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Blood vascular cells and lymphatic endothelial cells (BECs and LECs, respectively) form two separate vascular systems and are functionally distinct cell types or lineages with characteristic gene expression profiles. Interconversion between these cell types has not been reported. Here, we show that in conventional in vitro angiogenesis assays, human BECs of fetal or adult origin show altered gene expression that is indicative of transition to a lymphatic-like phenotype. This change occurs in BECs undergoing tubulogenesis in fibrin, collagen or Matrigel assays, but is independent of tube formation per se, because it is not inhibited by a metalloproteinase inhibitor that blocks tubulogenesis. It is also reversible, since cells removed from 3D tubules revert to a BEC expression profile upon monolayer culture. Induction of the lymphatic-like phenotype is partially inhibited by co-culture of HUVECs with perivascular cells. These data reveal an unexpected plasticity in endothelial phenotype, which is regulated by contact with the ECM environment and/or cues from supporting cells.
Higher animals possess two separate circulatory networks, the blood and lymphatic vascular systems, which have distinct but interdependent functions. In the developing mammalian embryo, the lymphatic vasculature appears after the formation of the blood vasculature and is believed to be of venous origin. In fact, blood vascular cells and lymphatic endothelial cells (BECs and LECs, respectively) share a common progenitor (Jain and Padera, 2003; Kubo and Alitalo, 2003). A subset of cells in the embryonic cardinal vein transdifferentiate and take on a lymphatic identity via the stepwise expression of lymphatic regulatory genes (Oliver and Harvey, 2002; Wigle et al., 2002). The Sox18 transcription factor specifies target cells in which expression of the Prox-1 transcription factor is induced (Francois et al., 2008), which, in turn, commits cells to differentiation into the lymphatic lineage such that this subset of cells gives rise to the entire lymphatic vascular network. This view is supported by data from mouse knockout studies, which show that the absence of Prox-1 expression leads to a failure to differentiate into the lymphatic endothelial cell lineage (Johnson et al., 2008). Once the two endothelial cells lineages are established, BECs and LECs are considered to be mature, non-interchangeable cell types that display characteristic profiles of gene expression (Hirakawa et al., 2003; Kriehuber et al., 2001; Podgrabinska et al., 2002).
In this study, we have identified an unexpected plasticity in the phenotype of human endothelial cells when analysed in several conventional in vitro models of angiogenesis. We show that blood vascular endothelial cells are able to adopt an expression profile resembling that of lymphatic endothelial cells in response to particular in vitro ECM environments, and this behaviour is reversible and independent of tubulogenesis. These studies shed new light on the environmental cues that regulate endothelial cell differentiation and lineage maintenance.
Human umbilical vein endothelial cells adopt a 'lymphatic' gene expression profile when cultured in 3D matrices
Endothelial cells form tube-like structures when cultured in 3D matrix environments and stimulated with pro-angiogenic growth factors (Fig. 1A–C). We performed a microarray analysis to compare gene expression in human umbilical vein endothelial cells (HUVECs) undergoing tube formation in a 3D fibrin matrix (Fig. 1B) with monolayer cultures (Fig. 1A). This analysis identified 169 genes that were upregulated and 554 genes that were downregulated during tubulogenesis (supplementary material Table S1). Unexpectedly, we observed altered expression of a number of genes that had been previously identified to be mainly or exclusively expressed in either BECs or LECs (referred to henceforth as BEC or LEC markers). A number of marker genes were examined further using quantitative real-time RT-PCR (qRT-PCR). These included the LEC markers: lymphatic vascular endothelial hyaluronan receptor (LYVE1) (Banerji et al., 1999); PROX1 (Hong et al., 2002; Wigle and Oliver, 1999); vascular endothelial growth factor-3 (FTL4) (Adams and Alitalo, 2007; Kaipainen et al., 1995); integrin α9 (ITGA9) (Huang et al., 2000; Mishima et al., 2007) and podoplanin (PDPN) (Hirakawa et al., 2003; Kriehuber et al., 2001; Petrova et al., 2002; Schacht et al., 2003). The BEC markers selected were: Lamin-B1 (LMNB1) (Podgrabinska et al., 2002), VEGFC (Hirakawa et al., 2003; Kriehuber et al., 2001; Makinen et al., 2001) and CD44 (Cao et al., 2006; Hirakawa et al., 2003).
Expression profiles of the markers were assessed in HUVECs undergoing tubulogenesis in either type I collagen or fibrin 3D angiogenesis assays, compared with HUVECs in standard monolayer culture conditions on plates coated with type I collagen. In both types of 3D matrix environment, the LEC markers LYVE1, PROX1 and FLT4 were upregulated, and the BEC markers LMNB1, VEGFC and CD44 were downregulated compared with monolayer culture (Fig. 1D–G). Another LEC marker, ITGA9, was upregulated in 3D collagen cultures, whereas no significant changes in expression were detected in 3D fibrin gels (data not shown). In contrast to the other LEC markers, expression of PDPN was not detected in either assay. The mRNA expression data were supported by the appearance of LYVE-1 protein consistently throughout the entire tubular network in HUVECs undergoing tube formation in 3D collagen gels, whereas only a few isolated cells were weakly positive for LYVE-1 in HUVECs cultured as a monolayer (Fig. 1H and supplementary material Fig. S1). We also observed VEGFR3 and nuclear Prox-1 staining in HUVECs that formed tubes (supplementary material Figs S2 and S5), and detected downregulation of Lamin-B1 by western blot (supplementary material Fig. S3). These data demonstrate that mature blood vascular endothelial cells alter their gene expression profiles upon culture in 3D matrices, where they transdifferentiate to a 'lymphatic-like' phenotype.
Transdifferentiation is not restricted to endothelial cells of fetal origin
We tested whether endothelial cells derived from other tissue sources showed gene expression changes in 3D culture. Committed adult human aortic endothelial cells (HAECs) showed similar changes in BEC or LEC marker gene expression during tubulogenesis in type I collagen as those observed in HUVECs (Fig. 2A,B). Once again, expression of the LEC marker podoplanin was not induced. Similar data were obtained with adult human dermal microvascular endothelial cells (HDMECs; data not shown), with the exception that podoplanin was expressed by these cells. However, data from these cells must be considered with caution because HDMEC preparations are known to contain variable proportions of LECs and BECs (Makinen et al., 2001). The finding that mature adult blood vascular endothelial cells are capable of adopting a lymphatic-like gene expression profile indicates that this plasticity is a property of many endothelial cell types.
Adoption of a 'lymphatic-like' phenotype is not dependent on a 3D environment
Induction of the lymphatic-like phenotype occurred in 3D culture in both matrix types tested, but not in cells in monolayer. We therefore investigated whether a 3D environment is essential for this process using the 2D Matrigel assay. In this assay, endothelial cells seeded on top of Matrigel migrated and aligned to form a network of cord-like structures within 24 hours (Fig. 2C). Analysis of expression of BEC and LEC marker genes showed that, similarly to the 3D tubulogenesis assays, HUVECs had upregulated expression of mRNA encoding LYVE-1, Prox-1, VEGFR3 (and integrin α9, data not shown) paralleled by downregulation of mRNA encoding lamin-B1, VEGF-C and CD44 over the course of network formation (Fig. 2D–E). Comparison of BEC and LEC marker expression in HUVECs cultured within 3D Matrigel versus cells seeded on Matrigel revealed similar patterns; indeed, induction of Prox-1 was strongest in cells forming alignments on top of this matrix. These data suggest that a 3D environment per se is not obligatory for induction of the 'lymphatic' phenotype, but the nature of the ECM is critically important.
Based on the results obtained when endothelial cells were grown on Matrigel, we next asked whether similar results could be obtained with cells grown in monolayer on top of collagen or fibrin gels. We observed that when seeded on top of collagen gels, HUVECs formed alignments in a manner comparable with cells seeded on Matrigel (Fig. 3A). By contrast, cells seeded on fibrin gels retained a cobblestone appearance resembling that of cells in standard culture (Fig. 3B,C). Expression analysis showed that cells seeded on collagen gels adopted the 'LEC' profile of gene expression, whereas cells seeded on fibrin retained the same profile as the standard 2D cells (Fig. 3D,E). The 'LEC' phenotype was induced more effectively when the cells were cultured in 3D collagen rather than on top of the same gel matrix (data not shown). These data provide confirmation that a 3D environment is not essential for the BEC to LEC transition, but they emphasise that the nature of the matrix is critical, because although monolayer growth on a dense collagen gel is permissive for the phenotypic change, growth on fibrin is not.
Adoption of a 'lymphatic-like' phenotype is independent of tubulogenesis and is reversible
We asked whether the 'LEC' phenotype was functionally linked with tubulogenesis by analysing marker expression in HUVECs cultured in 3D collagen in the presence of the broad-spectrum matrix metalloproteinase inhibitor BB-94 (Fig. 4A–D). This inhibitor blocks tube formation by inhibition of key enzymes required for ECM digestion and remodelling, such as MT1-MMP (Hiraoka et al., 1998; Lafleur et al., 2002). We observed that upregulation of expression of LEC markers and downregulation of BEC markers was largely unaffected in cultures in which tubulogenesis was inhibited, although a reduction in the level of FLT4 induction was detected. These data show that HUVECs undergo the transition to a 'lymphatic' phenotype when cultured in conditions conducive to tube formation, even when the tubulogenesis is inhibited.
We also tested whether induction of the 'LEC' expression profile might be reversed after transfer of the cells from a 3D environment back to monolayer culture. HUVEC were suspended within 3D collagen gels for 24 hours and then the gels were digested with collagenase and the cells returned to monolayer cultures and maintained for a further 48 hours. As previously observed, 3D culture led to induction of expression of the LEC markers LYVE1, PROX1 and FLT4, and the downregulation of the BEC markers LMNB1 and VEGFC. Following replating in monolayer culture, expression of each of these markers demonstrated full reversibility, returning to levels that were comparable with those observed in control monolayer samples at the assay end point (Fig. 4E,F). These data demonstrate that the pattern of BEC and LEC marker gene expression is directly regulated by the extracellular environment of the cells.
Adoption of the 'lymphatic-like' phenotype is partially inhibited by co-culture with perivascular cells
Blood vessels in vivo have an extensive basal lamina and are in close contact with smooth muscle cells or pericytes, whereas lymphatic vessels have little basement membrane and largely lack pericyte coverage (Pepper and Skobe, 2003). We hypothesised that the culture of isolated BECs in direct contact with the ECM in the absence of perivascular cells might create a microenvironment more similar to that associated with lymphatic vessels and thus encourage the cells to take on LEC-type behaviour; conversely, co-culture of HUVECs with perivascular cells might favour the maintenance of the 'BEC' phenotype. We therefore compared marker gene expression levels in HUVECs cultured in 3D collagen gels either as monocultures or co-cultured with murine perivascular cells (Brachvogel et al., 2005; Brachvogel et al., 2007). By using species-specific qRT-PCR probes we were able to determine expression levels in the human cells only. HUVECs cultured in the presence of perivascular cells showed markedly reduced expression of mRNA encoding LYVE-1 and Prox-1 compared with monocultures, whereas VEGFC mRNA expression increased (Fig. 5A,B). These results demonstrate a reversal of the expression of several BEC and LEC marker genes compared with the pattern seen upon monoculture in 3D matrices, such that induction of the 'lymphatic' phenotype was at least partially inhibited. LYVE-1 protein was clearly expressed throughout the tubular networks in HUVEC monocultures, and strongly downregulated in HUVEC and perivascular cell co-cultures (Fig. 5A,B; supplementary material Fig. S4). However, not all the BEC and LEC markers followed the same pattern, because co-culture with perivascular cells resulted in increased expression of FLT4 (Fig. 5B; supplementary material Fig. S5), and decreased expression of LMNB1 (Fig. 5B) compared with levels in HUVEC monocultures, suggesting that these genes respond to the matrix environment rather than the presence of pericytes. Interestingly, we saw a similar overall trend in the effects on BEC and LEC marker expression (namely, decreased LYVE1 and increased VEGFC and FLT4) when we used siRNA to reduce induction of PROX1 expression in endothelial cells seeded on Matrigel (supplementary material Fig. S6).
The culture of endothelial cells within or on top of 3D matrices such as type I collagen, fibrin and Matrigel provides simple, popular models of angiogenesis (Auerbach et al., 2003; Goodwin, 2007; Phung and Dass, 2006; Ucuzian and Greisler, 2007). The present work reports that when blood vascular endothelial cells such as HUVECs and HAECs are cultured under such conditions, they show gene expression changes that are characteristic of a shift to a lymphatic phenotype. The lymphatic-like phenotype was independent of tubulogenesis and fully reversible upon transfer of cells from 3D to monolayer culture on collagen-coated tissue culture plastic, suggesting that interactions between endothelial cells and matrix account for the alterations in gene expression observed. This was further reinforced by the observation that HUVECs adopt the lymphatic-like phenotype when cultured on top of Matrigel or a dense collagen gel, but not when grown on top of a fibrin gel. Also, co-culture of HUVECs with pericytes partially prevented the development of the LEC-like phenotype. These findings indicate that the concept of LECs and BECs as committed, differentiated lineages needs to be revised, and demonstrate the importance of contact with different extracellular matrices and perivascular cells in regulating endothelial cell phenotype and lineage specification.
Endothelial cells of different tissue origins represent different functional cell types, with characteristic gene expression profiles (Chi et al., 2003). We considered the possibility that, because HUVECs are of fetal origin, they might show increased phenotypic plasticity compared with adult endothelial cells. However, blood vascular cells of adult origin such as HAECs and HDMECs showed the same induced changes in gene expression, indicating that ability to adopt a lymphatic-like phenotype is a common property of cultured BECs. We also considered it possible that the HUVECs used in our initial experiments might contain a subset of LECs, which came to be the dominant cell type in the culture conditions we used. However, lymphatic vessels are absent from the umbilical cord, and moreover, the lack of expression of podoplanin in all our angiogenesis models using HUVECs and HAECs confirms the absence of a fully differentiated LEC subpopulation. By contrast, HDMECs that are known to contain an LEC subpopulation (Makinen et al., 2001) did indeed express podoplanin. Rather, these data, and in particular the induction of expression of Prox-1, imply an ability of BECs in 3D culture as a whole to undergo a type of transdifferentiation in which cells adopt a more lymphatic-like phenotype. But it is also clear from the absence of podoplanin expression in HUVECs undergoing 3D tubulogenesis that the cells do not undergo full transdifferentiation from a BEC to an LEC phenotype. This might imply that the cells adopt an intermediate phenotype that shows characteristics of both LECs and BECs, or it might reflect the fact that podoplanin expression is a late marker of lymphangiogenesis, which is consistent with the proposed role of podoplanin in later stages of lymphatic vascular patterning (Schacht et al., 2003). It is possible that HUVECs commence BEC-to-LEC transdifferentiation in the 3D culture models, but the process is not complete in the timeframe of the assays.
Adoption of the 'LEC' phenotype could be correlated with the tendency of the endothelial cells to form tube or cord structures. However, our findings show that induction of the 'LEC' phenotype is neither a consequence of tubulogenesis per se, nor dependent upon it. Rather, the nature of the cell's extracellular environment determines the induction of the 'lymphatic' phenotype. Importantly, the phenotype is reversible upon returning cells from 3D to standard monolayer culture. This reversibility shows that the 'LEC' expression profile is not the result of simple culture-induced de-differentiation, a possibility that needs to be considered because cultured endothelial cells are known to lose their lineage specificity (Amatschek et al., 2007), and low-level LYVE-1 expression has been reported in late-passage HUVECs (Banerji et al., 1999).
One of the most interesting observations from the present study is that inclusion of pericytes in the HUVEC tubulogenesis cultures blocked the changes in expression of key LEC and BEC markers such as LYVE-1, Prox-1 and VEGF-C. This indicates that signals provided by perivascular supporting cells help to reinforce the BEC phenotype. It is interesting that co-culture of HUVECs with perivascular cells resulted in a largely similar effect on marker expression to that observed in cells following knockdown of Prox-1. This suggests that signals elicited by interactions between endothelial cells and pericytes act via Prox-1 to regulate endothelial cell lineage specification. The converse might also apply, namely that Prox-1 expression in endothelial cells influences endothelial cell to pericyte communication. Supporting this notion, ectopic expression of Prox-1 in isolated blood vascular endothelial cells has been shown to convert the transcriptional programme of the cells toward the lymphatic phenotype by upregulating expression of LEC-specific marker genes, as well as downregulating expression of a large number of BEC-specific transcripts (Mishima et al., 2007; Petrova et al., 2002). Furthermore, constitutive Prox-1 expression is required for maintenance of LEC lineage specificity because conditional inactivation of Prox-1 in mice resulted in loss of phenotypic identity in lymphatic vessels and adoption of blood vessel characteristics, including abnormal coverage by pericytes (Johnson et al., 2008).
An unexpected result of both co-culture of HUVECs with pericytes and knockdown of Prox-1 was the marked induction of VEGFR3 expression. This is surprising given that VEGFR3 is directly regulated by Prox-1 (Hong et al., 2002; Mishima et al., 2007; Petrova et al., 2002). However, VEGFR3 is expressed in the absence of Prox-1 before lymphatic vascular formation (Kaipainen et al., 1995) and is re-expressed in blood vascular endothelium in some conditions (Tammela et al., 2008; Valtola et al., 1999). Furthermore, in our system, in both PROX1 siRNA-treated samples and under co-culture conditions, expression of the VEGFR3 ligand VEGF-C was increased relative to controls, which could create an autocrine stimulatory circuit.
Recently, the concept of endothelial plasticity, and lymphatic endothelial plasticity in particular, is receiving increasing attention (Bixel and Adams, 2008). Our findings add a novel and unexpected aspect to this discussion because we show that normal, committed blood vascular cells have the capacity to undergo aspects of a BEC to LEC transition in response to their ECM environment or the presence of perivascular supporting cells. The effects that we have observed on the expression profiles of endothelial cells grown in specific matrix environments strongly indicate a role for adhesion molecules, particularly integrins, in the control of the endothelial cell phenotype. Mechanotransduction of forces applied to cells by ECM environments of differing stiffness is known to have a profound impact on the specification of cell identity (Butcher et al., 2009), and it will thus be interesting to explore the effects of matrix density on the BEC and LEC expression profiles.
The question arises as to whether the artificial in vitro ECM conditions that we have studied relate to environments that EC experience in vivo. The 3D fibrin assay models an early phase of capillary formation in vivo, when the parent vessel dilates and extravasated fibrinogen is converted to fibrin, forming a provisional scaffold for assembly of the new vessel (Senger, 1996). The type I collagen model is more representative of later stages when EC in vivo migrate away from the parent vessel invading collagen-rich tissue stroma. By contrast, Matrigel might reflect the complex matrix environments of pathologies such as cancer and thus be relevant to the sprouting of tumour neovessels. The BEC-LEC plasticity that we have seen might be reflected in other recent observations from both in vitro and in vivo systems. For instance, expression of lymphatic marker genes in BEC has been seen in vitro in response to treatment with inflammatory cytokines such as interleukin-3 (Groger et al., 2004) or lysophosphatidic acid (Lin et al., 2008). Another recent paper described the presence of a subset of dermal blood capillaries expressing LYVE-1 and podoplanin in chronically inflamed skin (Groger et al., 2007). Likewise, the LEC marker VEGFR3 is seen on tumour neovessels and its inhibition attenuates blood vessel formation (Tammela et al., 2008).
In conclusion, our study has revealed an unexpected plasticity in the phenotype of endothelial cells when cultured in 'traditional', well-established in vitro angiogenesis assays, which suggests that these types of monoculture assays might in fact represent processes involved in lymphangiogenesis, rather than blood vessel formation.
Materials and Methods
ECM components, angiogenic factors, and other reagents
Plasminogen-depleted fibrinogen was obtained from Calbiochem (Merck). Growth-factor-depleted Matrigel™ and high concentration rat-tail type-I collagen solution were purchased from BD Biosciences. Calf skin type-I collagen for coating tissue culture surfaces was purchased from Sigma. VEGF and basic FGF were purchased from PeproTech EC. Antibodies used were a polyclonal goat anti-human LYVE-1 [R&D Systems (AF2089)], polyclonal rabbit anti-human antibodies to VEGR3 (Abcam, ab27278), Prox-1 (Abcam, ab11941) and Lamin-B1 (Abcam ab16048), and a mouse anti-human PECAM/CD31 (555444, BD Pharmingen). Polyclonal anti-GAPDH was from Cell Signaling Technology (Danvers, MA). Secondary antibodies included a FITC-conjugated polyclonal donkey-anti goat secondary antibody (Abcam), a goat anti-mouse Alexa-Fluor-488-conjugated secondary antibody (Invitrogen), donkey anti-mouse Cy2 or Cy3 (Jackson ImmunoResearch.) The MMP inhibitor batimastat (BB-94) was a gift from British Biotechnology.
Cells and cell culture
Primary human umbilical vein endothelial cells (HUVECs), primary adult human aortic endothelial cells (HAECs), and adult human dermal microvascular endothelial cells (HDMECs) were obtained from TCS Cellworks, except for the gene array studies for which the HUVECs were purchased from Clonetics. Cells were grown flasks coated with 60 μg/ml type-I collagen (Sigma), using medium and reagents provided by the manufacturer. Cells were grown at 37°C and 5% (v/v) CO2 and used between the first and fifth passages for all experiments. For experimental purposes, endothelial cells were grown in parallel with the 3D gel cultures in serum-containing endothelial cell culture medium, supplemented with 25 ng/ml VEGF and FGF-2. Murine MII perivascular cells have been described previously (Brachvogel et al., 2005; Brachvogel et al., 2007). These cells were originally identified from mice carrying a targeted insertion in Annexin V and were purified from heterozygous mice carrying the Anxa5-lacZ fusion gene (Brachvogel et al., 2005). The cells were routinely maintained on tissue culture plastic in Dulbecco's Modified Eagle's Medium (high glucose with GlutaMAX, Invitrogen), supplemented with 10% fetal bovine serum at 37°C and 5% (v/v) CO2. Pericytes were used between passages 35 and 40.
EC culture within or on top of 3D gels
Collagen gels (1.6 mg/ml) were prepared by mixing rat-tail type-I collagen solution (BD Biosciences) with 10× 199 medium (Sigma), 25 ng/ml VEGF and FGF-2 (PeproTech) and (where indicated) BB94 (a gift from British Biotechnology) or vehicle control (DMSO). The desired concentrations of Matrigel (BD Biosciences) and plasminogen-free fibrinogen (Calbiochem) were obtained by diluting stock concentrations with serum-free endothelial cell medium and supplementing with 25 ng/ml VEGF and FGF-2.
For 3D cultures, endothelial cells were resuspended in either 2.5 mg/ml plasminogen-depleted human fibrinogen (1.5×106 cells/ml), 5 mg/ml growth factor-depleted Matrigel or 1.6 mg/ml type-I collagen (both 1.25×106 cells/ml). Thrombin (0.5 U/ml; Sigma) was added to the fibrinogen mixture to enable polymerisation. Collagen gels were polymerised by adjusting the pH to 7.5 using 1 N sodium hydroxide. Cultures were prepared n 24-well plates and allowed to polymerise at 37°C and 5% (v/v) CO2 for 30 minutes. Where indicated, 200 μl growth-factor-depleted Matrigel (7.9 mg/ml) was polymerised in wells before addition of endothelial cells to upper surface of the matrix (2×104 cells/gel). Serum-containing culture medium was added to the top of the wells. VEGF and FGF-2 were added to the culture medium at a concentration of 25 ng/ml. Where indicated, BB94 or vehicle control were also added to bathing medium. Gels were then incubated for several days at 37°C and 5% (v/v) CO2.
For experiments in which cells were cultured on top of collagen and fibrin gels, the matrices were prepared as described above, and polymerised in six-well plates at a volume of 500−700 μl per well. 1.75×105 cells/gel were added to the upper surface of each gel, in 1 ml serum-containing culture medium supplemented with VEGF and FGF-2 at a concentration of 25 ng/ml. Gels were then incubated for several days at 37°C and 5% (v/v) CO2.
Collagen, fibrin and Matrigel gels were digested for cell harvesting using (respectively): 0.1% collagenase (Worthington), 5 mg/ml trypsin (Sigma) or Dispase (BD Biosciences) as per the manufacturer's instructions. Cells were then recovered from the digested gels by centrifugation at 1200 r.p.m. for 5 minutes.
HUVEC and perivascular cell co-culture
HUVECs were cultured in 3D type I collagen gels prepared as described above, either as monocultures (8.33×105 cells/ml) or in co-cultures with murine perivascular cells (1.6×105 cells/ml) at a 5:1 ratio. Cultures were stimulated with VEGF (10 ng/ml) and PDGF-B (10 ng/ml) added to the culture medium. Gels were cultured at 37°C and 5% (v/v) CO2 for 72 hours. For RNA expression analysis, gels were digested as described above, and HUVECs isolated from mixtures of HUVECs and perivascular cells using CD31-conjugated Dynabeads (Invitrogen) as per the manufacturer's instructions. Briefly, cell pellets isolated from collagen gel were mixed with 1 ml PBS containing 0.1% BSA and 50 μl Dynabeads-CD31, and incubated at 4°C for 20 minutes with gentle tilting and rotation. The HUVECs binding Dynabeads-CD31 were separated by magnetic force and RNA was extracted.
RNA extraction
Total RNA was extracted from endothelial cells using High Pure RNA extraction kit (Roche), RNeasy mini columns (Qiagen), or TRIzol reagent (Life Technologies), all as per manufacturer's instructions and quantified using an ND-1000 spectrophotometer (Nanodrop Technologies). 0.1−1 μg total RNA per sample was reverse transcribed to cDNA using random hexamers (Amersham Pharmacia), Superscript II reverse transcriptase (Life Technologies), dNTP mix (Roche) and RNase inhibitor (Promega) as per manufacturer's instructions
Protein expression analysis by western blotting
Monolayer cultures were harvested by rinsing cells and scraping into 1 ml of PBS. Cells were harvested from Matrigel by digestion using Dispase, as described above. To obtain sufficient protein for western blotting, cells harvested from ten separate Matrigel assays were combined into one sample. The cells were pelleted by centrifugation and pellets were resuspended in lysis buffer (10 mM Tris-HCl, pH 7.6, 10 mM NaCl, 3 mM MgCl2 1% NP40, supplemented with Roche Complete Mini EDTA-free protease inhibitor tablets. Samples were left on ice for 1 hour with occasional mixing. Cellular debris was pelleted by centrifugation, and the protein content of the soluble fractions was measured with a BCA protein assay kit according to the manufacturer's instructions (Thermo Scientific, Rockford, IL). For western blotting, samples separated by SDS–PAGE on a 10% resolving gel were transferred to PVDF (polyvinylidene difluoride) membranes (Bio-Rad). Protein bands were detected by incubation with the appropriate primary antibody followed by horseradish peroxidase (HRP)-conjugated secondary antibody (DAKO).
Microarray analysis of differential gene expression during HUVEC tubulogenesis
HUVECs were embedded in 3D fibrin matrices and cultured as described above. For 2D cultures, 2.5×105 HUVECs were seeded on top of 1 ml fibrin gels (prepared as described above). For 3D cultures, a maximum of four 300 μl gels were pooled and homogenised per sample. The cDNA for gene array analysis was prepared from 20 μg of total RNA. For each sample, two cDNA preparations were made and coupled to either Cy3 or Cy5 dyes (Amersham Biosciences, Little Chalfont, UK) in 0.1 M Na2CO3 pH 9.0 for 1 hour at room temperature. The coupling reaction was then quenched with 2 M hydroxylamine for 15 minutes and the labelled cDNA purified with Amicon Microcon-30 concentrators. Quantification of labelled cDNA was performed with an ND-1000 nanodrop spectrophotometer with readings at A260, A550 and A650. 25 pmol of dye labelled cDNA was used for each microarray probe.
Human 19K glass slide 'oligo arrays' were obtained from the Australian Genome Research Facility (AGRF, Parkville, Australia). Slides were first blocked in 0.1% SDS at 95°C for 1 minute, followed by a 1 minute wash in 5% ethanol and a further 1 minute wash in distilled H2O, and dried. Slides were then prehybridised with 10 mg/ml BSA, 25% formamide, 5× SSC and 0.1% SDS for 45 minutes at 42°C, and then hybridised with 25 pmol of each labelled cDNA. For each comparison (i.e. 2D vs 3D), sample A labelled with Cy3 was mixed with sample B labelled with Cy5. A dye-swap hybridisation control on a separate slide was also performed for each sample pair, with sample A labelled with Cy5 and sample B labelled with Cy3. 25 pmol of both A and B samples were mixed with Cot1 DNA (5 μg/μl), Poly A DNA (10 mg/ml), salmon sperm DNA (10 mg/ml) in hybridisation buffer (25% formamide, 5× SSC and 0.1% SDS) for a total reaction volume of 60 μl. Samples were heated at 100°C for 2 minutes and hybridised to glass slide microarrays overnight at 42°C. The next day, arrays were washed for 10 minutes in 2× SSC, 0.2% SDS, for 10 minutes in 2× SSC, and then for 10 minutes in 0.2× SSC. Glass slides were then dried by centrifugation at 500 g for 3 minutes and scanned with a GenePix 4000B scanner (Molecular Dynamics, Amersham). Gene array images were analysed using the GenePix Pro 4.0 software. Local background correction was performed and data normalised according to the median of ratios. A gene was considered differentially regulated if the ratio between sample pairs A and B was >1.5 or <0.67.
TaqMan real-time PCR analysis
TaqMan PCR procedures were carried out as described previously (Nuttall et al., 2004). The primer and probe sequences used are listed in supplementary material Table S2.
Immunostaining of monolayer and 3D collagen cultures
Samples were washed in PBS, fixed with 80% methanol, 20% DMSO overnight at 4°C, rehydrated in 50% methanol in PBS, 20% methanol in PBS and PBS-T (PBS, 0.1% Tween-20) for 1 hour each and then were incubated with blocking solution (PBS, 10% fetal calf serum, 5% BSA) overnight at 4°C. Primary antibodies including goat anti-human LYVE-1 at 2 μg/ml), mouse anti-human PECAM/CD31 at 1 μg/ml, rabbit anti-human VEGFR3 at 4 μg/ml, rabbit anti-human Prox-1 at 10 μg/ml were added into the samples incubated in blocking buffer overnight at 4°C and then washed six times for 1 hour each in TBS-T (TBS, 0.1% Tween 20). Corresponding secondary antibodies conjugated with Cy2 or Cy3 (Jackson ImmunoResearch) were incubated in blocking buffer overnight at 4°C and washed as described above. For secondary antibody control, there were no primary antibodies added before incubation with same secondary antibodies. After nuclei staining, samples were mounted in Gelvatol and examined by fluorescence microscopy (SteREO LumarV12 and Axioplan2; Carl Zeiss, Germany). Pictures were captured with Axiovision software (version 4.5). Antibodies used in double staining procedures have been tested for minimal cross reactivity and spectral overlap.
Two pre-designed siRNA duplexes targeting PROX1 and a negative control siRNA duplex were purchased from Ambion (UK). HUVECs were seeded in collagen-coated six-well plates and transfected the following day with the various siRNAs at concentrations of 150–200 nM, as indicated. Oligofectamine and Opti-MEM (Invitrogen, UK) were used for delivery of the siRNAs as per manufacturer's protocol. Cells were cultured for a further 48 hours before seeding onto Matrigel layers (5 mg/ml). Serum-containing culture medium was added to the top of the wells. VEGF and FGF-2 were added to the culture medium at a concentration of 25 ng/ml, and cells cultured for a further 24 hours at 37°C and 5% (v/v) CO2. Samples were harvested as described above at the indicated time points.
Statistical analyses
TaqMan expression data were analysed using one-, two- or three-way ANOVA tests as described in the text. P<0.05 was considered significant.
This work was supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC), the Big C, Norfolk Fundraisers and the EU Framework Programme 6 Cancerdegradome Project LSHC-CT-2003-503297. L.S.C. was a recipient of a BBSRC PhD studentship. Z.Z. and E.P. were supported by the British Heart Foundation, Project PG/06/071/21115T. E.W.T. and M.A.L. were supported by the Victorian Breast Cancer Research Consortium, Australia.
↵* Present address: Amgen, 1 Amgen Center Drive, Thousand Oaks, CA 91320, USA
Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.064279/-/DC1
- Accepted July 16, 2010.
- © 2010.


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11 Dec 2017 23:17
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Don't be afraid to misplace. It is instinctive to wish to playact against opponents World Health Organization are at or infra your accomplishment layer. Afterward all, it's no amusive to constantly lose! In that respect is, however, a John Major disfavour to this strategy - thither is no incentive to amaze meliorate. When you are playacting against citizenry who are improve than you, you wish memorise from your mistakes and be on their charge in no metre.
Switch in your video recording games when you are through playacting them. Many retailers proffer special discounts or credit towards your following leverage when you deal your games in. You lav beget the adjacent halt you wishing for a low-pitched toll when you do this. Later all, you don't require the games erst you beat out them.
Don't be shy around downloading games digitally! While this distribution channel experient a moderately rocky start, the kinks possess been ironed stunned. Agen Judi togel , downloading your games (legally) is a a good deal faster and to a greater extent commodious path to have admittance to them. It's besides a cracking means to bent onto them; with virtually reputable services, you're allowed to download a lame complete and terminated again. This makes it soft to recuperate from a computing device nuclear meltdown without fussing with a bundle of DVD-ROMs.
Modernistic television games are non without their dangers, and playing them obsessionally might be the matchless to concern most all but. If you lie with that a protagonist or phratry appendage is a gamer, be on the sentinel for signs that they're disbursal an immoderate total of clock playacting. If they're having bother confluence their obligations at school, work, or in their social circle, it May be clock time to present them some how they make do their gambling meter.
Call back almost what old age your kids make to be ahead they bottom spiel games rated M, or age. Virtually video recording halt systems put up be frozen so that full-grown games are plugged. It is more than unmanageable to do that on a computing machine. Be mindful of monitoring your child's gambling practices.
Don't pitch proscribed video games that are dated or senescence. You rear end trade wind them in for recognition. It is imaginable to purpose the money you fix from trading old games to corrupt fresh ones.
Practice picture games to plain a tighter kin. Level with altogether the video recording games proscribed today, it is unusual for adults to manoeuvre video games with their children fifty-fifty though this pot be fun for whole. Games that concentrate on Education Department and mob natural action are in force choices for everyone to wreathe consume put together in the eve.
Take the Metacritic mark of whatsoever picture halt you're reasoning around purchasing prior to buying it. More or less games are hard discounted for a reason, they aren't whatsoever sport. This means you've otiose your money in so much a leverage. Checking retired Metacritic scores of every gage you're considering buying butt assist.
Don't purchase games for your kids that are not long time pertinent. This agency that you testament require to mark off the eld grade on the straw man of the punt to learn if it is correct for your children. Fury tail be a primary winding focalize of about games, substance you May non deprivation to leverage them.
Whether you ilk to flirt on a console table or your cellphone phone, video games come out to be everywhere today. You no longer deliver to go to the colonnade to take on the better titles, or else you potty equitable dally they at dwelling. You don't regular bear to go to a store to grease one's palms them! What a cosmos we live on in.

subset: commoncrawl


text:
Cheryl Cole has jetted off for a holiday with two of her Girls Aloud bandmates, as she agonises over the future of her relationship with cheating husband Ashley.
The pop singer has told friends that she will make her final decision while away in Thailand with Nicola Roberts and Kimberley Walsh.
Cole has been left distraught by claims that Ashley betrayed her by sleeping with glamour model Brooke Healy in 2006 and single mum Aimee Walton in 2007.
"She has been through the most gut-wrenching time and she needed a break," a friend told The Mirror. "She had to get away from the twin influences of Ashley and her family. Ashley has been phoning her constantly but she hasn't been taking his calls.
"Her family are also putting her under pressure of a different kind. They feel that she should just dump him and move on. But the girls in the band have been non-judgmental, which is exactly what Cheryl needs right now. They have all told her they have her unwavering support whatever she decides.
"She still hasn't decided what she is going to do. It's the biggest decision of her life."


text: Job Discussion Forums :: View topic - Hamburg - is it do-able? Get TEFL Certified & Start Your Adventure Today! Job Discussion Forums"The Internet's Meeting Place for ESL/EFL Students and Teachers from Around the World!" FAQ Search Memberlist Usergroups Register Profile Log in to check your private messages Log in Hamburg - is it do-able? Job Discussion Forums Forum Index -> Germany View previous topic :: View next topic Author Message AllieJoined: 15 Sep 2005Posts: 32Location: UK Posted: Mon Feb 23, 2015 6:29 pm Post subject: Hamburg - is it do-able? I'm a Brit, with a CELTA, 5 years of teaching experience and fluent German. I lived in Hamburg over 10 years ago and really want to go back and live there. I wasn't teaching when I was there so I don't have any teaching experience in Germany. I will be the bread-winner for my family - husband and 6 year old. We don't live a fancy lifestyle. Is it realistic to think I would get enough work for us to live in Hamburg? I'm thinking of doing a MA in linguistics with TEFL to improve my chances. Is it work paying to do my MA? Should I forget Hamburg or is it worth taking the risk? Thanks for any advice! Back to top artemisiaJoined: 04 Nov 2008Posts: 875Location: the world Posted: Tue Feb 24, 2015 9:27 am Post subject: There are other posters with more up to date information than I have. However, TEFL has never really been well paid work in Germany unless you can build up a network of contacts, get to know the market and start working for yourself. That usually requires a fair bit of time in a place and many teachers slowly build these networks after going the language school route first. On the plus side, you won't have any visa issues and you do have the language and a CELTA, but local networks are very important. Germans place high value on recommendations and will often stay loyal to the business connections they already have, even if offered a cheaper provider. They also like people with direct experience in the business world. The other way to better paid work is a proper uni job but for that you'd definitely need a postgrad degree - probably higher than MA - and significant tertiary experience. They've always been incredibly competitive jobs to get. But it may still be possible to get sessional teaching work though universities. You should be able to get work in language schools and possibly even companies directly if you start putting together an offer and approaching businesses. I'd suggest smaller companies at first as the large ones will almost certainly already be set up. There are threads here on starting your own business (as opposed to working as a freelancer), but in the end it comes down to supporting a family and covering costs like health insurance. I think it is a risk but can't say if it's worth it. If you're determined to give this a go, my main suggestion is that you try this out on your own first. You can approach schools in advance, but you really have to be in Germany to get work. If your husband (and/or other family members) can take care of your child in the UK, check how practical it is firsthand before the family is uprooted. I'd also give this plan a time limit. Another possibility to consider is translation work. Back to top JNJoined: 17 Jan 2008Posts: 214 Posted: Tue Feb 24, 2015 10:47 am Post subject: I'd agree to everything that Artemisia says. It sounds like the state of things now. I don't have anything to add. Back to top AllieJoined: 15 Sep 2005Posts: 32Location: UK Posted: Wed Feb 25, 2015 7:02 pm Post subject: Many thanks for taking the time to reply. It's not what I was hoping to hear but I've very grateful for the honest opinion. Back to top HodJoined: 28 Apr 2003Posts: 1613Location: Home Posted: Wed Feb 25, 2015 9:59 pm Post subject: It sounds like you know Hamburg better than any of us. The market there will be freelance work via a school or as you speak German, unlike most TEFLers there, you can get your own work which will pay well. You probably know that Germans love their six weeks of annual holidays topped up with Krankheitsurlaub so you need to factor this into your earnings. Why not contact a school such as IH Hamburg? They won't be anything special, but they'll offer you a set amount of freelance hours per week which is a good start. As a freelancer, much to IH and co's chagrin, you aren't in theory supposed to work for only one supplier, so you are legally free to seek other work. Health insurance rules changed dramatically in 2009, and anyone resident in Germany must have proper health insurance, which isn't cheap. Employers pay about half these costs, but the freelancer will have to pay the lot. There are a few international policies which are just as good and cheaper, but you need specialist advice from a broker. Check Toytown. Health insurance and Hamburg's high demand for rental property aren't insurmountable problems, and your German ability will at least make the latter a lot easier. If you're not totally skint and can pay a rental deposit and a few month's expenses, I'd be tempted to give Hamburg a go. Back to top Display posts from previous: All Posts1 Day7 Days2 Weeks1 Month3 Months6 Months1 Year Oldest FirstNewest First Job Discussion Forums Forum Index -> Germany All times are GMT Page 1 of 1 Jump to: Select a forum Where to Begin----------------General DiscussionNewbie ForumCurrent Events ForumTechnology ForumOnline Teaching Forum Africa Forums----------------General Africa Forum Asia Forums----------------CambodiaChina (Job-related Posts Only)China (Off-Topic)Hong KongIndiaIndonesiaJapanMalaysiaPhilippinesSingaporeTaiwanThailandGeneral Asia ForumVietnam Caribbean Forums----------------General Caribbean Forum Europe Forums----------------Czech RepublicFranceGermanyGreeceItalyMacedonia and the BalkansPolandRomaniaRussia & C.I.S.SpainTurkeyUkraineUnited KingdomGeneral Europe Forum Latin America Forums----------------ArgentinaBrazilChileColombiaCosta RicaMexicoMexico (off-topic)PeruGeneral Latin America Forum The Middle East Forums----------------BahrainEgyptIsraelKuwaitOmanQatarSaudi ArabiaUnited Arab EmiratesGeneral Middle East Forum North America Forums----------------General North America Forum Oceania----------------Australia/New Zealand You cannot post new topics in this forumYou cannot reply to topics in this forumYou cannot edit your posts in this forumYou cannot delete your posts in this forumYou cannot vote in polls in this forum This page is maintained by the one and only Dave Sperling. 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text: PLOS Medicine "Diet-related noncommunicable diseases (NCDs) are increasing rapidly in low- and middle-income countries (LMICs) and constitute a leading cause of mortality. Although a call for global action has been resonating for years, the progress in national policy development in LMICs has not been assessed. This review of strategies to prevent NCDs in LMICs provides a benchmark against which policy response can be tracked over time. (...) This review indicates the disconnection between the burden of NCDs and national policy responses in LMICs. Policy makers urgently need to develop comprehensive and multi-stakeholder policies to improve dietary quality and physical activity."


text:
How to create transparent image background?
There are two ways you can use to create a transparent background:
Using the Wand tool (fuzzy selector). Simply activate Wand and tap the background area excluding the object you want to keep. Keep doing this until all the background is selected. Press Delete to remove it and save the image as PNG and that's it.
1.1 Tutorial link: http://docs.gimp.org/en/gimp-tool-fuzzy-select.html
Use the Lasso or Free Select tool. This time, use polygonal selector and start from a point for the object you want to keep. Go round its margins and click each time to create anchors. At the end click the starting point. Press CTRL + X to Cut, then create a blank layer and use Paste. Save as PNG and that's it.
2.2 Tutorial link: http://docs.gimp.org/en/gimp-tool-free-select.html


text:
Free Consultation: (602) 266-2667Tap to Call This Lawyer
Alan M SchiffmanSchiffman Law Office, P.C.
- Social Security Disability, Workers' Compensation, Insurance Claims ...
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Claimed Lawyer Profile
Since 1975 Alan M. Schiffman has been representing injured and disabled workers and their families regarding claims for workers' compensation, Social Security, long-term disability and personal injury matters. He and the other attorneys in the firm are dedicated to providing the highest quality service to those in need. So, if you find yourself in need of legal assistance, contact us - we are here to help. Check out our website at www.schiffmanlaw.com for detailed background information regarding us.
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- J.D. (1975) | Law
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- State University of New York - Buffalo
- B.A. (1972) | Political Science
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- Pennsylvania State University - University Park
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Professional Experience
- National Organization of Social Security Representatives
- Current
- Arizona Association Of Justice
- Current
- Arizona Association of Lawyers for Injured Workers
- Current
- Workers' Compensation Specialist
- State Bar of Arizona
Professional Associations
- State Bar of Arizona  # 004257
- Member
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Jurisdictions Admitted to Practice
- Arizona
- 9th Circuit
- U.S. Supreme Court
Free Consultation
Free consultations are available regarding workers' compensation, Social Security Disability, Long-Term Disability and personal injury matters.
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Most cases are handled on a percentage fee basis. Social Security matters are usually handled under the Social Security Administration's fee agreement procedure.
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- Insurance Claims
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Additional Practice Area
- Long Term Disability
Websites & Blogs
- Website
- Schiffman Law Office, P.C. website
Contact & Map


text:
Being good stewards is one of BASC's core company values. Not only do we strive to manage resources efficiently to create and preserve value for our customers, but we also take steps to care for our environment. BASC breaks it down into three categories: living green, learning green, and working green.
Visit the College Sustainability webpage for more information on campus-wide initiatives.
Living Green
We help our campus community members live green and reduce their carbon footprint through the following initiatives:
- Dining Services offers customers a Refillable Mug Program at The Square and cafes. It is estimated that the participants in this program have collectively diverted approximately 1,750 paper cups from the landfill each year.
- Many of the to-go foods are packaged in recyclable containers enabling our customers to recycle them.
- Recycling bins are available. Remember to recycle properly to avoid contaminating the recycling stream.
- Dining Services offers a number of plant-based menu items in all dining locations across campus. Eating more plant-based meals is a great for your health and the planet!
- Dining Services sources local foods whenever possible. Approximately 90% of the dairy products served on campus are produced locally and about 12-15% of produce is sourced locally through our prime vendors.
- Our prime food vendor, Sysco, works in collaboration with the World Wildlife Fund (WWF) to improve the sustainability of its seafood procurement. Through this partnership, Sysco works with its supply chains to transition fisheries and aquaculture farms globally to more sustainable and responsible resources. Look for sustainable seafood menu items we serve that have the WWF seal of approval.
- Each recipe has a specific portion for served items. In the dining halls, customers can always ask for more (or less – especially when wanting to sample a food item first). Portions are in place to help eliminate food waste. Customers are encouraged to take only what they know they'll eat and come back for seconds.
- The Barnes & Noble College Bookstore offers students used and rental book options. These are great ways to save money and reduce your carbon footprint!
Learning Green
BASC takes an active role in informing and educating our community about sustainability initiatives
- BASC worked collaboratively with BSG to inform students about impact of plastic straws on our planet and successfully phase them out on campus. Straws were eliminated from the dining halls in the fall of 2018. Beginning in the fall of 2019, all plastic straws have been eliminated from dining locations. It is estimated that this will divert approximately 300,000 straws from the landfill each year.
- BASC has partnered with the student club, ECOS, to:
- Educate students about the importance of bees in food production during the Fall 2018 Be Our Guest event at Brockway and Harrison dining halls. Local bee keepers also participated in this educational dinner. The event earned a prestigious Loyal E. Horton gold award from the National Association of College & University Food Services.
- Educate the campus community about proper recycling in a program called, Trash Talkers. View our Trash Talker video here.
- In 2015, BASC hosted a Harvest Celebration in Brockway and Harrison dining halls that featured many foods sourced locally. This event helped to inform the campus community about what is (and isn't) local to our area as well as what's in season during the fall. This event earned a prestigious Loyal E. Horton bronze award from the National Association of College & University Food Services.
- In 2012 and 2013, BASC held two Farm to Table Dinners where students traveled to Robb Farms located in Spencerport to watch our chefs cook using ingredients produced from the farm and dine in the middle of an apple orchard.
- Members of the BASC team work collaboratively with student groups, faculty, and departments on educational initiatives. Examples include, sharing information with the Greenhouse Living & Learning Community and sharing initiatives with the Sustainability in Recreation class.
Working Green
We implement sustainable business practices whenever feasible to be good stewards of resources and care for our environment.
- We use a sophisticated Food Management Software system to input recipes, manage inventory, and project the quantity needed for each recipe. This enables Dining Services to most accurately prepare the appropriate quantity of food for any given meal, and thus reduce food waste.
- Leftover food from serving lines or food that never left the kitchen that meets quality and food safety standards is properly stored and served at a next meal.
- Pre-consumer food waste (egg shells, coffee grounds, vegetable peels, etc.) and food that doesn't meet quality and food safety standards is composted via our partnership with Natural Upcycling. It is estimated that 85,000 lbs of scraps are diverted from the landfill each year through this program. Watch our Natural Upcycling video to learn more.
- At the end of the semester, foods set to expire before reopening are donated to local food banks.
- The dining halls are tray-less. Tray-less dining helps to reduce food waste by discouraging customers from taking more than they can eat. Plus, it saves resources from not having to wash trays.
- In partnership with the National Humane Society, our chefs and team members received plant-based culinary training to increase the number of delicious plant-based menu items served on campus. Learn more here.
- A fryer oil filtration system is used in Brockway and Harrison Dining Halls, and The Square, to extend the life of the oil. Once the oil has reached its maximum usage, BASC then recycles the fryer oil via a third-party vendor who converts it to bio-diesel, animal feed, and other uses.
- Our refuse vendor, Suburban Disposal, uses collection trucks that use cleaner burning natural gas.
- BASC has a shut-down schedule for our equipment over breaks to reduce energy usage.
- After equipment has exceeded its useful life or needs replacement, we aim to replace it with energy efficient models. For example, all laundry and vending machines on campus are energy star rated.
Frequently Asked Questions
Does Dining Services buy local foods?
BASC contracts with Giambrone, a Rochester produce food distributor, for fresh produce. Giambrone provides local produce whenever possible. There are signs in the dining halls that communicate weekly local produce. Local farms include Kirby's Market, Garden Farm, Hill-View Farms, Sodoma Farms, Intergrow, Smith's Farm Market, and many others. Approximately 90% of the dairy products served on campus are produced locally and about 12-15% of produce is sourced locally through prime vendors.
Does Dining Services have a compost program?
Dining Services partners with Natural Upcycling to compost pre-consumer waste. Vegetable scraps, egg shells, and coffee grounds are collected from Brockway Dining Hall, Harrison Dining Hall, and The Square at the Union to turn it into a renewable resource - such as electricity or natural gas – through a process called anaerobic digestion. It is estimated that 85,000 lbs of scraps are diverted from the landfill each year through this program. Watch our 0 to learn more.
Are energy efficient appliances used in Dining Services?
When equipment has exceeded its useful life or otherwise needs replacement, BASC aims to replace it with energy efficient models. Replacement kitchen equipment, including stoves, ovens, steamers, and refrigeration, are sourced not only for quality of construction, but also energy efficiency.
In 2012 the refrigeration units in Brockway were converted from a water-based cooling and heat transfer system to a Glycol-based closed system, dramatically reducing water usage while increasing energy efficiency.
Are environmentally-friendly disposable containers used in eateries on campus?
Nearly all of the to-go containers used by Dining Services are recyclable. Please remember to remove any food debris before recycling these containers. In addition, some disposable containers like portion cups and straws in the dining halls have been eliminated altogether to help reduce waste.
What happens to the used fryer oil on campus?
In Brockway and Harrison Dining Halls the fryer oil is filtered to extend the life of the oil. Once the oil has reached its maximum usage, BASC then recycles the fryer oil via a third-party vendor.
Why are the dining halls tray-less?
Tray-less dining helps to reduce food waste by discouraging customers from taking more than they can eat. Plus, it saves resources from not having to wash trays. Both dining halls have experienced reductions in food waste as a result of not using trays. In fact, after the initial switch to tray-less Harrison Dining Hall was able to eliminate two of their six refuse pickups per week. Brockway Dining Hall is currently at two pickups per week.
What is the Refillable Mug Program?
Dining Service's Refillable Mug can be purchased at the BASC Business Office or any cafe on campus for only $5! This mug saves you 20% off on the 16 oz. price when purchasing fountain beverages (soda, juice, Revolet), coffee or tea, hot chocolate or cappuccino from the machine at The Square or Eagle's Nest. This mug may not be used in Brockway or Harrison Dining Hall.


text:
3-Piece Teacher Appreciation Gift Set: Teaching is a Work of Heart Tote Bag, Water Bottle and Pen with Case. A fabulous thank you teacher gift to give that special teacher who's enlightened your child's mind and touched their heart. Tuck the pen and water bottle inside the tote and give this heartfelt teacher appreciation gift to your child's school, dance, or piano teacher to thank them for their hard work this year!
Set includes:
TOT085 - Teaching Is a Work of Heart Tote Bag
WBT121 - Teaching Is a Work of Heart Glass Water Bottle


text:
Telling Tales
children's book illustrator Peck puts career on display
Beth Peck, a well-known children's book illustrator who lives in Menomonie, is showcasing some of her best work in an exhibit at the Eau Claire Regional Art Center's Janet Carson Gallery called Tell Me A Story, which runs weekdays through March 10.
"I think people don't realize how much planning and sketching goes into making a picture book. I think this is what is so much fun to share with people." – Beth Peck, illustrator
Peck has illustrated more than 20 children's books by a variety of authors, including the likes of Truman Capote and Edna St. Vincent Millay. "I often chose the pieces for the show based on how much I learned doing them," Peck said. "They are the ones where I learned the most. Where the journey and the process was the most interesting. I think people don't realize how much planning and sketching goes into making a picture book. I think this is what is so much fun to share with people. And, it isn't just me. All illustrators put an incredible amount of research and planning and thought into making a picture book."
The process of becoming a working children's book illustrator was not a one-step process for Peck. "I did not feel ready to be an illustrator right out of college – I had graduated Rhode Island School of Design in the Illustration Department," she explained. "I grew up just outside of New York City, so I continued to take classes. I took figure drawing classes once a week for 10 more years at the Art Students League and the National Academy of Design. I studied for two years with children's book illustrator Uri Shulevitz, who taught from his New York City apartment. I also studied painting and drawing for two years in the private home studio of Burton Silverman. I did all this to become better at drawing. While I was doing this I started to bring my portfolio to publishers and Harper and Row gave me my first children's book, Sarah and the Dragon. I also started to freelance for a children's book publisher doing work that was called paste-up and mechanicals—when everything was done by hand, not by computer." After the success of that first book, several others followed.
There is an interesting story behind the favorite book Peck has illustrated. "I love the Truman Capote stories – they are so well-written – but I also had so much fun doing a book about baseball," she said. "Truman Capote was no longer alive, but I can tell the story of how I came to illustrate two his stories. Just after college I took a night class at the School of Visual Arts in N.Y. The teacher gave us an assignment to read The Grass Harp by Truman Capote and illustrate a scene from the story. I absolutely loved the story and on my own I went on to read everything by Truman Capote. The only thing I had read by him was In Cold Blood. Well, I read A Christmas Memory and The Thanksgiving Visitor and fell in love with these stories. And because these stories were about childhood, I thought they would be wonderful to illustrate. Ten years later, when I had more practice being an illustrator, and my first full-color picture book had been so nicely reviewed in the New York Times, I think it gave me a little courage to ask the publisher if I could illustrate these Truman Capote stories because no one had ever done so. So that is how I came to illustrate two of Truman Capote's stories."
Peck is not currently working on any children's books, but you can learn about her and the classes she offers at bethpeckillustrations.com.


text: The AP reports: Dwyane Wade's legs were sore, Udonis Haslem had a bad back and Shawn Marion was too hurt to play. Even at the end of a seven-game, 12-day road trip, the Heat still had enough to pull through. Wade had 32 points and racked up most of his 10 assists while helping thwart Oklahoma City's fourth-quarter rally as the Heat beat the Thunder 104-94 Sunday night to head home on a positive note… Yakhouba Diawara scored 14 points after starting in place of Marion (groin), and Jamaal Magloire and Mario Chalmers each added 13 points for Miami. Haslem had 11 points and a season-high 15 rebounds. Kevin Durant led Oklahoma City with 31 points, Jeff Green scored 22 and Russell Westbrook added 14 points and eight rebounds.


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Many people may not be familiar with the name Micromax, and for good reason. Micromax is India's homegrown brand and the largest smartphone maker in the country, surpassing even giants like Samsung and now it looks like U.S. is the next market they are looking to enter.
However, there are a few obstacles that Micromax needs to overcome despite how successful they are. First of, the name Micromax is an almost unknown brand to U.S. smartphone users and establishing themselves as a worthy competitor to companies like Apple and Samsung will not be an easy feat. Secondly, taking an example of other successful brands outside of the U.S. like Xiaomi and Huawei(who still have not entered the U.S. market), it opens up the risk of being sued, not immediately but further down the line.
Micromax was reportedly "considering launching its premium smartphone line called the Yu in the U.S. sometime next year", but Rahul Sharma, co founder of Micromax and also the man who runs the Yu brand said they have not decided to do so as of yet, according to The Information. The Yu device, which is sold in India for about $200 runs on CyanogenOS, which is a custom version of Android and the revenue gained from that is at an estimate of about $1.5 billion at the end of the fiscal year in March.
At this rate, Micromax is expected to reach at least $2.25 billion by the end of the fiscal year in March 2016. They have even mentioned plans to release up to 48 new phones per year, all of which will be priced up to $150. This is not only a bold plan but also a very difficult task to fulfill and keep up, but if Micromax pulls this off and is able to enter the U.S. market with their plan, it will be interesting to see how the smartphone market will change with the added variety and what the competition plans to do to ensure they remain on top. This might also spur companies like Huawei, Xiaomi and ZTE to follow suit and start selling devices in the U.S.


text:
Tweet This
There is always a well-known solution to every human problem—neat, plausible, and wrong – H.L. Mencken
It's a useful adage to apply to a whole range of difficult topics, not least energy policy. Investing in 'clean coal' for instance sounds like a perfectly simple policy win for the United States. 'High-efficiency, low-emission' (HELE) advanced coal technologies promise to revive a floundering U.S. industry and promote energy independence, potentially with zero emissions, using carbon capture and storage (CCS) technology.
Fellow Forbes contributor Jude Clemente certainly thought so, putting front and center the International Energy Agency's (IEA) championing of the issue and pointing out that this approach was also originally a part of President Barack Obama's first-term energy policy.
Yes and no.
First, let's get facts straight. 'Clean coal' for some may conjure images of artisanal, all-natural and American-made goodness, which is certainly the vision the Trump administration has been keen to promote. The reality is whilst the idea of capturing all the emissions from coal is technically feasible, the costs today are still astronomical, doubling a typical power station's capital and operating costs. The track record for storing emissions safely at any meaningful scale is also far shakier than not generating them in the first place.
An important context is missing here, and that's what has happened between 2008 and now. Rewind a decade, and a myriad of energy technologies were vying for attention, with policymakers around the globe wary of placing bets. Nuclear, clean coal with CCS, and renewables were all potential pathways to a secure, affordable and low-carbon emission future, and policy initiatives both in the U.S. and Europe took a cautious, 'all of the above' approach to tackling the issues of the day.
What has happened since then is nothing short of a landslide in favour of renewables. Solar technology costs have fallen over 85% since 2009. In the same period, coal (not clean coal mind you – just your regular polluting kind) remained flat, and nuclear costs have actually trended upwards.
Suddenly, the playing field looks very different.
Wind and solar in North America are now the cheapest forms of energy bar none on a levelized cost basis; cheaper than gas, nuclear or coal. Solar prices are continuing to drop without a clear end in sight, based on a learning curve of prices falling 20% for every doubling of installed capacity. With only 2% of U.S. energy coming from solar so far, this is a seismic shift that's far from over.
It's a trend that the IEA woefully misunderstands, as their annual World Energy Outlook projections embarrassingly demonstrate.
The momentum of this disruption curve is important, because it illustrates the futility of the clean coal movement catching up. There simply isn't an experience curve for clean coal that matches anything like what we are seeing in renewables.
Research into HELE coal could nudge the efficiency of coal up a point or two, but also reveals the incremental nature of such improvements. Specifically, as the high efficiency part that acronym means coal plant efficiencies of 40% rather than high 30s, it is not going to move the needle on cost competitiveness.
What's also forgotten is the low emissions part of the acronym – adding carbon capture – reduces the overall efficiency of such coal plants drastically, typically by around 10%. Stopping all the SOx, NOx, particulates and CO2 is a big energy drain in itself. Scrubbing the emissions means burning a lot more coal in the first place.
With renewables a clear leader on cost and carbon issues, the final line of argument for clean coal comes to energy security. Wind and sun, it is rightly pointed out, are intermittent sources of energy – as if this is some kind of 'gotcha' moment for the renewables industry that no one has considered. The impact of intermittency, it is claimed, is not factored into the cost of these technologies.
Again, to return to Mencken's opening witticism, the truth is a little more complex. The electricity system has always required a good degree of backup, particularly as large, centralised nuclear and fossil fuel plants present large and hard-to-absorb losses of generation when they do fail. This security has typically been met by a large fleet of fast starting, low efficiency gas 'peakers,' historically running rarely, expensively and only for short windows.
But here again, technology has provided us smarter, cleaner and cheaper ways to manage this issue.
A complementary trend to the decline in renewables prices has been a similar decline in the cost of energy storage technology, with lithium ion battery costs declining 80% since 2010 alone.
The declines have been so pronounced that prominent analysts like Wood Mackenzie increasingly note that battery energy storage is cheaper that fossil fuel peakers on both a capital and operating cost basis.
The evidence of this trend is already mounting. The world's largest lithium ion battery to date is now under construction in California, having been selected in a head-to-head procurement competition against gas peaker plants. In 2018 and again in 2019, the combined package of renewables and storage set record low prices in Arizona, Nevada and Hawaii, beating out fossil fuel alternatives.
Simply put, all of this points to an exciting and mounting trend. Renewable energy coupled with energy storage is already cheaper, cleaner and as reliable as conventional alternatives – even as the cost trends are still sharply downwards.
As this happens, longer and longer duration energy storage is becoming economic versus conventional thermal solutions, allowing utilities to take a tiered, portfolio approach to meeting peak demand with batteries. Utilities are also recognizing the benefits of super-fast responding, digital technologies for overall grid stability – avoiding the fuel costs, direct emissions and years of planning and permitting that come with traditional fossil fuels.
Our efforts should not be focused on incremental investment into old technology like coal, or even gas as a bridge fuel, but into removing the barriers to rapid electrification, the acceleration of renewables and transforming the grid with energy storage.
I'm a Forbes 30 Under 30 honoree, technology futurist and entrepreneur recognized for my work in energy and sustainability. I've dedicated my career towards building a…
I'm a Forbes 30 Under 30 honoree, technology futurist and entrepreneur recognized for my work in energy and sustainability. I've dedicated my career towards building a sustainable energy future and inspiring the next generation of sustainability leaders. As a founding member of Fluence, a global energy storage technology and services company backed by Siemens and AES, I'm at the forefront of the transformation of how we power our world. My experience includes instrumental roles in Europe's first commercial battery storage array as well as one of the largest battery storage fleet transactions in the world. Prior to this, I completed a doctorate focused on the impacts of renewable intermittency on energy markets and masters in engineering at Durham University. I've worked and traveled in over 50 countries and across diverse industry, policy and academic sector roles, including the fossil fuel industry and U.K. Parliament.


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Determining When Nobody Is Logged In
Use the server manager to determine if a given PC is connected to a server. Network connections are only restored when a user logs in.
Cheack Users who current In Use Domain
Open the Server Manager of the Domain, then open the View and select the Servers only, then choice that Server (PDC or BDC(s)) which you want to work on and double click to open it(or, go to Computer, open Properties...), then open the In Use. You can see who are current login your this Domain and what are they doing there.


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As fierce Santa Ana winds whipped the wildfires outside of Los Angeles, stirring exactly the kind of infernos that scientists expect in a hotter, drier California, President Donald Trump was gloating over the new allies he has won in his epic battle to block that state's efforts to fight climate change.
California has been a world innovator in crafting environmental policy, and its pioneering approach to the difficult issue of carbon emissions from cars helped put the United States on course to cleaner, more efficient vehicles. But a multi-pronged assault by the Trump administration now seeks both to hobble California's climate efforts and to shred the state's reputation as an environmental leader.
In its latest move, the Trump administration doubled down on its fight to eliminate the state's carbon emissions rules for vehicles. It had already revoked California's authority to set higher standards. Now it was pressuring carmakers to take its side in California's lawsuit over the move. When GM, Fiat Chrysler and Toyota made the surprise decision to comply last week, Trump tweeted: "California has treated the Auto Industry very poorly for many years, harming Workers and Consumers. We are fixing this problem!"
The attack isn't just on auto standards. On Sunday, Trump tweeted a threat to cut U.S. aid for fighting California wildfires, reiterating his previous false claim that they are due to the state (which owns of just 2 percent of the forest land within its borders) failing to "clean" its forest floor. Trump's Justice Department sued the state a week earlier, arguing that California exceeded its authority when it launched a cap-and-trade agreement with Quebec to lower fossil fuel emissions.
At the same time, Trump's Environmental Protection Agency has threatened to use its authority under the Clean Air Act to withdraw federal highway funds from the state over air pollution, and to initiate enforcement action for water pollution violations. In a flourish, the EPA blamed the latter on human waste and needles from homeless people in San Francisco.
Those warnings, issued directly from the desk of EPA Administrator Andrew Wheeler, were at odds with the agency's hands-off approach under Trump. At a time when EPA is weakening environmental standards on both air and water pollution and enforcement activity is at its lowest level in a decade, the agency was invoking one of the most severe penalties available, for a state that has invested billions in improved air quality and has among the best compliance records in the nation on water pollution.
California's governor has called the moves "pure retaliation," while former federal officials say that the unusual nature of the enforcement actions, and the management of the cases by top Trump appointees in Washington, reeks of political score-settling.
Together, the Trump administration's actions try to deliver the message that California is a laggard in addressing visible air pollution and sewage, and that the state's aggressive focus on global warming is a misguided distraction at best.
But the Trump administration's California narrative falls apart with a cursory review of the science and the state's record as a national leader in environmental protection. And it remains to be seen whether it has the legal arguments to persuade the courts, which will have the final say.
How Did We Get Here?
This is not the first time that politicians have sought to caricature California as a victim of its own progressive policies.
Former Vice President Dick Cheney famously used California's electricity crisis of 2001 as a pretext for expanded U.S. oil and natural gas production. Only later did it become clear that California's power shortages were created, not by conservation-oriented policies, but by profit-hungry energy traders like Enron who took advantage of the state's experiment with deregulation.
The same administration also sought to block California's effort to regulate greenhouse gas emissions from passenger vehicles, but the auto industry's collapse in 2008 forced it into the arms of the federal government for help. President Barack Obama's administration brokered an historic $80.7 billion bailout of the carmakers, which included an agreement to put California's standards into effect nationwide. It forced a gradual fleet-wide improvement in fuel economy that by 2025 would have cut 6 billion tons of carbon dioxide, the biggest step any nation has taken to reduce greenhouse gases.
Soon after Trump was elected, the carmakers sought to partially undo that deal—they still wanted a nationwide standard, but more flexibility. The Trump administration, however, sought a head-on battle with California that the automakers had long said they hoped to avoid.
Launching such a war runs counter to the Trump administration's stated goal of giving more environmental authority to the states. Just last month, for example, the Trump EPA delegated increased authority to North Dakota for oversight of oil and gas sites, and waste facilities, and power plants.
But EPA Administrator Wheeler has portrayed California as a different case—a rogue state that needed to be reined in.
"We embrace federalism and the role of the states, but federalism does not mean that one state can dictate standards for the nation," Wheeler said in a speech to the National Automobile Dealers Association just before announcing that California would be stripped of its greenhouse gas emissions authority on vehicles.
The following week, Wheeler took two surprising steps. He threatened to withhold California's federal highway funds for "failing to carry out its most basic tasks" under the Clean Air Act to control air pollution in its cities. Wheeler then fired off a letter to California Gov. Gavin Newsom, threatening enforcement action over what he said was his state's "failure to protect Californians from degraded water" due to human waste from homeless people in its cities. The EPA followed up with a notice of violation to the San Francisco Public Utilities Commission.
California's Environmental Record
Although the allegations by the Trump administration each contain a grain of truth, they paint a misleading picture of California's record on clean air and water.
California does indeed have the largest backlog of Clean Air Act implementation plans, but that is in part because the state has 35 regional authorities responsible for pollution regulation. In many of the backlogged cases, California is awaiting EPA action on proposed implementation plans, state authorities pointed out in a letter to Wheeler.
As the nation's most populous state and the largest by far in the size of its economy (by GDP it is larger than all but four nations), California indeed has more metropolitan areas than any other state that fail to meet the national air quality standard for smog. But California, so prone to poor air quality in the valleys between its coastlines and mountain ranges, has been ahead of the nation in setting standards to control air pollution, greatly reducing it over the past 50 years.
"California historically has had the worst air quality in the nation," said Paul Billings, senior vice president for advocacy for the American Lung Association. "Also, since the late 1950s, no state has done more to reduce air pollution than the state of California. And they have done it as their population and economy have grown."
Ironically, the greenhouse gas standards that Trump is seeking to overturn would have the benefit of further improving urban air quality. The rules are designed to drive greater adoption of electric cars, which would further reduce the fossil fuel pollutants that are precursors to smog.
The Trump administration's attacks belie its stated concern on clean air, argues Billings. "It seems to be politically motivated and about scoring points in some game and not about trying to improve the air quality in California," he said.
The threat to withhold federal highway funds from California was a shock for those familiar with EPA history, since that is a rarely imposed nuclear option in Clean Air Act enforcement.
"Sanctions are appropriate when a state is basically refusing to do what the law requires," said Janet McCabe, who served as acting assistant administrator in charge of the air pollution program at EPA during the Obama administration. "Almost all the time, states are working very hard to implement their responsibilities under the Clean Air Act. It's hard, and it takes a long time. As long as a state is working in good faith, I would say that is not a sanction situation."
Similarly, the EPA's allegations and threatened action against California on water pollution run counter to the administration's hands-off approach. EPA enforcement actions, inspections and penalties all are at their lowest levels in a decade under the Trump administration. Moreover, 45 states have a higher percentage of water systems or dischargers in significant violation of federal water standards than California, according to an analysis of EPA's own data by Cynthia Giles, a guest fellow at Harvard Law School who headed the EPA enforcement office during the Obama administration.
"You target California despite the fact that California has one of the best compliance records in the country," Giles said in a letter to Wheeler.
Political Appointees Spearheading Attacks
The EPA notice of violation to San Francisco came only days after the city's water board adopted a more stringent water treatment permit, aimed at eliminating discharges of partially treated wastewater to the ocean. The EPA and California authorities had been working together on an enforcement action on San Francisco's sewage system, but the EPA dropped the joint effort.
"This development is confounding, and contrary to [a] long history of productive state-federal cooperative enforcement," California Environmental Protection Secretary Jared Blumenfeld wrote in an Oct. 25 letter to Wheeler. Wheeler had reportedly bypassed the EPA's West Coast team to handle the actions against California from headquarters in Washington, D.C.
Likewise, the Justice Department action seeking to dismantle California's cap-and-trade agreement with Quebec is being advanced by an unusually limited team of lawyers, pointed out Sean Hecht, a former deputy attorney general for California who is co-executive director of the Emmett Institute on Climate Change and the Environment at the University of California, Los Angeles, Law School.
That case is being led by Trump's appointee to Justice's Environment and Natural Resources Division, Jeffrey Bossert Clark, a former lawyer for oil giant BP who has characterized Obama-era greenhouse gas regulation as "reminiscent of kind of a Leninistic program." Only members of Clark's own team are listed on the case. It doesn't list members of the civil division of the Justice Department, who would ordinarily handle foreign treaty or compact cases, nor personnel from the local U.S. Attorney's office.
"The idea the case 'isn't political' seems fanciful," Hecht wrote on UCLA and Berkeley's Legal Planet blog.
Newsom, a Democrat, has described the Trump administration's moves as "pure retaliation" against a state that has sued the Trump administration 60 times.
Nearly 600 former EPA officials also signed a letter to the House Committees on Oversight and Government Reform and Energy and Commerce calling for a probe into whether the agency's recent actions were improper uses of its authority to punish a state for opposing Trump's political agenda.
California Sens. Dianne Feinstein and Kamala Harris have asked the EPA inspector general for an investigation into "whether the White House pressured the agency to abuse its law enforcement authority to single out California and the city of San Francisco." San Francisco is home to House Speaker Nancy Pelosi, the Democrat who is now presiding over Trump's impeachment inquiry.
Turning to the Courts
It will likely be the courts that decide the outcome of the Trump administration's battles with California. And on the state's greenhouse gas emissions standards for autos, the legal hurdle that the Trump administration faces is high.
When Congress originally passed the Clean Air Act in the 1970s, lawmakers gave California authority to set its own standards, seeking not to disrupt the steps the state already had taken to address pollution under then-Gov. Ronald Reagan. All the state needs to do to obtain its "waiver" to act is show a compelling need, and that its action is not arbitrary or capricious and is at least as protective as the federal standard. Until Trump, no administration had ever revoked a California waiver that already has been granted.
"To revoke a waiver that is already issued is a huge leap legally, and is sort of ironic in the light of other instances where the administration is taking the position of deferring to the states, and giving them more responsibility and autonomy," said McCabe.
The Trump administration plans to make the legal argument that the greenhouse gas emissions standards are, in effect, fuel economy standards—and that Congress prohibited states from setting their own fuel economy standards when it established a national auto efficiency program in 1975.
Two federal courts—in Vermont and in California—rejected that legal reasoning during the George W. Bush administration.
Trump's rollback of greenhouse gas standards could work to the automakers' short-term benefit, since their less fuel-efficient SUVs and pickup trucks are their most profitable vehicles.
In the long-run, though, automakers risk getting left behind in the competition to meet growing global demand for electric vehicles, which explains why Ford, Honda, BMW and Volkswagen struck a deal with California in July to voluntarily implement annual fuel economy improvements across their fleets even if the Trump administration weakened standards. The deal reportedly enraged Trump, and his administration has threatened them with antitrust action over the agreement.
When asked how it answers the charges that it is engaging in a political vendetta against California, the Trump EPA returned to its message on the environmental shortcomings of the state that is fighting the administration to keep environmental protections in place.
"Highlighting that California has very serious environmental failures both with air and water quality is not a political issue, and unlike previous Administrations, under this Administration the EPA is acting to protect public health and the environment for all Americans," said Corry Schiermeyer, EPA's associate administrator for public affairs in an email.
From a purely political standpoint, Trump has little to lose in warring with California. It resonates with the president's base, if the response from his Twitter followers is any indication. "California is such a mess @potus!," tweeted @carrieksada. "Fires everywhere. And they're worried about exhaust fumes."
Trump lost the state in 2016 by 30 percentage points, but he won the election without the nation's largest pot of electoral votes.
Of course, the state was not always so politically lopsided. California was the home of two Republican presidents elected in the past 50 years. But as Reagan's daughter Patti Davis wrote of her state last week: with wildfires advancing on her father's presidential library in Simi Valley, that California is gone.
Top photo: House Speaker Nancy Pelosi, a California Democrat, meets with President Donald Trump at the White House. Credit: Brendan Smialowski/AFP/Getty Images


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David Michael BlanchardEmployment and Civil Rights Lawyer for Michigan's Workers
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Focus: Helping people get back to work, stay at work, fighting wage theft and preventing retaliation and wrongful termination. Blowing the whistle on Medicare fraud and other taxpayer fraud. Legal advice; counseling; trial lawyer. David Blanchard's job is to take the case to court and fight for the civil rights of human people. David Blanchard is a respected trial lawyer with a reputation for fighting injustices regularly heaped upon honest hard-working individuals and small business owners. He is recognized for his aggressive, goal-oriented advocacy and advice. David Blanchard received his B.A. in political science and a J.D. cum laude from the University of Michigan Law School. David Blanchard primarily practices in the areas of civil rights, commercial and employment litigation, and felony criminal defense. Blanchard handles all employment related matters, from harassment, discrimination or retaliation lawsuits to medical leave, benefits issues and severance negotiations. He appears in both state and federal court, including the Michigan Court of Appeals and the Federal Court of Appeals for the Sixth Circuit. David Blanchard is a past president of the Michigan Employment Lawyers Association and of the Washtenaw Association for Justice. He currently serves on the executive board of the Michigan Association for Justice. He has presented as faculty to the State Bar of Michigan Labor and Employment Section and to the National Employment Lawyer Association. David Blanchard's specialties include: Qui tam/False Claims Act, Whistle-blowers, Overtime pay, Sales Commissions, Professional Compensation, Employment Agreements, Non-Compete/Trade Secret Disputes, Retaliation, Discrimination, General Counsel and Advice.
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Apr 30
Posted by Wonder Russell
In February, dynamic filmmaking duo Kris & Lindy Boustedt emailed me about a new short film they'd written, Ten Years Later. They wrote it with Lisa Coronado and I in mind to play sisters who meet again after ten years – ten years my character has spent rotting in prison.
Lisa and I were honored (and not a little intimidated!) and started working on the relationship between the two women. The journey took me to L.A. and even to Dallas where I continued to delve deeper into "Alice," building her rage and thirst for revenge.
We shot the film over the second weekend in April, and it was an incredible experience. I've never been asked to play such a dark character, and the challenge was very rewarding. Kris and Lindy's faith in me spurred me to break through my own resistances to the material – I wanted to make them proud.
TEN YEARS LATER – Teaser from First Sight Productions on Vimeo.
Ten Years Later is an origin story for Alice, who shows up in their feature film, ASHLAND, which we hope to shoot later this year. In Ashland, Alice is remorseless and vicious; Ten Years Later shows you how she became that way.
I could not have brought everything I am to this project without Kris and Lindy's unswerving belief that I could handle this character, my coach Steven Anderson's unflinching homework assignments that gave me nightmares, and my lovely friend Lisa's unwavering commitment to giving her all. I haven't worked with a more competent and talented set of professionals. Thank you.
Ten Years Later will be released later this Summer.
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Tags: AGYST, Arts, ashland, filmmaking, first sight productions, kris boustedt, lindy boustedt, lisa coronado, Movies, short film, teaser trailer, ten years later, Vimeo
Nov 1
ASHLAND is the third feature film by filmmaker duo Kris & Lindy Boustedt (This Is Ours, The Summer Home, Perfect 10). I'm honored to be part of it.
As things progress, I'll update you on the role I have in the film. Check out the whole team and our respective projects. We are going to blow the roof off 2013.
Tags: acting, ashland, directing, feature film, filmmaking, indie film, indiefilm, kris and lindy boustedt, mood reel, teaser
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