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Epigenetic inheritance refers to the transmission of modified genetic material from one generation to the next. These "epialleles" are not caused by mutations in the DNA sequence, but instead by covalent modification of chromatin and DNA, guided by developmental and environmental cues. In general, epigenetic modifications that are programmed during development must be reset in the germline, so that the zygote is restored to pluripotency and can once again initiate embryonic development. For example, imprinted genes in the mouse are expressed predominantly from either the paternal allele or from the maternal allele in the diploid embryo, and so must be reprogrammed in the germline depending on its sex (Bartolomei and Ferguson-Smith, 2011). Indeed, the mouse genome undergoes several rounds of DNA methylation, demethylation and repair as germ cells differentiate, as well as in the embryo after fertilization when imprinted genes are largely immune (Bartolomei and Ferguson-Smith, 2011; Feng et al., 2010; Popp et al., 2010). For this reason, epigenetic inheritance is thought to be rare in mammals, and is generally restricted to non-essential genes.
Flowering plants are an important exception to this rule, as epigenetic modification during development can be inherited for hundreds of generations with dramatic developmental consequences (Cubas et al., 1999). The first (and most common) examples of epigenetic inheritance in plants involved transposable elements (TE), which can regulate nearby genes, and undergo epigenetic switches during development, resulting in the inheritance of epialleles (Martienssen et al., 1990; McClintock, 1965). As in mammals, epigenetic inheritance of transposon activity in plants involves DNA methylation (Becker et al., 2011; Cubas et al., 1999; Martienssen and Baron, 1994; Schmitz et al., 2011). Imprinted genes tend to be flanked by transposable elements, whose methylation can influence their expression (Radford et al., 2011). However, imprinting in plants is largely restricted to the extra-embryonic endosperm, a terminally differentiated tissue within the seed, so that imprinted chromatin and DNA modifications need not be removed once they are established (Feng et al., 2010; Jullien and Berger, 2009; Raissig et al., 2011). The extent of reprogramming in the plant germline thus remains an important question.
Unlike mammals, which set aside their germline in early development, flowering plants give rise to germ cells during post-embryonic growth and development, in some cases many years after embryogenesis is complete. The pollen mother cell (PMC) on the paternal side and the megaspore mother cell (MMC) on the maternal side are specified from somatic cells in developing flowers (Boavida et al., 2005). In the anthers, the PMC undergoes meiosis resulting in four haploid microspores. Each microspore subsequently undergoes an asymmetric division to differentiate a larger vegetative cell and a smaller generative cell, which represents the male germline (Figure 1A). The vegetative cell exits the cell cycle into G0, while the generative cell undergoes a further symmetric division to produce two identical sperm cells that are surrounded by the vegetative cell (Berger and Twell, 2011).
The most conspicuous evidence of reprogramming in the plant germline is that the vegetative nucleus (VN) of the pollen grain has completely decondensed heterochromatin, in contrast to the tightly condensed chromatin found in sperm cell (SC) nuclei (Figure 1A). Heterochromatin in plants is mostly occupied by TEs and repeats (Lippman et al., 2004). TE repression is important for genome integrity and mutants in DDM1 (DECREASE in DNA METHYLATION 1) and MET1 (DNA METHYLTRANSFERASE 1) have reduced DNA methylation levels resulting in up regulation of TEs (Lippman et al., 2004). MET1 maintains CG methylation, and its activity in the germline impacts epigenetic inheritance (Jullien et al., 2006; Saze et al., 2003). In plants, CHROMOMETHYLASE3 (CMT3) maintains CHG methylation, guided by histone modification, and cytosines can also be methylated in an asymmetric CHH context guided by RNA interference (RNAi) (Law and Jacobsen, 2010). RNA-directed DNA methylation (RdDM) requires the DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2), and the RNA polymerase IV and V subunits NRPD1a, and NRPE1a, which are involved in production and utilization of 24nt siRNA (Haag and Pikaard, 2011). These mechanisms interact, so that RdDM is required to remethylate TEs in ddm1 mutants. TEs without matching siRNA cannot be remethylated even when DDM1 function is restored through crosses to wild-type plants (Teixeira et al., 2009).
Loss of heterochromatin in the vegetative nucleus of the pollen grain is accompanied by the loss of DDM1, the activation of TEs, and the production of a novel class of 21nt siRNAs which accumulate in sperm cells (Slotkin et al., 2009). However, while some TEs and repeats were found to be demethylated in the VN, others were hypermethylated so that the role of DNA methylation in pollen reprogramming was unclear (Schoft et al., 2011; Schoft et al., 2009; Slotkin et al., 2009). We set out to determine the dynamics of DNA methylation during pollen development, via bisulfite sequencing of genomic DNA from Arabidopsis microspores, and from their derivative sperm and vegetative cells (Figure 1A). We found that symmetric CG and CHG methylation were largely retained in Arabidopsis pollen. However, CHH methylation was lost from at least 1500 TEs, mostly long terminal repeat (LTR) retrotransposons, in microspores and sperm cells. In the VN, more than 100 DNA transposons and non-LTR retrotransposons were targeted for CG demethylation by DNA glycosylases. Many of these transposons, including those that flank imprinted genes, gave rise to 24nt siRNA in sperm cells where DNA glycosylases are not expressed. Recently discovered recurrent epialleles were pre-methylated in sperm cells guided by a similar mechanism. Thus reprogramming of DNA methylation in pollen contributes to transposon silencing, the transgenerational recurrence of epialleles, and imprinting of maternally expressed genes.
Sequencing of the methylome from individual pollen cell types presents a significant challenge, especially in Arabidopsis where pollen yields are limiting. Sperm cells and vegetative nuclei were isolated using Fluorescence Activated Cell Sorting (FACS), through the use of cell specific promoters driving the expression of Red and Green Fluorescent Protein (RFP and GFP) (Borges et al., 2012). Microspores were obtained from young flower buds through a combination of mechanical filtration and purification through FACS, taking advantage of their small size and autofluorescent properties (Borges et al., 2012). Genomic DNA was isolated from each nuclear fraction, treated with sodium bisulfite and sequenced at 7–17x coverage (Table S1). To test whether each cytosine was methylated, the proportion of methylated reads to unmethylated reads was compared to the background error rate using a binomial test for each cytosine with sufficient coverage. The data was plotted as a heatmap on all five chromosomes, compared with the methylome of somatic cells from leaves (Figure 1B).
We observed a strong enrichment of DNA methylation in the pericentromeric heterochromatin in pollen (Figure 1B) resembling methylation profiles obtained previously from somatic cells (Cokus et al., 2008). The observed maintenance of symmetric CG and CHG methylation in pollen is consistent with expression of the maintenance DNA methyltransferases MET1 and CMT3 during microspore and generative cell division (Honys and Twell, 2004). Strikingly, however, CHH methylation in microspores and sperm cells was lost from pericentromeric retrotransposons and satellite repeats, and subsequently restored in the VN (Figure 1B).
To identify regions of the genome subject to differential methylation, we first identified Single Methylation Polymorphisms (SMPs) in a pairwise fashion (VN vs. microspore, SC vs. microspore, and VN vs. SC). Using the SMP information we next identified differentially methylated regions (DMRs). For CHH methylation, DMRs were defined as regions containing at least five SMPs, each < 50bp apart and containing a minimum of ten methylated cytosines. For CG and CHG methylation (which were far less variable), DMRs were defined as regions containing at least three SMPs, each < 50bp apart and containing at least five methylated cytosines. For each putative DMR the methylation calls were pooled across the whole region and then tested using Fisher's exact test.
We found that almost all DMRs corresponded to intergenic regions and transposable elements, and strikingly, that almost all CHH DMRs were hypomethylated in sperm cells while CG DMRs were hypomethylated in the VN (Figure 2A). We found that 2270 CHH DMRs overlapped with 1781 different TEs, including 1483 LTR/Gypsy elements and 139 DNA transposons (Figure 2B, Table S2). Pairwise comparisons of VN vs. microspore and VN vs. SC yielded similar results (Figure 2A, ,B;B; Table S2) indicating that these retrotransposons were similarly unmethylated in microspores. An example of an Athila LTR retrotransposon, in which CHH methylation is reduced in microspores and sperm cells, is shown in Figure S1.
We uncovered 221 CG hypomethylated regions (CG DMRs) in the VN relative to the SC (Figure 2, Table S2) that overlapped with 109 different TEs (Table S2), including AtMu1a (At4g08680), as previously reported (Schoft et al., 2011; Slotkin et al., 2009). 29 of these TEs were RC/helitrons, 34 were DNA/MuDR transposons, and the remainders were mostly non-LTR retrotransposons (Figure 2B). A similar trend was observed in a pairwise comparison between VN and microspores (Figure 2A, ,B)B) and there was a high degree of overlap between CG DMRs in the VN in both pairwise comparisons (Figure 2C). In contrast, CG methylation was very similar in SC and microspores with only a very few loci demethylated in microspores (Figure 2A). These same loci (15/21 DMR) were also demethylated in the CHG context in microspores relative to VN and SC (Figure 2A). CG DMRs in the VN and CHH DMRs in SC did not overlap (Figure 2C), suggesting that differential methylation might be due to differential expression of DNA methyltransferases and DNA demethylases in each pollen cell type.
The DNA glycosylase DEMETER (DME) is expressed in the VN, along with its homologs ROS1, DEMETER-LIKE2 (DML2) and DML3 (Schoft et al., 2011). DME is required for the demethylation of transposons and repeats that surround the imprinted Maternally Expressed Genes (MEGs) MEDEA (MEA) and FLOWERING OF WAGENINGEN (FWA). These genes are normally expressed from the maternal allele in the endosperm, but are also expressed in the VN of the pollen grain (Schoft et al., 2011). In order to determine whether CG DMRs in the VN were targets of DNA glycosylases, we performed pairwise analysis of CG DMRs between VN and SC, between endosperm and dme mutant endosperm (Hsieh et al., 2009), and between WT inflorescence and ros1/dml2/dml3 mutant inflorescence (Lister et al., 2008). Using the same DMR analytical pipeline, we found 267 targets of ROS1/DML2/DML3 (RDD) in inflorescence, and 121 targets of DME in the endosperm (Hsieh et al., 2009; Lister et al., 2008). Of the 221 DMRs hypomethylated in the VN, 134 DMRs were targets of RDD, and 48 were targeted by DME (Figure 2C). This accounts for 83% of all the DMRs which show decreased CG methylation in the VN compared to SC (Figure 2C). Similar values were obtained for CG DMRs between VN and microspore (Figure S2A). DME is only expressed in the VN of pollen and in the central cell of the female gametophyte, while ROS1, DML2 and DML3 are widely expressed in somatic tissues as well as in the VN. However, none of these genes are expressed in sperm cells (Schoft et al., 2011). Hence, DNA demethylases are responsible for the loss of CG in the VN.
The overall level of CHH methylation in microspores was approximately half the level found in the inflorescence (Table S1), as if reductional division during meiosis was not accompanied by RNA directed DNA methylation (RdDM). CHH methylation in sperm cells was further reduced, and the remnants were observed on both DNA strands (Table S1), likely reflecting random segregation of unmethylated strands after meiosis (Schoft et al., 2009). We hypothesized that loss of CHH methylation in the SC could be the result of differential expression of proteins required for CHH methylation. The DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2), a homolog of mammalian Dnmt3, is required for CHH methylation, guided by 24nt siRNA (Cao and Jacobsen, 2002). We constructed a DRM2-GFP transgene fusion driven by the DRM2 promoter that was introduced into plants. We found that the DRM2-GFP fusion protein was barely detectable in microspores, but accumulated prominently in the VN at the bicellular stage (Figure 3). Very low levels were detected in the generative cell and in mature sperm cells (Figure 3), implying that the male germline has only a limited capacity for de novo CHH methylation which would account for progressive loss of CHH methylation from microspores to sperm cells.
CHH methylation of retrotransposons is guided by 24nt small RNA (Haag and Pikaard, 2011; Law and Jacobsen, 2010). In sperm cells, CHH methylation is sharply reduced (Table S1; Figure 1B) and several genes required for 24nt siRNA biogenesis are no longer expressed in mature pollen (Grant-Downton et al., 2009; Honys and Twell, 2004; Pina et al., 2005) or sperm (Borges et al., 2008). CHH methylation is restored in the embryo (Hsieh et al., 2009; Jullien and Berger, 2012), and therefore must occur during or after fertilization. 24nt siRNA accumulate to high levels in the seed, and are maternal in origin in the seed coat and the endosperm (Lu et al., 2012; Mosher et al., 2009). Therefore, maternal 24nt siRNA might guide restoration of CHH methylation to incoming retrotransposons from sperm. To test this idea, we examined the size distribution of small RNA in sperm cells (Slotkin et al., 2009) and in seeds (Lu et al., 2012) corresponding to CHH DMRs in pollen (Figure 4). We found that DMRs that had lost CHH methylation in sperm cells matched both 21nt and 24nt siRNA in sperm cells but matched mostly 24nt siRNA in seeds (Figure 4). Thus retrotransposons that lost CHH methylation in sperm cells would be remethylated in seeds, guided at least in part by maternal 24nt siRNA, and high levels of RdDM activity during embryogenesis (Jullien and Berger, 2012).
In somatic cells, the activity of DNA glycosylases such as ROS1, DML2 and DML3 results in loss of siRNA production as well as loss of DNA methylation, so that RDD targets tend to gain small RNAs in rdd mutants (Lister et al., 2008; Ortega-Galisteo et al., 2008). Sperm cells do not express DME, ROS1, DML2 and DML3 resembling rdd mutants in this respect, and we found that many DMRs that lost CG methylation in the VN accumulated siRNA in sperm cells (Figure 4). As many of these CG DMR flank imprinted genes (Gehring et al., 2009), we examined methylation patterns in repeats flanking the imprinted Maternally Expressed Gene (MEG) SUPPRESSOR OF DRM2/CMT3 (SDC) and the imprinted Paternally Expressed Gene (PEG) PHERES1 (PHE1) (Figure 5A). SDC is expressed when flanking repeats are unmethylated (Henderson and Jacobsen, 2008), but PHE1 is only expressed when a tandem repeat downstream of the coding sequence is methylated, likely because methylation prevents inhibition by the MEA/FIS2 Polycomb Group (PcG) complex (Makarevich et al., 2008). We found that tandem repeats flanking both genes lose CG methylation in the VN (Figure 5A). However, CHH methylation was only detected at SDC and not at PHE1. Furthermore, SDC accumulated 24nt siRNA in sperm cells (Figure 5A) unlike PHE1. The siRNA accumulated to even higher levels in total pollen grains, indicating they may (also) be generated in the VN.
We extended these observations to a larger number of putative imprinted genes (Gehring et al., 2011; Hsieh et al., 2011; McKeown et al., 2011; Wolff et al., 2011) filtered to include only experimentally validated PEGs and MEGs, resulting in 28 imprinted loci (12 MEGs and 16 PEGs) that passed our filter for methylation calls and had a TE within 2kb of the coding sequence (Table S3). All 28 TEs lost CG methylation in the VN relative to the progenitor microspore, but interestingly only those surrounding MEGs were targeted by siRNA and CHH methylation in pollen (Figure 5B). We plotted the size distribution of siRNA corresponding to CG DMRs, and found that while CG DMRs accumulated both 21 and 24nt siRNA in sperm cells, MEGs and PEGs accumulated only 24nt siRNA in sperm cells and in seeds (Figure 4). siRNA levels for MEGs were much higher than PEGs in sperm cells and in seeds, but not in total pollen (Figure 5C). We conclude that 24nt siRNA from repeats surrounding MEGs accumulate preferentially in sperm cells. It is possible that these are derived from the VN, resembling 21nt siRNA in this respect (Slotkin et al., 2009).
In plants, epigenetic changes in gene expression are frequently inherited from one generation to the next, and gains and losses of DNA methylation arise as spontaneous epigenetic variation (Martienssen and Colot, 2001). In two recent studies, more than 100 loci (DMRs) were found to gain DNA methylation sporadically in young leaf tissue after 30 generations of inbreeding by single seed descent (Becker et al., 2011; Schmitz et al., 2011). Methylation gains were recurrent, occurring at the same loci in multiple independent lines, leading to the proposal that methylation gains and losses might be pre-programmed in the germline (Schmitz et al., 2011). Among 100 hypervariable loci that gain methylation, we identified several ROS1/DML2/DML3 (RDD) targets that were completely re-methylated in rdd mutants compared to wild-type (Lister et al., 2008). Most of the remaining hypervariable loci already showed high methylation levels in wild-type inflorescence tissue (Lister et al., 2008). Remarkably, we observed that 56 of these 100 variable DMRs were hypermethylated in wild-type sperm cells. An example of a RDD target, corresponding to one of the hypervariable epialleles, is shown in Figure 6. This target is contained within a COPIA LTR retrotransposon (Atg409455) that is heavily methylated at CG sites in sperm cells, and less so in the microspore and VN (Figure 6). Further examples are shown in Figure S3. DME, ROS1, DML2 and DML3 are expressed at low levels in the microspore (Honys and Twell, 2004), and high levels in the VN (Schoft et al., 2011), accounting for differential CG methylation observed in sperm. Importantly, CG methylation found in sperm cells was removed in the embryo, reflecting the restoration of ROS1 activity after fertilization (Figure 6).
In mammals, 5-methylcytosine occurs mainly in symmetric CG dinucleotides, and is depleted in male primordial germ cells by loss of DNA methyltransferases and by active demethylation (Feng et al., 2010; Popp et al., 2010) resulting in TE activation (Castaneda et al., 2011). Methylation is restored in mature round spermatids (Feng et al., 2010; Popp et al., 2010) and then extensively modified by hydroxylation just before fertilization (Salvaing et al., 2012; Zhang et al., 2012). Further rounds of methylation and demethylation occur in the blastocyst and early embryo (Feng et al., 2010) resulting in a complex pattern of DNA methylation that is reset in each generation (Bartolomei and Ferguson-Smith, 2011). In pollen, we have found that symmetric CG and CHG methylation are largely retained in the germline (Figure 7). This may account for the prevalence of epigenetic inheritance in plants, compared with mammals. Strikingly, however, asymmetric CHH methylation of transposons is reduced in the microspore, accompanied by down regulation of the RdDM methyltransferase DRM2, a homolog of the mammalian Dnmt3 (Figure 7). CHH methylation is restored in the embryo, and may reflect an ancient mechanism for transposon recognition.
The loss of asymmetric CHH methylation in sperm cells means that paternal retrotransposons are delivered to the zygote stripped of CHH methylation. Restoration of DNA methylation in the embryo (Hsieh et al., 2009), indicates that CHH methylation must occur during or after fertilization, when the RdDM pathway is active (Jullien and Berger, 2012) (Figure 7). We demonstrate that 24nt siRNAs in seeds match retrotransposons that have lost CHH methylation in sperm (Figure 6). CHH methylation is restored in seeds (Hsieh et al., 2009), guided by these 24nt siRNAs (Jullien and Berger, 2012). It has been proposed that most 24nt small RNA in seeds are maternal in origin, especially in the seed coat and the endosperm (Mosher et al., 2009), and target retrotransposons (Lu et al., 2012). We can speculate that paternal retrotransposons that have lost CHH methylation, but do not match maternal siRNA, might escape silencing immediately after fertilization (Josefsson et al., 2006).
DRM2 expression is restored in the VN, and retrotransposons are remethylated in these companion cells (Figure 7), most likely at the bicellular stage when DCL3 and other components of the 24nt siRNA biogenesis pathway are expressed (Grant-Downton et al., 2009). However, TEs are strongly activated in the VN and give rise to mobile 21nt siRNA that accumulate in sperm cells (Slotkin et al., 2009). CHH methylation by RdDM would not be expected to prevent transcription in the absence of the chromatin remodeler DDM1 (Teixeira et al., 2009), which is not expressed in the VN, accounting for transposon activation (Slotkin et al., 2009). Loss of chromatin remodeling can result in transposon transcription even in the presence of DNA methylation (Lorkovic et al., 2012; Mittelsten Scheid et al., 2002; Moissiard et al., 2012; Vaillant et al., 2006). Furthermore, the VN undergoes extensive histone replacement, with the loss of many canonical histones including the centromeric histone CENH3, which may contribute to transposon activation (Berger and Twell, 2011; Schoft et al., 2009). It is possible therefore that CHH methylation in the VN compensates for the loss of pericentromeric heterochromatin (Schoft et al., 2009).
Although CG methylation was globally retained, a subset of DNA transposons, some non-LTR retrotransposons, and intergenic regions lost CG methylation in the VN (Figure 7). These transposons are targets of the DNA glycosylases DME, ROS1, DML2 and DML3, which are expressed in the VN. In sperm cells, these enzymes are not expressed, and 24nt siRNA corresponding to some of their targets accumulate, resembling ros1/dml2/dml3 triple mutants in this respect (Lister et al., 2008). This is particularly true for transposons that flank imprinted genes which are expressed from the maternal allele in the endosperm (MEGs), and imprinting at the SDC locus is lost in the endosperm when inherited from mutant pollen impaired in RdDM (Vu et al., 2012). These results indicate that 24nt siRNA in sperm cells contribute to RdDM and transcriptional silencing before fertilization in at least some cases (Figure 7). Like 21nt siRNA, these specific 24nt siRNA may also be derived from the VN, although this has not been tested directly (Figure 7). In this way, imprinted genes are protected from the global loss of methylation, reminiscent of mammalian imprinted genes, which regain methylation in the germline before fertilization (Feng et al., 2010).
Many imprinted genes are expressed in pollen (Table S3), and dme mutants are transmitted poorly because of defective pollen germination (Schoft et al., 2011). Similarly, ros1 mutants exhibit severe fertility defects after 3 generations of inbreeding (Gong et al., 2002). It is likely therefore that the targets of DME and ROS1 play a role in fertilization when the vegetative nucleus supports pollen tube growth (Berger and Twell, 2011). Silencing in sperm cells would restrict expression to the pollen tube, as well as resulting in imprinting in the endosperm. A small number of target were also demethylated in the microspore, and may have a function earlier in pollen development (Figure S2B).
Similar silencing mechanisms may account for the methylation we observe in sperm at hypervariable epialleles. These epialleles acquire heritable methylation sporadically on inbreeding, prompting speculation that they might be reprogrammed in sperm (Becker et al., 2011; Schmitz et al., 2011). Some of these epialleles are silenced in ros1/dml2/dml3 mutants, and many correspond to TEs (Schmitz et al., 2011). We show that these variable epialleles are indeed methylated in sperm cells, and that many of them are methylated already in the inflorescence (Lister et al., 2008). Sperm cells do not express ROS1 and its homologs, accounting for higher methylation of RDD targets in sperm, and providing a mechanism for gain of heritable methylation if 24nt siRNA accumulate after fertilization to prevent demethylation by ROS1.
When transposon methylation is lost, it can be regained through RNAi (Teixeira et al., 2009) which seems to occur stepwise in subsequent generations consistent with its occurrence in the germline (Teixeira and Colot, 2010). Loss and gain of class II DNA transposon activity in maize occurs over generations (McClintock, 1965), during development (Li et al., 2010; Martienssen and Baron, 1994; Martienssen and Colot, 2001) and is inherited in the germline resembling the epialleles recently described in Arabidopsis (Becker et al., 2011; Schmitz et al., 2011). Our results suggest that similar epigenetic mechanisms silence epialleles and imprinted genes in pollen, which escape reprogramming in subsequent generations because of the retention of DNA methylation in sperm.
A detailed protocol for isolation of sperm cells, vegetative nuclei and microspores by Fluorescence Activated Cell Sorting (FACS) will be published elsewhere (Borges et al., 2012). In brief, open flowers from transgenic plants expressing MGH3p-MGH3-GFP (MGH3/HTR10, At1g19890) and ACT11p-H2B-mRFP (ACT11, At3g12110) transgenes were collected into a 2mL eppendorf tube. The tissue was vigorously vortexed in Galbraith buffer (45mM MgCl2, 30mM Sodium Citrate, 20mM MOPS, 1% Triton-100, pH to 7.0) for 3 minutes to release mature pollen (Galbraith et al., 1983). This crude fraction was then filtered though a 30 micron mesh into a tube containing 100uL of glass beads, and vortexed for additional 3 minutes in order to break the pollen cell wall. Sperm cells and VN were then isolated by FACS based on their distinct fluorescent signals (Borges et al., 2012). In order to isolate microspores, young flower buds were gently ground in a mortar and pestle in pollen extraction buffer (PEB: 10mM CaCl2, 2mM MES, 1mM KCl, 1% H3BO3, 10% Sucrose, pH 7.5) in order to release the spores (Becker et al., 2003). This crude fraction was initially filtered through Miracloth to remove larger debris, and concentrated by centrifugation (800g, 5 min). The resulting pellet enriched in pollen spores was resuspended in 1–2mL of PEB, and filtered through a 20 micron mesh before FACS. Microspores were sorted based on their small size and autofluorescent properties (Borges et al., 2012).
Genomic DNA was isolated from approximately 600,000 sperm cells, 300,000 vegetative nuclei and 1,000,000 microspores isolated by FACS (Borges et al., 2012) and fragmented by Covaris in 10mM Tris-HCl, pH 8.0. Fragments were end repaired, A-tailed and ligated to methylated Illumina adaptors. Ligated fragments were bisulfite treated using the EZ DNA Methylation-Gold Kit (Zymo), and PCR enriched with Expand High-Fidelity Polymerase (Roche). Amplified fragments of 340–360bp were size selected by gel extraction, and sequenced on an Illumina GAII platform as paired end 50 nt (PE50) reads.
To test whether each cytosine (covered by at least four reads) was methylated, the proportion of methylated reads to un-methylated reads was compared to the background error rate using a binomial test. The background false positive error rate (sequencing errors + conversion errors) was calculated using reads mapping to the unmethylated chloroplast genome. The number of methylated cytosines was calculated independently for each library. Correction for multiple testing was performed using Storey's q-values (Storey and Tibshirani, 2003) with an FDR of 0.05. The circos plot (www.circos.ca) was calculated as follows: The mean methylation across 10kb windows was calculated separately for each methylation context. Heatmaps were scaled based on the maximum level of methylation found within each methylation context across all tissues tested (CG: 0 to 0.95, CHG: 0 to 0.83 and CHH: 0 to 0.34).
For each pairwise comparison (VN vs. SC, VN vs. microspore and SC vs. microspore) the union of methylated cytosines were tested for SMPs using Fisher's exact test. For CpG and CHG (symmetrical) contexts, reads from both strands were used. For CHH (non-symmetrical) contexts, each strand was interrogated independently. Correction for multiple testing was performed using Storey's q-values. For CHH methylation a FDR of 0.05 was used. For CG and CHG methylation an FDR of 0.1 was used to reflect the more subtle changes in methylation expected.
For CHH methylation, putative DMRs were defined as regions containing at least five SMPs each < 50bp away from its neighboring SMP and containing a minimum of ten methylated cytosines. For CG and CHG methylation, putative DMRs were defined as regions containing at least three SMPs each < 50bp from its neighboring SMPs and containing at least five methylated cytosines. These regions were tested using the sum of reads (methylated and un-methylated) across the region using Fisher's exact test.
DMR were detected in published genome-wide methylation profiles using the same pipeline. This analysis uncovered 1624 CG DMRs between ros1/dml2/dml3 inflorescence and wild type inflorescence (Lister et al., 2008), and 171 DMRs between endosperm vs. dme mutant endosperm (Hsieh et al., 2009). This combined list was compared to regions of differential methylation between VN vs. SC and VN vs. microspore. We found an overlap of 131 of the 221 (60%) CG DMRs observed between SC and VN, and 83 of 164 (51%) CG DMRs observed between SC and microspore. When the list was refined to include only TEs, the overlap was (85%) as described in the text (Figure 2B).
Small RNAs from sperm (Slotkin et al., 2009) and seed (Lu et al., 2012) were collapsed and mapped to the Arabidopsis genome (TAIR10). All 20–25 nt smallRNAs overlapping CHH and CG DMRs, plus TEs within 2kb of MEGs and PEGs were then identified. The size distribution of those overlapping small RNAs was then calculated for each genomic feature. In purified SC, the median number of reads mapping to MEG TEs is 10.53 (Q1: 0, Q3: 46.07) per MEG and a median of 0.0 (Q1: 0.0, Q3: 6.142) for PEGs. In total pollen, the median number of mapping reads to MEG TEs is 21.34 (Q1: 8.326, Q3: 36.876) per MEG and a median of 13.4519 (Q1: 3.6158, Q3: 33.703) for PEG TEs. In seeds, the median number of mapping reads to MEG TEs is 9.764 (Q1: 5.633, Q3: 41.012) per MEG and a median of 3.755 (Q1: 0.0, Q3: 7.041) for PEG TEs.
pDRM2-DRM2:GFP was generated as described (Jullien and Berger, 2012). At least ten transgenic lines were analyzed and showed a consistent pattern of expression of the fluorescent reporter. Three complementing lines were used for further detailed analysis. The expression pattern of DNA methyltransferases in pollen was observed using a laser scanning confocal microscope Zeiss LSM510.
Figure S1. Loss of CHH methylation at the pericentromeric LTR retrotransposon Athila6A. A genome browser view of an Athila element from chromosome 4. Methylated cytosines in CG and CHH contexts are shown wild-type (INF) and rdd mutant inflorescence (rdd INF), microspore (MS), vegetative nucleus (VN), sperm cells (SC) and Embryo. CG methylation is maintained in the three cell types throughout pollen development relative to the wild-type inflorescence. CHH methylation is reduced in the microspore and sperm cells, but restored in the VN in the embryo. Small RNAs reads corresponding to SC and seed are presented. 21nt small RNA were detected in sperm cells as previously reported (Slotkin et al., 2009).
Figure S2. Demethylation in the vegetative cell and in the microspore. (A) CG DMRs in the vegetative nucleus (VN) vs microspore (MS) (blue) overlap with DMRs detected between WT endosperm and dme endosperm (green), which are targets of DEMETER (Hsieh et al., 2009), and with DMR detected between WT inflorescence and ros1/dml2/dml3 inflorescence (Lister et al., 2008), which are targets of ROS1 and its homologs (orange). These targets lose methylation in the VN. (B) CG DMRs in the MS vs sperm cells (SC), these regions lose methylation before pollen mitosis I and are predominately targets of ROS1 and its homologs.
Figure S3. Examples of hypervariable epialleles corresponding to targets of the DNA glycosylases ROS1, DML2 and DML3 (RDD). Genome browser views of two genes that overlap hypervariable epialleles (Becker et al., 2011; Schmitz et al., 2011) corresponding to targets of the RDD glycosylases ("RDD targets"). Methylated cytosines in CG and CHH contexts are shown in microspore (MS), vegetative nucleus (VN) and sperm cells (SC), as well as in inflorescence (INF) and inflorescence from triple ros1/dml2/dml3 DNA glycosylase mutants (rdd INF), and embryo. RDD targets gain methylation in rdd inflorescence (Lister et al., 2008), but are unmethylated in embryo and wild-type inflorescence. These genes are fully methylated in SC and in the VN but not in MS cells. In both examples, CG methylation in the WT inflorescence is also increased suggesting methylation may have already occurred during flower development. The presence of siRNA may protect the allele from ROS1 activity after fertilization, resulting in the inheritance of DNA methylation.
Table S1. Sequencing statistics, reads, coverage depth and coverage width. % mC is tabulated in each sequence context, CG, CHG and CHH, where H = A,C or T.
Table S2. Differentially Methylated Regions in each sequence context, annotated by Transposable Element class.
Table S3. Imprinted genes used in this study. Validated Maternally Expressed Genes (MEGs) and Paternally Expressed Genes (PEGs) were filtered for adequate bisulfite sequencing coverage. Most are expressed during pollen development, either in mature pollen (MP), in sperm cells (SC), microspores (UNM) and/or bicellular pollen (BCP). Signal expression values are shown along with absence/presence calls (Borges et al., 2008; Honys and Twell, 2004).
Table S4. DMRs corresponding to hypervariable epialleles. Hypervariable epiallelic loci that gain methylation in inbred lines derived by single seed descent (Becker et al., 2011) are tabulated according to genome co-ordinates, methylation levels in leaf, sperm cells, vegetative nuclei and microspores.
We thank Michael Regulski, Jude Kendall, Evan Ernst, Emily Hodges and Antoine Molaro for advice with bisulfite sequencing and analysis, Vincent Colot, Arp Schnittger and Daniel Bouyer for helpful discussions and Shiran Pasternak for help with graphic presentation. This work was supported by NIH grant R01 GM067014 to RM, grants PTDC/AGR-GPL/103778/2008 and PTDC/BIA-BCM/103787/2008 from Fundação para a Ciência e a Tecnologia (FCT) to JDB and FCT grant PTDC/BIA-BCM/108044/2008 to JAF. FB and PJ were supported by Temasek Lifescience Laboratory. JPC was supported by a graduate student fellowship from NSERC and by a grant from the Fred C. Gloeckner Foundation, F. Borges was supported by a FCT PhD fellowship SFRH/BD/48761/2008, and FVE was funded by a Herbert Hoover postdoctoral fellowship from the Belgian American Educational Foundation.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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New Chemtrail Poll: Do you believe in Chemtrails?
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Lol...a conspiracy within a conspiracy!!!
How terrible it must be for you to live in such an evil world...
It's not like any other missions have failed....
And whenever they do fail...it must be part of some plot...
You are pathetic.
Anonymous Coward 17975050
Third time's the charm . . . incompetence personified . . . seems beyond even the government to use the same failed company . . . unless you have a reason to make all climate analysis systems fall into the ocean . . . LoL!!!!!!
link to www.youtube.com
Both the rocket and the satellite were built by Orbital Sciences Corp. of Dulles, Va. . . . and they still have the contract for the next launch . . . !!!!!
link to www.nytimes.com
link to www.spacenews.com
link to www.spacenews.com
Fri, 4 March, 2011
Taurus Rocket Fairing Glitch Dooms NASA's Glory Mission
By Turner Brinton
NASA's Glory climate-monitoring satellite
Enlarge Image
Updated at 1:13pm
WASHINGTON — NASA's $424 million Glory climate observation satellite was lost in a March 4 launch attempt from Vandenberg Air Force Base, Calif., after the shroud designed to protect the spacecraft during its climb to orbit atop a Taurus XL rocket failed to separate, agency officials said.
The Taurus XL, built by Orbital Sciences Corp. of Dulles, Va., was making its return to flight two years after a similar fairing problem destroyed NASA's Orbiting Carbon Observatory (OCO) satellite. After replacing the fairing-separation mechanism thought to be the culprit in the OCO launch failure, the government-industry team believed the Glory launch had "an acceptable level of risk," Rich Straka, Orbital's deputy general manager for launch operations, said during a March 4 press conference at Vandenberg.
After an extensive investigation of the OCO launch failure, a decision was made to replace the Taurus XL's fairing-separation mechanism, a combustion system designed to create hot gas that pushes the pistons that eject the two halves of the nose cone, said Ron Grabe, the executive vice president and general manager for Orbital's launch systems group. It was replaced by a system that uses a bottle of cold, pressurized nitrogen to drive the pistons, he said. The cold gas system was successfully used in all three launches of the Orbital-built Minotaur 4 rocket last year, he said.
Meanwhile, Orbital is building an OCO replacement for NASA and in June was awarded a $70 million contract to launch the satellite on a Taurus XL. Depending on the findings of Glory's Mishap Investigation Board, the agency may have to change that plan, said Michael Luther, deputy associate administrator for programs in NASA's Science Mission Directorate.
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Reason for reporting:
Hillary Clinton will nominate justices to the Supreme Court who are prepared to overturn Citizens United."
Day After Clinton Cash Film's Go Worldwide, CEO Forced To Step Down
Ancient 'toilet paper' proves that Silk Road travelers spread parasites as they traveled
Sheriff David Clarke Says the DNC 'Seems to Be About Embracing Criminality'
Why News Junkies Are So Glum About Politics, Economics, and Everything Else
Here's What Happened When Democrats Recited 'The Pledge Of Allegiance' At The DNC
Superhuman Tech? Most Fear Worst..
Wikileaks Proves Primary Was Rigged: DNC Undermined Democracy
WIKILEAKS JULIAN ASSANGE Says He Has NEW Emails That Should Indict Hillary…And Why He's A Trump Fan
Undercover Video: Hillary Clinton Delegate Explains Her Deceptive Propaganda to Ban Guns
Is State Dept. Is Protecting Hillary Rotten Clinton's Presidential Bid?
Robots, drones fill police ranks
Companies around globe packing earth orbit with satellites to meet communications demands
Rise of black nationalist groups
Report: Mr. Robot's one-time only VR experience will return next week
'Kill More Cops' Graffiti Mural Appears on Freeway
DNC erects four-mile eight-foot tall fence around Philly convention site
Should the Postal Service Handle Your Loans as Well as Your Mail? Elizabeth Warren Thinks So.
HERE IT IS=> Detailed List of Findings in Wikileaks DNC Document Dump
BREAKING: That Was Fast! Hillary Immediately Brings Disgraced DNC Chair Onto Her Campaign! Do you think that Hillary has something to hide?
According to Soros, there's about to be an all-out class war riots, police state and civil war for America
Torrential Downpour To Kick Off Hillary's #DNC Election Steal
Montel Williams Goes Off On Bill O'Reilly For Flipping The Script On Their Interview
Dennis Miller, "Any Vote for Anyone That is Not Trump Is a Vote for Hillary"
Protester shouting 'DNC Leak!' ejected from Hillary Rally
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ETA - your driving, walking and public transit assistant
iOS iPhone Navigation
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We hope you're enjoying ETA. If you are then please consider leaving us a review. If you're experiencing any problems at all please reach out on email email@example.com or Twitter @whatsmyeta.
148Apps —ETA Review


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Skip to comments.Hillary rejected VP slot to ready her own 2016 run
Posted on 08/17/2012 9:18:11 AM PDT by null and void
Secretary of State Hillary Clinton was pressed by her husband and a top Obama aide to consider replacing Vice President Joe Biden just a couple of weeks ago, claims the author of the New York Times bestseller "The Amateur."
But Clinton, exhausted from four years of international travel and diplomacy, shrugged off the suggestion to lay the groundwork for her own 2016 bid with her husband at her side, according to author Ed Klein.
"As recently as a couple of weeks ago, the White House was putting out feelers to see if Hillary Clinton was interested in replacing Joe Biden on the ticket," Klein told Secrets. "Bill Clinton, I'm told, was urging his wife to accept the number two spot if it was formally offered. Bill sees the vice presidency as the perfect launching pad for Hillary to run for president in 2016."
He made similar comments Thursday night to CNBC's Larry Kudlow. The White House has dismissed speculation of a Clinton for Biden swap despite a string of recent gaffes by the vice president.
Klein, whose book is No. 2 on the NYT bestseller list, quoted unnamed sources who revealed that top Obama aide Valerie Jarrett put the vice presidency on the table during a lunch with the secretary of state. "The lunch was ostensibly about policy issues, but the subject of the vice presidency came up," he said. "Hillary told Valerie Jarrett that she was not interested in running as Obama's vice president."
Klein said she cited two reasons: If elected, she didn't want to be tied to Obama's left-leaning politics in her own 2016 bid. Second, if Obama loses, she would be tarred as a loser.
New on Friday: Klein told Secrets that Bill Clinton is working fast to get a 2016 Hillary for President campaign up and going. His sources told him that Clinton is sniffing around for a major donor to offer up a jet for the potential candidate to use. Also, Klein said, Clinton is looking for somebody to take over the Clinton Global Initiative "so that he can devote his full time to Hillary's campaign."
Klein has sources deep in the Clinton camp and he said that they said she is eager for a rest followed by a makeover. "She clearly is exhausted. She needs to lose weight and get her energy back for a four-year slog."
He added that Obama has never talked to Clinton about replacing Biden. The reason, said Klein, is that Obama was afraid she would turn him down and Bill Clinton would leak the snub to the media.
I think we can bank on a replacement for Biden being in the works!
It is very true that the press has been giving unusual coverage to his gaffes...and they wouldn't do that without permission from "above."
Yeah, but running as a Widow would be a resume enhancer.
If I'm Bill C, I hire a really good food-taster. Pronto.
However, she'd NEVER be an appealing choice for ME. On her own Evita has way TOO much baggage and even if I was Liberal, I couldn't seriously consider someone who has reinvented herself so many times.
Which Evita avatar will be running for President, I wonder?
the Vice President is chosen as assasination insurance, Palins comment was merely tongue in cheek taunting
And if she does help take the Kenyan down (and I never thought I'd say this) GOD bless her!
Wrong. The VP is chosen as impeachment insurance.
As such the choice is decidedly not someone an enemy of America would want to keep out of the Oval Office, quite the opposite.
Isn't Moochelle running in 2016?
Then you are wise, Skywalker, because the bitch be running with scissors NOW.
But for comic relief, I have to note this quote: "If elected, she didn't want to be tied to Obama's left-leaning politics in her own 2016 bid."
Valerie Jarrett. She's co-president already.
Hillary hasnt had any ideas since her healthcare debacle of 1994.
By then all that will remain of the miserable old battle-axe will be one big wrinkle.
If Romney is President she can run as the conservative candidate...
Obama and Hillary...Napolean and Snowball.
Hillary Clinton will be almost 70 years old in four years. She will never be elected president. If Romney wins, I look for him to serve only four years because he'll also be about 70 when his first term is over. He'll hand the mantle off to Paul Ryan, who could be elected at the age of 47, serve eight years as president and still be in his mid-50s when his two terms are done. It's time for old foggies to get out of the way and make room for the new generation of republican leaders. Sarah Palin could also be elected president and still be in her 40s.
THAT will be the debate to watch! Can't wait.
Hil's a proper 'Rat. All rats instinctively understand, you abandon sinking ships.
just comparing AGES, Donna....
as age defines the presidency.
I give the horrible human being slack, just one time to make a point about age, and I get criticized for it...sheesh ; )~
I called this one yesterday. Why would she want to tie herself to his policies even if they are hers also?
Uh, she's already tied to the King's policies. (just like Condoleeza Rice was tied to Bush's policies) She's been tied to the King's policies for 4 years, now. So the excuse that she turned it down because she didn't want to be tied to the King's policies doesn't wash.
we have to start to hang the yoke of Obama's failures on her neck as soon as we can.
So what do all of those who insisted that the King would NEVAH toss Biden under the bus have to say now?
now that I think about it, she will pretty much be the last of the Democrat Leadership Council's stars that still hold some sway. After she goes off to the speakers circuit, there will be nothing but mini-Obama's waiting in the wings and there will not be the Chicago protection racket to cover for them.
I think she will be on the ticket.. It's just being played down to make the SURPRISE at a later date that much more powerful.
Call me a misogynist but the thought of an old man running the country makes me feel safe. The thought of an old women (especially Hillary) running the country makes me
disgusted with the voters! crazy!
True, Hitlary and Mittens are the same age! Both were born in 1947.
Ugh, between the gingivitis/alco-breath, onion pit-stench, BO, and yeast infection...
Hosatbich down!
Misogynist ? I bet you would be first at the polls to vote if Sarah were the republican candidate. I know I would.
Not misogyny but anti-Marxist
Sorry, I don't support Feminism - all those isms are the same to me.
Synonyms: Feminism, Marxism, Communism, Socialism, Fascism, Islamism, Darwinism
We agree 7 outta 8
I believe the creator used whatever method he so chose and left it for man to figure it out... Along with learning how to make everything from fire to landing on the moon.
She should also have to explain:
4. The murders of our servicemen in Afghanistan by our alleged allies.
Notice how quiet the MSM has been about these atrocities? If GWBush were still president, they would be howling over the "failed" policy of
Vietnamization Afghanization. But it's Obama's failed policy, so shhhhhh...
Oh and btw, the reason these Afghan "allies" are murdering our folks? It's because our brilliant President Obama announced a withdrawal date.
What our CIC Odumbo didn't realize, or didn't care, was that his certain withdrawal date forced Afghans to take a side. And why would they pick our side? We won't be there to defend them.
Feminism, all the isms, don't leave room for God.
The clock is ticking.....she has only 16 days to join the ticket
Hillary, the American public does not want to see their President scowling and sneering at them every time said President gets behind a microphone. Just go away.
Sounds hugh and series!
It is widely believed that she rejected the VP spot because it might mean she must wash her hair on a more frequent basis.
I think she may be positioning herself to be the next Secretary General of the UN.
She prefers her own left-leaning politics.
"She's not aging quite as gracefully..."
True, and old fat thighed women are perceived as cronelike and witchy by our culture, whereas maturing men such as Reagan can pull off the persona of attractive wise men who maintain an aura of power and ability. Maybe it is an unfair gender thing, yet it is reality and Hillary will not appeal to any male voters by 2016, young or old. (And probably not too many women either, other than perhaps the few aging die hard feminist types, themselves witchy and haglike.)
She's benefited a lot from being in the "non-partsian" position of SOS. Being Osama's veep or failed veep running mate hurts and doesn't help her if she wants to run in 2016.
Quite frankly Obama winning isn't good for her. Her best hope is Romney is in and not popular.
With her as Veep?
That would depend entilerly on subsequent events...
As I've been saying for many years, I'm worried about VICE President Clinton.
Think of it:
Two years and one day into her term the first slotter gets arkincided
Under Article XXII, she can still run for two full terms as the incumbent
The Glorious Hillary! Decade begins
Hillary uses the Patriot Act to it's fullest extent, and beyond
At the end of the Glorious Hillary! Decade there is a National Emergency "temporarily" delaying the elections
Under the pressure of the National Emergency, the 1st, 2nd and 22nd amendments are repealed
By the time the National Emergency is over all the 'improved' source code for the touch screen electronic balloting will be in place......
Just as an aside, with the Clinton's well known love of 'ethnics', Obama is very expendable, isn't he?
1. She will take time off to rest and lose weight.
2. She will then become the DNC chair...giving her a stage and TV time until...
3. She announces she's running for POTUS at the appropriate time..
Yep she has aged quite a lot in the past twenty years.
Assuming he'll win is one thing but win and then die?
If he wins it will be because Romney blows it and he will be unpopular in 2016, she would do well to be as un-associated from him as she can be, being his veep would be the worst place for her.
And being a failed veep nominee could be almost as bad especially if the reintoduction of her own scandals becomes a factor in the election. I believe that is the primary reason Osama didn't make her his VP to begin with, he didn't need her baggage on top of his own.
It's a clinton thang.
She can't assume he'll win, but she can be certain he will die on cue.
Was that clear enough, or do you need me to draw you a picture?
It's a clinton thang.
She can't assume he'll win, but she can be certain he will die on cue.
Was that clear enough, or do you need me to draw you a picture?
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text:
In his latest radio and TV advertisement, Mar Roxas portrays himself as "hindi abusado", "hindi ma-drama", and most importantly "hindi magnanakaw". This is a clear swipe at his front-running rivals in the presidential race. 487 more words
Tags » Franklin Drilon
The controversy over the "zona libre" issue for the position of Vice Governor of Iloilo will put Mar Roxas in a no-win situation. It will cause the "unity ticket" that he and Franklin Drilon put together in Iloilo province to crumble before the campaign period for the presidency could even start. 431 more words
Definitely not world class
It's been three months since the last meeting in the Iloilo leg of the APEC 2015 was held at the Iloilo Convention Center, but the grossly overpriced building — originally estimated to cost only P200 million — is far from finished. 802 more words
Drilon promises increase of budget for state universities
Gracing the opening program of Bulacan State University's (BulSU) 111th founding anniversary, Senate President Franklin Drilon assured the increase of budget for state colleges and universities through the General Appropriations Act at the BulSU Activity Center, December 3. 243 more words
Drilon to Umali: Shut up, blabbermouth
"Mind your own business."
Senate President Franklin Drilon called Oriental Mindoro Representative Reynaldo Umali a "blabbermouth" for "interfering in the internal affairs of the Senate." 353 more words
Gone is the rule of law
I just received word that the Ombudsman, in less than one month, dismissed my motion for reconsideration in the graft complaint I filed against Senate President Franklin Drilon and several other respondents in connection with the Iloilo Convention Center project anomalies. 341 more words
Paradigm shift
After spending two months in San Pedro City, Laguna as guest of Kap Jun Ynion, I saw concrete proof that "zero corruption" does translate into better public services and greater efficiency in governance. 1,068 more words


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HOME NFL New York Giants Gear New York Giants Blankets, Bed & Bath New York Giants Blankets, Bed & Bath Sleepwear (12 items) Product ID: 857020
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- Quality embroidery
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women r as there is in smallervsizes I'm usually a size 9 (UK), unless I'm getting some close fitting (generally smart) shoes, but for both pairs of my barefoot style Merrells I have size 8.5 and there's still plenty of room for my feet to stretch out/expand.These are great casual footwear and very comfortable for barefoot shoes. As else commented, they have a slightly thicker heel than the more minimalist running shoes but these are still very light and thin.Recommended if you're looking to 're-train' your feet...:-) I am normally size 8 but have wide feet, so initially felt an 8and hal Abu Dhabi yles and shapes of clothes. These pictures are of idealized women with no lines or wrinkles, abundant hair, and slim, straight figures. And this undercuts the whole premise of the book, which is that you don't need to look like a young, slim supermodel in order to be gorgeous. The drawings say otherwise.Whyever didn't the author include some photos, say, of the fabulous 95-year-old Iris Apfel, or Helena Bonham-Carter, or Vivienne Westwood? Here truly is a case of where one picture would be worth 1,000 words - at least.I've solved this problem for myself, though. In the last few years I've collected a number of photographs of beautiful older women, looking like individuals and ageing naturally. Among them are actresses Liv Ullman, Marina Vlady and Anna Orso; Abba singer Agnetha; and 60-plus supermodels Daphne Selfe and Ing-Marie Lamy. Google them yourself, and you'll see why they are such great role-models for us older women.And if I can do it, why couldn't the author?Could she not be bothered? Or was there a problem with copyright? Either way, I felt cheated, and that's why I've give kitesurfing or kiteboarding away from my feet. I was really disappointed with these as I wanted a pair of fitflops for ages and having tried on a different model in the shop I was expecting them to be really comfy. These were really hard under foot and the band across was too tight. The toe post also felt like it would rub if I walked anywhere in them. Sent them back. Amazon return was easy as always. Absolutely great shoes, I live and walk for 20 miles a day in them , they are so comfortable and my legs are now so tight. I recommend them to all, and they can look classy too for evening times. ..arrived on time ,fit perfect ,and so comfy as with all the fit flop range i have bought lots of pairs :) i got these in black so classy on ,a bargain price ,thank you AMAZON I really must stop buying FF's but they are so good and these are no exception. Nice colour and very comfortable. well I don't like it so returning it. 3 reasons: the color is different from photo: darker blu supra vaider nd seller Arrived in good time but was quite disappointed by them! One sock had very tight elastic top n one was slack These socks are a great quality and wash really well. Still going strong and no holes yet. Thank you great. Great socks just as the picture.lasted nearly 2 school terms. Arrived quickly. Great quality and colour. Def recommend I ordered these shoes for my daughter to dress up as Dorothy in, but the 3 was too large. I contacted the seller and returned the shoes by post. They happily exchanged for a smaller size. The shoes themselves are lovely, very sparkly and will look great with the Dorothy dress. Lovely shoes, but big made. We have put them by for future use, so nothing lost. Beautiful and sparkly. Lovely sparkly shoes my daughter has worm them several times and the glitter has stayed on. Lovely shoes. Arrived promptly. A specific request from my 6 yr old goddaughter. Not sure of sizing as Christmas present so won't know until later. However I have bought these for her big sister so they have clearly made an impact. Was really pleased with the shoes and they arrived on time my daughter was delighted with them! Beautiful little shoes thTat my grandda.
best gadgets 6 - 8 and I am size 7.1/2 so they should be a really good fit, but they're too small and won't stay over my heel. They get even worse after washing. I had some much cheaper ones from another company that lasted me a very long time, but these are destined for the dustbin quite shortly. I'll probably only get 2 or 3 uses out of each pair before they get dumped.Disappointing!! I gave them 2 stars as they could have been even worse. I won't buy them again.
Pro:They are ok,Comfortable and they do the jobCons: (for me)The thing is that I bought this socks because they were completely black. I don't like the fact that you can see the big white letters in the backI think that the seller should include a clear picture black pair to really anchor the shoes in place while hitting the dance floor and after a thorough road testing on a long night out with plenty of dancing and walking, i got not discomfort from rubbing or the shape of them around my foot and they didnt damage the shoes at all, the straps stayed in place and are strong and have many years of use ahead of them.i wouldnt hesitate to recommend this item to a friend because they actually WORK 10/10
These came today and they are absolutely great. I have to wear a heel lift in one of my shoes and without straps cannot keep the shoe on when I am walking. I always buy ankle strap shoes but the styles are few and far between as I cannot walk in high heels so the maximum heel height has to be 2.1/2 ins. I can now buy a pair of normal court shoes and put these straps on and fasten it across my foot and keep the shoe in place. I don't want to wear the strap as shown in the picture, just across the instep of my foot so I will have to cut a bit off the length and punc no no pro o tie a bow with. Need to be tied quite tightly to prevent them slipping off the back legs whilst walking.
I must say that I really, really do love these shoes, as silly as they are I LOVE EM :)
I love these more than my cat!
very cool
they don't fit they are the right size but fall of heap of dump
There is no better book that captures the love affair and passion of sneakerheads.
He's a true sneaker head and wa a kid in the candy store when he opened this. Once opened. He couldn't put it down. So much history
Really interesting book showing the EARLY days of the sneaker game, being 38 I remember quite a bit of these shoes worn by my uncles and even som vivienne westwood sale t another fab pair of Flys for me! Sooo comfy and able to wear them straight away. Love the colour combination and bought them in the burgundy and mushroom combo too - gorgeous! Already have these in 3 other colours but with a sore toe these are so good and smart for going out, can't see past them and definitely feel it is money well spent as they last for ages. Always very much admired by all when i wear them - keep up the great work Fly! Until the next time...!!
Ordered these shoes and based on comments opted for the size 4. These were a little big as I am usually size 3.5 but with a heal fitting they are now perfect! They are the comfiest shoes I have ever bought - it is like wearing your favourite pair of slippers.
online shop products getting water logged.Unlike some products that apply a stain resistant coating these do definately waterproof.I can't say how long the coating will last, but I rarely have to re-treat my waterproofs and fleece more than every other month.Excellent product, I liked it so much I bought a 1ltr refill bottle.
I bought these for my son who is in the army over in Afghanistan. His boots where getting really stinky and he said these have helped
My teenage son complained about my shoes!!!!!! These have solved the problem, and they smile at me at 5.30 in the morning before I go to work.
They are O.K. they help with the pong. my bag smells better for them. not great but worth a try.
great at keeping the smells at bay
Purchased for my daughter who is a size 5 but way too big so I kept them for myself, although I'm usually a size 6 but they fit with plenty of room. Haven't worn them as yet but am sure they will be comfortable.
I am happy withle. I bought the size that I normally use in Fitflop, and let them stand for an entire weekend with shoe stretch blockers in before I put them on. It causes them to expand.I wish that FitFlop would make the same model in other materials - eg. crocodile embossed leather.On the negative side, I could do without the red color on the back of the shoe.
Im fairly consistently a size 6 and if I ever need another size, its normally a 6.5 or 7. My other Fitflops are all size 6 but these are huge! I cant walk in them because they fall off my feet. Unfortunately my son destroyed the box so I wasnt able to return them but even with a 1 size up spacer in the toe, they are still too big!
Shoes look great and fit is excellent. Plenty of room inside to move your toes about. and very comfortable as well. I would highly recommend this product. They look great as well. Good price too
Good hArdwearing comfy shoe but the size does come up big so no no pro ble. Sadly they were worn for a extended period and have seen 'action' so can't be replaced.If you get the size right, there is no doubt these are a great shoe. The quality is obvious, the sole is comfortable and the support means they are easily worn for hours on end, just make sure you get the right size !
I've recently bought my second pair of Merrell Intercept trainers. This time I went 1.5 sizes up as their sizing is somewhat er..wrong! I think the conversion from foreign (US?) to UK is wrong.It is an absolutely brilliant shoe though. Real leather with very tough materials inside and on the sole.Thi supra vaider n from these with little shock absorbency.I am hoping once they soften a little the ride will be better.
I bought these for the gym as i am recovering from a snapped Achillies heal and now have a torn cartlidge in my right knee...Read the Reviews and it was a toss up by these or slightly dearer brooks,and went for these,and i am glad i did..They are so comfortable and light and great for the gym..I don't do running but i guess they would be excellent for this too..I am made up with them....
The shoe's upper mesh is breathable and comfortable—I never had any rubbing or developed any hot spots which i had in other branded trainers. The shoe tends to have a foot hugging feel through the heel and mid foot and a slightly wider forefoot. Overall its a very comfortable shoe and will suit those with a wider forefoot.
looking for new trainers to replace my 12 month old Asics GT 1000 and ordered these as they were good value and free delivery - they arrived extremely quickly and are as expected, good fit and great value
I have had a pair of these before and because they were so good and comfortable I ordered another pair and was not disappointed. I would highly recomme.


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P.O. Box 458 110 First Street East Chester MT 59522 406.759.5445 http//www.libertycountylibrary.org
Monday – 11:00 a.m. to 6:00 p.m.
Tuesday thru Thursday – 9:00 a.m. to 4:30 p.m.
Friday – 9:00 a.m. to 3:00 p.m.
Services available at no charge:
11 Public Computers & WIFI
MontanaLibrary2Go for downloadable e-books and audiobooks
Books for all ages as well as magazines, genealogy, Montana collection & reference books
Audiobooks and dvd movies.
Meeting room is available to small groups
Other services:
Printer, Copy and Fax Machine
Interlibrary Loans
Espresso Bar (All proceeds go towards the improvement of the library)
Library Director:
Teresa Fenger
Librarian Assistants:
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Library Board Members:
- Peg Duffin, Chairwoman
- Penny Walston
- Gail Matkin
- Linda Backen
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text:
I am desperately trying to find a way to sort a List of strings, where the strings are predefined identifiers of following form: a1.1, a1.2,..., a1.100, a2.1, a2.2,....,a2.100,...,b1.1, b1.2,.. and so on, which is alread the correct ordering. So each identifier is first ordered by its first character (descending alphabetic order) and within this ordering descending ordered by consecutive numbers. I have tried sortWith by providing a sorting function specifying the above rule for all two consecutive list members.
scala> List("a1.102", "b2.2", "b2.1", "a1.1").sortWith((a: String, b: String) => a.take(1) < b.take(1) && a.drop(1).toDouble < b.drop(1).toDouble)
res2: List[java.lang.String] = List(a1.102, a1.1, b2.2, b2.1)
This is not the ordering I expected. However, by swapping the ordering of the expressions, as
scala> List("a1.102", "b2.2", "b2.1", "a1.1").sortWith((a: String, b: String) => (a.drop(1).toDouble < b.drop(1).toDouble && a.take(1) < b.take(2)))
res3: List[java.lang.String] = List(a1.1, a1.102, b2.1, b2.2)
this indeed gives me (at least for this example) the desired ordering, which I do not understand neither.
I would be so thankful, if somebody could give me a hint what exactly is going on there and how I can sort lists as I wish (with a more complex boolean expression than only comparing < or >). A further question: The strings I am sorting (in my example) are actually keys from a HashMap m. Will any solution effect sorting m by its keys within
m.toSeq.sortWith((a: (String, String), b: (String, String)) => a._1.drop(1).toDouble < b._1.drop(1).toDouble && a._1.take(1) < b._1.take(1))
Many thanks in advanced!


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Frameworks Software
Hot topics in Frameworks Softwareocr character recognition using matlab handwriting recognition handwriting recognition code handwriting ocr java handwriting recognition source code hex editor intelligent character recognition face recognition using backpropagation neural network artificial intelligence projects in java
Allows editing of any size file in hexadecimal, decimal and ASCII. A great tool that allows you to edit and analyze the contents for either the data or resource fork of any type of file. 68K, PPC and Carbon run on MacOS 7-9 and X.920 weekly downloads
Neuroph OCR - Handwriting Recognition
Neuroph OCR - Handwriting Recognition is developed to recognize hand written letter and characters. It's engine derived's from the Java Neural Network Framework - Neuroph and as such it can be used as a standalone project or a Neuroph plug in.122 weekly downloads
Nord Modular Editor
This project develops an open source editor for Clavia Nord Modular and Micro Modular synthesizers.48 weekly downloads
XLL4J is a framework for developing excel addins in Java without the need to compile any C++ code.15 weekly downloads
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BioAPI Java
A reference implementation of BioAPI2.0 is being developed.10 weekly downloads
Android Bangla Or Bengali Font Render
This is to help developer to render complex bangla or bengali font for android.6 weekly downloads
Xml Data Objects: A framework to declaratively parse XML17 weekly downloads
xdoclet and Prj Extensions for NetBeans
NbXDoclet for NetBeans project helps to develop applications based on xdoclet. It contains templates for Hibernate. This project also implements Project Extensions (natures).6 weekly downloads
CArtAgO is a framework for programming and executing virtual environments in multi-agent programs.9 weekly downloads
A Java Distributed Shared Memory Middleware8 weekly downloads
help you monitor file or directory's modifications. Simply use in java application. this implementation is multi-thread safe.8 weekly downloads
JME SOAP Server
SOAP-Server framework for Java Micro Edition (JME). Use this framework to run simple web services on mobile devices, e.g. PDAs. Supports SOAP 1.2 (without attachments). Runs on MIDP2.0/CLDC1.1 or CDC compatible VMs with sufficient memory.7 weekly downloads
Concurrent Building Block
Project provides a set of concurrent building blocks (Java & C/C++) that can be used to develop parallel/multi-threaded applications. Components are grouped into 4 categories: 1.Data Structures 2. Parallel Patterns 3.Parallel functions 4.Atomics and STM6 weekly downloads
Java Utilities
A set of common reusable code that I use, manage and maintain. It is generalized and uses alot of extensible interface based API's leaving expansion to the basic concepts in these libraries almost limitless.6 weekly downloads
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A Java class library that supports building 3D applications. Wraps the functionality of Direct3D.6 weekly downloads
Richfaces Maven Project Eclipse Plug-in
Eclipse plug-in for Richfaces Web project with maven support. Follow the instructions in the Readme.txt. The plug-in & maven archetype are also available at:- http://code.google.com/p/richfaces-maven-plugin/ . More at http://www.w3avatar.com3 weekly downloads
TCR Neuroph -Text Character Recognition
TCR Neuroph - Text Character Recognition is java tool developed to recognize scanned text , using Java Neural Network Framework - Neuroph5 weekly downloads
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Measurement science-based performance & conformance testing framework1 weekly downloads
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The Eigenbase Project is an open-source platform for building advanced data management systems, and is backed by a non-profit organization which designs, develops, and promotes it.4 weekly downloads
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Although many research efforts have been devoted to network congestion in the face of an increase in the Internet traffic, there is little discussion on performance improvements for endhosts. We propose a new architecture, called scalable socket buffer tuning (SSBT), to provide high-performance and fair service for many TCP connections at Internet endhosts. SSBT has two major features. One is to reduce the number of memory accesses at the sender host by using some new system calls, called simple memory-copy reduction (SMR) scheme. The other is equation-based automatic TCP buffer tuning (E-ATBT), where the sender host estimates 'expected' throughput of the TCP connections through a simple mathematical equation, and assigns a send socket buffer to them according to the estimated throughput. If the socket buffer is short, the max-min fairness policy is used. We confirm the effectiveness of our proposed algorithm through both a simulation technique and an experimental system. From the experimental results, we have found that our SSBT can achieve up to a 30% gain for Web server throughput, and a fair and effective usage of the sender socket buffer can be achieved.
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Is polyandry less common than multiple wives?
View Single Post
Senior Member
Join Date: Dec 2011
Posts: 712
I am bisexual. Back in the day, I thought it would be lovely to have a couple. My experience back then was with men who had way higher sex drives then I did. I thought if I shared a man with a woman, it would work out to match my drive better, and I wouldn't have to be the only one satisfying him. Although I probably imagined her a dynamo, because I would have enjoyed sex with her too.
A hundred years later, here I am in a vee with two men. One of them is slightly long distance; and both of them have much lower sex drives than I do.
But I love them both beyond reason. I fell in love with both of them after being friends with both of them. (long story can be found elsewhere, life stories and blogs)
I think that's why I wasn't in poly before. I wasn't seeking out a configuration, but these two men showed up and I was ready.
Love is that condition in which the happiness of another person is essential to your own...
Robert A. Heinlein
Me: female, bi, (formerly hinge of a vee)
with FirstBoyFriend (FBF)(moderately long-distance)
and no longer with CurrentBoyFriend (CBF)(who now lives in a house far away-with stairs I can't climb)
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Nokia 7610
- Nice design, good screen, fun software, great camera
- Unconventional keypad design; no shielding for camera
Bottom Line
A nice design, good software and a great camera make this a very good pick if you're seeking a phone with a bit more but don't want to carry around a full PDA.
Price$ 669.00 (AUD)
Best Deals (Selling at 1 store)
The Nokia 7610 is a nice looking phone, with unconventional curves and a white and grey styling that will appeal to many phone connoisseurs. It has a very prominent screen, capable of 176 x 208 display and very passable audio quality.
The number pad is a little compact for our liking, with buttons that appear to be more designed for style than function. They're not square, but thin and curved, which takes some getting used to. The directional keypad, used for navigating through Symbian OS 7.0s Series 60 operating system, was a little stiff, and we constantly found the cursor going in unwanted directions.
The OS itself is colourful and fun, packed with features and designed for usability. We've always found that getting stuff done in Symbian OS was fast, easy and intuitive, and the 7610 is no exception. It has a media player that supports, among other things, MP3 audio and MPEG-4 video, a calendar, contacts, games, XHTML browser and messaging and email. Symbian OS also supports Java. The accompanying PC synching software, Nokia PC Suite, is also quite decent.
The phone only has 8MB of internal memory, but also comes with a 64MB Reduced Size MMC--which can be upgraded if you need extra storage space. Given the high quality of the camera in the phone, you might very well need it.
Perhaps the 7610's biggest appeal is its camera. No more 640 x 480 shots with this phone. The camera in the 7610 is capable of 1 megapixel shots, resolutions up to 1152 x 864, making it comparable with the Sony Ericsson S700 and other advanced camera phones. They were decent quality shots, too, and there was no delay when taking photos (although there is a rather nasty delay when saving photos). They weren't equivalent in quality to shots from a real camera, of course, but good nonetheless. The phone, fortunately, will automatically resize photos that are larger than 100KB in order to squeeze them down to the maximum size supported by MMS (Multimedia Messaging Service). It also has a night camera mode and even some rudimentary software for combining captured video clips for sending over MMS.
In some aspects of its design, Nokia has favoured form over function, but overall the 7610 is a fun phone, with a great camera, good software and USB and Bluetooth connectivity.
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Monday, July 04, 2016
Spiced Rhubarb Crumble
This makes a pretty small crumble so go ahead and double the amounts if you wish. It has quite unusual flavors, but is really delicious. Serve it with vanilla ice cream!
Spiced Rhubarb Crumble
250 g rhubarb, cut into small pieces
2 tbsp sugar
1 tsp cinnamon
1/2 tsp ginger
zest from 1 orange
For the crumble:
150 ml oats
75 g sugar
125 ml flour
3 tbsp golden syrup
handful of crushed walnuts
0,5 tsp baking powder
0,5 tsp flaky sea salt
75 g butter
Bake at 200°C for about 20-25 minutes.
Saturday, July 02, 2016
Cookbook watch: Sweden junior master chef
When I had kids, I really wanted them to be into food. The kind of kids who'd gladly eat everything and enjoy cooking. Are we there? Not quite. But maybe, some day...
Thursday, June 30, 2016
Spiced meatballs with broadbeans, roasted carrots and lemon-chili yogurt
This is loosely based on a recipe from the Jerusalem cookbook, although I skipped a lot of the suggested ingredients and adapted it to my own liking. And, I'm afraid, I didn't measure the spices. Just add a LITTLE bit. The cookbook called for a mix called baharat that I can't find here, so I used most of the spices included instead. Just a little will do.
I love that cookbook though, by the oh-so-talented Yotam Ottolenghi and Sami Tamimi. I highly recommend it. And I highly recommend this dinner. We'll be making it again, even though the kids wrinkled their noses and proceeded to eat rice and soy sauce. (Well, Titus had one meatball, and I think he rather liked it, too.)
Spiced meatballs with Broad Beans
for the meatballs:
500 g ground beef
1 egg
100 g breadcrumbs
1 onion, finely chopped
2 garlic cloves, minced
3 tbsp fresh coriander, minced
black pepper
for the broad beans
6 garlic cloves, sliced
6 spring onions, sliced into 2 cm pieces
250 g broad beans, blanched and shelled (or bought ready-shelled, frozen - that's what I did)
500 ml chicken stock
Juice of one lemon
salt, black pepper
Mix everything together, and roll into walnut-sized meatballs. Fry in batches, in plenty of olive oil until browned on each side, for a few minutes. They don't need to cook through. Set aside. Clean the pan.
In the same pan, now that it's clean, heat some more olive oil. Fry the garlic and spring onions for a few minutes. Add the broad beans, and about 100 ml of chicken stock, and most of the lemon juice but not all of it. Add salt and pepper and let it cook for a few minutes. Then place all of the meatballs back in the pan, add the rest of the stock, cover with a lid and let this cook for about 10-15 minutes. If it feels too runny, remove the lid and let some of the juices reduce. When ready to serve, season with the rest of the lemon juice, and perhaps a little bit more salt or pepper.
Roasted carrots
4 carrots
olive oil
Cut the carrots into batons - I usually cut them into about 10-cm lengths, and halve or quarter those, depending on thickness. Drizzle with plenty of oil and roast in a 225°C oven (lower if you have a convection oven - mine broke!) for about 25 minutes. Make sure they don't burn but do let them roast properly.
Lemon Chili Yogurt
1 tbsp fresh coriander, finely chopped
zest from 1 lemon
1 tsp lemon juice
1/2 tsp sambal oelek or similar chili paste
3 tbsp thick yogurt, Greek or Turkish
1-2 tbsp wate
Stir together everything but the water. Add that gradually, stirring to get the thickness you want.
Serve everything with rice, or bread if you prefer. We also added crumbled feta cheese on top.
Friday, June 24, 2016
Happy Midsummer!
Monday, June 13, 2016
Amazing halloumi toast
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