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Verizon cuts Internet for repeat media pirates
updated 03:25 pm EST, Wed January 20, 2010
Verizon first major to disconnect for piracy
Verizon today admitted that it has disconnected some users whose connections have repeatedly been seen carrying pirated material. The provider's spokeswoman, Bobbi Henson, wouldn't say how many or after how many notices but said Verizon has "cut some people off" in small numbers. It had already been sending notices on a wider level since April and for the RIAA in particular since November.
Henson is quick to add that Verizon doesn't monitor its DSL and fiber optic services itself and that it rarely needs more than a single notice. Often the notice is sent to a parent that doesn't realize one or more of their children is pirating material and has the activity put to an end before a second warning is necessary.
An example message forwarded to CNET also reveals that Verizon still refuses to personally identify customers without a legal order and is only notifying the account associated with a given IP address at the time of the supposedly illegal trading.
Even so, the news contradicts Verizon's previous inclination to avoid cutting off service. Potentially, it also hints at Verizon having reached an agreement with music labels and studios where others haven't in the past. Insiders have noted that the RIAA's tentative deals have been "scarecrow" agreements meant to spook pirates through the press rather than any necessary guarantee of action. Verizon isn't legally required to obey cutoff requests.
Larger providers like Comcast still aren't known to disconnect users but indirectly curb piracy by placing caps or throttling service, discouraging pirates from having an always-open BitTorrent client or downloading particularly large material like HD videos and software.


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Just wanted to say a brief 'hey' - It's taking a lot of effort, but I'm working on getting in some semblance of shape after about 10 years of being mostly inactive. (Some climbing and lifting, but I'm ashamed to admit to basically 0 cardio.) Anyway, I'm already enjoying the fresh air and exercise, and I'm really glad I found this forum - seems like a good group.
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Cysteine peptidases in the two-spotted spider mite Tetranychus urticae are involved in essential physiological processes, including proteolytic digestion. Cystatins and thyropins are inhibitors of cysteine peptidases that modulate their activity, although their function in this species has yet to be investigated. Comparative genomic analyses are powerful tools to obtain advanced knowledge into the presence and evolution of both, peptidases and their inhibitors, and could aid to elucidate issues concerning the function of these proteins.
We have performed a genomic comparative analysis of cysteine peptidases and their inhibitors in T. urticae and representative species of different arthropod taxonomic groups. The results indicate: i) clade-specific proliferations are common to C1A papain-like peptidases and for the I25B cystatin family of inhibitors, whereas the C1A inhibitors thyropins are evolutionarily more conserved among arthropod clades; ii) an unprecedented extensive expansion for C13 legumain-like peptidases is found in T. urticae; iii) a sequence-structure analysis of the spider mite cystatins suggests that diversification may be related to an expansion of their inhibitory range; and iv) an in silico transcriptomic analysis shows that most cathepsin B and L cysteine peptidases, legumains and several members of the cystatin family are expressed at a higher rate in T. urticae feeding stages than in embryos.
Comparative genomics has provided valuable insights on the spider mite cysteine peptidases and their inhibitors. Mite-specific proliferations of C1A and C13 peptidase and I25 cystatin families and their over-expression in feeding stages of mites fit with a putative role in mite's feeding and could have a key role in its broad host feeding range.
Keywords:Comparative genomics; Cystatins; Cysteine peptidases; Feeding; Tetranychus urticae; Thyropins
The two-spotted spider mite, Tetranychus urticae, is one of the main pests of agricultural crops due to its broad host range. This polyphagous species feeds on more than 1,100 plant species, from which about 150 are of great economic value. Thus, it represents a very important pest for field and greenhouse crops, ornamentals, annual and perennial plants all over the world. T. urticae has one of the smallest known animal genomes of about 90Mbp with over 18,000 genes identified. Genome features, such as large expansion of gene families associated with digestion and detoxification of plant secondary compounds are consistent with the spider mite's wide host feeding range [1].
Regarding mite digestive physiology, mites use both extracellular and intracellular digestion, with the latter occurring in gut wall-derived epithelial cells that ingest and digest food particles that can be free floating [2,3]. The midgut is the site for synthesis and secretion of digestive enzymes and absorption of nutrients. Processed food and epithelial cells pass into the posterior midgut, are subsequently compacted in the hindgut and excreted as faecal pellets [3]. Mite species that feed on plants rely mostly on cysteine peptidase activities for the digestion of dietary proteins [4,5].
Cysteine peptidases are enzymes that hydrolyse peptide bonds using a catalytic cysteine. The MEROPS database contains all the existing peptidases grouped in clans [6]. Clans represent one or more families that show evidence of their evolutionary relationship by their similar tertiary structures, or when structures are not available, by the order of catalytic-site residues in the polypeptide chain and often by common sequence motifs around the catalytic residues. At present, among the 72 families of cysteine peptidases identified, 43 are included in 8 clans exclusively formed by cysteine peptidases, 13 are distributed in three clans that comprise peptidases with different catalytic mechanisms, and 16 are not enclosed in any determined clan. Most of the cysteine peptidases characterized in arthropods belong to the papain-like family (C1A), although members of the legumain (C13), calpain (C2), caspase (C14) or separase (C50) families have also been reported.
Arthropod papain-like cysteine peptidases are homologous to mammalian cathepsins, which are present in lysosomes, but can also be localized in extracellular spaces [7]. In mites and insect species belonging to the orders Coleoptera, Hemiptera and Homoptera most C1A peptidases, particularly cathepsins L and B-like, are involved in the digestion process [8-10]. Besides, they are implicated in other physiological processes in arthropods, such as embryogenesis or metamorphosis [11-14]. For T. urticaein vitro assays determined that major protease activity in extracts relies on papain-like cysteine type protease activity [4], which match up with the proliferation of this gene family in the spider mite genome [1]. In addition, a multigene family of legumain genes was also found in the spider mite genome [1], which could have a role in feeding similar to that observed for a legumain peptidase related to the digestive process of the hard tick Ixodes ricinus[15].
The most known inhibitors of C1A and C13 peptidases are the members of the I25 cystatin superfamily. In arthropods, the two ancestral eukaryotic lineages of the cystatin superfamily, stefins (I25A) and cystatins (I25B), have been reported [16]. Stefins are single copy intracellular inhibitors without disulphide bridges. Cystatins undergo frequent duplication events during evolution, and have a signal peptide and one or two disulphide bridges. The inhibitory consensus motif for C1A peptidases is formed by a conserved glycine residue in the N-terminal region, a QxVxG motif in the central region of the polypeptide and a tryptophan in the C-terminal region [17], although proteins with variants in these motives are also active as inhibitors, such as the sialostatins from ticks that lack the conserved tryptophan residue [18]. The inhibitory activity against C13 peptidases is achieved by an asparagine included in the consensus motif S/T-N-D/S-M/I/L based on vertebrate and plant cystatins [19,20], although the ability to inhibit legumains by variants of this motif has not been tested. Some studies have suggested that cystatins from arthropods have functions related to the control of endogenous proteolysis, the balance of host–vector immune relationships, innate immunity, or antimicrobial defence [21-24]. In addition, two other types of cysteine peptidase inhibitors, the propeptide regions of C1A peptidases (I29) and the thyropins (I31) have also been reported in arthropods [25,26]. However, with the exception of the cystatins that contributes to blood feeding in ticks by suppressing host immune response [27], their potential role in the feeding process of arthropods has yet to be investigated.
Comparative genomic analyses provide valuable insights into the conservation and evolution of protein families, which could aid to elucidate issues concerning their function [28]. Thus, we have performed a comparative study of the gene families of cysteine peptidases and their inhibitors in representative species of different taxonomic groups belonging to Arthropoda. This analysis has been focused in those cysteine peptidases potentially involved in T. urticae feeding [4]. Results indicate mite-specific proliferations of C1A and C13 cysteine peptidases and their inhibitors I25B cystatins, as well as a correlation between the in silico expression of specific cystatins with putative digestive cysteine peptidases, providing evidences for their involvement in the feeding process of the spider mite.
C1A and C13 cysteine peptidases and their inhibitors in fully sequenced arthropods
As previously described, a proliferation of both C1A and C13 cysteine peptidases was detected in the genome of the two-spotted spider mite [1]. To obtain further insights on the genomic content of proteins putatively related to the proteolityc digestive process of T. urticae, we extended the search of members from these peptidase families and their inhibitors to other species of arthropods, whose genomes have been completely sequenced and annotated, and drafts of these sequences are available on the web. The selected species were: two acari, T. urticae and Ixodes scapularis (black legged tick); one crustacean, Daphnia pulex (common water flea); and ten insect species, the dipterans Drosophila melanogaster (fruit fly) and Anopheles gambiae (African malaria mosquito), the lepidopteran Bombyx mori (domestic silkworm), the coleopteran Tribolium castaneum (red flour beetle), the hymenopterans Camponotus floridanus (carpenter ant), Apis mellifera (honey bee) and Nasonia vitripennis (jewel wasp), the hemipterans Rhodnius prolixus (kissing bug) and Acyrthosiphon pisum (pea aphid), and the phtirapteran Pediculus humanus corporis (human body louse).
The results obtained from genome extensive searches compared with the location of each species in the phylogenetic tree of arthropods are summarized in Figure 1. All the species have C1A cysteine peptidases, although the highest number of genes was found in T. urticae when compared with all the insect and even the other acari species analysed. Likewise, from the C13 family of peptidases, a strong proliferation of legumains was detected in T. urticae, 19 genes, against a maximum relative to the three genes encoding legumains in the herbivore pea aphid. On the contrary, a unique gene encoding a C13 GPI:protein transamidase was present in all species analysed. Differences in the number of cysteine peptidase genes could be hypothetically correlated with differences in the number of their putative inhibitors. Thus, we look for the gene content of both, the cystatins (I25CPI), which are putative inhibitors of C1A and C13 peptidases, and thyropins (I31Thy), which are putative inhibitors of C1A cysteine peptidases. As for C1A papains, the number of cystatins was considerably higher in T. urticae and in the crustacean D. pulex than in other insect species, whereas the number of thyropins was only slightly higher. Interestingly, I. scapularis, the other chelicerata species used in this study, showed a similar number of both cysteine peptidases and inhibitors than the insect species analyzed. As clade-specific proliferations were previously detected for C1A cysteine peptidases and C13 legumains [1], next analyses were focused to the evolutionary features of cysteine peptidase inhibitors.
Figure 1. Number of cysteine peptidases and their inhibitors in selected arthropod species. Schematic evolutionary tree of fully sequenced arthropods including for each species the number of inhibitory cystatin (I25CPI) and thyropin (I31Thy) domains and the C1A (papain-like, C1APap) and C13 (legumain-like, C13Leg and GPI:protein transamidases, C13GPI) cysteine peptidases, putative targets of both inhibitors. MYA, million years ago.
Gene content evolution of I25 cystatins in arthropods
Arthropod members of the cystatin superfamily belong to the stefin and cystatin lineages. Stefins were not present in the insect species and one stefin encoding gene was found in the genomes of the mite T. urticae and in the crustacean D. pulex. Architectures for proteins containing domains of the cystatin lineage vary among different clades. Three different architectures were detected (Figure 2A). Proteins including a unique cystatin domain were only detected in the annotated genomes of the two species of acari analysed, the crustacean, the dipteran species, and in P. humanus. Multicystatin proteins containing only cystatin domains, whose number range from two to eight, were only present in D. pulex and R. prolixus. In contrast, multicystatin proteins (two to twelve domains) containing an additional C1A cysteine peptidase domain were found in the crustacean D. pulex and in all the insect species analysed, with the exception of R. prolixus. Interestingly, these multicystatin-C1A peptidase proteins were not present in the acari species.
Figure 2. Evolutionary features of cystatins in arthropods. (A) Number of cystatin domains in each species distributed in base of their protein architecture. In brackets the number of repeats in multicystatin domains. CPI, single cystatin domain; MultiCPI, multicystatin domains. (B) Phylogenetic tree using the selected cystatin sequences from the different arthropod species. Coloured triangles show species-specific gene proliferations. In brackets the number of proteins in each subtree.
To understand how the cystatin lineage has evolved in the different arthropod clades, the individual I25B domains were aligned by MUSCLE (see Additional file 1A). Extensive amino acid differences avoid the construction of a robust phylogenetic tree using all the cystatin sequences. Thus, sequences contributing to extensive gaps in the conserved regions of the alignment were discarded and a phylogenetic tree constructed by the maximum likelihood PhyML method (see Additional file 2A). The corresponding schematic cladogram is shown in Figure 2B. As highlighted, clado-specific proliferations are detected, supported by approximate likelihood-ratio test values (aLRT) higher than 80%. This cladogram suggests that the evolution of the cystatin family in arthropods is the result of extensive duplications from the ancestral genes probably determined for specific features in each clade.
Additional file 1. Alignments performed using the MUSCLE program of the amino acid sequences corresponding to the proteins used in this study. (A) Alignment of cystatin domains from selected arthropod species. (B) Alignment of thyropin domains from selected arthropod species. (C) Alignment of T. urticae cystatin domains.
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Additional file 2. Complete phylogenetic trees of the cystatin (A) and thyropins (B) domains from selected arthropod species.
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Gene content evolution of I31 thyropins in arthropods
As for the cystatin family, we performed a phylogenetic analysis of the individual thyropin domains to know how this family has evolved in the different arthropod clades. The I31 domains were aligned by MUSCLE (see Additional file 1B), and a phylogenetic tree was constructed by the maximum likelihood PhyML method (see Additional file 2B), after discarding sequences contributing to extensive gaps in the conserved regions of the alignment. The corresponding schematic cladogram is shown in Figure 3A. A search for additional domains in proteins containing thyropin regions gives a broad combination of different domains (see Additional file 3). The repertoire of additional domains includes the serine peptidase inhibitory domains Kazal 2, Kunitz and Antistasin (Ant), the peptidase related inhibitory domain WAP (whey acidic protein), the calcium binding domain SPARC (secreted protein acidic and rich in cysteine), and the putative GTP-binding domain nuc121. As remarked, groups including thyropin domains associated to a second peptide domain and supported by approximate likelihood-ratio test values (aLRT) around or higher than 80% were found. When we analysed the composition of these groups, we observed that they are composed by proteins belonging to different arthropod species (Figure 3B). Proteins with thyropin domains associated to SPARC, WAP-ATN or Kazal domains are shared by insect, crustacean and acari species. A comparison of the cladograms showed in Figures 2B and 3A implies a different evolutionary process from cystatins and thyropins, with a small weight of clade-specific proliferations in the evolution of thyropins.
Additional file 3. Domain architectures of the proteins with thyropin domains from the selected arthropod species.
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Figure 3. Evolutionary features of thyropins in arthropods. (A) Phylogenetic tree using the selected thyropin sequences from the different arthropod species. Coloured triangles show thyropin gene proliferations associated to other protein domains. In brackets the number of proteins in each subtree. (B) Presence (+) or absence (−) of thyropins members of each subtree in the different arthropod species. WAP, whey acidic protein; ATN, antistasin; SPARC, secreted protein acidic and rich in cysteine.
Structural features of T. urticae cystatins
Since the cystatin lineage had a clade-specific pattern of proliferations, they could be related to the inhibition of the expanded groups of C1A and C13 cysteine peptidases of T. urticae. Thus, we analysed in more detail the sequence-structure relationships in this family of proteins. To find how spider mite cystatins are grouped, their I25B domains were aligned by MUSCLE (see Additional file 1C), and a phylogenetic tree was constructed by the maximum likelihood PhyML method. Additionally, a representation of the alignment was done including the location of the key residues involved in cysteine peptidase inhibition, and of the secondary structures predicted in their tri-dimensional conformation (see Additional file 4). Four different groups were detected, which had specific features in their amino acid sequences (Figure 4A) and a different number of protein members. Whereas groups 1 and 3 consisted of 1 or 2 genes, groups 2 and 4 have been extensively expanded in restricted scaffold regions probably due to recent gene duplication events. Group 1 is formed by TuCPI-1, which is the only T. urticae cystatin sequence that have the consensus motifs for C1A peptidase inhibition: a G in the amino acid part of the protein, the conserved QxVxG in the first loop and a W in the second loop. Besides, it has the consensus motif including the asparagine responsible to legumain inhibition as well as the four cysteine residues involved in disulphide bridges. Group 2 is constituted by cystatins TuCPI-2 to −6, -8, -15, and −18 to −22. Most members of this group retain the asparagine putatively involved in C13 inhibition surrounded by variants of the consensus inhibitory sequence, and all conserved motifs for C1A inhibition with the exception of the tryptophan in the second loop, which is replaced by several different amino acids. In contrast, most of these proteins have a conserved tryptophan residue four amino acids before the expected location of this. Group 3 is composed by TuCPI-7 and −12, and its members are similar to that of the second group, with the new conserved tryptophan residue, but they lack the cysteine residues that form the second disulphide bridge and the asparagine important for legumain inhibition. Finally, group 4 including TuCPI-9 to −11, -13, -14, -16, -17, and −23 to −25, is the most striking clade. The proteins that belong to this group do not present the conserved residues involved in C1A inhibition, neither the G nor the QxVxG or any of the tryptophans in the C-terminal part of the molecule. However, they maintain the four cysteine residues and have several conserved motifs in their sequences, as an YNK motif after the putative α-helix location. Besides, some of its members have an asparagine residue in the location where it is involved in legumain inhibition.
Additional file 4. Alignment of the different four groups of cystatin sequences from T. urticae showing conserved motifs and structural features.
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Figure 4. Evolutionary features of cystatins inT. urticae. (A) Phylogram of T. urticae cystatin sequences showing the presence of functional conserved residues in the four cystatin groups deduced from the phylogenetic tree. Amino acid sequences responsible of the inhibitor-C1A enzyme interaction are shaded in green. Putative sequences involved in C13 legumain inhibition are coloured in purple. Cysteine residues involved in disulphide bridges are in blue. (B) Homology models of T. urticae cystatins created using SWISS-MODEL. The residues in the region that interacts with C1A peptidases are coloured in green. The residues that could be involved in legumain inhibition are coloured in purple. Cysteines are coloured in blue. Red, α-helix; yellow, β-sheets.
To determine how amino acid differences can influence the tri-dimensional structure of the cystatins, the structures of the TuCPI-1, -3, -7, and −13 proteins, representatives of each group, were modelled using the crystallographic structure of the cystatins from chicken egg (1YVBI), soft tick (3L0R), and human cystatin F (2CH9) (Figure 4B). TuCPI-1 and −7 aligned to chicken egg cystatin at sequence identities of 30% and 23%, and with Q-MEAN Z-scores of −3.36 and −2.87, respectively. TuCPI-3 and −13 aligned to soft tick cystatin and human cystatin F at sequence identities of 28% and 22%, and with Q-MEAN Z-scores of −2.32 and −1.76, respectively. These results imply relatively accurate models for T. urticae cystatins. From models, strong differences in the putative region for C1A peptidase inhibition were observed. The consensus amino acid residues in TuCPI-1 fit with a canonical interaction with C1A peptidases. On the contrary, the conserved tryptophan in TuCPI-3 and −7 located at the end of the third β-sheet could be involved in a distinct interaction with these peptidases leading to changes in their inhibitory specificity. In the case of TuCPI-13, the lack of conserved residues in the domain responsible to C1A peptidase inhibition could mean a lack of inhibition to these peptidases. TuCPI-1 and −3 also have an asparagine in the loop after the conserved α-helix that could be involved in legumain inhibition. This asparagine was absent in TuCPI-7 and −13. However, four group cystatins have at least two different conserved motifs (YNK and SKPY) at the spatial region where the C13 inhibitory activity is achieved. A role for the asparagine of the YNK motif in legumain inhibition could be hypothesized, although the conserved motifs could be alternatively related to a different function.
Expression profiling of cysteine peptidases and inhibitors
To correlate genomic proliferations to gene expression, an in silico analysis of transcriptome expression was performed using the RNA-seq information available at the BOGAS T. urticae database. Most genes for cysteine peptidases and inhibitors had transcriptomic data. Only the stefin and seven cystatin genes had not RNA-seq information available. Figure 5A shows the developmental mite stages in which the highest level of expression was detected for each gene analysed. Genes belonging to the C1A cathepsin L and B, C13 legumain and I25 cystatin groups were more expressed in the feeding stages of development, mainly in the adult phase. These genes had a wide expression range, having many genes expression values over 100 and even over 1000, but also lesser than 10 (Figure 5B). Furthermore, the comparison of the expression levels between adults and embryos showed that more than 75% of the cathepsin B-like, cathepsin L-like, legumain and cystatin genes with an expression value higher than 5 were significantly overexpressed in adults (Table 1). On the contrary, genes included in the C1A cathepsin O, C13 GPI:protein transamidase, C2 calpain, C14 caspase, C50 separase and I31 thyropin groups, did not show a specific developmental pattern (Figure 5A), having most of them a similar level of expression in all the mite stages analysed (data not shown), with normalised expression values among 10 and 100 (Figure 5B). Likewise, none of the genes for these families was significantly more expressed in adults than in embryos (Table 1).
Figure 5. Expression profiling of cysteine peptidases and their inhibitors inT. urticae. (A) Number of genes for the different cysteine peptidase and inhibitor groups assigned to the developmental stage (embryo, larvae, nymph and adult) in which their highest expression was detected. (B) Number of genes for the different cysteine peptidase and inhibitor groups assigned to the intervals of normalized expression values (0–10, 10–100, 100–1000, >1000) in which their highest expression was detected, independently of the mite developmental stage analysed. (C) Number of genes for the different cystatin groups (Groups 1 to 4, see Figure 4) assigned to the developmental stage in which their highest expression was detected. (D) Number of genes for the different cystatin groups assigned to the intervals of expression values in which their highest expression was detected, independently of the mite developmental stage analysed.
Table 1. Expression of cysteine peptidases and inhibitors in adults against embryos
A similar analysis was conducted for the four groups of spider mite cystatins. The different groups have specific expression patterns (Figure 5C, D). TuCPI-1, the only protein in group 1, was highly expressed in all developmental stages (data not shown) with its maximum expression in nymphs. Members of group 2 showed the highest expression values and were most abundant in adults and nymphs. Conversely, group 3 cystatins presented the lower level of expression, with one gene mostly expressed in embryos and the other in larvae. Finally, the members of group 4 had, in general, low expression values, but had an interesting developmental pattern, with a weaker expression in adults or embryos than in nymphs.
Cysteine peptidases are crucial in different arthropod physiological processes [11-14], including the digestion of dietary proteins in mites and some insect species [8-10]. The dominant cysteine peptidase activity detected in body extracts of T. urticae points out their main role in proteolytic digestion after feeding [4,5]. In addition, the proliferation of C1A papain and C13 legumain families in the recently annotated genome of T. urticae and changes in expression detected in these peptidase genes after host change suggest their implication in the feeding process and, most probably, on the ability of mites to feed on the large number of plant hosts [1].
The broad multigene family of papain-like cysteine peptidases and the unusual proliferation of legumain peptidases in T. urticae were found to have no counterparts after extending the analysis to the annotated genomes of arthropods available so far, including ten insect species belonging to different orders, the crustacean D. pulex, and the acari I. scapularis. Gene expansions in C1A cysteine peptidases were also found, though in a lower extent, in insects such as the aphid A. pisum and the beetle T. castaneum, which also rely on cysteine peptidases for proteolytic digestion [29,30]. On the contrary, none of the insects analyzed belonging to the orders Diptera (D. melanogaster and A. gambiae), Lepidoptera (B. mori), Hymenoptera (C. floridanusA. mellifera and N. vitripennis) and Phthiraptera (P. humanus), with a digestive system mostly based on serine peptidases [31], presented this extensive proliferation of cysteine peptidases. Hemipterans (R. prolixus) and ticks (I. scapularis), that in addition to cysteine peptidases use serine and/or aspartyl peptidases for proteolytic digestion [31], did not also have an expansion of their cysteine peptidases. In the case of the crustacean D. pulex, in which digestion commonly relies on trypsins and chymotrypsins [32], proliferation of cysteine peptidase genes could be associated to specific adaptations to the lifestyle of a planktonic filter feeder in a highly variable aquatic environment. Thus, we could conclude that, in general, extensive C1A cysteine peptidase duplications in arthropods are correlated to their corresponding diet and could be related to nutritional functions.
Large-scaled gene amplification for legumains is a striking feature restricted to T. urticae. Legumains, also called asparaginyl endopeptidases, have been involved in the degradation of host hemoglobin in ticks [9,33]. A legumain from Ixodes ricinus is located intracellularly in the vacuoles of gut epithelial cells where digestion occur throughout the whole duration of feeding [15], and contributes directly to the cleavage of hemoglobin and/or the processing of other gut peptidase zymogens [34]. In most acariform mites, digestion is considered to be composed of an extracellular phase achieved by enzymes secreted to the lumen of the midgut, followed by an intracellular one that can be performed in free floating cells [2,3,35]. Legumains could be involved in the intracellular phase by processing some other peptidases or by a direct action on the plant feed proteins. Large expansion of this family in T. urticae could be related to the broad range of C1A cysteine peptidases that need to be activated for digestion or to their direct role on host selectivity by processing plant toxic proteins.
Massive gene expression data have been previously used to detect different peptidases in the gut of insects [30,36-38] and ticks [39]. Since digestive proteolytic enzymes should be abundantly generated to deal with the higher volume of food that must be hydrolysed in free living developmental stages, genes involved in the proteolytic digestion are expected to be expressed at higher rate in free leaving stages comparing to embryos. Transcriptomic data analysis in T. urticae indicates that most cathepsin B and L peptidases and legumains are expressed at a higher rate in larvae, nymphs and adults, confirming their putative role in proteolytic digestion. On the contrary, other gene cysteine peptidase families like caspases, separases, calpains and GPI:protein transamidases have similar levels of expression in the four developmental stages analysed, supporting a role for these genes in some other endogenous processes across the whole life of the spider mite.
The activity of this extended number of peptidases must be regulated in the acari. Among cysteine peptidase inhibitors, the cystatin, thyropin and C1A cysteine peptidase propeptide families have been previously described in arthropods. Cysteine peptidase propeptides are inhibitory domains that are included in all the C1A peptidases analysed in this study. They have a conserved role in the control of C1A peptidase activity before the inhibitory domain is released and the peptidase becomes active [40]. The T. urticae genome does not contain small propeptide-like genes similar to those present in Bombyx[22].
Remarkably, the expansion in peptidase genes of the families C1A and C13 in T. urticae is accompanied by a proliferation of cystatin inhibitors putatively targeting both enzyme families. The physiological functions of this extended number of I25 cystatins in T. urticae may be related to their inhibitory activity on specific C1A and C13 peptidases. In insects, cystatins have been associated to processes related to the regulation of endogenous protease activity, such as insect morphogenesis and development [23,41], and/or in the inhibition of heterologous cysteine peptidases, e.g. during insect immune response and plant feeding [21,42]. Moreover, cystatins have been previously related to blood-feeding in ticks [27,43,44], where they are expressed in salivary glands and the midgut contributing to suppress host immune response. The fact that cystatins have evolved differentially in arthropod clades implies a specific function for the members of the proliferated groups in each clade. Thus, besides their potential implications in some other physiological processes, spider mite cystatins could have a role in mite feeding by regulating their own digestive cysteine peptidases, by inhibiting cysteine peptidases of the host after feeding, and/or by contributing to counteract host defence mechanisms. Several results support the putative involvement of cystatins in mite feeding: i) transcriptomic data analysis indicate that group two and four cystatins are consistently detected at greater levels in larvae, nymphs and adults relative to embryos; ii) the amino acid sequences for several spider mite cystatin groups have diverged deeply from consensus cystatin motifs related to cysteine peptidase inhibition. In fact, group 4 cystatin genes code for a new type of cystatins with no known paralogs in living organisms that putatively contain C13 peptidase binding domains, but do not contain canonical C1A peptidase binding domains. These results support an evolutionary scenario in which cystatin family may have evolved in the spider mite to control the proliferation of divergent C1A cysteine peptidases and C13 legumains involved in protein digestion. Alternatively, cystatins in T. urticae may have evolved as specific adaptations to the lifestyle of an extremely polyphagous species to deal with highly variable resources.
On the contrary, limited data on the physiological roles for thyropins are available. Thyropins are thyroglobulin domains capable of exhibiting inhibitory activity, which has been reported mainly against C1A cysteine peptidases [45]. No arthropod thyropins have been characterized to date, although some members are present in the sialotranscriptome of several ticks [46,47]. The possible implication of thyropins in the control of peptidases involved in feeding comes from the reduction of cysteine peptidase activity and deleterious effects on larval growth when the thyropin equistatin isolated from sea anemone Actinia equine was introduced in the diet of the insects T. castaneum and Leptinotarsa decemlineata[48,49]. However, the parallel evolution of thyropins, with complex architectures shared by different arthropod clades, and their transcriptional pattern, having most of them a similar level of expression in adults and embryos, suggest a common conserved role that could be in some cases related to feeding, but no specific for the spider mite nutritional aspects.
Comparative genomic analyses have provided valuable insights into the conservation and evolution of cysteine peptidases and their inhibitors in T. urticae. A phylogenetic analysis of these gene families in representative species of different arthropod taxonomic groups has allowed us to state that clade-specific proliferations are common to C1A papain-like and C13 legumain-like peptidases, as well as to the I25 cystatins, whereas the I31 thyropins are evolutionarily more conserved among arthropod clades. Extensive duplications and transcriptomic data for spider mite C1A and C13 peptidases support their role in proteolytic digestion. The expansion of the I25 cystatin family of inhibitors and their highest expression in feeding stages suggest a role for some cystatin members in mite feeding by regulating endogenous or exogenous peptidases and/or by contributing to counteract host defence mechanisms. In conclusion, mite-specific proliferations of both peptidases and their inhibitors are in accordance with mite's feeding features and support a key role for these proteins in allowing the broad plant host feeding range described for the two-spotted spider mite.
Sequence searches
Blast searches for cystatins, thyropins and cysteine peptidases were performed in publicly available genome databases. Sequences for Tetranychus urticae were obtained at the BOGAS (Bioinformatics Online Genome Annotation System) website [50]. Sequences for other arthopods were identified by searching the current genome releases at: the ant Fourmidable database [51]; the AphidBase [52]; the invertebrate vectors for human pathogens VectorBase [53]; the Daphnia wFleaBase [54]; the FlyBase [55]; the Bombyx mori SilkDB [56]; the BeeBase [57]; the wasp NasoniaBase [58]; and the BeetleBase [59]. Blast searches were made in a recurrent way. First, a complete amino acid arthropod sequence from data banks corresponding to a protein of the family was used. Then, the protein sequences of each arthropod species were used to search in the species. Finally, after an alignment of the proteins found in arthropods, the conserved region surrounding the catalytic sites from the species most related was used to a final search in each arthropod species.
Information about gene models for all these proteins is compiled in Additional file 5.
Additional file 5. Information about gene models and accession numbers corresponding to the proteins (cystatins, thyropins, C1A peptidases and C13 peptidases) used in this study.
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Domain architecture prediction
Amino acid sequences for arthropod proteins putatively including at least one cystatin or thyropin domain were subjected to a sequence search in the Pfam database v 26.0 [60] to know the combination of domains within each protein. From these results, the domain architecture of each protein was manually schematized.
Protein alignments and Phylogenetic trees
Alignments of the amino acid sequences were performed using the default parameters of MUSCLE version 3.8 [61]. Sequences with extensive gaps were manually excluded from phylogenetic analysis using the multiple alignment editor Jalview version 2.7 [62]. Phylogenetic and molecular evolutionary analyses were conducted using the programs PhyML 3.0 and MEGA version 5.0 [63,64]. The program PROTTEST (2.4) was employed for selecting the model of protein evolution that fits better to each alignment according to the corrected Akaike Information Criterion [65]. The parameters of the selected models were employed to reconstruct the displayed clan CD cysteine peptidases trees by means of a maximum likelihood PhyML method using a BIONJ starting tree. The approximate likelihood-ratio test (aLRT) based on a Shimodaira-Hasegawa-like procedure was used as statistical test for non-parametric branch support [66]. All families were also analysed with the Maximum parsimony and the Neighbour-Joining algorithms, and with different gap penalties. No significant differences in the tree topologies were detected.
Molecular modelling of T. urticae cystatins
The three-dimensional structures of the T. urticae cystatins were modelled using the standard automated routine of SWISS-MODEL program [67]. The known crystal structures of the cystatins from chicken egg (PDB identifier 1YVBI), soft tick (3L0R), and human cystatin F (2CH9) were used to construct the homology-based models. The template structures were selected on the basis of highest sequence similarities. Models were evaluated with the QMEAN Z-score for predicting the absolute quality of a model [68]. The Swiss-PdbViewer program [69] was used to generate the single images of protein models.
In silico transcriptome expression
The transcriptomic information available at the BOGAS T. urticae website [50] was used to the developmental expression analyses. The protocol to normalized read counts of RNA-seq Illumina reads has been previously described [1]. To determine significant differences in the levels of gene expression between spider mite embryos and adults, we defined as differentially expressed genes that for which the false discovery rate (FDR) corrected p-value was ≤ 0.05 and for which the fold change was ≥ 2 (either up- or down-regulated).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MES, PH-C, VG and MM carried out the sequence recovery and analysis. ID, FO, MG and MM designed the study and carried out the interpretation of the results. MM drafted the manuscript. All authors read and approved the final manuscript.
The financial support from the Ministerio de Educación y Ciencia (AGL2011-23650) and Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-046) is gratefully acknowledged.
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Paul Sturrock wrote:What configuration do you want to use? You can either use a generator and delegate to something else to create an id or you can define the id as "assigned" and assign it yourself, you can't do both.
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Gary "Mike" Smothers, 58, of Iowa City, passed away on Sunday, April 21, 2013, at Iowa City Rehabilitation and Care Center in Iowa City. Visitation will be held on Friday, April 26, 2013 from 1:00 p.m. - 2:30 p.m. at the Sorden-Lewis Home for Funeral and Cremation Services in Lone Tree. Funeral services for Mike will follow visitation at the funeral home at 2:30 p.m. Rev. Susan Barnes will officiate. Burial will be in the Lone Tree Cemetery. A memorial fund has been established at the funeral home in memory of Mike. Online condolences for the Smothers family may be sent to www.lewisfuneralhomes.com.
Gary Michael Smothers was born on April 27, 1954, in Iowa City, Iowa the son of Gary and Sue (Abrams) Smothers. He was a 1972 graduate of Iowa City High School. In 1974, he was united in marriage to Marilyn Horrabin, and they later divorced. Mike worked for Smothers' Insurance as an insurance agent, and later Evan's Distributing. He enjoyed dancing, listening to country music, talking with friends, and was a hard worker. Mike had a great personality, and loved to laugh.
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Mike was preceded in death by his paternal and maternal grandparents.


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Gerry Lopez opines on "the drop" and takes us behind the scenes on his quest for the ultimate drop, heliboarding in British Columbia.
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By year-end, New York will award millions of dollars for programs that aim to reduce the need for jail beds but Dutchess County isnt in the running.
County criminal-justice leaders in charge of the jail, probation, mental hygiene and the public defenders office said they didnt apply for grant money available for programs that reduce crime, rehabilitate offenders and provide alternatives to detention. The deadline to apply was Monday.
Dutchess is considering whether to build a multimillion-dollar facility to house its overflow of inmates being boarded in other counties jails. In recent county jail studies, programs that reduce incarceration including those that could be funded by the state grant were proposed.
County spokeswoman Colleen Pillus said Dutchess is supporting community agencies that applied for the local assistance grant. The short grant time period made it beneficial for the county to play a collaborative role, she said.
The state Division of Criminal Justice Services wants to fund programs that reduce crime, recidivism and victimization, and limit the use of costly jail and prison beds to those who pose a risk to public safety. The 18-month grant of up to $300,000 has two annual renewal options. It is not a matching grant.
In total, $5 million is available via competitive evaluation of applicants by the state. Objectives include providing counties with more services for jail detainees and sentenced offenders, and targeting communities with a demonstrated need, plus delivering quality therapy and workforce development programs.
While Pillus declined to name which organizations or describe the programs, one agency said it applied: Exodus Transitional Community Inc. of Poughkeepsie.
Suggestions by the state overlap types of initiatives that Dutchess criminal-justice leaders said are needed in the 2012 Needs Assessment report. They include transitional housing, curfew monitoring and case management.
For example, the report on jail crowding suggested, A (mental health) crisis center and other diversion program could be considered when other funding opportunities become available.
New York is prioritizing applicants in communities with many individuals in jail and/or sentenced to prison. Dutchess annual jail population, including inmates housed in other counties, jumped 24 percent from 2002 to 2011.
Mary Ellen Still, who leads the Criminal Justice Council, which authored the report, and heads the county probation department, said a lot of services and programs already address the needs identified in the grant.
Once the county gets a program up and running, sometimes the grant runs out, she said. Depending on the length of the grant, it may benefit the county to partner with an agency rather than hire employees.
Dutchess Jail Administrator George Krom said his department wasnt applying but was interested in partnering with Exodus, an organization that serves parolees returning to the community.
Exodus Associate Director Diana Ortiz said it applied for grant money for the job preparation-training program called Ready Set, Work! which the state touts as one focus in the call for grant applicants. The jail is a partner in its grant proposal in the sense that it will provide classroom space and inmates, she said.
For more than a year, Exodus has worked with state parolees returning to Dutchess thanks to a state grant of $100,000 a year, which barely covers costs, Executive Director Julio Medina said.
My staff 60 percent is formerly incarcerated. We do this work from a different (vantage) point, he said. We have walked that walk.
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Deborah Meaux, LAE President
- Filed Under
Members of the Louisiana Association of Educators believe that Gov. Bobby Jindal's recent executive order has taken the necessary steps to ensure that Louisiana Superintendent of Education John White and the Board of Elementary and Secondary Education follow a transparent, competitive procurement process when obtaining academic assessments for Louisiana students.
Several reports have indicated that White ignored state law when he used a "no-bid" contract to commit Louisiana to using the PARCC assessment.
LAE, like the governor, believes that if BESE and the Louisiana Department ...


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Several eye-catching vehicles, such as this hot rod, will be on display during the Sixth Annual Miller's Swap Meet & Cruise-In, which takes place Saturday at the Ross County Fairgrounds.
- Filed Under
If you're a car enthusiast and looking for a restoration project to tinker around with during the winter season, one local event is offering possibly the last opportunity to do so before the snow starts to fall.
The Sixth Annual Miller's Swap Meet & Cruise-In is set to fill the Ross County Fairgrounds with race cars, rat rods, motorcycles, hot rods, classic cars, go karts and other types of "wheels" Saturday, and several vendors will be selling tools, parts and other merchandise in two large, heated buildings. Gates open at 9 a.m. and close at 4 p.m. for the rain-or-shine event. General ...


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Manitowoc IY-0606W-SPA-310 Water-Cooled 700 LB Half Dice Cube Ice Machine w/ SPA-310 Hotel Dispenser.
Indigo Series Ice Maker, Produces Half Dice Style Ice, 700 LB production/24 hours. 30-inch wide unit has self-contained, water-cooled condenser. 208-230 Volts, 60Hz, 1-phase, 10.7 amps. Paired with 30-inch wide Hotel ice Dispenser with Push Chute Ice Dispensing and Integrated Ice Storage Bin with 180 pound capacity. 115 Volts, 60Hz, 1-Phase. Ice Dispenser includes 6-foot power cord. 82 inches total height.
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November 28, 2015
Athletics, academics, art, and spirit. This week's Grand Valley Power Student of the Week does it all.
Grand Junction High School Senior, A.J. Bush is in several school and community activities including swim, orchestra, Celtic dancing, National Honors Society and Fellowship for Christian Athletes.
A.J. Bush focuses her time to becoming a well–rounded person and helping others do the same. While she has a full schedule, A.J. says she is just doing what she loves.
A.J. says "I like to say that I have just as much free time as any one else and I just chose to use it by being active because that's what I like to do."
A.J. says she loves helping people and spreading the word of God.
She plans to become a pastor and she will also continue playing music through her life and in the church.
While A.J. has been applying to several colleges, she isn't sure where she will end up yet, but she plans to major in Sociology and minor in Music.
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Address: Joe Crowley Student Union, 3rd floor, on the University of Nevada, Reno campus
Phone: 775-682-5626
Hours: 11 a.m. to 8 p.m. Monday through Friday; 11 a.m. to 4 p.m. Saturday
Price range: $5.99 to $11.99; happy hour menu, $3 to $5
Children's menu: No, but will accommodate
On the Web: www.cantinadellobo.com
A generous variety of Mexican dishes from appetizers to desserts, including chingalingas, 11 different burritos, huevos rancheros and fried ice cream.
Mesquite cheeseburgers or chicken sandwiches are offered "for the gringo."
Plenty of tables, bright color scheme and big-screen televisions.
Taco Tuesdays with all-you-can-eat tacos for $7.99 and an invitation to enter the taco-eating challenge to be El Taco Champion.
If you haven't been to the Joe Crowley Student Union on the University of Nevada, Reno campus, then you're missing one of the city's most beautiful -- and functional -- new buildings.
In addition to being a bustling spot for UNR students, faculty and staff, the Joe houses several eateries including, way up on the third floor, Cantina del Lobo.
Because I'm always looking for an excuse to get together with my college freshman, I recently make plans to meet him, his roommate and another friend at the Mexican restaurant for dinner.
Cantina del Lobo is owned by the same folks who own Cantina Los Tres Hombres on Victorian Square in Sparks. The campus menu is similar to the menu at the Sparks location, with the main difference being that no alcohol is served at Cantina del Lobo.
Another difference is that no children's menu is offered, but according to manager Hillary Smith, the staff is happy to accommodate young children with a single taco or other smaller portions.
Most of the time, however, the children who dine at Cantina del Lobo are in the 18- to 22-year-old range with hearty appetites.
Getting sauce-y
We start our dinner with two appetizers: nachos and chingalingas.
The nachos get mixed reviews -- plenty of guacamole, but we aren't crazy about the sauce. On the other hand, the chingalingas -- bite-size pieces of chicken rolled in pastry and deep fried -- are very tasty.
According to the menu, they were once featured in Bon Appétit magazine. Because it's happy hour, the appetizers are half price, a bargain at $4 each.
For dinner, we head in four different directions.
Aaron has arroz con pollo suizas, an unusual choice that he says is delicious but too much to finish. Luke goes for a couple of taquitos, which disappear before I can ask for a bite.
Max says his taco and sopa (Mexican meatball soup) are good, but he could have eaten more.
I'm a little underwhelmed by my legumbres picadas (vegetables sautéed in picado sauce), mostly because I don't like the picado, which we figure is probably the same sauce that's on the nachos.
But I appreciate that fact there are several healthy options like this dish on the menu.
When it's time for dessert, I'm stunned to discover that half our party has never tried fried ice cream, so I insist we split an order. Vanilla ice cream, rolled in All-Bran and coconut, quickly deep fried and topped with whipped cream -- what could be better?
To keep things fun, Cantina del Lobo has daily events.
On Totally Board Mondays, the restaurant serves half-price appetizers and shows snow and skate videos. All-you-can-eat tacos for $7.99 are served up on Tuesdays. According to Smith, Taco Tuesday lunches are the restaurant's busiest time of the week.
Wii Wednesdays also are popular, featuring Wii tournaments, Rock Band and Wii Sports. Open Mic Thursdays and Fajita Fridays (buy one fajita get half off any entrée) round out the week.
Another Friday bonus: Wear a Cantina del Lobo shirt and get free chips and salsa.
Bring along a pocketful of quarters for the meters, and park either in the lot across from Lawlor Events Center or behind Lombardi Recreation Center.


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On paper, The Witch's Sabbath sounds like a promising gem of a film. A coven of four witches must sacrifice 666 souls to their dark lord before the coming of Halloween. In return for their tribute, the witches will retain their immortal lives. In an effort to acquire the sacrifices needed to sate their evil bloodlust, the coven operates a local strip club and subtly lures its patrons to their doom. Did I mention the best part? In order for the ancient evil to consume the souls of the victims, the sacrifices must remain alive while the head is severed from the body.
I know, you're all thinking what I was when I sat down to view this beauty. "Strippers in Service of Satan" that's a magic combination, lets get it on! Well you'd be half right. While there is more than enough blood and boobs to keep your average 13-year old glued to the tube. The film as a whole suffers from some truly indescribable bad acting, most of which is perpetrated by Eli James, who embraces his characterization of Seth like some scary-ass outtake from frat boys gone wild. But, lets forget about the acting for a second, after all this isn't The English Patient. Lets talk about butchery and breasts for just a minute. Like all psychopathic stripper serial killer films these are the three things that should be taken seriously.
Boobs –let's call it two dozen, frankly I lost count. Virtually everyone in the film whips the sisters out for at least a dance or two. Most notably, scream siren Syn DeVil (Is this the best name ever, or what?) gives us all a proper show by highlighting her generous assets in some wonderfully revealing domination duds. In fact, I'm hard pressed to recall even one scene where DeVil keeps her robe on. Hey, I'm not complaining here, and neither were the victims. Well, they weren't complaining before she decapitated them. Come to think of it, they weren't complaining after she decapitated them either. So, that brings us to the blood.
Blood – There are gallons of blood spilt in this film. I mean ridiculous amounts of the old Kayro. I genuinely suspect that all the money the producers saved by hiring frat boy up there was spent acquiring the copious amounts of carnage that we see on the screen. You've got heads ripped right off, spinal cords torn out, even a lesson on the dangers of French kissing your would-be killer. In fact, some of the effects are so good that it almost pains me that this film is not better overall. So in the immortal words of AC/DC, if you want blood…you got it.
Now, before you all run off to the local video store and plunk down your begged or borrowed cash to pick up this flick, I would be remiss if I didn't tell you about the films illustrious cameo. Adult film Godfather Ron Jeremy (if you don't know who this is, I've gotta ask, how old are you?) makes a tragic-comic appearance as a door-to-door bible salesman (Yeah, you read that right). Not only does Ron's bible salesman scare the hell out of me on a whole moral level I never even knew I had, his character doesn't even know the correct words to the "Our Father". Ron also demonstrates true horror film logic by not being sharp enough to get the hell out of the creepy old house when his bible bursts into flames. Needless to say the only head Ron's getting in this film is the one the witches are serving up to Satan.
There is also a bit of a twist ending, which is hinted at a few times during the (I use this term loosely) plot. Unfortunately, the biggest problem is that after the twist, the film never really explains the foreshadowing. Of course that may just be me. After all, by that time I was almost blind from all the T&A. After the film, I took a cold shower and tried to contemplate the best way to describe the violation that I felt while watching this trashy blood feast. Alas, I'm at a near total loss, so I'll leave you with that nefarious task and just simply say, The Witch's Sabbath is a devilishly sordid masterpiece of sin. Whether or not that's a good thing or bad thing is still up in the air.


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As the seasons change, so do our bath and body needs. With a wide variety of comforting fall scents, like apple and pumpkin, and moisturizing skin care products to help your skin make the transition from summer to autumn, there's something for everyone at SJ Creations!
We've compiled a list of our favorite bath and body products for autumn. Check out our suggestions and then leave us a comment with your favorite SJ Creations product for fall!
"I like to moisturize my feet after a hot shower in the evening and slip on a cozy pair of socks! It is very relaxing and keeps my skin nice and soft." – Evencia, National Sales Manager
For an earthy, mild fragrance, olive oil is the way to go. It's both calming and refreshing, the perfect moisturizing body butter to use after a long, hard day to give your skin a quick pick-me-up.
"I love Aloe and Clover hand wash. The scent is nice and clean and the artwork with the butterflies is darling. The brown bottle really dresses up my bathroom to match my bronze accents." – Ashley, Art Director
A fragrant wash to cleanse your hardworking hands while you breathe in the delightful scent of Aloe & Clover. Your hands will look and smell fresh and clean.
"Not only does this apple hand wash smell delicious, but the apple shaped bottle perfectly accents the kitchen. Although it's great year-round, the autumn harvest makes this apple scent extra sweet." – Abby, Social Media
Enjoy the fresh scent of Apple as you cleanse your hands with this Apple Shaped hand wash. The unique shaped design will bring a little fun and color to your bathroom or kitchen!
"My skin is normally very dry, but it gets even worst with the cold weather fall brings. The "fantastic trio," as I like to call it, always leaves my skin feeling fresh and moisturized." – Megan, Graphic and Web Designer
Organic nourishing face serum provides peptides that stimulate skin to help rebuild collagen and aids with anti aging. Organic daytime face moisturizer and rejuvenating eye cream each deliver time release liposomes to enhance moisture penetration.
We've told you our favorites; now you tell us yours! Leave us a blog comment with your favorite SJ Creations product for fall and tell us why you like it, and you could win a $25 gift card to SJ Creations.
Each comment during September will be entered in our blog comment contest and one winner will be selected at the end of the month! Good luck!


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George Imoto, peering through big round glasses, distributed handfuls of salad into more than 40 waiting bowls at the Petaluma People Services Center's Senior Caf? Volunteering there is just the latest way the 87-year-old is helping feed Petaluma seniors.
The Penngrove resident began volunteering with Meals on Wheels shortly after he retired. One day a week for nearly 21 years, he delivered meals to homebound seniors. He stopped only after he fell and injured himself while making deliveries.
Shortly after that, he decided he should no longer drive on account of his age, but he soon resumed volunteering, this time at the Senior Caf? A neighbor and close friend, Mary McCarthy, drives him now.
When asked how long he plans to continue, he said, "Until I can't move."
"He's running circles around me," McCarthy said with a laugh as she watched Imoto add tomato slices to the salads.


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NOTE: this event is PAST. Please contact organizer to
see about their next year's event
Date: March 7, 2013
Time: 5:30 PM
Location: Ferrari Maserati of Vancouver
Ticket Cost: $100
Registration Link: Grape Juice: A Wine Auction benefitting Big Sisters of BC Lower Mainland
Organizing Group
Group Name: Grape Juice
Twitter Account Name: @GrapeJuiceEvent
Event Information
Five years ago, McNeill Nakamoto Recruitment Group and MAC Marketing Solutions created the Grape Juice Wine Auction to raise funds to support our Little Sisters in our mentoring programs. This event has come a long way in a very short time – growing from $12,000 to a whopping $83,000 raised in 2012!
Grape Juice 2013 will held at the swanky Ferrari Maserati of Vancouver where guests will be able to bid on wines from around the world while sampling wines from Marquis Wine Cellars, Laughing Stock Vineyards, and Ricossa, Wines of Piedmont and culinary creations.
Easiest way to donate wine to Grape Juice? Call in your order to Marquis Wine Cellars, pay by phone, and they deliver to Big Sisters! View the Marquis Suggested Wine List here. Please also fill out our donation form below.
Click here for the Grape Juice Auction donation form.
View the Grape Juice Sponsorship Opportunities here or contact Fleur Cooper.
For event updates & photos visit Grape Juice on Twitter and Facebook!


text:
November 29, 2015
WACO, Texas -- A judge has denied Texas' request to prevent the federal Health and Human Services Department from cutting off funding for the Texas Women's Health Program.
U.S. District Judge Walter Smith in Waco on Friday denied the state's motion for a preliminary injunction. Without it, Medicaid funding for the state program that provides cancer screenings and other services to 130,000 low-income women likely will expire Dec. 31.
State lawmakers want Planned Parenthood removed from the program and have banned any clinic affiliated with abortion providers from taking part. Federal officials have responded by denying Medicaid funding for the program.
A state health commission spokeswoman said Friday that an entirely state-funded version of the program will go forward without Planned Parenthood at the start of next year.
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Wedding Dresses near bluffton,ga
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J & A Auto Electric
(513) 732-3965
Hours of Operation
|Mon - Fri:
|9:00am to 6:00pm
Message from the Shop
Hybrids to Cadillac's we service them all. We test and repair using the latest Technology and Equipment. We can upgrade your cars computer with our factory scan tools. If your tired of the "GUESSING" game bring it to us. Our motto is " TEST, DON"T GUESS".
Of course if you don't want to pay for diagnosis we do offer a monkey with darts throwing at a parts chart or you can try your luck with a roll of the diagnosis dice.
Don't want to pay for a tow? We can come to you and diagnose your cars problem. $80.00 plus repair up to 35 miles.ASE Master TechAdvanced Level SpecialistASAHybrid Vehicle and Electric car trainingTech writer for Auto Inc magazine
We currently heat with recycled oils. We are in the process of getting solar panels to generate a portion of our electricity. We have an oil water separator to capture our waste water and remove any oil.
Auto Makes We Service
|Check Engine Light
|Mobile Repair
|Muffler & Exhaust
|Oil Change & Lube
|Roadside Assistance
|Scheduled Maintenance
|Service & Repair
|Timing Belts
|Tire Sales/Repair
|Child Friendly
|Early Bird Dropoff
|Heated Waiting Room


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The Future of Trade Block Monetary Union
This article is about the analyzing of the future of trade block monetary union focusing mainly on NAFTA, ASEAN, and EU. http://vidshare.vidzy.me/?videoId=kl1ujzRidmU http://vidshare.vidzy.me/?videoId=Py_IndUbcxc http://vidshare.vidzy.me/?videoId=okJL4×8NWrs http://vidshare.vidzy.me/?videoId=cCgrEhIovaM http://vidshare.vidzy.me/?videoId=LiwSUxszuN0 http://vidshare.vidzy.me/?videoId=gG_dA32oH44 http://vidshare.vidzy.me/?videoId=4TPwN4l7YvA http://vidshare.vidzy.me/?videoId=zv3bYWuUUng [:D]
The Future Of Trade Block Monetary Unions
Khath Ourng
Jones International University
The Gold Standard Online University
Course Title: Leading from Global Perspective
Course Number: BC-607
Professor: Dr. Miro Smriga
Theme: The World IS Flat
The world is flat (Friedman, 2005). Technology and the internet, economy, politics, war, people immigrating, intelligent draining, and environmental concern has mobilized and integrated the world to be flattened known as the global village. Laptop and desktop and smart phone such as iPhone, iPod, iPad, and Android combined with the internet have eliminated distance and shorten time to allow people from the corner of the world to communicate face-to-face via Skype in no time. Poor economy, politics, and war in certain areas have been the main issue for people immigrating. The economy of the country cannot support people living standard. The reasons can be the land fails to produce food for people's consumption. The people are not fitted with the politics of the country. The leader might be so powerful dominating the nations.
The civil war or the war between nations might force the people to be homeless, cannot live within the country for many reasons: food, clothes, shelter, and life threatening. Uneven intelligent has forced employers to seek intellectual employees from other countries in two ways: bring the intelligent employees into the countries or outsource the intellectual works to where the employees live. Finally, the high concern for the global warming has motivated people to think and act globally. They mobilize and build a coalition with other nations to take care of their world. These elements are the sources of integrating the global village.
Definition: What is globalization? Globalization is "the growing liberalization of international trade and investment, and the resulting increase in the integration of national economies" (Griswold, 2000). Multi-national corporations, where they sign the contract for fair trade, free marketing, tax-less trading of goods and services and so forth.
List of regional coalitions or organizations
NAFTA – North American Free Trade Agreement (Canada, Mexico, and America) (Wikipedia & Ask.com, 2012)
ASEAN – Association of South-East Asian Nations (Philippines, Brunei, Cambodia, Indonesia, Lao, Myanmar, Singapore, Thailand, and Vietnam) (ASEAN Secretariat, 2009)
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I agree with state Sen. joe Bolkcom on huge tax giveaways, citing Gov. Terry Branstad and Orascom. I hope Bolkcom felt the same about Obama giveaways to Solyndra and many other "green" projects that went bankrupt while sticking taxpayers with the loss instead of the private investors.
When Obama does it, it's an "investment", when Branstad does it, it is a giveaway.
I don't recall a Bolkcom column on Obama waste on half-billion dollar escapades. At least Orascom has not yet gone bankrupt.
Palmer Holden
Iowa City