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Ro ribonucleoprotein particles (Ro RNPs) consist of a non-coding Y RNA bound by Ro60, La and possibly other proteins. The physiological function of Ro RNPs is controversial as divergent functions have been reported for its different constituents. We have recently shown that Y RNAs are essential for the initiation of mammalian chromosomal DNA replication, whereas Ro RNPs are implicated in RNA stability and RNA quality control. Therefore, we investigate here the functional consequences of RNP formation between Ro60, La and nucleolin proteins with hY RNAs for human chromosomal DNA replication.
Methodology/Principal Findings
We first immunoprecipitated Ro60, La and nucleolin together with associated hY RNAs from HeLa cytosolic cell extract, and analysed the protein and RNA compositions of these precipitated RNPs by Western blotting and quantitative RT-PCR. We found that Y RNAs exist in several RNP complexes. One RNP comprises Ro60, La and hY RNA, and a different RNP comprises nucleolin and hY RNA. In addition about 50% of the Y RNAs in the extract are present outside of these two RNPs. Next, we immunodepleted these RNP complexes from the cytosolic extract and tested the ability of the depleted extracts to reconstitute DNA replication in a human cell-free system. We found that depletion of these RNP complexes from the cytosolic extract does not inhibit DNA replication in vitro. Finally, we tested if an excess of recombinant pure Ro or La protein inhibits Y RNA-dependent DNA replication in this cell-free system. We found that Ro60 and La proteins do not inhibit DNA replication in vitro.
We conclude that RNPs containing hY RNAs and Ro60, La or nucleolin are not required for the function of hY RNAs in chromosomal DNA replication in a human cell-free system, which can be mediated by Y RNAs outside of these RNPs. These data suggest that Y RNAs can support different cellular functions depending on associated proteins.
Citation: Langley AR, Chambers H, Christov CP, Krude T (2010) Ribonucleoprotein Particles Containing Non-Coding Y RNAs, Ro60, La and Nucleolin Are Not Required for Y RNA Function in DNA Replication. PLoS ONE 5(10): e13673. https://doi.org/10.1371/journal.pone.0013673
Editor: Anja-Katrin Bielinsky, University of Minnesota, United States of America
Received: June 28, 2010; Accepted: October 6, 2010; Published: October 27, 2010
Copyright: © 2010 Langley et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Cancer Research UK (project grant C1471/A8448; URL: http://www.cancerresearchuk.org/). A.R.L was supported by a Research Studentship from the Medical Research Council (MRC; URL: http://www.mrc.ac.uk/index.htm), and H.C. was supported by an Undergraduate Research Bursary from the Nuffield Foundation (URB/33112; URL: http://www.nuffieldfoundation.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Ro ribonucleoprotein particles (Ro RNPs) are soluble complexes in vertebrate cells which are detected by autoimmune antibodies from patients suffering from systemic lupus erythematosis or Sjögren's syndrome [1], [2]. Ro RNPs consist of a structured non-coding RNA termed Y RNA, which is bound by the 60 kDa autoantigen protein Ro60, the 50 kDa autoantigen protein La, and possibly other proteins. The physiological function of Ro RNPs is controversial as divergent functions have been reported for its different constituents.
Y RNAs are RNA polymerase III transcripts of about 100 nucleotides, which fold into characteristic stem-loop structures [3], [4]. They are evolutionarily conserved in vertebrates [5], [6]. Human cells express four Y RNAs (hY1, hY3, hY4 and hY5) [1] and the nucleotide sequence and domain structure of the representative hY1 RNA is shown in Fig. 1. Y RNAs have an essential function for the initiation step of chromosomal DNA replication in mammalian somatic cells [7], [8], [9], [10]. Knockdown of hY RNAs by RNA interference results in the inhibition of DNA replication and the cytostatic arrest of cell proliferation [7], [8], [10]. Additionally, depletion of hY RNAs from human cell extracts specifically inhibits the initiation step of chromosomal DNA replication in a mammalian cell-free system [7], [9]. This inhibition can be overcome by the addition of pure Y RNAs. Recently, we have shown that an evolutionarily conserved double-stranded RNA motif present in the upper stem of vertebrate Y RNAs is essential and sufficient for their function in DNA replication in mammalian cells [10]. These data indicate that Y RNAs are functionally active, but it is unknown if any of the associated Ro RNP proteins play a role in DNA replication.
The most stable secondary structure of hY1 RNA is shown as determined by the Mfold v3.2 RNA algorithm. The four conserved key structural elements, including specific protein binding sites, are indicated.
Ro60 protein binds to a highly conserved site on the lower stem of the Y RNAs [11], [12], [13], which is distinct from the domain required for DNA replication [10]. Ro60 has been proposed to function as a scavenger for mis-folded RNAs in a quality control pathway for non-coding RNAs [13], [14], [15]. Y RNAs have been implicated to play a regulatory role in this process as structural studies have indicated partially overlapping binding sites on Ro60 for misfolded RNAs and Y RNAs [13], [16]. This function of Ro60, however, is not essential as Ro60 knockout mice are viable and, furthermore, derived embryonic stem (ES) cells show no major proliferation defects [17], [18]. Ro60 knockout cells also show reduced levels of Y RNAs, thus implicating Ro60 in RNA stability control [17], [18], [19], [20], possibly by providing protection from exonuclease digestion. Additional studies have demonstrated that Ro60 binding is a prerequisite for the export of Y RNAs from the nucleus to the cytoplasm [21], [22].
La protein is required for the accurate and efficient termination of RNA polymerase III transcription and binds the 3′ polyuridine tail of newly synthesised RNAs in the nucleus [23], [24], [25], [26], [27]. Unlike most RNA polymerase III transcripts, mature Y RNAs retain a 3′ polyuridine tail and thus Y RNAs are believed to maintain a stable association with La [1], [2], [28], [29]. La has also been implicated in many other aspects of non-coding RNA biogenesis, such as protecting newly synthesised RNA against exonuclease digestion [27] and the nuclear retention of RNAs [21]. In addition to Ro60 and La, several other proteins have also been reported to interact with a subset of hY RNAs including the multifunctional protein nucleolin, which associates with the pyrimidine-rich loops of hY1 and hY3 RNA, but not with hY4 or hY5 [15], [30], [31].
Here, we investigate the functional consequences of RNP formation between Ro60, La and nucleolin with hY RNAs for chromosomal DNA replication. Our data show that RNPs containing Y RNAs, Ro60, La or nucleolin are not required for chromosomal DNA replication in a human cell-free system, and that that Ro60 and La proteins do not inhibit this function.
hY RNAs are present in several distinct RNP complexes in human cytosolic extract
To confirm that Ro60, La and nucleolin stably interact with hY RNAs, we first immunoprecipitated these proteins from HeLa cytosolic cell extract, and then analysed the protein and RNA compositions of these immunoprecipitates (Fig. 2). Anti-Ro60 antibodies precipitated both Ro60 and La, and anti-La antibodies also precipitated both of these proteins (Fig. 2A, bottom panel). Neither anti-Ro60, nor anti-La antibodies co-precipitated nucleolin under these conditions. Conversely, anti-nucleolin antibodies precipitated nucleolin from the extract, but did not co-precipitate La (Fig. 2B). To test whether the co-precipitation of Ro60 and La is mediated by RNA, we treated the extract with RNase A prior to immunoprecipitation. We have previously shown that this treatment reduces the levels of hY RNAs in the extract by three to four orders of magnitude [7]. RNAse treatment did not abrogate the co-precipitation of Ro60 and La (Fig. 2A, bottom panel), suggesting that these proteins either interact directly, or that the close proximity of the binding sites for Ro60 and La on the Y RNA protects the underlying associated RNA fragment from degradation. In any case, these data indicate that Ro60 and La proteins are present in the same complex, while nucleolin is present in a separate complex.
The indicated proteins were immunoprecipitated (IP) from HeLa cytosolic extract and associated proteins and RNAs were analysed by Western blotting and qRT-PCR, respectively. (A) Protein analysis of Ro60 and La IPs. Apparent molecular weights of the precipitated protein bands are shown, the asterisk indicates the IgG heavy chain. As a reference, 10% of the input cell extract was loaded on the gel. Where indicated, extract was treated with RNase A prior to IP (RNase). (B) Protein analysis of nucleolin (NCL) IPs. (C) RNA content analysis of the Ro60, La and nucleolin (NCL) IPs. The relative amounts of all four hY RNAs and 5S rRNA in the indicated immunoprecipitates relative to mock immunoprecipitates were determined by qRT-PCR. Mean values of two independent experiments are shown.
Next, we analysed the RNA content of the immunoprecipitates by quantitative reverse transcription-PCR (qRT-PCR). We determined the relative amounts of the four hY RNAs, and of 5S rRNA as a specificity control (Fig. 2C). All four hY RNAs were enriched more than 500 times in anti-Ro60 immunoprecipitates, compared with mock immunoprecipitates, whereas 5S rRNA was enriched only about 6 fold. In anti-La immunoprecipitates, hY1 and hY3 RNA were enriched 70–200 fold, hY4 and hY5 RNA were both enriched more than 500 times, whereas 5S rRNA was enriched only about 4 fold. In anti-nucleolin immunoprecipitates, hY1 RNA was enriched more than 500 times, while hY3, hY4, hY5 RNA and 5S rRNA were enriched between 15–70 times. We conclude that Ro60 and La stably interact with all four hY RNAs in cytosolic extract, while nucleolin interacts preferentially with hY1 RNA (and to a lesser extent with the other three hY RNAs and 5S rRNA).
Taken together, these data indicate that hY RNAs are present in several distinct types of RNP complexes in the cytosolic HeLa cell extract. First, all four hY RNAs are present in [Ro60/La/hY RNA] RNP complexes. Second, hY RNAs and 5S rRNA are also present in distinct [nucleolin/hY RNA] RNP complexes, which form preferentially with hY1 RNA. Thus, in the next experiments, we investigated the functional requirement of these hY-RNP complexes for chromosomal DNA replication.
[Ro60/La/hY RNA] and [nucleolin/hY RNA] RNP complexes are not required for the reconstitution of chromosomal DNA replication in vitro
Y RNAs are essential to functionally reconstitute the initiation of DNA replication in a human cell-free system [7]. In this system, isolated late G1 phase template nuclei initiate DNA replication when incubated in cytosolic extracts from proliferating cells [32], [33]. Nucleolytic degradation of hY RNAs present in the extract results in an inhibition of the initiation step of chromosomal DNA replication, which is negated by the re-addition of purified Y RNAs [7], [9]. We therefore used this system to investigate whether [Ro60/La/hY RNA] RNPs and/or [nucleolin/hY RNA] RNPs are required for chromosomal DNA replication.
First, we immunodepleted these complexes from the cytosolic extract and then tested the ability of these depleted extracts to reconstitute DNA replication in vitro (Fig. 3). Ro60 and La were both depleted specifically by La-specific antibodies, while the level of nucleolin was unaffected (Fig. 3A). Conversely, nucleolin was specifically depleted by the anti-nucleolin antibodies, while the levels of Ro60 and La were unaffected (Fig. 3B).
Immunodepletion of hY RNPs from cytosolic HeLa cell extracts. (A) Depletion of [Ro60/La/hY RNA] RNPs with La-specific antibodies, and (B) depletion of [nucleolin/hY RNA] RNPs with nucleolin-specific antibodies. Ro60, La and nucleolin (NCL) were analysed in the depleted extracts by Western blot analysis. Ponceau stains are shown as loading controls. For mock depletions, unspecific mouse IgG2a antibodies were used alongside purified mouse monoclonal anti-La antibodies, while pre-immune rabbit serum was used alongside the anti-nucleolin rabbit serum. (C) Functional reconstitution of chromosomal DNA replication in the immunodepleted cytosolic extracts. Late G1 phase template nuclei from mimosine-arrested human EJ30 cells were incubated in the indicated depleted extracts. Replication buffer and non-depleted cytosolic extract (cyt) served as negative and positive controls, respectively. Mock M IgG and R IgG indicate unspecific mouse and rabbit antibodies, as in panels A and B. Proportions of replicating nuclei were determined by immunoflorescence microscopy. Mean values of two independent experiments are shown.
We then tested the ability of these depleted extracts to reconstitute the initiation of DNA replication in vitro. When non-depleted cytosolic extract was used as a positive control, 63±4% of nuclei replicated in this extract, compared with 5±4% in buffer alone (Fig. 3C), in agreement with a previous characterisation of this system [32]. Mock depletion of the cytosolic extract with unspecific antibodies did not affect its ability to initiate DNA replication as 61±5% and 59±4% of nuclei replicated in extracts mock-depleted with either mouse or rabbit IgGs, respectively (Fig. 3C). Specific depletion of either [Ro60/La/hY RNA] RNPs or [nucleolin/hY RNA] RNPs resulted in 61±4% and 59±3% of nuclei replicating, respectively. Therefore, depletion of either of these hY RNA-containing RNPs from cytosolic extract has no affect on the initiation of DNA replication in this system, suggesting that they are not required for DNA replication. Furthermore, these results show that the cytosolic protein pools of Ro60, La and nucleolin are also not essential for DNA replication, whether or not these proteins are stably bound to hY RNAs.
We have previously reported that Y RNAs act redundantly with each other in the initiation of chromosomal DNA replication [7], [9], due to an evolutionarily conserved double-stranded RNA motif present in all vertebrate Y RNAs that is essential and sufficient for DNA replication in this system [10]. Therefore, depletion of either [Ro60/La/hY RNA] or [nucleolin/hY RNA] RNPs would still be consistent with redundant roles of either RNP complex in DNA replication. To exclude this possibility, we co-depleted both [Ro60/La/hY RNA] and [nucleolin/hY RNA] RNPs from cytosolic extract (Fig. 4). Ro60, La and nucleolin proteins were all depleted by >99% as determined by Western blot analysis (Fig. 4A). We next determined by qRT-PCR the relative amounts of all four hY RNAs and 5S rRNA remaining in the extract after this co-depletion (Fig. 4B). The relative level of hY1 RNA was reduced to 75% of that seen in the mock-depleted extract, while the relative levels of hY3 and hY5 were reduced to 38%. The levels of hY4 RNA and 5S rRNA, however, were only marginally affected. Taking into account the absolute amounts of these hY RNAs in this cytosolic extract [7], this co-depletion equates to an overall reduction of hY RNAs to about 50% (i.e. from 125±50 nM of hY1-5 RNAs in the undiluted extract [7] down to about 62±25 nM). We conclude that only about half of all hY RNAs in this cytosolic HeLa cell extract are present in RNP complexes with Ro60, La or nucleolin.
[Ro60/La/hY RNA] and [nucleolin/hY RNA] RNPs were co-depleted with La- and nucleolin-specific antibodies. (A) Protein levels of nucleolin (NCL), Ro60 and La in the depleted extracts were analysed by Western blotting. Mouse IgG2a antibodies and pre-immune rabbit serum were used together for the mock depletion. (B) Analysis of hY RNA and 5S rRNA levels remaining in the depleted extract. Proportions of the indicated RNA amounts remaining in the extract after [Ro60/La/hY RNA] and [nucleolin/hY RNA] RNP co-depletion were determined by qRT-PCR. Data are shown as percentages of the mock depletion, after normalisation to HPRT mRNA. (C) Functional reconstitution of chromosomal DNA replication in the co-depleted extract. Percentages of replicating nuclei were determined as described for Fig. 3C. Mean values of two independent experiments are shown in panels B and C.
We then went on to test the ability of this hY RNP-depleted extract to reconstitute the initiation of DNA replication in vitro (Fig. 4C). Neither mock depletion, nor [Ro60/La/hY RNA]/[nucleolin/hY RNA] RNP co-depletion inhibited the initiation of chromosomal DNA replication in this system as 59±1% and 60±5% of nuclei of nuclei replicated, respectively. We conclude that hY RNAs remaining in the depleted extract, which are not associated with either Ro60, La or nucleolin, are sufficient to reconstitute the initiation of chromosomal DNA replication in vitro.
Ro60 or La proteins do not inhibit chromosomal DNA replication in vitro
The experiments described above exclude an essential stimulatory function for the major Y RNA-binding proteins Ro60 and La in chromosomal DNA replication in vitro. It nevertheless remains a possibility that these proteins may negatively regulate DNA replication. Ro60 and/or La may sequester Y RNAs and prevent them from functioning in chromosomal DNA replication. To address this possibility, in the last set of experiments, we purified recombinant human Ro60 and La proteins and then tested if they inhibit hY RNA-dependent initiation of chromosomal DNA replication in vitro.
Recombinant human Ro60 was expressed as a poly-histidine tagged protein in E. coli [34], and purified to >99% using Ni-NTA agarose chromatography (Fig. 5A). Recombinant human La was expressed as a chitin binding domain fusion protein in E. coli [34], and purified to >98% homogeneity by affinity chromatography on chitin-agarose, followed by elution of the full-length human La protein after intein-mediated self cleavage (Fig. 5B).
(A) Protein analysis of purified Ro60 by SDS-PAGE and silver staining is shown on the left panel and Western blot analysis on the right panel. Positions of the molecular weight markers (kDa) are indicated. (B) Protein analysis of purified La by SDS-PAGE and Coomassie staining is shown on the left panel and Western blot analysis on the right panel. (C) Formation of [Ro60/La/hY RNA] RNPs from purified components in vitro. 32P-labelled hY1 RNA was incubated with purified Ro60 and/or La at the indicated molar ratios in the presence of a 10-fold molar excess of competitor tRNA. Free RNA was separated from RNPs on native 8% polyacrylamide gels, and radiolabelled RNA was visualised by autoradiography. Positions of free and protein-complexed hY1 RNA are indicated. (D) Functional reconstitution of chromosomal DNA replication in the presence of excess recombinant Ro60 and/or La. Template nuclei were incubated in limiting cytosolic extract (40 µg cyt) with recombinant proteins as indicated. Replication buffer and non-limiting cytosolic extract (cyt) were used as negative and positive controls, respectively. Proportions of replicating nuclei were determined as described for Fig. 3C. Mean values and standard deviations of n = 15–18 independent experiments are shown.
Before using these proteins in replication assays, we first conducted RNA band-shift assays to test their ability to bind purified hY1 RNA (Fig. 5C). Recombinant Ro60 and/or La were incubated with 32P-labelled hY1 RNA in the presence of a ten-fold molar excess of unlabelled competitor tRNA to inhibit unspecific binding. Results were analysed on native polyacrylamide gels, followed by autoradiography. Ro60 and La both resulted both in a pronounced upwards shift of the hY1 RNA band in the gel. A four-fold molar excess of Ro60 protein resulted in a shift of about 30–40% of the input hY1 RNA (Fig. 5C, lane 4), whereas equimolar amounts of La were already sufficient to shift >90% of the input hY1 RNA (Fig. 5C, lane 6). This result suggests that La has a higher binding affinity to Y RNAs than Ro60, or that Ro60 may only bind to a fraction of the Y RNAs, such as misfolded ones. Furthermore, addition of Ro60 and La together resulted in a super-shift of the hY1 RNA band above the position obtained with either protein alone (Fig. 5C, compare lane 12 with lanes 4 and 8). These results demonstrate that both recombinant Ro60 and La bind hY1 RNA to form ternary [Ro60/La/hY RNA] RNPs in vitro, indicating that these recombinant proteins are functionally intact.
In the last set of experiments, we investigated the effects of recombinant human Ro60 and La proteins on the initiation of chromosomal DNA replication in vitro. We tested for potential negative effects of these proteins under conditions of limiting cytosolic extract, which allow a more sensitive detection of an inhibition of DNA replication than saturated conditions. The addition of 40 µg of cytosolic extract resulted in only about half the maximal number of nuclei replicating in vitro (33±9%), whereas addition of >100 µg led to the saturation of the system with the maximal number of nuclei replicating (54±5%; Fig. 5D), in agreement with a previous characterisation of the system [32]. We thus titrated recombinant Ro60 or La into the cell free system in the presence of 40 µg of extract. First, we determined by Western blotting that 40 ng and 60 ng of endogenous Ro60 and La were present in the cytosolic extract per 40 µg of total protein, respectively. (This is equivalent to 160 nM and 300 nM of endogenous of Ro60 and La, respectively, whilst all four hY RNAs together are present at 125±50 nM in the extract [7]). Pilot titrations indicated that addition of up to 1 µg of either recombinant Ro60 or La to these conditions did not inhibit DNA replication (data now shown). This result indicates that molar excesses up to 25- or 17-fold of either Ro60 or La protein over total hY RNAs, respectively, do not inhibit the initiation of DNA replication in this system. We finally compared directly the addition of Ro60, La and both of these proteins together to the system (Fig. 5D). Neither the addition of Ro60, La, or of both proteins together at 350 ng had any statistically significant effect on the proportion of nuclei replicating (34±8%, 34±7%, 30±9%, respectively; two-tailed t-tests, unequal variance: P>0.3, n = 15). We conclude from these data that more than 10-fold molar excesses of recombinant Ro60 and/or La proteins over the levels of endogenous hY RNAs do not inhibit chromosomal DNA replication in vitro.
Y RNAs are essential for the initiation of chromosomal DNA replication in mammalian somatic cell nuclei [7], [9]. They were originally discovered as the RNA constituent of soluble, predominantly cytoplasmic, RNPs containing Ro60, La and nucleolin proteins [1], [2], [28], [31], [35]. As it was unknown if these Y RNA binding proteins also play a role in DNA replication, we investigated their function in this process. Our results demonstrate that all four hY RNAs are present in two distinct types of RNP complex in a human cytosolic extract: [Ro60/La/hY RNA] RNPs and [nucleolin/hY RNA] RNPs. We have shown that these RNPs are not required for chromosomal DNA replication in a cell-free system. In fact, immunodepletion of these RNPs removes only about half of the soluble hY RNAs from the extract. As specific degradation of Y RNAs by targeted ribonucleases inhibits DNA replication [7], [9], we conclude that the hY RNA pools, which are not associated with either Ro60, La or nucleolin are sufficient for the initiation of chromosomal DNA replication in vitro. Conversely, we have also demonstrated that an excess of exogenous Ro60 and La does not inhibit DNA replication in vitro. These results indicate that RNP formation between Y RNAs and Ro60, La and nucleolin is not required for Y RNA function in DNA replication, and that these RNPs may thus perform unrelated functions in the cell.
Distinct hY RNA-containing RNP complexes are present in cytosolic extract
Antibodies against Ro60 or La specifically precipitated Ro60, La and all four hY RNAs, indicating the presence of [Ro60/La/hY RNA] RNP complexes in the HeLa cytosolic extract and confirming previous reports [28], [31], [35], [36]. Degradation of the RNAs present in the extract did not abrogate the co-precipitation of Ro60 and La, also in accordance with a previous report [31]. This is most likely due to the close proximity of the binding sites for Ro60 and La on the Y RNA, thus protecting the associated RNA from degradation.
While anti-nucleolin antibodies precipitated nucleolin and all four hY RNAs, they did not co-precipitate Ro60 or La. Conversely, anti-Ro60 and anti-La antibodies did not co-precipitate nucleolin. Furthermore, our results show that nucleolin is in a complex with 5S rRNA, suggesting that it associates with additional RNAs, as well as with hY RNAs. In contrast to our results, Fouraux and colleagues have reported that anti-nucleolin antibodies precipitated only hY1 and hY3 RNA, but not hY4 or hY5 RNA, and that nucleolin co-precipitates with Ro60 and La [31]. These discrepancies regarding nucleolin-RNPs may be due to the use of different RNA detection methods (qRT-PCR and Northern blotting, respectively) and to the use of different antibodies.
[Nucleolin/hY RNA] RNP complexes are not required for DNA replication
Nucleolin interacts with pyrimidine-rich stretches in the loop domain of hY RNAs [31], [36]. We have previously shown that deletion of the entire loop domain does not negate the function of Y RNAs in DNA replication [10]. Here, we have shown by immunoprecipitation experiments that the [nucleolin/hY RNA] RNP complexes are not required for DNA replication in vitro, thus independently confirming that the interaction between nucleolin and Y RNAs is not required for the function of Y RNAs in DNA replication.
As result of heat shock or DNA damage, nucleolin rapidly re-localises from the nucleolus to the nucleoplasm and forms a complex with the DNA replication initiation factor RPA [37], [38], [39], [40]. This complex formation sequesters RPA and inhibits the initiation of DNA replication and the G1 to S phase transition. It therefore remains an interesting possibility that nucleolin (with RPA) may also sequester Y RNAs under stress conditions in the intact cell, thus inhibiting the function of Y RNAs in DNA replication under these conditions.
[Ro60/La/hY RNA] RNP complexes are not required for DNA replication
The main observation of our immunoprecipitation experiments and in vitro DNA replication assays is that RNP formation between hY RNAs, Ro60 and La is not required for Y RNA function in DNA replication. A potential caveat of these in vitro results is the lack of confirmation by an independent experimental approach (e.g. a combined protein knockout in vivo, followed by DNA replication assays). However, our data do directly confirm predictions derived from deletion experiments of the respective protein binding sites from Y RNAs. Ro60 binds to a highly conserved site on the lower stem of Y RNAs [11], [12], [29], while La interacts with the 3′ polyuridine tail [28], [29]. Deletion of these binding sites does not inhibit the function of Y RNAs during chromosomal DNA replication in vitro and in vivo [7], [10]. In contrast, a separate double stranded RNA domain in the upper stem of vertebrate Y RNAs is essential and sufficient for their function in DNA replication [10]. Therefore, either immunodepletion of the entire [Ro60/La/hY RNA] RNP, or deletion of the underlying protein binding sites from the hY RNAs have no effect on Y RNA function in DNA replication. We therefore conclude that Y RNA function in chromosomal DNA replication and Ro RNP function are two separable and independent functions of Y RNAs.
This separation of function allows the reconciliation of controversial reports in the literature. Consistent with our Ro60 immunodepletion data, Wolin and colleagues have reported that Ro60 knockout mice are viable and that derived embryonic stem cells show no major proliferation defects [17], [18]. The levels of mY1 and mY3 RNA in Ro60-knockout cells, however, are reduced, yet no major defects in DNA replication or cell proliferation have been reported [17], [18], [19], [20]. As such, these latter observations could be interpreted as evidence against an essential role for Y RNAs in DNA replication. In contrast, our published data rule out this interpretation, because we have demonstrated that nucleolytic degradation of Y RNAs directly inhibits DNA replication in vitro and in vivo [7], [8], [9], [10]. Like the depletion of Ro RNPs in Ro60 knockout cells, the immunodepletion of Ro RNPs from cell extracts would thus remove a particular Ro RNP-associated subpopulation of Y RNAs with no function in DNA replication, whereas nucleolytic degradation would remove all Y RNAs indiscriminately. Taken together, these data allow the conclusion that those Y RNAs which are not contained in [Ro60/La/Y RNA] or [nucleolin/Y RNA] RNPs constitute a functional relevant pool for DNA replication, while those Y RNAs present in these RNPs may be involved in unrelated processes such as non-coding RNA quality control or RNA stability [14], [17], [19], [20].
Here we have determined by quantitative RT-PCR that about 50% of the hY RNAs in the HeLa cytosolic extract are present in [Ro60/La/hY RNA] or [nucleolin/hY RNA] RNPs and about 50% are therefore outside of these RNPs. This is in contrast to an earlier report [41], showing by Northern blotting that the majority, if not all, cytoplasmic Y RNAs are bound by Ro60 and La. It is possible that these differences arise from different sensitivities of the two RNA detection techniques used. Alternatively, different cells and culture conditions may produce different expression ratios between Y RNAs and Ro60 or La proteins, resulting in different proportions of Y RNAs inside and outside of Ro RNPs. In support of the latter, we have shown that Y RNAs are significantly up-regulated between 5-13-fold in human cancer tissues, compared to normal tissues [8]. This observation suggests that highly proliferative conditions correlate with high Y RNA expression levels, which may result in higher proportions of Y RNAs outside of Ro RNPs.
We do not currently know much about the association of these functionally active Y RNAs with other proteins outside of [Ro60/La/Y RNA] or [nucleolin/Y RNA] RNPs. As it is highly unlikely that the Y RNAs exist as naked RNAs without associated proteins, we therefore speculate that these Y RNAs can associate with proteins involved in the initiation of chromosomal DNA replication. We are currently testing this hypothesis by identifying and characterising novel Y RNA-protein associations in our laboratory and preliminary results show that this is indeed the case. The results of this study will be reported elsewhere.
We found that the addition of more than a 10-fold molar excess of Ro60 and La proteins over the endogenous Y RNA pools did not inhibit the initiation of DNA replication. If one assumes that this extra protein is able to associate with hY RNAs in the extract, one can thus not rule out entirely that Y RNAs may support DNA replication even when associated with Ro60 or La. This scenario is, however, unlikely because La or Ro60 are already present in higher or equimolar amounts to endogenous Y RNAs in the extract, respectively, whilst immunodepletion of more than 99% of total La and Ro60 proteins only removes half of the soluble Y RNAs. It is therefore likely that excess Ro60 and La would be either outcompeted on the Y RNAs in the extract by other Y RNA-binding factors involved in DNA replication, or be bound by additional RNAs with no function in DNA replication, or remain uncomplexed.
An independent precedence for selective targeting of Y RNAs to a cellular pathway independently of Ro RNP formation has recently been reported for retrovirus assembly [19]. Mouse Y RNAs are selectively encapsidated into Moloney murine leukaemia virus (MLV) virions in Ro60-knockout cells as effectively as in wild-type cells, despite significantly reduced intracellular Y RNA levels in the knockouts [19]. By analogy, it is therefore likely that a proportion of cellular Y RNAs is specifically and efficiently recruited for chromosomal DNA replication without involving Ro RNPs. The molecular mechanisms involved in the recruitment of these Y RNAs to intranuclear sites where DNA replication initiates are currently under investigation in our laboratory.
Materials and Methods
Cytosolic extract of asynchronously proliferating human HeLa cells was obtained from Cilbiotech (Mons, Belgium). Extract was diluted in replication buffer (20 mM K-HEPES pH 7.8, 100 mM K-acetate, 1 mM DTT, 1 mM EGTA) and pre-depleted with protein G-agarose or protein A-agarose (both from Roche). (Where applicable, pre-depleted extract was treated with a final concentration of 0.3 mg/ml RNase A (Roche).) Pre-depleted extracts were incubated with the following primary antibodies: anti-60 (Euro-Diagnostica), anti-La (Santa Cruz sc-80655), and anti-nucleolin (a gift from Professor Hans Stahl, University of Homburg/Saar. Germany). Pre-immune rabbit serum and unspecific IgG2a antibodies from murine myeloma (Sigma M5409) were used as negative controls. Antibodies were then precipitated from extracts with protein G-agarose or protein A-agarose (both from Sigma). Protein content of immunoprecipitates was determined via SDS-PAGE followed by Western blot analysis using the same primary antibodies as used for immunoprecipitation. RNA composition was determined by quantitative reverse transcription-PCR (qRT-PCR) following phenol/chloroform extraction and ethanol precipitation.
Immunodepletion of cytosolic extract
Primary antibodies as used for immunoprecipitation were coupled to protein G-agarose or protein A-agarose beads by incubation in replication buffer. Unspecific IgG2a antibodies from murine myeloma and/or pre-immune serum were used for mock depletions. These antibody-coupled beads were then incubated with cytosolic extract in replication buffer to specifically deplete proteins from the extract. Following three rounds of depletion, protein content of extracts was determined via SDS-PAGE followed by Western blot analysis. RNA composition was determined by qRT-PCR following phenol/chloroform extraction and ethanol precipitation.
Quantitative reverse transcription-PCR (qRT-PCR)
cDNA libraries were synthesised from RNA preparations of immunoprecipitates or cell extracts using random primers (Promega) with the SuperScript II Reverse Transcriptase kit according to the manufacturer's protocol (Invitrogen). These cDNA libraries were then used as the templates for qRT-PCR on the iCycler iQ platform as described [7]. For RNA-specific cDNA amplification, pairs of primers for hY1, hY3, hY4, hY5, 5S and hypoxanthine ribosyltransferase (HPRT) were used as described [7].
The relative amount (AR(IP)) of each hY RNA and 5S rRNA in immunoprecipitates compared with mock immunoprecipitates was calculated from the threshold cycle (CT) of each cDNA amplification by using the following equation: AR(IP) = 2(CT of mock immunoprecipitate – CT of specific immunoprecipitate). Identical amounts of starting extract and of protein A or G agarose were used.
In cell extracts, the relative amount (AR(E)) of each RNA was first calculated from the threshold cycle (CT) of each cDNA amplification by using the following equation: AR(E) = 2−ΔCT = 2−(CT of test RNA – CT of reference RNA). The reference RNA was HPRT mRNA. After immunodepletion of cytosolic extract, proportions of the relative amounts of each RNA were normalised against the mock depleted extract using the following equation: proportion of AR(E) = 2−(ΔCT of immunodepleted extract – ΔCT of mock depleted extract) X 100%.
Expression and purification recombinant Ro60 and La
For expression of Ro60, the E. coli strain C43 was transformed with the pET28a/his-Ro60 expression vector [34] and grown at 37°C in Lauria broth (LB) to an A600 of 0.5. Isoproypylthiogalactoside (ITPG) was added to a final concentration of 0.4 mM, the temperature was lowered to 25°C and growth continued overnight. All purification steps were conducted at 4°C. Bacteria were harvested by centrifugation, washed in PBS, and resuspended in 30 ml protein purification buffer (20 mM KPO4, pH 7.8, 300 mM NaCl) with 0.2% Triton X-100 and lysed by sonication. The lysate was clarified by centifugation for 20 minutes at 25,000×g. Imidazole was added to the supernatant to a final concentration of 10 mM, which was then loaded onto Ni-NTA-agarose (Qiagen), pre-equilibrated in purification buffer containing 10 mM imidazole. The column was washed with 40 ml purification buffer containing 150 mM imidazole and Ro60 protein was then eluted with purification buffer containing 600 mM imidazole. The buffer of the eluted protein fractions was exchanged for protein storage buffer (50 mM KPO4 pH 7.8, 300 mM KCl, 1 mM EGTA, 0.5 mM DTT, 10% glycerol) using a PD10 column according to the manufacturers protocol (GE Healthcare), before flash freezing in liquid nitrogen and storing at −80°C.
For expression of La, E. coli C43 were transformed with the pTWIN1/La expression vector [34], grown at 37°C in LB to an A600 of 0.05. ITPG was added to a final concentration of 1 mM, the temperature was lowered to 26°C and growth continued for 20 h. All purification steps were conducted at 4°C. Cells were washed and lysed in 20 mM Tris-Cl pH 7.0, 500 mM NaCl, 1 mM EDTA, 0.1%Tween 20, 20 µM PMSF by sonication on ice. The lysate was clarified by centifugation for 30 minutes at 20,000×g and loaded onto chitin beads (NEB) and washed in 50 mM Tris-Cl pH 8.8, 1 M NaCl, 1 mM EDTA, 20 µM PMSF. On-column cleavage was induced by addition of elution buffer (50 mM Tris-Cl pH 8.8, 500 mM NaCl, 40 mM DTT, 1 mM EDTA). Eluted protein fractions containing La were pooled, exchanged to protein storage buffer, flash frozen in liquid nitrogen and stored at −80°C.
Concentrations of Ro60 and La proteins in human cytosolic extract were determined by serial dilutions of pure proteins alongside of cytosolic extract, followed by Western blotting.
RNA band shift assays
Human Y1 RNA was synthesised with SP6 RNA polymerase from recombinant DNA templates in the presence of α-32P UTP as described [7]. For band shift assays, 80 ng of radiolabelled hY1 RNA was incubated in band shift buffer (10 mM Tris-HCl pH 7.5 100 mM NaCl, 3 mM MgCl2, 1 mM DTT) with recombinant Ro60 and/or recombinant La at molar RNA to protein ratios of 1∶0, 1∶1, 1∶2 or 1∶4, and a molar RNA to protein to protein ratio of 1∶4∶4. Competitor tRNA (Roche) was also added to reactions at 10 fold molar excess of the hY1 RNA to inhibit unspecific binding. Reactions were incubated for 15 minutes at 37°C before being loaded onto native 8% polyacrylamide gels (acrylamide:bisacylamide ratio of 180∶1), which were run at 4°C. Following electrophoresis, gels were dried and radiolabelled hY1 RNA was visualised by autoradiography.
Cell Culture and DNA replication reactions in vitro
Culture of human EJ30 bladder carcinoma cells, cell synchronisation in late G1 phase by mimosine, and preparation of template nuclei were performed as described [32], [42]. Analysis of DNA replication reactions were performed as described [7], [32], [43], [44].
We would like to thank Clara Collart for critical reading of this manuscript, and Hans Stahl (Homburg/Saar) and Günter Steiner (Vienna) for reagents.
Author Contributions
Conceived and designed the experiments: ARL TK. Performed the experiments: ARL HC CPC. Analyzed the data: ARL TK. Wrote the paper: ARL TK.
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WILLIAM R. BRODY 303Assert Fact and Proclaim Truth
The True Meaning Of Eternity
Good morning. To our honorary degree recipients and our new members of the Society of Scholars, to our Trustees and alumni, faculty and staff, to our parents, family members and friends, but most of all, to our brand new graduates, I bring greetings on behalf of all of the Johns Hopkins University.
There are three classes of professions who wear robes as a symbol of their special role in our society.
The first are professors, who seek and teach truth.
The second are judges, who dispense justice.
The third are clergy, who contemplate eternity.
Every year at this time, they are joined by a fourth group, the new graduates.

subset: commoncrawl


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May 28, 2013 Agenda
7:00 p.m.
Commissioners Meeting Room
Charles County Government Building
Public Hearings:
Docket #1306 – La Plata Memorial Gardens
Action requested: Appeal of Zoning Officer Decision
Minutes to be approved: April 23, 2013
Orders to be signed:
Docket #1307 – Weatherly Farm, LLC
Docket #1309 – Febrey & Liza Pinggoy
Agenda is tentative and subject to change.
*Agenda items that cannot be completed by 10:30 p.m. will be rescheduled at a future meeting date.
*Portions of this meeting may be in closed session.
*All items scheduled to be discussed or heard at this meeting shall be rescheduled in the event the notified meeting is canceled due to inclement weather or acts of nature beyond the control of the County.


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Keep History Alive Fundraiser
We have a limited number of tickets still available for the event, so stop by the museum for tickets. The items for the silent and live auction have been coming in and we have put together a preliminary catalog. There will be about 80 items total with plenty of business gift certificates and good, travel experiences, sporting event tickets, antiques and collectibles and artwork. There is something for everyone! Click here for the preliminary catalog.


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One of the oldest amusement/theme parks in the city, the VGP Golden Beach Resort is perhaps the most popular landmark along Chennai's East Coast Road. Located at the edge of the city in Injambakkam, the VGP Universal Kingdom amusement park attracts large crowds on the weekends and boasts several new and exciting rides and attractions. The resort is also home to a number of restaurants and drinking options and is one of the most visited commercial attractions in the city.
VGP Universal Kingdom, East Coast Road, Injambakkam, Chennai, Tamil Nadu, India, +91 76678 47847
If you are an art lover driving along the ECR, then the Cholamandal Artists' Village is one stop you should definitely make. One of the most important art destinations in the city, it is impossible to talk about the artistic heritage and culture of Chennai without mentioning Cholamandal, the largest artists' commune in the country. The sprawling campus was founded in 1966 and was the centre of the Madras Art Movement that lasted from the 50s to the 80s. The Village is also home to a gallery that houses some of the important artworks to have come out of Chennai's art scene.
Cholamandal Artists' Village, East Coast Rd, Cholamandal Artists Village, Injambakkam, Palavakkam, Tamil Nadu, India, +91 44 2449 0092
Driving along the ECR is in itself quite a pleasurable experience. But if you're looking for some excitement and speed without endangering your life, your best option is to stop by one of the several offroad attractions along ECR. The most popular among these are Kart Attack and ECR Speedway for go-karting and Offroad Sports ECR for everything else from Jet Skiing and ATVs to even paintball.
Popularly known as Croc Bank, the Madras Crocodile Bank Trust is not only one of the best-known attractions along ECR but is also an extremely important wildlife venue in all of Chennai. Declared a medium zoo in the 90s, the sprawling Croc Bank campus is home to over 2,400 crocodiles and over 4,000 reptiles in total, making it one of the largest reptile conservatories in the country. While visiting the Croc Bank just to witness the majestic reptiles is a worthwhile affair, a highly recommended activity is visiting it in the late evening for the night safari, where visitors can tour the Croc Bank with guides and torches in the dark and witness the crocs at their wildest best.
Madras Crocodile Bank Trust, Post Bag No 4, Mahabalipuram, Tamil Nadu, India, +91 90800 00123


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We all love Banh Mi, you know… those cheap Vietnamese sandwiches that are available at almost every street corner in Vietnam. The following is our list of the best Banh Mi sandwich eateries in and around Saigon:
Address: Hem 41, An Duong, District 5
Review: The Banh Mi Hanoi cart across from An Duong Plaza is the tastiest sandwich we have ever had in Vietnam, and very cheap as well!
Address: 26 Lê Thị Riêng, Bến Thành, 1, Hồ Chí Minh, Vietnam
Open hours: 3:30 pm – midnight, daily
Price: 32,000 VND ($1.50) – this is one of the most expensive in Saigon, but it's also one of the most meat packed, so it's easily worth the price.
Review: This is a meaty expensive banh mi. 2nd best in flavor. like bahn mi sub. ou see many people get line to wait for their turn to buy. Really yummy and I love them the best. ( not far from New world hotel and Ben Thanh Market)
Address: 122 Bùi Thị Xuân, Phạm Ngũ Lão, 1, Hồ Chí Minh, Vietnam
Review: 3rd best, traditional bahn mi sandwich.
Address: 52 Nguyen Van Trang, District 1, Close to Pham Ngu Lao
Price: 15,000VND
Review: A good all around Banh Mi sandwich close to the backpacker district. They make their own bread!
Address: near Ben Thanh Market
Price: 17,000VND and up.
Review: Basic Banh Mi vendor that also makes their own bread. Consistent and reliable


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Voting is your Superpower...don't let anything keep you from using it!
Facts on the new Voter ID Law
Starting in 2020, all voters will be asked to show an acceptable form of ID in order to vote. We want EVERYONE to be able to participate in next year's elections – so make sure that you, your family and your friends are prepared!
Driver's License or Non-Operator ID Card
- Must be unexpired OR expired for one year or less
- REAL-ID is not required.
- If from another state, voter must have registered in NC within 90 day of election
Approved College/University Student ID
Military ID or Veterans ID
- May be expired or have no expiration date
Approved State/Local Government or Charter School Employee ID
US Passport or Passport Card
- Must be unexpired OR expired for one year or less
Tribal Enrollment Card
- Must be issued by a federally recognized or state-approved tribe
- May be expired or have no expiration date
If you are over 65 years of age: You may use ANY acceptable type of photo ID listed above that has been expired for ANY LENGTH OF TIME provided that the ID expired after your 65th birthday.
How to get a FREE Voter ID Card
If you don't have an approved ID to vote, do not worry. You can quickly and easily get a FREE one!
- Go to the county Board of Elections, at 201 N Chestnut St, Winston-Salem, any time before the end of onestop early voting.
- Board of Elections office is open M-F, 8 am - 5 pm.
- You will not need to show any documents to get a Voter ID card, however, you MUST BE A REGISTERED VOTER.
- If you are not registered, you can register to vote and get an ID at the same time.
Frequently Asked Questions about Voter ID
When is Photo ID required?
- Photo ID is NOT required for elections in 2019.
- REAL-ID is not required.
- Starting in 2020, you WILL be asked to show photo ID if you vote in person.
- If you vote absentee by mail, you will still need a photo ID and will be asked to include a copy of it with your absentee ballot.
- If you do not have an acceptable ID, you may sign an affidavit declaring you have a qualifying exception.
If I don't have an ID, can I still vote?
- All registered voters will be allowed to vote with or without a photo ID.
- You can vote a provisional ballot if you do not have an acceptable ID. The provisional ballot will count if you:
- Bring acceptable photo ID to the county Board of Elections no later than 10 days after the election OR
- Sign an affidavit declaring you have qualifying exemption from the requirement.
What are the qualifying exemptions?
- Reasonable impediment that prevents you from presenting a photo ID when you vote in person OR enclosing a copy of your ID when you vote absentee by mail;
- Religious objection to being photographed;
- Declared natural disaster occurring within 100 days before the election


text:
You are essential to our health care team.
See our patient resources for a variety of helpful tools
Learn more about specific conditions we specialize in treating.
Exceptional resources for exceptional health professionals
Research that brings the latest advances to our patients
Learn in a culture of compassion, innovation and excellence.
Find a location for you and your family near work or home
Charles and Anne Duncan Centennial Clinical Academic Scholar in Endocrinology, Department of MedicineAssociate Professor of Clinical Medicine, Academic InstituteHouston MethodistWeill Cornell Medical College


text:
When uploading new documents to ShareFile, a notification
email is sent to all users who have permissions to that folder. It would be extremely helpful if we could customize that email message before it goes out with some specific details about the file that is being uploaded or modified. Seems most users would prefer calling to find out what the changes to the document are rather than opening the file to review it. We may be able to provide all of that information ahead of time which would eliminate unnecessary phone calls and emails.


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- Uzoma Asagwara has been elected into Canada's legislature in the Manitoba province
- The Nigerian-Canadian said that she is glad that the new black representatives in the province are a reflection of the minorities in the community
- It should be noted that she was among the three black people that were elected in the legislature in its 150-year history
Uzoma Asagwara, a Nigerian-Canadian, has been elected into the country's legislative seat for the New Democratic Party.
Her election was a historical mark in the country as she was the first qu*er black person to be elected in Manitoba.
CBC News also reported that she was one of three black people in the 150-year history of the Manitoba legislative house.
As the results were coming in on Tuesday, September 10, Asagwara told CBC News that elected officials in the province should really portray the people and communities that are in Manitoba.
She said that making the elected officials reflect the people is the only way to make sure the people's voices are heard in the province.
It should be noted that Asagwara has worked in Canada as psychiatric nurse, and for long has been a community activist in Winnipeg.
Reacting to the result, the first black Winnipeg councilor said he was glad that immigrants and refugees will now see themselves in the new representatives.
Uzoma said she is grateful for everyone who has supported them long before the election. The Nigerian-Canadian is not only a politician and activist, she is also an athlete.
Meanwhile, Legit.ng earlier reported that Prime Minister Boris Johnson appointed Nigerian Olukemi Olufunto Badenoch as Children and Families' minister, as part of his government reshuffle.
PAY ATTENTION: Download our mobile app to enjoy the latest news update
The 39-year-old Badenoch is a British Conservative politician and Member of Parliament (MP) for Saffron Walden.
NAIJ.com (naija.ng) -> Legit.ng We have upgraded to serve you better
Top 4 Most Influential Nigerians in the World: Nigerians in Top Positions on Legit TV


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The embrace of a more fluid form of masculinity shows that many Chinese are frustrated with the traditional ideas pushed by the establishment.
Mr. Cai belongs to the tribe of "little fresh meat," a nickname, coined by fans, for young, delicate-featured, makeup-clad male entertainers. These well-groomed celebrities star in blockbuster movies, and advertise for cosmetic brands and top music charts. Their rise has been one of the biggest cultural trends of the past decade. Their image — antithetical to the patriarchal and stoic qualities traditionally associated with Chinese men — is changing the face of masculinity in China.
Innocent as they may seem, the little fresh meat have powerful critics. The state news agency Xinhua denounces what it calls "niangpao," or "sissy pants," culture as "pathological" and said in an editorial last September that its popularity is eroding social order. The Beijing newspaper's decision to include Mr. Cai in its profiles apparently prompted the Communist Youth League to release its own list of young icons: patriotic athletes and scientists, whom it called the "true embodiment" of the spirit of Communist youth.
The government attacks on this evolving idea of masculinity have triggered a strong counter-backlash from fans of the celebrities. And in online essays and posts, defenders of the young men make clear that their preference is more than a youthful countercultural fad. At its heart, the embrace of a more modern, less rigid form of masculinity represents frustration with traditional ideas of manhood.
"The ridiculous condemnation of 'sissy pants' men shows the gender ideology of a patriarchal society that equates toughness with men and fragility with women," a journalist who goes by the name Wusi wrote in an online essay in September, voicing a widely shared opinion.
The official push of traditional masculinity — including reinvented school curriculums and the sponsorship of boys-only clubs — is motivated in part by worries that the decades-long one-child policy produced a generation of timid and self-centered male youth ill equipped to fulfill their social responsibilities.
And in the context of China's increasing power, the establishment's preoccupation with promoting old-fashioned, Hollywood-style manliness also has a political message. Just as patriotic intellectuals a century ago argued that national strength derives from the virile energy of the youth, present-day Chinese nationalists see their ambitions take the shape of a macho willingness to fight for righteous causes.
This vision is on display in the 2017 action thriller "Wolf Warrior 2." The movie, featuring a former People's Liberation Army soldier caught in an African civil war, showed him putting the lives of local civilians above his own while single-handedly beating American-led mercenaries. The goal of the story, said Wu Jing, its director and lead actor, in media interviews, is to "inspire men to be real men." The movie went on to become China's top-grossing film in history.
There is little question about who in real life is meant to best personify the masculine chauvinism characterizing the official line today: Take a stroll down a city street or switch on the television at news hour — and you are greeted by the face of President Xi Jinping with a perennial look of self-assurance and determination.
How the Patriarchy Protects Itself
The Brett Kavanaugh circus shows us how little has changed
Why Trump Reigns as King Cyrus
The month before the 2018 midterms, a thousand theaters screened "The Trump Prophecy," a film that tells the story of Mark Taylor, a former firefighter who claims that God told him in 2011 that Donald Trump would be elected president.
At a critical moment in the film, just after the actor representing Mr. Taylor collapses in the flashing light of an epiphany, he picks up a Bible and turns to the 45th chapter of the book of Isaiah, which describes the anointment of King Cyrus by God. In the next scene, we hear Mr. Trump being interviewed on "The 700 Club," a popular Christian television show.
As Lance Wallnau, an evangelical author and speaker who appears in the film, once said, "I believe the 45th president is meant to be an Isaiah 45 Cyrus," who will "restore the crumbling walls that separate us from cultural collapse."
Cyrus, in case you've forgotten, was born in the sixth century B.C.E. and became the first emperor of Persia. Isaiah 45 celebrates Cyrus for freeing a population of Jews who were held captive in Babylon. Cyrus is the model for a nonbeliever appointed by God as a vessel for the purposes of the faithful.
The identification of the 45th president with an ancient Middle Eastern potentate isn't a fringe thing. "The Trump Prophecy" was produced with the help of professors and students at Liberty University, whose president, Jerry Falwell Jr., has been instrumental in rallying evangelical support for Mr. Trump. Jeanine Pirro of Fox News has picked up on the meme, as has Ron Dermer, the Israeli ambassador to the United States, among many others.
As the Trump presidency falls under siege on multiple fronts, it has become increasingly clear that the so-called values voters will be among the last to leave the citadel. A lot of attention has been paid to the supposed paradox of evangelicals backing such an imperfect man, but the real problem is that our idea of Christian nationalism hasn't caught up with the reality. We still buy the line that the hard core of the Christian right is just an interest group working to protect its values. But what we don't get is that Mr. Trump's supposedly anti-Christian attributes and anti-democratic attributes are a vital part of his attraction.
Today's Christian nationalists talk a good game about respecting the Constitution and America's founders, but at bottom they sound as if they prefer autocrats to democrats. In fact, what they really want is a king. "It is God that raises up a king," according to Paula White, a prosperity gospel preacher who has advised Mr. Trump.
Ralph Drollinger, who has led weekly Bible study groups in the White House attended by Vice President Mike Pence and many other cabinet members, likes the word "king" so much that he frequently turns it into a verb. "Get ready to king in our future lives," he tells his followers. "Christian believers will — soon, I hope — become the consummate, perfect governing authorities!"
The great thing about kings like Cyrus, as far as today's Christian nationalists are concerned, is that they don't have to follow rules. They are the law. This makes them ideal leaders in paranoid times.
This isn't the religious right we thought we knew. The Christian nationalist movement today is authoritarian, paranoid and patriarchal at its core.
- They aren't fighting a culture war.
- They're making a direct attack on democracy itself.
They want it all. And in Mr. Trump, they have found a man who does not merely serve their cause, but also satisfies their craving for a certain kind of political leadership.
The Patriarchy Will Always Have Its Revenge
I want to burn the frat house of America to the ground... I was riveted by the hearings, and Professor Hill's testimony about how her old boss, the Supreme Court nominee Clarence Thomas, behaved — the references to pornographic movies, to his own sexual prowess, the way he would ask her out, again and again, and not take no for an answer... It's one thing to say #MeToo, but if I find out it's them, too, I can picture myself hunting down the man who hurt them and dismembering him with my fingernails and burning the whole world down... When Clarence Thomas won his seat, I felt like someone had taken an eraser to the core of my being, and had rubbed a bit of me away. I felt diminished, a little less real, and, certainly, a lot less likely to be believed if I had anything to say about male colleagues... Bill Cosby was found guilty. Harvey Weinstein is going to trial. Les Moonves lost his job as chief executive of CBS, even if a CBS board member, Arnold Kopelson, said, "I don't care if 30 more women come forward and allege this kind of stuff.".. One by one, like bad dreams, the #MeToo men have come back from the allegations against them, having suffered — if that's even the right word — the equivalent of a misbehaving child's timeout.
.. Matt Lauer is swanning around Upper East Side steakhouses, reportedly assuring fans that soon he'll be "back on TV." Louis C.K. returned to the stage. John Hockenberry is telling his story in Harper's Magazine, and Jian Ghomeshi is telling his in The New York Review of Books.
.. Women aren't supposed to want revenge any more than we're supposed to be angry. It's not socially approved, not attractive, not ladylike. We swallow our pain and keep our own behavior exemplary while excusing the bad behavior of others, knowing, from examples like Professor Hill's, what could happen if we speak up, and what we stand to lose.
.. There are famous novels, canonical plays, entire genres of movies centered around men seeking revenge (the "Iliad," "Hamlet," every western ever). There aren't many stories about men righting their wrongs; even fewer about women making men sorry.


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Job description
At Arup, we design and deliver ground-breaking icons through our work in infrastructure, building design and specialist technical services. Independent in ownership and spirit, we're a global firm of designers, planners, engineers, consultants and technical experts who use our skills to make a positive difference in the world.
Due to our global reach and multi-disciplinary approach, Arup has been successful in the assessment and development of major sites for more than 30 years. We typically take on challenging sites and through the application of our skills, and collaboration with colleagues and other professionals, we prepare the sites to be consented and enabled for a range of end uses including small to large mixed-use developments, including housing and employment uses, public spaces, urban and transport related infrastructure and energy generation and distribution. We also provide support on a wide range of projects in the built environment including designing external drainage, utilities, highways and public realm for hospitals, sports stadia, educational and commercial buildings.
Current projects are varied, exciting and often high profile. They include the design of external works for major developments and of infrastructure across the UK, international projects, and site development projects on brownfield land where our integrated approach can radically influence what is built and where thus improving the quality of peoples' lives and of the local environment.
As one of the world's leading building and infrastructure design consultancies, we offer clients the innovation and technical know-how to continually deliver their projects on schedule and on budget, but without compromise.
About the role
We are currently recruiting for Civil Summer Students to join our well-established team across the UK. This is an excellent opportunity for students that are enthusiastic and knowledgeable about the Infrastructure sector to join a multi-disciplinary team for 4-8 weeks (dependent upon location) this summer.
You'll help us to deliver effective and professional services for a range of varied and high-profile clients. While you'll have a great deal of responsibility from day one, we'll give you the chance to take on more as you progress and broaden your skills.
Depending on performance, there may be an opportunity to receive a permanent offer of employment upon graduating.
What we offer
There are infinite possibilities with Arup. You'll receive an insight of the opportunities that a challenging, fulfilling career provides, and:
- A competitive salary
- An interesting and varied workload where you'll make a tangible difference to the world
- A collaborative, intelligent and friendly team in an excellent working environment
- A challenging workload that will stretch your mind and make the most of your skills
- The support you need to develop your skills
What we are looking for
We're looking for graduates with curiosity, personal drive and technical knowhow.
- Demonstrate strong motivation and passion for a career in civil engineering
- Ideally graduating in Summer 2021 or 2022
- Interest in and application of digital skills, such as computer coding for design automation and problem-solving is an advantage.
- Strong A-levels (or equivalent) at Grade B or above
- Studying a BEng or MEng in civil engineering
- On track to achieve a 2.1 or above, as proven through the modules that you have completed to date
Accepted degree subjects
Additional job details
- Location
- Cardiff
- Sheffield
- Solihull
- Salary
- Competitive salary
How to apply
Click Apply to start your application now. This job will be available on Prospects until 27 January 2020
Don't forget to mention Prospects to employers when you contact them.
Closing date: 27 January 2020


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Unlike Oculus, which charges a fee for each Rift devkit, the Vive will be completely free for qualified developers.
Developers looking to get their hands on a devkit for Valve's recently revealed Vive VR headset will be pleased to learn that Valve will be providing the devices absolutely free of charge to whoever passes its qualification standards.
So far, Valve and HTC have offered kits to a handful of specially chosen developers, including Owlchemy Labs (Aaaaaa! For The Awesome), Bossa Studios (Surgeon Simulator, I Am Bread), Fireproof Games (The Room), and Cloudhead Games (The Gallery). Valve's Doug Lombardi told Ars Technica that a sign-up page for aspiring devs would be coming over the next week, allowing all devs to submit themselves to Valve's qualification process.
Approved developers will get a Developer Edition kit pf the Vive that "will be free, at least initially," Lombardi said. Those kits will start shipping later in the spring as part of an "ongoing effort" to get the development hardware out widely ahead of the headset's planned 2015 consumer launch.
For comparison, Oculus is currently asking devs to fork over $350 for the latest version of its Rift devkit.
Valve's scheme comes with its own set of pluses and minuses compared to how Oculus is doing things. For example, offering the devkit as a paid product means that both members of the public and the press can easily get their hands on it, but offering it for free to approved developers means that devs will have more of an incentive to program games for the device.
Either way, it won't be long before we see the Vive in action for ourselves.
Source: Ars Technica


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Last week the Global Awareness Committee started what will hopefully become an annual fundraising event: The Hunger Banquet. The idea of this event is that ticket holders are allocated either a large and delicious dinner or a bowl of rice and an apple – or a plain but wholesome dinner – according to luck, just as in real life some of us are born into relative prosperity and others into poverty.
We were then also lucky enough to listen to Beth John from Christian Aid who spoke about malnutrition as part of a larger humanitarian crisis in places like South Sudan and Columbia.
All the tickets sold out and we managed to raise £455 to go towards Médecins Sans Frontières, the NGO Global Awareness is currently supporting. This is a great step, and along with the avocados and fruit being sold at breakfast, and the clothes we're sending to the refugees in Calais, Bedales is increasing the positive impact it has on the global community.
More photos can be found here.
By Hebe Bartlett, 6.2


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Lavenders sweet, woodland scent calms and soothes everything from headaches and tension to rashes, burns and insect bites. This moisturizing lotion is made from the world's finest organic lavender and skillfully blended for the most effective results!
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Now you can take it anywhere! This intensely rich creme is packed with shea butter and will leave your skin saying Oooh la la! Paraben free - we added natural extracts and oils of almond, apricot and vanilla. You (and your skin) are sure to fall in love with this super hydrating crème!Available in 2 oz.
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You have found the best natural deodorant in the world! Its scent certainly tells the story. Crisp, clean and natural. Grapefruit seed extract gently destroys bacteria, the real cause of odor. What a great alternative to the chemical-ridden deodorants we see on the big-store shelves. Does it work? Absolutely. Try a few sprays under your arms and on your feet to stop odor dead in its tracks! Also works fabulously for these alternative uses: deodorizing shoe's, deodorizing trash cans and diaper pails. Gallon
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tag:blogger.com,1999:blog-31155409.post6327997889480038522..comments2015-09-17T07:41:44.110+10:00Comments on Real UFOs - The latest UFO videos and News from all over the world ufo video photos ovnis ufos ovni: Dr. Carl Sagan knew UFOs were firstname.lastname@example.com:blogger.com,1999:blog-31155409.post-18306427980107716632011-12-16T06:58:54.664+11:002011-12-16T06:58:54.664+11:00It is obvious our Government has aerial vehicle pr...It is obvious our Government has aerial vehicle programs it wishes to remain secret so the UFO conspiracy-by-shadow-government-story works quite well in sending people off on wild goose chases while they work. It also seems to prepare the general public for facing the reality that something is going on and needs to be addressed leading to the inevitable funding of the military to protect us from evil alien invasion. The downed drone in Iran tells you everything you need to know about U.S. government motives. Instead of worrying about UFO's we should be trying to travel the stars and become them.Dave email@example.com:blogger.com,1999:blog-31155409.post-65377545104985307342011-09-22T09:55:22.362+10:002011-09-22T09:55:22.362+10:00Dr. Sagan said, on many occasions, that it was pos...Dr. Sagan said, on many occasions, that it was possible that extraterestials may have visited us thousands of years ago...
And they may visit us again thousands of years from now....
 But they are not visiting us now...
 I always wondered why he was so adamant about that... Now I guess we know19ken48http://email@example.com:blogger.com,1999:blog-31155409.post-61204156331953978812011-09-22T09:49:40.954+10:002011-09-22T09:49:40.954+10:00On many of Dr. Sagan's visits to the Tonight S...On many of Dr. Sagan's visits to the Tonight Show, he would say he believed extraterestials may have visited us thousand's of years ago, and they may come back thousand's of years from now... But, they are not visiting us now...
 I always wondered why he was so adamant about that... Now I suppose we know why19ken48http://firstname.lastname@example.com:blogger.com,1999:blog-31155409.post-82340427619367582312010-05-13T01:08:51.249+10:002010-05-13T01:08:51.249+10:00No, I think that Dr. Jonathan Reed met w/ Ivan Mar...No, I think that Dr. Jonathan Reed met w/ Ivan Marx & Professor Irwin Corey regarding his "alien encounter".

The only alien encounter experienced by Dr. Reed is with a 16 yr old Mexican girl in El email@example.com:blogger.com,1999:blog-31155409.post-57235834573117242582010-05-11T13:57:36.083+10:002010-05-11T13:57:36.083+10:00Dr. Jonathan Reed may also have met with Sagan and...Dr. Jonathan Reed may also have met with Sagan and received advice from him in regards to his alien firstname.lastname@example.com


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Stressing yourself when you are in debt will do more harm than good not only to your financial health but also to your mental and physical health. Ideally, when you are in debt, you must admit it instead of ignoring it. This will in turn help you to remain stress free. When you have no stress in your mind you will be able to think logically and take the right financial decisions.
You may face problems paying your medical bill or the monthly loan statements, credit card balances and others but all that requires coming out of it and restoring peace in mind is a proper plan. Such a plan will help you to deal with your debt and in turn stay healthy.
If you are prudent, strategic and organized, there is no way debt stress can affect your health, mentally or physically, no matter how large a debt you have. It will not affect family relationships, social bindings or your professional career as well.
With a few easy and simple steps, you can avoid it all. To start with you must admit that you are in debt. This is when you will feel that almost half of the stress is gone. Running away from debt, not attending the collection calls or not responding to the mails will not do any good to your financial as well as physical health. You will experience anxiety and depression, two most significant cause of a large number of health issues.
Instead, you should think proactively so that you can come out with an effective solution to prevent your debt from building up. Do not let debt affect your thought process as that will result in irrational decision.
Have courage to speak up
You will see that when you admit your debt, things will become easy. This is how.
- You will have the courage to talk about it with others. More often than not, they will come up with effective solutions and suggestions that will help you in attaining a stress free, if not debt free life. It is true that you may feel it a bit difficult to discuss about your finance with your close ones, but then you can always talk to the professional debt counselors for that matter.
- You will never be late to respond to your debt and thereby prevent it from affecting your health further resulting in some chronic health problems. You will be surprised to know that debt stress and anxiety may lead to diseases such as blood pressure, insomnia and even heart diseases and stroke.
- Stop ignoring your monthly bills. Instead, call up your creditors and tell them about your hardship, your current condition and your future plans. Be honest and reasonable and do not make any promises that you cannot keep. Often, the creditors will suggest plans that will help you to deal with your debts.
- You will get enough confidence when you admit your debts and take proactive measures. You can then look for other alternative ways to repay your debts such as by taking out a debt consolidation loan or opting for a debt settlement. However, you are suggested to research well and read the debt consolidation ratings to find the best agency to work with to avoid future complicacies.
- Set aside some money every month apart from being diligent to pay off at least the minimum payments of one or two of your outstanding bills. You will see that even if you can clear a single debt of a small amount you will get a feeling of achievement. This will change your perspective about debt and finance management.
All these proactive steps will help you to bring down your debt to a manageable level. You will have the moral boost to continue dealing with your debt more effectively and head on.
Get help to address stress
Managing debt is a difficult process and most borrowers find it demoralizing while continuing with their debt management plans. The best and most effective way to deal with debt and take control of the deteriorating mental health is to get help to manage your debts, existing as well as the new ones more efficiently.
You will come across a large number of charitable organizations and nonprofit credit counseling agencies that will be more than happy and ever ready to help you out from such stressful situations that is slowly but surely affecting your health. You can search for them in the internet or look up in the debt directory to find some reliable names.
If you want a peaceful state of mind then you will have to address stress and debt with a little bit of belief in you and following a few simple and disciplined steps. These agencies will tell you a lot about such steps that may include:
- Staying fit and healthy – This is the primary key to lessen stress and worries that are usually associated with debt. Eating a balanced and healthy diet will also provide you with the extra energy required to do some extra work to earn a little bit extra that you can put towards your debt repayment.
- Psychological aspects – Take a look at the emotional and psychological aspects when you are depressed. This will help you a lot to deal with your impulsive spending habit and prevent it from becoming an addiction.
Last but not least, you must stake the safest path to be free from debt stress by making sure that you do not take on a debt when you know you cannot afford to repay it on time. This is especially required when you are prone to use your credit cards too often and sometimes unnecessarily. This will help you to keep your debt manageable at all times which in turn will keep you healthy, mentally and physically. Remember, taking on debt is very easy but getting out of it may take time and a lot of discipline but is not impossible at all. The bottom line is: admit debt and believe in yourself!


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Make a list of your favourite things by clicking on the heart. This will come in handy when placing your orders.Start your favourites list now New to Abel & Cole? Sign up here
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Order now for deliveries from 27th January
At this time of year our tenderstem broccoli is from a farming cooperative in Murcia, Spain. They've been growing organic veg about as long as we've been packing it in boxes, and specialise in brassicas and lettuce. They are ambassadors of the organic ethos and like us, want to help everyone, everywhere to eat organic.
Abel & Cole handles celery, celeriac and nuts on site. Due to our packing process and re-use of boxes there is a risk of cross-contamination from these, and all other allergens.


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Ready to Get Grilling? A Grill Master's Checklist
That itch to be outdoors is getting harder to ignore as the weather warms up! There are an endless amount of activities to keep you outdoors – hiking, camping, swimming, and barbecuing! Our Grill Masters are soon to be cleaning off their grills to get to cooking. Here is a brief checklist to make sure
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How to Pack a Cooler with Dry Ice
Are you ready to go on a weekend getaway with family or friends? Memorial Day is right around the corner! Knowing how to pack a cooler, using dry ice, can ensure your perishables stay cold longer. Dry ice proves to be a superior option than regular ice, especially when going on an extended trip to
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Betting for a Cause at St. Catherine's Annual Gala
St. Catherine's annual SCCCript & SCCCreen Gala took place on Friday, May 3rd at the Franklin Plaza in Troy, NY. This year's Gala was Casino Royale themed, with a cut-out of 007 himself greeting you at the door! There was quite the assortment of activities ready for guests' entertainments. Plus, an endless amount of ways
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Starting a Customer Appreciation Tradition
What a successful 1st Customer Appreciation Day! On Thursday, April 18th Noble Gas Solutions hosted its first ever Customer Appreciation event at the newly renovated Showroom in Albany, NY. The event gave customers a chance to peek into the inner workings of their reliable gas supplier! Did you miss April's event? Don't worry – we
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Internship Opportunity: Operations Management
We are looking for a motivated, detail-oriented, and collaborative individual to join our Noble Team this Summer as an Operations Management Intern! This individual would be working closely with our Operations and Customer Service teams to improve upon current processes, roll-out new procedures, and assist in completing ongoing projects. We are seeking out an individual
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Job Posting: Customer Service Representative – Albany, NY
Position Title: Customer Service Representative – Warehouse Specialist Reporting To: Branch Manager Position Overview Assist with all store location activities to include warehousing, merchandising, customer service and sales. Responsibilities Provide warehouse support for shipping & receiving, inventory stocking, cleaning, labeling, or other tasks. Restock store merchandise as needed. Arrange stock on shelves or racks in the
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Safety Tips: MIG Welding
MIG welding is a very popular form of welding. Welders can be easily trained to produce quality welds that are both fast and inexpensive. Like all welding, steps must be taken to ensure the safety of workers and people passing by. Follow these easy steps to avoid injury in the workplace. Proper Clothing The proper
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SAVE THE DATE: Customer Appreciation Day
Noble Gas Solutions will be hosting its first ever Customer Appreciation Day! We're taking a moment to say "Thank You" to our loyal customers for always choosing Noble Gas Solutions. SAVE THE DATE and join us for this FREE Event on Thursday, April 18th at our Albany Showroom! Noble Gas Solutions will be providing lunch
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Meet our Newest Driver – Tony!
In the month of March, Noble Gas Solutions was proud to welcome Anthony (Tony) DeRusso to our Noble Team as a Driver! Tony has been in training for the past couple of weeks, learning from our team of drivers. He has been attentive throughout his training and eager to improve with the help of his
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Store Remodel for Customer Experience in Albany
This past month, our Albany Store has been undergoing a major remodel. Noble Gas Solutions values the customer experience. After all, it's what the Noble Difference is all about! What's New? We have made it easier for our customers to walk in and shop around our beautiful showroom! This face-lift actually started from the bottom
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Deaths Death Notices
CARPENTER, Donald F.
CARPENTER - Donald F. January 20, 2018. Beloved husband of 63 years to Betty "Elizabeth" L. (nee Neubecker) Carpenter; devoted father of Dawn (Anand) Choudri, Sue (John) Freeman-Russo, and Tina (Gregory) Anzalone; dearest grandfather of Steve (Jackie) Freeman, Jessica (Vincent) Ricchiazzi, Jennifer (John) Marsella, Christine Choudri, Aja Anzalone, and Anthony Anzalone; loving great-grandfather of Kaylie, Sophia, Peyton, Mason, and Carter; survived by two sisters, Shirley and Rose Marie; also survived by many loving nieces and nephews. The family will be present Wednesday and Thursday from 2-4 and 7-9 PM at the (Amherst Chapel) AMIGONE FUNERAL HOME, INC., 5200 Sheridan Drive (corner Hopkins Road) where prayers will be offered on Friday at 9:45 AM, followed by a Mass of Christian Burial at 10:30 AM from St. Mary's RC Church (Swormville). If so desired, donations may be made in Donald's memory to Hospice Foundation of WNY, 225 Como Park Blvd., PO Box 590, Cheektowaga, NY 14240-0590. Share condolences online at www.amigone.com