text:
- Research article
- Open Access
Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders
© The Author(s). 2017
- Received: 24 August 2016
- Accepted: 24 February 2017
- Published: 14 March 2017
Orb-web weaving spiders and their relatives use multiple types of task-specific silks. The majority of spider silk studies have focused on the ultra-tough dragline silk synthesized in major ampullate glands, but other silk types have impressive material properties. For instance, minor ampullate silks of orb-web weaving spiders are as tough as draglines, due to their higher extensibility despite lower strength. Differences in material properties between silk types result from differences in their component proteins, particularly members of the spidroin (spider fibroin) gene family. However, the extent to which variation in material properties within a single silk type can be explained by variation in spidroin sequences is unknown. Here, we compare the minor ampullate spidroins (MiSp) of orb-weavers and cobweb weavers. Orb-web weavers use minor ampullate silk to form the auxiliary spiral of the orb-web while cobweb weavers use it to wrap prey, suggesting that selection pressures on minor ampullate spidroins (MiSp) may differ between the two groups.
We report complete or nearly complete MiSp sequences from five cobweb weaving spider species and measure material properties of minor ampullate silks in a subset of these species. We also compare MiSp sequences and silk properties of our cobweb weavers to published data for orb-web weavers. We demonstrate that all our cobweb weavers possess multiple MiSp loci and that one locus is more highly expressed in at least two species. We also find that the proportion of β-spiral-forming amino acid motifs in MiSp positively correlates with minor ampullate silk extensibility across orb-web and cobweb weavers.
MiSp sequences vary dramatically within and among spider species, and have likely been subject to multiple rounds of gene duplication and concerted evolution, which have contributed to the diverse material properties of minor ampullate silks. Our sequences also provide templates for recombinant silk proteins with tailored properties.
- Theridiidae
- Latrodectus
- Steatoda
- Silk glands
- Gene expression
- Silk proteins
- Spidroin gene family
Major ampullate silk has been the primary focus of silk research because it is relatively easy to harvest and has tensile strength rivaling that of steel [6, 7]. However, all silk fibers have impressive and unique material properties [8]. For instance, minor ampullate silk of the orb-web weavers Argiope trifasciata, Argiope argentata, and Nephila clavipes have higher extensibility and lower strength than major ampullate silks from the same species, yet are equally tough [8–12]. Additionally, minor and major ampullate silks display different physical properties when wet. Supercontraction in water occurs with A. trifasciata and Nephila inaurata major ampullate silks, which allows them to become more extensible while maintaining their high strength [13]. However, when minor ampullate silks from the same species are exposed to water, supercontraction does not occur and the mechanical properties do not change [13]. Despite having consistent behaviors within species whether wet or dry, mechanical properties of minor ampullate silks varied more among species than did the properties of major ampullate silks [14, 15]. The consistency of minor ampullate silks' mechanical properties when wet or dry, as well as their increased extensibility relative to draglines and greater variability among species, could allow for different applications of minor ampullate silk that would not be feasible for major ampullate silk. The material properties of minor ampullate silks made by cobweb weavers have yet to be measured, but given the different functions of this silk in cobweb weavers compared to in orb-web weavers, we predict divergence in the mechanical performance of these fibers.
The variation in physical properties between major and minor ampullate silks and among minor ampullate silks of different species likely results from sequence differences among their component proteins, termed spidroins (spider fibroins). Spidroins are extremely large (>200 kDa) and are made almost entirely of a highly repetitive region, which is flanked by short, conserved carboxy (C)- and amino (N)-terminal regions [16–21]. The repetitive region of many spidroins contain numerous subrepeats of short amino acid sequences, called motifs [2]. Motifs found frequently in spidroin repetitive regions include GGX (where X refers to a subset of amino acids), (GA)n where n ≥ 2, An where n ≥ 4, and GPG [2, 18]. An and (GA)n form β-sheets, GGX forms 31-helices, and GPG forms β-spirals [2, 14, 18, 22–26]. Secondary structures of β-sheets likely confer strength, and β-spirals contribute to elasticity [18].
Major ampullate silks of all araneoid spiders examined thus far are made up of two proteins, MaSp1 and MaSp2, which are distinguished by a high GPG content in the latter but not the former [27, 28]. Similarly, N. clavipes minor ampullate silk is made up of two proteins, MiSp1 and MiSp2, although they are not as distinctly different from each other as MaSp1 and MaSp2 [29]. All described minor ampullate spidroins (generalized as MiSp) have consensus repeats that are primarily made up of An, (GA)n and GGX motifs disrupted by serine and threonine-rich "spacer" regions [2, 14, 18, 29, 30]. The functions of the spacer regions are not well understood, but artificially made recombinant proteins composed solely of spacer regions tend to form oligomers and only recombinant MiSp proteins that include at least one spacer, repetitive region, and the C-terminal domain can form fibers [31]. Both MaSp1 and MiSp are predicted to be composed of β-sheets connected by helical regions and amorphous chains [2, 14, 18]. The higher tensile strength of major ampullate silk may result from longer β-sheets made up of An repeats, in contrast to shorter β-sheets made up of An and (GA)n repeats in minor ampullate silk [14].
The large size and repetitiveness of spidroins make spidroin-encoding genes extremely difficult to sequence completely. The only full-length sequence of MiSp known is for Araneus ventricosus [30]. Furthermore, even partial MiSp sequences have been characterized from only a few orb-web weavers [29, 30]. The full length sequence shows that MiSp repeat units are not as similar to each other as are the repeats of MaSp1 or MaSp2 repeats; and that A. ventricosus MiSp possesses exactly two spacers that interrupt the glycine and alanine rich motifs [30]. The generality of these features for other MiSp-encoding sequences needs to be tested.
The only known sequences of MiSp in a cobweb weaver are partial sequences from Latrodectus hesperus [5, 32]. The known repetitive regions are similar between L. hesperus and orb-web weavers' MiSp, with the main difference being length of spacers [30, 32]. However, because cobweb weavers use minor ampullate silks for prey wrapping, while orb-web weavers use them in the auxiliary spiral of their web [5], we predict different selective pressures on cobweb weaving MiSp that could consequently alter secondary structure and physical properties.
Here, we characterize complete or nearly complete MiSp sequences from five cobweb weaving spider species: L. hesperus, L. tredecimguttatus, L. geometricus, Steatoda grossa, and Parasteatoda tepidariorum (Theridiidae). We also measured mechanical properties of minor ampullate silks in L. hesperus, L. geometricus, and S. grossa. Our goals were to determine the extent of variation in MiSp sequences and minor ampullate silk properties within cobweb weaving spiders, compare this variation to that found in orb-web weaving spiders, and identify any relationship between sequence and material property variation across both groups. We found evidence for at least two MiSp loci in each cobweb weaving species, and that one copy is more highly expressed than the other in minor ampullate glands in L. hesperus and L. geometricus. Phylogenetic analyses suggested extensive gene duplication and concerted evolution of MiSp-encoding loci contributed to variation in MiSp sequences. Finally, we found significant differences in mechanical properties among minor ampullate silks that were partially explained by variation in MiSp sequences.
Multiple loci encode MiSp in cobweb weavers
We employed a series of molecular experiments to identify MiSp-encoding sequences in our five species of cobweb weavers. Additional file 1: Figure S1 provides an overview of these various methods.
L. hesperus
Spidroins included in phylogenetic analyses of N- and C-terminal sequences
Spidroin name
N-term accession
C-term accession
Bc fibroin1
Bothriocyrtum californicum
Kh MaSp1
Kukulcania hibernalis
Dc MaSp
Diguetia canities
Dc MaSp-like
Diguetia canities
Ds MaSp2
Deinopis spinosa
DQ399328, DQ399329a
Aap MaSp
Agelenopsis aperta
Ud MiSp
Uloborus diversus
Mg MiSp
Metepeira grandiosa
Lh MiSp
Latrodectus hesperus
Aap TuSp1
Agelenopsis aperta
Aa TuSp1
Argiope argentata
Ab TuSp1
Argiope bruennichi
Nct TuSp1
Nephila clavata
Lh TuSp1
Latrodectus hesperus
At MaSp2
Argiope trifasciata
Nc MaSp2
Nephila clavipes
Ni MaSp2
Nephila inaurata madagascariensis
Nc MaSp1a
Nephila clavipes
Nc MaSp1b
Nephila clavipes
Lh MaSp1
Latrodectus hesperus
Lh MaSp2
Latrodectus hesperus
Lg MaSp1
Latrodectus geometricus
Ea MaSp
Euprosthenops australis
Nc Flag
Nephila clavipes
Ni Flag
Nephila inaurata madagascariensis
Av Flag
Araneus ventricosus
Lg MaSp2
Latrodectus geometricus
Ds fibroin1a
Deinopis spinosa
Lh AcSp1
Latrodectus hesperus
Lg AcSp1
Latrodectus geometricus
Lh MiSp_v1
Latrodectus hesperus
Lh MiSp_v2
Latrodectus hesperus
Lg MiSp_v1
Latrodectus geometricus
Lg MiSp_v2
Latrodectus geometricus
Lt MiSp_v1
Latrodectus tredecimguttatus
Lt MiSp_v2
Latrodectus tredecimguttatus
Sg MiSp_pseudo
Steatoda grossa
Sg MiSp
Steatoda grossa
Pt MiSp
Parasteatoda tepidariorum
Ncr PySp
Nephilengys cruentata
Nc PySp
Nephila clavipes
Nc PySp2
Nephila clavipes
Lh PySp
Latrodectus hesperus
Ud AcSp1
Uloborus diversus
Na MiSp
Nephila antipodiana
Nc MiSp1
Nephila clavipes
Ncr MiSp
Nephilengys cruentata
Ds MiSp
Deinopis spinosa
Ad MiSp
Araneus diadematus
Av MiSp
Araneus ventricosus
Pb MiSp
Parawixia bistriata
Ad MaSp1
Araneus diadematus
Ad MaSp2a
Araneus diadematus
Ad MaSp2b
Araneus diadematus
Ncr MaSp_like
Nephilengys cruentata
Na MaSp1
Nephila antipodiana
Ud MaSp1
Uloborus diversus
Ud MaSp2a
Uloborus diversus
Ud MaSp2b
Uloborus diversus
Ncr Flag
Nephilengys cruentata
Ncr TuSp
Nephilengys cruentata
Ds TuSp1
Deinopis spinosa
Ud TuSp1
Uloborus diversus
Na TuSp1
Nephila antipodiana
Primer sets designed to be specific to Lh MiSp_v1 versus Lh MiSp_v2 successfully amplified genomic DNA of three single individuals. Variant-specific PCR products were 99.3% identical to the targeted variant, but only 86.3% identical to each other on average at the 616 bp C-terminal/ 3'UTR region. Sequence chromatograms of Lh MiSp_v1 and MiSp_v2 amplifications exhibited double peaks in both directions in at least one individual (10 double peaks in one of three individuals amplified with Lh MiSp_v1 specific primers and one double peak in all three individuals amplified with Lh MiSp_v2 specific primers). The pattern of double peaks in one individual but not others for the Lh MiSp_v1 specific PCR product is consistent with allelic variation for Lh MiSp_v1 and confirms that Lh MiSp_v1 and Lh MiSp_v2 represent separate genomic loci.
L. tredecimguttatus and L. geometricus
Because these species are diploid, we expect no more than two alleles per locus within a single individual. Thus each species may possess as many as six loci encoding MiSp (11 variants identified in each species). However, because some PCR or cloning error could produce slight variants among sequences (despite the use of a proofreading enzyme) we conservatively considered the two distinct clusters to represent separate loci and completely sequenced one representative from each cluster (Fig. 2). We designated the completely sequenced clones from L. tredecimguttatus as Lt MiSp_v1 and Lt MiSp_v2, and the completely sequenced clones from L. geometricus as Lg MiSp_v1 and Lg MiSp_v2. Designation of MiSp variants with 1 or 2 is not intended to imply orthology among species, but simply reflects different genomic loci within species.
S. grossa
TOPO cloning and end sequencing of an ~5000 bp S. grossa PCR product that was generated with primers targeting complete MiSp resulted in ten identical clones from a single individual. When translated, these ~800 bp end sequences aligned well with our Latrodectus MiSp sequences, but contained numerous stop codons. This variant is likely a non-functional pseudogene, designated Sg MiSp_pseudo.
PCR amplification using primers designed from our ~3 kb C-terminal encoding S. grossa MiSp cDNA (designated Sg MiSp) failed to find any further variants. In contrast, TOPO cloning and sequencing of 20 N-terminal and adjacent repetitive MiSp encoding PCR products for a single S. grossa individual resulted in 20 unique sequences, each different from our ~500 bp N-terminal encoding cDNA. The clones (designated by clone numbers) grouped into three clusters (Additional file 1: Figure S3); within a cluster pairwise differences among sequences were <0.9% on average (0.13–1.5%) while between clusters pairwise differences were much greater (average pairwise distance = 10%). Nucleotide differences within a cluster could reflect alleles or multiple loci, but could also be attributable to PCR error because amplification was performed with Taq polymerase alone, which does not have proofreading capability, in contrast to the high fidelity polymerases used to amplify the Latrodectus species (see Methods). Conservatively, the three clusters represent three true variants. Thus there is a minimum of two loci that encode MiSp in S. grossa.
P. tepidariorum
We found partial length N and C-terminal MiSp-encoding cDNAs from P. tepidariorum (designated Pt MiSp, see Table 1 for accessions) that were nearly identical (>98%) to the 5' and 3' ends, respectively, of an Augustus-predicted gene model on Scaffold 853 in the publicly available genome. This gene model was predicted to span 10292 bp, but included four gaps (4353 bp of assembled sequence, 5939 bp of unknown). Characterization of a TOPO clone containing an ~2.3 kb PCR product for P. tepidariorum MiSp (KX584004) resulted in a sequence that was 100% identical to the 104 bp of overlap with the C-terminal encoding region of Scaffold 853 and filled 2236 bp of the last gap. We refer to the combination of our TOPO clone with Scaffold 853 as Pt MiSp variant 1 (Pt MiSp_v1). The fully sequenced TOPO clone included a 1175 bp long intron with canonical donor and acceptor sites identified by aligning with our C-terminal encoding cDNA using SPIDEY [33]. We also found BLASTN alignments of this 2.3 kb TOPO clone to three other scaffolds (954, 3280, and 3758) that may represent additional MiSp-encoding loci. The C-terminal encoding region of our TOPO clone aligned with 91% identity on Scaffold 954. The intron sequence aligned with 95% identity to Scaffold 3280 and 99% identity to Scaffold 3758. The repeat region flanking the intron (including 100 bases of intron) aligned with 89% identity to Scaffold 3280 and 90% identity to Scaffold 3758. The presence of two C-terminal encoding sequences that differ by 9% and are placed on separate scaffolds suggests that at least two loci encode MiSp in P. tepidariorum.
MiSp loci are expressed at different rates in cobweb weaving spiders
MiSp transcript abundance based on read counts in minor ampullate, major ampullate, and total silk gland RNA-seq libraries
L. geometricus
L. hesperus
RNA-seq Library
Total readsa
Ratio 3'd
Total readsb
Ratio 3'd
Minor 1
Minor 2
Total Silk
Major 1
Major 2
Variable genomic structure of MiSp
All MiSp genes characterized from Latrodectus lacked introns interrupting coding sequence. While only L. hesperus MiSp_v1 included both the start and stop codons, the other MiSp sequences missed no more than 300 bp of the N or C-terminal coding regions on either end. Our P. tepidariorum MiSp genomic TOPO clone that includes the C-terminal encoding region has an intron of 1175 bp. For comparison, orb-weaver A. ventricosus MiSp has a large, single intron of 5628 bp [30].
Inspection of the L. hesperus fosmid genomic clone revealed a TATA box approximately 50 bases upstream of the full-length L. hesperus MiSp coding region. The nucleotide motif CACG found in all previously characterized spidroin genes was also found in L. hesperus MiSp, 11 bases upstream of the TATA box. The P. tepidariorum sequence on Scaffold 853 in the i5k genome possessed a TATA box 63 bases upstream of the MiSp coding region and a CACG motif 10 bases upstream of the TATA box. The A. ventricosus sequence has a TATA box 63 bases upstream of the full-length MiSp coding region, and a CACG motif 30 bases upstream of the TATA box [30]. Polyadenylation signals were found 110 bases downstream of the stop codon for L. hesperus MiSp_v1, 242 bases downstream of the stop for P. tepidariorum MiSp_v1, and 82 bases downstream of the stop for A. ventricosus MiSp [30].
MiSp amino acid variation within and among species
Cobweb weaver MiSp length is highly variable within and among species (815 aa to 2751 aa, Additional file 1: Figure S4). For comparison, the completely sequenced L. hesperus MaSp1 is 3129 aa and MaSp2 is 3779 aa, and A. ventricosus MiSp is 1,766 aa [30]. Glycine, alanine, and serine are the three most abundant amino acids for all MiSp variants, showing similar composition percentages to L. hesperus MaSp1 and MaSp2. Serine is found in both the spacer regions and the repeat regions, while glycine and alanine are predominantly found in the repeat region only. Proline is found in very low abundance for both L. hesperus MiSp variants, both L. tredecimguttatus MiSp variants, L. geometricus MiSp_v2, and P. tepidariorum MiSp_v1, but is more abundant in L. geometricus MiSp_v1 and is the fourth most abundant amino acid in S. grossa MiSp, surpassing L. hesperus MaSp2 proline composition (Additional file 1: Figure S4). Codon usage is variable among MiSp loci and species, but there is an overall bias toward A or T in the third position of the codon, especially for alanine and glycine (Additional file 1: Table S1).
Material properties of minor ampullate spidroins correlate with MiSp sequence variation
Properties of minor ampullate silk for cobweb and orb-web weaving species
Diameter (μm)
Young's Modulus (GPa)
Strength (MPa)
Extensibility (ln mm/mm)
Toughness (J/cm3)
Latrodectus hesperus
1.12 ± 0.47
3.9 ± 2.9
245.4 ± 120.5
0.57 ± 0.02
66.7 ± 46.4
0.07 g
Latrodectus geometricus
1.09 ± 0.29
2.6 ± 1.3
174.4 ± 17.6
0.54 ± 0.02
43.6 ± 20.1
3.48 h
Steatoda grossa
1.07 ± 0.26
2.1 ± 0.7
251.3 ± 106.2
0.74 ± 0.02
57.3 ± 20.7
12.79 i
Nephila clavipes a
2.5 ± 0.2
3.0 ± 0.6
346 ± 53
0.30 ± 0.15
0 j
Nephila clavipes b
960 ± 50
0.25 ± 0.03
0 j
Nephila inaurata c
1.8 ± 0.1
11.2 ± 0.7
1500 ± 200
0.46 ± 0.05
300 ± 50
Argiope trifasciata d,e
0.69 ± 0.10
8.9 ± 0.05
751.5 ± 40
0.44 ± 0.04
150 ± 12
Argiope trifasciata c
1.8 ± 0.1
10 ± 0.4
1040 ± 6
0.45 ± 0.02
240 ± 20
Argiope argentata e
1.1 ± 0.7
10.6 ± 1.2
923 ± 154
0.33 ± 0.03
137 ± 22
Araneus diadematus f
0 k
Araneus gemmoides b
1400 ± 100
0.22 ± 0.07
Phylogenetic relationships of spidroins among orb-web and cobweb weaving species suggest multiple duplication events
Our characterization of complete or almost complete encoding sequences for the minor ampullate spidroin (MiSp) in multiple cobweb weaving spider species (Theridiidae) demonstrate the presence of at least two MiSp loci in each species. The substantial variation of MiSp sequences within and among species and its relationship to variation in material properties has multiple implications for molecular evolution, spider ecology, and biomimetic applications through recombinant DNA technology.
Two MiSp-encoding loci have also been documented in the golden orb-weaver spider, Nephila clavipes (Nephilidae), and therefore the presence of multiple MiSp-encoding loci in spider genomes most likely dates minimally back to the common ancestor of theridiids and nephilids (e.g. Araneoidea). Our reconciliation of gene trees with species trees suggests even older duplication events (Fig. 7). However, the maintenance of two loci appears to involve complex molecular evolutionary processes including intergenic concerted evolution of the loci within species, and multiple gains and losses of individual MiSp copies. Although our reconciliation analyses inferred independent duplication events within each of our cobweb weaving species, the grouping of MiSp loci within species based on the terminal domains (Figs. 6 and 7), could also result from intergenic concerted evolution of the N- and C-terminal encoding regions. We favor the latter hypothesis because of the dramatic differences in the repetitive region within some species (Fig. 3). Concerted evolution of the N and C-terminal encoding regions could occur through non-homologous recombination between the loci, facilitated by their similar sequences, as proposed for the major ampullate spidroin paralogs, MaSp1 and MaSp2, of multiple species [2, 32, 41–43]. Within theridiids, the rate of concerted evolution appears to be faster than speciation, since relationships among each species' pair of MiSp loci reflect species relationships (Figs. 6 and 7). Intergenic concerted evolution of terminal-encoding regions could be favored by selection because these regions are involved in assembly of multiple MiSp monomers into polymers and the conversion of the protein complex from a liquid to a solid [31, 44]. Highly similar terminal regions may be necessary for polymers to form.
Outside of theridiids, relationships among MiSp N and C-terminal domains are not congruent with species relationships. The low posterior probabilities and bootstrap support for these incongruous relationships suggest there is limited phylogenetic signal retained in the MiSp C-termini at the distant time scale of divergence of theridiids from other araneoid families (~170 million years ago, [36, 38, 39]). However, the consistent grouping of Nephila and theridiid MiSp C-terminal domains to the exclusion of araneid MiSp C-terminal domains suggests that nephilid and theridiid MiSp sequences are derived from a different ancient copy than the araneid MiSp sequences, as supported by our reconciliation analyses (Figs. 6 and 7). The loss of functional MiSp loci through multiple single nucleotide mutations is further supported by the presence of a MiSp pseudogene in the S. grossa genome. Many additional losses were inferred by our reconciliation analyses (Additional file 1: Figure S8), but because of incomplete spidroin sampling for most species we do not feel confident in estimating the extent of spidroin gene loss.
We found extensive variation in the MiSp repetitive regions between loci within a genome and among species (Fig. 3). MiSp variants in L. hesperus and L. tredecimguttatus, L. geometricus MiSp_v2, P. tepidariorum MiSp_v1, Nephila MiSp 1 & 2, and araneid MiSp sequences are similar in terms of amino acid motif composition. S. grossa MiSp and L. geometricus MiSp_v1 are especially divergent, with the difference between variants within L. geometricus being extremely striking (Fig. 3). The divergent repeats have an increased proline content, which likely occurred independently in S. grossa MiSp and L. geometricus MiSp_v1, based on gene tree relationships (Figs. 6 & 7). The mutation of alanine or glutamine codons to a proline codon requires only a single base change and the GPG motif is frequently found as GPGA or GPGQ, indicating that it could have evolved from (GA)n or (GQ)n motifs, which are the most common motifs in all other MiSp sequences (Fig. 3). Intragenic concerted evolution could rapidly proliferate these mutations throughout a single gene as has been proposed for other spidroin paralogs [16, 17, 45, 46]. The near identity of spacer sequences within each of our cobweb weaver MiSp variants (Fig. 4) supports the hypothesis that intragenic concerted evolution is common, as also found in orb-web weaver MiSp [30, 31]. However, intragenic concerted evolution must be offset by some other molecular processes, since the remainder of the repetitive regions are not as homogenized as the spacers (Fig. 3). The simple amino acid motifs found in MiSp such as (GA)n are encoded by microsatellite-like sequences and could thus be prone to high rates of slipped strand mispairing. It is also possible that excessive homogenization of MiSp repetitive regions adversely affects its function and is eliminated by selection.
The high proportion of the GPG amino acid motif in S. grossa MiSp correlates with higher extensibility in S. grossa minor ampullate silk fibers (Fig. 5c, Table 3). GPG content is probably not the only predictor of extensibility, however, since Latrodectus minor ampullate fibers were more extensible than orb-web weavers' even though GPG content for L. hesperus is similar to the orb-web weavers. Furthermore, we did not find a significant difference in extensibility between L. hesperus and L. geometricus minor ampullate silk fibers despite L. geometricus MiSp_v1 having a relatively high percentage of GPG motifs (Fig. 3). The MiSp_v2: MiSp_v1 transcript ratio in L. geometricus suggests that the GPG-rich MiSp_v1 is in lower abundance in L. geometricus (Table 2), which could explain why there is not a significant increase in extensibility of its minor ampullate silk fibers in comparison to L. hesperus. Cobweb weaving spiders use MiSp in their prey wrapping silks [5], and evolution of extensibility-conferring motifs could potentially allow for capturing different prey types by S. grossa compared to other cobweb weavers, although little is known about the ecology or diet of this species. It is also possible that L. geometricus modulates expression of its two MiSp-encoding loci in response to prey availability. Experimentally manipulating prey type has been shown to affect proline content of major ampullate fibers in Nephila, potentially as a result of plastic changes in relative expression levels of the proline-poor MaSp1 and the proline-rich MaSp2 [47, 48]. Variation among individuals in MaSp1 and MaSp2 transcript abundance has also been demonstrated for black widows [49]. Analogous experiments have not been done for minor ampullate fibers. However, the variation in MiSp_v2: MiSp_v1 ratios that we found between L. geometricus individuals (Table 2) suggests plasticity of expression is possible.
Our six complete or nearly complete single exon MiSp-containing clones can also serve as templates for recombinant proteins. Due to the dramatic differences in length and amino acid content of some of the proteins encoded by these loci, it may be possible to spin artificial fibers with custom-made properties. For instance, the GPG-containing L. geometricus MiSp_v1 could be used to make more extensible fibers, while the longer alanine-rich L. hesperus MiSp_v1 may make stiffer fibers.
We found that intragenic concerted evolution within MiSp-encoding genes likely led to rapid proliferation of proline replacements for alanine or glutamine in MiSp protein sequences independently in at least two species. For one species, the proliferation of proline coincides with higher extensibility of minor ampullate silks. This could allow cobweb weavers to access new prey types or the ability to modulate mechanical properties of their silks through altering expression levels of MiSp gene copies. Our multiple nearly complete MiSp sequences also provide various templates for tailored biomimetic applications through recombinant DNA technologies.
Taxon sampling
Individual L. hesperus were collected in Riverside, California and Tucson, Arizona. L. geometricus individuals were collected in San Diego, California. L. tredecimguttatus and S. grossa were obtained from SpiderPharm (Yarnell, Arizona). P. tepidariorum were obtained from a laboratory culture founded with spiders collected near Cologne, Germany and purchased from SpiderPharm.
Genomic library clone
We screened an L. hesperus genomic library with PCR for clones containing MiSp (see [16] for library construction and screening protocols). Primers used in screening were designed from the C-terminal encoding region of a partial-length L. hesperus MiSp cDNA (HM752571; primers listed in Additional file 1: Table S3). An ~49 kb MiSp containing clone was shotgun sequenced and assembled to 8× coverage by Qiagen (Hilden, Germany).
PCR amplification and cloning of Latrodectus genomic MiSp
We amplified full-length L. hesperus MiSp from genomic DNA using primers designed from the 3' UTR of the C-terminal encoding MiSp cDNA (HM752571) and the N-terminal region of another partial MiSp cDNA (HM752570). We amplified genomic MiSp from L. tredecimguttatus and L. geometricus first using primers designed from L. hesperus MiSp N and C-terminal cDNAs and then with internal species-specific primers (primers in Additional file 1: Table S3). Genomic PCR was performed with 1 unit of AccuPrimeTM Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. Cycling conditions were 40 cycles of 94 °C for 30 s, 50–60 °C (depending on primer pair) for 45 s, and 68 °C for 10 min.
PCR products were gel-excised and cloned using the TOPO®-TA Cloning® Kit (Invitrogen). Clones were screened by PCR amplification of MiSp N and/or C-terminal encoding region, and MiSp positive clones were sequenced with Sp6, T7, M13F, or M13R universal primers. End sequencing of TOPO clones indicated that, within both L. geometricus and L. tredecimguttatus, clones clustered into two distinct groups (Additional file 1: Figure S2). For both L. tredecimguttatus and L. geometricus, the longest clones representing each cluster were completely sequenced. End sequences and restriction digest patterns of L. hesperus TOPO clones were very similar. Thus, one clone was chosen for complete sequencing. Clones chosen for complete sequencing were subjected to random transposon insertion with the GPS-1 Genome Priming System (New England Biolabs, Ipswich, MA). Position of transposon insertion was mapped with restriction digests and clones were sequenced from both ends of the transposon. Sequences were assembled with Sequencher v. 4.9 (GeneCodes).
Steatoda grossa and P. tepidariorum cDNA libraries
We constructed cDNA libraries from S. grossa total silk glands and P. tepidariorum major and minor ampullate silk glands following procedures detailed in Garb et al. (2010). In brief, total RNA was extracted with TRIzol® (Invitrogen) and the RNeasy Mini Kit (Qiagen, Valencia, CA). mRNA was isolated with oligo-(dT)-tagged magnetic beads (Invitrogen). cDNA was synthesized with SuperScript®III (Invitrogen), fractionated by size with ChromaSpin 1000 columns (Clontech, Mountain View, CA), blunt-end ligated into pZErOTM-2 (Invitrogen), and electroporated into TOP10 Escherichia coli cells (Invitrogen). Approximately 2000 colonies per library were arrayed and stored at −80 °C. The libraries were screened by gel electrophoresis of plasmid DNA [50]. Plasmids with inserts >500 bp were sequenced with T7 or SP6 universal primers. We identified silk protein-encoding sequences by conceptual translations and comparisons to published sequences with BLASTX [51].
An S. grossa cDNA clone (designated Sg MiSp) with a 3 kb insert containing the C-terminal encoding portion of MiSp was completely sequenced by random insertion of transposons with the EZ-Tn5 < Tet > kit (Epicentre). Transposon mapping, sequencing, and assembly were performed as for TOPO clones above.
Amplification and cloning of S. grossa and P. tepidariorum MiSp
We attempted to amplify complete MiSp encoding sequences from S. grossa genomic DNA using primers designed from conserved regions of Latrodectus MiSp N-terminal encoding sequences and from the C-terminal encoding region of the S. grossa MiSp cDNA (Additional file 1: Table S3). S. grossa genomic DNA was amplified with 1 unit of Phusion® High-Fidelity DNA Polymerase (New England Biolabs), 200 μM each dNTP, 0.5 μM each primer, 1X Phusion® GC buffer, and 3% DMSO. Cycling conditions were 40 cycles of 98 °C for 5 s, 58 °C for 15 s, and 72 °C for 2.5 min. PCR products were gel-excised and cloned using the TOPO®-Blunt Cloning® Kit (Invitrogen). End sequencing of these clones revealed numerous premature stop codons and thus clones were not completely sequenced.
In order to obtain longer N-terminal MiSp encoding sequences from S. grossa, we amplified S. grossa genomic DNA using forward primers designed from the conserved region of Latrodectus MiSp N-terminal encoding sequences or from the S. grossa N-terminal MiSp cDNA, and a reverse primer designed from the repetitive region of the S. grossa C-terminal cDNA (Additional file 1: Table S3). Genomic DNA was amplified with 1 unit GoTaq Polymerase (Promega), 1X GoTaq Buffer, 200 μM each dNTP, 0.5 μM each primer, and 1.5 mM MgCl2 with 45 cycles of 30s at 94 °C, 45 s at 54.2 °C or 61 °C, and 1.5 or 2 min at 72 °C. PCR products from the two amplifications were separately gel-excised and cloned using the TOPO®-TA XL PCR Cloning ® Kit (Invitrogen). TOPO clones from each reaction were amplified with M13 forward and reverse primers and sequenced with T7 or SP6 universal primers.
A MiSp gene was identified in the P. tepidariorum genome by BLASTN searches of our N- and C-terminal encoding MiSp cDNA sequences against the genome assembly [52] generated by the i5K initiative [53]. We found alignments of both cDNAs to a region on Scaffold 853 that included an Augustus-predicted gene model (aug3.g7951.t3). Using the genomic sequence, we designed a reverse primer in the C-terminal encoding region and a forward primer in the adjacent repetitive region. Genomic DNA and cDNA were amplified as for S. grossa N-terminal encoding region but with 40 cycles of 30s at 94 °C, 45 s at 60.1 °C and 1 min at 72 °C. PCR products were gel-excised and sequenced. Genomic PCR products had sizes ranging from 250 bp to 2500 bp, while cDNA PCR products had sizes ranging from 250 bp to 1200 bp. Those bands that were too large to directly sequence were TOPO cloned using the TOPO-TA PCR Cloning Kit (Invitrogen) and completely sequenced by random insertion of transposons as for S. grossa TOPO clones above. Completed TOPO clones were compared to the P. tepidariorum genome by BLASTN.
Amino acid sequence characteristics
Amino acid composition and codon usage were calculated with codonW [54]. Kyte-Doolittle hydropathy plots [55] with a window size of seven were generated online [56]. Amino acid motifs GGX, An (n ≥ 4), (GA)n (n ≥ 2), and GPG were identified by word searches in Microsoft Word. Spacer regions were defined manually as regions that were not in the N or C-termini and were low in alanine and glycine and were manually aligned. Two authors (JVH and CYH) independently assigned spacer regions. Tandem repeats were identified by XSTREAM v1.4.8 using default parameters for moderate repeat degeneracy and high significance [57].
Transcript abundance estimation
Transcript abundance was estimated by aligning raw RNA-seq reads [58] (SRR1539569 and SRR1539569 for L. hesperus and L. geometricus respectively) to our full or almost full-length MiSp paralogs found in L. hesperus (Lh MiSp_v1 and MiSp_v2) and L. geometricus (Lg MiSp_v1 and MiSp_v2) using Bowtie 2 with default parameters, which searches for multiple alignments for each read but reports the one best alignment [59]. RNA-seq reads were generated for mRNA isolated from major and minor ampullate glands of ~30 adult females and from all the silk glands ("total silk") of a single adult female for both L. hesperus and L. geometricus. SAMtools v1.1 was used to report the number of sequence reads matching our reference MiSp sequences [60]. To compare transcript abundance between loci within the same library, we first compared the total counts of reads aligned to the entire reference sequence. We did not correct for gene length because many of our libraries were heavily 3'-prime biased. However, to ensure that differences in gene length did not dramatically affect our estimates of the ratio of one MiSp locus to another, we also compared the counts that aligned to the last 500 bases of coding sequence (Table 2).
Relationships among spidroin paralogs
We added our theridiid and A. ventricosus [30] MiSp N and C-terminal coding sequences to an alignment of 29 spidroin termini analyzed previously (see [17]). N-terminal sequences were not available for many spidroins and thus we added 25 additional spidroin sequences to the C-terminal alignment to more comprehensively represent spidroin gene family diversity in our analyses (Table 1). Additional C-termini of spidroin paralogs were chosen for species for which MiSp has been characterized. The expanded amino acid alignment was manually edited and used to guide the nucleotide alignment in SeaView v. 4.2.7 [61, 62].
We conducted heuristic searches for maximum parsimony (MP) and maximum likelihood (ML) trees based on amino acid and nucleotide alignments in PAUP* v4.0b10 using tree bisection reconnection branch swapping and 1000 (MP) or 10 (ML) replicates of random stepwise addition of taxa [63]. Support for clades recovered in MP analyses was evaluated with 1000 bootstrap pseudoreplicates and 10 random addition sequences per pseudoreplicate.
Bayesian analyses were carried out with MRBAYES v.3.1.2 [64, 65]. Optimal models of evolution were determined for nucleotide sequences with JMODELTEST v0.1.1 [66] and for protein sequences with PROTTEST v2.4 [67] for N and C-termini separately. Combined analysis of nucleotides employed a model partitioned by N and C-termini. Combined analysis of amino acids employed a mixed model, which allowed estimation of the optimal model of protein evolution during the Bayesian analysis. Default priors and Metropolis coupled, Markov-chain, Monte Carlo sampling procedures were executed for two independent runs of 10 million generations each, sampled every 1000th generation. Convergence was assessed every 1000th generation and the posterior distribution was considered adequately sampled if the standard deviation of split frequencies of these two runs was below 0.01. The mygalomorph spidroin, Bothriocyrtum californicum fibroin1 (Bc fibroin1), was set as the outgroup in all analyses.
Gene tree – species tree reconciliation
We inferred gene duplication events by reconciling each of our gene trees with two species trees (Fig. 1) using Notung v. 18.104.22.168 [68]. Species trees were based on published phylogenies as follows. Relationships among families were based on Garrison et al. [38] and Dimitrov et al. [39]. These studies agreed on all family-level relationships except the placement of Deinopidae and Uloboridae. The former placed Deinopidae sister to the RTA-clade (represented here by E. australis and A. aperta) with Uloboridae sister to Deinopidae plus the RTA-clade. The latter switched the placement of Uloboridae and Deinopidae. For relationships among theridiid species we used Liu et al. [35] and Garb et al. [34]. Relationships among nephilid species were based on Kuntner et al. [69]. Relationships among araneid genera were based on Dimitrov et al. [39] with relationships for Argiope species from Cheng and Kuntner [70].
Mechanical properties of minor ampullate fibers
We obtained minor ampullate silk from L. hesperus, L. geometricus, and S. grossa adult females within one week of being received or collected. Individual spiders were anesthetized with CO2 for 2–5 min. We then secured the spider ventral side up to a stereo microscope stage using Scotch ® tape, exposing the spinnerets. We manually pulled silk emanating from the minor ampullate spigots. Single fibers were taped to m-shaped cardstock (2.54 × 7.62 cm), with a single silk fiber suspended across each of the two rectangular notches (1 cm wide × 1.5 cm deep) in a collection card. Fibers were secured onto the cards using cyanoacrylate glue. We collected four to ten minor ampullate fibers from each of seven (L. hesperus and S. grossa) or eight (L. geometricus) individuals per species.
We measured silk fiber diameters using polarized light microscopy (as described in [6]). Tensile tests were conducted with a NanoBionix tensile tester (formerly MTS Systems Corp., Oak Ridge, TN; currently Keysight Technologies, Santa Rosa, CA) [6]. In brief, each m-shaped card was cut in half, such that only one fiber segment was tested at one time. Each silk sample was mounted onto the NanoBionix and extended at a rate of 1% per second until failure. We defined strength as the true stress of the fiber at breakage. True stress is the force applied to the fiber divided by its cross-sectional area calculated from the original cross-sectional area assuming constant volume of the fiber during extension [71]. We defined extensibility as the true strain at breakage. True strain is the natural log of the ratio of the instantaneous length of the fiber to the original gage length of the fiber. We also measured stiffness or Young's modulus, the initial slope of the true stress-true strain curve, and toughness, the area under the true stress-true strain curve. Force-extension data were visualized using Testworks 4.0 software (MTS Systems Corp.). We tested for differences among species for the four tensile properties using multivariate analysis of variance (MANOVA). We used Tukey's test for post-hoc pairwise comparisons. All statistical tests were carried out with SPSS 13.0 (SPSS Inc, Chicago, IL).
We compiled published tensile data for minor ampullate silks and fit the relationship between proportion of GPG amino acid motifs and extensibility to linear models implemented in R for each species for which at least one MiSp sequence and minor ampullate fiber tensile data were available. Minor ampullate silk was assumed to be composed only of MiSp and for each species all available MiSp motif percentages were averaged to estimate composition. To account for phylogenetic relationships and evolutionary distances among species we calculated phylogenetic independent contrasts using APE [72] implemented in R. We used estimates of divergence time from three published molecular clock studies [35, 38, 39] for branch lengths (see Additional file 1: Figure S6).
We thank Stephen Richards and colleagues at Baylor College of Medicine's Human Genome Sequencing Center for access to the P. tepidariorum genome. We also thank Alistair McGregor, Mario Stanke, and colleagues for annotating this genome. Students in Genetics Lab (BIOL 221) at Washington and Lee University assisted with collecting sequence data.
This work was supported by the National Science Foundation (IOS-0951086 to NAA, IOS-0951061 to CYH), National Institutes of Health (F32 GM78875-1A to NAA; 1F32GM083661-01 and 1R15GM097714-01 to JEG), Army Research Office (W911NF-06-1-0455 and W911NF-11-1-0299 to CYH), and Washington and Lee University through Lenfest Summer Fellowships to NAA and Summer Research Scholarships to JVH and AKL. ERB was supported in part by a grant to Washington and Lee University from the Howard Hughes Medical Institute through the Precollege and Undergraduate Science Education Program (52007570).
Availability of data and materials
Sequences generated for this study are available in GenBank (Accessions: KX584003 - KX584055). Accessions for previously published sequences are reported in the manuscript text and in Table 1.
Authors' contributions
CYH and NAA conceived the study. JMV, ERB, JEG, EES, and NAA generated sequence data. AKL, MAC, SMC, and CYH measured tensile properties of minor ampullate silk fibers. JMV, ERB, MAC, THC, and NAA analyzed the data. JMV and NAA wrote the manuscript. All authors edited the manuscript and read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Not applicable.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors' Affiliations
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As a new player you can have the opportunity to train yourself in various casino game types by playing for free in many casino websites. By continually playing you can be able to understand the working structure of each game type. After understanding the maxbet Game basics by gaining experience you can be able to build your own game strategy which helps you in increasing the winning choices. This makes the casino gaming to be more interesting and thrilling to many of the people. Without this they feel difficult to win in the game which makes them to get frustrated in online casino gambling.
In many casino websites you are asked to download their casino software in your computer or in your tablet. You can freely download the software of reputable casino websites as it will be free of harmful programs. Downloading and playing will be a better option if you are not having the fast internet connection. Another interesting way of play is the playing of flash game types. This needs the players to play the game in the online version and also you should have the flash players installed to support these game types.

subset: commoncrawl


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Aelters Ardchilds' Com.Undo
PowerBook jester Aelters apparently left DAT Politics because the loopy French laptop quartet weren't wacky enough. This should give you an inkling of the fractured digital dementia splattered all over Ardchilds' Com.Undo's 16 tracks, which find Aelters blowing mocking raspberries at "serious" techno and IDM tropes. While Ardchilds' may strike some as trifling and absurd, the disc contains flashes of irreverent brilliance. Spluttering, rough-hewn abstract techno tracks like "Starapeakotop" and "Porkfesonli" take bytes out of Cylob's hard drive while "Hombrero"'s industrial clank and pile-driver funk rhythms bump rambunctiously like late-'90s Si Begg. If nothing else, Aelters's spazztronics forever torch the misconception that electronic music is a strictly humorless endeavor.


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Crispy Sea Salt & Vinegar Cauliflower "Popcorn"
March 18, 2017 | Cooking time:
need a healthy TV snack for tonight?
1 large head cauliflower, outer leaves removed
1/2 teaspoon sea salt
Freshly ground black pepper, to taste
1/4 cup + 1 tablespoon apple cider vinegar, divided
3 tbsp coconut oil, melted
- Preheat the oven to 180c and line a baking tray.
- Cut the cauliflower into small bite sized florets and combine in a bowl with 1/4 cup ACV, coconut oil and salt and pepper
- lay out on a baking tray and bake 30 minutes or until super crispy!
If you want a bit more of a vinegar TANG, drizzle with the remaining ACV and season again if desired


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My WheelTug
Contact Us
About WheelTug
When WheelTug began developing its e-taxi solution over a decade ago, the idea of using electric taxi systems was not new - but it was considered by some to be unworkable. With our first proof of concept test in 2005 we established that not only was it workable, but that the economic potential of an e-taxi system made it a goal worth pursuing.
We spent the next few years researching and fine-tuning our designs, overcoming many technical challenges in the process. In 2010, we performed our second set of ground tests - further expanding our understanding of taxi requirements for narrowbody jets, and proving that WheelTug could operate in snow and slush in the process.
In 2012 we performed the first ground tests of our in-wheel motor design. It was a smashing success that has led us to today: finalizing the design of our production e-taxi system.
After more than a decade of effort, our team of dedicated professionals and our world-class development partners are excited to bring WheelTug to a plane near you in the very near future.
Want to Know More?
Our Partners
include a number of industry leaders in their areas of expertise.
WheelTug Customers
currently represent over 1,000 aircraft from more than 20 airlines.
If you have comments or additional questions, please don't hesitate to
contact us
You may also
find a local representative
Contact Us
Corporate Information
Video Library
Upcoming Events
WheelTug plc claims exemption from Section 12(g) of the Securities Exchange Act of 1934, as amended (the "Exchange Act"), pursuant to Rule 12g3-2(b) of the Exchange Act.
Accordingly, WheelTug plc has posted information on this site in compliance with Rule 12g3-2b.
Copyright © 2019 WheelTug plc -
Forward Looking Statement/Legal Disclaimer


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Did you know that eggs are an aphrodisiac?
Spice up your Valentine's Day with this natural aphrodisiac
Whether they are poached, scrambled, fried or hard boiled, eggs can spice up your St Valentine's Day as they provide the perfect combination of vitamin B5 and vitamin D. Vitamin B5 helps mental clarity and is needed for the production of sex hormones, including oestrogen and testosterone, whilst vitamin D is essential for reproduction.
Here are three recipes you might want to try on Valentine's Day - wink, wink, nudge, nudge!
Click here for the banana, blueberry and almond pancakes recipe.
Click here for the fried eggs with spiced avocado recipe.
Click here for the orange and cinnamon crème brulee recipe.
Recipes courtesy of eggs.org.au


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Best Tuscan
Countryside Retreat
Castel Monastero
This luxury hotel is set in a former medieval village with a rich history. Originally built in the 11th century as a monastery, it later became a castle and residence for the noble Chigi family. The hotel has been updated with modern amenities and sophisticated decor, and provides a relaxed, chic setting. The 74 guest rooms are dispersed throughout the different buildings and chock-full of charming historical touches. Surrounded by vineyards and the beautiful Tuscan countryside, the hotel is isolated, but there are many amenities at the hotel to keep guests occupied, such as a full-service spa, a Gordon Ramsay restaurant, a fitness center, and two outdoor pools.
Honorable Mention:


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Our Vision
At the 4Cs Multi Academy Trust we must ignite the student's imagination and their active, willing and enthusiastic engagement in their learning.
Our vision is to: ensure that every student succeeds; build on what learners already know; make learning vivid and real; make learning an enjoyable experience; and enrich the learning experience that we offer.
The Trust's approach should enable students to encounter and begin to explore the wealth of human experience through induction into, and active engagement in, the different ways through which humans make sense of their world: through knowledge, intellectual, moral, spiritual, aesthetic, social, emotional and physical experiences. As well as through language, mathematics, science, the humanities, the arts, religion and other ways of knowing and understanding and act upon it.
'Creative Thinking' is at the heart of the Trust's approach to learning. We aspire for the students to explore, negotiate, discover and experiment. We want them to speculate, empathise, reflect, collaborate, cooperate and persevere. Students need to show initiative, and demonstrate leadership, teamwork, flexibility, integrity and imagination.
The Trust wishes to encourage curiosity, courage and constancy.
To facilitate this aspiration, the most important gift we can provide is that of confidence.


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PMI-LA offers many benefits to our members:
- Monthly networking opportunities
- Volunteer opportunities
- National and local job leads from recruiters seeking candidates from our membership pool
- Discounts to monthly dinner meetings
- Invitations to conferences and expert presentations
- Discounts on Saturday seminars and professional development courses
- PMP certification prep workshops
- Ongoing membership support
- Project Management mentoring programs
- Ongoing training in project management and related fields
Already a chapter member? Be sure to login to access your benefits!
Not a chapter member (yet)?
- Join the Chapter
- Attend in-person events
- Become a Volunteer
- Be a Presenter
- Be a Sponsor
- Study for your PMP exam
- Earn PDUs


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My first day at Girls Grammar was different from anything I have ever experienced. Within ten minutes of entering the school I had not only been introduced to my entire cohort and both English teachers, and had the amazing realisation that everyone had name badges so it was impossible for me to get their names confused, but also been told that I was pretty and literally dragged into the group of friends I still had when I graduated. I was the only new student that day and felt like a shiny new toy in the best way possible.
By my second day, I thought I had everything sorted, except it was sports day; the one day when nobody wears a name badge. Determined not to let anyone realise that I'd forgotten all their names, I stuck to the one person whose name I could remember and somehow, despite being outwardly against any form of sport, ended up in every single event. As I sprinted the end of the 1500 m (despite being anti-athletic I was still fiercely competitive), I experienced the moment which summarised Girls Grammar for me; despite having not so much as spoken to any of the other year levels, they had all learnt my name and cheered me to the finish line. That was the moment when I knew I was home.
Before being at Girls Grammar, I had been at a school with an entirely different approach to learning. Despite having been one of the brightest students, and multiple complaints being made, I had been told to bring a book to class in lieu of doing any form of extension work. Conversely, within two weeks at Girls Grammar, despite having joined in Term two, I was boosted up a full grade in Mathematics and offered extension texts in English. The teachers never drew attention to this, most of my Year level never even knew, but for the first time in my schooling life I felt that the teachers were teaching me, rather than merely teaching a group of faceless students.
By the time I reached Year eleven, I had almost forgotten the girl I had been before. Instead, I felt valued and confident and, most importantly, like I was worthy of whatever I got. That might not sound like something world changing, but for a person who had previously been belittled by teachers and led to believe that she wasn't smart enough, it changed my outlook on life.
I graduated Girls Grammar as the Dux of the school and the top or equal top student in five of my six subjects and as one of two OP 1 students and I owe that in no small part to the passion and determination of my teachers, but that is not what I feel I graduated with. I graduated as someone who had previously been terrified of public speaking but this year stood up in front of all Year 11 and 12 students to compete in the Valedictorian competition. I graduated as someone who had once considered herself unworthy of authority, but stood for and was awarded a Prefect role and loved every minute of it. I graduated as someone who used to come home every day angry or upset but who hadn't had a truly bad day since 2011. So, thank you Girls Grammar, not just for helping me to reach my academic goals but also for allowing me to change from who I was into who I wanted to be.
Nicola Cole
2013 has once again proved to be a highly successful year for Girls Grammar students with the Year 12 graduating class performing exceptionally well with OP scores. Special congratulations go to OP1 students Nicola Cole and Emilia Festa. Approximately 20% of the graduating class achieved an OP score between 1 and 3 which is significantly above the state average of 10%. Girls Grammar students also performed above state averages in the other benchmarks of OP 1-10 and OP 1-15. These results are made possible due to the wonderful achievement of the girls in the QCS test where 25% of students achieved and 'A' compared to the state average of 16%.
These results follow on from our students' achievements on Years 3, 5, 7 and 9 NAPLAN testing where 100% of students in Years 3, 5 and 7 achieved at or above national standards – the only school in Central Queensland to achieve such a result. Our Year 9 students also scored significantly above state and national averages.
"These significant academic achievements are a demonstration of Girls Grammar values in action. We provide learning in a caring, small yet academically rigorous environment," says Principal Ms Melinda Scash. "Our students are going from strength to strength with their academic achievements which is testament to their continued hard work and efforts of our dedicated staff."
Recent media coverage has highlighted that our girls have performed exceptionally well in the 2013 NAPLAN tests. We are proud of the efforts of our students who always approach assessments with calmness and an appropriate level of seriousness and perspective.
100% of our Primary and Year 7 students tested at or above the National standards which is achieved by few schools nationally. This success is a recognition of the individual academic care afforded to Girls Grammar students in small classes, with personalised programs and specialist teachers. Our Year 9 cohort also achieved to a very high level and ranks as a leader in the region in literacy and numeracy.
Whilst we are proud of the results, we also acknowledge that they need to be viewed in context. NAPLAN results provide a rich source of data which provides teachers with insight into consolidation and extension opportunities for your daughters.
We congratulate all students on the way they managed the challenge of the National testing and look forward to continuing to build on these strong academic results.


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Scottish couples trying to start a family are set to receive three cycles of IVF treatment on the NHS.
The Scottish Government announced the move yesterday to offer three cycles – which is more than is currently available in some parts of England and Wales.
Public health minister Aileen Campbell defended the spending at a time of pressure on accident and emergency departments and waiting times.
Ms Campbell said: "We have always had a commitment towards ensuring there is as equitable access to IVF as there possibly can be and we had a number of recommendations presented to us that we accepted from the national infertility group.
"One of these included ensuring that couples could get access to three cycles of IVF. That is today what we are taking forward."
She said: "The very positive announcement of allowing couples who are desiring to have a family to get access to three cycles has been welcomed across the different fertility organisations."
The Scottish National Party minister said money had been "put aside" to fund the IVF programme. Asked how she squared this with other demands on funding in the Scottish NHS, she said: "We have got a fairly strong record across Scotland on our NHS. Some of our A&Es have outperformed the lion's share of England's A&Es. We are outperforming A&Es across the UK and have done for some months. We have good investment levels in the NHS and we have record numbers of staff. "We have got a commitment to our NHS that we will invest more money in our NHS than any of the other parties that stood in the election."
From 1 April, new patients referred for IVF treatment may be eligible for three full cycles rather than two, after the change was recommended by the National Fertility Group in its report to ministers last year.
Gwenda Burns, branch co-ordinator for Fertility Network Scotland said: "This is wonderful news for both patients and campaigners: for the one in six couples in Scotland who face the devastation and heartbreak of fertility struggles it is a fantastic beacon of hope; for Fertility Network UK it is the news we have been working towards with the National Infertility Group, since the latter's inception in 2010."
Scottish Labour's health spokesman Anas Sarwar said: "Scottish Labour argued in its manifesto for an increase in the number of full cycles of IVF available to couples from two to three. This is a very welcome and long-overdue announcement from the Scottish Government."


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Arif Castles, remote and peaceful in spectacular setting, is an ideal venue for conferences and meetings.
"Darbar Hall" Seats upto 120 guests in theatre style, the "Roof Top" seats up to 60 guests in theatre style. The seating in both the halls can be arranged in a variety of configuration to suit your business needs.
Meeting facilities with all the latest communications and audio-visual equipments are available for hire by the day:
An outdoor dance and dine area with a live bonfire.
The coffee shop is located on the lobby floor and it offers a range of snacks and drinks.
The restaurant at Arif Castles offers a selection of Continental, Indian and Chinese dishes and is open for lunch and dinner.
24 hour in-room dining is available throughout the hotel.


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In a previous post, we told you about how to stay current with New Jersey Legal (& Local) News. Whether using print or electronic versions of legal newspapers and periodicals, staying current with current events may give you an edge in practice.
Legal newspapers or periodicals will inform you of relevant court decisions, the emergence of new laws, changes to existing laws, and may also include substantive legal articles written by experts in their fields (and sometimes, gossip).
Another way to stay current is to have some of that information emailed directly to your inbox. Justia can supplement your daily intake of legal news and analysis. Justia users can subscribe to receive daily newsletters of opinion summaries for the Supreme Court, Circuit Courts of Appeal, and all state supreme courts. That means that it can be as easy as a free and simple registration to stay on top of 3rd Circuit Court of Appeals and NJ State Supreme Court opinions (and more). Once you register, you can elect to receive daily opinion summaries by jurisdiction and/or practice area. Each summary is followed by a link to view the case; users will be taken to the case page to find the opinion summary followed by the full-text opinion (HTML or PDF).
Justia also offers a daily newsletter of their Legal Analysis and Commentary column: Verdict.
Verdict columns cover a broad range of legal subjects, including constitutional law, international human rights, new technologies, discrimination, family law, law and economics, defamation, consumer issues, child sexual abuse and animal rights.
These commentaries are written by today's legal experts.
Subscribe today!
Written by CDS.


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A tracking device that measures changes in temperature, humidity and light.
Exporters had no way of tracking their goods and perishables while in transit
The transportation of perishables is a crucial component that supports large export industries all over the world. The problem for owners is that once containers are locked and loaded for shipping, they have no way of tracking what happens to the goods in transit, yet they foot the bill for damages, theft, contamination etc until the shipment reaches its destination. The founders of MOST realised that the Internet of Things could
solve this multibillion-dollar uncertainty. MOST created a tracking device that can be placed in shipping containers or transportation vehicles. Each device has the ability to measure temperature, humidity and light, as well as shockwaves. Working with our client in Sweden, BAS ITG, MOST needed a sensor-software and hardware platform to transmit the data from the device to a central repository.
Now, MOST's clientele are able to track all of their shipments simultaneously from one dashboard
We overhauled the original backend and CMS, extracting the information methodically and displaying it via a user-friendly interface. Our team also created instant tracking facilities, set up alert systems as well as user credential processes. We simplified the complex reporting systems by breaking each component down to highlight the nature of each route, the unique container identification number, trip duration and routes travelled. Each device is fitted with a sim card so
that when the device is out of range, the data can still be collated, recorded and transmitted once signal is restored. We also implemented SMS notifications so that owners receive alerts if temperature, light or humidity changes beyond an acceptable range. Now, MOST's clientele are able to track all of their shipments simultaneously from one dashboard, giving them access to important information about their goods in transit, including its location at any point throughout the journey.


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Coffee beans sing distinct tune
Crackling sounds could be key to automated acoustic roasters
Preston S. Wilson/UT-Austin
The snap-crackle-pop of coffee beans could tell automatic roasters when to turn down the heat.
Hot beans sing a distinct ditty that reveals their stage in the roasting process, mechanical engineer Preston Wilson of the University of Texas at Austin reports in the June Journal of the Acoustical Society of America.
Java roasters know to listen for some basic sounds, commonly called "first crack" and "second crack," but until now no one had measured and analyzed these noises, Wilson says. He roasted a small batch of green coffee beans — an espresso blend — in an electrically heated drum roaster and recorded the crackling sounds as the beans got toasty.
The first crack noises, which sound like popping corn, ring out between 400 and 600 seconds after roasting begins. Those crackles are louder, deeper and less frequent than the second chorus of cracks at 620 to


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Bergere French Painted Carved Kidney Dressing Table
Product Description
Beautiful cream painted furniture with just a handful of quality french style shabby chic pieces!
Stunning detail to this furniture range, with just a few pieces of unusual furniture.
The fine detail is finished in a cream painted colour!
The ornamental carvings, cabrio curves and femininity throughout are the hasllmark of Bergere! French styled furniture, the bergere design is inspired by the lavish, romatic flourishes and decoration that typified the the 18th century French Rococo style!
Height: 62" - 157cmWidth: 36" - 91cmLength: 16.5" - 41cm
Available to order
Home Delivery
FREE UK mainland delivery on orders over £300
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Oriental Living is the leading supplier of furniture and home décor in the south of Thailand and has a wide range of furniture for holiday homes and villas, hotels, restaurants and real estate projects.
Full container loads, or smaller quantities, for private and joint-venture projects both domestic and international, can be shipped at very reasonable rates. Products are transported directly from our premises with packaging carefully controlled by our team of professionals. Direct from factory sales receive discounts to ensure that buyers receive worthwhile and competitive prices. We have shipped furniture and home accessories for projects in Italy, the United Kingdom, Germany, Russia, Hong Kong, Malaysia and many other countries.
"Our aim is to supply your beautiful properties with high quality products at affordable prices and a luxury level of service that you can most rely on."


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Oct 23, Rossi Hair Care Reviews
We're starting to get some great reviews for our products. Here's a review from blogger Online Mummy who's tried both our Jojoba & Coconut Shampoo and Shea Butter & Olive Conditioner. We've kept a simple 100% natural formulation with high quality ingredients which are great to use and they both have a beautiful natural fragrance. See the complete review here


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Reduce sourcing time and effort by quickly finding similar suppliers.
The Supplier Discovery module gathers supplier capability data from numerous public and proprietary sources and maps them to your specific industry and regions. The result is the ability to quickly identify potential suppliers to augment existing capacity or replace lost capabilities.
Prospective suppliers can be identified by specific parts they produce, manufacturing processes and capabilities, quality criteria, location, and so on. This module puts global sourcing information at your fingertips, dramatically reducing emergency sourcing cycles.
Imaging that a hurricane is bearing down on one of your key suppliers. Within a few clicks, you're able to identify what that supplier is producing and quickly identify similar suppliers that are outside the path of the hurricane.
In addition to mitigating or negating supply chain disruptions, Supplier Discovery also can help when you need to accommodate a demand surge. With just a few clicks, users can easily see a ranked list of qualified manufacturers that have the right capability and capacity at the right time.


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WFDF World Ultimate Club Championships go live
03 July 2010The WFDF World Ultimate Club Championships in Prague, Czech Republic is under way.
WFDF Guts Committee Reformed
27 June 2010The WFDF Guts Committee has been reformed under the direction of Chair Ryan Scott. http://www.wfdf.org/index.php?page=structure/committees/index.htm#guts
World Ultimate Teams Archive Website Launched
17 June 2010
WFDF Approves Contribution to Ultimate Peace
05 June 2010WFDF Approves Contribution to Ultimate Peace
New WFDF Provisional Memberships Announced
29 May 2010The Argentina Flying Disc Association and the Ukraine Frisbee Federation proposals for Provisional WFDF Memberships have been approved by the WFDF Membership.
UPA changes name to USA Ultimate
27 May 2010
WFDF Ultimate Rules Website Launched
10 May 2010The WFDF Ultimate Rules Committee has created a new website which contains a wealth of information.
WFDF Ultimate Rules translated into French
02 May 2010
Page 9 of 22


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As one of the country's leading authorities in strategic planning, Wipfli LLP's strategic advisors work with numerous organizations on all facets of strategic planning, providing detailed comparisons and quantitative benchmarks to any size organizations.
We take pride in our creativity and our track record in developing and delivering innovative and strategic solutions to clients, which result in high-performing organizations—as defined by both earnings and leadership. We constantly research and explore trends and technical issues to develop innovative approaches not typically considered by other consulting firms.
Our reputed process treats every organization as a unique entity. Repeat engagements that span multiple decades affirm the value of our expertise and "holistic" approach. The customized strategic plan developed ensures careful crafting of the right strategy solutions. The customized strategic planning process typically includes the following:
- Fact finding
- Comprehensive financial analysis (including custom peer group analysis)
- GPS Survey™ (online key stakeholder assessment)
- Conference calls with Chief Executive Officer and/or committee
- Facilitation of on-site strategic planning meeting
- Development of strategic plan
The process typically includes the following focus areas; however, depending on unique circumstances, the planning process can be "focused" on specific areas:
- Mission, vision, and core values of the organization
- Internal and external analysis (including SWOT)
- Strategic priorities
- Financial performance targets
- Goals and strategies
- Action plan(s)
Our strategic advisors are also available to provide implementation assistance, should it be requested. Our approach is efficient, concise, results-oriented, and practical. We are interested in only customized solutions that can be effectively implemented and tied to bottom line results, as opposed to theoretical recommendations that inevitably end up being ineffective and sitting on a client's bookshelf.
Featured Thought Leader
Deron J. Kling
Deron Kling is an experienced consultant and leader focused on helping Wipfli's clients plan for the future and reach their goals. His background includes over 20 years as an organizational leader and management consultant.


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He's the guy who played the most recent Superman, but I liked him more in Scott Pilgrim. Either way, he has some monstrous brows. Or, I should say "had."
I will be taking a 3-week break after this post. Sorry everyone! Please keep sending your emails and everything though! Bye for now!
Saturday, September 17, 2011
Brandon Routh
3 comments:
- AnonymousSeptember 22, 2011 at 10:11 PM
You need to do all the presidential candidates and all the various Law and Order people. FUn Stuff!ReplyDelete
Um, I REALLY think you need to take Darren Criss's eyebrows off. He has super distinct eyebrows and would look super weird without them!!!ReplyDelete
- AnonymousApril 9, 2012 at 12:21 AM
He looks like a serial killer on the right!ReplyDelete
Subscribe to: Post Comments (Atom)


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RSA researcher Eli Marcus is said to have found a new Trojan, made up of around 25,000 lines of fresh code.
With most malwares based on proven platforms, entirely new code is said to be a rarity. This new Trojan, known as "Pandemiya" is capable of stealing data from forms, creating fake web pages and taking screen shots to send back to the botmasters.
The software is modular, pervasive and unique thanks to its ability to inject itself into all new processes via the Windows security registry function: CreateProcess API.
Eli Marcus said: "The advent of a freshly coded new trojan malware application is not too common in the underground." He also added that the modular approach means Pandemiya could become "more pervasive in the near future."
Marcus speculates that the Trojan was relatively unknown until now, due to its high price and new status, in comparison to the likes of Zeus and Citadel.
Return to internet news headlines
View Internet News Archive


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