text:
DivIVA is a conserved protein in Gram‐positive bacteria and involved in various processes related to cell growth, cell division and spore formation. DivIVA is specifically targeted to cell division sites and cell poles. In Bacillus subtilis, DivIVA helps to localise other proteins, such as the conserved cell division inhibitor proteins, MinC/MinD, and the chromosome segregation protein, RacA. Little is known about the mechanism that localises DivIVA. Here we show that DivIVA binds to liposomes, and that the N terminus harbours the membrane targeting sequence. The purified protein can stimulate binding of RacA to membranes. In mutants with aberrant cell shapes, DivIVA accumulates where the cell membrane is most strongly curved. On the basis of electron microscopic studies and other data, we propose that this is due to molecular bridging of the curvature by DivIVA multimers. This model may explain why DivIVA localises at cell division sites. A Monte‐Carlo simulation study showed that molecular bridging can be a general mechanism for binding of proteins to negatively curved membranes.
There is a Have you seen ...? (August 2009) associated with this Article.
DivIVA is conserved in Gram‐positive bacteria. Secondary structure predictions show that the protein mainly forms α‐helices (Figure 1A), and as DivIVA has some sequence similarity with tropomyosin, it is assumed that it forms coiled coils (Edwards et al, 2000). Biochemical and electron microscopic studies have shown that the purified protein forms multimers and assembles into large ordered lattices (Stahlberg et al, 2004). Despite the sequence conservation, the functional role of this protein varies between different bacterial species. In Bacillus subtilis, mutations in divIVA result in elongated cells that occasionally divide aberrantly near existing cell poles to produce minicells (Cha and Stewart, 1997). Fluorescence microscopy studies have shown that DivIVA is located at mid‐cell during cell division and at matured cell poles, and that it is responsible for the polar localisation of the division inhibitor, MinC/MinD (Edwards and Errington, 1997; Marston et al, 1998). The first step in cell division in most bacteria is the polymerisation of the tubulin‐like protein, FtsZ, into a ring‐like structure (the Z‐ring) onto which the cytokinesis apparatus assembles. In rod‐shaped bacteria, MinC/MinD prevents polymerisation of FtsZ close to cell poles (Margolin, 2001; Hale and de Boer, 2002). When MinC/MinD is delocalised, as a consequence of inactive DivIVA, cell division is largely inhibited and, in addition, cells can divide aberrantly close to cell poles, producing small anucleate minicells. DivIVA also plays an important role during sporulation. Incorporation of DNA into the polar prespore compartment is achieved by anchoring one chromosome copy to the distal pole of the prespore compartment. One of the proteins involved in this process is RacA, and DivIVA is responsible for the polar localisation of RacA (Ben‐Yehuda et al, 2003; Wu and Errington, 2003).
DivIVA homologues are also present in Gram‐positive cocci such as Streptococcus pneumoniae and Staphylococcus aureus (Figure 1A), although these bacteria do not contain a Min system and do not sporulate. In S. pneumoniae, deletion of divIVA results in a severe cell division defect, and two‐hybrid experiments suggested that DivIVA interacts with a number of cell division proteins (Fadda et al, 2003). It is surprising that in S. aureus, a divIVA deletion shows no apparent phenotype (Pinho and Errington, 2004). In the filamentous branching actinomycete, Streptomyces coelicolor DivIVA has an important function in tip growth and branching (Flardh, 2003; Hempel et al, 2008). Recently, it was suggested that a cellulose‐synthase‐like protein, involved in tip growth, may interact with DivIVA (Xu et al, 2008). In the non‐sporulating, non‐branching actinomycetes, such as Corynebacterium glutamicum and Mycobacterium tuberculosis, DivIVA is also required for polar growth (Kang et al, 2008; Letek et al, 2008). In all bacteria investigated so far, DivIVA localises to division sites and/or polar regions. It is therefore assumed that DivIVA functions as a scaffold that helps to localise other proteins to specific regions within the cell.
Despite the importance of DivIVA for cell morphology in such a diversity of Gram‐positive bacteria, little is known about the mechanism responsible for the localisation of DivIVA. An earlier mutagenesis study showed that mutations in the conserved N terminus of B. subtilis DivIVA destroy its polar localisation (Perry and Edwards, 2004). These mutations likely block interactions of the protein with a specific polar target. As the target might be a membrane‐localised protein, we examined the affinity of DivIVA for isolated cell membranes. It is surprising that it appeared that DivIVA has a general affinity for the phospholipid fraction of the cell membrane. Using cell shape mutants, we show that DivIVA preferably binds to strongly curved cell membranes. On the basis of electron microscopy (EM) results and earlier published data, we propose a general model for binding of proteins to negatively curved membranes.
DivIVA binds to membranes
A simple explanation for the localisation of DivIVA would be that this protein is targeted by another protein; possibly a membrane protein. To examine this, we isolated cell membranes from a B. subtilis strain lacking DivIVA (B. subtilis 3310) and mixed these membranes with purified DivIVA‐GFP. This mixture was then loaded onto a sucrose step gradient. After centrifugation, fractions from the gradient were analysed by western blotting using a GFP‐specific antibody. As shown in Figure 2, the addition of cell membranes resulted in shifting a part of the DivIVA‐GFP fraction towards the bottom of the gradient, where the membranes accumulated (Figure 2 B). When no membranes were present, DivIVA‐GFP remained in the top fraction of the sucrose gradient (Figure 2A). The reaction mixture contained an excess of BSA (1 mg/ml), suggesting that the interaction of DivIVA‐GFP with the membranes was specific. This was further supported by an experiment in which the binding buffer and gradient contained a high concentration of salt (0.5 M NaCl, Figure 2C).
To ascertain whether DivIVA‐GFP interacted with membrane‐bound proteins, we treated the membranes with Proteinase K. To our surprise, this treatment had no effect on the binding of DivIVA‐GFP (Figure 2D). The amino‐acid sequence of DivIVA does not show an apparent membrane‐interacting domain. Nevertheless, the lack of any effect of Proteinase K suggested that DivIVA might interact with the lipid fraction of the cytoplasmic membrane. To test this, we examined the association of DivIVA with lipid vesicles constructed from pure phospholipids. As shown in Figure 2E and F, purified DivIVA‐GFP bound efficiently to liposomes as well. It was possible that the GFP moiety somehow stimulated the interaction with phospholipids, therefore we purified wild‐type DivIVA and repeated the experiments. Pure DivIVA (D) also showed a clear interaction with liposomes (Figure 2G and H). As a final control, we tested purified GFP. This protein (G) did not show any interaction with liposomes (Figure 2I and J). In the sucrose gradients that contained DivIVA or DivIVA‐GFP, the liposomes appeared to aggregate. Clustering of liposomes by these proteins was in fact easily detectable by light microscopy (Supplementary data).
As an independent way to test the affinity of DivIVA for lipids, we turned to surface plasmon resonance (SPR). Liposomes were adsorbed onto an L1 sensor chip and purified GFP, DivIVA‐GFP, or DivIVA was injected. As shown in the sensograms of Figure 3A, the addition of GFP yielded only a small response (a), likely due to a buffer effect given that the signal dropped back to the baseline when the GFP injection ended (b). In contrast, the magnitude of the response was strong when DivIVA‐GFP was injected (Figure 3B), and the signal remained when the flow of DivIVA‐GFP ceased, indicating that the fusion protein strongly interacted with the phospholipid membranes (the initial sharp decrease was due to buffer effects). Induction of DivIVA (Figure 3C) also resulted in a strong response and, together with the low off‐rate, confirmed that this protein binds specifically to liposomes. Owing to the complex oligomerisation characteristics of DivIVA, it is difficult to deduce kinetic parameters from these sensograms. However, the difference in off‐rates between DivIVA and DivIVA‐GFP suggests that the GFP‐tag influences membrane binding.
The N terminus of DivIVA contains the membrane‐targeting domain
DivIVA contains several amphipathic helices that stimulate multimerisation by means of coiled‐coil interactions (Muchova et al, 2002; Rigden et al, 2008). However, amphipathic helices can also interact with lipid bilayers, and, for example, the cell division proteins FtsA and MinD bind to membranes by means of amphipathic helices at their C termini (Szeto et al, 2002; Hu and Lutkenhaus, 2003; Pichoff and Lutkenhaus, 2005). Possibly, the association of DivIVA with membranes is mediated by an amphipathic helix as well. To rate the potential amphipathic helices in DivIVA, we used the program LOCATE (Jones et al, 1992). Figure 1B shows the outcome of this algorithm. The putative amphipathic helices with the highest scores span amino acids 22–41, 102–118, and 145–154 (Figure 1B). Of these three sequences, the helix at amino acids 102–118 seemed least likely, as this region showed less sequence conservation than the others (Figure 1A). To test whether the N‐ or C‐terminally located amphipathic helices were required for membrane binding, we made several deletion constructs. Initial attempts using a DivIVA‐GFP fusion resulted in inclusion bodies or very low protein yields. Therefore, we used maltose‐binding protein (MBP) as a tag, as this protein is very soluble and easy to purify. As shown in the sensograms of Figure 3D, MBP does not bind to liposomes. However, when MBP was fused to DivIVA, the purified fusion protein did bind to the sensor chip (Figure 3E). Two deletion mutants were constructed, namely ΔN‐DivIVA‐MBP and ΔC‐DivIVA‐MBP, in which the N‐terminal 40 amino acids or the last 20 amino acids of DivIVA are lacking. Gel filtration experiments showed that neither deletion interfered with the oligomeric state of the protein (Supplementary data). As shown in the sensograms of Figure 3F and G, the absence of the predicted C‐terminal amphipathic helix did not affect binding to liposomes, whereas the absence of the predicted N‐terminal amphipathic helix abolished binding. The fusion to MBP reduced aggregation of DivIVA and this made it possible to test membrane binding using a simple sedimentation protocol (Figure 3H). Furthermore, membrane binding was abolished by the N‐terminal deletion but not by the C‐terminal deletion. When we looked at the effect on liposomes with light microscopy, it was clear that the N‐terminal deletion abolished clustering of liposomes, whereas purified ΔC‐DivIVA‐MBP was still able to aggregate liposomes (data not shown). These results suggest that the N‐terminal amphipathic helix is the membrane‐targeting domain.
The first 60 amino acids of DivIVA are sufficient for membrane binding
As described earlier (Edwards et al, 2000), DivIVA fused to GFP accumulates at the polar periphery of Escherichia coli cells (Figure 4A). We used this as an assay to further analyse the membrane‐targeting domain. Short amino‐acid stretches of the N terminus of DivIVA were fused to GFP, and the fusion proteins were expressed in E. coli. As the N‐terminal amphipathic helix spans amino acids 22–41 (Figure 4E), we fused the first 40 or 50 amino acids of DivIVA to GFP. Both fusions resulted in a diffuse fluorescence signal in E. coli (Figure 4B, and data not shown). Western blot analysis showed that the fusions were stable; hence, the diffuse GFP signal was not a consequence of proteolytic cleavage (data not shown). However, when the first 60 amino acids of the N terminus were fused to GFP, a peripheral fluorescence signal appeared (Figure 4C). Expression of this fusion protein in a B. subtilis strain lacking divIVA resulted in a peripheral fluorescence signal as well (Figure 4D), suggesting that the N terminus contains the membrane‐binding domain. However, when this truncation was purified and mixed with liposomes, no clustering was observed (data not shown). To test whether the large amphipathic helix within the N‐terminal 60 amino‐acid domain is involved in membrane binding, we changed one of the hydrophobic amino acids into a hydrophilic glutamate residue (Figure 4E). When valine 25 was mutated into a glutamate, a completely diffuse fluorescence signal was observed in E. coli. The same occurred when leucine 29 was mutated into a glutamate (data not shown, results comparable to Figure 4B). Western blot analysis indicated that these point mutations did not affect the stability of the fusion protein in E. coli (data not shown).
In E. coli, the MinCD proteins oscillate between the cell poles in less than a minute (Hu and Lutkenhaus, 1999; Raskin and de Boer, 1999). The fluorescent signals in Figure 4A and C are unequally distributed between the cell poles, which could indicate oscillation of the GFP‐fusion proteins, as well. However, time‐lapse experiments of DivIVA‐GFP‐expressing E. coli cells show no oscillation of the fluorescence signal, but indicate that DivIVA‐GFP continues to accumulate as the cell poles age (Supplementary data). This is possibly due to the inert nature of cell poles compared with the continuously growing lateral wall (Lindner et al, 2008).
EM analyses of DivIVA‐membrane interactions
There are several examples of peripheral membrane proteins that cause deformation of phospholipid bilayers, MinD is one such protein (Hu et al, 2002). Whether the binding of DivIVA influences the shape of lipid membranes was tested using EM. We first examined liposome–DivIVA mixtures by negative staining EM. As shown in Figure 5A–C, the presence of DivIVA seemed to result in liposomes stacked on top of each other, which gives a multilayered appearance (Figure 5C). Some ruffled edges can be observed on the top layers (Figure 5B), but this could be an artifact due to drying and was not observed in isolated liposomes. As the stacking of liposomes in negative staining EM obscured the observation of a possible deformation of liposomes, we fixed the samples and prepared them for thin section transmission electron microscope (TEM). Furthermore, the clustering of liposomes by DivIVA was apparent (Figure 5E). Often the DivIVA complexes that held liposomes together appeared as cruciform‐like structures (Figure 5E–H). Figure 5I shows part of a large liposome onto which a cluster of DivIVA molecules is attached via short stalks. Despite the clear association between DivIVA and liposomes, the TEM micrographs showed no indication that DivIVA deforms lipid membranes.
DivIVA accumulation at negatively curved membranes
In B. subtilis, the brightest DivIVA‐GFP signal appears at cell division sites in a ring‐like pattern that does not follow the constriction of the Z‐ring (Harry and Lewis, 2003). This is also illustrated in Figure 6A. In this case, we used deconvolution of a Z‐stack of images to increase the resolution. At the site of cell division (bands across cells in the membrane image), DivIVA‐GFP gives a dumbbell‐shaped fluorescence signal that is indicative of a ring‐like accumulation of the fusion protein. Interestingly, this is also the region where the membrane is most strongly curved. So, although DivIVA itself does not induce curves in lipid membranes, it might be that the protein has a preference for strongly curved membrane regions. There is currently no established method to test for binding to negatively curved membranes. As an alternative, we looked at the localisation of DivIVA‐GFP in aberrantly shaped cells. First, we re‐examined an old observation that suggested that DivIVA‐GFP tends to accumulate at protrusions formed in cells that contain an mreB mutation (Hamoen and Errington, 2003). These protrusions form strongly negative curved membranes, and indeed, as shown in Figure 6B, the strongest GFP signals colocalised with the bulges in the deformed mreB mutants. Thus, the preference for negatively curved membranes does not seem to be restricted to cell division sites. To test this further, we analysed the situation in E. coli. In wild‐type E. coli cells, DivIVA‐GFP accumulates primarily at the poles and forms clear fluorescent arcs that follow the curvature of the poles (Figure 6C). There are E. coli mutants that are devoid of murein hydrolases necessary to hydrolyse the peptidoglycan of the division septum. These mutants cannot separate after division and form long chains of cells (Heidrich et al, 2001). A consequence of this is that the poles of the cells are not round but flat, and that a relative sharply curved membrane is generated at the transition from a pole to a lateral wall (see model in Figure 6E). Expressing DivIVA‐GFP in such an E. coli mutant resulted in strong dumbbell‐shaped fluorescence signals at the fused cell poles, as shown in Figure 6D; thus, an indication for a ring‐like accumulation pattern. Hence, also in E. coli, DivIVA seems to accumulate at sites where the cytoplasmic membrane is most strongly curved. When we expressed the 60 amino‐acid N‐terminal domain of DivIVA (fused to GFP) in this E. coli mutant, the fluorescent signal marked the cell membrane, and no dumbbell‐shaped accumulation was observed (data not shown).
DivIVA recruits RacA to the membrane
DivIVA is required for the localisation of proteins, but so far there is no biochemical data showing a direct interaction between DivIVA and other proteins. We were unable to detect any effect on the lipid‐binding affinity of purified MinD by DivIVA (data not shown). Very recently, it was reported that MinJ (YvjD), a previously unknown transmembrane protein, is required for MinD localisation (Bramkamp et al, 2008; Patrick and Kearns, 2008), which explains our failure to show a direct interaction between DivIVA and MinD in vitro. Another potential target for DivIVA interaction is the chromosome segregation protein, RacA (Ben‐Yehuda et al, 2003). We first used a bacterial two‐hybrid assay to obtain more evidence for a direct interaction between DivIVA and RacA. Such a test gave a negative outcome in case of MinD and DivIVA (data not shown). We tested different combinations of both C‐ and N‐terminal fusions, and low‐ and high‐copy vectors (Supplementary data), and found a positive interaction between a DivIVA‐adenylate cyclase T25 fragment fusion on a low‐copy plasmid and an adenylate cyclase T18 fragment‐RacA fusion on a high‐copy plasmid (Figure 7A). Encouraged by this result, we purified RacA as a fusion with MBP. We used density gradient flotation experiments to test whether DivIVA would stimulate binding of RacA to lipid membranes. MBP‐RacA was mixed with DivIVA and liposomes, and loaded at the bottom of a sucrose gradient. A high concentration of BSA (0.5 mg/ml) was present to ensure specificity. After centrifugation, fractions were loaded onto a protein gel and analysed by western blotting using RacA‐specific antibodies (Figure 7B). In the absence of DivIVA, a small amount of RacA could be detected in the phospholipid fractions. However, the presence of DivIVA led indeed to a substantial increase in the amount of RacA in the lipid fraction.
DivIVA plays a structural role in the development of Gram‐positive bacteria by recruiting proteins to cell division sites and cell poles. How this process operates is unknown. Here, we show that the localisation of DivIVA to the cell periphery involves a direct interaction with the lipid bilayer, and that the protein can stimulate the binding of other proteins, such as RacA, to the lipid membrane. In addition, we show that DivIVA accumulates where the cytoplasmic membrane is most strongly curved. Very recently, it was shown that the B. subtilis protein, SpoVM, uses the curvature of membranes as a cue for its localisation (Ramamurthi et al, 2009). SpoVM is a small peripheral membrane protein that is involved in the spore coat assembly. The protein accumulates only at the spore membrane and not at the cytoplasmic membrane of the mother cell. SpoVM makes this distinction by having a stronger affinity for the positive curvature of the spore membrane compared with the negative curvature of the mother cell membrane. Hence, SpoVM is an example of a protein that prefers the convex side of curved membranes, whereas DivIVA is an example of a protein that prefers the concave side.
A deletion analysis of DivIVA showed that the first 60 amino acids are sufficient for membrane binding. The amino‐acid alignment of different DivIVA molecules (Figure 1A) suggests that these 60 amino acids form a distinct domain, as DivIVA sequences from both M. tuberculosis and S. coelicolor contain large non‐conserved inserts immediately after this region. Amino acids 22–41 are predicted to form a long amphipathic helix (Figure 4E). Such amphipathic helices form the basis for coiled‐coil interactions; however, an extensive mutagenesis study of DivIVA from Enterococcus faecalis showed that the N‐terminal domain is not required for oligomerisation (Rigden et al, 2008). There are also several examples of amphipathic helices that bind specifically to lipid bilayers (Szeto et al, 2003; Pichoff and Lutkenhaus, 2005). Therefore, the 20 amino‐acid long amphipathic helix at the N terminus of DivIVA would be a good candidate for the membrane‐targeting sequence. In fact, the hydrophobic and hydrophilic phases of this amphipathic helix are highly conserved in different DivIVA species (Figure 4E). Replacing some of the hydrophobic residues in this helix abolished the interaction with the cell membrane.
Despite the fluidity of the lipid bilayer, phospholipids are not homogenously distributed in the cytoplasmic membrane (Fishov and Woldringh, 1999; Mileykovskaya and Dowhan, 2000; Kawai et al, 2004). Using fluorescent dyes with preference for certain lipids, it was shown that the concentration of cardiolipin (CP) and phosphatidylethanolamine (PE) is increased at the septal and polar regions of B. subtilis (Kawai et al, 2004; Nishibori et al, 2005). Given that DivIVA binds to lipids, a striking possibility would be that the localisation of this protein depends on the localisation of certain lipid molecules. The synthesis of CP or PE can be blocked in B. subtilis without clear adverse effects on growth (Kawai et al, 2004; Nishibori et al, 2005). We have deleted genes involved in the biogenesis of these lipids but found no effect on the localisation of DivIVA (Supplementary data). We have also introduced a mutation that blocks the synthesis of another major lipid component, phosphatidylglycerol (Kobayashi et al, 2003), but again the septal and polar localisation of DivIVA remained undisturbed (Supplementary data). It seems therefore less likely that DivIVA binds a specific lipid species that would accumulate at cell division sites and cell poles.
Both in B. subtilis and in E. coli, DivIVA tends to accumulate at sites where the membrane is most strongly curved. As the two bacteria are evolutionary rather distinct, it seems that this tendency could be an intrinsic property of DivIVA. The Bar domain is a typical example of a protein structure that binds specifically to curved membranes. This conserved domain is found in proteins that are involved in vesicle formation and membrane remodelling (Peter et al, 2004). Bar domains bind to curved membranes by forming crescent‐shaped dimers that fit the curvature. In fact, when Bar domains are added to liposomes, they deform the membrane, which results in long tubular‐shaped liposomes (Peter et al, 2004). It should be mentioned that Bar domains bind to the outside of vesicles, thus to positively curved membranes, whereas DivIVA is enriched at negatively curved membranes. Nevertheless, if DivIVA binds to curved membranes in a similar manner, it seems likely that DivIVA deforms spherical liposomes, as the lipid bilayer will try to wrap around curved DivIVA complexes. However, our EM data showed no indication for membrane deformation. We have also tested whether the affinity of DivIVA depends on the diameter, thus curvature, of liposomes, but we found no differences (Supplementary data). It seems that the accumulation of DivIVA is not a consequence of a possible curved shaped of DivIVA multimers.
In one of the most detailed studies on DivIVA oligomerisation, it was shown that DivIVA forms doggy bone‐like structures of ∼22.4 nm in length, which assemble into large lattices (Stahlberg et al, 2004). These doggy bones appear to be composed of 6–8 DivIVA subunits. In the TEM pictures of Figure 5, the crucifix‐ and stalk‐like protein structures span on average 25 nm, which is almost the same size as doggy bones. These are remarkably large structures that in length surpass the diameter of ribosomes (∼20 nm). Possibly, this is one of the key factors that contribute to binding of DivIVA to negatively curved membranes. We propose that (i) the mutual interaction between DivIVA oligomers (doggy bones), (ii) their affinity for membranes and (iii) their large size stabilises DivIVA clusters by 'bridging' opposing membranes. This 'molecular bridging' is schematically depicted in Figure 8A. For simplicity, the DivIVA oligomers are depicted as spheres. The spheres can interact with each other and with the cell membrane. Three different situations are depicted: a cluster of eight spheres that freely floats in the cytoplasm, the same cluster but bound to the lateral (flat) cell membrane and a cluster that is localised in the corner formed by a curved membrane. The spheres at the periphery of these clusters (transparent spheres) have fewer interactions and are more prone to detach and diffuse away. Binding of the cluster to the membrane decreases the exposed surface and stabilises the cluster, and this is most noticeable when the membrane is strongly curved. In fact, the transition from the lateral to the perpendicular membrane region can be rather gradual, as the spheres will bridge the groove by forming a cluster. This model could explain the accumulation of DivIVA in a ring‐like formation at the site of cell division, without the need for either intrinsic curvature of the protein, binding to specific lipid molecules or the presence of other proteins.
The relative simplicity of the molecular bridging model meant that we could apply a whole‐cell Monte‐Carlo simulation to test it. The Monte‐Carlo algorithm employed attempts to move one molecule at a time, by a small amount in a three‐dimensional space. The new interaction energy experienced by the particle is then computed and compared with its old energy, and the move is subsequently accepted or rejected according to the standard Metropolis test (Allen and Tildesley, 1989; Binder and Heermann, 2002). This procedure is used to guide the system to the correct Boltzmann distribution in equilibrium. Provided that the moves are small enough and that the acceptance probability remains high, it has been shown that this Monte‐Carlo dynamics corresponds well with Brownian or molecular dynamics (Whitelam and Geissler, 2007). In our simulation, DivIVA oligomers (doggy bones) are represented as diffusing spheres with a 12.5‐nm radius. The bacterial cell is represented as a cylinder with a length of 4 μm and a diameter of 1 μm. The lateral membrane and the membranes at the sides (ends) of the cylinder are joined by a smooth region with variable radius of curvature Rc. The spheres experience a mutual interaction energy of Vpp, and are also attracted to the membrane via an interaction potential Vpm (see Supplementary data for details). For simplicity, the two potentials have been chosen to have constant magnitude (Epp and Epm, respectively) over their interaction ranges, which we take to be equal to rint for both interactions. The magnitude of Epp and Epm was chosen in the range 1.5–6 kBT (equivalent to 1–4 kcal/mol), which is in the range of typical weak protein–protein attractions. Figure 8B shows an example of a simulation. The simulation lasted 30 million iterations, which roughly corresponds to a 30‐s time interval (see Supplementary data on how to map simulation into real time). In this specific case, the spheres were allowed to make no more than eight interactions, which is a reasonable assumption considering the large lattices that are formed by doggy bones (Stahlberg et al, 2004). According to our TEM data, the DivIVA structures seem to bridge 2–4 membranes. Therefore, we also limited the possible interactions of the spheres to two membrane contacts in this simulation. The membrane curvature at the cylinder ends has a radius of 50 nm, which is roughly the measure of curvature found at cell division sites in B. subtilis (Supplementary data). Clearly, under these simulation conditions, the spheres accumulate at the ends of the cylinder, where the membrane is most strongly curved. Thus, the cooperative binding enables the spheres to accumulate at curved membranes that have a considerably larger radius than the radius of the spheres themselves. Figure 8C shows that this accumulation depends on the measure of curvature in a highly nonlinear way. This might explain why the localisation of DivIVA‐GFP at division sites of E. coli is not as distinct as that of B. subtilis. In contrast to B. subtilis, E. coli does not make a cross‐wall when dividing, but division occurs by constriction of the whole lateral wall. As a consequence, the curvature of membranes at E. coli division sites is less pronounced. When DivIVA‐GFP is expressed in the fission yeast, Schizosaccharomyces pombe, the protein accumulates at division sites as well (Edwards et al, 2000). Although S. pombe cells are much larger than B. subtilis cells, EM images of S. pombe show a sharp angle between the septum and the lateral wall (Sipiczki and Bozsik, 2000; Osumi et al, 2006), and we estimated a curvature with radius of about 60 nm (Supplementary data), which is close to the radius measured for B. subtilis division sites.
We have repeated the Monte‐Carlo simulations with different parameters, binding conditions and number of spheres, and a more detailed description of these simulations can be found in the Supplementary data. In most cases, a comparable result was achieved indicating that the postulated molecular bridging is a relatively robust mechanism for the localisation of proteins to negatively curved membranes. As a proof of principle, we have also simulated molecular bridging in a hemispherical cell, in analogy with dividing cocci. To show the general principle of the mechanism, we allowed this time only four contact sites, and no constrains in protein–lipid interactions. As shown in Figure 8D, a clear localisation in the equatorial ring is observed that is reminiscent of the localisation of DivIVA in S. pneumoniae and S. aureus cells (Pinho and Errington, 2004; Fadda et al, 2007).
It should be emphasised that we have made several approximations in our modelling, and caution should be exercised in translating these simulation results into the cellular reality. One of the main shortcomings of simulating spherical particles is that the binding sites are not defined at fixed positions on the surface. In case of the DivIVA doggy bones, it seems that only the ends are forming the contacts. In an attempt to account for this, we modelled doggy bones as a rigid rod‐shaped stack of four smaller spheres, whereby the top and bottom spheres make the interactions either with other doggy bones or with the membrane (see Supplementary data for details). Using these rod‐shaped structures in the Monte‐Carlo simulation resulted in a clear accumulation of protein at the edges of the cylinder (Figure 8E), and it appears that in this case, the localisation would persist to an even larger radius of curvature (Supplementary data). Although more research is required to confirm molecular bridging as a mechanism for DivIVA localisation, it will be interesting to see whether the principles can be applied to other proteins that accumulate at negatively curved membranes.
Materials and methods
General methods
A detailed description of strains, plasmid construction, protein purification and bacterial two‐hybrid assay can be found in the Supplementary data.
Membrane and liposome preparations
B. subtilis cell membranes were isolated based on Henstra et al (1996). An overnight culture of B. subtilis 3310, lacking divIVA (divIVA∷Tet, minCD∷Km) (Edwards and Errington, 1997), was used to inoculate 500 ml LB medium. The culture was grown at 37°C to the end‐log phase. Cells were harvested, washed in ice‐cold Wash buffer (20 mM Tris–HCl pH 8, 200 mM NaCl) and stored at −80°C. Cells were resuspended in 10 ml ice‐cold KPi buffer (50 mM KPi pH 7.5, 5 mM MgCl2, 0.5 mM DTT) containing PMSF, protease inhibitor (Complete Mini, Roche), Benzonase (Merck) and RNase. The cells were broken using French Press, which produces inside‐out vesicles, and cell wall debris was removed with a 2 × low‐spin centrifugation (SW51, 10k r.p.m., 10 min, 4°C). The supernatant was carefully collected with a pipette, and membranes were gathered following two subsequent high spins (SW51, 35k r.p.m., 1 h, 4°C). The membrane pellet was resuspended in 0.4 ml KPi buffer without DTT, aliquoted, frozen in liquid N2 and stored at −80°C.
Liposomes were prepared as described by Avanti Polar Lipids. E. coli polar lipid extract (Avanti Polar Lipids) dissolved in chloroform was desiccated in a rotary evaporator, followed by vacuum excitation (4 h). Lipids were resuspended in 50 mM Tris‐acetate (pH 7.5) by vigorous vortexing (20 min), and sonication (2 × 5 min) in a bath sonicator. Aliquots were covered with Argon and stored at −80°C. For interaction studies, liposomes were mixed with the specified buffer and freeze‐thawed several times followed by extrusion through a 0.1‐ or 0.4‐μm filter (Avanti Polar Lipids).
Density gradient centrifugations and sedimentation experiments
For interaction studies with cell membrane preparations, a step gradient of 10, 20, 30, and 70% sucrose was used. After centrifugation, membranes were visible at the 30–70% sucrose interface. When liposomes were used, a step gradient of 5, 10 and 20% sucrose was used. After centrifugation, liposomes were visible at the 10–20% sucrose interface. Centrifugation was carried out for 2 h (25k r.p.m., 25°C) in a Beckman Rotor SW50.1/55 using 0.8 ml tubes (5 × 41 mm) and suitable adapters. Purified proteins (approximately 0.1–0.5 mg/ml) and membranes (1 mg/ml liposomes) were mixed in 50 μl binding buffer (20 mM Tris–HCl pH 8, 5 mM MgCl2, 200 mM KCl, 1 mM DTT, 1 mg/ml BSA) and incubated for 30 min at 30°C before loading onto sucrose gradients. The sucrose gradients were prepared using the binding buffer. After centrifugation, the gradients were fractionated in 50 μl samples that were analysed by western blotting using GFP‐ or DivIVA‐specific antisera.
Sedimentation experiments were performed in the same binding buffer as used for density gradient experiments with the omission of BSA. Purified proteins (0.2 mg/ml) were mixed with liposomes (1 mg/ml) and after incubation for 30 min, the samples were centrifuged (Beckman TL‐100 rotor, 80k r.p.m., 15 min, 30°C). Pellet and supernatant fractions were analysed by SDS–PAGE and Coomassie staining.
For the RacA interaction experiments, density gradient flotation was used. MBP‐RacA was mixed with or without liposomes and DivIVA in the presence of 0.5 mg/ml BSA and 20% sucrose in buffer (20 mM Tris–HCl pH 8, 200 mM KCl, 2 mM MgCl2 and 0.2 mM DTT). Before adding MBP‐RacA, the mixtures were preincubated for 15 min at room temperature. After 1 h incubation at room temperature, the solutions were loaded into 0.8 ml centrifuge tubes (5 × 41 mm). Further, 100 μl of 15 sucrose, 10 sucrose, 5 sucrose, and 0% sucrose in the buffer was loaded. Gradients were centrifuged at 25k r.p.m. at 30°C for 2 h in a Beckman Optima MAX Ultracentrifuge using an MLS 50 rotor. After centrifugation, the gradients were sampled in five fractions, which were analysed by western blotting using RacA‐specific antibodies.
SPR measurements were carried out on a Biacore 2000 system (Biacore AB) using L1 chips at 25°C. Loading of liposomes on the L1 chip was carried out as described (Anderluh et al, 2005). An L1 chip was washed 3 × 1 min wash with isopropanol, 50 mM NaOH (3:2 (v/v)) at a flow rate of 30 μl/min. Liposomes (1 mg/ml prepared in the running buffer and extruded through a 0.1‐μm pore filter) were loaded in 15 min at a flow rate of 2 μl/min, and excess lipid was washed 3 × 1 min with injections of 100 mM NaOH at 30 μl/min. The integrity of the lipid layer was checked using a 1‐min injection of BSA (0.1 mg/ml). Protein samples were first dialysed for 3 h in the running buffer (20 mM Tris–HCl pH 8.0, 150 mM KCl, 5 mM MgCl2, 1 mM EDTA) before injection. A 1‐min injection of 100 mM NaOH at 30 μl/min was used to disrupt lipid–protein interactions.
Fluorescence microscopy
For fluorescence microscopy, the divIVA‐gfp region of plasmid pSG1612 (Edwards et al, 2000) was isolated (XhoI × SpeI) and cloned into pSG1154 (Lewis and Marston, 1999), resulting in plasmid pDG7. This plasmid replicates in E. coli and integrates in B. subtilis. The divIVA‐gfp fusion includes the divIVA promoter, which is also active in E. coli. The different deletions were made by PCR using pDG7 plasmid as a template (pDG13=DivIVa aa 1–40 ‐GFP, primers LH16 and GFP7, pDG15=DivIVA aa 1–60 ‐GFP, primers LH20 and GFP7). The point mutations were made by PCR using pDG15 a as template (pDG23=V25E, primers LH110 and LH111, pDG26=L29E, primers LH112 and LH113). Cultures were grown in PAB at 30°C. Samples were taken at exponential growth, and mounted onto microscope slides coated with a thin layer of 1.5% agarose. Membranes were stained with Nile Red or FM5‐95. Images were taken with a Zeiss Axiovert 200M coupled to a CoolsnapHQ CCD camera, and using Metamorph imaging software (Universal Imaging). For deconvolution, about 15–20 Z‐stacks were obtained with 0.1‐ to 0.2‐μm intervals. Two‐dimensional deconvolution (nearest neighbour) calculations were performed using Metamorph with the following settings: filter size 9, background subtraction 97% and result scale intensity of 2.
Liposomes (0.1 mg/ml) with and without DivIVA (0.01 mg/ml) were incubated in the binding buffer (20 mM Tris–HCl pH 8.0, 150 mM KCl, 5 mM MgCl2, 1 mM EDTA). Aliquots of liposomes with and without DivIVA were loaded onto glow‐discharged formvar‐coated 400‐mesh copper grids and negatively stained with 1% (wt/v) aqueous uranyl acetate. Excess stain was removed by blotting with a filter paper, and the grids were air‐dried. For thin section, TEM samples were fixed for 40 min on ice in a freshly prepared mixture of 1% glutaraldehyde, 1% osmium tetraoxide in 100 mM phosphate buffer (pH 7.0). The pelleted samples were washed in distilled water and sequentially treated with 1% aqueous tannic acid (1 h at 23°C) followed by 1% aqueous uranyl acetate (1 h at 4°C), dehydrated and embedded in Agar 100 resin. The thin sections were viewed in a Zeiss Omega 912 electron microscope equipped with an in column 2K Proscan CCD camera.
Supplementary data
Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org).
Supplementary Information
Supplementary Information [emboj2009129-sup-0001.pdf]
We thank the members of the Centre for Bacterial Cell Biology for helpful advice and discussions, and in particular Robyn Emmins for help with the bacterial two‐hybrid assays. Richard Losick kindly provided RacA antibodies, and Waldemar Vollmer E. coli strains. Simon Cockell is acknowledged for his support with initial modelling attempts. This research was supported by an EMBO Long‐Term Fellowship, a Wellcome Trust Research Career Development Fellowship (LWH), a grant from the UK Biotechnology and Biological Science Research Council (JE and LWH) and a grant from the Engineering and Physical Sciences Research Council (DM).
This is an open‐access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.
- Copyright © 2009 European Molecular Biology Organization


text:
Why can't tin box manufacturers give customers the first time quotation, what are the factors related to the price of tin?
Often encountered customers on the whatsapp ,skype or call to consult the tin box, some customers want to know the price to the size. The customer can not understand why I gave you the size, the number of why can not give me a rough price! At this time, we will ask the customer several questions. For example, how many are customized? What are the requirements for printing and so on? However, even if we know this, we cannot give the price immediately. Of course, we can also tell the customer a price irresponsibly, but this is a disrespect to the customer.
In fact, it takes a little time for the clerk to quote by factory. The calculation price of the tin box manufacturer is calculated based on tinplate raw material cost, labor cost, packaging cost, transportation cost, and auxiliary material cost of the tin box. These costs are also due to geographical factors. There will be fluctuations in the difference, which will affect the price of the entire tin box, so a price will take into account many factors.
Materials, the tin box packaging materials are most commonly used in tinplate and frosted iron, the price of the two materials is not the same, frosted iron due to different materials and processes, want to slightly higher prices for tinplate. In the customization process, the materials are selected according to the needs of the packaging, and the material is a factor that determines the cost of the tin box.
Iron box structure, the common iron box structure has a can, two-piece cans, three-piece cans, multi-piece cans, etc., the more the number of iron material, the higher the production cost; and the shape, round, square, The prices of rectangular and other conventional sizes will be lower than those of complex, can-shaped and heart-shaped products.
For printing, choose the iron material and choose the printing process. The printing plate can be divided into two types: single-sided or double-sided, and the cost of single-sided printing is slightly lower than that of double-sided printing. The cost price of multi-color printing relative to four-color printing will be relatively high. Complex production processes, such as engraving, stretching, craters, etc., have high production costs. In respect of oiling, it is generally light matte oil and gold oil. In the case of special requirements, the choice of pearl oil, explosive oil, wrinkle oil and other oil prices also directly affect the cost of tin.

subset: commoncrawl


text:
Week 23 Menu
Here's my menu for the week:
Monday: crockpot chicken stew
Tuesday: sweet and sour meatballs and rcie
Wednesday: parmesan chicken and baked potatoes
Thursday: tamale pie or Chicken Taco Cornbread Wedges with Ranchero Cilantro Drizzle
Friday: sloppy joes
Saturday: clam chowder
Sunday: chicken enchiladas
Now it's your turn! What's on your menu this week? Post your menu in the comment section. Or just post what you had last week. Have a good week!


text:
Silver Ring Size 5
To show the impression of a more lustrous, silver ring is completed with a glossy or satin finish paint. This silver ring laser-etched engraving so impressed charming. Features silver rings are also environmentally friendly, namely none fade, oxidation, rust or allergies (nickel and lead free).
Measure Ring Size
Another way is if you have a ring that has the size of your stay in the ring diameter measuring middle ring fitting measured with a ruler. The average diameter 1.8 to 2.4 cm in men and women from 1.5 to 1.9 cm. The last way that came to a store of gold or silver shop local then you measure with the tools in the shop and obtained the ring size, size men between 16-22 and women 11-15.


text:
When the cleaning solution comes in contact with the carpet or rug, it is anywhere between 65 to 121 degrees Celcius, according to the heat available from the cleaning unit. For instance, in a modern truck-mounted carpet cleaning machine, water can be heated to 150 degrees (C), but after passing through high pressure steel braided hose and several manifolds, the water loses some heat.
The typical cleaning method involves a preconditioning of the soiled surface with an alkaline (7 or above on the pH Scale) agent, followed by light agitation with a grooming brush and appropriate dwell time. Next, the carpet and rug surface is passed over several times with a cleaning tool to thoroughly rinse out the preconditioner and, using an acetic acid solution, lower the pH of the fibres to a neutral state. Finally, the surface is dried sufficiently to avoid any possibility of saturation, leaving you with a nice clean carpet.
M&R Carpets are specialists in all forms of carpet cleaning. So if you're in the need for the best carpet cleaning pretoria has to offer – contact us now


text:
Play Crazy Kings \r
A NEW KIND OF TOWER DEFENSE!\r
Tower Defense like youve never seen it before. As well as placing towers, you take control of your very own avatar to save the day. Are you ready for a brand new take on Tower Defense?\r
Loot your way through hundreds of levels and build your very own army. Every item can be combined to create bigger, better and badder towers.\r
But dont forget your avatar! You can customize and upgrade your fighter with incredible weapons, armor and special items to become the most powerful warrior in the land!\r
Or, why not borrow your friends avatars to lay waste to the merciless minotaur army? The more friends you make, the stronger you become!\r
With 200 levels to play through, have you got what it takes to defend the kingdom from the Crazy Kings?\r
*WHY YOULL LOVE CRAZY KINGS*\r
CLASSIC GAMEPLAY WITH A TWIST – Strategically place your towers and use your heroes to respond to changes on the battlefield.\r
LOOT, LOOT AND MORE LOOT! – Win loot cards from every battle and use them to power up your avatar!\r
BUILD AND UPGRADE – Level up your army from basic, rare, and epic, all the way up to legendary!\r
200 FREE LEVELS – Hundreds of awesome levels set across snowy tundra, arid desert, rolling hills and more!\r
FORGE ALLIANCES – Borrow your friends avatars to crush those Crazy Kings!\r
*PLAYER REVIEWS*\r
"Forget about old-school tower defense! Crazy Kings is completely different!"\r
"I cant believe how addictive it is! My avatar is super-powered now!"\r
Download now and get ready to fight! The Crazy Kings are coming!\r
PLEASE NOTE: Crazy Kings is completely free to play, but you can also purchase some items within the game for real money. Just disable in-app purchases on your device if you dont want to use this feature.\r
Mantis Shrimp Web SiteCrazy Kings Support\r
Whats New in Version 1.4.2\r
YAY! ITS HERE: DOWNLOAD THE NEW CRAZY KINGS UPDATE NOW!\r
- Device Sync. Play on multiple devices using the same Facebook-login!\r
- Native iPhone 6(+) support. Crazy Kings looks even better on the newest iPhones!\r
- Arena Tickets change reverted. Thanks to your feedback, weve switched back to 3 arena tickets! Its still possible to buy tickets one at the time instead of refilling the tickets fully.\r
- More levels & enemies! The Bird World now comes with 10 more levels to test even the best of players!\r
- Bug fixes, level tweaks and more!\r
Please Subscribe! ►\r
VISIT US: \r
FOLLOW US: \r
LIKE US: \r
WATCH US: \r
Google Plus \r


text:
BATON ROUGE � The extra month of food-stamp benefits approved for Terrebonne last week will be added to residents� cards tonight, a state spokesman said.
Last week, the U.S. Department of Agriculture extended a second month of food stamps to seven parishes that were impacted by Hurricane Ike, which flooded lower Terrebonne and parts of south Lafourche in mid-September.
If the benefits have not been added to your card by Friday morning, call 1-888-524-3578.
Both Terrebonne and Lafourche were among the parishes approved for a month of disaster food stamps after Hurricane Gustav, which made landfall Sept. 1 near Cocodrie.
Though Terrebonne residents who were issued food-stamp cards after Gustav were approved for a second month following Ike -- which hit about 12 days after Gustav -- the USDA has yet to extend the additional month of benefits to neighboring Lafourche.
The state Department of Social Services lobbied the federal government to approve a second month of disaster food stamps because of the hardship caused by Ike�s flooding following on the heels of Gustav�s destructive winds.
Residents who were approved for Gustav were told not reapply following Ike, because the benefits would be automatically added to their cards, pending USDA approval.
The state is still hoping to get Lafourche and the other six parishes who were eligible for Ike food stamps approved for a second month, the spokesman said.
�We�re continuing to work to get additional parishes approved,� Department of Social Services spokeman Trey Williams said, adding that the department is sending additional economic and storm-damage data to the federal government.
Food-stamp money to be added to accounts tonight
BATON ROUGE � The extra month of food-stamp benefits approved for Terrebonne last week will be added to residents� cards tonight, a state spokesman said.


text: Choosing Between Gas and Wood Burning Fireplaces Published on January 20, 2016 A fireplace adds personality and value to your home. When a person imagines their dream house, it typically includes a comfy home that keeps you and your family warm in the winter, either with a smoothly running heating unit or snuggling around a cozy fire. It's hard to resist the allure of of a crackling fire, but how do you decide between a gas or wood burning fireplace? There are many aspects to consider when deciding how to keep your home nice and toasty. Aesthetics and Efficiency Wood: A wood burning fireplace typically wins in the sensation category. You get the crackle. You get the pop. You get the chance to roast marshmallows indoors. Something you don't get is a powerful heating source. Wood fires normally receive up to a 15% efficiency rating, considerably lower than a furnace that has consistent service performed. They do generate a lot of heat, but most of that heat is lost up the chimney. Wood burning fireplaces not only lose the heat coming from the fire, but it also pulls warm air from other parts of the house up the chimney.Gas: There have been many artistic advances in gas fireplaces. The flames have become more realistic and some versions offer varied height adjustments. The types of logs used in gas fireplaces now have the look of the real thing and come complete with glowing embers, which don't need you to wait while they burn out. You can merely switch your gas fireplace on and off which gives you more control over your home's temperature and frees you from having to look after your fire. The lack of fire stealing oxygen gives gas fireplaces a 75% to 99% efficiency rating. Picture the level of comfort you could have when you incorporate that with a fully maintained furnace. Air Quality and MaintenanceWood: Air quality is crucial to homeowners and homebuyers. Burning wood causes air pollution in and outside your home and the smoky wood aroma that a wood burning fireplace exudes could be a health hazard. Wood also creates a byproduct called creosote that lines the coating of the chimney and must be removed by a chimney sweep. Much like furnaces that should have furnace service completed consistantly, gas fireplaces also require regular cleanings of cinders and spent logs.Gas: Gas fireplaces require a little bit of dusting every now and then and are virtually maintenance free. It is recommended that you get your gas fireplace cleaned and adjusted annually by a professional to keep it running both safely and effectively. If you want to convert your wood burning fireplace into a gas fireplace or you need to schedule annual furnace service, please call today at 613-707-2421 or schedule an appointment on our website. Our experts will have you snuggled up and warm in no time. Back To BlogContact Us


text:
Author: HP Newquist
Teen Reviewer: Mikaela Palandra
Rating: 4/5 stars
From ancient mythology to blood transfusions today, blood plays a huge role in our lives. And that's exactly what this book is about. It takes you through time, back when people thought releasing blood could cure illness to the past hundred or so years when we have actually discovered what blood's all about. The author explores the relevance of blood, clears up some common misconceptions, and covers a broad number of topics relating to blood. It provides you with plenty of interesting facts on the incredible liquid that flows through you constantly, keeping you alive.
This non-fiction book provides a span of information on blood. It does have its fair share of plain old facts, but it also talks about blood in animals and mythology, things you may not be as familiar with and I found to be the most interesting parts. The book keeps from being dull for too long with random facts, like explanations on blood types. The author does a good job with getting you to understand basic information about what's going on. It's an interesting read, exposing you to a lot of new information without being overwhelming. The layout is not too dense, which makes it a less intimidating read. I would have enjoyed a bit more information on the understanding of blood nowadays than the basic description, but it got across the facts well, and it does teach you something new. Basically it's a good, fun book, especially for someone who finds science as fascinating as I do.


text:
Let your search for a real estate professional end here. Please explore the resources within my website, you will find everything you need to buy or sell a home as well as learn about the market value of homes. You can search for homes with detailed descriptions, photos, community and school information and so much more.
I am ready to assist you with all of your real estate needs. My approach is customized for each client; my solutions are never one-size-fits-all! Please view this website as an introduction of my services and capabilities in this local area. Give me a call or send an email when you are ready to visit some homes or to schedule a free home buying or selling consultation. I look forward to working with you!
Click here for client testimonials...hear what my clients have to say!
Spectacular panoramic View, saddleback mountain City light and Ocean view. One bedroom with full shower on the main floor. Huge backyard with Pool and Spa. High ceiling, light and bright with bright morning sun. The Kitchen with the view of the pool, new Cabinets and appliances, with easy access to the family room. Formal living and formal dining with high ceiling and designer colors. Fire place in living room and master bedroom. Walk in closet with double sink in master bedroom with new cabinets and under construction shower. Attached three car garage with built in storage cabinets. Freshly painted exterior. Single loaded street in the most desirable area in Laguna Niguel with a very short drive to the beaches and five star hotels. Many hike and bike trials in the area. Professionally maintain landscaping, and professionally maintained pool and spa.


text:
Triple Play Of The Day
PISCATAWAY, N.J.-- Well, I've heard before that seeing a triple play in a given inning is 1 in 10,000. In that case, all 1,024 of us in attendance at Rutgers today should go out and buy a Mega Millions lotto ticket. In the top of the 6th, the Scarlet Knights executed a 5-4-3 triple play. A hot shot down the line was snared by the third baseman Tim Querns who stepped on the bag, threw to Mike Bionde at second, who relayed on to Tom Edwards to complete the feat. The only announcement about it was that it was the first ever at Bainton Field.
That crowd i mentioned was a record for Rutgers. A gorgeous day and having Notre Dame in town led to it, but fans were treated to a see-saw battle in the rubber match of the three game set in which the Fighting Irish took with a 6-4 final.
-Rutgers' star SS Todd Frazier had a tough day at the plate a day after beaking the Rutgers single-season and career home run records. I will have a feature on Frazierr this week.
-Rutgers had one of the strangest field I have ever seen. The Scarlet Knights used a 1.25 million dollar donation to install FieldTurf, but instead of just installing it where the grass would be, they put it where in the basepaths as well! Meaning that the only way to make the equipment manager unhappy with you is to slide into home. The rubberized carpet (by the way, the basepaths are a brown color), is a cause for some funky hops and some interesting slides, but it makes total sense. Last Friday when it rained all over the state, Rutgers was still able to get out and practice despite most of the area being under water.
-Rutgers may cause some problems for teams in the postseason if they can come out of Coney Island. They have a tough lineup 1-9 (some real big guys, including the junior Frazier who is slated as a first round draft pick WHEN he decides to leave after his junior season at the end of the year), and a pitching staff that can cause some problems. Casey Gaynor threw seven and two-thirds innings for RU, and only made one or two mistakes. Gaynor gave up five hits and four runs and received a no-decision.
-Rutgers DH Jon Gossard hit a clutch two out homerun to tie the game in the 8th inning for Rutgers, after Notre Dame had taken the lead on a two run base hit in the top half of the inning. But it was a tie game for only a short while. Rutgers reliever Chris Lillis gave up a two run triple in the top of the ninth that sealed the deal. Despite sthe Knights getting two runners in scoring position in the bottom half before Sam Elam struck out Tom Edwards to end the ballgame.
Notre Dame struggled out of the gate this year, and we'll see if they can get the ship totally turned around before they return to the NY area at the end of the month.


text:
To re-brand an upper mid-end real esate, apartment development, located within Ho Chi Minh City's up-and-coming District 2.
red repositioned Imperia An Phu as a premium residential location where modern families can grow. red created the brand logo, identity system, advertising and marketing collateral to communicate the benefits and 'reason-to-believe' of this new living environment encouraging the audience to 'crown their life with excellence'.
Contact us to discuss how we can help you to build your brand


text:
A man is driving near Seattle when he comes upon another man who is pushing an air plane down the road.
"What's wrong with the plane?"the man in the car asks. "Nothing" replies the man pushing the plane.
"Where are you pushing the plane to?"
"Why don't you fly it?"
"I don't know how to fly a plane."
"Why don't you learn to fly or hire a pilot?"
"Pilots cost money and I don't have time to learn to fly. Now if you'll excuse me I have to get back to pushing this plane."
How many time have you heard (or said) "We don't have time to do it right"? Yet it seems there's always time to take the long road or worse yet, do it twice.
Every company has air plane pushers. In the haste to accomplish some goal, people fail to develop the methods and document the procedures for the task. They concentrate on getting it done rather than how to get it done properly and efficiently. This is the single biggest squandering of company resources there is, replacing efficient methods with more humans and the associated labor costs. That's great if you're a philanthropist, not so great if you're trying to maximize profits.
Failing to plan causes a hemorrhage of money. Instead of letting out a bit of blood and suturing the wound with nice clean stitches, a dirty band aid is put on the gash and it's allowed to bleed slightly forever, with all the puss and infection that come with such methods. Eventually this leads to a hospital visit and possibly an amputation.
Technophobia, ego, narrow mindedness, failure to adapt, and simple lack of education or experience all play a part in creating air plane pushers. Often it starts innocently enough. A quick solution is needed for some information requirement, then as the company grows these Git'er Done fixes remain, detaching information from the one version of the truth and causing employees to back-track, re-research, guess and duplicate effort.
How many air planes are being pushed around your company?
Gotta run - I'm off to flight school then my shrink to work on that fear of heights


text:
On the replica watches internet shops will aswell be area to acquirement commodity abreast from replica watches broad appearance handbags. The average from the accomplished care to replica watches run out in the aisle of the stop from rolex replica the countenance while the end acquire to be short. Expertise is apparent and progressed in to louis vuitton replica the item, they every individual acquire a adventure to swiss replica watches acquaint on their actual own, authoritative them exclusive. Due to the accurate blueprint of the three-stage event, specific triathlon wetsuits had been advised in the backward 1980s to accommodate abundant allowances to triathletes for replica watches uk the pond date of the race.
|Bottle tops
|I found these bottle tops on the streets of London over several months.I photographed and enlarged them so that they are no longer rubbish but reveal different - almost human - characteristics.
Price: A4 £30 each unframed
3 for £75
A3 £60 each unframed
3 for £150
A2 £70 each unframed.
3 for £170


text:
In recent years, I've mainly worked as a freelance editor for legal associations and law firms. My most recent client was the Association of Costs Lawyers, where I edited the organisation's bimonthly magazine.
In recent years, I've mainly worked as a freelance editor for legal associations and law firms. My most recent client was the Association of Costs Lawyers, where I edited the organisation's bimonthly magazine. Before that, I edited the annual review of CMS, a pan-European law network.
I've also provided writing, editing and blogging services for LexisNexis Martindale-Hubbell. Here, I mainly worked on in-depth research reports, covering topics as diverse as law firms' use of social media to why corporate counsel select and retain specific legal practices. My journalistic articles on these topics have appeared in legal trade magazines around the world, including Asian Legal Business, The Lawyers Weekly (Canada), Top Legal International (Italy) and Law Business Review (UK).
On several occasions, I've provided freelance support for contract publishers and PR companies who required specialist legal copywriting services. One of my most recent clients was Rostrum Communications, where I helped draft "though leadership" articles for a major international law firm. I've also worked with Words Work on a legal case report project for the Office of Fair Trading, and with Prima PR & Marketing in providing editorial coverage the LawNet legal network. Other communications clients have included Coast Communications on behalf of the Professional Services Marketing Group.
Finally, I've also worked on projects with a strong academic flavour. Recent clients have included the UK's Legal Services Board and a consortium of universities.
Between December 2000 – March 2007 I worked for The European Lawyer Ltd. I initially wrote features for The European Lawyer magazine, covering developments in the European legal market.
Between March 1999– December 2000 I worked as a project manager at hemscottlegal.com, an innovative direct recruitment website for junior lawyers.
From September 1998 – February 1999 I worked in Nabarro's marketing office on a short-term assignment to boost the firm's media profile. My main role was to produce news release for the non-legal trade press.
I worked at Chambers & Partners between September 1996 – August 1998. I wrote legal and law firm management article for The Commercial Lawyer, and carried out research for the Chambers & Partners Guide to the Legal Profession.
Specialist areas
I am a freelance legal journalist, researcher and and copywriter, based in Cardiff, Wales. I have 20 years' experience of legal publishing. During that time, I have held a wide variety of editorial and project management-related positions. I hold two law degrees, and recently completed a PhD which explored the geographies of large law firms. I specialise in writing about the UK, EU and US legal markets. I mainly write about the management and strategies of law firms and in-house legal departments, but can also cover technical legal issues if required. Besides conventional legal journalism, I also handle legal PR and corporate report work. This work includes drafting lawyer biographies, and also writing and editing in-depth research reports. I am actively looking for short-term freelance editorial / copywriting opportunities in the legal sector.


text:
Eyes are one of the important organ in our body. Eyes are almost the most important part of our life; even you are able to read this page because of your eyes. So proper vision standard is the utmost requirement of India Defence forces. Maximum permissible vision defects differ from force to force like for a fighter pilot there would be no compromise in case of eye defects.
For a candidate to be declared fit he/she must posses eyesight within permissible limit and also there should be no Night Blindness, Squint or Colour blindness.
Visual Standards for IAF
Friends first of all we would like to tell you that there are two type of Rejection by medical boards one is TR (Temporary Rejection) and another is PR (Permanent Rejection). For example if you are overweight then you will be Temporary Rejected and if you reduced your weight in the given time then you will be considered fit and let's say if you have Color Blindness then you will be Permanently Rejected as it is not curable. If you are medically unfit then you will be given time of about 42 days to prove your medical fitness, for that you have to appeal to the Board. President of the board (Medical) will help you in Appealing for a Review Medical Exam. A Fee of Rs40. will be charged for filling appeal, Thereafter you have to report to Medical Board within the stipulated period i.e., 42 days.
If there is a strong family history of Myopia, particularly if it is established that the visual Defect is recent, if physical growth is still expected, or if the fundus appearance is suggestive of progressive Myopia, even if the visual acuity is within the limit prescribed, the candidate should be declared unfit.
Radial Keratotomy, Photo Refractive Keratotomy / Laser in Situ Keratomileusis (PRK/LASIK) surgeries for correction of refractive errors are not permitted for any air force Duties. Corneal Topography may be done in suspected cases. Candidates having undergone cataract surgery With or without IOL implants will also be declared unfit.
IAF has different visual standards for different branches. Check below the eyesight limits for different branches of IAF.


text:
Bye-Bye Google Reader, Hello Feedly
Google has decided to pull the plug on Google Reader. As of July 1st, we will no longer be able to enjoy our favorite blogs through Google Reader. If you are like me and depend on Google reader, I have found a solution for you. Drum roll please…
It's Feedly! I've been using Feedly for a little while and what I like the most about it is the ability to change the way you view your feeds. Check out these options:
I'm a visual person so magazine and card views are my favorites. With both options, you get a small preview. If you're interested in reading the entire post, click on the title and it instantly pops up. If there are posts you are not interested in, click on "mark as read" and they disappear. There are a lot of options with Feedly and it's easy to use. The best part is it's available on iOS, Android, and through a web browser so you can use it on your phone, tablet, and computer. If you're interested, import your Google Reader feeds into Feedly Cloud and you'll be seamlessly moved over.
When I first heard that Google Reader was retiring I was not at all thrilled. However, I think Feedly will fit my needs perfectly!
1 comment
I am making the switch to Feedly too. I like it, especially the different views. Definitely helped me get caught up on blog posts when I fell behind. Loved reading through your post, especially since my little blog was featured in so many pictures. LOL. I must have posted at just the right time. :)ReplyDelete
Subscribe to: Post Comments (Atom)


text:
Every year since 2004, HIMSS has honored an individual HIMSS chapter member as the Chapter Advocate of The Year, a prestigious and widely recognized award presented to the chapter advocate who demonstrates exceptional leadership advocating for health IT. This year at HIMSS17, chapter advocates, state officials and other leaders met to honor the achievements of Richard Moore, an advocacy chair, for the Central & Southern Ohio Chapter of HIMSS, as the 2016 Chapter Advocate of The Year.
Richard Moore has over 30 years of experience in the healthcare industry. He has successfully led the design, development and implementation of multiple modernization projects for both the public and private sectors. While Richard is the current president of eHealth Ohio, Inc., a non-profit regional affiliate of the Workgroup for Electronic Data Interchange (WEDI), a standards development organization to the Secretary of the Department of Health and Humans Services, Richard maintains an active role in HIMSS at the chapter and national levels.
He is the past president, VP/treasurer, past and present advocacy chair, of Central & Southern Ohio HIMSS; Past HIMSS RHIO/HIE liaison for the State of Ohio; and HIMSS National Chair for the RHIO Liaison Roundtable. He also serves as a regional vice-chair for the HIMSS Chapter Advocacy Roundtable.
Richard's advocacy efforts and leadership as a central figure with the Central & Southern Ohio Chapter propelled the Ohio chapters to the forefront of leading change in health IT in the state. His efforts to establish productive relationships with state elected officials have been extremely fruitful, as evidenced by several achievements, including the establishment of the reoccurring Ohio Healthcare Information Technology (OHIT) Day with recognition by the Governor's office via a Proclamation for the OHIT Day for consecutive years.
We are also grateful to Richard for creating a local chapter award program to recognize Ohio state legislators who have championed health IT.
The HIMSS Government Relations team continues to be inspired by his innovative efforts to include legislators in chapter events by working with the HIMSS Innovation Center in Cleveland to connect the state capital with the HIMSS Innovation Center in 2014, 2015, and 2016 through videoconferencing.
We also recognize Richard's outstanding collaboration with the Health Policy Institute of Ohio towards the creation of the State of Ohio Health Information Technology Roadmap, which became the basis of Governor Ted Strickland's Executive Order creating the Ohio HIE, and ultimately, CliniSync.
Because of the generous dedication and support over the years of volunteer chapter advocates like Richard Moore, , HIMSS continues to advance healthcare and lead change in the industry among states and at the national level.
Thank you Richard!


text: Olivia Thristan Wellies Wishlist | The festival edit! to Read ( Words) I am yet again making another shoe wishlist, despite purchasing 4 pairs in the last week I am considering purchasing even more, I can't help it! I recently came across the website Shoetique and felt they had so many amazing wellies I would want to purchase. I would make a wishlist for you lovelies and go for a feel festival feel as I know that a few festivals are coming up soon, like Leeds and Reading and V Festival. As I am not going to any festivals I decided I probably shouldn't purchase the wellies even though if it was acceptable to walk around in them 24/7 I actually would. Who knew that wellies came in so many awesome shapes and sizes, I am totally loving wellies which are bright and fun as when else can you wear them right? One // Two // Three // Four Do you enjoy reading wishlist posts? This is a sponsored post, all thoughts and words my own* Olivia Thristan 1 Comments


text:
Many of us suffer from allergies, especially in this day and age where you'll find thousands of harmful, untested chemicals, on the shelves. A question to ask yourself – is why do people continue to purchase ...
You are here: Home / Posts tagged "Allergies"
Browsing: Allergies
On 29 Oct 2018, In Health and Wellness, by Melanie Vollick, 7,702 Comments
Write Way Communications
Write Way Communications offers a variety of writing services most suited to your needs. We help you get heard, while producing outstanding written products that reflect your professional image, which is so critical in today's competitive marketplace.
"Melanie Vollick has an incredible grasp of the English language and uses it to produce very well written material for her clients in a way that demonstrates a deep respect for her clients personal styles and needs. Melanie's ability to research the topic she is working on goes above and beyond what is expected so that her work is up to date, and clear and concise. I highly recommend this talented and hard working person." November 5, 2011
- Roxann Campbell , Owner, U-Pick Parties (business partner)
- Roxann Campbell , Owner, U-Pick Parties (business partner)
Published Articles
Journey's Blend CaféDecember 3, 2017, 9,441
A day in the life of living greenDecember 21, 2011, 9,356
Profile – Paul Jones: DrummerDecember 18, 2018, 1,497
Roundabouts: to love or to hate!November 29, 2018, 7,210
Résumé Tips – White SpaceNovember 29, 2018, 3,883
black top hgh kits:
You have noted very interesting details! ps nice website.
CollingwoodStaynerfeaturedmelanie vollickwasaga beachwrite way communicationswrite wayontarioMusicianrestaurantblue mountainswriting servicesresume writingproofingbarriecoffee shopGreen CleaningDrummerPaul JonesDockdrum kitedenvale aerodromeairplanesbistro 26runwaysaircraftdiane babeckasrescuejohn nicolspeak fmmorning show hostradio broadcastingsimon williamscancerrunsearsgreat canadian runcancer foundationnational kids cancerCollingwood skating clubfigure skatinghemphemp productshealthomega oilshemp oilLinkes Local Artsgallerybaittacklefishingcamping gearmaria lealhemp seedontario grownjudy garbuttnutritionhealth foodsuper foodomega 6fatty acidshealth problemsroundaboutssustainableaccidentssustainable communityyieldapproachenterexitbatsbat conservationpesticidesbat factsbrown batbig brownmosquitoesnight patrollershibernatingfrank sloaninventorglobal inventiontoiletflushing systemventing systemgreen toiletplumbingtorontowritereditingcolumnistsocial mediaphotographytechinical writingbusiness writingGreenpeaceArctic ThirtySave Arcticpeaceful protestrelease prisonersresume helpcover letterscommunicationscoffeefair tradeorganichome madenancy cookseyWildlifeSerenityCleaning CompanyEco FriendlyHand MadeAllergiesJourneys BlendCafeCommunity ChurchDog CompostingPet WasteCompostingShaggy ManeMushroomsInky CapEdible MushroomsFreelance Writertoronto writersfeatured. writing mistakeswriting service. editingflying chestnuteugeniaclean greenAutumn Mushrooms
Write Way Communications
Business Phone:705-422-2075
Copyright © 2011 Write Way Communications
↑ Scroll to top


text:
Beat Coastal, The Sequel September 29, 2008Posted by Warren in Uncategorized.
Yes, it's college football season again. And yes, while everyone else pays attention to the Bowl Series schools, I'm watching 1-AA — the championship series. I'll get into the whole Bowl game vs. playoffs debate later on, promise.
This season, my own alma mater Liberty University has been on a roll. They've got the longest winning streak in 1-AA football, and are one of a very few teams that are still undefeated. Of course, two Division II schools at the beginning of the season will do that for you, so that's not a real big bragging point for me.
But the win this past weekend on the road against Youngstown State is a bragging point. My only gripe is that the win should have been bigger — Liberty led 28-14 going into the 4th quarter and let the Penguins come back. Of course, Youngstown State led 14-0 in the first half, and let Liberty come back and tie the game by halftime. Liberty won in the final second of the game, thanks to a short field goal.
HUGE win for the Flames' program. Their goal this season is to make the playoffs, which is actually almost as arbitrary as making a bowl game. The Big South doesn't get an automatic bid yet (2010 is the first year they're eligible), so Liberty has to play their way in. And that leaves them at the mercy of three different polls and a playoff selection panel that historically hasn't been very fond of Uncle Jerry's kids. But a win on the road in Youngstown is a major feather in their cap, and something the playoff folks and the pollsters cannot ignore. The Penguins haven't lost a non-conference home game since 2004, and Liberty got clobbered the last time they played there.
Which brings me to this week's game. Liberty heads to Conway, South Carolina to play Coastal Carolina. A team we beat last year at home. THE team to beat in the Big South if the Flames want to repeat as Big South champs. I'm tempted to do a makeover on the blog this week, going to the red and white Beat Coastal color scheme that LU had last season. If I have the time, I may do that.
Follow me on Twitter — I tend to live Tweet Liberty's games as I listen to the internet audio feed. Go Flames!!


text:
Police and schools join forces for parking campaign
Police are working with Rutland's schools to encourage parents and residents to think about where they park.
Officers are taking specially-made signs around the county to try to raise awareness of the importance of parking safely around schools.
The signs, bearing the message "Think before you park - you could endanger a child's life" are being used in the mornings and afternoons when parking problems are most common.
They are also involving pupils in the scheme so the message is spread even further.
PC Laurie Appleton was in Langham working with the village school's junior safety officers on Monday. He said: "We're trying to educate parents and residents about safe parking.
"It's mostly to do with inconsiderate parking at peak times. Concerns can come from the schools themselves, from local residents trying to go about their normal life and occasionally from parents who point out regular offenders."
Rutland County Council controls parking enforcement generally, although police can deal with people parking on zig-zag lines or parking dangerously.
PC Appleton said the problems were typically worst at the start of September, when several family members drop new starters off during their first couple of weeks at school.
Police hope to visit every school in Rutland, having already been to Cottesmore and Exton. So far the reception to their campaign has been largely positive.
PC Appleton said: "Generally if we have to speak to parents they are very apologetic. Once we explain the health and safety side, and that it's their children and friends who are at risk, they are pretty good.
"They are not always thinking of the bigger picture."
Officers hope to order more signs so they can leave them with as many schools as possible.
"The more signs that we get, the further our message can get," said PC Appleton.
Register or log in via Facebook, Twitter, LinkedIn, Google+ or your Iliffe Media account to post comments.
Iliffe Media does not moderate comments. Please click here for our house rules.
People who post abusive comments about other users or those featured in articles will be banned.
Thank you. Your comment has been received and will appear on the site shortly.
Terms of Comments
We do not actively moderate, monitor or edit contributions to the reader comments but we may intervene and take such action as we think necessary, please click here for our house rules.
If you have any concerns over the contents on our site, please either register those concerns using the report abuse button, contact us here.


text:
Osta also provides translation services. We do translations of documents, contracts, websites or any other materials into the language of your choice.
These documents can be informal, or formal, and include an official translation document, if required. We have direct translation ability for Korean, Chinese, English and Japanese, and are able to provide translation services for most other languages upon request.
Why use OSTA to represent your products?
OSTA's extensive human and business network, distribution channels, import/export and marketing experience can all be yours. We have already done all the hard work for you. We have the connections and know who to talk to and where to go to get things done. We speak your language and those of your potential buyers. We have local knowledge of all the markets we operate in, and can put your product in the right place at the right time for maximum profit.
Many of our clients do not have the time or the resources to employ someone full time to research new international markets, contact buyers, negotiate price and handle all the orders. Not to mention the costs associated with traveling around the world, hotels, food, translators and re-marketing products for a new market. We can perform all of these tasks for you at a much lower cost with much more benefit. The question is therefore not why should you use OSTA, but why aren't you working with us already?


text:
What? Me? Thankful for food...not a bit, you can't tell by looking at me, and sorry folks you WILL not be telling by looking at my plate this Thanksgiving holiday. Yes, I am just a wee bit on a dieting trend and I'm going to be a braggart- doing very well at 15lbs lost since Labor Day! Yes, I will eat, but only one plate and ONLY the foods that I absolutely MUST have. Turkey breast, cornbread dressing with a little giblet gravy, green beans, a roll and a salad! I will also make my famous Crow Bars-my family can't live without these! Now there is a story behind these. Don't I always have a story? Six years ago when Jason finally got brave enough to call and ask me out, I was busy making these for my staff at the bank, I of course had to change the name from K-Bars to Crow Bars once we married and he got a great many sampling of them! Whoever said the way to a man's heart was through his stomach must have known what they were talking about, because believe me, I am thankful for so many great and wonderful meals I have shared with my husband these past six years, and look forward to many, many more years of meals!
Crow Bars
1T butter
1/2 C white sugar
1/2 c white syrup
3/4 c peanut butter
3 c crushed cornflakes
1pkg milk chocolate chips
1pkg butterscotch chips
Bring butter, sugar and syrup to boil. Add peanut butter and cornflakes, mix well. Press into 8 1/2 x 11 pan. In double boiler melt chocolate chips and butterscotch chips. When melted pour over cereal mixture. Let cool. Do not Bake! Cut into squares. Can be doubled, but I do not double the chocolate chip mixture, it is too rich! Use a big cookie sheet if you double!
Now it's your turn to share what food you are thankful for and link up at The RHOK, thank you Mrs. Priss for hosting and having everyone share their great recipes today!


text:
You ARE being watched, US. Since 9/11 Homeland Security has pumped an enormous amount of money into public surveillance technologies (online and off). Yet, as most recent studies are showing, the presence of this surveillance does nothing to reduce crime or make people more safe. So, what is this surveillance being funded for?
To help ask this question, and to bring the public's attention to the rise of a surveillance society, the American Civil Liberties Union has setup an educational website. Check it out: http://youarebeingwatched.us


text:
One of the worst things about eating gluten free is feeling totally awkward when you go out for food with friends or family. I'm happy to go anywhere, if I can't eat somewhere I'll just eat beforehand/after and go along and be social. However, it really is a good feeling when you find somewhere where the gluten free options are awesome, and chances are, everyone else is happy to eat there. Nando's is one of those places. Praise the chicken gods who brought us Nando's – perfectly spiced chicken with awesome sides for a reasonable price – and practically all gluten free (yeah, you heard me.)
Obviously, the bread dishes aren't gluten free – Nando's hasn't stretched to gluten free burger buns just yet, but all the sauces (except Perinaise) are free from gluten and the sides are not contaminated with the evils of flour-batter (onion rings, I'm looking at you.)
Everyone who visits Nando's semi-regularly will know that sometimes it's pretty good and sometimes it's absolutely incredible. You never had a terrible Nando's – well, I haven't – but also those 'absolutely incredible' ones are a rare treat. Most of the time, Nando's is just a place to go when you can't agree on anywhere else and your feet are hurting from trailing Eldon Square/you don't have much time before the cinema. For us gluten free-eaters, Nando's is a great place to get filling and tasty food in a relaxed environment without having to be totally awkward for once. Those of you who has sat sifting through a giant ring binder of 'allergen info' will know what I'm talking about.
Generally my order at Nando's stays the same – boneless chicken thighs in mango and lime marinade, spicy rice and peri peri chips with chilli jam on the side. We usually order water because we don't drink soft drinks, but this time I treated myself to a Savanna Dry cider – the perfect chilled mid-week tea.
There's not much to say about the food that you won't already know, Nando's portions are hit and miss but, as you can see, the grill chef was feeling particularly friendly on this day. Price-wise, it's not bad. It's about the same you'd pay at Wagamama or Handmade Burger Co – not as cheap as somewhere like Wetherspoons. But for great gluten free food, often the price is one of the lesser important things on your mind.
All in all, Nando's is a great choice for any coeliac or gluten free-eater who is sick of being totally picky or awkward in front of friends/family and just wants a decent meal that they can trust.
Overall scores:
Service: 3/5 (It is what it is)
Availability of GF food: 4/5
Taste: 5/5
Value for money: 4/5


text:
Carlisle Sedation Dentist
Attending regular dental appointments is crucial for patients of all ages – but many people avoid theirs time and time again for reasons related to fear and anxiety. At Flenniken Family Dentistry, our team understands these emotions well. However, the last thing we want is for dental damage and decay to go untreated. That's why we strive to create an enjoyable and comfortable environment for patients in Carlisle, PA, and sedation dentistry is a big part of that goal. With sedation dentistry, patients let go of negative emotions and feel truly relaxed as we improve their oral health and happiness. If you're interested in undergoing sedation during your care with Dr. Neil Flenniken, please feel free to contact our office today!
Nitrous Oxide
Do you suffer from mild to moderate feelings of dental fear or anxiety? Nitrous oxide could be the right choice for you. Also known as "laughing gas," it is given to patients through a nasal mask for the length of the procedure inducing feelings of comfort, relaxation, and possibly even euphoria. Best of all, these effects wear off quickly after the gas is removed, which means patients resume their daily schedules immediately.
Conscious Oral Sedation
Patients with moderate dental phobia may find that the use of nitrous oxide doesn't relax them enough. In these cases, we typically recommend oral conscious sedation. Our Carlisle office prescribes you a sedative. We provide information on when to take that sedative for optimal effects. Once a trusted escort has brought you to our practice, we ensure your comfort and begin the procedure. The effects of oral conscious sedation aren't strong enough to put patients to sleep, but patients still experience a state of deep relaxation. Many don't even remember their procedure once it's complete. Your escort needs to take you home and monitor you for the rest of the day or until the effects of the medication wear off.
IV Sedation
Dr. Flenniken is proud to be certified in this advanced sedation option for patients with moderate to severe dental phobia. IV sedation has an effect that's similar to oral conscious sedation. The main difference is that the medication is administered intravenously, which allows our team to closely monitor your care and adjust dosage if needed. Many patients relax so completely they aren't even aware they're undergoing dental work. Contact our Carlisle area office today if you'd like to learn more.
More to Explore
Preventive Dentistry Laser Dentistry Restorative Dentistry


text:
"Mrs. Human, could you take a look at my food bowl. It seems that some of my wonderful tasty hard vitamin pellets have fallen out of the bowl."
"No problem Tabby, I will have a look later."
"Later does not exist in meow Mrs. Human. They are laying on the floor and it makes the general impression of my food corner untidy. We must have order in our lives you know."
"I did not know you were so fussy Tabby."
"That has nothing to do with being fussy. We felines are clean and everything has its place. You could also refresh my water. I noticed there is some residue in the water bowl, You can clean it and give it a polish. I like to see my reflection when drinking water."
"Yes Tabby, later."
"Mrs. Human later does not exist in meow, as I already said. What is so important that I have to wait for my usual punctual attendance."
"Tabby, I am cleaning your recycling tray. Afterwards I will immediately attend to your request for tidying the pellets and of course refresh your water. Anything else Tabby?"
"You may report back in an hour, I am now taking a refreshing sleep to recover from the hard work I had to finish today."
"I didn't notice Tabby, probably too busy attending to your other needs."
"I had to sharpen my claws and that can be very exhausting, do not forget I have four paws and each claw on each paw must have individual attention to keep them clean. I am now worn out. Before you go, perhaps you could fill a second bowl with some tuna fish, but be careful that you do not spill any."
"Yes Tabby, of course Tabby."
"Just a moment, Mrs. Human, where are you going?"
"Tabby, I am now so worn with all your various tasks, have to take a rest."
"OK, let's say an hour. In the meanwhile I will paw a list in case that anything else comes to my mind. Order must be, Mrs. Human."


text:
Archive for May 2005
Protected: Last night's beauties…
Blankety blank blink….
I'm posting this from Bryant park in the slanting afternoon sunlight, because I can't get my wireless router at home to stop going blinkblink blink, which means that it isn't connected, which means that I can't log on from home. While I enjoy being all urb/techie/yuppie sitting at a little green table and typing, it still has me pissed that I can't figure out this simnple connection problem.
In other news, T and I just had a wonderful trip though the Met, soaking in the pleasures of growing up in an empire that has plundered the world's treasures so that it can display them. We started off marvelling at Roman glass (how can you not admire a society where some one took the trouble to immortalise butt-fucking on the side of a goblet?) and moved on to "Adorning the World", Art of the Marquesas Islands, where I decided that I want to make a long pole topped with a pompom of my own hair, just like they did. We soldiered on to the Diane Arbus show, important and pleasureable, but so packed that people were litterally lining up and proceeding around the rooms in a kind of zombified shuffle, which made looking at art seem like one of the stupidest things you could ever do. I've never experienced anything like it. Afterwards we rested in the company of a giggle inducing Ascension: an awestruck crowd craning to look up at christ's delicately bloody feet just slipping out of the top of the picture and then repaired to the roof garden to take in some not very good Sol Lewitt's. There was another massive line to get back down again, but luckily that scored us a spot on the frieght elevator, exposing on the way down all the service infrastructure that makes the Met such a tidy and classy experience. A perfect corrective.
A slight detour to the shop and then out onto the street, where the weather was still glorious, which lead us to try our luck in an upper east side resturant. The gods were with us and we had a resonable and toothsome dinner. And here I am with the breeze blowing up my shorts.
And now off to home. Hopefully I can figure out what the right combination of cord jiggles is to make my network come to life again. In the mean time, I'm going to stroll downtown. This is the weather and the mood I've been waiting months for.
Saturday in bed…
The first weekend where I don't have an obligation to go into the office, or do something else work related for months. I'm in shock. And while I'm watching the silly denoument of Deepstar Six just now, I soon will be turning off the tv and walking out into the sunlight.
There's a great deal that I haven't written about and that I feel I should. At least now I'm feeling the pressure slowly lifting.
Coming this Sunday…
As part of the New York Erotic Art Fair
"Tom of Finland's Importance to Culture"
Panel co-sponsored by The Center and moderated by one of its Directors, Robert A. Woodworth. The panel includes Thomas Woodruff painter, illustrator, Chair of the Illustration and Cartooning Dept., School of Visual Arts; Nayland Blake, sculptor, writer, Faculty and Chair ICP-Bard Program, Bard College; David Humphrey, sculptor, painter, Guggenheim Fellow, Fellow of the National Endowment of the Arts; Michael Kirwan, illustrator, graphic homo-sociologist, photographer, writer, philosopher; and Durk Dehner, cofounder and Director, Tom of Finland Foundation.
(Included with fair admission.)
The Lesbian, Gay, Bisexual and Transgender Community Services Center
208 West 13th Street, New York, NY 10011
The fair is all weekend and luckily the rest of the panel is so high powered that I can get by with barely uttering a peep.


text:
1 ripe avocado, chopped3 teaspoons sour cream1 clove garlic, crushedJuice of 1/2 lime2 scallions, choppedSalt and pepper, to taste1/4 teaspoon red chili flakes
Blend all ingredients.
Serve with chips.
Makes about 1 cup.
To Search---First choose a Category from the drop-down list below,
enter an ingredient name or the name of a recipe your'e looking for in the
Search Box. Click the Search Button! Search Results will come from the
Category you Selected.


text:
Quick Links
Check out all of our new Blazer LED's!
Call or email us! (410) 758-1333 email@example.com
2023A West Street, Annapolis, MD 21401
514 Solomon's Island Rd N
Prince Frederick, MD 20678
1201 Shopping Center Rd
Stevensville, MD 21666
(410)-758-1333 (Prompt #1)
We have a huge selection of bearings, seals, hubs, electric and hydraulic brake parts, disc brakes, surge brake actuators, jacks, hitches, couplers, leaf springs, lighting, hardware, electrical, breakaway kits, wheels and tires, axles, and boat trailer parts. We carry parts from some of the top brands in the industry, such as Dexter Axle, Bulldog, Tie Down Engineering, Truck-Lite, Buyers Products, Kodiak, and many more! Looking for a specific part? Use the search box at the top of the page!
Click here for our walk in store locations
Have a technical question or need help figuring out exactly what you are in need of? Feel free to give us a call or send us an email!
(410) 758-1333
Business Hours 7:30 am to 5:00 pm EST
Monday - Saturday


text:
Saturday, October 9, 2010
I keep having memories of events from the early days of dad's brain injury (memories I haven't thought about in forever) and I remember the feeling of this incredible oppressive presence - I guess you'd call it evil (which sounds a bit dramatic, but that's what it was, the sense that there was this bad, very very bad, heaviness all around). In the early weeks after Iris was born, this raccoon made its way into our garage and I could hear it bumping around down there while I was up in the middle of the night nursing her. And it became this perfect allegory for that presence: this lumbering, hunched invader. Mac trapped it (peanut butter) and drove it to the edge of town - and that was that: it was gone. I still love thinking about that. All of the love that this family is being surrounded by just has to be a bulwark against that presence, that bad. All of the local businesses have signs up for this little boy, there are thousands and thousands of visits to the blog that his parents are running, dozens of people go to see them every day, and hundreds of people write in the guestbook daily. Surely, hopefully, this will protect them, give them moments of peace and safety within all the new things to understand. And in the midst of it all, the will of this little boy, making his way back.
So the image is of a dream vision that Jean Thenaud has of the terrain governed by Prudence (who is the rather incredible babe in the middle of the image - yes, that's milk). It's the main image of my conference paper which needs to be done by Thursday morning (I leave Thursday afternoon). I've been living with this image and thinking about it for weeks now, and feel ready to write, even as I've felt completely split between thinking about this little boy (something I can't do nearly enough about), and thinking about work (something I should be doing so much more about). Dream allegories are one of the most popular genres in medieval writing - a whole lot of works gets done when the Dreamer has his out-of-body experience. Thenaud articulates (in thousands of pages) his longing for Jerusalem (and this is what I want to write about in here soon soon). What I love is that, no matter how far away they go, or how complex their allegorical dreamscape is, these dreamers always make their way back.
Subscribe to: Post Comments (Atom)