text:
- Research article
- Open Access
Functional divergence of the NIP III subgroup proteins involved altered selective constraints and positive selection
- Qingpo Liu1Email author and
- Zhujun Zhu1Email author
© Liu and Zhu; licensee BioMed Central Ltd. 2010
- Received: 2 March 2010
- Accepted: 20 November 2010
- Published: 20 November 2010
Nod26-like intrinsic proteins (NIPs) that belong to the aquaporin superfamily are unique to plants. According to homology modeling and phylogenetic analysis, the NIP subfamily can be further divided into three subgroups with distinct biological functions (NIP I, NIP II, and NIP III). In some grasses, the NIP III subgroup proteins (NIP2s) were demonstrated to be permeable to solutes with larger diameter, such as silicic acid and arsenous acids. However, to date there is no data-mining or direct experimental evidences for the permeability of such larger solutes for dicot NIP2s, although they exhibit similar three-dimensional structures as those in grasses. It is therefore intriguing to investigate the molecular mechanisms that drive the evolution of plant NIP2s.
The NIP III subgroup is more ancient with a divergence time that predates the monocot-dicot split. The proliferation of NIP2 genes in modern grass species is primarily attributed to whole genome and segmental chromosomal duplication events. The structure of NIP2 genes is relatively conserved, possessing five exons and four introns. All NIP2s possess an ar/R filter consisting of G, S, G, and R, except for the cucumber CsNIP2;2, where a small G in the H2 is substituted with the bulkier C residue. Our maximum likelihood analysis revealed that NIP2s, especially the loop A (LA) region, have undergone strong selective pressure for adaptive evolution. The analysis at the amino acid level provided strong statistical evidences for the functional divergence between monocot and dicot NIP III subgroup proteins. In addition, several SDPs (Specificity Determining Positions) responsible for functional specificity were predicted.
The present study provides the first evidences of functional divergence between dicot and monocot NIP2s, and suggests that positive selection, as well as a radical shift of evolutionary rate at some critical amino acid sites is the primary driver. These findings will expand our understanding to evolutionary mechanisms driving the functional diversification of monocot and dicot NIP III subgroup proteins.
- Whole Genome Duplication
- Zingiber Officinale
- Codon Site
- Amino Acid Property
- Subgroup Gene
NOD26-like intrinsic proteins (NIPs) are plant specific integral membrane proteins, belonging to the aquaporin water channel superfamily. NIPs can be traced back to the early developmental stage of primitive land plants [1], indicating an important role during their evolution. Amongst plant aquaporins, only NIPs have the glycerol transport activity [2]. It was reported that there are 9, 13, 9 and 6 NIP genes encoded in the Arabidopsis, rice, sorghum, and Cucumis sativus genomes, respectively [1]. The divergence and proliferation of NIPs may be an adaptive response to an ever-changing environment [3].
Recent experimental evidences suggest that NIPs could perform a diverse range of functions, including a wider range for selectivity [4, 5]. It was demonstrated that two constriction points within the pore, referred to as the conserved dual NPA motifs and the aromatic/arginine (ar/R) selectivity filter, primarily determine the substrate selectivity of plant aquaporins [4]. However, it appears that the ar/R filter, which consists of four amino acid residues from helix H2, helix H5, and loop LE1 + LE2, plays a crucial role in determining the substrate selectivity of NIPs [6]. Based on difference in the ar/R filter motif and on our phylogenetic tree reconstruction, it is evident that NIPs should be divided into three distinct subgroups [1, 7]. The properties of the four residues making up the ar/R filter are remarkably different, leading to the significant variation of permeation ability for the proteins within each subgroup. In comparison to the NIP I and NIP II subgroups, NIP III proteins possess the largest constriction size of the pore (≥6Å), which allows larger solutes such as silicic acid (diameter 4.38Å) to permeate [6, 8].
With a few exceptions, the function of most of the NIP III subgroup proteins remains unknown. Of plant aquaporins, rice genes OsNIP2;1 (Lsi1) and OsNIP2;2 (Lsi6), and barley HvNIP2;1 (HvLsi1), which belong to the NIP III subgroup and localize in the plasma membrane, were demonstrated to function as a transporter of silicon across the biomembrane [9–11], a compound that can enhance the resistance of plants to biotic and abiotic stress. Moreover, it was found that rice OsNIP2;1 could be permeable to water, urea, boric acid, and silicic acid, but not glycerol. However, competition experiments indicated that this gene is a highly specific transporter of silicic acid [6]. When expressed in oocytes, both OsNIP2;1 and OsNIP2;2 were shown to have transport activity for arsenite but not arsenate [12, 13], with the former gene having a greater role in the major pathway for arsenite uptake [13]. In addition, the rice OsNIP2;1 was found to be responsible, at least partly, for the permeability to methylated arsenic species MMA and DMA [14]. All three arsenous acids are slightly smaller than silicic acid [13, 14], which may explain the permeability of OsNIP2;1. Given that arsenite and glycerol are structurally similar [12], it is surprising that rice OsNIP2;1 does not transport glycerol [6, 13], and suggests that other structural features may be involved in the process of efficient substrate discrimination [6, 12].
NIP genes generally exhibit a low expression level, and often show a tissue- and/or cell type specific expression pattern [1, 15, 16]. The rice gene OsNIP2;1 is specifically expressed in roots, and the expression level can be transiently enhanced during the heading stage [10, 17]; whereas OsNIP2;2 is expressed in both the shoots and roots, and its expression level in roots is much weaker than that of OsNIP2;1 [11]. The difference in expression pattern is consistent with their distinct silicon transport functions [10, 11]. Moreover, it was observed that the accumulation of silicon in different rice cultivars varies extensively, which might result from the differences in the expression of OsNIP2;1 and other silicon transporter genes in rice roots [17]. The barley HvLsi1, like rice OsNIP2;1, is specifically expressed in the basal root, and a weak correlation between silicon uptake and the expression level of HvLsi1 in eight tested cultivars was observed [9]. Moshelion et al. [18] examined the expression pattern of 33 aquaporin genes using macro-array hybridization, and found that ZmNIP2;1 is weakly expressed in maize suspension cultured cells. However, the function of ZmNIP2;1 remains to be further characterized precisely. In addition, the expression of rice OsNIP2;1 can be regulated by abiotic stresses. For instance, OsNIP2;1 expression is down-regulated during periods of dehydration and in response to the presence of ABA [19], but is induced by salt stress [20].
Homology modeling suggests that NIP proteins share a common general three-dimensional structure [7]. The ar/R filter of Cicer arietinum CaNod26 and Cucurbita pepo CpNod26 (defined as CaNIP2;1 and CpNIP2;1 respectively in this study) consists of Gly (H2), Ser (H5), Gly (LE1) and Arg (LE2) [7], identical to those NIP III proteins reported in grass species [6, 7], and suggests that they also function in silicon uptake and/or translocation [7]. However, for NIP III proteins in dicot species there is no any experimental evidence for the function in silicon uptake and/or translocation, although previous research revealed that the C. pepo CpNIP2;1, which is prominent in leaf veins, could catalyze the transmembrane flow of urea and water, but not glycerol [21]. Therefore, it is intriguing to further investigate whether dicot NIP III genes can transport larger solutes like silicic acid. Here we demonstrated shifts of selective constraint and positive selection may have been involved in the evolution of the NIP III proteins, which may correspond to functional differences observed between dicot and monocot NIP III genes.
Results and discussion
Phylogenetic analysis of the NIP III subgroup proteins
The protein and CDS sequences of approximately 20 plant genomes, as well as the cDNA and genomic DNA sequences of six other plant species (Eragrostis tef, Festuca pratensis, Elaeis oleifera, Musa acuminate, Zingiber officinale, Curcuma longa) that belong to Poaceae, Arecaceae, Musaceae, and Zingiberaceae, respectively, were searched. We found that there is at least one NIP2 gene presenting in all examined plant species except for two Arabidopsis spp. (Additional file 1). In monocotyledonous plants, there are two copies of NIP2 encoded within each grass species, except for maize, where three NIP III proteins were characterized [22]; while only one NIP2 gene (or fragment) was identified in Elaeis oleifera, Musa acuminate, Zingiber officinale and Curcuma longa. However, in the most tested dicotyledonous plants, besides G. max and cucumber, only a single copy of NIP2 gene was identified. In addition, a homologous NIP2-like protein was found in the licophyte Selaginella moellendorffii and the gymnosperm species Pinus taeda and Picea sitchensis. These results imply that NIP2 genes are relatively important for grasses.
In the monocot-specific clade, two subclades, based on the support of bootstrap values (91 and 99), were further presented: the NIP2;1 and NIP2;2 subclades (Figure 1). Within each subclade, the orthologous NIP2 genes derived from rice, maize, sorghum, barley, wheat, switchgrass, and B. distachyon were tightly clustered together, a result suggesting that the proliferation and diversification of NIP2s in Poaceae occurred before the divergence of grasses. Since previous studies revealed that the modern grasses diverged from a common grass ancestor [24], and a whole genome duplication (WGD) event was estimated to have occurred about 70 Mya [25, 26], we reason that the duplication of NIP2 genes in grasses may result from the corresponding WGD. To validate this, we analyzed the syntenic relationship between the chromosomes where the two NIP2 genes are located. We observed that three gene pairs including SbNIP2;1/SbNIP2;2, BdNIP2;1/BdNIP2;2 (see Additional file 3) and OsNIP2;1/OsNIP2;2 are located into chromosomal regions that were supposed to have undergone large-scale segmental duplications [1, 27]. In maize, ZmNIP2;2 and ZmNIP2;3, sharing an identity of 93.3% and 91.9% at the protein and DNA sequence level respectively, are closely phylogenetic related (Figure 1), although they are located in different chromosomes (6 and 9). Therefore, the production of ZmNIP2;2 and ZmNIP2;3 was probably through the recent WGD event occurred in maize [26, 28] after its divergence with sorghum from a common ancestor ~12 Mya [29].
4DTv distance (D4DTv) between paralogous NIP2 genes in monocot and dicot plants
Gene pair
Gene pair
O. sativa
Z. mays
T. aestivum
H. vulgare
B. distachyon
G. max
P. virgatum
C. sativus
S. bicolor
Sequence characteristic analysis
In grasses, the orthologous NIP III subgroup proteins are significantly homologous to each other (Figure 1). The average identity of sequences for the NIP2;1 and NIP2;2 subclade proteins is 85.9% and 87.5% respectively, indicating that the NIP2 orthologs within each subclade can perform the same or similar functions. In agreement with this postulation, barley HvNIP2;1 (HvLsi1), like rice OsNIP2;1 (OsLsi1), was demonstrated to function in mediating the influx of silicic acid in roots [9, 10]. In addition, we calculated the D4DTv values for the paralogous genes in grasses, and found that with one exception (ZmNIP2;2/ZmNIP2;3 with a D4DTv value 0.128), other paralogous gene pairs are highly diverged, having a D4DTv value not less than 0.26 (Table 1). Therefore, the sequence variation should be partly responsible for their functional diversification, as indicated by the functional studies of OsNIP2;1 and OsNIP2;2 [10, 11, 13].
In contrast to the high similarity in grasses, large sequence variation was observed among dicot NIP2s that share an average identity of 61.6%. In addition, we found that the overall average identity between monocot and dicot NIP2s is decreased up to 60.4%. Nonetheless, both monocot and dicot NIP2s show similar gene structures, possessing five exons and four introns (Additional file 4). Although the introns vary extensively in length, three exons (from 2 to 4) are remarkablely conserved in tested plants (see Additional file 4), suggesting that strong functional constraints should impose on the corresponding exonic regions. In the two soybean GmNIP2s, both the exons and introns are slightly different in length, supporting of their recent expansion. However, substantial variation is found in the intron lengths, especially in the second and fourth introns for the ZmNIP2;2/ZmNIP2;3 gene pair, implying that their diversification may be ongoing. The similar cases were also observed in cucumber (CsNIP2;1/CsNIP2;2) and other paralogous gene pairs in grasses.
Positive selection in the NIP2gene sequences
Results of positive selection analysis using a variety of codon substitution models
Omega distribution
Positively selected sitesa,b
M0 (one ratio)
ω = 0.197
M3 (discrete)
0.97% sites: ω = 1.85;
99.03% sites: 0.01<ω < 0.80
1162.2 (M3 vs M0)
p < < 0.01
M7 (β)
ω = 0.243
Not allowed
M8 (β+ω > 1)
1.72% sites: ω = 1.54; 98.28% sites: 0.0004<ω < 0.76
10.2 (M8 vs M7)
p < 0.01
The comparison of M3 vs M0 reveals that ω is not uniformly distributed along the NIP2 coding DNA sequences, and about 0.97% codon sites may be under the influence of positive selection (ω = 1.85). Similarly, compared with M7 model, the M8 model suggests that ~1.72% of codons fall in a category with estimated ω value 1.54, a result indicative of strong positive selection.
Using a variety of codon site models, we demonstrated the influence of positive selection in the evolution of NIP III subgroup genes (Table 2). Compared with the helix regions, the N- and C-terminus are highly divergent in NIP2 proteins (see Additional file 6). Nevertheless, it was found that plant aquaporins were regulated by phosphorylation within the cytosolic termini and loop regions [35, 38]. We used the NetPhos2.0 Server [39] to predict any possible serine and threonine phosphorylation sites in NIP2 proteins, and indeed found many predicted candidates in the corresponding regions (Additional file 7). These results suggest that the N-terminus and loop regions are functionally important, although they are not conserved as much as the helix regions. The site 77 is located in the loop between helix H1 and H2 (LA). In rice OsNIP2;1, the residue at the corresponding site is Ser (S), which was predicted to be a Serine phosphorylation site with high possibility (score = 0.89; Additional file 7); whereas in other NIP2s, the site is occupied by variant amino acids with different physiochemical properties (Figure 2). Therefore, positive selection in these regions may act as one major determinant in driving the functional divergence of NIP2s.
To test whether variable ω ratios are present among lineages, we performed the likelihood ratio test (LRT) to compare the two extreme models: the one-ratio model that assumes a unique rate ratio for all branches, and the free-ratio model that assumes an independent ω ratio for each branch [33]. The log-likelihood value under the one-ratio model is -11945.3, while the value is -11796.9 for the free-ratio model. Twice the log likelihood differences, 2Δℓ = 296.8, is strongly statistically significant (p < 0.01), revealing a heterogeneous selective pressures among lineages. We further observed that some branches of the NIP III gene phylogeny include several internal branches having ω >1 (Figure 1), showing strong evidence for adaptive evolution.
In a gene family, the fate of new genes produced by duplication would either evolve a new function under positive selection, or be lost during evolution [40]. As reported, plants have evolved more NIP proteins with multi-functions [1, 16, 22]. We revealed that positive selection was involved in the functional diversification of NIP subfamily genes [1], as well as in the evolution of NIP III subgroup genes (Figures 1 and 2, and Table 2). The grasses encoded two or three NIP2 genes, where the NIP2;2 gene has evolved new functions differentiating from that of NIP2;1s, which may represent an evolutionary advantage for grasses to uptake and translocate silicon efficiently.
Functional divergence analysis of NIP2 proteins
The Gu (1999; 2006) methods [41, 42] implemented in DIVERGE2 [42] were used to evaluate Type-I (shifted evolutionary rate) and Type-II (altered amino acid physiochemical property) functional divergence between gene clusters of interests in the NIP III subgroup. The advantage of these methods is that they use amino acid sequences, and thereby is not sensitive to saturation of synonymous sites [41, 42].
Based on the distinct expression patterns and functions of OsNIP2;1 and OsNIP2;2 [10, 11, 13], we supposed that functional divergence in NIP2s should have occurred after gene duplication in grasses. Unexpectedly, we found no statistical evidence for Type-I functional divergence between NIP2;1 s and NIP2;2s in grasses, because the coefficient of Type-I functional divergence was insignificant (θI = 0.086 ± 0.145; LRT = 0.44; p > 0.05). However, when the posterior probability (Qk) of divergence was determined for each site by DIVERGE2 [42], 7 sites with high probability (0.9 > Q k > 0.8) were identified to be Type-II functional divergence related (Additional file 8), indicative of a radical shift of amino acid properties [42]. It appears that site-specific changes of amino acid physiochemical properties may act as one of the major evolutionary powers in driving the functional divergence of NIP2 proteins after their duplication in grasses. Of the seven sites identified, 5 of them are located in helixes and two in loop regions (LB and LE) (Additional file 8). Further, we observed that although the three sites located in helix H2 (V/T), H5 (A/P), and LE (G/A) are physically close to the four residues of the ar/R filter (G, S, G, and R) in the amino acid sequences, only G/A in LE is predicted to spatially interact with R in LE2. In addition, we found that the two residues in LB (A/S) and LE (G/A) are not only physically adjacent to the dual NPAs, but spatially interact with the third residue (A) in the NPA motifs. LB and LE are functional loops that participate in the formation of the aqueous pore [35], implying that the A/S and G/A residues should be structurally important. Moreover, a contact between LE (G/A) and helix H3 (S/A) was also observed. Therefore, the radical change of physiochemical property of the 7 sites may be the primary contributor to the functional divergence of NIP2s in grasses.
We further employed the DIVERGE2 to assess the coefficients of Type-I and Type-II functional divergence (θI and θII) between monocotyledonous and dicotyledonous NIP2s. We found that the null hypothesis (no functional divergence) can be strongly rejected, because the θI value is statistically significant (θI = 0.145 ± 0.041; LRT = 12.6; p < 0.01). This indicates that shifted selective constraints must strongly operate on some amino acid sites in monocot and dicot NIP2s, and thereby leading to a lineage-specific functional evolution after their divergence from an ancient common ancestor. Furthermore, two critical amino acid sites (CAASs) were predicted to be highly Type-I functional divergence related (Q k > 0.9), where one site is located in LA, and the other in the helix H4 (Figures 2 and 3). The CAAS in LA (76L) is proximal to the identified positively selected site 77 S. Using InterMap3 D [43], the two sites are predicted to spatially contact with each other, and have probably coevolved during evolution. In dicot NIP2s, the CAAS in H4 is invariant Val (V), whereas it is substituted with Ile (I) in some monocot NIP2s. Nevertheless, no obviously statistical evidence for the Type-II functional divergence was found (θII = -0.030 ± 0.096; p > 0.05). Together, these observations indicated that amino acid site-specific shifts of evolutionary rate and changes of amino acid property should not uniformly act on the NIP2s after the split of monocots and dicots.
Determination of functional specificity positions among orthologous NIP2 proteins
Specificity determining positions (SDPs) in the monocot and dicot lineage-specific NIP2 proteins
Alignment position
Position in
Mutual information
Bernoulli estimator
In addition, we noticed that when using OsNIP2;1 as the reference sequence, the residue at the site 84 was invariant Gln (Q) in monocots including Musa acuminate and Elaeis oleifera, except for the Zingiber officinale ZoNIP2, where the corresponding site was occupied by Ala (72A) (Additional files 1 and 6). Whether the substitution of Q with A in ZoNIP2 is sequencing or assembling error is unknown. However, if the residue 72A in ZoNIP2 was replaced by G, this site was also predicted to be a SDP with high confidence (p value = 0.000; Table 3). Moreover, it was found that the residue 84Q lies on the channel side (Additional file 9), and exhibits an interaction with the first residue of the ar/R filter in H2. This suggests that the site 84Q may be involved in the formation of a functional pore, although the ar/R residues are identical in rice OsNIP2;1 and cucumber CsNIP2;1 (Figure 2 and Additional file 5). If it was true, the Z. officinale ZoNIP2 gene, like dicots, may perform a dissimilar function from other monocots.
Notably, we found that the predicted SDPs for the NIP III subgroup are mainly distributed in two spatial regions. As discussed above, the site 84Q was postulated to be involved in the formation of the channel (Additional file 9). By contrast, most of other SDPs lie on the surface of the protein and likely participate in establishing the tetrameric structure.
The current NIP III subgroup proteins were originated and diverged from an ancient common ancestor that emerged before the divergence of monocots and dicots. Subsequently, monocot- and dicot-specific expansion of NIP2 genes occurred. Plant NIP2s show relatively conserved gene structures, each containing five exons and four introns. With only one exception (CsNIP2;2), the ar/R filter of NIP2 proteins consists of G, S, G, and R.
All tested grasses encoded at least two NIP2 proteins in their genomes. These paralogous NIP2s were probably produced via the whole genome duplication (WGD) or segmental chromosomal duplication event occurred in the common ancestor of modern grasses. The proliferation and diversification of NIP2s in grasses may be, at least partially, responsible for their highly efficient influx of silicon in roots and then transporting them out of xylem. By contrast, most of tested dicot plants have only one NIP2 gene. Due to the small constriction size of the pore, CsNIP2;2 might be unable to transport silicon, although the cucumber genome encoded two NIP2 genes. In particular, Arabidopsis spp. should have lost their NIP III subgroup genes during evolution.
The NIP III subgroup genes have experienced strong positive selection and diverged in function between monocots and dicots. Several SDPs were identified to be responsible for the determination of functional specificity of monocot and dicot NIP2 proteins. These findings provide deeper insights into understanding the evolutionary mechanisms of NIP III subgroup proteins and their functional diversification.
Sequence data
The rice (OsNIP2;1 and OsNIP2;2), sorghum (SbNIP2;1 and SbNIP2;2), maize (ZmNIP2;1, ZmNIP2;2 and ZmNIP2;3), barley HvLsi1 (HvNIP2;1), C. arietinum CaNIP2;1 and C. pepo CpNIP2;1 proteins that belong to the NIP III subgroup were collected according to the published literatures [1, 7, 9–11, 22]. The Ricinus communis RcNIP2;1 (AN: EEF27965) was identified on the basis of a BLASTP search against the nr database with e value 0.01 in National Center for Biotechnology Information (NCBI). These proteins were used as query to search against the amino acid and DNA sequences of the Brachypodium distachyon, Triticum aestivum, Hordeum vulgare, Vitis vinifera, Carica papaya, Medicago truncatula, Glycine max, Lycopersicon esculentum, Populus trichocarpa, Cucumis sativus, Panicum virgatum, Mimulus guttatus, Prunus persica, and Manihot esculenta genomes using BLASTP and TBLASTN programs, respectively. In addition, an exhaustive search against the cDNA and/or genomic DNA sequences of the genomes Eragrostis tef, Festuca pratensis, Elaeis oleifera, Musa acuminate, Zingiber officinale, Curcuma longa, Picea sitchensis, and Selaginella moellendorffii were performed also using BLASTN and TBLASTN programs, respectively. Programs InterProScan [45] and ConPred II [46] were utilized to detect conserved domains and predict the putative transmembrane regions (TMs), respectively. FgeneSH http://linux1.softberry.com/ was employed to predict the gene structures of candidates identified.
Multiple sequence alignment and phylogenetic tree reconstruction
The NIP III subgroup protein sequences were aligned using the program L-INS-i implemented in MAFFT v6.6 [47], with the parameters: Scoring matrix for amino acid sequences, BLOSUM62; Gap opening penalty, 2.0; and Gap extension penalty, 0.2. The resulting protein alignment was subsequently employed to generate the codon-alignment of corresponding coding DNA sequences using a custom PERL script. In the codon-based alignment, the codon sites at which most sequences have data except for one or two sequences were kept while sites at which all sequences except for one or two have alignment gaps were removed. The phylogenetic trees were reconstructed with MEGA v4.0 [48] using the Minimum Evolution (ME) and Neighbor-joining (NJ) methods with the parameters of pairwise deletion of gaps/missing data and the p-distance substitution model where only the transversions were taken into account. The reliability of interior branches was assessed with 1000 bootstrap resamplings. Phylogenetic trees were displayed using MEGA v4.0 [48].
Tests of positive selection
We employed the CODEML program implemented in the PAML v4.4 software package [33] to test the hypothesis of positive selection in the NIP III subgroup genes. In the analysis, two pairs of models were contrasted to test for heterogeneous selective pressures at codon sites. First, models M0 (one ratio) and M3 (discrete) were compared, using a test for heterogeneity between codon sites in the dN/dS ratio value, ω. The second comparison was M7 (beta) vs M8 (beta+ω >1); this is the most stringent test of positive selection [49]. M0 and M7 belong to null models that do not allow for any codons with ω >1. In such comparisons, positive selection is indicated if the models that allow for selection (M3 and M8) are significantly better than the null model (no selection) in the likelihood ratio test (LRT). When the LRT suggests positive selection, the Bayes empirical Bayes (BEB) method was used to calculate the posterior probabilities that each codon is from the site class of positive selection under models M3 and M8 [50].
To test for heterogeneous selective pressures among lineages [33], models of variable ω ratios among lineages were fitted by maximum likelihood (ML) to the NIP III subgroup sequence alignment. The ratio of nonsynonymous-to-synonymous for each branch under two models (one-ratio and free-ratio for branches) was calculated, and the two models were compared using the LRT test to see whether the ω ratios are different among lineages. Accordingly, positive selection is indicated if the free-ratio model that allows for selection is significantly better than the one-ratio model (no selection) in the LRT analysis.
Estimation of functional divergence
The software DIVERGE2 [42] was employed to detect functional divergence between clusters of interests in the plant NIP III subgroup. The coefficients of Type-I and Type-II functional divergence (θI and θII) between any two interesting clusters were calculated. If θI or θII is significantly >0, it means site-specific altered selective constraints or a radical shift of amino acid physiochemical property after gene duplication and/or speciation [41, 42]. Moreover, a site-specific profile based on the posterior probability (Q k ) was used to predict critical amino acid residues that were responsible for functional divergence. In this analysis, large Q k indicates a high possibility that the functional constraint (or the evolutionary rate) and/or the radical change of amino acid property of a site is different between two clusters [41, 42].
Analysis of specificity determining positions
The SDPpred [44] was utilized to identify the specificity determining positions (SDPs) that determined differences in the functional specificity of homologous proteins. Based on a given multiple alignment, SDPpred computes Z-score for each alignment column. The higher Z-score, the more likely the position is a true specificity determinant. The Bernoulli estimator was incorporated into SDPpred to automatically produce a recognition cutoff (B-cutoff) to evaluate the significance of the Z-scores in order to assess whether the observed Z-score is sufficiently high to indicate an SDP. Positions scoring higher than this cutoff are predicted to determine the specificity.
Homology molecular modeling
The homology models of NIP III subgroup proteins were generated with HHpred [51] using the method of HMM-HMM comparison of the queried protein and the templates deposited in the PDB database. The generated models were prepared and viewed with UCSF Chimera [52], and the critical amino acid residues identified were mapped onto the corresponding structures accordingly.
4DTv calculation
4DTv distance (D4DTv) that stands for fourfold synonymous third-codon transversion was calculated to assess the genetic distances between paralogous pairs. In such analysis, the paralogous proteins for each species were pairwise aligned using MAFFT v6.6 [47], and subsequently the corresponding codon-alignment was created according to the resulting protein alignment using a custom PERL script. Based on these alignments, we firstly identified the conserved fourfold degenerate amino acids. And then the corresponding codons were extracted from the codon-alignment and used to calculate the 4DTv distance between each aligning pair. D4DTv ranges from 0 for recently duplicated peptides, to ~0.5 for paralogs with an ancient evolutionary past.
We thank the anonymous reviewers for their valuable and constructive suggestions for the manuscript, and Dr. Zhongyi Cheng (University of Chicago) for his helping to polish and improve the English language. This work was financially supported by grants from the Zhejiang Provincial Natural Science Foundation of China (No. Y3090152), National Natural Science Foundation of China (No. 31000170), the Foundation of Zhejiang Educational Committee, China (No. Y200805229), the intramural fund from Zhejiang A & F University to Q. Liu (No. 2010FR060), and the Special Fund for Grade B Innovative Research Team from Zhejiang A & F University to Z. Zhu.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Wow, these guys know how to keep busy. It was only a few days ago that I told you about the Interspire Shopping Cart and now they've ordered another review for another software package.
Today, we cast the spotlight on Interspire Website Publisher, a complete content management system that is supposed to be superior in a number of ways over the competition. Better still, it's been designed so that even "non-technical users" can "create and maintain fully functional websites."
It's not like the Interspire Website Publisher is completely new either, because this is apparently the fifth major release of the online software package. They've improved on it in just about every way and they're more excited than ever to help you build your website. Let's see what it's got.
The Robust Yet Intuitive CMS for Everyone?
A lot of us have come to rely on WordPress for our CMS needs, but the free platform may prove inadequate for certain users with certain needs. Those who need a simple yet robust content management system may find that WordPress lacks in the support department or that it may lack in certain functionality. That's where a paid solution like Interspire's comes into play.
Checking out the feature set, you'll discover a series of included templates, for example, so you won't need to buy a custom theme. Other highlights include SEO customization, custom content types, content expiry options, one click lead capture forms, users in multiple groups, logo editor, podcasting support, and a completely modular architecture.
Whether you're building a company website, developing an online newspaper, or promoting a podcast, it appears that Interspire Website Publisher will be able to handle your needs.
Just as with the Shopping Cart I reviewed a few days ago, the Interspire Website Publisher has a feature video that describes some of its key components. You can also try the online demo for a more hands-on approach.
A Glance at the Dashboard
One of the provided reasons as to why you may choose Interspire as your content management software provider is that they have "the easiest to use content management software in the world." For blogging veterans, WordPress may seem reasonably easy to use, but the Interspire solution is supposed to be even easier. It's all menu-based with a lot of dragging and dropping. That includes the actual design and layout as well.
The Interspire Website Publisher dashboard looks a lot like other Interspire products, taking on the same general appearance. You can see a few more shots of the dashboard on this page. Each tab tackles one aspect of running a website, like generating content, managing comments, and handling categories. There is also a handy image manager.
What Does an Interspire-Generated Site Look Like?
Building a new website using the Interspire Website Publisher is done through a WYSIWYG drag-and-drop style interface. You can use the provided templates as a launching point or you can import an existing design.
Awesome Cuisine is one of Interspire's satisfied customers and you can see a shot of their website below.
Other good examples include Caribbean Bug and Red Tag Vacations. These sites have all decided to go with the private label approach and Interspire has no problem with you removing the "powered by" link in their designs.
Get 20% Off with Coupon Code
Just as with Interspire's other professional software, the pricing for Website Publisher is not for the feint of heart. Even so, Interspire has done a good job with creating a system that "just works" while providing enough features to manage a fairly advanced website.
The regular prices for the Professional Edition and Enterprise Edition are $395 and $695, respectively. Based on this comparison chart, the main difference is just the number of admin user accounts.
Regardless of which version you choose, Interspire Website Publisher 5.0 can be had for 20% off if you use this coupon code: MXXHC93876KAFXU. Special savings for John Chow dot Com readers!

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The Intuit PaymentNetwork lets you make and receive online payments that are quick, easy, and secure. Click the Online Invoice Payments button in the Customer area of the QuickBooks Home Page to sign up for the service and pick the bank account … [Read more...]
Intuit PaymentNetwork: Getting Paid and Paying Bills Online
Video: Receiving payment and depositing a check
In this video, watch how Wally tracks a payment he's received from his client, Hannah. Video script Last week, Wally the Web Designer sent one of his customers, Hannah, an invoice for the work he did to redesign her web site. Today he got … [Read more...]
Video: Using estimates, invoices, and receive payments to get paid
In this video, you'll see how Wally the web designer creates an estimate, invoices, then receives payment for work that he does. Video script: Web Designer Wally is looking for work! Hannah, who owns a magic shop called Hannah's Illusion … [Read more...]
Video: Creating an invoice in QuickBooks 2013
In this video, watch how Wally uses QuickBooks for Mac to invoice a customer for work he's done for her. Video Script Wally's finished his work on the new web site he designed and built for Hannah's Illusion Supplies. He'd really like to get paid … [Read more...]
Time sheets and progress invoicing
Progress invoicing is a great way to invoice your customers over time for work you do for them. And many times, that work involves billable time. So how do you apply that billable time to a progress invoice? A couple of things to understand Before … [Read more...]
So many choices, Part 1: Customer transactions
This article is Part 1 of a two-parter on the transactions you can use in QuickBooks. Part 2 covers vendors and other transactions. This one covers customers. QuickBooks has lots of different transactions you can use to run your business. Which … [Read more...]
Bad debt: What to do when you don't get paid
Is there anything worse than a customer who doesn't pay you? OK, maybe a bounced check, but not getting paid at all is just so bad. Then you've got that invoice or statement just hanging around in QuickBooks. Well, I can't get your customers to pay … [Read more...]
Video: Receiving payments in QuickBooks 2012
Your customer has sent you a payment. Sweet! Now it's time to record that payment in QuickBooks. In this video, I'll show you how to use Receive Payments to enter money a customer has paid you. Video script: Hi, I'm Ro with the QuickBooks for … [Read more...]
Video: Creating a sales receipt in QuickBooks 2012
You're probably already familiar with sales receipts. Just about anytime you buy anything, you get a record of what you've bought and what you paid for those items. In QuickBooks, you can create sales receipts for your customers to track a … [Read more...]
Video: Progress invoicing in QuickBooks 2012
Editor's note: This shows you progress invoicing in QuickBooks 2012, but if you have QuickBooks 2011 or earlier, there's a manual method for invoicing over time. To use this feature, be sure you have Estimates and Progress Invoicing turned on in … [Read more...]
|You can also post your own question to the QuickBooks for Mac community.


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Product Description
Ampeg PF-210HE Portaflex Series Bass Cabinet - Drawing from the classic design of Ampeg's legendary Portaflex cabinets, the PF-210HE combines vintage styling with the performance of a modern 450W cab. The dual Eminence® 10˝ drivers push out true Ampeg tone. Since it's a Portaflex, you can attach any PF Series head to the top, flip it inside and hit the road Ampeg style.
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- Frequency response: 53 Hz - 17 kHz
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- Dimensions (H x W x D inches): 23 x 21 x 14
- Handling weight: 48 lbs / 21.6 kg
Product Reviews
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It's a SHOCKING FACT that 90% of all people starting to learn the piano give up after only four months!At the London Piano Institute, we offer ONLY the highest quality piano coaching FOR YOU with a virtuoso piano teacher!The primary reason why people give up is primarily due to the quality of individual piano lessons. At the London Piano Institute, we offer lessons of exceptional quality and we keep YOU EXCITED about playing the instrument!
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A Camden Jewel
August 10, 2011
The Camden Public Library in Camden, Maine is a magnificent example of stately architectural and landscape design, and of the citizenry prioritizing the most important building in town. When visiting a few weekends ago, I was struck by the 1996 addition to the library, and how the architect, John Scholz, approached the design with a careful hand. The addition was built UNDER the existing library and grounds, with a new lower level entry at the side street.
Street view
Architect's renderings of the grounds (from the library's website)
Corner view
New entry
The existing library and its views across the Olmstead-designed Harbor Park to the harbor is breathtaking and unchanged, aside from a lantern-like glass building on the lawn that serves as an oculus/skylight for the new spaces below.
From Library
Toward library from Amphitheater
Because of the glass entry to the street, the oculus and roof windows, the new lower level spaces are light-filled and pleasant. The interior spaces are elegantly finished similarly to the historic library above.
First Floor
Lower Level
Detail of oculus
From the library's website:
"On March 23, 1896, the citizens of Camden voted to establish a free public library to be known as the Camden Public Library. The proud townspeople of Camden raised the money to build this library through various fundraising efforts. No assistance was provided by library philanthropist Andrew Carnegie. Mary Louise Curtis Bok donated the land for the library in 1916. Parker Morse Hooper and Boston architect Charles G. Loring offered building plans. The cornerstone was laid on August 17, 1927 and the Library opened its doors on June 11, 1928 with Miss Katherine W. Harding serving as the first librarian.
In 1996 the library underwent a great expansion under the south lawn. The opening of this Centennial Wing allowed the library to accommodate larger collections and computer-based technology without compromising the scenic or historic value of the original building."
Most of our existing New England libraries are too small for current requirements and many of them could use an update and more space. There are a lot of examples of modern buildings being stuck on these old venerable structures, or even of libraries moving to new buildings on the outskirts of town, where parking and land is available. Because the addition was funded by the citizens and businesses of Camden, there were no federal or state mandates on parking, number of meeting rooms, etc. It was such a pleasure to see Camden's solution, which demonstrates pride in the historic structure, and an optimism for the new.
A brief history of the library and adjacent grounds also from the Camden Library website:
"Constructed in 1928, The Camden Public Library, the only library in the village of Camden, Maine, sits at the highest point on Main Street. Architects Parker Morse Hooper and Charles Greely Loring chose to position their building close to the street, under the shade of existing elms and maples – a more direct relationship with its surrounding built environment rather than its larger landscape scenery.
The grounds of the Camden Public Library create a distinctly unique, highly articulated series of landscape experiences, the centerpiece of which is a public outdoor garden amphitheater. Designed by the renowned landscape architect Fletcher Steele, this landscape is one of his best works of art. It was designed and constructed between 1928 and 1931, and funded by local patron of the arts, Mary Louise Curtis Bok. Steele's landscape design is an important transitional composition that blends elements of the traditional Neoclassical with the 'new' ideas of the French Moderne (Art Deco) and successfully marries the ideals of the Renaissance Italian garden theater with the richness of Maine's native landscape. The popularity and unique qualities of the amphitheater immediately led to the christening of the site as the "Camden Amphitheatre".
The library building is a long rectangular Colonial Revival structure whose primary axis runs southwest to northeast, parallel to Camden's Main Street. From the rear of the library, the back door opens onto a secondary axis, linking the library to the Amphitheatre's central lawn at an oblique angle. The primary axis for the Amphitheatre runs north to south, aligned with the primary views to the harbor. Experts have celebrated the use of this bent axis as one of the first steps in landscape architecture's move from Classical Revival to French Moderne (Art Deco).
Across Atlantic Avenue, the two-acre Harbor Park, designed by the Olmsted Brothers between 1928 and 1935, continues and extends the views from the Amphitheatre toward the harbor and its busy waterfront. This park retains its own, aesthetically distinct design vocabulary, and remains a separate yet intimately connected, companion to the Amphitheatre. The park was designed in concert with the Amphitheatre, though its naturalistic design and informal planting program contrast significantly with the structured design of the Amphitheatre.
The Camden Amphitheatre retains its historic integrity, setting, original materials and the quality of original workmanship and design. Fieldstone, brick, grass and native trees and shrubs weave their way throughout the Amphitheatre, and wrought iron rails, light standards, gates and arches add grace and French-inspired Art Deco overtones to the carefully executed, highly detailed landscape design.
For more information, visit the library's website, or the library itself! The Camden Library, Camden Harbor Park and Amphitheatre are located just off Route 1, on Atlantic Avenue, in downtown Camden.


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Many of the seafood items - lobster, shrimp, crabs, scallops and even clams and mussels - are available for "mail order" home delivery. Places like Lobster Anywhere (www.LobsterAnywhere.com) or The Crab Place (www.CrabPlace.com) can have fresh, delicious seafood products delivered directly to your door.
For the non-seafood lover (who may be) in the group, there are usually an assortment of "other" foods. These usually include french fries and onion rings to go with the seafood. You will also find a hamburger or cheeseburger and maybe even a steak sub.
What else comes out of the crab steamers? All sorts of good eats - for one example - steamed shrimp...and more...of course...
*Note, please, that seafood and other products are not actually "sold" on this site - SouthBaltimore.com. This resource is provided solely as a convenient tool for our Maryland seafood-loving readers and does not specifically recommend or endorse any particular product(s) or merchant(s). If you do order products after clicking through to a product supplier we may be compensated with a small commission. Don't worry that you may pay more by clicking here - any such commissions are paid by the vendor(s).


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All Films.All Above Board.NOW INCLUDES TV SHOWS & TV LISTINGS
Dalton Trumbo writes this classic Western starring Glenn Ford and Jack Lemmon. After losing all his money in a high-stakes poker game, cattleman Tom Reece (Ford) signs over a piece of his business to hotel clerk Frank Harris (Lemmon) in exchange for a one thousand dollar loan. Frank has long dreamed of being a cowboy, and also sees the deal with Tom as a chance to win the hand of the woman he loves, ranch owner's daughter Maria Vidal (Anna Kashfi). However, not only does Maria's father forbid them from seeing each other again, life on the ranch also proves to be very different to what the young tenderfoot had expected.


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Proclamations: Bring attention to the pain community!
Members and volunteers are encouraged to request ceremonial proclamations from state and local politicians. By submitting an already crafted template, individuals can easily request the proclamation in support of pain awareness month. By specifying the number of Americans enduring chronic pain, the cost incurred by the nation as a result of chronic pain, and how increased awareness about the effects of chronic pain can result in better outcomes for pain warriors. The increased exposure these proclamations receive will increase the chances for a future where pain warriors are supported, funded, and recognized instead of stigmatized.
Please plan to submit these requests before the end of July to ensure your local offices have ample time to respond. Late requests will be accepted, as pain awareness is an ongoing effort; however, submitting the request early ensures the proclamation will be completed and available in time for the start of Pain Awareness Month.
Be sure to check the location specific guidelines prior to submitting any proclamations. If you have any questions or need help obtaining specific information, please contact the Proclamations team at firstname.lastname@example.com for assistance.
How to submit a proclamation request for the month of September:
Local Proclamations:
Download the attached template and follow the instructions specified by your local government. Each municipality may have a different process: you may need to upload the file through an online form, email the file to the specified contact, or you may have the ability to submit a printed copy in person. When submitting your proclamation request, simply ask the official to show support for their constituents living with chronic pain conditions by completing the ceremonial proclamation for September's nationally recognized Pain Awareness Month. Notify the U.S. Pain Foundation once you have requested a proclamation and provide updates as the status of your request changes.
State Proclamations:
In order to avoid multiple proclamation requests being sent to the same state official, please express your interest in obtaining a state proclamation by completing the attached form. Once it has been determined that no other requests have been sent, you will receive a response with the appropriate template and directions on how to proceed. Even if your state has been submitted, your voice can be heard! Forms will be provided along with instructions for encouraging state officials to support constituents living with chronic pain conditions by signing the previously submitted proclamation.
Please notify U.S. Pain with any status changes or feedback:
Keeping us apprised of the status (submitted, pending, approved, denied, received hard copy) will allow U.S. Pain Foundation to better assist you with present and future Pain Awareness Month activities. Once you receive the hard copy, submit a photograph of the pain warrior/proclamation here so we can celebrate your efforts by sharing the image on our social media. All hard copies are to be mailed to our main office:
U.S. Pain Foundation
Attn: Lori
670 Newfield St., Ste. B
Middletown, CT 06457
Retaining the hard copies allows U.S. Pain to highlight your accomplishments for the pain community on social media, websites, and at awareness events that take place in your respective state or geographic location.


text: Blog Posts in December, 2014 Xarelto Linked to Life-Threatening Bleeds Posted By Maloney & Campolo || 23-Dec-2014 Life threatening bleeds, strokes, and even death may be associated with the popular blood-thinning drug, Xarelto. Since 2011, Xarelto has been prescribed to those experiencing leg and lung blood clots in relation to knee and hip surgeries and irregular heartbeats. Protect your parents and grandparents! If a loved one has been prescribed Xarelto, contact an injury attorney at Maloney & Campolo ... Continue Reading


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As Apple Inc. AAPL reported a sharp drop in iPhone sales, analysts in Asia were pointing to buying opportunities before the next version's launch. Read how iPhone sales were beset by 'rumors.'
A 26% fall in smartphone sales to 26 million units in the quarter ended June, as consumers held off purchases in wait for the iPhone 5, pulled down the Apple stock 5% in U.S. after-hours trade. In Asia, shares of most of its component suppliers fell by a smaller margin.
While iPhone assembler Hon Hai Precision Industry Co. dropped 4%, shares of Samsung Electronics Co. (a rival smartphone maker as well as a parts supplier to Apple) fell 0.3%, Foxconn Technology Co. shed 1.4% and Catcher Technology Co. lost 1.8%. Read MarketWatch First Take on why the iPhone 5 had better be good.
Shares of some firms in the Apple supply chain even rose, with Pegatron Corp. up 1.3% and Simplo Technology Co. 1.5% higher. Read more about impact of Apple results on Asia.
As the iPhone 5 is widely expected to be launched in time to capture a share of the consumer wallet during the crucial October-December period, analysts reckon Apple's supply chain will have to begin their own shipments before that.
"We view the muted July as an attractive entry point, especially considering the volume ramp-up beginning in August," Fubon Research analyst Jeff Pu wrote in a report.
Your browser does not support iframes.
And expectations are high. Really high.
Barclays analysts Kirk Yang and Ben Reitzes say market expectations that Apple will sell 43 million iPhones in the December quarter are "simply too low" and that the results offer a chance to buy its supply chain on the cheap.
Taiwan's Hon Hai, Apple's exclusive assembler for the iPhone, is aggressively building production capacity at Zhengzhou in China's Henan province to meet increased demand expected in the fourth quarter, they said.
-- V. Phani Kumar


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The world is now 'even more determined' to challenge climate change - Merkel
German Chancellor Angela Merkel responded to U.S. President's decision to withdraw from the Paris accord saying the decision will 'not stop all of us dedicated to the protection of our planet earth'. Rough cut (no reporter narration).
ROUGH CUT. NO REPORTER NARRATION. Germany's Chancellor Angela Merkel pledged her country's continued commitment to the Paris Climate Agreement in the wake of U.S. President Donald Trump's withdrawal. "The decision of the U.S. President to withdraw from the Paris Climate Agreement is very regrettable, and I'm expressing myself in a very reserved way when I say that," she said in a short statement to the German media on Friday (June 2), adding that the deal was needed to protect the environment. She added that the "decision can and will not stop all of us dedicated to the protection of our planet earth". She pledges the unwavering support of Germany to face "one of the challenges of humankind."


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Thinking of travelling for the holidays? Want to enjoy a relaxing and fun filled holiday experience? Then get in touch with Jovee Travel.
We can provide you with the best holiday packages at competitive prices with complete satisfaction. Whether you are travelling independently, with family or friends, we can help you create the perfect holiday package that fits your travelling needs and budget. We will help you cross the top tourist attractions off of your bucket list.
From Adventurous sites to historic marvels of the world, we can help you arrange the best holiday to any destination your heart desires. Need some inspiration? Join any of our existing tours with regular departures throughout the year.
Our Team is as passionate about travelling as you are. With over 35 years of experience in the travel industry and over 100 countries visited by our team and clients, we can help you plan one adventure of a lifetime!


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Humanitarian Aid & Relief
Humanitarian Aid & Relief
Afghanistan 2014 Flood Relief
Afghanistan 2014 Flood Relief
Water was provided though the purchase and distribution of the Sawyer PointONE Filter. This is a portable filter, easy-to-use, and reliable for the vulnerable families affected by flood disasters. Sawyer filters are certified for ABSOLUTE microns making it impossible for harmful bacteria, protozoa, or cysts like E. coli, Giardia, Vibrio cholerae and Salmonella typhi (which cause Cholera and Typhoid) to pass through.
Kuchi Distribution
Kuchi Distribution
Zaragan is a village located in Balkh district with population of approximately 60 households; they came from the village of Dawlat Abad and moved into this area. For this growing community, the stream is the main source of drinking water.
The distribution of Sawyer filters in this village took place on July 23, 2014 for 18 households. The JDA team traveled back to Zaragan to monitor and evaluate the impact of using the filters and found an interesting story.
Salhi Mohammad living in Zargaran, has a-three-years old son who became seriously sick with diarrhea and vomiting. Salhi spent 7000 AFs (two months' salary) in doctors and medicines. He visited 4 to 5 doctors within a week, but his son did not get better. After he received the Sawyer filter from JDA, he said;
"...I never thought that this tiny thing
could give such a big result. I will take care of my filter as I take care of my life. This acts incredibly and saves my family's life and money."


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Sarah Marquis is a Swiss national, who began adventuring in her 20s. National Geographic named Sarah an 'Explorer of 2014'. She has been profiled in The New York Times Magazine and National Geographic. In solo expeditions she has hiked the Pacific Crest Trail as well as across Australia and South America.
Published: March 2016
The epic story of one woman's 16,000 kilometre, three year trek from Siberia to Australia.


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A diverse collection of poetry, short stories, song lyrics, novel extracts and illustrations from some of the most important creators today.
This anthology for upper high school students follows on from the success of the collection Things a Map Won't Show You, which was aimed at younger readers. Susan La Marca and Pam Macintyre have gathered an exciting mix of original and pre-published works that cover themes of Indigenous issues, friendship, migrants in Australia, environmental impact, dystopia, sci-fi adventure and death.
The anthology includes original stories from: Davina Bell, Meg Caddy, Shivaun Plozza, Peta Freestone, Scot Gardner, Barry Jonsberg, Meg McKinlay (with illustration by Kyle Hughes-Odgers), Gayle Kennedy, Shaun Tan, Felicity Castagna, Amie Kaufman and Leanne Hall. Other contributors are Margo Lanagan, Ali Cobby Eckermann, Andrea Hirata, Arwa Abousamra, Trudy White, Alice Pung, Tony Birch, Wil Wagner & Lizzie Wagner, Courtney Barnett and Kate Miller-Heidke.
Where the Shoreline Used to Be is a collection of outstanding short stories, illustrations, poems, essays, and memories from some of Australia's best known writers. Contributors include Shaun Tan, Alice Pung, Leanne Hall and Tony Birch, alongside up-and-coming new writers like Indonesian author Andrea Hirata, Palestinian-Australian author Arwa Abousamra, and siblings Will & Lizzie Wagner.
The stories in Where the Shoreline Used to Be are diverse and varied - Some are funny, some are poignant, a few are fantastical, absurd or surreal. They raise questions and promote discussion about a broad range of issues including indigenous issues, identity, culture, sexuality, religion, bullying and race.
The stories are punchy and quick to read, but invite further consideration by readers; whether about the subjects and themes touched on in the narrative or the narrative style itself.
Amie Kaufman's family anecdote about the summer her father adopted a baby roo is laugh-out-loud funny, while Gayle Kennedy's verse story about a Koori teen's experience shopping for a birthday pre-sent is poignant and thought provoking. Barry Jonsberg's story about The Pale Man is creepy and at-mospheric with a twist at the end, and Arwa Abousamra's intensely personal story about her experi-ence as an immigrant child is even-handed and sympathetic.
Another great selection of works suited to all secondary students.
Reviewed by Lian


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|Bennett Samuel Tramer
|Known aliases:
|538 Market Road, Haddonfield, Illinois
|Known Relatives:
| Carl Tramer (father)
Bonnie Tramer (mother)
|June 20, 1961
|October 31, 1978 10:05 PM (age 17)[1]
|Portrayed By:
|Jack Verbois
|First Appearance:
|Halloween II
Bennett "Ben" Samuel Tramer (June 20, 1961 - October 31, 1978) is a character that is first mentioned in the original Halloween and appears in Halloween II, played by Jack Verbois.
Bennett Tramer was born to Carl and Bonnie Tramer on June 20th, 1961 in Haddonfield, Illinois. He is stated to have been liked by Laurie Strode. Annie Brackett learned of this from Laurie, after which she called him regarding the high school dance.
In Halloween II, Ben was chased by Dr. Loomis and Sheriff Brackett, who thought that he was was Michael Myers because he was wearing a similar mask. As Ben crossed a street without looking both ways as he was distracted by the panic, a police vehicle rammed him to a parked van, causing a fiery explosion and instantly killing him.
He later was taken to a coroner's department where Graham did dental tests to determine whether he was Michael or not. After inspection, it was revealed that he was not, as his teeth did not match those of someone who would be 21 like Michael Myers. Loomis and the policemen then realized that he was an innocent civilian and Michael was still out there, which was later confirmed to everyone on the news.
Ben was buried in Mt. Sinclair Cemetery on November 4th, 1978.
Trivia Edit
- Ben's social security number was 135-77-1386.[1]
- Ben's corpse was attended by Graham from 10:45 PM on October 31, 1978 to 11:50 PM on the same night.[1]
- He is the only person in the film to have died from a cause other than being murdered by Michael Myers.
Appearances Edit
- Halloween (mentioned)
- Halloween II


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At what point do you start marketing?
Does it work?
Do you know what to do next?
Do you have the time?
Does it work?
Do you know what to do next?
Do you have the time?
Welcome to the Marketing Orange© approach to making marketing work – for you and your business.
Marketing Orange© gets under the skin of your operation, to identify a way forward and develop confident, creative marketing processes, to grow your client base, profit and work satisfaction. Using a phased approach, and keeping simplicity as key, we unlock a practical, realistic way forward so you can achieve the results you need.
Marketing is defined by the UK's Chartered Institute of Marketing as 'the management process responsible for identifying, anticipating and satisfying customer requirements profitably".
The problem is, it's complex. Easy to talk about but tricky to coordinate and deliver. Marketing is so all encompassing, it's like finding a needle in a haystack sometimes. But the devil's in the detail, and so we start from within, take a step back, and find out what's going on inside, to get the right message to send outside….
There are so many aspects to consider – objectives, goals and targets; research and analysis; customer engagement, journey and touchpoints; campaign implementation and internal processes; monitoring and measurement,
… not to mention ongoing customer care, communications, new business development… if you don't plan it, write it down, you wouldn't be human if you didn't get lost.
Do you have time? The reality is that taking care of the day-to-day running of the business leaves little room for planning and coordinating effective strategies for a strong customer base.
So it makes sense to have a committed partner on board to focus, strategize and plan. Then by getting the right message out, clearly, to the right target audience, we create value and growth.
So, if that sounds like something you'd like to chat about, just call or email today – and look forward to feeling much better about your marketing approach.
Because marketing, done right… does work ©
fabulous clients, lovely feedback
We have enjoyed working with Susan, and it's been a great opportunity for us to step back and thoroughly review our marketing activities. We are confident that our plans going forward will now leverage cost and time savings. This has been a worthwhile exercise and definitely good value for money.
Susan and I did an Unpeel session which was hugely successful. On the back of that, I hired Susan to work on my marketing and help me make my business more visible in Scotland and the UK.
Susan gave us a good grounding on what was needed to improve our marketing success and once we started to implement her ideas, we saw an increase in turnover. She gave us the confidence & tools to carry the marketing plan forward.
Susan has been a wonderful help to my business. Already I can see a return on investment… & have no hesitation recommending Susan Branigan Marketing to anyone who feels their business has lost its way a little bit and needs some strategic help.


text: Hi, I spotted the Roadster driving around at Le Mans on Thursday last week and then a few minutes later parked up in the paddock. I wasn't too sure who was in it, I was too busy reaching for the camera phone. I never did see it again either (was going to try for a test ride!). I was surprised by how *totally* silent it was. Was anyone else there? Anyone get any pics? Dave


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Way back on July 10th, I threw a baby shower for my sister-in-law Katie and her first baby, Gloria. I know - that was a long time ago. Better late than never, right?
As soon as Katie told me she was pregnant, I began mentally planning her shower. I was so excited to do this for her. I had the basic framework of the shower planned but couldn't really go all out until Gloria was born and we found out that it was a girl!
I had big plans for this shower, and most of them came to fruition, but the first trimester of my pregnancy got to me, so I didn't finish everything that I had envisioned but I still think it all turned out great.
I wanted to make/have things for the shower that Katie could take home as a reminder of the shower, and to use in Gloria's nursery.
I enlisted the help of a super talented scrapbooker that I work with, the uber-fantastic Hayley to create this:
It turned out way better than the version that I created in my mind. It created such a wonderful centrepiece for the room.
And even better is that Katie was able to use this for Gloria's nursery. Fantastic.
I bought the birth announcement off an Etsy shop called Tilly Claire's. I absolutely loved it, and was excited that it was another item for Katie to take home after the shower.
Here are some more pictures:
Terrible lighting on this picture, but I wanted to show you that I made a cake! With pink icing! And put it on a table next to three different kinds of Jelly Belly's! Yum.
And look at three quarters of the room. I had to try to squeeze around 30 people in a room that was not big enough for 30. We all made it work though. Too bad I forgot to take pictures when there were people in the chairs... :(
That 'G' in the bottom right corner? I did that. Well, I bought a cardboard letter, and then painted it, and mod-podged fabric scraps on the top. Another decorative touch that Katie got to take home.
|The loot!
The beautiful momma lounging in the midst of all Gloria's new stuff!
And now for my favorite part - the quilt and baby photo!
Little Gloria is such a gift to our family and I was blessed to be able to throw this shower for Katie. It was such a wonderful day that was spent with family and friends enjoying each others company and good food (a big thanks to my other sister-in-laws Linda & Nicole for helping with the food, along with my mom and mother-in-law).
I love babies. Good thing that in 4 1/2 months I'll have my own!


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Destroying books since 1999.
This could have easily been another 5 star book. Easily! But Perry had to go fuck it up.
Yes, I'm blaming this on her. Dex was incredibly different in this one, I could never imagine him being so emotional and kind. And she's too much of a whiny bitch to accept it. Yes, I know she's hurt. Yes, I know she finds it difficult to trust Dex, but after all he's done to her I would expect a different reaction. It's unfair. She's behaving like a total bitch without a good reason.
Is this because it's Perry's time to be the pursued one? Is this the reason why she's playing so hard to get? Too hard to get, if you ask me. It's pretty much a turn off. I hope she'll snap out of it in the coming book.


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When a friend of family member moves away for a new job, a marriage, or simply to experience a different city, it's never easy to say goodbye. You can help to make the bittersweet move just a little easier on your friend by hosting a low-key farewell luncheon. Invite close friends or family members to celebrate the new chapter in your friend's life. Remember to keep the luncheon simple, as moving preparations can be overwhelming for some people. Here are a few ideas to get you started:
- For fun, choose themed foods that represent their current home state and their new place of residence. For example, if your friend is leaving Philadelphia for Texas, serve mini cheesesteaks and BBQ sliders. Or, choose to have your friend's favorite local restaurant cater your event.
- Use maps of your friend's new city to wrap vases of flowers or candles. Place these on the table for a fun centerpiece.
- Ask guests to send a photo of themselves with the guest of honor to you in advance so that you can create a small photobook. At the party, have each guest sign their well wishes by their photo.
- Create a trivia game with questions about your friend's new city or state to help them feel better acquainted with their new surroundings.
- Present a meaningful gift to your friend, like handmade map coasters to represent their old hometown and new hometown, a state-shaped cutting board to use in their new home, or a set of note cards to send updates after the move.


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Patrick Poole catalogues the incidents of Islamist terrorism in 2009, and concludes with a disturbing prediction about the year ahead.
What 2009 has taught us is how rapidly the nature of the threat from Islamic jihadists is evolving and how utterly unable our government is to recognize and respond to that threat. Sadly, I predict that the coming year will show us how bold, aggressive, and inventive our enemies have become and how little we have learned from the events of the past year.


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Girls soccer fights hard against St. Mary's Academy
Hang on for a minute...we're trying to find some more stories you might like.
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JV girls won 3-0 over St. Mary's Academy. Varsity girls fought hard and were up 2-0 at the half, but St. Mary's came back to win 3-2.


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Building Information Management (BIM) Co-ordinator
- Recruiter
- Fulcro
- Location
- Bidford-on-Avon, Alcester, Warwickshire
- Salary
- Competitive
- Posted
- 29 Sep 2018
- Closes
- 27 Oct 2018
- Ref
- Sector
- Engineering, Automotive, Electrical, Industrial, Mechanical
- Function
- Health & Safety, Maintenance
- Hours
- Permanent
Building Information Management (BIM) Co-ordinator
An innovative team of design technologists, engineers and creative thinkers who thrive on delivering solutions that are faster, better and more value for our clients. Our core activities are 3D/4D and 5D digital prototyping, Building Information Management (BIM) and 3D design detailing for construction, fabrication and assembly.
We work on the UK's and World's largest construction projects including Stadia, Iconic residential developments, flagship commercial buildings and Infrastructure projects.
We are growing and looking for talented individuals to join our team.
What's the job?
The role requires diligence and best practice to be applied to the creation of digital prototypes and the federation of wider discipline digital models. It is also a requirement that the role assumes responsibility for the creation and maintenance of Fulcro procedural best practice and synchronisation across Fulcro's other regional offices and teams.
- Review 3D Models from consultants and contractors
- Conduct clash detection and visual walkthroughs using NavisWorks Manage
- Audit and review models in Autodesk Revit
- Report Writing
- Hold weekly coordination meetings with project team members
- Early design review and constructability analysis
- Manage / Update BIM Project Documents
- 3D Model Quantity Extraction / Verification
- 4D Scheduling / Simulation
- Model Authoring
Do you have some or all of these qualities?
- An in-depth working knowledge of current BIM versions with emphasis on project sheet set up.
- Strong written and verbal communication skills.
- Ability to use time productively, maximise efficiency and meet challenging work deadlines and goals.
- Ability to take on additional responsibilities as needed as well as determining and manage priorities with minimal guidance
- Must possess and maintain exceptional work ethic as well as uphold company values and demand the highest standard of conduct from yourself and others.
- Essential that you are a self starter and possess the drive to pursue continue gaining knowledge on BIM.
- Ability to write and present information
- Able to positively lead and influence others to achieve results that are in the best interests of Fulcro
- Autodesk AEC Collection, particularly, Revit, AutoCAD and NavisWorks
- Solibri
- Microsoft Office
- EDMS / CDE experience


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Titanic Museum
2134 Parkway
Pigeon Forge, TN
I have been fascinated with the story of Titanic since I was a young girl, so while I was driving down Parkway checking out attractions I wanted to see, this imposing landmark beckoned me in.
There are costumed "crew members" to greet you, and while it's a self-guided tour, there are also staff around to answer questions, replay passenger experiences, and just add to the whole authentic feel of the tour.
You are issued a boarding pass and ticket with the name of a real passenger. At the end, you find out a little more about yourself and if you were one of the survivors or not.
The exhibits are so well done, they help immerse you in the experience, where you can climb the Grand Staircase, stroll the hallways, and take a peak inside to see what the interior rooms were like.
The Grand Staircase is an exact replica and it alone took $1 million to recreate from the actual blueprints. I hung out here a lot, walking up and down the stairs, looking up at the milkglass domed skylight and imagining myself in "Rose" clothes and big hat!
No photos are allowed to be taken inside, so those above are from their website. But you can get your picture taken and they impose it onto the Grand Staircase, Captain's Bridge and stateroom.
I wished I had dressed up better for the occasion!
You can stroll the deck under starry skies, try to keep your balance on the sloping decks demo, and feel how terribly cold the water was in which they were plunged.
It's an impressive museum and they vary themes and special exhibits periodically. During my visit they were featuring the iconic "Titanic violin" that belonged to and was played onboard by bandleader Wallace Hartley. One of the most moving parts of the survivors' story to me was how the musicians played on to the end, and how Hartley led the eight member band as the ship sank, all going down with the ship. A newspaper at that time reported "the part played by the orchestra on board the Titanic in her last dreadful moments will rank among the noblest in the annals of heroism at sea." Hartley's body was found "fully dressed with his music case strapped to his body."
I had the chance to meet the Captain – Edward J. Smith
(portrayed by Lowell Lytle).
This man is fascinating in his own right. He's an actor who plays the part of Capital Smith all over the globe. He was part of the expedition in 2000 where he spent 11 hours in a Russian deep sea submersible viewing the historic ship and its debris field. He told me of that experience, "This was not a movie – this was the real thing. No one can see Titanic with their own eyes and not be changed." His friendliness and eagerness to talk to guests about the museum and what it recreates was a real treat at the end of my journey.
So I definitely recommend checking this out when you're in Pigeon Forge – it's hard to miss!


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Prime Minister
Saturday، 19 September 2015 12:00 AM
Profile of Prime Minister Eng. Sherif Ismail
Eng. Sherif Ismail has been assigned by President Abdel Fattah El Sisi to form a new government on September 12, 2015.
Eng. Sherif Ismail was born in 1955. He graduated from Faculty of Engineering, Ain Shames University in 1978.
Ismail had worked for Enpi Company from 1979 till 2000 and occupied the post of director general of technical affairs and board member of Enpi.
Ismail also worked as undersecretary for the petroleum ministry from 2000 till 2005 and a board chairman of Egas from 2005 till 2007.
The newly appointed premier also occupied the post of board chairman of Ganoub el Wadi (South Valley) Oil Holding Comany from 2007 till 2013. He was later appointed as petroleum minister in outgoing prime minister Ibrahim Mahlab's government in 2013.


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Youth/HS: 2016 Dennis Gile Memorial Weekend QB Camp
2016 Dennis Gile Memorial Weekend QB Camp
When: May 28-29th (Sat/Sun)
Where: Scottsdale, Arizona
Hours: 8-4 P.M.
This QB camp will be only for high school quarterbacks. If you are currently a rising 8th grader you may attend as well, but we (Dennis Gile QB Academy) will need to review your film.
The camp will be taught by QB Coach Dennis Gile and former NFL quarterbacks Charlie Frye and Craig Nall. Please see the invitational video below for more information on the camp.
This is going to be a family oriented QB camp + the most elite training in the country your son will experience.
Information on hotel reservations (Discounted from $399 to $179)
For additional information regarding the camp, or the Dennis Gile QB Academy, you can check out qbacademy.com.
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A long-time reader writes:
World-class whiner Barack Obama has complained to Nicolas Sarkozy that "I have to deal with [Benjamin Netanyahu] every day." But if Obama is dealing daily with Netanyahu is it because he "has to"? Or is it because he has chosen to coerce or browbeat Israel into taking steps that satisfy the American president's agenda, but are at odds with Israel's perception of its interests?
I believe that if Obama halted his efforts at coercion and browbeating — efforts that have produced nothing positive for any party — he could deal with Netanyahu on a far less regular basis. Moreover, he would probably find the occasional dealings less aggravating.
I assume that Obama's left-wing ideology prevents him for taking this happy path. But one might have hoped that Obama would be disciplined enough to refrain from complaining to one foreign leader about the natural consequences of his attempts to dictate security policy to another foreign leader.


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Kilner Butter Paddles (Set of 2) OOS
- Product Code:
- 68859
- Set of 2 Butter Paddles by Kilner
- Make and mould your own homemade butter
- Comes with a recipe for roasted red pepper flavour butter
Average customer review
- Unrated
- |Add your review
This is part of the…
Make, mould and shape your own butter with this set of 2 Kilner Butter Paddles. This pair of essential equipment pieces for a homemade butter enthusiast is both stylish and practical. With markings of the traditional Kilner logo, feel like a professional even on your first attempt.
With deep groves, these paddles are perfect for removing any excess buttermilk, and if you're after a new taste experience then why not try out the roasted red pepper flavour butter recipe which is included.
Care & Use: Handwash only
Includes recipe for roasted red pepper flavour butter
Delivery: For most items we aim to deliver within 5 working days and use a secure courier. Small or lightweight items may be sent via Royal Mail.
Beds and mattresses are delivered directly from our suppliers and will take between 14 - 24 working days. Once your order has been dispatched you will be sent an email detailing your delivery method.
Please note: delivery of some items may take longer should we need to source it directly from a supplier, although we will always contact you in this instance. Please refer to our delivery and returns page for more information.
Returns: We will refund any item you are not completely happy with*. Just send it back to us, unused, within 28 days of receipt. Refunds will be issued via your original method of payment.
*Please note: For hygiene reasons we cannot accept returns of bed care, beds and mattresses. This does not affect your statutory rights.
You might also be interested in...
Kilner Thermometer


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Subscribe to IRMOS News
Join the IRMOS Group in LinkedIn
At a Glance
End Results
Post Production
Virtual and Augmented Reality
News and Events
Public Deliverables
IRMOS Newsletter
Contact us
IRMOS Newsletter Issue N°4 - 27 September 2010
The fourth issue of the IRMOS newsletter is now available at: http://irmosproject.eu/Files/IRMOS_Newsletter_Issue4.pdf IRMOS has entered its third and last year and has already produced and successfully demonstrated a first integrated prototype. During the past months, IRMOS has participated in several events, which have all contributed to increase the impact of the project on industry and research communities. In this newsletter we have selected the most representative ones: The 2nd IRMOS Project Public Seminar "The Real-Time QoS Enabled Cloud", the 2nd Summer School on Service and Software Architectures, Infrastructures and Engineering and the IRMOS Tutorial at WATERS 2010. As special topic of this newsletter issue, you can find The IRMOS Cloud Solution. It introduces the IRMOS approach and technical solutions as a new generation of Service Oriented Cloud Computing environment. Moreover, you can find a list of the main events where IRMOS will be present at in the near future, and where you will have the opportunity to discover more about the project. IRMOS is a research project partially funded by the European Commission under the Seventh Framework Programme FP7/2007-2011, ICT-2007.1.2. If you are interested in the project you can find further information at http://www.irmosproject.eu You can subscribe to the IRMOS Newsletter at http://www.irmosproject.eu/NewsLetter.aspx Keep yourself informed about the IRMOS latest news through our RSS News Feed, http://irmosproject.eu/News/rss.aspx and join the IRMOS Group in LinkedIn http://www.linkedin.com/groups?mostPopular=&gid=2099046


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Day in the life (4)
This post is part of a one week series detailing the life of a post-doc on her way to academia (next summer). Next week I will begin a series on writing your first research paper.
Checked my cells. Look good, adhesion still a problem.
Also, checked polymer adhesion to substrate. Interesting pattern, adhesion is good, declines, then improves. Will repeat taking more pictures in the middle phase.
Talked to boss about poor cell adhesion. Came up with totally new plan for that experiment, ordered supplies.
Ate lunch while reading scary CNN article about bird flu.
Get babysitter for this weekend. Think I have bronchitis, make Dr's appt.
Plan out new cell experiment. Repeat polymer adhesion experiment.
Work on paper.


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VYBZ KARTEL APPEAL: Lawyers drop cell phone bombshell about Kartel voicenote
Entertainment News Written by claude mills Tuesday, 17 July 2018 12:17 font size decrease font size increase font size 0
Samuels had been making her submissions in the murder appeal filed by the two entertainers against their conviction and prison sentence. She said evidence presented during their 17-week murder trial, which ended in April 2014, showed that the metadata was created on July 6, 2011, six weeks before prosecutors claim Williams was killed.
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You may want to upgrade your browser. We have detected you are using Internet Explorer 10 or earlier. It is recommended that you update your browser to the latest version to get a better user experience. Find out more
Phone number: 02 4645 4000
Email: firstname.lastname@example.com
Website: www.campbelltown.nsw.gov.au
Community festivals and events are a vital part of community life.
Event organisers wishing to conduct a major event within the City of Campbelltown on Council owned/managed land must submit an application under Section 68 of the Local Government Act 1993, where such an event is not otherwise authorised under a Development Consent. An event application fee(s) may also apply (see Council's Fees and Charges for details).
Council is supportive of the conduct of outdoor events as they contribute significantly to the City's sense of place and community. Our event approval process is aimed at ensuring that events are well organised, can be conducted in a safe, responsible manner, and are an enjoyable and positive experience for the community.
Applicants are encouraged to read through the attached guide(256KB, PDF) before completing an outdoor event application. The purpose of this guide is to outline the outdoor event approval process and the information required to accompany an application to hold an outdoor event(221KB, PDF).
Good to know: