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Peaceful and serene. Just what we were looking for. A quiet countryside bed/breakfast. Thank you, Tim for giving us just that. Location is tucked in the countryside surrounded by the vineyards. Away from the hustle/bustles but 10 minutes from all the tourist attractions. Our stay was like visiting a home of a friend/family. We explored the grounds and enjoyed R&R... More
- Also Known As:
- Country Inn Calistoga
- Calistoga Country Inn


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The American Mountain Theater in Elkins has scheduled a series of Sunday afternoon Southern gospel and bluegrass shows that begin at 3:30 p.m. Sunday and will feature the Sexton Trio.
The shows continue through Oct. 13 and will feature a variety of acts. The Hopper will take the stage Aug. 18, the Gaither Homecoming on Sept. 15 and Karen Peck & New River on Oct. 13.
For more information, visit www.americanmountaintheater. com or call 800-943-3670.

subset: commoncrawl


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March 15, 2014
A hour and a half bus ride and not enough seats and kids walking a mile to the bus stop. They're all problems the Lincoln County School system is trying to iron out this first year of consolidation. Parents vented their concerns about the bus route issues at the Lincoln County Board of Education meeting Tuesday night.
Ever since Harts, Duvall, Hamlin and Guyan Valley High Schools consolidated this year, the big concern with parents is the bus routes and how long it will take their kids to get home. We heard from parents and students about a problem they hope the county will quickly resolve.
It's takes more than a hour for these Lincoln County High students to get home. They even have to switch buses to make sure they all get a seat.
Parents told Lincoln County Board of Education members how they felt about the longer bus routes and cutting out some bus runs is forcing kids to walk almost a mile up a hollow to their home. The concern here is safety.
Amber Cyfer wiped tears away during the meeting. She has a one mile walk after getting off the bus.
Cyfers said, "Walking up that hollow frightened me."
George Scarberry comments, "All of us have girls. Next thing you know something nasty will happen."
The county's transportation director says he's working on getting students as close to their homes as possible and still get them home in a timely manner. There are 48 different bus routes in Lincoln County and each one has three to five different hollows extending from it and it could take 15 minutes to drive up a hollow if you're bussing small children.
Dr. Anne Seaver, "We know we have problems, but everyday we've alleviated the crowding. All children will have seats on buses.
As for the amount of time kids have to be on a bus, board members say their hands are tied.
Carol Smith, "Not anything we can do about the time. We knew that when we were forced into consolidation."
Dr. Seaver says the board contacted the Transit Authority to see if they could run routes to help shorten the rides for students, but according to state law that's prohibited.


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Copyright © 2012 The Authors All rights reserved.
Cell Reports, Volume 2, Issue 3, 695-704, 20 September 2012
High-Throughput Screening for Bioactive Molecules Using Primary Cell Culture of Transgenic Zebrafish Embryos
Haigen Huang1, Anne Lindgren1, Xinrong Wu2, Ning-Ai Liu3 and Shuo Lin1, ,
1 Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095, USA
2 Liu Hua Qiao Hospital, Guangzhou 510010, China
3 Department of Medicine, Cedars-Sinai Medical Center, University of California, Los Angeles, CA 90048, USA
Graphical Abstract
► Primary embryonic cell cultures from lineage-specific zebrafish lines respond to exogenous chemical stimuli ► High-throughput screening in primary cells can identify proangiogenic chemicals ► Proangiogenic compounds identified from zebrafish screen can be validated in vivo and in mammalian cells
- Transgenic zebrafish embryos expressing tissue-specific green fluorescent protein (GFP) can provide an unlimited supply of primary embryonic cells. Agents that promote the differentiation of these cells may be beneficial for therapeutics. We report a high-throughput approach for screening small molecules that regulate cell differentiation using lineage-specific GFP transgenic zebrafish embryonic cells. After validating several known regulators of the differentiation of endothelial and other cell types, we performed a screen for proangiogenic molecules using undifferentiated primary cells from flk1-GFP transgenic zebrafish embryos. Cells were grown in 384-well plates with 12,128 individual small molecules, and GFP expression was analyzed by means of an automated imaging system, which allowed us to screen thousands of compounds weekly. As a result, 23 molecules were confirmed to enhance angiogenesis, and 11 of them were validated to promote the proliferation of mammalian human umbilical vascular endothelial cells and induce Flk1+ cells from murine embryonic stem cells. We demonstrated the general applicability of this strategy by analyzing additional cell lineages using zebrafish expressing GFP in pancreatic, cardiac, and dopaminergic cells.
In the past decade, the zebrafish has emerged as a viable model organism for bioactive small-molecule discovery. Using zebrafish, it is now possible to assess the specificity, efficacy, and toxicity of small molecules in the context of live animals. Zebrafish and mammals have a similar genetic and tissue/organ makeup. Thus, small molecules discovered in zebrafish screens often have analogous effects in mammalian systems, and vice versa (Lally et al., 2007; Murphey et al., 2006; Patton and Zon, 2001; Peterson et al., 2000, Peterson et al., 2004; Stern and Zon, 2003; Zon and Peterson, 2005). For instance, a chemical screen targeting the Bmp pathway used a chemical library of 7,500 compounds to identify dorsomorphin, a molecule that has conserved inhibitory activity on Bmp type I receptors alk2, alk3, and alk7, and has dorsalizing effects on zebrafish embryos (Yu et al., 2008). Although they are impressive in terms of screening capacity as a vertebrate model, zebrafish embryonic assays still cannot match the scale and speed of cell culture assays. To overcome this limitation, we developed an automated analysis of cultured primary cell differentiation as a strategy to preselect bioactive candidate compounds for further analysis (Figure 1). This strategy uses genetically stable, lineage-specific green fluorescent protein (GFP) transgenic zebrafish embryos at the blastula/gastrula stages to generate pluripotent primary cells, and allows them to differentiate in vitro. This culture system is equivalent to mammalian embryonic stem cells and can be used to screen tens of thousands of small-molecule compounds or peptide agents for effects on GFP-expressing/differentiating cell populations. Positive compounds from this primary screen can then be rescreened using mammalian assays and intact embryos.
Reproducible Fluorescent Protein Expression of Transgenic Lineages in Primary Cell Culture
To visualize the differentiation of different cell lineages marked by fluorescent protein expression in primary culture, we used several transgenic lines expressing GFP or red fluorescent protein (RFP) in various cell types representing the mesoderm, endoderm, and ectoderm. These fish included stable transgenic lines expressing GFP or RFP under the regulatory sequences of flk1 (Kdrl) for vascular endothelial cells, gata1 and scl for hematopoietic cells, cmlc2 for cardiac myocytes, mlc for skeletal muscle, insulin for pancreatic endocrine beta cells, and vmat2 for dopaminergic/vesicular monoamine transporter 2-positive neurons. Embryos from each of these transgenic lines were collected at late blastula/early gastrula stages and dissociated to generate primary cells. The dissociated embryonic cells, which initially expressed no fluorescent proteins due to their undifferentiated status, were plated into multiwell plates and allowed to grow in L15 basic medium for 5–9 days. Most of the primary cells became attached to the bottom of the plate within 24 hr of culture. At the same time, a small but relatively stable percentage of cells in culture started to express transgenic fluorescent protein. Under our culture conditions, lineage-specific cells acquired characteristic features of cellular differentiation.
For vascular endothelial cell differentiation, flk1-GFP transgenic embryonic cells initially showed GFP expression after 24 hr of culture and individual cells appeared scattered. At day 2, flk1-GFP-expressing cells became elongated and clustered together to form network-like structures. At days 3 and 4, flk1-GFP positive cells formed more extensive tube-like networks. These GFP-expressing cells underwent apoptosis and disappeared after 6 days of culture (Figure 2).
For hematopoietic cell differentiation, we used two fluorescent proteins to colabel different stages of differentiation. Gata1 is a marker for erythroid progenitors and differentiated primitive red blood cells, whereas Scl, a bHLH transcription factor, is expressed in both hematopoietic and vascular progenitor cells, as well as in some neurons (Zhang and Rodaway, 2007). Double-fluorescent transgenic embryos carrying transgenes for gata1-dsRed and scl-GFP were dissociated at the late blastula stage to allow visualization of hematopoietic cell differentiation in primary culture. We observed scl-GFP and gata1-dsRed expression from day 2 to day 9 of primary culture (Figure S1). Consistent with gene expression profiling in vivo, scl-GFP and gata1-dsRed were coexpressed in round-shaped hematopoietic progenitor cells (Figures S1A–S1C, A for bright field, white arrowheads) and were also uniquely expressed in some non-round cells (Figure S1B, red arrowheads), which are presumably Scl-positive neuronal cells and Gata1-positive red blood cells (Figure S1C, blue arrowheads). Of note, gata1-dsRed-expressing round-shaped cells survived until day 9 and clustered into colony-like structures (Figures S1D and S1F, white circle and red cells) in which some cells showed colocalized expression of gata1-dsRed and scl-GFP (Figures S1E and S1F, white arrowheads).
For myocardial differentiation marked by cmlc2-GFP expression, cultured primary cells began to express GFP at day 2 and continued until day 5. Some of the cells became contractile, resembling cardiac cells (Figures S1G–S1J). The skeletal-muscle-specific mlc-GFP maintained its expression from day 2 to day 4 and showed a morphological resemblance to skeletal muscle cells (Figures S1K, S1L, and S1N, and bright field M for N). For pancreatic beta cell differentiation, marked by insulin-GFP expression, low-frequency but clearly detectable GFP-positive cells became visible after 24 hr of culture (Figure S2A). Treatment of these primary cells with 1 μM retinoic acid (RA) increased the number of insulin-GFP expressing cells by ∼10-fold (Figure S2C) compared with the control (B) at day 2. This low frequency of GFP-positive cells in untreated primary cells provides a consistent baseline for detecting agents that can increase the differentiation of pancreatic beta cells, as demonstrated by the effects of RA treatment (Figure S2). For vmat2-GFP-positive neurons, ∼2% of cells were GFP-positive after 48 hr of culture (Figure S2D). Basic fibroblast growth factor (bFGF) promoted the generation of vmat2-GFP-labeled neurons and suppressed the differentiation of pigment cells (Figures S2E and S2G) compared with the untreated control (Figures S2D and S2F). There is a lack of zebrafish lines with faithful GFP expression specifically in dopaminergic neurons. Thus, our vmat2-GFP line can be useful for studying in vitro differentiation of dopaminergic neurons.
Response of Endothelial GFP Expression to Regulatory Agents in Primary Cell Culture
The FGF and vascular endothelial growth factor (VEGF) pathways are critical for vasculogenesis and angiogenesis in both development and pathological conditions (Cross and Claesson-Welsh, 2001; Poole et al., 2001). Recombinant FGF has been reported to induce VEGF expression and proliferation in human umbilical vascular endothelial cells (HUVECs) via both autocrine and paracrine mechanisms (Parsons-Wingerter et al., 2000; Seghezzi et al., 1998). To investigate whether exogenous growth factors can promote endothelial differentiation in zebrafish primary embryonic cell culture, we supplemented the culture medium with recombinant bFGF and VEGF. At 100 ng/ml, bFGF significantly stimulated flk1-GFP expression at day 1 of culture (Figures 3A–3D). Moreover, recombinant human VEGF121 significantly stimulated endothelial growth (Figure 3; p = 0.0007), consistent with previous reports that recombinant VEGF121 remarkably enhanced sprout formation and vessel length in HUVECs (Nakatsu et al., 2003). SB-431542 is a potent and specific inhibitor of the transforming growth factor-β (TGFβ) superfamily type I activin receptor-like kinase (Alk) receptors Alk4, Alk5, and Alk7, which are involved in many biological activities, including cell growth, differentiation, migration, survival, and adhesion. Furthermore, the TGFβ/ALK5 pathway was found to inhibit endothelial cell migration and proliferation in vitro (Goumans et al., 2002). SB-431542 therefore acts to stimulate proliferation, differentiation, and vessel formation in endothelial cells derived from embryonic stem cells or from fetal mouse metatarsals (mouse metatarsal assays), when treated alone or in combination with VEGF (Liu et al., 2009; Watabe et al., 2003). Similarly, we observed that SB-431542 stimulated tube formation in flk1-GFP-labeled endothelial cells in primary culture at day 5 when cultured alone (Figures 3G and 3I, lane 2) or with VEGF (Figures 3H and 3I, lane 4). Compared with DMSO, SB-431542 increased the total tube lengths 3-fold and the number of branch points 2-fold. This finding suggests that SB-431542 can be used as a positive control for identifying other small molecules that can promote vascular differentiation in zebrafish primary cell culture. To validate that the increased GFP expression represents endogenous gene expression, we analyzed flk1 mRNA by quantitative PCR (qPCR), and found that SB-431542 increased its level by 2.2-fold.
The above results suggest that cultured primary cells are capable of responding to positive stimuli under culture conditions. We next tested whether flk1-GFP expression in primary culture would respond to inhibitory molecules. SU5416, a potent inhibitor of VEGFR2 signaling, has been widely used to inhibit angiogenesis in vivo and in vitro (Fong et al., 1999; Serbedzija et al., 1999). Consistent with previous reports, SU5416 markedly inhibited the growth and migration of endothelial cells labeled with flk1-GFP in primary culture (Figures S3A–S3D). In addition, SU5416 suppressed bFGF-induced endothelial cell growth at 24 hr after culture (Figures S3E–S3L), indicating that the FGF signaling pathway promotes proliferation and differentiation of endothelial cells through the VEGF signaling pathway. Overall, these studies demonstrate that primary cell cultures from transgenic zebrafish embryos expressing cell lineage-specific GFP or RFP can be used to test either inductive or inhibitory agents, ranging from small molecules to proteins, for the discovery of potential therapeutic drugs.
High-Throughput Screening for Proangiogenic Compounds in flk1-GFP Primary Culture
Investigators have made a tremendous effort to identify angiogenesis inhibitors for their potential application in cancer therapeutics. However, much less attention has been directed to the identification of proangiogenic drugs for treating diseases such as chronic wounds and peripheral arterial and ischemic heart diseases. In such conditions, the therapeutic goal is to stimulate angiogenesis to improve the delivery of survival factors to sites of tissue repair, mobilize regenerative stem cells, and ultimately restore function to the tissues. With the use of our flk1-GFP cultured cell assay, we were able to perform a large-scale screen for proangiogenic small molecules. Primary cells prepared from flk1-GFP transgenic embryos were seeded onto 384-well plates and treated with compounds. Images of flk1-GFP-labeled endothelial cells were taken with an automatic fluorescence analyzer at day 5 of culture. A representative image of a 384-well plate is shown in Figure S4, with column 2 as a positive control and column 23 as a negative control. We observed a clear difference in tube-like endothelial cells between the two columns (Figure S4). We developed an algorithm that measures the total tube length and branch points to identify the positive compounds (see Experimental Procedures ; Table 1). The relative ratios of total tube length and branch points between experimental compounds and controls were used to determine whether a compound was positive for promoting angiogenesis. For each plate, the average value of the column containing SB-431542 served as a positive control.
Table 1 |
Chemicals | SB | A48 | Nd | DTR | SST | A46 | A36 | A33 | A93 | A79 | Lh | ZDM | A69 | A54 | ||
Ratio in total tube length | 1 | 1.5 | 1.5 | 1.5 | 1.6 | 1.7 | 1.7 | 1.7 | 1.7 | 1.8 | 1.8 | 1.8 | 1.8 | 1.8 | ||
Ratio in branch point | 1 | 1.6 | 1.6 | 1.7 | 1.5 | 1.7 | 1.7 | 1.8 | 1.8 | 1.6 | 1.6 | 1.7 | 1.9 | 2 | ||
Chemicals | SB | A39 | A80 | A14 | Gh | ADT | Fh | MH5 | A62 | A86 | A65 | Dip | Rl | Ph | ||
Ratio in total tube length | 1 | 1.8 | 1.8 | 1.9 | 1.9 | 1.9 | 2 | 2 | 2.1 | 2.1 | 2.3 | 2.4 | 2.5 | 2.6 | ||
Ratio in branch point | 1 | 2 | 2.1 | 1.7 | 1.8 | 2.1 | 1.8 | 2 | 1.5 | 2.3 | 2.5 | 3 | 2.8 | 2.4 | ||
Chemicals used: A14, AST 6156114; A33, AST 6953386; A36, AST 6541036; A39, AST 06978839; A46, AST 6978646; A48, AST 5546348; A54, AST 7153654; A62, AST 6417262; A65, AST 6953065; A69, AST 5062069; A79, AST 6291379; A80, AST 6014780; A86, AST 6978186; A93, AST 5557493; ADT, androsterone; Dip, dipyridamole; DTR, DTR815; Fh, fenoterol hydrobromide; Gh, guanfacine hydrochloride; Lh, levamisole hydrochloride; MH5, methylhydantoin-5-(D); Nd, nimodipine; Ph, papaverine hydrochloride; Rl, rolipram; SB, SB431542; SST, succinylsulfathiazole; ZDM, zimelidine dihydrochloride monohydrate. |
Four commercially available libraries (Biomol, Prestwick, Microsource, and Tar) containing 12,128 compounds were selected for screening. Small molecules that induced total vascular tube lengths and number of branch points equal to or greater than those observed with SB-431542 treatment were considered as positive hits, which yielded 165 candidate compounds. These candidates were rescreened and ∼63% of them still passed the initial screening criteria. Candidates that demonstrated 1.5-fold greater vascular tube lengths and numbers of branch points than SB 431542 treatment were selected for further analysis. This more-stringent criterion yielded 26 compounds (Table 1). Figure 4 shows representative images of enhanced endothelial proliferation and tube formation by candidate compounds at day 5 of culture.
Validation of Candidate Compounds in Mammalian Cells and In Vivo
To determine whether the candidate compounds also promoted the growth of mammalian endothelial cells, we examined their activities in HUVEC assays. We tested 23 commercially available candidates via a thiazolyl blue tetrazolium bromide (MTT) assay and found that 11 of them enhanced the proliferation of HUVECs by 19%–60%, with a p value of 0.02–0.000001 (Figure 5A). The structures and names of these compounds are listed in Figure S5. Additionally, these compounds enhanced the migration of HUVECs (Figures 5B–5E; only data for A65 are shown). Akt (protein kinase B) signaling plays a major role in regulating endothelial cellular proliferation, survival, migration, and adhesion, and its activation by phosphorylation stimulates proliferation through multiple cell cycle targets (Chen et al., 2008; Manning and Cantley, 2007). Hence, we selected six candidate compounds (A65, A86, dipyridamole [Dip], fenoterol hydrobromide [Fh], guanfacine hydrochloride [Gh], and succinylsulfathiazole [SST]) and investigated their ability to modulate the phosphorylation levels of Akt. HUVECs were treated with these candidate compounds, followed by western blotting analysis using an anti-phospho-Akt antibody. For all six compounds, we observed increased phosphorylation of Akt compared with DMSO-treated cells (Figures 5F, 5G, and S6A), suggesting that the proangiogenic activities of these compounds involved Akt signaling pathways. As a confirmation, we cotreated the cells with wortmannin, a well-established inhibitor of the PI3K/Akt pathway, and observed reversal of the proliferation effects of HUVEC by the selected compounds (Figure S6B). Because Akt-dependent phosphorylation has been implicated in activating endothelial nitric oxide (NO) synthase (eNOS), which was found to be involved in cardiovascular homeostasis (Dimmeler et al., 1999), we sought to determine whether any of the tested compounds exert their proangiogenic activity through NO production. We conducted the same HUVEC proliferation assays with selected compounds, adding the eNOS inhibitor L-NAME (Hood and Granger, 1998). This assay showed that the proproliferative effect of SST, Dip, Gh, and Fh on HUVEC could be reversed by eNOS inhibition (Figure S6C), suggesting that the Akt-eNOS pathway is involved in the mechanisms of action for these compounds.
To determine whether these compounds could also induce mammalian endothelial differentiation, we examined the ability of the compounds to increase the number of Flk1+ cells generated from differentiating murine embryonic stem cells (mESCs). We tested the 11 compounds that were able to promote the growth of HUVEC cells by adding the compound to mESCs on day 2 of differentiation and examining the level of Flk1 expression by flow cytometry on day 4 of differentiation (Figure 5H). Contrary to its role in the promoting the proliferation of endothelial cells, SB431542 inhibited the differentiation of mESCs into Flk1+ cells. This result was expected because inhibition of TGFβ signaling promotes self-renewal and inhibits differentiation in pluripotent cells (James et al., 2005). The 11 compounds discovered in this work were all able to enhance the percentage of Flk1+ cells over that of control conditions. A36, nimodipine (Nd), and SST were all very consistent in their ability to increase the percentage of Flk1+ cells by 50% (Figure 5H). Overall, these findings validate that the effects of the small molecules identified from zebrafish embryo primary cell assays are largely conserved in mammalian endothelial cells.
Intussusceptive angiogenesis, a mode of blood vessel formation and remodeling, occurs in lung and eye vasculature by internal division of the preexisting capillary plexus without sprouting (Djonov et al., 2000; Makanya et al., 2009). Therefore, we next sought to investigate whether selected candidate compounds could enhance angiogenesis in adult zebrafish, as a test of proangiogenic activity in vivo. To that end, we treated flk1-GFP transgenic adult fish with selected small molecules and then observed the retinal vasculature after eye dissection (Cao et al., 2010). As shown in Figure 6, two selected drugs—SST and Dip—significantly enhanced retinal angiogenesis as compared with the DMSO control treatment (n = 22 retinas). SST and Dip increased the number of blood vessels by 27.2% (p = 0.0216, n = 12 retinas) and 34.3% (p = 0.0220, n = 13 retinas), and the vascular density by 49.8% (p = 0.0090, n = 12 retinas) and 50.9% (p = 0.0250, n = 13 retinas), respectively.
The zebrafish has been established as an excellent vertebrate system for studying embryonic development, modeling human diseases, screening for pharmaceutical drugs, and dissecting biological pathways. A key bottleneck in using zebrafish whole animals for drug screening is the limitation of throughput. In this work, we developed a primary-cell-based approach that significantly expands the utility of this valuable model organism by taking advantage of the vast collection of transgenic zebrafish expressing GFP or RFP in specific tissues. Similarly to immortalized-cell-based high-throughput screening, our approach offers the advantage of high throughput, high content (with multicolor transgenes), and high efficiency afforded by the screening of small molecules. Given the fact that several pairs of fish can produce thousands of eggs, a sufficient supply of embryos to generate primary cells is never a limiting factor. A trained person can easily set up three 384-well plates per day. As shown above, because primary cells differentiate in a time frame similar to embryonic development, this method captures a biological process in a way that is comparable to the zebrafish in vivo screening system but can be scaled up significantly. In addition, because it is difficult to visualize and quantify cells that are deeply embedded within the zebrafish embryo, such as pancreatic β cells, whole-embryo compound screening is often inefficient. The use of primary cultured cells overcomes this limitation by allowing direct visualization and statistical analysis of the cells of interest.
We have shown that 11 out of 26 chemicals were capable of enhancing the proliferation and migration of HUVECs, indicating that a reasonable number of small molecules identified in our zebrafish primary-cell-based screen could be translated into the human endothelial system. In addition, these compounds were able to promote the endothelial differentiation from mESCs. For those compounds that failed to promote mammalian endothelial cell proliferation and differentiation, it is likely that the properties of their target proteins differ somewhat between zebrafish and mammalian species. Further validating our finding that 11 compounds showed positive hits in both zebrafish and mammalian cells is the fact that several of these compounds were previously implicated in promoting vascular formation. Finger and Dunnett, 1989 reported that Nd, a calcium channel antagonist and a dilator of blood vessels, promoted vascularization in suspension cell grafts in rats, although the drug had no lasting effects on vascularization in intact brains. The authors proposed that Nd may increase endothelial survival, reduce loss of existing blood vessels, and stimulate existing vessels in the nearby area to grow into the graft. Dip, a compound that inhibits thrombus formation when given chronically, and causes vasodilation when given at high doses over a short time, was reported to induce capillary growth in normal and hypertrophic hearts of rabbits (Torry et al., 1992). It was also observed to enhance ischemic angiogenesis in the diabetic hind limb by preferentially promoting endothelial proliferation in mice (Pattillo et al., 2011). It is believed that elevated oxidative stress and reduced NO bioavailability are hallmarks of endothelial cell dysfunction and cardiovascular pathology during diabetes in both humans and animals. Therefore, Dip was further characterized to enhance ischemia-induced angiogenesis and arteriogenesis by increasing nitrite/NO production and decreasing oxidative stress (Pattillo et al., 2011; Venkatesh et al., 2010). Guanfacine, a α2-adrenoceptor agonist and vasodilator, was shown to prevent the cardiovascular effects of hemispheric ischemia by inducing a surge of luminal NO in rats (Figueroa et al., 2001; Saad et al., 1986). Fh, a β2-adrenoceptor agonist, was also found to induce NO production and eNOS phosphorylation in endothelial cells (Figueroa et al., 2009); however, whether it could stimulate angiogenesis was not reported. Nonetheless, these earlier reports are consistent with our finding that the proproliferative effect of SST, Dip, Gh, and Fh on HUVEC was reversible by eNOS inhibition. SST, an antibacterial drug, has not previously been reported to enhance endothelial cell growth. It will be interesting to determine whether this drug can be used to treat vascular-deficiency diseases. It is worth mentioning that five of these 11 compounds are FDA-approved drugs that can be used directly in humans. For the rest of the compounds, further studies of efficacy, pharmacokinetics, and safety are needed to determine their application to angiogenesis-deficiency diseases.
Experimental Procedures
Preparation of Zebrafish Embryo Primary Cells
We used the following transgenic zebrafish lines or promoters: gata1-dsRed (Long et al., 1997), scl-GFP (an enhancer trap line identified in-house) (Wen et al., 2008), cmlc2-GFP, mlc-GFP, flk1-GFP, insulin-GFP (Huang et al., 2001), and vmat2-GFP (Wen et al., 2008).
On day 1, ∼30 pairs of transgenic fish were set up with dividers in the afternoon and released together to mate early the following morning. Collected eggs were incubated at 32°C in fish water containing 1X antibiotics (Antibiotic-Antimycotic Solution; Cellgro). Later, at the gastrulation stage, embryos were cleaned several times with Holtfreter's solution that was prewarmed at 32°C.
Embryos were allowed to grow continuously to 80% epiboly/tailbud stage or stage of interest at 32°C. In the meantime, 20 μl of Leibovitz L15 medium (phenol red free, 5% fetal bovine serum [FBS], 1% antibiotics) was added to each of 384 wells of the Greiner μClear Black Plate (T-3037-9, ISC Bioexpress; Greiner Bio-One) using a multichannel pipette in a sterile tissue culture hood. To each well, compound (normally 10 μM) was then individually added via an automated liquid handler. The entire columns of 2 and 23 for a 384-well plate were reserved as positive (2 μM SB-431542) and negative (1% DMSO) controls, respectively.
Cleaned embryos were combined and treated in 8 ml Holtfreter's solution containing 3 μg/ml pronase for ∼8 min. This allowed removal of chorions after several washes with Holtfreter's solution. Dechorinated healthy embryos were transferred to a clean tissue culture dish. To produce enough cells for one 384-well plate, ∼1,600 embryos should be prepared.
The embryos were then bleached in 0.04% sodium hypochlorite (Cat. 23930-5; Aldrich) for exactly 3 min followed by five washes in PBS. The embryos were then transferred to a 1.5 ml Eppendorf tube, excess PBS was removed, and the embryos were homogenized with blue pestles (Fisher Scientific). After homogenization, cell aggregates were transferred to a tube containing 6 ml of 0.05% trypsin (Invitrogen) and incubated at 37°C for 10 min. Then an equal volume (6 ml) of medium containing 10% FBS was added to the tube to stop trypsin digestion. The cell suspension was gently mixed and then centrifuged at 800 rpm for 4–6 min at 4°C (Allegra 25R centrifuge; Beckman). The cell pellets were resuspended in the culture medium (18 ml for every 1,600 embryos) and 40 μl cell suspension was distributed into each of 384 wells using a multichannel pipette. Cells were grown at 32°C without CO2. At day 5, fluorescence of cultured tg(kdrl:gfp) cells was imaged using the ImageXpress Micro Screening System (Molecular Devices) equipped with LHS-H100P-1 camera (Nikon, Japan). The image of each well was analyzed with Angiogenesis Tube Formation (RD-1) of High Content Image Processing Software to generate data regarding the tube length and branch points (MetaXpress; Molecular Devices).
Endothelial Proliferation and Migration Assays
HUVECs were purchased from Invitrogen (C-015-5C) and cultured in the endothelial cell growth medium (MV2 plus complete supplement, C-22221 and C-39221; PromoCell) at 37°C and 5% CO2. For the proliferation assay, HUVECs were seeded at a density of 6 × 103 cells/well in 96-well plate in normal growth medium for 24 hr. The medium was then changed to 0.5% (v/v) FBS containing MV2 for serum starvation for 24 hr. Cells were stimulated with hVEGF165 (#8065; Cell Signaling) and small molecules for 72 hr (Holmes et al., 2010), and the number of viable cells was determined using the MTT assay (Twentyman and Luscombe, 1987). For the MTT assay, 2 ml fresh MTT (M2128; Sigma) solution was prepared per 96-well plate at 5 mg/ml in PBS, and 20 μl MTT solution was added to each well. The plates were put on a shaker at 150 rpm for 5 min to mix the MTT thoroughly into the media. Plates were incubated (37°C, 5% CO2) for 1–5 hr to allow the MTT to be metabolized. The media were discarded, and formazan (MTT metabolic product) was dissolved in 200 μl DMSO. The plates were put on a shaker at 150 rpm for 5 min to mix the formazan thoroughly into the solvent. Optical density was recorded at 570 nm and subtracted with background at 630 nm. Optical density is directly correlated with cell quantity. For the migration assay, HUVECs were seeded at a density of 5 × 104 cells/well in 24-well plate in normal growth medium for 48 hr. The media were then changed to 1% (v/v) FBS containing MV2 for serum starvation for 24 hr. A scratch was introduced to the cell monolayer using a sterile 200 μl pipette tip followed by incubation with hVEGF165 (#8065; Cell Signaling) and small molecules for 16–24 hr (Holmes et al., 2010). The migration distance was measured in micromolars for comparison.
Akt Phosphorylation Assays by Western Blotting Analysis
For Akt phosphorylation assays, 5 × 105 HUVEC cells/well were seeded in a six-well plate and cultured in the medium MV2 plus supplement for 24 hr, followed by 12 hr starvation in the medium MV2 plus 0.5% FBS. After starvation, cells were treated with DMSO or compounds in the same medium for 8 hr, followed by total protein preparation. Phospho-Akt (S473) antibody and total Akt (C67E7) antibody were purchased from Cell Signaling (p-Akt #9271 and Akt [pan] #4691), and protein gels were purchased from Thermo Scientific (Cat. 25200-25244). The Akt kinase assay was performed according to protocols provided by the manufacturers.
Growth and Differentiation of mESCs
iER71 mESCs were maintained in an undifferentiated state on a mitomycin C inactivated CF1 MEF feeder layer. Cells were grown in KO-DMEM (Invitrogen), 15% FBS (Hyclone), 1× NEAA, 1× penicillin/streptomycin/L-glutamine, 55 μM β-mercaptoethanol (all Invitrogen), and 1,000 U/ml LIF (Millipore). Differentiation was induced by plating 1 × 105 cells/well of a six-well plate in Iscove's modified Dulbecco's medium (Invitrogen), 15% FBS (Hyclone), 1× penicillin/streptomycin/L-glutamine and 55 μM β-mercaptoethanol (both Invitrogen), and 50 μM ascorbic acid. Compounds were added on day 2 of differentiation.
Flow Cytometry
Cells were harvested by 0.25% trypsin digestion on day 4 of differentiation. Immunolabeling was performed with 1:100 PE conjugated anti-mouse Flk1 (KDR; BD-PharMingen) in PBS with 1% BSA and analyzed on a BD LSRFortessa cell analyzer.
Retinal Angiogenesis Assay
Individual flk1-GFP transgenic adult fish (14 months old) were incubated in 60 ml fish water containing 0.5% DMSO and selective compounds or only 0.5% DMSO for 72 hr, followed by eye dissection under a Zeiss dissection microscope (Cao et al., 2010). Images were obtained and analyzed on an Axioplan 2 Zeiss microscope with fluorescence. Blood vessel branches within a circle with a radius of 90 μm from the optic disc were counted with the use of OpenLab software. Total vascular density was measured as percentage of area covered by fluorescent vessel within the same circle centering at the optic disc per eye.
We thank Zahra Tehrani and Matthew Veldman for proofreading the manuscript, Anqi Liu and Yuan Dong for zebrafish husbandry, and Robert Damoiseaux for preparation of chemical libraries. This work was supported in part by a research grant from the National Institutes of Health (DK54508 to S.L.).
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Publication Information
Received: March 12, 2012
Revised: June 1, 2012
Accepted: August 15, 2012
Published online: September 20, 2012


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I would think her moaning and saying "Oh yes, harder..." might be an indication that she was a willing participant?:p
I'm not defending the boy by any means, and I'm sure if I were the father I'd be mad as hell, but IMO when he continued to chase the boy down the street, that was where he crossed the line. I'm sure the daughter was yelling "Dad, stop!" or at least saying SOMETHING that would indicate that she had him there willingly.
IDK, I'm 22 years old, so maybe my sense of things is different, but I just think the Dad went too far. And the fact that the daughter and the bf had dated for over a year and he didn't know shows that he was a bit out of touch with the daughter. Or maybe she was going behind his back because she knew he would go nuts over any bf she had. Kids are like that, the parent says "No dating, period!" so the kid decides to go behind said parent's back. That sounds like what happened here.
Statutory Rape Is Illegal Sexual Activity Between Two People When It Would Otherwise Be Legal If Not For Their Age: In accordance with the FBI definition, statutory rape is characterized as non-forcible sexual intercourse with a person who is younger than the statutory age of consent.
The boy is lucky to be alive. And, no, you hardly think.
Last edited by wineslob; 09-17-2008 at 03:44 PM.
I was under the impression that statutory rape was when one person was over the age of consent, and the other under the age? Does that apply if they're both 18? I believe AZ has a clause where if the two parties are within 18 months age of each other than its not statutory rape.
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2:51 am
Mon May 14, 2012
Budget Woes Could Close Philly's Problem Schools
By Jeff Brady
Originally published on Mon May 14, 2012 4:44 am
Philadelphia's school district plans to close a quarter of its school buildings in coming years to eliminate a huge budget hole. But parents and activists don't trust the decision-makers. Many of them suspect the plan is a ruse to force charter schools and privatization on the district.


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Dad on the run.
Just stopping in for a quick hello. Wanted to show everyone what I built!!!
This is 199 online
And I built my own for 25 bucks getting my stuff from wal-mart
Pretty freaking awesome if you ask me!
My parents said I could be anything when I grew up, so I decided to be Awesome!
She posted already! She finished. Will keep the time secret so she can reveal when she posts here.
First HM: Covenant Health Knoxville HM 4/2013 2:24
Second HM: North Shore HM, Highland Park, IL 6/2013 2:20
Third HM: Hard Rock HM, Nashville, TN 11/2013 2:20
Fourth HM Michelob ULTRA Miami HM 2:31
I race in SparkleSkirts
jblackjr you build a TRX?! awesome!!!
PRs: 5K- 28:16 (5/5/13) 10K- 1:00:13 (10/27/13) 4M- 41:43 (9/7/13) 15K- 1:34:25 (8/17/13) 10M- 1:57:23 (6/15/13) HM- 2:21:47 (10/12/13)
I started a blog about running :) Check it out if you care to
Barking Mad To Run
On Saturday, did my 24th race of 2013: http://www.runningahead.com/groups/BF_and_Beyond/forum/Post/c5227e2719cc460cba9f51a07e3d0ae4#focus
Followed that up on Sunday morning by doing 3.5 miles around Lake Elmendorf Park, a local San Antonio park. Never been to this park in all the time I've lived here, to took a few photos along my route.
As I started out, I was greeted by the sight of all these birds - egrets, maybe? - in a couple of trees by the lake.
Some nice views from the park trail of the lake and of some of the buildings of Our Lady Of The Lake University
Some murals along some of the stonework near the park trails. I suppose some folks would call this 'graffitti" but it looks like quite well-crafted art-work to me.
Right before I got back to where my car was parked, I passed the trees where the birds were and found that the back of the trees were just as full of birds as in the first photo I took of the front of the trees.
In the past, the West Side of San Antonio has had a reputation of being somewhat of a high-crime area, and a not-so-safe area to be in. Yet for my run this morning, I did not find that so at all. I found it to be very pleasant and the people/residents I encountered along my route were quite friendly. I even got a few 'high-fives" along the way for my "I BEAT CANCER! NEVER GIVE UP!" shirt. This is a very nice little park and I was surprised that more people were not out there taking advantage of its good trails. Definitely will run here again in the future sometime.
"Do what you can, with what you have, where you are." Theodore Roosevelt
Miles to go
Hey guys! Finished in 1:56:00. Those last 3 miles were HARD! I never had the same spring in my step that I had for my 10 miler, but I got it done! Splits are ugly, though. Had a nice lunch with the family and just ate all the calories I burned in my race in cookies & cream mousse.
RR later, I think there is some celebrating to do at my MIL's house.
PRs: 5K: 25:35 / 10K: 53:03 / 10mi: 1:26:15 / HM: 1:55:02
Upcoming: 10 mi. 3/15, HM 4/13
Bad Ass
Great job!
Damaris, Marathon Maniac, Ultra Runner
Next: Space Coast Marathon
"The most powerful weapon on earth is the human soul on fire."
Ya it was pretty easy. First time working out on it today and all I can say is wow. The handles are from my bow flex and they worked out really well. Other wise you cabundled your own or buy some from Walmart for like 10 bucks a piece.
Haven't left yet, so I'll post my splits. They are pretty revealing...
1- 8:47
2- 8:49
3- 8:41
4- 8:45
5- 8:53
6- 8:34
7- 8:37
8- 9:07 (whole mile was uphill)
9- 8:42
10- 8:53
11- 9:01
12- 9:21
13- 9:22
0.7- 8:13 pace
Yay for Zelanie!! Scotty you havefun 24 races already this year?!! Damn!
Yeah Zelanie! Awesome race--you got your sub-2:00 with plenty to spare!
Future Races:
Melanie - you ran a great race. Under two hours for your first HM - hurray.
Coralie - yes, I guess they were right to cancel the race. That would not have been fun.
Scotty - for some reason, the photos don't show for me. Still, it sounds like a nice park.
Smaller By The Day
Awesome job Zel! I'm just checking in real quick. It's P3's birthday, and I'm just along for the ride. We're about to head out on our bicycles. No running today. That feels weird.
Hey guys! Finished in 1:56:00. Those last 3 miles were HARD! I never had the same spring in my step that I had for my 10 miler, but I got it done! Splits are ugly, though. Had a nice lunch with the family and just ate all the calories I burned in my race in cookies & cream mousse. RR later, I think there is some celebrating to do at my MIL's house.
Weight 100 pounds lost
5K 31:02 Sept. 2012 / 23:36 Sept. 2013 (Same Course)
10K 48:59 April 2013
HM 2:03:56 Nov. 2012 / 1:46:50 March 2013
MARATHON 3:57:33 Nov. 2013
St. Jude's Hero Profile
Happy birthday p3!!!
Happy Birthday, Pew Pew Pew!
Anson, tell Marla I am singing ** Happy Happy Happy Birthday**.
Zel, YOU DID AWESOME!!
Ginny, I can't see the pics either, but I'm on my phone.
4 miles done.
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From Larry Brown Sports:
Andrew Bynum's health has prevented him from playing this season, but his hair has managed to keep him in headlines.
The Philadelphia 76ers center was spotted watching his team play on Wednesday while sporting the 'do you see above. I really don't even know how to describe the thing.
Earlier this season, Bynum was sporting the Don King look.
But this new look? How do you even describe it? Is he going with the Ike Turner cut? Is it an ode to The Beatles? Is it taken from Moe of the Three Stooges?
I think Bynum does this intentionally just to mess with peoples' heads, kind of like the way he was so insistent on shooting threes with the Lakers. He just likes stirring it up.
Here are other looks at the 'do:
And courtesy of Beyond the Buzzer, we have the evolution of Andrew Bynum's hair:
Photos via SB Nation, @Jose3030
The Evolution of Andrew Bynum's Hair
Sixers' Bynum has a setback
The Evolution of Andrew Bynum's Hair (Photos)
Sixers Andrew Bynum Gets A Perm
Andrew Bynum YOLO FRO T-Shirt
Bynum Has 'Setback' In Recovery From Knee Injury
76ers C Andrew Bynum Now Has the Most Amazing Hair in Professional Sports
The only thing better than Andrew Bynum's YOLO fro is Charles Barkley in YOLO fro
Packers Corner Comes Out as a Transformer; Bynum Gets Silly
Andrew Bynum's Hair Is Pretty Epic
Andrew Bynum's Ongoing Injury Provides Opportunity for Jrue Holiday to Emerge as Leader for Sixers
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Honoratus served as guardian in a Warsaw friary already in 1860. He dedicated his energies to preaching, to giving spiritual direction and to hearing confessions. He worked tirelessly with the Secular Franciscan Order.
The failed 1864 revolt against Czar Alexander III led to the suppression of all religious Orders in Poland. The Capuchins were expelled from Warsaw and forced to live in Zakroczym, where Honoratus continued his ministry and began founding 26 male and female religious congregations, whose members took vows but wore no religious habit and did not live in community. They operated much as today's secular institutes do. Seventeen of these groups still exist as religious congregations.
The writings of Father Honoratus are extensive: 42 volumes of sermons, 21 volumes of letters as well as 52 printed works on ascetical theology, Marian devotion, historical writings, pastoral writings — not counting his many writings for the religious congregations he founded.
In 1906, various bishops sought the reorganization of these groups under their authority; Honoratus defended their independence but was removed from their direction in 1908. He promptly urged the members of these congregations to obey the Church's decisions regarding their future.
He "always walked with God," said a contemporary. In 1895 he was appointed Commissary General of the Capuchins in Poland. Three years before he had come to Nowe Miasto, where he died and was buried. He was beatified in 1988.
Honoratus worked very zealously to serve the Church, partly by establishing a great variety of religious congregations adapted to the special circumstances of Poland in those years. He could have retreated into bitterness and self-pity when the direction of those congregations was taken away from him; that was certainly a "perfect joy" experience. He urged the members of these groups to obey willingly and gladly, placing their gifts at the service of the Good News of Jesus Christ.
The author of the Joshua series, Fr. Joseph Girzone, retells the ancient stories of Jesus in ways that make them fresh and accessible.
Why did the pope choose the name Francis? Find out in this new book by Gina Loehr.
Learn how the life and teachings of St. Teresa Benedicta of the Cross (Edith Stein) serve as a guide for women's unique vocations today.
Find the real highway to freedom using the road map of Christ's two laws and the Ten Commandments.


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Dr. Bronner's Magic Organic Shaving Soap Gel Lavender -- 7 fl oz
After running out of my favorite shave lotion from Trader Joes (none in my state) I wanted a good, natural replacement. Tried this shave gel out and while it wasn't the cream I'm used to, I am totally in love. Very non-drying, light pleasant fragrance, and provides a fantastic smooth shave. Definitely a new fan!
May 25, 2013
Awesome shaving soap!
I realized how disgusting the barbasol I had been using to shave with smelled and that I was otherwise using very natural cosmetics, so I decided to give this a try. I really like it, it's very effective and makes for smooth shaving and a little bit goes a very long way. It is very liquid-y, though, so I store it upside down (cap side up, leaned against the wall of the medicine cabinet) so that it won't leak everywhere.
April 18, 2013
I am hooked on Dr. Bronner's products......this one is great!! The color is a little strange but it works!! Smells fantastic and gives me a closer shave than any other product I tried! No irritation either, and I am super sensitive.
October 17, 2009
Not the greatest shaving cream...
It is no better than using the Dr. Bronner's liquid soap to shave your legs. I appreciate the quality ingredients this brand uses but I just don't think this is all that useful a product...my boyfriend tried it on his face and agreed...it is quite liquid like the soap.
June 10, 2009
takes some getting used to
it took me a few uses to realize that this product would lather up. now that i know that, it works pretty decent. might not be the best shaving cream ever, but it's organic, it beats shaving with plain soap, and it does the job.
May 29, 2009


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The Bridge, Lakeshore Technical College's new intranet site, is now accessible to all LTC staff. In addition to providing current LTC news, the quick launch toolbar (found on the left side of the page) provides quick access to commonly used applications, forms, and websites that were previously located on this page. Click here to access
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Fankhauser goes 3-for-3 with an RBI.
Both Amu and Nieto turn in solid pitching performances.
Utes down Idaho State and Cal State Fullerton.
Utes tie with Cal Poly, 1-1.
Utes defeat Long Island and Nevada.
Jordan's 2013 Game-by-Game Stats
Jordan Notes: Will serve as a team captain ... has a great mind for the game ... trustworthy with the ball in her hands on defense ... triple-threat at the plate ... a quiet, but extremely competitive player. 2013: Pac-12 All-Academic Second Team ... had a Pac-12 season-high nine assists at Utah Valley (3/5) ... started 28 of 32 games that she has played in at third base ... registered hits in first three games of the season ... singled and scored a run against Seattle (2/9) ... stole a base against Arkansas (2/10) ... hit a double against Idaho State (2/15) ... had a base hit and run scored at UNLV (2/17) ... went 3-for-3 and scored twice against Colorado State (3/22) ... registered base hits against Nevada (2/23), Cal State Fullerton (3/9), Bethune-Cookman (3/10), No. 5 California (3/23) and No. 17 Arizona (3/28) ... laid down sacrifice bunts against Cal Poly (2/24) and at Oregon State (4/13).
2012: Had five hits in nine at-bats for a .556 average ... scored five runs ... connected on a double versus Southern Utah (3/21) ... hit a single against Drexel (2/24) ... had a pinch-hit single against Utah State (3/28) ... crossed the plate as a pinch runner against Cal St. Northridge (2/19) and Colgate (3/12) ... had three assists from third base against North Dakota (3/4) ... appeared as a pinch runner in three other games ... appeared as a pinch runner against San Diego State (2/10), UC Santa Barbara (2/11), No. 15/19 Hawaii (4/3) and No. 23 Oregon State (4/29) ... made a pinch hit appearance at No. 15/15 UCLA (4/21) ... knocked a pinch hit single and scored a run during first game of Weber State doubleheader (4/25) ... stole second base of career against Oregon State (4/28) ... crossed the plate during 6-0 win over No. 15/17 Stanford (5/10).
2011: Made 23 appearances with five starts, mainly at second base ... went 2-for-5 with four RBI and two run scored against Hampton (3/19) ... had a three-game hit streak with a single in each game she played in from March 22 through April 6: George Mason (3/22), Utah Valley (4/5) and Weber State (4/6) ... drove in two runs versus Weber State (4/6) ... scored a run and stole first career base at Colorado State (5/6) ... had a hit at CSU (5/7) ... Spring 2011 Academic All-MWC selection ... 2010-11 MWC Scholar Athlete.
High School/Travel Ball: Second-team all-state selection ... all-state tournament team member ... all-region team honoree ... all-county team ... served as captain of Utah Sting club team ... four-time letterwinner ... fields right and bats left ... also played volleyball and basketball ... academic all-region selection in volleyball as a libero ... recognized on the honor roll and Citizenship Honor Roll ... member of the Service Club.
Personal: Full name is Taylor Lynn Jordan (formerly Austin) ... married Zach Jordan last summer ... majoring in Human Development and Family Studies ... parents are Trevor and Kimberly Austin ... mother received All-American consideration in swimming at Ohlone College ... father is a graduate of the University of Utah ... born June 28, 1992.


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Walt Frasier live on stage off broadway college tour new york |
Walt Frasier live on stage off broadway college tour new york |
CLICK TO SEE FULL RESUME JOIN ME ON MYSPACE for more up to date news March 2008 NEWS The Laugh Factory is now the TIMES SQUARE ARTS CENTER/COMEDY CLUB. Our show has moved to the 140 seat off-broadway cabaret space. We have been packed this month thanks to a number of student tour operators using our show as the new hot spot for off-broadway group sales. NEW DINNER THEATER TICKETS AVAIL Filmed Commercial for VH1/NINTENDO - Should be on cable Late April 2008. I play a security guard cheering on Tina Yothers using teh new Wii FIT. Very Farley - like homage. HAPPY ANNIVERSARY - Laurice and I celebrate 15 years together at the end of this month. April 2008 NEWS Ohio Bound - EIGHT IS NEVER ENOUGH! has a series of shows and workshops for the National Guard. Funny Stuff Online Jimmy's Reviews We really need to shoot more of these. Film and Theater reviews by Jimmy - the 300lb., 5yo critic. WHERE's WALTER Mockumentaries and reality style comedy webisodes THE JOKE Me, George Lopez, Nat & Alex Wolf, and the rest of teh NICK Naked Brothers' Band Cast tell the nastiest joke of all time (Kids version of the Aristocrats) NAT IS A STAND UP GUY Online copy of my NBB episode PRESS ABC NEWS CLIP is now ONLINE Featured in Time Out NY Kids NEW YORK TIMES review of Warrior Sister and my most villainous role. TIME OUT NY photo on a holiday feature of NY Santas. Coming soon. If anyone has the article from West Side Spirit about our 2004 political show, please send it to me. I misplaced it before scanning and uploading. |
NAKED BROTHERS BAND ON NICKELODEON "Nat is a Stand-Up Guy" "The Joke" |
LATE NIGHT WITH DAVID LETTERMAN ON CBS Appearing on 9 episodes with Paris Hilton, Adam Sandler, Donald Trump, etc |
HAIR TRAUMA On the WE Network December 2007 Ep 2 October 2006 Ep 3 ALSO FALL 2007 SUPER AMAZING INVENTIONS NOW ON SUPER DELUXE Commercials Currently Airing VH1/Nintendo w/Tina Yothers Culligan Water Insight Cable |
Study Voice I am once again taking on new students that want to learn to sing. HERE ME SING CLICK HERE SEE WALTER LIVE ON STAGE OFF-BROADWAY Friday-Monday 8:30pm Sat 3, 8:30, 11 |
Above PHOTOS (L to R): Live Web Cast fromSoho Studio on PSEUDO.COM (Spring 2003), At the NY Improv (Christmas 2005), "ROB THE VOTE" Caroline's on Broadway (11/1/04), Rascal's Comedy Club, Montclaire Jersey (10/04), Laugh Factory Times Square (Summer 2006) BELOW PHOTOS (L to R): Live at the Knitting Factory (Spring 2004), Belmont Raceway Family Days, Long Island (Summer 2004), College Misericordia, Wilkes Barre PA, (Spring 2004), w/Vic Martino (Sopranos) in Patterson NJ (9/2/07), Fleet Week Special Performance (Spring 2006), Kraine Theater (Fall 2003) |
Online Video Series Short Film Series Clips of Live Shows Behind the Scenes Footage |
WALT FRASIER Click to Contact via email OFFICE 212-568-6560 |
LATE NITE AT THE LAUGH FACTORY Friday/Saturday 11pm ABSENT MINDED COMEDY |


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Jw Marriott Los Angeles L.A. Live
Jw Marriott Los Angeles L.A. LiveDog-Friendly Hotels900 West Olympic BlvdLos Angeles,California 90015Call 866-676-3077 To Book This Hotel Get Map or Directions In the heart of Downtown Los Angeles, just steps from STAPLES Center, JW Marriott Los Angeles L.A. LIVE Hotel fashions a new standard for luxury. Our stunning 54-story masterpiece hotel is the focal point of L.A. LIVE, offering guests a front-row seat to this emerging entertainment, sports and dining epicenter. With convenience to the LA Convention Center, guests at our hotel also enjoy an unmatched location for attending events at STAPLES Center, Nokia Theatre, Club Nokia, the GRAMMY Museum, Conga Room, Regal Cinemas, ESPN Zone, and much more. Hosting many of L.A.'s most fashionable events, our luxury hotel boasts 100,000 square-feet of contemporary, state-of-the-art Los Angeles meeting and event space. Entertainment events at L.A. LIVE are complemented by the hotel's lavish Ritz Carlton Spa, a rooftop pool and bar, dining options such as Wolfgang Puck's WP24 and LA Market by Kerry Simon, modern yet comfortable guest rooms, and luxurious suites. Entertain your senses at JW Marriott Los Angeles.Dogs of all sizes are allowed. There is a $125 one time pet fee.
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This fabulously creamy dessert is fat free, thanks to skim milk and fat free Greek yogurt.
Tangy Raspberry Semifreddo
Sure to dazzle guests, this gorgeous raspberry dessert gets its subtle tang from our Greek yogurt.
Strawberry Dulce de Leche Trifle
Trifle is always a popular holiday dessert- it feeds a crowd and is made well ahead of time, with no last minute preparation. Frozen berries are an economical alternative to the usual fresh berries- and they are always ripe.


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Iris Tyler Grey Leather Wristlet
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The wonderful city of Berlin is one of the busiest and most beautiful cities in all of Germany. This great city is home to a huge number of museums that contain historical pieces and facts surrounding numerous places and events in the long and interesting history of Berlin. One of the most interesting and informative of all these museums is the East Side Gallery (Berlin Wall). This huge open air gallery is the largest of this type of gallery in all of the world.
The East Side Gallery is home to and preserves the longest of the remaining sections that is left of the Berlin Wall. This section of the wall located in the museum is the piece with some of the most famous pieces of art work that was put on the wall some time ago. This art was recently restored to fix the deterioration that was done due to it being in open air.
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Create funny images with Make Me Bald
With many photo-editing applications on the market, it's hard to choose one that is legitimately useful or at least amusing. Make Me Bald is neither. It functions poorly and features are limited.
This app loads easily and it's quite straightforward due to its uncomplicated nature. The application initially hesitated when opened, but eventually opened to a stock image. A universal menu button at the bottom of the screen allows you to take a picture from within the application. We tested this app on three different devices -- Samsung Galaxy Nexus, Acer Iconia A100, and Asus Transformer Pad -- … Read more


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February 2014
Welcome, guest
Fri Sep 9, 2011
Bank of America Merrill Lynch has hired Michael Terry as global head of capital introductions. Based in New York, he will report to Jon Yalmokas, head of US prime brokerage sales.
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This iReport is part of an assignment:
'Superstorm' Sandy: Your stories |
A Sandy Story - 3 weeks later
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iReport welcomes a lively discussion, so comments on iReports are not pre-screened before they post. See the iReport community guidelines for details about content that is not welcome on iReport.


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Many state and local governments as well as utility companies offer incentives for energy efficiency and use of renewable or green energy sources...
In the past, the hard part has been finding these programs, as there wasn't a consolidated area with pointers to which programs were offered. North Carolina State University, together with the Interstate Renewable Energy Council and the North Carolina Solar Center, have created a huge database of incentives. This database is called "The Database of State Incentives for Renewables and Efficiency" or DSIRE.
The database is organized by state, allowing you to quickly see what programs are available in your area. The database shows energy incentives that range from incentives that discount your electric bill, to rebates on energy-efficient appliances, and even loans for items like solar panels. There's a ton of resources for every area, with over 50 programs listed for Texas, more than that for California, and something for every state.
See the database at dsireuse.org
Related Tips
I've check the database and it's quite cool, very accessible and very easy to navigate like looking for the designated are and it's available program. Thanks for sharing this very informative post and I am pretty sure that all consumers would love to check program available in their area. - buy you tube views
Re: Find energy rebates and electric bill discounts
I think if people switched all their appliances to energy star and lighting to CFL's at the same time and saw the actual savings it'd be more incentive for others to do the same. We were looking for frigidaire parts to fix an AC and decided to replace them with newer models.


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An adult 21 years or over must be present to sign for delivery of alcoholic beverages. Order tracking numbers will be emailed to you upon shipment, so you'll know when to expect delivery. We offer the following options for your convenience:
Ground: Delivery generally between 1 and 7 business days once the package leaves our warehouse.
3 Day: Delivery to businesses by 4:30 pm and to residences by 7:00 pm in 3 business days once the package leaves our warehouse.
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Overnight p.m.: Next business day delivery by 3:00 pm to most U.S. addresses ( by 4:30 p.m. to rural areas) once the package leaves our warehouse.
Overnight a.m.: Next business day delivery by 10:30 am to most U.S. addresses once the package leaves our warehouse; Remote areas by noon, 4:30 pm or 5:00 pm; Saturdays by noon, 1:30 pm or 4:30 pm. In extreme heat, ice-packs will be added.
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Red wines that are more fruit-forward and lighter in tannin and body.
Medium bodied reds that go down easy, with smooth tannins and supple fruit.
Wines where earthy and/or spicy dominate the flavors – typically medium to full body.
Full bodied wines that have concentrated fruit and are higher in alcohol and/or tannins. Some need age.
The Hyland unicorn was chosen for the Shiraz label due to its beauty and strength. The 2006 Hyland Shiraz is an elegant, medium-bodied wine with lifted aromas of ripe berry and spice integrated with savory, smoky notes. The middle palate is loaded with silky oak tannin to balance the rich fruit flavors. The finish is long and juicy with just a hint of oak. The Hyland Shiraz is best enjoyed over the next 1-5 years.
"The 2006 Shiraz Hyland offers up aromas of pepper, blueberry, and plum. This is followed by a medium- bodied, stylish wine with meaty flavors, good grip and depth, and a lengthy finish. Drink it over the next four years.
Penley Estate has turned out four exceptional values in the 2006 vintage. " 90 Points The Wine Advocate
"Deep ruby. Red- and blackcurrant aromas are complicated by cigar box and floral qualities. At once sweet and spicy, with suave red and dark berry flavors, silky tannins and good mineral lift. Nicely balanced shiraz with excellent purity and finishing vivacity." 90 Points International Wine Cellar
Established in 1988, by Kym Tolley, a direct descendant of the famous Penfold and Tolley winemaking families - the name Penley is an amalgam of the first three and last three letters from each title. Tolley has over 25 years of winemaking experience, including being involved in the making of many vintages of the famous Penfolds Grange. The estate is located in the heart of...Read More About Penley Estate
Located on the Limestone Coast of South Australia, Coonawarra is a pretty flat area, except for the vineyards, which are somewhat elevated. That, along with the proximity to the ocean and clouds that like to hang around, Coonawarra never gets too hot. There is a small strip of soil, about nine miles long and two miles wide, called Terra Rossa, or red soil/earth. It is the...Read More About Coonawarra
(seh-RAH/shee-RAHZ) It's a Smokin' Grape Syrah and Shiraz - same grape, different name. It's a popular and adept variety, growing in multiple regions and creating many different styles of wine. The home base of Syrah is the Northern Rhone, where it creates the exclusive wines of Hermitage and Cote Rotie. On the less pricy side, the Rhone makes Syrah-based wines in Crozes...Read More About Syrah/Shiraz
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Expert in Sales and Marketing and business plans Long, successful career as a recognized expert in sales and marketing, manufacturing and distribution in both product and service oriented businesses. I have lead major corporations as CEO, CFO, COO, or Industry Consultant, so I am known and recognized for what it takes to build successful companies.During my career I have lead companies to achieve 1.5 billion dollars in annual volume, yet be assured I know what it is to be a startup. Numerous industry awards and recognition attest to my expertise.

 St John's University BBA

Ask SCORE
This column is brought to you by the Merrimack Valley Chapter of SCORE, with nearly 70 current and former business executives available to provide free, confidential, one-on-one business mentoring and training workshops for area businesses. Call 603-666-7561 or visit merrimackvalley.score.org for information on mentoring, upcoming workshops and volunteer opportunities. SCORE is a national, non-profit organization and a resource partner of the U. S. Small Business Administration. Have a question you'd like answered in this column? E-mail it to firstname.lastname@example.com, with "Ask SCORE" in the subject line.
40 plus years in the field of business credit. Former Chief Operating Officer of the nations largest commercial collection agency, Dun & Bradstreet Receivables Management Services. Executive Director of the Commercial Collection Agency Association a section of the Commercial Law League of America. Seminar leader and author of several Dun & Bradstreet Business Education courses on credit decision making, cash flow, budgeting and strategic planning.
MBA, St. John's University, in Economics.
United States Navy, Civil Engineer Corps W.L. Gore & Associates Procurement Leader Purchasing Manager, Capital Equipment
M.E.,Environmental, - Old Dominion University M.S., Business Management - State University of New York B.S., Chemical Engineering - Cornell University Registerd Professional Engineer Certified Purchasing Manager
Lee worked 44 years in Technical marketing field for Caterpillar, Inc., most recently as Senior Marketing Professional working with independent dealers identifying improvement opportunities with their product support operations. This also involved training of service managers and first line supervisors with the business management of the product support operations. Lee was invovled with product development team for introduction of new and updated products.
Lehigh University - BSME
Experienced General Manager with a successful track record driven by business development, building strong teams, exceptional communication skills, strategic planning, sales leadership and management, operations management, mergers and acquisitions, branding and marketing, and execution excellence.
Enthusiastic leader with perspective gained from a variety of management roles within Fortune 500 consumer products companies, start-up organizations, and turn-around situations.
In 2004, I acquired Johnson Pools with a business partner. Since that time, we grew the company to be one of the leading design and build firms for high end residential outdoor living environments. The company's offerings include design, construction, service and retail operations. Through a focused strategic initiative, the company expanded its service area, product emphasis, and branding to grow through an historic recession.
During the period from 2000 through 2003 served as the CEO, President and Director of two subsidiaries of Magic Software Enterprises, LTD. (Nasdaq: MGIC) as well as on the senior management team of the parent company. Brought in to perform a strategic and financial turn-around for the North American operations of global publicly traded software and consulting company. The turn-around was successful and the stock price enjoyed unprecedented growth.
From 1993 to 2000 I served in multiple senior management positions within venture capital start-up companies in physician practice management and healthcare technology management, from business planning and funding, through execution of plans. Identified potential acquisition candidate organizations, performed due diligence, and acquisition negotiations. In each case I executed the strategy of making regional acquisitions and achieved synergies by standardizing processes, practices, and technology focused on the needs of the customer. Each of these companies was sold as part of the initial strategic plans with the venture capital investor partners.
Prior to the above, served in various sales management, marketing, and training and development roles including building the global training department of a Fortune 500 company.
Professional Experience
Managing Member
Right At Home, LLC dba Johnson Pools and Spas
2004– Present (8 years)
Johnson Pools is a member of the Association of Pool and Spa Professionals and The Aquatech Society, and has been an established quality pool design and building company for over 45 years. The staff is comprised of trained professional managers, sales executives, professional staff, job site supervisors, project managers, service technicians and retail staff. The company's outstanding reputation, professional team and high quality projects, are its greatest assets. Services include 3-D design and planning, project management, and specialized customer service, and a concentration on sales based upon a thorough communication with the customers and all related parties involved in every project.
CEO President and Director – Magic Software Enterprises, Inc. and CoreTech Consulting Group
Magic Software Enterprises, LTD
2000– 2003 (3 years)
Magic Software Enterprises, Ltd (NASDAQ: MGIC) was a global provider of application platform and business integration solutions. In addition, the CoreTech division, delivered quality IT staffing, consulting, and project solutions in distributed computing and client / server migrations, serving primarily the highly regulated pharmaceutical industry. The company's philosophy was to provide value-add services to companies by helping them leverage technology to enhance business processes.
I was brought in to perform a strategic and financial turn-around after the parent company purchased several operations throughout North America. I evaluated all operational units, developed strategic plans, divested un-profitable operating units, and managed on-going operations headquartered in Pennsylvania, California and Montreal, with annual revenues in excess of $25 million. During my tenure the stock price quintupled. After leading the successful turnaround of the business I decided to leave so that I could pursue purchasing my own company.
Senior Vice President and Chief Operating Officer
Continuum Healthcare, Inc.
1998– 2000 (2 years)
This was a start-up organization providing health promotion, wellness and fitness management services to corporate clients such as BNSF Railroad and UPS. The company was funded by Alex Brown ($25 million initial round). We first acquired a leading medical management company in the Atlanta area. Based on our developed strategy for new workflow system processes, we then built a new proprietary information management system, in conjunction with Medical Advisory Board, which enabled us to reduce timeframe for turnaround of exam results and overall costs. The company was sold as a part of the initial strategic plan with our capital partner.
Corporate Vice President of Sales and President of Medical Services Group, Inc.
Atlantic Health Group, Inc.
1995– 1998 (3 years)
This was a start-up organization providing medical services in both physician practice settings and through information technology to initially serve the members of our capital investor, Liberty Mutual Insurance Company ($20 million). Acquired medical practices in key markets where Liberty Mutual had its largest markets (Boston, Atlanta, etc.) Reduced the overall cost of workers compensation to the local insured of Liberty Mutual, by 15%. Reduced the average time for return to work by 50%. Although we were funded by LMI, we served the needs of all employers in our given markets.
Additionally, we acquired Medical Services Network, a national network of physicians focused on occupational health. We began to transform the organization to provide services and manage outcomes through a medical network rather than brick and mortar locations. This proved a model that was efficient, reduced costs and expanded the service area and size of the customers served. The company was sold as a part of the initial strategic plan with our capital partner.
Chief Operating Officer and Vice President of Sales
Occupational Health Resources, Inc.
1993– 1995 (2 years)
This was a start-up Physician Practice Management company funded by Tullis-Dickerson Partners ($5 Million). Through seed funding, acquired physician practices and physical therapy centers between Maine and Georgia. I was responsible for overall operations of the medical facilities, medical staff, and sales team. Prior to the sale of the company, we managed 15 locations along the east coast. The company was sold as a part of the initial strategic plan with our capital partner.
Director - Corporate Training & Development
Director of Sales
Manager - New Business Development
Black & Decker
Key Account Manager
Retail Sales Manager
District Salesperson

 Education

 University of South Florida 1976-1980

John Cunningham is a N.H. business lawyer whose practice is focused on LLC law and tax. He chaired the N.H. Business and Industry Association committee that drafted the Revised New Hampshire Limited Liability Company Act, a radical revision of New Hampshire LLC law that went into effect on January 1st. LLCs are, by a wide margin, the entities of choice for N.H. business start-ups.
Review the 15 key areas of the Business Check list before your start your business.
BS Engineering, USMA, West Point, NY
MS Civil Engineering, Construction, Stanford University
MBA, USIU, Mexico City Campus
MA Political Economy of Latin America, National Autonomous University of Mexico, Mexico City
Defense Language Institute, Spanish Language Course
Inter-American Defense College Strategic Planning Course


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Okay so a friend of mine rooted my droid and i have simply stunning v4.6
how do i get v4.8?
i guess i should have watched him do it but i was too preoccupied.
Okay so a friend of mine rooted my droid and i have simply stunning v4.6
how do i get v4.8?
i guess i should have watched him do it but i was too preoccupied.
Follow this link and the instructions included http://www.ssandroid.com/ssandroid/v...&t=800&start=0
It might help to know what recovery you have installed also...SPR or CWR you can find out by turning your device off and powering up by holding down the X and Power Key until a recovery screen comes up and then see if it says Clockwork or Sir psycho's recovery
Last edited by tp7six; 10-13-2010 at 05:29 PM.
recovery is clockwork.
You can follow the link and install just as described with clockwork
i don't really see any instructions...im so confused.
The easiest thing to do is download rom manager and install ss 4.8 from there. You will need the free version and the paid version. You can do it manually but it's quite easy via rom manager. Just go to download rom and click on chevys section and download 4.8.
i do have rom manager on here...but it's giving me the force close error. this is obnoxious. i really don't wanna leave 4.6 on here.
annnd nevermind! got it...i used the kernel 1.0...hope thats right?
Last edited by dana6097; 10-14-2010 at 09:56 AM.
yea download rom manager then dl ss 4.8 right to phone >go into rom manger hit install clockwork manager then in the same menu click install from sd card and check both boxes taht come up (backup rom and wipe data and cache) and thats a wrap
i did ittt!!! yayyy!! haha thank you!


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Crispy Chick'n Patties by Gardein$5.49Purchase Crispy Chick'n Patties by Gardein
IMPORTANT SHIPPING INFORMATION FOR THIS ITEM – PLEASE READ BELOW! There's no better way to make THE best vegan chicken sandwiches than with these great patties from Gardein! As with all Gardein meat alternatives, these are as close to the real thing as you'll find, but without the cruelty, animal ingredients, cholesterol and other unwanted ingredients. Each package contains 4 breaded patties that heat up perfectly in the oven or on your stovetop, giving you a great high-protein meal in minutes. 12.4 oz. (352g) package is $5.49.
Purchase Crispy Chick'n Patties by Gardein Other products in the category Food, Sweets and Beverages / Meat Alternatives:
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