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Hsp-90 and the biology of nematodes
Nik AIIN Him1,
Victoria Gillan1,
Richard D Emes2,
Kirsty Maitland1 &
Eileen Devaney1
Hsp-90 from the free-living nematode Caenorhabditis elegans is unique in that it fails to bind to the specific Hsp-90 inhibitor, geldanamycin (GA). Here we surveyed 24 different free-living or parasitic nematodes with the aim of determining whether C. elegans Hsp-90 was the exception or the norm amongst the nematodes. We combined these data with codon evolution models in an attempt to identify whether hsp-90 from GA-binding and non-binding species has evolved under different evolutionary constraints.
We show that GA-binding is associated with life history: free-living nematodes and those parasitic species with free-living larval stages failed to bind GA. In contrast, obligate parasites and those worms in which the free-living stage in the environment is enclosed within a resistant egg, possess a GA-binding Hsp-90. We analysed Hsp-90 sequences from fifteen nematode species to determine whether nematode hsp-90s have undergone adaptive evolution that influences GA-binding. Our data provide evidence of rapid diversifying selection in the evolution of the hsp-90 gene along three separate lineages, and identified a number of residues showing significant evidence of adaptive evolution. However, we were unable to prove that the selection observed is correlated with the ability to bind geldanamycin or not.
Hsp-90 is a multi-functional protein and the rapid evolution of the hsp-90 gene presumably correlates with other key cellular functions. Factors other than primary amino acid sequence may influence the ability of Hsp-90 to bind to geldanamycin.
Heat shock protein 90 (Hsp-90) is essential in all eukaryotes because of its role in chaperoning proteins such as kinases, growth factors and other signal transduction proteins [1]. As knock out of hsp-90 is generally lethal, specific inhibitors have been important for defining the cellular functions of Hsp-90. Of these, the best characterised is geldanamycin (GA) a naturally occurring ansamycin compound produced by Streptomyces hygroscopicus [2]. The mode of action of GA is well documented and the crystal structure of yeast Hsp-90 complexed to GA has been solved [3]. Most eukaryotic Hsp-90s are susceptible to inhibition with GA and will bind the drug in pull-down assays using GA linked to a solid support [4]. The exception to this rule is Hsp-90 (DAF-21) from the free-living nematode Caenorhabditis elegans (Ce), which fails to bind to GA in pull-down assays [5] and which shows no apparent ill-effect when maintained on a lawn of S. hygroscopicus. In contrast GA binds to Hsp-90 from the parasitic nematode Brugia pahangi (Bp) and is lethal to all life cycle stages [6]. The inability of Ce-Hsp-90 to bind GA was proposed to represent an example of adaptive evolution, as both C. elegans and the Streptomyces species that synthesizes GA are soil dwelling and so may share the same ecological niche [5]. In this study we adopted an integrated approach to investigating the possibility that nematode hsp-90s may have undergone adaptive evolution that influences GA-binding. We surveyed Hsp-90 from a range of free-living or parasitic nematodes for their ability to bind GA, and combined these data with statistical methods to compare substitution rates using maximum likelihood (ML) models of codon evolution [7, 8], in an attempt to identify if significant evidence for adaptive diversifying evolution (dN/dS >1) relating to GA-binding exists. Specifically we investigated whether hsp-90 from GA-binding nematodes has evolved under differing evolutionary constraints compared to non-GA binding nematode species
Nematodes are an extensive group of animals that have evolved to colonise most ecological niches. Many nematodes are important parasites of humans, domestic animals and plants and are responsible for considerable economic losses worldwide [9]. Since the sequencing of the C. elegans genome, the genomes of several additional free-living nematodes have been sequenced, including two other species of Caenorhabditis (briggsae and remanei) and Pristionchus pacificus [10], a species that associates with beetles. Draft genome sequences are available for C. brenneri and japonica, and for the parasitic species Trichinella spiralis [11], Brugia malayi [12], Haemonchus contortus http://www.sanger.ac.uk/Projects/H_contortus/ and Meloidogyne hapla [13] and incognita [14], plant parasitic nematodes. Furthermore, sequencing projects are planned for several other important parasitic species http://www.sanger.ac.uk/Projects/Helminths/. In addition EST projects have provided an abundance of sequence information on the phylum [15]. A phylogeny of the nematodes based on the small subunit ribosomal DNA sequences has been proposed [16] in which the nematodes are divided into five clades. Each of these clades contain parasitic species, suggesting that parasitism has arisen on multiple occasions during the evolution of the phylum. C. elegans is grouped in clade V, along with many important parasites of veterinary significance, while the filarial nematode Brugia malayi lies in clade III, as do some important ascarid parasites of humans and animals.
While free-living nematodes, such as Caenorhabditis species, often exhibit relatively simple life histories developing from egg to adult in one environment, parasitic species display a huge diversity in life cycles. Some parasitic species are transmitted direct from host to host and many of these have a free-living stage within the environment (e.g. the Trichostrongyles), while others, such as the filarial nematodes are obligate parasites. These worms have no free-living stages and are transmitted from host to host by the bite of an infected intermediate host, such as a mosquito. Likewise, the clade I species Trichinella spiralis is permanently parasitic and transmitted by ingestion of infected tissue. In this study, we set out to investigate whether the life history of a species was correlated with the ability of Hsp-90 to bind GA by analysing an additional twenty-four species of free-living or parasitic nematode, belonging to four of the five clades. These data were combined with an analysis of hsp-90 DNA sequences from a range of nematode species, in an attempt to determine whether the hsp-90 genes of GA-binding nematodes have significantly different ratios of non-synonomous (amino acid-changing) to synonomous (amino acid-retaining) nucleotide substitutions compared to non-binding species.
GA pull-down assays
The ability of nematode Hsp-90 to bind to GA was assessed using a standard pull-down procedure in which GA bound to a solid support was used to select Hsp-90 from worm extract. The presence of Hsp-90 in the pull-down was assessed by immuno-blotting. Prior to use in pull-down experiments, extracts of each worm were probed with an anti-B. pahangi Hsp-90 antibody or with AC88, a monoclonal antibody to Hsp-90, to confirm that Hsp-90 could be detected on a blot. Extracts of three species failed to react with either antibody over a range of antibody concentrations and exposure times: Trichinella spiralis, Trichuris muris and Anguillicolla crassus. In addition, extracts of adult worms of Haemonchus contortus did not react with either antibody, despite the inclusion of hsp-90 sequences in the Haemonchus EST database with reasonable overlap to the 3' end of Bp-Hsp-90 that was used to make the antibody [6]. Extracts of adult Haemonchus were prepared in the presence of an additional protease inhibitor (E44) or directly by boiling in SDS-PAGE sample cocktail. Only when worms were extracted directly into sample cocktail could Hsp-90 be detected, suggesting that the aqueous extract may be subject to proteolytic degradation. In contrast, a soluble extract of the L3 stage reacted well with the Brugia antibody and was used in subsequent pull-down assays.
Nine clade V nematodes were tested in the GA pull-down assay, five of which were free-living. These included C. elegans N2, C. elegans JT6130, a daf-21 mutant which carries a point mutation in hsp-90 (E292 to K), conferring a temperature-sensitive dauer-constitutive phenotype [17], Caenorhabditis briggsae (result not shown), Oscheia tipulae and Pristionchus pacificus. In addition four parasitic species were tested: Teladorsagia circumcincta, Nippostrongylus brasilensis, Heligmosomoides polygyrus and H. contortus. Hsp-90 from all clade V species reacted positively with the anti-Brugia Hsp-90 antibody (Figure 1, panels a and c), but none bound to GA in the pull-down assay, whether from free-living or parasitic species (see Figure 1, panels b and d). Blots were exposed for extended periods of time but no positive reaction was observed, while the control B. pahangi lysate always yielded a strong positive result. A similar picture was observed with clade IV species, where Hsp-90 from Strongyloides ratti, two species of the plant parasite Globodera and the free-living Panagrellus redivivus all failed to bind GA (data not shown). For clade III nematodes, the situation was more diverse with Hsp-90 from all five filarial worms tested B. pahangi, Acanthocheilonema viteae, Dirofilaria immitis, Onchocerca ochengi and Litomosoides sigmondontis (result not shown) binding to GA (Figure 2, panel c). Hsp-90 from three ascarid species (Toxocara cati, Ascaris suum and Parascaris equorum) also bound to GA, but Hsp-90 from the related anisakids (Anisakis simplex and Pseudoterranova decipens) did not (Figure 2, panel d, lanes 9 & 10) while A. crassus failed to react with either antibody (Figure 2 panel c, lane 11). Where a positive signal was obtained in the pull-down assay, experiments were repeated in the presence of excess GA to block free binding sites. In all cases free GA blocked binding of Hsp-90 to the GA beads demonstrating the specificity of the interaction (data not shown).
Hsp-90 from clade V nematodes does not bind GA in a solid phase pull-down assay. Extracts of various nematodes were analysed by immuno-blots (panels a and c) and by GA pull-down assay (panels b and d). Equivalent amounts of protein (determined by Bio-Rad protein assay) were analysed by SDS-PAGE on 10% gels and transferred to NCP. Blots were probed with 1:5000 rabbit anti-Brugia Hsp-90 antibody followed by 1:10,000 HRP-labelled anti-rabbit IgG. Bound antibody was detected by chemiluminesence. GA pull-down assays (panels b and d) were carried out with 500 μg of worm lysate using control or GA beads. Control beads were negative in all cases and are not shown. GA pull-down assays were analysed by SDS-PAGE and immuno-blotting as described above. Lane 1, B. pahangi, lane 2, C. elegans N2, lane 3, C. elegans daf-21 mutant, lane 4, O. tipulae, lane 5, P. pacificus, lane 6, B. pahangi, lane 7, T. circumcincta, lane 8, N. brasiliensis, lane 9, H. polygyrus, lane 10, H. contortus.
Hsp-90 from some clade III nematodes binds to GA. Extracts of various nematodes were analysed by immuno-blot (panels a and c) and by GA pull-down assay (panels b and d). Equivalent amounts of protein (determined by Bio-Rad protein assay) were analysed by SDS-PAGE on 10% gels and transferred to NCP. Blots were probed with 1:5000 rabbit anti-Brugia Hsp-90 antibody followed by 1:10,000 HRP-labelled anti-rabbit IgG. Bound antibody was detected by chemiluminesence. GA pull-down assays (panels b and d) were carried out with 500 μg of worm lysate using control or GA beads. Control beads were negative in all cases and are not shown. GA pull-down assays were analysed by SDS-PAGE and immuno-blotting as described above. Lane 1, B. pahangi, lane 2, A. viteae, lane 3 D. immitis, lane 4, O. ochengi, lane 5, B. pahangi, lane 6, T. cati, lane 7, A. suum, lane 8, P. equorum, lane 9, A. simplex, lane 10, P. decipens, lane 11, A. crassus.
Two species belonging to clade I were tested, T. muris and T. spiralis but neither reacted with the anti-Hsp-90 antibodies. For these species and for A. crassus, GA pull-down assays were analysed by Coomassie Blue staining. For T. spiralis only, the GA pull-down assay revealed a band of approximately 83 kDa that was not present using control beads. The binding to GA beads of the protein isolated from the T. spiralis extract was competed by pre-incubation of the extract with excess free GA. The band was excised from the gel, digested with trypsin and subjected to mass spectrometry at the Sir Henry Wellcome Functional Genomics Unit, University of Glasgow. MASCOT searches revealed multiple peptide hits to Hsp-90 from a variety of species with a high degree of significance (P < 0.05). Thus T. spiralis Hsp-90 binds to GA, while the GA-binding status of A. crassus and T. muris Hsp-90 remains uncertain. Table 1 summarises the data from the GA pull-down experiments.
Table 1 Summary of nematode species analysed and GA-binding properties
Bioinformatic analysis of nematode hsp-90s
Hsp-90 sequences from a variety of GA-binding and non-binding species of nematode were either retrieved from public databases or were cloned and sequenced as described in Methods. An alignment of the predicted amino acid sequences is given in supplementary data [see Additional file 1] and highlights the degree of conservation of Hsp-90, particularly over the N-terminal ATP-binding domain. Table 2 shows the pairwise similarity and identity between sequences used in the bioinformatic analyses described below. Full genomic and cDNA sequences were available for six species, C. elegans, C. briggsae, P. pacificus, H. contortus, B. pahangi, and T. spiralis allowing the comparison of hsp-90 gene structure between the different species. Exon-intron boundaries were mapped by comparison of genomic and cDNA sequences and are shown graphically [see Additional file 2]. Three introns are present in C. elegans and C. briggsae and the position of each is conserved. These are also perfectly conserved with those of H. contortus (equivalent to introns 1, 5 and 8 of Hc-hsp-90), although here the Hc-hsp-90 gene contains a total of eight introns. B. pahangi contains the largest numbers of introns (eleven), the position of three of which are conserved with H. contortus. A single intron (number 8 in Hc-hsp-90) is conserved in five of the six species analysed, the exception being T. spiralis.
Table 2 Pairwise percent similarities (bold) and identities (italic) between Hsp-90 peptide sequences of fifteen different nematodes calculated under the BLOSUM62 matrix image, generated using MatGAT [45].
Identifying sites and lineages subject to adaptive evolution
For the gene-wide analysis, using either set of nested models of codon evolution (M1/M2 or M7/M8) did not identify any evidence of positive selection that was consistent across the phylogenetic tree (see Table 3). However, using the branch-sites methods which allow evolutionary rates to vary across the tree did show some interesting findings. Three branches showed significant evidence of adaptive evolution (dN/dS > 1) after Bonferroni correction (see Figure 3, labelled A, B, C). Along each of these branches, sites were detected with posterior probabilities > 0.95 of belonging to a dN/dS > 1 class (see Table 4). Along branch A (LRT of dN/dS > 1, p < 0.01) a single site 659S was identified (amino acid positions correspond to full length B. pahangi Hsp-90 AJ005785). On branch B (LRT of dN/dS > 1, p < 0.05), three sites with dN/dS > 1, 297S, 373S and 464S, were detected. On branch C, nine sites were detected 21S, 29S, 165V, 312S, 458S, 472Q, 485A, 525S and 584S. Importantly ten of these thirteen sites are serine residues which when examined are perfectly or largely conserved across all species in the alignment [see Additional file 1]. The identification of these ten sites as dN/dS > 1 results from the serine being encoded by either codons TCn or AGy. As the codon model of evolution assumes that mutations within a codon are independent, the change from TCn-AGy is parsimoniously interpreted as two separate non-synonymous substitutions (Z. Yang personal communication): e.g. TCC (Ser)->ACC (Thr)->AGC (Ser). The biological interpretation of these results suggests that the conservation of serine at these positions is functionally important and that this may be evidence for divergent evolution with rapid convergence back to the functionally stable serine residue. However, this cannot be proven by comparison of the extant species available to us and these sites are not considered further here. The remaining codons 165V, 472Q and 485A are inferred to be evolving rapidly along the lineage separating the plant parasitic nematodes from the animal parasitic and free-living species studied (branch C, Figure 3). Site 472Q is located in the middle region of the Hsp-90 protein and the codon is a conserved cysteine in the four plant parasitic nematodes and glutamine in all other species tested. Site 485A is also located in the middle region of the protein and is glutamine in the plant parasitic species and alanine in all other species. Only a single site, 165V, is predicted to be evolving under positive selection and is also located in the N terminal GA binding region of Hsp-90. Site 165V is a valine residue in the GA-binding species (L. sigmodontis, B. pahangi, A. sum and T. cati) but also in the non-GA binding P. pacificus and H. contortus. The residue is a phenylalanine in C. elegans and tyrosine in other Caenorhabditids analysed. To determine if these initial findings are consistent with the theory that GA-binding is associated with rapid diversifying evolution of the hsp-90 gene, we used a modified branch-site approach [18] to a priori separate the phylogenetic tree into two groups, GA-binders (B. pahangi, L. sigmodontis, A. suum and T. cati) vs non-GA-binders. Repeating the branch-sites analysis failed to provide significant evidence to suggest that an elevated dN/dS ratio associated with adaptive evolution was present in either of the two groups (see Table 5).
Unrooted phylogenetic tree showing relationship of hsp-90 sequences from fifteen different nematode species. The tree was generated using phyML [37] under the HKY substitution model. 1000 bootstrap replicates were performed and node labels represent percent bootstrap support. Branches A, B and C were shown to have significant evidence of positive selection. Scale bar shows substitutions per site.
Table 3 Statistical analysis of sites subject to adaptive evolution using nested models of codon evolution (M1/M2 or M7/M8)
Table 4 Statistical analysis of branches showing significant evidence of adaptive evolution
Table 5 Branch-site analysis of hsp-90 sequences grouped according to GA-binding status
The results of our analysis of Hsp-90 from a range of parasitic and free-living nematodes has revealed an interesting heterogeneity in the ability to bind GA. None of the free-living species tested, nor any of the parasitic species which have free-living larval stages in the environment bound to GA. These included animal parasites belonging to clade IV, such as Strongyloides, and those such as Haemonchus and Nippostrongylus belonging to clade V. These parasites are generally gut-dwelling and are transmitted between hosts when eggs, passed out in the faeces of the infected animal, hatch to release free-living larval stages. Following a period of development in the environment, the L3 stage infects a new host. The plant parasites tested, Globodera spp., also failed to bind GA. These are commonly known as potato cyst nematodes, and live in the soil where the infective second-stage juvenile worms (J2) attack the roots of susceptible plants. A cyst containing eggs is formed from the body of the dead female worm and under appropriate conditions these will hatch to release the J2 stage worms. These penetrate the roots, start to feed and eventually develop into adults.
The clade III nematodes analysed produced an interesting dichotomy in results, containing both GA-binding and non-binding species. Hsp-90 from the five filarial parasites assayed bound to GA; filarial worms are obligate parasites, transmitted between hosts by the bite of an infected intermediate host, such as mosquitoes or other arthropods. In keeping with this result, Hsp-90 from another obligate parasite T. spiralis also bound GA. T. spiralis is a clade I species commonly transmitted via ingestion of infected muscle containing the encysted L1 stages. Thus neither filarial worms nor T. spiralis have free-living stages in the environment.
The clade III Ascarid parasites also provided an intriguing comparison, as those worms which bound GA (Toxocara, Ascaris and Parascaris) all have free-living stages outside the host, but are enclosed within a thick impermeable egg-shell while in the environment. Transmission to the new host is by ingestion of the egg containing the infective larva or via a paratenic host (in some species such as Toxocara). Interestingly, in the original report on the isolation of GA [2], the in vivo efficacy of GA against the mouse pinworm Syphacia obvelata is recorded, suggesting that this parasite may also express a GA-binding Hsp-90. In keeping with this observation, pinworms belong to clade III (Oxyuroids) and are transmitted by ingestion of a resistant egg containing the infective larval stage.
Two of the three clade III species associated with the marine environment (Anisakis and Pseudoterranova) failed to bind GA. Both parasites have complex life cycles involving one or more intermediate and/or paratenic hosts. The free-living larval stages are ingested by marine crustaceans, which are then eaten by fish, which are subsequently ingested by marine mammals such as seals (Pseudoterranova) or cetaceans (Anisakis). A. crassus belongs to the superfamily Dracunuloidea and parasitises the swim bladder of eels, but also has a free-swimming larval stage. As extracts of A. crassus (and T. muris) failed to react with either antibody, the GA-binding status of these species remains unresolved. An additional proviso of our data is that in some cases, larval stages were analysed and it is possible that larval and adult worms may display different GA-binding characteristics.
Our data suggest that differences in the ability of nematode Hsp-90 to bind to GA are correlated with particular life histories. Only obligate parasites or those which have free-living stages in the environment enclosed within a protective egg-shell possess a GA-binding Hsp-90, while Hsp-90 from free-living nematodes or from those parasitic species that have free-living larval stages in the environment do not bind GA. The adaptive evolution hypothesis would argue that organisms sharing the same niche as the GA-producing Streptomyces should fail to bind GA and the results from the pull-down experiments broadly support this hypothesis. In order to further explore this theory, we analysed hsp-90 sequences from fifteen different species of nematode in an attempt to identify codons that have undergone adaptive Darwinian evolution within the GA-resistant species. The results of these analyses provided strong evidence for rapid adaptive evolution along three separate lineages and identified a number of residues showing significant evidence of adaptive evolution. However, the model suggests that ten of the thirteen codons identified as sites of adaptive evolution had undergone at least two separate non-synonomous substitutions resulting in the conservation of a serine residue, implying that these sites may be critical for Hsp90 function. In mammalian Hsp-90α and β, both serine and threonine residues are known to be targets of phosphorylation [19, 20], a modification that is essential for some functions of Hsp-90 [21, 22]. Of the remaining three residues (165V, 472Q and 485A) showing significant evidence of adaptive evolution (posterior probability > 0.95 of dN/dS > 1), only a single site, 165V, was located in the amino terminal section of the Hsp-90 protein, known to be the position of GA binding. However, variation at this site does not differentiate between GA-binding and non-GA binding species. The residue is a valine in the GA binding species, but also in the non-GA binding species P. pacificus and H. contortus. Comparison of the amino acid sequences of Hsp-90 from GA-binding species and non-binding species identified only four amino acid positions that are conserved within either the GA-binding or the non-binding species, but that differ between the groups. These are: K259 (K GA-binders, T non-binders), F447 (F GA-binders, Y non-binders), F475 (F GA-binders, Y non-binders), V624 (V GA-binders, I non-binders) (all amino acid positions correspond to full length B. pahangi Hsp-90 [Genbank:AJ005785]). However, none of these substitutions reside in the N terminal region. Comparison of each of these positions in Brugia and Saccharomyces cerevisiae, which binds GA [23], shows that the equivalent amino acids in yeast are conserved with the non-GA-binding nematode species. Thus whilst evidence for rapid diversifying selection exists in the evolution of the hsp-90 gene (as evidenced by the significance of the branch-site tests) and most likely occurred early in nematode divergence, we have been unable to prove with confidence that the selection seen correlates with a change in the GA-binding function of Hsp-90. As Hsp-90 is a multi-functional protein in eukaryotic cells, the observed rapid evolution of the hsp-90 gene likely correlates with some other key function, rather than the ability to bind GA.
In S. cerevisiae, point mutations in hsp-90 alter the sensitivity to GA and to Radicicol, an alternative inhibitor of Hsp-90 function [23]; e.g. A587T and T101I are hypersensitive to GA, with T22I, G313S and E381K showing a lesser effect. However, additional factors are also known to influence sensitivity to GA in yeast, including the presence of co-chaperones such as Sti1p (mammalian Hop) and Sba1p (mammalian p23). Yeast deficient in Sti1p or Sba1p show increased sensitivity to GA [23, 24]. C. elegans expresses homologues of both co-chaperones [25] and a recent study identified STI-1 as an Hsp-90 interacting protein in C. elegans [26]. However, whether sti-1 null animals have an altered response to GA has yet to be explored.
While analysis of daf-21 mutants and RNAi studies in C. elegans clearly show that Hsp-90 is essential in the worm, C. elegans is not susceptible to GA and, indeed, in the micro-environment of a Petri dish, can grow perfectly well when fed on a culture of S. hygroscopicus [5]. However, it remains unclear whether C. elegans would interact with the GA-producing S. hygroscopicus in the environment. Although both organisms may be present in the soil, whether they share the same spatial and temporal niches is unknown. Indeed, the ecology of C. elegans in the wild is not well understood, despite its extensive use in the laboratory [27]. Although C. elegans can be found in soil, it appears that the predominant stage observed in the wild is the dauer larvae, typically found in organic-rich environments such as compost, or in association with invertebrates such as snails and isopods [28]. Dauer stages are resistant and non-feeding and the natural food source of C. elegans in the wild is unknown. C. elegans possesses a sensitive chemosensory system that, at least in the laboratory setting, allows the worm to differentiate between bacterial food sources and potential pathogens [29]. Thus in the environment, worms could potentially avoid bacteria producing noxious compounds.
Many Hsp90 inhibitors are natural compounds produced by micro-organisms and recent studies have provided an intriguing insight into the mechanisms by which the organisms that produce Hsp90 inhibitors can themselves survive. For example, in the yeast Humicola fuscoatra, which produces Radicicol, a single conserved amino acid substitution (L33I) in Hsp-90, is sufficient to alter the binding of Radicicol, without influencing the ability to bind ATP or GA [30]. Here, a leucine to isoleucine substitution alters the size of the hydrophobic drug-binding pocket increasing the binding of H2O molecules and hydration of the drug. Such findings serve to emphasise that very subtle alterations in amino acid residues can have major implications on drug binding efficacy and highlight the importance of understanding the structural basis of drug binding, which is not always apparent from analysis of DNA sequences.
Our results demonstrate that C. elegans Hsp-90 is not unique in its low affinity for GA; despite a relatively high level of sequence conservation, nematode Hsp-90s show functional diversity in relation to GA-binding. The ability to bind to GA is associated with the life history of a species; free-living nematodes and those parasitic species which have a free-living larval stage in the environment do not bind GA, while obligate parasites or those parasitic species which have free-living stages enclosed within a resistant egg, do bind GA. As S. hygroscopicus, the micro-organism that synthesizes GA, is found in soil, and as Hsp-90 is an essential molecule in all eukaryotes, these observations could be consistent with adaptive evolution of hsp-90 sequences. Analysis of hsp-90 sequences from fifteen different nematode species provided evidence of rapid diversifying selection along three separate branches. However, the selection observed did not correlate with the ability to bind GA or not.
Worm material
The following species of free-living nematode were used C. elegans N2 strain, C. elegans strain JT6130, which carries a point mutation in daf-21 (hsp-90), Caenorhabditis briggsae, Oscheia tipulae, Pristionchus pacificus (all clade V, obtained from the C. elegans Genetics Center) and Panagrellus redivivus (clade IV). Parasitic species analysed included Trichinella spiralis and Trichuris muris (both clade I), and the following clade III species: Brugia pahangi, Dirofilaria immitis, Onchocerca ochengi, Acanthocheilonema viteae, Litomosoides sigmondontis, Toxocara cati, Ascaris suum, Parascaris equorum, Anisakis simplex, Pseudoterranova decipens and Anguillicola crassus. Clade IV species included Strongyloides ratti, Globodera rostochiensis and G. pallida. Clade V species included Nippostrongylus brasilensis, Heligmosomoides polygyrus, Teleodorsagia circumcincta and Haemonchus contortus. Mixed life cycle stages were used for all the free-living nematodes while for parasitic species, usage was determined by the availability of particular life cycle stages; in most cases adult males and females were analysed with the following exceptions: Globodera sp. J2 stages; H. contortus, L3; T. spiralis L1 muscle stage larvae; A. simplex and P. decipens L3. S. ratti adults were free-living rather than parasitic females [31] and for O. ochengi only male worms were used.
Maintenance of nematodes
C. elegans N2 strain, C. briggsae, O. tipulae and P. pacificus were maintained at 20°C on NGM plates with OP50 as a bacterial food source [32]. Worms were washed off plates with M9 buffer (3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4 in 1 litre ddH2O) and collected by centrifugation. P. redivivus worms were maintained at room temperature on plates containing an oat-based medium. The cooked oats were poured into plates and the worms placed on the surface. As their population blooms, the worms move onto the lid, from where they can be collected by rinsing the lids with M9 buffer. Worms were then washed several times in M9 buffer by centrifugation.
Adult stages of all parasitic species were obtained from their respective hosts and frozen as soon as possible at -80°C. As no signal on a western blot could be obtained using anti- Hsp-90 antibodies to probe extracts of adult H. contortus, L3 of this species were obtained by harvesting eggs from infected sheep faeces and allowing these to develop [33]. For Globodera species, cysts were rehydrated in tap water for several days and then hatched in tomato root exudate (kindly provided by J. Jones, Scottish Plant Breeding Institute, Invergowrie). Cysts were checked daily for hatched J2 stages. These were removed by pipetting, washed in ddH20 and purified from debris by flotation on 60% sucrose. For T. spiralis, muscle-stage larvae (L1) were obtained by digestion of infected mouse muscle using standard methods [34]. Larval stages (L3) of A. simplex and P. decipens were obtained from infected fish.
Preparation of worm lysates
Lysates of worms were prepared in ice-cold TNES buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM EDTA, 1% Igepal CA-630). Protease inhibitors were added prior to use (Complete Mini Protease Inhibitor Cocktail, Roche Diagnostics). Worms were ground to a fine powder using a pestle and mortar containing liquid Nitrogen. Extracts were resuspended in 1.0 ml TNES, the mortar was washed out with 0.5 ml TNES, and the extract was pelleted at 14000 g for 15 minutes at 4°C. The supernatant was retained and assayed for protein concentration at OD 595 nm using the Bio-Rad Protein assay. Lysates of D. immitis and O. ochengi were prepared using a small glass homogeniser containing 100 μl of ice-cold TNES and the supernatant processed as described above.
Preparation of GA solid support and binding assay
1 mg GA (InvivoGen) was derivatised using 1,6-hexanediamine to yield 17-hexamethylenediamine-17 demethoxygeldanamycin [4]. After aqueous extraction, the mixture was dried under nitrogen then re-dissolved in 400 μl of DMSO. The product was incubated with 400 μl of washed Affi-Beads (Bio-Rad) for 4 h in the dark at room temperature. Beads were washed in TNES and then were rocked overnight at 4°C in 1.0 ml of ethanolamine (1 M, pH 8.3) to block residual binding sites. Prior to use, beads were blocked in 1% BSA in TNES for 1 h at room temperature. Control beads were incubated with DMSO alone.
300-500 μg of total protein in a volume of 300 μl was mixed with 50 μl packed volume of GA or control beads for 2.5 hours at 4°C. The beads were washed extensively in TNES by centrifugation and bound proteins were eluted by boiling in SDS-PAGE sample cocktail (0.125 M Tris, pH 6.8, 0.2 M dithiothreitol, 4% SDS, 20% glycerol, 0.042% bromophenol blue). Aliquots of the input fraction and protein eluted from control and GA beads were run on 10% SDS-PAGE gels and analysed by western blotting. Blots were probed with a 1:5000 dilution of an antibody raised to a C-terminal fragment of recombinant B. pahangi Hsp-90, as detailed previously [6], followed by 1:10000 dilution of HRP-labelled anti-rabbit IgG (Sigma). Bound antibody was detected by chemiluminescence using the Super Signal ® West Pico Kit (Thermo Scientific). If no signal or only a weak signal was observed, blots were repeated using the AC88 Hsp-90 monoclonal antibody (a kind gift of D. Toft) as the primary antibody at 1:500 dilution followed by anti-mouse IgG (Sigma) at 1:10000. AC88 recognises an epitope in the C-terminus of Hsp-90 and reacts with Hsp-90 from many different organisms. In all experiments, a lysate of B. pahangi was used as a positive control. The specificity of GA binding was determined in subsequent experiments by pre-incubating worm extracts with 20 μM GA prior to incubation with the GA beads.
Sequence and bioinformatic analysis
hsp-90 sequences from B. pahangi [GenBank:AJ005785], H. glycines [GenBank:AF461150], M. hapla [GenBank:AAT35588], C. elegans [GenBank:C47E8.5] and C. briggsae [GenBank:CBG040560] were obtained from the NCBI. C. brenneri, C. japonica and C. remanei hsp-90 sequences were obtained by carrying out a tBLASTn search at http://www.nematode.net using the Ce-DAF-21 amino acid sequence to retrieve full length cDNA sequences for each Caenorhabditis species.
The full-length sequence of T. spiralis hsp-90 was identified by a tBLASTn search of the available genome sequence at http://www.genome.wustl.edu with the Ce-DAF-21 amino acid sequence. A significant match was obtained to contig 9.2, the sequence of which was obtained from Amy Yu at Washington University School of Medicine. For P. pacificus, the full-length hsp-90 sequence was obtained by tBLASTn search of the sequence available at http://www.pristionchus.org using the Ce-DAF-21 amino acid sequence, resulting in the retrieval of contig 4.9. For Trichinella and Pristionchus, intron and exon sites were identified using Genewise in EXPASY, allowing assembly of the full-length predicted cDNA sequence. For H. contortus, the hsp-90 sequence was assembled by carrying out a tBLASTn search at http://www.sanger.ac.uk using the Ce-DAF-21 amino acid sequence to retrieve contig _0076156, as detailed previously [35]. The sequence was submitted to GenBank [GenBank:FJ717747].
For A. suum a partial hsp-90 sequence was obtained by carrying out a tBLASTn search at http://www.nematode.net using the Ce-DAF-21 amino acid sequence and retrieving EST sequence kj94a07.y1, which contained 768 bp of 5' A. suum Hsp-90 sequence. A primer was designed to the 5' end of the gene, 5'-ATGTCTGAGCAACAGCCTGAAGGA-3' and used in combination with a series of degenerate primers, designed over the conserved MEEVD region, to amplify the full length hsp-90 coding sequence from adult A. suum cDNA. One 3' degenerate primer was more successful than others and was used in all subsequent reactions, 5'-ANTCNACNTCNTCCATNCGNGANGC-3'. Full length A. suum hsp-90 was cloned into pCR2.1 TOPO (Invitrogen) and sequenced by automated DNA sequencing using vector and gene specific primers (MWG Biotech) and assigned an accession number [GenBank:FJ824589].
For L. sigmodontis and T. canis partial hsp-90 sequences were obtained by carrying out a tBLASTn search at http://www.ebi.ac.uk using the Bp-HSP-90 amino acid sequence. A 721 bp EST was retrieved which contained 676 bp of 3' L. sigmodontis hsp-90 sequence. As the 5' sequence was missing, an SL1 primer was used (5'-GGTTTAATTACCCAAGTTTGA-3') along with a primer designed to the 3' end of the L. sigmodontis sequence, 5'-TTAATCAACTTCTTCCATCCTCG-3'. The full length L. sigmodontis hsp-90 was amplified from adult L. sigmondontis cDNA, cloned into pCR2.1 TOPO (Invitrogen), sequenced as above and assigned an accession number [GenBank:FJ824588].
For T. canis, an EST of 647 bp was identified containing the 5' region of the gene. As the only biological material available was from the sister species T. cati, PCR was carried out using a primer designed to the 5' end of the T. canis hsp-90 sequence (5'-ATGTCCGAGTTTCAACAACAACCAG-3') together with the degenerate 3' primer described previously (see above) on cDNA extracted from adult T. cati worms. The full-length T. cati hsp-90 was cloned into pCR2.1 TOPO (Invitrogen), sequenced as above and assigned an accession number [GenBank:FJ824590]. All sequences were compiled from multiple reads in both directions and line-ups and annotations were carried out using Contig express and Vector NTI.
Partial hsp-90 sequences of X. index (1325 bp), G. pallida (685 bp) and G. rostochiensis (1394 bp) were obtained by carrying out a tBLASTn search at http://www.ebi.ac.uk or http://www.nematode.net using the Bp-HSP-90 amino acid sequence. For those species for which both full-length cDNA and genomic hsp-90 sequences were available, intron/exon boundaries were identified in genomic DNA sequences using Genewise in EXPASY and their positions mapped onto cDNA sequences.
Peptide sequences were generated from translated nucleotide sequences of hsp-90 from seventeen species of nematode C. remanei, C. briggsae, C. brenneri, C. elegans, C. japonica, P. pacificus, H. contortus, L. sigmodontis, B. pahangi, A. suum, T. cati, M. hapla, Heterodera glycines, G. pallida, G. rostochiensis, T. spiralis and Xiphinema index. These were aligned using Prank [36] and the aligned peptide sequence was back translated to generate an aligned nucleotide sequence. A phylogenetic tree, generated with phyML [37] under the HKY substitution model [38], was used as the starting phylogeny for the codeML program of the Phylogenetic Analysis by Maximum Likelihood (PAML) suite [39]. A phylogenetic tree generated under the M0 model inferred that branches leading to two of the species, T. spiralis and X. index, exhibited much larger divergence (> 3 substitutions per codon). To avoid potential problems of saturation at high divergence these species were removed from further analyses. Sequences from the remaining fifteen species were realigned with Prank and a maximum likelihood phylogenetic tree was again generated using phyML. To determine if evidence for adaptive evolution (dN/dS >1) exists in these sequences, three tests of selection were conducted to compare the ratio of non-synonymous to synonymous substitutions (dN/dS). (1) Site analysis using a likelihood ratio test (LRT) to compare the codeML nested models M1/M2 and M7/M8 which assume a uniform evolutionary rate across all branches of the tree [40, 41]. (2) Branch-sites methods, which allow for differential rates of evolution across different lineages [7, 8]. Using the branch-sites methods each individual branch was analysed. (3) Species were also grouped into GA-binders or non-binders and compared using the branch-sites method, as such comparisons can improve the power of analysis if the underlying biological assumption to group the branches is sound [18]. In all cases multiple independent runs of the CodeML program were conducted (minimum triplicate) to ensure convergence of maximum likelihood estimates of parameters. Where multiple tests were conducted, the type I error rate was controlled by Bonferroni correction [42] (See Table 3). Hsp-90 amino acid sequences from nematode species used in the bioinformatic analysis were also aligned using ClustalW [43] and coloured using the default residue groupings of CHROMA [44].
Hsp:
GA:
geldanamycin
PAGE- sodium dodecyl sulphate-polyacrylamide gel electrophoresis
Ce:
B. pahangi
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This work was supported by grants to ED from the Wellcome Trust and the BBSRC. NH was supported by an award from the Government of Malaysia. The work would not have been possible without the assistance of many nematode biologists who generously donated material for analysis, including Prof J. Allen, University of Edinburgh, Dr I. Coombs, University of Glasgow, Dr J. Foster, New England BioLabs, Dr Colin Fleming, Agri-Food and Biosciences Institute, Belfast, Prof R. Grencis, University of Manchester, Prof W. Harnett, University of Strathclyde, Dr J. Jones, Scottish Crop Research Institute, Invergowrie, Prof D. Knox, Moredun Research Institute, Dr C. Lawrence, University of Strathclyde, Prof R. Maizels, University of Edinburgh, Dr B. Makepeace, Liverpool School of Tropical Medicine, Dr S. Martin, University of Aberdeen, Prof J. Matthews, Moredun Research Institute, Mr J. McGoldrick, University of Glasgow Veterinary School, Prof M. Viney, University of Bristol. We also thank Professor David Toft for the kind gift of AC88 antibody. We acknowledge Kerry O'Neill and Margaret McFadyen for general laboratory assistance, Gary Saunders for help with scaffolding the H. contortus sequence, and our colleagues Dr Collette Britton and Prof. Mark Viney (University of Bristol) for critically reading the manuscript and their helpful discussions. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR).
Parasitology Group, Institute of Comparative Medicine, School of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, UK
Nik AIIN Him, Victoria Gillan, Kirsty Maitland & Eileen Devaney
The School of Veterinary Medicine and Science, The University of Nottingham, Sutton Bonington Campus, Sutton Bonington, Leicestershire, LE12 5RD, UK
Richard D Emes
Nik AIIN Him
Victoria Gillan
Kirsty Maitland
Eileen Devaney
Correspondence to Eileen Devaney.
NH carried out the pull-down assays; VG identified and sequenced hsp90 genes; KM helped with the pull-down assays; RDE undertook the dN/dS analysis; ED conceived and coordinated the study. NH, VG, RDE and ED contributed to the manuscript. All authors provided comments on the manuscript and read and approved the final draft.
Nik AIIN Him, Victoria Gillan, Richard D Emes contributed equally to this work.
Comparison of Hsp-90 sequences from fifteen nematode species used for phylogenetic analysis
Additional file 1: . Consensus coloured alignment of nematode Hsp-90 amino acid sequences using default residue groupings of CHROMA. Sequence positions are numbered according to available protein data; see Methods section of manuscript for details of accession numbers. Positions of protein domains were determined by alignment of S. cerevisiae Hsp90. (PDF 129 KB)
Genomic structure of nematode
Additional file 2: hsp-90 genes. The figure shows intron positions in genomic DNA sequences of hsp-90 from B. pahangi, H. contortus, C. elegans, C. briggsae, P. pacificus and T. spiralis. Introns were mapped by comparison of cDNA and genomic DNA sequences. Intron positions were marked by hand and introns with common positions are shaded. Numbers refer to the length of cDNA sequences of each gene. (PDF 24 KB)
Him, N.A., Gillan, V., Emes, R.D. et al. Hsp-90 and the biology of nematodes. BMC Evol Biol 9, 254 (2009). https://doi.org/10.1186/1471-2148-9-254
Parasitic Species
Adaptive Evolution
tBLASTn Search
Obligate Parasite


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You are here: Home / Blogs / Get to Know the BOSS Act of the British Virgin Islands
Get to Know the BOSS Act of the British Virgin Islands
In order to comply with its obligation under the Exchange of Notes Agreement which it entered into with the UK back in April 2016, the British Virgin Islands (BVI) has enacted Beneficial Ownership Secure Search System Act 2017 (BOSS Act). The Act came into effect on 30 June 2017. The BOSS Act was almost immediately amended by the Beneficial Ownership Secure Search System (Amendment) Act 2017, which also came into force on 30 June. The BVI is an important offshore jurisdiction and international tax and law enforcement authorities believe that the corporate veil is used for fraudulent practices. The enforcement of the act is aimed to clamp down tax evasion, money laundering, and terrorism financing.
The Exchange of Notes Agreement facilitates the sharing of information relating to beneficial ownership between the BVI and the UK government. The BOSS Act paves way for the setting up of a safe system for storing, retrieving and providing access to such information. The Beneficial Ownership Secure Search System, known as BOSS System, will enable the authorities to have a searchable database of BVI entities and their ownership details.
The BOSS Act co-exists with the Anti-Money Laundering Act (AML Act); therefore, the entities are required to comply with the KYC obligations as prescribed under the AML Act. It is of interest to note that the AML Act requires entities to collect and record KYC on natural persons who directly or indirectly control 10% or more of the shares or voting rights of a legal entity.
Related article: Company Setup Requirements for British Virgin Island »
A beneficial owner is the natural person who ultimately owns or controls a corporate or legal entity and include, though not limited to:
a natural person who ultimately owns or controls, whether directly or indirectly, twenty five or more per cent of the shares or voting rights in the legal person;
in the case of a legal person, a natural person who otherwise exercises control over the management of the legal person;
in the case of a legal arrangement
the partner or partners who control the partnership;
the trustee or other person who controls the legal arrangement; or
the settlor or other person by whom the legal arrangement is made.
In case of insolvent entities that are undergoing liquidation, administration or administrative receivership, the natural person who is appointed as a liquidator, administrator or administrative receiver of the corporate and legal entity; or
in the case of a receiver being appointed over twenty five or more per cent of the shares or voting rights in a corporate and legal entity, the creditor who appoints the receiver; or
in the case of a shareholder who would otherwise be a beneficial owner but is deceased, the natural person acting as an executor or a personal representative of the deceased's estate.
Where two or more persons hold any qualifying interest jointly, whether as owners or tenants, then each of them is a beneficial owner.
It must also be noted that a person shall not be treated as beneficial owner merely for having the benefit of a security interest of the qualifying share or voting rights or for having commercial exposure to the financial performance of a registrable entity pursuant to financial derivatives or similar contractual arrangements.
What is a registrable entity
A registrable legal entity in relation to a corporate and legal entity is a legal entity which:
would be a Beneficial Owner of the corporate and legal entity if it were an individual; and
one or more of the following applies to it:
it is a legal entity which is an exempt person;
it is a legal entity the securities of which are listed on a recognised stock exchange;
it is a licensee or a foreign regulated person; or
it is a sovereign state or a wholly owned subsidiary of a sovereign state
Registrable Information
A Registered Agent must maintain the following information in the BOSS Register:
The particulars of each corporate and legal entity including
the name, including alternative names;
the incorporation number or its equivalent;
date of incorporation;
status;
registered address; and
With respect to each beneficial owner of the corporate or legal entity:
date of birth; and nationality.
With respect to each registrable legal entity of the corporate and legal entity:
details of the registrable legal entity as outlined in section A above
jurisdiction in which the registrable legal entity is formed;
the basis or bases upon which the legal entity is designated as a registrable legal entity;
where the registrable legal entity is a foreign regulated person, the name of the jurisdiction of regulation and the name of the foreign regulator; or
where the registrable legal entity is a sovereign state or a wholly owned subsidiary of a sovereign state the name of that sovereign state and (if applicable) wholly owned subsidiary.
With respect to an exempt person
the details of the exempt person as outlined in subsection (3)(a); and
the basis or bases upon which the exempt person is designated as
Who is exempted from BOSS?
An exempt person is a person who meets one or more of the following conditions:
a corporate and legal entity which is recognised, registered or otherwise approved as a mutual fund under the Securities and Investment Business Act, 2010 including an approved fund, an incubator fund, a public fund, a professional fund and a private fund;
a corporate and legal entity the securities of which are listed on a recognised stock exchange;
a licensee;
a corporate and legal entity which is a subsidiary of a corporate and legal entity that falls within paragraph (a) or (b); or
a corporate and legal entity exempted by regulations.
Confidentiality and Security of Data
It is not accessible to the general public. It is accessible only by designated persons who have passed security-vetting tests, have made an oath of confidentiality and are considered fit and proper by the BVI authorities. Such persons are bound by confidentiality.
The designated persons can have access to the system only from designated secure premises and a secure IT system within BVI.
The designated persons shall search the system only on formal requests made by senior authorities from the following BVI government bodies:
the Financial Investigation Agency,
the Financial Services Commission,
the International Tax Authority, or
the Attorney General's Chambers.
Other Key Points
The obligation of providing ownership details and any changes thereof shall rest on the BVI entities.
The Registered Agents (RA) shall take reasonable efforts to identify the Beneficial Owners and registrable entities of each corporate and legal entities for which they act as Registered Agents.
Registered Agents are not required to identify the Beneficial Owners who own the interest, directly or indirectly, in a registrable entity of a corporate/legal entity that is identified by the agent.
The RA should maintain information collected in the database for a period of five years following the dissolution of the entity or for five year after the entity ceases to be a corporate/legal entity.
The entities that have ceased to exist, or were struck off before 1 January 2016 need not comply with the BOSS Act.
No change shall be recorded in the case of a beneficial ownership when a bona fide dispute over the beneficial ownership of the said interest is under adjudication by a court or tribunal.
Failure to comply with the obligation is an offence that would attract a fine of up to US$250,000 or an imprisonment for a period of up to five years or both. In addition, providing false information can lead to a fine of up to US$75,000, up to 5 years' imprisonment or both.
An entity shall notify the registered agent of information relating to beneficial ownership or any changes to the information relating to the beneficial ownership within 15 days of identifying such person or entity. Penalties for not informing changes in the relevant information could be up to US$10,000.
Our Comments
The BOSS Act is not going to significantly impact the prevailing information sharing procedure. The RAs are already required to maintain a register of Beneficial Owners and BVI authorities can still request and gain access to the information contained in the register. Hence, the RAs need not take any additional efforts to meet their obligations under the new compliance system; however, the Act would streamline the establishment of a centrally-maintained database that is accessible in a secure environment by designated persons. Such a single platform will ensure that the information is reported promptly and are subjected to reporting, security and confidentiality standards.
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Trains run to schedule, but buses are prone to unexpected delays, so much so that two might arrive together. Well, here we are with that syndrome in the music of Cape Breton, Nova Scotia: we arrive at 2016 with not one, but two major books on its music culture: Liz Doherty's Cape Breton Fiddle Companion, and Mats Melin's One with the music: Cape Breton Step Dance Tradition and Transmission.
Why the two volumes come out so close together, and at this time, is of course that music, and writing about it, are more like the roads than railways, subject to interruption in the form of declines, revivals, unexpected turns and temporal hazards, and human decision-making. Both these books have had a lot of the unexpected to contend with, but their writers have somehow found and negotiated space amid high-pressure teaching of traditional music and dance, performance, travel, research and academic administration.
it has to be said, perhaps most amazingly, both of them are compiled by scholars based in universities here on the island of Ireland where they are teachers in Traditional music studies. Liz Doherty is perhaps the longest-serving lecturer in this field in Ireland at this point, and was the youngest to gain such a role when she began third level teaching 22 years ago in 1994; she has now been nine years in University of Ulster. Mats Melin has taught for 21 years; he is from Sweden, but his interest in Scottish dance took him to Glasgow in 1995, and he has been in University of Limerick for eight years. There is something in this of course, because of the strength of the resurgence of Traditional music in Ireland – its track record for more than a century, peaking, somewhat surprisingly, in the Pop era after the 1950s. Indeed, we already have quite a stimulus to new writing in the form of the considerable body of academic and practical literature on Traditional music, all of which can be accessed on the shelves at the State-financed ITMA archive in Dublin. We have taught the music formally up to post graduate level too, most seriously since the 1980s.
Canada of course has done all of this as well (Liz's book lists 440 publications) if perhaps not with quite the same cultural meaning or political impetus. But the music of Canada's fiddlers has been part of the soundscape of that country's social life and broadcasting for all of the 20th century, with many popular stars.
In that light, the compilation of these books by Limerick and Derry city lecturers and performers is a comment on the opportunities created by the mature out-looking of a confident – and State supported – Traditional music in both parts of Ireland rather than it being a failure of Cape Bretoners.
But even that does not strictly apply, for neither of these writers is quite an "outsider" at all.
Liz and Mats have in fact utterly absorbed themselves in the aesthetic and social fabric of Cape Breton music-making and dance, making it their cultural homeland for a quarter of a century; in a way they are the inverse of (but no different to) the many Cape Breton music exiles who spent much of their working and music lives in cities in Canada or the United States.
each presents a quite amazing narrative e of a small island community of only c. 140,000 people which has evolved intricate music and dance forms and styles which are now core to Cape Breton's identity and form a central plank of its tourism, a considerable economic boost to an economically-marginal region of Canada.
You read Mats and Liz as voices from within the music and dance communities, contemporaneous and historical at one and the same time. Their lists of performer biographies shows the particular relevance of music and dance to Cape Breton's local communities – receding, as everywhere in the mid-20th century, but reviving with determined commitment after the 1970s. Indeed, as documented in the books, the symbiosis of dance and music on Cape Breton island itself has retained an efficacy long after it has slipped in the Scottish place of origin of Cape Breton's music traditions. Particularly strong in this is the role of the piano: the appropriation of the supreme emblem of Western-music rationalisation by a music of the people, its many facets documented indeed in Liz's book.
Mats Melin's work is a tremendous window on every possible dimension of step dance and social dance: Transmission, teaching, meaning, aesthetics, tradition, dance structure, and biographies on key figures. Quotations from stylists reflect the breath, depth and time-span of his investigation; they are warm and powerful. Mats's own observations are no less profound, notably his assessment of the aesthetic space dance represents for its performers: "a home from home where one can feel spiritually refreshed or feel rooted".
He observes two styles of learning: the visual and the kinaesthetic, watching and feeling. In this regards he cites dance teacher Minnie Macmaster who would hold the hands of learners: "so they could feel the beat through my hands". And Harvey Beaton, who considers that: "Dancing should be a personal language … subtlety, rather than fancy steps".
Mats remarks on one dance class being held on a wooden bridge, so the dancers could feel the vibrations of their actions, and quotes anecdotes about dancers who, when travelling, might stop at a wooden bridge to have a bit of a dance, to feel the dance; He tells us too of lumbermen who when felling large trees would try out a dance on a freshly cut stump.
This draws in gender, change over time from this robust masculinity to the fact that dance in the home was in the past taught by the women, and in the 19th century classes were run by specialist male dance teachers, but now it is dominated by women.
Dance is not separated from music however, and their confluence and interconnectedness is noted in Mats' observations on the vocabulary, dancers' aesthetic keywords such as 'timing', which enabled: "ease of dancing and creating a lift that matches the swing of the music". He notes too the overlap of the kinaesthetic and the aural – speaking of how the varying volume of stepped-out rhythms were a mnemonics patterning, remarking on the superiority of particular tunes for which a dancer would have a special "take". His observations on changes since the 1970s are related to this – the disappearance of those very tune-specific dances, along with increasing complexity which is paralleled by weakening of aesthetic concepts.
Liz Doherty's volume also summarises social dance practices in Cape Breton, but as an encyclopedia: with compact entries in a massive four hundred pages of data on fiddlers, fiddling style, tunes, fiddle history, revival, professionalism and change – a profound document with 900 individual topics.
The Fiddle Companion encompasses repertoire, aesthetic absorption, dedication, regional pride, professionalism, technology, and ongoing composition that bridges the music of this small place to mainstream Popular entertainment. There is much here for performers, lovers and students and teachers of any music form, since well ahead of the post-1977 fiddle-revival period there is a well-established professionalism shown among Cape Breton musicians, some of whom were nationally regarded in Canada. The exile community in the USA and Canadian cities are in themselves a tremendous story, and the legacy, influence – and often family trees – are traced through from vaudeville and 78rpm recording to modern-day world touring bands and digital transmission. We appreciate too from this book the importance of family reputation and influence, with documentary on families of musicians over several generations, many of them key players in modern time: there are some 900 indexed names, quite a statistic for a music from a population of 140K playing a derivative of Scottish Traditional music.
Yet this is not nostalgia, or a document of past glory, it is about a contemporary music form that is part of the modern-day commerce of professional music making as much as it is about cultural identity and preference: broadcasting, recording, migration and media are a binding medium in this text, rendering understandable the huge array of performers, their family histories, instrumental preferences, fiddle and piano technique and range of tune types, making the huge spectrum, technology and aesthetic preferences understandable, a comprehensive narrative that fills the mind with a sense of richness among economic poverty, worth in the hardship of Cape Breton as a developing region, and cultural and economic value on the music in today's ruthless world. This book is nominally about Cape Breton, but like Mats Melin's it is also fundamentally about the artistic integrity of a regional artform, the human need to connect with relevant music-scapes which have long historic roots, the psychological value of music in modernity.
Both these books are vital reading for the understanding of any regional Traditional music. They have much to say to Irish musicians in particular, because we in this country have also had a lengthy, extensive Traditional music revival that spans casual, functional dancing in kitchens to display on university platforms.


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Written by Michael Simons on July 10, 2019 . Posted in Building in Costa Rica, Costa Rica Living, Costa Rica Service Providers, EX PATS in Costa Rica, Newsletters
Tank Tops Flip Flops Newsletter edition no. 134
Costa Rica Renewable Energy Solutions–Solaris Energia Renovable–EXPATS in Paradise with Glen Nickerson
Glen Nickerson originally set up shop in Costa Rica in the Central Valley in 2006. As he got to know the country and start expanding his business he soon found out that it was much better to live and do business at the beach areas. He fell in love with the Playa Hermosa, Guanacaste area and bought a nice condo within walking distance to the beach. He has continuously looked at ways that he could bring in his engineering know how into a practical energy saving business in Costa Rica. We are glad he and his wife are members of the community and look forward to see their business grow. Congratulations Glen on being another successful EXPAT in paradise. We had Glen replace all the light bulbs in our RE/MAX Tres Amigos office with energy efficient LED bulbs and we have seen a huge savings on our monthly electric bill. Thanks again Glen!
Here is the Story of Glen Nickerson and how he started Solaris Energy here in Costa Rica
Describe your Business and explain what makes your Business unique and better than other similar businesses
My name is Glen Nickerson and I am CEO and Business Development Director of Solaris Energy. Solaris is a Costa Rica renewable energy solutions provider. We help our customers reduce their energy bill by auditing the home or hotel and creating a plan, for example, to change lighting to LED technology. Solaris offers the highest quality and certified products from USA, Germany, Canada and China. We have top efficiency solar panels and we utilize the most efficient LED lighting with up to 160 lumens per watt. We have electric bicycles utilizing Samsung and Panasonic lithium batteries.
Check out this video where Glen describes how he came up with the idea and built his business in Costa Rica
Solaris is a one stop shop for LED lighting, solar panels, air conditioners, and solar hot water heaters, as well as home storage battery banks and powerful electric bicycles with lithium batteries. We have found that as customers become more aware of energy usage, their patterns typically change. Coupled with the solutions we install, customers can achieve a significant reduction in their monthly power bills. The most satisfying part is that we are enabling a low carbon footprint for Costa Rica with our solutions.
In this video Glen showcases some of the products he carries
Give us some background of yourself, describe why you decided to come to Costa Rica and how long you have been here
I'm a professional mechanical engineer from the USA (Florida, California, and Texas) and I worked for 40+ years as a utility design engineer and facility operations manager for large companies Motorola and AMD. After completing an MBA at the University of Texas, I also started up many ventures including financial planning, marketing, and business development. I came to Costa Rica in 2006 to find the next chapter for my family. I was amazed to find there weren't any solar panels on rooftops, and that LED lighting was not being utilized anywhere. Failing to settle on a traditional retirement routine, I polished up on my Spanish and started another business, focused on renewable energy and conservation methods. For the last six years, the energy company has grown and now we have 5 employees along with other collaborators.
Tell us what you like about Costa Rica, Guanacaste and this area in particular
Costa Rica has so much to offer, it has great people, clean air, pure water, all the foods you can imagine. I like to explore new areas and find new places. Depending on what time you get up, you can reach most any part of Costa Rica you desire in a day. The Guanacaste area has hundreds of beaches and mountains with lots of adventure opportunities, so I love the idea of being able to work and play, help people and still have time for a cold brew at the beach around sunset. Regarding the cost of living, depending on your lifestyle, it's still good compared to North American standards. Especially if you own your vehicle and your home, the rest can be handled easily.
What do you do for recreation, hobbies, etc.
I like boating, river rafting, zip lining, mountain biking, meeting new people, exploring and bringing new technology to Costa Rica. I like visiting new places, finding new beaches and waterfalls, and seeing what kinds of opportunities for adventures and hotels are available. I also can't resist evaluating the lighting fixtures of any place I go and talking with the owners!
Glen tells us why and how he came to Costa Rica and why he likes the Guanacaste area
Describe your experience with Michael Simons, Tank Tops Flip Flops and/or RE/MAX
Searching the RE/MAX website and all the home listings was how I began the search. Then I called on Michael Simons to assist with comparisons and we toured the beach areas. Michael was very knowledgeable about the entire region, the best spots, the best restaurants and shops, and he helped us find our beach condo. Mr. Simons was great…he truly helps people make a change…he is a problem solver as well as a generous volunteer with his many community activities.
To Contact Glen directly go to his website solarisenergycr.com or Facebook page facebook.com/SolarisenergiaCR He will definitely show you the options for Costa Rica renewable energy and how to save a ton off your electric bill.
COME ON DOWN BABY!
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<!DOCTYPE html>

    <meta charset="utf-8">
    <title>Device_Vestel</title>
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Audio

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Device_Vestel Class

                            Extends Device

                            Defined in: src\core\devices\vestel.js:6

This is extendable class for Vestel group devices

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                                    Device_Vestel

                                    constructor

                                    createVideoElement

                                    initEvents

                                    initPlayerClass

Constructor

Device_Vestel

                                ()

                                        Defined in
                                src\core\devices\vestel.js:6

Methods

createVideoElement

                                    ()

                                        Boolean

                                            Defined in
                                    src\core\devices\vestel.js:25

Abstract Player createVideoElement function.

Returns:

                                                        Boolean:

true

initEvents

                                    ()

                                        Object

Inherited from
                                            Device:
                                    src\core\device.js:29

                                        Events are based on class. So it have to be constructed.

Returns:

                                                        Object:
                                                    It returns Events class

initPlayerClass

                                    ()

                                        Object

Inherited from
                                            Device:
                                    src\core\device.js:42

                                        This function stands for initializing Player class
                            It uses currentDevice and config info (extended during device extending)
                            It pass Events to Player class to use same initialized class

Returns:

                                                        Object:
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My heart pours out to Meili, too, and the adversity with which the peasant women in China must live. This is a huge reason I am so engrossed in reading foreign fiction; they point to a way of life utterly new to me. The horror makes me simultaneously guilty and thankful for what I have in America.
Another great review, this sounds excellent.
Just finished this one – I wouldn't say I enjoyed it as such (how could you?), but it is a good book. More importantly though, this is a great example of what we were talking about earlier in the IFFP v BTBA discussion. This is a book about an issue, and I expect it to do well.
Chinese fiction is area where I'm oddly ignorant, so thanks for this excellent review Stu – must investigate further!
Great review, Stu. I haven't read Ma Jian (or much other Chinese fiction) before now so I'll be coming to this one quite fresh, so to speak.
I've read his Red Dust, there's a review at mine (as well as a few other Chinese books). He's an interesting writer.


Silver Color and Brushed elegant finish.
4 AC Power US Type, 4 RJ45, 2 VGA, 4 HDMI, 2 USB, Silver.
Cut out size: 257mmx166mm (10.11''x6.54'').
Depth of the av box: 3.15inch.
Conference Table Connectivity av Box YV-210S – Ideal for Conference Room Tables – SILVER COLOR.
Connectivity Box YV-210S – This conference table box provides multiple av connectivity and power options for your presentation needs. Tilt up, and connect your HDMI, VGA, USB, network, or video inputs. Conference table box power can also be added to charge your laptop or smart phone. We can do customizations! Choose from all different insert options we have in stock and receive just like you want it!
Connectivity Box YV-210S – Presenters can conveniently share content from their own device with the conference table connectivity box. The Yolkvisual YV-210s offers stylish integration into your modern boardroom or classroom with a discreet hideaway panel. For added convenience the YV-205 features a replaceable modules for you to have what you need where you need when you need it. HDMI, VGA, USB Type C, USB Data, AC Power, Audio Miniplug, Audio Canon, RJ45, RJ11 and more.
2 AC Power US Type + 1 HDMI + 2 RJ45 + 1 USB.
2 AC (fixed) + 3 module spaces to choose from.
Cut out size: 5.27'' x 4.6''.
Black Color and Brushed elegant finish.
Dual side connectivity av box.
Conference Table Connectivity av Box YV–210B – Ideal for Conference Room Tables – BLACK COLOR. Conference Table Connectivity av Box YV–210B.
Black color Part n YV-550.
Cut out size: 5.83'' x 4.80'' or 14.8cm x 13.5 cm.
Package dimension 12 x 7.10 x 6.9 inches.
Conference Table Cable Management Box – Cubby style YV-550 – BLACK COLOR.


Through partnerships with commercial river outfitters who are respected for their years of experience, focus on safety, and commitment to every detail that ultimately makes up the overall experience, GOALS is proud to offer river expeditions on several stretches of river, each with its' own unique personality. From the red sandstone desert to the coastal rain forest in the western US - and from Patagonia to Nepal, we're proud to dip oars in some of the most revered rivers on Earth. Please click below to learn more about each one.
Ruby & Horsethief Canyons of the Colorado River in Colorado & Utah.
Westwater Canyon of the Colorado River in Utah.


Rick Mandelson has broad industry sector experience and keen insight into the day to day needs of small to medium business owners and individuals, which is of immense value to clients.
Providing accountancy and taxation services to clients in the Southern Highlands, nationally and around the globe.
Keeping abreast of economic trends in Australia, having ongoing business interests in overseas markets and simply enjoying his craft, means that his clients gain the benefit of strategic and up to date information, coupled with the latest technology and communication to ensure responsive service.
The unpredictable nature of small to medium enterprises means vigilant monitoring and a flexible attitude.
There are a myriad of ways in which Rick can provide helpful and timely advice and be an excellent sounding board at the time of decision making.
Not just at year end, when the task of returns to be submitted are being completed, but through the life of the business enterprise, it's ups and downs and ever changing market forces.
This approach is as vital for individuals as it is for businesses as a change of circumstances can often reveal a need to review and reshape personal matters to suit the current legal and financial situation.
Clearly enjoying the role of working with his clients to meet their goals and providing the knowledge gained from practical hands on experience, Rick is always ready to actively listen and to provide possible solutions or initiatives for consideration.
Depending upon the nature of our client's specific needs we can provide a referral to a specialist, for example to Key Computers for IT and computer systems support, Sandra Bowley for Centrelink matters or Elizabeth Wilson, Finance Broker for other financial advice.
For business owners, or those looking for start – up planning and advice, you may find some helpful insights in the articles written by Rick that focus on improving the odds for success and thinking of starting your own business?


text:
Prossos - Prynear
Prough - Provasty
Prouix
Vernon A Prouix
Vernon A Prouix (born 1920)
Jefferson County, New York United States
Vernon A Prouix of Jefferson County, New York United States was born c. 1920. Family, friend, or fan, this family history biography is for you to remember Vernon A Prouix.
Biography ID: 153766911
Vernon A Prouix Vernon Prouix (born 1920)
Vernon Prouix's Biography
Family, friend, or fan this Collaborative Biography is for you to show & tell Vernon's life so that he is always remembered.
About Vernon
This section is to introduce Vernon Prouix with highlights of his life and how he is remembered. Select the pencil to add details.
White, Citizen
Where was Vernon born and where did he live?
2 Years Of High School
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Semiskilled Painters, Construction And Maintenance
Share highlights of Vernon's life. Experiences, organizations, & how he spent his time.
Military serial#: 32377666 Enlisted: July 6, 1942 in Syracuse New York Military branch: Branch Immaterial - Warrant Officers, Usa Rank: Private, Selectees (enlisted Men) Terms of enlistment: Enlistment For The Duration Of The War Or Other Emergency, Plus Six Months, Subject To The Discretion Of The President Or Otherwise According To Law
Living status
Vernon's average age compared to other Prouix family members is unknown.
The average age of a Prouix family member is 82.
Who is Vernon Prouix to you? Share memories and family stories, photos, or ask questions.
Find records of Vernon Prouix
Find records of Vernon
Vernon's immediate relatives including parents, siblings, partnerships and children in the Prouix family tree.
Vernon's Family Tree
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Source(s): National Archives: WWII Enlistment Records: 1938 - 1946
1920 World Events
Add Vernon's birthday or the date he died to see a list of historic events that occurred during Vernon's lifetime. Refresh the page for new events.
In 1920, in the year that Vernon A Prouix was born, in September, a bomb exploded in the J.P. Morgan bank building in New York City, killing 30 people immediately - 8 later died due to their injuries - and injuring another 200. Killing more people than the 1910 bombing of the LA Times (the deadliest terrorist act up until then), no one took responsibility and the perpetrators were never found. Italian anarchists were suspected of the bombing.
In 1955, in January, President Eisenhower sent direct aid to South Vietnam. In February, U.S. advisors were sent to train troops.
In 1971, in March, Intel shipped the first microprocessor to Busicom, a Japanese manufacturer of calculators. The microprocessor has since allowed computers to become smaller and faster, leading to smaller and more versatile handheld devices, home computers, and supercomputers.
In 1980, on April 24th, a rescue attempt was begun in the Iranian Hostage Crisis. The attempt failed and 8 US servicemen were killed. Eight helicopters had been sent for the mission, but only 5 arrived in operating condition., Since the military had advised that the mission be aborted if there were fewer than 6 helicopters, President Carter stopped it. Upon leaving, a helicopter collided with a transport plane and the men were killed.
In 1990, after 27 years in prison, Nelson Mandela, the leader of the movement to end South African apartheid was released on February 11th 1990.
Other Prouix Family Biographies
Irene (Prouix) Campbell
Rose Prouix
Mar 11, 1892 - May 1985
Papaskeena Prouix
Oct 14, 1898 - Jul 1970
Joseph A N Prouix
Born c. 1920
Help paint a picture of Vernon so that he is always remembered.
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We are thrilled to present Dr. Jeffrey M. Reutter with the 2015 Lifetime Acheivement Award!
In his 45-year career, Dr. Reutter has worked on Lake Erie to combat algal blooms, control invasive species, report climate change, and reduce chemical and phosphorous pollution. Reutter's work helped Lake Erie recover in the 1990s, and he remains dedicated to ensuring a healthy, safe Lake Erie for all.
Before his well-deserved retirement in 2015, Dr. Reutter directed the Ohio Sea Grant College Program, including Stone Lab, the Center for Lake Erie Area Research (CLEAR), and the Great Lakes Aquatic Ecosystem Research Consortium at Ohio State University.
At Stone Lab, Dr. Reutter and others hosted and taught hundreds of schoolchildren, graduate students, scientists, hunters and anglers, journalists, elected officials, industry representatives, environmentalists, and other interested members of the public.
Dr. Reutter also created and maintained a science, technology, engineering, and math program for youth with more than $1.3 million in endowments for scholarships and research equipment.
On Saturday November 14, 2015, we will present Dr. Reutter with the Lifetime Acheivement award at the OEC's Green Gala. We can't think of anyone more deserving! Please join us at that Gala to celebrate Dr. Reutter's enormous contributions to the health of our beloved Lake Erie.
Register today and meet Dr. Reutter in person!
Meet John Funk, our 2015 Excellence in Journalism Award Winner!


Arcobaleno means the rainbow in Italian, but if you've looked at Savinelli's colorful Arcobaleno Series you may have guessed that already! Vibrant greens, blues and browns pair with both smooth and rustic finishes to adorn many classic Savinelli shapes in a playful way. If you are looking to add a little whimsy to your afternoon smoke one of these Italian crafted smoking pipes could be just the piece you're after. Fans of the Alligator series will love the spin on old classics that a striking finish offers here. Choose your favorite shape and color below, we dare you to try to pick out just one favorite!


Carriage House Exterior Lights - This is the latest information about Carriage House Exterior Lights, this information can be your reference when you are confused to choose the right design for your home.
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