text:
- Research article
- Open Access
Adaptive evolution of the matrix extracellular phosphoglycoprotein in mammals
© Machado et al; licensee BioMed Central Ltd. 2011
- Received: 31 March 2011
- Accepted: 21 November 2011
- Published: 21 November 2011
Matrix extracellular phosphoglycoprotein (MEPE) belongs to a family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs) that play a key role in skeleton development, particularly in mineralization, phosphate regulation and osteogenesis. MEPE associated disorders cause various physiological effects, such as loss of bone mass, tumors and disruption of renal function (hypophosphatemia). The study of this developmental gene from an evolutionary perspective could provide valuable insights on the adaptive diversification of morphological phenotypes in vertebrates.
Here we studied the adaptive evolution of the MEPE gene in 26 Eutherian mammals and three birds. The comparative genomic analyses revealed a high degree of evolutionary conservation of some coding and non-coding regions of the MEPE gene across mammals indicating a possible regulatory or functional role likely related with mineralization and/or phosphate regulation. However, the majority of the coding region had a fast evolutionary rate, particularly within the largest exon (1467 bp). Rodentia and Scandentia had distinct substitution rates with an increased accumulation of both synonymous and non-synonymous mutations compared with other mammalian lineages. Characteristics of the gene (e.g. biochemical, evolutionary rate, and intronic conservation) differed greatly among lineages of the eight mammalian orders. We identified 20 sites with significant positive selection signatures (codon and protein level) outside the main regulatory motifs (dentonin and ASARM) suggestive of an adaptive role. Conversely, we find three sites under selection in the signal peptide and one in the ASARM motif that were supported by at least one selection model. The MEPE protein tends to accumulate amino acids promoting disorder and potential phosphorylation targets.
MEPE shows a high number of selection signatures, revealing the crucial role of positive selection in the evolution of this SIBLING member. The selection signatures were found mainly outside the functional motifs, reinforcing the idea that other regions outside the dentonin and the ASARM might be crucial for the function of the protein and future studies should be undertaken to understand its importance.
- Amino Acid Level
- Tree Shrew
- Amino Acid Property
- Strong Positive Selection
- Sibling Family
Dentin, one of the major mineralized tissues of teeth, is deposited by odontoblasts, which synthesize collagenous and non-collagenous proteins (NCPs) [1, 2]. Among the NCPs, there is a family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs) consisting of dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), integrin-binding sialoprotein (IBSP), matrix extracellular phosphoglycoprotein (MEPE, also known as OF45) and osteopontin (SPP1) [3]. These genes share common genetic and structural features, including a small non-translational first exon, a start codon in the second exon and a large coding segment in the last exon (although exon number varies among the different genes) [4]. The entire SIBLING protein family likely arose from the secretory calcium-binding phosphoprotein (SCPP) family by gene duplication, since this cluster of genes encodes proteins with similar molecular-structural features and functions [5].
Members of this gene family are encoded by a compact tandem gene cluster (located on chromosome 4 q in Human and 5 q in mouse) characterized by: (i) common exon-intron features, (ii) the presence of the integrin-binding tripeptide Arg-Gly-Asp (RGD) motif that mediates cell attachment/signaling via interaction with cell surface integrins [4], and (iii) post-translational modifications of conserved phosphorylation and N-glycosylation sites [4]. In humans, the MEPE protein (525 amino acids) is encoded by four exons with a 1960 bp transcript with two N-glycosylation motifs (at residues 477-481), a glycosaminoglycan (SGDG) attachment site at residues 256-259, and the RGD cell attachment motif at residues 247-249 [6]. The RGD motif has a similar function in other members of the SIBLING's (DSPP, DMP1, IBSP, and SPP1) [7]. The protein MEPE has several predicted phosphorylation sites/motifs for protein kinase C, casein kinase II, tyrosine kinase, and cAMP-cGMP-dependent protein kinase and a large number of N-myristoylation sites that appear to be also a feature of the RGD-containing proteins [7]. The acidic serine-aspartate-rich MEPE-associated motif (ASARM motif) occurs at the C-terminus in MEPE (residues 509 to 522) [7] and when phosphorylated this small peptide can bind to hydroxyapatite and inhibit mineralization [8].
The basic MEPE protein was first cloned from a tumor resected from a patient with tumor-induced osteomalacia (OHO) [7, 9], which is associated with hypophosphatemia and is caused by a renal phosphate wasting. The MEPE gene is also up-regulated in X-linked hypophosphatemic rickets (XLH or HYP-osteoblasts) and OHO-tumors [7, 10–14]. Under normal conditions it is expressed primarily in osteoblasts, osteocytes, and odontoblasts [13].
Targeted disruption of the MEPE gene in mouse causes increased bone formation and bone mass, suggesting that MEPE plays an inhibitory role in bone formation and mineralization [15]. In humans, MEPE inhibits mineralization and is also involved in renal phosphate regulation [16, 17]. The inhibition of mineralization and phosphate uptake are related with the protease resistant small peptide ASARM motif located near the end of the protein [7, 17]. However, the MEPE protein has dual functions depending on the proteolytic processing. When the protein is cleaved by cathepsin B or D into several fragments, the small peptide ASARM is released [18] and when phosphorylated, this small peptide can bind the hydroxyapatite crystal and inhibit mineralization [8]. By contrast, when fragments containing the RGD motif are released and the ASARM is not degraded by proteases, mineralization is accelerated [19]. The influence of MEPE-ASARM peptides in the modulation of mineralization is due to a protein-protein interaction with PHEX, an X-linked phosphate-regulating endopeptidase homolog (also called the minhibin model) [17]. PHEX is also expressed in osteoblasts, osteocytes and odontoblasts and the protein interacts with MEPE, protecting it from the proteolytic process (from cathepsin-B) and preventing ASARM from being released into blood circulation [8]. Most of the disorders associated with MEPE result from a malfunction of this PHEX-MEPE interaction, which in turn leads to an increase of ASARM blood levels.
The majority of mammalian genes are strongly conserved in the coding sequence [20, 21]. Genes carrying signatures of selection may be involved in adaptation and functional innovation, and often have elevated ratios of nonsynonymous/synonymous nucleotide substitutions (dN/dS) in their coding regions [22]. However, evolutionary rates of nuclear and mitochondrial genes are not equal in all the mammalian lineages [23]. For example, while rodents tend to accumulate more mutations in nuclear genes than humans [24], the differences between the rates in the two lineages seems to be smaller than the generation time difference [23].
Since MEPE protein has an important role in the regulation of the skeleton mineralization process and since the mineralized tissue is a critical innovation in vertebrate evolution, the evolutionary study of this developmental gene could provide valuable insights on the adaptive diversification of morphological phenotypes in mammals. As the MEPE gene has been suggested to be under selection [25], our objective was to undergo a thorough analysis to evaluate signatures of positive selection using both a gene-level and protein-level approaches. We assessed the evolution of the MEPE protein-coding gene in 26 mammalian species, from Hyracoidea to Primates, showing that while four regions/motifs in the MEPE gene have a high degree of conservation, the majority of the coding region has a fast evolutionary rate, especially in rodents and tree shrews. Indeed, evidence of strong positive selection (gene and protein-level) was found in 20 amino acids that encompass MEPE protein, highlighting the role of molecular adaptation in the functionality of this gene.
Presence of the MEPE in vertebrates
Twenty-six mammalian MEPE sequences were retrieved from the GenBank and Ensembl databases, comprising eight different mammalian Orders (Additional file 1: Table S1). In addition, sequences of the putative MEPE orthologue, Ovocleidin-116, were obtained from the available bird genome projects (Gallus gallus, Taeniopygia guttata, Meleagris gallopavo) for comparative purposes. For the majority of the mammals considered in this work, the MEPE gene encompasses four exons that encode a transcript that varied from 1272 bp in Ochotona princeps to 2030 bp in Pan troglodytes. Some of the smallest reported transcripts may be incomplete, as in the case of O. princeps, which is missing a stop codon. The absence of the ASARM motif in the MEPE's C-terminal in some species (Equus caballus, Ochotona. princeps, Otolemur garnetti and Pteropus. vampyrus) also suggests that those genes were not fully annotated. Thus, we performed a detailed search in databases for those species using TBLASTN [26], which led to the identification of the ASARM in E. caballus, but not in O. princeps, O. garnetti and P. vampyrus (in these cases, the missing end portion of the protein corresponds to the end of the contig available in the database). However, several stop codons are present between the end of the present sequence and the putative ASARM motif in the E. caballus sequence and therefore it was not included in subsequent analyses.
Sequence analyses
Functional motifs
The cell attachment region, RGD, situated near the center of the MEPE protein, is fully conserved in 20 of the 26 mammalian species (Figure 2). However, some changes are observed in Tursiops truncatus, Procavia capensis, the bats Pteropus vampyrus and Myotis lucifugus, and in the rodents Dipodomys ordii and Spermophilus tridecemlineatus (Figure 2) and it is likely that such amino acids changes in the RGD motif may have functional relevance. Moreover, the RGD motif is also present in other genes of this gene-cluster family.
The SDGD is completely conserved among all the mammals, reinforcing the premise that this peptide region is, along with RGD, important to the MEPE function. These two motifs constitute the dentonin region, which was not detected in any of the others members of the SIBLING protein family. The chicken and the turkey MEPE orthologues appear to be exceptions, since they do not have the cell-adhesion motif, RGD, but contain the glycosaminoglycan-binding motif, SGDG. In these species we found a HGD near the SGDG motif, suggesting that RGD is replaced by HGD (Additional file 4: Figure S2). A similar change from RGD has been described in other members of the DSPP orthologues (e.g. in rat, Rattus norvegicus, the RGD replaces the HGD) [38]. Nevertheless, in zebra finch (T. guttata) we found the RGD motif but not the SGDG region (Additional file 4: Figure S2). The ASARM motif is highly conserved within the 21 mammals for which ASARM is annotated (average above 85%), although the Bottlenose dolphin (Tursiops truncatus) has a similarity of only 59.1%. Pairwise similarity among birds was 79.9% (among the three avian species), but on average only 27.3% similarity was observed between birds and the mammalian ASARM. Moreover, in birds this motif is capped at the C-terminal by 21 (G. gallus, M. gallopavo) to 24 (T. guttata) amino acids, and this region shows 77.2% similarity between G. gallus and M. gallopavo but less than 40% between these two species and T. guttata, showing that this region in birds is probably less constrained than the ASARM.
Rodentia and Scandentia selection signatures
Results from the RRTree test comparing substitution rates in Rodentia, Scandentia and the other mammals.
Rodentia and Scandentia (n = 6)
Other Mammals (n = 20)
< 0.01
Selection signatures at the codon level
PAML results of MEPE for the 20 mammalian species (excluding ambiguity data).
Model 0
ω = 0.46086
Model 1
p0 = 0.63812 p1 = 0.36188
Model 2
ω0 = 0.27631 ω1 = 1.00000 ω2 = 1.00000
p0 = 0.63812 p1 = 0.27244 p2 = 0.08944
M2 vs M1
Model 7
p = 1.06299 q = 1.09727
Model 8
p0 = 0.86974 p = 1.58369 q = 2.27462
(p1 = 0.13026) ω1 = 1.29913
M8 vs M7
The evaluation of positive selection using the model implemented in Single Likelihood Ancestor Counting (SLAC) showed three sites under selection, one of those sites being similar to that retrieved with model M8 in PAML. Since SLAC tends to be quite conservative, we also estimated the selection signatures using the Fixed Effects Likelihood (FEL) model, which is assumed to be more powerful than SLAC [42, 43]. The FEL model revealed a total of 23 sites under selection using this model, including a mutation in the highly conserved small peptide ASARM (from aspartate to glicine) (Figure 2). Such a radical change in ASARM was only observed in a few species and further studies are needed to better document its frequency across mammals. All the sites presented in the model implemented in Datamonkey have a significance value (p < 0.10) in FEL and SLAC, which is an accepted level of significance for the test of those models [42]. When we use a significance threshold of 0.05, the number of positive selected amino acids decreased to 14 in FEL and zero in SLAC, meaning that 9 sites (out of the 23 detected with a significance level of 0.10) had less evidence of being under strong positive selection. However, these positions may still be indicative of selection signatures. Recombination can affect several analyses, including phylogenetic reconstruction and analysis of positive selection [44]. Therefore, we assessed gene recombination using GARD implemented in the Datamonkey web-based server [43] and repeated the selection analysis including and excluding recombination in the dataset. Partitioning the data did not change the conclusions of the positive selection analyses (data not show), suggesting that recombination is not significantly affecting the MEPE gene evolution.
No additional sites were found using the SLR [45], but six sites under selection in the previous analysis were also statistically supported. Overall, across MEPE, 32 of the 525 sites (referenced to the length of the human MEPE) were under positive selection; additionally six sites were supported by more than one codon analysis (9 in PAML, 23 in FEL, 3 in SLAC, and 6 in SLR) (Additional file 6: Table S4).
Selection at the amino acid level
Selection models that use dN/dS ratios to detect selection are generally not sensitive enough to detect subtle molecular adaptations [46]. It is therefore necessary to employ alternative criteria within generally conserved protein-coding genes or within proteins with strict motifs intermixed with regions under fast directional evolution. Therefore, we used TreeSAAP [47], which evaluates destabilizing radical changes at each site, and an empirical threshold of change in three properties was applied as evidence that a site is under positive (or negative) selection.
MEPE properties under positive selection determined in TreeSAAP.
Equilibrium constant (ionization of COOH)
Isoelectric point
Power to be at the C-terminal
Power to be at the middle of alpha-helix
Power to be at the N-terminal
Solvent accessible reduction ratio
Turn tendencies
At the amino acid site level, MEPE has 181 sites (33.8%) under positive selection in at least one property. Although applying the empirical threshold of at least three properties showing signatures of positive selection the number of sites is reduced to 41 (7.6%) (Additional file 7: Table S5). The majority of these 41 sites are located in the N-terminal region of the protein and the dentonin region (68% of the positive selected sites). The alternative calculation method was performed using CONTEST and estimates of variation in amino acid charge and volume revealed 79 sites with signatures of positive selection for at least one of the amino acid properties (Additional file 8: Table S6). However, after the Bonferroni and False Discovery Rate (FDR) correction, only one site showed positive selection. This site, located at position 354 in the alignment (position 349 in the human sequence), corresponds either to lysine or glutamate and was not detected by TreeSAAP. The ancestral protein reconstruction in TreeSAAP, based on the baseml implemented in PAML, shows that glutamate is present in the common ancestor of non-Afrotheria mammals, suggesting that the radical change to lysine occurred in Cetartiodactyla, Perissodactyla and in at least one representative of the Lagomorpha.
Based on selection analyses at the protein level across MEPE, 42 of the 525 sites (human MEPE as reference) were under selection at the amino acid level (41 detected with TreeSAAP and 1 with CONTEST).
Selection at codon and amino acid level
Directed evolution analysis (DEPS)
MEPE evolution has disproportionally accumulated serines, threonines (potential phosphorylation target residues), arginines, alanines and valines, as all these amino acids showed directional evolution in the DEPS analysis (with a P-value < 0.01) (Additional file 9: Table S7). The MEPE protein had 14 sites under directional selection (Additional file 10: Table S8), seven of which are amino acids that tend to increase the disorder/unstructured probability of the regions. Additionally, eight of these 14 sites had a tendency to change to amino acids that are potentially phosphorylated residues, particularly at positions 496 and 503 (505 and 512 positions in the alignment), since these sites are relatively near the ASARM motif and the cleavage site by cathepsin-B.
Selection Signatures and the MEPE structure
The MEPE protein belongs to a category of proteins classified as "intrinsically unstructured/natively disordered", with 53.8% and 55.8% of the human and the mouse MEPE constituted by amino acids that are associated with disorder/unstructured regions, respectively. This is reinforced given that most of the protein (around 78.8%) is disordered at a 0.05% false positive rate. Interestingly, the ASARM motif has a high content of amino acids disorder promoters while the other functional motifs (such as RGD and SGDG) incorporate regions that are structured (Additional file 11: Figure S3). The protein has a high percentage of the amino acid aspartate, which characterizes the proteins of the SIBLING family. Given the importance of disorder/order in MEPE, we analyzed the implications of selection signatures relative to the protein structural differences, and found that sites 75-Ser, 127-Glu, and 481-Arg (human MEPE as reference) are under positive selection and have a higher number of non-synonymous mutations towards codons that encode the amino acids disorder promoters.
MEPE in the Tetrapods
Given the absence of the MEPE gene in fishes and amphibians, its origin likely coincides with the divergence of amniotes, when mineralization [11, 48] and phosphate regulation [49] had a crucial role in species survival and diversification. SPP1 diverged from SPARCL1 (secreted protein acidic cysteine-rich like 1) and both are expressed in bone, participating in the bone formation (as an inhibitor of mineralization in SPP1) [50]. Therefore, the presence of SPP1 in fishes with a broader tissue expression pattern [51] suggests that SPP1 might also have similar functions to MEPE. Remarkably, after duplication, the genes were conserved during evolution and probably have differentiated to assume various functions related with tissue mineralization specificity. Recently, it was proposed that SPP1 is a more-powerful inhibitor of mineralization than MEPE [52]. This suggests that after the emergence of the complete SIBLING family in vertebrates, some functions were possibly shared among genes, notably because MEPE is absent in fishes.
The MEPE gene has similarities with other SIBLING genes, suggesting that it originated through a duplication event from another member of the gene family [5], but different dynamics of gene duplication and gene loss have occurred among lineages (e.g. absence of MEPE - Figure 1). The five genes of the SIBLING family are present in therian mammals and reptiles, but birds only have four genes (IBSP, SPP1, DMP1 and MEPE/OC-116), while fish only have two genes (SPP1 and DSPP-like). The DSPP orthology in fishes is controversial [51]. However, despite the low similarity, DSPP starmaker was identified as a functional orthologue [31] clearly associated with DSPP [32]. The presence/absence of various SIBLING family genes in vertebrates suggests that despite the crucial role of MEPE in mammals, birds and reptiles, its function may have been compensated in other taxa by other genes of the family. For example, in fishes a duplicated copy of SPP1 has not been described, suggesting that the fish SPP1 orthologue may have had a similar function to MEPE since SPP1 and MEPE, interact with PHEX [52]. The release of the ASARM from the MEPE protein and the phosphorylation of this motif lead to an inhibition of mineralization [48]. Similarly, the ASARM from SPP1 inhibit the mineralization [52]. Moreover, the ASARM from SPP1 is potentially phosphorylated and can interact with the hydroxyapatite crystals leading to a negative regulation of mineralization [52]. Although SPP1 has an ASARM motif near the center of the molecule, it does not have the full dentonin region (just the RGD motif). Moreover, the SPP1-ASARM has been described as a more-potent mineralization inhibitor than the MEPE-ASARM [52]. However, the knockouts of SPP1 and MEPE in mice have different phenotypes. MEPE knockouts have increased bone mass and inhibition of age-related bone loss [11] while SPP1 knockouts cause a resistance to bone loss and trabecular bone mass [53].
Functional Conservation
The functional motifs of MEPE (RGD, SGDG and ASARM) are highly conserved among the studied mammals. In the SIBLING proteins the first coding exon encodes the signal peptides [4, 54], as is observed in MEPE. The RGD motif is a common feature of all member of the SIBLING family, remaining functionally preserved after the tandem duplication that gave rise to all the members of this gene family [4]. Surprisingly, birds do not have a complete dentonin region (RGD and SGDG), although the high conservation observed among mammals suggests that this region has an important role in the function of the protein. In fact, in mammals the gene function apparently depends on the full dentonin region, as the RGD motif alone does not enhance an optimal adhesion on biomaterial surfaces in osteoblast [55]. However, when SGDG is close to RGD the mitogenic activity of dentonin increases, while the presence of only the SGDG motif promotes the cell proliferation [56]. In mammals, MEPE is involved in bone formation and osteoblast proliferation [19, 56], while in birds it is involved in egg-shell formation [57]. This functional divergence may explain the sequence differences observed between the two lineages, particularly reinforced by the absence of the full dentonin region in birds. The ASARM motif is also highly conserved among mammals, but shares less than 50% similarity with the avian ASARM. Moreover, we have not detected a similar cathepsin-B cleavage site near avian MEPE-ASARM and this motif is capped at the C-terminal by 21 to 24 amino acids. Amino acids towards the C-terminal after the ASARM motif are also observed in marsupials [25]. Despite the lower similarity with the mammalian ASARM and its different position, the high conservation within birds suggests that this motif continues to have a crucial role. The changes are probably not due a relaxation of selection, but instead may have an adaptive role. In mammals, the cathepsin-B cleavage site is crucial for the function of MEPE, since this small peptide only interferes with hydroxyapaptite crystals when released [17]. Therefore, birds are also expected to have a mechanism for cleavage of ASARM. MEPE has not yet been annotated in a monotremata, no significant matches were found in a representative species of this group, the platypus (Ornithorhynchus anatinus). Nevertheless, the discovery of this gene in egg-laying mammals would be of great relevance to understanding the functional differences between mammals and birds.
The coding region of MEPE that flank the motifs described above is less conserved, but retain considerable phylogenetic signal across species. The human MEPE sequence has a high similarity with the great apes and with the genus Macaca (Cercophitecidae), even in the non-coding regions (M. mulatta). To a lesser degree, human MEPE also has some significant similarities in the non-coding regions with the genes of the lower primates (M. murinus and O. garnetti). MEPE appears to be particularly conserved among primates, in both coding and non-coding regions. The intronic conservation could provide valuable information about the role of non-coding sequences in the regulation/functionality of this gene. Despite the accelerated evolution in rodents, intronic conservation allowed us to reconstruct a well-supported species phylogeny from intronic sequences (even including the rodents sequences) with similar results as those obtained from MEPE coding regions.
Several human diseases increase MEPE expression [7, 14, 58], which may imply functional constraints in the gene even at the intronic level. Previous studies have demonstrated that highly conserved intronic regions are correlated with functional constraints and can be evidence of a hidden class of abundant regulatory elements [59]. Recently, a SNP in the region 7 kb 3' of the gene was associated with osteoporosis, a disease characterized by reduced bone mass and microarchitectural deterioration of bone tissue that reduces bone strength and leads to an increased risk of fracture [60]. These findings suggest that intergenic regions can also be important in gene function and may cause significantly different phenotypes. We hypothesize that intronic regions can also lead to significant differences at the expression level and ultimately to differences in phenotypes. This is consistent with our findings that there are strong evolutionary constraints in the MEPE intronic region.
Selection signatures and conservation
Within mammals, MEPE in rodents is evolving faster, presenting a high amount of transitions and transversions. A similar trend is also observed in the tree shrew T. belangeri. However, since we only had one MEPE sequence from the order Scandentia we were unable to infer if this pattern is species-specific or if it is typical of this order. The increased number of substitutions in rodents was expected as previous studies have shown that rodents tend to accumulate more mutations in the coding regions [24, 61]. We hypothesize that the observed differences in these two orders have resulted from either a divergent functional role or simply a relaxation in Darwinian selection. It is not known if the function of the rodent MEPE is similar to that in humans [62], but all the functional motifs are conserved and the signatures of positive selection or the differences observed were only detected outside of these important motifs. It is clear that positive selection may have an important role in the functional divergence of homologous proteins during adaptation to different habitats [63]. Indeed, selection may be episodic as positive and negative selection shifts over time across different lineages, reinforcing the importance of comparing sequences that have diverged within appropriate time frames [64]. The branch-site model, using rodents as foreground branches and allowing ω ratio variation not only between the branches but also among sites, identified 12 sites with strong signatures of positive selection. This suggests that the rodents and probably Scandentia may have lineage-specific selection differences in MEPE, not only in the magnitude of the selective pressure found in the branch, but also in the number of sites under selection. The acceleration of the substitution rates in rodents and the tree shrew potentially compromises the assessment of positive selection by increasing the number of synonymous mutations and because this heterogeneous site selection is observed in only two of the eight orders evaluated (i.e. Rodentia and Scandentia). The results may also be biased by the mixing of species with long and short generation times [61], as well as the related long-branch-attraction effect in phylogenetic reconstruction. Therefore, we did not include the rodents and the tree shrew in the site analysis.
The evolutionary analyses of mammalian MEPE codons (excluding the rodents and the tree shrew) found 32 sites under positive selection at codon level, and remarkably three were in functional regions of the protein, positions 6-Val and 11-Phe (Signal Peptide) and position 517-Gly (ASARM motif) (Figure 2).
Recent methods for investigating selection in protein coding genes have focused on evaluating the type of positive selection detected (directional or nondirectional, stabilizing or destabilizing), determining the presence of purifying selection, and interpreting how selection affects overall protein structure and function. Amino acid substitutions have different effects on a protein depending on differences in physicochemical properties and their position in the protein structure [65]. Here, we performed multiple analyses to differentiate among the different types of selective pressures acting in MEPE at the amino acid level. The evaluation of the amino acid physiochemical properties changes in the mammalian MEPE identified 37 more sites (36 using TreeSAAP and one using CONTEST) with selection signatures compared with the results retrieved using codon models. This shows that total reliance on models based on dN/dS using codon models may not detect some important sites with signatures of selection, often because a single adaptive mutation may occur in a small number of species, resulting in an omega lower than one. By contrast, these could also primarily be amino acid stabilizing rather than destabilizing changes, and a ω > 1 may not always be indicative of adaptive evolution.
Combining all the selection analyses, we found 69 amino acids with evidence of positive selection (20 well-supported by both codon and amino acid level approaches) (Additional file 12: Figure S4). Three clusters of positively selected sites revealed three new motifs that likely have a functional role, SEASEN (75-80), LNXEXS (96-101) and ENT (170-172) (Figure 10), using the human protein as site reference.
Selection analysis of MEPE in TreeSAAP using amino acid destabilizing properties revealed that the structural properties tend to be more affected by positive selection than the chemical properties. This suggests that the flexible and intrinsically unstructured nature of MEPE is linked to its multiple biological roles. The ASARM motif shows a "high tendency" to be a "disordered region and highly acidic", although the conformation of ASARM should be dependent on the phosphorylation level [66]. The ability to bind to hidroxyapatite is also correlated with phosphorylation state and PHEX cleavage of MEPE is dependent on the Serine phosphorylation status [8]. Moreover, our results shows that the protein tends to accumulate numerous residues with potential phosphorylation sites and this can be important to the folding/function of the protein. Proteins fold to minimize their free energy, although the structure also reflects an organization that can allow the recognition of a ligand or a transition state [67]. In fact, there is a balance between protein function and stability, and most of functional sites are non-optimal in terms of stability. If a residue is replaced by another residue, the protein activity will be reduced but the stability will be increased [67]. In MEPE we detected 75 sites with a Γ lower than -2 kcal/mol, indicating that a large number of sites in MEPE are non-optimal and therefore possibly involved in protein function. Moreover, 13 of those sites showed signatures of selection in one analysis, and sites 55, 127 and 276 in both codon and amino acid level analyses. Proteins have different secondary-structures and physicochemical properties and roles that help determine their evolutionary flexibility [68]. Thus, amino acids that comprise disordered regions, such as random coils, are more likely to be under positive selection than expected from their proportion in the proteins, compared with the residues in helices and β-structures which are subjected to less positive selection [68]. Indeed, when we compare the evidence of positive selection with the protein secondary structure in MEPE we observed that the number of sites under selection in the random coils and disordered regions are slightly higher than expected. This suggests that a high number of sites probably have a functional role or are at least relevant to an increase in MEPE protein flexibility.
Presently, most of the research on MEPE has centered on the biological role of the RGD and ASARM regions. However, our comparative study of mammalian MEPE orthologues revealed that the protein has lineage-specific properties (e.g. biochemical, evolutionary rate, intronic conservation), and that outside these two well-described motifs there are 69 sites (20 with high confidence level) under positive selection and of probable functional relevance. As positively selected sites might be either near catalytically important regions of the proteins [69] or be functionally relevant sites [70, 71], these sites are good candidates for mutagenesis and structural studies to determine the functionality of MEPE relative with the other SIBLING proteins.
MEPE is found in reptiles, birds and mammals (eutheria and metatheria), and to date has not been identified in monotremes. The description and study of MEPE in other taxonomic groups will be crucial to fully understanding the differences reported in avian and mammalian orthologues, and the adaptive significance of these differences. The absence of this gene in some vertebrate lineages suggests that SPP1 might partially cover the functions of MEPE in those groups. MEPE retains a strong phylogenetic signal at both coding and non-coding regions in mammals, probably due to in the functional relevance of these regions. Nevertheless, the gene is highly variable, particularly in the largest exon outside the functional motif, while other regions appear to be under strong positive selection. We found 20 sites with a significant signature of positive selection at both nucleotide and amino acid level complimentary analyses (in addition to other 69 sites with evidence of selection at either the nucleotide or the amino acid level). The analyses identified three motifs (LNXEXS, SEASEN and ENT) with selection signatures suggesting important adaptive functions. We also showed that Rodentia and Scandentia have an accelerated evolutionary rate with a unique evolutionary pattern. Finally, we showed that MEPE tends to accumulate amino acids that promote "disorder" and that present potential phosphorylation targets, supporting the contention that other regions outside the dentonin and ASARM might have crucial functional roles and demonstrating the need for future studies to understand the importance of these regions.
Comparative genomic analyses
MEPE nucleotide sequences were retrieved from GenBank and ENSEMBL. We aligned 26 MEPE sequences representing eight orders of mammalian species and produced two different alignments, one including all species and another excluding rodents and the tree shrew due to its nucleotide saturation bias. Given the low similarity between the avian and the mammalian sequences, the avian sequences were excluded from phylogenetic and selection analyses. BLAST searches were used to retrieve non-annotated sequences from several mammalian genomes. All the alignments were performed after the translation of nucleotides to amino acids and the corresponding alignments were back-translated to nucleotides. The alignment were performed in ClustalW [72] implemented in BIOEDIT v7.05 [73], MEGA4 [74] and LAGAN [75]. Sliding-window percent amino acid and nucleotide identity, and % GC content were calculated in Swaap 1.0.3 [76]. Saturation plots (including or excluding the third-coding position) and the estimated pI (excluding indels) were assessed in DAMBE [77]. Conservation in the coding and the non-coding regions was assessed using mVISTA [78].
Phylogenetic analyses
We used Modelgenerator version 0.85 [79] to determine the optimal model of sequence substitution for our protein dataset, employing the Jones-Taylor-Thornton (JTT+I+G) substitution model. MrModeltest 2.3 [80] was employed to determine the optimal model of sequence substitution for our coding sequence dataset, employing the General-Time-Reversible (GTR+I+G) substitution model with the invariant site plus gamma options (five categories). Bayesian inference methods with Markov chain Monte Carlo (MCMC) sampling were performed in MrBayes [81, 82]. The analysis was run for 5, 000, 000 generations with a sample frequency of 100 and burn-in was set to correspond to 25% of the sampled trees. The Maximum-Likelihood (ML) phylogenetic tree was constructed in PHYML [83], under the best-fit model for nucleotides and amino acids, 1000 bootstrap replicates and the NNI branch search algorithm. The parameters used in the tree reconstructions were set to: (i) Nucleotides: GTR+I+G with 6 substitution rate parameters and gamma-distributed rate variation with a proportion of invariant sites; (ii) Amino acids: JTT+I+G. A neighbor-joining tree was conducted in MEGA 4 [74] using the complete deletion of ambiguous data and the maximum composite likelihood option. The topologies were tested in TREE-PUZZLE [84] to identify the tree that best fits the alignment, using three tests: KH, SH and ELW. A phylogenetic signal test was performed in TREE-PUZZLE [84] using the implemented methodology [85].
Detection of positive selection
Gene-level analyses
Positive selection analyses were performed in the Eutherian mammals (the closely-related taxa) to avoid nucleotide saturation and base-compositional bias. We assessed positive selection using primarily a gene-level approach [65] based on the ratio (ω) of nonsynonymous (dN) to synonymous (dS) substitutions rate (i.e., ω = dN/dS), implemented in PAML v4.3 [86] and in the web-based program SELECTON [87, 88], PAML uses LRT to compare two nested models, a model that does not allow, and a model that allows, sites categories > 1 (null versus positive selection, respectively). Here, we used three LRTs based on site-specific models comparing the nested models: M1a-M2a, M7-M8 and M8a-M8. The first LRT was performed comparing M1a (nearly neutral: p0, p1, ω0 < 1, ω1 = 1, NS sites = 1) against M2a (positive selection: p0, p1, p2, ω0 < 1, ω1 = 1, ω2 < 1, NSsites = 2); the second LRT was comparing M7 (beta: p, q, NS sites = 7) with M8 (beta & ω: p0, p1, p, q, ωs > 1, NS sites = 8). The third LRT was between M8a (beta & ωs = 1: fix omega = 1, omega = 1, NS sites = 8) and M8. However, a significant LRT only demonstrated that the selection model is more suitable than the neutral model; it does not provide any indication of the sites under selection [89]. This can be accomplished through an Empirical Bayes (EB) approach to calculate the posterior probability (PP) that a given site comes from the class with ω > 1. Sites presenting a PP above the defined cut-off value (e.g. p > 95%) [90] are inferred to be under positive selection. A robust method was used to accommodate the uncertainties in the MLEs of parameters in the ω distribution, designated by BEB [90]. This approach was shown to be reliable in both small and large data sets, and also to have a good resolution power for identifying individual sites under positive selection, especially in large data sets or with strong selective pressure. We also performed an analysis using the branch-site model A [91] (model = 2 NSsites = 2), including and excluding the rodents and tree shrew as foreground branch, allowing the ω ratio vary both among sites and among lineages. The branch-site test 2 was performed using the null model, ω2 = 1 fixed (using the parameters fix omega = 1 and omega = 1). The sites under selection in the foreground branches were obtained after calculating probabilities of site classes using the BEB procedure.
Although the PAML models [86] allow for variation in the non-synonymous substitution rate, the synonymous rate is fixed across the sequence. To overcome that specificity, we used SLAC and FEL [92] for detecting positive selection while allowing variation in synonymous rate. SLAC is a heavily modified and improved derivative of the Suzuki-Gojobori counting approach [42, 93] that maps changes in the phylogeny to estimate selection on a site-by-site basis. SLAC calculates the number of non-synonymous and synonymous substitutions that have occurred at each site using ML reconstructions of ancestral sequences [42, 93]. The FEL model estimates the ratio of nonsynonymous to synonymous not assuming an a priori distribution of rates across sites substitution on a site-by-site analysis [93]. The SLAC and FEL methods were implemented using the web interface Datamonkey [94]. Since recombination in the gene can bias the analysis [44], we also re-run SLAC and FEL in Datamonkey using the GARD method [95], allowing each calculated partition to have its own phylogenetic tree.
Additionally, we used the LRT based analysis as implemented in the SLR (Sitewise Likelihood-Ratio) software package [45]. This method assumes that substitutions (both synonymous and non-synonymous) can occur independently with every other site, modulating substitution rates as a continuous-time Markov process. The LRT on a site-wise basis is performed testing a null model (neutrality, ω = 1) against an alternative model ω ≠ 1.
Protein-level analyses
We performed multiple analyses to differentiate the different types of selective pressures acting in MEPE: (i) positive versus negative selection, and (ii) stabilizing (selection that tends to maintain the overall biochemistry of the protein) versus destabilizing selection (selection that results in radical structural or functional shifts in local regions of the protein). These analyses provided insight into the structural and functional consequences of the residues under selection [46]. We used TreeSAAP v3.2 [47] and CONTEST [96] implemented in IMPACT [97] to detect selection signatures at the amino acid level. In TreeSAAP positive destabilizing-selection is detected based on the properties changes with significantly greater amino acid replacements than would be expected under neutrality for magnitude categories +7 and +8 (i.e., the two most-radical property-change categories). Within TreeSAAP, 31 amino acid properties were evaluated across the phylogenetic tree to identify the specific amino acid residues within each region that showed evidence of positive destabilization for each property. The baseml implemented PAML [86] is used in TreeSAAP [47] to reconstruct ancestral character states at the nodes on the MEPE phylogeny.
To test if evolutionary rates varied between lineages we used the relative-rate test, weighting by the predefined tree topology, as implemented in RRTree [98]. To detect directional selection over the tree or a large number of substitutions towards a particular residue in a maximum likelihood context we used the directional evolution in protein sequences (DEPS) analysis to identify statistically significant directional changes in amino acid residue frequencies [99].
MEPE Three-Dimensional Structure Modeling
To determine the position of the positive selected amino acids when the protein is folded, we modeled the three-dimensional (3D) structure of MEPE. Protein structure prediction can be approached in three ways: (i) comparative modeling, (ii) threading, and (iii) ab-initio folding. For MEPE, the first two methods, which build a protein model by aligning query sequences onto solved template structures, were not feasible. Thus, the only practical strategy was to run the I-TASSER [100] to obtain an ab-initio 3D model of MEPE. The model obtained using the Homo sapiens sequence had a TM Score of 0.46 ± 0.15 and a C-Score = -2.18. To accurately infer the correct topology, the model should have a C-score above -1.5, varying from [-5; 2] [100]. A TM score above 0.5 means that the obtained topology is not random [86]. Results using the sequences of the rock hyrax (out-group), the dog (i.e. one of the species showing differences in the pI) and the mouse (which demonstrates accelerated evolution) all had similar C-scores and the 3D structures similar to the results retrieved for the human MEPE, suggesting that the biochemical differences in the composition of the amino acids that constitutes the different orthologues are not imposing significant differences in the folding of the protein.
Structural analyses
To assess the surface exposure of the amino acids in the protein structure, we used the GETAREA 1.1 [101] web-based program based on the atom coordinates of the PDB file. This provides an estimate of the solvent exposure based on the ratio of the side-chain surface area to "random coil" value per residue, performing an analytical calculation of solvent accessible surface area residues. These are considered to be solvent exposed if the ratio value exceeds 50% and to be buried if the ratio is less than 20% [101]. Since MEPE has been described as an intrinsic unfolded protein, we also used the Protein DisOrder prediction System (PrDOS) server [102] to predict natively disordered regions of a protein chain based on the composition of the amino acid sequence. Protein stability was calculated with the PoPMuSiC 2.1 web server [103] using the MEPE PDB file previously obtained in I-TASSER to calculate the sites Γ considering all the possible mutations in each site. The secondary structure was visualized in POLYVIEW [104].
The authors acknowledge the Portuguese Fundação para a Ciência e a Tecnologia (FCT) for financial support to JPM (SFRH/BD/65245/2009) and the project PTDC/BIA-BDE/69144/2006 (FCOMP-01-0124-FEDER-007065) and PTDC/AAC-AMB/104983/2008 (FCOMP-01-0124-FEDER-008610). This work was further supported by a grant from Iceland, Liechtenstein and Norway through the EEA Financial Mechanism and the Norwegian Financial Mechanism. We thank Siby Philip from LEGE/CIIMAR for discussion and helpful suggestions. Comments made by the anonymous reviewers improved a previous version of this manuscript.
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text: Photo Gallery 2016 Warrior Run 2016 Warrior Run 5k at Bell Tower Park in Mariemont, OH. In support of the James W. Miller Memorial Fund. 2016 Harvest Hustle 2016 Harvest Hustle 5k at New Horizons Rehabilitation center in Sunman, IN. Serving individuals with disabilities in southeastern Indiana since 1968. 2016 Mini Heart 2016 Mini Heart 5k in downtown Cincinnati, OH. Benefits the American Heart Association, whose mission is to build healthier lives, free of cardiovascular diseases and stroke. 2015 Go Santa Go 2015 Go Santa Go 5k at Summit Park in Blue Ash, OH. This event is a great way to get into the "Holiday Spirit" and serves various charities. 2015 Fighting Hunger 2015 Fighting Hunger 5k at Mathew 25 Ministries in Blue Ash, OH. Benefits Hunger 5K Food Drive, which collects canned and nonperishable food for local food pantries and shelters. 2015 Cheetah Run 2015 Cheetah Run 5k at the Cincinnati Zoo in Cincinnati, OH . Proceeds benefit the Cincinnati Zoo, one of the oldest non profits in the city. Share this:

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- Utah, United States
Salt Lake City, often shortened to Salt Lake or SLC, is the capital and the most populous city in the state of Utah. With an estimated population of 189,314 in 2012, the city lies in the core of the Salt Lake City metropolitan area, which has a total population of 1,175,905. Salt Lake City is further situated in a larger urban area known as the Wasatch Front, which has a population of 2,350,274. It is one of only two major urban areas in the Great Basin (the other being Reno, Nevada), and the largest in the Intermountain West.
Before Mormon settlement, the Shoshone, Ute, and Paiute had dwelt in the Salt Lake Valley for thousands of years. At the time of the founding of Salt Lake City, the valley was within the territory of the Northwestern Shoshone; however, occupation was seasonal, near streams emptying from Canyons into the Salt Lake Valley. The land was treated by the United States as public domain; no aboriginal title by the Northwestern Shoshone was ever recognized by the United States or extinguished by treaty with the United States.
The first US explorer in the Salt Lake area is believed to be Jim Bridger in 1825, although others had been in Utah earlier, some as far north as the nearby Utah Valley (the Dominguez-Escalante expedition of 1776 were undoubtedly cognizant of the Salt Lake valley). U.S. Army officer John C. Frémont surveyed the Great Salt Lake and the Salt Lake Valley in 1843 and 1845. The Donner party, a group of ill-fated pioneers, had traveled through the Great Salt Lake Valley in August 1846.
Salt Lake City has a total area of 110.4 mi2 (285.9 km²) and an average elevation of 4,327 feet (1,320 m) above sea level. The lowest point within the boundaries of the city is 4,210 feet (1,280 m) near the Jordan River and the Great Salt Lake, and the highest is Grandview Peak, at 9,410 feet (2,868 m).
Museums and the arts
Salt Lake City is home to several museums. Near Temple Square is the Church History Museum; operated by The Church of Jesus Christ of Latter-day Saints, the museum contains collections of artifacts, documents, art, photographs, tools, clothing and furniture from the history of the LDS Church, which spans nearly two centuries. West of Temple Square, at the Gateway District, is the Clark Planetarium, which houses an IMAX theater, and Discovery Gateway, a children's museum. The University Of Utah campus is home to the Utah Museum Of Fine Arts as well as the Natural History Museum Of Utah. Other museums in the area include the Utah State Historical Society, Daughters of Utah Pioneers Memorial Museum, Fort Douglas Military Museum, the Social Hall Heritage Museum, and The Leonardo, a new art, science and technology museum.
Performing arts
Salt Lake City provides many venues for both professional and amateur theatre. The city attracts many traveling Broadway and Off-Broadway performances which perform in the historic Capitol Theatre. Local professional acting companies include the Pioneer Theatre Company, Salt Lake Acting Company and Plan-B Theatre Company, which is the only theatre company in Utah fully devoted to developing new plays by Utah playwrights. The Off-Broadway Theatre, located in Salt Lake's historic Clift Building, features comedy plays and Utah's longest running improv comedy troupe, Laughing Stock.
Main sights
Salt Lake City is the headquarters for The Church of Jesus Christ of Latter-day Saints (LDS Church) and has many LDS-related sites open to visitors. The most popular is Temple Square, which includes the Salt Lake Temple (not open to the general public) and visitors' centers that are open to the public, free of charge. Temple Square also includes the historic Tabernacle, home of the World-famous Mormon Tabernacle Choir. The modern LDS Conference Center is across the street to the north. The Family History Library, the largest genealogical library in the world, is located just west of Temple Square. It is run by the LDS Church and is open to the public and free of charge. Adjacent to Temple Square is also the Eagle Gate Monument.
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Category Archives: BAKING
butterscotch pie
I love two things most of all about chess pie — that sweet, buttery baked custard pie well-known across the South but whose reach can be tasted in everything from Canadian butter tarts to Brooklyn-ish Crack Pies — one, that… read more
No-Bake White Chocolate Raspberry Cheesecake
This No-Bake White Chocolate Raspberry Cheesecake is one of the best no-bake cheesecakes I've made so far and it's very easy to prepare. The raspberry heart shapes on top makes it ideal for Valentine's Day but is perfect for any… read more
Almond Coconut Cake (Raffaello cake)
This Almond and Coconut Cake is one of the best cakes I've made lately and it is inspired by the Raffaello truffles, which are a great combination of a smooth milk cream filling around almonds,… read more
an easier way to make cookies
Pretty much everything terrible about making cookies comes down to one thing: deciding you want a cookie and realizing that the expanse between now and when you get to eat said is unfairly wide.
For example:
• Butter needs to
Lemon Poppy Seed Pound Cake
This Lemon and Poppy Seed Pound Cake is a nice cake, moist and lemon, perfect for breakfast or coffee / tea.
While still warm pound cake brushed with a sweet lemon syrup, which brings more moisture and flavor to it.… read more
Saucy Cranberry Maple Pudding Cake
- For the filling
- 2 1/2 cups (9 oz) fresh or frozen thawed cranberries
- 1/3 cup plus 1 TBSP maple syrup (grade A or grade B dark amber is fine)
- 1/4 cup water
- For the topping
- 2/3 cup (3 oz)


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2000 Ford 24.7 concept station wagon
publicity photos courtesy by
Mariusz Szewczyk
Introduced at the 2000
Northern American Auto Show
in Detroit, MI USA
Want more info? Here's the
press release
The good: Ford produces a concept station wagon
The bad: the styling
The ugly: all over!
Copyright© 2000
Steve Manning
All Rights Reserved
Information in this document is subject to change without notice.
Other products and companies referred to herein are trademarks or registered trademarks of their respective companies or mark holders.
Return to www.stationwagon.com home page.....


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August 1
Annual Report
Each year we report to the Ministry of Education about how well we have met our key student progress and achievement targets (called Analysis of Variance). We also report on how we have spent our KiwiSport funding.
You can download and view these reports. If you have any comments or queries about the information in these, please don't hesitate to speak to Shelley Wilde (Principal).


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Michael Bird and Nik Sproal of Social Garden sit down to discuss the barriers that are often faced when sales and marketing teams come together, highlighting the benefits that a sales and marketing alignment strategy can provide in generating revenue and improving customer journeys. Watch the video below!
If you'd like a hand getting started with your sales and marketing alignment, check out our ebook, "3+ Questions to Start Your Sales and Marketing Alignment Process"
Hey, my name's Mike, I'm the CEO at Social Garden. I'm here with our Client Service Director, Nik Sproal. And today we're going to have a quick chat about marketing and sales alignment and how we can get this two business functions in the property sector working together to deliver a much better kind of customer journey and overall customer experience. So, in terms of what you've been seeing work across, as you said, you've worked at Stockland, Investa, across all kind of the major corporate property developers. And then, obviously here at Social Garden. What are some things that you're seeing work well when sales and marketing teams kind of team up? And what are some of the barriers to that right now?
Look, I think some of the key things you really see is that when sales and marketing work well together, not only does it drive the sales process and make a smoother process and make sure that I guess you're maximising your sales opportunities, it also actually really helps with other elements of the business like driving the product mix, driving the pricing, driving all the element that ultimately make a successful campaign in the long term. And also deliver a project that actually delivers profit and delivers consistent sales results. By having those teams working together it can have a really powerful insight to drive a whole multitude of touchpoints within the project not just the sales/marketing experience themselves.
Yeah, I mean, so many times when we first go in and start working with the client there's kind of like an invisible wall or sometimes a literal wall that sits between marketing and sales. When marketing is kind of responsible for brand and for generating leads and they kind of chuck them over the fence, and in sales either is loving it and they're accepting the leads and they want more and they kind of blame not having more on marketing. Or there's kind of an issue with lead quality, so the two functions historically, start to have kind of clashed heads -
But certainly when we look at the why the consumer journey had changed, and there is so much of the decision making process is happening online before a prospect ever engage a salesperson. Really marketing is significantly in a more responsible for that revenue piece.
And for driving the customer experience and making sure that the sales team have really good qualified enquiries to deal with.
Absolutely, I think one of the key things we've seen change is that marketing were probably always held responsible for softer metrics around click-through rates and impressions and so forth. But by moving that further down the funnel and making marketing more responsible for cost-per-lead, cost-per-appointment attended and so forth, it provides comfort for the sales team that we're driving the right quality of enquiries and it ultimately makes the marketing team have I guess, a seat at the table in terms of making those decisions and being involved in those elements that drive the revenue piece.
Yeah, I think it's just so important that it's really clear upfront before going into a - whether it's a new apartment project, or land project - whatever it might be. Really important that, what is determined to be a marketing lead and what is a sales lead, is super important. Because if you don't have that clearly defined upfront it's just gonna automatically, like create friction even before you even begin. So, by getting everyone kind of around the table at the same time, having marketing talking to sales, thinking about what are the qualifying questions that a prospect needs to answer in order for that person to be ready to speak to the salesperson. I think having that clear is a good start.
I couldn't agree more Mike. I think what you see, is we do start to drive a higher volume of leads, what the sales team are looking for is markers that indicate to them where a prospect is at in their journey and if we can build models around that whether it be a progressive profiling model--
Do you want to just talk about what progressive profiling is.
Yeah, absolutely. Obviously, as you were talking about marketing lead versus the sales lead, the marketing lead might be simply providing a base level information to get people to move into the CRM.
So for example, they might download an information pack from the lead capture page or from a website.
Absolutely. So, a sales lead is someone who's actually provide information about what sort of product they're looking for? Do they have finance in place? How much money do they have to spend? What sort of—how many homes or how many rooms they're looking for? So it really provides a much more granular level of detail around who they are, where they're at in the journey and what they're seeking. So, by being able to provide or capture that information during the lead capture phase, we're actually giving the sales team almost to leg up to actually start their conversation with that buyer based on relevant information that's pertinent to that discussion. So it becomes very personalised and very one-to-one rather than a simplified process that replied to every single enquiry.
Okay, cool. So that's our episode on marketing and sales alignment in the property sector. If you're interested in learning more, just feel free to download our guide, Sales and Marketing Alignment with in Property and we'll chat to you soon. Thanks!
: Thank you.
The Urban Developer is proud to partner with Social Garden to deliver this article to you. In doing so, we can continue to publish our free daily news, information, insights and opinion to you, our valued readers.


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We recommend you consult our professional financial adviser, Tania Tonkin, who will be pleased to assist you.
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Orchardleigh House Wedding Photography
The beautiful Mel & Thomas were married in Somerset on the wonderful Orchardleigh House estate in June, after days of beautiful weather, the skies weren't looking too special however as the morning progressed so too did the weather.
The ceremony was held at the gorgeous church at Lullington, my favourite church in this area, its light and has a beautiful atmosphere, Brian was to take the service, a lovely minister, he is always full of fun and his address is always a pleasure to listen too and is guaranteed to have the guests smiling as he speaks from the heart, with many experiences for the Bride and Groom.
Melanie and her girls were getting ready for the big day in the Benita suit at Orchardleigh, a stunning room, just perfect for the Bridal preparations.
Thomas and the lads were down stairs helping with the preparations for the reception room, both the Bride and Groom were very relaxed however there was a buzz of excitement throughout the house as all the guests anticipated the event.
We unobtrusively captured the morning and Tracy and I left for the church to ensure we captured the guests, family and of course Thomas and Mel's arrival.
The ceremony was lovely, two reading from close Family, a very warm and romantic service.
When we arrived back to Orchardleigh the sun was shining, the light was beautiful, I love photographing wedding's in the sunshine, the colors, contrast making for stunning photography.
I took the happy couple for a stroll around the grounds, capturing those natural reportage images.
Melanie & Thomas asked to have a few formal groups which we took in different locations on the estate, I have had the privilege of providing my wedding Photography at this beautiful house since the beginning of its journey as a wedding venue, I live 5 mins away from this wedding venue, I know the house and grounds as if it was my second home.
The evening was beautiful, warm with the sunshine, this gave everyone the perfect opportunity to enjoy the breathtaking grounds.
We stayed through until Melanie & Thomas danced for their first dance and then we left this wonderful wedding and made our short trip home.
Melanie and Thomas were heading off to the USA to spend three wonderful weeks touring the country, we wish them both all the love and luck in the world.
I have added a small selection of photographs from this wonderful wedding at Orchardleigh House.


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Ready to add myPharmacyConnect to your pharmacy?
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