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Diverse cell junctions with unique molecular composition in tissues of a sponge (Porifera)
Jennyfer M. Mitchell1 nAff2 &
Scott A. Nichols ORCID: orcid.org/0000-0002-4765-754X1
The integrity and organization of animal tissues depend upon specialized protein complexes that mediate adhesion between cells with each other (cadherin-based adherens junctions), and with the extracellular matrix (integrin-based focal adhesions). Reconstructing how and when these cell junctions evolved is central to understanding early tissue evolution in animals. We examined focal adhesion protein homologs in tissues of the freshwater sponge, Ephydatia muelleri (phylum Porifera; class Demospongiae). Our principal findings are that (1) sponge focal adhesion homologs (integrin, talin, focal adhesion kinase, etc.) co-precipitate as a complex, separate from adherens junction proteins; (2) that actin-based structures resembling focal adhesions form at the cell–substrate interface, and their abundance is dynamically regulated in response to fluid shear; (3) focal adhesion proteins localize to both cell–cell and cell–extracellular matrix adhesions, and; (4) the adherens junction protein β-catenin is co-distributed with focal adhesion proteins at cell–cell junctions everywhere except the choanoderm, and at novel junctions between cells with spicules, and between cells with environmental bacteria. These results clarify the diversity, distribution and molecular composition of cell junctions in tissues of E. muelleri, but raise new questions about their functional properties and ancestry.
Beyond simply gluing cells together, cell adhesion molecules are dynamically regulated during development and cell migration, spatially regulated in polarized tissues, and involved in cell signaling and mechanotransduction [1,2,3,4,5,6,7]. Consequently, myriad adhesion mechanisms have evolved to function in different contexts in animals (e.g., [8]). Of these, two predominate: (1) the adherens junction, which is involved in cell–cell adhesion and is composed of cadherin receptors, p120-, α- and β-catenin, and (2) focal adhesions, which are involved in cell–extracellular matrix (ECM) adhesion and composed of protein such as integrins, vinculin, paxillin, talin and focal adhesion kinase (FAK).
The molecular components of both the adherens junction and focal adhesions are widely conserved in animals, and some of their components have origins outside of animals [9,10,11,12,13]. However, experimental studies of cell junction composition and function are largely restricted to bilaterian animals, such as the roundworm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and vertebrates. Recent studies demonstrate conserved roles for adherens junction proteins in cnidarians, as well [14,15,16].
Organisms of critical importance for reconstructing early steps in the evolution of animal cell adhesion mechanisms are the sponges (Porifera). They are one of the most phylogenetically divergent groups of animals [17, 18], their anatomy is fundamentally different from other animals [19], and there are long-standing questions about the structure and homology of their tissues compared to epithelia in other animals [20, 21]. It has been argued that cell adhesion in sponges (class Demospongiae, in particular) relies primarily upon an extracellular proteoglycan complex termed the Aggregation Factor [22,23,24,25,26,27,28]. Antibodies raised against the Aggregation Factor have been reported to block reaggregation of dissociated cells [29], and purified Aggregation Factor can mediate adhesion between beads in cell-free assays [30]. Consequently, the integrity of demosponge tissues is thought to depend upon the interaction of cells with the Aggregation Factor, an ECM component, rather than through cell junctions like those found in epithelia of other animals [31, 32].
The singular importance of the Aggregation Factor has been challenged by sequencing studies that have revealed conserved homologs of genes encoding adherens junction and focal adhesion proteins in diverse sponges [33,34,35]. Moreover, there is mounting experimental evidence that these proteins have conserved adhesion roles in sponge tissues. In the homoscleromorph sponge Oscarella pearsei (formerly O. carmela), a homolog of vinculin (common to adherens junction and focal adhesions in bilaterians) was detected at cell–cell and cell–ECM adhesions, and was found to interact with actin and talin in vitro [36]. Also, a yeast two-hybrid screen revealed conserved interactions between the adherens junction component β-catenin with a classical cadherin [37]. Likewise, in the freshwater demosponge Ephydatia muelleri, both a classical cadherin and α-catenin were detected as co-precipitates of β-catenin (Emβ-catenin), which localized to actin plaques at cell–cell contacts that resemble adherens junctions (Fig. 1) [38]. These data indicate that adherens junction and focal adhesion proteins may have conserved functions in sponge tissues.
(artwork in b adapted from Schippers and Nichols (2018), and in c adapted from Mitra et al. (2005))]
Adherens Junction- and Focal Adhesion-like structures in tissues of Ephydatia muelleri. a Top: Cells of the apical endopinacoderm contain bundles of actin filaments that culminate in dense plaques resembling spot adherens junctions at points of cell–cell contact. Bottom: Cells of the basopinacoderm contain bundles of actin filaments that resemble stress fibers of focal adhesions (white arrowheads). b Cross-sectional diagram of the juvenile E. muelleri body illustrating the distribution of Emβ-catenin (blue arrows indicate the direction of water flow in the aquiferous system). c Schematic illustration of the molecular organization of a focal adhesion. Proteins highlighted in red are the subject of the current study [FAK = Focal Adhesion Kinase
However, the study of Emβ-catenin has also revealed new peculiarities of cell adhesion in demosponges. Cells of the basopinacoderm (the tissue at the interface with the substrate) contain actin bundles that Max Pavans De Ceccatty [39] described as "devices for cell-to-substratum attachment." If the mechanisms of cell–substrate adhesion in sponges are conserved with other animals, one might expect that these are integrin-based focal adhesions. Instead, they were found to stain positive for Emβ-catenin, an adherens junction component [38].
To better understand the composition and organization of cell junctions in demosponge tissues, we examined the endogenous interactions and distribution of the focal adhesion proteins vinculin (Vcl), focal adhesion kinase (FAK) and integrin-β (ITGB) in E. muelleri. We found that this species has diverse adhesion structures composed of adherens junction and focal adhesion proteins, but these proteins are not as strictly partitioned to cell–cell versus cell–ECM junctions as they are in epithelial tissues of bilaterian animals. Instead, these proteins are often co-distributed in both contexts. Moreover, E. muelleri has specialized junctions not found in other animals; including cell–spicule junctions and cell–bacteria junctions. These data contribute to an increasingly complex narrative about the ancestral diversity and organization of cell junctions and their roles in early animal tissue evolution.
BLAST search [40] of the E. muelleri transcriptome [41] revealed highly conserved homologs of the primary protein components of focal adhesions. We detected seven integrin-β homologs, six integrin-α homologs, two talin homologs, and one homolog each of vinculin, focal adhesion kinase, and paxillin (Additional files 1, 2); integrins were numbered to reflect their relative expression levels, not to indicate their orthology to integrin subfamilies in other animals. We characterized the distribution of select focal adhesion proteins in E. muelleri tissues by co-immunoprecipitation and immunostaining with custom antibodies against EmVcl, EmFAK and EmITGB1.
Focal adhesion proteins co-precipitate from cell lysates
To test for conserved functional interactions between focal adhesion homologs in E. muelleri, we used co-immunoprecipitation (co-IP) coupled with liquid chromatography–mass spectrometry (LC–MS/MS) to identify proteins that co-precipitate with EmVcl, EmFAK and EmITGB1 from whole-cell lysates (Fig. 2). Anti-EmVcl recognized a specific band of the expected size by Western Blot of E. muelleri lysates used as input for immunoprecipitation. This band was depleted in the flow-through/unbound fraction and enriched in the precipitate elutions (Fig. 2a). Analysis of the immunoprecipitate identified this protein as EmVcl, and there were few detected co-precipitates (Fig. 2a′; Table 1; Additional file 3). Pre-adsorption of anti-EmVcl with 5 µg of the recombinant antigen completely abolished immunostaining signal (Additional file 4: Figure S1).
Immunoprecipitation results. a–c Western blot analysis of immunoprecipitation samples. Bands corresponding to the predicted molecular weight of each target protein are indicated with a red asterisk. Anti-EmITGB and Anti-EmFAK lost activity upon cross-linking to the resin, so precipitates were co-eluted with the antibody and gel slices excluding the antibody fraction were analyzed by LC–MS/MS (red boxes = gel fraction analyzed by LC–MS; blue boxes = antibody heavy chain). Equivalent gel slices were analyzed from the IgG negative control sample. a′–c′ Scatter plots showing the abundance of E. muelleri proteins detected in each precipitate relative to the IgG control. The proteins with the greatest abundance in each precipitate are indicated, with the target antigen highlighted in red. All samples were filtered to reflect only those hits within the 95% confidence interval, and represented by at least 5 unique peptides (input = whole-cell E. muelleri lysates, FT = lysate flow-through/unbound fraction, W = 1 M LiCl wash, P1-P4 = precipitate fractions 1–4)
Table 1 Abundance of focal adhesion proteins in immunoprecipitates
Anti-EmITGB1 had low affinity for multiple proteins in denatured E. muelleri lysates as detected by Western Blot, none of which matched the expected molecular weight of EmITGB (Fig. 2b). Immunoprecipitation with anti-EmITGB was hindered by cross-linking to the agarose resin, but was successful if the antibody was not cross-linked. Multiple bands were detected by Western Blot of the precipitate, one of which potentially corresponded to the expected size for EmITGB1 in the precipitate, eluted fraction 2. Analysis by LC–MS/MS of the high-molecular weight fraction of EmITGB1 precipitates detected each of EmITGB1, EmITGB2 and EmITGB4. Of these, EmITGB1 was most abundant. This antibody is subsequently referred to anti-EmITGB, generally, to reflect this cross-reactivity with other paralogs.
In contrast to the EmVcl co-IP, many other proteins were detected as co-precipitates of EmITGB1 (Table 1; Additional file 5). The most abundant protein in the sample was EmITGA1 (it was also detected at very low levels in the IgG control). Integrin-β is well known to heterodimerize with integrin-α, so it is probable that EmITGA1 is highly represented in the sample because it heterodimerizes with the multiple EmITGB paralogs recognized by the antibody. Other than EmITGB, the most highly abundant protein in the precipitate was a phosphodiesterase (possibly PDE8). This protein was nearly equally abundant to EmITGB and has a predicted molecular weight of ~ 86 kDa. PDEs are known regulators of cell adhesion, and direct interactions with integrins have been characterized. Other known focal adhesion proteins detected in the sample include EmFAK, EmTalin2, EmITGA1 and EmITGA3, strongly supporting conserved endogenous interactions between focal adhesion proteins in E. muelleri. Immunostaining signal was abolished upon preadsorption of anti-EmITGB with 1 µg of the injected antigen (Additional file 4: Figure S2).
Figure 2c illustrates that anti-EmFAK recognizes multiples bands in denatured cell lysates by Western Blot, including a band of the expected size. This band was depleted in the flow-through/unbound fraction, and enriched in the precipitate. Like anti-EmITGB, anti-EmFAK activity was disrupted by cross-linking, so immunoprecipitates were co-eluted with the uncrosslinked antibody. High-molecular-weight gel slices were used for LC–MS/MS rather than the entire antibody-saturated precipitate. In the fraction analyzed by proteomic analysis, EmFAK was found to be highly enriched in the anti-EmFAK precipitate, and absent from the IgG negative control sample (Fig. 2c′; Table 1; Additional file 6). Preadsorption of anti-EmFAK with 10 µg of the recombinant antigen fully abolished immunostaining signal (Additional file 4: Figure S1).
These data strongly support that all three antibodies used in this study specifically bind to their expected targets under native conditions in E. muelleri tissues and lysates. Only EmVcl gave robust and specific signal under denaturing conditions (Western Blot). These data also support that EmITGB functions as part of a complex with the focal adhesion proteins EmITGA, EmFAK, EmTalin and EmPDE8 [42]. Neither EmVcl nor EmPaxillin (a constitutive focal adhesion component in bilaterian animals) was detected in precipitates.
Focal adhesion-like structures may function in substrate attachment
To determine whether focal adhesion-like structures in the basopinacoderm (Fig. 1) are involved in cell–substrate attachment, it was important to distinguish whether they form at the interface between the tissue and the substrate (coverslip), or between the tissue and the mesohyl (ECM-filled interior of the sponge). To test this, we used Total Internal Reflection Fluorescence (TIRF) Microscopy [43, 44] and found that junction-associated stress fibers [45] were within 100–200 nm of the coverslip, near the substrate–adjacent cell membrane (Fig. 3a)—consistent with a role in cell–substrate attachment. We further reasoned that shear forces associated with water flow and turbulence should lead to an increase in the number of focal adhesion-like structures, as mechanical tissue stress induces focal adhesion formation in cultured vertebrate cells [46]. To test this, we grew sponges in dishes on a rocking platform for comparison with sponges grown in dishes on a stable platform. As shown in Fig. 3b, a 43% increase was detected in sponges grown on a rocking platform (n = 12, p value = 0.0058).
Actin stress fibers in the basopinacoderm may be associated with cell–substrate adhesions. a Total Internal Reflection Fluorescence (TIRF) imaging was used to determine the subcellular localization of focal adhesion-like structures in the basopinacoderm. b Their abundance was quantified in individuals grown on a stable surface and compared to individuals grown on a rocking platform
Three different types of focal adhesion-like structures in the basopinacoderm
Further examination of focal adhesion-like structures in the basopinacoderm revealed three distinct categories (Fig. 4). The first category included the actin filaments detected by TIRF at the substrate–adjacent cell membrane (Fig. 4a), which we termed 'ventral adhesions'. The second category resembled ventral adhesions, but with one or both ends terminating at a membrane invagination or vesicle-containing bacteria (Fig. 4b). We termed these 'bacterial adhesions'. A third category of actin filaments was found to span vertically from the substrate–adjacent (ventral) cell membrane to the mesohyl–adjacent (dorsal) cell membrane. These actin filaments formed prominent plaques on the dorsal surface of the cell (Fig. 4c) that we termed 'dorsal adhesions'.
Three types of focal adhesion-like structures in basopinacoderm. a–c Basopinacocytes (cells of the substrate-attachment epithelium) are drawn at the top in profile view, with the mesohyl interface (dorsal surface) at the top and substrate interface (ventral surface) at the bottom. Bundles of actin filaments were found at a ventral surface, and b were sometimes associated with membrane invaginations containing bacteria (white arrowheads). c Actin staining, colored to depict pixel depth within the confocal stack. White arrows indicate dorsal adhesions, from which actin filaments descend ventrally within cell (a, b: red = actin; cyan = DNA)
Immunostaining of adhesion proteins at these three different categories of focal adhesion-like structures revealed that they were compositionally distinct. Only EmVcl was found to be associated with ventral adhesions (Fig. 5), whereas both Emβ-catenin and EmVcl were consistently detected at bacterial adhesions (Fig. 6), and only EmITGB was detected at dorsal adhesions (Fig. 7). The staining patterns of EmFAK were inconsistent in the basopinacoderm, and difficult to discern due to high levels of cytosolic staining. For example, EmFAK was not usually detected at focal adhesion-like structures (Figs. 5, 6, 7), except rarely at bacterial adhesions (Additional file 4: Figure S2).
Immunostaining of ventral adhesions in the basopinacoderm. Both Emβ-catenin and EmVcl exhibited faint cell boundary staining (black arrowheads), but only EmVcl was detected in association with ventral adhesions (white arrowheads) (a–d antibody staining only; a′–d′ antibody = green, DNA = blue, actin = red; scale bars = 10 µm)
Immunostaining of bacterial adhesions in the basopinacoderm. Both (a) Emβ-catenin and (b) EmVcl were detected at the interface of stress fibers and membrane pockets containing environmental bacteria. Neither (c) EmFAK nor (d) EmITGB were detected at these structures (but see text for further discussion of EmFAK) (a–d antibody staining only; a′–d′ antibody = green, DNA = blue, F-actin = red; scale bars = 10 µm]
Immunostaining of dorsal adhesions. Neither (a) Emβ-catenin, (b) EmVcl, nor (c) EmFAK were detected at mesohyl-interfacing plaques (white arrowheads) of dorsal adhesion stress fibers. Visible staining of Emβ-catenin corresponds to a bacterial adhesions (black arrowhead) and of EmVcl corresponds to ventral adhesions (Asterisk). In contrast, d EmITGB was highly enriched at dorsal adhesions (a–d antibody staining only; a′–d′ antibody = green, DNA = blue, F-actin = red)
Cell junctions at the spicule interface
In many sponges, spicules are a unique component of the ECM. Spicules are siliceous skeletal elements that act as tent pole-like tissue supports. Specialized transport cells attach to spicules and move them into position [47] where they are anchored by collagen at rosette-shaped clusters of cells in the basopinacoderm [47].
We consistently detected cell junctions at the interface of cells and spicules, which stained positive for both Emβ-catenin and EmVcl. Again, EmFAK had a low signal–noise ratio, making it difficult to determine if it was present at these structures. EmITGB was not detected (Fig. 8). It was unclear if the cells forming these structures were transport cells, basopinacocytes, or a different cell type altogether.
Immunostaining of cell–spicule junctions. a Emβ-catenin and b EmVcl localize to cell junctions at the interface with spicules (dotted lines mark spicules). c EmFAK was possibly enriched at these structures, but only marginally above background levels (arrowheads), whereas d EmITGB was not detected at all (a–d antibody staining only; a′–d′ antibody = green, DNA = blue, F-actin = red; scale = 10 µm)
No evidence for focal adhesion-dependent cell migration
Focal adhesions have well-characterized roles in the migration of cultured vertebrate cells, where they provide traction needed for movement across two-dimensional surfaces. However, movement in three-dimensional environments is often less dependent on integrin-mediated adhesion [48]. We have previously shown that migratory cells in the three-dimensional environment of the mesohyl (Video in Additional file 7) of O. pearsei stain positive for vinculin [36], but neither EmVcl, EmFAK, nor EmITGB was detected in migratory cells of E. muelleri (Additional file 4: Figure S3).
Both Emβ-catenin and focal adhesion proteins are present at cell–cell junctions
We previously reported cortical staining of Emβ-catenin in the basopinacoderm, choanoderm, and the apical endopinacoderm (the inner tissue layer of the sponge surface; see Fig. 1) [38]. As shown in Fig. 5a, Emβ-catenin was again detected at the cell cortex in the basopinacoderm, but we also detected EmVcl (Fig. 5b) and less frequently EmITGB (Additional file 4: Figure S4). This staining was generally of low intensity and patchy, and we wondered whether this might reflect the developmental stage of the immature juvenile tissues examined. To test this, we grew sponges for an additional 3 weeks and found markedly elevated levels of cortical staining and robust adherens junction-like structures that were EmVcl-positive (Additional file 4: Figure S5). This result indicates that low-intensity cortical staining of EmVcl (and other antibodies) may reflect the early developmental stage of the tissues and that cell–cell adhesions strengthen over time.
We detected focal adhesion proteins at cell–cell junctions in other tissues, as well. The most conspicuous cell–cell junctions in E. muelleri are found in the apical endopinacoderm at points where actin tracts align between neighboring cells (Fig. 1b). Emβ-catenin was previously detected at these structures [38], and we found that EmVcl, EmFAK and EmITGB were also constitutively present (Fig. 9). Cortical staining of EmITGB was also detected in an adjacent tissue, the exopinacoderm (Fig. 9d)—this is the outermost tissue and is so close in proximity to the apical endopinacoderm that they cannot be separately resolved by confocal microscopy.
Immunostaining of cell–cell junctions in the apical endopinacoderm. Emβ-catenin, EmVcl, EmFAK and EmITGB were detected at probable adhesion plaques where F-actin tracts align between adjacent cells (white arrowheads). Low-intensity staining of EmITGB was also detected at the cell cortex (black arrowheads) of the apical exopinacoderm (a–d antibody staining only; a′–d′ antibody = green, DNA = blue, F-actin = red; scale = 10 µm)
The detection of EmVcl and EmFAK at cell–cell junctions is not unprecedented. Applied force on E-cadherin has been shown to lead to phosphorylation of human vinculin at Y822 and recruitment to the adherens junction [49, 50]. However, alignment of EmVcl to human vinculin revealed low conservation in the region that contains Y822 (Additional file 4: Figure S6), making it difficult to predict if this mechanism for regulating vinculin function is conserved in E. muelleri. But, EmVcl was not detected as a co-precipitate of Emβ-catenin [38], nor did we detect adherens junction proteins as co-precipitates of EmVcl (Additional file 3).
Like vinculin, focal adhesion kinase has also been reported to function in contexts other than focal adhesions, including at the adherens junctions of vascular endothelia, where it directly binds VE-cadherin and phosphorylates β-catenin in response to VEGF activation [51]. However, EmFAK was not detected as a co-precipitate of Emβ-catenin [38], whereas it did co-precipitate with EmITGB (Table 1). We treated sponges with 5-µM FAK inhibitor 14 and found that this treatment abolished FAK staining at cell–cell junctions, but detected no other effects on the formation or molecular composition of adhesion structures in the apical endopinacoderm (Additional file 4: Figure S7).
The only tissue where we did not find evidence of co-distributed adherens junction- and focal adhesion proteins at cell–cell contacts was the choanoderm (Additional file 4: Figure S8). In this tissue, Emβ-catenin alone was detected [38].
Most animal cell adhesion proteins evolved early, concurrently with or before the transition to multicellularity [9, 12, 31, 34, 35, 37, 52,53,54]. However, our understanding of how these proteins functioned ancestrally, and when they were organized into interacting complexes (i.e., cell junctions) is limited. We examined the interactions and distribution of focal adhesion proteins in tissues of E. muelleri. We found that they co-precipitate as a complex from cell lysates and that EmVcl, EmFAK and EmITGB localize to apparent cell junctions, supporting the conclusion that they have conserved adhesion roles. However, we also detected a critical difference in the spatial distribution of these proteins in E. muelleri tissues compared to epithelia in other animals. Rather than being restricted to focal adhesion-like structures at cell–ECM contacts, they were also detected at adherens junction-like structures at cell–cell contacts, and were often co-distributed with the adherens junction protein Emβ-catenin.
In a prior study, we reported a similar anomaly: Emβ-catenin localizes to focal adhesion-like structures in the basopinacoderm of E. muelleri [38]. Here, we found that these structures can be parsed into structurally and compositionally distinct categories: ventral, dorsal, and bacterial adhesions. When taken into consideration, we found that Emβ-catenin was not actually associated with either ventral or dorsal adhesions. Instead, these structures, respectively, stained positive for EmVcl and EmITGB, consistent with their homology to focal adhesions in bilaterian tissues. The absence of EmITGB staining at EmVcl-positive adhesions does not necessarily indicate the absence of integrins altogether, as our antibody recognized only three of seven identified paralogs. In the future, the focal adhesion protein talin may serve as a more universal marker of integrin-distribution, as it is constitutively present at all integrin-based adhesions in other animals, and was detected as a co-precipitate of EmITGB from E. muelleri lysates.
The previously reported Emβ-catenin staining at focal adhesion-like structures in the basopinacoderm is actually restricted to bacterial adhesions. In this context, Emβ-catenin is co-distributed with the focal adhesion protein homologs, EmVcl and (sometimes) EmFAK. The functional significance of these junctions is unknown, but intriguing. In natural environments, bacterial biofilms are abundant and often provide settlement cues for the larvae of aquatic animals, including sponges [55]. Thus, it seems plausible that bacterial adhesions could be involved in environmental sensing through integrin-mediated signaling. An alternative possibility comes from the observation that the bacteria at these structures are encapsulated in membrane invaginations or vesicles. Most sponge cells are phagocytic [56], and perhaps bacterial adhesions are involved in phagocytosis. This could be an undescribed mode of feeding, or function in the uptake of intracellular symbionts or pathogens. There is precedent for such a mechanism in vertebrates, where integrins are known to be involved in phagocytosis of particles, including microorganisms as part of a pathogen defense system [57, 58]. A difference is that β-catenin is not detected at these structures in vertebrates.
In the future, it will be interesting to identify the bacterial species at these structures, track their fate in sponge cells, and test for bacterial adhesions in attached larvae undergoing metamorphosis. Animals evolved in an environment dominated by bacteria [59], and a compelling hypothesis is that cell adhesion molecules may have first evolved to mediate interactions with bacteria [53].
A mixture of adherens junction and focal adhesion proteins was also detected in adhesion contexts other than bacterial adhesions. Specifically, focal adhesion proteins were co-distributed with Emβ-catenin at cell–spicule junctions, which essentially have the same composition as bacterial adhesions, and cell–cell junctions. The latter had previously been interpreted as probable adherens junctions [38], but until the role of focal adhesion proteins (particularly integrins) at these structures is clarified, this conclusion is less certain.
The co-distribution of Emβ-catenin with EmVcl, EmFAK and EmITGB could indicate that these proteins are part of a common adhesion complex in E. muelleri tissues, but our immunoprecipitation results do not support this view. Focal adhesion proteins co-precipitate as a complex, just as adherens junctions proteins were found to co-precipitate [38]; each to the exclusion of the other. Also, Emβ-catenin alone was detected at cell–cell contacts in the choanoderm, and focal adhesion proteins alone were detected at ventral and dorsal adhesions in the basopinacoderm. This indicates that these protein complexes are functionally separable and may have discrete roles, even where they are co-distributed.
Full characterization of demosponge cell junctions will require further identification of associated adhesion receptors (e.g., cadherins), and integration of these data with models of Aggregation Factor-mediated cell adhesion. One clue to how these adhesion systems may interact is that the Aggregation Factor has reported RGD motifs, leading to the hypothesis that it may activate integrin signaling [31, 60]. But, the Aggregation Factor has predominantly been studied in vitro, in cell dissociation/re-aggregation assays. Its distribution is not well characterized in intact tissues.
An important consideration is that, until recently, hypotheses about the evolutionary origin of animal cell adhesion mechanisms have been inadvertently biased toward bilaterian models. As we begin to examine cell adhesion in non-bilaterian lineages, there appears to be more mechanistic diversity than anticipated. For example, the cnidarian Nematostella vectensis has a conserved classical cadherin/catenin complex [16], but β-catenin is not always detected at cadherin-positive cell–cell adhesions in tissues [15, 61]. Likewise, sequence analyses of ctenophores indicate that they lack conserved cadherin/β-catenin interaction motifs [52], and a recent study indicates that β-catenin is altogether absent at cell–cell contacts in Mnemiopsis leidyi [62]. The molecular composition of cell junctions in placozoans is entirely uncharacterized, but from an ultrastructural perspective they resemble adherens junctions [63]. Placozoans altogether lack cell–ECM junctions and a basal lamina [64]. A comprehensive understanding of the timing and sequence of cell junction assembly and the evolution of epithelia will require detailed studies of adhesion in diverse non-bilaterian tissues.
This study supports that adherens junction and focal adhesion proteins functioned in adhesion and tissue organization in the last common ancestor of sponges and other animals. This stands in apparent contrast to studies which have emphasized the Aggregation Factor as the predominant adhesion mechanism in demosponges. At least in E. muelleri, tissues appear to be organized much more like epithelia in other animals than previously appreciated. However, in contrast to cell adhesion properties that E. muelleri shares in common with other animals, we also discovered new differences. Adherens junction and focal adhesion proteins are not strictly partitioned into compositionally distinct cell–cell and cell–ECM junctions; rather, they are often co-distributed. Furthermore, the discovery of specialized cell–bacteria junctions raises new questions about the functional significance of these structures for sponge physiology (environmental sensing, feeding, symbiosis, or pathogen defense), and possibly about the ancestral role of cell adhesion molecules in animals. If the interaction of cell adhesion proteins with bacteria is an ancient feature of animal biology, bacterial adhesions in sponges may provide clues to the nature of these interactions.
Identification of focal adhesion protein homologs in E. muelleri
Representative sequences of the focal adhesion proteins integrin-α, integrin-β, vinculin, talin, focal adhesion kinase and paxillin were retrieved from Uniprot [65] and used to query the Ephydatia muelleri transcriptome [41] by BLAST search [66] to identify candidate sponge homologs. The putative domain composition of E. muelleri sequences was then annotated using HMMER [67] and SMART [68] web-servers. E. muelleri vinculin was previously distinguished from its close paralog α-catenin by phylogenetic analysis [36].
Ephydatia muelleri gemmules were collected from "upper" Red Rock Lake, Colorado, USA (40.0802, − 105.543) in early October. This lake is several hundred meters southwest of Red Rock Lake, Boulder County and is unnamed. Gemmules were stored in autoclaved lake water, in the dark at 4 °C. Before plating, gemmules were washed in 1% hydrogen peroxide for 5 min, washed three times in autoclaved lake water and grown at room temperature.
Cloning and recombinant protein expression
The coding sequence of target antigens was amplified by polymerase chain reaction (PCR) from an E. muelleri cDNA library using Phusion High-Fidelity DNA polymerase (NEB). Primer sequences and amplicons are specified in Additional files 1, 2. PCR products were cloned into pET28a (Novagen), pET28 His6 Sumo TEV LIC (1S) #29659 or pET His6 GST TEV LIC (1G) #29655 (Addgene) for expression.
Expression constructs were validated by Sanger Sequencing (Eurofins) and transformed into a protease deficient Escherichia coli strain (Rosetta 2(DE3), Promega). For expression, a single colony was grown in Luria Broth at 37 °C to an OD600 between 0.4 and 0.6, and then induced with 300 mM of isopropyl-1-thio-β-d-galactopyranoside (IPTG) for 3–5 h at 30 °C. Bacterial pellets were collected by centrifugation, resuspended in 1 × PBS pH 7.4 on ice. Cells were lysed by the addition of 1 mg/mL lysozyme and 0.2 mM phenylmethanesulfonyl fluoride (PMSF), incubation at room temperature (RT) for 15 min, then sonication for 4 × 30 s. Bacterial debris was removed by centrifugation and the supernatant incubated with either with HisPur Cobalt or Nickel Resin (Thermo Fisher Scientific) for His-tagged proteins or GST-agarose resin (Thermo Fisher Scientific) for GST-tagged proteins, for ~ 18 h at 4 °C on a tube rotator. The resin was collected by centrifugation, and washed in either 1× PBS pH 7.4 (His-tagged proteins) or 50 mM Tris, 1 M NaCl, pH 8.0 (GST-tagged proteins). After washing, purified recombinant protein was eluted by the addition of either 150 mM imidazole (His-tagged proteins) or 10 mM reduced glutathione (GST-tagged proteins).
Polyclonal antibodies were generated in rabbit against His-EmVcl, His-EmFAK and GST-EmITGB1 (Syd Labs) recombinant proteins. For affinity purification, two columns were made: (1) whole E. coli lysates, and (2) 6–10 mg of recombinant protein. Each was covalently bound to 1 mL of AminoLink Plus Coupling resin (Thermo Scientific, Cat#20501) according to the manufacturer specifications. Anti-sera were passed over the E. coli column to remove antibodies against bacterial proteins; then the flow-through was incubated with the antigen-coupled resin for 1 h at RT under rotation. This column was washed with 12 mL of AminoLink Wash Buffer and the antibodies were eluted with 500 µL 0.1 M glycine HCl, pH 2.5. The pH of eluted fractions was adjusted to neutral by adding 30 µL of 0.75 M Tris–HCL pH 9.0. Antibody titer was quantified by spectrophotometry (A280) and by visual comparison to BSA standards via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS/PAGE). The specificity of each antibody was validated by Western Blot, Immunoprecipitation coupled with LC–MS/MS, and by pre-adsorption with the injected antigen prior to immunostaining.
For each Western Blot, ~ 100 gemmules were grown in petri dishes with lake water containing 100 µg/mL ampicillin for 6–13 days at RT. Juveniles were scraped with a razor into 4× SDS-PAGE reducing loading buffer (1 M Tris, pH 7.0, 20% SDS, 20% Glycerol, 0.02% bromophenol blue and 2.5% β-mercaptoethanol), vortexed and boiled at 95 °C for 3 min. Proteins were separated by SDS-PAGE on a 10–12% gel, and transferred to a PVDF membrane (Millipore) at 350 mAmp for 30 min. Membranes were blocked for 1 h at RT in 5% nonfat milk in 1× PBST, pH 7.4 (0.05% Tween 20) and then incubated with affinity purified antibodies (1 mg/mL stocks) against EmVcl (1:3000), EmFAK (1:1000) and EmITGB (1:1500), in blocking solution for 1 h at RT and washed twice in 1× PBST pH 7.4. After 45 min of incubation with the secondary antibody (Alexa488® Goat Anti-Rabbit IgG Antibody; Life Technologies, 1:1000 dilution) at RT, membranes were washed in 1× PBST pH 7.4 and imaged using the Molecular Imager FX ProPlus (BioRad).
Immunoprecipitation and mass spectrometry
Affinity-purified antibodies were coupled to agarose A/G using the Pierce Crosslink CoIP Kit (Thermo Scientific Cat #26147). A control IP was performed using rabbit IgG (I5006, Sigma-Aldrich). For EmVcl, cell lysates were prepared by combining 1.1 mg of frozen adult tissues with 1.8 mL Pierce Lysis buffer (Thermo Fisher Scientific) containing Complete Mini Protease Inhibitor Cocktail (Roche, EDTA-free), Aprotinin and Leupeptin (1 mM). Lysates for EmFAK and EmITGB1 IPs were prepared by scraping ~ 300-week-old sponges into 1 mL Triton Lysis Buffer [TLB; 20 mM HEPES, pH7.4, 150 mM NaCl, 1 mM ethylene glycol tetraacetic acid (EDTA), 10% Glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM DTT, protease Inhibitor cocktail (ROCHE), 1 mM Aprotinin, 1 mM Leupeptin]. Different tissue sources and lysis buffers were used as the technique was optimized over the course of the project. Samples were vortexed 15 s and returned to ice for 2 min; this was repeated 3 times. Samples were further homogenized by hand (Argos Tech. A0001) for 30 s, and debris and gemmules were removed via centrifugation at 13,000×g for 10 min at 4 °C. 350 µL of the lysate was diluted with an additional 200 µL of lysis buffer and combined with the antibody-coupled agarose at 4 °C for 1.5 h. After collecting the flow-through (FT) and completing the washes recommended by the manufacturer, an extra wash using 1 M LiCl solution was performed to remove any non specific proteins. Finally, precipitates were eluted with Pierce Low pH Elution Buffer (Cat#21004, ThermoFisher) and neutralized with 1 M Tris–HCl pH 9.0 (Cat#42020208-1, Bioworld). 20–25 µL aliquots of these precipitates were mixed with 5 µL 4× SDS-PAGE loading buffer containing freshly added 20% 2-mercaptoethanol (BME), boiled for 3 min, and then loaded on a 12% gel for SDS-PAGE. One gel was used for Coomassie staining, and a replicate was analyzed by Western blot.
EmVcl precipitates were directly sent for further analysis by LC–MS/MS. EmFAK and EmITGB1 precipitates were excised from an SDS-PAGE gel to separate the precipitate from the co-eluted antibody. LC–MS/MS was performed by the Proteomics Core Facility, University of California, Davis for EmVcl and CU-Anschutz Proteomics Core Facility for EmFAK and EmITGB1. Results were analyzed using the software Scaffold (v3.1).
Ephydatia muelleri juveniles were grown from gemmules for 5–7 days on No. 1.5 uncoated dishes (MatTEK) or on glass coverslips. Tissues were fixed in 4% Formaldehyde in 95% cold EtOH for 30 min–1 h at 4 °C. Juveniles were then washed three times with 1× PBS pH 7.6, and incubated in blocking buffer (3% BSA in 1× PBST pH 7.4) overnight at 4 °C. All antibody preparations were titrated to determine their optimal working concentration, from 1:250 to 1:5000. After incubation, samples were washed three times with 1× PBST and then incubated for 45 min with secondary antibody (Alexa488® Goat Anti-Rabbit IgG Antibody; Life Technologies, 1:500 dilution), plus Alexa Fluor568® Phalloidin (Life Technologies, 1:80) and Hoechst (33,342, 1 µg/mL) at RT. Samples were washed once in 1× PBST and twice in 1× PBS pH 7.6 and preserved for imaging using anti-fade mounting medium (0.1 M Propyl gallate, 1× PBS pH 7.6 and 90% glycerol). Confocal Images were acquired on an Olympus Fluoview FV3000 confocal laser scanning microscope using either a 20×/0.85 NA, 60×/1.4 NA or 100×/1.4 NA objectives, and processed using FIJI [69]. Neither brightness nor contrast was adjusted in the antibody channel. Immunostaining results were validated by secondary-only control and by pre-incubating each antibody with its corresponding antigen for at least 1 h at 4 °C before staining (Additional file 4: Figure S1).
Quantification of focal adhesion abundance
Single gemmules (n = 12) from E. muelleri were placed in 3 mL of lake water in No. 1.5 uncoated dishes (MatTEK) and allowed to attach (3 days). After attachment half were transferred to a rocking platform for 24 h, while the others were left on a steady surface. All individuals were fixed and stained with anti-EmVcl and phalloidin, and the basopinacoderm imaged as described. Focal adhesion-like structures were counted in each of three image stacks per individual and their abundance in each treatment was analyzed using a one-way ANOVA with single factor of treatment in R studio [70].
Pharmacological inhibition of FAK
Five-day-old juveniles were treated with 5 µM FAK Inhibitor 14 (Sigma-Aldrich) for 4 h at RT in the dark. Treatment was removed and tissues were immediately fixed, immunostained, and imaged as described. The effects of FAK inhibition on cell motility are described in the Additional file 4: Figure S7.
Sequences and accession numbers for all genes and proteins analyzed in this study are made available in the online Supplement. Custom antibodies are available on reasonable request.
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The authors would like to thank Jeff Colgren for thoughtful discussions and critical input throughout the course of the project.
This work was supported by NASA Grant 16-EXO16_2-0041 (S.A.N.).
Jennyfer M. Mitchell
Present address: University of Colorado, Anschutz Medical Campus, 12801 E. 17th Ave. RC1S, 11401G, Aurora, CO, 80045, USA
Department of Biological Sciences, University of Denver, 2101 E. Wesley Ave. SGM 203, Denver, CO, 80208, USA
& Scott A. Nichols
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JM and SN were involved in all aspects of experimental design, data analysis and interpretation, and writing of the manuscript. JM performed protein expression and purification, antibody affinity purification and immunoprecipitation, drug treatments, and immunostaining/imaging. SN performed also performed immunostaining/imaging. Both authors read and approved the final manuscript.
Correspondence to Scott A. Nichols.
Additional file 1. DNA transcripts of E. muelleri focal adhesion homologs
Additional file 2. Predicted peptide sequences of E. muelleri focal adhesion homologs
Additional file 3. EmVcl coIP results
Additional file 4. Supplementary Figures
Additional file 5. EmITGB1 coIP results
Additional file 6. EmFAK coIP results
Additional file 7. Timelapse video of migratory cells within the mesohyl
Mitchell, J.M., Nichols, S.A. Diverse cell junctions with unique molecular composition in tissues of a sponge (Porifera). EvoDevo 10, 26 (2019) doi:10.1186/s13227-019-0139-0


Tom O'Keefe is a social media personality and internet entrepreneur living in Boston, Mass.
In 2008, Tom O'Keefe founded BostonTweet as a way to create awareness for local business during the recession. Today, BostonTweet reaches 145,000 Bostonians via Twitter and Instagram with news and pictures about life in Boston.
In 2014, Tom created #DownloadBoston as a way to create awareness for Boston-based consumer startups. In 2015, Tom will launch Flutter, a platform to experience good.
Previous to 2008, Tom created a handful of internet startups; WiredAlumni, ResearchConnect, Bizak, and infoMedMD. And even further back, Tom graduated from Boston College with a degree in economics.
Use your voice to get things done.
Spritz is focused on text streaming technology and its integration into modern communication.
Bootstrap Compost is Greater Boston's premier year-round residential and commercial food scrap pickup service.
Bridging the communication gap between patients and their caregivers.
No surges. Not even during prime time.


text:
The Stone Fields in the Schmale Heath and Extension () is a nature reserve (Naturschutzgebiet), 199 hectares in area, on the German island of Rügen in Mecklenburg-Western Pomerania. It includes large flint fields (), a representative section of the Schmale Heath and a separate extension with near-natural coastal dunes. The reserve was created on 27 April 1935. Two extensions of the nature reserve area came into force in the years 1959 and 1990.

The areas are located seven kilometres north of Binz between the Kleiner Jasmunder Bodden and the Baltic Sea. The reserve is assessed as in good condition. The high numbers of visiting tourists in the flint fields causes disturbances, though, to the fauna.

The nature reserve is accessible via a footpath from Neu Mukran.

In 2009 the nature reserve areas were taken over by the German Federal Environment Foundation (Deutsche Bundesstiftung Umwelt). According to EU law, the nature reserve is part of the Special Area of Conservation known as the Kleiner Jasmunder Bodden, Jasmund Peninsula and Schmale Heath (Kleiner Jasmunder Bodden mit Halbinseln und Schmaler Heide).

References

Literature

External links

Ordinance for the extension of the nature reserve
Information from the M-V state portal
M-V environmental map portal with geodata (nature reserve boundary, biotope mapping etc.)

Nature reserves in Mecklenburg-Western Pomerania
Geography of Rügen
Binz

redpajama_set_name: RedPajamaWikipedia


As the South Pole quests of Robert Scott and Roald Amundsen are commemorated, exploration and expeditions continue above, on and below the Earth's surface and waters. Assisted by cutting-edge technology and photography, 21st-century scientists and their teams plan for all stages, collaborate during the voyage and document their discoveries. Articles focus on Antarctica and other areas of recent discoveries. A rich variety of activities, lesson ideas, resources and suggested readings cross many disciplines in science, mathematics, technology and the fine arts in this curriculum guide.
Younger students are introduced to new plant and animal species and scientific exploration through two KidsPost articles. These articles are found in the KidsPost — New Species collection.
"This sea grass is REALLY old," a February 2012 article, explains how the age of sea grass discovered in the Mediterranean Sea is determined. "Really Old Sea Grass" provides study questions to use with the article.
Read the January 1, 2012, KidsPost article, "New animal species are found in Asia." The short news article reports that more than 208 new species have been found in the Mekong River region. A map reading exercise and discussion questions are provided.
To learn about more new species read "From global sea census, a treasure trove." Post science writer Juliet Eilperin reports on the ten-year study of discovery, Census of Marine Life.
To learn more about tigers and rare and endangered animals, review the previous Post INSIDE curriculum guide Sumatran Tiger.
Acquiring new riches, finding a Northwest Passage and claiming land for the sponsoring country motivated many early explorers. Captain James Cook took scientists and artists, including botanist Joseph Banks, aboard the Endeavor when he explored the South Pacific in 1768. This is just one of many voyages, individuals and discoveries that tell the story of exploration.
"Early Europeans Who Explored the Americas" lists explorers and the dates of main expeditions. This activity includes questions to guide students' research. The instructions on the activity sheet are left to the minimum so teachers can add their own criteria and expected outcome.
"Word Study: Discover the Roots of Explore and Adventure" ties to the theme of this Post curriculum guide. Although the lead of "Far below, new species emerge" is the springboard from which students are introduced to the etymologies, the Word Study can be used with several of the articles and suggested activities in this curriculum guide.
• "Far below, new species emerge": Who are the explorers in this expedition?
• "Exploring the abyss": As filmmaker James Cameron solo dives to the ocean floor in Deepsea Challenger, is he an example of an explorer or an adventurer?
• "Man on a mission": Enric Sala is a "National Geographic explorer — one of the few lucky souls who launch expeditions financed and documented by one of the nation's most venerable institutions." What is the job of a 21st-century explorer?
• "Fantastic Voyage": Matt Rutherford has a goal for his voyage. What is it? In what ways is Rutherford an explorer? An adventurer?
The assignment teachers give to use the "We Explore" activity sheet may vary. Teachers may ask students to research one explorer — perhaps from those listed in "Early Europeans Who Explored the Americas." Students could research an early and a more recent explorer in order to compare and contrast the expeditions. The assignment may be to draw conclusions after classmates have presented reports on different explorers.
Antarctica is a special continent in many ways. Familiarize students with its geographic location, climate and inhabitants before reading many of the articles in this curriculum guide.
• Which islands are the closest to Antarctica?
• Which South American country is closest to Antarctica?
• Are the geographic and magnetic South Pole at the same location?
• In what mountain range is the Beardmore Glacier located?
• For whom were the following locations named: Bellingshausen Sea, Amundsen Sea, Scott Island, Ross Ice Shelf, Shackleton Ice Shelf and Vinson Massif.
As two polar expeditions are commemorated, historians, scientists and the general public are considering the success and failure of both. In the one hundred years since British naval Capt. Robert F. Scott and Norwegian explorer Roald Amundsen set their sights on the South Pole, who has had a lasting influence?
After students have read the article, teachers could ask students to answer the questions and do the activities found in the worksheet, "He lost the race to the South Pole but made discoveries for science," found in Antarctica and Scott resources.
For more information on the fifth largest and coldest continent, its explorers and the work being done there, review the previous Post INSIDE curriculum guide, Antarctica.
Just as the United States has events taking place over four years to commemorate the Civil War, a series of world-wide initiatives commemorate Captain Robert Falcon Scott's 1910-13 expedition. These events will provide opportunities to study Antarctica then and now, to explore the impact of this and other expeditions, and to investigate expeditions that feature scientific study and discoveries.
In addition to The Washington Post article, a number of online sources may be used to augment students' understanding of the quest to be first to reach the South Pole. Eyewitness accounts are riveting. Scott's Last Expedition, Volume 1, his journals from the polar project, may be downloaded at no charge at Project Gutenberg. If teachers wish to focus on the final days, "Doomed Expedition to the South Pole, 1912" provides the last day's of Scott's expedition to the South Pole as recorded in his journal.
Herbert Ponting, the expedition's official photographer, taught Scott to use the camera. His and Scott's photographs have been acquired by the Scott Polar Research Institute. Students could be asked to review the photographs and to write about what it would be like to live there for months.
Teachers may also wish to separate the class into different exploration camps. For example, Scott, Amundsen, Ross and Shackelton. Do the research, then debate the importance of each expedition, including the personality and role of the expedition's leaders and impact of society and world conditions on the expedition.
Commemorative exhibitions are planned to remember events, provide perspective and to educate new generations. "Commemorate the Scott Expedition" provides guidelines for creating an exhibit of the Scott Expedition.
Teachers should decide if this project will be done as a mental exercise or if students will create an exhibition — a collection of dioramas, a display case or a virtual exhibit. The previous Post NIE curriculum guide, Museum Musings provides more information on setting up a display.
Students should locate Trieste, in the northeast corner of Italy. Then find Villagio del Pescatore nearby. This is where the story of Antonio, a dinosaur, took place.
A geology student who had been searching in the small fishing village in 1994 made this important find. Newspapers in Italy and around the world covered the story.
In March 2012, students from four third-year classes at Scuola Nazario Sauro di Muggia wanted to meet Tiziana Brazzatti, the graduate student who had made the discovery in a town near their school. All of these students are interested in journalism. Guilia Basili, who hopes to be a journalist, and her classmates read more about Antonio, wrote interview questions and arranged an appointment after school. Their interview was conducted in Italian and translated by Lilia Ambrosi, their teacher, with help from the students who had met with Brazzatti.
• How do the students' questions work together to unfold the story of the geology student and the dinosaur?
• Were Tiziana Brazzatti's descriptions helpful? Explain your answer.
• Do the math to convert meters and centimeters into inches and feet. Now write a comparison of Antonio with an object to communicate the dinosaur's size.
• What information did students find most interesting? Were they surprised that someone so young could make such an important discovery?
• What did the photographs and illustration add to the interview and story of the discovery?
The illustration is by paleoartist Tullio Perentin whose work includes paleontology projects. Teachers may discuss with students how a major in art can be combined with an interest in science. Career opportunities would include science research illustrator, textbook illustrator, museum and exhibit display artist and paleoartist.
To read about an earlier find in Italy, visit the previous Post INSIDE curriculum guide, The Science in Discovery. Otzi, a frozen mummy, was discovered in 1991; in 2001, x-ray technology revealed his cause of death.
Which modern explorer would students like to interview? Might this be an astronaut, a scientist doing laboratory work to find a cure, or someone finding ways to produce food in arid environments, for example? After the class has discussed the possibilities, conduct research to learn more about the individual and his or her area of expertise.
Write interview questions, arrange an appointment and conduct the interview.
The interview may be written in Q&A format or as a narrative. Perhaps students could record the interview to prepare a two- to five-minute video or podcast. Or students could take photographs to prepare a photo gallery with captions and voiceovers.
When weather permits, scientists from across the globe return to Antarctica and various research stations to work. For more than two decades Russian scientists have been drilling into the ice sheet in order to reach Lake Vostok. This exploration is accompanied with interest and controversy.
Read "In Antarctica, drilling into the past" and the February 7, 2012, follow-up article, "Russians drill into lake below ice of Antarctica." "Research | Lake Vostok," an e-Replica activity, suggests ways to return to the story when weather conducive to research returns to Antarctica.
In the Sports section on February 7, 2012, Dave Sheinin reported an attempt to circumnavigate the Americas solo and non-stop. "Fantastic Voyage" is the story of Annapolis sailor Matt Rutherford.
A map of the sea route and vocabulary found in the article is provided in Fantastic Voyages. Before reading the article, familiarize students with the vocabulary and discuss the route Rutherford has taken.
• Which continents is Rutherford circumnavigating?
• Which are the most treacherous geographic locations on the route?
• Rutherford has the advantage of modern technology on his solo voyage. How have they fared in their combat with natural forces?
• How does Rutherford confront solitude?
• What have been some of the "payoffs" or benefits of the voyage?
• What record has he broken? Was this a goal of his?
• Why might readers value Herb McCormick's evaluation of Rutherford?
• What reason(s) does Rutherford have for undertaking this voyage?
For enrichment, teachers might review a previous guide, Circumnavigation that focuses on an around the globe sailing race.
Science and photography are linked to document methods, collect data, record discoveries, display landscape, capture weather conditions, render the night sky and reveal deep-sea life. Use "Photography and Science: Seeing to Believe" to introduce the uses of photography in science to illustrate, explain and document. Discuss how the photographs illustrate the concepts. What photographs might students take to illustrate, explain and document science projects? Their current science lesson?
"Shining Strands" is an example of a photo essay. Post photographer Linda Davidson through photographs and text explains the appearance of gelatinous strands on the Mattawoman Creek. Through her photo essay, she relates the natural event to science (spring) and government (a recent denial to those wanting to build a highway through the Mattawoman watershed).
• Davidson took wide, medium and close-up shots. How do they work together to tell the story visually?
• In what ways do these photographs illustrate, explain and document natural phenomena?
• What does the caption add?
• What does the text explain?
The Washington Post photo galleries are another source of photographs to illustrate, explain and give evidence of photographers communicating science concepts to readers. Arcus: Internet Media Archive is another resource for photographs. The site is well organized and gives an opportunity to teach students how to appropriately credit their work.
In the spring of 1940, John Steinbeck and his business partner in Pacific Biological Laboratory embarked on the Western Flyer. During the two-month trip, they collected samples and conducted research. A year later Steinbeck wrote Sea of Cortez in which he tells of the expedition in the Gulf of California. Edward Ricketts contributed the 300-page scientific appendix.
Steinbeck's non-fiction work is just one of the books, essays and poems suggested in "American Sea Writing." The works are varied to meet different reading levels, genre and periods of American cultural development.
Teachers may ask students to write a book review similar to those written for Book World. A format and guidelines for writing a book review are found in the past Post guide Reviewing a Whirl of Books.
For more on the relationship of the sea, science and literature, review the previous Washington Post INSIDE curriculum guide The Sea — Rich and Strange.
Latin has been used for at least 400 years to name plant species. The Washington Post's Adrian Higgins reports on the recent decision to allow botanists as of January 1, 2012, to use English to name new species. Read "Veni, vidi, vici — and now history: Botany's new rules allow English to replace Latin in describing species," originally published on January 19, 2012.
• Who made the change to relax the rules that have been in effect since 1908 and actually used since the Renaissance?
• What are the arguments for continuing to use Latin?
• What are the arguments for allowing English to be used?
• If botanists spoke French, German or Chinese, would they be at an advantage or disadvantage with the rule change?
• Which argument do students find most convincing?
Teachers might bring in five to eight different plant specimen. Place the Latin name of each on tags. Ask teams of students to match the scientific name on the tag to the plant. Give one team the name of each plant in English. Are students any more successful in pairing the plant and its name in English?
The British Antarctic Survey has used radar and data from decades of polar expeditions to create the "most accurate and detailed map of the 'white continent' ever made." Study "Beneath the ice, a continent reveals its highs and lows" How might this information be used?
Lisa Wu, director of the Oceanography & Geophysical Systems Lab at Thomas Jefferson High School for Science and Technology in Alexandria, Va., has prepared a student activity, discussion questions/quiz and two sets of notes for teachers. Whether for the classroom or for individual student enrichment, the resources are diverse and offer many ways to stimulate student interest.
• Students may be given "A Few Questions for You: Exploring Some Ocean Web Sites" to accompany the assignment.
"Create a Geocache," a NOAA Ocean Explorer lesson, gives another way for middle and high school students to use technology (GPS devices) to find and to bury caches and to report (online and within class groups) discoveries. Students are encouraged to sketch a map during the exploration. This will give students practice in latitude, longitude and map reading while having an adventure.
Lisa Wu, director of the Oceanography & Geophysical Systems Lab at Thomas Jefferson High School for Science and Technology in Alexandria, Va., has prepared "Teacher Notes: High Tech Treasure Hunting or It's the Journey, Not the Destination." Refer to these for more ideas to use this resource.
A past Washington Post NIE guide, Putting Yourself on the Map, includes a KidsPost geocache article and activity.
Students may be surprised to learn of the wide variety of career paths that lead to involvement in and contribution to science-focused expeditions. "Explore Careers" lists more than 30 fields in which students may major. The activity sheet also suggests several Web sites to visit for internships and volunteer opportunities.
In the 100-year-anniversary of the sinking of the Titanic, filmmaker James Cameron engaged in a solo dive in Deepsea Challenger to the bottom of the Mariana Trench. Read "Exploring the abyss."
• What was Cameron's goal for the dive?
• What did he observe?
• What problems did he incur?
• What does the 1960 dive of the Trieste have to do with Cameron's dive?
• Which frontier does Cameron hope to "open up" to science?
• Evaluate Cameron's dive — exploration or adventure, success or failure, way to get personal attention or greater public awareness?
Do you wonder what it would be like to visit Earth's North and South Poles today? It is perhaps as simple as a click on PolarTREC. Here you can visit a virtual base camp, join live webinars and question explorers during the actual expedition, find resources for the classroom and materials that students can use to do their own research.
For example, you can meet the expedition team, find out where they are, what they are doing, and read their on-going journals as the expedition is happening. You can also read archived expeditions that have happened in the past. Because these are treks to both polar regions, expeditions will include Arctic and Antarctic explorations.
In 1981, Fondation Cousteau (later Equipe Cousteau) was established in France. From this base of supporters, Jacque Cousteau launched a worldwide petition campaign in 1990 to save Antarctica from mineral exploitation. His effort was successful: The Antarctic continent is protected for at least 50 years.
Teachers may wish to review the vocabulary in In the Know before students begin reading the article written by a former oceanographer. These terms appear in the article.
• Locate the Southern Ocean on a map. Which countries and continents touch it?
• What does it mean to be "geologically active"?
• How do species survive in the depths of oceans?
• Who are the contemporary patrons of explorers?
• Several science-related professions are presented in the article. What does each of these do: biologist, deep-sea biologist, geochemist, geologist, oceanographer?
1. Teachers will tell students how to indicate the countries; all are named on the map provided.
2. Answers will include providing a place for animals to survive.
3. Teachers will need to explain that Myanmar and Burma are names of the same country.
4. An endangered animal is at risk of becoming extinct.
4. Answers will vary based on current conditions. Productivity will change seasonally. It is influenced by sunlight, angle to the Earth, duration of daylight, rainfall and nutrients.
5. Answers will vary depending on student selection.
World History: Students describe the rise of English Colonial Empires.
Science: Develop explanations that explicitly link data from investigations conducted, selected readings and, when appropriate, contributions from historical discoveries.
Life Science: Recognize and describe that evolutionary change in species over time occurs as a result of natural variation in organisms and environmental changes.
b. Recognize that adaptations may include variations in structures, behaviors, or physiology, such as spiny leaves on a cactus, birdcalls, and antibiotic resistant bacteria.
Explain that technology incorporates human knowledge into physical hardware that will eventually respond to some human need or desire.
Latin: The student will reinforce and broaden knowledge of connections between Latin and other subject areas.


First time negotiating a lease and learned a ton here thanks to this website.
Incentives: $7250 (5k lease credit, 1k college grad, 750 loyalty, 500 OL). Planning to submit the $500 BMW CCA rebate.
Do you mind pm'n me this dealers information?
That looks like an awesome deal below the 1%. Good job and enjoy the car!
Can you also let me know which dealer in FL?
Would also be interested in the dealer info.
same, dealer info if you'd be so kind.
Judging by how many people asking, seems S.Florida and Florida in general could really use a lease broker. Why are deals in this state just so bad? California's a bigger market and yet has killer deals.
@mani_is_kool and @Benedetto both work Florida.
Why are deals in this state just so bad?
Was going to mention @mani_is_kool as well if you are looking for a broker in FL.
I too had the notion that FL in general was terrible to lease in, and to look for something in GA. Turned out that they were actually harder to negotiate with.
Could you share dealer info via PM?


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Eairlay History, Family Crest & Coats of Arms
The name Eairlay is rooted in the ancient Anglo-Saxon culture. It was a name for someone who was a male who was of manly character. The surname Eairlay originally derived from the Old English word Eorlic which referred to one who displayed manly characteristics. The surname Eairlay belongs to a broad and miscellaneous class of surnames. Nickname surnames referred to a characteristic of the first person who used the name. They can describe the bearer's favored style of clothing, appearance, habits, or character. Often nicknames described strong traits or attributes that people wished to emulate in a specific animal.
Early Origins of the Eairlay family
The surname Eairlay was first found in Somerset where one of the first records of the name was John de Earlea who was listed in the Pipe Rolls of 1162. A few years later, William de Erlega was listed in the Pipe Rolls of Sussex in 1190 and John de Erley was listed in the Pipe Rolls of Berkshire in 1230. [1] Bardsley claims the name was a local name "of Earley, a parish in the diocese of Oxford." [2] He quotes another contemporary source "Lower says 'A liberty in the parish of Sonning, co. Berks.' " [2] The latter Berkshire local was confirmed by Reaney. The Hundredorum Rolls of 1273 list the following found in Somerset: Clemens Erleghe; Phillip Erlee; and Warin Erlee. [2] The Berkshire local is the only one we could find today.
Early History of the Eairlay family
This web page shows only a small excerpt of our Eairlay research. Another 105 words (8 lines of text) covering the years 127 and 1273 are included under the topic Early Eairlay History in all our PDF Extended History products and printed products wherever possible.
Eairlay Spelling Variations
Eairlay has been spelled many different ways. Before English spelling became standardized over the last few hundred years, spelling variations in names were a common occurrence. As the English language changed in the Middle Ages, absorbing pieces of Latin and French, as well as other languages, the spelling of people's names also changed considerably, even over a single lifetime. Many variations of the name Eairlay have been found, including Early, Erlegh, Erlee, Erly, Earley, Erle, Erley, Arlie and many more.
Early Notables of the Eairlay family (pre 1700)
More information is included under the topic Early Eairlay Notables in all our PDF Extended History products and printed products wherever possible.
Migration of the Eairlay family to Ireland
Some of the Eairlay family moved to Ireland, but this topic is not covered in this excerpt.
Migration of the Eairlay family
In an attempt to escape the chaos experienced in England, many English families boarded overcrowded and diseased ships sailing for the shores of North America and other British colonies. Those families hardy enough, and lucky enough, to make the passage intact were rewarded with land and a social environment less prone to religious and political persecution. Many of these families became important contributors to the young colonies in which they settled. Early immigration and passenger lists have documented some of the first Eairlays to arrive on North American shores: Robert Early who settled in New England in 1633; and Thomas Early who settled in North Carolina in 1700; Edward Erle settled in Virginia in 1635; Michael Earley settled in Philadelphia in 1868.
Hundredorum Rolls
Pipe Rolls
^ Bardsley, C.W, A Dictionary of English and Welsh Surnames: With Special American Instances. Wiltshire: Heraldry Today, 1901. Print. (ISBN 0-900455-44-6)
Eairlay (English)

redpajama_set_name: RedPajamaCommonCrawl


In the current climate of ever-increasing surveillance, some say UK-based VPN providers may not be able to protect your privacy effectively.
We take a look at the effect of snooping on VPN provision in the UK, and USA - can we trust our VPN's to keep us anonymous?
…Still, everyone likes practical gifts, right?
EU-US Privacy Shield Draft – Lipstick on a Pig?
PayPal have stated that they will begin blocking payments to VPN providers who promote activities they don't condone - such as unblocking worldwide streaming content like netflix, or P2P file sharing. What's prompted this unwelcome move and how will it affect VPN users going forward?
All you online dating fans – remember to use protection… Get a VPN!
Once again Netflix have been making noise in the news about restricting VPN access to their content. We've heard this all before of course - but what's the truth about the so-called clampdown, and what does it mean for those of us who want to use VPNs to stream geo-restricted video in the future?
We've Improved the VPNs.co.uk Comparison Tool – check it out!
VPNs.co.uk Enters the Blogosphere… Welcome to our brand spanking new blog section!


Flying direct between two destinations is not only the fastest route. It is often here you find the cheapest tickets flights. From Campo Grande you can fly directly to Sao Paulo, Cuiaba, Curitiba, Brasilia,Bonito and many other destinations.


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FAIR Weekly Roundup
In a piece for The Bulwark, Cathy Young details "What Cancel Culture Is and Isn't," by first highlighting several prominent examples of cancel culture enacted by conservatives, such as past attempts to boycott Ellen DeGeneres and the Dixie Chicks.
Young then hones in on her central thesis: that although there's nothing new about people from across the political spectrum attempting to deplatform or "cancel" one another, "progressive cancel culture has a much broader reach." According to Young, this is because "it does not simply punish opposition," but frequently targets other progressives and even politically neutral individuals for "accidental transgressions against the new norms of identity-based social justice."
Young then cites recent examples of cancellations ordered by Left-wing progressives, pointing out that although the total number of cancellations may seem small, the damage they cause is disproportionate because "they add up to a social climate of intimidation." Young believes that "the liberal and centrist pushback against the 'new puritans' is not only healthy but essential."
For the Heterodox Academy blog, psychologist Dr. Nicole Barbaro wrote an article asking whether "the centrality of the peer-reviewed publication in the academy impact[s] the type of knowledge that is produced." According to Barbaro, the answer is "yes," and she cites the way academic incentive structures "shape what research questions scientists ask and the type of knowledge that is ultimately published in the world's top science journals."
One of these problematic incentive structures centers around grant funding. In short, the career advancement of scientists is often predicated on securing large and highly competitive research grants. However, the types of research questions that are typically funded are those that "align with the strategic goals of that agency, specific divisions, and programmatic initiatives." Because of this, the questions studied are often narrow in scope.
Barbaro also believes the centrality of research within the academy also impacts knowledge production more generally. This is because in order for a grant to be deemed "successful," grantees must publish their findings in academic journals and present their research at conferences. Barbaro believes that "this cycle of funding and publishing further entrenches priority areas of research and continues to constrain open inquiry of research areas that may not fall within the scope of funders and journal editors."
In a whistleblower report for City Journal, Christopher F. Rufo obtained a cache of internal documents on the latest Fortune 100 company to subject their employees to neo-racist diversity training.
In the report, Rufo revealed that the AT&T Corporation has created a radical "racial reeducation program" called Listen Understand Act, which teaches AT&T employees that "American racism is a uniquely white trait" and that "Black people cannot be racist."
Resources in the program are overtly political, promoting causes such as reparations, defunding the police, and many controversial aspects of trans activism. The education materials also emphasize concepts such as "whiteness," "white privilege," and "white fragility." According to Rufo, AT&T did not respond when asked for comment.
In Persuasion, Trent Colbert, the student at the center of a recent debacle at Yale, provides his behind-the-scenes perspective on what happened and why he refused to apologize to an angry activist mob.
In the age of social media, even the most innocuous of comments is liable to trigger anonymous social media flash mobs fixated on ruining someone's life, reputation, or career. Colbert believes that this phenomenon, which is becoming all-too-common, can only be halted by refusing to cave in to demands for an apology when there is truly nothing to apologize for.
I don't believe that the now-common ritual of compelled apology, complete with promises to "grow" and "do better" (their words, but ones I'm sure you've seen many times before) helps anyone, or is even intended to. If we continue to indulge this culture of performative denunciation, the very idea of an apology will lose its meaning.
For UnHerd, new contributing editor Tomiwa Owolade asks "What does it mean to be black?" and explains how "being reduced to a label deforms your humanity."
Owolade highlighted the patterns that run throughout old poems, as well as recent books, that reveal the expectations that are placed upon people because of their skin color. From so-called "savages" once put on display in human zoos for European patrons, to the modern state of race-based tokenism where people of color are expected to perform to fulfill certain political narratives, Owolade believes that when one's group identity is given more importance than their individuality, they can never be fully free and seen for who they are.
Labels should be used, if they are to be used, as the start of someone's identity, not the full definition of it. They should be used to open the doors to a deeper understanding of who that person is, rather than perceived as the only thing that matters. The alternative is race representatives, people who are exhibited, or exhibit themselves, to a white audience, to be gawked at and cuddled, and are expected to possess a morality befitting a child.
FAIR Board of Advisors
In The New York Times, FAIR Advisor Zaid Jilani reviews FAIR Advisor John McWhorter's new book Woke Racism. Jilani explained how his family, with his parents from Pakistan, had grown up with nothing but optimism for America. Though far from perfect, American society had, for the most part, accepted them with open arms.
But Jilani notes that in recent years a new narrative has been growing that denies progress and asserts that "nonwhites are little more than virtuous victims cast adrift on a plank in an ocean of white supremacy." Jilani explains how McWhorter's book aims to address how this pessimistic worldview has captivated so many and has become implanted within our major institutions.
According to McWhorter, this new ideology, which he calls "Third Wave Anti-racism," has literally become a new religion, and that "the only story they want us to tell is one where whites are the villains and minorities are the victims."
Read Jilani's full review of McWhorter's book here.
For Commentary, FAIR Advisor Wilfred Reilly wrote a piece pointing out that narratives portraying cultural conflict as being between white conservatives and progressive minorities are a gross distortion of reality. For example, data reveals that those most fervently championing a range of "progressive" issues, such as policing politically correct language, defunding the police, affirmative action, and limiting gun rights, are themselves disproportionately white.
Reilly believes that this is caused by a form of political opportunism, where radicals are attempting "to use the genuine plight of poor minorities as a wedge to force through changes to American society—changes that most people of color do not ourselves want."
Instead of being galvanized by oversimplified political narratives, Reilly believes that we should take time to consult the relevant data and actually ask people directly what they believe: "If you want to know what individual members of any particular group happen to think, ask them—not those who claim to speak and act in their best interest."
FAIR Advisor Steven Pinker recently attended a speaking event for UnHerd, in London, to promote his new book, Rationality: What It Is, Why It Seems Scarce, Why It Matters.
In conversation with Freddie Sayers, Pinker answered questions on the prevailing trends of irrationality, intelligence, gut feelings, mythology, and conspiracy theories. Pinker argues that we are losing our ability as a society to think clearly and reason effectively. However, he believes hope is not lost so long as we're able to maintain the mental "mechanisms of rationality" that help identify our irrational blind-spots and work to overcome them.
If you're not allowed to broach a hypothesis in the first place, then there are possible solutions that you could never discover, because even considering it might be criminalised. So cancel culture, abrogations of academic freedom and free speech are irrational because they disable mechanisms of rationality.
Watch the full event here.
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Paramahamsa Yogananda
The prophecy of Lahiri Mahasaya came true when Mukunda Lal Ghosh was born to Gyana Prabha and Bhagabati Charan Ghosh on January 5, 1893. Mukunda would later become adored worldwide as Paramahamsa Yogananda, a prophet for people stuck in the mire of delusion. An outpouring of Mukunda's unquenched thirst and unmitigated love for God surged so intensely in him, even in his early childhood, that he posted letters to God in heaven earnestly waiting for His reply. Groomed and bred on the anvil of the saintly care of his pious parents, the spiritual spark in him grew day by day until it became an all-radiant and effulgent light.
His strong and indomitable willpower, dynamic personality, divine love and high state of spiritual experience enabled him to be a spiritual beacon, blazing the path of Kriya Yoga for millions of people in the West. That he was divinely ordained to do so was evidenced by the prophecy of Swami Vivekananda: "I have laid the foundation of spiritualism, but after me a mahayogi will come who will teach you how to realise God through yoga." Yoganandaji's use of language in both his speeches and writings was so forceful that others could easily perceive the miraculous play of gurus and God within him. He blended Swami Vivekananda's clarity, Shankaracharya's wisdom and Chaitanya's love.
After a decade and a half of ceaseless spiritual work in the USA, Yoganandaji retuned to India in 1935 to the unspeakable exhilaration of his beloved Gurudev. During his stay, many great personalities such as Ramana Maharshi, Anandamoyee Ma, Kashimoni Devi, the saintly wife of Shyamacharan, and Mahatma Gandhi came in contact with him. Understanding the significance and universality of Kriya Yoga as the scientific foundation of all religions, Mahatma Gandhi and his disciples in Wardha Ashram took initiation from him in 1935. During his stay in Kolkata, Paramahamsa Hariharananda took initiation into second Kriya from him. His Gurudev Shriyukteshwarji bestowed on him the highest monastic title of paramahamsa during this period.
When Yoganandaji was at the Kumbha Mela, he received several urgent calls from his Gurudev to return immediately to Puri. Unfortunately, Paramahamsa Yogananda planned to combine his return to Puri with the foundation day of Puri Karar Ashram on the 22nd of March, the Vernal Equinox Day. Delaying his journey back to Puri caused him to miss the conscious departure of his beloved Gurudev on March 9. It is very difficult to describe the bereavement of a realised yogi who loses the physical company and guidance of his Gurudev. A mesmerizing and miraculous rendezvous with his beloved Gurudev took place some days later at the Regent Hotel in Mumbai on the eve of Yoganandaji's return to the West. Shriyukteshwarji materialised in blood and flesh before Yoganandaji and gave a thrilling account of his sojourn in hiranyaloka.
Yoganandaji's life was eventful, miraculous, monumental and epoch-making. On October 6, 1951, he wrote to Paramahamsa Hariharananda, "My life is a whirlwind of activity and struggle with work." A gifted orator and singer, he inundated the hearts of millions with the water of divine love. His published books, such as The Autobiography of a Yogi, Whispers From Eternity, The Divine Romance, and Science of Religion, stand as a glowing testimony to his boundless brilliance, erudition and experience with the Self. On March 7, 1952, when a banquet was given for his Excellency Mr. Binay R. Sen, former Ambassador from India to the USA, Yoganandaji, in midst of his speech, with a blessed smile, attained mahasamadhi to the amazement and stunning silence of all singing a beautiful song in paeans of India's eternal glory:
'Where Ganges, woods, Himalayan caves, and men dream of God;
I am hallowed, my body touched that sod.'
The mysterious event of his physical body remaining ensconced in eternal peace without the faintest shred of evidence of decomposition for twenty days leaves us awestruck and inspires us to experience the deathless state.


This will be the cover for the first Studio Morran calendar (Camilla's eighth!). I hope you'll like it. I'll send it to the printer this week. I'll let you know when it will be ready as soon as I know.
I love the colors and shadows and light. It reminds me of the Canadian painters: the group of seven. I love them too.
Love, love, love it Camilla!
I put this blog in my favorites also! bravo camilla + morran !
Like? Make that looooove... as always :) xox!
super lovely camila! i hope i can buy one this year. have a super week!
Simply beautiful, I can wait to order it!
Thank you Camilla for making our lifes so colorful!
Have a lovely day and a great weekend!
Looking really good as your work always does!
all the words are allready taken above, beautiful, lovely... Colourful! I'll by it!
This is so beautiful! I can't wait.
Gorgeous! I absolutely cannot wait!
so beautiful! just love it!


Apple has a new patent application published today by the USPTO (via AppleInsider), which details a stylus with a nib that can be extended to take advantage of multi-touch capabilities for a wider brush stroke, and that has built-in light sensors so that it can act as essentially an eye-dropper tool for the real world, capturing colors from physical objects to use in digital painting.
The patent details a number of different options for a stylus tip or nib, including one which has variable sizing to determine how much contact it has with a device screen. Extending the nib could allow a user to do broad, edge strokes for wide lines, for instance, while shortening the tip would allow for drawing fine lines. It would be partially flexible to allow for it to bend when in use, which could make drawing varying line widths more natural for artists used to traditional media.
Other designs include tips that can be changed out for drawing with different kinds of digital brushes, and a twist on the old select-a-pen idea where a group of fibrous strands makes up the nib. The strands could be reconfigured using a changeable exit on the stylus tip, meaning it could shape one continuous brush, a spread out arrangement of individual strands, or various other permutations.
Apple's stylus also details a nib with light sensors to detect visual feedback from the screen of a device like an iPad, and to communicate back to it via light as well, allowing the stylus to quickly change line width, color info, brush pattern and more on the fly instantly without any kind of wireless (i.e., Bluetooth) connection. The light sensors could also be used to pick up color information from real-world objects, so that you could sample the specific hue of your apple, for instance, and use that in your digital painting.
Other sensors, including accelerometers and gyroscopes, could be included according to the patent to give a better idea of the angle of the pen to change input type, too. These kinds of things are used in some Wacom stylus devices for input on their digital drawing tablets.
Apple has never been too keen on the stylus. Steve Jobs famously said "if you see a stylus, they blew it" during a Q and A for iOS 4, and when he unveiled the first iPhone in 2007, he asked derisively "Who wants a stylus?" But the landscape has changed, and sophisticated stylus devices that offer highly-tuned input for drawing and fine detail work could be a great boon to those who want to use iOS devices for digital creation.
Image depicted is a micro stylus, not an Apple creation.