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Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency.
To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-γ, MIF, TGF-β1 and TNF-α mRNA. We showed that the beta-2 microglobulin (β2-MG) gene was suitable for data normalisation since the level of β2-MG transcripts in naïve PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-β1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-β1 mRNA in response to LPS.
The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications.
Cytokines are a family of low-molecular weight proteins secreted by various cell types, with pleiotropic functions and constitute a tightly regulated network that plays a central role in the immune system. Cytokines, classified into different groups such as interleukins (IL), interferons (IFN), colony-stimulating factors (CSF), tumour necrosis factors (TNF), tumour growth factors (TGF) and chemokines are implicated in the differentiation, proliferation, migration and effector functions of immune cells. Interacting one with the others, they have polarizing effects on the target cells and are pivotal in tuning immune responses [1]. Therefore, it is rather the make-up of cytokines milieu that influences the immune response rather than the action of a single cytokine. Numerous studies indicate that the clinical and/or immunological status depends on the balance between pro-inflammatory cytokines and their regulatory counterparts [2]. Thus, cytokine profiling should be achieved through analysis of simultaneous quantification of a pattern of cytokines including pro and anti-inflammatory cytokines [2,3]. Moreover, recent reports have highlighted the need for clinical immuno-monitoring of patients to adapt treatment or prevent relapses [4-6]. Thus, analysis of the cytokine pattern is central not only in the definition of the immunological status of patients but also in the study of the pathophysiological pathways as well as the cellular subpopulations involved [7,8].
Cytokines are often produced locally so that the concentration of circulating cytokines in the plasma is usually low. Their half-life and turnover may vary complicating the delineation of informative cytokine profiles. Although transcription of messenger RNA is not strictly correlated to protein secretion and activity, detection of cytokine RNA by real time PCR is now considered a reference technique for analysis of small-size samples with high sensitivity [9]. It can be used on its own or to validate and complement information obtained with other techniques such as micro-arrays [10,11].
The already available techniques, which offer a so-called "absolute quantification" of the target cytokine mRNA, achieve quantification by reference to an external standard curve based on serial dilutions of a known amount of the corresponding cDNA [12]. Moreover, to allow for comparison between experiments, data are normalized by reference to an internal standard, which is an endogenous gene for which the number of copy per cell is supposed constant under different experimental conditions [13,14]. The term of "absolute" quantification is not completely appropriate since these techniques neither control for the variable efficiency of the RT step nor take it into account in their measurements [15,16].
In the present study, we first show that the efficiency of the RT step depends on the target mRNA and on the experiments and that these variations have critical impact on the reliability of mRNA quantification. To overcome this, we describe here CyProQuant-PCR, a new technique for absolute measurement of cytokine mRNA based on an external RNA standard curve. Primer pairs have been designed for allowing amplification of a set of cytokine mRNA using the same conditions both in terms of thermocycling parameters and master mix components, a prerequisite for multiple cytokine mRNA measurements with high throughput.
In the present paper, we describe i) the construction of the synthetic RNA standard, ii) the primer pairs specific for the following human cytokines IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-γ, and for the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ), the β2-microglobulin (β2-MG) and the ubiquitin-C (UBC) to be used as internal standards and iii) the conditions for efficient real time amplification of multiple cytokine specific mRNA. The technique was validated using in vitro stimulated PBMC and its intra and inter-experimental variability were assessed. Finally, CyProQuant-PCR was used to quantify TGF-β1 transcripts in small blood samples from children with acute Plasmodium falciparum malaria.
Primer design and validation
Primer pairs were designed from published genomic sequences using Primer Express software (Applied Biosystems), except for the UBC and the YWHAZ genes for which the primers had already been described [17]. When possible, the following criteria were applied. The percent of G+C content was kept in the 20–80% range and runs of an identical nucleotide were avoided. The five nucleotides at the 3' end had no more than two G and/or C bases and the melting temperature was kept between 58 and 60°C. Among the primers proposed by Primer Express software, we selected forward and reverse primers amplifying a product spanning one intron and not leading to the amplification of pseudogenes or other related genes to secure primer specificity for target cDNA. The specificity of the amplification was assessed for each gene by electrophoresis and dissociation curve analysis as shown in Figure 1A and 1B. The absence of any contaminating bands corresponding to genomic DNA amplification on agarose gel as well as the presence of a unique peak on the dissociation curve validated the specificity of the primer pair for the target cDNA. PCR products were systematically sequenced after cloning and showed more than 98% identity with the expected sequence (data not shown).
Figure 1. Primer validation. A. Agarose gel electrophoresis of several CyProQuant-PCR products generated by amplification of a pool of cDNA from PBMC stimulated in vitro by LPS or PHA. Scale is shown in base pairs (bp). B. Dissociation curve analysis of these CyProQuant-PCR products. Negative derivative of the fluorescence is plotted against temperature. The single peak shows that SYBR Green fluorescence detects only the specific CyProQuant-PCR product. C. Amplification plots and standard curve resulting from the amplification of a range of β2-MG external RNA standard using CyProQuant-PCR.
Primer concentrations were optimised to determine the minimum primer concentrations giving the lowest threshold cycle (CT) and the maximum signal-to-noise fluorescence ratio (ΔRn) while minimising non-specific amplification (data not shown). Among the final concentrations of 50, 300 and 900 nM tested for each primer, the optimal final concentration was set at 900 nM for every primer. The primer pairs are shown in Table I.
Table 1. Primer sequences used for Cyproquant assays Table shows position of amplification product within cDNA sequence (upper line) and within genomic sequence (lower line). GenBank accession numbers are indicated.
Standard curve design
Generation of the RNA standard curve
To generate the standard RNA corresponding to each target sequence, gene specific primers were fused in their 5' end to the RNA polymerase T7 promoter sequence. The PCR performed with these modified primers pairs lead to a larger PCR product with the T7 promoter sequences upstream and downstream from the specific amplicon. The in vitro transcription gave a synthetic RNA, which was assessed for its integrity and clonality by electrophoresis (data not shown). The molecular mass of each RNA standard was calculated on the basis of its sequence and solutions ranging from 101 to 1012 copies of standard RNA were made.
These serial diluted solutions were reverse transcribed and the cDNA amplified in duplicate to generate a standard curve by plotting the threshold cycle (CT) against the logarithmic value of the starting RNA copy number for each dilution. Figure 1C shows an example of these curves for β2-MG. Every curve generated a dynamic range of a least 6 orders of magnitude. This allowed for a reliable and reproducible quantification of cellular mRNA sample.
Comparison between CyProQuant-PCR and DNA standard based approach
CyProQuant-PCR was compared to the classical approach based on a DNA standard curve for the quantification of TNF-α, IL-1β and IFN-γ transcripts. DNA templates for RNA transcription of TNF-α, IL-1β and IFN-γ were used to generate a range of concentration from 10 to 1012 copies/μl. We thus disposed of DNA and RNA ranges stemming from the same sequence. In parallel, a range of cDNA was also generated from the RNA standards. Ranges of RNA standard were reverse transcribed and then amplified in parallel to the cDNA and DNA ranges by real time PCR under the same conditions (same mix, same final volume, same PCR plate). The slope of the standard curves generated by these three ranges as well as the corresponding efficiency are shown in Table 2. The data show that the RT-PCR efficiencies from CyProQuant-PCR are lower than the PCR efficiencies from cDNA or DNA ranges for the three genes. Since the PCR efficiencies of the cDNA and DNA ranges are similar for each gene, this discrepancy is due to the RT efficiency, which is lower than 100% (from 75% for IFN-γ to 98% for TNF-α). In addition, data show that RT efficiency displays intergenic variation with a lower efficiency for IFN-γ compared to the two other cytokines (Table 2).
Table 2. Comparison between CyProQuant-PCR and DNA standard-based approaches Efficiencies (E) are deducted from the slopes (S) of the standard curves based on E = 100*(10-1/S - 1). The deducted RT efficiency from the CyProQuant-PCR is calculated by dividing the RT-PCR efficiency from the RNA range (ERT-PCR) by the PCR efficiency from the cDNA range (EPCR).
In order to show that these intergenic differences were not due to interassay variation, we compared amplification efficiencies of TNF-α, β2-MG, MIF and UBC from i) a single cellular RNA sample, ii) a pooled RNA sample containing standard RNA for each of the targets and iii) a pooled DNA sample corresponding to the cDNA obtained from the pooled RNA sample containing standard RNA for each of the targets. Results obtained are summarised in Table 3 and show that RNA amplification efficiencies vary from one gene to the other but are not affected by the molecular context since they do not differ between the single cellular RNA sample and the pooled RNA sample. Moreover, since the amplification efficiencies of the pooled DNA sample have been normalised to 100%, the difference of amplification efficiency between the RNA samples and the pooled DNA corresponds to differences in RT efficiency.
Table 3. Intergenic difference of RT efficiencies is not due to interassay variation. Serial 1:10 dilutions of a single cellular RNA sample, of a pooled RNA sample and of a pooled DNA sample were used to generate standard curves for different targets (TNF-α, β2-MG, MIF and UBC). Slopes were indicative of amplification efficiencies, which were normalised to 100% for the pooled DNA sample for easier comparison to amplification efficiencies of the two other samples.
Altogether, these data demonstrate that RT efficiency displays intergenic variations that are not due to interassay differences.
Impact of RT efficiency variability on transcripts quantification reliability
RT efficiency has critical impact on transcript quantification as shown in Table 4. Indeed, if we compare CyProQuant-PCR or DNA standard based approach for quantification of a known number of TNF-α, IL-1β and IFN-γ transcripts, results show that DNA standard based approach underestimates of around two logs the number of transcripts. Moreover, this underestimation varies according to the transcript studied and the experiment. CyProQuant-PCR is thus more precise and more reliable than the classically used DNA-based approaches.
Table 4. RNA and DNA standard-based quantification of TNF-a, IL-1β and IFN-γ transcripts Fifty million copies of TNF-α, IL-1β and IFN-γ standard RNA were amplified in parallel of a range of RNA standard (CyProQuant-PCR) and DNA standard. Copy numbers were directly deduced from the cycle at threshold. *For TNF-α, the experiment was repeated at two days interval.
Taken together, these results support the use of RNA as external standard for reliable and reproducible quantification of transcripts.
RT-PCR efficiency is similar for sample and RNA standard
Since RT-PCR efficiency varies, absolute mRNA quantification can only be reliably obtained if the external RNA standard and the cellular RNA are retro-transcribed and amplified with the same efficiency. This was secured by comparing the standard curves obtained after amplification of a range of 10X serial dilution of cellular RNA and β2-MG external RNA standard. The slopes obtained were of -3,307 and -3,305 for the cellular RNA and for the external RNA standard, respectively. This corresponds to efficiencies of 100,6% and 100,7% respectively (data not shown).
Endogenous standard, normalization and reliability of the technique
The choice of a stable expressed endogenous gene to be used as an internal standard is a prerequisite for accurate RT-PCR expression profiling. This has to be adapted to the clinical situation and the tissue of origin of the samples. Since our purpose was to establish a technique to be used with peripheral blood leukocytes, we tested three genes reported in stable amounts in leukocytes: the β2-MG, the UBC and the YWHAZ [17]. We compared the stability of the amount of transcript under different conditions of activation. Total cellular RNA was extracted on two different days from the same two aliquots of PBMC stimulated for 3 hours by LPS. Thus, reverse transcription was realised on RNA extracted from the same number of cells. The three endogenous gene transcripts were amplified by CyProQuant-PCR in the same plate in duplicate using the same PCR master mix. Table 5 shows the mean of the results obtained for the two extractions. The data show that β2-MG transcripts are stable with less than 12% of variation whereas UBC and YWHAZ transcript levels showed up to 47% variability depending on the in vitro conditions. Moreover, based on the approach recently described by Pachot et al. [18], we assessed the inter-individual variability of the β2-MG basal level using whole blood samples from 6 different healthy donors. All CT values were within 19 and 21,86 cycles, which corresponds to a variation of less than 5 times in gene expression for an overall RT-PCR reaction efficiency of 1,81 (slope = -3,867). This is considered as acceptable for a reference gene [18]. β2-MG was thus chosen as an internal standard gene for future experiments. The same conclusions were drawn using PHA (phytohemagglutinin) as a stimulant (data not shown). In addition, these data validate the reliability and reproducibility of our RNA extraction protocol.
Table 5. Expression stability of endogenous standard genes under non-normalised conditions After 3 hours of stimulation with LPS, PBMC from healthy donors were split in 2 identical aliquots and total cellular RNA was extracted at 2 days interval. The same volume of RNA was reverse transcribed and β2-MG, UBC and YWHAZ transcripts were amplified by real time PCR using CyProQuant-PCR technique. Results show the mean values obtained for the 2 extractions in arbitrary units.
Validation of the CyProQuant-PCR technique
Human TNF-a transcript quantification: comparison of CyProQuant-PCR to TaqMan®
Since TaqMan® technology developed by Applied Biosystems is considered as the "gold standard", we compared CyProQuant-PCR to TaqMan® for the quantification of TNF-α transcripts in isolated monocytes stimulated for 6 hours with LPS. Total RNA was extracted from 106 cells and 10X serial dilutions were prepared and reverse transcribed. The resulting cDNA were amplified in duplicate on the same plate in the same thermocycling conditions using either the TaqMan® commercial kit (Applied Biosystems) or CyProQuant-PCR primers with SYBR Green PCR master mix (Applied Biosystems). Figure 2 shows the curves obtained using the two techniques on the same samples. This indicates that CyProQuant-PCR is as good as TaqMan® in terms of sensitivity and even more efficient: 105% (slope -3,198) for CyProQuant-PCR versus 80 % (slope -3,908) for TaqMan®.
Figure 2. Comparison to TaqMan® technology. Total RNA was extracted from isolated monocytes stimulated for 6 hours by LPS, serial-diluted 1:10 and retro-transcribed to generate standard curves by plotting the CT against the concentration of this cellular RNA in arbitrary units. TNF-α transcripts were amplified by real time PCR using either (A) the TaqMan® commercial kit and the TaqMan® universal PCR master mix or (B) our primers and the SYBR Green PCR master mix. Data represent the standard curves obtained for the two techniques, their slopes and the deducted efficiencies.
Quantification of TNF-α and MIF by CyProQuant-PCR and ELISA
To validate our approach, we measured the levels of TNF-α and MIF transcripts and secreted proteins by CyProQuant-PCR and ELISA respectively. PBMC from healthy individuals were stimulated by LPS or SAC for 3, 9 and 18 hours. Figure 3 shows an increase of TNF-α transcripts after 3 hours of stimulation that precedes protein secretion. In contrast, MIF transcripts were constitutively present at the steady state and LPS or SAC stimulation did not significantly modify the level of transcripts although it induces the release of substantial amounts of MIF proteins. This is in agreement with reports showing that MIF exists within cells under homeostasis both as a preformed protein ready to be secreted and as a messenger RNA, which can then be rapidly translated in the absence of induced transcription [19].
Figure 3. TNF and MIF transcription and secretion kinetics. PBMC from 2 donors were stimulated for 3, 9 or 18 hours (T3, T9 and T18 respectively) with LPS or SAC. TNF-α and MIF transcripts were quantified using CyProQuant-PCR and the corresponding secreted proteins were measured by ELISA. Transcript numbers are normalised for one million copies of β2-MG RNA relative to the level of transcripts in un-stimulated cells (fold increase). Secreted proteins are expressed as pg/mL for one million living cells.
Early cytokine profiling of PBMC from healthy donors stimulated with LPS or SAC
To validate the panel of primers designed for CyProQuant-PCR, we assessed the early cytokine response as well as the kinetics of expression for PBMC from two healthy donors under stimulation with LPS or SAC. Figure 4 illustrates CyProQuant-PCR detection of IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, MIF, TGF-β1 and TNF-α transcripts for one donor. No significant increase in IL-15, IL-18, MIF or TGF-β1 transcripts was detected. Both stimulants induce a similar kinetics of transcript accumulation for IL-1β and IL-10. In contrast, IL-4 and IL-13 transcripts peaked very early (2 hours) under LPS stimulation but not under SAC stimulation. IL-12p40 transcripts also accumulated earlier under LPS stimulation compared to SAC but was detected later on. The amount of TNF-α transcript was sustained under SAC stimulation compared to LPS. Detection of the corresponding proteins for TNF-α, IL-10 and IL-12p40 confirmed the mRNA profile observed (data not shown). The amplitude of the response differed somewhat between the two donors but the kinetics obtained for each cytokine were similar (data not shown).
Figure 4. Early cytokine production kinetics by PBMC stimulated in vitro with LPS or SAC. Total cellular RNA was extracted from PBMC from 2 donors stimulated for 0.5, 1, 2, 3, 6 or 9 hours with LPS (blue bars) or SAC (red bars). Cytokine transcripts were quantified using CyProQuant-PCR. The results are shown for one donor and were expressed in mRNA copy numbers calculated relative to un-stimulated cells, after normalisation against β2-MG (fold increase).
Application: Use of CyProQuant-PCR to quantify TGF-β1 transcripts in malaria patients
Before the application to clinical samples, the experimental reproducibility of CyProQuant-PCR was evaluated using a range of TGF-β1 and β2-MG RNA standards. Inter-experimental variability was assessed from eight independent experiments. The coefficients of variation were satisfactory whatever the starting copy number of RNA standard, ranging from 0,39 to 1,07% and from 0,91 to 1,2% for TGF-β1 and β2-MG respectively (Table 6). TGF-β1 transcripts were then quantified after LPS stimulation of PBMC from malaria patients and asymptomatic controls. Figure 5 shows a significant difference in the amount of TGF-β1 transcripts between patients and asymptomatic controls only after LPS stimulation (p = 0,036).
Figure 5. TGF-β1 transcripts quantification in malaria patients and asymptomatic controls TGF-β1 transcripts were quantified without stimulation and after 22 hours of LPS stimulation of PBMC harvested from asymptomatic controls (n = 5) and from patients suffering from acute malaria (n = 20). Results are normalised for one million copies of β2-microglobulin. Whiskers indicate data range, boxes extend from the 25th to the 75th percentile and the horizontal lines show the median. Asterisk indicates significant higher value in malaria patients compared to asymptomatic controls (p = 0.036) in the capacity of response to LPS.
Table 6. Inter-experimental reproducibility of the quantification of a range of TGF-β1 and β2-MG RNA standards A set of TGF-β1 and β2-MG RNA standards ranging from 103 to 1010 copies was amplified by RT-PCR. Coefficients of inter-experimental variation were determined from eight different experiments and calculated for threshold cycles.
In this paper we have described a new technique, CyProQuant-PCR, for absolute quantitative profiling of human cytokine mRNA using real time RT-PCR. Although real time PCR is now becoming a popular technique, it still requires improvement for proper quantification. All the techniques for absolute quantification available so far use a DNA standard curve assuming that RT efficiency is constant and approaches 100% [12]. We show here that RT is not 100% efficient, and more importantly, that its efficiency changes from one gene to another and from one experiment to another. We thus designed primers and standard RNA to be used in such a way that all cytokine mRNA of interest as well as three housekeeping genes could be amplified using the same conditions (thermocycling parameters and buffer). This technique is rapid, reliable and reproducible. When compared to the TaqMan® commercial kit, which represents the "gold standard", CyProQuant-PCR was as sensitive but less expensive and more flexible. Indeed, for a given gene, the design of the probe might be quite difficult and, with judicious selection of primer pairs, comparable sensitivity can be achieved with the use of SYBR Green [20].
A major reason for not using an RNA standard curve is its poor stability due to its sensitivity to RNase degradation. In our hands, we did not find any detectable degradation when keeping the standard in concentrated aliquots (stock solution at 1000 μg/mL) in RNase-free water at minus 80°C and avoiding freeze-thawing cycles. However, for a greater stability, we incorporated modified dNTP, 2'-Fluoro-dCTP and 2'-Fluoro-dUTP, which have been reported to decrease the sensitivity of the in vitro transcribed RNA to specific RNases [21,22], and tested the effect of this incorporation on the RT-PCR efficiency. We did not find significant difference in the efficiencies (100% for the non-modified IL-4 standard versus 104% for the 2'-Fluoro-standard) (data not shown).
The methodology described here was easily and successfully applied to the quantification of several cytokine genes. The quantification is reliable on 7 to 8 logs with a sensitivity ranging from 1000 to 100 copies depending the cytokine. It was first used to determine the magnitude and the kinetics of early induction of cytokines mRNA upon PBMC stimulation using bacterial derived materials. This showed that CyProQuant-PCR is powerful enough to detect early on modest cytokine induction such as IL-4. Such a tool is useful to decipher the kinetics of cytokine response involved in physiopathological pathways but also as a read-out to measure minor immune responses to specific antigens [23].
We finally applied CyProQuant-PCR to measure the level of TGF-β1 transcripts in asymptomatic controls and malaria patients after LPS stimulation. Interestingly, we observed that cells from malaria patients have a significant higher capacity to respond to LPS compared to controls. The increased TGF-β1 transcript accumulation by patients' cells, suggests that an inadequate production of TGF-β1 might play a role in malaria pathogenesis as already proposed [24]. Further work is needed to elaborate on this finding. This first study provided the proof that CyProQuant-PCR is readily applicable to small clinical samples from paediatric cases. This opens the possibility to further quantify the cytokine imbalance associated with malaria pathogenesis and generate a disease cytokine signature, a prerequisite for novel therapeutic interventions targeting cytokine gene expression. Areas of application include both infectious and non-infectious diseases, as well as chronic inflammatory diseases such as rheumatoid arthritis or sarcoidosis and acute diseases such as sepsis or malaria. Beside its importance in patient immuno-monitoring, cytokine profiling is also a major tool to study specific immune response against antigens for design and testing of immuno-modulatory drugs or vaccines.
In conclusion, we provide here CyProQuant-PCR, a simple technique for genuine absolute quantification of cytokine mRNA using SYBR Green® which is as sensitive as the TaqMan® technique. Because the parameters of amplification are identical for all the cytokines developed, CyProQuant-PCR is readily automatable notably for 384-well plates and might allow multiple cytokine profiling of samples of very limited size at a relatively high throughput. CyProQuant-PCR opens the possibility to use cytokine mRNA measurements in clinical studies not only for an increased knowledge but also to help clinicians in patients' stratification and treatment decision.
Healthy human PBMC: isolation and in vitro stimulation
Blood was collected from healthy donors at the French Blood Bank. Peripheral blood mononuclear cells (PBMC) were isolated by density separation over Ficoll Hypaque and washed two times in RPMI 1640 (Gibco BRL, Invitrogen, Cergy Pontoise, France). Cells were re-suspended in RPMI 1640 (Gibco BRL, Invitrogen, Cergy Pontoise, France) supplemented with 2 mM glutamine (Gibco BRL, Invitrogen, Cergy Pontoise, France) and 10% AB+ human serum (French Blood Bank) at 2.106 cells/mL and either used directly for RNA extraction or cultured in duplicate with or without LPS (10 ng/mL, E. Coli O111: B7, Sigma, L'Isle d'Abeau Chesnes, France) or SAC (0,0075%, PanSorbine Cells, Calbiochem, La Jolla, CA, USA). After incubation, cells were washed with PBS and re-suspended in RNA-PLUS (Q-Biogene, Illkirch, France) for RNA isolation.
Malaria patients and asymptomatic controls
Twenty children admitted during the high malaria transmission season of 2001 to the emergency room at the Department of Child Health, Korle-Bu Teaching Hospital, Ghana were included. Five asymptomatic controls matched to patients for age, residence location and time of sample collection were enrolled. The general inclusion and exclusion criteria were as described by Kurtzhals et al. [25]. Parents or guardians signed informed consent forms. The study received ethical clearance from The Ethics and Protocol Review Committee at the university of Ghana Medical School and the Ministry of Health. Total cellular RNA was extracted from PBMC recovered from 500 μL of blood following supplier's instructions (RNA PLUS, Q-Biogene, Illkirch, France) after 22 hours of incubation at 37°C, 5% CO2 with or without LPS (10 ng/mL, E. Coli O111: B7, Sigma, L'Isle d'Abeau Chesnes, France).
RNA isolation
RNA was extracted following supplier's instructions, re-suspended in 60 μL of RNase-free water (Ambion, Huntingdon, UK) and quantified spectrophotometrically at 260 nm.
Oligonucleotide primers were synthesized at Eurogentec (Saraing, Belgium). To validate primers, a pool of cDNA from healthy human PBMC stimulated for 6 and 12 hours with LPS (10 ng/mL, E. Coli O111: B7, Sigma, L'Isle d'Abeau Chesnes, France) and PHA-L (10 μg/mL, Sigma-Aldrich, Lyon, France) was used. Analysis of the amplicons was assessed by 4% agarose gel electrophoresis and dissociation curve studies using Dissociation Curve Software (Applied Biosystems, Foster City, CA, USA). PCR products were cloned into pCR2.1 vector using Original TA cloning kit (InVitrogen, Cergy Pontoise, France) and sequenced (Genome Express, Meylan, France).
Construction of the external DNA and RNA standards
PCR products generated by each primer pairs were column-purified (Nucleospin, Macherey-Nagel, Hoerdt, France) and quantified spectrophotometrically at 260 nm. The molecular weight of the standard DNA was calculated by N*487-[(N-1)*175] were N is the number of bases composing the standard DNA. Stock solution of 1012 copies of standard DNA /3,85 μL were made in Tris-EDTA buffer (Ambion, Huntingdon, UK), split in single-use aliquots and stored at -80°C in safe-lock tubes. External DNA standard range was made extemporaneously by 1:10 serial dilutions in water.
Gene specific primers were fused on their 5' end to the sequence of the RNA polymerase T7 promoter to generate modified primers. These primers were used to amplify a gene specific PCR product flanked by transcription initiation sites. Five hundred nanograms of this construct were in vitro transcribed (MegaShortScript, Ambion, Huntingdon, UK). The standard RNA generated was purified (MegaClear, Ambion, Huntingdon, UK) and loaded on a 4% agarose gel for electrophoresis. The concentration of the standard RNA was determined spectrophotometrically at 260 nm. The molecular weight of the transcript was calculated by N*500-[(N-1)*175] were N is the number of bases composing the standard RNA. Stock solution of 1012 copies of standard RNA /3,85 μL were made in RNA storage solution (Ambion, Huntingdon, UK), split in single-use aliquots and stored at -80°C in safe-lock tubes. External RNA standard range was made extemporaneously by 1:10 serial dilutions in water.
Reverse transcription
For CyProQuant-PCR assays, 100 ng of total cellular RNA from PBMC and serial dilution of external RNA standard were reverse transcribed simultaneously in a parallel procedure using Reverse Transcription TaqMan reagents (Applied Biosystems, Foster City, CA, USA) on a MasterCycler Gradient (Eppendorf, Le Pecq, France). The final volumes were set at 100 μL for the cellular RNA samples and 50 μL for the external RNA standard range. The thermocycling parameters were as follows: 25°C, 10 min.; 48°C, 60 min. and 95°C, 5 min. cDNA were immediately used for PCR amplification.
Real-time RT-PCR and quantification of transcripts
Reverse-transcribed standard RNA and cellular RNA were amplified simultaneously on the same PCR plate on an ABI Prism 7700 (Applied Biosystems, Foster City, CA, USA). An aliquot of 5 μL of the RT reaction was amplified in duplicate in a final volume of 30 μL of SYBR Green PCR Master mix (Applied Biosystems, Foster City, CA, USA). Thermocycling conditions were 50°C for 2 min., 95°C for 10 min. and 40 cycles of [95°C/15 sec.; 60°C, 1 min]. The sample target RNA copy numbers were calculated using SDS 1.9 Software (Applied Biosystems, Foster City, CA, USA). The baseline fluorescence was set manually to correct for differences in initial cDNA concentration and the threshold was positioned at a fluorescence level that was 10 times higher than the background signal. Target mRNA copy numbers in cellular samples were calculated based on a standard curve generated by SDS 1.9 Software (Applied Biosystems, Foster City, CA, USA) by plotting cycles at threshold (CT) against the logarithmic values of the starting RNA standard copy number.
ELISA tests
TNF-α and MIF secreted proteins were quantified by sandwich ELISA following supplier's instructions (Bio-Source, Clinisciences, Montrouge, France and R&D, Lille, France respectively). Results are expressed as pg/mL for one million living cells.
Statistical analysis
Tests for significance were done using Stata software (Stata Corporation, College Station, Texas, 77845 USA) by Kruskal-Wallis rank test.
Patent application
Results disclosed in this manuscript have been protected in French patent application FR0408645.
Authors' contributions
PB developed the entire technique. IV did the in vitro stimulation experiments. DJ gave technical assistance. SL helped in RNA extraction of malaria samples. JCB introduced PB to molecular biology techniques and provided critical advices. BDA designed and conducted the study that yielded the malaria samples. OMP revised the manuscript and supported the work. CB conceived the strategy and coordinated the study. PB and CB drafted the manuscript. All authors read and approved the final manuscript.
This work was supported by the European Commission program INCO-DC (Grant n°IC18-CT-980370), by the WHO/TDR/MIM project 980037 and by the program DVPI of the Pasteur Institute. Philippe Boeuf was supported by a PhD fellowship from the PAL-PLUS program (French Ministry of Technology, Research and National Education) and from CANAM (Caisse Nationale d'Assurance Maladie et Maternité des Travailleurs non Salariés des Professions non Agricoles).
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Overbergh L, Giulietti A, Valckx D, Decallonne B, Bouillon R, Mathieu C: The use of real-time reverse transciptase PCR for the quantification of cytokine gene expression.
J Biomol Tech 2003, 14:33-43. PubMed Abstract | Publisher Full Text
Rajeevan MS, Ranamukhaarachchi DG, Vernon SD, Unger ER: Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies.
Methods 2001, 25:443-451. PubMed Abstract | Publisher Full Text
Nomura I, Gao B, Boguniewicz M, Darst MA, Travers JB, Leung DY: Distinct patterns of gene expression in the skin lesions of atopic dermatitis and psoriasis: a gene microarrays analysis.
J Allergy Clin Immunol 2003, 112:1195-1202. PubMed Abstract | Publisher Full Text
Ramos-Payan R, Medina-Aguilar M, Estrada-Parra S, Gonzalez-y-Merchand JA, Favilla-Castillo L, Monroy-Ostria A, Estrada-Garcia CE: Quantification of cytokine gene expression using an economical real-time PCR polymerase reaction method based on SYBR® Green I.
Scand J Immunol 2003, 57:439-445. PubMed Abstract | Publisher Full Text
Radonic A, Thulke S, Mackay IM, La d O, Siegert W, Nitsche A: Guideline to reference gene selection for quantitative real-time PCR.
Biochem Biophys Res Commun 2004, 313:856-862. PubMed Abstract | Publisher Full Text
Bustin SA: Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.
J Mol Endocrinol 2000, 25:169-173. PubMed Abstract | Publisher Full Text
Stordeur P, Poulin LF, Cracium L, Zhou L, Schandené L, de Lavareille A, Goriely S, Goldman M: Cytokine mRNA quantification by real-time PCR.
J Immunol Methods 2002, 259:55-64. PubMed Abstract | Publisher Full Text
Ginzinger DG: Gene quantification using real-time quantitative PCR : An emerging technology hits the mainstream.
Exp Hematol 2002, 30:503-512. PubMed Abstract | Publisher Full Text
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real time quantitative RT-PCR data by geometric averaging of multiple internal control genes.
Genome Biol 2002, 3:34.I-34.II. BioMed Central Full Text
Pachot A, Blond J-L, Mougin B, Miossec P: Peptidylpropyl isomerase B (PPIB): a suitable reference gene for mRNA quantification in peripheral whole blood.
J Biotechnol 2004, 114:121-124. PubMed Abstract | Publisher Full Text
Calandra T, Froidevaux C, Martin C, Roger T: Macrophage migratory inhibitory factor and host innate immune defenses against bacterial sepsis.
J Infect Dis 2003, (Suppl 2):S385-90. PubMed Abstract | Publisher Full Text
Marino JH, Cook P, Miller KS: Accurate and statistically verified quantification of relative mRNA abundances using SYBR Green I and real-time RT-PCR.
J Immunol Methods 2003, 283:291-306. PubMed Abstract | Publisher Full Text
Meis JE, Chen F: In vitro synthesis of 2'-fluoro-modified RNA transcripts that are completely resistant to RNase A digestion using the DuraScribe T7 transcription kit.
Heidenreich O, Eckstein F: Hammerhead ribozyme-mediated clivage of the long terminal repeat RNA of human immunodeficiency virus type I.
J Biol Chem 1992, 267:1904-1909. PubMed Abstract | Publisher Full Text
Housseau F, Lindsey KR, Oberholtzer SD, Gonzales MI, Boutin P, Moorthy AK, Shankara S, Roberts BL, Topalian SL: Quantitative real-time RT-PCR as a method for monitoring T lymphocyte reactivity to full-length tyrosinase protein in vaccinated melanoma patients.
J Immunol Methods 2002, 266:87-103. PubMed Abstract | Publisher Full Text
Omer FM, Kurtzhals JAL, Riley EM: Maintaining the immunological balance in parasitic infections : a role for TGF-β?
Parasitol Today 2000, 16:18-23. PubMed Abstract | Publisher Full Text
Kurtzhals JAL, Adabayeri V, Quarm-Goka B, Akanmori BD, Oliver-Commey JO, Nkrumah FK, Behr C, Hviid L: Low plasma concentrations of interleukin 10 in severe malarial anaemia compared to cerebral and uncomplicated malaria.
Lancet 1998, 351:1768-1772. PubMed Abstract | Publisher Full Text


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Jerheme Wayne Urban
Position: WR
Height: 6-3 Weight: 212 lbs.
Born: November 26, 1980 in Victoria, TX
High School: Stroman
College: Trinity (TX)
Weighted Career AV (100-95-...): 11 (6614th overall since 1950)
+ Injured Reserve (finger): is on injured reserve (updated January 08, 2011)
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subset: commoncrawl


text:
Resources and Publications
Resource and Publication
Policymakers should take measures to reduce spam and improve cybersecurity on the Internet.
ITIF proposes a solution to consistently fund next-generation clean energy breakthroughs.
ITIF, Heritage, and CAP propose a set of nonpartisan policy proposals for reforming the National Laboratories.
All but "final mile" mail delivery should be opened up to private competition.
ITIF presents a rebuttal of the Economic Policy Institute's analysis of the STEM workforce.
3D printing is a new technology that raises old policy questions.
This fact sheet lists 53 documented economic benefits of IT, from jobs and output to competitiveness and...
A robust reauthorization of the America COMPETES Act can boost U.S. economic competitiveness and growth.
Policy decisions about IT in the vehicle should be driven by a broad government mandate to foster innovation...
While a modest proposal, E-labeling is a useful step towards more innovation, lower costs, and better design for the Internet of Things.
The House Science Committee should pass legislation to bolster U.S. manufacturing competitiveness.
China's solar trade policy hampers the clean energy economy and energy innovation.
Despite new statistics from the Census Bureau, wage and unemployment data show that we still suffer from a STEM shortage.
Data can be a key to enhancing global development.
Companies continue to face challenges securing IP rights in India, despite campaign pledges.
India simply cannot usher in a new, innovative economy if it continues to revert back to these same tried-and-failed mercantilist practices.
The U.S. Ex-Im Bank is vital for supporting exports by Boeing to markets where finance would be otherwise unavailable.
We must improve our innovation foundation or risk falling further behind economically.
ITIF president Rob Atkinson will present at Leading Edge Forum.
The Politico Pro team presents an in-depth conversation with special guest Rob Atkinson, President,...
Presentation on adoption of IP practices and trade strategies in developed and developing nations at the CES-...
ITIF Senior Analyst Daniel Castro will be moderating the panel "Academics and Technologists Look at the...
ITIF Senior Analyst Stephen Ezell presented on the future of advanced manufacturing at the AAAS annual...
From an economic policy perspective, the focus should not be on intangible capital vs. tangible capital.
ITIF president Rob Atkinson will present on a panel at 2:35 PM on the effects of technology on industry.
Rob Atkinson will present a pragmatic road map for how the United States can regain an innovation advantage...
"Technologies that Energize Us" Panel: A Conversation with Matthew Stepp, Senior Policy Analyst, on...
Stephen Ezell, author of "Innovation Economics," will host a reception with the Policy Exchange in...
ITIF files comments on effective universal service under a new Communications Act.
ITIF argues the FCC should develop flexible regulations under section 706.
ITIF cautions the FCC against encouraging inefficient use of public resources on municipal broadband...
ITIF filed comments with the FCC supporting a combined, larger company that can better serve consumers with...
ITIF files comments advocating for a flexible interconnection regime with the House Energy and Commerce...
The DOJ should rework these consent decrees to modernizing the way music copyrights are licensed in the...
Government data policy needs to balance privacy with innovation.
The Center for Data Innovation's Recommendations to the Senate Finance Committee on Health Data...
Rob Atkinson filed comments to the U.S. Senate Committee on Judiciary's Subcommittee on Antitrust,...
Bipartisan effort will be key to saving Social Security.
Conventional economics provides an increasingly poor guide for economic policy and should be replaced by an evolutionary economics framework.
Rob Atkinson contributed the chapter "Public Policy, Innovation and New Technologies" to the textbook
"Guide to U.S. Economic Policy."
Robert Atkinson contributed the chapter "Innovation in Cities and Innovation by Cities" for the book "Creating Competitiveness: Entrepreneurship and Innovation Polices for Growth."
In a chapter for the new book Practicing Sustainability, Rob Atkinson wrote about sustainability from an innovation economics perspective.
"Innovation Economics: The Race for Global Advantage." This new book delivers a critical wake-up call: a fierce global race for innovation advantage is under way and the United States is running the risk of losing.
Daniel Castro contributed a chapter titled "U.S. Federal Cybersecurity Policy" in the book "Cybersecurity: Public Sector Threats and Responses."
Rob Atkinson contributed a chapter titled chapter "Who's Who in Internet Politics" for the book "The Next Digital Decade: Essays on the Future of the Internet."
Supply-Side Follies methodically debunks the common assumptions of conservative economics and demonstrates why it is a flawed doctrine that is setting up the U.S. for a major economic downturn in the near future.
Taking into account the historical record, the book discusses the shortcomings of prevailing liberal and conservative economic doctrines and lays out a new growth economics agenda aimed at maximizing the productivity and innovation-enhancing forces of the New Economy.


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Kill The North Stonington Mountain Lion! (And Other Phrases I Hope People Never Utter)
Every time there's a report of a dangerous, predatory animal prowling local woods or waterways I fervently wish that two things don't happen:
Most people were so revolted by California Chrome co-owner Steve Coburn's comments last Saturday after his horse failed to win the Triple Crown they launched a blistering attack on Twitter — the 21st century version of tar-and-feathering — that eventually led to his tearful apology a couple days later on national television.
Not me.
I stood up and cheered when I watched Coburn launch his televised rant against rival horse owners at the end of the Belmont Stakes on Long Island, calling them "cowards" because unlike California Chrome most of them had skipped the Kentucky Derby and The Preakness, and therefore were well-rested for the 1.5-mile race.
"Right on, brother!" I cried.
You see, I make similar excuses any time I finish far back in the field.
Sure, I've crossed the finish line in first place in my division in various kayak and running competitions over the years, but whenever I fail to win, place or show I'm ready with an explanation.
"Everybody else cheated!"
Take the Boston Marathon in April.
Just because I hadn't run a qualifying marathon in a fast time the mean-spirited killjoys of the Boston Athletic Association gave me a ridiculously high bib number that forced me to start the 26.2-mile
running race in a corral of casual joggers more than an hour after the Kenyans and other elite runners took off. I hadn't even reached Heartbreak Hill, for God's sake, when Meb Keflezighi sprinted across the finish line on Boylston Street!
I experienced a similar situation a few years ago in New York, during the Mayor's Cup New York City Kayak Championship, a 28-mile race around Manhattan that attracted some of the world's best paddlers.
Strolling around the 79th Street Boat Basin on the Hudson River an hour or so before the race started to scope out the competition, I spotted Greg Barton, winner of four Olympic medals, including two golds, stretching his muscles next to a sleek surfski.
"Nice boat," I said. "Good luck."
"Thanks," he said. "You too."
Had I known that race organizers would have allowed Barton and his big-shot cronies to take off well ahead of the other paddlers — including my buddy Ian Frenkel and me in our tandem — I wouldn't have been so polite.
"Hey, Barton!" I would have sneered. "How about starting back with the rest of the paddlers, you thing you're so tough!"
Even though Ian and I took first place in the two-man sea kayak division, Barton and his pals had already circled Manhattan by the time we worked our way up the Hudson, across the Harlem, down the East, around the Battery and back up the Hudson. What's more, we had to wait nearly an hour during the awards ceremony for them to claim our prize.
While Barton and other elite paddlers walked away with thousands of dollars in swag and appearance fees, Ian and I took home a couple of crappy T-shirts and a faux-gold medal.
If somebody aimed a camera at me and thrust a microphone in my face then I would have spewed a string of epithets that would have made Coburn's tirade seem like a hissy fit.
In that race Ian and I were divisional winners in the tandem sea kayak class; I savored my most significant outright victory years earlier at a so-called Hash race, the brainchild of a British club, the Hash House Harriers, whose members identify themselves as drinkers with a running problem. Officials lay out the course in secret, marking the route with ribbons and signs marked on the ground in flour. Everybody starts at the same time, and a runner in the lead is supposed to shout, "On! On!" if he finds himself on the right trail, or "Go back!" if it turns out to be a dead end.
Anyway, I was running a hundred yards or so behind the lead pack with less than a quarter-mile to the finish, and watched them dart left at an intersection.
Ordinarily I would have followed, but realized I had only once chance to win, and turned right. I was correct: Directly ahead, the finish line beckoned!
"On! On!" I shouted. Well ... maybe I whispered.
The others quickly realized what had happened and reversed course, but they were too late.
I sprinted across the line and claimed my prize, a can of corned beef hash.
"You cheated!" one of the trailing races whined.
"Next time, run faster" I sneered.
Sortsmanship-smortsmanship. Legendary sportswriter Grantland Rice had it backwards: It doesn't matter how you play the game, as long as you win. And if you don't win, at least you can be a sore loser.
Every time there's a report of a dangerous, predatory animal prowling local woods or waterways I fervently wish that two things don't happen:
This week's cold snap has spread a shimmering, silken coating over ponds and lakes throughout the region, creating some of the best conditions for skating in years, so you must get out now and enjoy it because who knows...
First of all, it's way too soon to start whining about the cold. Let's all agree that by March, if we're still getting hammered by ferocious winds, driving snow and frigid temps, then a little complaining might be in...
The problem with New Year's resolutions is that people, even — make that especially — hard-core adventurers/endurance athletes tend to set the bar way too high: bike across the country, qualify for the Hawaii...
When those uninitiated in the longstanding New Year's Day tradition of running from Mystic to Fishers Island Sound and leaping into the icy water ask, "How can you stand it? Doesn't it hurt?" we stoic veterans...
While kayaking the 341-mile Erie Canal from Buffalo to Albany a few years ago I spotted another paddler a mile or so away headed in my direction. Having encountered only one or two other kayaks nearly a week into what turned out to be an...
When I pried a rock up with a mattock the other day, rushing to finish a small retaining wall before the ground froze, I inadvertently disturbed a hibernating spotted salamander's winter home.
Isn't life great?! Gas is so cheap now I can get rid of my puny, poky, fuel-efficient econobox and get behind the wheel of an auto with plenty of ponies under the hood that any American would be proud to drive, just like the good...
A brisk autumn breeze scattered crimson maple leaves that fluttered from trees lining a pond glittering in the morning sun as my friend Bob Graham and I loped along a narrow path the other morning.
After my overwhelming victory last year in a rake vs. leafblower contest I hoped I'd heard the last of those infernal, noisy, polluting contraptions — but while out for a run the other day, savoring the fall foliage, a familiar whine as...
Good morning! I hope you all had a good night's sleep, enjoyed the griddle cakes and are eager to work off those calories.
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User:JjjenkinEdit This Page
From FamilySearch Wiki
AncestorStalker.com is the place to go for professional genealogical research. If you are just starting your tree or scratching your head on what to do next, contact us and we'll discover what your best path is!
I volunteer time transcribing cemeteries, freely-accessible images online and volunteer as Administrator of over a half a dozen Y-DNA projects through http://www.familytreedna.com.
Member of national and local Association of Genealogical Professionals, historical and genealogical societies and other organizations to keep us on top of the game.
Maryland and DNA specialist for Global Alliance of Genealogy Professionals
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Opened in 2004, Snafu is located in the Castlegate area of Aberdeen on what is known locally as the Granite Mile.
View the venue listing
- Event admin /
- mrpeace
- Update this event
Line-up /Ben UFO (Hessle Audio)
Ennayess (DSR Audio/Dubpressure)
D. Peace (Submission)As one of the co-founders of Hessle Audio, Ben UFO has been responsible, alongside Pearson Sound and Pangaea, for some of the most varied and ground breaking releases of recent years. Having become immersed in the still emerging dubstep scenes in London and Leeds in 2005, his musical taste was shaped primarily by the DMZ and Sub Dub nights taking place in those cities. However, as tastes have shifted and scenes have fragmented, Ben UFO's approach to the music and to Hessle Audio itself has developed.
As BPMs have dropped and influences have broadened, so too has the vision of Ben UFO's DJing. He remains one of the few DJs in the UK to have made an impact without a production career to fall back on, and as such his reputation is solely the result of his skill as a selector and as a mixer. As a result he remains in a position to fully explore sounds both old and new, across the worlds of house and garage, dubstep and grime, and further into the past. This level of commitment to the dance music of the last two decades has led to a collection of records which reflects the position London has long held as a city responsible for the constant development of forward thinking music. -
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More on Snafu
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Partial presents: Hessle Audio Takeover
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Fri, 13 Feb 2015
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Nightvision presents Musika 8th Bday with Green Velvet & Eats Everything
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Purple Love - 3rd Base


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Menert persevered, and the work has clearly paid off. His involvement in the developmental years of Pretty Lights proved to be inspirational. "I was able to find another path," he points out, "because of what Derek paved with being able to travel around and tour."
In the four years since he set out on his solo venture, Menert says, the best thing about it has been the grassroots growth of his fan base. "Meeting fans, crashing on couches and getting rides to the airport from people's roommates," he says. "That's what it's all about."
And the success he's had so far — he's currently headlining a national tour with support from fellow locals AC Lao and Mikey Thunder — only seems to push him harder to stay on his path. "Every day is therapy for me," he says, "because music and orgasms are the two times where things don't bleed into my consciousness."
Free of doubt and worry about living a sketchy lifestyle, Menert takes time now to appreciate the fruits of his labor, be it a fan complimenting his music or a potential collaboration on a new song. "When people tell me they like my music, I am really grateful," he says. "This music is my soundtrack for what I go through and have gone through.
"I reflect on the things that I regret and the things that brought shame to my family, and there was nothing for me to be proud of," he concludes. "But for all the ugly shit that I've done, this is something beautiful that I created."


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Leather bustier. Note: This is the same pattern as Satin bustier #123
burda style magazine patterns FAQ
Stretch suede
Sep 30, 2012, 10.16 PMby Gurtlamorous
can the skirt pattern which comes with this purchase be used aswell?
Add to Favorites 70 people added it
Produced by burda style magazine
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Why you should wear clothing made to fit YOU! (In GIF's)
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Fashion & Trends
Vacation planned for the winter? Sew up these style to take away with you and also get 20% off!
We have just extended registration to accommodate a few more students


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All publications are available online. You will need Adobe Acrobat Reader to view most of the files. Contact NCCF to request a copy of our most recent publications or call 252-393-8185.
Coastal Review Online, the N.C. Coastal Federation's online news service, is moving to its own website on Feb. 9. Its new address will be www.coastalreview.org. Please be sure to change your bookmarks so you don't miss great stories about our coast. The federation's website will still be here but with a completely new design full of stunning photos, video and up-to-date information about all our projects and work to protect the beautiful N.C. coast. Be sure to come back and pay us a visit.
Our Mission: To provide people and groups with the assistance needed to take an active role in the stewardship of North Carolina's coastal water quality and natural resources.


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Hey Massy!
It depends on which kind of music you're doing. Yes, the TR-808, TR-909 are
still among my electronic favourites, though I couldn't name soundfonts, since
I have them in my Roland synth. But there are so many different styles of
kits. So what are you doing: Ballad, techno, hiphop,...?
Warmly yours
Such Is Life: Very Intensely Adorable;
Free And Jubilating Amazement Revels, Dancing On - FLOWERS!====== Find my music at ======
"If you live to be 100, I hope I live to be 100 minus 1 day,
so I never have to live without you." (Winnie the Pooh)
Linux-audio-user mailing list
LINUX® is a registered trademark of Linus Torvalds in the USA and other countries.
Linuxaudio.org logo copyright Thorsten Wilms © 2006.
Hosting provided by the Virginia Tech Department of Music and DISIS.


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Just off the slopes! Loved the food, drinks, and atmosphere!! Service was amazing. We had a large group and everyone was well taken care of.
Spaces inside are quieter and more suitable. Food for all tastes. My favorite goulash soup Apfelstrudel mit und vanilla Souce. During lunch'm very busy, but there's room for everyone! :)
Register now for free — and start getting automatic notification of new reviews, responding to traveler feedback, adding new photos to your listing and much more.


text:
Car Problem Reports
Nissan Sentra burned out ingition coils
Some Nissans with the 2.5 have multiple ignition coil failures.
The reason is the negative battery cable is corroded at the mounting at the bell housing on the transmission.
the solution is to replace the entire negative cable with a factory upgraded cable and replace the coils as well.Flag ThisLike | 1 person likes this1


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Join Meir Panim's campaign to "light up" Chanukah for families in need.
Marriage is under assault again in this country, as fewer adults choose to tie the matrimonial knot while the Left continues to lend civil and economic credence to unions of same-sex partners.
According to the Census Bureau, the percentage of married adults in the U.S. has dropped steadily over the past few decades, from 72 percent in 1970 to around 54 percent last year. Part of that decline can be attributed to the delay in getting married, as people spend more time pursuing higher education and establishing themselves in their careers.
Additionally, a large component of this decline stems from the fact that many choose not to marry at all. And those who do are far less likely to remain wedded for the balance of their lives than in past decades.
In a recent CBS News poll, seven in ten Americans said the institution of marriage is weaker now than it was 20 years ago, despite the fact that research shows married people tend to live longer and are generally healthier, wealthier, and happier.
The reasons for this shift are many, including the sense that many of the traditional benefits" of marriage – companionship, financial security, the ability to have and raise children – can be achieved in today's society without the so-called burdens of a longstanding relationship.
Of course, the question is not merely whether to wed or not to wed. In recent years, the question has increasingly centered on the fundamental definition of marriage as the civil union of a man and a woman.
To date, five states and the District of Columbia have approved gay marriage, while others permit civil unions of same-sex partners. Under pressure from the White House, the military recently repealed its longstanding policy of "don't ask, don't tell."
President Obama advanced the cause even further when he crossed into the judicial realm by announcing the Executive Branch would no longer oppose court challenges to the Defense of Marriage Act, a 15-year-old law that prohibits the federal government from recognizing same-sex marriages for purposes of taxes, social security and other programs. According to Attorney General Eric Holder, the law's definition of marriage as between a man and a woman is "unconstitutional."
Naturally, the administration's position, not to mention its timing, came under fierce attack. Lamar Smith, the Texas Republican who chairs the House Judiciary Committee, called the Justice Department's decision to stop defending the law "the real politicization of the Justice Department," lamenting that "the personal views of the president (can be made to) override the government's duty to defend the law of the land."
While politicians attacked the law largely for political considerations, organizations like the Agudah took exception to Obama's decision on religious and moral grounds, calling it, among other things, "a provocative step toward undermining our nation's traditional values and moral fiber."
While one can certainly appreciate and unequivocally support the aforementioned position regarding same-sex marriage, one wonders why this issue regularly garners so much of our time and energy.
After all, in what sense does such legislation change the current reality? Certainly the absence of such legal backing has not served as much of a deterrent until now.
Further we live in a society that routinely flaunts norms and values wildly inconsistent with our Torah lifestyle. Why have our leaders seen fit to speak out publicly and repeatedly against this particular issue?
I believe the reason has nothing to do with attempting to impose any meaningful change on the status quo. Rather, it is because we maintain that recognizing and affirming same-sex marriage pose a fundamental threat to the very fabric of human society.
According to our tradition, non-Jews as well as Jews have found such anti-family conduct to be on a level of complete abhorrence, comparable to cannibalism.
Ulla said, [there] are thirty commandments [comprised in the seven Noachide precepts] which the sons of Noach took upon themselves, but they [only] observe three of them . They do not draw up a kesubah document for males, they do not weigh flesh of the dead in the market, and they respect the Torah.
[Chulin 92a-b] Obviously, this in no respect implies the nations of the world have historically abstained from homosexual practices. To the contrary, it was a central element of many ancient civilizations. Still, they stopped short of legitimizing their desires in the form of marriage.
About the Author: Rabbi Naphtali Hoff is an executive coach and president of Impactful Coaching & Consulting (www.ImpactfulCoaching.com). He can be reached at email@example.com.
If you don't see your comment after publishing it, refresh the page.
Comments are closed.
Obama's Syrian policy failures created the current situation in the Golan Heights.
Our journey begins by attempting to see things differently, only then can we be open to change.
Despite Western 'Conventional Wisdom&PC,' the Arab/Israeli conflict was never about the Palestinians
Confrontation & accountability, proven techniques, might also help dealing with religious terrorists
In fact, wherever you see soldiers in Paris today, you pretty much know you're near Jewish site
Inspired by the Perek Shira pasuk for "small non-kosher animals" we named the bunny "Rebbetzin Tova"
The abuse following publication proved a cautionary tale: no one followed in Peters's footsteps
Plainly, there is no guiding hand dictating choices across the board.
How many sites that tell you to check your politics at the door have 10,000 likes?
In this particular case, the issue was whether the Arkansas prison system could prohibit, for security reasons, a devout Muslim's maintaining a beard of a certain length as a matter of religious practice.
While we recognize the Republican Jewish Coalition is hardly a non-partisan outfit, a snippet from a statement the group released is worthy of note:
"These are good matzah balls," my aunt Robertine would say, but her sister Irma would counter "No, not compared to Mama's. They were always so light yet they never fell apart."
Despite the 2005 Koby Mandell Act no Palestinian implicated in harming an American has been charged
The NY Times suggestion that HaMavesar cropping women from a photo is Israeli censorship is absurd.
He ruthlessly crushed the revolt, and, despite lacking official Roman sanction, ordered the rebel leaders put to death without trial.
Where did this incredible strength come from? What drove these Jews, who had nearly lost all of their national identity and spiritual connectivity, to risk their lives by standing up against one of the strongest and most fearsome governments of its time?
Of all the Jewish holidays, I would say Sukkos is far and away the least appreciated.
While there is no formula that will work for everyone, there are some strategies that if followed carefully and consistently can help our children – and us – gain the most from the upcoming school year.
"If Israel's offering of land, economic improvements, and even autonomy will not help, what will?"
For breaking his oath of allegiance, Tzidkiyahu was forced to witness the death of his sons before he himself was blinded and exiled to Babylon.
It's as if Hamas has pulled a page out of Pharaoh's handbook.
As a guide to others and a foremost member of the Great Assembly, Ezra provided strong leadership and a moral conscience to a people that had lost its way.
Printed from: http://www.jewishpress.com/indepth/opinions/in-defense-of-traditional-matrimony/2011/04/13/
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Founded in 1965, AFP DC is proud to serve our more than 1,000 members who are dedicated to fundraising and advancing the spirit of philanthropy. Our Chapter members – executive directors, development directors, consultants, grant writers, fundraising volunteers, and foundation executives – cover the range of DC Metro's vibrant nonprofit community.
AFP DC strives to provide you the tools and connections you need to excel in your job!
- Professional development conferences and workshops
- Breakfast and lunch learning sessions on diverse fundraising topics
- Roundtable discussions
- A sound mentoring program
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Pac-12: Tom Tunnicliffe
California needs to prove Pac-10 isn't a one-team show
Posted by ESPN.com's Ted Miller
TUCSON, Ariz. -- You'd never hear it from Pac-10 administrators, but the conference sure could use a California victory at Arizona tonight.
No. 25 Cal (4-1, 2-0) is the conference's only other ranked team besides USC and its only unbeaten team in conference play.
Also, the Bears lone defeat at Maryland doesn't look so bad these days, particularly after the Terrapins whipped No. 21 Wake Forest 26-0 Saturday and are now 5-2 overall (we'll just ignore the 31-zip loss to Virginia).
California has won four of five in the series but its last defeat at Arizona in 2006 -- a 24-20 heartbreaker -- ended up costing the then-eighth-ranked Bears their first Rose Bowl berth since 1958.
Bears coach Jeff Tedford used that defeat -- and the wild post-game celebration -- to motivate his team this week.
One guy who talked a lot about that game is Cal quarterback Nate Longshore. He's making his second consecutive start tonight after replacing Kevin Riley, but he threw three interceptions in the 2006 defeat
For Arizona, it's all about stopping the run.
As noted in today's Arizona Daily Star, the Wildcats have allowed more total rushing yards in their two defeats (507) than their four wins (316).
But New Mexico and Stanford featured 230-pound power backs. Cal offers a pair of speedy scatbacks in Jahvid Best, who returns after dislocating his elbow on Sept. 27 against Colorado State, and Shane Vereen. They both tip the scales under 200 pounds.
Also of note: Wildcats quarterback Willie Tuitama needs just 30 yards passing to eclipse Tom Tunnicliffe as the program's all-time passing leader. Tuitama enters the game with 7,589 career passing yards.
Heat won't be much of a factor. It's presently clear and 90 degrees, and it will cool considerably by the 7 p.m. kickoff (tonight's low is 58).
Arizona is tough at home, though. It has upset a top-25 foe in Arizona Stadium every year since Mike Stoops become head coach in 2004.
And the last victim was Cal.
Pac-10 lunch links: 2007 Huskies-Beavers was weird
Posted by ESPN.com's Ted Miller
You know in some cultures Thursday is no different than Friday. If you were still in college, you'd know this.
- Arizona quarterback Willie Tuitama should finish rewriting the school's passing record book against California on Saturday, eclipsing Tom Tunnicliffe, who's best known for starting a nine-game unbeaten streak against rival Arizona State.
- Arizona State's touted but struggling cornerback Omar Bolden got benched for a half against USC and then came back strong.
- While California running back Jahvid Best appears ready to play Saturday at Arizona, it wasn't a good day of practice -- two starters are hurt.
- Oregon is using the bye week to find an identity.
- For folks who don't remember how strange Washington's visit to Oregon State last year was, read this.
- Stanford's running game is a peach... it's the bees knees.
- UCLA's defensive shake up after poor tackling at Oregon isn't dramatic but it's something with Stanford coming to town.
- This USC injury report includes another plot turn in receiver Vidal Hazelton's quest for a redshirt year that has been met with resistance from coaches.
- When did the downfall start for Washington under Tyrone Willingham? Perhaps here.
- Will Washington State go all Stanford-2007 on USC?


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Though anguished lead single/album title track Crown of Thorns met a warm reception in the Booth, readers may nonetheless be relieved to know that its brooding tone doesn't pervade Dilated Peoples vet Rakaa Iriscience's entire freshman solo set. In contrast, the album's latest leak, Aces High is a gritty, hard-grinding joint drenched in Cali sunshine. Here, production heavyweight The Alchemist cooks up a soulfully chugging chopped-sample backdrop for rhymes by Rakaa and a few fellow Golden State elites. In addition to D.P. partner-in-rhyme Evidence, Booth-acclaimed buzzmaker Fashawn and Likwit Crew veteran Defari join the headliner to represent their home streets, stake their claim on the regional scene and, of course, wax poetic about the virtues of Cali kush ("¡Viva la cannabis!") What more could a West Coast devotee ask for? For more from Rakaa and his distinguished cast of collaborators, grab a copy of his debut solo full-length, Crown of Thorns, when it hits record store shelves and online retailers July 20.
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Death at campground in Portage ruled drowning
By AMy Lavalley Post-Tribune correspondent August 20, 2014 12:33PM
Updated: September 22, 2014 12:27PM
PORTAGE — Porter County Coroner Chuck Harris ruled Wednesday that a Portage man died after drowning at a lake in a local campground.
The body of Charles Gritton, 52, was recovered Tuesday afternoon by dive teams with the Portage, Chesterton and Porter fire departments after a search of Lake Minnehaha at Yogi Bear's Jellystone Park Camp Resort, 5300 Old Porter Road.
"I found nothing anatomically wrong with him but his lungs had water in them that would be consistent with drowning," Harris said, adding results from a toxicology screening will not be available for six weeks.
Harris did not have an estimate of how long Gritton was in the water before divers recovered his body.
The dive teams were called to the campground on the city's north side at about 3 p.m. after a 911 call from Gritton's girlfriend, who said the pair rented a campsite Monday evening and she had not heard from Gritton for several hours.
Gritton last spoke with his girlfriend at 10:30 a.m. Tuesday and told her he was going for a walk and a swim. Rescue crews found a car at the campsite with Gritton's clothes and wallet inside.
Porter Fire Department search dog Harley sniffed around the lake and found a possible point of entry for Gritton into the water. Divers searched the far west end of the lake in an organized pattern and, after about 90 minutes, found Gritton 110 feet from the shore in water that was about 20 feet deep.
The Portage Police Department assisted with the investigation.


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My First Crayola Washable Easy-Grip Markers are easy to hold but hard to roll! Markers in use stay in hand while others stay on the table. The broad nib is great for easy, colorful scribbles and fabulous first marks. Includes 3 Easy-Grip Markers.
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Dr Astrid Wingler
Molecular Plant Physiology
Our research focuses on the role of sugar signalling in plant development (in particular leaf development and senescence) and in the perception of environmental signals. For example, my group exploits natural genetic variation to investigate the genetic basis and molecular mechanisms that regulate leaf senescence. In particular, we investigate how sugar signalling integrates environmental signals, such as temperature. Most of this research is conducted with the model species Arabidopsis, but we also use a perennial relative of Arabidopsis, Arabis alpina, to explore how senescence is regulated by environmental conditions in perennial plants.


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Gerber FlashlightsGerber has developed flashlights along with the evolution of the LED. As the technology has gotten brighter and better, Gerber flashlights have evolved as well with more powerful and less expensive models. |
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Handmade Craft Ideas from 2010: A Year of Free Crafts
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Mosaic tiles very beautiful and can be turned into many different designs. Design your own with this craft project video. Heidi Borchers shows you how to construct your own masterpiece.
Use the tiles as a backsplash for your kitchen or make them into individual art pieces. Start experimenting with different colors, patterns, and designs to create the look you want. Grout comes in different colors, so don't be afraid to try them all!
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Find the greatest Motorola Droid Bionic accessories on the market here at AccessoryGeeks! We have a huge selection of Droid Bionic accessories that will enhance the use of your phone.
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Protect your Motorola Droid Bionic XT875 with an ultra sleek Motorola Droid Bionic XT875 Rubberized Hard Case - Solid White!
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I have a small question that I can't get an answer to. I assume everybody heard about rel canonical,.. ok.
So my issue is.. I have Page A, and Page B. I have to put a rel canonical tag on Page B, so that it points to Page A.
However, since it is the exact same page(dynamic content), if I put a rel canonical tag on Page B, it also appears on page A.
So basically, Page A is also pointing to Page A.
Did anybody had the same issue?.. It is ok for the canonical page to have a 'link rel="canonical"' pointing to itself?
Thank you very much !
if(strstr($_SERVER['REQUEST_URI'],"PageB.html")) { $AddTheRel="<rel link here>"; }
OR if you are using Query_Strings, maybe like this:
if(strstr($_SERVER['QUERY_STRING'],"PageB")) { $AddTheRel="<rel link here>"; }
ADDED: Sorry if I sound blunt, it's late here. =)


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AGROpreneurship Home Page
Sonoma County Beginning Farmer and Rancher Training Program
The University of California Cooperative Extension in collaboration with Santa Rosa Junior College, Small Business Development Center, and the Latino Service Providers, will provide training and a full range of supportive tools and resources to beginning farmers and ranchers in Sonoma County.
UCCE Livestock & Range Social Media Methods Evaluation
Please help me by completing the UCCE Livestock & Range Social Media for Program Delivery Survey at: http://t.co/C9koJHUPp5 Social media for UCCE Extension delivery is generally faster and less expensive than traditional methods that...
Food Safety Modernization Act Comments Due December 15, 2014
The Food Safety Modernization Act In late September, 2014, the FDA released a modified set of draft regulations for the Food Safety Modernization Act's (FSMA) Produce Rule and the Preventive Controls for Human Food Rule; they had been revised...
Observations on some behaviors of the Bagrada bug
Understanding the behavior of a pest is very important in developing appropriate control strategies. Information on feeding, host searching, migratory, and reproductive behavior of the invasive Bagrada bug is very limited in published...
Sonoma County AGROpreneurship Facebook Postings
Great public meeting to discuss the concept of an incubator farm in our county! More to come...
Shirlee Zane
Over 80 people turned out at Crane Melon Barn tonight to discuss starting an incubator farm in the Third District, rural Rohnert Park,...I hosted this meeting and champion the proposal...local production of food to feed our County's residents and nurturing the next generation of Farmers is near and dear to my heart!
Posted 1 day ago
We lost a good cheese maker, water buffalo guru, and very savvy marketer to cancer. He will be missed. this picture is from a cheese/dairy tour we did with our Agropreneurs at Ramini Mozzarella
Three farm tour in west sonoma/marin
Craig Ramini explaining his transformation from high tech to dairyman at Toluma Farms and Tomales Farmstead Creamery.
BFRs visit goats, water buffalo & chickens
Posted 7 days ago
Young Farmers & Ranchers! check them out!
Sonoma-Marin Young Farmers & Racnhers - 2015 Leadership Team
Posted 15 days ago


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Mary-Sues: Part 1Mary-Sues: Things You Need to Know and What to do if you see ThemMary-sues, Martha-sues, Marty-stus, Larry-Stus and Gary-Stus, we will all come across them at one point. Most of us have heard of them, some have been violently accused of making them via flaming, and others are still naïve to the terms. While people who have been on writing sites for years absolutely loathe them, most are inconsistent with an all-around definition. A majority claim that Mary-Sues are characters that are absolutely perfect in every shape, form and personality, while others just say that they are characters that are just too powerful, unique, or are so clichéd from past characters, and a few say they are self-inserts no matter how well-developed they are. Some on fan fiction sites even say that all Original Characters or Fan Characters (OCs) who are paired with a canon character or just take the spotlight are Mary-Sues. On the other side of the cr
The Mary Sue: Chapter 12Chapter 12: How to not be a "Mary Sue"In the previous 11 chapters of The Guide to The Mary Sue we have discussed the different traits of the characters classified as "Mary Sue", and their effect on literature as a whole. The "Mary Sue" is a disease that has been spreading throughout the world of literature since the inclusion of appealing male characters in stories. While some Mary Sues have become socially accepted, in some cases, even worshiped, i.e. Bella Swan in Twilight , there are some that have become the bane of the fanfiction community.In this chapter, our personal researcher into the world of the "Mary Sue", has some things she would like to say about how to avoid being THAT fanfic writer.We've all been there. Looking through the pages of a book, slowly falling for [insert name of attractive male character here], and we have all wanted to escape this reality and live in the world that books and other pieces of literature have created. As someone who once s
How to weed out a Flippy-Sue.Alright, so I'm writing this to help turn Flippy-Sue's into actual characters.So, I'll round it off with a few simple numbers, if you have interest in a specific point, you can locate it that way; somewhat like a guide.And I know there are probably a billion other guides that link to Flippy-Sues, but there can never be enough. Each number will also have a sort of added information-rant, I end up going off subject on the correct topic, so I may as well warn the reader.Guide by numbers:1. Making your story.2. Creating the characters image and personality.3. How not to make a Flippy-Sue, or a Sue in general.4. How to make sure your opinion of the original character links well with reality.5. Accepting a fellow writers point of view.Down to the point:1) Making your story:From experience, it is easy to think up a million stories and plots, but it's tedious to get them down on paper and on the computer. So let's break it down;---- For starters, if you are new to the writing side


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This no-rinse cleaner removes tough stains such as grease, lipstick, crayon, black heel marks, pencil marks and smoke film from almost any washable hard surface. Quart container with trigger sprayer.
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22-17074 - Anti-Fatigue Floor Mat Roll - 32 Square Feet
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28-1265 - 3V-12V 500mA Universal AC Adapter with 6 Tips
Universal Voltage Output AC Power Adapter Handy 500mA adapter is a must for the work bench or tool box. Choose from multiple DC voltage outputs and multiple, right-angle DC plugs.
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This summer, visit Melbourne's inner-city oasis. More than 10,000 plant species from around the world are presented in a kaleidoscope of colour and texture. Sweeping lawns, tranquil lakes and majestic trees are home to an amazing range of wildlife.
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Optics Planet features a distinct variety of Tripod Accessories. We're currently offering a free gift with a order of Tripod Accessories from Vanguard OpticsPlanet is a major retailer and we're working very hard to give you the top prices on Tripod Accessories. On our website, you can find customer testimonials, how-to guides and videos which will help guide you with your order. If you're continue to be undecided, you'll find product pros available that could speak with you on the phone, email and also by internet chat. Customer care is vital to us therefore we would like you to have a happy purchasing experience. So surf our variety of Tripod Accessories from Vanguard below and discover the perfect Tripod Accessories available for you. This free gift opportunity may perhaps change so that you should act now! Optics Planet is recognized for microscopes, hunting gear, military and eyewear . At Optics Planet, you'll find free shipping for the majority of orders over $29.95. So commence browsing while this offer lasts!


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Linda G. Green is a senior editor for Pool & Spa News and Aquatics International. A native of the Pacific Northwest, she obtained an Associate of Arts degree in journalism with highest honors and a Bachelor of Arts degree in Mass Communications (minor in German) with high honors. Her career has included work on daily and weekly newspapers, consumer magazines and, since moving to Los Angeles, trade publications. She finds the world of pools, spas and aquatics fascinating, and is so glad to be a part of this community.
Email: email@example.com Twitter: @LindaGreen_HW
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The Pool & Spa Show, powered by NESPA , is in Atlantic City, N.J., Jan. 27-29, 2015
The 2014 Canadian Pool & Spa Conference & Expo had something for everyone
Meet the 2014 Pleatco Perfect Guy/Gal and Spa Guy
Spas can boost a person's happiness level
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Thank you for your interest in The Foundation of Dixie Regional Medical Center. Our hope is to serve you and your family with helpful information as you learn about important life decisions involving family, healthcare, finances and estate planning. Thank you for taking the time to explore the benefits of gift planning. If you would like additional information please email us by clicking here or call us at 435-632-6827. Click here to see our Mission Video.
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