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Neonatal immune challenge by administration of lipopolysaccharide (LPS) produces enduring alterations in the development and activity of neuroendocrine, immune and other physiological systems. We have recently reported that neonatal exposure to an immune challenge by administration of LPS results in altered reproductive development in the female Wistar rat. Specifically, LPS-treated animals exhibited diminished ovarian reserve and altered reproductive lifespan. In the current study, we examined the cellular mechanisms that lead to the previously documented impaired ovulation and reduced follicular pool. Rats were administered intraperitoneally either 0.05 mg/kg of LPS (Salmonella Enteritidis) or an equivalent volume of non-pyrogenic saline on postnatal days (PNDs) 3 and 5, and ovaries were obtained on PND 7. Microarray analysis revealed a significant upregulation in transcript expression (2-fold change; p < 0.05) for a substantial number of genes in the ovaries of LPS-treated animals, implicated in immune cell signaling, inflammatory responses, reproductive system development and disease. Several canonical pathways involved in immune recognition were affected by LPS treatment, such as nuclear factor-κB (NF-κB) activation and LPS-stimulated mitogen-activated protein kinase (MAPK) signaling. Quantitative Real-time PCR analysis supported the microarray results. Protein expression analysis of several components of the MAPK signaling pathway revealed a significant upregulation in the expression of Toll-like receptor 4 (TLR4) in the neonatal ovary of LPS-treated animals. These results indicate that neonatal immune challenge by administration of LPS has a direct effect on the ovary during the sensitive period of follicular formation. Given the pivotal role of inflammatory processes in the regulation of reproductive health, our findings suggest that early life immune activation via TLR signaling may have significant implications for the programming of ovarian development and fertility.
Worldwide there is a trend for declining fertility. This is particularly apparent for the female population, whereby 10.9% of women suffer from impaired fertility and 6% are infertile (Martinez et al., 2012). Decreased female fecundity has been related to advanced maternal age but the increasing representation of young women in this category (Martinez et al., 2012), suggests a more complex epidemiology. Accumulating evidence points to the critical role of the early life environment in the establishment of reproductive health. Substantial epidemiological and experimental evidence indicates that adult physiological function and health status may have their origins in the early developmental period (Barker and Osmond, 1986; Barker et al., 1989; Barker, 1990). Plasticity during the perinatal period allows adequate adaptation of an organism to given environmental conditions and predicts later life functioning. While physiological plasticity holds a beneficial value for the developing organism, exposure to adverse environmental conditions during critical maturational periods, may result in alterations to the normal developmental trajectory, increasing susceptibility to physiological malfunction and pathology in later life. The process by which early life environment can have permanent effect on physiological systems has been described as perinatal programming (Welberg and Seckl, 2001; Davies and Norman, 2002).
One of the major physiological systems that undergoes development and maturation during foetal and early postnatal life is the immune system. Due to functional immaturity of the neonatal immune system in both animals and humans, there is increased susceptibility to infections and a lower response to immunogenic stimuli when compared to that of the adult (Vosters et al., 2010). The impaired immune responses, upon exposure to infectious agents, during the perinatal period have been associated with the decreased capacity of the neonate to develop mature protective T helper type 1 immune responses (De Wit et al., 2003). As such, low levels of proinflammatory cytokines have been detected in response to immune stimulation by LPS and Staphylococcus epidermidis as documented in neonatal rats and mice, as well in human cord blood (Angelone et al., 2006; Hartel et al., 2008; Hodyl et al., 2008). The development of the immune system is well-established to be dependent on the immune, autonomic and endocrine signals that it receives early in life (Holladay and Smialowicz, 2000; Zakharova, 2009; Fagundes et al., 2012). This physiological programming of the immune system and its relationship to later life pathology has been associated with a number of long-term health outcomes mediated by inflammatory pathways including, a predisposition to asthma, allergies, autoimmune diseases, metabolic disorders, cardiovascular diseases, multiple sclerosis, and more (Zakharova, 2009; Fagundes et al., 2012), depending on the timing and the extent of early life immunogenic exposure.
Programming of the immune system has been investigated using various animal models. Experimentally-induced early life immune activation is commonly achieved by administration of lipopolysaccharide (LPS) [derived from Salmonella Enteritidis or Escherichia (E.) coli], the cell wall of Gram-negative bacteria, in rodents. LPS is recognized by the Toll-like receptor 4 (TLR4) that is expressed by several cell types, such as monocytes, macrophages, adipocytes as well as gonadal supporting cells (i.e., ovarian granulosa and testicular sertoli cells) (Medzhitov, 2001; Richards et al., 2008). The impact of neonatal LPS exposure has been examined on a variety of physiological and behavioral outcomes, such as adult immune responses (Boisse et al., 2004; Spencer et al., 2006; Walker et al., 2009b; Mouihate et al., 2010), metabolism (Walker et al., 2006; Iwasa et al., 2009a), brain morphology (Bilbo et al., 2008; Amath et al., 2012; Sominsky et al., 2012b), stress responsivity, and anxiety-like behaviors (Walker et al., 2009a, 2012; Sominsky et al., 2013).
The reciprocal relationship between the hypothalamic-pituitary-gonadal (HPG) axis and the immune system has an extensive impact on development and functioning of both systems, as well as on health outcomes. Oestrogen and androgen receptors are expressed on immune cells (Tanriverdi et al., 2003), and these in turn have been shown to populate all reproductive organs (Seamark et al., 1992). Recently, increasing attention has focused on the effect of neonatal immune challenge on reproductive development and functioning. Activation of the immune system during neonatal life has long-term consequences for reproductive functioning (Marchetti et al., 2000; Morale et al., 2003). Experimental evidence has indicated altered puberty onset, diminished HPG axis activity and impaired sexual behavior in both male and female rodents neonatally exposed to bacterial endotoxin, LPS, in particular on PNDs 3 and 5 (Iwasa et al., 2009b,c; Knox et al., 2009; Walker et al., 2011; Wu et al., 2011; Sominsky et al., 2012a), suggesting that these changes may be attributed to the critical window of reproductive development. Supporting this assertion, the critical period for programming of pubertal onset in the female rat has been determined to be before 7 days of age. LPS treatment on PNDs 3 and 5 has been shown to result in a significant delay in puberty, while LPS administered on PNDs 7 and 9 or PNDs 14 and 16 produced no such effect (Knox et al., 2009). Furthermore, neonatal LPS exposure has been demonstrated to aggravate LPS-induced suppression of LH pulse frequency in adulthood, when compared to neonatal saline-treated group (Li et al., 2007). Similarly, neonatal LPS-treated female rats exhibited prolonged oestrous cycle in response to LPS injection in adulthood, while no such response was observed in neonatal saline-treated animals (Iwasa et al., 2009c).
Neonatal LPS exposure has also been reported to produce alterations in gonadal morphology. Reduced gonocyte populations in neonatal testes, as well as increased epithelial disorganization and delayed spermatogenesis in adulthood were demonstrated in male rats neonatally exposed to LPS (Walker et al., 2011). Robust alterations in gonadal morphology in response to neonatal LPS challenge have also been documented in female animals. Diminished follicular reserve has been detected in the ovaries of LPS-treated female rats (Wu et al., 2011), with reduced population of primordial follicles being evident at 2 weeks of age, after LPS exposure on PNDs 3 and 5 (Sominsky et al., 2012a). This later finding corresponded with advanced senescence and poor pregnancy outcomes in LPS-treated females (Sominsky et al., 2012a), suggesting a prolonged and more robust effect of an immune challenge on female reproductive lifespan. Of particular relevance to the model of neonatal immune challenge, is that the initial follicular assembly and maturation are known to be dependent on local inflammatory agents, such as growth factors and cytokines (Dissen et al., 2002; Skinner, 2005; Schindler et al., 2010). Even after puberty, when ovarian function is largely governed by the HPG hormones (McGee and Hsueh, 2000), ovulation resembles an inflammatory process, as constituted by increased vasodilation and hyperaemia of ovarian follicles, and their ability to produce cytokines, chemokines, and prostaglandins (Espey, 1980; Richards et al., 2002). This concept is further strengthened by the abundance of macrophages, principal innate immune cells, in the ovary, contributing to the regulation of ovarian function (Wu et al., 2004). Furthermore, ovarian granulosa and cumulus cells exert several immune-related functions, including the expression of TLR4, which during ovulation respond to endogenous ligands, resulting in the release of pro-inflammatory cytokines, such as IL-6 (Richards et al., 2008).
While immune-regulation of ovarian function is crucial for normal fertility, any alteration in this delicate relationship may lead to ovarian dysfunction and subfertility. Common ovarian diseases, such as polycystic ovarian syndrome (PCOS) and endometriosis, have been associated with increased levels of follicular and serum pro-inflammatory cytokines, constitutive of chronic low grade inflammation (Wu et al., 2004). The direct impact of LPS exposure on ovarian function has been previously assessed in-vitro, and was reported to result in diminished follicular function (Herath et al., 2007) and impaired oocyte meiotic competence (Bromfield and Sheldon, 2011). No studies have investigated in vivo the mechanisms that underpin the effect of peripheral LPS exposure on ovarian development during the neonatal period. In the current study, we aimed to identify the ovarian pathways that lead to the previously documented impaired ovulation and reduced oocyte development, in the model of dual LPS exposure on PNDs 3 and 5 (Knox et al., 2009; Wu et al., 2011; Sominsky et al., 2012a). In order to characterize the cellular mechanisms that underpin the immediate effect of peripheral LPS exposure on PNDs 3 and 5 on ovarian development, we examined its effect on the ovarian transcriptome on PND 7. Given that in the rat, the assembly of primordial follicles is not established until PND 3 (Rajah et al., 1992; Skinner, 2005), we propose that an immune challenge by administration of LPS at this time point may directly intervene with the formation and establishment of the finite follicular pool via activation of inflammatory pathways.
All animal experimental procedures were conducted with the approval of the University of Newcastle Animal Care and Ethics Committee (ACEC). Seven experimentally naive female Wistar rats were obtained from the University of Newcastle animal house and mated in the University of Newcastle Psychology vivarium. Animals were maintained under normal housing conditions at 21–22°C, under a 12 h light/dark regime. At birth (PND 1), whole litters were randomly allocated into either LPS (4 litters; litter size M = 13, SD = 1.8) or saline-treated conditions (3 litters; litter size M = 15, SD = 0). As documented previously (Walker et al., 2004, 2006, 2009a,b, 2010; Sominsky et al., 2012a,b), on PNDs 3 and 5 pups were briefly removed from their home cages, weighed and administered intraperitoneally with either LPS (Salmonella enterica, serotype enteritidis; Sigma-Aldrich Chemical Co., USA, dissolved in sterile pyrogen-free saline, 0.05 mg/kg) or an equivolume of saline (Livingstone International, Australia). No significant differences in neonatal weight were observed when assessed on PND 3, PND 5, and PND 7 in the female offspring. We have previously reported neonatal LPS treatment to produce variable effects in regards to the neonatal weight gain, inducing weight loss (Walker et al., 2004; Sominsky et al., 2012a), weight gain (Walker et al., 2011) or no significant change (Walker et al., 2009a). Litter size and male-to-female ratio were not significant covariates to this analysis, confirming that these factors did not contribute to the possible developmental effects of litter.
On PND 7, female pups were euthanized by rapid decapitation. Ovaries were collected, frozen on dry ice and stored at −80°C until further analysis. For microarray and qRT-PCR analyses, ovaries were obtained from 5 animals per treatment group (derived from 2 LPS and 2 saline-treated litters). For western blotting analysis, ovaries were obtained from 4 animals per treatment group (derived from 2 LPS and 1 saline-treated litters). An additional subset of ovarian samples was used for the exploratory immunohistochemical analysis. Ovaries were obtained from 3 LPS and 3 saline-treated animals (derived from 2 LPS and 2 saline-treated litters) on PND14. This later time point was chosen to ensure a complete structural representation of the neonatal ovary, as at this age the ovary has been shown to contain antral follicles, which are completely absent prior to 12 days of age (Carson and Smith, 1986).
RNA was isolated from ovaries using RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA) in accordance with the manufacturer's instructions, with slight modifications. Briefly, ovaries were homogenized in lysis buffer (RNeasy buffer RLT) using a basic plastic homogenizer. The homogenized RNA was then passed five times through a 20 gauge needle. Lysate was centrifuged at 13,000 rpm for 3 min at 25°C. The supernatant was collected, 350 μl 70% ETOH added and mixed. The solution was then transferred to an RNeasy spin column and placed in a 2 ml collection tube. Following 20 s centrifugation at 10,000 rpm the flow through was discarded. Seven hundred micro liters of wash buffer (RNeasy buffer RW1) was added to the RNeasy spin column, centrifuged for 20 s at 10,000 rpm, following which the flow through was discarded. This step was repeated twice with 500 μl of RNeasy buffer RPE. RNeasy spin column was placed in a new 1.5 ml collection tube, add 30 μl RNase-free H2O were added directly to the column membrane and centrifuged for 1 min at 10,000 rpm to collect RNA. RNA concentrations were determined using spectrophotometer, NanoDrop 2000c (Thermo Fisher Scientific, Wilmington, DE USA) (refer to Table S1 for RNA quality information). The extracted RNA was used for microarray analysis and for verification by qRT-PCR.
Total RNA obtained from neonatal ovaries (1–2 μg) was submitted to the Australian Genome Research Facility (AGRF). Microarray analysis was performed using Agilent SurePrint G3 Rat GE 8x60K Array platform. Total RNA obtained from neonatal ovaries was quality ascertained on the Agilent Bioanalyser 2100 using the NanoChip protocol (Table S1). A total of 1 μg was labeled using the Ambion Total Prep RNA amplification kit (Applied Biosystems—AMIL1791). The quantity of amplified product was ascertained using the Agilent Bioanalyser 2100 using the NanoChip protocol. For the Agilent 8x60k chip format, 600 ng of amplified cRNA was used for the Cy3 coupling process using a ULS labeling kit (Kreatech EA-006). After coupling the dye, the reaction was cleaned using the KREApure columns in the kit. The Cy3 labeled cRNA samples were then fragmented using the Ambion fragmentation kit (Applied Biosystems—AM8740). The fragmented cRNA was quality checked on the Agilent Bioanalyser 2100 using the NanoChip protocol. The fragmented Cy3 labeled cRNA was then prepared for hybridization to the Agilent 8x60k Rat array using the GX hybridization kit (Agilent—5188-5242) where each hybridization reaction had a final volume of 50 μl. The Agilent hybridization chambers were prepared according to manufacturer's instructions. Each sample was loaded onto the Agilent hybridization gaskets slide which is placed into a hybridization chamber. The 8x60k Rat array was carefully lowered onto the gasket to create a sealed array for each sample. The hybridization chambers were then placed in a rotating hybridization oven at 65°C for 17 h. After hybridization, the chip was washed using the appropriate protocols as outlined in the Agilent manual using the GX washing buffers (Agilent—5188-5327). Upon completion of the washing, the chips were then scanned in the Agilent DNA Microarray Scanner (High Resolution). The scanner operating software, Agilent Scan Control, converts the signal on the array into a TIFF file which can be used for subsequent analysis. The experiment was conducted in three biological replicates, which consisted of ovarian tissue obtained from five animals per treatment group. Differentially expressed genes were determined by a twofold difference and a significance level of p < 0.05. Ingenuity Pathways Analysis (Ingenuity Systems, Redwood City, CA) software was used for further analysis to identify canonical signaling pathways affected by LPS treatment. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE46436 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE46436).
Reverse transcription was performed with 1 μg of total RNA using a SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen Corp., Carlsbad, CA, USA), according to manufacturers' instructions, by combining the components [5× VILO reaction mix (4 μ L), 10× SuperScript enzyme mix (2 μ L), RNA sample (up to 1 μg), diethyl pyrocarbonate (DEPEC) treated water (to 20 μ l)]. Tube contents were gently mixed, incubated at 25°C for 10 min, and then incubated at 42°C for 60 min. The reaction was terminated at 85°C at 5 min. qRT-PCR was performed using SYBR Green PCR Master Mix (Invitrogen, Carlsbad, CA, USA) on a 7500 RT-PCR Fast instrument (Applied Biosystems, Foster City, CA, USA). Primer sequences were designed using the Primer3 software (http://frodo.wi.mit.edu). Sequence specificity was tested using the Basic Local Alignment Search Tool at NCBI (Altschul et al., 1997) and primer pairs were obtained from Invitrogen (custom DNA oligonucleotide synthesis service). PCR efficiency for each pair of primers was determined by a standard curve method, where C(t) values for serial dilutions are related to the logarithm of the dilution factor, and the slope is a measure for reaction efficiency. Information regarding primer sequences, including product size and PCR efficiency, is listed in Table S2. The 25 μl PCR mixture consisted of 12.5 μl SYBR Green PCR Master Mix, 9.5 μl water and 2 μl of each primer was added to 1 μl of the cDNA template (10 ng/ml). All reactions were performed in duplicate under the following conditions: 95°C for 20 s and 40 cycles of 95°C for 3 s and 60°C for 30 s. Melting curve was determined under the following conditions: 95°C for 15 s, 60°C for 1 min, 95°C for 15 s and 60°C for 15 s. The data were normalized to an endogenous control, β-actin. A relative quantitative measure of the target gene expression compared with β-actin mRNA was analysed using the equation 2−ΔΔC(t), where C(t) is the threshold cycle at which fluorescence is first detected as statistically significant above background, and presented as a fold increase relative to the saline control.
Protein was extracted using RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8, 1% Triton X-100), supplemented with a protease inhibitor cocktail (ProteCEASE, G-Biosciences St. Louis, MO, USA). Protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Quantified aliquots were separated by electrophoresis and transferred onto a nitrocellulose Hybond C-Extra membrane (Amersham) prior to blocking for 1 h in 5% skim milk powder in TBST (0.1% Tween-20), as previously described (Sobinoff et al., 2010). After blocking, the membranes were incubated overnight at 4°C with goat polyclonal TLR4 (L-14) (diluted 1:200; Santa Cruz, sc-16240); goat polyclonal PKCβ1 (C-16) (diluted 1:500; Santa Cruz, sc-209); or rabbit polyclonal anti-JNK1 (diluted 1:2000; Abcam, ab10664) primary antibodies. Following washing and incubation with horseradish peroxidase-conjugated donkey anti-goat secondary antibody (Santa Cruz, sc-2020) at a 1:3000 dilution or goat anti-rabbit secondary antibody (Millipore DC03L) at a 1:2000 dilution for 1 h at room temperature, proteins were visualized using an ECL Detection Kit (GE Healthcare Life Sciences) according to manufacturer's instructions. The membranes were then stripped of primary and secondary antibodies in Western Re-Probe (G-Biosciences) according to the manufacturer's instructions at room temperature for 1 h and reprobed using a mouse monoclonal anti-α-tubulin (Sigma, T5168) as a loading control. The densities of TLR4, PKCβ1 and JNK1 bands were visualized using ImageQuant LAS 4000 imager (GE Healthcare Life Sciences). The protein bands were measured using MultiGauge V3.0 (Fuji, Stamford, CT, USA) software and were expressed as the ratio to α-tubulin.
Immunohistochemistry was used to localize TLR4, PKCβ1, and JNK1 proteins in the neonatal ovaries and for proliferating cell nuclear antigen (PCNA) detection of primordial follicle activation and growth. Ovaries were obtained from LPS and saline-treated animals on PND14. Tissue was fixed in Bouin's fixative solution for 4 h, washed four times in 70% ethanol dehydrated, embedded in paraffin and sectioned at 4 μm. Slides were dewaxed in xylene and rehydrated with subsequent washes in ethanol. Antigen retrieval was carried out by microwaving sections for 3 × 3 min in Na Citrate buffer (10 mM Sodium Citrate, pH 6) for TLR4, PKCβ1, and PCNA, and in Tris buffer (50 mM, pH 10.6) for JNK1. Slides were allowed to cool before being blocked in 3% BSA/Tris-buffered saline (TBS) for 1 h at room temperature. Sections were incubated overnight at 4°C with anti-TLR4 (diluted 1:100; Santa Cruz, sc-16240); anti-PKCβ1 (1:200, Santa Cruz, sc-209) and mouse monoclonal anti-PCNA (diluted 1:200; Merck KGaA, NA03T). For detection of anti-JNK1 (diluted 1:500, Abcam, ab10664) sections were incubated for 2 h at room temperature. The above primary antibodies were used for Western blotting prior to their application for the immunohistochemical analysis, ascertaining antibodies specificity (Kurien et al., 2011). Slides were then washed in TBS containing 0.1% Triton X-100, and incubated with the appropriate fluorescent-conjugated secondary antibodies (Alexa Fluor 594 donkey anti-goat IgG; Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG; Invitrogen; 1:200 dilution). Sections were then counterstained with 4#-6-diamidino-2-phenylindole (DAPI) for 2 min, mounted in Mowiol (4-88, Sigma), viewed using an Axio Imager A1 fluorescent microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY) under fluorescent optics and pictures taken using an Olympus DP70 microscope camera (Olympus America, Center Valley, PA).
Bouin's fixed sections were dewaxed and rehydrated as mentioned above. Slides were then boiled in Tris buffer (50 mM, pH 10.6) for 20 min and treated with 20 μg/ml Proteinase K for 15 min in a humidified chamber. TUNEL analysis was then performed as previously described (Sobinoff et al., 2010, 2011, 2012) using an In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics Pty Ltd), according to the manufacturer's instructions. Slides were then counterstained with DAPI for 5 min, mounted in Mowiol, and viewed using an Axio Imager A1 fluorescent microscope (Carl Zeiss) under fluorescent optics and pictures taken using an Olympus DP70 microscope camera (Olympus).
Statistical analyses were conducted using the Statistical Package for the Social Sciences for Windows, Version 18 (SPSS Inc.). Data were analysed using analyses of variances (ANOVA) design. The significance level was set at p ≤ 0.05.
Administration of LPS on PNDs 3 and 5 caused a significant change in ovarian gene expression for 712 genes, representing 2.4% of the total number of genes present on the array, with significant upregulation of 710 genes (Figure (Figure1;1; Table S3). Further functional analysis using Ingenuity Pathway Analysis software identified these significantly altered genes as components of several molecular networks implicated in cell signaling, immune cell trafficking, inflammatory response as well as reproductive development and disease (see Table Table1).1). These results suggest a robust impact of peripheral immune challenge on a variety of molecular mechanisms regulating ovarian development and function. No distinct function has been assigned to the two significantly downregulated genes, SBK2 and TEDDM1.
Further functional assessment of differentially regulated genes revealed several canonical pathways involved primarily in immune recognition and inflammation were activated in response to neonatal treatment [e.g., virus entry via endocytic pathways; nuclear factor-kappaB (NF-κB) activation; mitogen-activated protein kinase (MAPK) signaling; pattern-recognition receptors signaling; MSP-RON signaling, whereby macrophage-stimulating protein (MSP), acts through the RON receptor tyrosine kinase, and plays a regulatory role in the inflammatory response], suggesting dysregulation of inflammatory processes in response to peripheral LPS exposure may occur also locally, in the ovary (Figure (Figure2;2; Table S4). Figures FiguresA1A1–A7 provide a graphical representation of the significantly altered genes in the top canonical pathways.
LPS-stimulated MAPK signaling pathway was chosen for validation of microarray results, due to its specific relevance to the current model of LPS challenge. qRT-PCR analysis confirmed significant upregulation of 8 out of 9 genes that were identified as components of this pathway, in the ovaries of LPS-treated animals (Table (Table2).2). Of these genes, upregulation of MAPK8/JNK1 has been previously associated with oocyte dysfunction (Sobinoff et al., 2010); PKCβ has been reported to be involved in oocyte activation (Carbone and Tatone, 2009); PIK3C2A has been implicated in proliferation of ovarian theca-interstitial cells (Kwintkiewicz et al., 2006); PIK3R1 and PKCZ have been associated with the incidence of PCOS (Diamanti-Kandarakis, 2008; Kim et al., 2009; Rivero et al., 2012); PKD3 has been implicated in organogenesis (Ellwanger et al., 2008); RELA and RRAS2 have been associated with ovarian tumorigenesis (Chan et al., 1994; Niesporek et al., 2007; Fan and Richards, 2010); and finally, increased expression of TLR4 (receptor for LPS) has been demonstrated to underpin impaired follicular growth and function (Herath et al., 2007).
To further validate the microarray results, immunoblotting analysis was performed to examine protein expression of three significantly upregulated genes (TLR4; PKCβ; MAPK8/JNK1) in the LPS-stimulated MAPK signaling pathway. Densitometry analysis of protein expression in the ovaries of LPS-treated rats, as compared to α-tubulin, revealed a significant increase of two protein bands associated with TLR4 expression [F(1, 5) = 10.2; F(1, 5) = 17.06, p < 0.05 for both]. No significant differences were evident in the expression of PKCβ1, however, a trend was observed in increased expression of JNK1 protein in the ovaries of LPS-treated animals [F(1, 5) = 4.12, p = 0.09; see Figure Figure3B].3B]. Representative immunoblots are presented in Figure Figure3A3A.
To detect localization of TLR4, PKCβ1 JNK1, in the neonatal ovary, observational immunohistochemical staining was performed in the ovaries of LPS and saline-treated animals, obtained on PND14. As demonstrated in Figures Figures4A4A,,B,B, TLR4 immunolabeling was detected in the oocytes. In the ovaries of LPS-treated animals TLR4 was also detected in ovarian blood vessels, suggesting activation of local immune cells. PKCβ1 staining was evident in the ovarian blood vessels, to a greater extent in the ovaries of LPS-treated animals (Figures (Figures4C4C,,D).D). JNK1 immunolabeling was detected in theca cells, in both treated and control samples (Figures (Figures4E4E,,FF).
LPS and saline-treated ovaries were also probed for markers of follicular activation and cell death. PCNA staining was equally detected in the granulosa cells and the oocytes of primordial, primary and secondary follicles in both treatment and saline control group. TUNEL staining did not reveal any significant apoptosis in either of the experimental groups (data not shown).
In this study we assessed, for the first time, the effect of immune challenge by peripheral administration of LPS on the ovarian transcriptome in neonatal rats. Microarray analysis revealed specific activation of immune-related pathways, such as virus entry via endocytosis, NF-κB activation by viruses, role of pattern recognition receptors in recognition of bacteria and viruses, LPS-stimulated MAPK pathway and MSP-RON signaling pathway. Exposure to LPS upregulated expression of genes implicated in cell signaling, immune cell trafficking, inflammatory response as well as reproductive development and disease, suggesting dysregulation of inflammatory processes in response to peripheral LPS exposure may occur also locally in the ovary, contributing directly to programming of reproductive health.
Given the specific relevance of LPS-stimulated MAPK pathway to the model of neonatal LPS exposure, microarray results were confirmed and elaborated on the altered components of this pathway. Activation of MAPK signaling by LPS binding of TLR4 results in transcriptional regulation and synthesis of inflammatory agents, such as tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and IL-6, as well as nitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) (Nagano et al., 2002; Kawai and Akira, 2007). Other downstream targets include phosphoinositide-3-kinase (PI3K), which stimulation enhances activity of protein kinase C (PKC) (Frey et al., 2006). In the current study, several subunits of these genes, identified with the MAPK pathway, were upregulated in the ovaries of LPS-treated animals.
PI3K has been reported to induce activation of the serine/threonine kinase Akt, also known as protein kinase B (PKB), which in turn stimulates the mammalian target of rapamycin (mTOR) protein. Overactivation of PI3K/Akt/mTOR signaling pathway, induced by exposure to carcinogens and xenobiotics, has been implicated in premature activation of primordial follicles, leading to early follicular atresia (McLaughlin and Sobinoff, 2010; Sobinoff et al., 2010, 2011). Activation of PI3K/Akt has also been associated with the development of PCOS (Lima et al., 2006). While in the current study immunohistological assessment for markers of follicular activation and atresia revealed no significant alterations were induced by neonatal LPS exposure, our previous findings have indicated diminished population of primordial follicles in the late neonatal period in the ovaries of LPS-treated females (Sominsky et al., 2012a). These data suggest that apoptotic processes may occur early on, during the acute response to the LPS immune challenge, hindering follicular development.
The current model of neonatal LPS administration induces an acute pro-inflammatory response which involves the release of pro-inflammatory cytokines (i.e., TNF-α, IL-6, IL-1β), and this response typically resolves within 3–6 h from the time of drug administration, followed then by an anti-inflammatory release of glucocorticoids (Walker et al., 2004, 2009b). This brief activation of the immune response has been recently shown to impact on the development of the immune, reproductive, endocrine, metabolic, nervous and other physiological systems (Walker et al., 2010; Wu et al., 2011; Amath et al., 2012; Sominsky et al., 2012a, 2013). In the rat, the primordial follicle pool undergoes its final stages of formation and assembly postnatally, continuing till approximately day 3 after birth (Rajah et al., 1992; Skinner, 2005). This process is considered to be gonadotropin independent, supported and guided by growth factors and cytokines, one of which is the proinflammatory cytokine TNF-α. Previous research has demonstrated that ovarian TNF-α in the neonatal rat has an important role in determining the finite size of the primordial follicle pool. It has been demonstrated in vitro that TNF-α can decrease the number of oocytes and primordial follicles, via induction of apoptosis (Morrison and Marcinkiewicz, 2002). The formation of primordial follicles is then followed by the initial event of follicular development—transition to the primary follicle stage (Fortune et al., 2000; Skinner, 2005). As opposed to the later stages of follicular development, the transition from primordial to primary stage is hormone independent (Skinner, 2005). Exposure to LPS in adulthood has been also shown to induce follicular atresia in vivo in mice (Bromfield and Sheldon, 2013) and rats (Besnard et al., 2001) and in vitro in cattle (Bromfield and Sheldon, 2013). Interestingly, LPS-induced atresia is particularly detrimental to the population of primordial follicles (Bromfield and Sheldon, 2013), indicating that depletion of the primordial follicle pool during early development would subsequently impact the ovarian reserve and result in impaired fertility in later life. To our knowledge, no studies have assessed the gene expression of ovarian inflammatory pathways which were examined in the current study. While it is likely that similar changes would be evident in response to adult LPS exposure, the impact of such changes during early development, when the ovary has not yet morphologically and functionally developed, has significantly stronger implications for the growing follicle populations. It is therefore plausible to suggest that the neonatal LPS treatment instantly interferes with the delicate process of primordial follicle pool assembly and the subsequent activation of follicular development, through stimulation of an acute pro-inflammatory response. While this might be a transient perturbation, it ultimately leads to long lasting alterations in the size of the follicular pool, as observed in previous studies (Wu et al., 2011; Sominsky et al., 2012a). Even though no evidence of primordial follicle activation or apoptosis was evidenced in the current study, these processes might have occurred at an earlier time-point. Therefore, further investigation is required to clarify the time-course of apoptotic processes leading to the diminished follicular reserve reported in later life following neonatal exposure to LPS.
Activation and growth of primordial follicles are stimulated by several signaling pathways, including the MAPK and PKC (Jin et al., 2005; Du et al., 2012). The expression of several PKC isoforms (PKC α, β, δ, and ζ) was previously identified in the rat ovary (Cutler et al., 1994). In the neonatal rat ovary, MAPK, and PKC signaling cascade is involved in maturation of primordial follicles (Du et al., 2012). In the current study increased gene expression of MAPK8 and PKCβ in the ovaries of LPS-treated animals was determined by the microarray analysis and further confirmed by qRT-PCR. No significant changes in the expression of MAPK8 and PKCβ1 proteins were evident at the same time-point. However, significant increase in protein expression of TLR4 was found in the LPS-treated ovaries, and was associated with the increased mRNA levels of this gene.
TLR4, pathogen-associated molecular pattern recognition receptor, is expressed by innate immune and tissue specific cells, and recognizes bacterial molecules (e.g., LPS) (Medzhitov, 2001). While TLR4 activation is critical for generation of both innate and adaptive immune responses, its inappropriate activation can be harmful (Peroval et al., 2013). TLR4s are present in the ovary and expressed by ovarian surface epithelial cells, granulosa/cumulus cells (Herath et al., 2007; Liu et al., 2008; Richards et al., 2008), as well as by ovarian macrophages (Zhou et al., 2009), and play an essential role in regulation of fertility, through the support of ovulation and sperm capacitation (Liu et al., 2008; Shimada et al., 2008). Ovarian TLR4 signaling is normally induced by endogenous ligands, one of such is hyaluronan (HA)-rich matrix (Richards et al., 2008). Its synthesis during the ovulatory process is initiated by the surge of luteinising hormone (LH). HA-rich matrix is then recognized by cumulus cells, inducing inflammation and expression of innate immune-related genes, leading to the release of prostaglandins, TNF-α, IL-6 and other cytokines, and chemokines (Liu et al., 2008; Richards et al., 2008). These inflammatory agents activate chemokine receptors present on sperm, inducing sperm capacitation and motility (Shimada et al., 2008). Even though regulated production of cytokines is essential for ovulation and successful fertilization, dysregulation of cytokine production can impair fertility (Richards et al., 2008). In human patients, the existence of endometriosis was found to be associated with increased levels of IL-6 in circulation and in the follicular fluid, and is proposed to be related to the impaired follicular development and decreased oestradiol production, which results in infertility (Garrido et al., 2000; Pellicer et al., 2000). Activation of ovarian TLR4 by LPS has been studied in various animal and in vitro models. For instance, impaired antral follicle growth and function, as well as suppressed oestradiol production were reported in response to uterine E. coli infection, intravenous infusion with LPS and following exposure of granulosa cells to LPS in vitro, in cattle and sheep (Battaglia et al., 2000; Herath et al., 2007; Sheldon et al., 2009). A recent study in adult mice has demonstrated that LPS-induced atresia of primordial follicles is mediated via TLR4, since no follicle atresia was evident in TLR4-deficient mice (Bromfield and Sheldon, 2013).
The long-term effect of neonatal LPS exposure on TLR4 expression has been previously assessed in adult rats. Increased TLR4 mRNA expression was evident in the spleen and liver, two primary organs responsible for the synthesis of pro-inflammatory cytokines (Tracey, 2002), of neonatally-treated animals (Mouihate et al., 2010). In addition, increased expression of COX-2, the principal target of TLR4 activation, in the liver of LPS-treated animals was associated with a transient rise in circulating prostaglandin and increased activation of the HPA axis, in response to adult LPS exposure (Mouihate et al., 2010). These outcomes suggest that early life exposure to LPS has a persistent programming effect on the neuroimmune axis and this occurs via activation of TLR4. The current findings report that postnatal LPS challenge results in increased mRNA and protein expression of TLR4 in the neonatal ovary. In addition to its role in the LPS-stimulated MAPK pathway, TLR4 was associated with a number of inflammatory pathways activated in response to the neonatal treatment, as revealed by the canonical pathways analysis (i.e., role of pattern recognition receptors, MSP-RON and NF-κ B signaling pathways). Given the important regulatory role of TLR4 in reproductive function, activation of the receptor and the subsequent pro-inflammatory cascade in early life are most likely to have significant effects on ovarian development. Imbalance in the internal ovarian milieu may potentially interrupt the delicate process of follicular formation and growth, compromising reproductive capacity. Importantly, the process of primordial follicle assembly and development is highly conserved between mammals, and similar regulation of this process is considered to occur in different species, including humans (Skinner, 2005). Thus, while further studies are required to elucidate the immune mechanisms involved in the impaired ovarian development and functioning, our results importantly suggest that exposure to an immune challenge in early life, resulting in activation of TLR-related inflammatory pathways, may have significant consequences for programming of reproductive health in later life. Moreover, given that alterations in TLR4 expression are associated with pathological outcomes of common bacterial infections, such as Escherichia coli and Chlamydia trachomatis, including impaired fertility (Herath et al., 2009; Laisk et al., 2010), the current findings provide a valuable insight into the link between early life infection and fertility.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
A graphical representation of the top canonical pathways that were significantly upregulated by neonatal LPS treatment, as identified by Ingenuity Pathway Analysis. Red shading indicates up-regulation of the gene. No color indicates genes that form the pathway and have no change.
The Supplementary Material for this article can be found online at: http://www.frontiersin.org/Neuroendocrine_Science/10.3389/fnins.2013.00100/abstract
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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I want to write phrase "Capital of A is B, or I'm mistaken?", but I'm not sure that the second part of thus phrase is correct. Should I write "... or I've made a mistake?", or "... or I'm wrong?"? What is the best?
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As the question stands, the OP asks if (forms of) mistake can be used as other than a noun (Should I write "... or I've made a mistake?").
In that sense, the "Capital of A is B, or I'm mistaken?" is correct in the use of mistaken as an adjective.
Incidentally, minor errors of grammar need correction:
What you're looking for is "...if I'm not mistaken," but it's rather a cliché. "...if I remember correctly" is not much better. "...I believe" is a bit more honest.
Expressing a little doubt in these clichéd expressions actually indicates certainty: you know you're not mistaken, and it's a false modesty.

subset: commoncrawl


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This Just In: Robert Pattinson is Single!
We give up.
While the most recent string of reports made it sound like Robert Pattinson and Kristen Stewart were absolutely, 100%, undoubtedly a couple - one that is "all smiles" on the set of Eclipse - the actor himself is now shooting down those rumors.


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Football players from Utah colleges drafted to the NFL in the 1970s
Round 16, Pick 416
Dallas Cowboys
Gordon Longmire was drafted by the Dallas Cowboys but never played a game in the NFL.
- Oldest first
- Newest first
- Most recommended
This is an interesting article, but it would be more meaningful if you included what they're doing now. Some are productive in later life, some are not.
- 9:54 a.m. Nov. 23, 2012
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Ray Groth became a successful high school coach. He was the head coach at
Skyline, Granger, Highland and Tooele High Schools and was an assistant coach at
a few others.
Gifford Nielsen I know became a broadcaster for the Oilers.
But yes, a where are they now thing would be interesting...
- 11:03 a.m. Nov. 23, 2012
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Great article. It must have taken a lot of work. The other comments seem to be
focused on what the article doesn't offer. My primary comment is to say
that what the article does offer, is quite interesting. I'm not going to
take a gift like this and wish it were something more. An article about where
are they now, for 76 players, would have taken a prohibitively enormous amount
of work.
Thanks for the piece. I also think it is a great thing to spotlight players from times gone by. At one time, each of those players was a star at the college level, and brought a lot of happiness to the people in this state. I think it is interesting that a couple of the players made a comeback during the strick season in the late 80s. I love second chances.
Looking forward to the next pieces in this series. I expect Marc Wilson to be in there, in the 1980s draftees. It's too bad we don't get to see the draftees from the 1960s, to read again about Merlin Olsen, that never gets old. At least he got a mention as Phil's brother, in this piece.
- 1:09 p.m. Nov. 25, 2012
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A couple of players who were left off the list. Walt Love 1973 Westminster College drafted in the 10th round by the New York Giants. Played one year before injuries kept him from continuing. Len Walterscheid 1975 drafted by Chicago Bears played three seasons I believe.
- 8:51 a.m. Nov. 26, 2012
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Ike Spencer is a successful and well-loved Jr High School Principal teaching in the Salt lake Valley.
- 7:19 a.m. Aug. 16, 2013
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Dave Costa, who played at U of U, had a long and illustrious career in the NFL.
The list keeps growing! Good stuff.
- 5:22 p.m. June 17, 2016
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Thought of another...Halvor Hagen, who played at Weber State, then went on to play in the NFL for 5 or 6 years, I believe. Might be the first NFL player born in Norway...and wasn't a kicker!
- 3:43 p.m. July 6, 2016
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Storms Roll Through As Long Stretch Of Hot Weather Begins Taking TollThis string of hot weather is creating plenty of challenges.
Report: Le'Veon Bell Facing 4-Game Ban For Missed Drug TestBad news for Pittsburgh Steelers fans...
Overdose Epidemic Has State Cracking Down, Pharmacists Now Asking For IDIf you've been to the pharmacy lately, you were probably asked to show your driver's license.
Gov. Wolf: Access To Medical Marijuana Will Come Sooner Than LaterThe department of Health says it'll take until early 2018 to make medical marijuana available to eligible patients, but Gov. Tom Wolf said it won't take that long to get access.
Lengthy Rehab Project On Pittsburgh's Sister Bridges Is About To BeginA total rehabilitation of Pittsburgh's iconic bridges spanning from Downtown to the North Shore is about to begin.
Psychologist Wants People To Remember During Tough Times, 'They Are Blessed'Pittsburgh's own Dr. Kevin Elko joined the "KDKA Morning News" to discuss his new book and the current amount of anger that a lot of Americans seem to be feeling.
Survey Finds 80% Of Drivers Have Experienced Road Rage Over The Last YearA new survey by AAA estimates that 80 percent of drivers have taken part in some form of "road rage."
Police Cracking Down On Speeding In Construction ZonePennDOT says that frustration is resulting in dangerous speeds in a work zone on Route 65.
Local Boy Chosen To Dance On Stage With Justin BieberHe's one of the hottest acts in music, but Justin Bieber is still willing to share the stage.
Couple Refuses To Sit Near Police At Restaurant, Officers Pick Up Their TabIt is a difficult time for the country as nerves between some in the community and police are frayed, but recently, four local officers handled an uncomfortable situation without any confrontation.
Fan Guidelines Announced For Guns N' Roses Show At Heinz FieldGuns N' Roses' "Not In This Lifetime Tour" will rock Heinz Field Tuesday night. If you're going to the show, there are some things to keep in mind.
Pittsburgh Officials Preparing For Bicentennial CelebrationThere are all kinds of events planned for the Bicentennial this weekend, and at a time when security is at the top of everyone's mind, city officials are making preparations to keep people safe.


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Clinical-Style Validation, Regulation, and Quality Control...
Co-Sourcing Improves Pharmaceutical Laboratory Productivity
Keep the Opportunities, Lose the Opportunists
Loss of Lives in Clinical Trials...
Ultrahigh field magnetic resonance spectroscopy (MRS) is one of those new techniques that is yielding higher resolution images, higher signal-to-noise ratios, and, sometimes, greater speed.
"Ultrahigh fields for magnetic resonance are being used to target molecules other than water," explains Jullie Pan, M.D., Ph.D., associate professor of neurosurgery at Yale University. We can now image biochemically important molecules such as lactate, glutamate, and GABA with an imaging resolution that is informative and pertinent to living tissue. Ultrahigh field MRS gives researchers a new slant on what they are looking at, how aggressive a tumor is, whether it's necrotic or proliferative, et cetera," she elaborates.
Her lab has had success using ultrahigh field MRS to study brain tumors and epilepsy. "The latter condition works with patients who are mainly MRI-negative but who are clearly suffering from a focal brain disease," she notes.
"The developments and implementation of ultrahigh field MRS imaging opens a distinctively functional and metabolic avenue to imaging the brain, with resolution comparable to that of a PET scan without the issue of radioactivity, which limits repeatability. There also is long-standing research interest in using MRS imaging to study muscle, liver, prostate, and other cancers."
In the 1990s, Dr. Pan says, typical field strength for human imaging was 1.5 Tesla. In fact, "whether 1.5 Tesla was the optimum field strength was being debated." Now, the preferred clinical imaging field strength for the human brain is 3 Tesla. However, at higher field strengths, "the size of the human body is closer to the wavelength of the radiofrequency signal at 7 Tesla," so the detector technology to optimally generate and detect the signals has had to evolve.
"For even higher magnetic fields such as 9.4 and 11.7 Tesla (which exist for human application), this area of work will continue. It's a long road." Nonetheless, while technical difficulties of detectors, homogeneity, and relaxation are major challenges, effective approaches are being developed, opening the way to take advantage of the ultrahigh field's higher signal-to-noise ratio for imaging of anatomy and biochemistry in human study.
At the A. I. Virtanen Institute, University of Eastern Finland, Olli Gröhn, Ph.D., professor of biomedical NMR, is investigating the application of spin-lock MRI contrast in vivo. "T1p, one of the spin-lock MRI contrasts, is currently used mainly for cartilage MRI. However, spin-lock contrasts seem to be more sensitive to tissue changes associated with cell death than conventional MRI."
During the next several years, he believes that applications of spin-lock MRI will expand to more neurological applications and are likely to include imaging for Parkinson disease and stroke as well as monitoring tumor treatment responses, "especially glioma gene therapy," he predicts. Already, researchers have successfully demonstrated the utility of spin-lock MRI contrast in these applications in clinical settings.
In earlier work, Dr. Gröhn and colleagues found ways to adjust the sensitivity of that technique. While investigating tissue changes associated with hyperacute stroke, he and researchers from the University of Eastern Finland (formerly the University of Kuopio), the University of Minnesota, Kuopio University Hospital in Finland, and Dartmouth Medical School, determined that the sensitivity of spin-lock MRI contrast could be modified by varying the spin-lock preparation blocks.
Traditionally, acute ischemia lesions have been predicted by their diffusion-perfusion mismatch. Dr. Gröhn and his collaborators, however, determined that using longitudinal relaxation time in the rotating frame obtained by on-resonance continuous wave spin-lock MRI "predicts the tissue outcome in the acute phase when therapies based on clot removal and/or neuroprotection are most likely to be beneficial."
The limiting factor for the spin-lock MRI contrast technique seems to be the high specific absorption rate (SAR) of energy, which may cause tissue heating. This is a particular concern when tissues within the brain are involved, he points out. That, therefore, is why clinical applications have so far concentrated on monitoring conditions outside the brain.
"Areas outside the brain are easier, as SAR limits are higher," Dr. Gröhn says. Despite that limitation, he and other researchers have used spin-lock techniques successfully for human brain imaging, allowing researchers to assess slow molecular motions that have an extended range of correlation times.
For more information on in vivo imaging, check out GEN's on-demand webinar entitled Applying Molecular Imaging in Drug Discovery and Development. The presentation explores how in vivo molecular imaging using multiple techniques can facilitate identification of novel therapeutics early in the drug discovery and development cycle. Featured speakers include imaging experts from Caliper Life Sciences, Millennium, and Abbott Laboratories. Listen to the archived webinar by visiting: www.genengnews.com/molecularimaging.
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MDMA (commonly known as the empathogen "ecstasy") is classified as a Schedule 1 drug, which is reserved for compounds with no accepted medical use and a high abuse potential. Two researchers from Stanford, however, call for a rigorous scientific exploration of MDMA's effects to identify precisely how the drug works, the data from which could be used to develop therapeutic compounds.Do you agree that ecstasy should be studied for its potential therapeutic benefits?
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text: Politics 21 January 2013 Watch: Obama's second inauguration Watch President Obama get sworn in for a second term. Print HTML › The YSL catwalk: it's not just women's fashion that has a problem with extreme thinness A vendor sells newspapers at the 57th Presidential Inauguration. Photograph: Getty Images Subscribe More Related articles France is changing: an army stalks the streets and Boris Johnson wanders the Tuileries The investigation into Australia's "Abu Ghraib" could neglect wider abuses in the Northern Territory Watch: Lame Bernie brocialists boo mentions of Hillary Clinton as they'd rather have Donald Trump for President


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YDM,If you anticipate that SLW is going parabolic in the next few days, do you intend to get right back into the front of the investment bus? - Just wondering.Cheers!
got to like this stock. bought some a few weeks ago at 15.34, thought it was stuck in the mud but now its on a roll.
My objectives are different than most. I have to live on this money right now.So I have to invest in flyer's with a bond investor mentality, hence my touting covered calls all the time.I may daytrade a portion of my stash but I don't feel comfy more than a few minutes at a time. If you're looking for Max Pain, the March call and put total is so close across three strike prices, I don't know where the smart money is anymore.Somebody is loading up in anticipation of option expiration and end of month investing. Up 3% in a no news, silver flatish day, I don't trust the obvious anomolies I can ascertain, so I will be nimble because it is easy to get crushed $1.50 in nothing flat, as we didn't "earn" the half buck yesterday so who knows what forces are at work.The market looks down today, overseas taking a fresh look at Greece for some reason it ignores every other day, and silver is flagging with overseas markets.CPI will provide fresh insight into inflation facing joesixpac, it needs to be a goldilocks number in order to continue the make believe rally.Tomorrow is quadruple witching, the original "March madness."I'll know more about today when the CPI comes out and premarket reacts. If at all, just toes in the water for me the best of times.
http://stockcharts.com/h-sc/ui?s=SLW:SLV&p=D&yr=0&mn=6&dy=0&id=p11763974812this chart shows the performance of slw relative to silver, which is a pretty decent leading indicator of the slw stock price. The ratio nearly broke down into a downtrend with the 10 ema falling to the 20 and the 50 ema, but it just bounced up off that conjuntion and is heading up, with plenty of room to run on the stocastics, and a trend strength as denoted by the adx, which is currently weak but getting stronger. Also the money flow relative to slv, has been very negative but its now trending in the right direction. So all and all things look pretty good for this stock and I am in accumulation mode.


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Product DescriptionCome into a very exciting entrance accented with our tissue red rope stanchion decorations. Accented with gold ends our tissue stanchion will stretch to 8 feet. Our red tissue stanchion is sold by the piece. Please order in increments of 1 piece.
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- I would buy this product again and again Review by Vicky the Merma
I love them ropes, beautiful color sturdy and good quality.
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- ok Review by JC
One of these was in good working order, the other had 4-6 flaws (where the tissue didnt attach correctly). We taped it up to make it work, but it certainly didnt look as good.
(Posted on 11/8/11)
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Just how far into the past do you personally go in coding support for legacy web browsers?
Personally, I code only as far back as about 5 years or so, particularly when it comes to Microsoft products. For example I am tending now to drop support for I.E. 8 and earlier and I tend to put something on the web page that says something along the lines of "You have a crappy old web-browser, upgrade it already!!"
Nevertheless, I still get emails to admin along the lines of "Your website doesn't work on my steam-powered computer running IE 1.0!"
I believe that trying to support everything since Noah's Ark was built is a waste of time. For example, if I went in to my local motor spares store and asked them if they had a windscreen wiper for a Model T Ford, they would probably laugh at me! Same philosophy goes for software.


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Men's Health
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Even if you've put off going to the doctor for your annual checkup, don't wait to get a screening test for the most common cancers that affect men -- colorectal, prostate, lung, and skin. They're easier to treat when you catch them early. Your doctor may recommend one of these tests, or a combo: ColRead Full Article
Will Testosterone Rev You Up or Stall You Out?
Testosterone replacement therapy may not be the fountain of youth for aging men. A large government-funded study published in the New England Journal of Medicine in February reports that testosterone, touted for its ability to jump-start libido, did little to improve a man's abilities in the bedroomRead Full Article
Fertility Facts for Men
Guys, don't take fertility for granted. If you want to father children, you need to pay attention to your own health, especially as you get older. "Your age, the foods you eat, the medications you take, and any medical issues you may have: All can affect the way your sperm function," says Bruce GilbRead Full Article
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It happens to a lot of guys, but few of them want to talk about it -- especially when "it" is a low libido. After all, virility plays a big role in our concept of manhood. There's this idea you're supposed to live up to: "Real men are always in the mood." But that's not true. Lots of men have low seRead Full Article
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It's the hormone that helps turn boys into men. During puberty, it brings about facial hair, a deeper voice, and bigger muscles. In grown men, it helps to control sex drive and make sperm. When your testosterone levels dip below what they should be, you can have problems with your sex drive and abilRead Full Article
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NEW SERVICE SPOTLIGHT: Sloan Fixtures Added To ATS Spec Service | Facility Executive - Creating Intelligent Buildings
Specs on the manufacturer's vitreous china fixtures are now available to users of Allied Technical Services
NEW SERVICE SPOTLIGHT: Sloan Fixtures Added To ATS Spec Service
Sloan, a manufacturer of commercial plumbing systems, and Allied Technical Services (ATS), a plumbing specification information system, have announced that Sloan's vitreous china fixtures are now included in the ATS specification services system.
The no-cost ATS specification service enables users to compile and review various combinations of commercial plumbing systems—with images, written specs, design details, and Revit files.
The inclusion of Sloan vitreous china fixtures on the ATS Spec U.S. website provides a broader product offering to facility managers and other specifiers who are working on plumbing building projects. Other Sloan products such as electronic faucets, flushometers, and sink systems are already part of the ATS service. In addition, through ATS and through Sloan's website, access to BIM models of Sloan's product lines is now available.
The vitreous china fixture line from Sloan includes:
High-Efficiency Toilets
High-Efficiency Urinals (seen here)
Bedpan Lug Bowls
Gravity Series Tank Toilets
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Topic Tags: construction-and-renovation, Plumbing, restrooms, Sloan
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Very cool. Just logged in and pretty easy. All of the fixtures and components were there, complete spec downloaded, and all eequired revit to match. I hope this site stays free to use. So far – as advertised.


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text: my names brandon, and im an alien. after flying around the universe on my unicorn for 1400 years i landed on earth, in a magical town called lubbock texas. i told someone about my unicorn that left me for a horse as soon as i got here. anyway they said i was crazy. Ive been battling against people who tell better lies than that in the psychology field ever since. a creative mind is not a disease and that's what the band shortbuSs is all about. in college i met my best friend clarence hes just as "crazy" as me. over the years we mastered many musical insturments and would love to play them for you.


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94 740i Transmission or possibly engine stuttering
I'm not sure how to explain this, so I'll just describe the problem.
I've got a German spec 1994 740i with an automatic transmission. Actually we just got it and I want to make sure everything is fine with the car.
The problem is when I initially start off to drive when I've just selected "D" from the shifter. If I press lightly on the gas pedal, for a smooth drive-off, the engine or transmission stutters. If I press a little harder, it drives fine. I don't understand it honestly. The dealer told me this was an "engine brake" and that it helps keep the car from rolling on inclines if you take your foot off the brakes.
Honestly, this is the only problem that I've found - other than the interior dash board lights aren't working, likely due to a blown fuse. If someone can tell me where, and which, fuse I should replace I'd appreciate it. The car runs fine other than that. Engine sounds great. The transmission doesn't make any noise and seems to transition through the gears just fine. Only when making the initial start, from a stop light for instance, does this problem happen (if it is a problem). If this isn't a problem, is there some way to disable it? It's a bit annoying honestly.
No tranny-error lights coming on at all..
94 740i Transmission or possibly engine stuttering
Hi Matt,
first of all welcome here in the E32 community. There are some other boards also specialized on the E32. Use my website below for information, there is also a download for the onwers manual and other tips and tricks.
click here
Wiring diagrams are also the based on production year.
"The problem is when I initially start off to drive when I've just selected "D" from the shifter. If I press lightly on the gas pedal, for a smooth drive-off, the engine or transmission stutters. If I press a little harder, it drives fine. I don't understand it honestly. The dealer told me this was an "engine brake" and that it helps keep the car from rolling on inclines if you take your foot off the brakes."
that is not normal. Maybe you clean the idle control valve and look for airleaks in the intake system.
"other than the interior dash board lights aren't working, likely due to a blown fuse. If someone can tell me where, and which, fuse I should replace I'd appreciate it"
Fuses are under the hood and under rear seat driver side (LHD). Use the correct wiring diagram and trobeshooting manual as listed on my website below, usually from around page 15 all relais and fuses are explained in detail, for what they are and where they are.
Under the cover of the fuse box in the engine room there is also a sticker explaining the fuses.
If only some do not work, replace them, info on my websote how to remove the steering wheel and the cluster.
Use online shops for parts such as www.autohausaz.com
much cheaper.
Another reason for the dashlight not on could be the dimmer switch. Did you turn that one down? Check that first before you start disassembling other things.
Study the owners manual in detail, buy a Bentley repair Manual for the E32, helps to save money.
Visit the other E32 boards as shown on my website, good info everywhere.
Shogun's E32 Tech Tips: http://tridem.han-solo.net/auto/erich/auto.htm
Re: 94 740i Transmission or possibly engine stutte
Thanks, that's great information.
I did get the dash lights working - the number 5 fuse was blown. Replaced with a new one and I was back in business.
I'll also look into this stuttering problem now that I know it's not normal.
I appreciate your help!!
Re: 94 740i Transmission or possibly engine stutte
Today, actually about a half hour ago, I finished cleaning the Mass Air Sensor, hoping this would have some positive result, but sadly none. I read several posts from other BMW owner's who said they had positive gains.
I guess my next option is to clean the idle control valve as mentioned above, but my car idles just fine, so I have my doubts.
Basically if I lightly put my foot on the gas pedal, and hold it, the engine needs to reach about 1500rpm before the car starts to move, which is a pronounced "stuttering". If I mash down on the pedal harder, then there seems to be a slight hesitation, but no real stuttering.
I'm beginning to wonder if this is a torque converter problem - the car shifts between all the gears just fine, and I don't notice any other problems after the initial start-off.
I think I'll replace all the spark plugs as well, just to eliminate the possibility.
Any other thoughts?
Re: 94 740i Transmission or possibly engine stutte
Just to update - I also found that selecting winter mode on the transmission doesn't show any of the problems that I have just using economy mode. Winter mode bypasses 1st gear, essentially making starts from 2nd gear instead.
Makes me think this problem is directly related to the transmission. I also took it to my local Stuttgart BMW dealer and asked a service tech to drive it. He also suggested the car has transmission problems, but the engine was fine. He didn't charge me either, which was surprising. Probably thought I was some broke bloat since I was driving a 13 year old car. :)
The car has been on the diagnostic computer recently as well, but nothing reported on the tranny; guessing because it's still working and hasn't actually failed.
ever changed transmission fluid and filter?
and checked the fluid level? That is not that easy on the 5HP30 tranny, as the filling and overflow is from bottom and the fluid must exactly have the correct temperature. Go to my website below, there is a link to Europeantransmissions.com, there have the detailed info on checking and refilling etc.Shogun's E32 Tech Tips: http://tridem.han-solo.net/auto/erich/auto.htm
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If You're Thinking of Living In/Lido Beach, L.I.;Bay, Ocean, Private Homes and Condos
By VIVIEN KELLERMAN
Published: July 14, 1996
LIDO BEACH in the 50's and 60's was synonymous with private beach clubs. There were five south of Lido Boulevard -- the Colony Club, the Coral Reef, the Monaco, the Sands and the Shelborne. They drew thousands of day-trippers who arrived early to use the cabanas, beaches, pools and courts and stayed late for the restaurants and nightclubs with big-name entertainers.
By the 60's, as people left the city for the suburbs, the private beach clubs on the Atlantic Ocean began to lose their popularity. They were replaced by condominiums slotted along a string of public parks and beaches.
The rest of Lido Beach, which sits between the ocean and Middle Bay, is a series of small neighborhoods mostly developed in the 50's and 60's. Lido Beach is on Long Beach Island, which stretches 10 miles from Atlantic Beach to Point Lookout.
"Lido Beach is a little island off the United States," said David Cohen owner of Century 21 Kaye Realty in nearby Long Beach. "It's probably the best-kept secret around."
Mr. Cohen said prices for single-family homes ranged from $250,000 to $1 million, with the Dunes, an area of about 300 houses on a private oceanfront beach, the most prestigious.
Home styles in the Dunes are eclectic and include Cape Cods, updated split levels and large contemporary homes on parcels that average 60 by 100 feet. There is no parking on the streets and photo identification is required to enter the guarded beach.
Even undeveloped land in this area is expensive. One 8,000-square-foot oceanfront lot is on the market for $800,000. About 40 percent of the houses in the Dunes are second homes, Mr. Cohen said.
The Dunes Civic Association has a membership of 140 families. A fee of $15 a year generally pays for events like a beach party at the end of the summer and an annual Halloween Party.
One of the condominium complexes on the ocean is Lido Beach Towers, a 184-unit gated community that was once the Lido Beach Hotel. It has a private beach, tennis courts, clubhouse, health club, billiard room and whirlpool. Prices range from $150,000 for a studio with no ocean view to $500,000 for an oceanfront duplex.
Another condominium, the 223-unit Lido Towne House, is a converted garden-apartment complex with a private beach and pool. Prices range from $125,000 for a one-bedroom apartment to $175,000 for two bedrooms. In general, oceanfront apartments are about $25,000 more.
On the bay side of Lido Beach is a collection of Cape Cods, split levels, high ranches and colonials priced from $250,000 to $350,000, with some homes directly on the bay going for as much as $900,000.
There are no rental apartments, but Patricia McDonald, owner of Lido Beach Realty, said the summer rental market for single-family houses was strong. Rents range from $7,000 to $10,000 a month.
MR. COHEN said that, as in many waterfront communities, obtaining property insurance had become an "ongoing battle." But, he said, everyone is getting insurance, even if the deductible is high. "People around here just know not to put in a claim for the small stuff," he said.
Stan Zimiles, an agent in Mr. Cohen's office, said that for a $400,000 home, full property insurance runs about $1,900 a year.
Stephanie and Mitchell Kaufman moved to the Dunes six years ago from Boca Raton, Fla. The couple, who have a son, Jared, 6, and a daughter, Blake, 3, built a four-bedroom, two-and-a-half-bath contemporary home with an ocean view. Because Mrs. Kaufman misses her family in Florida, the couple plan to move and have put the house on the market, for $665,000. Selling the house, she said, is the hardest thing she has had to do.
"People don't believe they can live like this on a private beach without having to go to the Hamptons," she said.
Lido Beach students attend public schools in Long Beach, a city eight minutes away. Long Beach has one pre-kindergarten center, four elementary schools from kindergarten through the fifth grade, one middle school and a high school, with a total enrollment of 4,600 students. The district has an alternative high school for students in danger of dropping out. Seventy students are enrolled in the program. The pre-kindergarten center, one elementary school, the middle school and the high school are in Lido Beach. The two-story modernistic high school is on prime bayfront property.
Superintendent Elliot Landon said that in 88 percent of the last year's graduates went on to higher education. One student, who created a computerized device to help the blind identify currency, was a winner in the Westinghouse Science Competition. This year the district had a Westinghouse semifinalist. Mr. Landon said enrollment in the district had been increasing about 3 percent a year for the last three years. Projections are that by 2005, enrollment should reach 5,000, he said. Three years ago, the district issued $10 million in bonds to expand. By this September, 12 classrooms and a second gymnasium will have been added to the elementary buildings.
John and Leslie Doody moved to the bay side of Lido Beach when Mr. Doody retired from his home-center store. Mrs. Doody, who had lived in and around the area most of her life, said she and her husband had not actually been looking for a house when they saw the for-sale sign on a "lovely" waterfront house.


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Star Wars Jedi Knight: Dark Forces II User Reviews
a GREAT game with Kyle KatarnThe good:
1. Great story.
2. All of the worlds had their own theme kind of thing.
3. Kept me playing for hours at a time.
1. The graphics weren't very good. Lot's of Pixelation. But it is an old game....
2. Cheesy cutscenes.
GREAT game. it had an awsome storyline, and it was easy to play. i mean it didn't take me long to learn the controls or the story. this is overall one of the best games i've ever played. i would really recommend this game to a star wars person.
i liked how it takes a while to beat the game. i like long games, and this one took me a while. it was also challenging. the 7 dark jedi, except yun, gave me a big challenge. i was really happy with level 6 and 13. 6 had a big tower, which was pretty cool, and 13 was in a big valley.
Star Wars Jedi Knight: Dark Forces 2The good:
The game has a very good selection of weapons and powers, and its levels are challenging enough.The bad:
The graphics are a little bad.Summary:
The graphics are a little off and when I have been playing a while it will get a little distorted, ex. my lightsaber lit up a different color than it should've been. I have also noticed that it is not very often that you get force stars, I am on the 5th level and only have two stars one of which I have already used on by one force power, "Speed".
The game is great I was just wondering if it were possible to get more stars at where I already am.
JK is the best man!!!The good:
This is brilliant!!!Summary:
Jedi Knight is the story after the sceond death star is destroyed, I a new dark jedi, Jerec, and his other six dark jedi have taken over the empire and are after the lost valley of the Jedi. You must stop them!!! This game includes an exallent story, clips that'll drive you mad! Multiplayer levels that will keep you fit! And brillant gameplay, the graphics depend on your pc, so they can also become ver, very good!!!
I have spent over two years free time on this game and now I am pro at it. If you every want to give me a battle, I am sometimes in the MSN gaming zone. My nick is TheShrimpKing!...
may the force be with youThe good:
-you choose which side of the force your on
-you get to configue the force powers
-controls are wacked(like the way you look with the mouse. down is up and up is down...)Summary:
this is a reall fun game. the graphics are a bit weak but the game is still fun. the great part of it is you can use the force you chock and move objects.
if your a star wars person this is a game for you.
Dark or Light?The good:
All of it, apart from the fact that it's on the boffy PC.The bad:
The boffy PC bit.Summary:
Which do you prefer: the Dark Side, or the Light Side? Choose in Jedi Knight: Dark Forces 2 whether to be an agent of evil or a true Jedi. And, if anyone was thinking of going Light, don't. Dark Side powers are better and you get to keep your own lightsabre, plus you loose that idiot ghost of Rahn.
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Published: October 11, 2003
ORANGE JUICE DROPS. -- Orange juice fell on a report that orange production in Florida will rise 24 percent. In New York, frozen concentrated orange juice for November delivery fell 5.15 cents, or 6.8 percent, to 70.45 cents a pound.
Graph tracks Orange Juice prices (near-month contract), since April 2002. (Source: Bloomberg Financial Markets). It also lists the 52-week highs and lows of selected commodities, along with their closes yesterday and their changes, yesterday, year to date, and over the past 52 weeks.


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"Carpool Karaoke" is arguably one of best running gags currently airing on late night television. Thank God, the first installment of the popular Late Late Night Show segment wasn't a sign of things to come.
Who signs up to participate for a new skit called "Carpool Karaoke" but refuses to sing, you ask? Only a diva like Mariah Carey would, that's who. James Corden reveals to Howard Stern that Mimi refused to sing during his talk show bit. "She came out and said, 'I'm not singing today.' And she went, 'We'll just drive around and have a chat." Hilarious!!!
Even though she never fully committed, at least the "#Beautiful" singer played along for a bit and sang a few bars. Thankfully, Jennifer Hudson was booked as the next guest and showed how amazing the segment could be. Til this day, watching her singing her order at a fast food drive thru still cracks me up.
Listen to Corden's interview below and learn more behind-the-scenes drama about launching "Carpool Karaoke."
For Mariah Carey, throwing shade is like breathing. I honestly do think she sometimes doesn't even know she's doing it. I can relate. I've often at times been accused of doing the same.
While visiting Andy Cohen on Watch What Happens Live last night, once again, Mimi was asked about that infamous "I don't know her" diss aimed at Jennifer Lopez. When Cohen asked if they knew each other, Carey replied "You know what? I'm very forgetful. Apparently, apparently, I'm forgetful."
She implies the two divas have never had a long conversation with each other and have only had a brief introductory meeting. Carey could've easily squashed all the beef when Andy asked her if she thought J. Lo was 'cool.' Apparently, she wasn't in the mood to extend an olive branch. Watch the video below to see how she answers that.
"You guys are living for this footage," says Mariah Carey in the clip. And you know what? The 46-year-old diva just might be right.
I wasn't very optimistic when E! first announced Carey's foray into reality. I was a little worried we were on the brink of another Whitney Houston in Being Bobby Brown or Britney and Kevin: Chaotic. That doesn't appear to be the case. Mariah's World looks like a fun, superficial and glamorous romp. Exactly what I want from a pop diva.
"I'm wearing these glasses because we're in fluorescent lighting, and I have a rule which states that I will not be seen in fluorescent lighting without sunglasses. I know it's very '90s," declares in the trailer.
The eight-part docuseries follows Carey as she prepares for her Sweet Sweet Fantasy European tour and upcoming wedding to fiancé James Packer. No premiere date has been revealed, but it's expected to debut later this year. Check out the first trailer for Mariah's World below.
Rihanna has 14 No. 1 singles to her credit. Cumulatively, they've spent a whopping 60 weeks in pole position. Thanks to "Work" logging another week at the summit of the Billboard Hot 100, the 28-year-old pop superstar now surpasses The Beatles for the second-most weeks at No. 1 all time. Only Mariah Carey stands in her way for chart supremacy. Mimi has 79 weeks under her designer glittery belt.
With "Work" ruling the chart for nine consecutive weeks at No. 1, the Drake-assisted smash is only one week away from tying "We Found Love" as her longest-running chart-topper. It has stiff competition on next week's chart from Desiigner's "Panda" which pounces from 5-2 this week. It'll be a tight battle for sure.
Justin Bieber also achieves a milestone on this week's chart. "Love Yourself" breaks the record for the most consecutive weeks logged in the top 10 from a song's debut with 22 weeks, passing his two prior singles from his album Purpose. "Sorry" and "What Do You Mean?" both spent 21 weeks in the Top 10.
Check out this week's Top 10 chart below.
Everything is definitely awesome today. The first teaser trailer for the highly-anticipated LEGO Batman Movie just arrived and it looks fantastic.
Will Arnett returns to voice the Lego Caped Crusader. Joining him for his latest adventure are Rosario Dawson, Ralph Fiennes, Zach Galifianakis, Michael Cera and Mariah Carey. Are you kidding me? Mimi is in this. I can't wait.
The Lego Batman Movie Synopsis
In the irreverent spirit of fun that made "The LEGO® Movie" a worldwide phenomenon, the self-described leading man of that ensemble – LEGO Batman – stars in his own big-screen adventure. But there are big changes brewing in Gotham, and if he wants to save the city from The Joker's hostile takeover, Batman may have to drop the lone vigilante thing, try to work with others and maybe, just maybe, learn to lighten up.
The LEGO Batman Movie rides into theaters on February 10, 2017. Watch below to see what's brewing in Gotham City.
What is Mariah Carey thinking? Someone of her stature should not be doing this. Both Whitney Houston and Britney Spears' foray into reality didn't exactly help out their respective careers. Fingers crossed Mimi has better luck.
Mariah's World is an eight-episode docu-series that will give her loyal Lambs VIP access to her world like never before. It'll follow her as she heads to the UK, Europe and Africa for her Sweet Sweet Fantasy Tour.
When not on stage, viewers will see Carey planning her upcoming nuptials to Australian mogul James Packer. Hopefully, her inner bridezilla doesn't come out. To be fair, this will be her third marriage. She's probably a pro by now. Read Carey's statement below.
London-based house DJ Sigala interpolates "Always Be My Baby" by Mariah Carey for his current single "Say You Do." If you haven't checked out the tropical house jam, definitely do. It's perfect to listen to while lounging poolside.
During a stop on BBC Radio's Live 1 Lounge, Katy B starts off covering Sigala's single before transitioning into a soulful rendition of the Carey classic. The 26-year-old songbird does the diva justice, even showing off some full-on diva arm movements. Check it out below.
It doesn't take a rocket scientist to know that Ben Affleck's new phoenix tattoo is beyond douchey. Jennifer Garner recently weighed in to Vanity Fair about her ex's new ink.
"You know what we would say in my hometown about that? 'Bless his heart.' A phoenix rising from the ashes. Am I the ashes in this scenario?" she said, later adding, "I take umbrage. I refuse to be the ashes."
The other Jennifer in Affleck's past also shared her thoughts on his back tat. During J. Lo's appearance on Watch What Happens Live, the American Idol judge played a round of "Plead The Fifth." Given that it was her third time playing the game, the 46-year-old superstar should've known not to pass on the first question. Really, she couldn't choose between her Idol colleagues who to Shag, Marry or Kill?
Naturally, Cohen had a couple of scandalous questions for the next two. He asked Lopez about Affleck's tattoo and why Jennifer was texting during Mariah Carey's Billboard Music Awards performance. Is there or isn't there a feud? Check out her response to both questions below.


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