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Cell Reports, Volume 2, Issue 6, 1571-1578, 29 November 2012
In Vivo Differentiation Potential of Epiblast Stem Cells Revealed by Chimeric Embryo Formation
Yali Huang1, Rodrigo Osorno1, Anestis Tsakiridis1 and Valerie Wilson1, ,
Corresponding authorGraphical Abstract
► Epiblast stem cells (EpiSCs) form chimeras when injected into postimplantation epiblast ► Embryonic stem cells do not form postimplantation chimeras ► EpiSCs do not integrate if they are injected after gastrulation
- Chimera formation after blastocyst injection or morula aggregation is the principal functional assay of the developmental potential of mouse embryonic stem cells (ESCs). This property, which demonstrates functional equivalence between ESCs and the preimplantation epiblast, is not shared by epiblast stem cell (EpiSC) lines. Here, we show that EpiSCs derived either from postimplantation embryos or from ESCs in vitro readily generate chimeras when grafted to postimplantation embryos in whole embryo culture. EpiSC derivatives integrate and differentiate to derivatives of all three embryonic germ layers and primordial germ cells. In contrast, grafted ESCs seldom proliferate in postimplantation embryos, and fail to acquire the identity of their host-derived neighbors. EpiSCs do not incorporate efficiently into embryonic day 8.5 embryos, a stage by which pluripotency has been lost. Thus, chimera formation by EpiSCs requires a permissive environment, the postimplantation epiblast, and demonstrates functional equivalence between this cell type and EpiSCs.
The preimplantation epiblast generates all embryonic cell types (reviewed in Gardner, 1998). When injected into blastocysts, embryonic stem cells (ESCs) derived from preimplantation epiblast cells (Brook and Gardner, 1997) integrate into the preimplantation embryo, generating chimeras with high efficiency. ESCs can contribute to all cell types in the body, including germ cells (Bradley et al., 1984), demonstrating their ability to undergo normal in vivo cell differentiation.
Clonal analysis shows that during gastrulation, single postimplantation epiblast cells can contribute to all three germ layers up to early gastrulation (Lawson et al., 1991; Tzouanacou et al., 2009). Together with the ability of pre- and postimplantation epiblast cells to form teratocarcinomas (tumors containing derivatives of all three germ layers and embryonal carcinoma cells) this shows that the epiblast before and after implantation can be considered pluripotent (Stevens, 1970). Epiblast stem cells (EpiSCs) are cell lines derived from the postimplantation epiblast (Brons et al., 2007; Tesar et al., 2007) or in vitro from ESCs (Guo et al., 2009). Like ESCs, EpiSCs express the core pluripotency genes Oct4, Nanog, and Sox2, and are able to form teratocarcinomas and differentiate into multiple lineages in vitro.
Despite these functional similarities, ESCs and EpiSCs differ in several distinct respects (for reviews, see (Chenoweth et al., 2010; Nichols and Smith, 2009, Nichols and Smith, 2011). EpiSCs and ESCs have distinct signaling requirements for self-renewal. Female EpiSCs exhibit X chromosome inactivation, whereas ESCs do not. The gene expression profiles and colony morphologies of the two cell types are distinct. Crucially, unless EpiSCs are selected for rare subpopulations that resemble very early postimplantation epiblast (Han et al., 2010) or are genetically engineered to express a high level of E-cadherin (Ohtsuka et al., 2012), they are unable to contribute to chimeras upon blastocyst injection or morula aggregation. Mouse EpiSCs are of particular interest because human ESCs share many of their distinctive properties (reviewed in Chenoweth et al., 2010). However, the lack of an in vivo assay for the potency of EpiSCs leaves the issue of whether these cells represent a true in vitro counterpart of an in vivo cell type in doubt. Alternatively, the preimplantation embryo environment may be incompatible with EpiSC survival and integration. The latter possibility implies that EpiSCs should integrate into the postimplantation embryo.
To address this issue, we grafted EpiSCs to postimplantation embryos that were then cultured in vitro. Mouse embryos undergo normal development in culture for periods of 24–48 hr (Copp et al., 1990), permitting analysis of cell integration in the epiblast. We find that EpiSCs, but not ESCs, efficiently form chimeras in gastrulation-stage embryos, colonizing somatic and germ cell lineages. The inability of EpiSCs to form blastocyst-injection chimeras is therefore explained by their incompatibility with the environment of the preimplantation epiblast, not an inability to differentiate in the context of the embryo.
Results and Discussion
EpiSCs Incorporate Efficiently into Postimplantation Embryos
Initial experiments to test the assimilation of EpiSCs into gastrulation-stage embryos using ultrasound-guided injection of EpiSC cell suspensions to the amniotic cavity in utero did not result in any chimeras at birth (data not shown). Similar injections of single-cell suspensions in ex-vivo-cultured embryos formed free-floating unincorporated clumps in the amniotic cavity (data not shown). In contrast, EpiSC clusters that were manually dislodged from the culture dish and grafted in the epiblast (Figures 1A–1C) survived, proliferated, and dispersed from the original graft site (Figures 1D, 1E, and S1A–S1D). This implies that EpiSC integration in embryos depends upon cell-cell contacts, and that single-cell suspensions are unable to form such contacts to integrate into the host.
To assess EpiSC incorporation in defined regions of the postimplantation epiblast, we grafted EpiSCs constitutively expressing green fluorescent protein (GFP) in four sites of gastrulation-stage (early-streak to late-streak) embryos: mid-anterior (MA), distal (D), mid-posterior (MP), and proximal-posterior (PP; Figures 1A–1C). Embryos were cultured in vitro and analyzed after 24–48 hr (Figures 1D and 1E). The distribution of donor cells was assessed by fluorescence microscopy (Figures 1D–1F).
Two EpiSC lines that ubiquitously express GFP (r04-GFP, derived from E6.5 epiblast, and C2, derived in vitro from ESCs) were injected. Two ESC lines (AGFP7, which ubiquitously expresses GFP, and TßC44cre6, which expresses GFP under Nanog regulatory control) were injected for comparison. Both ESC lines efficiently form chimeras following blastocyst injection (Chambers et al., 2007; Gilchrist et al., 2003). To determine the number of cells that initially grafted into the embryos, the embryos were fixed immediately after grafting. An examination of confocal z-stacks (Figure 1C) showed that 12 ± 2.83 cells/embryo were grafted (range: 10–16 cells; nembryos = 4).
A total of 91 embryos received a graft of either EpiSCs or ESCs. Most of these (n = 81) developed normally and contained labeled cells after culture, verifying that the grafting technique is efficient. The remainder either did not contain labeled cells after culture (n = 4) or developed abnormally (n = 6). These embryos were excluded from the analysis. Seventy-three embryos received grafts of cells carrying ubiquitously expressed GFP; 55 of these embryos were analyzed after a 24 hr culture, and the remainder were analyzed after 48 hr (Figure S1E). To describe the distribution of donor cells in the host after culture, we scored wholemount embryos for the approximate number of donor cells that had dispersed from the graft site, and the spread of donor cells within the host (Figures 1D–1I and S1E). We compared EpiSC and ESC grafts, using the number of dispersed cells and their spread as a semiquantitative assessment of donor cell contribution. Following culture, an embryo was considered to be a bona fide chimera if the following criteria were met: (1) at least 12 donor cells had clearly dispersed from the graft site, indicating that dispersed cells had survived and/or proliferated in the host; and (2) dispersed donor cells were not restricted to one area and had spread over >1/4 of the embryo's anteroposterior length, indicating robust intermixing with the host cells.
The profiles of the contributions made by in-vitro- and embryo-derived EpiSCs to embryos were similar (Figure S1E), and therefore we combined those data (Figures 1G–1I). Both EpiSC lines incorporated efficiently into the D, MP, and PP regions (Figures 1H and 1I). In the D region, 17 of 18 embryos contained ≥12 dispersed donor cells. Donor cells spread over >1/4 of the embryo's length in 19 of 22 embryos (Figure 1H). Similarly, in the MP and PP regions, 21 of 23 embryos contained ≥12 dispersed donor cells, which spread over >1/4 of the embryo's length in all 21 embryos (Figure 1I). Hence, the overwhelming majority (82%–91%) of the embryos grafted with EpiSCs in D, MP, and PP regions formed bona fide chimeras (Figures 1H and 1I). Moreover, after 24 hr of culture, 20 chimeras (74% of those injected in D, MP, and PP regions) contained 24–96 dispersed cells, indicating cell proliferation in the host (Figures 1H, 1I, and S1E). After 48 hr of culture, more than half of the embryos injected into the D, MP, and PP regions contained >96 dispersed donor cells, distributed throughout >1/2 of the embryo's length (Figures S1A–S1D, showing two embryos containing ∼300 graft-derived cells), indicating further proliferation and dispersal between 24 and 48 hr. Therefore, EpiSC readily proliferate and disperse from regions that are in the primitive streak (MP and PP regions) or are recruited there during culture (D region).
EpiSCs that were grafted in the MA region showed less cell dispersal. Just under half (47%) of the embryos contained ≥12 dispersed donor cells after culture. Donor cells extending for >1/4 of the embryo's length were seen in only 20% of the embryos (Figure 1G). Fate maps indicate that few, if any, cells in the MA region will be recruited to the primitive streak by the mid-streak to late-streak stage (Lawson et al., 1991). The primitive streak is a site of extensive cell rearrangement and may provide grafted cells in these regions with a greater opportunity for cell intercalation with the host than cells grafted at the MA site. Alternatively, the high levels of Nodal and fibroblast growth factor (FGF) signaling in distal and posterior regions of the embryo may favor EpiSC growth because these pathways are important for EpiSC maintenance.
To determine whether the dispersed donor cells differentiate to acquire the identity of their immediate host-derived neighbors, we sectioned cultured embryos and assessed them for cell integration. Double nuclear and actin filament staining revealed that the morphology of the EpiSC-derived donor cells was similar to that of their neighbors (Figure 2B). Moreover, immunohistochemistry showed that in all assays of somatic lineages (n = 27; Table 1), both in-vitro- and embryo-derived EpiSC descendants expressed markers characteristic of their host location, i.e., T (Brachyury) in the primitive streak and emergent mesoderm (Wilkinson et al., 1990; Figure 2C), Tbx6 in the paraxial presomitic mesoderm (Chapman et al., 1996; Figure 2D), AP-2α (Tcfap2a) in the surface ectoderm (Arkell and Beddington, 1997; Figure 2E), Sox2 in the neurectoderm (Avilion et al., 2003; Figure 2E), Foxa2 in the floor plate and endoderm (Sasaki and Hogan, 1993; Figures 2F and 2G), and Cdx2 in the allantois and posterior mesoderm (Beck et al., 1995; Figure 2H). Therefore, EpiSCs integrate efficiently into all three germ layers within 24–48 hr. Notably, T (Wilson et al., 1995), Foxa2 (Dufort et al., 1998), and Cdx2 (Chawengsaksophak et al., 2004) are required for correct differentiation at this stage, and thus failure to express these markers would preclude functional differentiation.
Table 1 |
Donor Cells | Stage of Recipients | Graft Site | Number of Sectioned Embryos | Number of Embryos with Incorporated GFP+ Cells | Number of Sectioned Embryos Containing Incorporated Cells in Tissue (Number of Embryos Confirmed/Number Tested by Immunohistochemistry) | |||||||
Surface Ectoderm (AP-2a) | Neural Ectoderm (Sox2, Foxa2) | Mesoderm (T, Cdx2, Nkx2.5, Tbx6) | Endoderm (Foxa2) | PS (T) | Allantois (Cdx2, Stella, TNAP) | |||||||
Embryo-derived EpiSC (r04-GFP) | M-LS | MA | 4a | 3 | 3 | 1 | ||||||
D | 9a | 9 | 1(1/1) | 8(4/4 Sox2, 2/2 Foxa2) | 8(2/2 T, 2/2 Cdx2) | 3 | 1(0/1 Stella) | |||||
MP | 4 | 4 | 1(1/1) | 1(1/1 Sox2) | 1(1/1 Tbx6) | 2(2/2) | 3(2/2 Cdx2, 0/1 Stella) | |||||
PP | 3 | 3 | 3 | 1 | 3(2/2) | 3(3/3 Cdx2) | ||||||
E-MS | PP | 4 | 4 | 2 | 4 | 4 | 3 | 2(1/2 Stella) | ||||
In vitro EpiSC (C2) | M-LS | D | 2b | 2 | 2 | 2 | 2 | |||||
MP | 4a | 4 | 4(1/1 Tbx6) | 2(2/2) | 4(1/1) | 4(1/2 TNAP, 0/1 Stella) | ||||||
ES | PP | 4 | 4 | 1(1/1 Foxa2) | 2(1/1 Cdx2, 2/2 Nkx2.5) | 4 | 2 | |||||
ESCs (AGFP7) | M-LS | D | 5a | 2 | 2c | 1(0/1) | ||||||
PP | 3 | 3 | 1+1c | 2(0/1d) | 2(0/1 Cdx2d) | |||||||
Brackets: number of sectioned embryos containing GFP cells that expressed the indicated marker/number of embryos tested. For each embryo, three or more sections were tested for immunoreactivity to any one antibody. Staging: ES, early streak; E-MS, early to mid streak; M-LS, mid to late streak.See also Figure S3. |
a One sectioned embryo contained a small unincorporated clump of cells (three or fewer sections). b Both embryos also contained small unincorporated clumps of cells. c GFP+ cells present in three or fewer sections. d GFP+ cells showed ectopic Sox2 and Oct4 expression, and only one cell correctly expressed Cdx2. |
To further demonstrate that EpiSCs form cell types that are functional at early stages, we injected them into the primitive streak of embryos in the early-streak stage, when cardiac progenitors are present in the primitive streak. After 48 hr in culture, at which point the heart is beating and required for normal development, we observed donor-derived cells in the heart of two chimeras (Figure 2I). These cells expressed the cardiomyocyte marker Nkx2.5 (Lints et al., 1993; Figure 2J; Table 1) at levels comparable to those observed in their neighbors, indicating differentiation of EpiSC toward cell types with an early functional role.
We also observed graft-derived alkaline phosphatase (TNAP)-positive cells with primordial germ cell (PGC) morphology at the junction between the allantois and posterior primitive streak (Figure 2K), conventionally designated as PGCs (Ginsburg et al., 1990). Furthermore, graft-derived cells in this location express the PGC-specific marker Stella (Figure 2L; Saitou et al., 2002). Specific antibody immunofluorescence of Stella was verified by the detection of Stella+, Nanog:GFP+ ESCs (Figure S2H) and Stella+, Nanog:GFP+ cells at the base of the allantois in embryonic day 8.5 (E8.5) transgenic embryos (Figure S2I). Thus, EpiSCs grafted in early- to mid-streak-stage embryos are not restricted to somatic lineages, consistent with earlier reports that EpiSC can undergo PGC differentiation in vitro (Hayashi et al., 2011). Nevertheless, not all graft-derived cells at the base of the allantois expressed PGC-specific markers (Figure S2J; Table 1), and only two of the seven embryos tested contained any putative PGCs, consistent with a short time window of PGC specification that is complete by early to midstreak stage, and the low abundance of PGCs in the embryo (Lawson and Hage, 1994; Tam and Zhou, 1996).
Sox2, which marks not only neural progenitors but also EpiSCs (Figures S2A and S2B), was downregulated in the vast majority of EpiSC-derived nonneural cells in the embryo (Figures 2E and S2B; five of five assays, comprising three or more sections per assay; Table 1). This indicates that dispersed donor cells lose EpiSC characteristics as they incorporate into the host. Subpopulations of EpiSCs in vitro also express T and Foxa2 (Figures S2C and S2D), but we did not observe ectopic expression of these markers in chimeras (12 of 12 assays; Figure 2C; Table 1). A small minority of EpiSCs in vitro (<1%) expressed Cdx2, AP-2α, and Nkx2.5 (Figures S2E–S2G), yet after injection into the embryo, most EpiSC correctly expressed these markers (11 of 11 assays; Figures 2E, 2H, and 2J; Table 1). AP levels in EpiSCs are uniformly low (Brons et al., 2007; Greber et al., 2010) and we did not detect Stella-positive EpiSCs in vitro (data not shown). In each case, EpiSC-derived cells expressed these markers in appropriate locations in host embryos (Figures 2K and 2L; Table 1). Therefore, given that only 10–16 cells were injected into the embryo, and most grafts produced cells that expressed markers appropriate for the site of integration, it is unlikely that grafted EpiSCs expressing these differentiation markers would have selectively integrated and/or expanded in the host embryos.
Because morphological integration correlated with functional integration (as verified by marker expression) in all embryos analyzed, we used morphological integration as a criterion to score the remaining sectioned embryos (Table 1). In all cases, the cellular distribution of dispersed EpiSCs in their host is consistent with the fate map of the epiblast (Table 1; Figure S3). Greater endoderm contribution is observed in embryos that received an EpiSC graft at the early-streak stage, consistent with the early segregation of endoderm (Tzouanacou et al., 2009). Thus, EpiSC chimera formation is efficient and follows the normal fates of cells at the graft site. Moreover, the similar chimera-forming capacity of embryo- and in-vitro-derived EpiSCs highlights that cell lines produced by these distinct routes share common functional properties. Because mouse EpiSCs also share features with human ESCs (Tesar et al., 2007), these data suggest that human ESCs also represent a true in vitro counterpart of an in vivo cell type.
Occasionally, clumps of cells were observed in sections of the embryo at the graft site (Table 1). Six out of 34 sectioned embryos contained such clumps, two of which were very small, extending over one to two sections. These clumps may represent a subpopulation of EpiSCs that are incapable of integrating into the host embryonic environment, or EpiSCs may require contact with neighboring host cells to ensure their appropriate differentiation. In support of the latter possibility, when we performed additional grafts containing larger numbers of EpiSCs (>12 cells per embryo, n = 4), this did not result in more extensive chimeras after 24 hr (in all four embryos, 24–48 dispersed cells extended between 1/4 and 1/2 of the embryo length). Instead, all embryos additionally contained partially integrated clumps. Importantly, Sox2 was ectopically expressed at low levels in the center of the clump but was absent in nearby dispersed cells (Figure S4A). This strongly suggests that the primary influence on EpiSC differentiation is dispersal from the grafted cell cluster, rather than variations in the abilities of subpopulations to integrate and differentiate. Additionally, unincorporated clumps were previously observed in orthotopic grafts of E7.5 epiblast (Beddington, 1981), indicating that even tissue from an origin equivalent to the host site can fail to incorporate, presumably because there are too many cells to be assimilated by the surrounding host cells.
ESCs Do Not Incorporate into E7.5 Embryos
The efficient integration of EpiSCs into embryos prompted us to compare them with AGFP7 ESCs in terms of their ability to form chimeras. The vast majority of ESC-derived cells typically remained as nonintegrated clumps either in the amniotic cavity (data not shown) or at the graft site (Figures 1F, left, and S4B). Only one of 13 embryos grafted with AGFP7 ESCs contained >12 dispersed donor cells extending >1/4 of the embryo length (Figures 1F, right, and 1I), suggesting that was a bona fide chimera. However, immunohistochemistry on sections of this embryo showed that nearly all ES-derived cells ectopically expressed the ESC markers Sox2 and Oct4 (Figures 2M and 2N). Moreover, differentiation markers were not upregulated appropriately: T and Foxa2 were not expressed, and only one cell expressing Cdx2 was detected (Figures 2O and 2P; Table 1). To further test whether ES-cell-specific gene expression persists in ESCs after grafting, we grafted TßC44cre6 ESCs, which contain a GFP transgene integrated at the Nanog locus (Nanog:GFP) in the MA, D, and MP regions of eight E7.5 embryos. If these donor cells incorporate into the host after culture, they should downregulate GFP in somatic tissue, since Nanog expression is restricted to the PGCs by around E8.0 (Hart et al., 2004; Yamaguchi et al., 2005). However, like the AGFP ESC grafts, Nanog:GFP ESCs generally formed small self-adherent clumps (in seven of eight embryos) after culture. One embryo contained apparently dispersed cells at the base of the allantois; because PGCs, like ESCs, express Nanog:GFP (Osorno et al., 2012), this embryo was uninformative. In two of eight embryos, dispersed GFP+ cells were also detected distant from the PGC location at the base of the allantois, indicating their failure to downregulate pluripotency markers (Figures S4C and S4D). Thus, unlike EpiSCs, donor ESCs incorporate very inefficiently into the host, and even when they do, they do not differentiate correctly.
EpiSCs Do Not Incorporate Well into E8.5 Embryos
The epiblast loses pluripotency at the beginning of somitogenesis (Osorno et al., 2012). However, axial progenitors in the node-streak border (NSB) contribute to two germ layer derivatives, neurectoderm and mesoderm, during organogenesis (Tzouanacou et al., 2009). The NSB can change the fates of E8.5 streak regions on heterotopic grafting (Cambray and Wilson, 2007), suggesting a strong inductive influence on grafted cells. To investigate whether EpiSCs can colonize older embryos, we grafted embryo-derived EpiSCs into the NSB of two to five somite-stage (E8.5) embryos (Figure 3A) and analyzed embryos cultured for 24–48 hr. Although the donor cells proliferated extensively in recipients (Figures 3B and S4E), the majority remained as discrete clumps in the medial somite area and ectopically expressed Sox2 (Figures 3C and S4F; n = 5). Despite expression of notochord marker Foxa2, the cells remained distinct from the endogenous notochord and did not express T (Figures 3D and 3E). Few dispersed cells were observed (Figures 3G and 3H). Thus, EpiSC integration is severely compromised in embryos later than E7.5, suggesting that the period of their compatibility with the host embryo corresponds to the period during which the postimplantation epiblast is pluripotent (Osorno et al., 2012)
In summary, both embryo- and in-vitro-derived EpiSCs are able to form chimeras and differentiate appropriately in postimplantation embryos, and thus can be considered functionally equivalent to postimplantation epiblast cells in vivo prior to somitogenesis. Therefore, although future technological advances are required to demonstrate the formation of liveborn chimeras, embryo grafting and culture provide an accessible means to test the differentiation capacity of both wild-type and mutant EpiSC lines in the context of normal early development.
Experimental Procedures
Mouse Husbandry and Staging Embryos
Wild-type, outbred MF1 recipient mice and Nanog:GFP [129-Nanogtm1(GFP-IRES-Puro)] females derived from TNG-targeted ESCs (Chambers et al., 2007) were maintained on a 12-hr-light/12-hr-dark cycle. Timed matings were set up overnight. Noon on the day of finding a vaginal plug was designated E0.5. At E7.0–E7.5, early-streak, mid-streak, and late-streak-stage embryos (Downs and Davies, 1993) were selected for grafting. At E8.0–E8.5, two to five somite-stage embryos were used for grafting.
Cell Lines
r04-GFP EpiSCs were derived as previously described (Tesar et al., 2007) from an E6.5 embryo of mixed genetic background. Tps/tb;HPRTTREdsRed2-C2 (C2) EpiSCs (which carry a doxycycline-inducible dsRed transgene not used in these experiments) were differentiated as previously described (Guo et al., 2009) from a feeder-free ESC line. Both EpiSC cell lines carry a random integration of a CAG-driven GFP, conferring ubiquitous GFP expression. Each line was passaged at least five times after genetic manipulation and before injection into the embryo. AGFP7 ESCs (Gilchrist et al., 2003) carry a randomly integrated CAG-GFP transgene and TßC44Cre6 (Chambers et al., 2007), and contain a GFP reporter integrated at the Nanog locus. TNG ESCs (Chambers et al., 2007) were used as a positive control for Stella immunocytochemistry.
Grafting Cells into Embryos and Embryo Culture In Vitro
Grafting was performed by hand in a dissecting stereomicroscope using a hand-pulled micropipette. ESCs or EpiSCs were scraped from culture dishes using a 20–200 μl pipette tip. The resulting cell clumps were placed close to the embryos, and a small clump was drawn into the micropipette by gentle suction. The embryo was held loosely in place with forceps while the micropipette was inserted into the region of interest to create an opening. Cells were then gently expelled as the micropipette was drawn out of the embryo, leaving a short string of cells lodged in the epiblast (Figure 1A). Embryos were cultured in 50% rat serum as previously described (Copp et al., 1990) except that E7.5 embryos were cultured in static four-well dishes (Nunclon) in an incubator at 5% CO2 in air.
Embryo Sectioning, Staining, and Immunofluorescence
After culture, embryos were fixed with 4% paraformaldehyde in PBS at 4°C for 2 hr (E7.5 + 24–48 hr culture embryos) or overnight (E8.5 + 24–48 hr culture embryos), embedded in 7% gelatin blocks, and snap-frozen in liquid nitrogen. The blocks were stored at −80°C and sectioned at 7 μm in a cryostat (Leica). AP staining was performed with a leukocyte AP kit (Sigma-Aldrich), and actin filament staining with phalloidin-Alexa 647 (Invitrogen) was performed according to the manufacturer's instructions. Immunohistochemistry was performed as described in the Extended Experimental Procedures. Immunocytochemistry was performed as described previously (Osorno et al., 2012).
Images of whole embryos were captured with the use of Volocity software (Perkin Elmer) on a digital camera (Qimaging) attached to a Zeiss Stemi SV11 or a Nikon NZ100 dissecting microscope. To count grafted GFP-labeled cells, whole embryos were imaged in a Leica DM IRE2 inverted confocal microscope (Leica Microsystems). Images were processed with Volocity software. A compound photomicroscope with fluorescence optics (Olympus BX61) was used to capture images of the sections. Images were processed with Adobe Photoshop software (Adobe Systems).
Immunohistochemistry and list of antibodies
Sections were blocked with 3% serum (Sigma) and 1% BSA (Sigma) in PBST (PBS/0.5%v/v Triton X-100 (Sigma)) for 1h at room temperature before incubating with primary antibodies diluted in blocking buffer at 4°C for 2 days in a humidified box. After three washes in PBST, sections were incubated with diluted fluorescence conjugated secondary antibodies at room temperature for 2h, followed by two washes in PBS and a final wash in PBS with 4',6-diamidino-2-phenylindole (DAPI) nuclear counterstain (Invitrogen). Sections were mounted using mounting media (Vector Labs). Primary antibodies, suppliers, catalog number and working concentration were as follows: AP-2α (DSHB; 5E4; 20 μg/ml); T(brachyury) (Santa Cruz; sc-17743; 1 μg/ml); Cdx2 (BioGenEx; MU392A-UC; 1:50); Foxa2 (Santa Cruz; sc-6554; 1 μg/ml); Sox2 (Santa Cruz; sc-17320; 1 μg/ml or Chemicon; AB5603; 5 μg/ml); Nkx2.5 (Abcam; ab35842; 20 μg/ml); Stella (1:2000;(Nakamura et al., 2007).
We thank Ian Chambers, Tilo Kunath, Sally Lowell, and Frederick Wong for critical readings of the manuscript; Carol Manson for help with animal maintenance; and Ronald Wilkie for technical assistance. This work was supported by the UK Medical Research Council (G080297), the Association for International Cancer Research (08-0493), and a China Scholarship to Y.H.
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Osorno et al., 2012 (2012). The developmental dismantling of pluripotency is reversed by ectopic Oct4 expression. Development 139, 2288–2298. CrossRef | PubMed
Saitou et al., 2002 (2002). A molecular programme for the specification of germ cell fate in mice. Nature 418, 293–300. CrossRef | PubMed
Sasaki and Hogan, 1993 (1993). Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development 118, 47–59. PubMed
Stevens, 1970 (1970). The development of transplantable teratocarcinomas from intratesticular grafts of pre- and postimplantation mouse embryos. Dev. Biol. 21, 364–382. CrossRef | PubMed
Tam and Zhou, 1996 (1996). The allocation of epiblast cells to ectodermal and germ-line lineages is influenced by the position of the cells in the gastrulating mouse embryo. Dev. Biol. 178, 124–132. CrossRef | PubMed
Tesar et al., 2007 (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199. CrossRef | PubMed
Tzouanacou et al., 2009 (2009). Redefining the progression of lineage segregations during mammalian embryogenesis by clonal analysis. Dev. Cell 17, 365–376. Abstract | Full Text | PDF (1677 kb) | CrossRef | PubMed
Wilkinson et al., 1990 (1990). Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 343, 657–659. CrossRef | PubMed
Wilson et al., 1995 (1995). The T gene is necessary for normal mesodermal morphogenetic cell movements during gastrulation. Development 121, 877–886. PubMed
Yamaguchi et al., 2005 (2005). Nanog expression in mouse germ cell development. Gene Expr. Patterns 5, 639–646. CrossRef | PubMed
Supplemental References
Nakamura et al., 2007 Nakamura, T., Arai, Y., Umehara, H., Masuhara, M., Kimura, T., Taniguchi, H., Sekimoto, T., Ikawa, M., Yoneda, Y., Okabe, M., et al. (2007). PGC7/Stella protects against DNA demethylation in early embryogenesis. Nat. Cell Biol. 9, 64–71..
Tam and Behringer, 1997 Tam, P.P., and Behringer, R.R. (1997). Mouse gastrulation: the formation of a mammalian body plan. Mech. Dev. 68, 3–25..
Publication Information
Received: January 9, 2012
Revised: July 10, 2012
Accepted: October 30, 2012
Published: November 29, 2012


text:
Farmer suicide: Data from the Queensland Suicide Register (QSR) and Psychological Autopsy case studies
File | Size | Format | |
66794_1.pdf | 14Kb | Adobe PDF | View |
Title | Farmer suicide: Data from the Queensland Suicide Register (QSR) and Psychological Autopsy case studies |
Author | Milner, Allison Joy; De Leo, Diego; Hawgood, Jacinta |
Publication Title | 4th Asia Pacific Regional Conference of the International Association for Suicide Prevention |
Year Published | 2010 |
Peer Reviewed | No |
Published | Yes |
Publisher URI | http://www.iasp.info/congresses.php |
Copyright Statement | Copyright remains with the authors 2010. The attached file is reproduced here in accordance with the copyright policy of the publisher. For information about this conference please refer to the conference's website or contact the authors. |
Publication Type | Conference Publications (Extract Paper) |
Please use this identifier to cite this record: http://hdl.handle.net/10072/37229
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subset: commoncrawl


text:
Definition of immaterialism in English:
Line breaks: im¦ma|teri¦al|ismPronunciation: /ɪməˈtɪərɪəlɪz(ə)m
- The belief that matter has no objective existence.More example sentences
- There is less agreement on Berkeley's argumentative approach to idealism and immaterialism and on the role of some of his specific arguments.
- In 1709 he issued An Essay towards a New Theory of Vision, followed in the next year by The Principles of Human Knowledge, the main exposition of his immaterialism.
- What had touched the world's hearts was the ethereal immaterialism of their secluded world and something primal in the music they sang.
- More example sentences
- He was an immaterialist: matter does not exist.
- Philonous needs to convince him (as Berkeley needed to convince his readers in both books) that a commonsensical philosophy could be built on an immaterialist foundation, that no one but a skeptic or atheist would ever miss matter.
- Rather, it's that the good arguments don't even get a hearing since they offer an immaterialist account, and immaterialist accounts can't really be knowledge.


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This item may be Check for Availability This title in other editionsDesign in Nature: How the Constructal Law Governs Evolution in Biology, Physics, Technology, and Social Organizationby Adrian Bejan
Synopses & ReviewsPublisher Comments:In this groundbreaking book, Adrian Bejan takes the recurring patterns in nature—trees, tributaries, air passages, neural networks, and lightning bolts—and reveals how a single principle of physics, the Constructal Law, accounts for the evolution of these and all other designs in our world.
Everything—from biological life to inanimate systems—generates shape and structure and evolves in a sequence of ever-improving designs in order to facilitate flow. River basins, cardiovascular systems, and bolts of lightning are very efficient flow systems to move a current—of water, blood, or electricity. Likewise, the more complex architecture of animals evolve to cover greater distance per unit of useful energy, or increase their flow across the land. Such designs also appear in human organizations, like the hierarchical "flowcharts" or reporting structures in corporations and political bodies. All are governed by the same principle, known as the Constructal Law, and configure and reconfigure themselves over time to flow more efficiently. Written in an easy style that achieves clarity without sacrificing complexity, Design in Nature is a paradigm-shifting book that will fundamentally transform our understanding of the world around us. About the AuthorADRIAN BEJAN is Duke University's J. A. Jones Professor of Mechanical Engineering.
J. PEDER ZANE is an award-winning columnist who has worked for the New York Times and the Raleigh News. What Our Readers Are SayingBe the first to add a comment for a chance to win!Product Details
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Radio stations: We're still relevant in the Internet age
LAS VEGAS--Over-the-air radio broadcasters have a plan to stay relevant even as their listeners continue to migrate to the Web.
Radio Heard Here, a new initiative from the National Association of Broadcasters (NAB) and the HD Digital Radio Alliance is focused largely on trying to convince the public that radio remains relevant.
The plan calls for a public-relations campaign, including video ads on YouTube, and a method to connect players online. But there's little real meat. In reality, it's a response to those skeptical about the industry's chances to survive in the Internet era who have lately … Read more


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I downloaded free android mobile phone adobe with the pdf files its on my cell but wont let me download the files on sites?
Can you please describe what steps you are taking to view files? Generally, you have to download the PDF's from within the browser. Once downloaded, you can open the files and a dialog will appear asking you to choose which PDF viewer to use. Adobe Reader should be on that list if it's installed. Further, you can open Adobe Reader directly, go tot he Documents tab and browse your SD card to the Downloads directory to open the file.


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Sigma 105mm f/2.8 EX DG OS HSM Telephoto Macro Lens, Nikon Fit
Click on the image to zoomProduct information
Sigma's medium telephoto macro lens incorporates Optical Stabilisation allowing the use of shutter speeds approximately 4 stops slower than would otherwise be possible. SLD glass provides good control of aberration and the floating inner focus system ensures high rendering throughout the focusing range.
The HSM (Hyper Sonic Motor) provides quiet and high speed autofocus. It is also possible to use full-time manual focus, even if the lens is set to the AF position. This lens has a rounded 9 blade diaphragm which creates an attractive blur to the out of focus areas.
When taking photos with APS-C sized digital SLRs, the (supplied) dedicated hood adapter, which extends the length of the hood can be attached. This gives more protection from harmful light that could negatively affect image quality.
Includes: hood, hood adapter, padded case, front and rear caps.
- Brand
- Sigma
- Dimensions
- L126.4 x Dia.78mm
- Eligible for International Delivery
- Lens Mount
- Nikon F
- Model name / number
- Sigma 105mm - Nikon
- Weight Information Unpackaged weight
- 725g
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Minute Beef Stroganoff
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1 pound sirloin steak Butter 2/3 cup water 1 can sliced mushrooms, drained 1 envelope onion soup mix 1 cup sour cream 2 tablespoons flour
Cut meat in strips diagonally across the grain. Brown quickly in butter; add water and mushrooms. Stir in soup mix. Heat to boiling. Blend in sour cream and flour; cook until thickened slightly. Serve over hot cooked rice or noodles.
Sue Walsh Bettendorf
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Discover more great organic beauty and skin care lines. Your package contains 3+ free hand-selected samples with every order. Enjoy!
A valuable group of compounds in grape seed, OPC (Oligomeric Proanthocyanidins) can renew the collagen fibres - the elementary tissues of our body. They are also strong antioxidants and can regenerate the epidermis and the connective tissues. Our Rich Grape Whip Moisturizer contains selenium and vitamin E, and is enriched with germ oils. By having regenerating, toning, tightening and wrinkle eliminating effects, it is effective for everyday application on flabby, aging, oil and water deficient skin types. Skin Types / Skin Conditions: sunburn dry skin wrinkles cellulite stretch marks and pregnancy skin care chapped skin and dry areas mature skin (or as a night cream for premature skin) deep layer oil- and water deficiency flabby, aging skin Effects: regenerates tightens tones charges skin with vitamins compensates water- and oil deficiency increases turgor wrinkle eliminator, wrinkle smoothing effect strengthens the keratin layer helps collagen formation normalizes the functions of sebaceous glands
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Federal Lawsuit Against NMSU Football Coach Settled Before Trial
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Las Cruces, NM – The Federal Civil Discrimination Lawsuit Filed by the ACLU of New Mexico against New Mexico State University Head Football Coach Hal Mumme was settled yesterday. The lawsuit, filed on behalf of four former Muslim players, claims they were subjected to a hostile environment towards Islam. As KRWG's Evan Woodward reports, both sides are content with the outcome.


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68 Prince St., New York, NY, 10012
nr. Lafayette St.
By kmi7 on 3/25/2009
Unfortunately, I haven't eaten here yet, but I stopped in while walking in Soho last weekend because of a beverage recommendation. I had the iced Cardamom Coffee and I am a convert!! I absolutely loved it. If only they had a location close to my work...this would be my morning coffee spot.
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By kris10 on 9/3/2010
I stopped in for lunch on a steaming hot weekday. It was certainly a hopping place but I scored a seat and sandwich in no time! I had a refreshing glass of watermelon juice and the curried chicken salad on 7 grain. It was just what I needed to beat the heat and satisfy my stomach. I can't wait to go back and try their other items. I had been meaning to try it out for some time now and I'm so glad I finally made it in.
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Adam Platt picks 2013's top dining destinations,
including Blanca, Mission Chinese Food, and Perla.
The best that the city's restaurants have to offer:
bar food, dumplings, soft serve, tongue, and more.
We live in a city full of small cheap-eats miracles,
including pork buns, Asian hipster grub, and pizza.


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Outside Scholarships
Students are encouraged to find additional sources of funding through outside scholarships. Below is a list of Websites with more information.
FastWeb is the nation's largest source of local, national, and college-specific scholarships.
Specific Opportunities
The following outside scholarship opportunities are organized by the month in which applications are due. Be sure to plan ahead and ensure the organization receives all required documents by the deadline.
Apply for the CampusRN - Massachusetts Nursing Scholarship.
Visit CampusRN to apply for the $1,000 scholarship or to search for thousands of Nursing Jobs and Healthcare jobs and scholarships.
American Chemical Society Scholars Program
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Open to high school seniors, first-year college students, as well as college sophomores and juniors intending to or already majoring in biochemistry. Must meet specific GPA and ethnicity requirements. -
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Educational scholarships are awarded to Hispanic students on a competitive basis and range from $250 to $1000. Applications are only accepted within the 45-day period prior to the deadline. That is, as of January 1 for the February 15 deadline, and September 1 for the October 15 deadline. -
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Since we are a Datatel School - it's a very good opportunity for our qualified students. Several Regis students have received this scholarship through the years. -
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Zuberi Lateef Brown struck, killed by car while walking in Bowie
February 7, 2013 - 05:20 am
Maryland State Police early Thursday morning responded to an accident on Route 301 where a pedestrian was hit and killed.
The incident happened around 1am along the southbound lanes of Route 301 at Point Ridge Drive in Bowie, Maryland. The victim, now identified as Zuberi Lateef Brown, 39 of Bowie, was walking northbound in the southbound lanes when he was hit by an oncoming car headed south.
The driver remained on the scene and police are still investigating the accident.
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Quartet Flipchart Stand
66" Height - Aluminum - Silver - View Complete Details
Product Number: QRT55E
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Product Details
Quartet Flipchart Stand - Heavy-duty easel features adjustable, tubular 1" aluminum legs that twist-lock into the desired height from tabletop to floor-standing position. Locking cross-braces and antiskid feet ensure stability, and adjustable display holders slide into the proper position. Conveniently downsize for storage and transport. Attachable Quartet pad retainers (50T and 51T) are available for flip chart use (sold separately). Easel adjusts from 38" to 66" high and supports up to 45 lb.
- 1" heavy-gauge tubular telescoping legs twist-lock into desired height, fold up to store or carry.
- Locking cross braces ensure stability.
- Adjustable display holders slide to proper position.
- Supports materials up to 48w x 36 h.
- Antiskid feet.
- Easel bag sold separately.
- Supports up to 45 lbs.
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The Enchanted Castle (Puffin Classics)by Edith Nesbit
Staff Pick Like the four children of Edward Eager's Half Magic who fell in love with this book, many of us on the Kids' Team were quite taken with it as well. Adventure, magic, and sibling squabbles are all told with a delightfully British sensibility. The Enchanted Castle, like Nesbit's other books, is a great way to introduce young readers to fantasy that isn't too dark or frightening. Synopses & ReviewsPublisher Comments:An invisible princess, a magic ring, and more adventures than you could dream of. This is what Gerald, Kathleen and Jimmy find when they stumble upon a mysterious castle. At first it all appears to be a lark. But the children soon discover they need all their bravery and ingenuity to contend with the castle's supernatural forces. Exquisite watercolor illustrations.
Synopsis:Gerald, Cathy and Jimmy wake a beautiful princess from her hundred year sleep in an enchanted garden. It's really only Mabel, the housekeeper's niece - but the garden really is enchanted, and the ring she slips on really is magic.
About the AuthorEdith Nesbit (1858 – 1924), was a mischievous, tomboyish child who grew up to be an unconventional adult. She and her husband were founder members of the socialist Fabian Society and their home became a centre for socialist and literary discussion. Their friends included some of the time's greatest writers and thinkers, including George Bernard Shaw and H. G. Wells. Everything about Edith showed her as a woman trying to break out of the mould demanded by English society at the time – she expressed her individuality through her clothes, hairstyle, lifestyle and her habit of speaking forcefully on almost any subject. She lived her socialism and late in life her charitable deeds brought her close to bankrupcy. E. Nesbit – she always used the plain initial for her writing and was sometimes thought to be a man – started to write for children after years of successful writing for adult magazines. She was asked to write about her childhood but instead of facts chose to describe her happy girlhood in fiction. The result was books still read today, firm bestsellers for decades. She was brilliant at combining real-life situations with elements of fantasy and humour. Films –such as The Railway Children - have kept her stories in the public eye and her magical fantasies, including Five Children and It, continue to delight each new generation of children. What Our Readers Are SayingAdd a comment for a chance to win!Average customer rating based on 1 comment:
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White Chocolate Raspberry Cheescake
Traditional, creamy cheesecake topped with a fresh raspberry puree.
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The world's best cheesecake just got even sweeter! This to-die-for cake is a decadent blend of luxurious white chocolate and our signature New York Cheesecake. The premium, all-natural cake is baked atop a chocolate cookie crust, then topped with a puree of locally grown raspberries. Simply divine!
Each cake is made by hand using pure cream cheese, fresh eggs, and real vanilla extract. Using locally sourced, top quality ingredients results in a cheesecake that has an unbeatable flavor and perfect texture. Measures 7" and serves 6.
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great taste, great packaged, fast delivery February 22, 2013
the ice packs worked awesome!!!!
the cake was kept very cool and could be enjoy at the right day.


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Lodge Pro-Logic P14W3 Cast Iron Wok, Black, 14-inch | |
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From Paris With LoveWMAG · VOGUE.COM · GRAZIA.IT Obviously the best is saved for last. Highlights from the streets of Paris during fashion week. ENJOY! The post From Paris With Love appeared first on Studded Hear...51
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NYFW STREETSTYLE ROUND UPSTREETFSN · VOGUE.COM · HARPER'S BAZAAR · ELLE · VOGUE UK My favourite streetstyle looks from a very cold NYFW, all wrapped up in a massive post just for you. The post NYFW STREETSTYLE ROUND UP...93
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CAMPCAMP LOOKBOOK PHOTOGRAPHER: JASON LEE PARRY STYLIST: PAUL BUI MODEL: CORA KEEGAN HAIR & MAKEUP: NATASHA SEVERINO Been dying to share this one with you. Camp is 2Bandits designer Tamar Wider's newest...65
MINIMALISECOVER FEBRUARY 2014 PHOTOGRAPHER: ANDREAS OHLUND STYLIST: EMELIE JOHANSSON MODEL: HANNA SORHEIM The post MINIMALISE appeared first on Studded Hearts.67
REVOLVE CLOTHING SPRING 2014EMILY RATAJKOWSKI FOR REVOLVE CLOTHING SPRING 2014 CATALOGUE This girl is so insanely hot. You can shop all the pieces here. The post REVOLVE CLOTHING SPRING 2014 appeared first on Studded Hea...138
AWAYGoing away to Thailand for a couple of weeks and I doubt a lot of blogging action will be happening while I'm over there. I'll try my best to update on Instagram and Tumblr so hit me up there instead....49
VACAY PACKINGROMPERS: ZIMMERMANN / MIGUELINA / DOLCE VITA BIKINIS: MIKOH / MARYSIA / T BY ALEXANDER WANG (↑ Click each item in picture to shop) Just finished packing for my trip to Thailand (leaving tomorrow!)....55


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Malaria in Pregnancy Pilot Projects Nationally Adopted in Kenya and Malawi
Following successful pilot programs led by the Council to prevent malaria among antenatal clients, Kenya and Malawi have developed national policies to prevent malaria in pregnancy.
In the most malaria-endemic areas of Africa, women in their first and second pregnancies have the highest risk of acquiring malaria and, consequently, of malaria-associated anemia and low birthweight. Two USAID-funded interventions aimed at strengthening efforts to prevent and manage malaria in pregnancy were pilot-tested at the district levels in Kenya (1998–2002) and Malawi (1998–2004).
The goal of the projects was to reduce malaria illness and death among children and mothers primarily by: (1) providing intermittent presumptive treatment (IPT) of sulfadoxide-pyrimethamine (SP) to pregnant women visiting antenatal clinics, and (2) promoting the use of insecticide-treated bed nets by pregnant women through price subsidization, aggressive promotion, and wide distribution.
In 2005 and 2006, the Council's FRONTIERS program conducted case studies to assess the sustainability of these initiatives, documented best practices for promoting their scale-up, and drew lessons for replication in other East and Southern African countries where malaria is endemic.
Findings demonstrated sustainability in pilot districts. Inventories show that most of the facilities surveyed (37 of 45 in Kenya and 26 of 29 in Malawi) were still offering malaria in pregnancy service two or more years after the pilot projects ended. The proportion of women sleeping under bed nets increased significantly by the time the evaluation study began (in Kenya from 3% to 58%, and in Malawi 81% of facilities provided nets).
In both countries, the programs have been scaled up, and IPT has been adopted as national policy. In Kenya, SP is now available over-the-counter, and bed nets are available at subsidized prices to facilitate access to them. In Malawi, national surveys show that a large proportion of women receiving antenatal care (90% of pregnant women) received first and second doses of SP in 2004, and use of bed nets by pregnant women doubled between 2004 and 2006.
National political commitment to a specific goal and to an evidence-based approach was essential to the success of both of these programs, which elicited high levels of support from development partners. Strong links within ministries of health and with district- and community-level organizations also enhance the potential for program sustainability.
No publications are listed
Project Stats
Reproductive Health
Maternal and newborn health
Safe pregnancy, antenatal, and delivery care
Duration: 1/1998 - 12/2004
Non-Council collaborators:
African Medical and Research Foundation
Blantyre Integrated Malaria Initiative
Government of Malawi
Kenya Ministry of Health
US Centers for Disease Control and Prevention
Get Involved
- Make a contribution to the Population Council
- Honor a loved one with a gift in his or her name
- Sign up to receive e-mail announcements


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Eriksen puts English clubs on alert
Premier League - 1 year ago
Watching: He's one of the hottest prospects in European football.
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Does anyone have any recipes for spray or something for insect control or repellent, on vegetable plants that won't harm the bees?
Does anyone have any recipes for spray or something for insect control or repellent, on vegetable plants that won't harm the bees?
Jimmy, there is still so much to be done!
Quick Lime...It helps the soil, too. Raises the PH.
I use Diatomaceous Earth for the garden pests,won't harm the bees and works fairly well against most pests.Lowes is on place that carries it.
http://www.lowes.com/pd_93648-316-HG-93 ... 151&rpp=24
Getting old is inevitable....Growing old is optional
Soak a few cigarettes in water overnight and filter it clean. The nicotine extracted into the water is poisinous to insects (like it is to people). Before the modern "fancy" sprays became accepted, nicotine was the method of choice for many garden extermination aplications.
Proud member of the Beekeeping Forums,
(middle-east division)
From the land flowing with milk and HONEY
If you have a Tractor Supply Store close, this might be worth looking in to:Originally Posted by Crackerbee
http://www.tractorsupply.com/red-lake-e ... b--1019864
Greg Whitehead
Euchee, TN
Watts Bar Weather
Greg's Bee Adventure
At this point, everything I do is an experiment!
Plant enough that the insects can have their share too.


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Honeyville's Wild Berry Smoothie Mix makes delicious smoothies in minutes. Simply add water and ice, blend and enjoy a creamy smoothie that is sure to keep you cool on a hot summer day. Our smoothie mixes are made with real fruit and have the perfect balance of flavors that blend together into a yummy frosty treat. You can add yogurt, milk, ice cream or even Honeyville's freeze dried fruit for an additional flavor boost. Try one of our great smoothie flavors today!
Shelf-Life: Wild Berry Smoothie Mix will store for 1 year in a sealed small can (oxygen absorber included) under ideal storage conditions (cool, dry place).
Instructions: Blend one scoop of smoothie mix to 4 ounces of water and 4 ounces of ice. Makes one 8 ounce serving.
Uses: Wild Berry Smoothie Mix can be used to make delicious smoothies or a flavor enhancer for protein shakes, milkshakes, and more.
Packaging: Wild Berry Smoothie Mix is sealed air tight in a small can and weighs approximately 15 ounces. A pack contains 6 small cans and weighs 90 ounces. Each can contains 15 servings. One serving size is 1 ounce.
Ingredients: Sugar, Non-Dairy Creamer (Coconut Oil, Corn Syrup Solids, Sodium Caseinate (a milk derivative) and/or Nonfat Milk, Dipotassium Phosphate, Mono and Diglycerides, Sodium Stearoyl Lactylate, Tricalcium Phosphate, Lecithin, Artificial Flavor), Natural & Artificial Flavor, Lactic Acid, Salt, Dried Strawberry with seeds, Dried Raspberries, Dried Blueberries, FD&C Blue #1, FD&C Red #40.
Allergen information: Contains milk. This product is produced on equipment that processes products containing soy, wheat, egg, milk, peanut, and tree nuts.


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I've been using Illustrator since version 8.0 and don't feel comfortable jumping over from version CS3 to CS4 or CS5, mainly because I find that "Smart Guides" (which is an option I relay on most of the time) have became less-Smart than they were in CS3.
I need total accuracy on may job, but somehow shapes do not snap the same way using smart guides as they did before. Can't tell exactly why this is but its an annoying thing! Don't know the reason why Adobe changed them in the first place they worked fine as they were.
First experienced this on CS4 and thought that this might be a bug that would be corrected later on but have noticed they the same way on CS5 afterwards.
Did anyone experience the same issue? Comments? Suggestions?
Thanks in advance.


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Saturday, June 30, 2012
Friday, June 29, 2012
crochet tutorial :: simple stars
These small stars are really easy to make! Each one has five points that are formed around a center ring.
Start by chaining 5:
Use a slip stitch to close your chain into a ring:
Chain 5 more stitches:
Single crochet in each stitch back down that chain so you end up back at the ring:
Then do one single crochet into the ring to make sure that little bit of crochet is snuggly attached to the ring. That's the first point of your star:
Chain 5 again. We're starting the second point of your star:
Single crochet in each stitch back down the chain so you end up back at the ring. Do one more single crochet into ring so that "arm" of the star is snuggly attached to the ring, just like before:
Continue in the same way, creating each point by chaining 5 and working back to the ring:
When you have made 5 points simply join the last point with the base of the first one with a slip stitch for a clean look:
Weave in your loose ends:
And your star is finished!
Read more...
Thursday, June 28, 2012
Sneak Peek!
I'm working diligently on a new crochet tutorial and a new soft sculpture (almost finished with both -
soooo close!).
Here's a sneak peek that pertains to both projects:
Cool, huh? I have to say that I'm extremely happy with this piece, it's going to be gorgeous.
{And Cornflower Blue Studio is featured on the Frankie Magazine website today - such an awesome Aussie magazine! Such an honor!}
Read more...
Wednesday, June 27, 2012
{homemade focaccia}
I made homemade focaccia this week and it was so good! I had never made it before, but it was really easy and I'll definitely be making it again - for some reason I thought it would be a difficult bread to make on my own. My husband got the recipe from an Italian friend who likes to make bread, too. The timing for this bread was perfect because my little rosemary plant on the front porch is getting big enough to start using in recipes!
Have ever tried making your own focaccia before? Did you use an herbs or toppings?
Read more...
Tuesday, June 26, 2012
feminism + zines
I'm so excited to be contributing to the Philly Feminist Zine Fest in August! I'm sending this little package which will be a prize in the raffle - two of my own zines and an embroidered design.
There's not much better than feminism and zines, 'tis true. I think the festival will be an amazing event, if you're in the Philadelphia area make sure to check it out!
I am a Woman
Phenomenal Woman,
that's me.
--Maya Angelou
Monday, June 25, 2012
Sunday, June 24, 2012
color story :: purple and green
I saw this beautiful landscape planting while on a walk yesterday - I just love the combination of deep purples with bright acid green! I would love something similar in my future yard.
Read more...
Saturday, June 23, 2012
Friday, June 22, 2012
*Inspire Someone*
A few weeks ago I posted with a theme about being yourself and I've been thinking about it ever since. It seems like the easiest thing in the world, but sometimes following our inner guide takes courage and guts.
I've made two new note designs to offer encouragement to those of who are blazing our own trail!
Read more...
sewing and embroidery,
Thursday, June 21, 2012
*movie date*
On Tuesday went on a movie date to see the documentary Marley. It was really hot outside, but I knew I'd be cold in the theater so I wore my giant scarf as an extra layer.
I love big scarves like this and I'm always trying out new ways to tie them - this video is my favorite place to find cool scarf ideas.
The movie was awesome! If you are interested in Bob Marley and love his music you'll love it, too!
personal style
Wednesday, June 20, 2012
Tuesday, June 19, 2012
Monday, June 18, 2012
DIY :: Fancy Fascinator Headband
In the summer I like to dress up my outfits with scarves and headbands in my hair. I had to idea to make a lacy fascinator headband - it turned out to be a really quick project and super easy.
To make this headband you'll need a large piece of lace (I cut mine from a vintage table runner I found at the thrift store). The piece I'm using happens to be a round medallion, but since you'll be mounting it to felt you could use any shape you like. I think a floral motif would be excellent!
You will also need a piece of felt, approximately 21 inches of soft elastic (I used thin lingerie elastic that has a lacy look, too), craft glue, scissors, and a needle & thread.
Trim your lace to the size and shape you want, then apply craft glue to the back and glue it onto your felt.
When the glue has dried cut your shape out of the felt.
Grab your elastic - 21 inches of elastic was the perfect size for my head. Double check this for your own head and alter the measurement if necessary.
Overlap the short ends of your elastic about half an inch, then stitch in place.
Glue the elastic across the back of your felt piece with the sewn edges int he middle to hide them.
Once the glue has dried add a line of simple straight stitches to reinforce the glue.
10 Simple Pleasures
I went for a nature walk first thing this morning and it made me think about all the simple things that make life so great. Nature walks are a perfect time to reflect and make mental lists of things you are grateful for - the sunshine and fresh air seem to be especially conducive to this kind of thinking!
Here is a list of 10 simple pleasures I compiled this morning on my walk:
fresh flowers from the farmer's market
going to the library and browsing for an hour, making a list of all the books you want to read
a cupboard full of herbal teas
taking walks in the morning or at dusk
weekend naps
baking cookies and eating some while they are still warm
watching birds and listening to their calls
collecting stones, pine cones, leaves, and shells for a nature basket
a signature scent, one you really love
music for whatever mood you're in
Sunday, June 17, 2012
bird watching
This weekend I've been identifying birds in my area - now that I'm paying attention I see cool birds everywhere! I've found three (beautiful!) Red-Headed Woodpeckers, a flock of Chimney Swifts, Wood Thrushes, Cardinals, and Finches. I think there are quite a few Mockingbirds in my neighborhood, but I still need to compare them more closely to my guidebook photos : )
Are any of you into birds? What bird guides would you recommend for beginners?
Read more...
Saturday, June 16, 2012
Friday, June 15, 2012
{week in review & summer necessities}
I'm planning a restful, restorative weekend for myself as a reward for working hard over the last few days. I have been cleaning out closets, cabinets, and drawers, deciding which items are more clutterful than useful : ) A charity pick-up is scheduled for tomorrow to cart away everything that needs to go. I also did some caulking in the bathroom, hand-washing of delicates (I actually love washing delicates by hand!), and a bunch of other home-keeping chores.
On Tuesdays we've been going to the summer Art House movie series and this week we saw A Separation - it was so good! If you are interested in foreign films this would definitely be a great one to watch.
I've also been making a list of necessities for the summer weather. I love this sunscreen, this swimsuit, and these comfy French shoes. I have my eye on this Italian summer scarf and these colorful sunglasses, too!
Read more...
personal style,
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Definition of Acetyl CoAAcetyl CoA: See: Acetyl coenzyme A. Source: MedTerms™ Medical Dictionaryhttp://www.medterms.com/script/main/art.asp?articlekey=32064 Last Editorial Review: 9/20/2012 Medical Dictionary Definitions A - ZSearch Medical Dictionary
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COLUMBIA — A Columbia man was held up at knife point by a group of boys early Friday morning.
Columbia man robbed at knifepoint
Friday, August 21, 2009 | 12:38 p.m. CDT
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If you'd like to read more about the value of being a member, read this column from the Missourian's executive editor, Tom Warhover.


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National Hockey League | |||||||||||||||||||||||||||||||||||||||||||||||||
· Top Story · Injuries · News · Current Odds · Live Odds · Previews · Recaps |
· Daily Schedule · Yearly Schedule · Standings - East · Standings - West · Scoreboards · Statistics · Teams |
· Transactions More Hockey: · College Hockey · MLH |
Devils' Henrique set to return Newark, NJ (Sports Network) - New Jersey Devils forward Adam Henrique will likely make his season-debut Thursday against the New York Islanders after recovering from thumb surgery. Henrique, a finalist last spring for the Calder Trophy as the NHL's top rookie, was playing for Albany of the American Hockey League during the lockout and suffered a left thumb injury during a late November game. The surgical procedure repaired a damaged ulnar collateral ligament. In 74 games for the Devils last season, Henrique posted 16 goals and 51 points. He added five goals and 13 points in 24 playoff games to help New Jersey to the Stanley Cup Finals. 01/31 11:29:23 ET |


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Haha! You're the first one to notice.
love the hair :)
amazing work
this is pretty neat
this is fucking awsome
i love this. so so so so much.
this is amazing. your so good
love this i would buy it. voted
this is brilliant voted
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now only two more votes :) Voted!
Love this, Voted!


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Whats the latest build number for Fusion 2013?
i'm running 220.127.116.11 from the Factory Design Suite Ultimate.
Is the Fusion Tech Preview on Labs any newer?
thats the latest version
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2006 OPEN FORUM Abstracts
Leak Compensation Comparisons in Mechanical Ventilation
Jessica Patnaude, RRT, Keisha Smith, CRT, Ryan Totten, CRT, Joe Warren,
CRT, & Aaron Light BSRT RRT Ozarks Technical Community College, Springfield, MO
Background: A bench test was performed comparing leak
compensation on the Maquet Servo i, Drager Evita 4, and the Viasys Avea. Using
the same ventilator circuit for each ventilator a leak was created by opening
the saline port on a Ballard suction catheter. Using the same ventilator
settings the peak pressure, minute volume, measured rate, and exhaled tidal
volume were measured. The intrinsic peep
was measured by a Hans Rudolph Series 1101 breathing simulator. This study will
confirm which ventilator tolerates a leak with a minimal change in parameters.
Method: A piece
of large bore tubing was connected to a Hans Rudolph Series 1101 breathing
simulator. Then a size 7.0 mm endotracheal tube was placed in the tubing and
the cuff inflated to make sure there was no leak. Each
ventilator was then attached to the endotracheal tube (ETT) using the same
ventilator circuit. A Ballard closed system catheter was also placed in line
with the circuit at the ETT. The
breathing simulator was set for a resistance of 7 cmH20/l/sec, compliance of 40
ml/cmH20, and a set breath rate of 12. All
ventilator parameters were set the same (VT 500ml, f 12, PEEP 5 cmH20, I.T. 1.2
seconds). The Viasys Avea and Maquet Servo I were placed in the pressure
regulated volume control mode of ventilation while the Drager E4 was placed in
CMV with auto-flow. The sensitivity on all ventilators was set for flow trigger. PIP, VE, measured f, exhaled VT and intrinsic PEEP was then noted without a leak. A leak was created by uncapping the saline
port on the Ballard suction catheter and then all parameters were then noted
with the leak.
Drager Evita 4 | Without Leak | With Leak |
Peak Pressure | 18 cmH2O | 18 cmH2O |
Minute Volume | 5.7 L/min | 5.2 L/min |
Measured Rate | 12 bpm | 12 bpm |
Tidal Volume Exhaled | 489 ml | 439 ml |
Intrinsic PEEP | 5 cmH2O | 4.2 cmH2O |
Viasys Avea | Without Leak | With Leak |
Peak Pressure | 18 | 18 |
Minute Volume | 6 | 6.5 |
Measured Rate | 12 | 19 |
Tidal Volume Exhaled | 460 | 350 |
Intrinsic PEEP | 4 | 4 |
Maquet Servo I | Without Leak | With Leak |
Peak Pressure | 19 | 13 |
Minute Volume | 5.7 | 5 |
Measured Rate | 12 | 14 |
Tidal Volume Exhaled | 493 | 378 |
Intrinsic PEEP | 5 | 3 |
Conclusion: The Drager Evita 4 ventilator performed the
best with minimal changes in measured parameters. The Viasys Avea showed signs of auto
triggering but did not decrease the peak inspiratory pressure in response to
the leak. The Maquet Servo i did not
perform as well as the other two ventilators. Not only did it auto trigger but
also the peak inspiratory pressure decreased in response to the leak. We
believe this drop in pressure is due to the ventilator sensing the leak was a
sign of improved lung compliance.


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Airline sued for asking men to change clothes before boarding
Passengers charge U.S. Airways with discrimination.
Two young African American men boarding a U.S. Airways flight were allegedly denied access to their seats until they changed out of casual attire. An airline employee at the gate told the two men they couldn't sit in first class wearing hoodies and jeans, New York Daily News reports, and were instructed to remove their baseball caps, put on button-up shirts and nicer shoes and change from jeans into slacks.
Miles and MacCraig Warren, brothers traveling from Denver to Los Angeles on their way home from a funeral in August, were "humiliated" but wanted no trouble. They returned to the gate in the required clothes only to find two other young men — one Caucasian, one Filipino — boarding first class dressed in hoodies and sneakers.
The Warrens filed a lawsuit in federal court this week charging the airline with discrimination and causing emotional distress. The brothers have said they're not after money but that "an apology would be nice."
Bing: Trouble with the Boeing 787?
U.S. Airways issued a statement saying it's taking the allegation seriously and that "we welcome customers of all ethnicities and backgrounds and do not tolerate discrimination of any kind." Their defense may hinge on the fact that the Warrens obtained their first-class tickets through a friend employed by U.S. Airways who provided them with a "buddy pass" — which, an airline representative said, would require the men to dress according to company guidelines for traveling employees. But that policy may not have been made clear to Miles and MacCraig before they were at the gate.
More from MSN Living: Is air travel safe?
The episode echoes another airline-attire incident we related last summer (along with other things that get people kicked off airplanes), though in that case a Spirit Airlines crew spirited a passenger off a flight when he became verbally abusive after being asked to lift up his sagging pants.
More from MSN Living: 50 Facebook dos and don'ts
Back in the Golden Age of air travel, passengers dressed in business attire to fly. Today, people travel in the same everyday duds they wear to Wal-Mart. Not a great look on some, but maybe if the airlines didn't treat us like we were in a bus station, we wouldn't dress like it. Whatever the airplane crew's perspective on proper attire may be, it should match passenger to passenger — regardless of where he or she is sitting, how much was paid for the ticket or whether a flight attendant is afraid of black people.
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War vet arrested and disarmed while hiking
'Frog whisperer' helps de-croak neighborhoods
Conspiracy theories Americans believe today
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Photo: U.S. Airlines planes / Kevork Djansezian/Getty Images
The employee who gave them their passes is probably going to get fired. These guys need to issue a HUGE public apology. I'm glad they're not my "friends"
UNBELIEVABLE! As one of my instructors in flight attendant training said, (paraphrased after 20+ years) "You are getting PAID for your time and you are flying for FREE. If (insert name of airline here) asks you to wear a giant bunny suit on the airplane, that's what you wear. Express yourself on your own time"
This was in regards to uniform regulations, but non-rev travel attire is the same thing. You are flying for FREE!!!
Hey! Would you put on a big yellow T-Shirt that says, "I LOVE USAirways" in exchange for a free ticket in First Class? Yes? They how about just donning business attire and a respectful, grateful attitude?
I've been in the Travel Industry for over 28+ years. Anytime we fly on passes or reduced rate tickets or on "Buddy Passes" (which all are an extended PRIVLEGE!). We always had to dress like you were representing the airline you were flying (their dress code). There's nothing wrong with that. There friend is the one to blame here not the airline. Quit wasting the courts time. It wasn't discrimination. It was a lack of communication from your friend.
inspire: live a better life
10 travel mistakes and how to avoid them
From international roaming fees to skipping travel insurance, these mistakes can put a damper on your next vacation.
Where in the world? Special places you've never heard of
Where in the world do you want to go? Somewhere unique? Any of the places described here are sure to make your next world-travel adventure truly memorable.
10 inspiring leaders and what we can learn from them
It's easy to lead, but it takes courage and conviction to be a truly great leader. Here are 10 inspiring leaders and what we can learn from them.
30 top trips (vacations) of a lifetime
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Daylight Saving Time: For or against?
The semi-annual glitch in your daily routine is back this weekend.
9 sneaky things everyone leaves out of their budget
You'll stave off credit card debt by the end of the year if you account for these often-forgotten expenses.
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Mark Zuckerberg has an even bigger effect on your life than you thought.
One hour a week is all you need to get more stuff done
Take this advice to finally tackle that nagging to-do list.
Search: Celebrating Doctor Seuss
The iconic storyteller would have been 110-years-old
Women who inspire us
Take the time to appreciate women's contributions to society. Here are some of the women who inspire us by their example.
Search: 'Brady Bunch' reunion
Cast of the iconic TV show reunites for Florence Henderson's birthday
The most inspiring Olympic moments for women
We couldn't be more excited to watch the best athletes in the world compete at the Winter Olympics in Sochi. This year, about 44 percent of the competitors are women according to the International Olympic Committee—which is awesome but wasn't always the case. Let's look back at some of the amazing women who paved the way with incredible, memorable feats of girl power.


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New York's iconic Guggenheim Museum is featuring an exhibition of Zaha Hadid's work. Zaha Hadid: 30 Years of Architecture will run until August 23rd and will focus upon her interdisciplinary approach to architecture in a wide range of media including painting, drawing, urban plans, proposals for international design competitions, scale models and documentation of projects currently under construction. The London based practice has seen spectacular growth in the last year with staff levels rising from 70 to over 150 and is still recruiting. Feedback from current job adverts on WAN have shown a tremendous interest in candidates wanting to work for Zaha.
Aquatics Centre
Al Wakrah Stadium
Heydar Aliyev Center
Library & Learning Centre, Wirtschaftsuniversität Wien
Serpentine Sackler Gallery
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The Brown College Associate of Applied Science in Network Development program is designed to help prepare students to evaluate, deploy and manage computer hardware, software and network components. The program emphasizes hands-on learning and computer-based training opportunities. Specifically, it familiarizes students with the installation and upgrading of hardware and software components.
Brown's AAS in Network Development's curriculum features instruction in LAN/WAN training. It includes instruction in network setup, configuration and management using industry standard client/server platforms. Additional topics include troubleshooting, help desk operations, end-user support and evolving technologies. Graduates will be expected to:
Courses in Brown's AAS in Network Development focus on helping students build the knowledge and skills necessary to seek entry-level employment designing, installing and maintaining networks. Classes are led by instructors, many with real-world working experience in the IT industry.
Other core requirements in this program include:
Course curriculum is subject to change.
Prospective areas for entry-level career opportunities for students who successfully complete the Brown College AAS in Network Development include:
By providing your mobile number, you agree to receive text messages from Brown College via its mobile text message provider. You may opt out of receiving messages by texting the word STOP to 94576, or simply reply with the word STOP to any text message you receive from Brown College.
While CEC or its mobile text message provider will not charge end users for receiving/responding to promotional messages, depending on the terms of your mobile phone plan, you may incur a cost from your mobile service carrier to receive and respond to any promotional text messages (standard messaging and data rates/fees and other charges may apply). Charges will appear on your mobile phone bill or will be deducted from pre-paid amounts. Current participating/supported carriers are: Alltel, AT&T, Boost, Cellcom, Cellular One, Cellular South, Cincinnati Bell, Cricket, Element Wireless, Golden State Cellular, iWireless, Metro PCS, Nextel, nTelos, Plateau Wireless, Sprint, T-Mobile, US Cellular, Verizon Wireless, Viaero Wireless, Virgin, and more.
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North Bay, Ontario // Shot: October 30, 2013
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The high winds sweeping across Lake Nipissing are bringing cooler temperatures than the mild ones earlier today. A female Common Merganser is not enjoying the wind which makes keeping its 'hair' in place difficult.
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7:52 am
Tue March 17, 2009
Newly Appointed NMSU Regent Excited to Work for His Alma Mater
By Evan Woodward
Las Cruces, NM – Last week after local pecan farmer Thomas Salopek declined his nomination to the New Mexico State University Board of Regents for personal reasons, Governor Bill Richardson chose Issac Pino, President of SunCor New Mexico in his place. KRWG's Evan Woodward spoke with Pino yesterday about his appointment.


text: VanderCook encourages you to consider how you can help support future music teachers who aspire to leave their own legacies in the VanderCook tradition. Support the annual Share Your Passion Campaign or make a gift to the Great Teachers Scholarship Fund, as part of your annual philanthropy. If you are interested in getting more information about making a gift to VanderCook, please contact Clarmarie Keenan, director of institutional advancement at (312) 788-1131 or email@example.com. You may also donate online through our partnership with justgive.org.


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The American Payroll Association (APA) and VISA are pleased to present a special eLearning webinar. There is no cost to view.
Earn 1 Recertification Credit Hour (RCH) by viewing this on demand webinar.
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Each live webinar is converted into a Webinar On Demand after the live presentation has been completed. The Internet streaming consists of the speaker's presentation and a PowerPoint, PDF, or other displays presented during the live webinar.
You will receive an e-mail with a link to the recorded webinar. Enter the order number provided in the e-mail. The program will begin to play as it continues downloading in the background. The process requires a high-speed internet connection and Windows Media Player 9 or later. Once the link is activated, you have 48 hours to view the program. The e-mail will contain links to the course material associated with the segments, if available.