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Conceived and designed the experiments: SJ YJC RP. Performed the experiments: SJ YJC. Analyzed the data: SJ YJC. Contributed reagents/materials/analysis tools: SJ YJC. Wrote the paper: SJ YJC RP.
Large-scale DNA deformation is ubiquitous in transcriptional regulation in prokaryotes and eukaryotes alike. Though much is known about how transcription factors and constellations of binding sites dictate where and how gene regulation will occur, less is known about the role played by the intervening DNA. In this work we explore the effect of sequence flexibility on transcription factor-mediated DNA looping, by drawing on sequences identified in nucleosome formation and ligase-mediated cyclization assays as being especially favorable for or resistant to large deformations. We examine a poly(dA:dT)-rich, nucleosome-repelling sequence that is often thought to belong to a class of highly inflexible DNAs; two strong nucleosome positioning sequences that share a set of particular sequence features common to nucleosome-preferring DNAs; and a CG-rich sequence representative of high G+C-content genomic regions that correlate with high nucleosome occupancy in vivo. To measure the flexibility of these sequences in the context of DNA looping, we combine the in vitro single-molecule tethered particle motion assay, a canonical looping protein, and a statistical mechanical model that allows us to quantitatively relate the looping probability to the looping free energy. We show that, in contrast to the case of nucleosome occupancy, G+C content does not positively correlate with looping probability, and that despite sharing sequence features that are thought to determine nucleosome affinity, the two strong nucleosome positioning sequences behave markedly dissimilarly in the context of looping. Most surprisingly, the poly(dA:dT)-rich DNA that is often characterized as highly inflexible in fact exhibits one of the highest propensities for looping that we have measured. These results argue for a need to revisit our understanding of the mechanical properties of DNA in a way that will provide a basis for understanding DNA deformation over the entire range of biologically relevant scenarios that are impacted by DNA deformability.
Although it has been known since the work of Jacob and Monod that genomes encode special regulatory sequences in the form of binding sites for proteins that modulate transcription, only recently has it become clear that genomes encode other regulatory features in their sequences as well. Further, with the advent of modern sequencing methods, it is of great interest to have a base-pair resolution understanding of the significance of the entirety of genomes, not just specific coding regions and putative regulatory sites.
One well-known example of other information present in genomes is the different sequence preferences that confer nucleosome positioning [1]–[3], with similar ideas at least partially relevant in the context of architectural proteins in bacteria also [4]. It has been shown both from analyses of sequences isolated from natural sources and from in vitro nucleosome affinity studies with synthetic sequences that the DNA sequence can cause the relative affinity of nucleosomes for DNA to vary over several orders of magnitude, most likely due to the intrinsic flexibility, especially bendability, of the particular DNA sequence in question [3], [5]–[8]. The claim that intrinsic DNA sequence flexibility determines nucleosome affinity has led not only to many theoretical and experimental studies on the relationship between sequence and flexibility [9]–[15], but also to the elucidation of numerous sequence "rules" that can be used to predict the likelihood that a nucleosome will prefer certain sequences over others (summarized recently in [2], [7]). For example, AA/TT/AT/TA steps in phase with the helical repeat of the DNA, with GG/CC/CG/GC steps five base pairs out of phase with the AA/TT/AT/TA steps, are a common motif in both naturally occurring and synthetic nucleosome-preferring sequences [3], [7]. Similarly, the G+C content of a sequence and occurrence of poly(dA:dT) tracts have been very powerful parameters in predicting nucleosome occupancy in vivo [2], [16]–[19]. Our aim here is to explore the extent to which these sequences, when taken beyond the context of cyclization and nucleosome formation to another critical DNA deformation motif, exhibit similar effects on a distinct kind of deformation.
There has been an especially long history of the study of these intriguing sequence motifs known as poly(dA:dT) tracts, in the context of nucleosome occupancy as well as many other biological contexts. Such sequences, composed of 4 or more A bases in a row ( with ) or two or more A bases followed by an equal number of T bases ( with ), strongly disfavor nucleosome formation, both in vivo [20]–[23] and in vitro [24]–[28], and are in fact thought to be one of the primary determinants of nucleosome positions in vivo [2], [21], with their presence upstream of promoters and in the downstream genes correlating with increased gene expression levels [20], [29], [30]. Poly(dA:dT) tracts show unique structural and dynamic properties in a variety of in vitro and in vivo assays (summarized recently in [21], [31]), with one of their hallmark characteristics being a marked intrinsic curvature [31]. There is evidence that poly(dA:dT) tracts may also be less flexible than other sequences [32]–[34], which is often given as the reason for their low affinity for nucleosomes, though there is some evidence that poly(dA:dT) tracts might actually be more flexible than other sequences [9]. It is clear, however, that some special property or properties of A-tracts leads them to be especially resistant to the deformations that are required for DNA wrapped in a nucleosome [21], [31], and, indeed, to their important functions in several other biological contexts as well [31].
In this work, we make use of sequences that, in the context of nucleosome formation and cyclization assays, appear to be associated with distinct flexibilities as a starting point for examining the question of what sequence rules control deformations induced by a DNA-loop-forming transcription factor, as opposed to those induced in nucleosomes. We have previously argued using two synthetic sequences that DNA looping does not necessarily follow the same sequence-dependent trends as do nucleosome formation and cyclization [35]. Here we expand our repertoire of sequences to specifically test the generalizability of three sequence features known to be important in nucleosome biology and cyclization. We focus in particular on the intriguing class of nucleosome-repelling, poly(dA:dT)-rich DNAs that are thought to be especially resistant to deformation, making use of a naturally occurring poly(dA:dT)-rich sequence that forms a nucleosome-free region at a yeast promoter [23]. We note that the poly(dA:dT)-rich DNA we use here differs from the phased A-tracts that have been extensively characterized in the context of DNA looping, both in vivo and in vitro [36]–[44]. Phased A-tracts contain short poly(dA:dT) tracts spaced by non-A-tract DNAs such that the poly(dA:dT) tracts are in phase with the helical period of the DNA, generating globally curved structures that are known to significantly enhance DNA looping [36]–[40]. The poly(dA:dT)-rich sequence we examine here contains unphased A-tracts that we do not anticipate to have a sustained, global curvature.
We compare the effects on looping of this poly(dA:dT)-rich DNA not only to the effects of two synthetic sequences we have previously studied, but also to those of two additional naturally occurring, genomic sequences: the well-known, strong nucleosome positioning sequence 5S from a sea urchin ribosomal subunit [45], which, along with the 601TA sequence we previously studied, contains the repeating AA/TT/TA/AT and offset GG/CC/CG/GC steps that are common in nucleosome-preferring sequences; and one of the GC-rich sequences that are abundant in the exons and regulatory regions (e.g. promoters) of human genes, and that correlate with high nucleosome occupancy in vivo [18], [19], [22]. The 5S sequence has been examined using both in vitro cyclization and in vitro nucleosome formation assays and, along with the two synthetic sequences E8 and 601TA [8], [46], can be used as a standard for comparison between our and other in vitro assays. The five sequences used in this work and their effects on nucleosomes are summarized in Table 1.
To measure the effect of these sequences on looping rather than nucleosome formation, we made use of a combination of an in vitro single-molecule assay for DNA looping, called tethered particle motion (TPM) [47]–[50], with the canonical E. coli Lac repressor to induce looping, and a statistical mechanical model for looping that allows us to extract a quantitative measure of DNA flexibility, called the looping J-factor, for the DNA in the loop [35], [51]. We have recently demonstrated [35] that this combined method offers a powerful and complementary approach to established assays that have been used to probe the mechanical properties of DNA, particularly at short length scales, to great effect, such as ligase-mediated DNA cyclization [8], [15], [52]–[57] and measured DNA end-to-end distance by fluorescence resonance energy transfer [34], [58]. In particular, using the Lac repressor as a tool to probe the role of DNA deformability in loop formation allows us to examine the effect of sequence on the formation of shapes other than the roughly circular ones formed by cyclization and nucleosome formation, which we have argued may be an important caveat to discovering general flexibility rules from nucleosome formation and cyclization studies alone [35].
Interestingly, we find that the poly(dA:dT)-rich sequence that strongly excludes nucleosomes in vivo [23] and that belongs to a class of sequences usually thought of as highly resistant to deformation is in fact the strongest looping sequence we have studied so far. Moreover, the 5S and TA sequences, which share sequence features important to nucleosome formation (see Figures S1 and S2 in File S1 and Ref. [7]) as well as trends in apparent flexibility in in vitro cyclization and nucleosome formation assays [8], [59], [60], behave very differently from each other in the context of looping. We also find that G+C content, a good predictor of nucleosome occupancy, is not likewise positively correlated with looping, and in fact our data suggest the G+C content and looping may be anticorrelated. Taken together, these results strongly suggest that very different sequence rules determine DNA looping versus cyclization and nucleosome formation, possibly because of the protein-mediated boundary conditions that differ between looping geometries and nucleosomal geometries, and that the biophysical characteristics of poly(dA:dT)-rich DNAs and their biological functions may be more diverse and context-dependent than has been previously appreciated.
Our experimental approach to examining the effect of DNA sequence on looping combines an in vitro single-molecule assay for DNA looping, called tethered particle motion (TPM) [47]–[50], with a statistical mechanical model that allows us to extract biological parameters from the single-molecule data [35], [51]. As shown schematically in Fig. 1(A), in TPM, a microscopic bead is tethered to a microscope coverslip by a linear piece of DNA, with the motion of the bead serving as a reporter of the state of the DNA tether: the formation of a protein-mediated DNA loop in the tether reduces the motion of the bead in a detectable fashion [47]–[50]. We use the canonical Lac repressor from E. coli to induce DNA loops. Because more readily deformable sequences allow loops to form more easily, we can quantify sequence-dependent DNA flexibility by quantifying the looping probability, which we calculate as the time spent in the looped state divided by the total observation time (see Methods for details).
More precisely, our statistical mechanical model (described in the Methods section) allows us to extract a parameter called the looping J-factor from looping probabilities [35]. The J-factor is the effective concentration of one end of the loop in the vicinity of the other, analogous to the J-factor measured in ligase-mediated DNA cyclization assays [52], [61], and is mathematically related to the energy required to deform the DNA into a loop, , according to the relationship:
where ( being Boltzmann's constant and the temperature). A higher J-factor therefore corresponds to a lower free energy of loop formation. In the case of cyclization, where the boundary conditions of the ligated circular DNA are well understood, the J-factor can be expressed in terms of parameters describing the twisting and bending flexibility of the DNA, and its helical period [10], [15], [62], [63]. However, in the case of DNA looping by the Lac repressor, where the boundary conditions are not well known (summarized in Fig. 4 of [35]), an expression for the looping J-factor in terms of the twist and bend flexibility parameters of the loop DNA has not been described. Nevertheless, by measuring the J-factors for different sequences, we can comparatively assess the effect of sequence on the energy required to deform the DNA into a loop, and thereby gain insight into the sequence rules that control this deformation.
Given that 5S and TA share both sequence features and similar trends in apparent flexibility in the contexts of nucleosome formation and cyclization [8], [59], [60], we expected these two sequences to behave similarly to each other in the context of looping. On the other hand, since poly(dA:dT)-rich sequences are supposed to assume such unique structures as to strongly disfavor nucleosome formation [21], [31], while high GC content is one of the strongest predictors of high nucleosome occupancy [17], [22], we expected these two sequences to behave very differently from each other in the context of looping. Given the common assumption that poly(dA:dT)-rich DNAs are highly resistant to deformation, we especially did not expect to observe much, if any, loop formation with the poly(dA:dT)-rich, nucleosome-repelling sequence.
As shown in Fig. 1, none of these expectations were borne out. TA and 5S do not behave similarly, nor do CG and poly(dA:dT) behave especially dissimilarly, nor does poly(dA:dT) resist loop formation. Moreover, the behavior of these special nucleosome-preferring or nucleosome-repelling sequences is dependent on the larger DNA context, in that the addition of the 36-bp bacterial lacUV5 promoter sequence to these roughly 100-bp loops changes the relative looping probabilities of the five sequences (see Methods for the rationale behind the inclusion of this promoter). Without this promoter sequence (Fig. 1(C)), the two synthetic sequences, E8 and TA, exhibit comparable amounts of looping, while the three natural sequences, including both 5S and poly(dA:dT), all loop more than either E8 or TA. With the promoter (Fig. 1(D)), however, TA loops more than E8, but 5S less than either E8 or TA. Both with and without the promoter the supposedly very different GC-rich and poly(dA:dT)-rich DNAs loop more than the random E8 sequence. The looping probabilities of the poly(dA:dT) sequence are especially surprising—instead of looping very little, as we expected, this sequence loops more than any other sequence without the promoter and a comparable amount to TA with the promoter.
These five sequences differ not only in looping probability, but also in the loop length at which that looping is maximal: the poly(dA:dT) sequence is maximized at 104 bp, the 5S and CG sequences at 105 bp, and the E8 and TA sequences at 106 bp. These different maxima could be explained by different helical periods for these five DNAs, though without more periods of data we cannot definitively quantify their helical periods. In the case of the poly(dA:dT) sequence, an altered helical period would not be unexpected, as pure poly(dA:dT) copolymers are known to have shorter helical periods (10.1 bp/turn) than random DNAs (10.6 bp/turn) [64], [65]. On the other hand, 5S exhibits the same helical period as E8 and TA in cyclization assays [60], so it is intriguing that its looping maximum occurs at a different length than that of E8 and TA, perhaps suggesting a different helical period in the context of looping than that of E8 and TA. The promoter does not appear to alter the maximum of looping for a given sequence. As noted above, it is difficult to use these looping data to comment further on other DNA elasticity parameters, in particular any sequence-dependent differences in torsional stiffness, but in Fig. S3 in File S1 we provide evidence that these sequences may share the same twisting flexibility, even if they differ in helical period.
The effect of the promoter on loop formation can be more clearly seen when looping J-factors are compared across sequences, instead of the looping probabilities. Because the no-promoter and with-promoter loops are flanked by different combinations of operators (Fig. 1(B); see also Methods), their looping probabilities cannot be directly compared. However, as described above and in the Methods section, we can use the statistical mechanical model that we have described for this system to extract J-factors from each looping probability [35]. These J-factors are shown in Fig. 2. Loop sequence can modulate the looping J-factor by at least an order of magnitude (compare the poly(dA:dT) J-factors to those of 5S with promoter or E8 and TA, no-promoter). The lacUV5 promoter has the largest effect on the TA and 5S sequences (though of opposite sign), but appears to have little effect on poly(dA:dT)-containing and E8-containing loops, and moderate effect on CG-containing loops. It is intriguing how large and diverse an effect the 36-bp lacUV5 promoter has on the roughly 100 bp loops we examine here; but one possible explanation for its minimal effect on the poly(dA:dT)-rich sequence, at least, compared to the others, is that the properties of A-tract structures tend to dominate over the properties of surrounding sequences [31]. We note that our results in [35] comparing the effect of sequence versus flanking operators on measured J-factors preclude the possibility that the differences between the no-promoter and with-promoter constructs are due to the difference in flanking operators. We also note that it is possible that the effect of the promoter stems not from the promoter sequence itself, but from the fact that the sequences of interest that form the rest of the loop are shorter when 36 bp of the loop are replaced by the promoter sequence. However, we consider this explanation to be less likely, because as shown in the left-hand panels of Fig. 1(C) and (D) above, we have measured the looping probabilities (and J-factors; see [35]) of more than two periods of E8- and TA-containing DNAs, allowing a direct comparison of loops that contain the same amount of E8 and TA both with and without the promoter (compare, for example, no-promoter loop lengths of 90 bp to with-promoter lengths of 120 bp). In this case we still find that without the promoter the J-factors of the E8- and TA-containing loops are indistinguishable, but with the promoter the TA sequence loops more than the E8 sequence, indicating that it is the promoter and not a shortening of some unique element(s) of the E8 or TA sequences that cause the difference in J-factors with versus without the promoter for these two sequences.
TPM trajectories not only provide information about the free energy of loop formation, captured by the J-factors discussed in the previous section, but also contain some information about the preferred loop conformation as a function of sequence, through the observed length of the TPM tether when a loop has formed. In fact, previous work from our group and others has shown that the Lac repressor can support at least two observable loop conformations for any pair of operators, with any sequence, because these conformations lead to distinct tether lengths in TPM [35], [37]–[39], [39], [40], [51], [66]–[68]. Although the underlying molecular details of these two looped states, which we label the "middle" ("M") and "bottom" ("B") states according to their tether lengths relative to the unlooped state, are as yet unknown, they must differ in repressor and/or DNA conformation in a way that alters the boundary conditions of the loop, since they are distinguishable in TPM. It has been proposed that the two states arise from the four distinct DNA binding topologies allowed by a V-shaped Lac repressor similar to that shown in the Lac repressor crystal structure [69], [70], and/or two repressor conformations, the V-shape seen in the crystal structure and a more extended "E" shape [39], [40], [66], [68], [71]–[73]. It is likely, in fact, that the two observed looped states are each composed of more than one microstate (that is, some combination of V-shaped and E-shaped repressor conformation(s) and associated binding topologies [69]). Even without knowing the details of the underlying molecular conformation(s) of these two states, however, we can use them to provide a window into the effect of sequence on preferred loop conformation.
In particular, by examining the relative probability of the two looped states as a function of both loop length and loop sequence, we can assess the contributions of sequence to the energy required to form the associated loop conformation(s). As shown in Figure 3, which of the two looped states predominates depends in a complicated way upon the loop sequence, the presence versus absence of the lacUV5 promoter, and the loop length. In [35], we showed that having E8 or TA in the loop region, over two to three helical periods, leads to alternating preferences for the middle versus the bottom looped state, with the middle state predominating when the operators are in-phase and looping is maximal, but the bottom state predominating when the operators are out-of-phase. The inclusion of the promoter in the loop increases the preference for middle state for out-of-phase operators. These trends are captured in the top left panel of Fig. 3.
These trends do not hold for the three genomically sourced loop sequences (CG, dA, and 5S). For the poly(dA:dT)-rich sequence, as with E8 and TA, the promoter increases the preference for the middle looped state for out-of-phase operators; for 5S, however, the presence of the promoter decreases the preference for the middle state. The preferred state of the CG sequence is mostly insensitive to the presence versus absence of the promoter. Both with and without the promoter, though, the middle state is generally preferred () at more loop lengths for the genomically sourced DNAs than for the synthetic sequences, insofar as we are able to determine from the lengths shown in Fig. 3. These results demonstrate a complicated dependence of preferred loop state on sequence that does not always follow overall trends in looping free energy: for example, 5S and TA are the two sequences that show the largest change in J-factor with the inclusion of the promoter, but E8 and TA are the sequences that show the largest change in preferred looped state with the promoter. However, the trend seen in the preceding section with CG and poly(dA:dT) having more in common than 5S and TA holds true for preferred loop conformation as well.
A different measure of loop conformation can be derived from the TPM tether lengths themselves—that is, from the measured root-mean-squared motion of the bead, , as in the example trajectory shown in Fig. 4(A), which exhibits three clear states, the two looped states and the unlooped state. Because of variability in initial tether length, even in the absence of Lac repressor, we calculate a relative measure of tether length for the unlooped and looped states, where the motion of each bead is normalized to its motion in the absence of repressor. We might expect, then, that in the presence of repressor, the unlooped state would fall at a relative of zero, and the looped states at negative values. However, as can be seen in the sample trace in Fig. 4(A) and in the lefthand panels of Fig. 4(B), the unlooped state in the presence of repressor is actually shorter than the tether in the absence of repressor (i.e., the horizontal black dashed line in Fig. 4(A) lies above the mean of the unlooped state in the blue data). In [35] we present evidence for this shortening of the unlooped state in the presence of repressor being due to the bending of the operators induced by the Lac repressor protein that is observed in the crystal structure of the Lac repressor complexed with DNA [70]. (We note that this is a Lac repressor-specific result; compare, for example, the recent results from Manzo and coworkers with the lambda repressor [74], where a similar shortening of the unlooped state is shown to be due to nonspecific binding. For example, the Lac repressor does not exhibit the dependence of the looped tether length on repressor concentration that is seen with the lambda repressor [35], [74]).
As shown in Fig. 4(B), the length of the TPM tether in both the unlooped and looped states is similar but not identical for the five sequences and eight lengths that we examine here. The most obvious modulation of tether length correlates with loop length, with the shortest unlooped- and looped-state tether lengths occurring near the maxima of the looping probability. We believe this modulation with length is due to the phasing of the bends of the DNA tether as it exits the repressor-bound operators in the looped state, or the phasing of the bent operators in the unlooped state. At the repressor concentration we use here, the unlooped state should be primarily composed of the doubly-bound state [35], meaning that the two operators are both bent by bound repressor. As shown schematically in Fig. 4(C), when these bends are in-phase, the tether length should be shortest (and also the looping probability is highest, because the operators are in-phase). A similar argument can be made for the modulation of the looped state, regarding the relative phases of the tangents of the DNA exiting the loop.
It is interesting to consider how the sequence of the loop might influence the length of the tether in the unlooped state, when no loop has formed; see, for example, the CG with-promoter versus 5S with-promoter sequences, where the latter is consistently longer than the former (Fig. 4(B)). We do not see a sequence dependence to tether length in the absence of repressor, ruling out the possibility of a detectable intrinsic curvature to the CG sequence. We speculate instead that CG alters the trajectory of the DNA as it exits the bend in the operators in the unlooped state, compared to the trajectory when the sequence next to the operators is 5S, leading to a consistent difference in unlooped tether lengths.
Interestingly, in contrast to its influence on preferred looped state (middle versus bottom), the promoter does not alter the length of the tether for a given sequence at a given loop length (see also the bottom left panel of Fig. S5 and Fig. S6 in File S1). On the other hand, as shown in Fig. 4(D), the poly(dA:dT)-rich sequence, noticeably more so than the other sequences, stands out as a sequence that does strongly affect the tether length of the loop, in that it mandates a very narrow range of tether lengths as a function of looping J-factor (related, for a particular sequence, to the loop length or equivalently the operator spacing). A similar but less pronounced trend can be observed for the unlooped state with the GC-rich sequence (Fig. 4(D)). The other sequences allow much more variability in tether length as a function of J-factor/operator spacing (see Figure S6 in File S1). This strong trend in tether length as a function of J-factor could be evidence of the formation of special, defined loop structures with the GC-rich and poly(dA:dT)-rich sequences that constrain the allowed loop conformations as a function of operator spacing more than the other sequences do.
Further computational and modeling efforts will be required to relate these data on tether lengths and preferred loop length to loop structure, similarly to how Towles and coworkers have used TPM tether lengths to show that different DNA loop topologies can explain the observed tethered lengths of the two looped states [69]. However, even without currently knowing the underlying molecular details causing these sequence-specific trends in tether length and preferred loop state, and therefore in loop conformation, it is clear that it is the loop sequence, and not the Lac repressor itself, that determines the loop conformation to a large degree. It has been shown recently that the Lac repressor is capable of accommodating many different loop conformations [40], which is consistent with the results we present here. We hope that computational and modeling efforts with these data, as well as continued efforts to use assays such as FRET to directly probe loop conformation [37]–[40], will shed light on this complex interplay between sequence and loop conformation.
In [35] we showed that the synthetic E8 and TA sequences show no sequence dependence to looping in the absence of the lacUV5 promoter but a nucleosome-like sequence dependence in the presence of the promoter. We hypothesized that perhaps the promoter alters the preferred state of the loop to one whose shape is more similar to that of DNA in a nucleosome or DNA minicircle formed by cyclization, leading to similar sequence trends with the promoter as with nucleosomes. We still attribute the difference in the patterns of sequence dependence that we observe between looping and nucleosome formation to the role of the shape of the deformation in determining the observed deformability of a particular sequence. However, we have shown here with a broader range of sequences that the role of the promoter in controlling loopability is more complicated than we had previously hypothesized. Neither with nor without the promoter does loop formation follow the sequence trends of nucleosome formation. As shown in Figure 5, if looping J-factors did follow the same patterns of sequence preference as do cyclization J-factors and nucleosome formation free energies, a plot of the looping J-factors versus cyclization J-factors for the various sequences we have studied here would fall on a line with a positive slope. We find that this is not the case; in fact, without the promoter there is perhaps a slight anticorrelation between looping J-factors and cyclization J-factors (and no discernible correlation with the promoter).
The strong correlation between a sequence's ease of cyclization and of nucleosome formation, as shown in Fig. 5(A), has been used to argue that nucleosome sequence preferences depend largely on the intrinsic mechanical properties of a DNA, particularly its bendability [8], though other mechanisms have also been proposed, such as that described by Rohs and coworkers, which depends not on sequence-dependent DNA flexibility but on sequence-dependent minor groove shape [75]. We have shown here that three sequence features that commonly determine nucleosome preferences, either through their effect on DNA flexibility or on other structural aspects recognized by the nucleosome, do not likewise determine looping, arguing for the need to identify a different set of sequence features that determine loopability. The most striking contrast between previously established sequence "rules" derived from nucleosome studies and the trends in looping J-factors that we observe here is that of the nucleosome-repelling, poly(dA:dT) sequence, which has the lowest looping free energy that we have quantified so far. Other in vitro assays predominantly show poly(dA:dT) copolymers to be highly resistant to deformations; for example, Vafabakhsh and coworkers recently used a FRET-based cyclization assay, analogous to traditional ligase-mediated cyclization assays, to show that poly(dA:dT)-rich sequences have cyclization rates significantly smaller than other sequences such as E8 and TA [34]. Although ease of cyclization is often equated with bendability, it appears that such observed bendability is more context-dependent than has been previously appreciated: that is, the simplest model that one would write down to describe the energetics of these different deformed DNAs would feature the persistence length as the governing parameter that is used to characterize bendability, and yet, the distinct responses seen in looping, nucleosomes and cyclization belie that simplest model. It will be informative to extend this study of an unphased poly(dA:dT) tract in DNA loops to include more sequences containing both pure poly(dA:dT) copolymers and naturally-occuring poly(dA:dT)-rich DNAs that exclude nucleosomes in vivo, in order to elucidate the precise role of poly(dA:dT)-tracts in determining looping. It is clear, however, that poly(dA:dT)-rich DNAs should not be exclusively thought of as stiff or resistant to bending in all biological contexts.
A second striking contrast between our results here and previously established rules for nucleosome formation concerns the role of G+C content in determining loop formation. The G+C content of a DNA is one of the most powerful parameters for predicting nucleosome occupancy in vivo [17], [19], with higher G+C content correlating with higher occupancy. However, as shown in Fig. 6, G+C content offers little predictive power for loopability, or is anticorrelated with looping. We note that a recent, systematic DNA cyclization study demonstrated a quadratic dependence of DNA bending stiffness on G+C content [15]. In our case of protein-mediated DNA looping, the looping J-factor contains contributions from protein elasticity in addition to those from DNA elasticity, and our DNA sequences contain A-tracts and GGGCCC motifs that were excluded in [15], making a direct comparison between our results and theirs difficult; but it is possible that the looping J-factor is neither correlated or anticorrelated with G+C content but instead depends quadratically on G+C content, as do cyclization J-factors. More data will be necessary to make a strong statistical statement about the anticorrelation or lack of correlation between the looping J-factor and G+C content, and to determine the form of the relationship between the looping J-factor and G+C content (e.g. quadratic versus linear), but we propose low G+C content as the starting point of a potential new sequence "rule" for predicting looping J-factors, and a fertile realm of further investigation. Finally, we have shown that the repeating AA/TT/TA/AT and GG/CC/GC/CG steps that characterize the 5S and TA sequences, as well as many nucleosome-preferring sequences, do not likewise determine looping J-factors, as these two sequences behave very differently from each other in the context of transcription factor-mediated DNA looping.
Here we have extended our previous work on the sequence dependence of loop formation by the Lac repressor to include three naturally occurring, genomic sequences that have either nucleosome-repelling or nucleosome-attracting functions in vivo, in addition to the two synthetic sequences we described previously [35]. We find that two sequences that share sequence features important to nucleosome formation and that share trends in observed flexibility in cyclization and nucleosome formation assays, the 601TA and 5S sequences, behave less similarly in the context of DNA looping than the two sequences that should have least in common, the GC-rich, nucleosome attracting sequence and the poly(dA:dT)-rich, nucleosome repelling sequence. 5S and TA share neither trends in looping free energy relative to the random E8 sequence, nor loop length where looping is maximal, nor preferred loop conformation, nor their response to the larger sequence context (as evidenced by the fact that the inclusion of the lacUV5 promoter sequence in the loop increases the looping J-factor for TA but decreases it for 5S).
We have also shown that a poly(dA:dT)-rich DNA that forms a nucleosome-free region in yeast [23] is actually extremely deformable in the context of looping by a transcription factor. The rest of the sequences show a range of J-factors that does not correlate with any observed trends in flexibility as measured by ligase-mediated cyclization assays, nor with the observation that high G+C content correlates with nucleosome occupancy [17]. The diversity of the effects on DNA looping that we observe with these five sequences (ten, if the inclusion of the promoter is considered to create a "new" sequence) underscores the necessity of a large-scale screen for sequences that control loop formation both in vivo and in vitro, much as has been done in the context of nucleosome formation to help establish the sequence-dependence rules of that field (for example, see [5], [59]).
Our work in no way undermines previous claims of the sequence dependence to nucleosome formation and/or occupancy either in vivo or in vitro; rather, it demonstrates that the "rules" of sequence flexibility derived from cyclization and nucleosome formation studies are inapplicable to DNA looping, possibly due to the difference in the boundary conditions and therefore DNA conformations involved in forming a protein-mediated loop versus a DNA minicircle or a nucleosome. It will be interesting to extend these studies of the role of sequence in loop formation to other DNA looping proteins besides the Lac repressor. As noted above, it has been shown recently that the Lac repressor can accommodate many different loop conformations [40]. The variety in tether lengths and preferred looped states that we observe are consistent with a forgiving Lac repressor protein. Nucleosomes, on the other hand, have a more fixed structure that should not be as accommodating to a range of helical periods and DNA polymer conformations (hence the hypothesis that poly(dA:dT)-rich DNAs disfavor nucleosome formation because they adopt geometry that is incompatible with the structure of the DNA in a nucleosome [21]). It would be informative to measure the looping J-factors of these same sequences with a more rigid looping protein. It will also be interesting to see if other bacterial promoter sequences have similar effect of altering the looping boundary condition as the very strong and synthetic lacUV5 promoter. In fact, the lacUV5 promoter should be a key starting point for identifying sequences that have a strong effect on looping, since it can have significant effects on the behavior of a loop, even when it comprises only one-third of the loop length.
The poly(dA:dT)-rich sequence (from Fig. 4 of Ref. [23]), GC-rich sequence (from "Human 2" at http://genie.weizmann.ac.il/pubs/field08/field08_data.html), and 5S sequences (from Fig. 1 of [45]) were cloned into the pZS25 plasmid used in [35], with these eukaryotic sequences replacing the E8 or TA sequences in that plasmid. In cases where the loop lengths used in this study were shorter than the 147 bp that are wrapped in nucleosomes, the corresponding looping sequences used in TPM were taken from the middle of these sequences (relative to the nucleosomal dyad); in cases where the nucleosomal sequences were shorter than the desired loop length, they were padded at one end with the random E8 sequence [8], [35], [60]. See Figures S1 and S2 in File S1 for details. As in [35], "no-promoter" loops were flanked by the synthetic, strongest known operator (repressor binding site) and the strongest naturally occurring operator ; "with-promoter" loops were flanked by and a weaker naturally occurring operator, , because these with-promoter constructs are also used in in vivo studies of the effect of loop architecture on YFP expression, in which case is a more convenient choice of operator than . Similarly, the motivation to include the lacUV5 promoter in the loop stems from parallel in vivo studies, in which the promoter is a natural part of the looping architecture. The promoter is included in the loop between the sequence of interest and the operator. Figures S1 and S2 in File S1 gives the exact sequences used in this work; Fig. 1(B) shows the TPM constructs schematically.
Cloning of the sequences of interest into the pZS25 plasmid was accomplished in either one or two steps. For the 5S sequences, oligomers were first ordered from Integrated DNA Technologies as single-stranded forward and reverse complements, consisting of 69 bp (for the "with-promoter" constructs) or 105 bp (for the "no-promoter" constructs) of the 5S sequence, plus the and / operators, and, where applicable, the lacUV5 promoter sequence. These oligomers were annealed and then ligated into the pZS25 plasmid at the AatII and EcoRI restriction sites that fall just outside the operators that flank the E8 or TA sequences in the original pZS25 plasmids [35]. Second, Quik-Change mutagenesis (Agilent Technologies) was performed to generate additional lengths (that is, to introduce insertions or deletions) of the 5S sequence from the initial 105 bp loop lengths. However, we found that this site-directed mutagenesis step generated distributions of products for the poly(dA:dT) constructs, possibly due to replication slipped mispairing over repetitive sequences [76]. Therefore all lengths of the poly(dA:dT) sequence, as well as of the GC-rich sequence, which also have the potential to contain such "slippery" regions, were created by ligation of synthesized oligomers into the pZS25 plasmid. All constructs were confirmed by sequencing (Laragen Inc.) to have clean sequence reads, and the approximately 450 bp digoxigenin- and biotin-labeled TPM constructs were created by PCR as described for the E8- and TA-containing constructs in [35], [51]. Sequences of TPM constructs were again confirmed by sequencing before use.
Tethered particle motion assays were performed as described in [35]. Briefly, linear DNAs, labeled on one end with digoxigenin and on the other end with biotin, were introduced into chambers created between a microscope slide and coverslip, with the coverslip coated nonspecifically with anti-digoxigenin. Streptavidin-coated beads (Bangs Laboratories, Inc) were then introduced into the chamber to complete the formation of tethered particles. The motion of the beads was tracked using custom Matlab code that calculated each bead's root-mean-squared (RMS) motion in the plane of the coverslip, and looping probabilities were extracted from these RMS-versus-time trajectories as the time spent in the looped state (reduced RMS), divided by total observation time. Similarly, the probabilities of the "bottom" versus "middle" states (see Results section) were defined as the time spent in a particular state, divided by the total observation time.
By measuring the looping probability of a construct at a particular repressor concentration, and using the repressor-operator dissociation constants for , and in [35], we can calculate the J-factor for that construct. All measurements in this work were carried out at 100 pM repressor, using repressor purified in-house. The relationship between the looping probabilities measured in TPM (), the repressor-operator dissociation constants for the two operators that flank the loop (, and ), and the looping J-factor of the DNA in the loop () can be described as
where is the concentration of Lac repressor, and and are repressor-operator dissociation constants of the two operators flanking the loop ( and or ). A similar expression can be derived for the J-factors of the individual "bottom" and "middle" looped states and is given in [35].
The J-factors plotted in Figure 5 are the maximum looping or cyclization J-factors over a particular period. Specifically, the looping J-factors used are those at 104 bp for dA, 105 for 5S and CG, and 106 for E8 and TA; the cyclization J-factors are for 94 bp of the E8, 5S or TA sequences and are taken from [60]. Although we are not directly comparing identical lengths between cyclization and looping, the general trends hold regardless of lengths chosen. In fact, identifying the loop length that corresponds to a particular cyclization length is difficult, given that the flanking operators for looping must be taken into account in some fashion. That is, for cyclization, DNA length is easy to compute—it is simply the length of the oligomer used in the ligation reactions. However, in the case of looping, it is unclear if the appropriate length for comparison is just the DNA in the loop (excluding the operators), or the length between the midpoints of the operators, or including all of the operators. Similarly, we are not comparing identical loop lengths across sequences; we chose to compare loop flexibility at the looping maximum for each sequence in an attempt to compare lengths at which the operators are most likely to be in phase, such that we are comparing only bending and not twisting flexibility. Finally, we note that here we are interested in the same kind of comparison that Cloutier and Widom were in Ref. [8], which was the inspiration for this figure; in [8], Cloutier and Widom compared cyclization and nucleosome formation free energies, even though the cyclization experiments were performed with roughly 100 bp DNAs and the nucleosome formation assays with roughly 150 bp DNAs. Likewise, we do not expect that the fragments of nucleosome-preferring or nucleosome-repelling sequences that we examine here in the context of looping will necessarily have exactly the same characteristics as the full-length nucleosomal sequences from which they were derived; but we are interested in comparing general trends in observed flexibility of these roughly 110 bp loops with those of roughly 100 bp ligated minicircles and of roughly 150 bp nucleosomal DNAs.
Supporting figures. Figure S1 "No-promoter" looping sequences used in this work. Figure S2 "With-promoter" looping sequences used in this work. Figure S3 Sequence-dependent twist stiffness. Figure S4 Looping probabilities and J-factors for the two looped states separately. Figure S5 Tether lengths of looped and unlooped states as a function of loop length and sequence. Figure S6 Tether length as a function of J-factor.
We are indebted to the late Jon Widom for the inspiration of this project and for his guidance, mentorship and friendship over many years. We thank Chao Liu, David Wu, David Van Valen, Hernan Garcia, Martin Lindén, Mattias Rydenfelt, Yun Mou, Tsui-Fen Chou, Eugene Lee, Matthew Raab, Daniel Grilley, Niv Antonovsky, Lior Zelcbuch, Matthew Moore, Ron Milo, Eran Segal, and the Phillips, Mayo, Pierce and Elowitz labs for insightful discussions, equipment and technical help; and Winston Warman at Transgenomic, Inc. (Omaha, NE, USA) and Jin Li at Laragen, Inc (Culver City, CA, USA) for special help with sequencing the poly(dA:dT)-rich DNAs.
The authors gratefully acknowledge the support of the National Institutes of Health (www.nih.gov, grant numbers DP1 OD000217A [Director's Pioneer Award], R01 GM085286, R01 GM085286-01S1, and 1 U54 CA143869 [Northwestern PSOC center]). SJ gratefully acknowledges the support of a graduate fellowship from the National Science Foundation (www.nsf.gov), and RP the support of the Fondation Pierre Gilles de Gennes (http://www.fondation-pgg.org/). 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.


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Murray State Racers vs. Eastern Kentucky Colonels Pick-Odds-Prediction - 3/7/14 OVC Semifinal
Eastern Kentucky Colonels (22-9) at Murray State Racers (18-10)
College Basketball: Friday, March 7, 2014 at 10:00 pm (Nashville Municipal Auditorium)
The Line: Murray State Racers +2 -- Over/Under: See the Latest Odds
The Eastern Kentucky Colonels battle the Murray State Racers in an Ohio Valley semifinal Friday.
Is Murray State's return to elite status complete? Close but this team isn't close to the one that challenged perfection a few years ago because their defense isn't nearly as good. However it's not really fair to compare those teams anyway but suffice to say the Racers have generally scored and defended well in conference play and are playing well at both ends of the floor. Except for the season ending 118-115 loss to Southeast Missouri State I would say we have a nicely balanced squad. With four DD scorers there are plenty of scoring options so opponents can't key on any one player so it's pick your poison with this group.
Jim Brown-USA TODAY Sports
Eastern Kentucky advanced to the Ohio Valley semifinals with an 84-76 decision over Southeast Missouri State on Thursday for win number four in a row. This looked like it was headed for the win column early with the Colonels up 14 at the break before a late Redhawks rally closed the gap. In the end, EKY had too much firepower on 53% shooting including 10-20 from distance while owning a 26-18 edge at the foul line. Corey Walden led the attack with 17 points and five yanks with Glenn Cosey tossing in 15 followed by Orlando Williams and Tarius Johnson with 14 and 12 points with Johnson snatching seven boards.
Both teams can score the basketball so the one that can get a few stops will likely move on but with no spread out I'll have time to look at this further so please check back...

subset: commoncrawl


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This study is provided by ICPSR. ICPSR provides leadership and training in data access, curation, and methods of analysis for a diverse and expanding social science research community.
Subjective Vulnerability: The Role of Disempowerment in the Utilization of Mediation Services by Women in a Northeastern Town of the United States, 1989-1990 (ICPSR 6357)
Principal Investigator(s): Northrup, Terrell A.; Segall, Marshall H.
This collection investigates gender differences as they relate to interpersonal conflict and feelings of vulnerability that arise in the conflict process. In Phase 1 of the study, 52 female and 48 male clients of a neighborhood mediation center were assessed on the number and kinds of strategies they utilized in dealing with conflicts before they consulted the mediation center, their feelings about the services rendered, the concerns, fears, and other affective states experienced in relation to their disputes, their feelings about the outcomes, to what they attributed success or failure, and how they felt gender had affected the conflicts. Phase 2 of the study was implemented as a follow-up to determine the generalizability of the findings of Phase 1. Respondents from the same geographic area in which the subjects in Phase 1 were drawn were assessed on three areas of concern in relation to a hypothetical conflict: feelings of vulnerability, aspects of mediation that seemed desirable to them, and concerns they might have if they were to use mediation services.
Access Notes
Data in this collection are available only to users at ICPSR member institutions. Please log in so we can determine if you are with a member institution and have access to these data files.
Study Description
Northrup, Terrell A., and Marshall H. Segall. Subjective Vulnerability: The Role of Disempowerment in the Utilization of Mediation Services by Women in a Northeastern Town of the United States, 1989-1990. ICPSR06357-v1. Ann Arbor, MI: Inter-university Consortium for Political and Social Research [distributor], 1995. https://doi.org/10.3886/ICPSR06357.v1
Persistent URL: https://doi.org/10.3886/ICPSR06357.v1
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This study was funded by:
- Fund for Research on Dispute Resolution (G1-88-02)
Scope of Study
- 1989-06--1990-08
- 1989--1990
Universe: Phase 1: Clients of voluntary neighborhood mediation centers that deal with interpersonal conflicts such as landlord-tenant, employer-employee, divorce, family, neighbor, and consumer-business disputes. Phase 2: Adult residents of a town in the northeastern United States.
Sample: Phase 1: 100 adult clients of a neighborhood mediation center. Phase 2: 100 adult residents of a town in the northeastern United States, randomly selected from the phone book from the five most predominant ZIP codes of the subjects in Phase 1.
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Original ICPSR Release: 1995-06-05
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12 Candy Bar Wrappers, Party Favors, Hot Pink and Black, Animal Print, Bridal Shower, Lingerie Shower, Bachelorette Party
Product Info
My fun animal print & sexy lingerie candy wrappers will make your milk chocolate bars perfect favors for your lingerie or bridal shower or your bachelorette party!
Printed on a beautiful white 48lb glossy paper. Personalized on the front area with whatever you wish!
The listing is for a set of 12 candy bar wrappers that will fit 1.55 oz regular size milk chocolate bars.
Candy is not included.
* 12 personalized wrappers
* 12 7.5" x 6" PINK OR BLACK OR HALF OF EACH COLOR foil sheets ( ♥ Select your choice from the drop down menu )
I'll include a glue stick and complete instructions with pictures for assembling your wrappers (don't worry, it's super easy to do). Wrap the foil sheet around your candy bar first, then the wrapper....simple!
1) Select your foils sheets color choice from the drop down menu
2) Select your quantity from the drop down menu. (Sold by the dozen. A quantity of '1" = 12 wrappers, "2" = 24 wrappers, etc.)
3) Add to cart.
4) Tell me what you want on your wrappers. Please include this in the "Note to Seller" box on the shopping cart page" box during the checkout.
I'll send you a proof within 24 hours.
♥ Ships in 3 business days after your purchase. I ship Monday - Friday. ♥
♥ This item ships by USPS Priority Mail (once in the mail, this is usually a 2-3 day delivery time) ♥
♥ Shipping upgrades are available in the checkout ♥
PLEASE NOTE: If you need something rushed, please contact me first. I will check my production schedule to see if I can fit your order in. Rush fee may apply.
♥ You are purchasing this item with the design pictured above. Design, color schemes, font style and font colors are exactly as shown here. If you would like any aspect of the design or color scheme changes from what is shown in the samples, please contact me *BEFORE* you place your order to see if it is possible. Custom design fee may apply, depending on your needs. My design fees start at $10. per quarter hour ♥


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Complaint Review: Contigo Limo
- Contigo Limo
San Francisco, California
United States of America
- Phone: 415-515-8844
- Web: contigolimo.com
- Category: Limousine Services
Contigo Limo Charged me for services they did not deserve San Francisco, California
I should've known the trip would go downhill from there. The limo itself was in terrible condition. The air conditioning would not turn on and we sweated the entire ride. The iPod plug-in, which I had called to confirm would be there, did not work and we had no music for our ride. A bunch of the colored lights were burnt out.
As for the trip, the limo driver was the rudest I have ever seen. Multiple times I saw him rolling his eyes when I asked him questions. I had never been to the area before and he wouldn't give me answers to any of my location questions. By driving 20mph under the speed limit, he made us miss 2 of our reservations.
On the way home, he told me if we went 5 minutes over our time limit, he would be forced to charge us an additional $150. Because he went so slow, we ended up not reaching our hotel (even though we had left 20 minutes earlier than anticipated to account for his slow driving). He let us out at a random street corner and we had to hail cabs back to our hotel.
This was honestly the worst customer service experience I have ever had. The unfortunate part was the amount of money that we had spent for such terrible service! Especially because it was a "mandatory tip ("company policy")" of $150!!! I tipped him $150 to be let out at a random street corner miles from our hotel!
When trying to contact the company after our "experience," their voicemail was full and they would not answer my phone calls. I would NEVER hire this company for anything again!
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This report was posted on Ripoff Report on 04/17/2012 08:10 PM and is a permanent record located here: http://www.ripoffreport.com/reports/contigo-limo/san-francisco-california-/contigo-limo-charged-me-for-services-they-did-not-deserve-san-francisco-california-870160. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year. Ripoff Report has an exclusive license to this report. It may not be copied without the written permission of Ripoff Report. READ: Foreign websites steal our content
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Attn. Harry Reid: Obama, Biden and Hillary Took Koch Money Too (w/ Video)
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Sen. Harry Reid has an addiction to Koch. I'm not kidding. Watch this video:
Now, here's comes the head exploding time:
During the past five congressional campaigns (2006-14) Koch money went to President Obama, Joe Biden, Hillary Clinton, Dianne Feinstein, Mark Pryor, Chuck Schumer and other members of Congress.×
101 Things All Young Adults Should Know
by Sir John Hawkins
John Hawkins's book 101 Things All Young Adults Should Know is filled with lessons that newly minted adults need in order to get the most out of life. Gleaned from a lifetime of trial, error, and writing it down, Hawkins provides advice everyone can benefit from in short, digestible chapters.Buy Now
The chart below represents direct donations to Democratic Party candidates from Koch Industries employees since 2006 according to OpenSecrets.org (2014 donations are based on FEC numbers through March 10) Retired Democratic Party members of Congress received another $291,000 but were not included in the chart.
See also: : Even Liberal Reporter Bob Woodward calls for full investigation of IRS after probes find IRS workers backed Obama while on the job
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Duane Lester
Duane Lester is co-founder of All American Blogger, and the primary writer. Following graduation, Duane entered the United States Navy as a journalist. He spent five years touring the world, reporting on local news and sports. Following his enlistment, Duane spent almost 10 years working with adjudicated youth in residential treatment environments. Duane discovered politics after September 11. He credits Erich "Mancow" Muller for opening his eyes to his conservative beliefs. Since then, Duane has devoured books and literature on politics, reading everything he can from Adam Smith to Larry Elder to Thomas Sowell. He refers to his style of politics as "conserva-tarian", a mixture of conservative and libertarian beliefs.


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Paper Chase is a research database designed to offer abstracts of research articles published in journals that have a highly rated impact factor as determined by ISI Impact Factor and PageRank. Abstracts are organized by date, with the most recently published papers listed first.
Paper Chase
Evolutionary relationship between the T3 chains of the T-cell receptor complex and the immunoglobulin supergene family.
Laboratory of Molecular Immunology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115.
Antigen receptors on the surface of the thymus-derived (T) lymphocytes are associated with small integral membrane proteins called the T3 (CD3) gamma, delta, epsilon, and zeta chains. After interaction of the T-cell receptor with antigen, the T3 proteins are believed to transfer an activation signal to the intracellular compartment. In previous studies, the human gamma, epsilon, and delta chains have been cloned along with the mouse delta chain, but a relationship between these sequences and known molecular families has not been established. We now report the molecular cloning and characterization of the murine T3-epsilon protein and a sequence and structural analysis of the relationships between all the T3 chains and the immunoglobulin superfamily. It is established that the T3 chains are immunoglobulin-related and a particular relationship to the neural cell adhesion molecule (N-CAM) is noted. This sequence relationship adds interest to previous findings that the T3 chains are genetically linked to N-CAM and Thy-1 antigen on band q23 of human chromosome 11.
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Gee WaWa Marla Distressed Goat Leather Military Natural Boots
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Another fabulous pair of goat leather GeeWawa boots for Fall or Winter. Gee Wawa's run true to size and this is a 7. The most fabulous style too. These are called the Kenya Distressed and they are what Gee Wawa calls white, but as you can see from the picture, they are more of a natural color. They are distressed goat leather so they will wear forever! These are a calf boot, 9" shaft, with a side zip cross over goat leather strap with antique gold "buttons". They have leather laces as well. Once you adjust, the beauty is you don't touch the laces again because of the side zip. The soles are leather with barely a heel -- less than an inch. And the heel back bottom of the heel has a man made inset piece for good traction. Boot out bland and boring with this cutting edge, almost military vibe style to them. These are new in the box with everything, care cards, extra laces etc. If you are familiar with Gee Wawa, you know their box alone weighs a good pound.
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I will be heading down to SXM this friday. I was there last year and got some good shots but I was wondering where this picture was taken from. I know Dan said it was the Maho Beach Hotel and Resort but I was wondering exactly what room or around abouts where it was taken from.
Photo © Dan Valentine


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For those ISVs who have been wanting to get a hold of Windows Vista SP1 for testing, you can find on MSDN later this month. There will be two versions, an update and a full install. The announcement will be made on the Windows Vista Blog in the next week or so. The full public release is set for mid-March.
There is an interesting post over at blogs.msdn.com


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Turkey Run Church is located near Fairfield County's border to Pickaway County. When dedicated in 1835, the church was officially named The Turkey Run Predestinarian Baptist Church and more than 500 people came from miles around to attend its dedication. Built of brick made and fired on the property, the church could accommodate nearly sixty worshippers on hand-grained pews cushioned by early quilts. Adjacent to the church is an early cemetery with the names of many settlers, the descendants of which live in Fairfield County today.
Directions and Map
Turkey Run Church is located at 11635 Lancaster-Circleville Road, SW, in Lancaster, Ohio.
From the corner of Main and Broad Streets in Lancaster, Ohio, head west on Main Street/Lincoln Avenue for about 1 mile. Turn right onto 188 West/Cedar Hill Road. Remain on 188 West for about 12 miles. The lane for Turkey Run Church will be on the left.
Location Features
- Nineteenth-century church made of bricks constructed and fired on site.
- Turkey Run Church contains retains its original pulpit and hand-grained pews which are a fine example of folk art painting.
- Early cemetery, dating back to 1812.
Visitors are welcome to tour the grounds of the church, which are open from sunrise to sunset. The church itself is currently closed for repairs and renovation.


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- Presentation
- HAL publications
- Activity reports
DIVERSE Research team
Diversity-centric Software Engineering
- Leader : Olivier Barais
- Type : Project team
- Research center(s) : Rennes
- Field : Networks, Systems and Services, Distributed Computing
- Theme : Distributed programming and Software engineering
- Partner(s) : Université Rennes 1,Institut national des sciences appliquées de Rennes
- Collaborator(s) : Institut de recherche en informatique et systèmes aléatoires (IRISA) (UMR6074)
Team presentation
Our main objective is to automatically compose and synthesize software diversity from design to runtime to address unpredictable evolutions of software intensive systems. We address this objective through 4 main research axis: software language engineering, software variability, software adaptation and software diversification.
Research themes
The research in DiverSE is organized around 4 research axis:
- Software Language Engineering to handle the diversity of languages used by the stakeholders involved in the construction of software intensive systems
- Software Product Lines to address the diversity of features required by the different customers of these systems
- Distributed architecture and deployment to handle the diversity of runtime environments in which software has to run and adapt
- Software diversity and testing to enhance the resilience of software.
These four axis share and leverage the scientific and technological results developed in the area of model-driven engineering in the last decade. This means that all our research activities are founded on sound abstractions to reason about specific aspects of software systems, compose different perspectives and automatically generate parts of the system.
International and industrial relations
International collaborations
- SINTEF, ICT (Norway): Model-driven systems development for the construction of distributed, heterogeneous applications. We collaborate since 2008 and are currently in two FP7 projects together.
- Universitée du Luxembourg, (Luxembourg): Models@runtime for dynamic adaptation and multi-objective elasticity in cloud management; model-driven development.
- Open University (UK): models@runtime for the Internet of Things.
Industrial collaborations
- Thales: systems engineering
- Obeo: domain specific modeling
- Orange: secure software engineering
Keywords: Software engineering Modeling Variability Diversity Adaptation Languages
Research teams of the same theme :
- ASCOLA - Aspect and composition languages
- FOCUS - Foundations of Component-based Ubiquitous Systems
- INDES - Secure Diffuse Programming
- PHOENIX - Programming Language Technology For Communication Services
- RMOD - Analyses and Languages Constructs for Object-Oriented Application Evolution
- TACOMA - TAngible COMputing Architectures
Team leader
Olivier Barais
Tel.: +33299847298
Tel.: +33299847437


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Seattle, Washington (PressExposure) July 29, 2009 -- Metal Wall Art, an online wall art company known for its excellent customer service, opens the gallery for its most elegant and never-before-seen metal wall decors to the public, but still maintains its commitment for lowest price guarantee in the market. The gallery has a wide-ranging variety, style and design: from Silver Grass Abstract to Colors of the Contemporary Universe to Framed Gingko Abstract.
Motivated by its objective to bring its customers an increasingly larger selection of metal wall art at the lowest possible prices, the company is bringing together these unique and eye-catching modern metal wall art for display and purchase. Ultimately, the goal is for the company to bring these personally handcrafted masterpieces to the homes. Each piece of contemporary wall art is made and signed by American artists themselves. Hence, customers always know that they get the best deal from Metal Wall Art's gallery.
As an example a piece at the gallery for top-quality metal wall decors includes "Abstract Autumn Whirlwind". This is a seven-piece wall sculpture that appears like an abstract s-shaped whirlwind shape and texture. It is a surreal take on autumn whirlwind indeed. Handcrafted and signed by an American artist, the artwork is pegged at $695 in the market. But the company offers it for only $329. The company suggests that the said artwork be displayed beneath track lighting to show off to best effect its most impressive and highly detailed features.
Aside from its variety and great display of contemporary wall sculptures, the company is also known for its highly competent and caring group of customer service specialists to handle all customer services needs. The service specialists are available from 8:00 am to 6:00 pm, PST, always ready to answer any questions customers have regarding the products as well as concerns or questions regarding the ordering and shipping process. Metal Wall Art can boast that its customer service specialists know the products they sell from top to bottom as most of their products are in the warehouse and not collecting dust in someone else's facility hundreds of miles away.
Metal Wall Art is offering an extensive and ever-growing variety of distinctive products in many different categories including Abstract, Animals, Floral, Leaves & Trees, Entertainment & Musical, Grapes & Wine, Panels & Plaques, Fleur De Lis, and Candle Art. The company's website makes its products easy to find using handy a search tool, or customers can shop by category, style, shape, or price. Above all, customers can be sure that their order will ship in top-quality condition and well within the allotted processing time. Once customers receive their order, it is guaranteed that they will be satisfied in every way or they will get their money back - no questions asked.


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I could go in to great lengths about my labor and delivery with my new little baby BUT I will try and keep it short.
On Tuesday, October 9, I began feeling some back pain at bed time. I didn't think much of it because for the last eight months I had been feeling back pain. I of course didn't really pay attention to the fact that these pains would come and go about every ten minutes or so. At around midnight/one I couldn't "sleep" any longer so I decided to wake up and do a little Facebook stalking. My husband was the voice of reason and said, "maybe they're contractions...perhaps you should start timing them." Contractions? Really? I hadn't actually thought about it. So, bring on the trusty App Store. They actually make apps that will calculate your contractions, time between, length, etc. Awesome! Sure enough they were coming at regular intervals...about every five minutes apart. About two hours later we decide we should probably make the 40 minute trek to the hospital. We didn't want to wait too long and find ourselves delivering a baby on the side of the Interstate.
We finally made it to the hospital around 4:15a on October 10. I was nervous because I didn't want to think that these contractions weren't actually doing anything. I didn't want them to check me and say, "well, you are only at one centimeter..." However, that wasn't the case. Yay! I was at five centimeters and could be admitted. Three blown veins later...I had my IV in and was waiting for the anesthesiologist to come in and do the epidural. I couldn't handle it. My husband said I probably could have. I was at seven centimeters and was doing...okay-ish. I wanted the drugs though. (Don't judge me.)
Two movies later (Date Night and Napoleon Dynamite) we were ready to push. Three contractions later and we had a little boy! We were both shocked. We swore it was going to be a girl, but God surprised us with another boy.
After our little boy was born there was excessive bleeding. The doctor was called back in and I was put immediately on some drugs to help slow the bleeding down. I'm so thankful that the nurses and doctor were on top of things. Unfortunately I had some sort of allergic reaction to one of the drugs. Nothing terribly serious but I broke out in a rash and my entire body itched. A small price to pay though...I'll take it! A few hours to recover and our whole family was moved to a new wing in the hospital.
This hospital room would be our new home for the next four days. If I had known that we would be staying so long, I would have brought more magazines to read :) Our first night stay was pretty uneventful. Our second day we were hoping to be discharged but after a meeting with the lactation consultant we decided we should get our baby's tongue clipped to help with feeding. Unfortunately the surgeon wouldn't be available to do that until the following morning. So we waited another night in the hospital. My husband had to take care of our other son, so I spent the night alone. A bit depressing, but what a great time to bond with my new boy!
The next morning the surgeon came and clipped the tongue. The whole process took maybe 15 seconds. We were ready to go when we found out that my son's bilirubin count was high. That meant another night in the hospital. He spent the entire day hanging out on the table. Now I know that this happens to a large percentage of newborns. However, the part I was most frustrated with came down to the table itself. We were supposed to have an additional light that goes over the baby as well. Of course none of the lights worked so they came up with just the lower light. I knew that it wouldn't work but there isn't much you can say to the nursing staff. I wasn't surprised when they checked his blood the next morning and it hadn't changed at all. Somehow, though, they were able to come up with a working overhead light the next morning. Another day at the hospital.
Finally the lights were doing there job. Another blood test showed that the number was getting a bit lower. He was moved off the lights and if he could maintain or decrease his number over the next eight hours he could be discharged. Much later that night we were given the green light! Numbers went down by a half point or so and we were allowed to leave! We couldn't pack up fast enough! We left the hospital and made the trek back home (but first we had to stop at Panda Express to appease the older son...well, and mommy too).
Now we are back home and enjoying the newness of everything again. I am completely blessed to have a wonderful husband and two amazing boys. I can't believe I'm outnumbered though.


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Older reviews
As always, True Beauty is the best -- most professional, caring staff and warm and relaxing environment. Thank you!(Anonymous, 1,307 days ago)
THERE WERE FOUR OF US THAT WENT ON FRIDAY FOR MY BIRTHDAY AND I JUST WANTED TO THANK EVERYONE FOR MAKING MY AFTER BIRTHDAY SO SPECIAL-WE ALL CAME OUT OF THE SPA SO RELAXED AND WANTED TO KNOW WHY WE DON'T DO THIS MORE OFFER. THE PEOPLE WERE GREAT, THE PRICE WAS RIGHT AND IT WAS TOTALLY WONDERFUL. THANK YOU ALL FOR MAKING THE DAY SO SPECIAL FOR ME AND EVERYONE ELSE. THANKS.(Diana C., 1,397 days ago)
Great experience, will be going back.(Anonymous, 1,541 days ago)
I went in with a Living Social and now I'm a client for life! Augusta gives the absolute BEST facials! She is friendly and knows her stuff. I usually cringe when I get extractions but she has a great touch and OMG, her massage is the BEST. I always leave relaxed, glowing and my skin looks and feels great for days! So glad I found her!! Lauren(Anonymous, 1,613 days ago)
I second this commentary. No other place gave me the satisfaction I have gotten when I personally met "Jackie" myself. I stopped by to check out the place and was I ever surprised ...!! A True Beauty Wellness Spa it says it all... Want to be treated with the Best professional and caring way. Well, for all if you Ladies & Gentleman " This Sacred & Beautiful Temple & Retreat Center will bring you to have only the Best from the very moment you walk in. Ann V(Ann V., 1,628 days ago)
This place is rocking with enthusiasm, great service, welcoming sinergy, my appt it happen to be when the Manager Jackie" process not only my voucher she made me feel like I was coming "home" it was such a great feeling, her genuine and welcoming knowledge was so seamless and courteous, professional and yet personal enough for me to want to have all my friends and acquaintances to experience this beautiful Temple where International Goddesses and cultures are openly viewed. I was transported by her to an extraordinary paradise. For me first contact is very important. This Testimonial is all about "Jackie" and the service I got under her umbrella was exquisite and I want to comeback soon for more..!! Thank you "Jackie" for giving me the BEST unexpected experience and for such an affordable cost. Sandra Lopez(Sandra L., 1,628 days ago)
Great experience- wonderful atmoshpere! Augusta and Halse are wonderful!(Anonymous, 1,741 days ago)
I went in for microdermabrasion and a chemical peel. The office is nicely appointed and relaxing. Telsha was fantastic. It was my first time so I was a bit nervous but she was very knowledgeable and so gentle I actually fell asleep on the table! I'd highly recommend this spa to anyone seeking some "me" time. I know I'll be going back.(Keith D., 1,891 days ago)
Next 10 reviews


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IndustriesFood and Beverage
The European Commission's Joint Research Centre (JRC) has released a study on the shortcomings in the regulation of food contact materials (FCM). The study is released through Mapping the industry and regulatory frameworks for food contact materials to support better regulation.
The study reveals these four shortcomings:
- a lack of common guidelines and transparency in how risk assessments are undertaken across EU member states;
- inconsistent national measures and standards on food safety requirements and good manufacturing practice (GMP);
- varying lists of authorized substances among member states and differences in how restrictions are imposed on chemicals; and
- a lack of methods to test for enforcement and compliance, which makes it difficult to demonstrate that national laws ensure food safety.
The study was released on January 18, 2017.


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PUPILS at a Wigan school put on their Sombreros and ponchos to help raise money for charity.
The Mexican-themed day at St Mark's Primary School in Newtown was all in aid of former pupil Bethany Sharkey, who is heading to the Central American country for the second time to help some of the poorest people in the city of Tijuana.
Pupils payed a donation to dress up in a Mexican theme and staff and pupils all enjoyed a chilli dinner as they look to help raise money for building materials for the project.
Bethany, 16, who is now at Winstanley College, travelled to Mexico last year with the Urban Saints charity.
She said: "It has been a fantastic gesture of support from everybody at St Mark's to help me raise the money I need to get building materials.
"When I went last year we helped build 12 houses for some of the poorest families in Tijuana. This year I'm going back to do more work there. I have almost reached my sponsorship target of £1,050, which helps buy the building materials we need."
St Mark's head, Glen Robinson, said: "Bethany is a great role model for our younger pupils and she even came in to lead an assembly on her work."


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This week we talk about what makes a mark, more on WWE Backlash, an interview with one half of LAX Hernandez and the Newsdesk! See much more at www.pwrshow.com!
Wednesday, April 29, 2009
Buddy Rose Found Dead
Breaking News on "Playboy" Buddy Rose
Posted by Dameon Nelson at 4/29/2009 02:34:00 PM No comments: Links to this post
Monday, April 27, 2009
Pro Wrestling Report Radio - April 26, 2009
This week we have Hernandez from TNA as a Guest, a full review of WWE Backlash, an in depth segment on Scott Steiner and so much more! See more at www.pwrshow.com!
Posted by Dameon Nelson at 4/27/2009 02:47:00 AM No comments: Links to this post
WWE Backlash Results
Dameon Nelson reviews WWE Backlash!
Posted by Dameon Nelson at 4/27/2009 02:24:00 AM No comments: Links to this post
Sunday, April 26, 2009
More Superstar Interviews!
The Pro Wrestling Report is pleased to announce the arrival of several major stars to PWR TV in the next few weeks! We kick it all off tonight LIVE on 540ESPN Radio with our first of two interviews with one half of LAX, HERNANDEZ! Then check out PWR TV each week for another NEW superstar interview. Only PWR brings you these stars in these exclusive interviews so be sure to head to the site for all the latest information
Posted by Dameon Nelson at 4/26/2009 08:54:00 PM No comments: Links to this post
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Around the Districts
Adamstown Lotto
Winning Numbers: 6-25-27-28. No Jackpot winner. One match 3 - Mick Doran. The next draw in the Adamstown Lotto takes place Monday 4th January 2016 in Cullens Pub. Jackpot €15,100. Do buy a ticket and be in with a chance. Wishing all our players, ticket sellers and draw workers a very Happy Christmas and many thanks for your support over the past year.
Childrens Choir
Lou Duggan & Michael Ridgeway have started a Children's Choir, they practice after 10.30am Mass on Sunday mornings in the church, if you would like your child to be part of this choir just be there at that time.
Horse Charity Ride
A Horse Charity Ride will take place on Sunday 3rd January 2016, starting in Camross at 10.00am. Suitable for all ages. Part of the day's proceeds will go to Adamstown National School. Further details later
Colaiste Abbain
Coláiste Abbáin, Adamstown recently held an information evening with regards to new entrants for the 2016-2016 school year. The meeting was very well attended and established that there is a significant interest in Coláiste Abbáin and the new facilities, which will come on stream after the Easter holidays 2016. With the opening of the new school in 2016, applicants to Coláiste Abbáin undoubtedly will increase.
We are currently enrolling for the 2016 First Year in-take and welcome completed application forms up to and including 23rd January 2016. Please note that the maximum number being enrolled into First year is 60 and the practice of "first come, first served" will apply.
The school will hold Assessment Day on Saturday 23rd January 2016 at 10.00am. Meanwhile should you have any queries please contact the main office on 053 9240564.
Prayer Group Notice
The Sunday night Prayer Group will take a break for Christmas and will resume on Sunday 10th January 2016.
Athletic Club News
Adamstown Athletic Club will have their annual awards night in the Community Centre on Sunday 3rd January 2016. We look forward to seeing everyone there on the night. The club will be hosting a fashion show in the Horse and Hound on Friday 19th February 2016. The event kicks off at 8pm sharp. The fashion show will feature the latest fashion from Lulu's Boutique, Si Jolie Boutique, The Loft Wexford , Wallaces Wellingtonbridge and French and Danish clothing lines provided by Helen. Entertainment on the night by DJ Liam Sharkey. Proceeds on the night will go to the Athletic Club and to Console. Tickets are €10 and are now on sale. Please support.
Sr Breda English R.I.P.
Sincere sympathy is extended to Paddy English and Family, Raheenduff, Adamstown on the death of his sister Sr Breda Mary English in South Africa. Formerly of Tomfarney Adamstown. Sympathy also to her brothers, sisters, relations and friends. May her gentle soul rest in peace.
Surgery Opening Hours
Tuesday 29th & Wednesday 30th December-Normal Hours. New years eve-open in the morning with Caredoc from 1pm. New Years Day (Friday)-Caredoc, Monday 4th January-Normal Surgery Hours Resume.
Pharmacy Opening Hours
Tuesday 29th, Wednesday 30th & Thursday 31st December-9am to 6pm. Friday 1st January-closed. Saturday 2nd January-10am to 2pm.
New Year's Day Swim
The annual Cahore New Year's Day Swim takes place at Cahore pier at 2 p.m. Get 2016 off to a refreshing start with a dip in the Irish Sea and support Cahore Inshore Rescue Service at the same time. Sponsorship cards are available from Gerry O'Loughlin on 087 2771473 or Brenda Kavanagh on 087 9022589.
Cahore Inshore lifeboat wishes all its friends a very Merry Christmas and a peaceful and happy New Year, and thanks them for their continued support.
Christmas lights
Eileen and Richard McDonald have bedecked their house at Clonevan, Ballygarrett, with their annual Christmas lights display. The lights are on from December 8 to January 8 after dark. All are welcome to go along to see them and all proceeds of donations go to the local church.
Mrs. Mary Breen r.i.p.
Everyone in Boolavogue was saddened to learn of the death of Mrs. Mary Breen (née Turner) of Grange, Boolavogue who died on Tuesday the 15th.of December. Her remains were removed to St. Cormac's church at 9.15pm on Wednesday the 16th. and interment in the adjoining cemetery followed Requiem Mass at 11.00am on Thursday morning.
Pre-deceased by her husband Martin and son Michael she is survived by her daughters Gretta and Kit, sons Pat, John-Joe and Martin brother Mick, grand-children and extended family. Sincere sympathy is extended to all from everyone in Boolavogue.
GAA Lotto
No-one had the four number drawn on Tuesday the 8th. of December. The numbers draw were 5, 8, 14 and 21 and while no one had all four, Bernie Murphy, James O'Loughlin and Helen Doolan had three of the numbers each and so shared the 'match-three' prize of €160.
Braveheart Runners
A 5 km walk/run in aid of the Irish Heart Foundation is being organised for Sunday the 3rd. of January, 2016. It will start from the Monageer/Boolavogue GAA complex in Monageer at 12.00 noon. Participants can register in the complex from 10.30am on the day of the run.
There will be a prize for the first man and the first woman home and numerous spot prizes. Refreshments will be provided afterwards. Come along and join in the fun and get the New Year off to a great start!
Split The Pot
Brother and sister, Brendan and Bernadette Carton won the 'Split-the-Pot' prize when the draw was made in the Monageer Tavern on Friday night, the 18th. of December. With almost 500 'festive-fiver' envelopes sold, Bernadette and Brendan share half of the pot, €1225 between them.
Coiste na nÓg would like to thank everyone who sold and bought envelopes and a special thanks to Séamus and Mary who hosted the event in the Monageer Tavern.
300-1 Club Draw
Winners of the most recent Kilmyshall 300-1 Club Draw : 1. Kathleen Sinnott, Ballypraecus; 2. Jim O'Reilly, Ballypraecus; 3. Janette O'Neill, Glasslacken; 4. Timothy Farrell, Kyle; 5. Noel Plunkett, Ballypraecus; 6. Annette & Michael Murphy, Ballyphilip; 7. Frances Cowman, Rossard; 8. Jim Kenny, Ballypraecus; 9. Colm & Linda Kehoe, Camteigue; 10. Mary Codd, Craan.
Clonroche Poulpeasty
Historical Society
Local historian Tom McDonald has another article published in the latest addition of Journal of Wexford Historical Society. The journal features an article on crime and anarchy in the Templeudigan area around 1820. Templeuidgan was part of the old parish of Kilegney until 1853 when it split into the modern day parishes of Rathnure and Cloughbawn.
Fitness Pilates
Thanks to all who attended Fitness Pilates throughout 2015. I would like to wish you all a Merry Christmas & a very Happy Healthy New Year. Classes will resume Monday 4th of Jan in Clonroche CC. Call or text Claire for more info on 0860766326-suitable for beginners.
Thank You
I would to extend my thanks to all who contributed to the parish notes during the year, to all the Club PRO's who send in updates on all their activities, this made my job very easy. Notes can be submitted by text, telephone or email deadline Friday morning 10.00am to firstname.lastname@example.com or 087 6341614 Best wishes for Christmas and and a very prosperous 2016
Riverchapel whist
Fr. Tom and the Parish Committee would like to take this opportunity to thank everybody who helped in any way possible at Riverchapel Whist over the past year. The Committee would also like to take this opportunity in wishing all a prosperous New Year.
Please note the Riverchapel Whist Drive will be closed on Thursday, December 31, and will reopen on January 7 2016 at 8.30 p.m. sharp. All will be made most welcome.
Réalt na Mara
Ballygarrett Réalt na Mara GAA Club would like to wish all their club members, supporters, team and pitch sponsors best wishes for 2016. We look forward to seeing everyone on and off the pitch for the 2016 season.
Congratulations to our Scor group who have qualified for the Leinster Final on January 3 which will be held in Westmeath. This is a great achievement for the group who represent both club and county.
Blotto: There was no winner in last week's Réalt na Mara Ballygarrett GAA Club Blotto draw. The numbers picked were: 19, 20, 26, and 28. The winner of the €30 Pettitt's voucher was Gerry Hunt. The €20 lucky dip winners were: Tom O'Loughlin; Fiona Cameron; Kieran Phipps; Stephanie Furlong; and Sean Breen. The five free tickets winner was Joan Casey. This week's jackpot rose to €3,500.
Ladies Club
Riverchapel Courtown Ladies Club will resume on Monday, January 11, at 7.30 p.m. in Riverchapel Community Centre. The club sends its thanks to Cllr Robbie Ireton for his help during the year. Thanks also to the club committee for all their help and the parish committee for the use of the hall. The club wishes everyone a peaceful new year.
Water safety
Wexford Water Safety will hold lifesaving classes in Courtown Adventure and Leisure Centre on Wednesdays from 6:30 p.m. to 8 p.m. for 10 weeks, starting Wednesday, January 13. The cost is €50pp. Limited numbers. Minimum age, 10 years. Must be able to swim. To register, text Áine on 087 2871670 with the child's name and age (and previous WS level achieved). Safety 1 to Rescue 1.
Meals on Wheels
The Meals On Wheels Gorey group is extending its service into the Courtown area. The service is a local registered charity operating for the last 40 years in the Gorey urban area. It works to support independent living and providing assistance to elderly people living at home
Are you interested and can you afford about an hour a week to support your community? If you can, contact Colin at 087 6392539. Access to your own transport and Garda vetting will be required.
Seal Dip
Seal Rescue Ireland is holding a Seal Dip fundraiser on Courtown's north beach at 1 p.m. on New Year's Day. Supporters can take a swim in the ocean to raise funds for Seal Rescue Ireland. Learn more or register at www.sealrescueireland.org/fundraisers/
Service times
The service times for the Ardamine Union of Parishes are as follows: Sunday, January 3: Monamolin 9 a.m. Morning Prayer; Ardamine 10.15 a.m. Holy Communion; Kilnamanagh 11.45 a.m. Morning Prayer.
Mass times
Mass times in our Lady Star of the Sea, Riverchapel are as follows: Saturday's Vigil Mass is at 6 p.m. Sunday Masses are at 9 a.m. and 11 a.m.
Davidstown/ Courtnacuddy
GAA lotto Draw results
Results of the lotto draw held on Monday 14th December. Jackpot €1950. Numbers drawn were 6-14-26 & 29. There was no jackpot winner and no match 3's. lucky dip winners were PJ Martin, Stonepark & Lilly Dunne O' Connor c/o Hugh Dunne. Sellers prize was won by Peg Jordan. Next draw is on December 28th and the next jackpot will be €2,100.
GAA Club Dinner Dance
Davidstown / Courtnacuddy GAA Club Dinner Dance is on 6th February with music by NERO and Disco till late. Tickets €25 each, 4 Course Meal, with County Final medal presentation & Raffle on the nightFor tickets contact; Majella Dunne 0876786154. Micheal Hayes 0861736235.Maureen Doyle 0868311225. Mick Murphy 0879912706.
Marshalstown/ Castledockrell
New Year Wishes
Get well wishes to all from the parish who are sick at the moment in hospitals, nursing homes or at home. Happy New Year to all readers of and contributors to these notes. Thank you for your support over the past year.
Wedding Bells
Congratulations to Mark Delahunty and Geraldine Hughes who were married recently. Wishing them health and happiness for the future.
Important Dates
February 10th Ash Wednesday, March 1st First Confessions in Castledockrell, March 3rd First Confessions in Marshalstown, March 27th Easter Sunday, April 23rd First Holy Communion in Marshalstown, April 24th First Holy Communion in Castledockrell, May 29th Patron in Marshalstown, July 1st Patron in Castledockrell.
Late Tom Bolger
Sympathy is extended to the family of the late Tom Bolger, Marshalstown who was laid to rest in Marshalstown Cemetery on Monday, 21st December following Funeral Mass in St. Joseph's Church. May he rest in peace.
Get On Track
Back by popular demand-Get On Track with Operation Transformation returns on Tuesday, Jan 5th in Farmleigh. Training sessions every Tuesday and Friday at 7:30pm. Registration at 7pm on the 5th! Professional trainer every night. Great value at only €40 for 8 weeks membership (that is only €2.50 per session) Age 16+. Christmas gift vouchers available, phone Louise on 0879001227.
We welcome into the Christian family baby Katie Marie Roche who was baptised recently and we congratulate her parents.
Story Telling
Next session of Story Telling is in Castledockrell Community Centre on Thursday, 21st January at 8p.m. Refreshments served. Raffle held on the night. Wishing all who attend Story Telling health and happiness for the new year. Thank you for your support over the past year.
Lotto Results
Marshalstown Castledockrell Lotto results for Thursday 17th December 2015. Winning numbers 21, 23, 29, 32. No Winner. Match 3 winners, Theresa Denton c/o the bingo, Patrick Kinsella, each wins €50. Next week's draw will be held in the community centre on Thursday 31st December 2015. Next week's jackpot will be €3,100. Happy Christmas and a happy new year to everyone. Thanks for your continued support.
Sympathy is extended to the family of the late Kathleen Comerford and to the family of the late Nancy Grannell who died recently. May they rest in peace.
Sincere sympathy is extended to the Hourigan family of Cull Na Tear on the death of their mother Jean who died on Saturday 19th December. Burial took place after 10 o'clock in Monamolin to Deans Grange Cemetery, Dublin on Tuesday 22nd December. May she rest in peace.
Happy birthday wishes go to Alas Earle, Cullentra who celebrated her birthday on 21st December.
ladies Club
The ladies Social club will reopen after Christmas on Wednesday 13th January.
Well wishes
Get well wishes go to Martha Laffan of Ballyedmond. This comes from all your family and neighbours
Carol singing
A big thanks to all who turned up to the Carl service on Saturday night in the street. A very big thanks to poor Santa who made sure every child got a present and to all his helpers. Thanks also to the Alley Bar for the use of their Lounge.
Wexford People