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Trappin is a multifunctional host-defense peptide that has antiproteolytic, antiinflammatory, and antimicrobial activities. The numbers and compositions of trappin paralogs vary among mammalian species: human and sheep have a single trappin-2 gene; mouse and rat have no trappin gene; pig and cow have multiple trappin genes; and guinea pig has a trappin gene and two other derivativegenes. Independent duplications of trappin genes in pig and cow were observed recently after the species were separated. To determine whether these trappin gene duplications are restricted only to certain mammalian lineages, we analyzed recently-developed genome databases for the presence of duplicate trappin genes.
The database analyses revealed that: 1) duplicated trappin multigenes were found recently in the nine-banded armadillo; 2) duplicated two trappin genes had been found in the Afrotherian species (elephant, tenrec, and hyrax) since ancient days; 3) a single trappin-2 gene was found in various eutherians species; and 4) no typical trappin gene has been found in chicken, zebra finch, and opossum. Bayesian analysis estimated the date of the duplication of trappin genes in the Afrotheria, guinea pig, armadillo, cow, and pig to be 244, 35, 11, 13, and 3 million-years ago, respectively. The coding regions of trappin multigenes of almadillo, bovine, and pig evolved much faster than the noncoding exons, introns, and the flanking regions, showing that these genes have undergone accelerated evolution, and positive Darwinian selection was observed in pig-specific trappin paralogs.
These results suggest that trappin is an eutherian-specific molecule and eutherian genomes have the potential to form trappin multigenes.
Trappins are a family of small secretory proteins that possess an N-terminal transglutaminase-substrate (TGS) domain and a C-terminal whey acidic protein (WAP) domain [1]. The TGS domain consists of repeats of six semi-conserved amino acids, KGQDPV, that act as anchoring regions. In this case, the lysine or glutamine residues of these regions are cross-linked with extracellular-matrix proteins by the action of transglutaminases, which helps trappin molecules to become concentrated at the site of action [2-4]. In contrast, the WAP domain is a four-disulfide core region and is defined by eight conserved cysteine residues. The WAP domain of trappin shows anti-proteolytic [4-6] and antimicrobial [7-9] activities that allow it to act as an innate immune defense molecule. In fact, trappin-2 displays antibacterial activities against Gram-positive and Gram-negative bacteria [7-9]; it also has antifungal activity [9], and the antimicrobial activity is independent of its antiprotease function [9]. The most well characterized trappin is human trappin-2, which is also known as elafin, skin-derived antileukoproteinase (SKALP), elastase-specific inhibitor (ESI), or protease inhibitor 3 (PI3) [1,10]. It has strong inhibitory activity against leukocyte and pancreatic elastases and proteinase 3 [4-6], and shows anti-inflammatory activity [11] as well. The antiproteolytic and antimicrobial activities of trappin-2 are quite similar to those of secretory leukocyte protease inhibitor (SLPI) [12,13], which consists of two WAP domains with the second WAP domain being highly homologous to the WAP domain of trappin-2. Trappin-2 is expressed in the trachea, lung, gut, epidermis, esophagus, vagina, and oral epithelia [2,4]. In these tissues, the expression is induced by proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF)-α [14,15].
The number of trappin genes varies among mammalian species. For example, humans and sheep have a single trappin-2 gene [16,17], while pigs have at least six: trappin-1, trappin-2, trappin-3, trappin-7, trappin-8, and trappin-9 [18,19]. At the other extreme are the mouse and rat, which lack trappin genes entirely [20], though the guinea pig has genes for trappin-12 and its derivatives caltrin II and seminal vesicle secretory protein (SVP), which lack TGS- and WAP-coding regions, respectively [21,22]. Despite the variance in copy number between the different mammalian lineages, all trappin genes are encoded by three exons. Exon 1 encodes a signal peptide, exon 2 codes for a TGS- and WAP-domains, and exon 3 encodes a 3' untranslated region [18,19,23]. While the exonic organization is highly conserved among various mammalian lineages, there is variation in the number of six-amino-acid repeats in the TGS domain [18,19]. Due to a point mutation of splicing site, guinea pig trappin-12 exceptionally lacks intron 2, which is present at the 3' noncoding region of the trappin gene [22]. A short interspersed element (SINE) is found in intron 2 of the trappin genes of the pig, wart hog, and collared peccary [18,19].
While we have mentioned several species that possess multiple trappin genes, it is not known if (1) these are exceptional cases or (2) trappin genes normally exist as a multigene family. In an attempt to find the answers, we analyzed genome databases developed by the Mammalian Genome Project http://www.broad.mit.edu/mammals/ webcite and identified six trappin genes from the nine-banded armadillo (Dasypus novemcinctus) genome. The nine-banded armadillo belongs to the taxonomic order Xenarthra. Because this lineage is believed to be one of the most ancient lineages of placental mammals [24], the analyses of armadillo trappin genes are quite interesting because the duplication and evolution of armadillo trappin genes are expected to have occurred independently from other species. In contrast, we identified a single trappin-2 gene from the genome databases of many species including the chimpanzee, rhesus macaque, bushbaby, dog, cat, horse, cow, European shrew, European hedgehog, megabat, and microbat. This fact suggests that trappin-2 is the ancestral form of trappin genes, and trappin-null species such as mouse and rat are exceptional. Finally, we identified anciently duplicated trappin-18 gene in Afrotheria such as the elephant (Loxodonta africana), tenrec (Echinops telfairi), and hyrax (Procavia capensis), and trappin-related genes in chicken and opossum, suggesting that the gene family originated as far back as more than 100 million years ago.
Identification of trappin, SLPI, and trappin-related genes in eutherian mammals, opossum, platypus, chicken, and zebra finch
To estimate the origin of trappin genes, we analyzed the genome databases of eutherian mammals, opossum, platypus, birds (chicken and zebra finch), Xenopus, fish (Danio rerio, Takifugu rubripes, Tetraodon nigroviridis, Gasterosteus aculeatus, and Oryzias latipes), sea squirts (Ciona intestinalis and Ciona savignyi), insects (Drosophila melanogaster, Anopheles gambiae, and Aedes aegypti), and Caenorhabditis elegans. Typical trappin genes were identified only in the mammalian species (Figure 1C-D). A single homologous gene was identified in chicken, zebra finch, and opossum, and multiple homologous genes were identified in platypus, (Figure 1A-B). The other species did not show any homologous genes to trappins except for other WAP-coding genes with a low homology (data not shown).
Figure 1. WAP domain of the trappin gene family. WAP domains of SLPI (A), trappin-related genes (B), trappin-2 (C), and the other species-specific trappin paralogs (D) are shown. Conserved and semiconserved residues among trappin-2 genes are indicated by light gray. Eight conserved Cys residues constituting the WAP motif signature sequence are shaded in black. The asterisk indicates the catalytically important Met residue. The variable region, which is thought to determine the specificity of WAP motifs, is boxed.
The presence of the SLPI gene was also analyzed using the genome databases, and a single orthologous gene was identified in all mammalian species except for the guinea pig and the rabbit (Figure 1A). In contrast, there are no clear direct orthologs for the SLPI gene in the genome databases of chicken, zebra finch, and opossum, and the trappin-homologous genes are also the most homologous to SLPI. In platypus, the above-mentioned trappin-homologous genes encode two-WAP-domain proteins and may be the paralogs of the mammalian SLPI gene.
The trappin-related genes in chicken, zebra finch, and opossum have a single WAP-coding region but lack a TGS-coding region. Only the WAP-coding region is similar to trappin and the SLPI genes, but the other flanking regions lack any significant similarity except for a weak similarity in the signal-peptide coding regions (data not shown). The deduced amino acid sequence of the WAP domains of the trappin-related genes are shown in alignment with those of mammalian trappin and the SLPI genes (Figure 1B). The catalytically important Met residue (an asterisk in Figure 1, A and 1C) is conserved in the opossum and platypus genes but not in the chicken and zebra finch genes.
Platypus SLPI genes show a stronger identity with trappin-2 (67%) than SLPI (55%) in the deduced amino acid sequences. The phylogenetic analysis for trappin, mammalian SLPI, platypus SLPI, and trappin-related genes of chicken and opossum is shown in Figure 2A. Trappin-related genes of chicken and opossum and platypus SLPI genes are not clearly categorized as trappin or SLPI.
Figure 2. Phylogenetic analyses of trappin genes. (A) Phylogeny of trappin, SLPI, and trappin-related genes. The WAP-coding region of each gene was used for the analyses. (B) Phylogeny of trappin genes. Noncoding region of the trappin genes that was used for the analyses. The phylogenetic trees were constructed using neighbor joining (NJ), maximum parsimony (MP), and maximum likelihood (ML) methods. The NJ trees (left) with bootstrap values for both the NJ and MP methods and ML trees (right) are shown. Trappin genes with short nucleotide sequences were not included in the analyses, because inclusion of short sequences reduces the reliability of the analyses when we removed all the sites containing missing data and alignment gaps prior to the calculation (called the complete deletion in MEGA software). Bars indicate 10% replacement per site. Species- or lineage-specific trappin multigenes are shaded in gray.
Identification of trappin-2 genes from various eutherian mammals
We analyzed genome databases for various eutherian mammals including human, chimpanzee, rhesus macaque, bushbaby, mouse, rat, rabbit, dog, cat, cow, European shrew, European hedgehog, microbat, megabat, nine-banded armadillo, sloth, elephant, hyrax, and tenrec. Only mouse, rat, and rabbit lack trappin genes in their genome databases, but the other species have at least one trappin gene in their genome databases (Figure 3). All the newly identified trappin genes consist of three exons like previously analyzed trappin genes: exon 1 encodes a signal peptide, exon 2 encodes the TGS and WAP domains, and exon 3 encodes the 3' untranslated region (data not shown). We aligned the amino acid sequences of the WAP domains of those genes, and categorized them into two groups: the first group contained the catalytically important Met residue (asterisk in Figure 1C) and was named trappin-2 (Met rule); and the second group lacked the Met residue and was named according to the order of discovery. Most animals have a single trappin-2 gene (Figures 1C and 3). This finding suggests that the trappin-2 gene is the ancestral form of the trappin genes. According to this definition, the previously reported porcine trappin-8 should also be renamed trappin-2b as it also has the Met residue. Tenrec trappin-18 also has the Met residue at the catalytic site. However, the phylogenetic analyses using nucleotide sequences of the noncoding regions (introns, exon 3 and 5' and 3' flanking regions) clarified that this gene is closely related with trappin-18 genes of elephant and hyrax. Therefore we call this gene tenrec trappin-18 as an exception to the Met rule.
Figure 3. History of the evolution of trappin genes in mammals. Phylogeny of mammalian species with the time scale was generated based on the works by Kumar and Hedges [31], Hasegawa et al. [48], Nishihara et al. [24], Hallstrom and Janke [49], and Arnason et al. [50]. Ranges of the estimated dates for gene duplications are indicated by open boxes. Bayesian estimations of duplications using the nucleotide and the amino acid sequences of trappin genes are indicated by arrows and arrow heads, respectively. The dates of duplication estimated by MEGA software using single reference point are indicated by asterisks. The dates of duplications individually estimated by MEGA software for each species are indicated by double asterisks. Mya, million years ago.
The average nonsynonymous and synonymous distances were calculated on 150 bp WAP-coding regions among the trappin-2 genes of the various species, trappin paralogs except for trappin-2, and all trappin genes (Table 1). In the WAP-coding region, the rate of nonsynonymous substitutions is lower than that of synonymous substitutions among trappin-2. The rates of nonsynonymous and synonymous substitutions are similar in the WAP-coding region among trappin paralogs except for trappin-2. These results suggest that the purifying selection is operating on the trappin-2 gene in various eutherian mammals.
Table 1. Purifying selection of trappin-2 genes.
Identification of novel trappin multigene families in armadillo and Afrotheria (elephant, tenrec, and hyrax)
Database analyses demonstrated the presence of six trappin genes in nine-banded armadillo (Dasypus novemcinctus), which were named trappin-2 and trappins-13-17 (Figures, 1 and 3). Afrotherian species such as the elephant (Loxodonta africana), tenrec (Echinops telfairi), and hyrax (Procavia capensis) had two trappin genes, which were named trappin-2 and trappin-18 (Figures, 1 and 3). We also found two novel trappin paralogs from the bovine genome database, and named them trappin-19 and trappin-20 (Figures 1D and 3).
Phylogenetic analyses of the noncoding regions of trappin genes from several mammalian species are shown in Figure 2B. All armadillo trappins-13-17 genes form a single branch with armadillo trappin-2 gene. Bovine trappin-19 and trappin-20 also share the same branch with bovine trappin-2. These results suggest that those genes are recently duplicated species specific paralogs. On the other hand, Afrotherian trappin-18 is divided near the root, suggesting that trappin-18 duplicated much earlier.
Estimations of the dates for the duplication of trappin multigenes
A linearized tree was constructed by using the nucleotide sequences of trappin multigenes and the dates of the duplications were calculated with MEGA software. When the divergence time between Primate and Artiodactyla (96.2 Mya) was used as a reference point, the date of the duplications of trappin genes of pig, cow, armadillo, guinea pig, and Afrotheria (elephant, hyrax, and tenrec) were calculated as 7.0, 8.8, 15.9, 79.0, and 161 Mya, respectively (asterisks in Figure 3).
We next calculated the date of duplications individually for each species using the taxon pair that was most closely related to the node of interest as a reference point. We found that some of the trappin gene subfamilies were relatively young. For instance, when the divergence time between sheep and cow (18.3 Mya) was used as a calibration point, the date of the duplication events giving rise to the pig and bovine trappin gene families were calculated as 7.8 and 9.7 Mya, respectively. Similarly, when the divergence time between human and armadillo (96.2 Mya) was used as a reference point, the date of the duplication event giving rise to the armadillo trappin gene family was calculated as 15.5 Mya. On the other hand, certain trapping gene subfamilies appear to be more ancient. For example, when the divergence time between primate and rodent (61.7 Mya) was used as a reference point, the date the guinea pig trappin gene subfamily was estimated to have originated 55.2 Mya, and when the divergence time between elephant and tenrec (48.6 Mya) was used as a reference point, the Afrotherian trappin trappin gene subfamily was calculated to have originated 91.9 Mya (double asterisks in Figure 3).
We also estimated divergence times using the Bayesian method implemented in the BEAST software package [25]. The date of the duplications of trappin genes of pig, cow, armadillo, guinea pig, and Afrotheria were calculated as 3.3, 12.6, 11.4, 34.7, and 244 Mya (arrows in Figure 3), respectively, when the nucleotide sequences of trappin genes were used for the calculation. When amino acid sequences were used for the calculation, the date of the duplications of trappin genes of pig, cow, armadillo, and Afrotheria were calculated as 49.7, 18.3, 50.9, and 154 Mya (arrowheads in Figure 3), respectively. Thus, the dates calculated for nucleotide and amino acid sequence data are very different. The former are similar to the dates generated using the linearized tree method in pig, cow, and armadillo, and the latter are similar to the dates generated using the linearized tree method in Afrotheria. In pig, cow, and armadillo, the protein-coding regions of trappin multigenes have evolved rapidly (Table 2), and the dates calculated for amino acid sequence data showed larger values. In Afrotheria, since amino acid sequences evolve more slowly than nucleotide sequences and are, therefore, less prone to homoplasy over the long evolutionary time frames being considered, it is reasonable to suspect that the dates generated using nucleotide sequence data are not as reliable as those generated from acid sequence data for this particular study.
Table 2. Accelerated evolution of trappin multigenes.
Synteny analyses around trappin genes
It has been demonstrated that the trappin-2 is mapped on the WAP four-disulphide core (WFDC) domain locus which contains a number of WFDC genes [17]. Moreover, the conserved synteny of WFDC loci has been studied for primates, rodents, and the dog [20,26]. To extend these studies, we analyzed the genes neighboring trappins in species that were included in our study. Most scaffolds containing trappin genes were too short to analyze. However, we could analyze genes neighboring to horse trappin-2, megabat trappin-2, bovine trappins, hyrax trappin-18 and elephant trappins (Figure 4A). As reported previously for the dog WFDC locus, the horse, megabat, and bovine trappin genes flanked WFDC5 in opposed directions, whereas WFDC12 and WFDC15 were not found between WFDC5 and trappin(s). The bovine trappin genes were tandemly arrayed, suggesting that these genes arose by tandem gene duplication. In contrast, hyrax and elephant trappin-18 flanked WFDC5 in the same direction and was mapped to the same locus as WFDC12. Harr Plot analyses demonstrated that human WFDC12 is highly homologous to the 5'-flanking region, exon 1, and intron 1 of hyrax trappin-18 (Figure 4C). Similar homology was observed between human WFDC12 and elephant trapin-18 (Figure 4D).
Figure 4. Evolutional relationship between trappin-18 and WFDC12. (A) Schematic illustration of conserved synteny around trappin(s). Illustrations of human, mouse, rat, and dog loci were generated based on the works by Clauss et al. [20] and Hurle et al. [26]. Black and gray arrows indicate genes and pseudo genes, respectively. KCNS, potassium voltage-gated channel, member 1 (B) The WAP domains of WFDC12 and the WFDC12-like peptide encoded by intron 1 region of trappin-18. Conserved and semiconserved residues are indicated by light gray. Eight conserved Cys residues constituting the WAP motif signature sequence are shaded in black. (C) Harr plot analyses of the hyrax trappin-18 in comparison with the hyrax trappin-2 and human WFDC12. (D) Harr plot analyses of the elephant trappin-18 in comparison with the elephant trappin-2 and human WFDC12. Exons are indicated by boxes. WAP-coding regions are represented by black boxes. Black circles indicate regions encoding WFDC12 like peptide.
Recently, Hurle et al. found that primate trappin-2 contains a pseudogene for WFDC12 in intron 1, and suggested that trappin-2 and WFDC12 have a common ancestral gene [26]. All trappin genes contained a pseudogene for WFDC12 in intron 1 (data not shown) except for Afrotherian trappin-18, which codes for a WFDC12-like peptide in intron 1 (Figure 4B).
Accelerated evolution of TGS and WAP coding region trappin multigenes in armadillo, cow, and pig and positive selection of the WAP-coding region of pig trappin paralogs
The average distances of the 5'-flanking region, exon 1, intron 1, exon 2, intron 2, exon 3, and 3'-flanking region among trappin multigenes for each species were calculated (Table 2, line A1-A7). In armadillo trappins, the average Jukes-Cantor (JC) distance between the exon 2 regions was 0.235 (Table 2, line A4), which is 4.4 times higher than that between the non-coding regions (0.054; Table 2, line B1). When we calculated the average Tamura-Nei (TN) distances with gamma correction, the value between exon 2 (0.442; Table 2, line A4) was also 5.6 times higher than that between the non-coding regions (0.079; Table 2, line B1). Fisher's exact test using the numbers of varied sites and common sites between the exon 2 regions (39 varied sites in 201 common sites) and those between the non coding regions (87 varied sites in 1691 common sites) demonstrated that the difference is significant (P < 0.01). A similar difference was not observed in the other regions.
In cow, the average distances between the exon 2 regions were 0.167 (JC method) and 0.299 (NJ method) (Table 2, line A4), and were 3.1 and 4.3 times, respectively, higher than those between the non-coding regions (Table 2, line B1; P < 0.01, Fisher's exact test). In pig, the average distances between the exon 2 regions (Table 2, line A4) were 12 and 16 times higher than those between the non-coding regions when calculated by the JC and TN methods, respectively (Table 2, line B1; P < 0.01). In contrast, there was no significant difference in the average distances between the exon 2 regions (Table 2, line A4) and those between the non-coding regions (Table 2, line B1) of elephant, hyrax, and guinea pig trappin genes. In pig, the average distances between the exon 1 regions and between the intron 2 regions of different genes (Table 2, lines A2 and A5) were also higher than those of the non-coding regions (Table 2, line B1) (P < 0.01).
Next, we calculated distance values for synonymous substitutions per site (ds) and non-synonymous substitutions per site (dn) for the signal peptide (pre peptide), TGS, and WAP coding regions (Table 2, line C1-E3), and compared against the average distance of the non-coding regions. In armadillo, dn of the TGS coding domain (P < 0.05) and both ds and dn of the WAP coding domain (P < 0.01) were significantly higher than the average distance of the non-coding regions. In cow, only dn of the TGS and WAP coding regions were significantly higher than the average distance of the non-coding regions (P < 0.01). In pig, both dn and ds of the signal peptide, TGS, and WAP coding regions were higher than the average distance of the non-coding regions.
To examine the presence of positive Darwinian selection, we compared dn and ds of the rapidly-evolved coding regions using Fisher's test. When the average values in each species were used for the analyses, we could not detect any statistically significant difference between dn and ds of all the trappin multigenes. However, the pairwise comparison matrix of dn and ds on the paralogs of each species demonstrated a dn/ds rate of 3.3 for porcine trappin-2 vs. trappin-3, 4.9 for porcine trappin-2 vs. trappin-9, and 5.2 for porcine trappin-8 vs. trappin-9 (P < 0.05) (Table 3). Because trappin-2 is the most conservative gene within the trappin family and porcine trappin-8 is the closest homolog of porcine trappin-2, these data indicate the positive Darwinian selection of porcine trappin-3 and trappin-9. Although the dn/ds rate between bovine trappin-5 and other bovine paralogs are as high as 2.8-5.8, the differences were not statistically significant (P = 0.07) (Table 3).
Table 3. Positive selection of species-specific trappin paralogs.
Evaluation of the quality of genomic sequence with low coverage
The nucleotide substitutions between seven known cDNAs and corresponding exons in the genome databases were calculated and shown in Table 4. Some of these substitutions may have occurred as a result of sequencing errors or site-specific polymorphism within each species. Yet, we can still infer that the average rates of sequencing errors are lower than the substitution rates. In low coverage genomic sequences of armadillo, rabbit, cat, and elephant, the average substitution rates of the seven genes were 0.10-0.49%. In high coverage genomic sequences of cow and human, the average substitution rates of the seven genes were 0.23 and 0.06%, respectively. These estimates are not substantially different, suggesting that artifacts due to errors or polymorphism are negligible. In the case for armadillo trappin genes, for example, the 129-bp WAP-coding regions and the ~2.1-kb entire genes may contain less than 0.4 and 6-base sequence errors, respectively. Among armadillo trappin multigenes, the WAP-coding regions and the ~2.1-kb entire genes have 8-50 and 96-165-base substitutions, respectively. Thus, the sequencing errors of genomic sequences with low coverage appear to be negligible, although the caveat remains that we can not negate a small number of possible errors.
Table 4. Nucleotide substitutions between known cDNA and corresponding genomic sequences
Origin of trappin gene
Computer analyses of genome databases revealed that typical trappin is a eutherian mammalian specific gene. The typical trappin genes were found only in eutherian mammals and not other species including Xenopus, fish, sea squirt, insects, and C. elegans. The trappin-related genes were found in chicken and opossum. The computer analyses also showed that most eutherian mammalian species have a single SLPI gene, and platypus has multiple SLPI genes. The trappin-related genes of those animals and platypus SLPI genes show strong similarity with trappin in the WAP domain only, but all the other regions have no significant homology. Therefore, these genes may relate with the ancestoral WAP domain of trappin. Interestingly, platypus SLPI showed higher homology to the WAP domain of trappin-2 than that of mammalian SLPI. This strongly suggests that the WAP domain of trappin and SLPI share a common ancestor.
Trappin is a protein that consists of TGS and WAP domains. SLPI is a two WAP-domain protein. The second WAP domain of SLPI and the WAP domain of trappin-2 are quite similar in their amino acid sequences and functions such as antiproteolytic and antimicrobial activities, suggesting an ancestral relationship. However, except for the WAP-coding regions, there is no significant homology in the nucleotide sequences between trappin and SLPI. Interestingly, trappin genes are known to have weak but significant similarity with other TGS genes in introns, TGS-coding, and noncoding regions (Figure 5) [27]. This mosaic pattern of homology in trappin genes indicates that trappin genes originated from the TGS gene and obtained a WAP domain possibly by exon-shuffling. Afrotherian trappin-18 codes for a WFDC12-like peptide in intron 1 region, and the other trappins contain a pseudogene for WFDC12 in intron 1. These results support the hypothesis of Hurle et al. [26] that trappin and WFDC12 are derived from a common ancestral gene which codes for both trappin and WFDC12.
Figure 5. Schematic representations of TGS, trappin, SLPI, and the trappin-related gene. (A) The three-exon structure of mammalian TGS and trappin genes and the four-exon structure of mammalian SLPI gene are shown. (B) Structure of the trappin-related genes of chicken and opossum (upper) and platypus SLPI gene (lower). The homologous regions are shown by dotted lines. The exons are shown by boxes. TGS- and WAP-coding exons are indicated by light and dark gray, respectively. The signal-peptide-coding regions and noncoding regions are indicated by black and white boxes, respectively. S, signal-peptide-coding region.
Evolution of trappin genes in eutherian mammals
Nineteen species of eutherian mammals were analyzed by a search for the presence of trappin genes within their genome databases, and the results were combined with those of previous experimental analyses of human [17,23], pig [18,19], wart hog [19], collared peccary [19], cow [28], sheep [16], and guinea pig [21,22]. In total, we could compare the trappin genes from 24 eutherian mammals (Figure 3). Within the 24 species analyzed, we could isolate trappin genes from 21 species. A single trappin-2 gene was found in 11 species, and multiple trappin genes were found in 8 species. These results indicate that trappin-2 is the most common and is an ancestral form while the other trappins are specie-specific paralogs. We could not find trappin genes in three mammalian species: mouse, rat, and rabbit. Our experimental analyses (data not shown) and the integrity of the genome databases of mouse and rat suggest that mouse and rat lack trappin genes in their genome [20]. In mouse and rat, other WAP-motif containing proteins such as SLPI and SWAMs may compensate the function of trappin. In the case of rabbit, it is not certain whether rabbit really lacks trappin genes or rabbit has a trappin gene that has not yet been analyzed by the genome project.
By computer analyses of genome databases, we found that the nine-banded armadillo as well as pig and cow also have recently-duplicated trappin multigene. The computer analyses of bovine genome databases also revealed two novel trappin paralogs and the sequences of the introns and flanking regions, which enabled the detailed evolutional analyses of bovine trappin multigenes. As previously reported porcine trappin multigenes, the WAP-coding regions of the trappin multigenes of armadillo and cow were shown to have evolved under accelerated evolution. Only dn was accelerated in the WAP coding regions of bovine trappins, and both dn and ds of the WAP coding regions were accelerated in armadillo and porcine trappins. The accelerated substitutions of non-synonymous sites of WAP-coding regions may be explained by positive Darwinian selection or relaxation of functional constraints, because we observed statistically significant positive selection of the WAP coding regions of porcine trappin-3 and trappin-9 but no significant difference between dn and ds of other trappins (Table 3). However, the question why synonymous substitutions are also accelerated can not be interpreted simply by the existence of positive Darwinian selection or relaxation of functional constraint. The mechanism whereby the synonymous substitutions are accelerated must be clarified by future studies.
The molecular clock and Bayesian analyses using the nucleotide sequences estimated the date of duplication as 11.4-15.9, 8.8-12.6, and 3.3-7.8 Mya for trappin multigenes of armadillo, cow, and pig, respectively. These results are consistent with previous experimental analyses demonstrating that the collared peccary that was separated from porcine 33 Mya [29,30], and sheep, which was separated from bovine 19.6 Mya [31], do not have trappin multigenes [16,19]. The findings of recently-duplicated accelerated-evolved trappin multigenes in three individual species demonstrate that mammalian genomes have the potential to form trappin multigenes in several million years. The selective pressure that formed the trappin multigenes may relate with some pathogens, and the variety of amino-acid sequences in the WAP-domain may contribute to the acquisition of antimicrobial activities for a large spectrum of pathogens. Tissue distribution of trappin paralogs in pig and cow has been shown to vary among genes: porcine trappin-2 is expressed in the trachea and the large intestine, porcine trappin-1 in the small intestine, bovine trappin-2 in the epidermis and the tongue, bovine trappin-4 in the trachea and the tongue, and bovine trappin-5 in the trachea [28]. Therefore, the selective pressures might also affect the regulation of the tissue-specific expression of trappin genes.
Our previous analyses revealed that guinea pig has a trappin-12 gene [22] and two derivative genes, SVP [32] and caltrin II [19]. SVP and caltrin II genes have significant homology with trappin including introns, noncoding region of exons, and flanking regions, but lack WAP and TGS domains, respectively. The molecular clock analysis estimated the date of the duplication of the guinea pig genes as 34.7-79.0 Mya. This date of duplication is much earlier than those of pig, cow, and armadillo.
In Afrotherians we found two trappin genes, trappin-2 and trappin-18, whose date of duplication was estimated as 91.9-244 Mya. This date is surprising, because it is earlier than the date of the periods of divergence of the major orders of eutherian mammals (70-10 Mya) [24,31], and suggests that the duplication of trappin-18 occurred in the ancestors of the eutherian mammals before the divergence of the species. In this context, most species lack trappin-18, however, only Afrotheria has retained the gene. The reason is still unknown, but it is conceivable that trappin-18 increases resistance to Afrotheria-specific pathogen. Another possible alternative explanation is that trappin-18 underwent substitutions at a faster rate per year than other trappin genes and that lead to the duplication time being overestimated.
• Typical trappin genes are only found in the genome sequences of eutherians but not in those of other vertebrate species.
• Trappin-2 is the most widely distributed and is the strongest candidate of the ancestral forms of trappin. Recently-duplicated species-specific trappin paralogs are present in the genomes of armadillo, pig, and cow, and the non-synonymous sites of those genes have undergone accelerated evolution as a result of positive Darwinian selection or relaxation of functional constraint.
• Synonymous sites of recently-duplicated trappin paralogs of armadillo and pig have also undergone accelerated evolution by unknown mechanisms.
• The anciently-duplicated trappin-18 gene is only retained in afrotherian species and is a fossil molecule of the trappin gene family.
Isolation of trappin genes from various animal species
The genome database of various species (URL: http://www.ensembl.org/index.html webcite) [33] were screened using the amino-acid sequence of human trappin-2. The exon-intron organization was estimated by comparing it with that of the human trappin-2 gene. The nucleotide sequences of the trappin genes were deposited in the DDBJ/EMBL/GenBank DNA databases as third party annotations (TPAs) under accession numbers BR000322 to BR000327 and BR000708 to BR000720.
Evaluation of the quality of genomic sequence with low coverage
To evaluate the quality of genomic sequences with low coverage, we compared known cDNA sequences with those of corresponding exons in the genome databases. We used cDNA sequences for SDHA, MDH2, ATP5B, GAPDH, SDHB, CS, and IDH1 which was determined by Kullberg et al. [34]. The corresponding exons of armadillo (2 × coverage), rabbit (2 ×), cat (1.87 ×), elephant (2 ×), cow (7 ×), and human (Genome Reference Consortium GRCh37 assembly) were isolated and the numbers of nucleotide substitutions between the sequences of cDNA and the genome databases were calculated for each species using MEGA software [35]. The sequences used for the analysis are shown in Supplemental Table S1 (see Additional file 1).
Additional file 1. Nucleotide sequences used for the evaluation of the quality of genomic sequence with low coverage. Accession numbers for the cDNAs (upper) and scaffold, contig, or accession numbers of the genomic sequences (lower) are shown. ND, no data.
Format: DOCX Size: 15KB Download file
Phylogenetic analyses
Nucleotide sequences of the WAP-coding regions of trappin, SLPI, and trappin-related genes were used to analyze their phylogenetical relationship. The introns, exon 3 (noncoding exon), and 3'-noncoding regions were used to analyze recent evolution of trappin genes in eutherian mammals. The nucleotide sequences were aligned using ClustalW software [36], and the best fit/gap placement was confirmed manually. Phylogenetic analysis was performed by the neighbor-joining (NJ) method [37,38] and maximum parsimony (MP) method [38] with 2,000 bootstrap replicates using MEGA software [35] or the maximum likelihood (ML) method with 200 bootstrap replicates using PHYML [39] plugin for Geneious software http://www.geneious.com webcite. The sequences used are as follows with the accession numbers in parentheses: human (Homo sapiens) trappin-2 (D13156) and SLPI (X04502); chimpanzee (Pan troglodytes) trappin-2 (XM_514671) and SLPI (DP000037); macaque (Macaca mulatta) trappin-2 (XM_00110935) and SLPI (DP000043); bushbaby (Otolemur garnettii)trappin-2 (BR000708) and SLPI (DP000040); mouse (Mus musculus) SLPI (AF002719); rat (Rattus norvegicus) SLPI (AAHX01026351); guinea pig (Cavia porcellus) trappin-12 (AB161363), caltrin II (AB161364) and SVP (U59711); European shrew (Sorex araneus) trappin-2 (BR000713) and SLPI (AALT01303048); European hedgehog (Erinaceus europaeus) trappin-2 (BR000714) and SLPI (AANN01307740); microbat (Myotis lucifugus) trappin-2 (BR000712) and SLPI (AAPE01410948); megabat (Pteropus vampyrus) trappin-2 (ABRP01168531) and SLPI (ABRP01290205); horse (Equus caballus) trappin-2 (XM_001503186) and SLPI (XP_001503242); dog (Canis familiaris) trappin-2 (BR000710) and SLPI (AAEX02024101); cat (Felis catus) trappin-2 (BR000711) and SLPI (AANG01238466); bovine (Bos taurus) trappin-2 (AJ223216), trappin-4 (AJ223217), trappin-5 (AJ233218), trappin-6 (AB011010), trappin-19 (BR000718), trappin-20 (BR000719) and SLPI (AAFC03003522); sheep (Ovis aries) trappin-2 (NM_001035224) and SLPI (AY346135); porcine (Sus scrofa) trappin-1 (D50320), trappin-2 (D50319), trappin-3 (D50321), trappin-7 (D50323), trappin-8 (D50322), trappin-9 (AB003285) and SLPI (NM_213870); elephant (Loxodonta africana)trappin-2 (BR000716), trappin-18 (BR000717) and SLPI (AAGU01360578); hyrax (Procavia capensis) trappin-2 (ABRQ01439157), trappin-18 (ABRQ01336046), and SLPI (ABRQ01352342); tenrec (Echinops telfairi) trappin-2 (BR000715), trappin-18 (AAIY01696839), and SLPI (AAIY01696839); wart hog (Phacochoerus aethiopicus) trappin-1 (AB003282) and trappin-2 (AB003281); collared peccary (Pecari tajacu) trappin-10 (AB003283); hippopotamus (Hippopotamus amphibius) trappin-11 (AB003284); nine-banded armadillo (Dasypus novemcinctus) trappin-2 (BR000322), trappin-13 (BR000323), trappin-14 (BR000324), trappin-15 (BR000325), trappin-16 (BR000326), trappin-17 (BR000327) and SLPI; sloth (Choloepus hoffmanni) trappin-21 (ABVD01210669) and SLPI (ABVD01323747); platypus (Ornithorhynchus anatinus) SLPIa (AAPN01348542), SLPIb (AAPN01336636), SLPIc (AAPN01050486), SLPId (AAPN01048517) and SLPIe (AAPN01030446); chicken (Gallus gallus) trappin-related protein (NC_006107); finch (Taeniopygia guttata) trappin-related protein (ABQF01028586); and opossum (Monodelphis domestica) trappin-related protein (BR000720).
Molecular clock analysis and Bayesian divergence time estimation
The introns, exon 3 (noncoding exon), and 5'- and 3'-noncoding regions of pig, cow, armadillo, guinea pig, and Afrotheria (elephant and hyrax) trappins were aligned, and a phylogenetic tree was constructed by the NJ method. A linearized tree was constructed and the dates of the duplication events of trappin genes were calculated by MEGA 3.1 using the divergence time between Primate and Artiodactyla (96.2 Mya) [40] as a calibration point for dating.
As an additional method to investigate divergence times, we used the Bayesian method implemented in the software package BEAST 1.4.8 [25]. To generate divergence times, the following nine fossil calibration points were taken from the work by Benton and Donoghue [40] and implemented as priors in the analysis of both DNA sequence and amino acid sequence data: (1) human-chimp: 6.5 Mya; (2) human-Macaque: 23.5 ± 0.5 Mya; (3) dog-cat: 43 ± 0.2 Mya; (4) cow-sheep: 18.3 ± 0.1 Mya; (5) cow-dog 96.2 ± 0.9 Mya; (6) human-cow: 96.2 ± 0.9 Mya; (7) human-armadillo: 96.2 ± 0.9 Mya; (8) tenrec-elephant: 48.6 ± 0.2 Mya; (9) human-opossum: 124.6 ± 0.1 Mya. The chains were run until convergence was reached (i.e., until the effective sample size for each parameter exceeded 200), which was 93 million states for the DNA sequence data and 10 million states for the amino acid sequence data. The HKY + gamma model was used for the analysis of the DNA sequence data, and the WAG model was used for the analysis of the amino acid sequence data. For both sequence data types, the birth-death speciation process was used as a tree prior.
Calculation of nucleotide substitution rates
Nucleotide sequences were separated into the following regions: the 5'-flanking region, exon 1, intron 1, exon 2, the WAP-coding region of exon 2, intron 2, exon 3, and the 3'-flanking region. These regions were aligned separately using ClustalW software. Jukes-Cantor (JC) distances [41] and Tamura-Nei (TN) distances [42] were calculated using MEGA software [35]. For the calculation of TN distances, we estimated the gamma shape parameter using MrBayes [43] plugin for Geneious software. Distance values for the synonymous substitutions per site (ds) and non-synonymous substitutions per site (dn) of the signal-peptide-coding region of exon 1, TGS- and WAP-coding regions of exon 2 were calculated using the modified Nei-Gojobori (NG) method [44]. Standard errors were computed using the bootstrap method [45] with 2,000 replicates. Fisher's exact test was used for the statistical analyses [46].
Synteny and Harr plot analyses
Synteny of neighboring genes of the trappin genes was investigated by surveying neighboring genes on horse genome cont2.26764 (AAWR02026765), megabat genome cont1.168530 (ABRP01168531), cow chromosome 13 (DAAA02036736) [47], hyrax genome cont1.336045 (ABRQ01336046), elephant SuperContig scaffold_19, human chromosome 20, mouse chromosome 2, rat chromosome 3, and dog chromosome 24. Harr plot analyses were performed at a 23/40 nucleotide stringency using Genetyx-win software (Genetyx Co., Tokyo).
List of abbreviations
TGS: transglutaminase substrate; WAP: whey acidic protein; SVP: seminal vesicle clotting protein; SLPI: secretory leukocyte proteinase inhibitor; Mya: million years ago; ds: distance values for synonymous substitutions per site; dn: distance values for non-synonymous substitutions per site; NJ: neighbor-joining; MP: maximum parsimony; JC: Jukes-Cantor; NG: Nei-Gojobori.
Authors' contributions
AK and SH planned and designed the study. AK, YF, and AR performed the analyses. AK, AR, and SH wrote the manuscript. All authors read and approved the final manuscript.
We thank Tomoko Okada for her secretarial assistance. This work was supported by the Ministry of Education, Culture, Sport, Science, and Technology of Japan (MEXT) 21st Century and Global Center of Excellence Program of MEXT.
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text:
The Partnership's UpSkill Houston initiative is the nation's first business-led, community-wide, integrated workforce effort. The initiative focuses on closing the skills gap in Houston by increasing the number of Houstonians trained for great careers across the region. The Partnership forecasts nearly 75,000 annual jobs openings in these "middle skills" careers that require more than a high school education but less than a four-year college degree.
UpSkill Houston has three key objectives:
Career Connections:
Other Resources
On November 12, GHP held the UpSkill Houston Workforce Summit which brought together industry, education and community leaders to discuss the middle skills workforce challenges and solutions for Houston and the nation, and provided an update on UpSkill Houston's progress and 2015 agenda.
The event featured a number of expert panels and a luncheon keynote from Joe Fuller of Harvard Business School, who discussed the U.S. Competitiveness Project and the role companies can play in creating supply-demand alignment. The U.S. Competitiveness Project is a research-led effort by Harvard Business School to identify practical steps to strengthen the nation's economy, with recent emphasis on beefing up job skills, K-12 education, transportation and infrastructure.
In addition, JPMorgan Chase released an important report, "Preparing Houston to Skill Up, Addressing the Skills Mismatch to Meet Employer Demand in High-Growth Industries."
Building a Demand-Driven Workforce System: A National Perspective
Fred Dedrick, Executive Director, National Fund for Workforce Solutions
JPMorgan Chase's "Closing the Skills Gap" Report
Dr. Gloria Mwase, Jobs for the Future
Workskills Gap In Houston: New Solutions from K-12
Dr. HD Chambers, Superintendent, Alief Independent School District
Accelerate Lone Star: Fast Track to Careers
Dr. Nadia Nazarenko, Lone Star College
United Way THRIVE Family Financial Stability Collaborative
Lynne Liberato, Board Member, United Way of Greater Houston
The New Evolution of Apprenticeship
Brandon Spence, Apprenticeship Consultant, South Carolina Technical College System
Project GRAD Houston Summer College Institutes
Dr. Ann Stiles, Executive Director, Project GRAD
U.S. Competitiveness: Building America's Middle Skills
Joseph Fuller, Senior Lecturer, Harvard Business School
Preparing Houston to Skill Up, Addressing the Skills Mismatch to Meet Employer Demand in High-Growth Industries
The Houston region has experienced incredible growth over the last several years. Led by a resurgence in energy, petrochemicals, manufacturing, life sciences, and construction, the region's economy has been widely acclaimed as a city of opportunity and a great place to do business.
While this is all largely true, leaders from across the business community have identified one of the region's most pressing issues that is critical to our continued success: workforce development. We must develop a qualified employee base properly trained for tomorrow's job needs or face an understaffed economy that will stifle the growth and vitality of our region. We must also ensure that young people in Greater Houston have the skills and opportunities to enter the workforce and build successful careers, raise families and prosper.
To address the challenge, the Greater Houston Partnership developed UpSkill Houston, a comprehensive, industry-led approach to bridge the gap and fill jobs in middle-skills occupations.
UpSkill Houston is an innovative blueprint for leaders from the business community, educational institutions and social service organizations to utilize as we lead this effort to build a quality workforce.
A number of reasons for the skills gap have been suggested by researchers investigating the issue. This includes changing skills required due to technological advancements, demographics, policies and priorities, culture and the fact that today's students often choose their field of study based on personal interest, rather than labor market information.
The Houston region's 1.4 million middle skills jobs are spread across 348 occupations. The largest portion of these –188 occupations– fall into the blue collar category, which includes production, transportation, and construction occupations. The 513,660 jobs in these occupations in 2012 represent almost 35 percent of the region's middle skills jobs.
The region's workforce challenge is compounded by the energy boom and the associated investment in the region.
Many of the region's largest manufacturers in terms of employment are connected to the Oil and Gas and Petrochemical sectors, however, within the broad Advanced Manufacturing sector, which includes mechanical, physical or chemical transformation of materials, substances or components into new products, middle skills account for as much as two-thirds of employment in some of the associated subsectors. The bulk of employment in Houston's healthcare industry is also comprised of middle skills workers. In fact, these occupations represent more than one-half of total employment across all the major subsectors for healthcare.
Other key sectors facing the middle skills challenge in the Houston region include ports and maritime, commercial and industrial construction and utilities.
Create sector councils to ensure the critical workforce needs and challenges of each sector are addressed.Strategy 2: Awareness Campaign
Develop and launch an aggressive campaign to change the awareness and perception of middle-skills career opportunities.Strategy 3: Basic Skills & Employability
Encourage the adoption of common tools and curricula to assess individuals and provide training to address weaknesses in basic skills and employability.Strategy 4: Coordination
Facilitate connections between stakeholders and the dissemination of information throughout the system.Strategy 5: Data System
Develop tools to collect better demand-side and supply-side data.Strategy 6: Supply-Side Synchronization
Work with education and training providers to establish networks to enable the efficient dissemination of information, sharing of best practices, and strengthening of industry partnerships.
If you would like to get involved, please contact Peter Beard, Senior Vice President, Regional Workforce.

subset: commoncrawl


text:
Border Patrol agents say that best way to watch the line is by air, but even that asset is wanting here.
The collapse in asset values since 2007 has busted the net worth of the median family all the way back down to early 1990s levels.
With Lehman, the asset quality was much worse and the hole much bigger.
A possession that can be turned into cash to cover liabilities.
Note: Commonly, the term denotes anything of value.


text:
Narrow your search
These $129 headphones from Plantronics promise long battery life, high-end stereo sound, and a flexible design.
Just as rugged as its predecessor, the new AW120 gives you a better lens for shooting underwater or in low-light conditions.
Designed to be mounted like a hanging lamp, the megaphone-shaped Spark is one funky-looking wireless speaker.
Joining the ranks of cylindrical portable Bluetooth speakers, the water-resistant Sony SRS-BTS50 hits stores this month for $130.
This update to one of the toughest point-and-shoots around is now ready to be mounted to your bike, helmet, or car.
Review: Arnie did say he'd be back -- but he's the best thing in this disappointing remix of the 1984 classic.
What?! Best Chromebook deal ever! And it probably won't last long.
Pivothead's pricey Durango video-recording eyewear is a fast, easy way to shoot Web-friendly POV movies.
Technically Incorrect: Building on the principle of evolutionary convergence, an academic says it's likely that aliens will resemble us.
In projectors designed for both the cinema and at home, lasers are starting to replace traditional lamps as the light source of choice. This is potentially awesome. Here's why.


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You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
33 year old man | Cypress, CA, USA | Seeking women 24-31 within 5 miles
If you could have a super power, it would be to ...
Hmm, so what can I say to a complete stranger that clicked on my profile based on one picture, and is now starring at these words on their computer screen clicking through the rest of the profile pictures. So let me start like this ... I will open the door for you and ask you how your day was, and really listen to what you are saying. I say please and thank you, and I can carry myself well in a causal and a business setting. I'm ambitious, driven, and I have big dreams. I know what I want out of life and I'm going for it. I know who I am and what I stand for and I am not scared to speak my mind. All my ducks are in a row, and now I'm looking for a LADY to grow with and share experiences. The most important things in my life are God, family, and business in that order. I have a heart to please God and I live a life of integrity and excellence as I live with passion and purpose to bless others. My family and friends have provided me with my roots, and my association allows me to expand my horizons. From my association, I have learned that the most important thing in life is the ability to connect with others and to listen to what is really being said. I believe that life is about how deep and meaningful of a connection I can create with another person. Connecting is something that comes easy to me. My family and friends would say that I am very outgoing, encouraging, and that I bring a lot of positive energy into a room. I would say that I am a very good listener and that I am not afraid to laugh at myself. If we met, I bet I could make you smile. =) I work in education and on investment projects. I am confident in my abilities and not scared to take calculated risks. Education is a huge part of my life. Not just formal but also informal. In May, I will be completing a doctorate program. I know that in the end, the program will not be about the diploma that I earned, but more about the commitment to the process and the person that I had become in order to reach my goal. Informally, I also mentor and am being mentored by people who have done what I want to accomplish. Life is a team sport, so I believe that it is important to surround myself around people who are not scared to tell me the truth. So what am I looking for? A LADY that is able to hold a meaningful conversation and has a great sense of humor. Someone who is not afraid to laugh at herself and is comfortable in her own skin. She is confident in her abilities, but not arrogant. She is classy and fun. Formal education is NOT as important as having common sense and the ability to carry herself well in public. I am looking for a lady who is able to be professional in a business setting, but can still be down to earth. LIFE is not about how many breathes you take, but how many moments take your breath away. If you enjoy the simple things in life like; laughing, genuine smiles, enjoying another person's company and getting to know how another person ticks. We should chat.
Alumni connections , Coffee and conversation , Business networking , Dining out , Hobbies and crafts , Movies/Videos , Museums and art , Exploring new areas , Nightclubs/Dancing , Performing arts , Playing cards , Playing sports , Political interests , Religion/Spiritual , Shopping/Antiques , Travel/Sightseeing , Volunteering , Watching sports , Wine tasting
Aerobics, Baseball, Basketball, Billiards / Pool, Bowling, Dancing, Other types of exercise, Running, Swimming, Walking / Hiking, Weights / Machines
I exercise 5 or more times per week.
Don't display my sign
I have Cats
My cat is entertaining, fun to watch and play with. She thinks she's a dog. We play fetch and she greets me at the door when I come home. The cutest thing is how she sprints to her carrier every time she hears me shaking a bag of treats.
Some other viewpoint
To tell you the truth, I believe that it is more about who you are with rather than where you are physically located. I would rather be surrounded around positive people in a place with good energy.
University of Southern California, Los Angeles, CA
I enjoy music with a good beat that makes you want to move your feet and dance, as if no one were watching you. I also enjoy being out and about with my family and friends or reading a book.
I'm taking dance classes in ballroom and salsa. It's a great addition to working out in the gym. Exploring new places on eat on YELP is always an adventure. I would say meeting new people, reading and learning new things round out my free time.
I read the Bible daily. Things become clear when the truth is right in front of me. Who knew? I also enjoy reading about personal development because there's always room to improve.
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|5'8" (172cms)
|5'0" (152cms) to 5'7" (170cms)
|Body type:
|Athletic and toned
|Slender, Athletic and toned
|No preference
|Auburn / Red, Black, Light brown, Dark brown, Blonde, Dark blonde
|No Way
|No Way
|Social Drinker
|Never, Social Drinker, Moderately
|Education / Teacher / Professor
|No preference
I work in education and I love what I do. I have invested a lot of time and energy into working with at-risk youth. Everyday is always a new adventure. I believe that I am blessed to have the opportunity to make an impact in the lives of my students.
|No preference
|Never Married
|No preference
|Have kids:
|No preference
|Wants kids:
|Definitely, Someday
|Asian, Latino / Hispanic, Middle Eastern, Native American, Pacific Islander, White / Caucasian
I'm Filipino.
|Christian / Catholic
|Christian / Catholic, Christian / LDS, Christian / Protestant, Jewish, Spiritual but not religious, Christian / Other
I believe that I reap what I sow. So, I live my life to the fullest and without regret. I am positive and I uplift people daily because I may never have that opportunity again. I believe that I can never say the wrong thing to the right person.
|PhD / Post Doctoral
|No preference


text:
News Archive for 2014
Systems Engineering Information Sessions
Cornell Systems Engineering announces fall dates for online information sessions. read more
Cornell Engineering and the Maker Pro Catalyst
Cornell's David Schneider talks about developing student makers within their engineering program. He also discusses the success of the Intel-Cornell Cup, a college-level embedded design competition... read more
O'Rourke elected to Royal Academy of Engineers
CEE's Thomas O'Rourke, the Thomas R. Briggs Professor of Engineering, has been elected to the Royal Academy of Engineering as an International Fellow. read more
Nozick reappointed to nuke waste board
Systems Engineering Professor Linda Nozick has been reappointed to the Nuclear Waste Technical Review Board by President Barack Obama. read more
Jery Stedinger elected to National Academy of Engineering
Jery Stedinger, professor of civil and environmental engineering, has been elected to the National Academy of Engineering. read more


text:
|Thread Tools
Hot wings in the crockpot and chips here tonight, with brownies for dessert. Yeah, junk food fest at our house tonight. :-)
Apartment Farm - the chronicles of my cooking, gardening, crafting and other such things.
Formerly known as "JessicaRenee". Single mama to Jude (Sept '09)!
For superbowl (um, I cannot get out of this in my house, lol), I made about 70 cream cheese stuffed jalapeno peppers wrapped in bacon, pizza stuffed mushrooms, clam stuffed mushrooms (about 30 of each), devils on horseback (SO GOOD), taco platter (layered thing w/cream cheese and sour cream seasoned w/tex mex flavors on bottom, refried beans, guacamole, salsa, lettuce, cheese, olives), and 2 kinds of chicken wings. All was grain free and not one person missed them. We ate leftovers the next night for supper.
The night before superbowl we had collard greens w/smoked pork, baked apples and butternut squash, smoked chicken legs, and oranges.
Last night we had chicken and veggie soup.
Tonight...not sure yet, but maybe breakfast for supper. I have some pork sausage and lots of eggs. Maybe I'll have one of the kids make some pancakes, too. Or I'll do biscuits and sausage gravy..
Happy Homesteading Homeschooling Homebirthing Beekeeping Dready (& a bit redneck even) Mama to 4 fab kids : dd (23), dd (13), ds (11), dd (5)
Last night was spaghetti, garlic bread, and green beans
The beans and sauce were home-canned from our garden. I chopped up the leftover zucchini and onions and smashed the leftover sweet potatoes from the night before and added them to the sauce. It was tasty!
Right now I have chicken broth going in the crock pot. I found a carcass in the freezer and had a bag of veggie trimmings. Tonight I will be Creamy Chicken and Rice Casserole with broccoli on the side.
Tomorrow will be spaghetti pie and some sort of veggie.
Mom to Sam (3/2007), Bekah (4/2009), Jedidiah (4/2012), and Caroline Grace (3/2014)
shrimp tacos with corn tortillas
homemade refried black beans
chopped lettuce, tomato and avocado
carrots and pears for kids
Tonight we're having buttered spaghetti. That is literally all, because we both have some sort of stomach nastiness. And honestly, I don't know if I can even eat that... actually, why am I on this thread tonight? Even when sick and can't tolerate food, I still think about food. :-)
Apartment Farm - the chronicles of my cooking, gardening, crafting and other such things.
Homemade pizza: just sausage on kids' side, sausage, peppers and mushrooms on dh's side (chopped sauteed spinach in the sauce)
Cobb salad for dh and I: mixed greens, baby spinach, chicken breast, bacon, eggs, tomato, avocado, blue cheese, warm vinaigrette
Carrots, celery and pears for kids
Trying something completely new! A spinach and potato dish from a new Spice cookbook I found. Sauteed onions, steamed potato, steamed spinach, chilies and garlic, shaped into a patty and topped with breadcrumb. Then you saute and serve with yogurt/cucumber sauce. Salad on the side.
We had crap from our country store last night. I had chili cheese fries (grain free, lol), and a salad. 2 kids had chili cheese dogs, my oldest dd ate what I did, my son had a chicken strip dinner (and half of my chili cheese fries, the little pig), and my dh had some of my salad, fries, and chicken strips. 2 kids had onion rings.
tonight we're going to try the new Thai place, I think. Then we're done eating out for awhile.
Happy Homesteading Homeschooling Homebirthing Beekeeping Dready (& a bit redneck even) Mama to 4 fab kids : dd (23), dd (13), ds (11), dd (5)
Chili fries and chicken tenders sound soooo good! Even though I still feel junky. Tonight I'm making smoked chicken in the stovetop cooker and buttered noodles. Not sure on the smoke yet - probably hickory. So benign for sensitive stomachs, but still a little flavor.
Apartment Farm - the chronicles of my cooking, gardening, crafting and other such things.
Tonight we are going out for Ruby's with MIL, it's one of her favorite places.
Wednesday: Salmon, roasted German Butterball potatoes, and a salad (my birthday dinner- YUM!).
Thursday: More chili and cornbread with friends, this time with roasted sweet potatoes and a salad.
Tonight: We had burgers, tater tots, and a salad again (oh and some left over sweet potatoes, too).
Tomorrow is farmer's market day.... so, we'll see what looks good!
We had the best chicken curry for lunch, and I just "winged it" so I hope I can make it again sometime. It had apples in it and it came out so deliciously sweet.
For dinner we are having breakfast: french toast casserole, fried apples (we have a lot of apples we need to use up), and bacon.
It's junk food night here, so fish and chips for everyone.
battered halibut
garlic fries
tator tots, pears, carrots and celery for kids
Fish and chips sounds so good! Tonight I'm making Spanish rice and cornbread. I've been needing a meatless meal lately, so it'll be nice and simple.
Apartment Farm - the chronicles of my cooking, gardening, crafting and other such things.
I'm at work. DH is making roast chicken, & cooking cauliflower & we'll have some salad and bread.
We ate at this lovely little restaurant down the street from us called Carnitas Snack Shack. They serve all local food and it's so good!
Tonight Whole wheat spaghetti & meatballs with salad
Mon: Cobb salad & homemade bread
Tue: Venison steak with red wine reduction, homemade gnocci in parsnip cream, and sauteed spinach with garlic...I love making gourmet food at home for cheap!
Wed: Taco-ish soup following this recipe except I use quinoa instead of cous cous & I have pinto beans that I cooked in the freezer to use up instead of kidney beans & homemade stock. Quesadillas & salsa & sour cream to go with it.
Thurs: Pork chops red cabbage & potatoes *if anyone has a good recipe I could use some ideas* I'm thinking of using balsamic in lightly sauteed cabbage with onions & garlic...
Fri: Chicken Cordon Bleu, asparagus and salad
Sat: We won't be home for dinner
We had curry last night- it was incredible- third time in the last week and a half we made it- this time we added broccoli- very very good.
I went to grocery store friday finally!!! I got salmon, tilapia, hamburger, a bag of shrimp, tortellini and stuffed shells and stuff to make french onion soup.
Cincinnati chili here tonight, except I though I had kidney beans but I do not... so I guess we'll just have it without.
Apartment Farm - the chronicles of my cooking, gardening, crafting and other such things.
sauteed kielbasa, red peppers, mushrooms and onions
mashed potatoes
broccoli and apples for kids
Chicken tetrazzini with peas and mushrooms.
MIL gave us 3 rotisserie chickens, we used the first on chicken curry, and we will use the last one on chicken enchiladas.
I loaned a friend some money and she just paid me back so I splurged and ordered a pizza... I can barely open my eyes right now I am so tired- not sure what is wrong with me....
For one- I take the 3 month pill and if I even take it a few hours late I get my period... I cant tell you how many times I have had my period the last month and a half- but its alot- I am going to look into differrent birth control- this is awful. I think it is really making me very tired and grouchy.
We are having a rather "processed" meal: Russian chicken (baked with onion soup mix, apricot jelly & 1000 island dressing), stove-top stuffing (whole-wheat, on sale), and baked acorn squash. It is SOOOO yummy, we won't have leftovers.
Check out my radio blog, Pagan Musings, and my writing (as Selina Wolfcat & Sarah Buhrman).
I'm a head-covering witchy mama to DS ('06) and DD ('10) with DH, Stormie, a Heathen breadwinning daddy.
Spinach and fresh pea soup (the greenest soup ever!), a ham steak, and cornbread. This was soooo good!
When I was pregnant my partner made this recipe for me over and over: http://altoonsultan.blogspot.com/2011/01/sweet-and-sour-red-cabbage.html
It is from a Deborah Maddison cookbook. He would slice pork into really thin pieces and add them near the end, before the vinegar, so they would still be tender. On several occasions we would have this over mashed potatoes. It is one of my favorites!
I have a half ham with the bone I need to use in the freezer, so I'm trying out a new Black Bean Soup recipe, I'm going to double it, start on the stove and finish in the CP:
Ham sandwiches
Carrots, celery and pears for the kids
I think I'll make some chocolate chip cookies, too
Tonight is tater tot casserole and applesauce
Mom to Sam (3/2007), Bekah (4/2009), Jedidiah (4/2012), and Caroline Grace (3/2014)
Tonight: baked chicken taquitos with guacamole and salsa, green salad and strawberries.
We never tried the Thai place yet. We'll see. NO telling if a Thai place in BFE rural Texas is gonna be any good, rofl!
We had grilled sausages (no msg, fillers, etc.)=polish, hot and spicy, and sun dried tomato/basil ones. Plus fresh brussels sprouts cooked in bacon grease and butter w/red onion, and fresh green beans sauteed in coconut oil, garlic, and sea salt. I had orange slices, peeled and dredged in unsweetened coconut to put on the table and forgot.
I stocked up groceries. Yesterday I made a huge batch of hot chocolate mix, a huge batch of "instant" peaches and cream oatmeal mix (used peach leather I found in the cabinet that I dehydrated 2 years ago, sealed in foodsaver bags), and a huge batch of cinnamon brown sugar oatmeal mix. Also made a big noodle stir fry and Asian roasted chicken legs for lunch/snacks. Today I made a big batch of pinto beans and put a bunch in a bowl of leftover rice w/some salsa and spices. My son is about to make a bunch of quesadillas w/that and cheddar. We'll bake them and them freeze them. Also making a batch of chili with pork stew meat and black beans (and some of the pintos) to serve over baked white and sweet potato chunks w/cheese and sour cream. Will have a salad on the side.
Wishing I had more brussels sprouts!
Happy Homesteading Homeschooling Homebirthing Beekeeping Dready (& a bit redneck even) Mama to 4 fab kids : dd (23), dd (13), ds (11), dd (5)
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text:
Waiting in a doorway was a Moroccan man with a handgun and two butcher knives.
There is a reason why I keep a bb gun in my nightstand that could easily be mistaken for a handgun.
As a result, people like Darrell Fuller can obtain anything from a handgun to a semiautomatic assault rifle.


text:
By: David
Filed under: Television Recap
Date: Aug 29, 2013
Impact Wrestling kicks off with The Aces & Eights lead by the president Bully Ray. Bully goes on about what happened to his brother Devon. He get's a word from every member of the Aces of Eights and finalizes his statement by saying Devon has always been a loser and the only man he needs on his side is Tito Ortiz.
Bully Ray wants to go give Tito his own colors until Anderson interrupts him objecting to that motion. Bully responds by saying he is the president of this group and will do what he pleases. He then wants his girl Brooke Tessmacher to come out. A&E music hits the arena and out comes Brooke Tess holding an Aces & Eights jacket. She hands the jacket to Bully Ray to give to Tito.
Bully Ray tells us that he heard his father in law Hulk Hogan is here tonight, and wants to introduce him to the only Brooke that mattered in his life.
Later in the show, Bully Ray, Brooke Tessmacher and Tito Ortiz are backstage. Bully says that he wants Tito to keep his eye on Mr. Anderson.
Hulk Hogan is in the ring and wants to bring out his next order of business, but Bully Ray along with Brooke Tessmacher make their way down to the ramp interrupting Hogan. "Dad! You're back!" says Bully Ray. He tells him a lot has happened since he has been gone. Hogan insists that he has been taking care of business and hands him divorce papers. Bully throws the papers down and says he doesn't care about divorce papers, or his daughter, claiming he just used her to get everything he wanted.
Hulk Hogan wants to give Bully Ray something, which is a title match against one of the Aces and Eights members in two weeks! Also, going to be defending his title next week against Sting! Brooke escapes the ring just in time as Sting and Bully Ray go at it.
By: David
Filed under: Exclusive
Date: Aug 27, 2013
Attention TessNation! Its about that time for another BrookeTess.com exclusive interview with the one and only Brooke Tessmacher! This interview is a little bit different from the last, not focusing so much on wrestling, but getting to know a little more about Brooke! In this interview, you will get to know what Brooke thinks about the latest trending topics such as Miley Cyrus, special diets, fashion icons and what she can't leave her house without! So if you're ready, here we go!
What are your other projects you're currently working on other than TNA Wrestling?
I am working on a huge supplement sponsorship to become the face of the brand. I can't let you in yet on who but looking forward to closing the deal with this wonderful company! Will be pretty exciting.
Growing up, did you ever expect yourself to be where you are currently?
What I have accomplished and the path I chose for my career is definitely not where I saw myself. I wanted to be a veterinarian, nurse, or forensic scientist….. Hummm how did I get here again? Lol.
If people didn't know you, what would you most like them to know about yourself, that someone might not be aware of?
I am deep down very very shy.
Would you consider yourself a sex symbol?
Me a Sex symbol? Of course. I have cashed in my entire career using my looks and cute hiney :).
Lady Gaga, Marilyn Monroe, Kate Moss are just a few of the fashion icons of today, who would you say is your current favorite style icon?
There isn't just one. I love J-lo's style, Beyonce takes risks, and Blake Lively is so classic.
Read the rest of this interview by clicking here!
That isn't all we have for you guys! Be sure to checkout these exclusive shots from one of Brooke's favorite photoshoot she has done! More shots will be released in the coming weeks, but for now enjoy these 4 amazing photos from the photoshoot!
By: David
Filed under: Videos
Date: Aug 23, 2013
By: David
Filed under: Television Recap
Date: Aug 22, 2013
Last week, two bullies joined forces. Now Tito Ortiz and World Heavyweight Champion Bully Ray have come to gloat and celebrate. The new champion rubs his championship win in the face of all the Impact fans. He fooled us all with Tito by his side. The Aces and Eights President turns the microphone over to Ortiz, who calls out Rampage Jackson. As his opponent for the November 2nd Bellator MMA Pay-Per-View, Jackson never stood a chance. The Huntington Beach Bad Boy promises to knock the Main Event Mafia member out every chance he gets until his big grand finale knockout November 2nd.
Bully says that while Tito's attack may have been a surprise, no one is prepared for the one coming next. Everyone knows that Ray has been chatting with Brooke every week. Let's bring her out and tell the world about the secret relationship the champion has been having with Brooke …Hold on. Not so fast.
\It's not Brooke Hogan. It's Brooke Tessmacher. This whole time, the "Brooke" he's been with hasn't been Hogan at all. He got us again. Ray continues.
"Now that I've got the hot Brooke with me, now that I've got the Huntington Beach Bad Boy with me, now that I've got my boy Tazz with me, does anybody doubt that Bully Ray can ever be stopped? There's only one thing left to do…"
With that, the multi-talented Brooke Tessmacher literally sucks Bully's wedding ring from his finger and spits it to the ground. So much for happily ever after.
By: David
Filed under: Exclusive,Photo Update
Date: Aug 7, 2013
View Photos: Knockouts Book Of Lingere > Click Here – Knockouts "Photo Book" > Click Here – ShopTNA.com: Click Here
Miss Tessmacher is featured in the new Special Edition Knockouts "Book Of Lingere". It includes photos from the recent Knockouts photoshoots including some outtakes of never before seen photos of the girls. Be sure to check out these HQ scans with special thanks to Todd J. Parrish for sending these to us!
By: David
Filed under: News Updates
Date: Aug 7, 2013
Brooke Adams / Miss Tessmacher is set to appear for Lucky 13 Promotions on September 21, 2013! Check out what Lucky 13 Promotions tweeted on their official Twitter Account.
Will have @BrookeTess with us Sept 21 Edison NJ @WrestleSpectac. Will have her figure – banner & photos. Will be doing Bikini photo ops.
— Lucky 13 Promotions (@L13Promotions) August 6, 2013
If you are planning on attending the event and would like to donate any photos you take to the site, go ahead and e-mail them to email@example.com! Full credit will be given to you!


text:
Kuala Lumpur: When Siti Nurfadhilah Naluwi from Kuala Lumpur paid for her shopping and dining using her CIMB Bank Debit MasterCard, it never crossed her mind that she would walk away with a brand new Ford Fiesta worth more than RM80,000.
Siti Nurfadhillah, together with other CIMB Bank customers, were recently rewarded at a prize presentation ceremony of the CIMB Bank Debit MasterCard "Get Social" Campaign. The campaign which ran from November 2011 to April 2012, allowed CIMB Bank customers who spent more than RM50 in a single receipt with their debit card the chance of winning fantastic prizes, including two grand prizes of Ford Fiestas. The campaign which ran in two phases, had earlier named Heyati Krystalyna @ Chang Pooi Yew of Johor Bharu as the first phase grand prize winner.
An excited Siti Nurfadhilah, after being chosen as the grand prize winner for the second phase, received the keys to her spanking new Ford Fiesta from Peter England, Head of Retail Financial Services, CIMB Bank in a ceremony held in Petaling Jaya today.
Apart from the grand prize winners, CIMB Bank also rewarded 120 monthly winners with prizes ranging from Samsung Galaxy Tabs (worth RM1,800), Jusco shopping vouchers (worth RM4,000) and holiday vouchers (worth RM6,000).
"This is just one of the many ways we are rewarding our customers. By using the CIMB Bank Debit MasterCard, customers are given exclusive benefits and privileges that allow them to enjoy savings, rebates and discounts, for themselves as well as their friends. We note that there has been a growing awareness among our customers who realise the convenience of carrying a single card when making purchases. Not only does the Debit Card allow customers the option of cashless transactions, it also provides customers the added advantage of allowing them to track their spending," explained Peter.
"It is encouraging to note that more than 500,000 cards have been issued within six months of its launch and an average spending of about RM600 per month, per active card. To drive more card issuance and usage, we will continue to come up with exciting campaigns such as the 'CIMB Loves New York' contest that we are currently running," added Peter.
The 'CIMB Loves New York' contest offers a chance to cardholders to be one of the 10 lucky winners for a 4D/3N trip to New York for two persons with RM10,000 worth of spending money. In addition to that, one of the 10 winners will also be in the running to experience a 'MasterCard Priceless Moments Collection New York City Shopping Celebration Package' for two which entails celebrity treatment all the way – from business class travel, chauffer-driven tours and shopping spree, luxury hotel accommodation and more. For an automatic entry, cardholders must spend at least RM2,000 over the period of 1 May to 31 August 2012 using the debit card, and earn additional entries by increasing their monthly deposit balances of RM500 over the same period. At the same time, cardholders can also look forward to exclusive one-for-one deals and a three percent cash rebate for the first three transactions within thirty days of the card issuance as a start-up offer.
CIMB Islamic also offers the CIMB Islamic Debit MasterCard for its savings and current account holders. For further details on the debit cards, customers may either call 1 300 880 900, log on to www.cimbank.com or www.cimbislamic.com, or visit any CIMB Bank and CIMB Islamic branches nationwide.


text:
Heidi Brooke's Profile
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Africa Smart Safaris Limited
best safari with the best price
Jan 11, 2013 02:44 am
Our safari was well organized with no hassle in Dar Es Salaam for an extremely reasonable price. We were very much looked after and cared for throughout the trip and had no complaints. The rooms/tents were clean and the food was really good.
We saw many animals and thoroughly enjoyed our safari. We would like to thank the staff of Africa Smart Safaris and Sinka court hotel for providing a memorable and fantastic trip.
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June 13th, 2012 #1Registered User
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Cartoonist/Illustrator Daniel Clowes' Work Process and Tools
I figure some here might be interested in this, I think there's interesting things here even if you're not a fan. This is from 'The Comics Journal' in 2003, it's just Daniel Clowes talking through his work process and approach.
Clowes, for those unaware, is a comic artist who is maybe best known for 'Ghost World.' He's also done covers for The New Yorker, the film poster for 'Happiness,' Criterion DVD covers and other such work. At the end are some of my most favourite examples of his work, just for those unfamiliar.
The rest of the pages you can see here, on this Clowes fan Tumblr:
You can also download as a cbz file here:
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- Dis is defined as apart, to refuse, to do or cause the opposite of.
An example of dis is disadvantage, which is an unfavorable situation.
- discount
- distance
- the god of the lower world; Pluto
- the lower world; Hades
Origin of DisClassical Latin contr. ; from dives, rich, translated, translation of Classical Greek Ploutōn, Pluto
- : also used as an intensifier [disannul]
- away, apart: dismiss, disperse
- to deprive of, expel from: disfrock, disbar
- to cause to be the opposite of: disable
- to fail, cease, refuse to: dissatisfy, disappear, disallow
- to do the opposite of: disjoin, disintegrate
- not, the opposite of, un-: dishonest, dissatisfied, displeasing
- opposite of, lack of: disease, disunion
Origin of dis-; from Middle English or Old French or L; Old French des- ; from Classical Latin dis- ; from Indo-European an unverified form dis- (; from an unverified form dwis-, twice, in two ; from base an unverified form dwi-: see bi-) from source Old English te-, Old High German zi-, Gothic dis-
or diss
transitive verbdissed dissed, diss·ing, diss·es Informal
To show disrespect to, often by insult or criticism: "[The network] is often dissed for going after older, less demographically desirable viewers" (Michael McWilliams).
Origin of disAfrican American Vernacular English, short for disrespect.
nounRoman Mythology
- The god of the underworld; Pluto.
- The underworld.
Origin of DisLatin Dīs, from variant of dīves, wealthy (from the belief that the underworld was the source of wealth from the ground); see dyeu- in Indo-European roots.
- Not: dissimilar.
- a. Absence of: disinterest.b. Opposite of: disfavor.
- Undo; do the opposite of: disarrange.
- a. Deprive of: disfranchise.b. Remove: disbud.
- Free from: disintoxicate.
- Used as an intensive: disannul.
Origin of dis-Middle English, from Old French des-, from Latin dis-, apart, asunder.
- plural form of DI
dis- - Computer Definition
Variant of di-
- twice, double, twofold: dichroism, dicotyledon
- Chem. having two atoms, molecules, radicals, etc.: diacid
Also dis-
Origin of di-Classical Greek di- ; from dis-, twice ; from Indo-European an unverified form dwis (from source Classical Latin bis, Middle High German zwis) ; from base an unverified form dwo, two