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IFN-ε is a unique type I IFN whose constitutive expression in lung, brain, small intestine, and reproductive tissues is only partially understood. Our previous observation that posttranscriptional events participate in the regulation of IFN-ε mRNA expression led us to investigate whether the 5′ and/or 3′ untranslated regions (UTR) have regulatory functions. Surprisingly, we found that full-length IFN-ε 5′UTR markedly suppressed mRNA expression under basal conditions. Analysis of the secondary structure of this region predicted formation of two stable stem-loop structures, loops 1 and 2. Studies using luciferase constructs harboring various stretches of IFN-ε 5′UTR and mutant constructs in which the conformation of loop structures was disrupted showed that loop 1 is essential for regulation of mRNA expression. Incubation of HeLa cell extracts with agarose-bound RNAs harboring IFN-ε loop structures identified importin 9 (IPO9), a molecular transporter and chaperone, as a candidate that associates with these regions of the 5′UTR. IPO9 overexpression decreased, and IPO9 silencing increased basal IFN-ε expression. Our studies uncover a previously undescribed function for IPO9 as a specific, and negative, posttranscriptional regulator of IFN-ε expression, and they identify key roles for IFN-ε stem-loop structure 1 in this process. IPO9-mediated effects on 5′UTRs appear to extend to additional mRNAs, including hypoxia-inducible factor–1α, that can form specific loop structures.
Viral invasion of mammalian cells is followed by uncoating of the viral particles, exposure of nucleic acids, and host cell–mediated replication of the viral genome (1). Recognition of viral nucleic acids by host intracellular receptors initiates a cascade of events that culminate in the production of type I IFNs and other cytokines (2, 3). Type I IFNs are encoded by multiple, usually intronless, genes, and, in humans, most IFNs (e.g., IFN-β, -ω, -κ, -ε, and 14 IFN-α species) map to chromosome 9 and are expressed in tissue-specific manners (4). IFN-ε, a recently identified member of this family, is expressed in specific tissues, including female reproductive organs such as the ovary and uterus (5). It appears to play a role in immunity and protection against viruses owing to its ability to induce a short-lived and localized mucosal immune response (6). Seminal plasma has been reported to upregulate expression of IFN-ε in cervical and vaginal tissues (7), a response that may represent an antimicrobial defense mechanism that evolved to fight infections. Importantly, a recent study demonstrated that the reproductive tracts of female mice deficient in Ifn-ε display increased susceptibility to vaginal infections (8), suggesting that IFN-ε is a cardinal type I IFN that plays key roles as a mediator of innate immunity.
We previously reported that, in human cervical cancer cells, IFN-ε mediates STAT1 phosphorylation in response to stimulation with TNF-α and presented evidence indicating that transcriptional and posttranscriptional mechanisms participate in the regulation of IFN-ε expression (9). Gene expression is regulated at the levels of replication, transcription, mRNA splicing, stability and translation, protein posttranslational modification and stability, and others (10). Posttranscriptional control of gene expression relies on specific RNA–protein interactions that stabilize mRNAs, promote targeted degradation, or prevent access of the ribosome to the translation start codon (10). Such interactions can be mediated by the 5′ or 3′ untranslated regions (UTR) of mRNAs (11, 12). Sequences located in 3′UTRs are thought to participate primarily in the regulation of mRNA stability. A number of unstable mRNAs, including those encoding cytokines, oncogenes, and transcription factors, harbor AU-rich elements (AREs) whose signature sequence (i.e., AUUUA) is located in the 3′UTR (13). The 5′UTR-mediated regulation of expression is associated with events related to the initiation of mRNA translation, although mRNA-destabilizing effects also have been noted (11). Mammalian 5′UTRs often form stable secondary structures such as stem loops positioned between the cap structure and the AUG codon, which can inhibit translation initiation; the extent of this inhibitory effect depends on the thermodynamic stability and position of the structures (14).
In this study, we investigated whether regions in the 5′ and 3′UTRs of IFN-ε mRNA regulate expression. Our motivation to conduct these analyses was based on previous studies showing that IFN-ε expression is regulated posttranscriptionally (9). We present novel evidence demonstrating that cis 5′UTR sequences negatively regulate constitutive IFN-ε expression. This effect is mediated by a 5′UTR stem-loop structure that, when disrupted, leads to enhanced mRNA expression. The effect is specific and involves direct or indirect interaction with importin 9 (IPO9), a molecular transporter and chaperone that belongs to the superfamily of karyopherins (15).
Materials and Methods
Cell culture and transfection
The human cervical cancer cell lines HeLa and HeLa S3 were purchased from American Type Culture Collection and were maintained in a 5% CO2 atmosphere at 37°C in DMEM (Life Technologies) supplemented with 10% FBS (ThermoFisher Scientific). Transient transfection was accomplished by plating HeLa cells at a density of 1.5 × 105
IFN-ε 5′UTR cloning
We cloned the 5′UTR of human IFN-ε using a 5′ RACE kit (Life Technologies). The template consisted of 1 μg total RNA from HeLa cells previously transfected with polyinosinic-polycytidylic acid (polyI:C; 100 ng, 6 h), a treatment previously shown to increase IFN-ε mRNA expression (9). We generated cDNA using SuperScript II reverse transcriptase (Life Technologies) combined with gene-specific primer 1 (5′-TTGCTTCATGTCGTTCAAGG-3′). We purified the cDNA and then added a poly (C) tail at the 5′ end using TdT. Initial amplification was accomplished by PCR (94°C, 30 s; 65°C, 30 s; 72°C, 3 min; 25 cycles followed by 10 min at 72°C) using gene-specific primer 2 (5′-TCTCCCAACCATCCAGAGAAA-3′). A second round of PCR (94°C, 30 s; 55°C, 30 s; 72°C, 3 min; 30 cycles followed by 10 min at 72°C) was performed using 2.5 μl of a 50× dilution of the initial PCR product combined with a nested gene-specific primer 3 (5′-GCCAGCAGCACCAACACAGT-3′). The forward primer (AUAP: 5′-GGCCACGCGTCGACTAGTAC-3′) was provided by Life Technologies. The generated 5′ RACE PCR product was gel purified, cloned, and sequenced at the University of Utah DNA Sequencing Core Facility.
Plasmid construction
Luciferase reporter constructs were generated in the mammalian expression vector pcDNA3.1/Zeo+ modified to express firefly luciferase cDNA [generated from pGL3-Basic (Promega) by digestion with HindIII and XbaI, as described by Dixon et al. (16)]. We used the 5′RACE PCR product as template to generate constructs harboring stem loops 1 and 2 (ε1 + 2), posterior stem loop 2 (ε2), or no stem loops (ε0), using Platinum Pfx DNA Polymerase (Life Technologies). The reverse primer harbored a 5′-NcoI site shown in lower case (5′-TTccatggTGAAGGTCAAATATGCTAC-3′), and forward primers included 5′-XhoI sites (XhoI-ε1 + 2, 5′-TGGctcgagTGCCTCAAGGAAAGCATACAA-3′; XhoI-ε2, 5′-TGGctcgagATGTACTCTGAACACCATGA-3′; XhoI-ε0, 5′-TGGctcgagACATTAGAAAACGAAAGCAAC-3′). The products were purified, digested with XhoI and NcoI, and then cloned into the pcDNA3.1/Zeo+-luciferase (pcDNA-luc) vector described above.
The 3′UTR of IFN-ε mRNA was generated by amplification using primers XbaI forward, 3′UTR (5′-AGtctagaGTGGAGGGACTAGAGGACTT-3′) and XbaI reverse, 3′UTR (5′-CCtctagaGAAGAATCAACCATATTAATG-3′). The product was purified, digested with XbaI, and then cloned into the XbaI site of pcDNA-luc.
A cDNA encoding full-length IPO9 was obtained by amplification of HeLa cell cDNA using Platinum Pfx DNA Polymerase. We used primers NotI-IPO9 forward (5′-CTTgcggccgcGATGGCGGCGGCGGCGGCAGCT-3′) and SalI-IPO9 reverse (5′-CTAGAgtcgacCTAACTAGTGTCCATTGATTC-3′). The amplified products were cloned into the NotI and SalI sites of the mammalian expression vector, p3XFLAG-CMV7.1 (Sigma-Aldrich).
Mutations in the loop and stem structures of IFN-ε 5′UTR were generated using a site-directed mutagenesis kit from Stratagene (La Jolla, CA). Our objective was to generate mutant constructs predicted to have significant destabilization of secondary structures while harboring minimal changes in primary sequence. To achieve this, we made use of CentroidFold, a software program that predicts the thermodynamic stability of RNA secondary structures. The primer sequences used to generate mutant IFN-ε loops and stem were as follows: 5′-GCATACAATGAATAAcTTATTaTctTACTTCTTCAAAATA-3′ and 5′-TATTTTGAGGAAGTAagAtAATAAgTTATTCATTGTATGC-3′ (loop 1) and 5′-GGGAACCTGAAAATCatAAcTGTAAACTTGGAGAA-3′ and 5′-TTCTCCAAGTTTACAgTTatGATTTTCAGGTTCCC-3′ (loop 2). Mutated nucleotides are shown in lower case font.
Quantitative RT-PCR
Total RNA was extracted using a RNeasy total RNA isolation kit with on-column DNase I digestion (Qiagen). We used 1 μg total RNA as template for first-strand cDNA synthesis, combined with oligo(dT) primers and Moloney murine leukemia virus reverse transcriptase (Life Technologies). An Opticon 2 Real-Time PCR System (Bio-Rad) was used for quantitative analyses of luciferase and zeocin expression. The sequences of the primers were as follows: IFN-ε forward, 5′-AGGACACACTCTGGCCATTC-3′; IFN-ε reverse, 5′-TTGCTTCATGTCGTTCAAGG-3′; Luciferase forward, 5′-ACGGATTACCAGGGATTTCAGTC-3′; Luciferase reverse, 5′-AGGCTCCTCAGAAACAGCTCTTC-3′; GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′; GAPDH reverse, 5′-ATGGTGGTGAAGACGCCAG-3′; CCL5 forward, 5′-CTACTCGGGAGGCTAAGGCAGGAA-3′; CCL5 reverse, 5′-GAGGGGTTGAGACGGCGGAAGC-3′; IL-6 forward, 5′-ATGAACTCCTTCTCCACAAGC-3′; IL-6 reverse, 5′-AAGAGCCCTCAGGCTGGACTG-3′; hypoxia-inducible factor (HIF)–1α forward, 5′-GAGAAATGCTTACACACAGAAA-3′; HIF-1α reverse, 5′-CGGTAATTCTTTCATCACAATA-3′; 18S rRNA forward, 5′-ACTCAACACGGGAAACCTCA-3′; 18S rRNA reverse, 5′-AACCAGACAAATCGCTCCAC-3′; Zeocin forward, 5′-GACTTCGTGGAGGACGACTT-3′; Zeocin reverse, 5′-GACACGACCTCCGACCACT-3′.
Amplifications were performed using iQ SYBR Green Supermix (Bio-Rad), according to the manufacturer's specifications. Cycling conditions were as follows: 50°C, 2 min; 95°C, 3 min; 40 cycles of 95°C (15 s) plus 58°C (30 s) plus 72°C (30 s). A melting curve was generated by acquiring fluorescence measurements while slowly heating to 95°C at a rate of 0.1°C/s. Melting curves and quantitative analysis of the data were performed using an Opticon monitor, version 3.1, as previously reported (17).
Reporter assays
Twenty-four hours after transfection with reporter constructs, we harvested cells using reporter lysis buffer and determined luciferase activity using a commercially available assay system (Promega). When indicated, the cells were cotransfected with a β-galactosidase cDNA (pSV-β-Gal). For normalization purposes, we assessed either total protein concentration in the cell lysates or β-galactosidase activity. The latter was determined using a chemiluminescence-based reporter assay (Roche Applied Science). The data presented are representative of at least three independent experiments performed in duplicate, and they are reported as the mean ± SD.
Identification of 5′UTR-binding proteins
HeLa S3 cells were grown in suspension in DMEM containing 10% FBS. A cytoplasmic extract was obtained by harvesting 5 × 108 cells in buffer A (10 mM HEPES-KOH [pH 7.5], 3 mM MgCl2, 14 mM KCl, 5% glycerol, 1 mM DTT, and 0.5% protease inhibitor mixture [Sigma-Aldrich]). Following brief incubation (15 min on ice), the lysate was homogenized with 20 strokes of a Dounce homogenizer and then subjected to centrifugation (5000 × g, 15 min). The supernatant was stored at −80°C in small aliquots.
We immobilized RNA on agarose beads essentially as described by Caputi et al. (18). Briefly, the vectors diagrammatically shown in Fig. 5A were linearized using NcoI and then were in vitro transcribed using T7 RiboMAX (Promega), following the manufacturer's recommendations. RNAs (500 pmol) were placed in 400 μl reaction mixtures containing 100 mM sodium acetate (pH 5.0) and 5 mM sodium m-periodate, and were incubated for 1 h in the dark at room temperature. RNAs were precipitated using ethanol, and then were resuspended in 400 μl 100 mM sodium acetate (pH 5.0). The 200-μl aliquots of adipic acid dihydrazide agarose beads (Sigma-Aldrich) were washed four times with 10 ml 0.1 M sodium acetate (pH 5.0). After the final wash, the beads were resuspended in 400 μl 0.1 M sodium acetate (pH 5.0), mixed with periodate-treated RNAs, and then rotated for 16 h at 4°C. Next, we washed the beads with 3 × 1 ml 2 M NaCl and then 3 × 1 ml buffer D (20 mM HEPES-KOH [pH 7.6], 5% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM DTT). We then mixed agarose-bound RNAs with HeLa S3 cytoplasmic extracts and rotated the mixtures for 2 h at 4°C. Beads were decanted (5000 rpm, 1 min) and washed four times with 1 ml buffer D. After the final centrifugation, agarose-bound proteins were eluted with 2× SDS sample buffer, subjected to electrophoresis on 10% SDS-polyacrylamide gels, and visualized with Coomassie Blue. We excised several bands of interest from the stained gel, and performed in-gel tryptic digestion, as described (19). The resulting tryptic peptides were dissolved in 20 μl 0.1% trifluoroacetic acid and then desalted using C18 ZipTip (Millipore, Bedford, MA), according to the manufacturer's recommendations. Next, the peptides were dissolved in 0.1% formic acid and subjected to nanoflow-liquid chromatography-tandem mass spectrometry analyses and identification, according to Ozaki et al. (20).
The 3′ end of IFN-ε mRNA harbors AUUUA pentamers that are not required for constitutive expression
Our first goal was to investigate whether, in addition to transcriptional regulation (9), posttranscriptional mechanisms regulate expression of the IFN-ε gene. We first hypothesized that one or more AREs might regulate the stability of IFN-ε mRNA under basal conditions. AREs are usually located in the 3′UTR of mRNAs (21), and sequence analyses revealed the presence of two AUUUA elements in the 3′UTR of IFN-ε mRNA (Fig. 1A). We cloned this region using RACE and then generated a mammalian expression vector in which the 3′UTR was cloned downstream of the firefly luciferase cDNA (Fig. 1B). Transfection of HeLa cells with this construct resulted in luciferase expression at levels similar to those achieved by a construct lacking the 3′UTR (Fig. 1C). These results suggest that the 3′UTR is not required for constitutive expression of IFN-ε.
Effect of the 5′UTR of IFN-ε mRNA on firefly luciferase production under basal conditions
We next focused our attention on the 5′UTR of IFN-ε mRNA. We cloned this region using RACE and found that it harbors 265 nt upstream of the human IFN-ε translation start site (Fig. 2A). We then generated a mammalian expression construct in which this region was inserted at the 5′ end of the firefly luciferase cDNA (5′-Luc; Fig. 2B). In addition, we created a construct that harbored both the 5′ and 3′UTRs (5′-Luc-3′) to assess potential synergistic effects (Fig. 2B). HeLa cells transfected with 5′-Luc expressed significantly lower levels of luciferase activity relative to total protein, compared with cells transfected with a construct that lacked UTRs (Fig. 2C). These results point at 5′UTR-mediated negative regulation of constitutive IFN-ε expression. Inclusion of IFN-ε 3′UTR did not affect 5′UTR-mediated effects (5′-Luc-3′; Fig. 2C), thus confirming lack of participation of the 3′UTR in posttranscriptional regulatory events.
The 5′UTR of IFN-ε mRNA does not affect mRNA stability
Regulation of mRNA stability is a common posttranscriptional mechanism to control protein levels in response to cellular needs. In previous work, we provided evidence suggesting that HeLa cells regulate IFN-ε expression by modulating mRNA stability (9). Thus, we hypothesized that the ability of the 5′UTR of IFN-ε to decrease luciferase mRNA levels (Fig. 2C) might involve destabilization of the mRNA. To test this, we compared the stability of IFN-ε mRNA in the presence and absence of the 5′UTR, under experimental conditions that precluded transcription, as previously described (9). We observed no 5′UTR-mediated effects on mRNA t1/2, suggesting that the 5′UTR is not involved in mRNA-destabilizing effects (data not shown).
The 5′UTR of IFN-ε mRNA is predicted to form stem-loop structures that affect constitutive mRNA expression
RNA secondary structures located between the cap structure and the initiation codon can inhibit translation initiation, and the extent of this effect depends on the thermodynamic stability and position of the structure (10). Based on findings presented in Fig. 2C, we hypothesized that defined regions in the 5′UTR of IFN-ε mRNA might adopt secondary structures that impact posttranscriptional events. We conducted computer-aided analyses of the 5′UTR of human IFN-ε mRNA using CentroidFold (www.ncrna.org/centroidfold), a web-based approach frequently employed as a tool to predict RNA secondary structures (22). This analysis identified two sequences potentially capable of forming stem-loop structures, as follows: loop 1 spanning bases 23–49, and loop 2 spanning bases 110–142 (Fig. 3). The formation of stem-loop structures was consistently predicted by multiple additional algorithms, including KineFold (http://kinefold.curie.fr/; Supplemental Fig. 1A), RNAfold (http://rna.tbi.univie.ac.at; Supplemental Fig. 1B, 1C), and Mobyle (http://mobyle.pasteur.fr; data not shown). Moreover, stem-loop formation at the 5′UTR of IFN-ε is predicted for a variety of primates (Fig. 4) (23), suggesting evolutionarily conserved functions for these regions. We used this information to generate firefly luciferase expression constructs in which IFN-ε mRNA 5′UTR sequences harboring 0-1-2 loops were cloned between the CMV promoter and luciferase cDNA (Fig. 5A), and then used these constructs to assess the impact of loop structures on posttranscriptional events. We found that the presence of loop 2 (ε2) did not affect luciferase mRNA expression compared with that resulting from a construct that lacked stem-loop structures (compare ε0 with ε2; Fig. 5B), suggesting that this structure does not regulate IFN-ε mRNA expression. In contrast, inclusion of loops 1 and 2 robustly decreased relative luciferase mRNA levels (compare ε0 with ε1 + 2; Fig. 5B). These combined observations suggest that loop 1, or the combined presence of loops 1 and 2, suppresses IFN-ε mRNA expression.
Effect of loop structures within 5′UTR of IFN-ε on constitutive mRNA expression
Our next goal was to investigate whether proper conformation of stem-loop structures is involved in the regulation of IFN-ε mRNA expression. To accomplish this, we generated a series of 5′UTR mutant constructs in which each of the predicted secondary structures was individually disrupted (Supplemental Fig. 2A, 2B). We found that disruption of loop 1 rescued luciferase mRNA expression to a level comparable to that of ε2, which lacks loop 1 (Fig. 6). In contrast, disruption of loop 2 had no effect compared with the control (ε1 + 2). These data are consistent with studies shown in Fig. 5, and they suggest that proper folding of loop 1 is essential for constitutive suppression of IFN-ε mRNA. Our results also suggest that loop 1, and not the combined presence of loops 1 and 2, is responsible for negative regulation of IFN-ε expression.
Identification of IPO9 as a protein that binds to IFN-ε 5′UTR stem-loop structures
We next sought to identify proteins that specifically interact with IFN-ε 5′UTR stem loops, and determine whether the identified candidates play a role in posttranscriptional regulation of IFN-ε expression. To accomplish this, we generated RNAs corresponding to IFN-ε 5′UTR harboring both loops (ε1 + 2) or no loops (ε0). We attached individual RNAs to agarose beads, incubated them with HeLa cell extracts, and then subjected bound proteins to SDS-PAGE. We found that removal of loops 1 and 2 robustly decreased association with three proteins of apparent molecular masses 68, 108, and 118 kDa (Fig. 7). We subjected the 100- to 130-kDa region of the SDS-PAGE gel shown in Fig. 7 to mass spectrometric analyses, and identified IPO9 (predicted molecular mass = 110 kDa), a member of the importin β superfamily of nuclear transport receptors, as a candidate involved in posttranscriptional regulation of IFN-ε expression. We were unable to identify the 68-kDa protein owing to technical difficulties.
IPO9 silencing enhances constitutive expression of IFN-ε mRNA
To investigate whether IPO9 functionally affects IFN-ε expression, we silenced its expression using siRNA (Fig. 8A) and found that decreased IPO9 expression enhanced constitutive IFN-ε mRNA expression (Fig. 8B). Stimulation with TNF-α modestly, but significantly, increased the level of IFN-ε mRNA, in agreement with our previous findings (9). However, IPO9 silencing restored IFN-ε mRNA to an extent equal to that observed in unstimulated cells (Fig. 8B). These results indicate that IPO9 suppresses constitutive, but not stimulated, IFN-ε mRNA expression. We next investigated whether IPO9 suppressed expression of other mRNAs and found that constitutive expression of HIF-1α, predicted to form a large secondary structure within its 5′UTR (Supplemental Fig. 3), was enhanced by IPO9 silencing, in a manner similar to that observed for IFN-ε (Fig. 8C). In contrast, the levels of constitutively expressed (housekeeping) gene GAPDH (24) and those of the chemokine CCL5 and the cytokine IL-6 were not affected by IPO9 silencing (Fig. 8D–F). Although the 5′UTRs of these mRNAs also are predicted to adopt secondary structures (data not shown), the stem loops formed by IFN-ε and HIF-1α appear to be unique.
Loop 1 is required for IPO9-mediated suppression of IFN-ε expression
To investigate whether the regulatory effects of IPO9 and loop 1 on IFN-ε expression are functionally related, we assessed the impact of IPO9 on inhibition of IFN-ε expression mediated by our 5′UTR constructs. We used a two-pronged approach whereby IPO9 was either overexpressed (Fig. 9A) or silenced (Fig. 9B). These studies showed that IPO9 regulates INF-ε mRNA levels in a loop-dependent fashion. Increased IPO9 led to marked reductions in the expression of a construct harboring loops 1 and 2 (ε1 + 2), but had no effect when loops 1 and 2 were deleted (Fig. 9A). Conversely, silencing IPO9 enhanced expression of ε1 + 2, but not that of constructs lacking loop 1 (ε0 and ε2; Fig. 9B). These results demonstrate that IPO9 regulates constitutive expression of IFN-ε mRNA through loop 1 within 5′UTR of IFN-ε. Loop 1 in IFN-ε and a stem loop in HIF-1α (candidate loop structure; Supplemental Fig. 3) share unique features; the stems are composed of 6–10 bp, and the loops harbor 5–7 nt. Whereas this requirement remains to be confirmed experimentally, it appears that the suppressive effect of IPO9 on mRNA expression is dependent on the formation of peculiar secondary structures within 5′UTRs.
IPO9 does not regulate IFN-ε expression at the promoter level
Our final goal was to investigate whether IPO9 transcriptionally regulates IFN-ε expression. To test this, we transfected IPO9-silenced and control HeLa cells with a previously described IFN-ε promoter construct, and then determined promoter activity, as before (9). We found that IPO9 silencing did not activate the IFN-ε promoter, in contrast to transfection with polyI:C, which was used as a positive control (Fig. 10) (9). We conclude that IPO9 does not regulate IFN-ε expression at the promoter level.
Previous studies have shown that IFN-ε expression is induced upon infection with Semliki Forest (5) and vaccinia (14) viruses, and in response to exposure of cervicovaginal tissues to seminal fluid (7). In addition, RNA interference–mediated silencing of IFN-ε significantly inhibits activation of STAT1 (9), a key transcriptional activator of antiviral, immune, and antitumorigenic responses (25). These observations point at the importance of IFN-ε in host immune responses such as those required to establish antimicrobial, antiviral, and antitumorigenic states in specific organs. The mechanisms that control IFN-ε levels in various tissues, including those of the female reproductive tract, are only partially understood. Previous work rightly focused on transcriptional initiation events shown to participate in the regulation of IFN-ε expression (5, 9). However, this is not the only mechanism whereby the levels of IFN-ε are regulated, as early evidence suggested that posttranscriptional mechanisms also are involved (9). In the current study, we investigated whether, and how, IFN-ε expression is regulated at the posttranscriptional level because constitutive expression of IFN-ε was recently shown to play essential roles in the protection of female reproductive organs from sexually-transmitted infections (8). We found evidence supporting a role for the 5′UTR of IFN-ε mRNA in regulation of expression under basal conditions.
The 5′UTRs often include cis elements that survey individual mRNAs and affect their stability. Examples include regions in the 5′UTRs of mRNAs encoding IL-2 and growth-related oncogenes (26, 27). Several RNA secondary structure prediction algorithms identified two potential stem-loop structures in IFN-ε 5′UTR, and the studies presented in this work show evidence supporting a role for loop 1 as a negative regulator of IFN-ε mRNA expression. This inhibitory effect does not appear to involve changes in mRNA stability, and, whereas the precise mechanism involved remains to be elucidated, thermodynamically stable stem loops have been shown to stall ribosomes and initiate endonucleolytic cleavage events (28).
The inhibitory effect exerted by IFN-ε 5′UTR on mRNA expression may be specific for certain cell types and most likely depends on expression of proteins that recognize defined structures or sequences located in the 5′UTR. Our studies identified IPO9 as protein that binds primarily to stem-loop structure 1 in IFN-ε 5′UTR and negatively impacts mRNA expression. To our knowledge, this constitutes the first report identifying IPO9 as a regulator of IFN-ε mRNA expression, a function suggesting roles additional to those previously reported. Importins, including IPO9, are known for their ability to mediate active transport through nuclear pore complexes (29) and effectively suppress the aggregation of their basic import cargoes in polyanionic environments (15). Importins exert their functions through protein–protein interactions; they recognize nuclear localization signals and prevent protein aggregation by shielding basic patches such as those found in a variety of ribosomal proteins (15). No studies to date have reported direct interactions between importins and nucleic acids, leading us to speculate that, in our studies, IPO9-mediated posttranscriptional effects on IFN-ε mRNA expression involve intermediates, such as unidentified RNA-binding protein(s). Our observation that IPO9 inhibited expression of IFN-ε and HIF-1α, but not that of other mRNAs, points at specific interactions limited to a subset of mRNAs whose 5′UTR fold into defined secondary structures such as IFN-ε loop 1.
In conclusion, we have identified a novel mechanism for posttranscriptional regulation of IFN-ε expression. A distinct stem-loop structure predicted to form in the 5′UTR directly or indirectly interacts with IPO9, a member of the importin family, negatively regulating IFN-ε mRNA expression under basal conditions. These observations provide a novel mechanism for regulation of IFN-ε, and describe a previously unidentified function for IPO9 in posttranslational, gene-specific regulation of expression. This regulatory mechanism also may be used to regulate the expression of other mRNAs, including HIF-1α.
The authors have no financial conflicts of interest.
We are indebted to Janeth C. Tayone for excellent technical assistance.
This work was supported in part by KAKENHI Grants-in-Aid for Scientific Research 20591934 and 23590560 (to T.M.) and by the Kanzawa Medical Research Foundation (to T.M.). Additional support was provided by National Institutes of Health Grants 5P01CA073992 and P30 CA042014 (to the Huntsman Cancer Institute for support of core facilities), and by the Huntsman Cancer Foundation.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- AU-rich element
- hypoxia-inducible factor
- importin 9
- polyI:C
- polyinosinic-polycytidylic acid
- siRNA
- small interfering RNA
- untranslated region.
- Received July 12, 2012.
- Accepted June 13, 2013.
- Copyright © 2013 by The American Association of Immunologists, Inc.


text:
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Censorship: No
Language : English
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Game Information:
Personal Trainer is a story about a guy who lost it all (job, home, fiance) after a life of doing good for others. He found alcohol, of course, to be the answer. Lucky his cousin Sophie offered her couch until he gets back on his feet. As fate would have it, an exciting opportunity presented itself.

subset: commoncrawl


text:
(UPDATE) Changes to our Grading System
Why grade?
This is part of the never-ending debate! Grading a boulder problem (inside or outside) gives people an idea of the difficulty of a climb. This makes it easier to find a problem at the right level for your session goals. It can also help to understand how likely you might be to fall off a problem, and it can be a way of measuring and recording progression over time.
The Arch sets climbs by circuits, where a certain colour of hold reflects a particular difficulty. We find this is striking, easy to follow and allows for consistency across our centres.
In 2008/2009 we were the first climbing centre we know of in the UK to use V grades. We adopted a simplified version of the American 'V' grading system without VB/V0-/V0+ that would be easiest to understand, and not be directly comparable to outdoor climbs in the UK, or to other climbing centres. We've recently decided that there are still ways to improve this system however, so are looking to implement some changes this year...
What are we changing?
- We had several circuits that included 3 'V' grades. We listened to feedback from some climbers who found that this is too broad a range of difficulty. With this in mind, most circuits will now focus on no more than 2 V grades. The yellow circuit for example used to be V2 to V4, whereas now it will only range from V3-V4. This "slim-lining" should help everyone get on problems at the right difficulty, and also help climbers in measuring their progression through the circuits.
- Narrower difficulty circuits will also help us spread out the grades a little more consistently, whereas before we had a higher concentration of climbs at around V3.
- White and Green spots are now ungraded "ladders" for learning, warming up & down climbing. Black climbs will stay at a V0 grade, but will offer more climbing variety than before.
- At the other end of the spectrum, the Green circuit will encompass 3 grades, with the addition of V9, so that there is more for climbers of all abilities.
- In addition to this, our team athlete Matt Cousins will be setting and inviting guest setters for a V9 and harder 'Project Circuit' of 5 climbs at each centre, set on a monthly basis, starting with Building One+. This will be featured in it's own monthly video once we get the ball rolling.
- At Acton, along with some other improvements which we'll post about soon, we're increasing the density of circuit climbs by about 15-20%, to fit in more problems of all grades. We're also working out a new schedule for the main stage (competition) wall that will include more frequent resets, and full on events with world cup style finals problems and holds.
- At North, along with some improvements which we'll also post about soon, we'll be adding a purple & yellow circuit to the mix.
The old system and new system are shown below...
How will it affect me?
There will be a small variation in grading as we roll out the new circuits at all the centres. Once we reach April 2019, we should be fully switched over to the new style. Our staff will be able to inform you which areas have been set with the new system, and we will be updating this article with a list as we go. In the meantime, please bear with us!


text:
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text:
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Room 611, 6/f Elite IND CTR,


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Lift tickets, ski rental, lessons, activities and more..
Best Accommodation For This Resort
Resort features
A stunning location overlooking the snow-covered scenery of the magnificent Mt. Bandai. This is a ski resort with a splendid view from the slopes at the top of the mountain. Many of the slopes have a gentle incline, making this an ideal place to enjoy at your own leisure with your family. Taking a stroll on the snowy mountain is the best, too! Located on the back of Mt. Bandai, this little-known gem is surrounded by large-scale ski resorts. You can enjoy the sense that you are on a private slope. There's also a variety of other things to enjoy, like the famous Yellow Hole Tour or various trekking courses. With a width of 200 meters, the slopes are wide and gentle.
Open season
Opening hours
Resort information
|〒 969-27011170-5 Hibara Obudairayama, Kitashiobara-mura, Yama-gun, Fukushima-ken
|Snow park information
|Kids' park
|Ski lodge
|Ski center in front of the main course (information, rentals, school, restaurants, shops, Kids' Room)
|Rescue facilities
|Patrol Center(ski Center2F)
|Childcare facilities
|Ski Center Restaurant
|Payment methods
|Snow trekking Toboggan Snowscoot Snow strider
Regular pricing
Official pricing listed by the ski resort
These prices do not reflect the discount. Prices may differ from WAmazing Snow.
〒 969-27011170-5 Hibara Obudairayama, Kitashiobara-mura, Yama-gun, Fukushima-ken
Transportation fares are approximate. The actual fare may be different.
Book Snow & Ski in Japan via WAmazing Snow


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Duke invested in a new clinical research management system (CRMS) to improve quality and support reworked business processes that create administrative simplicity and operational excellence. The CRMS is the central tool to track the clinical research operations portfolio. Its integration with the electronic IRB system (a 1:1 match on protocols, including exemptions) is critical to understanding the diverse clinical research activities across the institution. This required Duke to: implement system-wide workflows, create pathways for heavy and infrequent users, and develop tools for monitoring non-compliance and data completeness. The CRMS has been integral in helping study teams use standardize workflows. Nearly a year post-go live, this presentation will review key success factors, decision points, challenges and successes of an enterprise-wide adoption of Forte's CRMS, OnCore.
Orla Mester, Senior Vice President, Services, Forte Research Systems, Inc.
Denise Snyder, MS, RD, Associate Dean for Clinical Research, Duke University Medical Center


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September 15, 2019 | 68° F
Use the fields below to perform an advanced search of The Daily Targum's archives. This will return articles, images, and multimedia relevant to your query. You can also try a Basic search
1000 items found for your search. If no results were found please broaden your search.
Sophomore outfielder Jawuan Harris hit three home runs in one game at USC Upstate and leads the team with seven on the season.
A Piscataway man died after jumping off of the Rockoff Hall parking deck on Saturday afternoon.
Senior Mike Carter is one of the many graduating seniors for the Rutgers baseball team.
An estimated 400 Rutgers students arrived at the Cook Student Center on Wednesday night to hear Bassem Youssef speak about producing a satirical television show following the Arab Spring in Egypt.
Senior defender Chris Groel leads the Scarlet Knight defense in ground balls and has seen the unit progress over the years into one of the best defenses in the country.
Sophomore outfielder Jawuan Harris recently moved into 10th place all time in Rutgers history for career stolen bases and will be key for the Scarlet Knights progressing this season.
Senior first baseman Tom Marcinczyk finished Tuesday's loss against Rider with two hits, reaching base safely three times in an otherwise anemic offensive display for the Knights.
Head coach Louis Levine saw his team finish in ninth place at this year's Big Ten Championships, hosted by the Scarlet Knights, one place better than the team's 10th-place finish last season.


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Episode 4.9 "Identity of 3-Losing Streak"
1st Half (1:14)
Seniors Trevor Waite, Joel Okafor and Evan Maxwell go inside the mind of a player in the middle of a losing streak.
2nd Half (8:53)
Coach Tonagel shares how he processes a losing streak as a coach and how to continue to find an identity of 3 regardless of an outcome.
Click here for Apple Podcasts
Click here for Anchor Podcasts


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After three towns suffered natural gas explosions, carriers are helping them get back on their feet. As three towns in continue to recover from several natural gas explosions, the Massachusetts insurance industry is trying to help. Certain specific insurance companies have stepped up to try to assist with the return to normalcy. The natural gas explosions rocked their communities as one person was killed and 25 were injured. Certain parts of the Massachusetts insurance industry have reached out to try to help those who are affected. The blasts occurred on…Read More
Tag: mema
MEMA releases flood insurance information for consumers
MEMA information could help consumers understand their policies and file claims The Mississippi Emergency Management Agency (MEMA) has released information that aims to help the victims of flood file flood insurance claims. Mississippi residents that have flood insurance receive their coverage through the National Flood Insurance Program (NFIP). The majority of the state's homeowners, however, do not have coverage that protects them from the damage caused by floods. Yet, many of these homeowners believe they are adequately protected by the coverage they receive through the state's wind pool policies. MEMA…Read More


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By Gwendolyn Audrey Foster
Black girls filmmakers not just deserve an viewers, Gwendolyn Audrey Foster asserts, however it can be crucial that their voices be heard as they fight opposed to Hollywood's buildings of spectatorship, possession, and the inventive and distribution features of filmmaking.
Foster offers a voice for Black and Asian ladies within the first precise exam of the works of six modern Black and Asian girls filmmakers. She additionally incorporates a distinctive advent and a bankruptcy entitled "Other Voices," documenting the paintings of alternative Black and Asian filmmakers.
Foster analyzes the foremost movies of Zeinabu irene Davis, "one of an increasing number of self sufficient Black girls filmmakers who're actively developing [in the phrases of bell hooks] 'an oppositional gaze'"; British filmmaker Ngozi Onwurah and Julie sprint, filmmakers operating with time and area; Pratibha Parmar, a Kenyan/Indian-born British Black filmmaker considering problems with illustration, id; cultural displacement, lesbianism, and racial identification; Trinh T. Minh-ha, a Vietnamese-born artist who revolutionized documentary filmmaking by way of displacing the "voyeuristic gaze of the ethnographic documentary filmmaker"; and Mira Nair, a Black Indian girl who concentrates on interracial identity.
Read or Download Women Filmmakers of the African and Asian Diaspora: Decolonizing the Gaze, Locating Subjectivity PDF
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Additional info for Women Filmmakers of the African and Asian Diaspora: Decolonizing the Gaze, Locating Subjectivity
Sample text
I had been muted. The daughter, as filmmaker, retrieves her mother's memories and sees through their lines the pain rendered by the beauty system that separated them yet gave the daughter a great deal of pleasure in the beauty of her own body. In a scene in which the pair go to a bathhouse, the daughter is forced to recognize that she has never understood how others might view her mother. As the mother falls asleep, her towel drops away from her missing breast and all of the women in the shower gaze on with looks of shame and horror.
The mother-daughter economy of the senses is the shared frame of The Body Beautiful. The fragmented narratorial style of the film marks a return to the senses, which "can never be a return to realism ... always mediated by memory and assembled from sensory and experiential fragments, this assemblage will always be an act of imagination-thus opposed to the reductions of realism" (Seremetakis 217). As the camera pans across the bodies of mother and daughter to reveal the scar of the mastectomy, the voice-over narrates the life of the filmmaker herself.
Davis playfully injects her own body into the text and encourages the viewer to read her as another suture to the text, just in case she missed the many autobiographical connections with the main character. As a filmmaker, Davis is obviously comfortable playing with the concept of the fourth wall, and with the "look back" of the camera/enunciator. Within this context of self-reflexive representation, the voice-over narrator's phrase, "They do connect: Body-image, dating, motherhood, tonight," takes on an extradiegetic quality.


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Our collection of free online strategy games is sure to be entertaining, while also giving you a mental workout. Try your hand at one of our different types of free online Sudoku games, like Daily Sudoku, Chess, or MathDoku, our take on the ever-popular game called KenKen.
The Best Free Strategy Games for PC Digital Trends.
At current, the developer Riot Games boasts over 110 million players who sign into the game at least once per month, making League not just the most popular game right now, but the most popular game of all time. Personally, Ive been doing battle on the Rift for almost four years now, and havent spent a dime. As long as youre happy learning the ropes on the stock rotation of free weekly champions, its only a matter of time before youll be able buy your favorites using the in-game currency to play as often as you like in ranked and unranked matches, online or off. Download League of Legends for free today. Starcraft II Arcade. If you ask anyone whos older than 12 what the quintessential real-time strategy game of our era is, Starcraft is the answer youll get.
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1672 Fairwood Ave, San Jose 95125 – IN CONTRACT!
Built by Eichler Homes in 1960, this iconic low gable atrium model in the beautiful tree-lined Fairglen tract retains many original details that appeal to mid-century modern enthusiasts. The updates that have been made fit tastefully in the originally intended "modern" form and function.
Eichler Features:
Beautifully remodeled low gable atrium Eichler in prime Fairglen location. 4 b/2b 1618, 6181 sf lot. Atrium w/ slate tiles, synthetic grass, water feature, sun shade & a variety of plants. Stunning great rm w/ dramatic brick fireplace lined by floor-to-ceiling windows. Original details: mahogany-paneling, brick fireplace, globe lighting, radiant heat. Sleek kitchen w/ euro cabinetry, quartz countertop, s/s appliances & custom hood. Other amenities include dual paned sliders & windows, AC & radiant boiler. Master bed w/ designer carpet & walk-in closet. Remodeled master bath w/ custom vanity & designer tiles. Inside laundry rm w/skylight. Guest bath w/shower over tub. 1 car garage w/original door & finished sheetrock. Tastefully landscaped 2 patios, expansive lawn & variety of shrubs & trees. Section 1 clearance. Tight-knit Fairglen neighborhood w/ block party/Eichler Home Tour. Highly desirable Willow Glen location Walk to parks, schools, shops. Award Winning Schools: Booksin, Willow Glen Middle & Willow Glen High.
As Eichler Specialists, the Boyenga Team was able to put this Eichler on the market and find a buyer in just 2 days! We were able to showcase this home during the San Jose Eichler Celebration event hosted by the Preservation Action Council of San Jose. To find out more about this annual Eichler Weekend Event, check out https://blog.chime.me/www-boyengateam-com/2019/03/28/eichler-weekend-event-with-the-boyenga-team/
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Canon PIXMA TS5040 top notch shade printing from the comfort of your very own home, with check and replicate attributes, Wi-Fi, cloud connectivity and also a simple 7.5 centimeters (3″) LCD existing– all had within a portable, space-saving body.Publish appealing images as well as crisp message– over USB cord, Wi-Fi or directly from your SD cards. Canon's PENALTY inkjet print advancement as well as 5 singular inks bring convenience, cost savings, and beautiful prints.
Experience a lean, space-saving kind element 40% smaller as compared to previous equal designs. Smart contrasting appearances, rounded edges, as well as tapering contours, make the printer's already-small framework show up also smaller sized. An exceptional printer in and out– as well as an elegant improvement to any kind of work workdesk or office.
Integrated Wi-Fi means you can publish from your smart phone, tablet or computer system utilizing help for Mopria ™ for Android, Apple AirPrint ™, Google Cloud Print ™, Windows 10 Mobile ™ as well as the Canon PRINT app. Publish your photos directly from Wi-Fi-enabled camera, or making use of the integrated SD card port.Every feature is within very easy reach with the manually adaptable front panel, 7.5 centimeters (3″) LCD present as well as the all-natural interface. The intense, basic buttons suggest quick ease of access to the printer's highlights, whether printing, duplicating or scanning.
Making use of PIXMA Cloud Link, accessed with the Canon PRINT app on your mobile phone or tablet, you could release from preferred social networks and cloud services consisting of Facebook, Instagram, Google Drive, Dropbox, OneDrive along with a lot more. You could also post checked papers as well as photos to Google Drive, OneDrive and also Dropbox *.
Running expenses won't break the bank, many thanks to the optional high-capacity Canon XL ink cartridges, letting you conserve as long as 30% per web page compared with standard matchings. The five solitary ink cartridges suggest you'll only change the shade that has actually run out.
Obtain motivated with CREATIVE PARK as well as CREATIVE PARK COSTS. Design as well as print greeting cards, schedules, picture collections along with great 3D paper craft pets, toys, as well as places frameworks.
Include brand-new life to your social media sites network breaks– accessibility Instagram making use of PIXMA Cloud Internet link after that publish your much-loved shots using Canon's 13×13 centimeters (5″ x5″) Image Paper As well as Glossy II square media.
Trick Functions:
- Use as well as share wirelessly
- Canon PRINT application
- Optional high yield inks
- Print from and check to the cloud
- Publish from SD flash memory card
- Huge, clear screen
- Publish 13×13 centimeters (5″ x5″) square photos
- Only change the ink that goes out
- 10×15 centimeters (4″ x6″) images in simply 39 secs
Canon PIXMA TS5040 Specifications:
- Features: Wireless Publish, Replicate, Scan, Cloud Link
- Publish Resolution: As much as 48001 x 1200 dpi
- Print Modern technology: 5 Person Ink Containers (PGBK, BK, C, M, Y) ChromaLife100 inks
- Mono Publish Speed: Approx. 12.6 ipm2
- Colour Publish Speed: Approximate. 9.0 ipm3
- Picture Print Speed: orderless 10x15cm: Approx. 39 seconds4
- Indeterminate Printing: Yes (A4, Letter, 20x25cm, 13x18cm, 10x15cm, 13x13cm).
- Two Sided Printing: Hand-operated procedure.
- Scanner Type: CIS flatbed picture as well as record scanner.
- Scanner Resolution (Optical): 1200 x 2400 dpi7.
- A4 Scan Rate: Approximate. 19 sec8.
- Scanning Deepness (Input/ Output): Colour: 48 little bit/ 24 bit as well as Greyscale: 16 bit/ 8 bit.
- Maximum Document Size: 216 x 297 mm.
Canon PIXMA TS5040 Software & Driver Setup
Procedure 1: Driver Setup of Canon PIXMA TS5040 via Free Download
Through this procedure, the setup of Canon PIXMA TS5040 is performed with a so-called Automatic Wizard as you eliminate CD,
However you have to;
- Provide Canon PIXMA TS5040 software file or its driver setup, which you can download from the link below.
- Have USB cable is essential for its function to connect both the printer device and the computer. USB cable is available in the printer box.
Afterward, pay attention toward the steps below to setup Canon PIXMA TS5040
- Switch ON both Canon PIXMA TS5040 and your computer.
- Only if it is requested, you can connect the USB cable of PIXMA TS5040.
- You can download the setup file, then install it anytime you want it by considering the step-by-step of the setup.
- Done with the setup, now you can pair Canon PIXMA TS5040 USB cable with your computer. The wizard then will proceed the USB cable to ensure that both devices are properly connected. Another step-by-step for the setup will appear.
- You have to follow the setup instructions to complete the setup.
- Finish with the setup, Canon PIXMA TS5040 is ready to use.
Procedure 2: To Setup Manually Canon PIXMA TS5040
Determine this procedure, "Add a printer" function that is an inbuilt feature of Windows is necessary.
Things to do:
- Download Canon PIXMA TS5040 software file, if you don't have on your system. Rest assured, the link is available below.
- You need Canon PIXMA TS5040 USB cable to proceed the set up.
Setup Instruction:
- Switch ON both Canon PIXMA TS5040 and your computer.
- Connect both devices above with Canon PIXMA USB cable
- Prior the setup, you have to extract the downloaded file that is stored in TEMP folder.
- If your device operating system is XP, hit "Fax and Printer" section. However, if you use Vista or Windows 7, find Control Panel, then hit "View Devices and Printer".
- Move your mouse, then hit "Add a printer" feature.
- Keep in mind that you only give the path to the aforesaid feature, only if the feature is customized and ready.
- The instruction will pop-up, to complete the set up, you need only to follow the instructions.
- You finish the installation, Canon PIXMA TS5040 will proceed your command smoothly.
Procedure 3: To Setup Canon PIXMA TS5040 via Software CD
The setting up of Canon PIXMA TS5040 with Automatic Wizard/Software CD
Ensure You Have:
- DVD/CD drive should be installed on your computer.
- Check your printer package to find Canon PIXMA TS5040 software CD.
- You need the USB cable that you can find within the package.
Comply the below mentioned instructions to finish Canon PIXMA TS5040 setup with Software CD.
- Make sure that your devices PIXMA TS5040, and computer are ON.
- Before, it's requested, connecting both devices using Canon PIXMA TS5040 are not allowed.
- Infix the Software CD of Canon PIXMA TS5040 to the computer DVD/CD drive. Follow the step-by-step instructions which are necessary for the setup.
- You can connect now the USB Cable of your printer Canon PIXMA TS5040 to your computer, when you are instructed to.
- It takes some time until PIXMA TS5040 USB cable is detected by your computer. Instructions for the installation will appear afterward, you have to execute each step to complete the installation.
- At the moment the installation is complete, you can print with your Canon PIXMA TS5040, immediately.
Windows 7 / Windows 7 (x64) / Windows 8.1 / Windows 8.1 (x64) / Windows 8 / Windows 8 (x64) / Windows Vista / Windows Vista (x64) / Windows XP / Windows XP (x64) / Macintosh / Mac OS X (10.5/10.6/10.7/10.8/10.9/10.10) / Linux.
Driver & Software Download For Canon PIXMA
Operating System
|TS5040 series Getting Started Guide
|TS5040 series Getting Started Guide
|ICA Driver (OS X 10.12/10.11/10.10/10.9/10.8)
|PosterArtist Updater (Windows 10/10 x64/8.1/8.1 x64/8/8 x64/7/7 x64/Vista/Vista64)
|PosterArtist Lite (Windows 10/10 x64/8.1/8.1 x64/8/8 x64/7/7 x64/Vista/Vista64)
|IJ Network Device Setup Utility (Windows 10/10 x64/8.1/8.1 x64/8/8 x64/7/7 x64/Vista/Vista64)
|TS5040 series MP Drivers (Windows 10/10 x64/8.1/8.1 x64/8/8 x64/7/7 x64/Vista/Vista64)
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Hotels in 50th Belfast Festival
Meet 92 travelers, 46 tour guides and discover 1,246 photos
Travelers are voting Armagh City Hotel, Carrickdale Hotel And Spa and Castle Leslie Estate as the best of 408 hotels in 50th Belfast Festival. There are 383 hotels in Dublin a city just 167 km from 50th Belfast Festival and 58 hotels in Donegal which is 176 km distant. The most popular of these are Fairways Hotel Dundalk in Dublin and Station House Hotel in Donegal. You can find 1106 Hotel in the country of Ireland and Mount Juliet is our members favorite Hotel in all of Ireland. Want more? Check out our map of hotels in 50th Belfast Festival.
Armagh City HotelTourist Essentials Hotels2 Friary Road, Armagh, BT60 4FR, Ireland
Location. Armagh City Hotel is located in Armagh, close to St. Patrick's Church of Ireland Cathedral, St. Patrick's Church, and St. Patrick's Trian…1
Carrickdale Hotel And SpaTourist Essentials HotelsCarrickcarnon, Ravensdale, Dundalk, Ireland
Location. Located in Ravensdale, Carrickdale Hotel and Spa is a family friendly hotel in the historical district and local attractions include…1
Castle Leslie EstateTourist Essentials HotelsCastle Leslieestate22317, Glaslough, 00000, Ireland
-Nestled On 1000 Acres Of Undulating Irish Countryside Dotted With Ancient Woodland And Glittering Lakes Castle Leslie Estate Is One Of The…1
Park Hotel DundalkTourist Essentials HotelsArmagh Road, Dundalk, Ireland
Location. Park Hotel Dundalk is located in Dundalk, close to Oriel Park and Proleek Dolmen. Regional points of interest also include King John's…1
Innisfree HouseTourist Essentials GuesthouseInnisfree House, Carrick road, Dundalk, Louth, Ireland
Innisfree House is a beautiful early Edwardian townhouse offering natural comfort with a warm welcome in relaxed traditional surroundings.
Greengates B&BTourist Essentials GuesthouseDublin Rd, Blackrock, Dundalk, Ireland
RAC 3 Diamond and Warm Welcome Award Winner. Orla O'Grady and family welcome you to their luxurious home just 1 km from the…0
Fairways Hotel DundalkTourist Essentials HotelsDublin Road 1, Dundalk, 00000, Ireland
Price and value are the focus for these properties, along with comfortable accommodations and professional guest services.1
Crowne Plaza DundalkTourist Essentials HotelsGreen Park Inner Relief Rd, Dundalk, 0000, Ireland
Location. Crowne Plaza Dundalk is located in Dundalk, close to Oriel Park. Additional area points of interest include Proleek Dolmen. Features.Dining…1
Keernaun HouseTourist Essentials GuesthouseDublin road, Blackrock, Dundalk, Louth, Ireland
Keernaun house is a guesthouse which is approved by the Irish Tourist Board and is a member of the Irish Hotel Federation.
Lynolan HouseTourist Essentials GuesthouseMullagharlin Rd, Haynestown Dundalk Co. Louth, Dundalk, Louth, Ireland
Located just outside Dundalk, host Evelyn Carolan has been offering top quality accommodation at Lynolan House for over 20 years.


text: Votavi is a social platform for polls and questions. Ask questions to your friends, your customers or anyone else. Use it for social media or use it on your website or blog. Compare any content like movies, pictures and shopping products and use Votavi to quickly create an opinion poll. Votavi Visit the Votavi platform. Support and tutorials Get help and look at tutorials to make interesting, fun and creative questions. Votavi for Business Here you can find out how to use Votavi for your business. In a fun and interesting way more interaction with your customers without invading their privacy. News Find the latest news and updates about Votavi.


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Will launch alongside 3rd Gen Threadripper.
AMD's most powerful APU gets an upgrade.
Guns. Lots of guns. Also, knights, horses and polygons. We look at the new Mount and Blade.
Paradox Interactive and Taleworlds have announced a new Mount and Blade title, With Fire and Sword.
September 16 2019 | 14:00
Copyright © 2000 - 2019, bit-tech.net. All rights reserved.


text: Former lieutenant general and a comrade of Tohdoh. Seeking to draw attention to the fight the Nippon Kaihō Sensen is attempting against Britannia, he took several Britannian citizens and students hostages at the Lake Kawaguchi Convention Center Hotel, while meeting Zero and Tōdō's strong disapproval as a result. (Source: Wikipedia)


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Jasic ARC 160 PFC Dual Voltage MMA Inverter Welder
The Jasic Arc 160 Dual Voltage is a cost effective, compact and highly portable 'plug and go' inverter Welding Machine. As with all Jasic welders, this machine comes with a market leading 5 year warranty.
The Jasic arc 160 welder benefits from an auto sensing facility, ensuring the correct 110/240 voltage is maintained automatically. The dual voltage function makes it perfect for on site tasks where 240V may not be allowed or available. Power factor correction (PFC) allows the unit to run over a wide voltage range meaning it will still run well on long extension leads or weak power supplies.
Being part of the 'Jasic Pro' range, this machine features IGBT Inverter technology meaning that the Welder may also be run by a generator. Auto-compensation for voltage fluctuations also protects the machine when being used with generators.
The machine has easy arc starting, for ease of use and resulting in minimal spatter. Hot start is a handy feature that temporarily increases the output current during the start of the weld. This momentary increase in power assists the ignition and quickly and reliably starts the arc. Hot start ensures an excellent arc ignition which helps prevent the electrode sticking and hence avoids metallurgical defects in the weld.
The Jasic Arc 160 is a highly efficent Arc welder with 40 Khz inverter frequency.
The Jasic 160 has a duty cycle of 160 Amps at 35 % capable of welding electrodes up to 4.0mm and weighs just 4.7 kg. This dual voltage inverter is all within a compact package, with rubber mouldings, striking looks and handle for ease of portability.
Jasic has a certified ISO 9001 quality management system and a product range that fully complies with CE, RoHS, C-TICK, CSA and CCC standard.
Package Includes: Inverter power source, Cable plugs, Operating instructions, 5 year extended warranty.
Not Included: MMA Leads, Mains plug, TIG torch & adaptor, TIG consumables, MMA Electrodes, Shielding gas, Filler wire, PPE (Welding helmet, gloves, etc)
Jasic 'Lift TIG' allows the user to TIG weld. If chosen from the above dropdown options, the TIG torch supplied would be a weldcraft type "Starparts" brand WP17 torch. Torch spares, TIG filler rods and tungsten electrodes are all available on the site. Feel free to call us if you are unsure what you would require.
TIG Torch will require an earth lead. If the machine, welding lead set and TIG torch is purchased, the TIG torch can be used with the earth lead from the MMA welding lead set.
If you would like to specify the MMA welding lead electrode holder type, please put a note when checking out. We are able to supply a twist type or crocodile type electrode holder (both the same price).
Supported by a 5 Year Manufacturers Warranty.
Wilkinson Star, the UK importer/distributor of Jasic machines also offer component level repair both in and out of warranty time frames. In the unlikely event of a machine going wrong in the 5 year warranty period, Jasic UK will arrange, at its expense to collect and return the equipment for repair. In our experience, warranty claims are carried out quickly and efficiently, minimising downtime.
Key Features
- Jasic Arc 160
- IGBT inverter technology for the professional user (Generator Friendly).
- MMA/DC Lift Tig operation.
- Hot start ignition with anti-stick.
- Self-adaptive arc force and voltage reduction device.
- 40 KHz inverter frequency for high efficiency.
- Compact size, lightweight and modern design
- Easy arc starting, minimal spatter.
- Stable and reliable arc.
- Auto compensation for voltage fluctuation.
- Advanced single PCB construction.
- 16 – 25 dinse sockets
Technical Data
Input Voltage: AC 230V (50/60 Hz) AC 115V (50/60 Hz)
Fuse Rating, slow 16A 32A
Input Power (kVA) 7.1 5
Current Range (A) 10 – 160 10 – 120
Duty Cycle @ 40'C 160A @ 35% 120A @ 35%
No-Load Voltage (V) 63
Efficiency (%) 85
Power Factor 0.70
Dimensions (mm) 313 x 130 x 250
Weight (Kg) 4.7


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THIS week's howler comes from Sports presenter Clare Balding.
Talking at half-time in the St Helens vs Warrington rugby league game about the players having difficulty in the heat handling the ball, she gaffed: "Balls sweat as well as hands."
G Hughes, from Christchurch, Dorset, wins #35 for that WiseCrack.
Send entries by email or on a postcard to: WiseCracks, The People, 1 Canada Square, London E14 5AP.


text:
New York weather forecast: SNOWSTORM to BURY NYC - schools closed and flights cancelled
NEW YORK CITY has been battered by heavy snow after a winter storm hit the northeastern part of the USA overnight.
Areas in New York City have been smashed by severe winter conditions as several inches of snow dropped overnight. Approximately five inches of snow were dumped by the storm in New York's Central Park on Monday. This is the largest snowfall total of the winter so far in the area.
The winter storm has caused widespread disruption to the Big Apple, with school closures and cancelled flights.
Mayor Bill de Blasio confirmed hundreds of schools will be shut down on Monday and also warned school buses would be significantly delayed because of the wintry weather.
On Sunday, the mayor said the city is expecting the "biggest snow accumulation we've had all year".
He added: "This is going to be a serious storm.
READ MORE: New Jersey weather ALERT: State of emergency DECLARED as storms drop eight inches of SNOW
New York weather forecast: A winter storm has brought widespread disruption to New York
"People should be ready for really tough conditions in the morning."
New York Governor Andrew M. Cuomo has also announced he would be deploying state resources, including thousands of plow trucks, specialised law enforcement vehicles and utility workers.
In a statement the governor said: "We have learned from experience that it is better to be over-prepared in these situations - if you're not prepared before the storm, it is too late."
Public schools in Jersey City and Boston will also be closed on Monday, with New Jersey, located just across the river from New York, expecting several inches of snow.
New Jersey Governor Phil Murphy declared a state of emergency, asking drivers to "stay off the roads so that we are able to deploy available resources to clean the snow."
In addition, more than 700 flights have been cancelled and another 1,200 delayed on Monday as snow, ice, and rain descended on the New York area.
The weather affected the city's three major airports and airlines including American, JetBlue, Delta, United, and Southwest have waived rebooking fees at affected airports.
But despite the significant snowfall across the large city, the New York subway is still running.
Although some lines are delayed, the subway seems to be functioning normally.
You can check if the delays are affecting your travel HERE.
The wintry weather began developing on Saturday, putting 43 million people under winter weather alerts.
The storm has already started to leave the northern East Coast, but is expected to hit people in areas from Colorado to Maine on Monday.


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Featuring : Contains mat, chalk, selection of marbles, chalk and miniature marble games booklet and Supplied in metal pocket-size tin. 97mm x 59mm x 23mm, this item is produced by Brimtoy. The Marble Shooting Games Marbles Mat Book In A Tin is now available to our visitors to order from the paddling pools shop at our great price.
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text: Marketing professional, software developer among hardest-to-fill positionsOne-third of hiring managers have positions that have been open for 12 weeks07/19/2013|hrreporter.com|Last Updated: 07/19/2013 More than one-third (35 per cent) of hiring managers in the United States currently have positions that have remained open for 12 weeks or longer, according to a poll by CareerBuilder. "Although the recession created an abundant pool of readily-available, unemployed talent that still exists today, employers are struggling to find new employees for technology-related occupations, sales, health care and a variety of other areas," said Brent Rasmussen, president of CareerBuilder North America. "The skills gap that exists for high-growth, specialized occupations will become even more pronounced in the years to come, prompting the need to place a greater emphasis on re-skilling workers through formal education and on-the-job training." Two in five employers (41 per cent) reported that they continuously recruit throughout the year, so that they have candidates in their pipeline in case a position opens up down the road, found the poll of 2,000 hiring managers in the U.S.Most difficult to fill jobs:• Marketing professional: 57,045 new jobs; 11.3 per cent growth • Software developer: 103,708 new jobs; 11.2 per cent growth• Machine operator/assembler/production worker: 135,363 new jobs added from 2010 to 2013; 9.9 per cent growthRELATED ARTICLESB.C. funds measures to help forestry workers hurt by downturnFederal response to 'gig' workers hindered by unreliable data: governmentGains in part-time, youth employment in August as wage growth slows• IT manager/network administrator: 48,709 new jobs; 7.5 per cent growth• Truck driver: 113,517 new jobs; 6.7 per cent growth• Nurse: 135,325 new jobs; five per cent growth• Engineer: 73,995 new jobs; 4.9 per cent growth• Accountant: 55,670 new jobs; 4.5 per cent growth• Mechanic: 53,002 new jobs; 4.1 per cent growth• Sales representative: 584,792 new jobs added from 2010 to 2013; 3.8 per cent growth.© Copyright Canadian HR Reporter, HAB Press. All rights reserved. Add Comment * Title * Name * Email Anonymous * Comment


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FORT WALTON BEACH � With the majority of scores in Wednesday�s Miracle Strip Invitational in at least the 80s, and a few players posting higher numbers than usual, Choctaw�s Amber Ritchie and Crestview�s Morgan Stutler went the opposite direction.
FORT WALTON BEACH � With the majority of scores in Wednesday�s Miracle Strip Invitational in at least the 80s, and a few players posting higher numbers than usual, Choctaw�s Amber Ritchie and Crestview�s Morgan Stutler went the opposite direction.
Ritchie turned in a lower-than-usual 77 to finish third. Stutler carded the best score of her young golf career: a 78, good for fourth.
Individually, the two were the cr�me de la cr�me of area golfers, finishing third and fourth respectively. But team-wise, Niceville once again was the best top to bottom.
PHOTOS: View a photo gallery from the invitational.
Niceville�s 329 �checks a box� on the team goals, coach Mitch Inness said, as it brings the tournament trophy �back to the team�s trophy case� after Fort Walton Beach won last year�s showcase.
The Eagles accomplished the feat with Kaide Farne, Lauren Miller and Sarah Pabst each shooting a 79 on the par-72 course and Meg Horrigan rounding out the team score with a 92. Madi Messmore (94) and Alex Messmore (98) also competed. Baker, Fort Walton Beach, Navarre and South Walton also were represented in the tournament, which continues today with the boys� rendition.
�I�m happy with three 79s,� Inness said of Niceville�s performance. �Regardless the team, three scores in the 70s says a lot. The sky is the limit with this group. This will be a special group this season.�
Inness did consider the Vikings� scores �a little higher� than he expected. Farne, a freshman, said the scores weren�t in the mid-70s due to the greens� rough conditions.
Then again, Ritchie and Stutler didn�t have much issue with them.
Ritchie�s 77 was the third-best individual round � behind Pensacola golfer and medalist Madison Pacheco�s 72 and Catholic golfer Abigail Kerrman�s 76 � and Stutler�s 78 was fourth. There were only nine below-80 scores out of the 61 golfers competing.
�This was my best round ever,� said Stutler, a senior who usually shoots in the low 80s. �I think (the high scores compared to my low score) was more of a mentality. It�s really hot and it�s more difficult to focus when it�s like that.�
Stutler, joined by Katy Dale (95) as Crestview�s only golfers, shot a 39 in each half of her round. Ritchie�s day was a little more up and down � or down and up. The junior started on hole No. 10 and shot a 41 with a double bogey on the 12th hole and three straight bogeys to end the back nine.
She rebounded with a 36 � all pars except a bogey on the par-3 No. 7 and birdie on the par-4 No. 8.
�The first nine holes played more difficult,� Ritchie said. �I was a little irritated. � Once I got to the back nine, I went with it.�
Milton�s 345 was second among team scores, and Choctaw (349) finished third with a school record, Indians coach Andy Snaith said. Pim Pim Panyaprachum (85), Holly Loiocano (87) and Lexi Gomez (100) rounded out the team scoring with Maggie Keeling (100) also playing.
�I�m proud of how they�re working hard, putting in the effort and gelling as a team,� Snaith said.
Catholic (358) finished fourth and Gulf Breeze (373) finished fifth. Fort Walton Beach (378) finished sixth with counting scores from Kristin Allen (90), Audrey Vaughn (92), Karina Caraballo (97) and Maddie Williams (99). Madison Torrey played but did not finish her round.
Chiles (410) finished seventh with Navarre (415), Pace (425) and West Florida (463) rounding out the team scoring. Navarre�s top four rounds came from Ailsa Clark (84), Sultana Pohlmann (98), Laurel Emerson (110) and Payton Evans (123).
Baker, Mosley, South Walton and Tate had individual golfers but not enough to compete as a team.
Miracle Strip Leaderboard
Madison Pacheco, Pensacola 72
Abigail Kerrman, Catholic 76
Amber Ritchie, Choctaw 77
Morgan Stutler, Crestview 78
Kaide Farne, Niceville 79
Miranda Holt, Tate 79
Vickie Kwak, Chiles 79
Lauren Miller, Niceville 79
Sarah Pabst, Niceville 79
Ashley Rogers, Pace 80
Levi Worrell, Milton 80
Ailsa Clark, Navarre 84
Hallie Rice, Milton 84
Katt Crowdis, Gulf Breeze 85
Pim Pim Panyaprachum, Choctaw 85
Chloe Dawson, Milton 86
Holly Loiocano, Choctaw 87
Kristin Allen, Fort Walton Beach 90
Bailey Berry, Gulf Breeze 90
Madelyn Dimitroff, Catholic 92
Meg Horrigan, Niceville 92
Conlan McKernan, Catholic 92
Audrey Vaughn, Fort Walton Beach 92
Sam Carver, Baker 93
Marybeth McGuire, Mosley 94
Madi Messmore, Niceville 94
Katrina Boteler, Milton 95
Katy Dale, Crestview 95
Lauren Ybarra, Milton 96
Karina Carabello, Fort Walton Beach 97
Alex Messmore, Niceville 98
Paige Mougey, Catholic 98
Sultana Pohlmann, Navarre 98
Kara Dorsey, Gulf Breeze 99
Kasey Richards, Gulf Breeze 99
Maddie Williams, Fort Walton Beach 99
Brittany Daigle, Pensacola 100
Lexi Gomez, Choctaw 100
Maggie Keeling, Choctaw 100
Marissa Jackson, Tate 101
Jessica Chorpening, Chiles 106
Elyzabeth Jackson, Chiles 107
Alex Page, South Walton 107
Laurel Emerson, Navarre 110
Willow Foxworth, West Florida 111
Mary Kate Blaylock, Pace 112
Hailey Tomas, West Florida 112
Frances Williams, Gulf Breeze 113
Ashley Andrews, Pace 114
Madison Mouhey, Catholic 114
Allison Beatty, Chiles 118
Josie Venable, West Florida 118
Raeanne Nolasco, Pace 119
Taylor Bensley, Chiles 121
Madison Gorman, Tate 122
Elizabeth Wright, West Florida 122
Payton Evans, Navarre 123
Jillian Shelby, Pensacola 123
Savannah Clark, South Walton 124
Savanna Calhoun, West Florida 125
Madison Torrey, Fort Walton Beach, DNF


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Welcome To Lyme Bytes!
April, 2014- HELLO ALL! I am no longer posting to this blog. For the latest on me and my work, I invite you to subscribe to my NEW blog: www.conniestrasheim.blogspot.com where I share my latest findings on how to heal from chronic illness involving Lyme and other conditions. Thanks!
Greetings and welcome to my Lyme disease blog, a comfy cozy (and sometimes crazy!) place for cutting-edge information, encouragement and insight into the fastest-growing epidemic disease in the United States. In this blog you will find everything from bug-killing strategies to immune system and hormone help, as well as lifestyle and spiritual suggestions for healing from chronic illness involving Lyme disease. The information contained within this blog is based upon my own healing journey and what I have learned over the past eight years as I have been diligently digging and researching my way back to a better state of health. May you find it to be a source of hope, inspiration and wisdom in your own journey towards wellness.
About the book:
Published August, 2009
Written by Connie Strasheim
Learn More - Bulk Orders - Table of Contents
Saturday, July 13, 2013
New Babesia-like Organism Identified, "FL1953 Protomyxzoa Rheumatica"
3 comments:
Isn't Dr Schaller a psychiatrist?
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Chevy Trucks for Sale near Bellevue
Chevy Trucks for Sale near Bellevue
When searching for the best trucks on the market, it is important to consider all factors. For example, you want to know that your new truck can be relied upon to perform as well as last for many years. If you are looking for excellent quality and reliability, then you will want one of our Chevy Trucks for Sale near Bellevue. At Evergreen Chevrolet, we carry new and used versions of the Chevy Colorado, Chevy Silverado 1500, Chevy Silverado 2500, Chevy Silverado 3500 and the Chevy Silverado Chassis Cab. We carry both new and used versions of Chevy trucks and other Chevrolet models.
New Chevrolet Silverado
When considering the 2016 Chevy Silverado, you may want to learn more about your powertrain options. Currently, the base model Silverado is equipped with a 4.3L V6 providing the driver 285 horsepower and 305 lb-ft of torque with which to work. Another engine option is the 5.3L V8 providing 355 horsepower and 383 lb-ft of torque. The top-of-the-line engine offering the most power and towing capacity is a 6.2L V8 providing 420 horsepower and 460 lb-ft of torque. Not only are you able to choose your engine and model, but there are also trim levels and packages to choose from with each of our Chevy Trucks for Sale near Bellevue.
2016 Chevrolet Colorado near Bellevue
If you are looking for a truck that can be used as an everyday commuter but is also capable of accomplishing hauling and towing jobs, the Chevy Colorado is a great choice from our Chevy Trucks for Sale near Bellevue. With a good amount of towing and hauling capability, the newest Colorado can help you with your latest project or most recent job. These trucks are a lot of fun to drive and very useful to own. With similar fuel ratings to some sedans, each tank of gas will get you as far as you could look for. With each model year the Colorado offers more and more, which is not to say the used models do not have plenty to offer. Your best bet is to take a closer look at what we currently have available to choose the right truck for you.
If you would like to take a closer look at any of these Chevy trucks, or any other Chevy car or SUV, stop by Evergreen Chevrolet at 1601 18th Ave. NW in Issaquah, WA. We are just a drive away from those in Woodinville, Bellevue, Issaquah and beyond.
© Copyright Content Motive, 2016.
All Rights Reserved. | Dealer website content by Content Motive


text:
Our collaborations with area companies are key to our success preparing Career Collaborative participants to secure quality jobs with benefits. We're grateful to Bank of America for presenting the financial literacy workshop "Economic Mobility Basics" to our participants on June 26, 2019. The session aimed to help them establish or rebuild good financial habits.
Led by Bank of America Vice President-Sourcing Manager John Bolognese, Merrill Lynch Financial Advisor Phillip Hardin, and James Kirkman, Bank of America Senior Vice President, Digital Channel Management, the workshop covered the basics of budgeting and banking, managing income and paychecks, and financial safety.
Career Collaborative's employer-partnerships offer a great opportunity to fill your staffing needs with competent, motivated employees, give back to your community, and further your company's efforts to make a real difference in people's lives. For more information on partnering with Career Collaborative contact us at info@careercollaborative org, 617.424.6616, or visit us online at careercollaborative.org.


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UPDATE: I have decided not to ship anything until the weather cools down (still pretty warm here in Arizona). If you are on the notification list, you will be notified as soon as I get ready to process and ship orders. :) Thank you for your patience. I am planning on taking pre-orders soon.