Custom Track View of DNase I hypersensitive sites (DHS) sites.

<p>DHS sites, Pol II, H3K20me1, H3K4me3, H3K4me2, H3K4me1, H2BK5me1 and H2A.Z along the FRA2 gene on Chr 2 were obtained from the UCSC Genome Browser.</p

Longitudinal Analysis of Vaginal Microbiome Dynamics in Women with Recurrent Bacterial Vaginosis: Recognition of the Conversion Process

<div><p>Bacterial vaginosis (BV) affects ∼30% of women of reproductive age, has a high rate of recurrence, and is associated with miscarriage, preterm birth, and increased risk of acquiring other sexually transmitted infections, including HIV-1. Little is known of the daily changes in the vaginal bacterial composition as it progresses from treatment to recurrence, or whether any of these might be useful in its prediction or an understanding of its causes. We used phylogenetic branch-inclusive quantitative PCR (PB-qPCR) and <i>Lactobacillus</i> blocked/unblocked qPCR (Lb-qPCR) to characterize longitudinal changes in the vaginal microbiota in sequential vaginal self-swabs from five women with recurrent BV, from diagnosis through remission to recurrence. Both patients with acute BV samples dominated by <i>G. vaginalis</i> recurred during the study with similar profiles, whereas the three patients with acute BV samples dominated by other anaerobes did not recur or recurred to an intermediate Nugent score. <i>L. iners</i> dominated remission phases, with intermittent days of abnormal microbial profiles typically associated with menses. The exception was a newly discovered phenomenon, a sustained period of abnormal profiles, termed conversion, which preceded symptomatic acute BV. Species known to have antagonistic activity towards <i>Lactobacillus</i> were detected in pre-conversion samples, possibly contributing to the decline in <i>Lactobacillus</i>. Lb-qPCR scores define two categories of response in the initial post-treatment visit samples; scores <5 may correspond with poor response to treatment or rapid recurrence, whereas scores >8 may predict delayed or no recurrence. Amsel criteria or Nugent scores did not have this potential predictive capability. Larger studies are warranted to evaluate the prognostic potential of detecting conversion and poor Lb-qPCR scores at the post-treatment visit of recurrent BV patients.</p></div

Shifts in dominant species common to conversions in P1 and P2.

<p>Panels represent qPCR data using Mycoplasmatales primers (top) or <i>Enterococcus</i> primers (bottom). Amplicons were sequenced to identify species, or in some cases were identified by their distinguishing melt curves that matched those of the sequenced products.</p

Microbial profiles of near-daily vaginal swabs from patients with histories of recurrent BV, characterized with 11 PB-qPCR targets (legend).

<p>Data are converted to % total titers and depicted on a log scale. Top panels of P1 and P2 show the expected rise to dominance of <i>Lactobacillus</i> after treatment, and conversions before acute BV. In both patients, sharp increases are seen in <i>G. vaginalis</i>, (2), <i>Prevotella</i> (4), <i>L. amnionii</i> (5), <i>BVAB2</i> (7), and <i>Mycoplasma</i> sp. (11). Patients P3 and P4, who did not recur, show sustained dominance of <i>Lactobacillus</i> after treatment, and their non-<i>Lactobacillus</i> populations remain generally at <1%, with frequent transient spikes. Red bar  =  menses;↓ =  coitus. Data is also presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082599#pone.0082599.s005" target="_blank">Table S4</a>.</p

STAT5 motif analysis of the mapped ChIP clones in activated CD4+ T cells.

<p>Out of the 105 binding sites for activated CD4+ T cells immunoprecipitated with anti-STAT5 Ab, 68% had TTN₅AA motifs and TTCN₃GAA sites were present in 26% of the target sites. The labels denote the category name and percentage.</p

The locations of putative STAT5 binding sites in activated CD4+ T cells.

<p>Mapping of STAT5 binding sites relative to annotated genes were categorized into four groups. Intergenic region denotes binding sites present greater than 10<10 kb upsteam denotes the region less than 10 kb upstream of the 5′ region of the nearest gene. Internal intron denotes introns other than intron 1 of the gene and intron 1 depicts the presence of a binding site within the first intron of the gene.</p

Profiles and treatment regimens for recurrent BV patients.

<p>Note: Day 0 refers to day of enrollment as a longitudinal patient. Treatments were all for 7 days. A =  Amsel criteria, number positive; N =  Nugent score. n/a =  not available. *  =  Clue cells present but not above 20%; **  =  not clinically confirmed.</p

PB-qPCR generated microbial profiles of acute and post-treatment vaginal swabs of patients with histories of recurrent BV.

<p>Data is also provided as Table; these and the conversions to relative titers as described [71]. Patient 1 (P1) was sampled at 3 separate acute BV (aBV) episodes; 3a and 3b are samples of the 3^rd episode taken 5 days apart. P1 and P2 recurred during the study; P3 and P4 did not. P5 responded poorly and was ultimately diagnosed with BV at an intermediate Nugent score, 4 (P5-iBV). uc  =  uncultured.</p

IL-2 regulates expression of FRA2.

<p>Quantitative RT-PCR was performed on PHA activated CD4+ T cells stimulated with or without IL-2 for different times to evaluate the expression of FRA2. Expression levels are presented as fold increase (logarithmic scale) and compared to the baseline levels (cells not treated with IL-2). 18 s was used as the housekeeping gene and served as the endogenous control. IL-2 stimulation strongly induced <i>FRA2</i> expression with two peaks observed at 4–6 hours and 24 hours post stimulation. This is representative of at least three independent experiments performed in triplicate.</p

Activation of the JAK3-STAT5 pathway is essential for the induction of FRA2 gene expression by TCR activation.

<p>CD4+ T cells were stimulated via the TCR ± the JAK3 inhibitor R333, RNA was prepared four hours post treatment. qRT-PCR analysis was performed to detect the relative expression of FRA2. FRA2 gene expression was induced by TCR activation and abrogated by R333 treatment. Shown is a representative experiment performed in triplicate and repeated three times.</p