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|NS Class 8600/8700/9400/9500
Two coupled VIRM units at Amsterdam Bijlmer ArenA
Downstairs interior of a VIRM train.
|In service
|1994 - present
|De Dietrich Ferroviaire
|Number built
|178 sets
|6 cars (8600/8700)
4 cars (9400/9500)
|8600/8700 - 129 (1st), 442 (2nd)
9400/9500 - 61 (1st), 330 (2nd)
|NS Reizigers
|Maximum speed
|160 km/h (99 mph)
|Power output
|2,388 kW (3,202 hp) (8600/8700)
1,592 kW (2,135 hp) (9400/9500)
|Electric system(s)
|1.5 kV DC Catenary; all units have extra space for equipment to operate on 25 kV AC Catenary
|Current collection method
|Track gauge
|1,435 mm (4 ft 8 1⁄2 in) standard gauge
VIRM trains, full name Verlengd InterRegio Materieel (Lengthened interregional rolling stock), are a series of electric multiple unit (EMU) double-deck trains operated by Nederlandse Spoorwegen or NS (Dutch Railways), the principal railway operator in the Netherlands. NS has 178 of these double-deckers — 98 four-carriage sets, and 80 six-carriage sets. The trains were built between 1994 and 2009 - for the most part by Talbot, part of Bombardier Inc., with some railcars built by De Dietrich.
The VIRM trains evolved out of the previously existing DD-IRM series (DubbelDeks InterRegio Materieel or Double-deck interregional rolling stock). The first batch of 81 VIRM trains was created by lengthening all the existing DD-IRM combinations by one or two railcars. Three-unit trainsets were augmented by one carriage to transform them into a four-unit VIRM, and the four-unit sets received two more carriages, to create six-unit VIRM trains.
- Regiorunner
- Dubbeldeksinterregiomaterieel
- Verlengd InterRegio Materieel
First generation[edit]
The first generation of DD-IRM were delivered from 1994 to 1996 as three and four car units. The three car units were delivered in the 82xx class and the four car units delivered as 84xx. The first batch of 290 double deck coaches (individual) were delivered. The first 47 were built by De Dietrich and from then on by Talbot.
As the coaches were individual but would run as a unit, they are individually numbered. Collectively they have a set number e.g., 8201, having coach number 380 8001. However, there are no double numbers, for example there can not be an 8201 and 8401 at the same time.
In 2000 an order was placed for new coaches. The 3 car sets were to be upgraded to 4 car sets, and the four car sets upgraded to 6 car sets. This is why they are now called VIRM (V = verlengd - extended). These were renumbered into the 94xx series and the 86xx series. These sets were formed between 2001 and 2005.
Second generation[edit]
The second generation of VIRM were new sets built to the 4 and 6 coach formations, at the same time as the first generation were rebuilt. These were numbered 95xx (4 car units) and 87xx (6 car units). These sets featured different seats, which were also in the new coaches placed inside the first generation sets.
Third generation[edit]
These sets were delivered from June 2008 and these are all 4 car units. They are numbered 9547 - 9597. These sets differ in that they do not feature the catering lift, meaning the stairs could be wider and the destination screens inside the coaches have disappeared, where in the future screens with more information will be put in place.
|vehicle type
|Short Trainsets
Long Trainsets
|8201...8281 (34)
8401...8481 (47)
|9401...9481 (34)
8601...8681 (47)
|9502...9525 (13)
8701...8723 (12)
8726–8746 (21)
|9547–9597 (51)
|mBvk1/2 290
|8501–8662 (162)
|8663–8712 (50)
|8713–8754 (42)
|8759–8861 (103)
|ABv3/4 380
|8001–8081 (81)
|8101–8125 (25)
|8126–8146 (21)
|8147–8197 (51)
|ABv5 380
|8201–8247 (47)
|8248–8259 (12)
|8260–8280 (21)
|ABv6 380
|8301–8381 (81)
|8382–8406 (25)
|8407–8427 (21)
|8428–8478 (51)
|mBv7 260
|8801–8847 (47)
|8848–8859 (12)
|8860–8880 (21)
- 34 trainsets in 9401–9481, no double numbering with 47 trainsets in 8601–8681
- 12 trainsets 8701...8723 only odd numbers[1] plus 13 trainsets 9502...9525 remaining numbers
- mBvk1/2 8755–8758 are reserve carbodies, 8755 has become replacement 8718
- mBvk1/2 8829 was built as a replacement vehicle for an older trainset
Different liveries[edit]
- 8733 - This featured adverts between the two decks, advertising that it was the 300th VIRM set built. Livery no longer carried.
- 9514 - This carries adverts for Lekker lezen in de trein, Comfortable reading in the train.
- 9525 - This wore the adverts for the NS/Olympic partnership.
- 9522 – This wore a white/magenta livery advertising the OV-Chipkaart.
- 9524 – This wore advertising liveries for Mamma Mia and The Lion King. As well as that, it wore a completely white/magenta livery, advertising the OV-chipkaart similar to unit no. 9522.
- 9520 - This carried an orange/blue livery advertising Queen Beatrix Wilhelmina Armgard resigning from the throne.
Services operated[edit]
The VIRM is now the main intercity train and can be seen across most of the NS network.
Green Train[edit]
In 2015, NS hopes to let 50% of their trains run on electricity powered by windmills. By 2018, all the trains should be running on "clean" electricity.[2] To celebrate this and the fact that the first Dutch railway line was opened 175 years ago, NS and Eneco Energy held a competition. Designers had to design a green train.
The winners were two students from the Technical University in Delft. Their names are Michiel van Sinderen and Steffen de Jonge. Their design was to have the train be checkered with small black and blue squares. From even a small distance the train appears to be green. Michiel van Sinderen said it was the most beautiful thing he had ever made.[3] This design was brought on VIRM number 9556. It was named De Groene Trein which means The Green Train. The difference between this train and other VIRMs is the paler shade of yellow and white arrows on the doors, similar to those on DDZ trains and modernized ICRm railway cars.
The train debuted on September 20, 2014. Its maiden voyage was an extra trip from Amsterdam Centraal to Haarlem. Now it can be spotted and ridden anywhere in the Netherlands.
Accidents and incidents[edit]
- On 21 April 2012, unit 8711 was one of the two trains involved in a head-on collision near Amsterdam Sloterdijk station.
External links[edit]
|Wikimedia Commons has media related to DD-IRM.


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Telephone: 81-78-306-3170; Fax: 81-78-306-3171
Tissue-Specific Stem Cells
Gli2 Is a Novel Regulator of Sox2 Expression in Telencephalic Neuroepithelial Cells†‡§
Version of Record online: 5 JAN 2009
DOI: 10.1634/stemcells.2008-0580
Copyright © 2008 AlphaMed Press
Volume 27, Issue 1, pages 165–174, January 2009
Additional Information
How to Cite
Takanaga, H., Tsuchida-Straeten, N., Nishide, K., Watanabe, A., Aburatani, H. and Kondo, T. (2009), Gli2 Is a Novel Regulator of Sox2 Expression in Telencephalic Neuroepithelial Cells. STEM CELLS, 27: 165–174. doi: 10.1634/stemcells.2008-0580
- †
Author contributions: H.T.: conception and design, manuscript writing, collection, data analysis and interpretation; N.T.-S. and K.N.: data analysis and interpretation; A.W.: provision of study material; H.A.: financial support, provision of study material; T.K.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript.
- ‡
Disclosure of potential conflicts of interest is found at the end of this article.
- §
First published online in STEM CELLSExpress October 16, 2008.
Publication History
- Issue online: 5 JAN 2009
- Version of Record online: 5 JAN 2009
- Manuscript Accepted: 28 SEP 2008
- Manuscript Received: 18 JUN 2008
- Neural stem cells;
- Sox2;
- Gli2;
- Hes5
- Top of page
- Abstract
- Supporting Information
Multipotential neural stem cells (NSCs) in the central nervous system (CNS) proliferate indefinitely and give rise to neurons, astrocytes, and oligodendrocytes. As NSCs hold promise for CNS regeneration, it is important to understand how their proliferation and differentiation are controlled. We show here that the expression of sox2 gene, which is essential for the maintenance of NSCs, is regulated by the Gli2 transcription factor, a downstream mediator of sonic hedgehog (Shh) signaling: Gli2 binds to an enhancer that is vital for sox2 expression in telencephalic neuroepithelial (NE) cells, which consist of NSCs and neural precursor cells. Overexpression of a truncated form of Gli2 (Gli2ΔC) or Gli2-specific short hairpin RNA (Gli2 shRNA) in NE cells in vivo and in vitro inhibits cell proliferation and the expression of Sox2 and other NSC markers, including Hes1, Hes5, Notch1, CD133, and Bmi1. It also induces premature neuronal differentiation in the developing NE cells. In addition, we show evidence that Sox2 expression decreases significantly in the developing neuroepithelium of Gli2-deficient mice. Finally, we demonstrate that coexpression of Gli2ΔC and Sox2 can rescue the expression of Hes5 and prevent premature neuronal differentiation in NE cells but cannot rescue its proliferation. Thus these data reveal a novel transcriptional cascade, involving Gli2 Sox2 Hes5, which maintains the undifferentiated state of telencephalic NE cells. STEM CELLS2009;27:165–174
- Top of page
- Abstract
- Supporting Information
The central nervous system (CNS) contains multipotent neural stem cells (NSCs) that are capable of self-renewal and give rise to neurons, astrocytes, and oligodendrocytes [1, 2]. Both extracellular and intracellular factors help to maintain NSC proliferation and multipotentiality; however, details of the mechanisms involved are not known.
Sox2 is a member of the SRY-related box (Sox) gene family, which encodes transcription factors with a high-mobility group DNA-binding domain [3]. Several lines of evidence suggest that Sox2 maintains the developmental potential and proliferation of NSCs [4–7]: neuroepithelial (NE) cells in the ventricular/subventricular zone (VZ/SVZ) express Sox2 but progressively lose it as the cells differentiate [4–7]. Overexpression of Sox2 in NE cells blocks differentiation, whereas overexpression of a dominant-negative form of Sox2 results in the loss of NSC markers and the acquisition of early neuronal markers [6, 7]. Together, these findings have suggested that Sox2 is required for NSCs to maintain their "stemness."
The way in which sox2 expression is maintained in NE cells remains unclear. We previously identified an enhancer (designated R1) that is required for sox2 expression in telencephalic NE cells. This R1 sequence associates with Brm-containing SWI/SNF complex, and the factors within this complex induce sox2 expression in cultured NE cells [8]. As it is unlikely that Brm recognizes a specific DNA sequence, we have attempted to determine what DNA-binding factor recognizes the R1 enhancer and regulates sox2 expression in telencephalic NE cells.
In the present study, we show that Gli2, a key mediator of sonic hedgehog (Shh) signaling, binds to the R1 enhancer in cultured NE cells, whereas two other mediators of Shh signaling, Gli1 and Gli3, do not. Overexpression of either a truncated form of Gli2 (Gli2ΔC) or Gli2-specific short hairpin RNA (Gli2 shRNA) in NE cells, in vivo and in vitro, inhibits the expression of Sox2 and other NSC markers, such as Hes1, Hes5, Notch1, CD133, and Bmi1; it also induces the premature differentiation of NE cells into βIII-tubulin-positive neurons in vivo. We present evidence that Sox2 expression is reduced significantly in the developing NE cells of Gli2-deficient mice. Moreover, we demonstrate that overexpression of sox2 in Gli2ΔC-expressing NE cells blocks premature neuronal differentiation and rescues the expression of Hes5, but not that of Hes1. Together, these findings reveal that Gli2 is a novel regulator of sox2 expression in telencephalic NE cells.
- Top of page
- Abstract
- Supporting Information
Animals and Chemicals
Animals were obtained from the Laboratory for Animal Resources and Genetic Engineering at RIKEN Center for Developmental Biology. Gli2 mutant mice were provided by J. Motoyama [9]. Chemicals were purchased from Sigma-Aldrich (St. Louis, MO http://www.sigmaaldrich.com), except where indicated.
NE cells were prepared from embryonic day (E) 14.5 mouse telencephalon and expanded in basic fibroblast growth factor (bFGF; 10 ng/ml; Peprotech, Rocky Hill, NJ, http://www.peprotech.com) as described before [10]. Transfection of NE cells was performed as described previously [8]. In brief, the cells (2 × 106) were transfected with the vector (10 μg) using the Nucleofector device (Amaxa Inc., Gaithersburg, MD, http://www.amaxa.com) and then cultured in bFGF. To examine the effects of the transgenes, the transfected cells were harvested, and 5 × 104 cells per well were recultured on poly-L-ornithine (15 μg/ml)- and fibronectin (1 μg/ml; Invitrogen, Carlsbad, CA, http://www.invitrogen.com)-coated cover glasses (f13 μm; Marienfeld, Lauda-Königshofen, Germany, http://www.marienfeld-superior.com).
In Utero Electroporation
In utero electroporation was performed using E14.5 ICR mice, as described before [11]. In brief, 1–2 μg of plasmid was injected into the left side of the lateral ventricle of the embryonic brain and electroporated to the cerebral cortex with an Electro Square Porator BTX 830 (BTX Harvard Apparatus, Holliston, MA, http://www.btxonline.com).
The dissected mouse brains were fixed in 4% paraformaldehyde overnight at 4°C. After fixation, the brains were cryoprotected with 12%–18% sucrose in phosphate-buffered saline (PBS) and embedded in OCT (Sakura Finetek, Torrance, CA, http://www.sakura.com). Coronal sections (10 μm thick) were prepared from the cerebral cortex. The sections were pretreated with 0.3% Triton X-100 in PBS for penetration and then treated with blocking solution (2% skim milk, 0.3% Triton X-100; PBS) for 1 hour and incubated with primary antibodies for 16 hours at 4°C. Immunostaining of the fixed cells was carried out as described previously [8]. The following antibodies were used to detect intracellular antigens: rabbit polyclonal anti-Sox2 (diluted 1:1,000; StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) (specificity of the Sox2 antibodies is shown in supporting information Fig. 1), rabbit polyclonal anti-Hes1 (diluted 1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), rabbit polyclonal anti-Hes5 (diluted 1:100; Abcam, Cambridge, United Kingdom, http://www.abcam.com), rabbit polyclonal anti-Gli2 (diluted 1:200; Abcam), rabbit polyclonal anti-CD133 (diluted 1:100; Abcam), mouse monoclonal anti-βIII-tubulin (diluted 1:200; Sigma-Aldrich), mouse monoclonal anti-5-bromo-2′-deoxyuridine (anti-BrdU; diluted 1:400; G3G4; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/∼dshbwww), rat monoclonal anti-green fluorescent protein (anti-GFP; diluted 1:1,000; Nacalai Tesque Inc., Kyoto, Japan, http://www.nacalai.co.jp/en), and rabbit polyclonal anti-GFP (diluted 1:1,000; Abcam). The antibodies were detected with Alexa 568-conjugated goat anti-mouse or rabbit IgG (diluted 1:200; Molecular Probes, Eugene, OR, http://probes.invitrogen.com) or Alexa 488-conjugated goat anti-rabbit or rat IgG (diluted 1:200; Molecular Probes). To visualize all nuclei, the cells were counterstained with Hoechst 33342 (1 μg/ml). Fluorescence images were obtained by using a Fluoview FV1000 confocal laser-scanning microscope (Olympus, Tokyo, http://www.olympus-global.com), and Sox2 intensity (arbitrary units) was analyzed by FV10-ASW, version 01.06.01.00 (Olympus).
For BrdU staining, 500 μl of 10 μg/ml BrdU was injected into the pregnant mice intraperitoneally, and the pups were dissected 1 hour later. For the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, In Situ Cell Death Detection Kit TMR red was used according to the supplier's instructions (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com).
Luciferase Assay
Luciferase assays were carried out as described previously [8]. In brief, NE cells were transfected with 1 μg of sox2-enhancer-firefly luciferase expression vectors and 0.04 μg of the internal control vector pEF-Rluc (gift from S. Nagata and K. Shimozaki, Osaka University, Osaka, Japan) using the Superfect Transfection Reagent according to the supplier's instructions (Qiagen, Hilden, Germany, http://www1.qiagen.com). After 24 hours, the activities of the two luciferases were measured using the Dual-Luciferase Reporter Assay System according to the supplier's instructions (Promega, Madison, WI, http://www.promega.com).
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation (ChIP) assays were carried out as described previously [8]. In short, NE cells were transfected with a control vector, pcDNA3.1-HisB-mGli1, pCMV/FLAG/SV-Gli2 full (both HisB-mGli1 and FLAG/SV-Gli2 expression vectors were gifts from H. Sasaki, RIKEN CDB, Kobe, Japan), or pcDNA3-FLAG-Gli3 using a Nucleofector device (Amaxa). On the following day, transfected NE cells were fixed and sonicated. The cell extracts were harvested by centrifugation and immunoprecipitated with anti-FLAG M2 antibody (1:100 dilution) or anti-His antibody (1:100 dilution; Santa Cruz Biotechnology), and protein G-Sepharose (50 μl of a 50% suspension; GE Healthcare, Little Chalfont, U.K., http://www.gehealthcare.com). The precipitated DNA fragments were purified by phenol/chloroform extraction and used for polymerase chain reaction (PCR) with primer sets for R1, R3, and Patched1 promoter (PTCH1). The following oligonucleotides were synthesized to amplify PTCH1: the 5′ primer was 5-CGCTCGCAAAAGGCGTCTCG-3, and the 3′ primer was 5-AAAGGCTGGAACTCCCGCCC-3. PCR conditions were 30 seconds at 94°C, 30 seconds at 61°C, and 30 seconds at 72°C. Cycle parameters were 50 cycles for R1, R3, and PTCH1.
Reverse Transcription-PCR Analysis
Reverse transcription (RT)-PCR was carried out as described previously [8]. The following oligonucleotides were synthesized. For sox2, the 5′ primer was 5-TTGATATCATGTATAACATGATGGAGAACGGAGCT-3, and the 3′ primer was 5-CTCCAGCCGTTCATCTGCGCGTA-3. For gapdh, the 5′ primer was 5-ACCACAGTCCATGCCATCAC-3, and the 3′ primer was 5-TCCACCACCCTGTTGCTGTA-3. For hes1, the 5′ primer was 5-CAGCCAGTGTCAACACGAC-3, and the 3′ primer was 5-TGATCTGGGTCATGCAGTTG-3. For hes5, the 5′ primer was 5-CGACTGCGGAAGCCGGTGGT-3, and the 3′ primer was 5-AGCAGCTTCATCTGCGTGTCG-3. For notch1, the 5′ primer was 5-AGCAGTCTGCCTGTGCACAC-3, and the 3′ primer was 5-AAATGCCTCTGGAATGTGGGT-3. For bmi1, the 5′ primer was 5-GGGTACTTCATTGATGCCAC-3, and the 3′ primer was 5-AGGTTCCTCTTCATACATGAC-3. For c-myc, the 5′ primer was 5-CACTGGTCCTCAAGAGGTGC-3, and the 3′ primer was 5-TGTCGACTTATGCACCAGAGTTTCGAAGC-3. For bcl2, the 5′ primer was 5-AGAATTCGCCACCATGGCGCAAGCCGGGAGAAC-3, and the 3′ primer was 5-TCTCGAGTCACTTGTGGCCCAGGTATGC-3. PCR conditions for sox2 and gapdh were 30 seconds at 94°C, 30 seconds at 58°C, and 40 seconds at 72°C, and cycle parameters were 45 cycles for sox2 and 35 cycles for gapdh. PCR conditions for hes1, hes5, notch1, and bmi1 were 30 seconds at 94°C, 30 seconds at 55°C, and 40 seconds at 72°C, with cycle parameters of 50 cycles for hes1, 40 cycles for hes5, 55 cycles for notch1, and 65 cycles for bmi1. PCR conditions for c-myc and bcl2 were 30 seconds at 94°C, 30 seconds at 59°C, and 50 seconds at 72°C, and cycle parameters were 40 cycles.
Statistical Analysis
Statistical analyses of Sox2 intensity and proportion of Hes1, Hes5, βIII-tubulin, TUNEL, and BrdU-positive cells from immunohistochemistry were carried out by analyzing at least four slide glasses from one embryo. At least 100 cells were counted from each slide, and statistical analyses were performed using Excel Stacel2 (Microsoft, Redmond, WA, http://www.microsoft.com). Similar results were obtained from three independent embryos, and representative results are shown in each figure.
- Top of page
- Abstract
- Supporting Information
Gli2 Is an Essential Factor for Sox2 Expression in Cultured NE Cells
We first examined whether the R1 enhancer contains any recognizable sequences for transcription factor binding and found two potential Gli-binding sites (Fig. 1A, GBS1 and GBS2). There are three mammalian Gli proteins (Gli1–Gli3) that are regulated by Shh signaling, which plays a crucial part in CNS development [12]. It has been shown that Gli2 and Gli3 are expressed in the VZ/SVZ and are required for the proliferation of NE cells [13–15]; although Gli1-deficient mice are capable of developing normally, both Gli2-deficient and Gli3-deficient mice die during the perinatal period, with severe defects in the brain [14, 16–18]. Gli2 is a transcriptional activator, and Gli3 acts primarily as a repressor, although it is converted to a weak activator in the presence of Shh [12, 19, 20]. Moreover, we found that Gli2 is strongly expressed in NE cells, most of which are positive for Sox2, in culture and in developing VZ/SVZ (supporting information Fig. 2). Together, these findings suggested that Gli2 may be a primary regulator of sox2 expression in NE cells.
To test this possibility, we transfected NE cells with either a pCMS-enhanced green fluorescent protein (EGFP) control vector or a pCMS-EGFP-Gli2ΔC (Gli2ΔC) vector. One day after transfection, the cells were fixed and immunostained for Sox2 and GFP; more than 95% of control vector-transfected NE cells were labeled for Sox2, whereas less than 20% of the Gli2ΔC-expressing cells were so labeled (Fig. 1B, 1C). The proportion of Sox2-positive cells among the Gli2ΔC-transfected cells had decreased during the culture period (Fig. 1C). To confirm that overexpression of Gli2ΔC prevents sox2 expression, we transfected NE cells with either the control vector or the Gli2ΔC vector, purified GFP+ cells by flow cytometry, and then examined sox2 expression by RT-PCR. As is shown in Figure 1D, overexpression of Gli2ΔC significantly inhibited sox2 expression in the cultured NE cells. As Gli2ΔC has been shown to interfere with the function of Gli2 and Gli3 [12, 19], we constructed three other vectors: a control vector encoding GFP and a scrambled shRNA (scrambled), a Gli2-specific shRNA expression vector encoding GFP and a Gli2-specific shRNA (Gli2 shRNA), and a Gli3-specific shRNA expression vector encoding GFP and Gli3-specific shRNA (Gli3 shRNA) (specificity of Gli2 and Gli3 shRNAs is shown in supporting information Fig. 3). We transfected the NE cells with these vectors, used flow cytometry to purify GFP+ cells, and then analyzed GFP+ cells for sox2 expression by RT-PCR as described above. Overexpression of Gli2 shRNA significantly knocked down sox2 expression in cultured NE cells, whereas overexpression of either scrambled or Gli3 shRNA did not, indicating that Gli2, but not Gli3, is required for sox2 expression in cultured NE cells.
Gli2 Associates with the R1 Enhancer of the Sox2 Promoter in Cultured NE Cells
To address whether Gli2 directly regulates sox2 expression in cultured NE cells, Gli-binding sites (GBS; Fig. 1A, GBS1 and GBS2) in the R1 enhancer were first examined to determine which is critical for sox2 expression. GBS1 and GBS2 sequences were replaced with random sequences, making mR1-GBS1 and mR1-GBS2, respectively; they were inserted into the pGL3 promoter vector, which contained a minimal SV40 promoter upstream of a firefly luciferase gene (Fig. 2A, left schematic diagrams). The NE cells were transfected with R1, R3 (which has no enhancer activity), mR1-GBS1, or mR1-GBS2, along with a vector (pEF-Rluc) encoding a sea pansy luciferase that we used previously [8]. Twenty-four hours after transfection, the cells were assayed for firefly luciferase activity and normalized against the sea pansy luciferase activity. As shown in Figure 2A (right panel), both the R1 and the mR1-GBS1 vectors produced significant luciferase activity, whereas neither the R3 nor the mR1-GBS2 vector did so, indicating that GBS2 is an essential site for sox2 expression in cultured NE cells. We also examined whether overexpression of Gli2ΔC inhibits the R1-dependent luciferase activity in cultured NE cells. The pGL3 control vector and R1 vector were transfected with either the pCMS-EGFP control vector or the Gli2ΔC vector into cultured NE cells, and their luciferase activity was assayed after 24 hours. As shown in Figure 2B, overexpression of Gli2ΔC strongly blocked the R1-dependent luciferase activity. Together, these results suggest that Gli2 targets GBS2 in the R1 enhancer and regulates sox2 expression in cultured NE cells.
To examine further whether Gli2 binds directly to the R1 enhancer in cultured NE cells, we used ChIP. NE cells were transfected with either a pcDNA3/FLAG control vector or a FLAG-tagged mouse Gli2 expression vector, and the cells were cultured for 24 hours. The cells were then fixed in formaldehyde and sonicated, cell extracts were incubated with an anti-FLAG antibody and protein G-Sepharose, and the precipitated DNA was analyzed using PCR. As shown in Figure 2C, FLAG-Gli2 associated with R1 but not R3. The other Gli members, Gli1 and Gli3, were also examined to determine whether they bind to the R1 enhancer in cultured NE cells as described above. Figure 2D shows that neither Gli1 nor Gli3 associated with the R1 enhancer in cultured NE cells, whereas all Gli proteins bind to PTCH1, which contains a definitive GBS [21]. Together, these data indicate that some Gli2 binds to the R1 enhancer in cultured NE cells, whereas Gli1 and Gli3 do not.
Because we showed previously that Brm, an SWI/SNF chromatin remodeling factor, is recruited to R1 and induces sox2 expression in NE cells [8], we examined whether Gli2 associates with Brm in the cells. We sonicated NSCs in a cell lysis buffer, harvested the extracts, treated them with either anti-FLAG or anti-Brm antibodies followed by protein A-Sepharose, and then analyzed the immunoprecipitates by Western blotting using anti-FLAG and anti-Brm antibodies. As shown in supporting information Figure 4, anti-Brm antibodies precipitated endogenous Gli2, whereas anti-FLAG antibody did not, indicating that some Brm and Gli2 are in the same complexes in cultured NE cells.
Gli2 Regulates Sox2 Expression in Developing Telencephalic NE Cells
We tested whether Gli2 is involved in sox2 expression in NE cells in vivo. We electroporated either the control GFP-expression vector or the Gli2ΔC vector into the NE cells lining the ventricle in E14.5 mice, fixed the transfected brains, sectioned them, and immunostained the sections for Sox2 and GFP. One day after the transfection, the Sox2-positive VZ/SVZ layer became significantly thinner in the brain transfected with Gli2ΔC compared with those transfected with the control vector (Fig. 3A, left panels), and 2 days after transfection, the Sox2-positive layer became disorganized in the Gli2ΔC-transfected brain (Fig. 3A, right panels). The Sox2 intensity in the Gli2ΔC-expressing cells significantly decreased to less than 50% of that observed in the control vector-transfected cells (Fig. 3B). A similar result was also obtained when Gli2ΔC vector was electroporated into the NE cells of E16.5 mice (data not shown).
To confirm that Gli2 is essential for sox2 expression in vivo, we electroporated either the control scrambled vector, the Gli2 shRNA vector, or the Gli3 shRNA vector into the NE cells and labeled sections of the brain for Sox2 and GFP after 2 days. As shown in Figure 3C and 3D, overexpression of Gli2 shRNA significantly decreased Sox2 expression levels in the developing NE cells, whereas either scrambled or Gli3 shRNA did not. Collectively, these data indicate that Gli2 plays an important role in sox2 expression in developing telencephalic NE cells, as well as in cultured NE cells.
Overexpression of Gli2ΔC Induces Cell Cycle Arrest and Cell Death in the Developing Telencephalic NE Cells
To analyze the fates of Gli2ΔC-expressing telencephalic NE cells, the cultured NE cells were transfected with either the control vector or the Gli2ΔC vector, and then their proliferation and cell death were examined. The transfected cells were cultured with BrdU for 6 hours and then immunolabeled for BrdU and GFP. None of Gli2ΔC-transfected cells were positive for BrdU, whereas 38% of control vector-transfected cells were BrdU-positive (supporting information Fig. 5A). To detect cell death, we performed a TUNEL assay and found that more than 16% and 32% of Gli2ΔC-expressing GFP+ cells were TUNEL+ at 24 and 48 hours, respectively, whereas less than 3% and 2% of control vector-transfected cells were TUNEL+ (supporting information Fig. 5B–5D). Moreover, we found that Gli2ΔC-induced cell death was blocked significantly in the presence of the pan-caspase inhibitor Z-VAD (supporting information Fig. 5B–5D).
We also examined the proliferation and cell death of Gli2ΔC-expressing NE cells in vivo. Two days after electroporation, BrdU was injected intraperitoneally into pregnant mice 1 hour before sacrifice, and brain sections of the embryo were immunostained for BrdU and GFP. As shown in Figure 4A and 4B, 69% of GFP+ cells were labeled for BrdU in the control vector-transfected brain, whereas 24% of GFP+ cells were labeled for BrdU in the Gli2ΔC-transfected brain. Moreover, the proliferating cell nuclear antigen (PCNA)-positive cell layer became thin in Gli2ΔC-transfected cells (supporting information Fig. 6A, 6B). Cell death in Gli2ΔC-expressing NE cells was examined using the TUNEL assay. As shown in Figure 4C and 4D, less than 0.2% of the control vector-transfected NE cells were TUNEL+, whereas 2.8% of Gli2ΔC-expressing cells were TUNEL+. These results suggest that overexpression of Gli2ΔC induces both cell cycle arrest and cell death in the telencephalic NE cells.
Overexpression of Gli2ΔC Induces Premature Neuronal Differentiation of Developing Telencephalic NE Cells
There is evidence that both Gli2 and Sox2 are involved in the expression of a number of NSC genes, including Bmi1 and Notch1 [22–24]. We therefore examined the relationship of Gli2, Sox2, and their target genes in NE cells. We transfected cultured NE cells with either the control EGFP-expression vector or the Gli2ΔC vector, cultured the cells for 2 days, purified GFP+ cells by flow cytometry, and then analyzed the expression of NSC genes in GFP+ cells by RT-PCR. As shown in Figure 5A, overexpression of Gli2ΔC significantly decreased the expression levels of sox2, hes1, hes5, notch1, and bmi1 in cultured NE cells. Using Gli2 shRNA and Gli3 shRNA, we confirmed that overexpression of Gli2 shRNA, but not that of Gli3 shRNA, inhibits the expression of same genes in cultured NE cells (Fig. 5B). This suggests that Gli2 may be coordinately stimulating the expression of a number of NSC genes.
We examined the differentiation of Gli2ΔC-expressing NE cells by immunolabeling in vitro and in vivo. Seven percent and 94% of control vector-transfected cultured NE cells were labeled for a neuronal marker, βIII-tubulin, and an NSC marker, Nestin, respectively, whereas 48% and 41% of Gli2ΔC-expressing cells were so labeled (supporting information Fig. 7A, 7B, 7D). In contrast, overexpression of Gli2ΔC did not have any significant effect on GFAP expression in cultured NE cells (3% of control vs. 4% of Gli2ΔC-transfected cells) (supporting information Fig. 7C, 7D). We confirmed that overexpression of Gli2ΔC induces premature neuronal differentiation in NE cells in vivo: 8% of control vector-transfected cells and 27% of Gli2ΔC-expressing cells were positive for βIII-tubulin (Fig. 6A, 6D). Moreover, we found that overexpression of Gli2ΔC decreased the expression of Hes1 and Hes5 in NE in vivo: 60% and 58% of control vector-transfected NE cells were labeled for Hes1 and Hes5, respectively, whereas 38% and 27% of Gli2ΔC-expressing cells were so labeled (Fig. 6B, 6C, 6E, 6F). In addition, we found that expression of CD133, a marker of NSCs, significantly decreases in Gli2ΔC-expressing NE cells (90% of control vs. 9% of Gli2ΔC-transfected cells) (supporting information Fig. 8). Taken together, these data suggest that overexpression of Gli2ΔC induces premature neuronal differentiation by inhibiting the expression of Notch effectors.
Reduction of the Sox2-Positive Layer in Gli2-Deficient Mice
To further confirm the results that the Gli2 function defects decrease Sox2 expression in telencephalic NE cells, we examined Sox2 expression in developing NE cells of Gli2-deficient mouse brain. We fixed E18.5 Gli2−/− brains, sectioned them, and immunostained them for Sox2, Hes5, and βIII-tubulin. As shown in Figure 7A, the Sox2-positive layer became extremely thin and the intensity of Sox2 immunostaining was significantly weak in Gli2-deficient brains, whereas Sox2 expression in Gli2+/− brains was similar to that in Gli2+/+ brains (data not shown). In addition, the βIII-tubulin-positive layer expanded into the VZ/SVZ of Gli2−/− brain, as seen in Gli2ΔC-transfected brains (Fig. 7A). The Hes5-positive layer also became noticeably thinner in Gli2−/− brain (Fig. 7B), consistent with premature neuronal differentiation in the VZ/SVZ of Gli2−/− brain. Thus, these data complement data that suggest that Gli2 is an important transcription factor for sox2 expression in telencephalic NE cells.
Overexpression of Sox2 Rescues Hes5 Expression in the Gli2ΔC-Expressing Telencephalic NE Cells
The finding that Sox2 is one of the downstream factors of Gli2 in telencephalic NE cells led us to examine whether Sox2 is involved in the expression of Gli2 target genes, including Bmi1 and Bcl2 [22, 25]. We transfected NE cells with either the scrambled vector or a sox2 shRNA that encodes sox2-specific shRNA and GFP, cultured the cells for 2 days, purified the GFP+ cells by flow cytometry, and then analyzed gene expression by RT-PCR. As shown in Figure 5B, overexpression of sox2 shRNA prevented the expression of sox2, hes1, and hes5 but not that of notch1, bmi1, or bcl2. Thus, Sox2 does not appear to regulate expression of the putative Gli2 target genes bmi1 and bcl2.
We next examined whether overexpression of sox2 could rescue the expression of Hes1 and Hes5 in Gli2ΔC-expressing NE cells. We electroporated pCMS-EGFP-Gli2ΔC-IRES-Sox2 (Gli2ΔC-Sox2) vector into developing NE cells and immunolabeled the brain sections for Hes1 and Hes5. Overexpression of sox2 slightly rescued the expression of Hes1 in Gli2ΔC-expressing NE cells; whereas 39% of Gli2ΔC-expressing cells were positive for Hes1, 43% of Gli2ΔC-Sox2-expressing cells were positive for Hes1 (Fig. 6B, 6E). In contrast, overexpression of sox2 strongly increased the expression of Hes5 in the transfected NE cells; whereas 25% of Gli2ΔC-expressing cells were positive for Hes5, 97% of Gli2ΔC-Sox2-expressing cells were found to be positive for Hes5 (Fig. 6C, 6F). Consistent with Hes5 upregulation in Gli2ΔC-Sox2-expressing cells, only 1% of Gli2ΔC-Sox2-expressing NE cells were positive for βIII-tubulin, compared with 26% for control vector-transfected cells (Fig. 6A, 6D). Moreover, we confirmed that overexpression of Hes5 is sufficient to block premature neuronal differentiation in Gli2ΔC-expressing NE cells (supporting information Fig. 9).
We also immunolabeled the brain sections for PCNA to examine proliferation of Gli2ΔC-Sox2-expressing NE cells. It was found that overexpression of sox2 could enhance the proliferative defects of Gli2ΔC-expressing NE cells (supporting information Fig. 6). Sox2, therefore, appears to play an important part in keeping NE cells undifferentiated, but it is not sufficient, in the absence of Gli2, to rescue the proliferative defect observed.
- Top of page
- Abstract
- Supporting Information
Many transcription factors have been shown to be involved in the maintenance of NSCs; however, the precise function of each one remains uncertain. We show here that expression of Sox2, an essential transcription factor for NSC maintenance, is regulated by Gli2, also an important transcription factor in NSCs, in the telencephalic NE cells. Since all three Gli family members have been shown to recognize the GBS and share redundant functions, it was speculated that Gli1 and Gli3 might also be involved in sox2 expression. However, our data clearly show functional differences between Gli2 and other Gli members in sox2 expression in telencephalic NE cells: Gli2 binds to the R1 enhancer that is essential for sox2 expression in telencephalic NE cells, whereas Gli1 and Gli3 do not. Overexpression of either Gli2ΔC or Gli2 shRNA decreased sox2 expression in telencephalic NE cells, whereas overexpression of Gli3 shRNA did not change sox2 expression. Furthermore, sox2 expression is greatly reduced in the developing telencephalic NE cells of Gli2-deficient mice. Many lines of evidence also suggest that each of the Gli members has specific functions. For instance, Gli1 and Gli2 are transcriptional activators, whereas Gli3 primarily acts as a repressor, although it can act as a weak activator in the presence of Shh [12, 19, 20]. Gli1-deficient mice are capable of developing normally, whereas both Gli2-deficient and Gli3-deficient mice die during the perinatal period with severe brain defects [14, 16–18]. Moreover, expression of both copies of Gli1 in place of Gli2 in mice cannot completely rescue the prenatal death observed in Gli2-deficient mice [26]. Thus it is of great interest to investigate the specific functions of each Gli transcription factor in NE cells.
The brain phenotype of Gli2-deficient mice is much milder than that seen in Sox2-deficient mice; the Sox2-deficient brain has cerebral malformations with parenchymal loss and ventricle enlargement, whereas the Gli2-deficient brain has thin telencephalic vesicles and a reduced tectum and cerebellum [14, 27]. These results suggest that transcription factors other than Gli2 are also involved in sox2 expression in telencephalic NE cells. Indeed, as shown in Figure 7, one cell layer lining the ventricle in Gli2-deficient brain is still immunolabeled for Sox2, although the signal is much weaker than that in Gli2+/− brain. In addition, it was shown that wnt signaling and the POU-domain transcription factors Brn1 and Brn2 also regulate sox2 expression in NE cells [28, 29]. Since sox2 expression in the developing CNS depends on a patchwork of separate enhancers, one of which contains the R1 enhancer [30], it is of interest to determine transcription factors that regulate sox2 expression in other brain regions.
We also showed that overexpression of Gli2ΔC in telencephalic NE cells decreases the expression of other NSC factors, including Notch1, Hes1, Hes5, and Bmi1, and induces premature neuronal differentiation. Moreover, coexpression of sox2 with Gli2ΔC in telencephalic NE cells rescues the expression of Hes5, but not Hes1, and prevents neuronal differentiation; this gene combination, however, does not rescue NE cell proliferation, suggesting that although Sox2 is crucial for the maintenance of NE cells, it might not be directly involved in their proliferation. This result is consistent with previous reports that enforced expression of sox2 in the developing NE cells maintains the characteristics of NSCs but also decreases their proliferation, although the way in which sox2 overexpression decreases cell proliferation remains unclear [7].
It has been shown that overexpression of Sox2 in NE cells can induce the expression of Notch1, which regulates Hes5 expression through the activation of RBPjk [23, 24]. However, coexpression of Gli2ΔC and sox2 in telencephalic NE cells was found to activate Hes5 expression but not Notch1 expression (our unpublished observations). Since the mouse Hes5 promoter contains a putative Sox2-binding site (−123 CTTTGTG −117), Sox2 might directly induce Hes5 expression in telencephalic NE cells. Together, these findings suggest that Gli2 stimulates the self-renewal of NSC by inducing the expression of Bmi1 and Hes1 [31, 32] and maintains the undifferentiated state of NSC by inducing the expression of Sox2.
There is growing evidence that all three Gli proteins are strongly expressed in malignant brain tumors [33, 34]. There is also evidence that brain tumors contain cancer stem cells, which are essential for both the development and recurrence of the tumors [35–38]. A recent study has revealed that cyclopamine, a chemical inhibitor of Shh signaling, can block the growth of gliomas, suggesting that Shh-Gli signaling could potentially be major targets for glioma therapy [39]. In addition, it was found that an active form of Gli2 induced sox2 gene expression in a keratinocyte cell line, which was used as a model for basal cell carcinoma [40]. It will, therefore, be of great interest to investigate whether Sox2 plays an important part in gliomagenesis.
- Top of page
- Abstract
- Supporting Information
For the success of CNS regenerative medicine, it is important to understand how proliferation and differentiation of NSCs are controlled. In the present study we have shown that the expression of sox2 gene, which is essential for the maintenance of NSCs, is regulated by the Gli2 transcription factor. Moreover we demonstrated that Sox2 negatively regulates neuronal differentiation of telencephalic NE cells but is not involved in their proliferation. Thus, we revealed a novel transcriptional cascade, Gli2 Sox2 Hes5, which is essential for maintaining undifferentiated state of telencephalic NE cells.
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- Abstract
- Supporting Information
We thank M. Raff for critical reading and comments on the manuscript; R. Lovell-Badge for mouse Sox2 5′ genomic DNA; J. Motoyama for Gli2-deficient mice; K. Shimozaki and S. Nagata for the pEF-Rluc vector; H. Sasaki for the pcDNA3.1-HisB-mGli1 and pCMV/FLAG/SV-Gli2 full vectors; K. Nakashima for technical advice on mouse NE cell preparation; T. Suetsugu, D. Konno, and F. Matsuzaki for technical advice on in utero electroporation; K. Miwa for purification of GFP+ cells by flow cytometry; H. Hiraga for critical reading of the manuscript; and members of our laboratory for their discussion and encouragement. H.T. was supported in part by the Uehara Memorial Foundation in Japan.
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- Abstract
- Supporting Information
The authors indicate no potential conflicts of interest.
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- Abstract
- Supporting Information
- 1Mammalian neural stem cells. Science 2000; 287: 1433–1438..
- 2The development of neural stem cells. Nature 2001; 414: 112–117..
- 3SOX2 functions in adult neural stem cells. Trends Neurosci 2005; 28: 219–221..
- 4A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature 1990; 346: 245–250., , et al.
- 5Sox2 regulatory sequences direct expression of a b-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo, revealing regionalization of gene expression in CNS stem cells. Development 2000; 127: 2367–2382., , et al.
- 6Vertebrate neurogenesis is counteracted by Sox1–3 activity. Nat Neurosci 2003; 6: 1162–1168., , et al.
- 7SOX2 functions to maintain neural progenitor identity. Neuron 2003; 39: 749–765., , et al.
- 8Chromatin remodeling and histone modification in the conversion of oligodendrocyte precursors to neural stem cells. Genes Dev 2004; 18: 2963–2972., .
- 9Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development. Development 1997; 124: 113–123., , et al.
- 10Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes Dev 1996; 10: 3129–3140., , et al.
- 11Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol 2001; 240: 237–246., .
- 12Gli proteins encode context-dependent positive and negative functions: Implications for development and disease. Development 1999; 126: 3205–3216..
- 13Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm- and mesoderm-derived tissues suggests multiple roles during postimplantation development. Dev Biol 1994; 162: 402–413., , et al.
- 14Hedgehog-GLI signaling regulates the behavior of cells with stem cell properties in the developing neocortex. Development 2004; 131: 337–345., .
- 15Sonic hedgehog controls stem cell behavior in the postnatal and adult brain. Development 2005; 132: 335–344., , et al.
- 16Diminished Sonic hedgehog signaling and lack of floor plate differentiation in Gli2 mutant mice. Development 1998; 125: 2533–2543., , et al.
- 17Gli2 is required for induction of floor plate and adjacent cells, but not most ventral neurons in the mouse central nervous system. Development 1998; 125: 2759–2770., , et al.
- 18Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation. Development 2000; 127: 1593–1605., , et al.
- 19Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: Implication of Gli2 and Gli3 as primary mediators of Shh signaling. Development 1999; 126: 3915–3924., , et al.
- 20Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 2000; 100: 423–434., , .
- 21Expression of the PTCH1 tumor suppressor gene is regulated by alternative promoters and a single functional Gli-binding site. Gene 2004; 330: 101–114., , et al.
- 22Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res 2006; 66: 6063–6071., , et al.
- 23SOX2 is a dose-dependent regulator of retinal neural progenitor competence. Genes Dev 2006; 20: 1187–1202., , et al.
- 24Role of Sox2 in the development of the mouse neocortex. Dev Biol 2006; 295: 52–66., , et al.
- 25Activation of the BCL2 promoter in response to Hedgehog/GLI signal transduction is predominantly mediated by GLI2. Cancer Res 2004; 64: 7724–7731., , et al.
- 26Gli1 can rescue the in vivo function of Gli2. Development 2001; 128: 5161–5172., .
- 27Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain. Development 2004; 131: 3805–3819., , et al.
- 28Conserved POU binding DNA sites in the Sox2 upstream enhancer regulate gene expression in embryonic and neural stem cells. J Biol Chem 2004; 279: 41846–41857., , et al.
- 29Convergence of Wnt and FGF signals in the genesis of posterior neural plate through activation of the Sox2 enhancer N-1. Development 2006; 133: 297–306., , et al.
- 30Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals. Dev Cell 2003; 4: 509–519., , et al.
- 31The bHLH gene hes1 as a repressor of the neuronal commitment of CNS stem cells. J Neurosci 2000; 20: 283–293., , et al.
- 32Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 2003; 425: 962–967., , et al.
- 33Hedgehog signalling in cancer formation and maintenance. Nat Rev Cancer 2003; 3: 903–911., .
- 34Brain as a paradigm of organ growth: Hedgehog-Gli signaling in neural stem cells and brain tumors. J Neurobiol 2005; 15: 476–490., .
- 35The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis. Development 2001; 128: 5201–5212., , et al.
- 36Cancer stem cells in nervous system tumors. Oncogene 2004; 23: 7267–7273., , et al.
- 37Brain cancer stem-like cells. Eur J Cancer 2006; 42: 1237–1242..
- 38Brain tumour stem cells. Nat Rev Cancer 2006; 6: 425–436., , .
- 39HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity. Curr Biol 2007; 17: 165–172., , et al.
- 40Overlapping and distinct transcriptional regulator properties of the GLI1 and GLI2 oncogenes. Genomics 2006; 87: 616–632., , et al.
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text:
Efficiency said to cut power use, costsBy STEPHEN SINGER
The Associated Press | December 13,2012HARTFORD, Conn. Improved energy efficiency will help keep electricity use flat in New England in the next decade, allowing customers to save on utility bills and power companies to scrap costly transmission upgrades, the grid operator said Wednesday.
The regions six states spent $1.2 billion from 2008 to 2011 to boost energy efficiency, ISO-New England told reporters in a briefing. Spending on energy efficiency is expected to increase to $5.7 billion from 2015 to 2021.
The Holyoke, Mass.-based grid operator said energy efficiency has more than doubled since 2008 in an annual auction to win commitments from generators and others for power available three years from now. The result is that electricity use previously projected to rise by 0.9 percent annually between 2012 and 2021 will instead be flat.
The increased efficiency also will help utilities save money by skipping transmission upgrades. ISO-New England said the region can defer 10 transmission upgrades that earlier studies showed were needed to ensure reliability. Deferring the upgrades will save an estimated $260 million, it said.
In 2010, New Englands six states sponsored more than 125 energy efficiency programs offering financial incentives to promote efficient electrical devices, ISO-New England said.
Stephen J. Rourke, vice president for system planning at ISO, said replacing incandescent lighting with compact fluorescent bulbs is the easiest and least expensive way to cut energy use. Savings are multiplied as apartments, office buildings, factories, schools, hospitals and other large energy users install efficient heating and air conditioning, he said.
Seth Kaplan, vice president for policy and climate advocacy at the Conservation Law Foundation, a regional environmental group, said the report shows that efforts to conserve energy work. He said the group welcomes the ISO-New England report.
This is a very big deal, he said. The system needs to know what the demand is.
Nationally, demand for electricity is leveling off as residential power use falls, experts say, reversing a long upward trend. More efficient lighting and electric devices are partly credited for the change. New homes also are being built to use less electricity, and government subsidies for home energy savings programs help older homes use less power. Rourke said the weak economy also has contributed to reduced electricity use.
New England used about 130,000 gigawatt-hours of electricity last year. One gigawatt-hour can serve about 1 million homes for one hour.MORE IN Vermont NewsPHILADELPHIA After standing at the podium to second the nomination of Sen. Full StoryBARRE A Middlesex man could serve as many as five years in prison if he fails to complete drug... Full StoryPHILADELPHIA With his presidential campaign officially over, independent Sen. Full Story
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Marrying Mattieby Tanya Hanson
View All Available Formats & Editions
Mattie Carter is hopelessly in love with the handsome horse doctor
Caldwell Hackett knows everything about horseflesh and nothing about women, yet he's managed to snare beautiful Mattie Carter's heart. With their wedding coming up, he's nervous about his inexperience in the bridal bed, but his lovely fiancée manages to ease his worries in just the right way.
Mattie Carter is hopelessly in love with the handsome horse doctor and knows this marriage will be wonderful, unlike her first that was fraught with her wealthy husband's infidelity. Eager to begin her new life with Call, Mattie is heartbroken when her former husband halts their vows, claiming to the whole church she's still his wife.
Can Mattie regain Call's trust? Can Call, whose livelihood is threatened when an epidemic hits the horses in Paradise, figure out the truth with Mattie's help? Or will these star-crossed lovers be destined to live apart?
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- 9781601548078
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- Publication date:
- 07/28/2010
- Pages:
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- Product dimensions:
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Paradise, Nebraska is anything but a paradise after Woodrow Carter arrives. Neighbor is set against neighbor. Gossip fuels more and more unrest creating a hostile environment for Mattie and Call as they try to unravel problems and mysteries that stopped their marriage and threatens their future. With Call's lifelong dream in danger of being destroyed after years of college and hard work, he labors until he is ready to drop trying to save sick animals and find what caused the malady. In spite of all he does, his integrity, ability, and efforts are questioned. Piled on top of his professional problems, his insecurities and fear of losing Mattie make Call misconstrue some of Mattie's actions. Mattie knows Call is the love of her life, but her past refuses to leave her in peace. As she sees the people of Paradise that she has come to care about being hurt because of her, she has to rethink her decisions. Mattie's intelligence, integrity, honesty, compassion, and unfaltering love for Call make her a heroine that wins the reader's heart. The secondary characters, some known from the first book of Paradise Brides, add depth to true friendships, while others add doubts about deeds and motivations. However, Woodrow P. Carter is the antagonist that wreaks havoc on the once peaceful Paradise. The damage he does is far worse than a tornado. Tanya Hanson gives the reader some heart-racing reading as she reveals bit by piece Woodrow's devious, demonic plans. Tensions run high in Marrying Mattie, keeping the reader turning pages. But it also gives the reader a sense of being in Paradise, Nebraska in the late 1800s when social mores, prejudice, and greed make some people do less than admirable things while others never falter in their steadfast friendship, good deeds, and most of all in their love. Marrying Mattie is a hard won happy-ever-after that captivates. Originally posted at The Long and Short of It Romance Reviews
Tanya Hanson has a flair for rancher stories. The descriptions are rich and the characters jump off the pages. Beautiful!


text:
Boris Johnson fails IQ test
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text:
A Colorado Springs woman is in jail after police say she killed her own mother.
11 News spoke to a family member who explained what happened leading up to the alleged murder.
Mara Jefferson said her sister is the suspect, 57-year old Rae Lena Stanley, and the victim is her mother, Charlas "June" Stanley.
Jefferson said she remembers her mother and sister's relationship was strained for years.
"As my sister's mental stability started getting worse [the relationship was] very unhealthy. My mom blamed herself for Rae's mental problems," she said.
Jefferson said her sister Rae suffers from schizophrenia and that she had left Cedar Springs Mental Hospital about a week and a half ago and was staying with her mother.
"[Charlas] loved her family, she loved her family dearly. She loved her kids," said Jefferson.
She said that night Charlas and Rae were fighting about family issues.
"I guess my mom was afraid and agreed to let Rae stay one more night but then the next day she had to go," said Jefferson.
Before that could happen, police say Stanley killed her own mother in her home.
"The took a lot of knives, bloody knives ... God knows I forgive her for what she did, but I no longer have a sister," Jefferson said.
Rae Stanley was arrested and is now facing First Degree Murder charges.
Officers found the suspect walking down the roadway near the home on Curtis Road just before 1:00 p.m. on Saturday.
They say Stanley was saying things that made them concerned and when they arrived at the house that's when officers found Charlas Stanley dead.


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MIDDLEBURY — Mary Hogan Elementary School directors on May 19 appointed Middlebury resident Victoria Jette to a board vacancy created by the recent resignation of incumbent Jim Callahan.
Callahan verbally resigned from the ID-4 school board on April 17, citing a disagreement with board Chairwoman Ruth Hardy. Callahan had been elected to a three-year term on the board on March 4 and attended only two meetings before resigning.
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MONTPELIER — Rep. Peter Welch hosted the 33rd annual Congressional Art Competition for high school students at The Gallery at Vermont College of Fine Arts in Montpelier on May 15.
Greta Suzette Exeter from Vergennes, a student at Vergennes Union High School, received Congressman Welch's choice award for her piece "Michelle Dockery."
VERGENNES — Vergennes workers and private contractors will undertake at least five significant projects between June and August, including paving two streets, improving two stretches of Main Street sidewalk, and making progress to solve the city's longstanding sewer-system overflow problem.
VERGENNES — Senator Patrick Leahy (D-Vt.) has announced a new $6,723,732 Defense Department contract with UTC Aerospace Systems based in Vergennes to develop and test improved guidance systems for U.S. munitions, which would allow greater precision and avoid damage to innocent bystanders and property.
I don't trust flowers.
There are grounds for my suspicion; flowering plants are proven masters of deception. For instance, the sundew uses sparkling droplets of sticky "faux dew" to ensnare and digest curious flies; bee orchids dupe male wasps into wasting their copulatory efforts on floral structures that look and smell like a female wasp. And what about humans? As I labor on behalf of flowers, fertilizing, tilling, watering and sweating, I sometimes wonder if I'm being led down the proverbial garden path. Exactly who's cultivating whom?
SHOREHAM — Expressing a desire to "recharge his batteries" and devote more time to the family farm, Rep. Will Stevens, I-Shoreham, announced this week that he will not seek a fifth consecutive two-year term in the Vermont House.
MIDDLBURY — The Russ Sholes Senior Center was a pretty big draw when it formed in Middlebury some 35 years ago. Dozens of seniors gathered regularly in the lower level of Middlebury's municipal building to share potluck meals, chat, and play cards and board games. A group of crafts enthusiasts —whom then-Town Clerk Richard Goodro fondly anointed the "sew-sew girls" — would also meet up to stitch and knit together, making clothes that they would donate to local charities.
VERGENNES — The keynote speaker at the service following the Vergennes Memorial Day parade will bring a lifetime of experience to the podium on the city green. Michael Wojciechowski, a Purple Heart recipient and highly decorated soldier, served in the Army National Guard for more than a quarter of a century — some of that in the hot spots of Iraq and Afghanistan.
He lives with his wife, Elizabeth, and family in Addison, and is a member of American Legion Post 14.
ADDISON COUNTY/BRANDON — The Spring Open Studio Tour over Memorial Day Weekend offers a great way for locals and visitors alike to see the Green Mountain State as well as the work of Vermont artist right where it is made. Bright yellow signs are placed along Vermont's roads to mark the way to studios of craftspeople and fine artists during the self-guided tour on May 24 and 25 at a time when the landscape is fresh and inviting and without the crowds of summer and fall.
RIPTON — Third- and fourth-graders from Mary Hogan Elementary School, under the guidance of teacher Steve Flint, released trout into the Middlebury River in Ripton on May 20. Earlier this year Flint began a program to raise trout from eggs in the school with the students.
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Sexiest Movies of 2010
Posted by handmeafork on Apr. 04, 2011
Elizabeth Rage takes you through the steps to create the Enchantress look f...
On this episode, Master Chalk Artist Chris Carlson takes on an epic Star Tr...
Get you geek on and come check out these hilarious clips!
Posted by handmeafork on Apr. 04, 2011


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Very interesting, I saw them as a salad at the store. F+L
This pancaking is simply rock that has formed on the sea floor (a few M years ago...) Then been uplifted at some point, then the weather & sea took it out on it.
Iron & concerete. Despite that the bridge did not last...
"Hey you, YES YOU, stop disterbing my sleep!! Yer I know its drizzling... it's the 'coast, what do you expect...? Let me go back to sleep"


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Odd request but I am trying to find a way to mute the speakers hooked up to a Sony ES5800 receiver, but NOT mute the preouts. I can not seem to find a device to connect inline between the speakers and the receiver. I also have to be able to mute or Turn off the speakers via 12v, ir, or rs232. Thanks
post #2 of 4 Old 10-03-2012, 02:44 PM
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Generally main zone audio is passed simultaneously to the speaker posts and pre-outs so muting the audio mutes both sets of outputs.
"JD" - Call 585-645-1006 or email firstname.lastname@example.com for AVR selection and pricing discussion 8am - 8pm EST daily (incl Sat/Sun)
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I think I found a way to turn off the speaker posts without turning off the preouts using 3x Altronix RB-5 relays and tying them to the 12v trigger
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Make sure whatever is going to driving the 12V trigger can supply 360 mA (3 x 120 mA) of current.
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Section 20
The bang-bang funnel controller: An experimental verification
Version of Record online: 3 DEC 2012
DOI: 10.1002/pamm.201210356
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Special Issue: 83rd Annual Meeting of the International Association of Applied Mathematics and Mechanics (GAMM), Darmstadt 2012; Editors: H.-D. Alber, N. Kraynyukova and C. Tropea
Volume 12, Issue 1, pages 735–736, December 2012
Additional Information
How to Cite
Hackl, C. and Trenn, S. (2012), The bang-bang funnel controller: An experimental verification. Proc. Appl. Math. Mech., 12: 735–736. doi: 10.1002/pamm.201210356
Publication History
- Issue online: 3 DEC 2012
- Version of Record online: 3 DEC 2012
- Abstract
- References
- Cited By
We adjust the newly developed bang-bang funnel controller such that it is more applicable for real world scenarios. The main idea is to introduce a third "neutral" input value to account for the situation when the error is already small enough and no control action is necessary. We present experimental results to illustrate the effectiveness of our new approach in the case of position control of an electrical drive . (© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim)


text:
Posted: 7/23/2012
This place is great. First off the train the Detroit Lions and Red Wings, which is awesome. AOS puts you in a one-on-one training session to see what works best for you and then puts you in groups training. Since becoming a member I am able to keep off the weight that I so badly wanted to get rid of. I highly recommend it to anyone who is looking to shed a couple pounds, or those who just love a good workout!


text:
|Publication number
|Publication type
|Application number
|Publication date
|Jun 23, 2009
|Filing date
|Jun 20, 2006
|Priority date
|Jun 20, 2006
|Fee status
|Publication number
|Walter Kenneth Porter
|Original Assignee
|Sip Home Systems, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (57), Referenced by (12), Classifications (17), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This present invention relates to structural insulated panels that are utilized in building construction and in particular to a building system employing structural insulated panels that are connected to rods or anchors embedded in a foundation or a load bearing footing.
The majority of houses constructed are stick built, i.e., typically constructed of 2 by 4 or 2 by 6 structural members and nails. Wallboard is typically attached to the structural members in forming the walls and ceilings of the stick built house. Structural Insulated Panel's (SIPs) are increasingly being used in building construction as an alternative to the stick built approach. SIP construction employs rigid outer facings attached to one or both sides of a light insulating foam core. High strength bonding of the outer facings to the inner core forms a structural I-beam in the form of flat panels. Previously mentioned wallboard panels as well as SIPs are attached to the 2 by 4 or 2 by 6 structural members by conventional connectors such as nails or screws.
SIPs are attached to the base (or bottom) and top plates forming part of the 2 by 4 or 6 lumber framework as well as to spaced studs extending between the base and top plates. Current building codes and engineering specifications often require a particular wall or wall section to be a "shear-wall". These walls or wall sections are typically connected to a foundation or footing by use of a "hold-down" or "tie-down".
High strength and/or reinforced metal rods are typically embedded in a foundation or load bearing footing to provide an anchor for securing a wall section to the ground. Anchors are utilized to counter the forces acting on a wall that will cause the wall to turn or tip over. Hold-downs or tie-downs are used connect a wall section to the ground and prevent a wall from overturning. The terms hold-downs and tie-downs are often used interchangeably and refer to the system of components use to secure the wall to the foundation or footing.
Hold-downs can take many forms. Some look like straps that emerge from the foundation and nail to the edge or face of a stud. Others connect foundation bolts or threaded rods to the studs via bolts or nails. The hold-down creates a path for forces to travel out of a shear wall and into other portions of the building.
SIP users often attach one or more panel sections to a threaded rod embedded in a foundation or footing as per design specifications. However, methods of attachment of the SIP to the embedded rod have proved time-consuming and costly for most users. Also, previous attachment methods have provided connections in the lower section of the panel, thus not taking advantage of the strength and shear-resistance of the entire panel.
Embodiments of the present invention address the aforementioned limitations of previous attachment methods and devices by providing an SIP panel with a structural chase to easily accommodate a connection for a rod or anchor embedded in a foundation or load bearing footing. The structural chase of embodiments of the present invention is formed by using structural members to create a cavity that extends the entire length of the panel.
Structural members commonly used to form the chase are solid wood, laminated veneer lumber (LVL) or any suitable structural equivalent utilized in building construction. Through utilization of a chase or cavity defined by structural members, embodiments of the present invention provide a lightweight, structural insulated panel capable of forming high strength connections to hold down anchorage attachments in a foundation or footing with a minimum of connectors within the panel. The structural chase described herein also increases the bending strength and the compressive strength of the panel.
Accordingly, it is an object of an embodiment of the present invention to provide a building system employing a modular structural insulated panel which reduces the need for timely and costly methods and devices to anchor a panel to the foundation or footing. It is a further object to provide a structural insulated panel designed to accommodate a reinforcing rod within a panel cavity while maintaining or increasing the structural and insulative properties of the panel.
It is also an object of an embodiment of the present invention to decrease the time and expense of anchoring a structural insulated panel to a foundation or load-bearing footing. Yet another object is to provide a structural insulated panel that can be anchored to a foundation and allow for accessible inspection and/or verification by a building inspector.
It is another object of an embodiment of the present invention to provide a structural insulated panel design that can meet the requirements of building codes including, but not limited to, the International Residential Code (IRC). At least some of these objects will be achieved by embodiments disclosed herein.
The character of the invention, however, may be best understood by reference to one of its structural forms, as illustrated by the accompanying drawings, in which:
Referring to
Embodiments of the present invention provide a structural insulated panel that can be quickly and easily anchored to an embedded rod in a foundation or load bearing footing without the need for time-consuming and expensive attachments. The unique design presented herein provides for a structural chase to house at least one embedded rod, an attachment element, and a connecting rod to anchor the panel to the foundation or footing.
An embodiment of the structural insulated panel of the present invention could also be utilized in a multi-story application (
An important characteristic of SIPs is their insulative properties. The unique chase in the SIPs of the present invention allows insulation in the chase itself without interfering with the structural rods and connector. An access hole can be cut through the outer member (OSB panel for example) as well as the rigid insulation lining the chase (
The structural members that define the chase are typically 2 by 4 or 2 by 6 lumber and can be comprised of solid wood or laminated veneer lumber (LVL). Any structural member will suffice. The chase's structural members also provide the panel with more resistance to compressive loads acting though the top plate or tensile loads through the metal rod anchored in the foundation or footing.
Structural members in building panels typically are comprised of engineered wood product. All engineered wood products utilize recycled or reconstituted wood materials. Structural composite lumber is a type of engineered wood product that includes laminated veneer lumber (LVL), parallel strand lumber (PSL), laminated strand lumber (LSL) and oriented strand lumber (OSL). Other common terms used to describe engineered wood products are oriented strand board (OSB), plywood and particleboard. Structural glued laminated timber (Glulam) is also considered an engineered wood product.
The connecting rods typically utilized are commonly referred to as embedded rods, allthread, and threaded rods. Any type of connector that can accept and couple threaded portions of the rods can be utilized within the chase. A washer and nut are commonly used to secure the rod to the top plate. Any suitable fastener and spacing component can be utilized within the scope of the embodiments of the present invention.
In some heavy duty applications, the chase can be defined by more than two structural members between the outer members of the panel. On tall buildings, there could be four structural members or struts (possibly two on each side of the chase) to provide a stronger panel.
When shear-walls are specified by building plans and/or design, a panel described herein would be utilized near one end of the shear-wall. The chase would typically be positioned next to the end or corner of the building. Another similar panel would be placed on the opposite end of the wall with the chase positioned near the building corner or the end of the wall. It should be further noted that application of the embodiments of the present invention are not limited to exterior walls. These embodiments can also be used for interior load bearing walls on a foundation or load bearing footing.
While the illustrations show one embedded rod in each chase, there may be applications where more than one embedded rod is required. In yet another embodiment, the panels could also comprise more than one chase if necessary.
Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It should be evident that this disclosure is by way of example and that various changes may be made by adding, modifying, or eliminating details without departing from the fair scope of the teaching contained in this disclosure. The invention is therefore not limited to particular details of this disclosure except to the extent that the following claims are necessarily so limited. Thus it is intended that the specification and examples be considered as illustrative only, with the true scope and spirit of the invention being indicated by the following claims.
|Cited Patent
|Filing date
|Publication date
|Mar 29, 1933
|Apr 13, 1937
|Bitting Inc
|Building structure
|Mar 26, 1962
|Jun 22, 1965
|Mitchell Reginald F
|Visual landing aid for airplanes
|Oct 16, 1964
|Nov 15, 1966
|Whelan Joseph A
|Prefabricated structure
|Sep 28, 1965
|Feb 20, 1968
|Ralph R. Berg
|Building system
|Feb 8, 1968
|Sep 2, 1969
|Grover Morgan
|Winch lock movable partitions
|May 7, 1970
|Nov 23, 1971
|Alvic Dev Corp
|System for connecting precast concrete slabs together
|Aug 10, 1970
|Dec 26, 1972
|Stahl Joel S
|Plastic plumbing wall
|Jan 29, 1975
|Apr 13, 1976
|W. H. Porter, Inc.
|Insulating structural assembly and stud member for forming same
|Oct 8, 1976
|Aug 30, 1977
|Lingle Cleo M
|Fastening means for frameless insulating panels
|Jul 23, 1976
|Jan 30, 1979
|W. H. Porter, Inc.
|Insulating panel construction
|Dec 29, 1977
|Feb 27, 1979
|Becker Otto A
|Prestressed hollow chamber composite structural element capable of supporting heavy loads
|Sep 26, 1977
|Apr 3, 1979
|Day Stephen W
|Composite wall panel assembly and method of production
|May 26, 1977
|Aug 7, 1979
|Smith Glenn W
|Insulated building panels
|Mar 31, 1986
|Aug 25, 1987
|Constro S.A.
|Set of modular building construction elements
|Jun 22, 1987
|Aug 23, 1988
|Seven S Structures Inc.
|Wall panel with foam insulation
|Dec 2, 1988
|Jun 26, 1990
|Porter William H
|Interconnecting panels
|Mar 19, 1992
|Jan 18, 1994
|Gulur V Rao
|Insulated wall system
|Jan 31, 1994
|Aug 29, 1995
|An Shun Enterprise Co., Ltd.
|Combination wall
|Mar 9, 1994
|Nov 14, 1995
|Lin; Yi-Chung
|Building construction of longitudinal and cross hollow components
|Jul 12, 1994
|Mar 12, 1996
|Porter; William H.
|Structural insulated panels with metal edges
|Jan 25, 1995
|Apr 15, 1997
|Nitto Boseki Co., Ltd
|Sound insulating wall and method of installing the same
|Aug 24, 1994
|May 13, 1997
|Porter; William H.
|Structural insulated panels joined by insulated metal faced splines
|Jun 14, 1995
|Oct 21, 1997
|Megawall Corporation
|Modular precast wall system with mortar joints
|Apr 9, 1996
|Dec 30, 1997
|Taraba; Emil M.
|Structural foam core panels with built-in header
|Jul 31, 1996
|Jun 16, 1998
|Richard; Michel V.
|Pre-insulated prefab wall panel
|Apr 12, 1996
|Jun 30, 1998
|Porter; William H.
|Electrical access in structural insulated foam core panels
|Jul 29, 1997
|May 4, 1999
|Josey; Gary L.
|Composite wall structure
|Jun 3, 1998
|May 9, 2000
|Mcclellan; Robert B.
|Construction of wall panel and panel structure
|Jun 25, 1998
|May 23, 2000
|Kaieitechno Co., Ltd.
|Connecting structure for concrete block and connector used therefor
|Oct 2, 1998
|Oct 17, 2000
|Crockett; David P.
|Wall unit structural system and method
|Nov 23, 1998
|Dec 26, 2000
|Roberts; Scott J.
|Reinforced foam block wall
|Dec 13, 1999
|Mar 6, 2001
|Peter William Schuyler
|Hold down device and method
|Nov 18, 1998
|Mar 27, 2001
|William H. Porter
|Asymmetric structural insulated panel
|Mar 1, 1999
|Apr 3, 2001
|William H. Porter
|Asymmetric structural insulated panels for use in 2X stick construction
|Oct 20, 1998
|Jun 5, 2001
|William H. Porter
|Building panels with plastic impregnated paper
|Nov 29, 1999
|Jul 3, 2001
|Todd Carper
|System and method for facilitating multiple applications on a smart card
|Apr 12, 1999
|Jul 10, 2001
|Frank J. Babcock
|Modular building construction and components thereof
|Nov 4, 1999
|Aug 7, 2001
|William H. Porter
|Structural insulated panels for use with 2X stick construction
|Jul 28, 1999
|Oct 16, 2001
|Hideo Matsubara
|Apparatus and method for forming precast modular units and method for constructing precast modular structure
|Oct 8, 1999
|Oct 30, 2001
|William H. Porter
|Structural insulated panel
|May 9, 2000
|Dec 11, 2001
|Kaieitechno Co., Ltd.
|Connecting structure for concrete block and connector used therefor
|Jun 21, 2000
|Jan 22, 2002
|Herman Miller, Inc.
|System of wall panels
|Dec 11, 2000
|Apr 9, 2002
|Hurri-Bolt, Inc.
|Anchor for a structural tie-down apparatus
|Jun 16, 1999
|Jun 25, 2002
|William H. Porter
|Reinforced structural insulated panels with plastic impregnated paper facings
|Jan 12, 2000
|Nov 19, 2002
|William H. Porter
|Structural wall panels
|Nov 2, 2000
|Feb 25, 2003
|William H. Porter
|Building panels with plastic impregnated paper
|Aug 16, 2001
|Jul 8, 2003
|William H. Porter
|Building panels with plastic impregnated paper
|Oct 31, 2000
|Mar 2, 2004
|William H. Porter
|Structural insulated panel building system
|Mar 13, 2003
|Feb 15, 2005
|Charles Starke
|Continuous structural wall system
|Jul 23, 2002
|Oct 4, 2005
|Espinosa Thomas M
|Fastener assembly serving as a product, or combined with other components as a product, allows automatic controlled movements in one direction and prevents movements in the opposite direction when forces are applied
|Aug 12, 2003
|Dec 19, 2006
|Commins Alfred D
|Continuous hold-down system
|Mar 24, 2005
|Jan 9, 2007
|Espinosa Thomas M
|Fastener assembly serving as a product, or combined with other components as a product, allows automatic controlled movements in one direction and prevents movements in the opposite direction when forces are applied
|Mar 11, 2002
|Sep 11, 2003
|Lstiburek Joseph W.
|Shear wall panel
|May 30, 2001
|Jun 3, 2004
|Tonny Bergqvist
|Building structure and modular construction method
|Aug 11, 2004
|May 10, 2007
|Miniter Frederick M
|Thermally insulating panel & wall constructed therefrom
|Aug 14, 1992
|Feb 17, 1994
|Siegfried Klett
|Erecting building walls with apertures from wall elements - involves spacing two wall elements with facing side plate edges for forming wall aperture
|Feb 26, 1990
|Sep 12, 1990
|Thierry Meunier
|Facade structure of the curtain wall type with tensioned cables or cords
|Citing Patent
|Filing date
|Publication date
|Jul 15, 2009
|Oct 16, 2012
|Richard Fearn
|Monopour form
|Sep 30, 2011
|Dec 10, 2013
|John Daines Chadwick
|Techniques for building construction using fabricated timbers
|Dec 9, 2013
|Feb 10, 2015
|John Daines Chadwick
|Techniques for building construction using fabricated timbers
|Jun 28, 2012
|Jul 12, 2016
|Nicholas Timothy Showan
|Method of erecting a building with wooden panels
|Jan 21, 2010
|Richard Fearn
|Monopour form
|Oct 9, 2008
|Apr 15, 2010
|Thermapan Structural Insulated Panels Inc.
|Structural Insulated Panel for a Foundation Wall and Foundation Wall Incorporating Same
|May 6, 2009
|Apr 28, 2011
|Jeffrey Robert Parker
|Wall System
|Nov 11, 2011
|Jul 19, 2012
|Kull Jeffrey J
|Modular habitat structure
|Aug 6, 2012
|Aug 1, 2013
|Sean Coyle
|Panel and a method for building panels
|Jun 28, 2012
|May 1, 2014
|Nicholas Timothy Showan
|Method of erecting a building with wooden panels
|Jun 28, 2010
|Dec 29, 2010
|Timeloc Pty Ltd
|Building construction
|Dec 18, 2014
|Jun 25, 2015
|Aus Group Alliance Pty Ltd
|Plastic panel and structures using the same
|U.S. Classification
|52/794.1, 52/295, 52/309.11, 52/127.7, 52/293.3, 52/309.9, 52/309.7
|International Classification
|Cooperative Classification
|European Classification
|Aug 15, 2006
Owner name: SIP HOME SYSTEMS, INC., CALIFORNIA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:PORTER, WALTER KENNETH;REEL/FRAME:018111/0718
Effective date: 20060616
|Feb 4, 2013
|Maintenance fee reminder mailed
|Jun 23, 2013
|Lapse for failure to pay maintenance fees
|Aug 13, 2013
|Expired due to failure to pay maintenance fee
Effective date: 20130623


text:
The Poetry of Dylan Thomas: Under the Spelling Wall (Hardcover)
Special Order
Published in anticipation of the centenary of the poet's birth, The Poetry of Dylan Thomas is the first study of poet to show how his work may be read in terms of contemporary critical concerns, using theories of modernism, the body, gender, the carnivalesque, language, hybridity and the pastoral in order to view it in an original light. Moreover, in presenting a Dylan Thomas who has real significance for twenty-first century readers, it shows that such a reappraisal also requires us to re-think some of the ways in which all post-Waste Land British poetry has been read in the last few decades.
About the Author
John Goodby, scholar, poet, and translator, is Senior Lecturer at the University of Swansea and author of The Poetry of Dylan Thomas: Under the Spelling Wall, Illenium, and A True Prize.


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Little Elm, TXUSA
Hometown: Tallahassee, FL
July 18, 2011
I am a consultant for Sogeti USA and reside within Tallahassee. A lot of my work involves SharePoint in some fit or fashion, so I'm always ready to learn more.
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iHerb Customer Reviews
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Anything I have been thinking about writing lately has been cheese ball when it comes to getting back to printmaking. The point of all of it is to declare to you that I am getting back into it after a bit of a hiatus…a decade long hiatus. 345 more words
Tags » Goose Barnacles
Storm Imogen - a sand eater
Storm Imogen has coincided with the new moon spring tides. The waves are being blown hard against the sand cliffs on Penrhos Beach, eating away the faces that had already become unstable from the incessant rains and winds. 191 more words
Goose Barnacles
What do you talk about in a good restaurant, if you do not talk about food? Or other restaurants? I cannot imagine.
Yesterday I was lucky enough to have lunch in… 489 more words
WP designer Brie says get up close and personal with your subject this week.
Percebes at the Festa do Marisco de Vigo
Percebes are tube-shaped crustaceans that cling to the granite rocks of the storm-bashed Galician coastline. 182 more words


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you are viewing a single comment's thread.
I am not in any way casting aspersions by this comment, but, if you are not aware of people pumping a stock because they are long or dissing a stock because they are short, in addition to a totally manipulated market by the hedgie's, MM's and short sellers, you will never win in this game. I am sure you are a kind person and I commend you for that, but please know this is a great big gambling casino and our Country is going down the tubes quickly because of American GREED. Good Luck.


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The HOM coordinator will confirm new events and schedule changes for the History of Medicine Society presentations and will post them as they occur. (more information).
All events will be held in room 2117 Medical Education Research Facility (MERF) unless otherwise specified. The following information is largely confirmed but actual details about the talks won't be distributed until a month or so before the talk.
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April 17th 2014
5:30 PM to 9:00 PM
Chelsea Piers – Sunset Terrace
At Pier 61
(West Side)
New York, NY 10011
$169.00 per person
including everything
Cocktail hour
Open bar the entire event and includes Bottled Beers, Red & White Wines, Soft Drinks and bottled Water
Clicking on link will take you to the ticketing page
Directions Cars & Taxis
Cars and taxis enter Chelsea Piers through the vehicular entrance via the southbound lanes of Route 9A, off the West Side Highway, at the entrance opposite 23rd Street. Taxi stands are located at the entrances to Pier 61 and Pier 60. Moderately priced on-site parking is available. Pick-up and drop-off locations are shown on the map above.
Public Transportation
The M23 bus crosses Manhattan on 23rd Street and stops directly in front of Chelsea Piers. The M23 connects with all of Manhattan's north-south bus routes and subway lines. Subway-bus connections are located at the following 23rd Street stations:
#6 - Park Avenue
W, N, R - Broadway/Fifth Avenue
V, F, PATH - Sixth Avenue
#1 - Seventh Avenue
C, E - Eighth Avenue
The M14-D, which crosses Manhattan on 14th Street, terminates at 18th Street and the West Side Highway across the street from Pier 59.
This was such a great event. Cant wait for the next!
1 · April 18, 2014
A special thank you to DW who's dedication, support and tireless effort helped make this event possible. It was great meeting so many of you and I look forward to seeing everyone in Denver # GOWEEDSTOCK!!!!
1 · April 18, 2014
Fantastic. Family business meeting!
1 · April 18, 2014
Working on afterparty for Bill C. Bill told me the night isnt going to be over till he says it is
He is taking us Bowling at Bowlmor and it is only $1.00 each for the first 50 people
(he said free but the website thinks its being robbed and wont release the ticket for less than $1.00)
Bowlmor lanes at the pier. All the cool kids will be there
0 · April 17, 2014
Was I one of the first 50
0 · April 17, 2014
https://twitter.com/DW_EVENTS for updates regarding tonight.
Update #1
Bill C from FITX wants to take us bowling afterwards, I'm planning for the first 50 dinner guests who have already bought tickets to go for free! Immediately following dinner at Bowlmor Lanes at the Pier
0 · April 17, 2014
I got mine printed out already
0 · April 17, 2014
If you have reced your tickets yet, please notify us. All tickets have been emailed for the 4/17 event. Please check spam to make sure it is not there. We found quite a few would up in spam or junk filters. IF YOU HAVENT RECEIVED YOUR TICKETS PLEASE NOTIFY US ASAP. ALL TICKET ORDERED THRU 5PM today have been sent out.
0 · April 15, 2014
Thank you
0 · April 15, 2014
Primary Ticketing for the event is coming to a close in the next few hours. If you havent bought your tickets yet or you need additional tickets, now is the time to purchase them. You can use this link to purchase and confirm
Be there or be square. All the cool kids will be there :-)
0 · April 14, 2014
Can't do a coast to coast trip this month. I look forward to all the post details.
0 · April 5, 2014
I'm hoping to organize some form of transportation Bos-NYC for the 4/17 event. Anyone interested? Message me on 420investors site
0 · April 3, 2014
Is anybody flying into Newark? Looking to ride share Wed morninh to Terra Tech in Belvidere N.J. from my hotel in Newark . Maybe we can book the same Newark hotel? I have not selected one yet. I am attending the dinner on Thursday night, so we can ride share to NYC also.?
0 · March 29, 2014
4/17 Thursday works for me; coming in from Eastern Pa; love to make a day trip to NYC and meet the gang. Looking forward to that date. First TRTC in NJ; next day NYC... Doesn't get any better... See you all then..
1 · March 23, 2014
Thank you to everyone for your continued support and patience as we work to bring this event to fruition . As both DW and myself have been asked by many members regarding transportation options to and from TRTC's open house . I have setup a poll to gauge interest in setting up a private 420 investor bus to the TRTC open house.
The Bus would depart from NYC 4/16 in the AM and return to NYC 4/16 in the PM. You can access the poll by clicking on the MORE tab located at the top of the page or by Heading to this URL. http://www.meetup.com/NYC-420-Investor/polls/1045092/
Once again thank you for your continued support. Gabriel
1 · April 3, 2014
DW your one bizzzy fellow. Hats off to you and GB for getting this venue up and running.
1 · April 3, 2014
Wish I was going but I will be in Denver. Looking forward to the reports; and I'm especially excited to here how the TRTC open house goes. Derek will be the future rock star of our awakening industry.
0 · April 1, 2014
hey: I feel like I missed the center of the messaging. Is this a buyer's club? A sampler? Just having drinks to fantasize about it? What is the basic thrust of the meet-up?
0 · March 24, 2014
Daniel, 4/17 is a Thursday. Doors open at 4:30 and there will be a 1.5 hour cocktail hour, meet & greet before dinner. It was chosen to accommodate as many as possible. Some of those that work in the city, can only come for drinks early and cant stay for dinner.
Seemed like the best start time to accommodate the most people. There is no requirement to be there as soon as the doors open but people can come right over after work (or not), Market closes at 4PM. Starts half hour after the market closes and since the market is closed on 4/18, no need to leave early if you dont have to.
0 · March 22, 2014
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Marvel Assembles an Official Title for Third "Avengers" Movie
Comic Books, Film
Start Time: 01:00PM End Time: 02:30PM
From the movies The Road to Perdition to the recent bloody romp, Red, both based on graphic novels, "funnybooks" are making a larger impact in the digital age. On Saturday, March 19, at Meltdown Comics on the Sunset Strip, the So Cal branch of the Mystery Writers of America will present a panel on the writing and production of crime comics. Panelists are: Joshua Hale Fialkov (Elks Run, Tumor), Deborah Vankin (Poseurs), and Doselle Young (Gangland, Authority), moderated by Gary (High Rollers) Phillips.
This event is free.
Meltdown Comics
7522 Sunset Blvd.
Los Angeles, CA 90046
More Info: http://www.meltcomics.com/
This event was submitted by CBR.
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text:
Research Article
Entry and competition in generic biologics
Version of Record online: 17 AUG 2007
DOI: 10.1002/mde.1352
Copyright © 2007 John Wiley & Sons, Ltd.
Managerial and Decision Economics
Special Issue: Economic and Policy Issues in the Pharmaceutical Industry
Volume 28, Issue 4-5, pages 439–451, June - August 2007
Additional Information
How to Cite
Grabowski, H. G., Ridley, D. B. and Schulman, K. A. (2007), Entry and competition in generic biologics. Manage. Decis. Econ., 28: 439–451. doi: 10.1002/mde.1352
Publication History
- Issue online: 17 AUG 2007
- Version of Record online: 17 AUG 2007
Funded by
- Genentech
- Abstract
- References
- Cited By
Patents for several blockbuster biological products are expected to expire soon. The Food and Drug Administration is examining whether biologics can and should be treated like pharmaceuticals with regard to generics. In contrast with pharmaceuticals, which are manufactured through chemical synthesis, biologics are manufactured through fermentation, a process that is more variable and costly. Regulators might require extensive clinical testing of generic biologics to demonstrate equivalence to the branded product. The focus of the debate on generic biologics has been on legal and health concerns, but there are important economic implications. We combine a theoretical model of generic biologics with regression estimates from generic pharmaceuticals to estimate market entry and prices in the generic biologic market. We find that generic biologics will have high fixed costs from clinical testing and from manufacturing, so there will be less entry than would be expected for generic pharmaceuticals. With fewer generic competitors, generic biologics will be relatively close in price to branded biologics. Policy makers should be prudent in estimating financial benefits of generic biologics for consumers and payers. We also examine possible government strategies to promote generic competition. Copyright © 2007 John Wiley & Sons, Ltd.


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Heartland Monitor Poll
The twelfth installment of the Allstate/National Journal Heartland Monitor polling series focused specifically on the different experiences and economic expectations of men and women in the changing economy. This survey catalogues experiences of men and women in their home, family, and professional life, and gathers perspectives on the idea of opportunity in society and the workplace in the present day and how opportunity has and will change across generations. The survey also measures Americans' opinions about the changing gender profile of the country's workforce and what factors contribute to the continuing wage gap.
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National Journal feature articles:
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Next Economy Supplement (PDF)
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Translation of Islam in Spanish:
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- They argue Islam is a religion of peace and that Islamic faith is not the cause of terrorism.
- It does not cover cults within other world religions such as Islam and Hinduism.
- Now we have to watch some other clerics on TV talking about Islam being a religion of peace.
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