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Conceived and designed the experiments: YR JDV NK SN. Performed the experiments: YR OM MDP BC CG MW. Analyzed the data: YR OM MDP CG FP BdM JDV NK SN. Contributed reagents/materials/analysis tools: MW FP HK. Wrote the paper: YR SN.
Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice.
We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.
Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis.
Spermatogenesis is a complex biological process that involves the proliferation and differentiation of diploid spermatogonia into mature haploid spermatozoa. This process is divided into three sequential phases: first, the mitotic proliferation of spermatogonia gives rise to primary spermatocytes. These then undergo two meiotic divisions to generate haploid spermatids. The final phase, spermiogenesis, involves the morphological metamorphosis of early round spermatids into mature spermatozoa. As might be expected, the production of mature sperm requires a highly specialized control program of gene expression at both the transcriptional and post-transcriptional levels [1].
Small RNA molecules are important regulators of mRNA transcription, stability, turnover, processing, storage and translation (for review see [2]). These small RNAs can be classified into different categories based on their biogenesis, mechanism of action and function, and include microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs) and piwi-interacting RNAs (piRNAs). There is increasing evidence that small RNA-directed gene silencing pathways are essential for normal spermatogenesis [3], [4]. Not only have a large set of miRNAs [5], [6] and piRNAs [7], [8], [9] been identified in male germ cells, but all essential members of the RNA interference (RNAi) machinery, including DROSHA, DICER1, AGO2 (EIF2C2), PIWIL1(MIWI), PIWIL4 (MIWI2) and PIWIL2 (MILI) to name but a few, are expressed in meiotic and post-meiotic germ cells [10], [11].
DICER1 is a conserved RNAse III endonuclease that is essential for the processing of several classes of small RNAs, including miRNAs and endo-siRNAs, as well as for the degradation of toxic transposable elements [12], [13]. DICER1 has been found in nearly all organisms, and multiple studies have shown its essential role in animal development [14], [15], [16], [17], [18], [19], [20]. The functional relevance of DICER1 and miRNAs in spermatogenesis is only just beginning to be unravelled. Selective ablation of Dicer1 in Sertoli cells leads to infertility, due to the complete absence of spermatozoa. More precisely, progressive testicular degeneration results from the defective maturation of Sertoli cells and their incapacity to properly support meiosis and spermiogenesis [21], [22]. However, the role of DICER1 in the male germ cell lineage in general, and specifically during spermatogenesis, is not as clear. Two groups have used Cre recombinase-expressing mice, TNAP-Cre (Tissue Non-Specific Alkaline Phosphatase) in an attempt to generate animals in which Dicer1 is absent in germ cells [23], [24]. Hayashi and colleagues found that germ cell-specific deletion of Dicer1 causes a defect in proliferation of male gonocytes and late adult infertility, likely due to spermatogenic arrest [23]. Maatouk and colleagues found that these mutant mice were subfertile, due to defects in both sperm motility and the transition from round to elongating spermatids [24]. Importantly, these results should be interpreted with caution as the TNAP-Cre transgenic mouse is neither fully penetrant (only ~50% of germ cells express Cre), nor specific to germ cells. It should also be emphasized that the expression of TNAP-Cre begins as early as E10 [25], so that the primary effects appear in the primordial germ cell (PGC) population. It is therefore difficult to precisely interpret the mechanism and timing of any spermatogenesis defects occurring in the adult.
To investigate the precise roles of germ-cell DICER1 during spermatogenesis and to overcome the above-mentioned caveats of previous studies, we developed a mouse model in which the Dicer1 gene was inactivated in a specific and fully penetrant manner in the male germ cell lineage. Our results indicate that Dicer1 is required for normal spermatogenesis, since the deletion of Dicer1 in male germ cells led to multiple defects in meiosis and spermiogenesis resulting in the absence of functional spermatozoa and complete infertility.
To investigate the functional relevance of DICER1 in spermatogenesis, we disrupted Dicer1 in the male germ line by combining a conditional Dicer1 allele (Dcr1fx) [15] with a Ddx4(MVH or Vasa) promoter-driven transgenic Cre line [26]. This Ddx4-Cre transgene has been reported to induce specific Cre-mediated recombination in >90% and >97% of spermatogonia by embryonic day (E)18 and postnatal day (P)3, respectively [26]. When Ddx4-Cre; Dcr1fx/wt males were mated with Dcr1fx/fx females, we observed that only ~11% of the offspring were Ddx4-Cre;Dcr1fx/fx mutant males, instead of the expected ratio of 25% (for details see Table S1). This discrepancy from the Mendelian ratio is a consequence of early recombination events at the 1- or 2-cell stage, as previously described [26], leading to global embryonic Cre-mediated deletion of Dicer1 and lethality around E7.5 [15], [27]. In the remaining viable Ddx4-Cre;Dcr1fx/fx males, Dcr1 exon 24, which encodes most of the second RNAse III domain [15], was efficiently and specifically deleted in the germ cell lineage: using elutriated fractions containing spermatocytes isolated from P60 control and mutant Ddx4-Cre;Dcr1fx/fx mice, we found that levels of Dcr1 transcripts that contained exon 24 were reduced by ~93%; a proportion corresponding to the purity of the spermatocyte fractions (Fig. 1A, B). Furthermore, qRT-PCR showed complete loss of miR-34c and miR-184, two miRNAs expressed specifically in spermatocytes/spermatids ([28], and H. Kaessmann, personal communication; Fig. 1C). Together, these data show a complete loss of Dicer1 and miRNA biogenesis in the male germ-cell lineage.
Ddx4-Cre;Dcr1fx/fx males were viable and grew normally displaying normal external genitalia, when compared to control littermates. At P60, testes in which Dicer1 was depleted in germ cells showed a reduction in size (compare Fig. 2A and B with C) and a 55% decrease in weight compared to control Ddx4-Cre;Dcr1fx/wt and Dcr1fx/fx littermates (107±7 mg versus 238±9 mg and 196±11 mg respectively, for details see Table S2 and Fig. 2G). Internal reproductive organs (i.e. seminal vesicles) were normally developed and, importantly, plasma testosterone levels were unchanged between experimental and control groups (Table S2). While control Ddx4-Cre;Dcr1fx/wt and Dcr1fx/fx males were fertile, Ddx4-Cre;Dcr1fx/fx mutant males were found to be sterile, although they were sexually active and produced vaginal plugs in female partners (data not shown). Histological analysis and sperm count in mutant individuals revealed that sperm production was drastically altered in the mutant testis. Indeed, mature spermatozoa were rarely found in the tubule's lumen due to a near complete absence of elongated spermatids (Fig. 2F). As a consequence, epididymal ducts were almost completely devoid of mature spermatozoa, whereas exfoliated germ cells were found in the lumen of mutant epididymides (data not shown). Finally, epididymal sperm count analysis revealed a ~99% decrease in the number of spermatozoa in Ddx4-Cre;Dcr1fx/fx males compared to control Ddx4-Cre;Dcr1fx/wt and Dcr1fx/fx littermates (0.141±0.041 *106/ml versus 8.39±0.5 *106/ml and 10.58±1.175 *106/ml respectively; Fig. 2H and Table S2).
Adult seminiferous tubules contain several types of germ cells; each cell type is layered within the tubule in a centripetal manner and germ cells are arranged in typical cellular associations within the seminiferous tubules known as stages of the seminiferous epithelium cycle. In mice, a spermatogenetic cellular association is divided into 12 stages (from I to XII; [29]). An in depth analysis of the mutant seminiferous epithelium histology and cytology revealed multiple defects. First, the relative proportions of the different populations of germ cells are clearly unbalanced (Fig. 2M, N and O). Using propidium iodide incorporation followed by flow cytometry analysis, we found that the proportion of haploid (1n) cells was reduced by ~30%, while tetraploid (4n) cells showed a 2.5-fold increase in Ddx4-Cre;Dcr1fx/fx males (Fig. 2I). This result corroborates our analysis of the mutant testis histology, in which almost no elongated spermatids were found (Fig. 2N). Secondly, vacuoles (Fig. 2M, arrows) were numerous in the mutant epithelium, likely due to germ cell sloughing, as were the Sertoli cell cytoplasmic extensions in the lumen (Fig. 2M, asterisk). As a possible consequence of these multiple cellular defects, we observed a reduction in the small diameter of mutant seminiferous tubules when compared to Dcr1fx/fx littermates (123±2 µm versus 167±8 µm, Table S2). Another important characteristic of mutant seminiferous tubules was the disturbance of the synchronization of germ cell associations (Fig. 2O). For instance, we observed leptotene spermatocytes (Fig. 2O, black arrowheads), which are ordinarily found in stage IX–X tubules, associated with round spermatids (Fig. 2O, red arrowhead), normally found in stage I–VIII tubules. These observations prevented the clear identification of specific tubule stages in Dicer1 mutant testes. Interestingly, although global germ cell organization was completely disturbed, Sertoli cell nuclear organization and number appeared normal (Fig. S1; Table S1).
The significant reduction of post-meiotic germ cells (Fig. 2I) in mutant seminiferous tubules suggested that germ cell survival may be affected in the absence of DICER1. A TUNEL assay revealed that apoptosis was 8-fold higher in mutant testes compared to control seminiferous tubules (115±8 apoptotic cells/20 tubules versus 15±1 apoptotic cells/20 tubules and Fig. 2T). Interestingly, apoptotic cells were not distributed evenly but usually localized in specific seminiferous tubules (Fig. 2Q and S). Histological analyses involving double immunostaining for TUNEL and a subset of germ cell markers (γH2AX (phosphorylated H2AX), H3K9me3 (tri-methylated Lysine9 Histone 3), alpha tubulin or pH 3 (phosphorylated-Ser10 Histone 3)), suggested that apoptotic cells mainly comprise pachytene spermatocytes (Fig. S2 and data not shown). Furthermore, aging mutant testes, at P180, showed high apoptotic cell numbers with extensive tubular defects, although Sertoli Cell Only (SCO) tubules were not observed (Fig. 3D and H). In short, our data indicate that depletion of DICER1 RNAse activity in the germinal compartment of the testis leads to significant defects in spermatogenesis, affecting both the survival and differentiation of male post-mitotic germ cells.
To further characterize the events that lead to infertility in Ddx4-Cre;Dcr1fx/fx mutant mice, we compared the development of control and mutant testes during the early phases of the first spermatogenic wave, from P5 to P21. No discernible abnormality was found in mutant testes during the mitotic phase of spermatogonia A (P5), when spermatogonia B enter meiosis I (P10) (Fig. S3), or when spermatocytes are in the zygotene/pachytene transition (P12) (Fig. 3A and E). The first histological abnormalities began to appear at P15 with the presence of germ cell sloughing and Sertoli cell cytoplasmic extensions (Fig. 3B and F). At this stage, apoptosis also increased by ~2.5-fold in mutant testes in comparison with control individuals (41±1 apoptotic cells/20 tubules versus 16±3 apoptotic cells/20 tubules respectively, Fig. 3J). By P21, in wild-type testis, meiosis I is complete and round spermatids begin to differentiate, which was not the case is in mutant testis. A difference in testis weight also became apparent (23.75±3.1 mg versus 51.11±6 mg respectively, Fig. 3I), which reflects seminiferous tubule deterioration and increased apoptosis (21±3 apoptotic cells/20tubules versus 8±2 apoptotic cells/20 tubules, Fig. 3J).
In order to better characterize the apoptotic cell population present within the developing tubules, we performed electronic microscopy (EM) of control and mutant testes at the end of prophase I (P21; Fig. 4A, B, C, D). In Dicer1-depleted germ cell individuals, most of the pachytene spermatocytes showed apoptotic features (e.g. misshapen nuclear membrane and fragmented heterochromatin, arrows and arrowheads in Fig. 4D, respectively), and those that survived prophase I rarely reached the metaphase step. In addition, metaphase plates appeared abnormal (data not shown). Zygotene (not shown) and pachytene spermatocyte nuclei in Ddx4-Cre;Dcr1fx/fx mutant tubules seemed to contain abnormally large perinuclear heterochromatin areas (arrowheads in Fig. 4B and C). We used ser-10 phosphorylation of histone 3 as a specific marker of cells in metaphase (i.e. spermatogonia and late meiotic cells; [11]) to compare mutant individuals to control littermates at P60. This revealed an increased number of cells in meiotic metaphase in the tubules of the mutant group (Fig. S4). These findings indicate that spermatocytes may be partially blocked in the late prophase I phase in Ddx4-Cre; Dcr1fx/fx mice.
Since pachytene spermatocytes seemed to be affected by the deletion of Dicer1, we analysed the expression of γH2AX at P12, during the zygotene to pachytene transition. During the normal zygotene phase, γH2AX localizes to the whole nucleus, but in the subsequent pachytene stage remains only in the XY body (or sex body) where X and Y-linked genes undergo transcriptional silencing [30]. At this stage, we found that the number of pachytene spermatocyte-containing tubules was reduced by ~2-fold (3.88±0.4 versus 8.16±0.1 XY body-positive tubules/20 tubules respectively; Fig. 4G) in mutants compared to control littermates (arrowheads in Fig. 4E and F). Chromosomal spread preparations from germ cells collected at P12 confirmed the reduced number of pachytene spermatocytes in mutant testes (Fig. 4 H, I, J), whereas, pre-leptotene and leptotene cells were more numerous compared to controls. No difference in the meiotic process was observed, such as synapsis (SYCP3 staining) or repair (DMC1 punctuate staining). Taken together, these data show that the loss of Dicer1 in germ cells severely impairs the first spermatogenic wave, by delaying the transition from the leptotene to the zygotene/pachytene stages of the first meiotic prophase, and by increasing apoptosis in mid/late pachytene stage spermatocytes.
To characterize the morphological defects in the developing germ cells after meiosis, we performed an EM study on testis sections at P60. The few spermatids found in Ddx4-Cre;Dcr1fx/fx mutant mice displayed various defects; the acrosome of round spermatids was fragmented in Ddx4-Cre;Dcr1fx/fx with multiple acrosomal granules tethering the nucleus (Fig. 5C, arrowheads) instead of a single granule found in control testes (Fig. 5 A, arrowhead). Interestingly, we found no apparent defects in the overall morphology and position of the chromatoid body, a cloud-like structure thought to be the site of RNA processing and/or storage (white arrows in Fig. 5A and B). The few remaining elongated spermatids of mutant animals showed abnormal head shape and chromatin condensation (red arrows in Fig. 5E and F). Acrosomes were also misshapen and fragmented (compare arrowheads in Fig. 5 E and F with D). Finally, mitochondria displayed abnormal shape and dilated cristae or intermembrane space, which appeared as a dense structure, compared to control spermatids (blue arrows, Fig. 5 D, E, F).
Since round and elongated spermatids showed significant defects in acrosome formation and nuclear condensation we analysed further the morphology of the remaining epididymal sperm (Fig. 2 H). All of the few spermatozoon-like structures found in the epididymides of P60 mutant individuals displayed drastic morphological defects of the head and flagellum (Fig. 5G, H, I, J). Using a dye specifically staining mitochondria, we evidenced a heterogeneous and aberrant localisation of these organelles in the mutant spermatozoa, with mitochondria surrounding the nucleus (arrows in Fig. 5L and M) or mislocalized within the flagella (arrowheads in Fig. 5M). In addition to the misshapen but normally-sized spermatozoa present in the mutant sperm, we were able to see what resembled small pin-head spermatozoa (Fig. 5N; α-tubulin red staining). Nevertheless, these structures were devoid of nuclei and harbour a faint and sparse mitochondrial staining. None of the mutant spermatozoa were motile when examined by visual inspection with a light microscope (data not shown). Our results indicate that functional DICER1 is required for normal spermiogenesis, since its ablation in germ cells leads to a reduced number of haploid germ cells and extensive defects in global sperm morphology.
In addition to its essential role in miRNA biogenesis, DICER1 has been implicated in the control of repetitive elements. The recent discovery that DICER1 is critical for cell survival by degrading toxic retrotransposon transcripts in retinal pigment epithelium [12] raised the possibility that DICER1 might fulfil the same function in the germ cell lineage. To investigate this possibility, we examined the expression levels of IAP (intracisternal A particle element), LINE1 (long interspersed nuclear element 1) and SINE B1 and B2 (short interspersed nuclear element B1 and B2) by quantitative RT-PCR in two groups of P60 elutriated germ cells (Fig. 6). While transcript levels for transposable elements LINE1 and IAP were unaffected, we observed a significant up-regulation of SINE B1 and SINE B2 transcripts in enriched populations of leptotene/zygotene/mid-pachytene spermatocytes (1.8-fold increase for SINE B1 and 1.5- fold for SINE B2) and late-pachytene/diplotene spermatocytes (1.8-fold increase for SINE B1 and 1.6- fold for SINE B2) prepared from mutant testes. Whether the ~1.5–2 fold increase in SINE transcripts affect germ cell survival and/or spermatogenesis remains an open question.
DICER1 is essential for the processing of several classes of small non-coding RNAs, including miRNAs and endo-siRNAs, and for the degradation of retrotransposon transcripts. In this study we used the Cre/Lox system to conditionally inactivate Dicer1 in male germ cells. We found that DICER1 is not required for spermatogonia stem cell renewal and mitotic proliferation, but is essential for the meiotic and haploid phases of spermatogenesis. The first defects appeared as early as P12 when we observed a delayed progression of prophase I, followed at P15 by an increase in apoptosis of mid/late pachytene stage spermatocytes. Not all spermatocytes died by apoptosis, as a small fraction of these completed meiosis. The transition from round spermatids to mature spermatozoa was also severely affected since the few spermatozoa that did form in mutant animals (at 1% of control levels) were immobile and misshapen, exhibiting irreversible morphological defects due to disturbance in acrosome formation, nuclear condensation in the few remaining round and elongated spermatids.
The loss of Dicer1 in male germ cells thus led to a complex and severe phenotype, which likely represents the sum of low impact defects that accumulate at various meiotic and post-meiotic stages. This type of phenotype, resulting from the cumulative impairment of several developmental cellular processes and increased cell death, has in fact been observed in numerous conditional Dicer1 knockouts [15], [16], [17], [31]. Conditional removal of Dicer1 in the male germ cell lineage led to various phenotypes whose severity depends on the efficiency of Cre recombination and/or the stage at which DICER1 has been inactivated. Initial studies using a Tnap-Cre transgene has been problematic due to early inactivation (E10) as well as the partial (50%) and non-specific Cre-mediated expression [25]. As a result of inefficient Cre recombination, Tnap-Cre;Dicerfx/fx mice were found either fertile or subfertile and it was a rather difficult task to evaluate precisely the impact of DICER1 on sperm production [23], [24]. The use of a specific, fully penetrant Cre line such as the Ddx4-Cre transgene was instrumental to investigate in depth the function of DICER1 in spermatogenesis. Interestingly, ablation of DICER1 in germ cells at different development stages using two different spermatogonia-specific Cre mice (the Ddx4-Cre and Ngn3-Cre) revealed a gradation in severity of the reproductive phenotype. Ddx4-Cre and Ngn3-Cre transgenes are both efficient at deleting DICER1 in gonocytes at ~P0 and in spermatogonia at ~P5–P7, respectively (Korhonenet al, co-submitted manuscript). However, germ cell-specific removal of DICER1 using a Ngn3-Cre transgenic resulted in a phenotype slightly milder than the Ddx4-Cre;Dicer1fx/fx described here. Both mutant males were infertile and exhibited similar defects in spermato- and spermiogenesis but the meiotic phenotype was more severe and precocious in Ddx4-Cre;Dicer1fx/fx animals with defects in prophase I progression and a lower number of remaining spermatozoa. Since small RNAs are stable molecules, we hypothesize that ablation of DICER1 in gonocytes at ~P0 will deplete the pool of the remaining miRNAs or endo-siRNAs more extensively than in P5–P7 spermatogonia thus affecting spermatogenesis and sperm production more severely.
There is no doubt that DICER1 is essential for spermatogenesis and sperm production. However, emerging from these findings is the essential question of which DICER1 activity is crucial for germ cell function. In other words, the relative contribution of miRNAs versus other DICER1-dependent non-coding RNAs to the phenotype remains unclear. DICER1 is essential for both endo-siRNA and canonical miRNA biogenesis, but not for piRNA production (for a review see [2]). Recent expression profiling analyses using RNA sequencing technology revealed that male spermatogenic cells expressed numerous classes of small non-coding RNAs including miRNAs and endo-siRNAs [5], [6]. Whether the infertility phenotype observed in our Ddx4-Cre;Dcr1fx/fx is consecutive either to the loss of endo-siRNAs and/or miRNAs is still uncertain. While revising this manuscript, it has just been reported that germ cell inactivation of Dicer1 or Drosha, an RNase III enzyme required for canonical miRNA biogenesis but not for endo-siRNA processing, both resulted in male infertility due to oligozoopsermia or azoospermia caused by constant depletion of phachytene spermatocytes and spermatids [6]. The similar phenotype observed in Drosha knockout testes when compared with the Dicer mutants suggests a role for miRNAs in regulating spermatogenesis. These results are different to those found in female germ cells, where both miRNAs and endo-siRNAs are also present in the developing oocytes [32], [33]. While mouse oocytes lacking Dicer1 arrest in meiosis I due to disorganized spindles and defects in chromosomal alignment [34], [35], oocytes deficient for Dgcr8, an RNA-binding protein that assists DROSHA in the processing of microRNAs, mature normally and do not impair early embryonic development [36]. This striking difference in phenotype between Dicer1- and Drosha-deficient oocytes suggests that endo-siRNAs, rather than miRNAs, are essential for oocyte maturation and the early stages of mammalian development [6]. Another important difference between male and female germ cells is the timing of defects. In oocytes lacking Dicer1 a specific defect is observed at metaphase, affecting either the spindle assembly checkpoint (SAC) and/or the anaphase-promoting complex (APC/C). Indeed, it was demonstrated that DICER1 is not required for female germ cell development but only for meiotic maturation [37]. Our results clearly show that unlike oocytes, defects in spermatogenesis appear earlier during prophase I.
The chromatoid body (CB) is a dynamic, cloud-like, dense structure located in the cytoplasm of round spermatids, and is thought to be the site of RNA processing and/or storage [38]. This cytoplasmic perinuclear organelle contains various types of RNAs, including piRNAs, mRNAs and miRNAs as well as various RNA-binding proteins or proteins involved in RNA processing pathway (e.g. DDX4, PIWIL1, PIWIL2, DICER1, tudor domain proteins such as TDRD6 and TDRD7; [39]). The potential role of the CB in the mRNA regulation [38], together with the multiple cumulative meiotic and post-meiotic defects found in Dicer1-depleted germ cells, suggested the hypothesis that the CB structure and/or its function may be affected in Dicer1-depleted germ cells. EM studies of testes at P60 revealed that the CB was present, and normally shaped in round spermatids lacking Dicer1, this suggests that DICER1 is not essential for the formation of the CB. Whether its molecular composition and/or its function in RNA regulation is affected in mutant germ cells still remains unclear.
The mouse and human genomes are crowded with transposable elements that account for 30 to 40% of their total size [40]. Different silencing mechanisms have been developed in germ cells to suppress their activities, including DNA methylation [41] and RNAi-triggered silencing (for a review see [42]). The large set of piRNAs expressed in spermatocytes highlights the importance of these small RNAs in protecting male germ cells from transposable elements [43]. While piRNA biogenesis requires neither DROSHA nor DICER1, there is substantial evidence that regulation of transposable element expression includes DICER1-dependent mechanisms [44], [45], [46]. Several retrotransposons from the MT and SINE families were shown to be significantly up-regulated in mouse oocytes lacking Dicer1 [34]. Recently, DICER1 was demonstrated to be involved in retrotransposon transcript degradation. Dicer1 ablation in retinal pigmented epithelium (RPE) induced accumulation of alu/SINE RNAs in both human and mouse cells, resulting in cytotoxicity and RPE degeneration [12]. Our genetic analysis suggests that the SINE family of retrotransposons is up-regulated in spermatocytes lacking Dicer1. Whether this 1.5- to 2-fold increase in SINE expression is sufficient to affect survival and/or specific stages of spermatogenesis remains unclear and requires further analysis. In this perspective, high throughput RNA sequencing combined with an in-depth differential proteomic analysis of Dicer1- and Dgcr8-depleted germ cells would be instrumental for a better comprehension of the roles and impact of small non-coding RNAs in the production of mature sperm.
Dcr1flox (Dcr1fx) and Ddx4:Cre (Mvh-Cre) mice were kindly provided by B.D. Harfe and D.H. Castrillon respectively, and were genotyped as described [15], [26]. To achieve selective inactivation of Dicer1 in germ cells, we mated transgenic male mice expressing Cre recombinase under the control of the Ddx4 promoter with female mice carrying two floxed Dicer1 alleles in order to generate 50% Ddx4-Cre;Dcr1fx/wt and 50% Dcr1fx/wt mice. Ddx4-Cre;Dcr1fx/wt males were mated with Dcr1fx/fx females in order to produce Ddx4-Cre;Dcr1fx/fx as well as Dcr1fx/fx and Ddx4-Cre;Dcr1fx/wt control littermates. The genetic background of these mice is mixed C57Bl/6J and SV129. Protocols for the use of animals were approved by the commission d'Ethique de l'Expérimentation Animale of the University of Geneva Medical School and the Geneva Veterinarian Office.
Male germ cells were obtained from P60 adult mice (n=3, each pool of germ cells was isolated from either four pairs of Ddx4-Cre;Dcr1fx/fx mutant testes or two pairs of Dcr1fx/fx control testes) by mechanical disruption and liberase treatment (Roche Applied Science). Cells were elutriated and separated into multiple fractions according to [47] and subsequently frozen for further analysis.
Total RNAs from elutriated germ cells were extracted using Trizol (Sigma-Aldrich) according to the manufacturer's instructions. For further quantification of repetitive elements associated RNAs, extracted RNA was subjected to DNAse treatment using the Turbofree DNA kit (Ambion). Quantification of transcript levels was performed using the Kapa Sybr Fast kit (Kapa Biosystems) with primers for Dicer1 already described in [48], SINE B1, B2 and LINE L1 RNA primers described in [49] and IAP primers sequences came from [50]. Taqman miRNA kit (Applied Biosystems) was used for quantification of miRNAs according to the manufacturer's instructions using primers for miR-34c and miR-184 (for probe sequences see Applied Biosystems website). Data were analyzed using the 2−ΔΔCt method as described in [51]; normalization was performed with 18s rRNA levels for repetitive elements and mRNA transcripts, whereas U6 small RNA was used for miRNA level normalization. Each assay was performed at least in three independent technical and biological replicates.
Blood was collected by cardiac puncture from P60 mice. Plasma samples were stored at-20°C and used subsequently to assess the levels of LH, FSH and testosterone. Hormone levels were measured in individual adult plasma samples (n=3–9) from P60 adult mice. LH was measured by RIA, using a commercially available kit supplied by IDS (LH RIA CT # AHR002, Liège, Belgium), whereas FSH was measured by IRMA, using a kit from the same supplier (FSH IRMA CT # AHR004). Testosterone concentrations were assayed using a kit from MP Biomedicals (Testo DA kit, CT number 07-189102, Eschwege, Germany). Intra- and inter-assay coefficients of variation (CVs) of all three assays were less than 5% and 10%, respectively.
Epididymal sperm count was performed with sperm extracted from the caudal epididymis and ductus deferens of adult (P60) male mice and was analyzed for its concentration as previously described [52]. Ploidy was assessed on Ddx4-Cre;Dcr1fx/fx and Dcr1fx/fx P60 testis using Propidium Iodide incorporation as described in [53]. Epididymal sperm from Ddx4-Cre;Dcr1fx/fx and Dcr1fx/fx was spread on SuperFrost Plus glass slides, fixed 10 minutes at room temperature (RT) with 4% paraformaldehyde (PFA), then permeabilized in PBS/triton-X100 0.5% and incubated for 1 hour at RT with anti-alpha-Tubulin (Abcam clone B-5-1-2, 1500). For fluorescent staining an Alexa594 secondary antibody (Invitrogen) was used for signal revelation; nuclei were counterstained using DAPI and Mitotracker (1 µM, Invitrogen) was used for mitochondrial staining.
Tissues were fixed overnight either in 4% PFA or in Bouin's fixative and embedded in paraffin. Five-µm sections were stained with haematoxylin and eosin (H&E) or processed for immunohistochemistry (IHC). For IHC analysis, PFA-fixed sections were incubated overnight at 4°C with the following antibodies: anti-GATA4 (Santa Cruz Biotechnology, sc-9053, 1500), anti-pH 3(Ser10) (Millipore, Cat#06-570, 1500), anti-γH2AX (Calbiochem, Cat#dr-1017, 1500) and anti-H3K9me3 (Millipore, Cat#07-523, 1500). For fluorescent staining, Alexa-conjugated secondary antibodies (Invitrogen) were used for signal revelation and sections were counterstained using DAPI. All images were obtained either with a Zeiss Axioscope microscope and processed using the AxioVision LE software.
Apoptotic assays were performed by TdTmediated X-dUTP nicked labeling (TUNEL) reaction using the Apoptag kit (Millipore) stained with either DAB chromogen and counterstained with eosin, or with Permanent Red or anti-Digoxygenin coupled to fluorescein and counterstained with DAPI. The percentage of apoptotic, TUNEL positive cells within seminiferous tubules was expressed as the average number of apoptotic cells within 20 seminiferous tubes. A minimum of 100 seminiferous tubules were counted per testis (5 sections/testis) and at least 3 animals per genotype per age were assessed.
Spermatocytes nuclear spreads were prepared and stained as previously described [54]. The primary antibodies used were: guinea pig anti-SYCP3 (gift from C. Heyting, 125 000), mouse anti-γH2AX (Upstate, 05-636, 125 000) and rabbit anti-DMC1 (Santa Cruz Biotechnology, H100, 1200). Secondary antibodies were AlexaFluor-488 and AlexaFluor594 conjugates (Molecular Probes).
Digital images were obtained by using a cooled CCD camera, Coolsnap HQ (Photometrics), coupled to a Leica DMRA2 microscope using the same exposure time for all acquisitions. Each colour signal was acquired as a black-and-white image using appropriate filter sets and was merged with Photoshop Imaging software.
P60 and P21 testes from Ddx4-Cre;Dcr1fx/fx and Dcr1fx/fx individuals were fixed in 0.1 M Na Cacodylate with 4% Glutaraldehyde in PBS. After fixation with 1% osmium tetroxide, the testes were embedded in epoxy resin (Glycidether 100, Merck). Selected areas were sectioned, stained with 5% uranyl acetate and 5% lead citrate, and visualized on a JEOL 1200 EX transmission electron microscope.
Sertoli cells organization and number are not affected in testes lacking Dicer1 in the germinal compartment. Gata-4 immunostaining (red) revealed the presence of Sertoli cells nuclei at the edge of tubules in Dcr1fx/fx (+/+) control individuals (A) as well as in Ddx4-Cre;Dcr1fx/fx mutant mice (−/−) (B), indicating that Sertoli cells organization is not affected by Dicer1 depletion in germ cells. (C) The number of Sertoli cells within seminiferous tubules is also unaffected (n=3 animals per genotype; a minimum of 20 tubules were analyzed per individual).
Apoptotic cell population display pachytene spermatocytes localisation in Ddx4-Cre;Dcr1fx/fx (−/−) tubules. At P60, TUNEL-positive cells (green) are found near late prophase I spermatocytes in Dicer1 mutant as shown by γH2AX staining of the XY body (red foci, white arrowheads in A and B). C and D show the localization of prophase I cells at the edge of the tubule (arrows) and the XY body in round spermatids (red foci, arrowheads) according the H3K9me3 immunostaining. TUNEL-positive cells localize between those two populations. E and F show the presence of few elongating spermatids stained using anti-alpha-tubulin antibody (red) which are reduced and disorganized (asterisks) in Dicer1-depleted germ cells tubules. Cross-sections were counterstained with DAPI.
The early spermatogenic phase is not affected in Ddx4-Cre;Dcr1fx/fx (−/−) individuals. H&E staining of P5 (A&B) and P10 (C&D) cross-sections from control (A&C) and −/− (B&D) seminiferous tubules. At these stages, no histological differences were observed between mutant and control testes.
Accumulation of metaphasic-like cell population in Ddx4-Cre;Dcr1 fx/fx (−/−)tubules. At P60, undergoing metaphasic cells are more abundant in Dicer1 mutant as shown by Histone H3Ser-10 phosphorylated (pH 3) positive cells (green, A and B). (C) shows a 2.5-fold increase of pH 3 positive tubules in −/− versus +/+ individuals. Cross-sections were counterstained with DAPI. Results are mean±SEM, *p<0.05, versus controls.
Frequency of Ddx4-Cre;Dcr1fx/fx obtained is lower than expected. Ddx4-Cre;Dcr1 fx/wt males were mated with Dcr1fx/fx females in order to produce Ddx4-Cre;Dcr1fx/fx (−/−) as well as Dcr1fx/fx (+/+) and Ddx4-Cre;Dcr1fx/wt (+/−) control littermates. The genetic background of these mice is mixed. The expected Mendelian ratio should be 25% for each genotype. Here, we obtained ~11% of Ddx4-Cre;Dcr1fx/fx, probably due to an early recombination event in some mutant embryos resulting in embryonic death.
Table comparing reproductive and endocrine measurements between Ddx4-Cre;Dcr1fx/fx (−/−) individuals compared to Dcr1fx/fx (+/+) and Ddx4-Cre;Dcr1fx/wt (+/−) control littermates.
We would like to thank Chantal Combépine, Nicolas Veillard and Mara Rodrigues for excellent technical assistance, Junior Reynoird for histology advice and Pierre Calvel for critical reading of the manuscript. We are grateful to D. Castrillon for the Ddx4:Cre transgenic mice and B.D. Harfe and M.T. McManus for the Dcr1 fx mice.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This project is supported by grant no. 3100A0-119862 to SN from the Swiss National Science Foundation. CG and BdM are supported by grants from the Centre National de la Recherche Scientifique and the Agence Nationale de la Recherche (ANR-09-BLAN-0269-01). NK and OM are supported by the Academy of Finland, Emil Aaltonen Foundation and Turku Graduate School of Biomedical Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
A paradise in Yosemite area! it is in the middle of the mountain, very quiet and very well equiped. The attention of the owner is excellent. We stayed there 2 nights and was awesome. Though it is not inside the park (have to travel 30 min, by car) it is a great place to stay!! We went June 2011
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com and Priceline so you can book your Bear Creek Cabins reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Bear Creek Cabins Hotel Mariposa

subset: commoncrawl


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text:
Iron Maiden Announce US tour
first date is CHARLOTTE NC holy fuck. and Alice Cooper! hope the tix aren't too expensive
Looks like I'm seeing them at Verizon again
And Coheed yes
hm.... not sure if i'd rather see coheed or alice
Fucking fuckerson....Come to St. Louis for once you dumb Brits.
Ending the tour in town while I will be in Minnesota. Fuckers.
jeez, these dudes can just sit back and chill and maybe tour 3 or 4 months out of the year and the rest of their lives are just gravy, guess they busted their ass for it i guess.
Sweet. Been wanting to see Iron Maiden for a bit; though given that it's Jones Beach the prices are probably going to start pretty high.
Coheed doesn't join until after Indianapolis. Noooooo
Prudential center july 2nd. wish it was coheed
Kinda annoyed that the hyperlink didn't work :/
"Coheed doesn't join until after Indianapolis. Noooooo"
The fuck is wrong with you? You're getting Alice fookin' Cooper!!
Also why are they touring? They aren't putting out a new album are they?
^^ they got millions of fans that would pay an arm and a leg to see them play 30 year old songs, they don't need a new album to tour.
"^^ they got millions of fans that would pay an arm and a leg to see them play 30 year old songs, they don't need a new album to tour."
This. not to mention they put on an insanely good show live.
I thoroughly recommend anyone to see them live, even in their 50s they are as good as ever.
They usually have great stage sets and stuff too.
Dammit, all of the east coast dates aren't with Coheed. I'd definitely go for Maiden and Alice, but seeing Coheed with them would have been sick
Digging: Relient K - Air For Free
sweet I still have yet to see these guys live, totally going
if they weren't playing at the worst venue in town, i'd go for sure.
Wooo Charlotte, NC as the first date. I'll go depending on how expensive the tickets may be. Thank god I won't have to see them with Coheed if I go.
They should do a world tour.
shitfuck doublepost
nice vancouver
i really should go, might be the last time i get a chance
Digging: Bones - PaidProgramming2
Rick Santorum must be opening
Damn, no minnesota
No Florida, boo! Guess I'll be shooting for a trip to Jones Beach.
Come back to Ireland! D:
August 1st!
• Michael Gira Accused of Rape• R.I.P. Lemmy• R.I.P. David Bowie• Scott Weiland dead at 48 • The Story So Far's vocalist dropk..• Rush drummer retires• Killswitch Engage debut new single• Brand new Brand New Single• Deftones stream new single• Riverside guitarist dies
• Coroner Enter Studio• Alcest unveil Kodama• First Joy Opposites (ex-FACT) • New Father John Misty single• 40 Watt Sun Album Announced• Russian Circles stream 3rd single• Bones releases PaidProgramming2• New Shadows (ex-FACT) Song• Disillusion Tease New Single• New A Day To Remember Song• New Alter Bridge Single• Nails on hiatus• Manson announces new album• Départe Unleash New Track• Dir en Grey Keep It Weird» see all news
Bands: A B C D E F G H I J K L M N O P Q R S T U V W X Y Z


text:
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text: On Sun, Feb 13, 2011 at 3:25 PM, Andrew Dalke <dalke@...> wrote: > On Feb 12, 2011, at 6:55 PM, Egon Willighagen wrote: >> In general I try to stick as close to what the file has to give, >> unless I know for sure how the format documentation expects reader to >> get more information (like the SMILES specification expecting readers >> to perceive aromaticity...) > > Really? I pushed hard to make sure the OpenSMILES specification does > not require aromaticity perception, but I haven't reviewed the entire > spec for a while. I said SMILES, not OpenSMILES :) > I believe this is sufficient to allow people to write simplified > SMILES readers which only preserve input aromaticity. Indeed. It works until people start complaining... :/ BTW, the CDK SMILES parser allows this now, I think... Egon -- Dr E.L. Willighagen Postdoctoral Researcher Institutet för miljömedicin Karolinska Institutet Homepage: http://egonw.github.com/ LinkedIn: http://se.linkedin.com/in/egonw Blog: http://chem-bla-ics.blogspot.com/ PubList: http://www.citeulike.org/user/egonw/tag/papers


text:
Article Tools
Recent crimes and emergencies
- Flood warning issued for Lackawanna, Luzerne counties until 11:15 p.m.
- Police: Man tried to flee drug arrest
- Case of teen accused of stabbing woman to death moves toward trial
- Scranton man arrested under suspicion of dealing drugs
- Two charged in city crack deal
- Pizza delivery man charged with lewdness, related charges
- Carbondale man facing child sex charges
Recent Abingtons News
- Duffy's goes mobile with brew trailer
- Pizza delivery man charged with lewdness, related charges
- Man accused of breaking into Clarks Summit home, assaulting woman
- First Yields: Waverly Community Garden begins harvesting produce
- Dalton man arrested for theft
- Correction - Abington Heights will save $700G
- Board approves transportation contract
Man admits to corrupting minors
SCRANTON - A Clarks Summit man admitted Tuesday to providing cigarettes to teenage girls and speaking with them about sexual topics, an assistant district attorney said.
Jennifer McCambridge said 47-year-old Stephen J. Cardamone faces up to seven years in prison after pleading guilty on Tuesday to one count of corruption of minors and two counts of harassment.
Mr. Cardamone initially faced additional felony charges on accusations he performed a sex act in front of a teen girl and had another teen girl perform a sex act in 2007.
Ms. McCambridge said a sentencing date hasd not yet been set. Mr. Cardamone remains free on $100,000 bail.


text:
Tea Towel Tuesday: Take 15% off 1+ yards of Linen Cotton Canvas Ultra Through 11:59 PM EDT July 26th.
Preview scale: 21.0 x 18.0 inches

Change DPI Base size is 150
Collection: Random
snake (757),
snakes (401),
serpent (236),
year of the snake (123)
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to add a comment.
modgeek says:
Thanks, Rhonda for your wonderful comment!
Posted over 3 years ago.
rhondadesigns says:
There is a marvelous feeling of vitality in this terrific sea of sinuous kraits!
modgeek says:
Thanks, Grace!
gracedesign says:
cool snakes!
modgeek says:
Thanks, chickoteria and zaira! So glad people like this one.
zapi says:
<3 love this!
chickoteria says:
woo...HOOO...out loud LOVE this...would love to see this in rayon...good luck in the contest!
modgeek says:
Thanks, Tara!
taramcgowan says:
This is a very fun design! I love the colors! -Tara
modgeek says:
Thanks, Maria, Raven, and Su for the comments! Su, I do not like snakes at all, but too think this print would be great for clothing. It could be pretty and terrifying at the same time.
su_g says:
This totally works! Pops all over! They are indeed really neat looking snakes, the pattern looks great in yardage and I find this a totally convincing fabric - I can imagine it used in both casual clothing and formal blouses, for instance.
ravenous says:
Ooo I like this!
mariafaithgarcia says:
Nice job!!!
© 2008-2015 Spoonflower Inc


text: Food is not bad, but not that great either. Average at best, but you do get a lot of food for the price. Pizzas are large in size, but can sometimes taste like cardboard. Sandwiches are a good size. The fries (with cheese) are really good here, but outside of the fries, everything else is either just okay, or a... More


text:
A Scene From Coachella 2015
After hologram Tupac, you know it's only a matter of time before this is what going to Coachella will look like.
Image Attached is FWD's weekly cartoon series.
Leslie Wood is a cartoonist. Perhaps you would like to follow her on Twitter?
- Hillary Clinton has officially accepted the Democratic party's presidential nomination with some fiery words for Donald Trump 🇺🇸
- A federal court struck down North Carolina's voting restrictions, ruling they intentionally made it harder for black people to vote.
- Four people in Florida are likely the first to contract the Zika virus from mosquitos in the US, the state's governor says.


text:
If you want a horse that is friendly and loaded with personality (not to mention cow!) then you need a foal out of then here is your guy: MRI Snips Spirit…$8,000
What a nice filly bred to work for you. Dam is blue roan retired rope horse. Our horses are raised in big pastures in eastern Montana. They know how to…SOLD
Well broke paint mare, very flashy. Easy to ride, neck reins, crosses water, moved cows, packed out game, easy to catch, been in the bob marshal, loads…SOLD
UPDATE!~Gypsi gave us a gorgeous black filly with a huge heart on her forehead, May 15th...from Greenbriar's Tequila Sunrise. Gypsi is a great trail horse…SOLD
Copper is a racing & rodeo bred gelding. His dam was a Montana HS Champion calf horse. He has Boon Dox John, Sun Frost, Shy Ole Man, The Ole Man & Quincy…SOLD
Iggy is a nice bridle horse who has traveled a lot of miles on a ranch. He is an all-around cow horse. He has been ridden all day in the mountains to rope…SOLD
This fabulous mare is an ABRA World Show Top 5 Winner in 2011 & 2014. She is an AQHA World Show Qualifier in 2012 & 2014. AQHA points 16.5. AQHA Amateur…SOLD
One of our best out of one of our best mares - a black mare of ideal conformation. This filly has many greats on her pedigree. Sire is blue roan. She…SOLD
Bayard, NE


text:
Hearing aids in 75214, Dallas, TX
We found 16 hearing clinics with 32 reviews within 10.4 miles of 75214, Dallas, TX.
Featured hearing professionals near you
Jill Copley, AuD, CCC-A, FAAA
Dr. Copley earned her Doctor of Audiology Degree from the University of Florida at Gainesville and (read more)
Laura Gentile, AuD, CCC-A
Laura earned her undergraduate degree from Stephen F. Austin State University, her Master's (read more)
Hearing Wellness Clinic @ Texas Institute for Sinus and Respiratory Disease Center
Heena Peshwani, AuD, FAAA
Dr. Heena Peshwani, Au.D is a licensed Audiologist. She earned her Bachelor's degree (read more)
Laurie Even, AuD, CCC-A, FAAA
Dr. Laurie Even received a Bachelor of Science in Communication Sciences and Disorders from The (read more)
Stephen Miller, AuD
Dr. Stephen E. Miller has his doctorate in Audiology and has been in private practice for (read more)
Johanna Whitson, AuD, CCC-A, FAAA
Johanna earned her Master of Science in Audiology from the University of Washington and her (read more)
Jeffrey Martin, PhD, CCC-A
Jeffrey is the Head of Audiology and a Clinical Assistant Professor at Callier Center. He earned his (read more)
Susan Deahl, MCD
Susan Deahl, MCD, practices at the Medical City Hospital in Dallas. She specializes in audiology (read more)
Jennifer Horak, AuD, CCC-A, FAAA
Dr. Jennifer Horak is a licensed audiologist and has been in practice since 1996. She earned her (read more)
Laura Trainor-Collins, AuD, CCC-A
Laura Trainor-Collins is a licensed audiologist in the state of Texas. She provides (read more)
Audiology Center of Garland - Office of James B. Maddox MD PA
Laura Douglas, M.S., CCC-A
Laura Douglas is a clinical audiologist with over 21 years of experience. Born and raised in the (read more)
Clay Mainord, Au.D., CCC-A
Dr. Mainord is both owner and Doctor of Audiology at Calvert Hearing Care. He earned his Bachelor of (read more)
Additional professionals near you
Hearing Diagnostic Center
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Dallas Ear Institute
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text:
John Snyder: HELLO
Opening Friday: Over the past quarter-century, artist John Snyder has dipped deep into American folk painting, pre-Renaissance Italian frescoes, arts-and-crafts porcelain, Hudson River landscapes, Buddhist cave painting and, most recently, African and Oceania imagery. From this heady stew of images and ideas he has concocted vast tableaux of allegorical figures caught up in enigmatic stories. Now settled near Decorah, Iowa, Snyder returns to Minneapolis for his first show in a decade, including woodcuts, drawings and paintings on new themes and moral conundrums. Nothing is simple or straightforward in his elaborate pictures, but their narrative power is always arresting. (Opening 6-9 p.m. Fri., free. Ends April 6. Noon-5 p.m. Wed.-Sat., Bockley Gallery, 2123 W. 21st St., Mpls., just west of Lake of the Isles near W. Franklin Av. 612-377-4669 or www.bockleygallery.com)
Mary Abbe
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Comments will be reviewed before being published.
- Hacked emails complicate Democratic National Convention
- Be wary of Trump's long list of promises
- Hillary Clinton looks to close trust deficit at convention
- Milone on the mound as Twins look to take series in Boston
- What happens to suburbs if Southwest light rail isn't built?
- Could the Mississippi River actually begin in South Dakota?


text:
Tea Towel Tuesday: Take 15% off 1+ yards of Linen Cotton Canvas Ultra Through 11:59 PM EDT July 26th.
Preview scale: 42.0 x 36.0 inches

Change DPI Base size is 150
Collection: A moonlit moth fandango
animals (20126),
stripe (9330),
butterfly (5907),
dot (3816),
moon (2715),
coordinates (1115),
spot (1112),
moth (572)
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joonmoon says:
Beautiful collection!!!
Posted over 4 years ago.
coggon_(roz_robinson) says:
moonlit moth fandango on cotton sateen
coggon_(roz_robinson) uploaded a fabric image for
coggon_(roz_robinson)'s design
A moonlit moth fandango :
coggon_(roz_robinson) says:
cotton sateen
coggon_(roz_robinson) says:
moon phase spots on cotton sateen
coggon_(roz_robinson) says:
Thank you pink, thank you Isabella!
inscribed_here says:
What a magical collection. The moon phase spots are inspired. Fantastic work!
pinksodapop says:

coggon_(roz_robinson) says:
Thanks Sheila, I'm so glad you like them - I did have to cheat with the lyrics of the song a little, but i think sky and moon are better than floor and face!
sheila's_corner says:
Great song reference... I love the moon dots! The stripes are great!!! Great everything!
coggon_(roz_robinson) says:
Thanks for your lovely comment, and congratulations on the arrival of luna! I have two daughters (2 years and 8 weeks old!) named after constellations, so really wanted to do something with stars in, but then this song got in my head and these patterns happened! I will order test swatches soon, and then i'll put them for sale!
s_lukjanovs says:
I love these so much! I have a new daughter named Luna, so I need these fabrics for sure. I hope you win, but if not, I hope you make them for sale! Great job, so lovely.
coggon_(roz_robinson) says:
a more detailed view!
coggon_(roz_robinson) says:
Thanks Rhonda - I have had the words stuck in my head all week, and the designs just seemed to come from that! Suggestions of how to work the theme into a quilt are very welcome. Happy New Year :-)
rhondadesigns says:
You have a lovely variety of patterns that are linked by the common theme! This would be a wonderful set of fabrics to use in a special quilt, especially of you could link the reference to the "Whiter Shade of Pale" into the design! (I just know that the song will be on replay in my head now!)
© 2008-2015 Spoonflower Inc


text:
Dovre Group
- 500 - 1,000 Employees
- Lansi-Suomen laani (fi), Vastra Finlands lan (sv), Finland
- 1977
- http://careerslip.com/dovre-group
Welcome to Dovre Group Career Portal
Dovre Group is a global provider of project management services. Dovre Group has two business areas - Project Personnel and Consulting - and employs over 500 people worldwide. Dovre Group is listed...
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Resume Reserve Vault (RRV)
At Dovre Group, we recruit from time to time from talents (that have shown interest in working with us) stored in our Resume Reserve Vault. If we do not have a vacancy that interests you but would like an opportunity to join us, please submit your resume for future job considerations.Click here to Submit your Resume to our Reserve Vault
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Creative SoundBlaster Audigy LS
In recent years sound has taken off, and Creative has always been at the forefront, offering quality options. The SoundBlaster name is one that people the world over recognise. I decided to upgrade my old SoundBlaster 16 that shipped with my PC, with the Audigy LS, a mid-range soundcard with some of the latest features. I'm not the most technical user, so this upgrade was very much a lesson in basic PC upgrading on the hardware side as well. The upgrade was performed twice - once in Windows98 and then in Windows XP, just for the sake of process comparison. The differences were surprising.
So what does the Audigy LS deliver? Well, if you start browsing the range of soundcards out there, you'll soon discover that there are many different trademarks and functions, making it a veritable minefield of choice. So let's start with the important parts: this soundcard costs £60 at the time of writing, and delivers Dolby 5.1 Digital Surround Sound, full 24-bit. For that price, you'll want some performance out of it. Our version was also bundled with Hitman 2, if that is an incentive to buy. So lets get down to the detail - how easy was the upgrade?
The supplied instructions and drivers support Windows 98 upwards. Following the instructions to the letter, the card was installed in Win98. Soon into the installation there was a problem, and a subsequent restart. The system we were installing in to had a TAD connection to the modem - during the change, the modem set up managed to get changed. Not a significant problem, but unexpected, and typical of W98. When installing the card in Windows XP there were no problems at all and it was smooth and easy. The supplied CD provided all the drivers and software.
The supplied software includes Creative Media Source player - another media player that will do all the things that the others will. It seems that everybody wants you to use their player. You also get the EAX console, which provides you with a range of audio features that some might find useful, and some not. EAX enhances the sound in many newer games, providing a more exciting 5.1 output. The EAX console can allow you to alter the audio as you like - bass boost, the ubiqutous graphic equaliser, and audio clean-up function, to remove noise from MP3s of old recordings, 'SVM' which will match the sound levels across music tracks and the EAX effects which gives a range of advanced audio effects, like reverb and so on.
This card is very much aimed at the media user who wants 5.1 sound, and listens to a stack of MP3s on their PC and intends on doing a hardcore amount of gaming. At the price, you need to have a decent set of speakers to support it and take advantage of the 5.1 - especially the advanced functions. There are a range of higher spec cards for those wanting 6.1 or 7.1 sound, but then you really do need suitable speakers, or you're just wasting your money on sound you'll never hear. Luckily there are plenty of cheaper cards on the market, and its best to select what you need, by what you want to do. Overall, I was impressed with what the card can do, despite the hiccups in Windows 98 installation. To alleviate sub-Windows XP issues it's best to check Creative's site for the latest drivers much like a graphics card.
Harman Kardon Go + Play review: Post-retro design, powerful audio
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- Harman Kardon Go + Play review: Post-retro design, powerful audio
- Plex now lets you play music on any Sonos device from the Sonos app
- Audio-Technica ATH-MSR7NC headphones review: Make some noise
- Deezer launches in the US: How does the music-streaming service work?
- Zemi Aria speaker review: Majors and minors
- Magnetic Bluetooth 4.1 Wireless Sport headphones deliver 5 hours of clear music and podcasts (37 per cent off)
- Cambridge Audio SE1 earphones give you Beryllium drivers for under £100, profits go to charity
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- Apple's 2016 iPhone could be called iPhone 6SE, not iPhone 7


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i never thought it would be possible to love someone as much as i love you...
Learn more at imjust-a-girl.tumblr.com
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my dream would be to get this to 1 million notes by the 1 year... (m.k.)
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crazy thing about dating... True story


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Zebra, deatil og back
Credit: Henrik Sorensen
Creative #: 140929986
License type: Rights-managedRights-managed products are licensed with restrictions on usage, such as limitations on size, placement, duration of use and geographic distribution. You will be asked to submit information concerning your intended use of the product, which will determine the scope of usage rights granted.
Collection: Iconica
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Zebra Deatil Og Back Stock PhotoAnimal Wildlife,Animals In Captivity,Black Background,Black Color,Color Image,Denmark,Design,Horizontal,Natural Pattern,No People,One Animal,Part Of,Photography,Side View,Striped,Studio Shot,White Color,ZebraPhotographer Henrik SorensenCollection: Iconica Photographer Henrik Sorensen - CopehagenDetail of zebras back, side view


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Welcome to Urban Photo Fragments. Here I shoot photos in the urban environment up close. My photography is fragments of the urban environment. Little glimpses into what is around us.
These photos make for interesting pieces that looks great as prints, wall art and much more. Be sure to check out my collection.


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Bella BriteAdditional Locations
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A picture of one of the male lions at the MGM in Las Vegas Nevada.
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|By Adine Deford
Article Rating:
|February 6, 2014 03:00 PM EST
AppZero, the fastest way to move enterprise applications, today announced the results of its first "State of Readiness for Windows Server 2003 End of Support (EOS)" survey. The 2013 results of more than 200 Fortune 1,000 companies show that 40% of respondents wish to move to the cloud as part of their EOS remediation efforts.
The full results of the survey are available here and indicate that with eighteen months until support ends, the majority of respondents still have significant numbers of machines running WS2003. Security compliance and vulnerability management are a major concern and many organizations are not aware of or prepared to deal with the impending challenge.
Microsoft has announced that End of Support for Windows Server 2003 is July 14, 2015 and recommends AppZero for moving a variety of applications from Windows Server 2003. Industry experts estimate that there are more than 12 million machines still running WS2003 that are at issue. Especially for those in regulated industries - pharmaceutical, banking, insurance, oil and gas - this will cause a heavy compliance burden.
"AppZero is the only technology that can "up-level" production Windows applications onto a newer operating system," said AppZero CEO, Greg O'Connor. "We conducted the survey to size the problem because the choice people make in remediating this issue can affect their datacenter strategy for the foreseeable future. We felt that a majority would want to move to the cloud and modernize in one step."
The survey provides insight into the pain points shared among the respondents and reveals clarity into the upgrade path the majority intends to follow. At the conclusion of the findings, AppZero shares recommendations that can be followed to protect from the risk of running an unsupported operating system.
According to new research by GigaOm, "While perhaps lacking the drama of Y2K mediation at the end of the last millennium, the Windows Server 2003 event touches on all of the same issues, including large mitigation expenses and soft risk factors."
"As with Y2K, the Windows Server 2003 end-of-support event is significant, but with proper planning can be managed smoothly, eliminating disruption. Careful consideration of the costs and risks involved will enable IT personnel to choose the mitigation methods most appropriate for the organization."
AppZero is the fastest, most flexible way to move server applications to and across any cloud or datacenter, without code change or lock-in. Encapsulating Windows applications in VM/OS-free packages, AppZero's patented software moves complex server applications with ease. AppZero allows you to modernize your infrastructure, moving from an old OS to a newer one with the click of a button - modernize and move to the cloud in one step. For more information, visit www.appzero.com and follow us on twitter @AppZero_Inc.
If you are within a stones throw of the DevOps marketplace you have undoubtably noticed the growing trend in Microservices. Whether you have been staying up to date with the latest articles and blogs or you just read the definition for the first time, these 5 Microservices Resources You Need In Your Life will guide you through the ins and outs of Microservices in today's world.
Jul. 30, 2016 04:30 AM EDT Reads: 4,178
Before becoming a developer, I was in the high school band. I played several brass instruments - including French horn and cornet - as well as keyboards in the jazz stage band. A musician and a nerd, what can I say? I even dabbled in writing music for the band. Okay, mostly I wrote arrangements of pop music, so the band could keep the crowd entertained during Friday night football games. What struck me then was that, to write parts for all the instruments - brass, woodwind, percussion, even k...
Jul. 30, 2016 01:45 AM EDT Reads: 2,380
This digest provides an overview of good resources that are well worth reading. We'll be updating this page as new content becomes available, so I suggest you bookmark it. Also, expect more digests to come on different topics that make all of our IT-hearts go boom!
Jul. 30, 2016 12:45 AM EDT Reads: 3,783
Keeping pace with advancements in software delivery processes and tooling is taxing even for the most proficient organizations. Point tools, platforms, open source and the increasing adoption of private and public cloud services requires strong engineering rigor – all in the face of developer demands to use the tools of choice. As Agile has settled in as a mainstream practice, now DevOps has emerged as the next wave to improve software delivery speed and output. To make DevOps work, organization...
Jul. 30, 2016 12:30 AM EDT Reads: 2,344
SYS-CON Events announced today that Isomorphic Software will exhibit at DevOps Summit at 19th International Cloud Expo, which will take place on November 1–3, 2016, at the Santa Clara Convention Center in Santa Clara, CA. Isomorphic Software provides the SmartClient HTML5/AJAX platform, the most advanced technology for building rich, cutting-edge enterprise web applications for desktop and mobile. SmartClient combines the productivity and performance of traditional desktop software with the simp...
Jul. 29, 2016 10:00 PM EDT Reads: 1,292
Internet of @ThingsExpo, taking place November 1-3, 2016, at the Santa Clara Convention Center in Santa Clara, CA, is co-located with the 19th International Cloud Expo and will feature technical sessions from a rock star conference faculty and the leading industry players in the world and ThingsExpo Silicon Valley Call for Papers is now open.
Jul. 29, 2016 08:00 PM EDT Reads: 2,749
There's a lot of things we do to improve the performance of web and mobile applications. We use caching. We use compression. We offload security (SSL and TLS) to a proxy with greater compute capacity. We apply image optimization and minification to content. We do all that because performance is king. Failure to perform can be, for many businesses, equivalent to an outage with increased abandonment rates and angry customers taking to the Internet to express their extreme displeasure.
Jul. 29, 2016 03:45 PM EDT Reads: 1,689
Right off the bat, Newman advises that we should "think of microservices as a specific approach for SOA in the same way that XP or Scrum are specific approaches for Agile Software development". These analogies are very interesting because my expectation was that microservices is a pattern. So I might infer that microservices is a set of process techniques as opposed to an architectural approach. Yet in the book, Newman clearly includes some elements of concept model and architecture as well as p...
Jul. 29, 2016 02:45 PM EDT Reads: 9,831
Ovum, a leading technology analyst firm, has published an in-depth report, Ovum Decision Matrix: Selecting a DevOps Release Management Solution, 2016–17. The report focuses on the automation aspects of DevOps, Release Management and compares solutions from the leading vendors.
Jul. 29, 2016 12:00 PM EDT Reads: 1,843
Adding public cloud resources to an existing application can be a daunting process. The tools that you currently use to manage the software and hardware outside the cloud aren't always the best tools to efficiently grow into the cloud. All of the major configuration management tools have cloud orchestration plugins that can be leveraged, but there are also cloud-native tools that can dramatically improve the efficiency of managing your application lifecycle. In his session at 18th Cloud Expo, ...
Jul. 29, 2016 11:30 AM EDT Reads: 1,306
SYS-CON Events announced today that LeaseWeb USA, a cloud Infrastructure-as-a-Service (IaaS) provider, will exhibit at the 19th International Cloud Expo, which will take place on November 1–3, 2016, at the Santa Clara Convention Center in Santa Clara, CA. LeaseWeb is one of the world's largest hosting brands. The company helps customers define, develop and deploy IT infrastructure tailored to their exact business needs, by combining various kinds cloud solutions.
Jul. 29, 2016 11:15 AM EDT Reads: 1,353
SYS-CON Events announced today that Venafi, the Immune System for the Internet™ and the leading provider of Next Generation Trust Protection, will exhibit at @DevOpsSummit at 19th International Cloud Expo, which will take place on November 1–3, 2016, at the Santa Clara Convention Center in Santa Clara, CA. Venafi is the Immune System for the Internet™ that protects the foundation of all cybersecurity – cryptographic keys and digital certificates – so they can't be misused by bad guys in attacks...
Jul. 29, 2016 09:45 AM EDT Reads: 1,493
No matter how well-built your applications are, countless issues can cause performance problems, putting the platforms they are running on under scrutiny. If you've moved to Node.js to power your applications, you may be at risk of these issues calling your choice into question. How do you identify vulnerabilities and mitigate risk to take the focus off troubleshooting the technology and back where it belongs, on innovation? There is no doubt that Node.js is one of today's leading platforms of ...
Jul. 29, 2016 08:30 AM EDT Reads: 667
Let's just nip the conflation of these terms in the bud, shall we?
"MIcro" is big these days. Both microservices and microsegmentation are having and will continue to have an impact on data center architecture, but not necessarily for the same reasons. There's a growing trend in which folks - particularly those with a network background - conflate the two and use them to mean the same thing.
They are not.
One is about the application. The other, the network. T...
Jul. 29, 2016 04:45 AM EDT Reads: 3,733
DevOps at Cloud Expo – being held November 1-3, 2016, at the Santa Clara Convention Center in Santa Clara, CA – announces that its Call for Papers is open. Born out of proven success in agile development, cloud computing, and process automation, DevOps is a macro trend you cannot afford to miss. From showcase success stories from early adopters and web-scale businesses, DevOps is expanding to organizations of all sizes, including the world's largest enterprises – and delivering real results. Am...
Jul. 29, 2016 04:45 AM EDT Reads: 2,381
This is a no-hype, pragmatic post about why I think you should consider architecting your next project the way SOA and/or microservices suggest. No matter if it's a greenfield approach or if you're in dire need of refactoring. Please note: considering still keeps open the option of not taking that approach. After reading this, you will have a better idea about whether building multiple small components instead of a single, large component makes sense for your project. This post assumes that you...
Jul. 29, 2016 04:15 AM EDT Reads: 4,289
The 19th International Cloud Expo has announced that its Call for Papers is open. Cloud Expo, to be held November 1-3, 2016, at the Santa Clara Convention Center in Santa Clara, CA, brings together Cloud Computing, Big Data, Internet of Things, DevOps, Digital Transformation, Microservices and WebRTC to one location. With cloud computing driving a higher percentage of enterprise IT budgets every year, it becomes increasingly important to plant your flag in this fast-expanding business opportuni...
Jul. 29, 2016 04:15 AM EDT Reads: 2,700
In his session at @DevOpsSummit at 19th Cloud Expo, Yoseph Reuveni, Director of Software Engineering at Jet.com, will discuss Jet.com's journey into containerizing Microsoft-based technologies like C# and F# into Docker. He will talk about lessons learned and challenges faced, the Mono framework tryout and how they deployed everything into Azure cloud. Yoseph Reuveni is a technology leader with unique experience developing and running high throughput (over 1M tps) distributed systems with extre...
Jul. 28, 2016 10:15 PM EDT Reads: 2,256
Sharding has become a popular means of achieving scalability in application architectures in which read/write data separation is not only possible, but desirable to achieve new heights of concurrency. The premise is that by splitting up read and write duties, it is possible to get better overall performance at the cost of a slight delay in consistency. That is, it takes a bit of time to replicate changes initiated by a "write" to the read-only master database. It's eventually consistent, and it'...
Jul. 28, 2016 08:30 PM EDT Reads: 2,365
Node.js and io.js are increasingly being used to run JavaScript on the server side for many types of applications, such as websites, real-time messaging and controllers for small devices with limited resources. For DevOps it is crucial to monitor the whole application stack and Node.js is rapidly becoming an important part of the stack in many organizations. Sematext has historically had a strong support for monitoring big data applications such as Elastic (aka Elasticsearch), Cassandra, Solr, S...
Jul. 28, 2016 07:15 PM EDT Reads: 3,977


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Austin job fair offers over 5,000 positions
Hundreds of employers are looking to fill thousands of jobs in the Austin area.
Hundreds of employers are looking to fill thousands of jobs in the Austin area.
"We don't want this to just be a one-time deal. We want this to be something that is awarded in her memory for 40 years from now."
More than 100 aftershocks from Thursday night's magnitude-6.5 earthquake continued to rattle the region as businesses and residents got a fu…
Since Union Pacific tracks are out of the question, the board is looking at using the I-35 corridor, the SH130 corridor, the abandoned MoKan…
The public can make name suggestions until 5 p.m. Friday either in person or online.
There is plenty to do this weekend, including a free military air show.
Go to SoLaStyle.com
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Austin-area teams struggle in the semifinals of the state soccer tournament
Go to KissNMakeup.com
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Go to texashumaneheroes.org
Working together, the agencies are bringing attention to the series of murders and hate crimes targeting LGBT Texans in Austin, Dallas, Hous…
Bernie Sanders is struggling to demonstrate how Hillary Clinton was influenced in her policies by donations from Wall Street, as he's often …
Visitors will be able to learn about sustainability before checking out the rock.
New rules require fingerprint background checks for drivers with ride hailing apps like Uber and Lyft, but an election in May could change t…
It's been six weeks of trial and error to get the model for Phillip's prosthetic legs just right.
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Comments about '5A high school football preview: Brighton at Fremont'
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Fremont is good enough to get to the turf, but it ends in the QF.
- 7:33 p.m. Oct. 27, 2011
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Fremont is good enough to go all the way with a good match first round. Bingham isn't the team it was last year but talk is cheap coming from the stands. We will just have to see which teams wants it bad enough stranger things have happened who would have thought Bingham would loose by 35 points to any team?
- 9:20 p.m. Oct. 27, 2011
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Brighton is playing well now, they'll give Fremont a game. If Fremont happens to get past Brighton they won't go any further. The best teams in the state by far are Lone Peak, Alta and Bingham.
- 10:52 p.m. Oct. 27, 2011
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Ya, Ya, thats all we ever hear!! Heard it last year, used to it this year, we shall see, we shall see!! Go Fremont!! Lets hope we happen to get by Brighton, we will worry about playing the best teams in the State when it is time for that!! Can't wait for the Bengal's return to Plain City!! Good luck to all the Region 1 teams!!
- 11:36 p.m. Oct. 27, 2011
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Alta who? They couldn't even get past the Region 1 4 seed!!! I'm sure glad Fremont could get past Brighton,the best part of Brighton's game were the guys wearing stripes!!! Region 1 is a lot tougher than given credit, Go Wolves, Titans & Knights!!! Show the rest of the state how football is played!!! Bring on the blue of Bingham, guess there will be no black this year!!! Great coach & team, but they have to come to our house!!!! Go Wolves!!!
- 9:02 p.m. Oct. 28, 2011
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[IMAGE]Union-Station-2.png[/IMAGE]The California High-Speed Rail Authority (CHSRA) has set aside $30 million in federal funding for property acquisition and railway development in the Los Angeles area. Officials indicate that some of the funds may be used to acquire the Los Angeles Union Station property — the region's primary transportation hub, where three high-speed rail segments will converge.The announcement follows several weeks of discussions with the Federal Railroad Administration (FRA), which is the funding agent, and the Los Angeles County Metropolitan Transportation Authority (Metro), which has been a planning partner with the CHSRA in Los Angeles County. This money is included in the recent grant agreement with the FRA outlining the use of federal funds under the American Recovery and Reinvestment Act (ARRA). The grant agreement also includes $500,000 each for station design in Merced and Bakersfield and $4.5 million total for station area planning in Fresno, Visalia/Kings, Bakersfield, Merced, Palmdale, Gilroy and San Jose, among others.The specific use of the funds is still being determined. However, officials from both the CHSRA and Metro indicate that some of the funds dedicated to Southern California may be used to acquire the Los Angeles Union Station property — the region's primary transportation hub, where three high-speed rail segments will converge. Using the funds to acquire the L.A. Union Station site creates independent benefit for existing transportation entities in the region. An L.A. Union Station revamp would improve operations and service for Metro light rail, Amtrak, Metrolink and the Metro bus system. "Even as we plan to begin construction in the Central Valley — the backbone of a statewide system — we must also steer startup funding in urban areas like L.A. to ensure that regional agencies can begin to set the stage for the arrival of high-speed rail while also benefitting existing infrastructure," said Roelof van Ark, CEO of the CHSRA.This infusion of funding will create immediate benefits in the Southern California region; however, the design and environmental impact review process being undertaken by the CHSRA is still underway, according to officials.
Design and environmental engineering teams have been engaged since 2007 with local communities and transportation agencies to design three segments in Southern California: Palmdale-Los Angeles, Los Angeles-Anaheim, and Los Angeles-San Diego, via the Inland Empire. That process is still underway, with Environmental Impact Reports yet to be released. The CHSRA is developing an 800-mile high-speed train system that will operate at speeds of up to 220 miles per hour, connecting the state's urban centers, including the Bay Area, Fresno, Los Angeles and San Diego.
The first phase of the project, projected to cost about $43 billion, will begin operation once the first operable segment (150-200 miles) is built, connecting the system to at least one major metropolitan center. The project is being funded through a voter-approved bond, public-private partnerships and federal grants.California has already secured more than $3 billion in federal funding, the most of any state in the nation and a welcome step toward a long-term federal commitment. This incorporates grants under the American Recovery and Reinvestment Act, as well as federal grants from other sources. Matched with varying levels of state funds, these dollars mean that more than $5.5 billion total is available to begin work on California's high-speed rail system.The federal grant agreement and other documents outlining the ARRA funds are available on the CHSRA's website. Another grant agreement pertaining to the High-Speed Intercity Passenger Rail Program FY 2010 funds, also to be directed to the Central Valley, is forthcoming.
The grants, which will be selected by the FRA and awarded and administered by the FTA, will help commuter railroads implement PTC, which prevents accidents and saves lives.
The project, which starts next week, will utilize Hyperloop Transportation Technologies crowdsourcing and community collaboration, while implementations will be financed by Deutsche Bahn.
A group argues that the flawed data suggested the move would not have an overly burdensome effect on low-income and minority riders and that it should have taken steps to curb those impacts before ending the service in March.
During the investigation, it was determined that the man was laying on his back in the middle of the inbound side of a track on the south side, when a northbound train came down the track because the system was running on a single track.
Will emphasize beautiful travel photography and long-form journalism to cast itself as an upscale brand and replace its current magazine, "Arrive," which is more focused on celebrities, what's-on content and regional destinations.
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