That it is used otherwise may be difficult even if you remove the ring and ring contraction is that it is the correct size is important.
The patient's complaint of keys associated with the current system to maintain an erection and VED is included.
Constriction ring load of 1) the cylinder becomes more complicated, and requires too much power.
2) From the cylinder, is painful to force the ring contraction on top of the base of the penis. View how to give dog erection.
Erection that persists too loose contractile ring 3) can not be. View vaccum erection.
Contractile ring is too tight and painful 4).
If you remove the 5) ring, which is clumsy, and in some cases painful.
History of the VED system
VED it has been prescribed by doctors to treat ED well for over 20 years. ED affects millions of people to various degrees, in order to maintain or achieve an erection sufficient for sexual intercourse, has been defined as non-persistent. In most cases, you have organic etiology associated with an underlying disease associated with social impairment and psychological break the usual damage, blood flow, nerve. The cause is as follows. Prostate and colon cancer, diabetes, chronic lung disease, coronary artery disease, arthritis, hypertension, neuropathy, quadriplegia, paraplegia, severe depression. Includes the treatment of ED, the VED but oral medication, injection therapy, hormone therapy, penile implant, and urethral suppositories. See natural ways to get erection. The vacuum erection device, to draw blood to the penis by, to create a vacuum, has been designed to produce an artificial erection. When the penis with blood, a special rubber band is placed at the base of the penis, vacuum equipment will be removed. This leaves a man with an erect penis engorged with blood. The band must be removed immediately at the end of sexual intercourse. Chronic long-lived, often organic ED is about 70% of patients who undergo the loss, or temporary (18 months from 6) part of the normal sexual function of treatment condition experience, of prostate cancer In addition,. Mimic the erection of the night will help for patients with prostate cancer treatment following ED temporary, many doctors, but shows a lot of research, atrophy with help, to avoid fibrosis and shortening of the penis oral medication (for example, antagonist PDE5) has been promoting early return to sexual function to formulate a comprehensive rehabilitation therapy combining the use of VED and every day in order to. And to treat patients after surgery for nerve damage is estimated clinical theory actively, is to stimulate the recovery of the nerve, reducing the extent of damage perhaps irreversible. The basic hypothesis, artificial induction of erections after surgery, increasing the possibility of maintaining the ED potentially, to reduce the cavernosal fibrosis in the absence of nocturnal erections, oxygenation of the tissue immediately is to promote. Regardless of VED, to reason, the ability to draw blood to the penis of neuropathy has become the center of penile rehabilitation protocol.
It is currently manufactured VED, which consists of a vacuum chamber at the open end as a closed end that is connected to the constriction ring placed in a vacuum chamber near the entrance to the open end, a vacuum pump. See natural way to improve erections. In order to achieve an erection, the penis is inserted into the open end of the vacuum chamber has been pushed to the abdomen in order to provide a hermetic seal. You maybe interested in what causes the male erection. Then, vacuum is generated in the chamber with a vacuum pump drive (battery) manual or electric. Partial vacuum in the chamber, causing blood flow to the penis to produce an erection in this way. See premature ejeculation . Spare ring contraction has been installed in the vacuum chamber in order to maintain an erection is forced to slide down the root of the penis. Contractile ring inhibits the blood flow from the penis to maintain the erection in this way, are placed at the root of the penis. After this, the vacuum is released, the chamber is removed from the penis erect.
A number of studies and research, and therapy, to reduce the psychiatric symptoms, self-esteem increases the vacuum constriction, indicating that the patient can help to improve sexual satisfaction. (Urology clinic "therapy in the management of erection suction device" Roy Witherington,, of 15 versus North America, No. 1, February 1988) from the DE Price. , Diabetes drug, "Managing impotence of diabetic men by vacuum erection therapy" in 1991; 967 from 964). From W. Meinhardt. "Negative pressure device for erectile dysfunction: What is it fail?"; Journal of urology year 1285, V. 149, PP.
There are a number of other patents have issued that are involved in the erectile dysfunction. U.S. patent. Been issued to Gamper et al. "Appliances, vacuum-powered external for the treatment of erectile dysfunction. Check out also reviews of herbal erectile dysfunction products entitled" 6248059 die, in order to reduce the diameter, attached to the ring to the other end, if necessary, of the cylinder is connected to one end, discloses a motorized or manual pump.
U.S. patent. Have been issued to the Osbon senior "auxiliary equipment erection" 4378008, the first entitled, includes a vacuum cylinder elongated to receive the organ of the male, the disclosure devices that are connected to the interior of the flexible conduit to the vacuum source by this can be displaced in order to cause the organ to the cylinder has been is erect, consisting of expansion. Rubber band, the opening end of the cylinder, to close in order to maintain the organ is attached to the valve through the base of the whereafter organ, erect in the cylinder partial vacuum easily the valve manually during the conduct of the adjacent To remove the cylinder to reduce the vacuum erection vacuum that may have been connected by flexible conduit can be, is captured, you can open. You maybe interested in why morning erection video.
U.S. patent. 4856498 die having the rights issued to Osbon "generation and vacuum constriction device to extend the efficacy of the man" is a hose to connect the pump chamber and a vacuum chamber, vacuum pump and the manual has been disclosed. U.S. patent. "Seal and contraction device combination" 5344389, entitled first. 6705987 first, entitled "constriction ring and seal penis" were issued were issued from Walsdo rf, to Anderson et al U.S. Patent discloses a combination of the seal ring that is inserted into the penis flaccid first have. Check out also erection suppressant.
U.S. patent. 6036635 first entitled "erection control system" which was issued to Y. Altshuler, discloses a penis-shaped vacuum chamber that conforms to the inconspicuous operation under the cloth of the user. To baffle and seal penis is adjustable to the length of the periphery of the penis of the user, without the additional support in the vacuum chamber, to provide attachment to hang up the penis of a user is placed detachably have. Are held by the belt for applying pressure in the radial direction of the ribbon-shaped inner constriction device was decided in advance, are wound in multiple turns through each other to form a cylindrical ring. Check out also sex en erection. Transfer device with the contractile apparatus dislodges loop by pulling the penis along with the seal and the baffle is placed detachably on top of the erect penis in a vacuum without the simultaneous release of an additional mechanism. Penis seal, baffle, belt is formed from a segment of the condom is placed detachably.
In a broad aspect, the present invention is a vacuum erection control system for the treatment of erectile dysfunction. Check out also reviews of herbal erectile dysfunction products. Vacuum erection control system, which contains a vacuum erection device including a vacuum chamber and a thin elastic sealing element. The vacuum chamber vacuum generating means, has a distal end operable for connection to the outside surface of the inner surface of the proximal end and open. Thin elastic sealing element is formed of elastic material having an end portion of the seal and the second end portion of the first seal. The first end portion of the seal is enlarged to place removable sealing the proximal end of the vacuum chamber. View medical time lapse erection. End of the second seal, has a central orifice to form a seal penis for the introduction of the penis. You maybe interested in condoms erection sealed. Elastic material, without losing the characteristics of this non-transparency, can be extended to more than double the original area of transparency and non-air significantly. End first seal is fixed to the proximal end of the vacuum chamber. View medical time lapse erection during operation, the penis of an operator is introduced through the opening of the center seal of the penis, a vacuum, in a vacuum chamber that is generated by the vacuum generation This means that it can be operably connected to. You maybe interested in herbal aphrodisiac . And the vacuum chamber, has been enhanced the progress of the penis toward the distal end of the progress of the seal penis toward the base of the penis commensurate like erection. You maybe interested in erection with a female doctor.
If the erection desired has been achieved, separation, clamp, in order to maintain an erection during the removal of the device erection vacuum, has been placed around the penis between the seal penis and the base of the penis constriction adjustable.
The present invention is compatible with complaints from patients of the key associated with the / VED existing technology systems. The present invention, the patient, providing a cost effective and low VED simpler and more options. Technology components that make up the present invention in addition to being an integrated system, since it is sold under the medical Pharma, the Classic Ultra trademark and, in order to improve functional and easy cost and operational effectiveness of current medical devices Pharma has backward compatibility can be used to present the existing VED system, the clinical assignee.
Figure. 1 shows the assembly, erection is a perspective view of a vacuum control system of the present invention.
Figure. 2 is an exploded perspective view including a clamp constriction in order to use it, vacuum erection of the control system of the present invention.
Figure. Figure 3 - 3 of 3 along the line, cross section, is a perspective view partially. A.
Figure. 4 is a cross-sectional view of the proximal end of the vacuum erection control system arranged in an inverted form before the operation, showing the seal element.
Figure. 5, in the first step of the operation is a cross-sectional view of a vacuum erection control system indicates the introduction of the penis. You maybe interested in condoms erection.
Figure. 6 shows the progress of the penis toward the distal end of the vacuum chamber.
Figure. Shows the application of the clamp constriction 7.
Figure. 8 shows the last step of the vacuum chamber has been removed. See viagra side effects four hour erection.
Referring to the drawings and character reference mark on it, Figures 1 and 2 as 10, shows, a vacuum erection control system of the present invention, which is commonly specified. Control system 10 erection vacuum as 12, includes a vacuum chamber that is specified in general, 16 to have a 14 distal end for connection operable to generate a vacuum of 18 proximal end open means the outer surface and inner surface. As 20, a thin elastic sealing element that is specified in general, which includes the first end 22 of the seal arrangement removable sealing the proximal end of the vacuum chamber. View medical time lapse erection 12. The second seal portion 24 has a central orifice 26 to form a seal penis for the introduction of the penis. You maybe interested in condoms erection sealed.
Vacuum chamber 12 is a tubular, are formed from plastic and preferably be able to mark to where the vacuum pump should be placed. Of the vacuum chamber 12 (ie, the end of the penis) may have the edge of the ridges and grooves help flare depending on the proximal, to hold the elastic sealing element 20 on top of the cylinder. Also 12 (ie cylinder) chamber, the patient can be to measure the length of his penis, there may be measurement markings along the side of the chamber 12. Also, if necessary, the chamber 12, it can be configured to have a slightly smaller opening to the proximal end of element 20 to accommodate the great seal.
Seal element 20 is formed of elastic material without losing the characteristics of this non-transparency, can be extended to more than double the original area and substantially impermeable to air. Seal is formed from a material is preferably a thin latex rubber. View erection big. Silicone seals are provided for patients with latex allergy. 20 seal elements that are defined by the distance between the second seal portion 24 and seal the first end 22, in the range of about 0.5 inches and 3.0 inches, has a length of unstretched. It is the width of about 2.5 inches. First seal portion 22 is preferably, as can be seen clearly in the figure, will be rolled back over itself in order to facilitate the application and removal from the vacuum chamber 12. Three. Sealing element, it is less than the thickness (50 mil) 50/1000 inch is preferred.
Vacuum generation, 16 is preferably electric (usually battery-powered) means that the vacuum pump is driven. See erectile dysfunction medication treatment. Alternate vacuum pump is a hand pump. Pump 16 is preferably, has a cycle of intermittent pump. It is also, preferably, has a warning of the timer. View erection lasts 4 hours. Intermittent cycle of the pump is started, based on the setting timer automatically stops the vacuum cycle. It is bad erection and patients is useful in certain actions fast and / or when the pump, to solve the problem of erection without result. In addition, the cycle of intermittent pump, it is possible to reduce the bruising and discomfort. Warning function of the timer (usually 10 minutes) You are using the beep button is pressed after the time set a warning to the patient that you have a certain period of time. Timer is in the / training practice session, it is useful for patients for use during the rehabilitation session penis.
As will be described in detail below, the clamp between the seal of the penis in order to maintain an erection during the removal of the erection vacuum erection and the desired base of the penis has been achieved constriction is commonly specified as 28 can be placed around the penis. Device (10). Clamp 28, as shown in Figure 1, for example, separation may be adjustable clamp constriction constriction. Check out also soft erection causes. Preferably with the patient squeeze the clamp to the desired level of closed 2 using the two ratchet system. The term "isolated", as used herein refers to both ends of the clamp 28. Clamp 28 is open in the figure, has been identified by the position of the separator. Two. By compressing the vein on both sides at the top of the erect penis and trap the closed clamp 28, similar to the ring contraction of the penis blood is currently being used. Although not shown in this figure, the clamp 28, for safety and convenience, you may have a release lever Quick & Easy. To remove, or to adjust the clamp 28 patients easily, you can use the lever. Clamp 28, to replace the constriction ring of the prior art applicator and contraction of the rubber ring, it is available again. Clamp 28, the option has a better control of the patient, less convenience, more pain, providing a potentially low cost. In addition, the clamp 28 is very simple to apply, is very intuitive to the patient. Check out also panethnic acid erections.
A reference to the drawings. 4 from the previous operation seal element 20 is placed in the form of an inverted within the cylinder 12. As shown in FIG. As shown in Figure 5, 32 penis has been introduced through the opening 26 the center of the seal penis and then, vacuum is generated by the vacuum generation, means that the 16 that can be operatively connected to from the vacuum chamber 12 The. Seal element 20, since the closed end of the cylinder 12, to contribute to the vacuum in order to ensure success, the erection of a superior This will create much more easily than conventional devices. And to press the cylinder against his abdomen and pelvis, it is no longer needed. Similarly, the patient does not require the insertion of the lubricating oil and rubber seal is no longer for closing the ends of the cylinder 12. You can avoid the inconvenience and cost of applying a lubricant as a result patients. In addition, the skin around the penis and pubic hair. You maybe interested in zoloft erections is kept from the suction is to improve the patient experience, reduce the bruising and discomfort organization, in an open cylinder 12.
Vacuum is created within the cylinder 12, 32 as the penis length and girth has grown into, seal 20 has spread can pass through opening 32 regardless of the penis. As of the penis, the path 32, the skin of the penis of a patient, you will receive a positive sense even seal the opening 26. Sense is to raise the erection of external patient, plays a role as a simulated potential to promote increased blood flow. Seal 20 is fixed at (the end or talent) ridge groove in the cylinder 12. No longer requires the insertion of the lubricating oil and rubber to use the seal of 20 patients. Erectile tissue destruction and no pressure for more effective, positive feedback sensational, low-cost, less confusion, no rubber insert, for the body, and no hair, bruising of the following: As a result, many, including the seal 20 provides the benefit of the patient. One of the advantages of the seal, by expansion in a vacuum chamber, still provides a comfortable fit snugly around the penis, the material is very thin, lies in the fact that it is very stretchy. Will be to minimize the contact area between the seal 20 effects, 32 and the penis of the "umbrella", as shown in the figure. See side effects of energy drinks erections. These effects will create a unique experience of the patient compared to the current VED options. Satisfaction and more effective.
Figure. Figure 6 shows an erection that will enhance the progress of the penis 32 to 14 toward the distal end of the vacuum chamber 12, has been commensurate progress of seal penis toward the base 20 of 32 penis 34.
Figure. Shows the application of the clamp 28 7 after constriction desired erection is achieved. Clamp 28, offers a unique option less convenient, more pain, for patients with good control, to provide a potentially low-cost system than the current VED.
Finally, it is shown in the figure. 8, in the vacuum chamber 12 will be deleted.
Clamp contraction is simply clamp after you have used.
Other embodiments and configurations may be devised without departing from the spirit of the scope of the present invention and the appended claims.


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Aries Narrow PendantThis product is no longer available.100% Price MatchHouzz |
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2011 design.
By Robert Abbey.
The Aries Narrow Pendant produces a unexpected star burst light pattern from it's perforated metal shade as light shines through. Utilizing 1 X 60W B10.5 Candelabra lamp socket, deep patina bronze metal finish, 6" diameter shade and supplied with 12 feet of black suspension cord. Dimmable with a Standard Incandescent Dimmer.
Dimensions: 6" D X 16.25" H X 154" Max Suspension Length
Lamp Type: INCANDESCENT
Bulbs: 1 X 60W 120V E26 (medium base) B10.5 torpedo incandescent lamp (not included)
Listing: UL , CUL
Manufacturer Specifications: Click to download specifications.
Instructions: Click to download instructions.
Model(s): 9006
Robert Abbey
At Robert Abbey, designs are a true passion. Robert Abbey works diligently to offer the most trend right merchandise with the highest quality standards at affordable prices. The Robert Abbey family owned and operated company has been designing and manufacturing fine lighting products since 1946 from their Robert Abbey design studio and factory in Hickory, North Carolina.
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Since joining Lincoln Laboratory in 2001, Dauler has focused his research on single-photon detectors, including Geiger-mode avalanche photodiodes and superconducting nanowire detectors, and their application to optical communications and quantum measurements. He is currently creating new types of detectors, including one based on quantum dots, that may enable higher-performance single-photon detection.
The Lincoln Laboratory nanowire photodetector arrays are advancing what is possible in optical communications. They are the fastest, most sensitive single-photon detectors available. They operate in the ultraviolet, visible and near-infrared spectral regions; provide the largest photon-counting rates; are relatively easy to fabricate; involve simple signal digital post-processing to obtain photon-number information; and detect light coming from a range of angles. In the future, nanowire photodetector arrays may be used for both ground-based and on-orbit laser-communications receivers in an optical deep-space communications network.
Geiger-mode avalanche photodiode two-dimensional arrays of ultrasensitive solid-state photodetectors enable three-dimensional laser radar imaging used in mapping terrain and construction sites, acquiring data for robotic vision, and imaging partially obscured objects. These arrays have also been employed in highly sensitive laser-communications receivers.
Dauler holds SB, MEng and PhD degrees in electrical engineering and computer science from MIT. He performed thesis work at Lincoln Laboratory under the MIT VI-A Master's in Engineering Program and was a participant in the Lincoln Doctoral Scholars Program.


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ICE finds challenges in technology unit sale
09 Dec 2013
Early efforts by IntercontinentalExchange, the new owner of NYSE Euronext, to offload NYSE Technologies businesses have met a cool reception from potential industry purchasers, including Hong Kong Exchanges and Clearing, Nasdaq OMX, Markit and Deutsche Börse.
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Robie House
But after visiting Robie House here as part of Road Trip 2013, CNET reporter Daniel Terdiman thinks he might have seen the best Frank Lloyd Wright building of them all.
With what is the quintessential example of Wright's Prairie Style of architecture, Robie House is a stunning work of art. Located at the University of Chicago, the house, completed in 1910 for businessman Frederick C. Robie, is in the middle of a major, multi-million-dollar renovation that should return it to its original glory.
Already, the building's exterior is done. But now, work is being done on the interior, although it's not clear when it will be finished.
July 8, 2013 4:00 AM PDT
Photo by: Daniel Terdiman/CNET
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Channel the perseverance and talent of your favorite Dolphins player, Reggie Bush, with this #22 authentic jersey by Reebok. It features a heavyweight mesh body and high-quality tackle twill numbers on the front and arms with Bush's name across the upper back and rib-knit sleeves wrapped with team-colored stripes. It's a faithful show of Dolphins pride that'll have you looking and feeling like the ultimate 12th man.
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Article ID: 2703309 - View products that this article applies to. SYMPTOMSConsider the following scenario:
CAUSEThis issue occurs because the media session in Lync 2010 overrides the screensaver configuration. This is an intended behavior in Lync 2010. RESOLUTIONTo resolve this issue, install the following cumulative update: 2701664 After the update is installed, the environment administrator must run the following command in Lync Server Management Shell to apply a client policy:
(http://support.microsoft.com/kb/2701664 /
Description of the cumulative update package for Lync 2010: June 2012 New-CsClientPolicyEntry -Name "UseDefaultWindowsScreenSaverPolicy" –Value "True" MORE INFORMATIONFor more information about the New-CsClientPolicyEntry command, go to the following Microsoft website: General information about the New-CsClientPolicyEntry command
PropertiesArticle ID: 2703309 - Last Review: June 16, 2012 - Revision: 2.0


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MP3 Insider Podcast 176: Cache is king
Donald and Jasmine throw out a few tasty leftovers from CES 2010, including an upcoming Slacker iPhone app update that will cache music offline. We check out some speaker light bulbs, an amplified helmet, and cross our fingers for XviD support on the Zune HD.
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Please save Chickshore
By the end of 2005 Chicksands north shore area is scheduled for demolition, this is due to insurance complications, and the FC are not prepared to sponsor the area and let it remain open. To help keep it going please sign up.
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Ok maybe this needs to be explained to you. Ricky is an atheist so that is his way to mock those who are seemingly doing something about it (praying) and getting into news because of it versus people who are actually doing something (sending money, collecting money, volunteering)
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March 2009 DirectX SDK Available!
The March 2009 edition of DirectX Software Development Kit (SDK) is out.
The March 2009 SDK includes an update to the Technical Preview for Direct3D 11, the latest version of Direct3D that provides support for
tessellation and general purpose GPU processing, as well as the Technical Preview for Direct2D and DirectWrite, the new Windows APIs for accelerated 2D text rendering.
This version of the DirectX SDK contains the following new things:
- Technical Previews of Direct2D, DirectWrite, and DXGI 1.1
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