text:
- Research article
- Open Access
Cell- and region-specific miR30-based gene knock-down with temporal control in the rat brain
- Beihui Liu1Email author,
- Haibo Xu1, 2,
- Julian FR Paton1 and
- Sergey Kasparov1Email author
© Liu et al; licensee BioMed Central Ltd. 2010
- Received: 11 May 2010
- Accepted: 6 December 2010
- Published: 6 December 2010
RNA interference (RNAi) emerges as a powerful tool to induce loss-of-function phenotypes. In the context of the brain, gene manipulation is best targeted to specific subsets of cells in order to achieve a physiologically relevant outcome. Polymerase II-based viral expression systems can be used to cell-specifically express constructs incorporating flanking and loop sequences from endogenous microRNA (miRNA), which directs the designed hairpins into the endogenous gene silencing machinery. While many studies have documented non-cell-selective gene knock-down in the brain, it has not been tested whether different cell types or different areas of the central nervous system (CNS) are equally amenable to this approach. We have evaluated this issue using a tetracycline (Tet)-controllable and cell-specific miRNA 30 (miR30)-based short hairpin (shRNA) interference system.
To achieve targeted expression two cell type-specific promoters were used; the enhanced compact glial fibrillary acidic protein (GfaABC1D) promoter and the enhanced human synapsin-1 (SYN) promoter. Powerful luciferase (Luc) and the neuronal isoform of nitric oxide synthase (nNOS) gene knock-down were achieved both in vitro and in vivo. Administration of doxycycline (Dox) abrogated gene silencing. However, the efficacy of gene knock-down in both neurones and astrocytes in the hippocampus (HIP) was lower than that in the dorsal vagal complex of the brainstem (DVC). This was not due to regional differences in the expression of the the key enzymes involved in miRNA processing.
The results from the presented experiments demonstrated that selective gene knock-down in subsets of brain cells is achievable. However, there are some presently unknown regional factors which affect either the processing of miRNA-based cassettes or their potency for gene silencing.
- PC12 Cell
- Shuttle Vector
- Relative Light Unit
- 1321N1 Cell
- miRNA Processing
RNAi is a conserved regulatory process that mediates sequence-specific post-transcriptional gene silencing in a variety of species [1, 2]. The RNAi technique has become a potent experimental tool for functional analysis of genes and is believed to have significant therapeutic potential [3–6]. In the context of the brain, where numerous cell types with different functions are intermixed, gene knock-down targeted to a cellular specified phenotype in most cases would be preferable, in order to achieve a physiologically or medically relevant outcome [7, 8]. Within vector-based RNAi systems, miRNA-based cassettes, in which shRNA is embedded in a precursor miRNA context, are particularly attractive [9–12]. Endogenous miRNAs are transcribed predominantly by polymerase II (Pol II) promoters as long primary miRNAs transcripts (pri-miRNAs) that are subsequently processed into precursor miRNAs (pre-miRNAs) and eventually incorporated into the RNA-induced silencing complex (RISC) to mediate silencing of the target gene ([9]). Vectors can be made to mimic naturally occurring miRNAs whereby a designed pre-miRNA is embedded in an authentic miRNA context enabling its entry into the endogenous miRNA/RNAi pathway [9, 11, 13, 14]. The value of this approach was demonstrated by Stegmeier et al who achieved an effective knock-down with only a single viral genome per cell [15]. This is an important feature given that it is hard to achieve high copy numbers in a single cell in the brain. Moreover, large libraries of pre-designed constructs of this type are commercially available from OpenBiosystems and Invitrogen. Typically, in these constructs the miRNA-like motifs are fused to the end of a sequence encoding a fluorescent protein, giving rise to a transcript which encodes both the pre-miRNA and the reporter gene. As this is a Pol II- driven system it can be controlled by a tetracycline (or its analogue Dox)-responsive promoter element (Tre) as used in many viral systems [16–18]. If expression of the transactivator such as the Tet-off transactivator (tTA) is cell type specific, this would allow targeting gene knock-down to a specific cell type. As the same gene frequently co-exists in both neurones and astrocytes in the brain and may have different or even opposite functions, cell-selective gene manipulation has obvious advantages. In addition, for most experimental and potential clinical applications, viral gene knock-down might be most advantageous if targeted to a selected brain region(s). It is usually assumed that the mechanisms of miRNA-mediated gene knock-down are ubiquitous and operate constitutively in various tissues [3, 19–21]. However, concerning the brain, it has not yet been tested whether the same gene knock-down cassette is equally effective in different areas of the central nervous system (CNS).
There are two main objectives for this study: Firstly, we asked whether cell-specific and Dox-controllable gene knock-down can be achieved in both astrocytes and neurones. Secondly, we compared the efficacy of gene knock-down in two very different areas of the CNS, an evolutionary "old" part of the brain within the medulla oblongata involved with the control of various autonomic functions and a much "younger" part of the brain, the hippocampus (HIP). To this end, we constructed a binary Dox-controllable and cell-specific miR30-based RNAi system to express shRNAs targeting a reporter gene for Luc and an endogenous gene for nNOS. Luc was chosen as it allowed a quantifiable assessment of the efficacy of gene knock-down at the protein level. Furthermore, Luc quantification is reliable and technically straightforward. nNOS was chosen as a typical neuronal protein which is expressed in both areas of the brain we chose to study at moderate levels [22–25]. In these constructs gene targeting sequences were embedded in the precursor miRNA context derived from miR30, one of the most well-studied miRNA in mammals. This construct was fused to the 3' end of the GFP sequence and placed under control of the Tre promoter within a lentiviral vector (LVV) backbone. To drive its expression in a cell-specific manner it was co-applied with the other LVV expressing Tet-off transactivator tTA using either GfaABC1D promoter for astrocytes or SYN promoter for neurones, both promoters were transcriptionally enhanced using previously published approaches [26]. This yielded in a binary system which was both, cell-specific and Dox-controllable.
We demonstrate that significant knock-down can be achieved in a cell-selective and Dox-controllable manner for both an exogenous and endogenous targeted gene. However, there are major regional differences in the efficacy of the same gene knock-down cassette, which cannot be explained by the differential expression of the main known proteins involved in miRNA processing.
LVVs used in this study
Plasmid construction
All the LVV plasmids were based on the improved lentiviral shuttle vector pTYF-SW-Linker backbone using standard cloning procedures [27]. To generate the LV-Tretight-GFP-miR30-shRNA/Luc shuttle vector, we excised the Tretight fragment containing the modified Tet-responsive promoter from pTRE-Tight-DsRed2 (Clontech) and inserted it into the pTYF-SW Linker and cloned, into the obtained vector, PCR product of GFP-miR30-shRNA/Luc cassette from pPRIME-CMV-GFP-FF3 (kindly provided by F. Stegmeier, Harvard Medical School) downstream of Tretight promoter. To construct the LV-Tretight-GFP-miR30-shRNA/nNOS shuttle vector, we replaced the Luc shRNA sequence in the LV-Tretight-GFP-miR30-shRNA/Luc shuttle vector with the nNOS shRNA. We obtained LV-Gfa ABC1D-Luc and LV-SYN-Luc shuttle vectors by replacing EGFP in pTYF-1×GfaABC1D-EGFP and pTYF-1×SYN-EGFP [26] with the Luc cDNA from the pGL3-Basic vector (Promega) respectively. LV-mCMV/GfaABC1D-tTA and LV-mCMV/SYN-tTA shuttle vectors were both produced by replacing EGFP in pTYF-mCMV/SYN-EGFP and pTYF-mCMV/GfaABC1D-EGFP [26] with the tTA cDNA from the LV-1×SYN-tTA shuttle vector [7].
LVV production
The LVV system used in this study is derived from HIV-1 and pseudotyped with the vesicular stomatitis virus coat [28]. LVV stocks were produced by transient co-transfection of the shuttle plasmids, the packaging vector pNHP, and the envelope plasmid pHEF-VSVG in HEK293FT cells. Viral concentration and titration were carried out as described earlier [28].
Cell culture and in vitro LVV vector transduction
The in vitro transduction experiments were carried out in a neurone-derived rat pheochromocytoma PC12 cell line and a 1321N1 glial cell line from human brain astrocytoma. PC12 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 5% horse serum. 1321N1 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS. The cells were split and plated in 24-well plates at a cell density of 5 × 104 per well with 0.5 ml culture medium. After 24 h, cells were transduced overnight with appropriate LVVs in the presence of polybrene (8 μg/ml). The ratio for three vectors in all cotransfections was fixed to 1:1:1 and the total viral MOI per well was fixed to 5. Cells were then washed in phosphate-buffered saline (PBS) and cultured in full media for a further 48 h. For each virus combination, 3 wells were transduced. At the end of the incubation, cells were washed and permeabilized with 100 μl of reporter cell lysis buffer (Promega) for Luc activity assay or 100 μl of radioimmunoprecipitation (RIPA) buffer (50 mM Tris, 1% NP-40, 1% Sodium Deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, pH 7.5) plus a protease inhibitor cocktail (Sigma) for western blot analysis respectively. The lysed samples can be kept at -80°C until processing.
In vivo stereotaxic injections
All experiments were carried out in accordance with the Animals Scientific Procedures Act 1986. The animals were housed individually, allowed normal rat chow and drinking water ad libitum, and kept on a 12-hour light/12-hour dark cycle. Briefly, adult male Wistar rats (200~250 g) were anaesthetized with ketamine and medetomidine intramuscularly and their heads mounted securely in a stereotaxic frame. LVV were microinjected stereotaxically into either the DVC including the hypoglossal motor nucleus as before [8] or HIP at the following coordinates: anterior, -4.4 mm; lateral, +3.2 mm; ventral, -2.5 mm from the surface of the dura as described in [29]. A total of six microinjections of viral vectors were made bilaterally for both DVC and HIP injections. In cases where three viral vectors had to be co-injected, their ratio was fixed to 1:1:1 and the total dose was 6 × 106 infection unit (iu) per rat. In cases where two viral vectors were co-injected, their ratio was fixed to 1:4 and the total dose was also 6 × 106 iu per rat. The injection rate was 0.5 μl/min and the injection needle was allowed to remain in situ for 5 min before being slowly retracted at the end of each injection. Dox was administered at a concentration of 2 mg/ml supplemented with 5% sucrose in the animals' drinking water as required. Seven days postinjection, rats were terminally anaesthetized (sodium pentobarbital, 100 mg/kg intramuscularly) and perfused through the heart with 0.1 M PBS (pH 7.4). For Luc activity assays, the brain tissue samples were removed and stored at -80°C until processing. After adding PBS buffer (100 μl PBS per 50 mg tissue), each sample was homogenized by sonication for 10 sec on ice, and then centrifuged at 13000 rpm at 4°C. In total, 10 μl of the supernatant at room temperature was used for the Luc activity assay. For nNOS western blot analysis, the brain tissue samples were removed and immediately homogenized with a manual homogenizer in RIPA buffer containing protease inhibitor cocktail. Total protein extracts were then kept at -80°C until further processing.
Luciferase assay
Luciferase assay was performed with a luciferase assay kit (Promega) in a single-tube luminometer as described [29]. The results are expressed in relative light units (RLU) per well of a 24-well plate for in vitro experiments or per rat region for in vivo experiments. Data are expressed as the mean ± standard deviation.
Western blot analysis
The nNOS western blot analysis was carried out as previously described [30]. Briefly, total protein was extracted from homogenized samples, followed by quantification with a BCA protein assay kit (Pierce). 20 μg of total protein per lane were separated on NuPAGE 4-12% Bis-Tris gels (Invitrogen) and transferred to PVDF membranes (Millipore). The membranes were blocked in 5% non-fat dry milk (NFDM) in Tris-buffered saline with 0.1% tween-20 (TBST) for 45 min, and incubated with polyclonal rabbit anti-nNOS antibody (Zymed) at 1:5000 in 3% NFDM-TBST or monoclonal anti-beta-actin antibody (Sigma) at 1:5000 in 1% BSA-TBST overnight. Following incubation with polyclonal swine anti-rabbit immunoglobulins/HRP (Dako) at 1:5000 in 3% NFDM-TBST or polyclonal rabbit anti-mouse immunoglobulins/HRP (Dako) at 1:10000 in 1% BSA-TBST for 90 min, the immunoreactions were detected with an Immun-Star Western chemiluminescent kit (Bio-Rad) and Amersham high performance autoradiography film (GE Healthcare). Scion Image software (Scion Corporation) was used to quantitatively compare the relative blot intensities.
Northern blot analyses
Total RNA was isolated from rat tissues seven days post-injection using mirVana Isolation Kit (Ambion). Fifteen micrograms total RNA were fractionated on a 15% denaturing polyacrylamide gel and blotted onto Hybond-XL membrane (Amersham). RNA was immobilized by UV crosslinking and baking for 1 hour at 80°C. Hybridization was carried out at 42°C using UltraHyb-Oligo Hybridization buffer (Ambion). Probes were labeled with 32P using T4 polynucleotide kinase (New England Biolabs). Membranes were washed twice in 2xSSC, 0.1% SDS and 0.2xSSC, 0.1% SDS at 37°C and exposed to film. Scion Image software (Scion Corporation) was used to quantitatively compare the relative blots intensities.
Real time RT-PCR
Total RNA was isolated from cell lines and rats using RNAqueous-Micro kit (Ambion) and treated with DNase I. RNA purity was verified by performing PCR on samples not treated with reverse transcriptase. Real-time RT-PCRs were carried out using a DNA Engine Opticon 2 system (MJ Research) and the QuantiTect SYBR Green RT-PCR kit (Qiagen), as described [31]. Expression of target genes was assessed in relation to a housekeeping gene (β-actin) using the comparative Pfaffl method [ratio = (Etarget)ΔCT target (control-treated)/(Eref)ΔCT ref(control-treated)] in each sample [32]. Fold differences against DVC were calculated (n = 5). PCR primers for rat Dicer, Argonaute proteins (Ago), Digeorge syndrome critical region gene 8 (DGCR8), Exportin 5 and Drosha were designed according to sequence information provided by NCBI with the accession numbers: XM_001069041, XM_001058231, NM_001105865, NM_001108789 and NM_001107655 respectively. Among the above, XM_001069041 for Dicer and XM_001058231 for Ago are predicted sequences by automated computational analysis using gene prediction method: GNOMON, supported by mRNA and EST evidence. Primer sequences are detailed in the Additional file 1.
Combinations of vectors used in this study
Viral combinations used in this study and their functions.
Vector combination
+ LV-Tretight-GFP-miR30-shRNA/Luc
+ LV-mCMV/SYN-tTA
Neurone-specific Luc knock-down system. Tet-off transactivator tTA is expressed from LV-mCMV/SYN-tTA which contains the bidirectional amplified SYN promoter to ensure high level of tTA. tTA binds to Tretight promoter in LV-Tretight-GFP-miR30-shRNA/Luc and activates the expression of shRNA/Luc.
Astrocyte-specific Luc knock-down system. Transactivator tTA is expressed from LV-mCMV/GfaABC1D-tTA which contains the bidirectional amplified GfaABC1 D promoter to ensure high level of tTA. tTA binds to Tretight promoter in LV-Tretight-GFP-miR30-shRNA/Luc and activates the expression of shRNA/Luc.
+ LV-Tretight-GFP-miR30-shRNA/Luc
Control combination used in Luc knock-down experiments for the neurone-specific system. Since there is no tTA expression in this combination, shRNA/Luc will not be expressed. So this combination marked the background expression level of Luc driven by SYN promoter.
+ LV-Tretight-GFP-miR30-shRNA/Luc + LV-GfaABC1D-WPRE
Control combination used in Luc knock-down experiments for the astrocyte-specific system. Similar as LVVs-miRLuc-control 1, there is no tTA expression in this combination; shRNA/Luc will not be expressed. Therefore this combination marked the background expression level of Luc driven by GfaABC1 D promoter.
+ LV-mCMV/SYN-tTA
Neurone-specific nNOS knock-down system. Mechanism is similar as LVVs-miRLuc-neurone.
LV-Tretight-GFP-miR30-shRNA/nNOS + LV-mCMV/GfaABC1D-tTA
Astrocyte-specific Luc knock-down system. Mechanism is similar as LVVs-miRLuc-glia.
LV-Tretight-GFP-miR30-shRNA/nNOS + LV-SYN-WPRE
Control combination used in nNOS knockdown experiments for the neurone-specific system. Mechanism is similar as LVVs-miRLuc-control1.
LV-Tretight-GFP-miR30-shRNA/nNOS + LV-GfaABC1D-WPRE
Control combination used in nNOS knockdown experiments for the astrocyte-specific system. Mechanism is similar as LVVs-miRLuc-control2.
LVVs-miRnNOS-negative control1
+ LV-mCMV/SYN-tTA
Negative control combination used in nNOS knock-down experiments for the neurone-specific system. Instead of producing shRNA/nNOS it produces shRNA/Luc which shouldn't work on nNOS knockdown.
LVVs-miRnNOS-negative control2
+ LV-mCMV/GfaABC1D-tTA
Negative control combination used in nNOS knock-down experiments for the astrocyte-specific system. Instead of producing shRNA/nNOS it produces shRNA/Luc which shouldn't work on nNOS knockdown.
Analyses of the functions of miR30-shRNA/Luc in vitro
Of note, the astrocytic system LVVs-miRLuc-glia was more effective than the neuronal system, LVVs-miRLuc-neurone. We therefore tested whether this could reflect a higher efficacy of the amplification strategy when applied to the GfaABC1D as compared to SYN promoter. To this end, we performed a real time PCR analysis of tTA expression level from LV-mCMV/GfaABC1D-tTA in 1321N1 cells and LV-mCMV/SYN-tTA in PC12 cells as compared to LV-GfaABC1D-tTA and LV-SYN-tTA respectively. LV-mCMV/GfaABC1D-tTA increased tTA expression 14.5 fold as compared to LV-GfaABC1D-tTA while LV-mCMV/SYN-tTA increased 7.6 fold as compared to LV-SYN-tTA (data not shown). Thus, higher efficacy of the astrocytic system is likely to reflect a higher level of tTA in glial cells and therefore a higher expression of the gene knock-down cassette.
Analysis of the effects of miR30-shRNA/Luc in vivo
Surprisingly, much weaker Luc knock-down was observed using both the neurone-specific and the astrocyte-specific systems, LVVs-miRLuc-neurone and LVVs-miRLuc-glia in HIP compared to DVC [Figure 3b(1), B vs A; Figure 3b(2), B' vs A' ]. Reduction in Luc levels with neuronal system was ~ 27% (5.5 × 104 ± 1.64 × 104 vs 7.52 × 104 ± 2.48 × 104 RLU per HIP) and ~ 35% with the astrocytic system (6.32 × 105 ± 2.43 × 105 vs 9.65 × 105 ± 3.20 × 105 RLU per HIP). Neither effect was significant (P > 0.05), although the trend was clear in both cases.
nNOS gene knock-down in vitro and in vivo
We first confirmed the efficacy of the anti-nNOS construct, LV-Tretight-GFP-miR30-shRNA/nNOS in PC12 and 1321N1 cells. An adenoviral vector (AD) AD-CMV-nNOS was used to induce high level of nNOS expression in both PC12 and 1321N1 cells since there are almost no endogenous nNOS expression in these two cell lines (unpublished observation). As shown in Figure 4 in the absence of Dox, both LVVs-miRnNOS-neurone and LVVs-miRnNOS-glia markedly knocked down nNOS in PC12 cells (~ 69% reduction in optical density, treatment 2 vs treatment 1, Figure 4a) and 1321N1 (~ 82% reduction, treatment 2 vs treatment 1, Figure 4b). This nNOS knock-down effect could be prevented by Dox (treatment 3 in Figure 4a and Figure 4b). It is important to note that anti-Luc construct, LV-Tretight-GFP-miR30-shRNA/Luc (treatment 4 in Figure 4a and Figure 4b), was without effect in either cell line, indicating that the nNOS knock-down was sequence-specific.
A substantial nNOS knock-down (~ 55% reduction) (treatment 2 vs treatment 1, Figure 4c) was observed when LVVs-miRnNOS-neurone was injected into DVC in vivo and consistent with the in vitro data this knock-down could be fully prevented by Dox (treatment 3). However, similar to the experiment with Luc, the nNOS knock-down in HIP (~ 35% reduction; treatment 2 vs treatment 1, Figure 4d) was noticeably weaker. Again, the anti-Luc construct LV-Tretight-GFP-miR30-shRNA/Luc (treatments 4 in Figure 4c and Figure 4d) did not trigger nNOS knock-down.
Analysis of miRNA processing enzyme expression in DVC and HIP
This study was designed to address two questions. Firstly, we wanted to test the efficacy of Pol II-based cell-type specific and Dox-controllable gene knock-down in the brain. Secondly, we wanted to find out whether the same knock-down system should be expected to operate with equal effectiveness in different parts of the CNS. The reason for posing this question is that the lower parts of the CNS, such as medulla oblongata, differ from the higher brain in many respects, including their cellular composition, embryonic origins and gene expression patterns. Neither of these questions has been addressed previously. Moreover, we wanted to assess the effect at the protein level rather than mRNA content since it remains unclear how these two readouts of gene expression are affected by different RNAi constructs, many of which are also thought to operate via translational repression rather than through mRNA degradation.
Lentiviral systems developed in the course of this study enable tight Dox-controllable and cell-specific miR30-based RNAi gene knock-down. Using the Tet system in these designs bring additional benefits because it not only allows switching off the knock-down effect but also simplifies re-targeting of the knock-down vector to different cell types, provided that a sufficiently cell type specific promoter is available. For the Tet system to operate it is essential to achieve high levels of Tet transactivator expression. Mammalian cell-specific promoters are seldom sufficiently powerful, so we have used the previously validated bidirectional TSTA [26] to enhance two such weak promoters here: the SYN and GfaABC1D. Interestingly, the degree of enhancement achieved using the same bidirectional TSTA strategy was higher for GfaABC1D as indicated by a much higher increase in tTA transcription from enhanced GfaABC1D compared to enhanced SYN (see results Analyses of the functions of miR30-shRNA/Luc in vitro). This correlated with the overall higher efficacy of gene knock-down achieved with the astrocyte-targeted system both in vitro and in vivo than the neuronal specific system.
Our experiments show that at least in the DVC we can expect a very significant knock-down of the target protein (~ 55%) with our approach. The efficacy of knock-down seems to correlate with the power of the vector used to express the tTA, making astrocytic system slightly more potent than the neuronal one. It is clear that there is no cross-talk between neurone- and astrocyte-targeted knock-down since when Luc is placed in the phenotype not targeted by the knock-down vectors there is no decrease in expression (see Additional file 2). This also strongly argues against the involvement of any non-specific factors such as local interferon induction. Removal of the knock-down response by Dox (Figures 2, 3 and 4) is another argument for the specific nature of this effect.
Both Pol II and Pol III promoters have been used to express shRNA for RNAi [33]. Unlike most small cellular RNAs, miRNAs are primarily transcribed by Pol II, which may reflect a need for more moderate, regulatable and cell-specific expression. In the context of this study, the Pol II driven production of miRNA-based shRNA allows cell-specific knock-down and exogenous control using the Tet-system. In addition, Pol II promoters in miRNA-based RNAi systems have no strict requirements for the transcriptional start site and termination signal so theoretically various Pol II promoters can be used [2, 9, 34]. Insertion of a GFP reporter gene upstream of miR-shRNAs is thought to have at least two advantages. Firstly, this allows monitoring of the shRNA production in individual cells. Indeed we have noticed GFP expression in our experiments (data not shown). Secondly, the expression of miR-shRNAs and protein mRNAs in a monocistronic transcript could lead to both effective processing of the miR-shRNAs and translation of protein from the mRNA [15, 35, 36].
It is important to realize that when Luc was used as a target, vectors to express Luc and the knock-down LVVs were applied together as a mixture. While they should have been internalized by the same population of cells in the target area it is possible that some cells would only take in one or two vectors, but not all three. Therefore, a possibility is that some of the residual Luc expression comes from those cells where the binary knock-down system was absent or did not assemble in full (for example, no LVV to express Tre was internalized). When nNOS is chosen as a target, the protein can only come from the cells which endogenously express it in the target area. It is likely that the level of most endogenous genes will be less than those expressed exogenously by a LVV. This could be one of the reasons why nNOS knock-down was evident not only in DVC (as was the case for Luc) but also in HIP (Figure 4c and 4d) although Luc knock-down was only evident as a trend in HIP (Figure 3b).
To our knowledge, this is the first study to evaluate the different efficacies of miR30-based RNAi system among different regions in the brain in vivo. At present we do not know what accounts for this difference. We hypothesized that lower efficacy of the tested constructs in HIP compared to DVC was a result of a lower level of expression of the components involved in the RNAi pathway. However, real-time RT-PCR analysis of Dicer, Argonaute, DGRC8, Exportin 5 and Drosha revealed slightly higher levels of all of these transcripts in HIP than in DVC. Hence, other untested or unknown components must account for the difference. RNAi is a dynamic process and its machinery depends on multiple components with only poorly characterized expressions patterns [37]. Moreover, novel components involved in posttranscriptional gene silencing are very likely to emerge. Accumulating evidence indicates that many miRNAs show distinct expression patterns in an organ or tissue-specific way [37–39]. Our constructs, following the design of Stegmeir et al. used flanking and loop sequences from an endogenous miR30 [37]. A recent study uncovered 44 miRNAs which exhibited marked differences in the level of expression between spinal cord, cerebellum and hippocampus in the adult mouse [40]. For example, miR-195, miR-497, and miR-30b were found to be enriched in the cerebellum whereas miR-218, miR-221, miR-222, miR-26a, miR-128a/b, miR-138 and let-7c were highly expressed in the HIP. Unfortunately, the DVC was not studied specifically. It is not impossible that in the brain there are not only regional differences in the levels of expression of the miRNAs but also of some other factors which favour the the production and maturation of specific miRNA. If that was the case, processing of knock-down constructs built using elements of some miRNAs could be more efficient in some parts of the brain than in others as we have found herein. This should be kept in mind for loss-of function studies in the rat brain.
In summary, we have demonstrated that efficient, cell-specific and Dox-controllable gene knock-down can be achieved in the rat brain although the potency of the knock-down using the same construct may differ in different parts of the CNS. This efficient gene silencing system will be a valuable resource for basic gene function study and potentially, for the development of gene-based therapeutics of the CNS. While this manuscript was under review, a study where we have used the system for neurone-specific knock-down of nNOS was published [41], demonstrating the practical value of this approach. In that study we demonstrated that nNOS plays a key role in several pathological processes triggered in motor neurones by axotomy. Critically, the knock-down in that experiment was performed in the same brainstem area where, as we show here, this type of RNAi has proven to be most effective. Commercial availability of miRNA-like hairpins might create a feeling that any such construct may be used with the same effect in any part of the brain. This study therefore alerts the readers to avoid such an assumption when planning loss-of function studies in the rodent brain. It also illustrates the fact that more research is needed to perfect and optimise the current strategies for RNAi-mediated gene knock-down in vivo.
S elective gene knock-down in subsets of brain cells is achievable; however, there are some presently unknown regional factors which affect either the processing of miRNA-based cassettes or their potency for gene silencing.
B Liu, S Kasparov and JFR Paton were supported by the British Heart Foundation RJ/07/006. H Xu was funded by the British Heart Foundation PG/06/085. JFR Paton was also in receipt of a Royal Society Wolfson Research Merit Award. The authors thank Samantha Lane and Fiona McBryde for their help in polishing the English of this paper.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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"What is most surprising is how easy it is to keep in touch with the teachers and how engaging they are."
Before I started this program, I was substitute teaching because I made some mistakes, along the way, in my professional teaching career where my bachelor's and teaching certificate didn't matter anymore. I felt utterly defeated, and knew I deserved better — that I could do better than letting all this negativity take me down. Then I thought back to that professor in community college, whose job I wanted, where he taught both English and creative writing. I love being creative, and I am a writer with many ideas and stories untold. I want to be like my professor, to have my own class, so I can portray my passion for creativity, so I engage students in conversations, so I can help them become better writers.
However, I needed my master's in English to complete this. I made a decision back in 2013, to go back to school and take online classes, so I could work while trying to reach my goal. I was apprehensive about it because I wasn't sure how challenging it be, and I wasn't sure if it would be like my other online classes where it was so difficult that it felt like a chore; I had no idea to what to expect.
But SNHU surprised me and is still surprising me: the staff responds quickly to my e-mails, and my advisor is always keeping in touch, helping me out with class choices and answering any questions I have. What is most surprising is how easy it is to keep in touch with the teachers and how engaging they are, how involved they are, and this is all through a computer, of all things. I never would have expected this from online classes, and yet there it is. It's easy to navigate, get your books, and even get stuff from the library sent to your house.
I didn't think you could do that, and I didn't think that something done exclusively online would make you feel like you are there, even though you may be in your bedroom in another state. The great thing about SNHU online is how understanding they are about technical issues and the flexibility, so I can work part-time while attending online classes full-time.
I have been looking for other part-time work besides substituting, but I also take care of my house where I live with my dad and my uncle, and all of us work together on cooking and keeping things in order. With my dad, he and I have been a team since my mom passed away, and while we don't always agree, we do agree about how important it is to achieve in education. I know he's proud of how well I am doing in school, and that I am succeeding in my classes. I think my mom would be proud of my accomplishments if she were alive today, and she would be supporting me because she always thought it was important to further one's education.
Though she is no longer here, I feel like she's watching over me and is probably encouraging me from the heavens. I have the support of my family who thinks it's great that I'm continuing my education to further my dreams. I also have the support of my current boyfriend, who believes that I can achieve my goals. These goals could not be met without SNHU, where there is such a supportive staff, and that plays a huge role in my own success. When I had a technology problem my first term that impacted my grade, my professor wrote a form to fix that grade, so I passed the class. So far, all my professors are easy to work with, and there were times that I felt really challenged, but I think the challenge is only preparing me for the workforce. I can only hope that when it is all said and done, I will achieve my goals to accomplish my dream and obtain a brighter future.

subset: commoncrawl


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Webster defines "coincidence" as "the occurrence of events that happen at the same time by accident but seem to have some connection."
Notice that coincidence is "by accident," but that the events can also "seem to have some connection."
Writer Haddon Chambers in the early twentieth century in his drama, "Captain Swift," spoke of "The long arm of coincidence." The English poet Lord Byron wrote, "'A strange coincidence' to use a phrase/ By which such things are settled now-a-days." Thomas Hardy, writing on the destruction of "The Titanic," likened the glacier and the giant liner being "on paths coincident—twin halves of one august event/'Till the Spinner of the Years/Said 'Now!'."
So far, nothing as cataclysmic as the wreck of the Titanic has occurred, and we would hope that it would not. But indeed, it was "the long arm of coincidence," and, we believe, intervention by the "Spinner of Years" that Bruce Dyer of Dalton and I came to have a telephone conversation a few weeks ago, and it was from him I began to learn of his beloved grandmother, the late Emma Lena Nix Dyer, about whom he wanted to know more information.
It happened like this, in a string of "brought together" coincidences (or, as Bruce and I now believe, it was no 'accident'). Someone in Dalton where Bruce Dyer owns and operates several carpet-tufting mills took him a copy of "The Union Sentinel." It was the issue when we were pleading with Dyers and Southers everywhere to attend the July 15 Dyer-Souther Reunion, and especially to attend the 3:00 p.m. ceremony that day that would review the contributions of inventor Micajah Clark Dyer (1822-1891) as a portion of Georgia Highway 180 was named in his honor.
"This has something about the Dyers," the donor of the paper said to Bruce Dyer. "I thought you would like to read it."
And with that coincidental giving of the paper, Bruce Dyer was furnished with my telephone number. This was after the reunion happening on July 15, but he called me and we talked a long time. He didn't know much about his connections to the Dyers, except that his grandmother, Emma Lena Nix had married David Marcus Dyer. Beyond that, he had no inkling of whether or not he was in the direct line of Micajah Clark Dyer. I told him I had a few resources I could check, and I would be back with him in a few weeks. With that one telephone conversation, we struck a rapport, and I wanted, if possible, to help Bruce Dyer, businessman, connect with his ancestors.
To make a long story short, I had great success in tracing Bruce Dyer's roots back to his great, great grandfather, Micajah Clark Dyer, and even beyond the inventor to the first known Dyers in the line. Furthermore, I was able to trace the roots of his grandmother, Emma Lena Nix, back to the earliest known Nixes in America. I had help, of course, from online sources, from genealogy buffs like myself, Linda Trader Jordan of Gainesville, Dr. Joe Turner of Gainesville, and the five books of genealogy my cousin Watson Benjamin Dyer published from 1967 through 1988, and a helpful tome, "The Nix Family Tree," published by Wanda West Gregory in 1980.
I could hardly pull the wonderful information together fast enough and send to Bruce Dyer. On August 28 I had the second telephone call from him, thanking me profusely for my efforts and assuring me that he and his two sons, Mark and Jeff, who are in the carpet business with him in Dalton, were delighted with the results of my findings and were trying to absorb the various family connections I had unraveled for them.
Since the subject of this column set out to be about Emma Lena Nix Dyer (1889-1955), I will continue with that subject, and a lofty subject she turned out to be, indeed.
Emma Lena Nix was born November 22, 1889 in Union County, Georgia, the fifth of eight children of John Wesley Nix (1-5-1863 – 10-13-1896) and Minty Lavada Reece Nix (2-12-1863 – 8-6-1933). Emma Lena married David Marcus Dyer (1885-?) on January 6, 1907 in Union County. The couple lived in the Owltown District of Union County. David ("Dave") was a son of Robert F. Dyer (1856 - ?) and Elizabeth Fortenberry Dyer (1856-?). And Robert F.'s parents were Micajah Clark Dyer (1822-1891) and Morena Owenby Dyer (1819-1892). Indeed, I had easily traced Bruce Dyer's lineage on the Dyer side back to the nineteenth century inventor of the "Apparatus for Navigating the Air." And on the Nix side, back through John Wesley, Archibald Carr, James, William "Grancer", and John Nix.
Bruce knew some information about his grandmother whom he loved dearly. He told me when they held her funeral in Whitfield County, Georgia following her death on July 12, 1955, that "crowds of people came out of the mountains (around Owltown, Union County) to pay their respects. She and David Marcus Dyer had lived in Owltown until they moved to Dalton about 1946. She had been a noted mid-wife in the era before doctors were readily available to attend births around the countryside in Owltown. Those who came to her funeral were some of the grown-up children she had delivered, and their elderly parents who appreciated what this good woman had done for their families.
Emma Lena Nix Dyer was a devoutly religious woman. She and her family attended the Church of God in the Owltown Community. One summer, a Rev. Woody was leading a revival, and Mrs. Dyer got "in the Spirit," and in her state of spiritual ecstasy, she walked around inside the church building with her hands raised, praising the Lord. Rev. Woody fell in behind her, and so did most of the congregation. When Mrs. Dyer sat down, the preacher went back to the pulpit and resumed his preaching and the congregation seated themselves and sat listening.
She did most of her trading with traveling peddlers who came by the Dyer house in Owltown. She saved up eggs, and caught fryer chickens to barter for goods from the peddler's wagon (or, in later years, his truck). She also was noted for the produce she canned from her garden and orchard, and often traded pickles, jams, jellies and vegetables for the peddler's wares. When the peddler got back to town, people rushed to his wagon to see what Mrs. Emma Lena Dyer had traded. They wanted first choice of her goods, knowing that they were "put up" with care and attention to detail.
After World War II, the Dyer family moved from Owltown District to Dalton, Whitfield County. David Marcus Dyer was a carpenter by trade. He worked for awhile in Atlanta, building houses, until they moved to Dalton. There both of Bruce Dyer's grandparents died and were buried. The lineages on "both sides" of this family, Nix and Dyer, show a line of stalwart pioneers, salt-of-the-earth people who lived by high moral and religious standards, treated their fellowmen with respect, and left a legacy of hard work and stability.
c 2006 by Ethelene Dyer Jones; published Aug. 31, 2006 in The Sentinel, Blairsville, GA. Reprinted by permission. All rights reserved.


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District 1 Councilman Rafael Espinoza is a neighborhood advocate and architect who has championed community input and collaboration for over a decade. Rafael took office with nearly 70 percent of the vote and a public mandate to provide meaningful representation to Northwest Denver by amplifying resident voice and ensuring residents had a say in the preservation and growth of their neighborhoods.
As a champion of community voice, Rafael has created an open Neighborhood Council for residents of Northwest Denver to help advise the councilman and take action on issues concerning them. Responding to resident concerns, he is working with communities to ensure growth is appropriate and conforms to neighborhood plans and existing character; improve safety through police and civilian relations; enhance primary education; enhance District 1 infrastructure, ensure housing is available for working families; improve parks, recreation and walkability; and champion community voice to bring about win-win solutions for all Northwest Denver stakeholders.
Rafael's success as a community advocate led residents to encourage his decision to run and eventually take office. Working in tandem with neighbors, Rafael helped negotiate community supported solutions to development in Jefferson Park, stopped excessive signage on Sports Authority Field; steered the committee to draft a 20-year vision for Jefferson Park; chaired a land-use committee that incorporated community input into a high-density apartment complex on Speer and 28th; and helped encourage small-business growth in his community while continuously protecting the interests of residents.
Rafael's ability to drive solution-based advocacy is shaped by his profession. With dual degrees in building science and architecture from Rensselaer Polytechnic Institute in New York, Rafael has worked for Fentress Bradburn Architects; the Colorado Department of Transportation; and Northeast Denver Housing Center on projects such as the State Capitol, Mile High Stadium, airports, affordable housing and numerous public and private designs. Using his past experience, Rafael considers the relationships between design, project budgets, regulatory compliance, development goals, neighborhood consensus and what does and does not improve the quality of life as he works with neighbors to grow and preserve Northwest Denver communities.
Rafael and his wife Kari, also an architect, live in a home they designed and built in Jefferson Park where they spend much of their free time cheering on the Colorado Rapids and trying to stop slugs from eating their garden. If you see them around town, please stop and say "Hi".
Land Use, Transportation & Infrastructure
Community planning, North Denver Cornerstone, Public Works, parks, cultural facilities and more…
Safety, Housing, Education & Homelessness
Safety, environmental health, Denver Health, human services, education, housing and more…
Learn more about other council committees.


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As the official unveiling of the 2014 Chevy Corvette C7 draws closer, GM is doling out choice bits of information piecemeal. While much of this information is sort of obvious, like gleaning aerodynamics data from their American Le Mans racing series, it does give us a look into just how much of an improved product GM hopes to deliver with the next Corvette. After years of anticipation, the next Corvette is almost here.
Last week GM released the first official video teaser, and they followed it up with a treatise on aerodynamics. Obviously wind resistance is a big deal in the world of high-speed racing, and GM gleaned information from its motorsports efforts to sculpt the 2014 Corvette C7. GM specifically mentions the front grille and radiator flow path as a way to reduce lift and drag.
But that's not the only motorsports-minded improvement. Rather than go for a grand-tourer, like the Corvette of the 70's, GM is pushing the 'Vette as a track-capable car. Air flow is designed to be as efficient as possible, while both the transmission and differential were given heat exchangers for all-day racing fun.
Aerodynamics is all well and good, but let's talk engine. The video is hosted by Jordan Lee, the lead engineer for the Corvette's new fifth-generation LT1 engine. Lee talks about the new suite of technologies going into the LT1 engine such as direction injection. One thing we couldn't help but noticed was that on the slideshow at the beginning of the video, one of the bullet points says that this new V8 is more efficient than a twin-turbo V6.
Sounds like GM was considering such a power plant. We're happy they stuck with the V8 though, and with over a month to go before the full reveal of the C7 Corvette, we're sure there will be plenty of teasers to mull over in the coming weeks.


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Ever wondered how the Bank of England gathers intelligence to inform its decision-making process?
Eight times a year, the Bank of England's Monetary Policy Committee ("MPC") meets to determine the important monetary decisions of the day for the UK, such as how to deal with interest rates; whether to pursue quantitative easing and other issues such as how to treat or support corporate bond lending.
Ahead of the meeting, Bank of England staff provide a fair amount of analysis to the MPC, including a published summary of reports compiled by the Bank's twelve regional agents, following discussions with around 700 businesses across the UK. The summary provides information on business conditions from firms across all sectors of the economy. The summary does not represent the Bank's views, or those of any particular firm or region.
Alongside this summary of reports, the Bank's agents also produce quantitative assessments of economic conditions, as seen from their region. These scores provide numerical measures of the intelligence that they gather from month to month, and cover some areas of the economy for which there are no official statistics. The MPC then uses the intelligence provided by the agents, alongside information from other sources, to help it understand and assess current economic conditions.
The reports are online at: http://www.bankofengland.co.uk/publications/Pages/agentssummary/default.aspx
As part of this intelligence-gathering process, Sherrards were honoured to be invited to meet with the Bank and provide our views on what is happening in the economy, what the trends are, what we are seeing from our interaction with our clients, friends and associates (both domestically and, indeed, internationally), and, of course, following the EU Referendum, and the very dynamic times that we live in.
Sherrards put forward a team consisting of Jean-Paul da Costa, Head of Corporate (speaking on corporate developments and Chinese investment into the UK); Geraldine Fabre, a dual-qualified English and French lawyer (speaking on French and European attitudes to inbound investment into the UK); Andrew Cooke (speaking from a perspective of high-tech industry); Antony Mao, a property specialist (speaking on what is happening in the UK property market, from both commercial and residential angles), and Paul Marmor, Head of International Services (speaking on the business development opportunities actively being pursued by the firm).
The information was provided at a high level, in general terms, with our team encouraged to speak openly, candidly and without reservation, whatever their political and economic views, which did make for a very fulfilling and engaging discussion. Whatever else, one common view which emerged from Team Sherrards is their support for the leadership that has been provided in these challenging times by the Bank's Governor, Mark Carney.
Lai Wah Co, Deputy Agent for Greater London for the Bank of England, comments: "Meeting businesses across the country is an incredibly important exercise for the Bank, and it really does assist us to bring first-hand intelligence straight to the top. The Sherrards team had a lot to tell us and were very helpful, and we are looking forward to continued co-operation."
Jean-Paul da Costa reflects: "To have the opportunity to provide the Bank of England with our insights on what we see happening on the ground in the UK's economy is certainly a rewarding experience."
Facts about the Bank of England
- The Bank was founded in 1694, and is the central bank of the United Kingdom (sometimes known as the "Old Lady of Threadneedle Street").
- The Bank's mission is to promote the good of the people of the United Kingdom by maintaining monetary and financial stability.
- The Bank has a range of tasks, from producing secure bank notes to keeping inflation low and stable, supervising banks and insurers and safeguarding the stability of the financial system.
- The Bank's Monetary Policy Committee, which sets UK interest rates, is made up of nine members including the Governor, the three Deputy Governors (monetary policy, financial stability, and markets and banking), with the Bank's Chief Economist and four external members appointed directly by the Chancellor of the Exchequer.
- The Bank has twelve agents based around the UK, whose primary role is to assess the economic and financial conditions affecting businesses in their areas. Each agent is a part of the Bank's "eyes, ears and voice" in the regions, collecting information on trends and developments across the country, as well as explaining the Bank's policy decisions to local businesses, industry and labour groups.


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The self-inflicted wounds Facebook received from its new privacy setup are getting deeper as some users pull their information, and others quit the social network altogether.
While I think that a lot of the criticism being leveled at Facebook is
hyperbole, Facebook's new privacy regime does represent an almost 180-degree turn for the
world's largest social network.
When Facebook launched, it was a private network reserved exclusively for students and alumni at a handful of schools. But over time, as it has opened up to the world at large, it has also become more public. The motivation for this is simple: it will be impossible for Facebook to grow into the company it needs to be financially as a closed, private network. Right?
Maybe not. Much of Facebook's popularity is derived from its private nature. Even as Facebook opened up its membership, it largely left in place controls making it possible for users to maintain their privacy. Those controls are now pretty much gone, replaced with a confusing array of options clearly designed to make it difficult for users to share less of themselves with the world.
But Barry Schnitt, Facebook's Director of Corporate Communications and Public Policy, has a solution for that: lie. That's right. He told the Wall Street Journal that users can falsify certain portions of their Facebook profiles.
TechCrunch's Jason Kincaid noticed the conflict between his comment and Facebook's terms of use, and reached out to Schnitt, who reiterated:
…you don't have to indicate your current city or you can indicate that your current city is "Atlantis", "Valhalla" or, again, anything you like. We hope people will use accurate information if they are comfortable doing so because that information helps them to be found by their friends, which is part of the point of joining the site.
This might seem like an innocuous comment but Facebook's fortunes might hinge on whether or not Schnitt truly represents with his position.
Here's why: Facebook is sitting on a gold mine. Although Facebook will reportedly pull in approximately $500mn in revenue this year, most of that from advertising, much of the Facebook gold mine hasn't been tapped yet.
The gold in that gold mine, course, is user data of various kinds, from profile details to the social graph. It has immense value because it gives and will give Facebook the ability to develop innovative solutions that connect advertisers to members of Facebook's massive audience. How'd Facebook acquire its gold mine? As my colleague Meghan Keane pointed out yesterday, users have felt comfortable sharing their personal information and content with Facebook. And for good reason: prior to Facebook's about-face, much of it was hidden from public view by default. Something unique to Facebook in the realm of major social networks.
But Facebook's new anti-privacy approach may very well kill the goose that laid the golden egg. If Facebook users think twice about sharing accurate information with Facebook, and go back and remove or falsify the information they've already shared, Facebook will suffer. But it's advertisers who may suffer even more. That's because the amount and quality of the data that Facebook can give them access to as they look to target Facebook users will decrease.
Sure, Facebook can still use basic geolocation tools to figure out that you don't like in Atlantis, for instance. But what happens when users start removing other small but valuable pieces of personal information? In the aggregate, it could hamper Facebook's quest to realize the social networking dream: the ability to build a complete portrait of each individual user which can be used by an advertiser to target him or her with pinpoint precision efficiently in a one-to-one fashion.
Facebook is still far from realizing that dream, but make no doubt about it: if any company has ever had the potential to pull it off, it's Facebook. But thanks to short-sighted thinking and confusion over its identity, that dream may remain just a dream. Better luck next time, advertisers. Facebook would rather own a stream than a gold mine.
Photo credit: Andrew Feinberg via Flickr.


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"Too many games released today," says Dr. Ray
Dr. Ray says there's too many games being released these days. So stop releasing them all at once, innit.
BioWare co-founder Ray Muzyka has said there's too many games being releasd onto the market these days.
"There are too many games released today," said Muzyka, speaking to Develop.
"It's interesting, because it's very, very busy – it makes it very hard as a player to keep up. The releases clump up – even though that is changing a little bit."
Muzyka says he plays games around two-three hours per night, but it still isn't enough!
"For us, we have to play our games, play competitor's games, play other relevant games and play the handful of games we just really want to play more of and finish.
"I try and play two-or three hours a night, but that's hard and it's not enough."
What is Ray's favourite game, though? Just wondering.
"Mine was probably the original System Shock. It was awesome. I finished that game four times in 10 years.
"I loved the way it was the first true 3D adventure. The shooter mechanic in 3D meant it was very hard to control in a way, but the richness of the world was amazing.
"Our IT guys hate us for it, but we use a fan website with patches to make sure System Shock will work on our current gen laptops."
Big Ray loves System Shock. Respect.
BioWare has its own line-up of titles all due out within the next year: Dragon Age II on March 11 in the UK and Star Wars: The Old Republic at some point in 2011.
A new project from the developer will be shown at the VGAs on December 11.


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Mickey and his pals are holding court! Are you ready for some of the fastest-paced, highest-flying, hardest-slamming basketball action you've ever seen? Disney Sports Basketball brings exaggerated moves and original locations to life as your favorite Disney characters take to the court.
Game features include 2-on-2 courtside action, 6 game modes including a full range of mini games and over 25 magic items to boost performance.
Disney Sports Basketball is a three-on-three basketball game featuring many of Disney's most beloved characters. It's also one of six current and future Konami/Disney sports games. Given my experience with this title, however, I'd much rather catch a Boat Trip/The Adventures of Pluto Nash double feature than play any of the other five. The ten-year-old NBA Jam is exponentially better than this. The two-year-old NBA Street is eight kajillion times better than this. I realize that Disney Sports Basketball is targeted at children, but even the foolish youth of America, despite their love of rap music and their chronic obesity, deserve a game that's fun, don't they? Read More »
Release Region: United States
Release Date: January 15, 2003
Publisher: Konami
Release Region: Japan
Release Date: Unreleased
Publisher: Konami
Also available on: Game Boy Advance
For more info on these technical requirements and features, please view GameSpy's Specs FAQ.
Disney Sports Basketball at IGN
Disney Sports Basketball at GameSpy
Disney Sports Basketball at GameStats


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In my first Chance course in the fall of 1991 Walton's book was the main text. We basically discussed the major ideas of that book in a group discussion, taking several chapters at a time. The class contained 12 students. We had begun the course with a unit on probability and coincidences. The students had some familiarity with sequences of chance events.
The core of the book is the first 20 chapters, roughly half the book. In fact, the first 3 chapters outline Deming's philosophy (the 14 points and the 7 deadly diseases), with the remainder of these 20 explicating it. I preceded our discussions by showing the ``Against All Odds'' video mentioned above. An easily digested introduction like this seemed to be a good idea and a short video or a short, non-technical article (like those mentioned in section 1 above) plays this role.
Deming's philosophy is radical, but the students tend to have spotty experience with the world of business and consequently may not find Deming easy to discuss. There are probably various ways to overcome this problem, but two come to mind. First you could have the students apply Deming's philosophy to a context they have all experienced, such as education. They have al l gone through 12 or more years of education and have pretty strong opinions about it. Ask them to break up in to smaller groups (say 4 or 5) and discuss a series of questions about education such as (i) who is the customer/producer in the education setting?, (ii) what are the (two or three) most important of Deming's points in the education setting and what has been your experience vis a vis them, and (iii) how could education improve its performance vis a vis these points?
Another way to draw on common experience is to have students visit some local industry. We did it as a class in the fall of 1991, visiting a local, successful manufacturer of sportswear. Having do ne this tour together enhanced our ability to discuss the book. A variation on this idea would be to have different students (working in small teams) visit different local industries.
We spent 6 2-hour periods on this unit. The first period was a general discussion of the first part of Walton (after viewing the video). The next period we did the red bead experiment and discussed it. (We even plotted the data, although the technical details of control charts were not the focus.) This was very enlightening. Even though they'd read about this experiment in Walton, actually doing it themselves was very revealing. The teacher got to ham it up, intoning in the manner of Dr. Deming himself. The third period was the plant tour, followed by the fourth period where we discussed the visit. (This discussion precluded our discussing the latter part of Walton, which is a series of case studies. We read them, but having a live example was much better.)
The fifth period we performed the funnel experiment (details below). This was less than successful. They saw through it; too many of them realized intuitively that the smallest variability would occur from the no-adjustment strategy. Our sixth and final 2-hour class period was a wrap-up discussion. I am sure one could make a successful unit using far less time than this. A discussion on the first few chapters of Walton as applied to education, perhaps augmented with the red bead experiment or a video might constitute a nice 2 to 3 hour introduction to Quality. Even though Deming is the focus of this unit it is probably a good idea to alert the students to the notion that total quality management (TQM) goes beyond Deming. The paper by Snee suggests this to some extent.
I gave one writing assignment in this unit. I asked the students to write a one page essay on how well the company we visited adhered to Deming's principles, realizing that these would necessarily be based upon first impressions, and that it would be impossible to speak to all points. Most students did a good job with this assignment and found it a useful exercise to tie the tour into the reading.
On the next interation of Chance, I would retain the red-bead experiment. I would focus the initial discussion by using small groups and trying out the discussion of education. I would arrange another plant tour (maybe more than one, or maybe have them do different ones in teams). I would forgo the funnel experiment until I come up with a better understanding of how to do it. I would also consider having the students begin the unit by actually going out and collecting some data to discover that variability affects quality. For example, something as simple as comparing the percentage of in-tact cookies in competing brands of animal crackers would be a good project (albeit somewhat expensive).
Red Bead Experiment
The red bead experiment (or parable of the red beads) is explained adequately in both Walton's and Deming's books. One can buy fancy kits to perform this experiment and one could spend some time at the workbench building a paddle to lift beads from a box 50 at a time, but I used lack of money, time and woodworking skill as motivation for the following quick and dirty solution. It worked fine.
I bought at the local discount store about $15worth of marbles that are used to line the bottoms of clay flour pots. These are approximately half an inch in diameter and conveniently (at my discount store, at least) came in two colors: black and clear. I bought 12 bags of black beads and 3 of clear. There are roughly 100 marbles per bag so my box contained 1200 black and 300 clear. (These numbers are not precise and I didn't bother to count out the beads exactly as this is not necessary for the experiment.) My experiment becames then the ``clear bead experiment''. Recall that for Deming the white beads are what we wish to produce, the red beads are defects, and there are roughly 20 per cent defects in the process.
The paddle conveniently was found on the games shelf of the Moore family home. Boggle is a commercially available word game in which 16 lettered dice are shaken into a 16 cell (4 by 4) paddle that fits perfectly my marbles. If one dips the Boggle paddle into the box of marbles and lets the excess marbles fall off one obtains a neat sample of 16 marbles. Sixteen is smaller than 50 and, yes, the normal approximation to the binomial may be inadequate at this level (for the control chart calculations given in Walton), but that is clearly not the point of this exercise. This set-up worked beautifully and provided us with a very lively class.
The Funnel Experiment
I include this description here because I adequately solved the losistics of how to set it up (maybe you can find improvements in this and I'd love to hear them) but was disappointed in how it went with the students and invite you to make it work and tell me about it. I think that my students in this particular class were a bit too sharp, or perhaps I should have performed the experiment before they knew as much as they did.
I enclose the relevant pages of Out of the Crisis to describe the goal and basic set-up. My main contribution is a few details on the set-up. I bought simple kitchen funnels at the local hardware store. I borrowed the holders from the physics department; they were clamp stands they use in a number of demonstrations. When I secured the funnels in the stands they still wiggled so I secured these with rubber bands. (The funnels were a bit cocked, which didn't matter. They were stable.)
The students dropped ball bearings (that barely fit through the funnel holes but didn't bind) through the funnel and onto a large piece of graph paper. (I photo-enlarged regular graph paper.) By putting a piece of carbon paper (yes, such stuff still exists) over the graph paper in the approximate area the bearing will land the dropped bearing makes a nice dark mark on the graph paper. Of course, you want to have someone snatch the bearing out of the air after just that first bounce so you don't get extraneous marks, but my students were talented enough to handle this and yours will be too. After each drop, write the number of the drop beside the last mark to keep track of the sequence of drops.


text: On 14 Sep 2011, at 10:11, Mallory van Achterberg wrote: > Is there a (decent, maintained) Perl-based e-commerce platform > out there? No. > I can find plenty of, for example, shops running Magento and I can > see (as a user) what all comes with that. Even themeing magento is a pain in the arse, let alone extending it. I ask for danger money to work with it and even still I get fed up of recruiters calling offering nearly-danger-money. It's horrible and hateful and I never want to work with it again. > Can someone point me to a site or resource that really compares > Perl e-commerce packages to these popular PHP ones? Something > that describes all that they come with and what merchants can and > cannot do with them, without having to actually install all of these > and try setting them up just to see? Like, a review site. Don't trust any of them. And then suffer the PHP and buy cubecart or similar (the open source ones all have their various major failing, mostly around security, which is what i expect most of these 'scripts' will suffer from). However, I do have a private project going on to create a large, open source perl e-commerce platform, it just hasn't gotten very far off the ground. My background is in e-commerce, so I've seen how most platforms fall apart :) /j


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Festive Rice Stuffing
Quick Facts
Servings: 8
Prep Time: 20 min.
Cook Time: 35 min.
Total: 55 min.
The tastes of tart green apples and sweet raisins complement the undertone of curry in this versatile stuffing. Use Wehani rice or any unseasoned rice blend.
- 1 1/2 cups Wehani or rice blend
- 3 cups water
- 3/4 tsp salt
- 2 tsp canola oil
- 1 cup diced onion
- 1 tsp minced garlic
- 1/4 cup diced celery
- 2 1/2 cups sliced mushrooms
- 2 Tbs medium curry powder
- 1/4 cup apple juice
- 1/2 cup raisins
- 1 1/2 cups diced Granny Smith apple
- Rinse rice and let drain. Bring water and salt to a boil in a medium saucepan. Stir in rice, cover, and return to a boil. Reduce heat to a simmer and cook until all the liquid is absorbed and the rice is tender. Transfer the cooked rice to a strainer and rinse quickly with cold water to stop cooking. Drain the rice thoroughly, then transfer to a mixing bowl.
- As the rice cooks, heat oil in a small skillet until hot but not smoking. Add onion and toss to coat with oil. Cook, stirring constantly for 1 minute. Reduce heat and continue to cook, stirring often, until onion is soft, about 8 minutes.
- Raise heat to medium. Add garlic, celery, mushrooms, and curry. Continue to cook, stirring frequently, until mushrooms have softened, about 8 minutes. Stir in apple juice and cook just to blend.
- Add the mushroom mixture along with the raisins and apples to the rice and stir to combine. Reheat stuffing in a 350°F (175°C) oven for 20 minutes before serving.
- Serve the rice as a side dish or as a stuffing for bell peppers, prebaked winter squash, or eggplant.
Nutrition Facts
Calories 203
Calories from Fat 15 (7%)
(3%)Total Fat 2g
(1%)Saturated Fat 0g
Polyunsaturated Fat 0g
Monounsaturated Fat 1g
(0%)Cholesterol 0mg
(10%)Sodium 228mg
(8%)Potassium 284mg
Total Carbohydrate 44g
(10%)Dietary Fiber 3g
Sugars 11g
Sugar Alcohols 0g
(8%)Protein 4g
* Percent Daily Values are based on a 2,000 calorie diet.
Nutrition facts are calculated by a food expert using nutritional values provided by the USDA for common products used as recipe ingredients. Actual nutritional values may differ depending on the amounts or products used and can be affected by cooking methods.
Copyright © 2019 Healthnotes, Inc. All rights reserved. www.healthnotes.com
Read our healthy recipe definitions.
Learn more about Healthnotes, the company.
The information presented here is for informational purposes only and was created by a team of US–registered dietitians and food experts. Consult your doctor, practitioner, and/or pharmacist for any health problem and before using any supplements, making dietary changes, or before making any changes in prescribed medications. Information expires December 2019.


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- Google Retail UK Blog - the new blog replacing this one with news, insights and opinion relating to the retail industry (also follow us on Twitter!)
- Inside AdWords - the official Google blog with news and information on Google AdWords
- Google Barometer Blog - recently launched, this blog focuses on UK insights, tips and tools for planning marketing campaigns
- Conversion Room - tips on tracking and improving conversions online
We hope you'll find some of the above blogs useful. Thanks for your time.


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Crystal Metallic Stardream Card Stock
Extremely versatile, these pure white metallic sheets of card stock are suitable for any kind of creative project! Their brilliant white glistening finish means you'll be able to match them with any color and make an impression! Whether you're looking for the right card for your wedding invitations or the perfect canvas for your business cards, you are sure to love these amazing covers! From 5.5x8.5 to 13x19, you'll surely find the perfect one for your project. Plus, with a thickness of 105#, these Stardream sheets will not only last a long time but will also give you the right support for your creative ideas!
Stardream Crystal Metallic Card Stock
Starting At:$7.89Choose Sizes Below
- 105lb white cardstock with glimmering sheen and smooth texture
- Available in 12 different sizes
- Can be ordered unpunched or 8 various punch styles
- Comes in packs from 4 to 15 sheets, depending on size


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Chicken Marbella with FYSIS Oregano & Bay Leaves
Gastronomic Explorations in London
A couple of days ago we had the opportunity to visit London. After a few hours of exploring the city, our friend Rebecca suggested teaching us how to cook one of her favourite dishes: Chicken Marbella with herbs – oregano and bay laurel! How could we refuse this offer of a home cooked meal, especially when we always want to try new recipes and explore new tastes?
During a fun night filled with laughter and good company, we used FYSIS Oregano and FYSIS Bay Laurel to prepare the dish. We let it marinate overnight to let the flavours infuse and impatiently awaited the following evening, when it would be ready to go in the oven.
The dish — Chicken Marbella — is delectable and definitely worth preparing for a special occasion. Below, you can find the ingredients and steps involved to enjoy this easy-to-make dish. Kali Orexi!
- 1/2 cup olive oil
- 1/2 cup red wine vinegar
- 1 cup pitted prunes
- 1/2 cup pitted Greek olives
- 1/2 cup capers with a bit of juice
- 6 FYSIS Organic Bay Leaves
- 1/4 cup FYSIS Organic Oregano
- 1 head of garlic, peeled and finely pureed
- Coarse salt and freshly ground black pepper, to taste
- 5 – 6 chicken legs
- 1 cup brown sugar
- 1 cup dry white wine
- 1/4 cup fresh Greek (flat-leaf) parsley or fresh cilantro, finely chopped
Day 1
Combine the olive oil, vinegar, prunes, olives, capers with their juice, bay leaves, garlic, oregano, salt and pepper in a large bowl. Add the chicken and stir until all chicken has been well covered with the sauce. Then cover the bowl and refrigerate overnight.
Day 2
- Preheat the oven to 175°C.
- Use one or two large shallow baking pans to arrange the chicken in a single layer, and spoon the marinade over it evenly and then sprinkle the brown sugar over the chicken pieces and pour the white wine around them.
- Bake for 50 minutes to 1 hour, basting frequently with the pan juices.
- Transfer the chicken, prunes, olives, capers, and sauce onto a bed of orzo or a pasta of your choice and serve!
Note: To serve the dish cold, cool to room temperature before transferring the pieces to a serving platter. If the chicken has been covered and refrigerated, reheat it in the juices, then allow it to come to room temperature before serving. Finally, spoon some of the reserved juice over the chicken and enjoy.


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Gov. Jerry Brown is continuing his public outreach efforts to call attention to California's water shortage in this fourth year of drought.
He met with local leaders in San Diego Tuesday and praised the city for hitting state-mandated water conservation goals in recent months.
Brown said those water savings will be crucial as the state continues to battle wildfires.
"In the next month or two you're going to see more fires," he said. "So we have to stay vigilant and we have to be prepared to make the investments needed so that we have the water when we need it, and we protect ourselves from forest fires or other fires that are connected to the drought."
California beat the governor's 25 percent water reduction mandate last month.


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eaton house
Eaton House is the new FinTech hub and work platform where sophisticated individuals meet. We are a Social Work Club – a hybrid between a private member's lounge and a workplace. In the heart of Hong Kong's financial district, we offer you an exclusive and refined workspace, great F&B offering and unique events programming. Eaton House is the progressive platform for thought leaders who believe in collaboration.


text: Terms of Use Privacy Policy Hide Diet & Eating Harmful Health Supplements Harmful Health Supplements Supplements That Can Damage Your Health Yohimbine Phenethylamines Geranium Any Ingredient With "Andro" In The Name Bitter Orange Germander Guarana Yerba Mate Extract Kratom Bael Tree Fruit Geranium John Travis July 17, 2014 Share Tweet Flip 0 Shares Several sport supplement products have tried to pass off a harmful off-patent drug known as DMAA, as a natural constituent of geranium or its extract. It is not related to geranium, and it has been associated with numerous adverse health events and even several deaths. Several professional and Olympic athletes have lost their eligibility to compete due to DMAA. Even though the FDA has banned this ingredient it may still show up in products in stores and online, so make sure to avoid it. Prev Next Recommended Reading Health & Sports News Trending News: Your Supplements May Contain Viagra, Prozac Or Worse, FDA Warns Health & Sports News Trending News: Kratom 'Is An Opioid,' According To The FDA Commissioner Fitness & Workouts Are Custom Supplements Worth The Money? We Tried Some To Find Out Health & Sports News Trending News: Boosting Your Testosterone Can Come With Risks, Research Says Health Issues Whether You Hit The Gym Or Not, These Supplements Deserve A Spot In Your Diet


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Photography by Hanna Putz
Hanna Putz is an Artist born in Vienna in 1987. She works primarily in the medium of photography. Her work has been exhibited in the U.S. and in Europe and is published in TAR, New York Magazine, Zeit Magazin, I-D,Wallpaper*,DUST Magazine and Dazed&Confused amongst others. She is a visiting lecturer at the University of Art and Design in Linz, Austria as well as at the Bauhaus University in Weimar, Germany.


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Last week, as part of their Mobile Economy Project, the Brookings Institution invited me to participate in panel discussing the universal impact of mobile technology. The event highlighted how mobile technology is affecting economies, politics, education, and healthcare, and only just getting started. In my remarks, I argued three main points:
- The spread and impact of mobile technology is primarily a global phenomenon. There are 6 billion mobile connections in the world, and 5 billion of these are located in the developing world. We need to keep this global reach in mind when discussing how best to leverage mobile technology and telecommunications infrastructure.
- Mobile technology isn't just a change agent (as the event's title stated) but a leapfrogging agent. Outside the developed world, mobile technology has provided an opportunity to skip steps in the technology development process; mobile phones have leapfrogged telephone landlines in the developing world and completely changed the velocity of money in places like Kenya, the Philippines and Tanzania.
- The mobile web is the next frontier of growth and usage. Forecasts for 2011 to 2016 estimate that the global mobile internet data traffic will grow 18 fold. This has tremendous implications and needs to be taken into account for growing an inclusive digital economy.
These three trends show mobile technology's impact as more than an access device to important information but increasingly an authoring device. And the important question that remains is how best to make mobile technology a tool for more meaningful and global contribution.
Check out a summary of the event "Cell-Phone Connectivity Changes Economies, Health Care, Lives Across Globe" in the National Journal and the full video of the event "Mobile Technology: A Change Agent in the United States and Across the Globe".


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Text Size Large SizeMedium SizeSmall Size Color Scheme Black SchemeWhite SchemeGrey SchemePaper Scheme
Angel of Mercy
PREVIOUSLY CALLED "Ashes and Dust" SPOILER WARNING!! DISCLAIMER WARNING (i only own the main character and her family...... mineminemine) A visitor comes to Forks. She's strange and not like the others, but what if something happened between her and Jacob, something that never should have. What if her past was entwined with Edward's? What if that gets between Bella's and Edward's relationship? And what if it starts a war between the vampires and werewolves so big that it might never end?
2. What Have I Done?
Rating 5/5 Word Count 1441 Review this Chapter
The Cullens were so welcoming. They let me stay for how ever long I want. Alice forced Bella and me to go shopping with her this weekend because she thought that we lacked a decent wardrobe.
" Alice you know I hate shopping," Bella complained. I noticed that she did that a lot.
" Yeah come on you just went with Rosalie this morning," I knew that I needed to go because I have nothing to wear but I still protested.
" I don't care. Bella you need to update and Asthe don't even get me started. You don't have any clothes."
"But what about the stuff I brought with me?"
" That trash? You have got to be kidding me. A homeless wouldn't be fit to wear it."
"We are going and that's final"
"You might as well," Edwards voice came from the top of the stairs, "if Alice want something she'll stop at nothing to get it."
"Gee, thanks for the support Edward," Bella said.
" I hate to say it but he's right, besides Alice probably had a vision about it anyway."
" Yay, thank you Edward"
Today was Sunday and it was my first day at the Cullen's house. Everyone treated me like I've been living with them my whole life. Surprisingly even Rosalie was nice to me. I still tried to avoid Edward because I didn't want to trigger any memories. In 1917 I met Edward for the first time. I was already a vampire then and he was human. My "parents" knew his because we also were one of the richest families in Chicago. They introduced us on March 3. I fell for him as any girl would and the next year, 5 weeks before the influenza hit, he proposed. Unfortunately by that time my family was wanted by the Volturi. Staying was not an option and a week later we left. I couldn't tell Edward anything; our entire family disappeared from the city. Four weeks after we left, the Spanish influenza swept through and the Masens were caught in it. The last news I heard of them was that both Elizabeth and Edward sr. were dead. What happened to their son I did not know… until now. I guess he doesn't remember anything because he never mentioned it when he got a chance. I'm thankful because I don't want to come between anybody.
The day crawled by and I got really bored. The Cullens left to go hunting and Alice was babysitting Bella at her house. I got the place all to myself. Why not go exploring, I thought, might as well check out the surroundings. Before leaving I decided to test out my latest ability. I have a ton of them. The new one allows me to mask my scent so no one will be able to tell if I'm a vampire or not. It also allows me to control my paleness. I can change the shade of my skin so that it's a little darker than Bella's. I probably wouldn't need it but just in case, you never know.
I left the house a few minutes later after I made the decision. As I was running in the forest I came across the strangest scent. It war repulsive and smelled sickly musky. Maybe I should turn around, was the first thought that went through my head. Curiosity got the better of me and I decided to follow the scent at a human jog, just for extra precaution since I was already using my ability.
About a mile later the smell got so strong that I had to stop breathing. I was in a small forest right outside of Seattle. The scent was comin from a clearing up ahead. When I entered it the firs thing I saw was a guy sitting in the middle of the clearing. As soon as I entered he turned his head towards me and we both froze. His face looked like it belonged to a seventeen year old but his body looked like it belonged to a 25 year old. He had dark, copper skin; he probably belonged to one of the Indian tribes that are around here.
The girl was breathtaking with electric blue eyes and brown hair so dark it almost looked black. She was amazing and for a second I forgot about everything and just stared at the girl. She was probably no older than 18.
" Are you lost?" I decided to speak.
"No, I was just exploring," her voice was like a bell, clear and strong. Perfect just like her.
"Where do you live? There are no roads leading to Seattle from here."
"I don't live in Seattle."
"Then where do you live?" There was something in her eyes and voice that made me think she was hiding a secret.
"In Forks."
Forks! I've never seen her there.
"Did you just move here?"
"Are you alone or do you live with somebody?"
"I live alone."
" You look a little young to love alone."
"No, I'm 18 and legal."
Should I be telling him this? He is a stranger after all. I couldn't stop myself I felt so at ease with him and every time I looked into his eyes I felt that my heart would skip a beat if it could.
"Well welcome to Washington then," he had a gentle voice and there was something strange in it. I came up and sat next to him.
"What's your name?"
"Astheroshe Saunière, and you?"
"I'm Jacob Black and I live on the Quileut Reservation. It's about northwest of Forks."
"What are you doing here then Jacob?
" Exploring." Haha funny, that was my response.
" Do you need a ride home?" he was offering me a ride. I wondered if I should go. Why not, if he's some criminal or something I can fix that.
"Sure, thanks."
" Come on the car is this way."
During the entire ride we talked about everything we could possibly think of. At some point we even talked about global warming. That was so random it was hilarious. By the time we were anywhere near Forks it was really late.
" Wow it's almost midnight aren't you tired? You've been driving for a while."
"No I'm ok, what about you."
"I think I'm gonna collapse," I lied
" Why don't you stay at my place? It's just at the next right. Forks is farther."
" Sure," I blurted out before I could even think about it. What would the Cullens say? I'm probably in so much trouble.
We arrived at a nice small house and as soon as we parked a man in a wheelchair came out of the house. I assumed it was Jacob's dad and I was right.
"Jake! Where have you been all day?" His dad had an expression of relief on his face.
"Relax dad. Listen, a friend of mine is staying over." I noticed that Jake didn't answer his question.
"Sure who is it?" As soon as he said that Jake motioned for me to get out of the car. When I came out of the car, I saw a look of nervousness, happiness, and worry pass across his face. He quickly composed his face back to that calmness he had before.
"Hello, what your name?"
" That's a very interesting name. Welcome to our home."
"Thank you"
Jacob said I could sleep in his room while he takes the couch.
" I don't think you'll like it there. The couch is really lumpy."
" 'Kay. Good night."
" Night," I heard Jake pause " Astheroshe?"
When I turned to face him, he was standing about an inch from me. He lowered his head so it was on the same level as mine. All I could do was breathe, not that I needed to. All of a sudden just when I thought he was about to leave, he put his hands on my face and our lips met. He kissed me softly, carefully like he was afraid I might break. If only he knew. I couldn't resist so I put my hands around his neck and pressed my body to his. When we parted we were both out of breath.
"Good night Astheroshe."
" Good night Jake."
I briefly kissed him one more time before he left the room. I wonder what I'm gonna tell Carlisle tomorrow. What have I done? I thought to myself as I layed on the bed quietly thinking of the consequences.
- Author
- vampire princess
- Rating
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- 27 Aug 07
- Updated
- 03 May 10
- Status
- In Progress


text:
both GP B and C seem to have the wrong titles and don't reflect the original meaning. B used to be: Indicate any limitations or constraints C used to be: address the impact of the option suggest: B: indicate limitations or constrains on the optional feature C: address the implication of the optional feature --lynne At 10:52 AM 11/16/2004, Karl Dubost wrote: >QA WG, > >Look at the title of 4.2C and tell me if you think it's wrong. > >4.2 Good Practice A: Make sure there is a need for the optional feature >4.2 Good Practice B: Clearly identify optional feature >4.2 Good Practice C: Indicate optional feature > (previous was: Indicate any limitations or constraints) > >-- >Karl Dubost - <http://www.w3.org/People/karl/>http://www.w3.org/People/karl/ >W3C Conformance Manager >*** Be Strict To Be Cool *** > > >Received on Tuesday, 16 November 2004 16:07:14 UTC
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