Searching for a Stochastic Background of Gravitational Waves with LIGO

The Laser Interferometer Gravitational-wave Observatory (LIGO) has performed the fourth science run, S4, with significantly improved interferometer sensitivities with respect to previous runs. Using data acquired during this science run, we place a limit on the amplitude of a stochastic background of gravitational waves. For a frequency independent spectrum, the new limit is $\Omega_{\rm GW} < 6.5 \times 10^{-5}$. This is currently the most sensitive result in the frequency range 51-150 Hz, with a factor of 13 improvement over the previous LIGO result. We discuss complementarity of the new result with other constraints on a stochastic background of gravitational waves, and we investigate implications of the new result for different models of this background.Comment: 37 pages, 16 figure

Transcriptome Profiling of microRNA by Next-Gen Deep Sequencing Reveals Known and Novel miRNA Species in the Lipid Fraction of Human Breast Milk

<div><p>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 (<em>p</em><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.</p> </div

21 potential novel miRNA species in human milk fat globules were identified from rhGH synchronized lactating women in our discovery set (n = 6) by massive parallel sequencing.

<p>12 of 21 (57%) species were thereafter confirmed in our robust validated set (n = 33) and are designated <b>in bold</b>.</p

Chromatograms of breast milk preparations.

<p>Shown above are chromatograms from RNA demonstrated in gel electrophoresis from Fig. 2. (<b>A</b>) & (<b>B</b>): Whole breast milk (lanes 1 & 2), (<b>C</b>) & (<b>D</b>): Lipid fraction (milk fat globules) of fresh breast milk (lanes 7 & 8), (<b>E</b>) & (<b>F</b>): Lipid fraction frozen breast milk (lanes 9 & 10). These panels demonstrate enrichment for miRNA (blue boxes) in Trizol-treated, mirVANA isolated samples, which minimized the content of ribosomal RNA.</p

Delta delta CT comparing between rhGH (normalization) cohorts and dietary manipulated cohorts.

<p>When normalized to rhGH synchronized lean subjects, no significant differences were observed among obese lactating women on either high glucose or high galactose dietary manipulations. However, novel- miR-67 and novel-miR-27 species were observed to vary significantly (p = 0.01) following introduction of a maternal high fat diet in lean subject cohorts. This data is represented as fold change of cohorts with ANOVA for significance in Table S19.</p

The 10 most abundant previously described (known) miRNAs in milk fat globules from human breast milk.

<p>The complete set of read counts with exact matches to known and previously characterized miRNAs are presented in Table S3. The number of targets predicted by TargetScan and miRanda for the 10 most abundant miRNAs are presented here. Gene symbols for targets predicted for all expressed miRNAs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050564#pone.0050564.s002" target="_blank">Supporting Information S1, Table S4</a>) and the top 10 expressed miRNAs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050564#pone.0050564.s002" target="_blank">Supporting Information S1, Table S5</a>) as well as enriched Gene Ontology (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050564#pone.0050564.s002" target="_blank">Supporting Information S1, Table S6, S8</a>) and KEGG Pathways (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050564#pone.0050564.s002" target="_blank">Supporting Information S1, Table S7, S9</a>) are presented in the supplemental materials.</p

Graphs of top 20 enriched GO terms for miRNA targets.

<p>The top 20 enriched GO terms based on FDR (FDR levels shown) were graphed for gene targets of the top 10 expressed known miRNAs (A) or the validated novel miRNAs (B). GO terms related to the regulation of transcription, metabolic processes, and biosynthetic processes were highly represented among the known miRNA targets and form a connected graph consisting of 19 of the top 20 GO terms (A). A connected graph of terms related to regulation of transcription and metabolic processes was also present for targets of the novel miRNAs (B). For the novel miRNA targets, 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. Graphs were generated using the Cytoscape <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050564#pone.0050564-Smoot1" target="_blank">[47]</a> Enrichment Map plugin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050564#pone.0050564-Merico1" target="_blank">[48]</a>.</p

Flowchart outlining the acquisition of breast milk samples into sets for discovery, interrogation and validation.

<p>Each of these cohorts has been previously described and well-characterized <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050564#pone.0050564-Maningat1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050564#pone.0050564-Mohammad1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050564#pone.0050564-Mohammad2" target="_blank">[32]</a>. Briefly, in the discovery set lean healthy women received weight-based recombinant human growth hormone for synchronization at 24 hours of study initiation for synchronization. The women continued to collect breast milk via pump every 3 hours for an additional 48 hours, and every 4 hours thereafter for a total of 96 hours studied. Breast milk samples used for the discovery set were collected at hour 24 and hour 96 (2 samples from each of 3 women, for a total of 6 samples). To both confirm and ascribe potential functional significance, two dietary manipulations were additionally employed in the validation set. These two cohorts were part of two crossover studies (whereby each subject serves as her own control following random sequence allocation) examining the effects of diet composition on milk quality, quantity, and carbohydrate expenditure. In the glucose-galactose cohort <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050564#pone.0050564-Mohammad1" target="_blank">[27]</a>, 7 obese subjects consumed a diet of 15% protein, 50% carbohydrate, and 35% fat under controlled GCRC conditions. After an overnight fast, women were randomized to receive an isocaloric drink every 3 hours comprised of either glucose or galactose, the sugars in which provided them with 60% of their daily estimated energy requirement. In the high carbohydrate-high fat cohort <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050564#pone.0050564-Mohammad2" target="_blank">[32]</a> 7 lean subjects were randomly assigned to either a high-carbohydrate, low fat diet (60% carbohydrate, 25% fat, and 15% protein), or 2) a low-carbohydrate, high-fat diet (30% carbohydrate, 55% fat, and 15% protein), the caloric value of which was tailored to the woman’s required total energy intake (1.3 times basal metabolic rate). Breastfeeding occurred every 3 hours followed by milk expression via electric pump.</p

Characteristics of women in discovery and validation sets.

<p>The discovery set was comprised of three women (1 Caucasian, 1 Hispanic/Mexican American, and 1 African American), rhGH synchronized at two time points, while the validation set arose from 19 lactating women (7 Caucasian, 8 Hispanic/Mexican American, and 4 African American). There were no statistically significant differences between the sets with respect to maternal age, height, BMI, or weeks postpartum. Weight was increased in the validation set by virtue of cohort design (p<0.01). However, this did not significantly confound composite BMI between the discovery and validation sets.</p

Electrophoresis of RNA extracted from breast milk samples.

<p>RNA integrity gels were employed in assessing for the integrity of RNA species. smRNA bands are shown, and ribosomal RNA identified with blue arrows. Lanes 1–4: Exosome precipitation with Exoquick system on whole fresh breast milk with a 2 hour precipitation (Lanes 1&2) versus precipitation overnight (Lanes 3 &4). Lanes 5&6: Trizol reagent smRNA preparation from lipid breast milk fraction. Lanes 7–10: mirVANA smRNA preparation from lipid fractions of fresh (Lanes 7 & 8) and previously frozen (Lanes 9&10) breast milk.</p