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Conceived and designed the experiments: KA EM MM PM M. Haymond. Performed the experiments: KA EM RH MM AB SP LS M. Hu CS PM PG M. Haymond. Analyzed the data: KA RH CS PG. Contributed reagents/materials/analysis tools: KA RH MM AB PM PG M. Haymond. Wrote the paper: KA EM RH MM M. Haymond.
While breast milk has unique health advantages for infants, the mechanisms by which it regulates the physiology of newborns are incompletely understood. miRNAs have been described as functioning transcellularly, and have been previously isolated in cell-free and exosomal form from bodily liquids (serum, saliva, urine) and tissues, including mammary tissue. We hypothesized that breast milk in general, and milk fat globules in particular, contain significant numbers of known and limited novel miRNA species detectable with massively parallel sequencing. Extracted RNA from lactating mothers before and following short-term treatment with recombinant human growth hormone (rhGH) was smRNA-enriched. smRNA-Seq was performed to generate 124,110,646 36-nt reads. Of these, 31,102,927 (25%) exactly matched known human miRNAs; with relaxing of stringency, 74,716,151 (60%) matched known miRNAs including 308 of the 1018 (29%) mature miRNAs (miRBase 16.0). These miRNAs are predicted to target 9074 genes; the 10 most abundant of these predicted to target 2691 genes with enrichment for transcriptional regulation of metabolic and immune responses. We identified 21 putative novel miRNAs, of which 12 were confirmed in a large validation set that included cohorts of lactating women consuming enriched diets. Of particular interest, we observed that expression of several novel miRNAs were altered by the perturbed maternal diet, notably following a high-fat intake (p<0.05). Our findings suggest that known and novel miRNAs are enriched in breast milk fat globules, and expression of several novel miRNA species is regulated by maternal diet. Based on robust pathway mapping, our data supports the notion that these maternally secreted miRNAs (stable in the milk fat globules) play a regulatory role in the infant and account in part for the health benefits of breast milk. We further speculate that regulation of these miRNA by a high fat maternal diet enables modulation of fetal metabolism to accommodate significant dietary challenges.
Breast milk is the ideal source of nutrition for mammalian infants [1], [2]. It is widely recognized that human milk-fed infants receive significant health benefits compared to those fed substitute milk preparations [3]. These benefits include a decreased risk of a wide range of diseases, including otitis media, meningitis, respiratory infections, sudden infant death syndrome, diabetes, cancer, and obesity, as well as higher neurocognitive behavior testing and intelligence scores [3], [4]. Moreover, the benefits of breastfeeding extend to mothers as well as mother-infant pairs [4]–[6]. For years, infant formula companies have struggled to replicate this unparalleled balance of lipids, oligosaccharides, and proteins necessary for infant nutrition. However, formula-fed infants do not share the same health benefits as those partially or exclusively breast-fed [3]. The mechanisms underlying these beneficial effects are poorly characterized but may be the result of biologic macromolecules, including growth factors, antibodies, and small nucleic acids [7]–[9]. Each of these macromolecules is largely underappreciated and likely are not readily duplicated in substitute milk preparations, as they may be species specific.
Small, noncoding RNAs (smRNAs), including 20–24 nucleotide (nt) microRNAs (miRNAs), are critical, highly conserved mediators of posttranscriptional gene regulation and modulate gene expression in eukaryotes [10]. miRNAs have been described as functioning transcellularly with ubiquitous expression in human tissues and fluids and have been previously isolated in cell-free and exosomal form from bodily liquids (serum, saliva, urine) [11]–[16] and various bodily tissues including mammary tissue [17]–[18]. miRNAs play pivotal roles in regulating diverse developmental processes by targeting mRNA for translational repression, cleavage, or destabilization [19]. Recent studies have revealed that transcellularly acting miRNAs may play salient roles in the development of diabetes [20], and cancer [15]. Transcellular acting miRNAs are also a current focus of cardiovascular and cell-to-cell communication research [12]. miRNAs are crucial mediators of immune cognition [21], [22], and recent reports in immune models have demonstrated that in addition to their characteristic composition of proteins, T cell–derived exosomes contain RNA that can be stably transferred to the antigen-presenting cell, as an example of cell-to-cell genetic transfer, to modulate functional cognate immune interactions [11].
With these findings in mind, we speculated that the miRNAs in breast milk modulate infant physiology, immunity, and feeding behavior. Several studies have demonstrated that miRNA is present in bovine milk [23]–[25] and in whole human breast milk, including within milk fat globules [7], [9], [17], [26]. However, to our knowledge, no studies have interrogated for novel miRNAs employing Next-Gen deep sequencing discovery pipelines. Given that array analysis interrogates miRNAs that have been previously described in other research findings, and therefore are likely to be missing miRNA species limited to newly interrogated tissues or fluids, we sought in the present study to take an unbiased sequencing-based approach to identify novel species in RNA-enriched milk fat globules. We reasoned that since miRNAs bind a large number of targets, extensive mapping of both known and novel miRNA species to the 3′ untranslated region (UTR) of gene targets may in part explain the potential significance of miRNAs in breast milk.
In the current study, we hypothesized that a massively parallel sequencing-based approach would not only definitively identify miRNA in breast milk, but also expand the number of known miRNAs in human milk and concomitantly enable the identification of novel miRNAs. These miRNAs, both previously described and novel, could thereafter be mapped to target gene pathways to reveal potential molecular and physiologic functions which might account for the observed nutritive, cognitive, and immunity-based benefits that breast-fed infants are known to receive. We further reasoned that the potential functional significance of those novel miRNAs could be surmised using quantitative measures in well-described cohorts of lactating women subjected to significant dietary manipulations.
We studied three cohorts of lactating women (Fig. 1). Following IRB approval, a total of 5 lactating women were admitted to the General Clinical Research Center at Baylor College of Medicine as outlined in Methods, and baseline breast milk samples were collected every 3 hours for the first 24 hours. Since multiple hormones, including growth hormone, prolactin, insulin, and steroid and thyroid hormones, are necessary to ensure successful lactation, we opted to use a previously well-studied supraphysiological dose of rhGH (0.1 mg/kg/day) in short duration as a means to normalize across our cohort in an effort to facilitate comparison among subjects [26]. In the discovery set, 6 breast milk samples were obtained from 3 women (mean age, 23.3+/−3.2 years; mean BMI, 25.2+/−0.1) of self-identified Caucasian (n 1), Hispanic (n 1), or African-American (n 1) race. In the validation set, 33 breast milk samples were obtained from 19 women (mean age, 27.3+/−4.1 years; mean BMI, 27.7+/−1.39) of Caucasian (n 7), Hispanic (n 8), or African American (n 4) race or ethnicity. There was an expected statistically significant difference in participant's weight between discovery (lean) and validation sets, as obese women were intentionally included by design of the original cohorts (notably, obese women studied in the glucose-galactose cohort) [27]. However, this difference did not manifest as a significant variation in BMI (Table 1).
We first characterized optimal enrichment of yield and integrity of smRNA macromolecules from distinct fractions and preparations of human breast milk. Compared to the yield of smRNA from whole milk (Fig. 2, lanes 1 and 2), the lipid fraction of human milk containing milk fat globules was enriched for smRNA species (lanes 3 through 6). Moreover, the Trizol and mirVANA RNA isolation procedures gave the highest purity of miRNA with the lowest level of ribosomal RNA (Fig. 3). Based on these findings, the mirVANA isolation of frozen lipid fraction breast milk samples stabilized with Trizol were used for all subsequent experiments with both our discovery and validation sets.
After small RNA sequencing with the Illumina GA-I Analyzer, a total of 124,110,646 usable reads were obtained from 6 breast milk samples (Supporting Information S1, Table S1). We processed these reads using our previously described pipeline [28]. Of these reads, 31,102,927 (25%) exactly matched to known miRNAs in miRBase 16.0, and 74,716,151 reads (60%) were matched using sequential relaxing (match to +4, and loose matches) (Supporting Information S1, Table S2). These matches corresponded to 308 of the known 1018 previously described miRNAs (Supporting Information S1, Table S3). The 10 most abundant of these previously described miRNAs are presented in Table 2.
To investigate the functional role of miRNA in human breast milk fat globules, we used TargetScan [29] and miRanda [30] predictions to identify miRNA gene targets. All miRNAs with an exact match to miRBase targeted 9074 genes predicted by both TargetScan and miRanda (Supporting Information S1, Table S4). The 10 most highly expressed miRNAs based on read counts targeted 2691 genes of interest that both algorithms predicted (Supporting Information S1, Table S5).
For genes targeted by all miRNAs, enrichment analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) [31] revealed 316 Gene Ontology (GO) terms (Supporting Information S1, Table S6) and 16 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (Supporting Information S1, Table S7) enriched at a false discovery rate (FDR) <0.01. Similar analysis of the 10 most commonly expressed miRNAs showed enrichment for 145 GO terms (Supporting Information S1, Table S8) and 8 KEGG pathways (Supporting Information S1, Table S9). The top 20 enriched GO terms based on FDR for gene targets of the top 10 expressed miRNA were visualized as a graph with the node size representing the number of genes present in the GO term and the edge thickness representing the overlap between genes in different GO terms (Fig. 4A). GO terms related to the regulation of transcription, metabolic processes, and biosynthetic processes were highly represented among the enriched terms and form a connected graph consisting of 19 of the top 20 GO terms. This pattern of GO term enrichment was also seen for genes targeted by all miRNAs with "regulation of transcription from RNA polymerase II promoter" being the most significantly enriched term with 533 genes and 285 of these genes were involved in positive regulation. Other significantly enriched GO terms were involved in axon- or neuron-related terms. Notable KEGG pathways showing enrichment were "Insulin signaling pathway" and "Axon guidance".
While no single immunity–related GO terms or KEGG pathways were significantly enriched at a FDR<0.01, DAVID clustering of GO terms or pathways showed a cluster of GO terms related to immune development and T and B cell function with an enrichment score of 2.17 (Annotation Cluster 52, Supporting Information S1, Table S10). Using the less stringent Benjamini multiple testing correction, we found that several of the terms in this cluster, including "immune system development," were significant at <0.01. DAVID clustering of KEGG pathways showed a cluster of immune cell signaling pathways with an enrichment score of 1.75 (Annotation Cluster 3, Supporting Information S1, Table S11).
After comparison to miRBase to identify known miRNAs and performing additional filtering, we found that 4759 reads corresponded to 21 potentially novel miRNAs (Table 3 and Supporting Information S1, Table S12, S13). TargetScan was used to predict gene targets for the 12 novel miRNAs that were validated (see Validation below). After filtering TargetScan predictions for those with a context+ score percentile of ≥90, 3554 gene targets were identified (Supporting Information S1, Table S14). GO enrichment showed 9 terms significantly enriched at FDR <0.01 (Supporting Information S1, Table S15), but KEGG Pathway enrichment analysis did not show any pathways enriched at the FDR <0.01 significance level (Supporting Information S1, Table S16). The top 20 GO terms associated with genes targeted by the novel miRNAs were visualized as a graph (Fig. 4b). Similar to the graph for known miRNA targets, a connected graph of terms related to regulation of transcription and metabolic processes was present. Connected graphs of terms related to the plasma membrane, "regulation of T cell receptor signaling pathway", and ion binding were also identified. The enrichment for "calcium ion binding" is of particular interest given the importance of calcium as a constituent of breast milk. With the exception of "integral to plasma membrane" and "intrinsic to plasma membrane", all GO terms showing enrichment for genes targeted by the novel miRNAs were shared with those enriched for genes targeted by all known miRNAs.
As a second means of identifying the potential functional significance of the putative novel miRNA species we discovered in human breast milk fat globules, we sought to interrogate the relative abundance of these miRNA species following maternal dietary manipulations in previously described and well-characterized obese and lean subject cohorts [27], [32]. Samples from the rhGH-treated cohort [7], [26] were used to normalize values and derive fold increase among each dietary manipulation. Specifically, read counts for all identified known miRNAs were quantile-normalized, and Pearson correlations were performed (Supporting Information S1, Table S17). Pearson correlations in pairwise comparisons of pre- and post-rhGH treatment sequencing data for the same subject ranged from 0.96 to 1.0, and correlations across the entirety of the cohort ranged from 0.95 to 1.0 for all known miRNAs identified (Supporting Information S1, Table S17). As a second means of determining appropriateness of normalization, the Linear Models for Microarray Data (Limma) R package [33] was also applied to the sequencing data after quantile normalization; no differential miRNA expression between the pre- and post- rhGH treatment sequencing data was identified at an FDR <0.01 (Supporting Information S1, Table S18). The lack of evidence of an effect of rhGH treatment on miRNA expression confirms the appropriate use of the rhGH cohort for our subsequent comparisons between dietary manipulation cohorts.
After we determined an appropriate normalization set, we then sought to derive individual and cohort variation with respect to body habitus (obese versus lean) and dietary manipulation (relative glucose-galactose, and high fat-high carbohydrate). We observed significant variations (delta delta Ct following normalization to rhGH) only with putative novel miR-67 and miR-27 and only in women who were on a high-fat diet (Fig. 5). When considered as means among groups, we similarly failed to observe a significant effect of body habitus (obese versus lean), nor by dietary cohort except with high fat diet intake and only among putative novel-miR-67 and novel-miR-27 (ANOVA p=0.01; Table S19). Novel-miR37 and miR-68 approached but did not reach significance (p=0.07 and 0.08, respectively) (Fig. 5 and Supporting Information S1, Table S19).
After we identified and validated novel miRNA in human breast milk, we next sought to determine whether these species are present in placenta. Like mammalian milk, the placenta is generated and only expressed during the interval of reproduction and has been found to play a pivotal role in modifying fetal growth and metabolism [34], [35]. Five of the novel miRNAs we identified were validated in both breast milk and placenta (novel-miR-62, −114, −118.2, −111, −120), and no miRNAs that did not validate in breast milk were validated in placenta (Supporting Information S1, Table S20). Although comparison in dietary cohorts could not be made, we found that the placental expression of detected novel miRNA in lean or obese subjects did not differ significantly (Fig. S1).
Researchers have understood for decades that human breast milk provides optimal postnatal nutrition for infants; however, the molecular mechanisms that enable the transfer of immunity- or metabolic- benefits previously described are incompletely understood. Given emerging evidence that transcellulary acting miRNAs are functionally present in serum, urine, and exosomes [12], [15], [20] and are crucial mediators of immune cognition [13], we sought to investigate both their presence and function in human breast milk. We found that not only are known and novel miRNAs present and stable in the milk fat globules, but that their measured expression is altered by a maternal high fat diet. Based on the findings of our target pathway analysis, we speculate that altered expression of miRNA species (from dietary manipulation) bears the potential to modify either mothers' or infants' metabolic pathways. This is consistent with the presumptive role of circulating miRNA biogenesis and secretion as pathophysiologic mediators, for which alterations in both concentration and composition of miRNA have been well correlated with cardiovascular morbidity and mortality [12], [15]. Zhou [36] recently speculated that miRNAs could be transferred through the gut in infants to aid in development of infants' immune systems; we suggest that breast milk miRNAs could similarly be transferred through the infant gut and modulate gene expression, a mechanism explaining epigenetic maternal effects of offspring phenotype.
We found that the lipid fraction of breast milk, including milk fat globules, contained the highest quantity of miRNA and yielded the purest miRNA fractions. Previous study of mammalian genomes suggested that the most highly conserved proteins in milk were related to secretory processes and were contained within the milk fat globules [37]. Conversely, the most evolutionarily divergent proteins were associated with nutritional biogenesis. We found a significant number of conserved and known miRNA species that affect a large number of genes in a focused arena of pathways (Supporting Information S1 and Fig. 4) in the milk fat globules secreted from mammary cells, suggesting that the process of miRNA excretion in breast milk confers a potential evolutionary advantage.
Since it is not feasible to directly measure nor visualize a human infants' physiologic responses along these miRNA-targeted pathways, we undertook a robust number of approaches aimed at elucidating and ascribing functional significance to both known and novel miRNAs we identified in breast milk. These included target gene pathway analysis of both known and novel miRNA species, analysis of specific and well-described perturbations in the maternal diet, and ascertainment of the presence of novel miRNA in another tissue unique to reproduction, the placenta.
With respect to the analysis of the mRNA transcripts that are targeted by these miRNAs, the 308 known miRNAs we found in breast milk target genes related to the regulation of gene transcription and metabolism as well as the establishment and refinement of immunity (Fig. 4A, Supporting Information S1, Tables S4 through S11). That the miRNAs in breast milk targeted these functional classes of genes suggests a molecular basis for the benefits experienced by milk–fed infants [3]–[6]. Additionally, many of the novel miRNAs discovered in our research target genes related to cancer. Target examples such as these suggest that miRNAs in breast milk could contribute to the decreased risk of cancer in humans that were milk-fed as infants [3]. Novel-miR-118.2 possibly targets odz, odd Oz/ten-m homolog 2 (Drosophila) (ODZ2); this family of proteins has been found in high quantities in central nervous system tissue, notably on brain axons, and is thought to be involved in cell-cell communication [38]. Based on these target predictions, it is interesting to further speculate that the presence of novel-miR-118.2 in breast milk may suggest a molecular mechanism for neurocognitive benefits derived from breast milk.
To more formally investigate the functional roles of these secretable miRNA species, we interrogated alterations in the relative composition of breast milk miRNAs in response to changes in the maternal diet. We used two previously described, well-characterized dietary manipulations in these experiments, in which breast milk samples from diet-varied women were normalized to rhGH-synchronized lean lactating women (Supporting Information S1, Tables S17 and S18). With respect to the appropriateness of our normalization cohort, Pearson correlations in pairwise comparisons between pre- and post- rhGH treatment sequencing data for the same subject ranged from 0.96 to 1.0, and correlations across the entirety of the cohort ranged from 0.95 to 1.0 for all known miRNAs identified. Moreover, Limma was applied to the sequencing data after quantile normalization and revealed no differential miRNA expression between the pre- and post- rhGH treatment sequencing data at an FDR <0.01 (Supporting Information S1, Table S18). These findings are consistent with Maningat's earlier study of this cohort [26], which demonstrated that although the short-term administration of rhGH induced DNA synthesis, there was no significant difference in milk protein gene expression or milk production between pre- and post-treatment groups.
We have previously demonstrated that high-glucose diets in obese lactating women are associated with higher endogenous fat mobilization and oxidation during meal absorption compared to women fed high-galactose diets, whereas milk production, energy expenditure, and carbohydrate oxidation are no different [27]. This is in contrast to earlier findings pertaining to high fat relative to high carbohydrate consumption in lean lactating subjects, whereby a high-fat diet increases milk fat concentration as well as energy output and expenditure [32]. In this cohort, a high fat diet (and therefore a low carbohydrate diet, as the two diets were isonitrogenous and isocaloric) resulted in the lactating mothers adapting to low carbohydrate intake by decreasing carbohydrate oxidation. While we did not directly assess the infants' physiology, our observations pertaining to miRNA excreted in the stable and transferable milk fat globules merit considerable attention.
Consistent with the overall effects of a high-glucose and –galactose diet on the physiology of the mothers and their milk, we did not observe significant variation in any of our novel miRNA species (Fig. 5 and Supporting Information S1, Table S19). However, the same is not true for women on a high-fat diet. Novel-miR-67 and -27 expression was significantly increased with high fat relative to high carbohydrate consumption (Supporting Information S1, Table S19) in the cohort as a whole, with as great as 6-fold effect sizes among individuals (Fig. 5). These findings suggest that breast milk has rich epigenetic potential in human infants. Alterations in the quantity and type of miRNA expression in breast milk could thus represent dynamic maternal regulation of infant gene expression based on changing environments and notably with significant maternal diet perturbations. Given that lactation helps mothers and offspring adapt to an ever-changing food supply, this would potentially provide an evolutionary advantage to the offspring [39]. Moreover, these findings are consistent with our prior observations in a non-human primate model on the impact of both the maternal and post-natal diet on modification in the offspring's hepatic epigenome [40], [41]. In line with previous research showing that human milk constitution varies with gestational age at delivery as well as through the first six months of life [42], it is logical to speculate that miRNA expression would also change throughout the timeframe of lactation until weaning and future studies will interrogate miRNA profiles by virtue of gestational age at delivery.
Further insights into the potential biologic significance of our identified novel miRNAs can be gleaned by their predicted targeted transcripts. TargetScan was used to predict gene targets for the 12 novel miRNAs that were validated revealing 3554 gene targets for those with a context+ score percentile of ≥90 (Table S14). The top 20 GO terms associated with genes targeted by the novel miRNAs were visualized (Fig. 4B) and similar to known miRNA targets, a connected network of terms related to regulation of transcription and metabolic processes was observed. With the exception of "integral to plasma membrane" and "intrinsic to plasma membrane", all GO terms showing enrichment for genes targeted by the novel miRNAs were shared with those enriched for genes targeted by all known miRNAs. Of noted interest, in addition to the transcription and metabolic regulatory network graphs of terms related to the plasma membrane, "regulation of T cell receptor signaling pathway", and ion binding were uniquely prevalent among targets identified with mapping of our novel miRNA species. The enrichment for "calcium ion binding" is of particular interest given the importance of calcium as a constituent of breast milk [43], and further underscore the likely functional role these novel species heretofore not identified play in both infant and mother.
Finally, we measured the presence and abundance of the novel miRNAs we discovered in placental tissue from both lean and obese subjects to investigate the function of these miRNAs (Fig. S1 and Supporting Information S1, Table S20). Of our 21 putative miRNAs, none of the 9 species not validated in breast milk fat globules could be validated in placenta. However, of the 12 validated in breast milk, 5 of 12 novel miRNAs were validated in both breast milk and placenta (Novel-miR-62, −114, −118.2, −111, −120; Supporting Information S1, Table S20). These findings suggest that there may be common expression of miRNA among tissues and fluids which are uniquely produced in women during critical periods of reproduction and development, and may play a regulatory role in both the mother and her infant. This early line of investigation deserves significant attention in future studies.
Our study had several potential limitations. Given the high financial burden of sequencing, we elected to derive limited discovery sets and more robust validation sets. Thus, we performed sequencing on 6 samples from 3 patients in the discovery set, which likely does not completely represent the rich genetic diversity of the human species. However, given that each of our samples came from patients of different races, and that the validation sets were representative of the discovery set with both lean and obese subjects (Supporting Information S1, Tables S17 and S18), the limit to diversity was theoretically minimized. Another potential limitation of the present study was that potentially important miRNAs might reside in the whey portion of breast milk, which was not represented in our analysis of only the lipid fraction. However, our study is one of few that examines the RNA transcriptome of highly conserved milk fat globules [7], as many other studies of miRNA in breast milk have either discarded the milk fat globules in preparation for study of the whey fraction of milk [8], [36] or analyzed whole milk [9], [17], [25]. Our findings are thus an important contribution to the existing literature. It is possible that novel or known miRNA may have been degraded in the thaw process prior to purification; however, in previous work by Kosaka [9], the stability of miRNA has been demonstrated in at least 3 freeze-thaw cycles without changes in expression levels and thus we predict that the thaw effect was minimal to negligible. Although we could not biopsy normal infant tissue to more accurately define the functional significance of the novel miRNAs we discovered, their relative abundance in milk from women of various diets may lend merit to their presumptive significance. Finally, small errors in sequencing, though rare in this shotgun sequencing method, may have caused us to misidentify novel miRNAs or fail to identify additional miRNAs. Future studies should consider these limitations and examine the presence of these miRNAs in other breast milk samples from women of varying races and environments, as well as examine profile differences between whey and lipid fractions and fresh versus previously frozen milk. It is imperative that future studies deeply explore the dynamics of miRNA expression due to changing environments. However, despite these limitations, our study, novel in both construct and analysis, makes a pivotal leap in potentially explaining the molecular mechanisms for the benefits milk-fed infants receive.
Our findings indicate that massively parallel sequencing paired with robust analysis in well-described cohorts serves as a valid and powerful means of identifying known and novel miRNAs in human milk fat globules and characterizing their composition and abundance following diet alterations in lactating women. These miRNAs target critical pathways of the regulation of transcription, metabolism, and immunity. Thus, our findings elucidate a novel arena of molecular mechanisms that might explain the well-known benefits of human breast milk for human infants. We speculate that miRNAs function as a previously unrecognized means of dynamic maternal regulation of infant gene pathways and opens an exciting subfield of epigenetics that warrants deeper exploration.
All study protocols and consents were approved by the Institutional Review Board at Baylor College of Medicine (Houston TX). Females between 6 and 12 weeks postpartum of term (>37 weeks gestational age) with healthy infants gave written informed consent for breast milk donation after the nature and possible consequences of the study were explained. Exclusion criteria were age <13 years, preterm delivery, any chronic illness including diabetes, and any relatives with diabetes. Study participants were admitted to the General Clinical Research Center and collected breast milk at semi-hourly intervals while breastfeeding their own infants. Samples for both the discovery and validation sets were selected from 3 studies in which breast milk was obtained after women who were exposed to various research conditions (Fig. 1).
A total of 5 lactating women were admitted to the General Clinical Research Center at Baylor College of Medicine. At 0800 hours on the first day of the study, each woman breastfed her infant every 3 hours, and after 24 hours, received weight-based recombinant human growth hormone for synchronization. The women continued to collect breast milk via pump every 3 hours for an additional 48 hours, then every 6 hours thereafter for a total of 96 hours studied. Breast milk samples used for the discovery set were collected at hour 24 and at hour 96 (6 samples from 3 subjects).
In addition to the 5 samples from the 5 women in the discovery set (obtained at hour 96), breast milk samples from two other cohorts of breastfeeding women were used for validation. These cohorts were part of two crossover studies examining the effects of diet composition on milk quality, quantity, and carbohydrate expenditure.
7 lactating women (1 African American, 2 Caucasian, and 4 Hispanic) participated in the study. For the 3 days prior to admission to the General Clinical Research Center, the women consumed a diet of 15% protein, 50% carbohydrate, and 35% fat. Upon admission and after an overnight fast, the women were randomized to receive an isocaloric drink every 3 hours (starting at 0900 hrs) made of either glucose or galactose, the sugars in which provided them with 60% of their daily estimated energy requirement. Breastfeeding occurred every 3 hours followed by milk expression via electric pump. Breast milk samples studied in this investigation were collected 6 hours after the isocaloric drinks were administered (1500 hrs). As part of the crossover design, each woman participated in each study (glucose or galactose based drinks) with a 1 week washout period in between.
7 lactating women (2 African American, 2 Caucasian, and 3 Hispanic) were randomly assigned to either a high-carbohydrate, low fat diet (60% carbohydrate, 25% fat, and 15% protein), or a low-carbohydrate, high-fat diet (30% carbohydrate, 55% fat, and 15% protein), the caloric values of which were tailored to the woman's required total energy intake (1.3×basal metabolic rate). Fructose content of both diets was constant at 20% of total carbohydrates. Each patient was provided complete meals/snacks in their home for the first 4 days of the study, thereafter they were moved to the General Clinical Research Center at Baylor College of Medicine, where they completed an additional 3 days of study consuming the same diets as was consumed during the previous 4 days. Breastfeeding occurred every 3 hours followed by milk expression via electric pump. After an overnight fast on day 3 in the hospital, small meals were provided every 15 minutes starting at 0900 hrs. Breast milk samples studied in our investigation were collected at 1600 hrs that day. As part of the crossover design, each woman underwent each 7 day diet (high carbohydrate or high fat) and milk collection with a 1 to 2 week washout period in between.
After infant feeding, breast milk was obtained from both breasts by a standard breast pump until the breasts were emptied. These samples were then stored on ice until centrifugation in RNAse free tubes at 4 degrees Celsius (3000 rpm for 10 minutes); thereafter the fat layer was transferred to a new RNAse free tube. TRIzol (Invitrogen Life Technology) was added to the samples for RNA stabilization, and the samples were stored at -80 degrees Celsius until RNA isolation.
For experiments determining the optimal enrichment and yield of smRNA from human milk, 3 different preparations methods were compared. For all 3 preparations, human milk was collected with standard breast pump and centrifuged at 2000 g for 10 minutes to remove large cellular debris. Preparations were analyzed using the Experion Standard-Sensitivity RNA Analysis kit (#700-7103, Bio-Rad USA); all run samples passed small RNA integrity parameters and were analyzed by gel electrophoresis.
The Applied Biosystems mirVANA protocol (AM1560) was used for breast milk samples from the discovery and validation sets. RNA libraries were created from the mirVANA isolated samples and sequenced using the Illumina 1G Genome Analyzer to generate 36 nt sequencing reads. These reads were used to identify miRNAs using our previously described pipeline [28]. miRNAs were mapped against the public database miRBase release 16 for identification of known miRNAs. Reads that did not map to known miRNAs were mapped to the human genome (NCBI 36/UCSC hg18). Reads mapping exactly to the genome along with 100 bp flanking sequence were folded as RNA using the Vienna package [44] to identify hairpins. Reads within hairpins were further filtered to remove miRBase release 18 miRNAs, snoRNAs, scaRNAs, repeats as identified by RepeatMasker [45], and reads with >90% GC content [46]. Remaining reads that were present in at least two of the sequencing runs were identified as putative novel miRNAs.
Gene targets for known miRNAs were identified using TargetScan [29] context+ scores and contributions for all conserved miRNA sites (http://www.targetscan.org/vert_61/vert_61_data_download/Conserved_Site_Context_Scores.txt.zip) and miRanda [30] good mirSVR score, conserved miRNA (http://cbio.mskcc.org/microrna_data/human_predictions_S_C_aug2010.txt.gz) target predictions. Targets predicted by both algorithms were identified to compile a stringent list of targets. This was done for both the top 10 most highly expressed miRNAs based on read count as well as all miRNAs that were identified in the sequencing data. The TargetScan tool was run on the putative novel miRNA sequences to identify targets for novel miRNAs.
Gene Ontology (GO) and KEGG pathway enrichment were performed using DAVID software [31]. Graphs of the top 20 enriched GO terms were generated using the Cytoscape [47] Enrichment Map plugin [48].
Placental tissue, previously acquired at time of delivery of lean and obese gravid women and immediately stored at -80 degrees Celsius, was homogenized in lysis buffer on ice prior to extraction, precipitation, and elution of miRNA as described in the manufacturer protocol (AM1560, Applied Biosystems), as to avoid release of RNAses from cellular compartments. Lean and obese subjects were defined by their pregravid body mass index (BMI), with a BMI >30 kg/m2 denoting obesity, and <25 kg/m2 denoting lean.
QT-PCR was used to validate miRNA sequences of interest in both breast milk and placental. For the breast milk, samples obtained from the discovery set (growth-hormone synchronized women) and validation set (women in the two crossover cohorts plus women in the growth-hormone synchronized cohort) were purified as noted for sequencing; placental samples as noted above. Primers and probes were designed with the custom TaqMan® small RNA assays kit (Applied Biosystems, Carlsbad, CA) for the detection of miRNAs. TaqMan® putative mature miRNA (pmm) probes were designed as follows (all 5′ to 3′): Novel-miR-102 TCCATATCCCAACCTGTCAGAGTCT; Novel-miR-79TTTTTTGCTGGAACATTTCTGG; Novel-miR-54TAAGTTATCAAGGCATGAGAGA; Novel-miR-114GTGCGTGGTGGCTCGAGGCGGG; Novel-miR-37TTGGAGAGTGGGCAGCAGAGA; Novel-miR-111TGGGCGAGGGCGGCTGAGCGGCT; Novel-miR-67TTGAGGGGAGAATGAGGTGGAGA; Novel-miR-109ACGCGATTTGTAGCACAGACA; Novel-miR-27TCTCACCTGGCATAAGCAATT; Novel-miR-120TCTAGCAAGGGGCAGCTGCAGA; Novel-miR-123AGCAAAGCAAAGCTCAGTTGGA; Novel-miR-44TCAGCTACTACCTCTATTAGGA; Novel-miR-112TGAGAAAATGTAGAACCAAT; Novel-miR-113CAGGGCCTGGAACTCCAATGGGA; Novel-miR-118.2TTGAACTCGAGTTGGAAGAGGCG; Novel-miR-135ATTAGGTAGTGGCAGTGGAA; Novel-miR-138TCTGCACTCTATACGACCCAG; Novel-miR-62TGAGAGGGAGAAGGGGCTGCAG; Novel-miR-68CTGGTGAGCCCTGTGCTGTTCCAGGA; Novel-miR-26GCGATTGTAGATGTTATTGAT; Novel-miR-126GGGACCCAGGACAGGAGAAG. Values were normalized by Livak and Schmittgen 2−ΔΔCT Method employing rhGH subjects for normalization and U6 snRNA (5′ GTGCTCGCTTCGGCAGCACATATACTAAAATTGGAACGATACAGAGAAGATTAGCATGGCCCCTGCGCAAGGATGACACGCAAATTCGTGAAGCGTTCCATATTTT3′) as an internal control. All data and statistical analysis was performed using Excel (Microsoft) and ANOVA (Partek). A p-value of <0.05 was considered statistically significant.
Non-significant fold change of variation in placental expression among lean and obese cohorts of validated novel-miRNA species. Graph of the placental expression fold changes for three miRNAs that were validated in both human placenta and breast milk. The miRNAs were extracted from placentas of both lean and obese patients. None of the p-values between the lean and obese groups were significant (novel-miR-62 p=0.567076, novel-miR-114 p=0.719292, novel-miR-118.2 p=0.750468).
Table S1. Summary of Sequencing Statistics. Table S2. Sequencing Statistics mapped to miRBase release 16. Table S3. Mapping frequencies for miRNA sequenced. Table S4. Gene targets for all expressed miRNAs with exact matches. Table S5. Gene targets for the top 10 expressed miRNAs with exact matches. Table S6. Gene Ontology classifications for genes targeted by all miRNAs. Table S7. KEGG pathway classifications for genes targeted by all miRNAs. Table S8. Gene Ontology classifications for genes targeted by the top 10 expressed miRNAs. Table S9. KEGG pathway classifications for genes targeted by the top 10 expressed miRNAs. Table S10. Gene Ontology term clustering as determined by DAVID for genes targeted by top 10 expressed miRNAs. Table S11. KEGG Pathway clustering as determined by DAVID for genes targeted by top 10 expressed miRNAs. Table S12. Putative Novel miRNAs. Table S13. Putative Novel miRNA hairpins (figures). Table S14. Validated novel miRNA TargetScan predictions. Table S15. Gene Ontology classifications for genes targeted by validated novel miRNAs. Table S16. KEGG Pathway classifications for genes targeted by validated novel miRNAs. Table S17. Pearson correlations between quantile normalized read counts from each sequencing sample. Table S18. LIMMA analysis for miRNA differential expression pre- and post rhGH treatment. Table S19. Fold change of putative novel miRNA species by virtue of maternal diet. Table S20. 21 novel miRNAs whose expression was measured in both human breast milk and placenta.
This work was funded by the National Institutes of Health (NIH) Director New Innovator Award (DP2120OD001500-01 KA with funded support to RH), National Institute of Child Health and Human Development (NICHD)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) #R01DK080558-01 (KA), the NIH Common Fund Human Microbiome-HMP (KA (NIHU54HG004969)), and the Burroughs Welcome Fund (Burroughs Welcome Fund Preterm Birth Initiative (KA)). 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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Sadda Haq 8th September 2015 Written Update Desitvbox, Sadda Haq 8 Sep 2015 Telly Update, Written Episode 8th September 2015, Sadda Haq Written Update 8th September 2015 Review, Sadda Haq 8 Sep 2015 Written Story.
Sadda Haq 8th September 2015 Written Episode Update
Scene 1
Ranawat sees his images in sanyu's hand. Sanyu suggests to ranawat I've always looked your decision. I wished to know about your school life. So I found this. Sir who Is that this Female? she appears to be wise and intelligent. Ranawt says i dont know. sanyu claims higher education authorities mentioned her name was sohani and he or she was your library and review partner.
Ranawat finds a virus in his notebook. He says how is this feasible. Ranawat claims why dont i try to remember what connection do i have with this Lady? He arrives and stares at notice board. You will find there's laser gentle on his again. Adda sees it and usually takes him apart to save lots of. She suggests i slipped sorry. he suggests why i come to feel like you rescued me from some thing? She states I had been going to journey. Ranawat states adda? He suggests do I'm sure you?He demonstrates her the faculty Image and says is you? Sohani? adda says no this isn't me. you have some confusion.
Sanyu claims randhir we will't endanger ranawat's daily life. You know very well what adda can do. Randhir says practically nothing will happen to ranawat believe in me.
Adda receives a contact from an individual. The person says why did you help you save ranawat? are you currently double crossing us? She states I'm sure we really have to get rid of him i will do it on right time. If he dies complete system can again hearth. Randhir and sanyu will shatter. If randhir uncertainties we've been driving ranaawat's Dying he will be out. We really have to regard the situation. he states just after this mission you may eliminate ranawat? She claims ok and hand up. Randhir and sanyu overhear all this and find out adda cupping her head in her arms.
Randhir states to sanyu we have endangered ranawat's everyday living what will we do now. We can't again off. Sanyu is in shock as well. Randhir states What exactly are you wondering? Sanyu suggests There exists a Resolution. Adda beloved ranawat. She was his junior. She beloved him and obtained in recruited in solution group. When ranawat remaining the group the adda plead them never to kill him. They deleted his memory. adda nevertheless has thoughts for him or she wont have saved him. Randhir says Now we have her weak point now. Sanyu suggests This really is risky. he claims we dont have an alternative choice. sanyu suggests what if we notify ranawat all the things in detail.
randhir takes sanyu to library. sanyu says exactly what is it? he says We've just one possibility only. Randhir tells her the strategy. sanyu states no we are able to't make this happen. he states we dont Use a option. sanyu suggests This can be dangerous.
Parth requires vid to library and states I like you. She states someone will come. He suggests you questioned me to strip now i wish to do a little something. I arrived when i was chaotic too. I go out of the way in which for You mostly. She states dont rely it now. He says i wont cause you to count anymore. Parth comes in his room in anger and suggests she only thinks about herself.
randhir says sanyu we should kidnap ranawat. sanyu claims we are able to explain to him. Randhir says He'll try out to unravel anything on his very own. sanyu states hear me. he shoves her sanyu falls down. he picks her up and suggests consider recognize. In case you dont assist me i will do this alone.
Sanyu locks randhir in his place. She says We've got to tell ranaawat. but this is right. Randhir open the doorway don't make this happen. sanyu leaves.
Sanyu goes to ranawat and says i want to tell you something. She tells him everything. She says this is situation and we dont know what to do.
Ranawat says sit here. he says you did what you had to now my turn. Randhir hits his head with a rod. Ranawat faints. sanyu says what have you done? He says you made me do it. sanyu says what now? he says we have to keep him in store. We will rope him there. They take ranawat to store room. Sanyu says to randhir you will blackmail adda? he says you wont involve yourself in this plan now. Can I trust you on that?
Randhir says to adda secret group didn't do this. I kidnapped ranwat. She says why? He says i know who was keeping him alive. Your game is over. Adda says secret group will kill you. Randhir says when they find out that you love him they will kill you. Adda says you have no idea of secret group's power. Randir says i had fit a tracker in your body? here are your movements. i can even kill you. I put a chemical in your blood i have the particle's radiated energy in my hands. Now you will live in secret group and work for me. Dont try to control sanyu and ranaawat's brain. He says postpone the blast. I dont want to do this. She says i have committed to them. Secret group never postpones missions. randhir says because till now ranawat was safe. Adda says you wont harm him. Randhir says try me.
Precap-randhir says 3 pm was your deadline. He is about to hit ranawat. Ada says i am ready to do anything you want. Adda says to sanyu your game is over. She shoots sanyu.
Written Update By Sahir


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Mazda Repair Questions? Ask Mechanics for Answers ASAP
Hello, there is not a one unit BCM on this vehicle, May I ask why the question and what is leading you to a BCM?
here is a link, what fuse?
unsure... hard with vehecle not near you and sort of guessing, I have to opt out and open the post to the other experts
Hi my name is ***** ***** I will be happy to assist with this today.In some cases I may need more info to assist better.I apologize for any delay.
Do you still need help with this here?
Let me know so we can continue,thanks Pete.


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Homeware Business with Good Family Life Balance
Auckland City
Retail General
Refer to Broker
About this business for saleHomeware Business with Good Family Life Balance
This Homeware Business is Located in the busy Mt Eden shop area, selling homeware, gift and bed linens with kiwi favourite. It is a good opportunity for business and living together with Auckland double Grammar zone.
Upstairs is currently empty with a 60 m2 one bedroom flat. Potential to rent or live in. Downstairs is as extra shop area with 2 storage rooms, 1 kitchen and 2 car parking.
The annual turnover is about $200,000.
If you are looking for a great balance between business and family life (children education) then the shop is your dream.
+64 9 9300200
021 202 0268
*Sellers Discretionary Earnings, also known as EBPITD (Earnings Before Proprietors Compensation, Interest, Tax and Depreciation). This is the estimated total discretionary earnings (profit) for a business, usually based on historical trading.


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Zoe`s Menu
All-Natural Dog Treats
Home-Made Gourmet Dog Biscuits
True Blue Roo
Chicken Fanciers
Beef Lovers Delight
Cheese & Vegie Munch
Doggy Zacs
Tuna Chews
Variety Biscuit Packs
Real Meat Mutfin Doggy Treats
This Months Money Saving Oppawtunity
Trial Packs
Gifts Bags
Available At
About Us
About Our All-Natural and Healthy Home-Baked Dog Treats
Is Grain Free Kibble Better
Too Much Salt
The Easy Way to Select the Healthiest Treats
What Is In That 'Can or Packet' Of Dog Food?
Contact Us
Too Much Salt
31 Jan 2017 2:46 PM -
Sodium Chloride / Salt.
Do you know how much salt is in the processed dog treats and pet food you may be feeding your dog or cat?
On a raw meaty diet they would naturally get all the salt they require, about o.4 percent, however some processed pet foods can have elevated levels high as 3 .0 percent.
Salt Water is around 2.6 percent.
Salt is commonly used as a preservative only some companies will not label it as such and then declare their products to be preservative free.
Check food labels
. Ingredients are usually listed by weight. Because dry food is lower in moisture, it may seem that it should have less sodium than wet food does, but this is not necessarily true. Whichever processed pet food you feed your furry pal, make sure that salt is never in the first five ingredients listed. Be suspicious of the packet or can that suggests you have a bowl of fresh water next to the feed bowl, this is usually an indication of a high salt content.
Pet food regulations have an established minimum for salt – but regardless of health
concerns there is NO established maximum of salt content in pet foods. And because there are no regulations holding manufacturers accountable, there is no consistency to the amount of added salt in pet foods.
At Zoe`s all our raw ingredients are
salt free
and we definitely do not add salt during any processing of our
home-made gourmet dog treats
and is why we can honestly declare our products to be
preservative free
and why they have a very short shelf life compared to commercially made dog treats and pet foods and the reason refrigeration is required to keep them in good condition while maintaining their maximum nutrients.
It is worth checking the ingredient labels on commercially made dog treats and foods you feed your dog and if there is more than .4 percent the salt content is too high.
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text: Maps in Strife. MAP29: Entity's Lair (Strife) MAP01: Sanctuary (Strife) MAP28: Alien Ship (Strife) MAP09: Castle: Programmer's Keep (Strife) MAP31: Training Facility (Strife) MAP06: Sewers (Strife) MAP04: Power Station (Strife) MAP12: The Temple of the Oracle (Strife) Pages in category "Strife levels" The following 31 pages are in this category, out of 31 total. A MAP30: Abandoned Front Base (Strife) MAP28: Alien Ship (Strife) MAP08: Audience Chamber (Strife)B MAP11: Borderlands (Strife)C MAP07: Castle (Strife) MAP09: Castle: Programmer's Keep (Strife) MAP13: Catacombs (Strife) MAP25: Catacombs: Ruined Temple (Strife)E MAP29: Entity's Lair (Strife)F MAP24: Factory: Conversion Chapel (Strife) MAP22: Factory: Forge (Strife) F cont. MAP21: Factory: Manufacturing (Strife) MAP20: Factory: Receiving (Strife) MAP15: Fortress: Administration (Strife) MAP16: Fortress: Bishop's Tower (Strife) MAP19: Fortress: Security Complex (Strife) MAP18: Fortress: Stores (Strife) MAP17: Fortress: The Bailey (Strife) MAP03: Front Base (Strife)M MAP14: Mines (Strife)N MAP10: New Front Base (Strife)O MAP23: Order Commons (Strife) P MAP04: Power Station (Strife) MAP05: Prison (Strife) MAP26: Proving Grounds (Strife)S MAP01: Sanctuary (Strife) MAP06: Sewers (Strife)T MAP27: The Lab (Strife) MAP12: The Temple of the Oracle (Strife) MAP02: Town (Strife) MAP31: Training Facility (Strife) Retrieved from "http://doom.wikia.com/wiki/Category:Strife_levels?oldid=38091" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Strife Stock levels


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Now that we know what the Common Core Curriculum is, what can parents and community members do to help their students to be prepared? Tune in as Mr. Yancey talks about some of the methods and activities that can be done to help students to be prepared. Before that, make sure you tune in to "Style Avenue" with Arielle. She will get you up to date on the fashions at the big events, as well as give you some suggestion on what you can do for your personal wardrobe. Feel free to call in with your questions and comments at (914)338-0665.
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Gone Girl by Gillian Flynn
My rating: 2 of 5 stars
There is a lot to like about Gillian Flynn, much to admire. But this book baffles me. It's being touted as summer/beach reading extraordinaire. It just made me miserable. It's like ill-advised sex with someone you don't know if you can trust. It's fun at first, then you start feeling nervous about it, and ultimately when it settles into what it always was anyway, it just makes you sick. Flynn is probably on a short list of writers who could make it as entertaining and impressive as it is. But when things are miserable all you see are the gimmicks and the woman behind the curtain. I kept thinking admiring Flynn's efforts behind the curtain would be enough. But at its end I was frustrated and glad it was over.
View all my reviews
ps. BUT, I still recommend her. Flynn is a smart, interesting weird. She's warped and dark and brave. Her first book Sharp Objects has stuck with me.


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Reference Number. 119253
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Jolly Kids Castles28 Benvenue Avenue, Eastwood
Tel: Click Here for Phone Number
Jolly Kids Castles is a New Famil run business, located in Southend on sea, Essex. We have many Themed bouncy castles such as Ben 10 and Hello Kitty. We also provide Adult inflatables, Slides, Obstacle Courses and Sumo Suits. We are always updating with new stock and our castles are all clean, approved and most are brand new. We cater for events, birthdays, fundays or any occasion to just have a day of fun !!! We also provide, Foil Balloons, Helium Balloons and Party Bags, that can be delivered with your castle. Our aim is total customer satisfaction and repeat happy customers.
Bouncy Castle Hire Leigh-on-Sea, Bouncy Castle Hire EssexTo add your free business listings see our Free Advertising page.
Jolly Kids Castles are listed in; Bouncy Castle Hire Directory : Bouncy Castle Hire in Essex : Bouncy Castle Hire in Leigh-on-Sea : Essex Business Directory : Leigh-on-Sea Business Directory : Bouncy Castle Hire Listings : Jolly Kids Castles
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The Guest of the Voice of Croatia / Gost Glasa Hrvatske
The Guest of the Voice of Croatia is a weekly program which brings to your home individuals, well-known or otherwise, from Croatia's social and political life. During the half-hour conversation they respond to questions about current events and also comment on various topics from their professional or academic fields. You can listen to this program on the Voice of Croatia every Wednesday at 12:05 CET. Thirty minutes, countless topics, dozens of questions and one guest: the Guest of the Voice of Croatia.


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Ariño Duglass has consolidated its position as a key player in cutting edge architectural glass in the United Kingdom through success and improvement across the last 10 years, generating trust and passion among our partners and clients.
In 1998, Ariño Duglass entered the UK market.
2007 was to prove the beginning of a fruitful decade, with the execution of the Curve Theatre project in Leicester. Since then, we have produced over 200,000 m2 of glass for a wide range of emblematic buildings in the UK, such as the roof light at Airport, the Olympic Aquatic Centre for London 2012 by architect Zaha Hadid and the Edinburgh Convention Centre. These 10 successful years in the UK culminate in 2017 with the inauguration of the renovated Centre Point, the first skyscraper built in London.
In the coming years, the presence of Ariño Duglass is assured in the United Kingdom, with participation in important projects such as Principal Place Tower (Norman Foster + Partners) and The Bower Old Street, both in London, and Sensor City, in Liverpool.
15-06-2017 - Hotel Grand Hyatt Bogotá (Colombia)
15-06-2017 - Stronger than ever 10 years of success in the United Kingdom
15-06-2017 - Singular buildings Diagonal 191


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#7 Erectile Dysfunction
The old saying is true. If you don't use it, you lose it.
#6 Bladder Problems
Women's pelvic muscles get stronger when they have s3x frequently. No sex means a weaker bladder
#5 Blood Pressure
S3x relieves stress. Not having s3x will result in high blood pressure.
#4 Natural Lube Production
Women who don't have enough s3x will stop producing as much natural lube. That could leave them pretty dry down there


text: Seller: buildingcharacter717 (917) 100%, Location: Lancaster, Pennsylvania, Ships to: US & many other countries, Item: 321478148315 QTY: 5 SIZE: Approxmately 5"w x 7/8"t -- Distance between holes: 4 1/4" MATERIAL: Metal CONDITION: As seen in photos. OTHER: Hardware included. SHIPPING: Please note, we ship only to the address provided by Paypal/Ebay -- Please make sure your address information is up to date. All of our packages are shipped with insurance. OR, Save the Shipping! Pick this item up locally at our brick and mortar store: BUiLDiNG CHARACTER 342 N. Queen Street, Rear Warehouses Lancaster, PA 17603 Condition: As seen in photos. Used. Vintage. Functional.


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Skyland Pines, 3550 Columbus Rd NE, Canton, OH 44705
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Pre-Dinner Technical Session (5:00 -
6:00 pm)
Akron-Canton Regional Foodbank:
we are the difference between hunger
and hope
Presented by
Leslie Letner
Leslie Letner is the Senior Manager, Corporate Relations and Special Events for the Akron-Canton Regional Foodbank
Feeding America's 2012 Food Bank of the Year,
the Akron-Canton Regional Foodbank was founded in 1982 and serves 8 counties, distributing emergency food to more than 500 member agencies who serve 263,200 clients each year. In 2013, the Foodbank distributed more than 24 million pounds of food throughout its network.
Leslie's tenure at the Foodbank, revenue for campaigns and events such as the Harvest for Hunger Campaign, Taste of the NFL and Hunger-Free Families has increased by more than 300% to $2 million annually. Leslie organizes and conducts large-scale fundraising events; including tasting events with over 500 guests in attendance, a grand opening gala with sold out attendance of more than 400 guests and a community food and funds drives raising millions of dollars and thousands of pounds of food for those in need each year.
Leslie is a graduate of the
Inaugural Leadership Stark County
Spotlight Young Professionals
program and a member of Leadership
Stark County Signature Program Class
28. She is also active in the community as a member of the Basic Needs Impact Council for the United Way of Greater Stark County, member of the Junior League of Stark County, OH, Inc., member of Torchbearers Akron, member of the Summer Safari Committee for the Akron Zoo, member of the Association of Fundraising Professionals Northeast Ohio Chapter and a Board member for Blick Clinic Akron.
After-Dinner Technical Session (7:00
- 8:00 pm)
Future CPA Topics
Presented by
Michael Elliott
Elliott is a graduate of
Franklin University,
majoring in Accounting and Business
Administration. Michael has been in
public practice since 2007, and is
currently serving as a Principal at
the firm Dittrick & Associates, Inc
and will be transitioning into the
role of Managing Principal within
the next 18 months.
his position, Michael serves the
needs of a wide variety of clients
including those in manufacturing,
agriculture, construction and
professional services. The bulk of
the firm's workload is in the fields
of taxation and consulting. In his
position as Principal, he is also
involved with the technological and
procedural operations of the firm.
Michael is a member of both the Ohio
Society of CPAs as well as the
American Institute of CPAs. He is a
2012 graduate of the AICPA's
Leadership Academy and has been
involved in several projects with
both the AICPA and the Ohio Society.
Currently, Michael serves on the
Public Relations Advisory Council of
the Ohio Society. He is also
involved with a local non-profit
Academy acting as a board member and
Michael has spoken at several
national conferences on a variety of
subjects including the dynamics of
being an entrepreneurial CPA as well
as the process of transitioning a
firm from current ownership to a new
generation of owner.
Outside of the office, Michael is
very involved with his church and
coaches youth basketball. In his
spare time Michael spends time with
his wife Kristen and daughter Ana.
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Please join us as we promote this mission in the
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Prime Central London – London Villa is a provider of housing solutions, operating across Central London. London Villa team of experts combines 25 years of local knowledge dealing with residential real estate in Prime central area of London: Mayfair, Knightsbridge, Marylebone, Notting Hill, Hyde Park, Kensington, and Chelsea. Our specialty lies within Residential Sector – whether you are in a search for fashionable penthouse or a spacious contemporary family home, period or new built block of apartments, individual or multi-unit property – London Villa Team is there for you.
Our continuing success has been built on delivering the finest possible service to tenants, landlords, buyers, and sellers thereby creating loyal and enduring relationships with all of our clients.
As highly experienced professional managers of tenanted buildings and apartments, we are probably the only UK letting agency to assure you of 100% occupancy, offering a five-day tenant-finding guarantee – and in 2013 alone, we welcomed over 500 new tenants. If you're an established landlord with several properties - or you have just purchased property in the London buy-to-let market – you can simply hand over your whole property management process to London Villa. As experts in central London property lettings, we can find you a reliable tenant from our established database of appliants. Among whom are international young professionals, mature students, corporate employees, families, and periodic visitors to London.
A wide range of lettings services
Our letting services include:
If you have employees relocating to London, you want to be sure that they can give of their best, safe in the knowledge that they're living in accommodation that reflects their status, their expectations and necessary comforts.
Our Corporate Relocation department can provide a full property-finding service throughout central London, and we'll be on hand to help you from the moment you start seeking accommodation on their behalf.
Looking after your employees' relocation needs
Working closely with you, we'll develop a brief as to the kind of property required – then, as soon as your employee is ready to start looking, we can escort them to a carefully chosen portfolio of central London properties, ensuring that they are accompanied by a knowledgeable individual who speaks their language.
We aim to provide the highest standard of property maintenance for our properties in Central London.
Whatever your property maintenance needs, our highly skilled tradesmen will be happy to help 24/7
Our skilled, experienced and trustworthy engineers work quickly and efficiently, minimising hassle
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We cover a range of reactive, planned and preventative maintenance services including: electrical, gas, plumbing and heating, roofing, glazing, carpentry, alarms and CCTV. All in all we can install, fix, upgrade and maintain your property, keeping it in tip top condition at all times.


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Top nearby In-Home Care Services
Reviews of Visiting Angels Homecare & Respite | Sunnyvale, CA
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From Visiting Angels Homecare & Respite
Are you an adult son or daughter seeking home care for your parent(s) in the San Francisco area? If your mother lives in California and you're in Arizona, how do you take care of her? If you're dealing with either of these questions, you've come to the right place. Visiting Angels provides Private Duty non-medical homecare for the elderly. We are also keenly aware that the adult children of our clients are challenged with the prospect of caring for their parents. We are here to help manage your loved one's homecare and ease this responsibility for you.
Sometimes, Assisted Living Facilities seem to be the only options for care. Visiting Angels' services allow people to maintain the independence of their daily routines and familiar surroundings. Having a caregiver come to your home is an excellent option for many families. Caregivers are able to provide meals, transportation, hygiene assistance and more.
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Thank you for visiting our website. Gaining insight on our services is the first step in making an important decision for your loved-one. We've included a lot of information on our site. However, we always welcome an opportunity to speak with you. So, please feel free to call us or stop by our office for more information.
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Dad's Kaluah
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Recipe by kfivejthreenine
This is my Fathers recipe. He has been making it for as long as I can remember. I'm pretty sure the original recipe came from his grandmother, so I think I can call it a "Family Recipe". We drink this liqueur with milk (add milk to taste). This is a large batch, to make less halve the non-alcoholic ingredients and use a fifth of vodka. Dad swears cheap vodka is best in this recipe.
See Discussion:
Ingredients Nutrition
- 6 cups sugar
- 6 cups water
- 2 ounces instant coffee granules
- 2 fluid ounces vanilla extract (scant 1/4 cup)
- 4 cups boiling water
- 1⁄2 gallon vodka (Crystal Palace)
Directions See How It's Made
- Heat six cups sugar and six cups water in large pan. Simmer until thick and syrupy. Thirty minuets or so maybe more maybe less. Meanwhile combine four cups boiling water with coffee and vanilla, stir to dissolve. The syrup is the tricky part if cooked too long it will be just fine as long as it is not burnt. Remove syrup from heat and add coffee mixture. If there are any chunks of hardened sugar the syrup has been cooked too long. To remedy this cook and stir over low heat until dissolved. Cool the end result until at least lukewarm before adding vodka. I place the pan in a sink full of ice water to cool faster. Once vodka is added this makes about 1 gallon. To make less halve ingredients and use one fifth (750 ml) of vodka.


text: Jeff Spaleta (jspaleta gmail com) said: > What we could use, is a technical mechanism in how codeina works that > a 3rd party repository can hook into to add additional codec offerings > via rpm packages. Once someone installs the livna-release package, it > would be great if the livna-release package could drop in the > necessary files on the filesystem so codeina would know how to request > package installation from livna. codeina works by reading /usr/share/codeina/xml/available-plugins.xml, which lists for a particular 'bundle' (i.e., tarball): - gstreamer element/mime type - descriptive info - EULA URI - arches, sha1sum, etc. - download link - price, if needed To change this list would reqiure respinning codeina. codeina, at the moment, has no mechanism for installing RPMs or similar packages. Bill


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At this time of year many of us are returning to work. Today's forum is called The Office and over the next hour we'll be talking about the design of the workplace: past, present and future.
This [series episode segment] has
Do you know this name, Chris Bangle? Car enthusiasts in the By Design audience will know him, in the world of car design he's a star, but all of you know his work.
The Pixel building, as it is known, is the new Melbourne city headquarters for the developers Grocon - known for many of Australia's major buildings. Eureka building on Melbourne's Southbank is one of their most prominent. This is considered one of the tallest buildings in Australia. The Pixel building, though, is small, and an experiment in all things green. The building's architects Studio 505 are one of Australia's most innovative and thoughtful firms, with the co-founder Dylan Brady coming out of LAB Architecture, the firm that designed Melbourne's Federation Square.
On his deathbed in a Paris hotel room, Oscar Wilde famously quipped: 'My wallpaper and I are fighting a duel to the death. One or the other of us has to go.' In Australia, since the 1840s, fashions in wallpaper have come and gone in Australia during our long, on-off love affair with wallpaper.
Where were you when the lights went out? For whatever reason they went out, you'll probably remember where you were when it happened because our electrically lit-up life has become so natural to us that when the lights go off, the darkness seems abnormal and memorable.
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text: i don't know what the hell Marvel is doing. its like every trailer they release is something revealing from the movie that you would rather see in the movie than from a trailer. one thing that really pissed me off is seeing the Downey/Iron man scene is a friggin commercial, and what made it worse is that is the first scene of the commercial. I would rather see this in the movie and say wow.


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|Modelling and Simulation
The design of a new or the extension of an existing container terminal depend on criteria related to productivity and efficiency. The different components of a container yard under operation build up a comprehensive dynamic system continuously changing. Apart from static considerations, it is then vital for the design stage to attach particular importance to these spatiotemporal dynamics. A simulation helps identify possible bottlenecks in terminal operation before the implementation of the aspired changes.
For simulation purposes, we prepare an individual model of your terminal for you. It considers local aspects and, moreover, the best possible optimisation strategies for all simulated parameters. This makes the results of the simulation independent and resilient. You thus obtain an abstract of the performance data you can expect.
Usually, a simulation aims to define the most efficient solution or to identify the limiting factor on the terminal. Thanks to this knowledge, HPC prepares tailor-made solutions that will optimise the terminal's productivity and efficiency.
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text: "Don't stand still for too long." —Game description Armored Archers are the armored variants of Drow Archers in Skylanders: Giants. This article is a stub. You can help Skylanders Wiki by editing this page. Drow Spyro's Adventure Drow Spearmen - Goliath Drows - Drow Witches - Drow Zeppelins - Blade Witches - Blitzer Bullies Giants Drow Lance Masters - Drow Archers - Armored Goliaths - Armored Archers - Armored Lance Masters SuperChargers Pint-Sized Prodders (Dark-Shard) - Rush Crushers (Shade) - Twilight Twisters (Abyss) - Witch Pitchers (Pitch-Black) Retrieved from "http://skylanders.wikia.com/wiki/Armored_Archers?oldid=77504" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Article stubs Enemies Skylanders: Giants Skylanders: Giants Enemies Drow


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Definition of a cult
My ex-career as a muslimah
Fringe benefits – Part 9 – Exercise and wear a regular swimsuit
May 14, 2015
Filed under:
Fringe Benefits
— evebitestheapple @ 9:21 pm
I was searching through my drawer for something and I came across an old Muslim outfit that I forgot about. Several years ago I went to the YMCA and signed up for membership for my kids and me when I was a Muslim. After getting accepted, we went to the Y several times a week and I used pretty much everything in the facility even though I wore a jilbab and hijab. I had ditched the niqab by this time because it became too much of a nuisance especially since I was newly single. Eventually I went and bought a x-large mens long sleeved shirt and baggy pants so I could have better workout wear without breaking the religious commandments of covering my awrah. This allowed my to spend plenty of time on the weight machines, free weight area, stationary cardio machines, indoor track, and the video game room. I was able to use everything EXCEPT the pool. This Y had a huge heated indoor lap pool, a jacuzzi, and a sauna. Every time we went I would feel the cool breeze of chlorinated water and long to feel the water on my skin once again. There was no way to escape it either because the building was designed to pass by the pool at every angle. Even in the dressing room the cool water beckoned with every beach towel flung over a changing stall. Working out on the weights didn't do much to quell my desire to jump in either. All of the sweating from deadlifts, shoulder presses, and seated rows with all of the clothes I had on just intensified my desire jump in just once. After awhile I started to feel like crack fiend that wanted just one hit. It was like thinking "the pool, it be callin me man, it be callin me".
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Nadirim PC
- Publisher: Twisted Tribe
- Release Date: Nov 7, 2011
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Jan 16, 2012If you're hoping for talent trees, intense spec choices or mind-blowing spell effects then keep walking, buddy. The talent trees from beta are gone for good, replaced by a wealth of skills which must be picked between as only a certain number may be taken into any given battle. What the game lacks in animation it really does make up for in graphics and gameplay but, if there's no way you can enjoy a game without enough flashes to give you seizures every twenty seconds, then Nadirim won't cut it for you.
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Ordinary 20A; Proper 15 Genesis 45:1-15; Romans 11:1-2a, 29-32; Matthew 15:21-28 Emotions reverberate through both the Old and New Testament readings for this week. "Joseph could no longer control himself." Or, "he kissed all his brothers and wept upon them." The Canaanite woman shouts, croaks, squawks and cries out in desperation. The disciples are irritated, anxious to have her sent away. Jesus appears indifferent at best, uncaring at worst. We've got grief, anger, relief, frustration, love, fear – the roiling gamut of human feelings are woven into these stories, and I find this messy display a relief. No earthy response to life's circumstances is considered unclean or unwelcome in the realm of the holy. God utilizes our whole selves, our whole lives, all our complex feelings and relationships to make manifest divine truth and will. Alleluia. Further, even Jesus has compassion fatigue, capitulates to cultural stereotypes and must be prodded into a transformation of the … [Read more...]
Protected: Songwriting with the Psalms (webinar replay)
This content is password protected. To view it please enter your password below: Password: … [Read more...]
10th Sunday after Pentecost — August 13, 2017
Genesis 37:1-4, 12-28: Romans 10:5-15; Matthew 14:22-33 Ordinary 19A; Proper 14 I once had someone tell me that he didn't believe in God. He didn't believe in God because to imagine that God cared about him, his daily life and his well-being was the height of self-aggrandizement. Further, he wouldn't want to believe in a God concerned with such trivialities. To believe in God, he posited, was to make himself too big in the face of clearly much bigger issues. I considered his perspective. Christianity has long denounced the sin of pride, and I suppose his argument at least took that human tendency seriously. Nonetheless, I responded to him that perhaps he wasn't making himself too big so much as making God too small. Could God not be capable of giving attention to both the personal and the cosmic? I thought of those lists of divine descriptors in several our confessions: omnipotent, all-powerful, eternal, maker of heaven and earth. The list, of course, goes on. Had I … [Read more...]
9th Sunday after Pentecost — August 6, 2017
Genesis 32:22-31; Matthew 14:13-21 Ordinary 18A; Proper 13 Jesus tries to get away, at least for a short time. He gets in a boat and goes to a deserted place. Jesus attempts to withdraw and then withdraw some more. He had just learned of the violent death of his cousin, John the Baptist. No wonder he wanted to escape. John knew Jesus like no other and now, because of his closeness to the Son of Man, John has been murdered. Oh, and right before Jesus heard of John's death, Jesus had been run out of his hometown. In the brief time of his earthly ministry, this had not been a season of affirmation and encouragement. What does St. Ignatius call this? Desolation? Who knows, of course, what Jesus was feeling when he got in the boat and sought to be alone. One can imagine sorrow and grief must have been part of the mix. Maybe even despair lurked as he thought about the people he'd grown up with ridiculing him and his cousin dead on account of remaining loyal. But he can't get … [Read more...]
The Dazzling Tonic of Silence
90-minute webinar presented by Charlotte Matthews Tuesday, October 10, 2017 at 2 pm ET (11 am PT) Live broadcast with on-demand replay – $29.95 (1-6 participants from the same church/ministry) Purchase the Webinar Have you been hungering for a way to invite more silence into your personal and professional life? In a world with a spinning array of distractions, silence is now on the endangered list. In both practical and conceptual ways, this webinar will help you and those in your life preserve this sacred element of life. This webinar will be helpful to anyone who feels busy, overwhelmed or burdened. If you are feeling like you want more time and spaciousness, this is the webinar for you. Silence is, indeed, the most straightforward route to insight. We know insight to mean deep understanding, perception and acumen. But dig deeper and you discover that the word comes from the archaic Scottish insicht: furniture or household goods. It was legal terminology found in … [Read more...]
The rainbow (September 3, 2017)
UNIFORM LESSON FOR September 3, 2017 Scripture passage and lesson focus: Genesis 8:20-22; 9:8-17 Our attention now shifts from Luke's account of God's providential guidance of the early church in the Acts of the Apostles to God's covenantal care for humanity and creation as described in the Hebrew Scriptures. We are moving from a sharply focused look at God's covenantal people in the first century A.D. to a much broader perspective on the renewal of humanity after the primeval flood described in Genesis 7. Rather than Luke's theologically interpreted history of the early church, we look at ancient Israelite traditions shaped by centuries of oral and written transmission filtered through differing theological perspectives. These traditions form the bedrock from which our Christian understanding of God and God's redemption of the world is derived. A central theme of the Hebrew Scriptures is a covenant that describes the relationship between God and human beings. Over the … [Read more...]
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> Baby&Kids
> Baby & Toddler Clothing
> Portable Baby Milk Powder Formula Dispenser Feeding Box Case
2 Colors: Pink,Blue( Two color for your choose)
Package included: 1pcs Milk Powder Dispenser
Closure type: Rotating snap-on lid
Easy-pour spout
Compact size,large capacity.
Easy to carry, portable, clean and sanitary.
Can be used to go out of milk powder/food storage
Brand New and High Quality.
Perfect for all mommies to take the milk powder outside for your baby.
Compact size,large capacity ,easy carry
Milk powder dispenser has 3 interlayer, convenient to carry
Please be sure to clean before use
Please do not use microwave to sterilize, you can put it in boiling water disinfection
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1.Please allow 1-3mm error due to manual measurement.
2.The colors may have different as the difference display,pls understand.Thank you!
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text:
Abhorrent enough that even Major General Curtis E. LeMay along with Admiral William D. Leahy, Admiral Chester W. Schwartz' robotics and computer science students built the silver plastic arm with a 3 D printer. Schwartz's students scale and modify open source designs with computer aid drafting (CAD) software. The prosthetic is printed in small plastic components and assembled using elastic string, glue and other materials, according to previous Enterprise reporting..
decorating tools Any prosecutor interested in the truth and in justice would have used all the tools at her disposal to find them. Ms. Mosby ignored them. The building was purchased by Triad Hunter LLC last year for $394,000 and was renovated at a cost of around $2.5 million. Nine apartment units and three suites will house employees and executives of the local oil and gas company. Three of those located on the second floor will be part of the tour, according to Kupsche.. decorating tools
silicone mould That can be accessed fully after the purchase. Speaking of popular games being free, The Pokemon Go, in contrast, was not pay to play game and all the profit was earned by the makers from the in app purchases made by the users. A section of Mario lovers have also expressed their negative reactions for the pricey availability of their favourite plumber game.. silicone mould
fondant tools Visit the bakery and gourmet center in B. A. Miller Auditorium and arts and crafts in the main lobby of Community Memorial Hospital. If you are planning to go simple, try to hide the fact that you're scrimping by making the snacks or dishes look fabulous. Instead of serving snacks, why not organize a chocolate holiday party? It will certainly work with the occasion and who could ever resist chocolate? A wine and cheese affair seems elegant and apt for intimate holiday events. Or you can do a party wherein everyone gets to cook his own food (of course, this would only work if you have the necessary equipment, such as smokeless grills, among many others).. fondant tools
bakeware factory The enemy withdrew, leaving bodies behind, but they were far from finished. At 0230, enemy mortars shelled Golf Company. Direct hits by RPGs destroyed a machine gun emplacement and several backup positions on the primary avenue of approach into the company position. bakeware factory
baking tools Cleveland is the country's in July capital. So says The Wall Street Journal in a piece that looks at how retailers and toymakers launched Christmas sales and promotions this month, hoping to boost sagging sales and help cash strapped consumers stretch out their holiday spending. The concept is spreading, The Journal says. baking tools
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May Seventeenth Two Thousand & Ten (PM) 05/18/2010
These days it seems I am constantly swimming in a pool of tissue; most likely because I am. Every year around March, my allergies start to flare up. The pain and agony usually last until July or so. I've tried every kind of medication and nothing works. I've even attempted to combine different options, mostly I am left with a sore nose, dry sinuses and still unable to get a measly stream of air through either nostril.
Tomorrow my new workout buddy is making her way to whole foods to get her own personal items. I conned her into picking me up some local honey and bee pollen. I can only pray that this will work. I feel as though it is my last option. And I desperately want to be able to enjoy the outdoors again. I mean it is summer after all. Why should I have to suffer through allergies AND the intense Texas heat? It just doesn't seem fair.
Universe, I am taking action. It's up to you to do the rest. Don't let me down.
The Roommate
Turns out my instinct was wrong. Perhaps it was actually just clouded by my overwhelming ability to think positively? Either way, my immediate reaction to the news was panic. All I could think of was the money we don't have. And then it hit me that I only thought that way because I have been doing it for so long; weird how patterns become habitual.
The House on Hall Street
The fact is that we do have money now. It's quite lovely. I decided to release my panic to The Universe and trust that there is a greater plan at hand. I mean, we already have the house on Hall Street waiting for us for a wee $500 a month. It will be nice to be able to create my own space again.
Oh, how I long to submerge my paintbrush in paint and fingers into pastels.
Soon, very soon.
I got the urge to ask a friend if they wanted to be my roommate. It was odd, came out of no where and left me feeling pleasant about the possible experience. Either way my friends boat goes, it's nice to know the offer was considered.
I asked the landlord if I could plant flowers, plants and create a garden. She said yes. For this, I am stoked. It will be a glorious garden. Tomatoes, tamitillos, jalapenos, peppers, and whatever else makes it's way into my shopping cart.
There is nothing as rewarding as planting something and watching it grow.
Pun intended.
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By Tom McMillan
The passengers of United airways Flight ninety three on September eleven, 2001 have earned their rightful position one of the pantheon of yank heroes. Flight ninety three provides a riveting narrative in accordance with interviews, oral histories, transcripts, recordings, own excursions of the crash web site, and voluminous trial proof made public purely in recent times. There is also lots of chilling new aspect for readers who think they be aware of the tale of the flight. using learn instruments that weren't on hand within the years instantly after the crash, the e-book deals the main whole account of what truly happened aboard United ninety three – from its not on time takeoff at Newark overseas Airport to the instant it plunged upside-down at 563 miles according to hour into an open box in rural Somerset County, Pennsylvania.
Quick preview of Flight 93: The Story, the Aftermath, and the Legacy of American Courage on 9/11 PDF
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The Third Coast: When Chicago Built the American Dream
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Paul Revere's Ride
Paul Revere's hour of darkness experience looms as a nearly legendary occasion in American history--yet it's been mostly neglected via students and left to patriotic writers and debunkers. Now one of many optimum American historians deals the 1st severe examine the occasions of the evening of April 18, 1775--what led as much as it, what relatively occurred, and what followed--uncovering a fact way more extraordinary than the myths of culture.
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After we ponder the major figures of early American background, we predict of explorers, or pilgrims, or local Americans--not livestock, or goats, or swine. yet as Virginia DeJohn Anderson finds during this brilliantly unique account of colonists in New England and the Chesapeake quarter, cattle performed a very important function within the settling of the recent international.
Mecca and Main Street: Muslim Life in America after 9/11
Islam is Americas quickest becoming faith, with greater than six million Muslims within the usa, all residing within the shadow of September 11. who're our Muslim buddies? What are their ideals and wishes? How are they dealing with lifestyles lower than the struggle on Terror? In Mecca and major road, famous writer and journalist Geneive Abdo bargains illuminating solutions to those questions.
Extra info for Flight 93: The Story, the Aftermath, and the Legacy of American Courage on 9/11
Peter Hanson positioned a moment name to his father in Connecticut at 9:00 a. m. cell documents later acquired via the 9-11 fee convey that the decision lasted 192 seconds—just greater than 3 mins. "It's getting undesirable, Dad," Hanson stated. "A stewardess used to be stabbed. they appear to have knives and mace. They stated they've got a bomb. It's getting very undesirable at the airplane. Passengers are throwing up and getting ailing. The airplane is making jerky pursuits. I don't imagine the pilot is flying the aircraft. i believe we're taking place.
On October 7, Jarrah took an unforeseen holiday from his education, touring from Atlanta to Frankfurt, Germany, after which directly to Bochum—where Aysel used to be dwelling and learning. He took Aysel on an aspect journey to Paris and spent a complete of twenty-three days overseas. this is able to be the 1st of 5 in a foreign country journeys Jarrah took in the course of a ten-month span among October 2000 and August 2001. All 5 journeys have been to Germany, and incorporated connections to Lebanon to work out his relations. In January he even introduced Aysel to Florida with him for a ten-day stopover at.
MM00704, conceal sheet of fax despatched to Ramzi Binalshibh from Florida Flight education heart, August 17, 2000. 36. stressed out $2,200 to the varsity as a deposit, assertion for the checklist, FBI Director Robert S. Mueller III, Joint Intelligence Committee Inquiry. 36. the opposite was once in October from Berlin, 11th of September and Terrorist shuttle, employees record of the nationwide fee on Terrorist assaults Upon the USA, pp. 13–14. 36. "Please recognize that we can't offer you a visa," McDermott, excellent infantrymen, p. 201.
They'd withstood layout controversies, land squabbles, investment shortfalls, and various different stumbling blocks. A extraordinary grassroots attempt that had all started within the bleak days after the crash with neighborhood citizens Judi Baeckel, John Chaves, Barbara Black, Donna Glessner, Kathie Shaffer, Chuck Wagner, Rick King, Wally Miller, and others was once approximately to culminate in a powerful tribute to at least one of the main heroic acts within the country's background. "Pearl Harbor didn't ask to be bombed," acknowledged Henry cook dinner, president of the Somerset belief corporation and a member of the degree II jury.
We're venerated to proportion the legacy of people that made a distinction, whose lives had nice which means . . . and who stay deeply overlooked. " the ultimate act was once a personal burial provider for the households at the morning of Monday, September 12. virtually from the time he'd arrived right here ten years past, Somerset County coroner Wally Miller thought of this position a cemetery, the ultimate resting spot of the passengers and workforce. Now he might make it professional. 3 caskets of intermingled yet unidentifiable human is still from Flight ninety three have been stored at a mausoleum in Windber, approximately twenty miles to the northeast.


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17 January 2017
Had the sashimi which was amazing, fresh quality.
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Beautiful sushi, great service and a great price
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amazing food with a great standard and price.
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