Raf‐1 protein kinase has been identified as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf‐1 is achieved by Ras.GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. We have used gene targeting to generate a 'knockout' of the raf‐1 gene in mice as well as a rafFF mutant version of endogenous Raf‐1 with Y340FY341F mutations. Raf‐1−/− mice die in embryogenesis and show vascular defects in the yolk sac and placenta as well as increased apoptosis of embryonic tissues. Cell proliferation is not affected. Raf‐1 from cells derived from raf‐1FF/FF mice has no detectable activity towards MEK in vitro, and yet raf‐1FF/FF mice survive to adulthood, are fertile and have an apparently normal phenotype. In cells derived from both the raf‐1−/− and raf‐1FF/FF mice, ERK activation is normal. These results strongly argue that MEK kinase activity of Raf‐1 is not essential for normal mouse development and that Raf‐1 plays a key role in preventing apoptosis.
The mammalian raf‐1 gene was first identified as the cellular homologue of v‐raf, the oncogene responsible for the induction of sarcomas in mice by the MSV3611 virus. Two other genes highly homologous to raf‐1 have been cloned: A‐raf and B‐raf. All three raf genes code for serine/threonine protein kinases and they share a high degree of sequence similarity. The N‐terminal region encodes the regulatory domain and binds essential cofactors including Ras while the C‐terminal region contains the catalytic kinase domain. Deletion of the N‐terminal regulatory regions of all three kinases gives rise to proteins that are constitutively active and are oncogenic in a wide variety of cell types. The kinase domain of B‐raf appears to be the most potent of the three in these assays (Pritchard et al., 1995). In the mouse, transcripts for all three raf genes are detectable in all cells (Storm et al., 1990; Barnier et al., 1995).
Of the three Raf isotypes, most biochemical studies have focused on Raf‐1. Inactive Raf‐1 is normally cytosolic, but Raf‐1 binds to Ras.GTP in vitro and in vivo and so translocates to the plasma membrane in the presence of active Ras (Marais and Marshall, 1996 and references therein). However, binding to Ras is not sufficient for full Raf‐1 activation (Traverse et al., 1993; Marais et al., 1995, 1997; Mason et al., 1999) and additional signals at the plasma membrane including phosphorylation are required (Marais and Marshall, 1996). Our previous studies in COS cells have shown that activation of Raf‐1 requires phosphorylation of Y340 and/or Y341. Substitution of these residues to phenylalanine, creating RafFF, blocks activation of Raf‐1 by oncogenic Ras and Src, and by ligand stimulation (Marais et al., 1995, 1997; Diaz et al., 1997; Stokoe and McCormick, 1997; Barnard et al., 1998). Recent data have also suggested that phosphorylation of Raf‐1 at serine 338 is required for activation, demonstrating that complex phosphorylation events take place within this region of Raf‐1 (Diaz et al., 1997; Barnard et al., 1998; Mason et al., 1999). However, the physiological importance of these phosphorylation events is unclear.
The principal functions of the Raf protein kinases appear to be participation in the highly conserved Ras/Raf/MEK/ERK intracellular signalling pathway (Marshall 1994). This pathway is activated by different classes of cell surface receptors including receptor tyrosine kinases (RTKs) and G protein coupled seven transmembrane receptors, all of which confer their biological effects through Ras (Dickson and Hafen, 1994; Marshall, 1994). ERK activation has been associated with many of the downstream consequences of Ras activation and the Raf proteins provide a vital link between activated Ras proteins and the ERKs. A variety of biochemical and genetic data point to the importance of Raf‐1 as a MEK activator. Activation of an inducible version of oncogenic Raf‐1 induces the rapid activation of MEK and ERK as well as immediate early gene expression in NIH 3T3 cells (Samuels et al., 1993; Kerkhoff and Rapp, 1997). Immunoprecipitated endogenous Raf‐1 can phosphorylate MEK1 and ‐2 in vitro (Howe et al., 1992; Kyriakis et al., 1992; Marais et al., 1998) and the Raf/MEK/ERK cascade can be reconstituted in vitro using proteins expressed in Sf9 cells (Macdonald et al., 1993). Kinase inactive Raf‐1 cannot activate MEK in this system. Finally, dominant‐negative Raf‐1 mutants block growth factor and oncogenic ras‐stimulated activation of ERKs in fibroblasts (Schaap et al., 1993; Chao et al., 1994; Troppmair et al., 1994).
Intriguingly, a number of observations do not entirely fit with the view that the endogenous Raf‐1 protein is a physiologically important MEK activator (Marais and Marshall, 1996). First, only a small proportion (<10%) of the entire cellular Raf‐1 is activated upon treatment of cells with growth factors (Dent et al., 1995; Reuter et al., 1995; Jelinek et al., 1996). Secondly, B‐Raf has been identified as the major MEK activator in neuronal cell types and NIH 3T3 cells, despite Raf‐1 also being expressed in these cells (Moodie et al., 1993, 1994; Catling et al., 1994; Jaiswal et al., 1994; Traverse and Cohen, 1994; Reuter et al., 1995). The kinase domain of B‐Raf is a considerably stronger activator of MEK and has a higher affinity for MEK than the kinase domain of Raf‐1 (Pritchard et al., 1995; Marais et al., 1997; Papin et al., 1998). Finally, Raf‐1 and ERK activation are not coincident in some circumstances (Wood et al., 1992, 1993; Kuo et al., 1996). A pool of Raf‐1 is also thought to be located in mitochondria (Wang et al., 1996) and an ERK‐independent role for mitochondrial Raf‐1 in apoptosis has been postulated (Neshat et al., 2000).
Many of the previous studies designed to address the involvement of Raf‐1 in mediating signals between Ras and ERKs have used antisense or dominant‐negative constructs overexpressed in tissue culture cell lines to inhibit its expression or activity (Kolch et al., 1991; Chao et al., 1994; Troppmair et al., 1994). These approaches have the disadvantage that they may sequester the function/expression of other Raf isotypes (A‐Raf and B‐Raf) or other Ras effectors. To achieve the required specificity, gene targeting has been used to ablate individual raf genes (Pritchard et al., 1996; Wojnowski et al., 1997, 1998). In this study, we describe the generation of Raf‐1 deficient mice (raf‐1−/−) as well as mice with a 'knockin' mutation of the endogenous 340/341 tyrosines to phenylalanine (raf‐1FF/FF). Although there is no detectable Raf‐1 activity in cells derived from either strain of mouse, ERK activation was not compromised in either. However, the raf‐1−/− animals die in embryogenesis due to vascularization defects and increased apoptosis, while the raf‐1FF/FF animals survive to adulthood and have an apparently normal phenotype. Our results show that the full‐length Raf‐1 protein is essential for normal mouse development and for protection against apoptosis, but they argue that Raf‐1 kinase activity towards MEK is not necessary for these processes.
The generation of the mutant mice is described in the Supplementary data (available at The EMBO Journal Online.) Both mutations were established on the mixed 129Ola/C57BL6 and 129Ola/MF‐1 backgrounds. For the raf‐1 knockout, raf‐1+/− animals were intercrossed and PCR genotype analysis was performed on tail DNAs from surviving offspring. No surviving animals with the raf‐1−/− genotype were obtained (n = 14 matings) but raf‐1+/− animals were born at the expected Mendelian frequency and were indistinguishable from raf‐1+/+ littermates. Therefore, raf‐1+/− intercrosses were set up and embryos were genotyped. On the 129Ola/C57BL6 background, at E9.5, a number of abnormal embryos were observed and these all PCR genotyped as raf‐1−/− (Table I). On the 129Ola/MF‐1 background, in early backcross generations, a phenotype similar to that on the 129Ola/C57BL6 background was observed (Table II). However, a number of normal embryos at E9.5–E10.5 also typed as raf‐1−/− (Table II). When the raf‐1 mutation was further backcrossed to the MF‐1 strain, raf‐1−/− embryos were observed at E12.5 up to birth but these were small and morphologically abnormal.
On the 129Ola/C57BL6 background, at E9.5, raf‐1−/− embryos were morphologically smaller and were reduced in size by approximately one‐third (Figure 1A and B). They were developmentally arrested, had fewer somites and were anaemic, but were still alive as judged by the presence of a regular heartbeat. Strikingly, all of the mutants lacked small and large blood vessels in the yolk sac (Figure 1C and D), as visualized by staining with an antibody to platelet endothelial cell adhesion molecule‐1 (PECAM‐1; Figure 1E). PECAM‐1 staining of the raf‐1−/− embryos also revealed abnormal vascular network formation (Figure 1F–I). In the head region, there was a reduction in the number of large and small blood vessels, the vessels were disorganized and there were no capillary sprouts into the neuroectoderm (Figure 1H and I). Haemorrhaging was observed in approximately one‐ quarter of the mutant embryos (data not shown). There was a significant reduction in the number and density of cells throughout the mutant embryos but cell size appeared larger (Figure 1J and K). Staining of the embryonic brain with the Ki67 antibody, a marker for cells in S phase, showed that there was no significant reduction in the numbers of proliferating cells in the raf‐1−/− embryos (Figure 1L and M). However, the terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assay indicated that there was an increase in the numbers of apoptotic cells throughout the mutant embryos (Figure 1N and O).
Phenotype of the 129Ola/C57BL6 raf‐1−/− embryos. (A) raf‐1−/− embryo at E9.5; (B) raf‐1+/+ littermate embryo at E9.5; (C) raf‐1−/− embryo at E9.5 in yolk sac; (D) raf‐1+/+ littermate embryo at E9.5 in yolk sac; (E) PECAM‐1 immunostaining of yolk sacs from wild‐type embryo at E9.5 (left) and from mutant embryo (right). (F–I) PECAM‐1 staining of raf‐1+/+ embryo (F and H) and raf‐1−/− embryo (G and I) at E9.5. (H) and (I) are magnifications of the embryonic brain regions. (J–O) Longitudinal sections through forebrain of raf‐1+/+ (J, L and N) and raf‐1−/− (K, M and O) embryos at E9.5. (J) and (K) were stained with haematoxylin and eosin, (L) and (M) were immunostained with a Ki67 antibody, and (N) and (O) were processed for TUNEL staining. Scale bars: A–E, 250 μm; F and G, 100 μm; H and I, 25 μm, J–M, 50 μm; N and O, 100 μm. NE, neural ectoderm; SE, surface ectoderm; Lu, lumen of prospective forebrain; MT, cephalic mesenchyme tissue.
On the 129Ola/MF‐1 background, the raf‐1−/− embryos died within a few hours of birth. They were 50–60% lighter in weight than the raf‐1+/+ embryos and they were anaemic (Figure 2A). The placenta was considerably smaller than the raf‐1+/+ placenta and was disorganized (Figure 2B and C). The spongiotrophoblast layer was considerably reduced in size, a number of the mesenchymal cells of the labyrinthine layer were undifferentiated and there were fewer blood vessels (Figure 2D and E). As with the 129Ola/C57BL6 raf‐1−/− embryos, there were fewer cells in the mutant liver than the wild‐type liver, but the mutant cells appeared to be greater in size (Figure 2F–K). In addition, there were notably fewer areas of haemopoiesis (Figure 2F and G). There did not appear to be any difference in the number of proliferating cells in the raf‐1−/− liver compared with the raf‐1+/+ liver (Figure 2H and I). There was also no significant difference in the number of TUNEL positive cells in the raf‐1−/− liver sections compared with the raf‐1+/+ liver sections (Figure 2J and K), indicating no significant difference in levels of spontaneous apoptosis. The fact that the raf‐1−/− livers are hypocellular is likely to have been caused by increased susceptibility to apoptosis at earlier developmental stages (see below).
Phenotype of the 129Ola/MF‐1 raf‐1−/− embryos. (A) Photograph of raf‐1−/− embryo (left) and raf‐1+/+ littermate embryo (right) at E15.5. (B and C) Cross‐sections of placentas from raf‐1−/− (B) and raf‐1+/+ (C) embryos showing the labyrinthine and spongiotrophoblast (Sp) layers. (D and E) Cross‐sections of labyrinthine layer of placentas from raf‐1−/− (D) and raf‐1+/+ (E) embryos. (F–K) Cross‐sections of liver from littermate raf‐1−/− (G, I and K) and raf‐1+/+ embryos at E15.5. These sections were either stained with haematoxylin and eosin (F and G), immunostained with the Ki67 antibody (H and I) or processed for TUNEL staining (J and K). Scale bars: B and C, 400 μm; D–K, 50 μm.
To assess the raf‐1FF/FF phenotype, raf‐1+/FF animals on the 129Ola/C57BL6 and 129Ola/MF‐1 backgrounds were intercrossed and PCR genotype analysis was performed on tail DNAs from surviving offspring. Of 148 animals assessed, 45 typed as raf‐1+/+, 67 as raf‐1+/FF and 36 as raf‐1FF/FF (n = 19 matings). Therefore, there was no significant difference in the ratio of raf‐1+/+ to raf‐1FF/FF animals surviving to adulthood (P = 0.15). The raf‐1FF/FF animals on both genetic backgrounds survived for >1 year, were normal in weight and had no behavioural abnormalities. T‐cell development was normal in these animals as judged by CD4 and CD8 staining of T cells (data not shown).
Mouse embryonic fibroblasts (MEFs) were derived from the raf‐1−/− and raf‐1FF/FF embryos and characterized for their ability to proliferate and undergo apoptosis in vitro. We consistently failed to culture raf‐1−/− MEFs from E10.5 on the 129Ola/C57BL6 background as most of the cells were dead following embryo homogenization. This may be because of the apparent spontaneous apoptosis of the raf‐1−/− embryos observed on this genetic background (Figure 1N and O). However, we successfully obtained raf‐1−/− MEFs from E14.5 embryos on the 129Ola/MF‐1 background. Raf‐1FF/FF MEFs were isolated from E14.5 embryos resulting from raf‐1+/FF intercrosses on both genetic backgrounds. Sibling raf‐1+/+ MEFs were also isolated in each case.
There was no detectable difference between the growth rates of the raf‐1−/− cells compared with raf‐1+/+ cells over 8 days in culture (Figure 3A), or the raf‐1FF/FF cells compared with the raf‐1+/+ cells (data not shown). Cell proliferation was measured by assessing bromodeoxyuridine (BrdU) incorporation in MEFs that had been made quiescent and then stimulated with 10% fetal calf serum (FCS). Again, the raf‐1−/− and raf‐1FF/FF MEFs showed no difference in their ability to undergo DNA synthesis as measured by this assay compared with raf‐1+/+ MEFs (Figure 3B).
Proliferation and apoptosis analysis of MEFs. (A) Growth curves of raf‐1+/+ MEFs (open circles) compared with raf‐1−/− MEFs (closed circles) over 8 days in culture are shown. (B) DNA synthesis of raf‐1−/− and raf‐1FF/FF primary MEFs compared with raf‐1+/+ cells induced by 10% serum. The data represent pooled data from four experiments of cells with each genotype. (C and D) Levels of apoptosis in raf‐1FF/FF cells compared with raf‐1+/+ cells (C) and in raf‐1−/− cells compared with raf‐1+/+ cells (D). Cells were either not treated (NT) or treated with serum‐free media (SFM), etoposide or anti‐Fas antibody for 20 h. The percentage of cells undergoing apoptosis was quantified by flow cytometric analysis of annexin V staining. Each experiment was performed seven times and the data show mean values ± standard deviation. (E) An example of flow cytometric analysis of annexin V staining of cells for data presented in (C) and (D). The percentage of annexin V positive cells is indicated. (F) Hoechst 33258 staining of apoptotic cells. raf‐1+/+ cells (left panels) and raf‐1−/− cells (right panels) were either untreated (top panels) or treated with anti‐Fas antibody (bottom panels).
Except for the C57BL6 raf‐1−/− MEFs, the primary MF‐1 raf‐1−/− and raf‐1FF/FF MEFs did not show evidence of spontaneous apoptosis under normal growth conditions (Figure 3C–E). Apoptosis was induced by treatment of raf‐1+/+, raf‐1−/− and raf‐1FF/FF MEFs with etoposide, anti‐Fas antibody or by serum withdrawal, and cell death was assessed by annexin V or Hoechst 33258 staining. The raf‐1FF/FF cells showed no increase or decrease in programmed cell death (PCD) upon treatment with these apoptotic agents compared with raf‐1+/+ cells (Figure 3C). In contrast, raf‐1−/− cells showed a significant increase in PCD (Figure 3D–F). Upon treatment with etoposide, the raf‐1+/+ cells showed 21.1% PCD whereas the raf‐1−/− cells showed 30.0% PCD (n = 7; 95% CI for difference 0.5–17.2%, P = 0.04; Figure 3D and E). Upon treatment with anti‐Fas antibody, the raf‐1+/+ cells showed 29.9% PCD whereas the raf‐1−/− cells showed 48.3% PCD (n = 7; 95% CI for difference 9.5–27.4%, P = 0.0007; Figure 3D and E). The raf‐1−/− cells did not show a significant increase in PCD upon serum withdrawal as this treatment induced 26.0% PCD for the raf‐1−/− cells and 18.7% for the raf‐1+/+ cells (n = 7; 95% CI for difference −6.8 to 21.4%, P = 0.284; Figure 3D and E). Hoechst 33258 staining confirmed that the raf‐1−/− cells were more susceptible to PCD induced by the anti‐Fas antibody than the raf‐1+/+ cells, as assessed by nuclear morphology (Figure 3F).
For measuring Raf kinase activities, primary MEFs were immortalized with the SV40 large T antigen and permanent cell lines were derived. Only cells expressing similar levels of the T antigen and with similar growth rates were analysed and compared (data not shown). We first compared the activities of the endogenous Raf proteins in raf‐1+/+ MEFs using the immunoprecipitation kinase cascade assay (Marais et al., 1997). For these initial studies, the conditions were optimized for B‐Raf activity, since B‐Raf is the most active isotype under our assay conditions (Mason et al., 1999). In agreement with our previous studies, B‐Raf had elevated basal activity in unstimulated cells, whereas both Raf‐1 and A‐Raf were inactive (Figure 4A). When stimulated with epidermal growth factor (EGF), B‐Raf activity in raf‐1+/+ cells was increased by ∼2‐fold and returned to basal levels by 60 min (Figure 4A). However, we did not detect any activation of Raf‐1 or A‐Raf in this assay (Figure 4A), despite numerous reports previously describing the activation of Raf‐1 in growth factor stimulated cells (Marshall, 1994 and references therein). We obtained similar results using two other B‐Raf‐specific antibodies, generated to peptides from different regions of B‐Raf, but did not detect B‐Raf activity with these antibodies in MEFs derived from B‐raf−/− embryos (Wojnowski et al., 1997; our unpublished data). However, despite this extremely strong kinase activity of B‐Raf, the protein could not be detected in these cells with these antibodies even by 35S‐labelling or by immunoprecipitation combined with western analysis (data not shown). By contrast, A‐Raf and Raf‐1 are present at high levels in MEFs as detected by western analysis.
Raf kinase activities. (A) Time course of activation of immunoprecipitated A‐Raf, B‐Raf and Raf‐1 in the kinase cascade assay in raf‐1+/+ cells following stimulation with EGF. The conditions for the assay were the same as those used for routinely measuring B‐Raf activity. (B) Time course showing the fold Raf‐1 activation in raf‐1+/+, raf‐1−/− and raf‐1FF/FF MEFs following EGF stimulation. Standard conditions for measuring Raf‐1 activity were utilized. (C) Time course showing the fold A‐Raf activation in MEFs following EGF stimulation. The conditions were the same as for the Raf‐1 assay. (D) Time course showing the fold B‐Raf activation in MEFs following EGF stimulation. The B‐Raf assay conditions were utilized. Each experiment was performed in triplicate and error bars show standard deviations.
The above results suggest that the specific activity of B‐Raf towards MEK as measured by this kinase cascade assay is far greater than either Raf‐1 or A‐Raf. To examine Raf‐1 and A‐Raf activation, we increased the sensitivity of the assay by using 10‐fold more lysate (1 mg) and by increasing the first incubation period from 15 to 30 min (Materials and methods). These have been developed as standard conditions for measuring Raf‐1 activity (Marais et al., 1997). Immortalized raf‐1+/+, raf‐1−/− and raf‐1FF/FF MEFs were made quiescent and stimulated with EGF, platelet‐derived growth factor (PDGF), phorbol 12‐myristate 13‐acetate (PMA) or lysophosphatidic acid (LPA) over a time course of up to 60 min. Raf‐1 kinase activity was stimulated 4‐ to 6‐fold in raf‐1+/+ cells following 2–5 min treatment with EGF and returned to basal levels within 60 min (Figure 4B). As expected, no Raf‐1 kinase activity was detected in the raf‐1−/− cells (Figure 4B). In the raf‐1FF/FF MEFs, no activation of Raf‐1 was detected in response to EGF; the level of activity observed was comparable to that in the raf‐1−/− cells and to background levels in assays performed without substrate. The same Raf‐1 activation results were obtained upon stimulation with PDGF, PMA and LPA (data not shown). In raf‐1+/+ cells, A‐Raf kinase activity was much lower than Raf‐1 activity and was stimulated only by 1.3‐fold upon 2 min treatment with EGF. Similar levels of A‐Raf activity were detected in the raf‐1−/− and raf‐1FF/FF cells compared with raf‐1+/+ cells (Figure 4C). We compared B‐Raf activation in the raf‐1+/+, raf‐1−/− and raf‐1FF/FF cells using the assay conditions described above for B‐Raf. The levels of basal B‐Raf kinase activity in the raf‐1−/− and raf‐1FF/FF cells were similar to that observed in raf‐1+/+ cells (Figure 4D). The time course of activation by EGF was similar in the three cell lines, although the level of induction of B‐Raf in raf‐1−/− and raf‐1FF/FF was elevated slightly compared with raf‐1+/+ cells.
To measure ERK activation, cells were made quiescent and stimulated with EGF and levels of phospho‐ERK were assessed. In the raf‐1+/+ cells, phospho‐ERK increased following 2 min of EGF treatment and continued to increase up to 5 min of treatment but reached basal levels after 60 min (Figure 5A). For the raf‐1−/− and raf‐1FF/FF cells, the level of phospho‐ERK was similar to that in the raf‐1+/+ cells (Figure 5A). ERK phosphorylation was assessed in cells that were made quiescent and then stimulated with EGF, PDGF, serum, LPA and PMA for 10 min (Figure 5B). In the raf‐1+/+ cells, ERK phosphorylation was induced following treatment with all stimuli. A similar level of ERK phosphorylation was observed in the raf‐1−/− and raf‐1FF/FF cells with all stimuli (Figure 5B). ERK activation was also measured by using myelin basic protein (MBP) as a substrate for immunoprecipitated p42ERK. As with the ERK phosphorylation data, ERK activation increased in all cells following 2 min EGF treatment and reached maximum at 10 min (Figure 5C). There was no difference observed in either the time course or level of ERK activity between the raf‐1+/+, raf‐1−/− or raf‐1FF/FF cells following EGF treatment (Figure 5C). ERK activation was also assessed upon stimulation of quiescent cells with sub‐saturating amounts of EGF. MEFs of each genotype were made quiescent and then stimulated with varying concentrations of EGF from 0 to 10 ng/ml for 10 min. With increasing doses of EGF, a greater level of ERK activation was observed in all cell lines (Figure 5D). However, no difference was observed in the level of MBP phosphorylation upon treatment with any given dose of EGF between the raf‐1+/+, raf‐1−/− and raf‐1FF/FF cells (Figure 5D).
ERK activation. (A) Stimulation of ERK phosphorylation in raf‐1+/+, raf‐1−/− and raf‐1FF/FF MEFs over a time course of EGF treatment. (B) Stimulation of ERK phosphorylation in raf‐1+/+, raf‐1−/− and raf‐1FF/FF MEFs following treatment with different stimuli for 10 min. The blots in (A) and (B) were incubated with an anti‐phosphoERK antibody (top panels) and an anti‐ERK2 antibody (bottom panels) to control for protein loading. (C) ERK activation in raf‐1+/+, raf‐1−/− and raf‐1FF/FF MEFs over a time course of EGF treatment as measured by the immunocomplex MBP kinase assay. (D) ERK activation in raf‐1+/+, raf‐1−/− and raf‐1FF/FF MEFs following stimulation with different concentrations of EGF for 10 min as measured by the MBP kinase assay.
The Ras/Raf/MEK/ERK cascade links the activation of cell surface receptors at the membrane with downstream events in the nucleus (Marshall, 1994). A wealth of biochemical data have indicated a role for the Raf‐1 protein kinase in this pathway (Dent et al., 1992; Howe et al., 1992; Kyriakis et al., 1992; Samuels et al., 1993). Genetic data in Drosophila and Caenorhabditis elegans have provided evidence that the Raf homologues in these species stimulate MEK and ERK activation and mediate many of the proliferative and differentiation signals induced by RTKs and Ras (Dickson and Hafen, 1994; Eisenmann and Kim, 1994). Inhibition of Raf‐1 in fibroblasts using antisense or dominant‐negative expressing constructs disrupts proliferation induced by serum, transformation induced by oncogenic Ras and growth factor activation of the ERKs (Kolch et al., 1991; Schaap et al., 1993; Chao et al., 1994; Troppmair et al., 1994). In this study we show that, although Raf‐1 is not the only physiological MEK activator, it is essential for mouse development.
The raf‐1−/− embryo phenotype reported here shows defects in vascularization and placenta development as well as increased apoptosis of many tissues. On the mixed inbred 129Ola/C57BL6 background there was an earlier lethal phenotype than on the mixed 129Ola/MF‐1 background. This strain‐specific phenotype is almost identical to that described by Wojnowski et al. (1998), who generated homozygous mice with a hypomorphic allele for raf‐1 that had 10% residual kinase activity on inbred (C57BL6) and outbred (CD‐1) genetic backgrounds. In the manuscript by Mikula et al. (2001), raf‐1−/− embryos were established primarily on the inbred 129Sv background and these also showed increased apoptosis of fetal liver cells and MEFs as well as similar placental deficiencies. However, those embryos survived to a later stage of development (E12.5–16.5) than the embryos on the 129Ola/C57BL6 background and they did not possess yolk sac vascularization defects. These differences in phenotype are likely to be attributable to different genetic modifiers in each strain, the nature of which is not known.
On the 129Ola/C57BL6 background, the most striking defect in the raf‐1−/− embryos is the absence of endothelial cells in the yolk sac and disorganization of blood vessels in the embryo (Figure 1E–I). The establishment and modelling of blood vessels is controlled by ligands such as vascular endothelial growth factors (VEGFs), angiopoietins and ephrins that modulate the activity of RTKs (Risau, 1997). Vasculogenesis appears to be mediated by VEGF through the VEGF–R2 RTK while angiogenesis is mediated by VEGF through the VEGF–R1 RTK as well as angiopoietins at later stages of development (Fong et al., 1995; Sato et al., 1995; Shalaby et al., 1995; Hanahan et al., 1997). The lack of vascularization in the yolk sac of the raf‐1‐deficient mice suggests a link between Raf‐1 and VEGF–R2 signalling and/or VEGF/VEGFR expression in vivo that cannot be compensated by other Raf proteins. VEGF–R2 has also been implicated in the determination of the haematoangioblast progenitor (Shalaby et al., 1995; Risau, 1997) and the data presented by Mikula et al. (2001) confirm that 129Sv/raf‐1−/− embryos have defects in early stages of haemopoiesis. In cultured endothelial cell lines, VEGF induces Raf‐1/ERK activities and stimulates mitogenesis (Takahashi et al., 1999). How ever, the fact that the raf‐1FF/FF mutant mice do not show defects in vasculogenesis and the fact that ERK activation is not disrupted in the raf‐1−/− MEFs argue that the role of Raf‐1 in this process is largely ERK independent.
The placental and yolk sac deficiencies could clearly contribute to the generalized growth retardation and developmental delay observed in the raf‐1−/− embryos on both genetic backgrounds (Figures 1 and 2). However, in addition to this, the raf‐1−/− embryos and MEFs clearly demonstrate increased levels of apoptosis, whereas there is no defect in the ability of these cells to undergo proliferation (Figures 1,2,3). Therefore, either endogenous Raf‐1 has no role in the regulation of the cell cycle or its function in proliferation is compensated by other MEK activators. A role for Raf‐1 in apoptosis has been indicated from a number of biochemical studies (Wang et al., 1996; Pritchard and McMahon, 1997; Lau et al., 1998) and Ras has been reported to induce apoptosis through activation of Raf‐1/ERKs (Dudek et al., 1997; Kauffmann‐Zeh et al., 1997; Downward, 1998). Activated Ras is also thought to suppress apoptosis through PKB/Akt and recently it has been shown that Akt can suppress Raf‐1 activity by direct phosphorylation of serine 259 (Rommel et al., 1999; Zimmermann and Moelling, 1999). The anti‐apoptotic protein Bcl‐2 has been shown to interact with Raf‐1 and this interaction appears to be independent of Raf‐1 kinase activity (Wang et al., 1994). One function of this interaction may be to target Raf‐1 to the outer mitochondrial membrane where it could promote resistance to apoptosis through phosphorylation of BAD, a pro‐apoptotic protein (Wang et al., 1996). This inhibition of BAD appears to require PI3‐kinase but is ERK‐independent (Neshat et al., 2000). The fact that the raf‐1FF/FF animals have no increased apoptosis also suggests that the role of Raf‐1 in suppression of apoptosis is ERK independent in vivo.
The raf‐1FF/FF animals have no detectable mutant phenotype although we cannot yet exclude the possibility that they are not more or less susceptible to stressful situations such as bacterial infections or tumorigenesis. In addition, the raf‐1FF/FF phenotype has so far only been examined on mixed genetic backgrounds and further backcrossing to the MF‐1 and C57BL6 strains may reveal phenotypic heterogeneity between strains, as has been revealed for the raf‐1−/− animals. The raf‐1FF/FF animals express a mutant version of Raf‐1 that shows no detectable activity towards MEK in the immunoprecipitation kinase cascade assay (Figure 4B), a result that supports our previous data in COS cells, demonstrating that overexpressed RafFF has virtually no MEK kinase activity (Marais et al., 1995). It is possible that RafFF possesses extremely low levels of MEK kinase activity that are beyond experimental detection but are sufficient to allow survival of the RafFF animals. This would then suggest that in some cell types, Raf‐1 is the limiting MEK activator, and would explain the difference in survival between the raf‐1−/− and raf‐1FF/FF animals. However, in the studies of Wojnowski et al. (1998), 10% residual Raf‐1 activity was found to be insufficient for mouse survival and we were unable to detect this level of Raf‐1 activity in the raf‐1FF/FF MEFs. We also observed no difference in the level or profile of ERK activation in the raf‐1−/− and raf‐1FF/FF MEFs compared with the raf‐1+/+ cells (as also shown by Mikula et al., 2001) and, furthermore, MEK activity was detected in both the raf‐1−/− and raf‐1FF/FF cells (data not shown; see also Mikula et al., 2001). Thus, Raf‐1 activity is not required for MEK and ERK activation, at least in some cell types, presumably because other Raf isoforms or MEK activators (such as the MEKK proteins) compensate for the loss of Raf‐1. We did find a slight increase in B‐Raf activity in the raf‐1−/− MEFs, which may in part compensate for the loss of Raf‐1. Taken together these data show that Raf‐1 is not required for ERK activation at least in some cell types and yet animals expressing RafFF, a version of Raf‐1 that is unable to activate MEK, survive.
Even in the raf‐1+/+ MEFs, the kinase activity of endogenous Raf‐1 towards MEK in the immunocomplex kinase cascade assay is difficult to measure unless sensitive assay conditions are utilized (Figure 4A and B). In contrast, immunoprecipitated B‐Raf from MEFs has extremely strong kinase activity towards MEK under much less sensitive assay conditions (Figure 4A), even though the B‐Raf protein is difficult to detect (data not shown). In B‐raf−/− MEFs, ERK activation was reduced compared with wild‐type MEFs (M.Hüser, unpublished; Wojnowski et al., 2000). Taken together, these data provide strong evidence that B‐Raf, rather than Raf‐1, is the primary Raf isotype that activates MEK/ERKs in MEFs, if not more cell types. B‐Raf regulation differs from that of Raf‐1. Unlike Raf‐1 and A‐Raf, B‐Raf does not require tyrosine phosphorylation for activation, and B‐Raf is reported to be activated by Rap1a, notably in PC12 cells (York et al., 1998) and by TC21 (Rosario et al., 1999). B‐Raf also has strong basal kinase activity towards MEK that is independent of Ras, although oncogenic Ras does stimulate B‐Raf activity further through a mechanism involving translocation of B‐Raf to the membrane (Marais et al., 1997; Mason et al., 1999). Thus, although the induction of B‐Raf activity by growth factors in MEFs appears to be weak (2‐ to 3‐fold), it is possible that it is the relocation of active B‐Raf to the plasma membrane in the presence of active Ras that gives the large induction of MEK/ERK activity.
What then is the role of Raf‐1? Our data imply that the MEK kinase activity of Raf‐1 is not essential for normal mouse development, but that the protein is required. A number of other substrates for Raf‐1 have been reported including the dual specificity phosphatase Cdc25A (Galaktionov et al., 1995) and the ankyrin repeat protein Tvl‐1 (Lin et al., 1999). Possibly, the raf‐1−/− phenotype may be manifested through disruption of the activities of these other substrates, whereas RafFF may be able to phosphorylate and activate these substrates. Alternatively, the main function of Raf‐1 may not be as a kinase. Raf‐1 forms part of a multiprotein complex in the cell including HSP90, p50 and 14‐3‐3 proteins, and binding of these scaffold proteins is thought to be important for maintaining the stability of Raf‐1 (Stancato et al., 1993; Wartmann and Davis, 1994; Reuter et al., 1995). Many of the proteins that interact with Raf‐1 also interact with KSR (kinase suppressor of Ras), a putative Ras‐effector protein kinase that was identified by genetic studies in Drosophila and C.elegans (Kornfeld et al., 1995; Sundaram and Han, 1995; Therrien et al., 1995). Studies in C.elegans have shown that the kinase activity of KSR is not important for its function (Stewart et al., 1999). Thus, at least in some circumstances, Raf‐1, like KSR, may not require its kinase activity for function, and like KSR, one of its main functions may be as a scaffold protein (Denouel‐Galy et al., 1998; Yu et al., 1998).
During evolution, three highly conserved raf genes have arisen from a single prototypic gene as a result of genome duplication followed by functional adaptation. Biochemical data indicate that the three mammalian Raf enzymes are differentially utilized and the results presented here suggest that the Raf‐1 protein has evolved such that its kinase activity towards MEK is not essential for mouse development, or that sufficient redundancy exists to overcome the need for Raf‐1 kinase activity, but not for Raf‐1 protein. A‐Raf has extremely weak, if any, activity towards MEK (Figure 4 and Marais et al., 1997) and is primarily located in mitochondria (Yuryev et al., 2000), suggesting that it too has evolved alternative functions. Interestingly, Drosophila raf is more homologous to B‐raf in many domains than A‐raf or raf‐1. It is now important to identify the non‐MEK kinase functions of Raf‐1 and A‐Raf.
Tissues and embryos were prepared, sectioned and then either stained with haematoxylin and eosin or processed for Ki67 immunostaining or TUNEL assay (Monkley et al., 2000). The Ki67 antibody was a rabbit polyclonal antibody supplied by Dianova (Germany). Embryos and yolk sacs for whole mount staining with PECAM‐1 were processed by the method described by Schlaeger et al. (1995).
Individual embryos were dissected and cut, washed several times with cold phosphate‐buffered saline (PBS) and then incubated at 4°C for 2 (raf‐1/C57BL6) or 6 h (raf‐1/MF‐1 and rafFF) in 0.25% (w/v) trypsin. An aliquot of Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS and 100 U/ml penicillin/streptomycin was added and the suspension was transferred to tissue culture plates and cultured. Primary cells were immortalized with the ZIPTEX virus expressing the SV40 large T antigen (Sladek and Jacobberger, 1992). Virus and cells were co‐incubated in 8 μg/ml polybrene for 4–24 h and immortalized cells were grown out by continuous culture.
For growth curves, 2 × 104 primary cells were plated and counted at 24 h intervals in triplicate using a haemocytometer. For DNA synthesis assays the method described by Treinies et al. (1999) was followed. To induce apoptosis, primary cells at 80% confluency on 6 cm dishes were treated with 50 μm etoposide, 50 ng/ml anti‐Fas antibody with 0.5 μm cycloheximide or serum‐free medium for 20 h in a 37°C humidifying incubator. Suspension and attached cells were collected, incubated for 20 min at 37°C in fresh media and stained with annexin V (Bender MedSystems). Stained cells were assessed by FACS analysis (Becton Dickinson). Alternatively, treated cells were stained with 5 μg/ml Hoechst 33258 in PBS for 1 h at 37°C. Data were analysed by using the two‐tailed unpaired t‐test.
MEFs were made quiescent by culturing in DMEM containing 0.5% (v/v) FCS for at least 24 h. They were stimulated by addition of 10 ng/ml EGF, 50 ng/ml PDGF, 40 μM PMA, 200 ng/ml LPA or 20% (v/v) FCS and incubated over a time course of 0–60 min. Protein lysates were prepared as described in Luckett et al. (2000) for the ERK analysis or Marais et al. (1997) for the Raf kinase assays. Western blots were prepared and processed as described previously (Luckett et al., 2000). Primary antibodies were a mouse monoclonal antibody against Thr202/Tyr204 phospho‐p44/42 ERK (New England Biolabs) and a rabbit polyclonal antibody for ERK2 (Leevers and Marshall, 1992). Raf proteins were immunoprecipitated for 2 h at 4°C from 0.1–1 mg cell extract with 2 μg of anti‐Raf‐1 antibody (Transduction Laboratories), 4 μg of anti‐B‐Raf rabbit polyclonal antibody (Mason et al., 1999), 2 μg of anti‐B‐Raf goat polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and 2 μg of anti‐A‐Raf mouse monoclonal antibody (Transduction Laboratories). p42 ERK was immunoprecipitated by using 5 μl rabbit anti‐p42ERK polyclonal antibody (no. 122; Treinies et al., 1999). The activity of each Raf protein was assessed by using the Raf kinase cascade assay using glutathione S‐transferase (GST)–MEK, GST–ERK and MBP together with [γ‐32P]ATP as sequential substrates (Marais et al., 1997). For B‐Raf, 0.1 mg of lysate was used and the first kinase incubation period with GST–MEK and GST–ERK was for 15 min whereas for Raf‐1 and A‐Raf, 1 mg of lysate was used and the first incubation period was extended to 30 min. ERK activity was measured using the MBP kinase assay (Samuels et al., 1993).
We thank Jim Norman, David Critchley, Gerry Cohen and Chris Marshall for critical reading of the manuscript, members of the Marais and Pritchard laboratories for their support, Lee Topping for help with statistics and the Division of Biomedical Services for their help. We also thank Phil Soriano for providing the ZIPTEX virus, Hugh Paterson for providing the DNA synthesis protocol and reagents, Martin McMahon for providing the C‐terminal Raf‐1 antibody and Rich Murray for providing targeting vectors. This work was supported by a Royal Society fellowship to C.P., BBSRC studentships to J.L. and K.M., and project grants from the Cancer Research Campaign and The Wellcome Trust. We thank these organizations for their invaluable support.
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text:
LIFT-UP looks to local farmers as food insecurity doubles
Thomas Phippen
tphippen@postindependent.com
Potter's farm co-owner Justina Potter feeds the cows treats on land owned by John Ward where the cows are currently grazing.
A cow on Potter's farm.
Potter Farms co-owner Justina Potter waits for more cows to come down from the hills on land owned by John Ward where the cows are currently grazing.
Cows walk down from the hillside to receive treats from Justina Potter
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The number of Garfield County families relying on food assistance has more than doubled in the past month, and LIFT-UP is relying on local farms to provide quality food to those in need.
"I love it, because it nourishes the community, as well as the economy," said Angela Mills, executive director of LIFT-UP.
Before the COVID-19 pandemic, and the related economic costs of lockdowns, LIFT-UP typically served 625 families per month. Now, they're serving close to 2,000 at mobile food pantries from Parachute to Carbondale.
The increased demand for food has brought in more funding, which has allowed LIFT-UP to expand an existing Farm to Food Pantry program, sponsored by a LiveWell Colorado grant, designed to help charities source food locally.
"As long as the funding is there, what we've been able to do is bring more farmers on board, and purchase more options from them," Mills said.
One of the existing local suppliers is Potter Farms in Missouri Heights, which supplies ground beef to LIFT-UP. Mills has had to increase orders of the pasture-raised beef in recent weeks to keep up with the need.
"We could go through 1,400 pounds of ground beef a week," Mills said.
Justina Potter, who runs the cattle ranch and farm operation with her husband, Ted, is grateful for the additional orders.
"We have a school and restaurants that we supply to. The school is shut down, and one restaurant is doing takeout here in Carbondale, but it's limited so that's reduced, and the other restaurant is shut down," Potter said.
As the weather warms for vegetable crops, LIFT-UP will have several local garden cooperatives providing fresh produce.
There are other benefits to sourcing food locally beyond nutrition and supporting nearby farms.
"So many people, I think, tend to think that they go to the grocery store, and it will always be there," Potter said.
Pandemic shopping has shown that one cannot always count on items being available.
LIFT-UP hasn't faced supply chain issues with produce or meat, but a few weeks ago cereal was hard to get, Mills said.
"I think that having folks understand how local agriculture and food production is tied to food insecurity is huge, and it's not a conversation we have a lot," said Sara Tymczyszyn, director of the fledgling Highwater Farm in Silt.
As a new producer in the Farm to Food Pantry Program, Highwater is gearing up to plant their first crops at the Silt River Preserve. The farm intends to donate 25 percent of their crop to LIFT-UP, and sell the remaining 75 percent.
Sourcing food for nutrition programs locally could avoid harm from disruptions to the broader supply chains.
"I think that's a benefit because when the bigger operations shut down, they have a place to get products for people in the community," Potter said.
Even if charities and average consumers get more of their food locally, it can be difficult to predict how much local farmers will need to plant to cover demand later in the season, according to Ben Armstrong of the Farm Collaborative at Cozy Point Ranch in Snowmass, which supplies cabbage to LIFT-UP.
"On the one hand, there's a lot of demand for local food because people are realizing the bigger supply chains might be at risk. On the other hand, we know that our normal markets are going to be out of whack," Armstrong said.
"All of us farmers feel very fortunate to still have work during this time. We're going to keep working as hard as we can to grow a lot of food, and hopefully, there will be some programs to get that into everybody's hands," Armstrong added.
AS OF TUESDAY, JAN. 19
El departamento de salud pública del condado de Garfield ofrece gratis pruebas de COVID-19 por la comunidad para Parachute, Rifle y New Castle
Valley View, Grand River suspend COVID-19 vaccine wait lists as demand heightens
Doctor's Tip: New recommendations for Vitamin B12 supplementation

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: What are good Information System design resources? After taking a class about information systems, I want to find additional resources for learning the various patterns, concepts, and examples of their implementation. The course did focus on Martin Fowler's Principles of Enterprise Application Architecture and had a project that attempted to implement some of them.
Basically, where can I learn more about Information System design and implementation?

A: Well, if you are looking for examples from a Coders point of view - this book - Core J2EE Patterns helped me a lot when I was first starting out developing Enterprise applications in Java.  It's examples are specific to Java, but they can easily be applied to most OOP languages.  Of course, there is always the Gang of Four book.
However, if you are looking for more high level concepts... go no further than Fowlers own Domain Specific Languages or something like this.  Also, it never hurts to read classics like Mythical Man-Month or CODE Complete to get overview of what it will take to design, develop and deliver enterprise solutions. (Even if those last two are not specific to enterprise development - but do apply).
Also, one last point - everyone should read this Peopleware.  It really brings home how without the right people, not design will ever be successfully developed and delivered.

redpajama_set_name: RedPajamaStackExchange


The U.S. Department of Justice seized phone records for several Associated Press reporters last year in a move that is raising concerns of government intrusion and overreach in private news gathering practices.
The Associated Press reported on Monday that records relating to 20 telephone numbers in New York, Washington, D.C. and Hartford, Connecticut were seized last year by the DOJ in what it believes is a criminal investigation into the leak of possible classified information.
The AP seems to think the seizure of records was prompted by a May 2012 story involving a terrorist plot that was foiled by the Central Intelligence Agency. Some of the phone records seized by the DOJ belonged to phone numbers associated with reporters who worked on that story.
The DOJ only recently notified the AP that its phone records had been obtained.
The AP's Chief Executive Officer Gary Pruitt has called for the DOJ to return the phone records and destroy any copies.
"The ability of journalists to develop and protect sources is vital to keeping the public informed about issues affecting their lives," the Guild said in a statement on Monday.
On Monday, the White House said it was unaware of the DOJ's attempt to collect AP phone records beyond what had been published in the media.


Jason and Sofia met 4 years ago from their wedding day, on the white sandy beaches of Cuba. I had so much fun with them during their engagement photo session that I couldn't help but look forward to their wedding day! My team and I captured both photography and videography of their big day.
We started off at Jason's place where he got ready with his groomsmen and family. His nephew the ring bearer stole the show in a few of the photos!
Then we headed over to Sofia's place where she got ready with her bridesmaids and family.
The ceremony was held at St Christopher's Church. All eyes (especially Jason's) were on Sofia as she walked down the aisle with her parents.
We went to the Adamson Estate for our creative photo session.
The reception was held at the Mississauga Grand.
Congratulations Jason and Sofia!! We loved capturing your wedding day!


If you love rose and violet creams, have you ever tried lavender and geranium? Our Floral Fondant Creams Chocolate Truffle box contains 8 delicately scented floral fondant creams wrapped in rich dark chocolate and presented in a pretty oval box. Who needs a bouquet of flowers when you could have your very own box of floral fondant creams! Each box contains 2 truffles of each flavour.
Allergy Warning: See ingredients. May also contain traces of nuts and milk as these products are handled in our factory. E124, E102 may have an adverse effect on activity and attention in children.
The key with lavender and geranium, indeed with all floral flavours, is to use high quality oils and essences in small doses.


text:
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Kennedys nets Meadowhall site for Sheffield United
We are pleased to confirm that we have secured a new retail site at the Meadowhall Shopping Centre for Sheffield United Football Club.
A nightmare on the high street
The retail sector has been hard hit over the last 10 years, with an endless list of casualties on the high street but why are we seeing a continual increase of brand name evaporation?
Development finance – lenders must give due consideration to due diligence
Development finance presents one of the more hazardous areas for lenders, due to the risks associated with providing funding at the outset of a project when redeveloping or erecting a new building.
Kennedys grows presence in Portugal with merger
We are pleased to announce that we have grown our presence in Portugal by merging with boutique Portuguese firm N. Maldonado Sousa & Associados. The merged firm will move into our existing Lisbon office in the centre of the city.
Cross-border success for Kennedys in food business acquisition
Commercial Brief May 2019: market insights
A summary of recent developments raising issues in relation to the regulation of the Internet of Things, ethical guidance for artificial intelligence, a consultation to reduce the cost of litigation, environmental considerations in central government contracts, a review of the Consumer Contract Regulations, a consultation on confidentiality clauses, greater protection for residential tenants and an awaited appeal to decide the extent of an employers' liability for their employees actions.
Welcome changes for 'Generation Rent'
With increasing numbers of people renting residential properties long term due to high house prices, the government has introduced legislation limiting the ability of landlords and letting agents to charge certain fees to tenants in the private residential rented sector.
If you build it (affordably), they will come – the answer to the UK's housing crisis?
Homelessness, housing benefit spend, the cost of the private rental market and the average price of houses are all at an all-time high. The number of people able to buy their own home, conversely, is at an all-time low. It is not difficult therefore to see why many have concluded that that there is a housing crisis in the UK.
Kennedys hires partner in Dubai to bolster international arbitration capabilities
We are pleased to announce that international arbitration specialist Celine Abi Habib Kanakri has joined our Dubai office as a partner. Joining from Baker McKenzie Habib Al Mulla, Celine's practice is focused on complex UAE and international arbitration across a range of industry sectors.


text:
Knotel is a flexible office platform that gets, builds and runs and optimizes space for companies. We provide workplaces to some of the world's largest companies and continue to scale globally to do so. Knotel is fast, flexible, full-service & great value
NC Contact Knotel with NOAH Connect!
Edward Shenderovich
Founded in 2016, Knotel has more than 3 million square feet across more than 200 locations in New York, San Francisco, London, Los Angeles, Washington, D.C., Berlin and Brazil. The complete office solution, Knotel brings tenants tailored space on flexible terms without all the fuss typically involved in long-term office leasing. As the leading disruptor in the real estate industry, Knotel provides short- and long-term office space for companies ranging from the small to the enterprise. Knotel offers workplace solutions designed to quickly and easily adapt to the needs of a wide range of office users from start-ups to globally recognized enterprise companies. Knotel's office leases for clients are shorter term than the traditional office lease and office operations are managed behind the scenes by Knotel staff, allowing decision-makers and employees to focus on their core business. Leveraging a team of experts in real estate brokerage, workplace experience and design, Knotel is equipped to identify and brand space for clients' unique corporate culture and business objectives within 30 to 60 days. Knotel was named a Business Insider Top 50 Startup and New York's Hottest New Workspace Model by AllWork. To learn more, visit knotel.com.
https://knotel.com/
Knotel is a company specializing in providing customized, fully managed office spaces for businesses to use as headquarters or new locations. It aims to offer a more scalable and flexible alternative to long-term leasing for business premises and allows its clients to make their spaces fit their company's brand, as well as to grow with their needs. Knotel has been innovating office spaces and offering a revolutionary and cost-effective Agile HQ model in cities all over the world. Property owners can partner with Knotel to improve their offerings and bring in the fastest and best revenue from their commercial spaces. Knotel, based in New York City, NY, USA, has been featured in the Wall Street Journal, the Financial Times, Forbes, and the Commercial Observer. As of May 2019, Knotel has spaces that businesses can use in over 150 world cities, including London, Berlin, Paris, Madrid, Toronto, Sao Paolo, San Francisco, Los Angeles, and NYC, where the business started. It has raised over $160 million USD in investment funding and has attracted members including SeatGeek, Omnicom, HotelTonight and Cheddar. It has also been named as a Top 50 Startup by Business Insider and was named the Hottest New Workspace Model in New York.
Sophie Higgins
Nico Kidel
Tom Dugarin
Jacob Lauer
Hamish Mathieson
Harriet Chambers
Niko Woischnik
Knotel - NOAH19 London
Knotel - NOAH19 Berlin


Details: The eXergy lead free bullet was designed from the ground up by Sellier and Bellot to be the best all-around lead free hunting bullet on the market. Designed to perform like the standard soft pint hunting bullets the eXergy has rapid, consistent expansion. Along with these characteristics the eXergy bullet offers near 100% weight retention along with extreme accuracy making it a perfect choice for loading in hunting ammunition.


It seems Alipay is taking China by storm.
Have are you currently an Alipay user?
Check out this video. Yes. It's actually possible to get around on some days in China without a wallet.
SG50 : Happy National Day to Singapore !
Being Singaporean, I am naturally proud that our island nation have come a long way to celebrate 50 years of independence.
Once again, Happy National Day to Singapore !
Bring an aviation buff, I cant help but to also showcase a photo of the Republic's flying acrobatics team, the Black Knights. These F16s, painted in the colours of the national flag, flew over the skies of Singapore in the run-up events leading to the main celebratory event on 9 Aug.
What a cute luggage bag! Any one want one of these ?
Here's Renee toying with Kindo.


text:
Lady Gaga's Humble Wedding Plans
Amanda Best February 23, 2015
Photo Credit: Helga Esteb / Shutterstock.com
Lady Gaga's massive engagement ring is anything but subtle, but the star could be planning a low-key courthouse wedding.
Gaga, who got engaged to boyfriend Taylor Kinney on Valentine's Day, had little to say about her wedding plans when asked about them on the Oscars red carpet, commenting that she had been too busy rehearsing for her (outstanding) The Sound of Music tribute performance.
But the star told Howard Stern in a radio interview back in November 2013 that she had no interest in planning a big wedding.
He gave me his heart on Valentine's Day, and I said YES!
A photo posted by @ladygaga on Feb 16, 2015 at 1:06pm PST
"The thought of a wedding to me is exhausting," she said at the time. "Why would I put myself through that?" She went on to describe how her "dream wedding" would go: "We get completely hammered, we get the worst dresses we can find and then we go to the courthouse." Only time will tell if Gaga has changed her mind about how she wants her nuptials to go down, but a quiet ceremony would match the couple's life together.
My favorite part of my engagement ring, Taylor and Lorraine designed "T❤S" in white diamonds on the band. He always called me by my birth name. Since our very first date. I'm such a happy bride-to-be! I can't stop smiling!! A photo posted by @ladygaga on Feb 18, 2015 at 4:01pm PST
"I wear his flannels and we eat pizza and we watch TV and then I play him my next single and ask him if he likes it, and that's our normal life," she told Ellen Degeneres in November 2013.
In the same interview Gaga described Taylor, who plays Kelly Severide on the show Chicago Fire, as a "hidden weirdo" and her best friend.
"He's extremely strange, actually, and we complement each other's weirdness."
"At the end of the day he's my best friend, and having your lover be your best friend, I mean, it's the best thing ever."
A photo posted by Taylor Kinney (@taylorkinney11) on Dec 8, 2014 at 4:49pm PST
Aaaw. So sweet!
Taylor Swift 'Elated and Relieved' over Apple Decision
Ed Sheeran Confirmed to Play Deadly Character!
8 Child Actors Who Actually Made It Big
Staff Writer 5 years ago
Ashton and Mila's Mysterious Marriage!
Rapper Troy Ave Arrested After Fatal Shooting at T.I. Concert
Liana Minassian 3 years ago
Amanda Best
Amanda is a proud New Zealander currently calling Canada home. She has a background in journalism, a love of words, cats and tea, and many lofty ambitions.


Steven Mead is widely regarded as one of the most successful professional euphonium soloists in the world today, performing over 75 concerts per year with some of the leading orchestras, wind bands and brass bands in the world. In recent years he has played solo concerti with symphony orchestras, including: Germany (Stuttgart Philharmonic Orchestra) Norway (Trondheim Symphony Orchestra) , Finland (Lahti Symphony Orchestra and Helsinki Philharmonic), Poland (Capella Cracoviensis), USA (Minneapolis Pops Orchestra) and the Japan Chamber OrchestraSteven has also appeared as soloist with many of the leading military bands in the world including Band of the Garde Republicaine (Paris, France), The Central Band of the RAF (London, UK), The Italian Police Band (Rome, Italy), The Dutch Marine Band (Rotterdam,Netherlands), The US Army Band (Washington DC, USA). He has been a featured guest at all the major brass and wind band festivals and low brass festivals, including several at European Brass Band Championships and National Brass Band Festivals as well as at similar events in Norway , the Netherlands , Belgium , Sweden , USA and Japan and the MidWest Band Clinic, Chicago.
There have been world premiere performances of over 250 solo works for euphonium including the premiere performances of euphonium works by Philip Sparke (all 3 of his concerti for solo euphonium), Martin Ellerby (Euphonium Concerto), James Curnow, Tadeus Kassatti, Torstein Aagaard Nilsen (Concerto for Euphonium and Orchestra), Robert Jager, Vladimir Cosma (Concerto for Euphonium and wind orchestra, and brass band), Rolf Wihelm (Concertino for Euphonium), Arthur Butterworth, John Reeman, Thomas Dos, Yasuhide Ito, Marco Putz, Rolf Rudin (Concerto for Euphonium and Symphony Orchestra) and Hermann Pallhuber (Bliss) for example.He is the most recorded euphonium artist in the world, with 62 CDs where he is featured as soloist or guest soloist, including the World of the Euphonium Series (five volumes) the Euphonium Magic series (three volumes) of multitrack recording, five CDs with the British Tuba Quartet (1991-2000) which he formed in 1991.
Steven is featured on 16 educational series books (all with CD) with the deHaske company.
Several of his recordings have won international awards, including British Bandsman Solo CD of the year 2006,2008 and 4Barsrest.com Solo CD of the Year 2006,2008 , Brass Band World Solo CD of the Year 2008, and the Roger Bobo International Recording Award 2006 and 2008. He formed his own recording company, Bocchino Music, order to retain full artistic control.
In recent years Steven has been involved in new creative projects as a soloist with leading chamber groups in Europe: Spanish Brass quintet – widely regarded as the leading Brass quintet in Europe today. A recently recorded an album includes two new works by leading Spanish composers.
Trombonisti Italiana – with this quartet of Italian trombonists, he recorded two albums, the second being 'Colours of the World' which was an original concept that sourced some previously unknown folk music from around the world. They have also given over 15 concerts in Europe.
Sound Inn Brass – Together they recorded two albums with this professional 12-piece symphonic brass ensemble based in Upper Austria. Several of the works recorded were first performances of original compositions an arrangements for euphonium and symphonic Brass ensemble. They have toured Europe together giving over 30 concerts in the last eight years and was recently been appointed musical director and conductor of this ensemble.
Classic Quintet – This collaboration with this woodwind quintet, based in Bolzano, Italy produced two albums over 20 concerts . Steven is the first euphonium player to develop repertoire for euphonium and woodwind quintet. Several of these works have now been published.
Steven is a founder member and solo euphonium of the Brass Band of Battle Creek, USA since 1992, (www.bbbc.net) the leading fully professional brass world in the world.
As well as teaching at the RNCM for over 20 years, in the role of Senior Tutor, he has sought to enhance brass pedagogy around the world. He has recently been appointed a Fellow of the RNCM, for his services to this hallowed institution. He was one of the very first euphonium teachers in higher education in the UK, first being invited to teach euphonium at the Royal Academy of Music by the then Head of Brass, Harold Nash, a position he held for five years, until deciding to concentrate his activities further north , firstly teaching at the Birmingham conservatoire of 12 years where he was also Director of Conservatoire Brass Band and Wind Orchestra for four years. Then Steven concentrated his teaching activities at the RNCM where he attracted students from many countries including, USA, Japan, Taiwan, Australia, Norway, Sweden, Switzerland, France and China. Many of these students have returned to their countries and won positions as teachers or as members of leading military orchestras.
Steve has also been involved with innovative euphonium education and the world , for example at specialist He directed the Euphonium Course at the new European Superior Institute for Bands , in Trento, Italy (2000-2006) , co-sponsored by the European Union. He has also given many master classes and workshops in Holland, Germany, Austria, Switzerland, Italy , Spain, Luxembourg, France, Norway, Sweden, Estonia, Russia, Denmark, Finland, USA, Canada, Japan, Singapore, Belgium, Australia, New Zealand, Slovenia, Lithuania and Poland.
Steve directed or co-directed all six of British National Tuba Euphonium Festivals, 1994/95/97/99/00/04 in Birmingham and Manchester as well as 3 Dutch Tuba Festivals and 2 Belgian Low Brass Festivals. Now he is the Artistic Director of the ITEC 2012, to be held in Linz, Austria, June 23-30, 2012.
He is proud of his work as an instrument consultant and clinician for the instrument company Besson for over 25 years and has been responsible for the development and design work for their three most recent professional model Euphoniums , the Besson GS model euphonium, the Besson Prestige and Sovereign Euphoniums, suggesting fourteen new design features that were accepted for the new Prestige range. Now he is involved with all stages of development work and subsequent testing, visiting the factory in Markneukirchen, Germany each month to play test their entire professional series production.
With the mouthpiece manufacturer Denis Wick – he originated the 'SM' and 'Ultra' ranges, much sought after and played by many of the leading players in the world. It is estimated that over 42,000 of these mouthpieces have been sold around the world since 1995. He has also recently designed the highly acclaimed SM Travel Mute.
Through his specialist websites www.euphonium.net and www.euphoniumstore.net he aims to not only inform the world about development of the euphonium but also facilitate the circulation of all newly published original music for euphonium and tuba with over 500 titles in the catalogue.


text:
Q: Sending a message to 'self' causes initialize method to be called? I have two classes Foo and Bar:
require 'pry-byebug'
require 'fileutils'

class Foo < Pathname
  include FileUtils
  def initialize(path)
    puts "Inside Foo init..."
    super
    puts "Side effect happening..."
  end

  def some_method
    puts "Inside some_method inside Foo..."
    basename.to_s
  end
end

class Bar < Foo
end

bar = Bar.new('bar')
# binding.pry
bar.some_method

This is the output:
Inside Foo init...
Side effect happening...
Inside some_method inside Foo...
Inside Foo init...
Side effect happening...

As you can see the "side effect" is happening twice. Taking a look at a pry-byebug session confirms:
Inside Foo init...
Side effect happening...

From: /Users/max/Dropbox/work/tmp/super_test/foo.rb @ line 23 :

    18: class Bar < Foo
    19: end
    20:
    21: bar = Bar.new('bar')
    22: binding.pry
 => 23: bar.some_method

[1] pry(main)> step

From: /Users/max/Dropbox/work/tmp/super_test/foo.rb @ line 13 Foo#some_method:

    12: def some_method
 => 13:   puts "Inside some_method inside Foo..."
    14:   basename.to_s
    15: end

[1] pry(#<Bar>)> step
Inside some_method inside Foo...

From: /Users/max/Dropbox/work/tmp/super_test/foo.rb @ line 14 Foo#some_method:

    12: def some_method
    13:   puts "Inside some_method inside Foo..."
 => 14:   basename.to_s
    15: end

[1] pry(#<Bar>)> step

From: /Users/max/Dropbox/work/tmp/super_test/foo.rb @ line 7 Foo#initialize:

     6: def initialize(path)
 =>  7:   puts "Inside Foo init..."
     8:   super
     9:   puts "Side effect happening..."
    10: end

So breaking it down:

*

*I instantiate bar which is an instance of Bar which inherits from Foo. Bar's superclass' initialize is called and the "side effect" happens. So far this is totally expected.

*I call some_method on bar who doesn't have it so ruby goes up and to the right and finds it inside of Foo

*Ruby hops inside of some_method and finds a method that's sending a message to self called basename

*Ruby goes back to Foo's' initialize method?...

Step 4 is catching me completely by surprise. Why would sending a message to self cause the initialize method to get called again? Is this documented anywhere? Is this expected?
Is it possible to control this? Or conditionally check if I'm inside the initialize method because I'm actually instantiating a class and not just randomly landing there? For example:
class Foo < SomeClass
  def initialize args
    @args = args
    if instantiating_a_class?
      puts "Side effect happening..."
    else
      puts "Don't do anything..."
    end
  end
end

A:
Why would sending a message to self cause the initialize method to get called again? Is this documented anywhere? Is this expected?

That's how basename is implement, it returns a new instance:
/*
 * Returns the last component of the path.
 *
 * See File.basename.
 */
static VALUE
path_basename(int argc, VALUE *argv, VALUE self)
{
    VALUE str = get_strpath(self);
    VALUE fext;
    if (rb_scan_args(argc, argv, "01", &fext) == 0)
        str = rb_funcall(rb_cFile, rb_intern("basename"), 1, str);
    else
        str = rb_funcall(rb_cFile, rb_intern("basename"), 2, str, fext);
    return rb_class_new_instance(1, &str, rb_obj_class(self));
}

The last line is equivalent to calling new.
You can verify this easily:
class Foo < Pathname
  def initialize(path)
    puts "initialize(#{path.inspect})"
    super
  end
end

foo = Foo.new('foo/bar/baz')
# prints initialize("foo/bar/baz")
#=> #<Foo:foo/bar/baz>

foo.basename
# prints initialize("baz")
#=> #<Foo:baz>

A: As you said it has to do with basename method. As it can be seen in the source code of the documentation, basename instantiates another object of the class.

A: Pathname instance methods usually return Pathname instances. To do so, they need to call initialize on the current class.
If you look at the sourcecode of basename :
 return rb_class_new_instance(1, &str, rb_obj_class(self));

If it's not the desired functionality of your Foo and Bar classes, you could stop inheriting from Pathname, and define a @pathname instance variable.
Finally, you probably don't want to create directories automatically in initialize as you proposed yesterday :
Getting the basename of file.txt could create a file directory.

A:
Step 4 is catching me completely by surprise. Why would sending a message to self cause the initialize method to get called again? Is this documented anywhere? Is this expected?

Well, yes. Methods are allowed to call other methods. That's pretty much the whole point of methods. basename returns a new Pathname object. So, how do you think it constructs this new Pathname object? Of course, it calls self.class::new (which is actually Class#new) which in turn calls Pathname#initialize.
This is what the implementation of Pathname#basename looks like in Rubinius's implementation of the Ruby standard libraries:

def basename(*args) self.class.new(File.basename(@path, *args)) end

The implementation of Class#new looks roughly like this:
class Class
  def new(*args, &block)
    # allocate a new empty object from the ObjectSpace
    obj = allocate

    # initialize it (must use send because initialize is private)
    obj.send(:initialize, *args, &block)

    # return object that was initialized
    obj
  end
end


text:
Gblog -- A Simple Python Blog
=======================

This is a simple blogging engine based on python 3 and uses MySQL to store posts. See [Demo](http://gongyusong.com)

Requirements
-------------

1. python 3
3. MySQL(mariaDB)

And the required Python packages (using pip or easy_install to install):

* tornado
* MySQL-python
* Markdown
* Pygments
* Unidecode

目前封装 mysql 接口的 python 官方包还不支持 python 3 的,可以使用第三方的项目 [MySQL-for-Python-3](https://github.com/davispuh/MySQL-for-Python-3) :

```
git clone https://github.com/davispuh/MySQL-for-Python-3.git
python3 setup.py install
```

Set Up MySQL
------------

Since it depends on MySQL, you need to set up the database schema. Connect to MySQL and create a database and user for the blog.

1. Create the database

```
# Connect to MySQL as a user that can create databases and users:
mysql -u root

# Create a database named "blog":
mysql> CREATE DATABASE blog;

# Allow the "blog" user to connect with the password "blog":
mysql> GRANT ALL PRIVILEGES ON blog.* TO 'blog'@'localhost' IDENTIFIED BY 'blog';
```

2. Create the tables in your new database (using gblog/data/schema.sql).

```
# use the provided /data/schema.sql file by running this command:
mysql --user=blog --password=blog --database=blog < schema.sql
```

Run the blog
------------

Install and run in test

```
python3.4 setup.py develop
gblog --port=3000
```

or you can use `nginx` and `supervisor` in production.

Configure
------------

The configure file `gblog.conf` contains main blog settings.

```
mkdir ~/.gblog
cp /path/to/gblog/data/gblog.conf ~/.gblog/gblog.conf
```

redpajama_set_name: RedPajamaGithub


text:
Watch the 11 p.m. news live right now on Local 4+
Expect More from Local 4 News at 11. Your source for Local information & breaking news across southeast Michigan plus 4Warn Weather providing you with accurate forecasts so you can plan your day.
Brandon Carr, Digital Content Producer
Tags: Detroit, Wayne County, Southfield
Crash closes all lanes of I-96 at Milford Road in Oakland County
Southfield man charged with armed robbery of 2 dollar stores in Detroit
Fernando Darryl Ford was given a $300,000 cash bond with GPS tether for both cases
Southfield man charged with armed robbery of 2 dollar stores in Detroit (Detroit Police Department)
DETROIT – Prosecutor Kym Worthy has charged Fernando Darryl Ford, 30, of Southfield with armed robbery of two dollar stores in Detroit.
The first incident occurred Friday (Jan. 13) at 7:10 p.m. in the 11630 block of Greenfield Road as Detroit police were called to the store for a reported robbery.
When officers arrived, the victim said Ford produced a handgun before robbing and fleeing the store.
The second incident occurred Friday (Jan. 13) at 7:45 p.m. in the 14610 block of McNichols Street as Detroit police were called again for another robbery.
A female employee said Ford entered the store as a customer before producing a gun, robbing the store, and fleeing on foot.
Investigation into a multitude of robberies since New Year's Day led to the 30-year-old man's arrest on Saturday (Jan. 21).
Read: Police: 20 dollar stores robbed at gunpoint in 3-week span in Detroit
Ford has been charged with two counts of armed robbery and one count of felony firearm.
He was arraigned on both cases on Tuesday (Jan. 24) in the 36th District Court and given a $300,000 cash/surety bond with a GPS tether for both cases.
A bond redetermination hearing is scheduled for Jan. 26 at 9:30 a.m. The probable cause conference on both cases is scheduled for Jan. 31 at 8:30 a.m., and the preliminary examination on both cases is scheduled for Feb. 6 at 8:45 a.m.
Brandon Carr
Brandon Carr is a digital content producer for ClickOnDetroit and has been with WDIV Local 4 since November 2021. Brandon is the 2015 Solomon Kinloch Humanitarian award recipient for Community Service.


Multiple sources of coldwater fish provide protein and essential amino acids for overall health and maintenance.
Grains have been replaced by lentils and sweet potatoes, selected for their nutritional value and low-glycemic properties. Low-glycemic carbohydrates, which slow or lower glucose release into the bloodstream, may lower the risk of diabetes and help prevent obesity.
Certified low glycemic and diabetic pet friendly by the GRI, a professional clinical research organization specializing in glycemic index testing.
Salmon oil and coldwater Atlantic herring are used as the primary sources of DHA and EPA omega-3 fatty acids, to contribute to a healthy skin and coat.
Powerful prebiotics & probiotics provide a greater absorption of nutrients and promote digestion, while glucosamine & chondroitin are incorporated to maintain joint health.
Ingredients:Deboned haddock, deboned cod, deboned flounder, deboned pollock, ocean whitefish meal, green peas, menhaden fish meal, sweet potatoes , lentils, canola oil (preserved with mixed tocopherols), sun-cured alfalfa meal, deboned herring, tapioca, natural fish flavor, salmon oil (source of DHA), coconut oil, pumpkin, butternut squash, carrots, spinach, broccoli, apples, pears, blueberries, cranberries, pomegranate, juniper berry extract, ginger, fennel, green tea extract, peppermint leaf, licorice root, turmeric, vitamins vitamin E supplement, ascorbic acid (vitamin C), vitamin A supplement, niacin, calcium pantothenate, inositol, riboflavin, pyridoxine hydrochloride, thiamine mononitrate, biotin, vitamin B12 supplement, vitamin D3 supplement, folic acid, minerals zinc oxide, zinc proteinate, iron proteinate, ferrous sulfate, copper proteinate, copper sulfate, manganese proteinate, manganese oxide, calcium iodate, sodium selenite, potassium chloride, dicalcium phosphate, lecithin, salt, chicory root extract, choline chloride, dried kelp, DL-methionine, yeast extract, glucosamine hydrochloride, taurine, yucca schidigera extract, L-lysine, rosemary extract , chondroitin sulfate, L-carnitine, dried Lactobacillus plantarum fermentation product, dried Enterococcus faecium fermentation product, dried Bacillus subtilis fermentation product, dried Bifidobacterium animalis lactis fermentation product, dried Lactobacillus casei fermentation product, dried Lactobacillus helveticus fermentation product, dried Bifidobacterium longum fermentation product, dried Lactobacillus acidophilus fermentation product.


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Shipping to Switzerland
If you have baggage that requires shipping to Switzerland, Tudor International Freight has a range of shipping and forwarding services to get your packages where they need to be. If you are shipping to Geneva or you would like some additional advice in respect of shipping to Zurich, please don't hesitate to contact our team. We are also shipping to Berne along with many other Swiss destination on a regular basis, and offer a door to door road freight service to most European destinations.
Air Freight Services to Switzerland
Air Freight Charges to Switzerland Airports
Berne (BRN)
Lugano (LUG)
The above costs are up to arrival at the airport only. They do not include ANY charges which may be incurred in Switzerland such as (but not only) terminal handling, documentation, customs clearance, examination, quarantine or inspection fees, customs duties, taxes or storage costs.
Tudor International Freight has been shipping to Switzerland for many years. If you have any specific queries or require some further advice, please contact us.
A fully completed Shippers Inventory in English is required if you are shipping to Switzerland.
We are not able to offer a sea freight service to this destination.
Road Freight Services to Switzerland
Road Freight Charges to Switzerland
First 30 Kilos
Additional Kilos
Berne by Road
Geneva by Road
Lugano by Road
Zurich by Road
The above costs include all related transportation services up to arrival at the consignee's door.
The packages will not be delivered inside of the consignee's home. The consignee must be at the delivery address to receive the goods when the packages are delivered and they may be required to retrieve the consignment from the delivery vehicle. If the shipment has been palletised the consignee may be required to unwrap the pallet on the vehicle to retrieve the packages.
Please note that if the delivery address cannot be accessed by the delivery vehicle, the consignee may be required to retrieve the packages from the vehicle at the closest point of access.
The above costs do not include ANY charges which may be incurred in Switzerland such as (but not only) terminal handling, documentation, customs clearance, examination, quarantine or inspection fees, customs duties, taxes or storage costs.
Door to Door by Air Services to Switzerland
Door to Door by Air Charges to Switzerland
Berne Door to Door
Geneva Door to Door
Lugano Door to Door
Zurich Door to Door
All door to door by air shipments are subject to a £35.00 handling and security charge in the UK in addition to the freight costs.
These fees, if applicable, must be paid by the consignee before the delivery can take place.
The consignee must be at the delivery address to receive the goods when the packages are delivered.
The above costs do not include ANY charges which may be incurred in Switzerland such as (but not only) customs duties, taxes, examination, quarantine or inspection fees. These fees, if applicable, must be paid by the consignee before the delivery can take place.
Excess Baggage Services to Switzerland
For those searching for excess baggage services to Switzerland, Tudor Freight International has over 20 years' experience in helping people ship their excess baggage to destinations throughout Switzerland.
We've helped a great range of people save thousands on expensive airline rates while delivering their excess baggage to destinations across the world.
Transit times to Switzerland
For those using our road freight service to Switzerland, transit time can take between seven and 12 days.
If you're looking to use our air freight service to Switzerland, the transit time for this service is seven days.