Truncated SRA RNA derivatives inhibit estrogen receptor-α-mediated transcription.

The steroid receptor RNA activator (SRA) is a long non-coding RNA (lncRNA) that acts as a putative coactivator for steroid receptor-mediated transcription. A recent study showed that SRA RNA can be structurally dissected into four domains comprising various secondary structures, but the contribution of each domain to the coactivation ability of SRA RNA was previously unknown. Here, we assessed the functional contributions of the various domains of SRA. We examined the effects of each domain on the coactivation of estrogen receptor-α (ERα)-mediated transcription of a luciferase reporter gene in HeLa cells. Then the detailed domain analysis was focused on domain III (D3) not only with the reporter gene in HeLa cells, but also with ERα-responsive genes in MCF7 breast cancer cells. Domain deletion analysis showed that the deletion of any domain decreased the luciferase activity, and that deletion of D3 caused the largest decrease. This D3 deletion effect was not recovered by co-expression of D3 alone; moreover, the expression of D3 fragments (particularly helices H15-H18, which are highly conserved across vertebrates) inhibited luciferase expression in HeLa cells. Moreover, a fragment containing helices H15-H18 reduced ERα-responsive gene expression in MCF7 breast cancer cells. Our findings indicate that D3 inhibited ERα-mediated transcription of a reporter gene in HeLa cells and that helices H15-H18, as a core element responsible for the D3-driven inhibition, reduced expression of ERα-responsive genes in breast cancer cells.

Vancomycin-resistant Enterococcus colonization in the intensive care unit: clinical outcomes and attributable costs of hospitalization.

BACKGROUND: The clinical and economic impact of vancomycin-resistant Enterococcus (VRE) colonization remains unclear. Little data are available on factors affecting hospitalization length of stay (LOS) and costs. This study aimed to estimate mortality, LOS, and hospitalization costs for VRE colonized patients compared with a matched hospital population. METHODS: We performed a retrospective propensity score matched cohort study comparing the outcomes of patients with VRE colonization with those of uncolonized subjects matched at the time they were admitted to the intensive care unit (ICU). Between January 2008 and December 2010, we obtained rectal swab cultures within 24 hours of ICU admission to detect VRE colonization. RESULTS: During the study period, 567 (7.2%) of the 7,703 patients were colonized with VRE. There were 199 VRE colonized patients compared with 199 uncolonized patients using the propensity score. VRE colonized patients when compared with uncolonized patients were likely to have a higher case fatality rate (24.6% vs 17.1%; OR, 2.35). Longer total admission days were observed in the VRE colonized patients (28.7 vs 21.4 days; multiplicative effect, 1.25; P = .004). VRE colonization is found to be a significant factor associated with increased ICU cost in the multivariable regression model ($6,065 vs $5,298; multiplicative effect, 1.22; P = .029). Multivariable analysis identified the factors affecting ICU cost as follows: VRE colonization (odds ratio [OR], 1.20; P = .038), ICU length of stay (OR, 1.93; P < .001), ICU type (OR, 1.51; P = .001), valvular heart disease (OR, 2.38; P = .27), hospitalization within 12 months (OR, 1.21; P = .037), and use of invasive devices (OR, 1.28; P = .017). CONCLUSION: Compared with a matched hospital population, VRE colonization was associated with increased mortality, LOS, and costs. Strict infection control programs, including preemptive isolation for a high-risk group, should be helpful.

Direct, sequence-specific detection of dsDNA based on peptide nucleic acid and graphene oxide without requiring denaturation.

Sequence-specific detection of double stranded DNA (dsDNA) is important in various research fields. In general, denaturation of dsDNA into single strands is necessary for the sequence-specific recognition of probes to target DNA, posing several drawbacks which decrease the efficiency as a DNA sensor. Herein, we report a direct, sequence-specific dsDNA detection system without requiring any thermal denaturing step. Our strategy utilizes peptide nucleic acid (PNA) and graphene oxide (GO) as a probe and as a fluorescence quencher, respectively. The PNA first binds to the end of dsDNA strand due to the relatively easily dissociable terminal base pairs of DNA duplex. Next, superior binding affinity of PNA towards complementary DNA induces branch migration for gradual strand replacement, resulting in the formation of PNA/DNA duplex. Unlike other dsDNA sensors based on complementary DNA probes, PNA in combination with GO enabled hybridization with the target sequence hidden as a duplex form without denaturing step and thus, the formation of PNA/DNA duplex was translated into selective fluorescence signal. Moreover, it provided tighter turn-on signal control with very low background signal and high sensitivity and sequence selectivity even in the presence of serum proteins.

Two tandem RNase III cleavage sites determine betT mRNA stability in response to osmotic stress in Escherichia coli.

While identifying genes regulated by ribonuclease III (RNase III) in Escherichia coli, we observed that steady-state levels of betT mRNA, which encodes a transporter mediating the influx of choline, are dependent on cellular concentrations of RNase III. In the present study, we also observed that steady-state levels of betT mRNA are dependent on RNase III activity upon exposure to osmotic stress, indicating the presence of cis-acting elements controlled by RNase III in betT mRNA. Primer extension analyses of betT mRNA revealed two tandem RNase III cleavage sites in its stem-loop region, which were biochemically confirmed via in vitro cleavage assays. Analyses of cleavage sites suggested the stochastic selection of cleavage sites by RNase III, and mutational analyses indicated that RNase III cleavage at either site individually is insufficient for efficient betT mRNA degradation. In addition, both the half-life and abundance of betT mRNA were significantly increased in association with decreased RNase III activity under hyper-osmotic stress conditions. Our findings demonstrate that betT mRNA stability is controlled by RNase III at the post-transcriptional level under conditions of osmotic stress.

RNA recognition by a human antibody against brain cytoplasmic 200 RNA.

Diverse functional RNAs participate in a wide range of cellular processes. The RNA structure is critical for function, either on its own or as a complex form with proteins and other ligands. Therefore, analysis of the RNA conformation in cells is essential for understanding their functional mechanisms. However, no appropriate methods have been established as yet. Here, we developed an efficient strategy for panning and affinity maturation of anti-RNA human monoclonal antibodies from a naïve antigen binding fragment (Fab) combinatorial phage library. Brain cytoplasmic 200 (BC200) RNA, which is also highly expressed in some tumors, was used as an RNA antigen. We identified MabBC200-A3 as the optimal binding antibody. Mutagenesis and SELEX experiments showed that the antibody recognized a domain of BC200 in a structure- and sequence-dependent manner. Various breast cancer cell lines were further examined for BC200 RNA expression using conventional hybridization and immunoanalysis with MabBC200-A3 to see whether the antibody specifically recognizes BC200 RNA among the total purified RNAs. The amounts of antibody-recognizable BC200 RNA were consistent with hybridization signals among the cell lines. Furthermore, the antibody was able to discriminate BC200 RNA from other RNAs, supporting the utility of this antibody as a specific RNA structure-recognizing probe. Intriguingly, however, when permeabilized cells were subjected to immunoanalysis instead of purified total RNA, the amount of antibody-recognizable RNA was not correlated with the cellular level of BC200 RNA, indicating that BC200 RNA exists as two distinct forms (antibody-recognizable and nonrecognizable) in breast cancer cells and that their distribution depends on the cell type. Our results clearly demonstrate that anti-RNA antibodies provide an effective novel tool for detecting and analyzing RNA conformation.