OTU richness estimates for each body site.

<p>Estimated richness calculated using CatchAll <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034242#pone.0034242-Bunge1" target="_blank">[16]</a> with both the V1–V3 and the V3–V5 tag data for each body site. Bars represent the upper and lower confidence bounds provided by Catchall. Both sets of rRNA tags provided similar estimates. The stool samples showed the most richness with the oral samples having the next greatest richness, followed by the skin and vaginal samples.</p

Relative abundance of OTUs.

<p>For each OTU, the relative abundance is plotted for each sample in which the OTU is present. The OTUs with the highest number of total sequences are ranked first and plotted at the leftmost side, and the OTUs with the lowest total number of sequences are ranked last and plotted toward the right. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034242#pone-0034242-g003" target="_blank">Figure 3A</a> shows the V1–V3 OTUs and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034242#pone-0034242-g003" target="_blank">Figure 3B</a> the V3–V5 OTUs. Even OTUs that are among the top 10 most abundant span at least 3 orders of magnitude of relative abundances from less than 0.01% to more than 10%.</p

Body site preference of distinct OTUs from individual taxa.

<p>The use of genus or family names alone can imply a broad colonization of one organism across body sites. Using 3% OTUs, we can discern a high degree of site specialization of distinct organisms within these taxonomic groups, especially within the oral cavity. Not all OTUs assigned to these taxa were graphed, and only OTUs with greater than 100 tags were included. The area under each curve sums to 100% of the occurrence of that OTU across the body sites. Body site labels are: saliva (sv), supragingival plaque (supp), subgingival plaque (subp), keratinized gingiva (kg), tongue dorsum (td), hard palate (hp), buccal mucosa (bm), palatine tonsils (pt), throat (th), anterior nares (an), stool (st), left and right antecubital fossae (laf, raf), left and right retroauricular creases (lrc, rrc), mid-vagina (mv), posterior fornix (pf), and vaginal introitus (vi).</p

Richness estimates for each body site.

<p>Estimated number of species for each body site using both the V1–V3 and the V3–V5 tags computed with CatchAll. Numbers in parentheses are upper and lower confidence limits. The stool samples have the highest estimate of total richness, followed by the oral sites, particularly the plaque and tonsils. The skin and the vaginal sites have the lowest estimated richness.</p

Core OTUs present in at least seven of the nine oral sites.

<p>While each 16S amplification (V1–V3 and V3–V5) has 6 core OTUs, there are differences between the amplifications, with Pasteurellaceae and Lactobacillales identified with the V3–V5 tags but not the V1–V3 tags, and <i>Leptotrichia</i> and <i>Granulicatella</i> (a member of the Lactobacillales order) identified with the V1–V3 tags but not the V3–V5 tags. <i>Fucobacterium</i>, <i>Gemella</i>, <i>Streptococcus</i>, and <i>Veillonella</i> were identified in both sets of OTUs.</p

Comparison of OTU abundance and prevalence across all samples.

<p>The OTU rank is defined by the total number of sequences across all samples, with the most abundant ranked first (on the left). In panel A, OTU prevalence, the fraction of samples containing that OTU, is compared to OTU rank abundance. The most abundant OTUs appeared in a higher percentage of samples than the less abundant OTUs. In panel B, the cumulative abundance of OTUs as a function of OTU rank abundance showing that the 100 most abundant OTUs accounted for almost all of the sequence reads in both the V1–V3 and the V3–V5 amplifications. Panels C and D (V1–V3 and V3–V5 respectively) show the OTU prevalence rank against the OTU rank, with the most abundant OTUs tending also to be the most prevalent OTUs.</p

Size of the 95% core microbiome by body site.

<p>The oral cavity sites show the greatest number of core OTUs with both the V1–V3 and the V3–V5 tags, followed by the stool, the anterior nares, then the skin and the vaginal sites. Core OTUs are defined as those OTUs appearing in at least 95% of all samples for a given body site. Body site labels in order are: saliva (SV), supragingival plaque (SUPP), hard palate (HP), palatine tonsils (PT), tongue dorsum (TD), throat (TH), buccal mucosa (BM), subgingival plaque (SUBP), keratinized gingiva (KG), anterior nares (AN), stool (ST), right (RAF) and left (LAF) antecubital fossae, left (LRC) and right (RRC) retroauricular creases, posterior fornix (PF), mid-vagina (MV), and vaginal introitus (VI).</p

PCoA of Biome Types in stool and vaginal midpoint samples.

<p>Panels A and B are principle coordinates analyses of stool samples based on the RDP taxonomy and using Morisita-Horn distance. With the V3–V5 data, the <i>Bacteroides</i>-dominated subjects are segregated from both the <i>Ruminococcus</i>-dominated samples and the <i>Prevotella</i>-dominated samples. The <i>Alistipes</i> and <i>Oscillibacter</i> samples overlap with the other biome types. The <i>Bacteroides</i> and Clostridiales show greater overlap with the V1–V3 taxonomy (Panel B), while <i>Prevotella</i> is still segregated but not well separated. With both V3–V5 and V1–V3 data, the intra-biome type distances are as great as inter-biome type distances. At the OTU-level (Panels C and D) the <i>Bacteroides</i> and Clostridiales biome types have much greater overlap. The Prevotellaceae biome type has complete overlap with the other two biome types in the V3–V5 OTU data (Panel C) but mild segregation with V1–V3. At the OTU-level, the intra-biome type distances are greater than the inter-biome type distances. Panels E and F are the PCoA results for the mid-vagina samples, V3–V5 and V1–V3 respectively. The V3–V5 OTUs did not differentiate the <i>Lactobacillus</i> species, but show that while most subjects fall under the <i>Lactobacillus</i>-dominated type, there are also types dominated by either Bifidobacteriaceae or other taxa. The V1–V3 OTUs separated the subjects by <i>Lactobacillus</i> sub-types. Not all of the subjects classified as Bifidobacteriaceae with V3–V5 had corresponding samples large enough (>1000 tags) to be included in the V1–V3 plot and vice-versa.</p

Developmental Exposure to Estrogen Alters Differentiation and Epigenetic Programming in a Human Fetal Prostate Xenograft Model

<div><p>Prostate cancer is the most frequent non-cutaneous malignancy in men. There is strong evidence in rodents that neonatal estrogen exposure plays a role in the development of this disease. However, there is little information regarding the effects of estrogen in human fetal prostate tissue. This study explored early life estrogen exposure, with and without a secondary estrogen and testosterone treatment in a human fetal prostate xenograft model. Histopathological lesions, proliferation, and serum hormone levels were evaluated at 7, 30, 90, and 200-day time-points after xenografting. The expression of 40 key genes involved in prostatic glandular and stromal growth, cell-cycle progression, apoptosis, hormone receptors and tumor suppressors was evaluated using a custom PCR array. Epigenome-wide analysis of DNA methylation was performed on whole tissue, and laser capture-microdissection (LCM) isolated epithelial and stromal compartments of 200-day prostate xenografts. Combined initial plus secondary estrogenic exposures had the most severe tissue changes as revealed by the presence of hyperplastic glands at day 200. Gene expression changes corresponded with the cellular events in the KEGG prostate cancer pathway, indicating that initial plus secondary exposure to estrogen altered the PI3K-Akt signaling pathway, ultimately resulting in apoptosis inhibition and an increase in cell cycle progression. DNA methylation revealed that differentially methylated CpG sites significantly predominate in the stromal compartment as a result of estrogen-treatment, thereby providing new targets for future investigation. By using human fetal prostate tissue and eliminating the need for species extrapolation, this study provides novel insights into the gene expression and epigenetic effects related to prostate carcinogenesis following early life estrogen exposure.</p></div

Morphology of MCF-7 cells grown in 2D and 3D cultures.

<p>MCF-7 human breast cancer cells grown in 2D monolayer culture for 3 days on poly-L-lysine coated coverslips and stained with periodic acid-Schiffs and hematoxylin (PASH) under low (A) and high (B) magnification. Cells exhibit typical cobblestone morphology, with several aggregates of cells (asterisks). MCF-7 human breast cancer cells grown in scaffold free non-adhesive agarose hydrogels for 7 days form microtissues with luminal spaces containing PAS-positive secretions (arrows) under low (C) and high (D) magnification. Scale bar = 50μm.</p