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Human phosphatase CDC14A regulates actin organization through dephosphorylation of epithelial protein lost in neoplasm
Edited by Thomas D. Pollard, Yale University, New Haven, CT, and approved April 11, 2017 (received for review November 24, 2016)
Human hCDC14A phosphatase has been shown to regulate cell migration and adhesion by modulating actin dynamics. Here we identify the potent tumor suppressor eplin as an hCDC14A substrate. Through dephosphorylation of serine residues 362 and 604 of eplin, hCDC14A promotes F-actin bundling and counteracts EGF-induced membrane ruffling. Cells lacking hCDC14A activity or eplin expression showed reduced E-cadherin levels and β-catenin retention at cell–cell junctions and formed more invasive colonies than wild-type cells. Expression of a phospho-inhibitory, but not a phospho-mimetic, eplin mutant rescued these phenotypes. Database analysis indicated that hCDC14A and eplin are frequently inactivated in human colorectal cancer and this is associated with poor prognosis. Therefore, the hCDC14A and eplin axis plays an important role in limiting tumor malignancy.
CDC14 is an essential dual-specificity phosphatase that counteracts CDK1 activity during anaphase to promote mitotic exit in Saccharomyces cerevisiae. Surprisingly, human CDC14A is not essential for cell cycle progression. Instead, it regulates cell migration and cell adhesion. Little is known about the substrates of hCDC14A and the counteracting kinases. Here, we combine phospho-proteome profiling and proximity-dependent biotin identification to identify hCDC14A substrates. Among these targets were actin regulators, including the tumor suppressor eplin. hCDC14A counteracts EGF-induced rearrangements of actin cytoskeleton by dephosphorylating eplin at two known extracellular signal-regulated kinase sites, serine 362 and 604. hCDC14APD and eplin knockout cell lines exhibited down-regulation of E-cadherin and a reduction in α/β-catenin at cell–cell adhesions. Reduction in the levels of hCDC14A and eplin mRNA is frequently associated with colorectal carcinoma and is correlated with poor prognosis. We therefore propose that eplin dephosphorylation by hCDC14A reduces actin dynamics to restrict tumor malignancy.
The highly dynamic nature of the actin network of nonmuscle cells contributes to its functions in cell migration, cell synaptic transmission, endocytosis, and the immune response (1⇓–3). It is therefore not surprising that deregulation of actin is associated with a range of human diseases, including leukemia, kidney disorders, and cancer development (4, 5). Long lists of actin-associated proteins from Rho GTPases, kinases, phosphatases, and actin-binding proteins regulate actin filament dynamics (1, 3, 4, 6⇓–8). Actin-bundling proteins such as α-actinin, fascin, and filamin cross-link actin filaments into bundles and play important roles in regulating membrane protrusions and cell mobility (9⇓⇓⇓–13). Cofilin severs existing actin filaments to generate more free ends and thus plays a critical role in actin dynamics (9, 10, 14, 15). The protein epithelial protein lost in neoplasm (eplin) bundles F-actin filaments and can control G-actin nucleation by regulating the activity of the Arp2/3 actin complex (16). Interestingly, the activity of eplin is regulated through phosphorylation by extracellular signal-regulated kinase (ERK) at serine residues 362 and 604 (17). However, the phosphatase that reverses these important phosphorylations is unknown.
In Saccharomyces cerevisiae, the dual-specificity phosphatase CDC14 counteracts cyclin-dependent kinase (Cdk1) activity to drive mitotic exit (18, 19). Therefore, CDC14 is essential for the viability of S. cerevisiae. In contrast, its ortholog, CDC14A, is not essential for the viability of either human or chicken DT40 cells. Surprisingly, cells in which the hCDC14A coding sequence had been disrupted did not show any obvious defects in cell cycle progression (18, 19). However, we recently reported that hCDC14A colocalizes with F-actin at the leading edge of cells and stress fibers where it regulates actin dynamics. This regulation is important for cell migration and cell adhesion (20).
Because there have been no systematic attempts to identify phospho-sites that are regulated by hCDC14A, we know relatively little about the identity of either the proteins that are dephosphorylated by hCDC14A or, indeed, the kinases that counteract hCDC14A in human cells. The only known substrate of hCDC14A that acts within the actin cytoskeleton is the protein Kibra (20, 21). However, considering the broad distribution of hCDC14A throughout the actin network, it is highly likely that hCDC14A dephosphorylates additional actin-associated proteins.
To identify hCDC14A substrates and decipher the molecular mechanisms by which hCDC14A controls cell migration, we combined phospho-proteome analysis with the biotin identification (BioID) proximity assay (22). These approaches identified the potent tumor suppressor eplin as a key actin-associated hCDC14A substrate. We show that hCDC14A controls actin-bundling activity of eplin to locally modulate actin rearrangements by counteracting EGF-induced ERK-mediated phosphorylation of eplin. Eplin also links F-actin to the E-cadherin–α/β catenin complex (23). Consistently, we found a reduction in the enrichment of α/β catenin at cell–cell junctions and decreased E-cadherin levels when either hCDC14A phosphatase activity was ablated or eplin was removed. This striking correlation indicates that hyperphosphorylated eplin lacks the ability to interconnect the E-cadherin–α/β catenin complex with F-actin. As down-regulation of hCDC14A and eplin is a common feature of colorectal cancer and is associated with poor prognosis, our data strongly suggest that loss of hCDC14A–eplin regulation may be a key step in driving the malignancy of invasive colorectal cancers.
Phospho-Proteome Analysis Revealed Multiple Actin Regulators as hCDC14A Substrates.
We applied affinity purification of phospho-peptides followed by large-scale phospho-proteomics to identify hCDC14A substrates. HeLaTetON-hCDC14A-YFP and TetON-YFP cells [stable integration into the flippase recognition target (FRT) site] were grown in heavy (Arg13C15N Lys13C15N) and light (Arg12C14N Lys12C14N) medium, respectively (Fig. 1A). After eight passages, addition of doxycycline (Dox) to the medium induced hCDC14A-YFP and YFP expression. Twenty-four hours later, proteins were extracted, the extracts were mixed at a ratio of 1:1, and, after phospho-peptide enrichment, peptides were analyzed by LC-MS/MS. Over 14,000 phospho-sites were identified in each of three independent replicate experiments. Surprisingly, only 68 phospho-sites (0.5%) belonging to 51 proteins (threshold = 0.5) were hypo-phosphorylated upon hCDC14A-YFP expression compared with the YFP control (Fig. 1B and Table S1). Of the 68 hypo-phosphorylated peptides, 65 (95.6%) contained phospho-serine (P-serine) residues and only 3 (4.4%) phospho-threonine (P-threonine), whereas P-serine represented 80.6% and P-threonine 18.6% of the global phospho-proteome of these cells. Thus, in line with in vitro data, in vivo hCDC14A also appears to preferentially target P-serine residues (24, 25).
Hypo-phosphorylated peptides were analyzed with the Perseus program to generate a consensus dephosphorylation motif for hCDC14A (Fig. 1C). This analysis is consistent with structural data (26) indicating that hCDC14A shares a common preference with cyclin-dependent kinases in being proline directed with a preference for lysine or arginine three residues after the phosphorylation site (25). The presence of proline residues at positions -1, -2, and +2 indicates that hCDC14A may also counteract other kinases such as ERK1/2 and glycogen synthase kinase 3 (GSK-3β) that emulate cyclin-dependent kinases in being proline-directed kinases yet have a preference for additional proline residues surrounding the phosphorylation site (www.phosphosite.org/). Consistent with hCDC14A's localization and function along F-actin, gene ontology enrichment revealed that the majority of hCDC14A substrates were either involved in the organization of the actin cytoskeleton or associated with adherens junctions and focal adhesion sites (Fig. 1D). Bioinformatic analysis using STRING and BioGrid revealed a molecular network of multiple actin regulators (Fig. 1E) that contained a high number of actin-bundling proteins such as eplin, drebrin, filamin, and supervillin.
To validate the data arising from the transient hCDC14A overexpression approach, we compared the phospho-proteomes of wild-type (WT) and hCDC14A hCDC14B double knockout (hCDC14A/B-KO) RPE1 cells. We used hCDC14AΔ/Δ hCDC14BΔ/flox cell lines acutely infected with AdCre for this experiment (hCDC14A/B-KO) to minimize adaptation in response to the absence of the hCDC14B gene. Forty-nine cytoskeletal proteins were hyper-phosphorylated in comparisons between the hCDC14A/B-KO and WT cells. However, in contrast to the hCDC14A overexpression screen, in which the vast majority of dephosphorylated sites were "pSP/pTP" (Fig. 1C; 84.1%), only 60% of hyper-phosphorylated sites of the hCDC14A/B-KO screen were pSP/pTP. The remaining sites were not flanked by a proline residue and so probably arose from adaptation to the consequences of the prolonged lack of hCDC14A activity, for example, as a secondary consequence of the long-term down-regulation of other phosphatase(s) or up-regulation of kinase(s). Of the pSP/pTP substrates, the spectrum of the interaction network of hyper-phosphorylated cytoskeletal proteins of hCDC14-KO cells was reminiscent of the hypo-phosphorylated proteins of TetOn-hCDC14A cells in being focused around eplin (Fig. 1 E and F and Table S2). Given that hCDC14A associates with the actin cytoskeleton (20) and that hCDC14B is enriched in the nucleus during interphase before associating with the mitotic chromatin (Fig. S1A), the lack of hCDC14A activity is probably responsible for the hyper-phosphorylation of actin-associated proteins in the RPE1 hCDC14A/B-KO cells.
hCDC14A Interaction Network by BioID.
One of the challenges for the identification of hCDC14A substrates is the transient nature of the interaction between the phosphatase and its substrate (27, 28). The BioID approach is therefore highly attractive for this task as it is able to identify weakly interacting proteins because it supports efficient enrichment by virtue of the fact that neighbors are identified as a result of their biotinylation by hyperactive promiscuous biotin ligase BirA that has been fused to the protein of interest (22). To confirm hCDC14A substrates, we performed a proximity interactor screen with hCDC14A-BirA-HA as bait (Fig. 2A). TetON-hCDC14A-BirA-HA and TetON-BirA-HA cells were analyzed following the experimental stable isotope labeling with amino acids in cell culture (SILAC) approach outlined in Fig. 1A. The biotinylation reaction was initiated by the addition of biotin at the same time as the TetON promoter was induced by doxycycline. After affinity purification of biotinylated proteins and mass spectrometric analysis, 46 proteins were significantly more biotinylated in the hCDC14A-BirA-HA cells than in the BirA-HA control (Fig. S1B; threshold = 2). Gene Ontology (GO) analysis of partners revealed an enrichment of proteins with functions in exosome formation at focal adhesion and cell junction sites (Fig. S1C and Table S3). The Kibra paralogue WWC2 was identified in the hCDC14A BioID (Table S3). The actin bundler eplin once again occupied a pivotal position in the hCDC14A interaction network (Fig. 2B).
Because eplin plays a crucial role in the regulation of actin dynamics and cell transformation, we focused upon understanding the relationship between eplin and hCDC14A. Eplin is expressed as two isoforms, α- and β-eplin, which have translation starts in exons 4 and 2, respectively (29) (Fig. S2 A and B). The actin-bundling activity of both eplin isoforms depends on the centrally localized LIM domain (exons 10–11) and two flanking regions (16, 29). Both isoforms contain the two previously characterized ERK phosphorylation sites at positions 362 and 604 (numbering refers to β-eplin). We began by monitoring the localization and biotinylation status of eplin in the hCDC14A-BirA-HA and BirA-HA cells. Eplin colocalized with hCDC14A-BirA-HA and the streptavidin signal at the cell leading edge (Fig. 2C), a colocalization that was absent from BirA-HA cells (Fig. 2C). Enrichment of biotinylated proteins by streptavidin pull-down showed that both isoforms of eplin were biotinylated in hCDC14A-BirA-HA but not in hCDC14B-BirA-HA or BirA-HA cell lysates, confirming the specificity of the interaction between hCDC14A and eplin (Fig. 2D). Moreover, the mobility of the upper band of α- and β-eplin increased upon hCDC14A-BirA-HA expression (Fig. 2D, lane 4), which is suggestive of an hCDC14A-dependent dephosphorylation of both eplin isoforms.
Together, the hCDC14A phospho-proteome and BioID analyses define an interaction network of hCDC14A with the actin-bundling protein eplin at its heart.
hCDC14A Dephosphorylates Eplin.
Next, we verified the interaction between eplin and hCDC14A by coimmunoprecipitation. We cotransfected flag-β-eplin and myc-hCDC14A in HEK293T cells and performed immunoprecipitation experiments. The phosphatase dead mutant (C278S) version of hCDC14A but not the WT hCDC14A or GFP was coimmunoprecipitated by flag-β-eplin (Fig. 3A). This preferential association of the mutant hCDC14A protein with eplin is consistent with the previous demonstration that CDC14C278S functions as a substrate trap that binds substrates tighter than the WT CDC14 (27). Thus, eplin and hCDC14A are present in common complexes.
Expression of hCDC14A in HeLa cells prompts the dephosphorylation of serine residues 362, 374, 604, and 609 of both isoforms of eplin (Fig. 1). These residues are conserved throughout mammals (Fig. S3A). To further confirm that these sites in eplin are regulated by hCDC14A in vivo, HEK293T cells were transfected with flag-β-eplin without (mock) or with myc-hCDC14A. Mass spectrometric analysis of the immunoprecipitated flag-β-eplin showed that phosphorylation of all four serine residues was strongly reduced by the expression of myc-hCDC14A (Fig. S3B). Thus, hCDC14A has the ability to dephosphorylate serine residues 362, 374, 604, and 609 of eplin in cells.
EGF treatment of cells activates ERK, which in turn phosphorylates α- and β-eplin at two serine residues at positions 362 and 604 (17, 30). Because both ERK sites were identified in the hCDC14A phospho-proteomic analysis (Fig. 1 and Fig. S3B), we asked whether hCDC14A could antagonize ERK phosphorylation of eplin. In logarithmically growing HeLa cells, both eplin isoforms were resolved as multiple bands in immunoblots. Expression of hCDC14A increased the migration of α- and β-eplin (Fig. 3B, lane 3), reflecting a reduction in phosphorylation of eplin. This dephosphorylation was not observed when the phosphatase dead hCDC14C278S was expressed (Fig. 3B, lane 7). EGF treatment invoked a reduction in the migration of α- and β-eplin (Fig. 3B, lanes 2 and 6). However, when cells were treated with EGF after hCDC14A induction, α- and β-eplin migrated at an intermediate position in between the hyper- and hypo-phosphorylated forms, indicating that there was an antagonistic impact of hCDC14A on the ERK phosphorylation events on eplin (albeit a partial one; Fig. 3B, lane 4). The diminished eplin phosphorylation did not arise from a reduction in ERK activity because the levels of active phosphorylated ERK that are universally used as indicators of ERK activity were unaffected by hCDC14A induction (Fig. 3B, P-ERK). As before, expression of hCDC14C278S had no influence upon eplin phosphorylation by ERK (Fig. 3B, lane 8). We conclude that hCDC14A dephosphorylates eplin to counteract phosphorylation of eplin by ERK kinase.
Given this striking in vivo correlation between hCDC14A and dephosphorylation of the ERK target sites in both eplin isoforms, we assessed the ability of hCDC14A to directly dephosphorylate these residues in an in vitro phosphorylation/dephosphorylation assay. Because α-eplin was expressed to higher levels in Escherichia coli than β-eplin, we used this protein as the substrate for this assay (Fig. S3 C–E). Phosphorylation of recombinant α-eplin by human ERK1 kinase invoked an upshift of the α-eplin band in the SDS/PAGE (Fig. 3C, lane 2). Incubation with the nonspecific alkaline phosphatase triggered a downshift of this eplin back toward the position at which the initial recombinant, nonphosphorylated α-eplin migrated. Thus, the mobility shift of recombinant α-eplin was a consequence of direct phosphorylation by ERK1. Importantly, incubation of ERK1-phosphorylated α-eplin with recombinant hCDC14A induced an identical downshift of the α-eplin band (Fig. 3C). Mass spectrometry analysis showed that the serine residues 362, 374, and 604 of eplin were phosphorylated by ERK and subsequently dephosphorylated of recombinant hCDC14A (Fig. 3C). For unknown reasons, the peptide carrying S609 was not identified in three repeated mass spectrometry analyses. We conclude that hCDC14A has the ability to directly dephosphorylate the ERK1 kinase target phosphorylation sites of eplin.
Dephosphorylation of eplin by hCDC14A implies that both proteins colocalize in cells. Indeed, in RPE1 hCDC14A-YFP stable cells, endogenous eplin colocalized with hCDC14A-YFP at the cell edge and along stress fibers (Fig. 3D) to suggest that hCDC14A dephosphorylates eplin at these locations.
hCDC14A Regulates F-Actin Through Dephosphorylation of Eplin.
The fact that eplin is an actin bundler (16) and the finding that eplin is an hCDC14A substrate (Fig. 3) raise the possibility that hCDC14A regulates actin dynamics through eplin. This model predicts that hCDC14A counteracts EGF-induced changes of the actin cytoskeleton. We tested this possibility by analyzing F-actin structures in HeLa TetON-hCDC14A cells by combining EGF-induced eplin phosphorylation with TetON-hCDC14A induction. EGF treatment of HeLa cells promoted the formation of large plasma membrane ruffles that are characterized by highly dynamic actin rearrangements in their lamellipodia (Fig. 4 A and B; EGF, Dox−) (31, 32). Dox-induced expression of hCDC14A inhibited this EGF-induced ruffling of the membranes (Fig. 4 A and B; EGF, Dox+) to suggest that hCDC14A can counteract EGF-induced changes of the actin cytoskeleton.
We next performed experiments designed to interrogate the role played by the EGF/hCDC14A control of eplin in this modulation of the F-actin cytoskeleton. HeLa cells were treated with or without EGF; cells were then transfected either with a mock control or eplin-GFP constructs alongside myc-hCDC14A as indicated. Overexpression of eplin-GFP induced the formation of robust stress fibers without the formation of lamellipodia (Fig. 4 C–F). Addition of EGF together with eplin-GFP expression promoted formation of lamellipodia and reduced the number of stress fibers (Fig. 4 C–F; compare eplin-GFP with EGF+eplin-GFP). These results are consistent with the previous observation that phosphorylation of eplin by ERK reduces its ability to bundle actin filaments (17). Consistently, expression of the phospho-inhibitory eplinS362/S604A promoted the generation of stress fiber but abolished lamellipodia formation even in the presence of EGF (Fig. 4 C–E). Thus, inhibition of stress fiber formation by EGF is mediated by ERK-induced phosphorylation of eplin. We also analyzed the impact of the additional phospho-sites in eplin (Fig. S4 A–C) and found that the inclusion of the phospho-blocking mutations S374A and S609A alongside S362A and S604A in the same molecule of eplin did not further influence the ability of eplin to induce stress fibers under EGF induction beyond the impact of the double S362A S604A change. Thus, phosphorylation of S362 and S604 alone is sufficient to inactivate the F-actin bundling activity of eplin. Finally, coexpression of hCDC14A and eplin-GFP in EGF-treated cells supported the formation of stress fibers and inhibited lamellipodia formation (Fig. 4 C–F). We conclude that hCDC14A counteracts ERK-induced inactivation of the bundling activity of eplin by directly dephosphorylating this key regulator of the F-actin cytoskeleton.
hCDC14APD and eplinKO Cells Display a Reduction in E-Cadherin and a Decline in the Enrichment of β-Catenin at Cell–Cell Adhesion Sites.
Eplin links the E-cadherin–catenin complex to F-actin (23). Down-regulation of eplin frequently occurs in cancer tissues and contributes to the epithelia-to-mesenchymal transition (EMT) that enhances cancer cell metastasis and chemo-resistance of tumors (23, 33⇓⇓⇓–37).
To determine whether eplin regulation by hCDC14A and EGF contributes to EMT, we knocked out the eplin gene in human colon cancer HCT116 cells using the CRISPR/Cas9 system (Fig. S2 B–D). Independent knockout clones were confirmed by sequencing of the chromosomal locus and immunoblot analysis with eplin antibodies (Fig. S2 C and D). Two independent eplinKO clones, eplinKO-23 and -33, showed an ∼50% reduction in E-cadherin protein levels (Fig. 5 A and B). The loss of linkage between the E-cadherin–catenin complex and F-actin has previously been shown to destabilize the E-cadherin protein at cell adhesion sites and promote a similar reduction in E-cadherin levels (38, 39). Furthermore, E-cadherin levels were also reduced in hCDC14APD (hCDC14A phosphatase dead version) cells (Fig. 5 A and B) that carried hyper-phosphorylated, inactive eplin (Fig. 1). Thus, hCDC14A-mediated dephosphorylation of eplin plays an important role in stabilizing the molecular linkage of cell–cell adhesion. This notion was further supported by the observation that enrichment of β-catenin at cell–cell junctions in hCDC14APD and eplinKO cells was decreased, even though total cellular β-catenin levels remained constant (Fig. 5 A–D).
To determine whether β-catenin mislocalization is a direct consequence of the hyper-phosphorylation of eplin, we expressed the eplinS362/S604 phospho-mimetic and -inhibitory forms in eplinKO and WT cells. Interestingly, eplinS362A/S604A restored the β-catenin organization as efficiently as WT eplin, yet eplinS362D/S604D failed to do so (Fig. 5 E and F). This result suggests that phosphorylation of eplin at S362 and S604 regulates recruitment of β-catenin to cell adherens sites.
The β-catenin/E-cadherin phenotype of hCDC14APD and eplinKO cells suggests that both hCDC14A and eplin stabilize the E-cadherin–α/β-catenin complex. Consequently, it can be anticipated that malfunction of either hCDC14A or eplin may eventually affect cell–cell adhesion and promote metastasis. We therefore used a soft agar tumor formation assay as a test of this hypothesis. Both HCT116 hCDC14APD and eplinKO cells formed tumor colonies with rough edges, whereas WT cells formed compact colonies with smooth edges (Fig. 5 G and H). In fact, both hCDC14A and eplin are significantly down-regulated in colorectal carcinoma (Fig. 5I) (20). This down-regulation is associated with poor survival in cancer patients (Fig. 5J) (20). Taken together, we suggest that hCDC14A regulates cell migration and adhesion through dephosphorylation of eplin (Fig. 5K).
We have previously shown that human CDC14A regulates cell migration and cell adhesion (20). One of the major bottlenecks for understanding the phenotypes arising from manipulation of CDC14A in functional studies of human CDC14 phosphatases has been the lack of a defined cohort of substrates. We have therefore combined phospho-proteomics and BioID analyses to generate a comprehensive list of the components of the molecular network regulated by hCDC14A. The actin regulator eplin occupies a pivotal position within this network (Figs. 1, 2, and 5K). It is well known that phosphorylation of eplin by ERK at S362 and S604 decreases its actin-bundling affinity (17). Strikingly, we find that dephosphorylation of these sites by hCDC14A regulates the balance between formation of stress fibers and membrane ruffling that is promoted by EGF stimulation (Fig. 4).
Eplin provides the molecular linkage between the actin cytoskeleton and the E-cadherin–catenin complex that is critical for stabilizing cell–cell adhesion and maintaining apical cell polarity (23) (Fig. 5). We recorded a reduction in β-catenin enrichment at cell–cell junctions in both eplinKO and hCDC14APD cells to indicate that the dephosphorylation of eplin by hCDC14A is required to stabilize this interaction (Fig. 5K). Moreover, the protein level of E-cadherin was significantly reduced in both cell lines, probably due to the destabilization of the E-cadherin–α/β-catenin–F-actin association (Fig. 5). Because of the prominent roles that E-cadherin plays in maintaining proper cell–cell adhesion, polarity of epithelial cells, and sequestration of the oncoprotein β-catenin at the plasma membrane, down-regulation of E-cadherin is a frequent event during carcinogenesis (40⇓⇓⇓–44). Loss of E-cadherin often leads to EMT and enhanced invasion and metastasis. This may explain why the eplinKO and hCDC14APD colonies in soft agar have rough edges (Fig. 5G) (20). Importantly, a reduction of hCDC14A and eplin levels is frequently observed in colorectal carcinoma and is associated with poor prognosis in cancer patients (Fig. 5 I and J) (20, 34, 35). Collectively, we propose that the balance of eplin phosphorylation that is maintained by the antagonistic activities of ERK and hCDC14A toward conserved phosphorylation sites on eplin plays a key role in regulating actin dynamics to modulate cell migration and adhesion of both normal and transformed cells (Fig. 5K). Thus, hCDC14A and eplin are important factors in restraining tumor aggressiveness.
Materials and Methods
Cell Culture.
HeLa FRT and HEK 293T cell lines were cultured in DMEM GlutaMax medium containing 10% FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 °C and 5% CO2. Stable cell lines were constructed as previously described (20).
Quantitative Phospho-Proteomic Analysis.
HeLa FRT YFP and hCDC14A-YFP cells were cultured in light and heavy SILAC medium, respectively. Proteins were extracted and mixed at a ratio of 1:1. Phospho-peptides were enriched as described (45). Peptide identification and quantification was achieved using the MaxQuant software package (126.96.36.199) (46). Results were analyzed using Perseus (47).
CRISPR/Cas9-Mediated Eplin Knockout.
Guide RNA (gRNA) targeting the exon 9 of eplin was designed using a web tool (crispr.mit.edu/) (48). The transfected cells were sorted via FACS and seeded on 96-well plates for single-clone selection. The clones were harvested and screened via genomic PCR and Western blot.
Details are in SI Materials and Methods including experimental procedures and reagents.
No human or animal clinical experiment was done and no ethics approval was needed for the study.
SI Materials and Methods
Antibodies and Reagents.
The following antibodies and reagents were used: Phalloidin–Atto 550 (Sigma, 19083, 1:500); recombinant human ERK1 (Sigma, E7407); DRAQ5 (Cell Signaling, 4084, 1:1,000); Streptavidin-Alexa647 [Thermo Fisher, S21374, 1:2,000 in indirect immunofluorescent (IF)]; mouse anti-GFP [Roche, 11814460001, 1:200 in IF and 1:1,000 in immunoblots (IB)]; rabbit anti–E-cadherin (24E10) (Cell Signaling, 3195, 1:100 in IF and 1:1,000 in IB); mouse anti–β-catenin (BD Bioscience, 610153, 1:1,000 in IF and IB); mouse anti-flag (9A3) (Cell Signaling, 8146, 1:1,000 in IF and IB); rat anti-HA (3F10) (Roche, 11867423001, 1:100 in IF and 1:1,000 in IB); rabbit anti-eplin (Bethyl, A300-103A, 1:100 in IF and 1:2,000 in IB); rabbit anti-ERK1/2 (137F5) (Cell Signaling, 4695, 1:1,000 in IB); rabbit anti–phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling, 4370, 1:1,000 in IB); and mouse anti-myc (9E10) (Santa-Cruz, sc-40, 1:100 in IF and 1:200 in IB).
Cell Culture.
HeLa FRT and HEK 293T cell lines were cultured in DMEM GlutaMax medium containing 10% FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 °C and 5% CO2. Stable cell lines were constructed as previously described (20). HCT116 WT and mutant cell lines were cultured in McCyo's 5A medium containing 10% FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin in 5% CO2 at 37 °C.
Quantitative Phospho-Proteomic Analysis.
HeLa FRT YFP and hCDC14A-YFP cells were cultured in light (Arg12C14N Lys12C14N) and heavy (Arg13C15N Lys13C15N) SILAC medium (Silantes), respectively. After eight passages, cells were expanded to 4- × 15-cm dishes for each condition. Proteins were extracted and mixed at a ratio of 1:1. Subsequently, phospho-peptides were enriched according to the strong cation exchange chromatography/immobilized metal affinity chromatography (SCX/IMAC) protocol as described (45). This resulted in 48 fractions (24 phospho-enriched + 24 phospho-depleted), which were analyzed by LC-MS using a Dionex UltiMate 3000RSLCnano HPLC system (Thermo Scientific) coupled to a LTQ Orbitrap Elite mass spectrometer (Thermo Scientific). Peptides were separated on a self-pulled and self-packed analytical emitter C18 column, using gradients of increasing amounts of acetonitrile (phospho-enriched = 90 min, phospho-depleted = 120 min), and measured in the mass spectrometer using a Top15 collision-induced dissociation high–low data-dependent acquisition strategy. Peptide identification and quantification was achieved using the MaxQuant software package (188.8.131.52) with its built-in Andromeda search algorithm (46). The results of the database search were analyzed using Perseus (47). Three replicates of the experiment were run, and Table S1 provides a short list of the results.
To investigate whether a wider range of substrates can be identified upon human CDC14A and CDC14B deletion, the global phosphorylation assay was performed using RPE1 WT and AΔ/Δ BΔ/flox (hCDC14A knockout; hCDC14B one allele KO and one allele floxed) cell lines acutely infected with AdCre. A label-free approach was used, and MS/MS analysis was done after phospho-peptide enrichment as described above (20). Three replicates for each cell type were run, and the result is shown in Table S2.
HeLa FRT hCDC14A-BirA-HA, hCDC14B-BirA-HA, and BirA-HA cells were cultured in medium containing 50 nM biotin. Doxycycline (1 µg/mL) was added to the medium to induce ectopic protein expression. Twenty-four hours later, cells were harvested and lysed using RIPA buffer [10 mM Tris–Cl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS and benzonase (Millipore)]. The lysate was centrifuged at 20,000 × g for 15 min, and the supernatant was harvested and incubated with streptavidin Sepharose beads (GE Life Science) at 4 °C for 3 h. Then the beads were collected by centrifugation and washed with RIPA buffer three times. Bound proteins were eluted with 2× Laemmli buffer containing 2 mM biotin at 95 °C for 10 min.
HeLa FRT cells or HCT116 cells were seeded onto wells or coverslip. When the cell confluency reached 60%, cells were transfected with appropriate plasmids using Lipofectamine 2000. For six-well transfection, 1 µg plasmid and 4.5 µL Lipofectamine 2000 were used. For 12-well transfection, 0.5 µg plasmid and 2 µL lipofectamine 2000 were used.
Cells on a coverslip were washed with PBS once and fixed with 3% paraformaldehyde for 10 min at room temperature. The sample was washed with PBS once and incubated in 10% FBS to block unspecific binding. Then the sample was incubated with primary antibodies for 1 h at room temperature, followed by washing three times with PBS. The sample was then incubated with secondary antibodies for 30 min at room temperature in the dark followed by washing three times with PBS. The coverslip was mounted with ProLong Gold Antifade Mountant (Thermo Fisher) and dried overnight at room temperature.
Eplin Purification.
N-terminally His10SUMO3-tagged, mouse α-eplin was produced in E. coli Rosetta cells (Merck Millipore) at 18 °C overnight and induction with 0.2 mM isopropyl β-d-1-thioglactopyranoside. After cell lysis and clarification of the supernatant, eplin was purified by Ni-NTA affinity chromatography (GE Healthcare). The N-terminal His10SUMO tag was cleaved by SenP2 protease and removed by reverse Ni-NTA chromatography. The resulting unmodified murine EPLIN was further purified by size-exclusion chromatography (Superdex 200 26/60, GE Healthcare). The purification was carried out using buffer A [50 mM Tris⋅HCl, 500 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine, pH 7.5] for elution from the Ni-NTA column, and buffer A containing 500 mM imidazol was used. The secondary structure of recombinant EPLIN was determined using circular dichroism (CD) spectroscopy (JASCO J-715, Spectra Manager II software for analysis). Zn2+-dependent changes in protein size were studied using size-exclusion chromatography (SEC 650, BioRad) in buffer A containing 0 or 10 µM ZnCl2.
In Vitro Phosphatase Assay.
Eplin (1 µg) was first phosphorylated by ERK (from Sigma) in the presence of 200 µM ATP in 25 µL kinase buffer (50 mM Tris⋅HCl, 10 mM MgCl2, 0.1 mM EDTA, and 2 mM DTT) at 37 °C for 1 h. Then the reaction was terminated by increasing EDTA concentration to 20 mM. For phosphatase assays, 5 µL of the terminated ERK kinase reaction product was added into 20 µL phosphatase buffer (30 mM imidazole, 1 mM DTT, 1 mM EDTA, 150 mM KCl, 1 mM MgCl2, and 25 mM K-Hepes). Recombinant hCDC14A or alkaline phosphatase was added to the solution and incubated at 30 °C for 1 h. The products were analyzed on immunoblots following SDS/PAGE. A fraction of the ERK phosphorylation and hCDC14A dephosphorylation reactions on eplin was analyzed by three independent mass spectrometry analyses.
CRISPR/Cas9-Mediated Eplin Knockout.
The gRNA targeting the exon 9 of eplin was designed using the web tool (crispr.mit.edu/) (48). The gRNA (GTTGAACCTCACTCTTAACC) was ordered as primer, inserted next to the U6 promoter in the scaffold vector pSpCas9(BB)-2A-GFP(PX458) (a gift from Feng Zhang, Broad Institute of MIT and Harvard, Cambridge, MA; Addgene plasmid #48138), and transfected into HCT116 cells using lipofectamine 2000. The transfected cells were sorted via FACS and seeded on 96-well plates for single-clone selection. The clones were harvested and screened via genomic PCR (forward 1: CTCCTCACTGAGAACCTGATTTC; reverse 1: ACCAGTAGACCAATGTCCAAAG; forward 2: GGCAGAGGGTAAGAAAGCCT; reverse 2: GACCAATGTCCAAAGGCTCAG) and Western blot.
E.S. is supported by Deutsche Forschungsgemeinschaft Grant DFG Schi 295/3-3. T.R. is supported by Collaborative Research Centre Grant SFB1036.
- ↵1To whom correspondence should be addressed. Email: e.schiebel{at}zmbh.uni-heidelberg.de.
Author contributions: N.-P.C. and E.S. designed research; N.-P.C., B.U., R.H., W.D., M.P., I.L., and P.K. performed research; T.R. contributed new reagents/analytic tools; N.-P.C., B.U., R.H., and W.D. analyzed data; and N.-P.C. and E.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1619356114/-/DCSupplemental.
- ↵.
- Blanchoin L,
- Boujemaa-Paterski R,
- Sykes C,
- Plastino J
- ↵.
- Tojkander S,
- Gateva G,
- Lappalainen P
- ↵
- ↵.
- Stevenson RP,
- Veltman D,
- Machesky LM
- ↵.
- Rognoni E,
- Ruppert R,
- Fässler R
- ↵.
- Zhou Y,
- Su J,
- Shi L,
- Liao Q,
- Su Q
- ↵.
- Hall A
- ↵.
- Sit ST,
- Manser E
- ↵
- ↵
- ↵
- ↵.
- Meyer RK,
- Aebi U
- ↵.
- Yamashiro S,
- Yamakita Y,
- Ono S,
- Matsumura F
- ↵
- ↵.
- Sumi T,
- Matsumoto K,
- Takai Y,
- Nakamura T
- ↵.
- Maul RS, et al.
- ↵.
- Han MY,
- Kosako H,
- Watanabe T,
- Hattori S
- ↵.
- Mocciaro A,
- Schiebel E
- ↵
- ↵.
- Chen NP,
- Uddin B,
- Voit R,
- Schiebel E
- ↵.
- Ji M, et al.
- ↵.
- Roux KJ,
- Kim DI,
- Raida M,
- Burke B
- ↵.
- Abe K,
- Takeichi M
- ↵.
- Bremmer SC, et al.
- ↵
- ↵.
- Gray CH,
- Good VM,
- Tonks NK,
- Barford D
- ↵.
- Lanzetti L, et al.
- ↵
- ↵
- ↵.
- Zhang S, et al.
- ↵.
- Isogai T, et al.
- ↵.
- Balaji K, et al.
- ↵.
- Liu R, et al.
- ↵.
- Collins RJ,
- Jiang WG,
- Hargest R,
- Mason MD,
- Sanders AJ
- ↵
- ↵.
- Ohoka A, et al.
- ↵
- ↵.
- Le TL,
- Yap AS,
- Stow JL
- ↵
- ↵
- ↵
- ↵.
- Umbas R, et al.
- ↵
- ↵.
- Oka H, et al.
- ↵
- ↵
- ↵
- ↵


text:
This review will focus on a 3 ounce/85 gram bag of the Duck Dynasty jerky brand name for their hickory smoked bacon jerky. The bag was purchased back in December 2013, at a ShopRite store in Lyndhurst, New Jersey.
The very large, successful private labeler Monogram Foods LLC. manufactures and distributes this jerky. They are located in Memphis, Tennessee, although they have Read More

subset: commoncrawl


text:
Northern Success Stories
Northern Development > Explore Our Region > Northern Success Stories
What Are People Saying in Valemount?
"When the Olympic/Paralympic Live Sites program was announced in 2005, Valemount's Council had a dream and saw the opportunity. But it was only with the contributions of Northern Development and the Columbia Basin Trust that this dream became a reality."Silvio Gislimberti
Economic Development Officer
Village of Valemount


text:
Hanging With The Girl-Kings Of One Of The World's Only Matriarchies
by Mallika Rao / 09/04/2014
Mawlynnong isn't like the rest of India, or the rest of the world for that matter. Tucked into the jungle of the northeast Indian state of Meghalaya, on the border of Bangladesh, the village is home to the indigenous Khasi people. Theirs is a matrilineal society, where women inherit land, children take their mothers' names, and men marry into their wives' family homes. The setting is also unique: lush, and so well-cared for, tourists across India visit to marvel at the lack of trash.
The inherent visual drama of Mawlynnong wasn't lost on Karolin Klüppel. Last year, the German photographer traveled there to photograph the girls of the village in their homes and outdoors.
In her series "Mädchenland," or "girl-land," Klüppel shows her subjects in classically girlish poses. She did this to highlight how adult they actually are. Indeed, the twist on the iconic visual of a child wearing oversized jewelry more fit for her mother — the necklace below is made of dried fish skeletons — is arresting.
Küppel describes the inverse at play as "a completely opposite impression… namely a certain elevation of the girls above childhood, a strong self-awareness and pronounced air of self-sufficiency."
Five Things We Know About Societies Run By Women
by Jill Hamilton / 2013/05/10
First, let's get one thing straight—there are exactly zero societies that are run completely by women. When women have power, they don't just take over the joint, refusing men an education and paying them 80 cents on the dollar. Nope, instead they do a bunch of girlie junk like keeping everyone happy with shared resources, stable families and good sex. Christopher Ryan, Ph.D., co-author of Sex at Dawn says, "Societies in which women's status is equal to or greater than men's tend to be characterized by less conflict, greater social harmony, and higher levels of sexual interaction." Happier in other words. Let us count the ways.
Matriarchy Is Not The Opposite Of Patriarchy
"In matriarchies, mothers are at the center of culture without ruling over other members of society," says Heidi Goettner-Abendroth, founder of The International Academy HAGIA for Modern Matriarchal Studies. "The aim is not to have power over others and over nature, but to follow maternal values, ie. to nurture the natural, social and cultural life based on mutual respect." In other words, if patriarchy is West Point, heavy on the rules and hierarchy, matriarchy is that alternative school where kids call teachers by their first names and play non-competitive games with the parachute.
Marriage Is Less Binding (If It Exists At All)
Contrary to every diamond ad ever, when women have a say in things, they aren't all that eager to jump into a long, forever marriage. There is a large overlap between the top ten countries with the highest gender income ratio and the highest divorce rates. Women with economic power aren't forced to stick it out in bad marriages. So they don't. Among the Khasi of northeast India, matrilocality (the practice of children living with the mother's family or clan) eliminates economic free fall or a jarring move if parents divorce. "No matter how many times the woman marries, her children will always remain with her," says Patricia Mukhim, a Khasi and editor of The Shillong Times. "And even if a man abandons a woman he has impregnated, the children are never 'illegitimate.'"
More Sex, Less Judgement
Empowered women can be, technically, sluttier – that is, they have can sex with a variety of partners of their choosing – but conversely, they're not treated as "sluts." Women in the Chinese matrilineal Mosuo society, for instance, get their own private babahuago (flower room) for receiving visits from lovers. No one worries about commitment since any resulting children are raised in the mother's house with the help of her brothers and the rest of the community. No one's judging the women harshly, and why should they? Everyone's too damn happy. Meanwhile, Iceland, considered to be one of the most feminist-friendly countries in the world, banned strip clubs, calling them an exploitative of women.
Fatherhood is Redefined Entirely
In matriarchal societies, men tend to get way more or way lessinvolved in parenting. The devoted dads of the Aka tribe in Africa are within reach of their infants 47% of the time and so dedicated they'll put a crying baby on their own nipple to pacify it, according to archaeologist Barry Hewlett. And in family-friendly Sweden, 85% of men take paid parental leave. By contrast, among the Mosuo there is a word for "father" but "it's nothing like our concept of what a father should be,"according to writer Ricardo Coler. "These duties are taken over by the mother or the family. Often, the women don't know which man is responsible for the pregnancy." And in at least 18 different South American tribes, there is shared paternity. They believe that each different man's sperm helps shape a baby and thus, each child has several "fathers."
The Gulabi gang is a group of female vigilantes that stalk the streets of India dressed in matching pink saris. They target rapists, shady government officials and abusive husbands, and exact their own brand of violent justice on them. The gang was founded in 2006 by Sampat Pal Devi, after she discovered a friend was a victim of domestic abuse and that the police were reluctant to get involved. Sampat Pal Devi gathered a group of neighbourhood women, taught them to fight with laathis (bamboo sticks), and led an attack on the alcoholic husband. Since then the group has grown to over 20,000 members over India.
Crime Plummets
Men commit far more violent crimes than women (about 90% of homicide and robbery offenders in the U.S. are men) but equality can dampen crime. "In more gender-egalitarian societies, there is much less crime by both women and men," says Jennifer Schwartz of Washington State University, Department of Sociology. "And in those societies, the crime gap between women and men is somewhat larger, that is, women participate even less in crime." It turns out that not even men like it when a bunch of jerky men are bossing everyone else around. "For most men, [egalitarian] societies are far preferable to those in which a few high-status men dominate everyone else, accumulating resources, power, and available women for themselves," said Ryan.
Amazons, 5th Century BC
Historians have some disagreement about whether the legendary female society of Amazons ever truly existed. Some theorize that they lived in what is now the Ukraine, some think near Syria. The historical record is spotty, but an archeological dig near the Russian steppes revealed some interesting things. Buried there were women warriors with weapons and armor, and nearby were men and children. This tribe could very well have inverted the basic gender roles that the rest of the world subscribed to, but it is unlikely that they are the Amazons of tale.
Vietnam, AD 40 to 43
One of the most important historical periods in Vietnamese lore is also the only time that the nation has been ruled by a pair of females. Trưng Trắc and Trưng Nhi were a pair of Vietnamese siblings who rebelled against the Han Dynasty occupation after Trắc's husband was killed. They led a revolution to take back the land of Nam Viet from Chinese occupation, after which the pair proclaimed themselves co-queens and ruled the troubled land for three years. Overcome by China in AD 43, Nam Viet was once again absorbed into China. The Trưng sisters committed suicide by drowning themselves in a river to preserve their honor, but today they are some of the liberated country's most respected historical figures.
Egypt, 1479 to 1458 BC
Ancient Egypt saw a diverse split in the genders of its pharaohs, with both men and women holding the throne for significant periods of time. But it's telling to learn that one of the greatest periods in the empire's history came under a woman—Queen Hatshepsut. She was dedicated to expanding her country's advances in construction, building more structures than any of her contemporaries. She also financed expeditions to foreign lands, one of which led to the transplanting of a grove of myrrh trees in Egypt. She was succeeded by her son, Thutmose III.
Matamba, AD 1626 to 1663
Traditionally, most of the native tribal groups of Africa were run by men. But a very interesting exception came in the 17th century with the rise of Queen Njinga, who lived in what is now Angola. The strong-willed Njinga's first taste of diplomacy came when her brother King Ngola Mbande sent her to negotiate a peace treaty with the invading Portugese. When Ngola Mbande died in 1624, instead of a traditional successor Njinga declared herself the regent. She then allied with neighboring states to drive out the Portugese. During her reign, she would continue to war with Portugal, even personally leading guerilla strikes on their ships. She died in 1663 and is remembered in modern-day Angola as one of the region's greatest heroes.
Trobriand Islands, Present Day
One of the most interesting matrilineal societies contains 12,000 indigenous natives of the Trobriand Islands, a series of coral atolls off the coast of New Guinea. The islanders are subsistence farmers, but they're all under the direction of womenfolk, who pass down inheritance through female lines, rather than male ones. Every year, males of the community are required to make an offering of yams to their sisters and mothers, according to Annette Weiner's The Trobrianders of Papua New Guinea. And babies aren't created by having sex with men in their religion, but rather by the ghost of an ancestor taking up residence in a woman's womb. On the island of Orango Grande, in the Bijagos Archipelago, off the coast of Guinea Bissau, there is a matriarchal society where women possess all the power, where they organise themselves into associations which manage the economy, social welfare and the law.
"Bijago matriarch and her family. It is women who impose sanctions, direct, advise and distribute goods, who are respected as the absolute owners of both the house and the land. Here it is the man who has the obligation to dress very well to attract the attention of a woman. Women hold the supreme power of divorce in marriage. Men are turned to only for the tilling of the fields, hunting monkeys and fishing."
6 Modern Societies Where Women Rule
by Laura Turner Garrison / July 23, 2012
By standard definition, a matriarchy is a "family, group or state governed by a matriarch." Anthropologists and feminists have since created more specific classifications for female societies, including the matrilineal system. Matrilineality refers not only to tracing one's lineage through maternal ancestry, it can also refer to a civil system in which one inherits property through the matriline. This often leads to the division of such societies into matrilineal clans, or "matriclans." Here are a few notable ones that still exist.
"Chez les Mosuo, la violence interpersonnelle est rare"
Living near the border of Tibet in the Yunnan and Sichuan provinces, the Mosuo are perhaps the most famous matrilineal society. The Chinese government officially classifies them as part of another ethnic minority known as the Naxi, but the two are quite different in both culture and language. The Mosuo live with extended family in large households; at the head of each is a matriarch. Lineage is traced through the female side of the family, and property passed down along the same matriline. Mosuo women typically handle business decisions and men handle politics. Children are raised in the mother's household, and take her name. The Mosuo have "walking marriages," in that there is no institution and women choose their partners by literally walking to the man's home. Couples never live together. Since the child always remains in the mother's care, sometimes the father plays little role in the upbringing. In some cases the identity of the father is not even known. Instead, the male's childrearing responsibilities remain in his own matrilineal household.
Living primarily in West Sumatra, Indonesia, at four million people the Minangkabau are the largest known matrilineal society today. In addition to tribal law requiring all clan property to be held and bequeathed from mother to daughter, the Minangkabau firmly believe the mother is the most important person in society. Upon marriage, every woman acquires her own sleeping quarters. The husband may sleep with her, but must leave early in the morning to have breakfast at his mother's home. At age 10, boys leave their mother's home to stay in men's quarters and learn practical skills. Men are always clan chief, but women select the chief and can remove him from office should they feel he failed to fulfill his duties.
"The introduction of Islam in the seventh century a.d. had the long-term effect of superimposing patrilineal institutions upon traditional matriliny. Most Tuareg today are bilateral in descent and inheritance systems (Murphy 1964; 1967). Descent-group allegience is through the mother, social-stratum affiliation is through the father, and political office, in most groups, passes from father to son."
The Akan are a majority in Ghana, where they predominantly reside. Most still adhere to the matrilineal social structure, despite pressure from the government. The Akan social organization is fundamentally built around the matriclan. Within this matrilineal clan, identity, inheritance, wealth and politics are all determined. All matriclan founders are female, but men traditionally hold leadership positions. Succession to inheritable appointments is still determined by the male's relationships to the women in his matriclan. Often, the man is expected to not only support his own family, but those of his female relatives.
The Bribri are a small indigenous group of just over 13,000 people living on a reserve in the Talamanca Canton in the Limón Province of Costa Rica. Like many other matrilineal societies, the Bribri are organized into clans. Each clan is made up of extended family, and the clan is determined through the mother/females. Women are the only ones who traditionally can inherit land and also the only ones endowed with the right to prep the cacao used in sacred Bribri rituals.
Much like their Khasi neighbors in the North-East Indian state of Meghalaya, the Tibeto-Burman-speaking Garos pass property and political succession from mother to daughter. The youngest daughter is typically named heiress and her marriage will often be arranged. Sometimes the family may have to physically capture the future husband. Once married, the husband lives in his wife's house. Should it not work out, the union is dissolved without social stigma. Marriage is not a binding contract, but one is expected to remarry after divorce. . If a Garos woman pursues her own mate, she plays aggressor and the male demurs. Parents must still sanction the union, as any match remotely inter-clan is forbidden.
The Nagovisi live in South Bougainville, an island west of New Guinea. Anthropologist Jill Nash reported Nagovisi society was divided into two matrilineal moieties, which are then divided into matriclans. Nagovisi women are involved in leadership and ceremonies, but take the most pride in working the land entitled to them. Nash observed that when it comes to marriage the Nagovisi woman held gardening and shared sexuality at equal importance. Marriage is not institutionalized. If a couple is seen together, sleeps together, and the man assists the woman in her garden, for all intents and purposes they are considered married.
MUST BE WILLING to SHARE
Residents Of Noiva Do Cordeiro, Almost All-Woman Town, Seek Bachelors
by Simon McCormack / 08/28/2014
According to the Telegraph, they have constructed a society in the southeast part of Brazil that is communal, egalitarian and almost all-female. But some of the women in the town say it would be nice to have a few more dudes in the mix. "I haven't kissed a man for a long time," Nelma Fernandes, 23, told the Telegraph. "We all dream of falling in love and getting married. But we like living here and don't want to have to leave the town to find a husband. We'd like to get to know men who would leave their own lives and come to be a part of ours. But first they need to agree to do what we say and live according to our rules." There are a few married women in Noiva Do Cordeiro, but their husbands work in cities no closer than 60 miles away and tend to only live in the town on weekends. Both the Telegraph story and other outlets portray the desire for more members of the opposite sex in the town as a desperate plea to men to help them "shoulder the burdens" of everyday life. But judging from what the residents have actually said, that doesn't seem like what's going on. "There are lots of things that women do better than men," Rosalee Fernandes, 49 said. "Our town is prettier, more organized, and far more harmonious than if men were in charge. When problems or disputes arise, we resolve them in a woman's way, trying to find consensus rather than conflict." So it appears more like these women have created a society of anarchist anthropologists' dreams and some of them would like to hang out with guys more, provided they don't tear the town's social fabric to shreds.
SIGNS YOU MAY ALREADY BE a WOMAN
If you're looking for a society run by women, you could be looking at one all around you — or at least the rapid evolution toward one. The potential evidence is everywhere, but before we go into any examples, let's take a step back and cover a few fundamental points in the debate. There are different ways the term matriarchy is defined, and it can get a little confusing. For example, some people consider matriarchies to mean women are the dominate gender in a society. They make the decisions, they drive the economy and they rule the politics — basically the flipside of patriarchies. But others expect matriarchies to manifest more as egalitarian societies. That means everyone stands on equal footing and works in partnership with one another. Experts who endorse this interpretation believe it's unfair to assume a matriarchy would look anything like a patriarchy in practice, for the very reasons a distinction can be made in the first place.
Management the Matriarchal Way
Let's start with the latter definition from the last page. Rather than imagining a matriarchy as a culture where women pin down men by the points of their stiletto heels, picture it as a cultural model where women simply occupy a central role in the society. Everyone is on even ground and decisions are reached by consensus. One excellent example of this is the Minangkabau people of West Sumatra. Around 4 million in number, the Minangkabau are an ethnic group that consider themselves a matriarchy, and this, combined with a philosophy called adat that emphasizes nurturing both people and nature, forms a core tenet of their society [source: Sanday]. How does this work? Women own the land, and it's passed down matrilineally from mother to daughter. Rather than women moving in with their husbands upon marriage, the men join their new brides' households. Women are considered central to the community, and older women even more so — they're regarded as the strongest pillars in the society. The men aren't oppressed; they're simply peripheral in familial organization. All the members of a family work for the betterment of everyone else.
But what about a society where females truly do dominate males? According to some, in many parts of the world that's exactly what's happening, even in the United States. The theory goes like this: As we've transitioned into a postmodern society, women seem more adaptable or more naturally suited for the changes and challenges that evolution involves. Perhaps success is due in part to talents such as social smarts and communication skills which are now more often valued in the workplace than the physical strength and stamina that originally propelled men to the top. Women today hold more managerial and professional jobs than they ever have (50 percent up from 25 percent in 1980). About three quarters of the approximately 8 million jobs lost in the latest recession were lost by men in blue-collar industries such as construction and manufacturing, and white-collar industries like finance. When it comes to growing sectors of the economy, men are leading in only 2 of the top 15 categories — janitorial and engineering [source: Rosin]. In terms of education, women are getting degrees three to two over men. In terms of money, women control more than half the wealth in the United States, and in terms of purchasing power, their control is even greater. Women generally manage some 80 to 90 percent of their families' buying power [sources: PBS, Pittsburg Post-Gazette].
Whether these statistics are signs of a domineering or egalitarian matriarchy is not yet completely clear. But it does appear that a strong social shift has been occurring and still is. Although a wage gap remains between the genders, women workers are slowly but steadily closing that divide. In the past few decades, women have taken the workplace by storm; they still largely rule the home; and they comprise a huge portion of the consumers pushing the demand side of the market as well. Even if that's not technically a matriarchy, it certainly doesn't sound like much of a patriarchy either.
How to Design a City for Women: An experiment in "gender mainstreaming"
by Clare Foran / Sep 16, 2013
In 1999, officials in Vienna, Austria, asked residents of the city's ninth district how often and why they used public transportation. "Most of the men filled out the questionnaire in less than five minutes," says Ursula Bauer, one of the city administrators tasked with carrying out the survey. "But the women couldn't stop writing." The majority of men reported using either a car or public transit twice a day — to go to work in the morning and come home at night. Women, on the other hand, used the city's network of sidewalks, bus routes, subway lines and streetcars more frequently and for a myriad reasons. "The women had a much more varied pattern of movement," Bauer recalls. "They were writing things like, 'I take my kids to the doctor some mornings, then bring them to school before I go to work. Later, I help my mother buy groceries and bring my kids home on the metro.'"
Women used public transit more often and made more trips on foot than men. They were also more likely to split their time between work and family commitments like taking care of children and elderly parents. Recognizing this, city planners drafted a plan to improve pedestrian mobility and access to public transit. Additional lighting was added to make walking at night safer for women. Sidewalks were widened so pedestrians could navigate narrow streets. And a massive staircase with a ramp running through the middle was installed near a major intersection to make crossing easier for people with strollers and individuals using a walker or a wheelchair. The decision to look at how men and women used public transit wasn't a shot in the dark. It was part of a project aimed at taking gender into account in public policy. In Vienna, this is called gender mainstreaming.
Gender mainstreaming has been in place in the Austrian capital since the early 1990s. In practice, this means city administrators create laws, rules and regulations that benefit men and women equally. The goal is to provide equal access to city resources. And so far, officials say it's working. Vienna has adopted gender mainstreaming in a number of areas of city administration, including education and health care policy. But nowhere has it had more of an impact than on the field of urban planning. More than sixty pilot projects have been carried out to date. As the size and scale of these projects increase, gender mainstreaming has become a force that is literally reshaping the city.
Urban planners have been melding mainstreaming and city design in Vienna for over two decades and they've gotten it down to something of a science. Before a project gets underway, data is collected to determine how different groups of people use public space. "There are so many questions that need to be asked," Eva Kail tells me. Kail has been instrumental in bringing gender mainstreaming to Vienna and currently works as a gender expert in the city's Urban Planning Group. "You need to know who is using the space, how many people, and what are their aims. Once you've analyzed the patterns of use of public space, you start to define the needs and interests of the people using it," she explains. "Then planning can be used to meet these needs."
Mainstreaming got off the ground in Vienna in 1991 when Kail and a group of city planners organized a photography exhibit titled "Who Owns Public Space — Women's Everyday Life in the City." It depicted the daily routines of a diverse group of women as they went about their lives in the Austrian capital. Each woman tracked a different route through the city. But the images made clear that safety and ease of movement were a priority for all of them. It sparked a media firestorm. "Newspapers, television and radio were all covering it and 4,000 people visited," Kail says. "At the time it was something completely new. But politicians quickly realized it was something people were interested in and they decided to support it." Soon after, the city green lit a series of mainstreaming pilot projects. One of the first to be carried out was an apartment complex designed for and by women in the city's 21st district. In 1993, the city held a design competition for the project, which was given the name Frauen-Werk-Stadt or Women-Work-City.
Women-Work-City has an on-site kindergarten (Image: Paolo Mazzoleni)
The idea was to create housing that would make life easier for women. But what exactly did that mean? Time use surveys compiled by Statistik Austria, the Austrian national statistics office, showed that women spent more time per day on household chores and childcare than men. Women-Work-City was built with this in mind. It consists of a series of apartment buildings surrounded by courtyards. Circular, grassy areas dot the courtyards, allowing parents and children to spend time outside without having to go far from home. The complex has an on-site kindergarten, pharmacy and doctor's office. It also stands in close proximity to public transit to make running errands and getting to school and work easier. "What made the project unique was that we worked to define the needs of the people using the space first and then looked for technical solutions," Kail says. "Very often it is the opposite, where technical or aesthetic solutions determine the end result."
Following completion of Women-Work-City, city officials turned their attention to Vienna's network of public parks and commissioned a study to see how men and women use park space. What they found was surprising. The study, which took place from 1996 to 1997, showed that after the age of nine, the number of girls in public parks dropped off dramatically, while the number of boys held steady. Researchers found that girls were less assertive than boys. If boys and girls would up in competition for park space, the boys were more likely to win out. City planners wanted to see if they could reverse this trend by changing the parks themselves. In 1999, the city began a redesign of two parks in Vienna's fifth district. Footpaths were added to make the parks more accessible and volleyball and badminton courts were installed to allow for a wider variety of activities. Landscaping was also used to subdivide large, open areas into semi-enclosed pockets of park space. Almost immediately, city officials noticed a change. Different groups of people — girls and boys — began to use the parks without any one group overrunning the other.
People have started to pay attention. In 2008, the United Nations Human Settlements Programme included Vienna's city planning strategy in its registry of best practices in improving the living environment. Vienna's park redesign project, along with a program to create a gender mainstreaming pilot district, has even been nominated for the United Nations Public Service Award, a badge of honor recognizing efforts to improve public administration. This change hasn't come without criticism, however. "When we came up with the idea for the exhibit "Who Owns Public Space" a lot of our colleagues thought it was ridiculous," Kail says. "Everyone we worked with had to give feedback. People said things like, "does this mean we should paint the streets pink?" "Gender can be an emotional issue," Bauer adds. "When you tell people that up until now they haven't taken the women's perspective into account they feel attacked. We still have people asking, 'Is this really necessary?'" Planners also run the run the risk of reinforcing stereotypes in attempting to characterize how men and women use city space. To distance themselves from this, city officials have begun to shy away from the term gender mainstreaming, opting instead for the label 'Fair Shared City.' Whatever its limitations, there's no question that mainstreaming has left an indelible mark on the Austrian capital. It began as a way to look at how men and women use city space differently. Today, however, mainstreaming has evolved into a much broader concept. It's become a way of changing the structure and fabric of the city so that different groups of people can coexist. "For me, it's a political approach to planning," Kail says. "It's about bringing people into spaces where they didn't exist before or felt they had no right to exist."
Malawi president Joyce Banda is from Matriarchal Chewa tribe
by Karen E. Klein / September 10, 2014
In 2009, when Amy Norman and Stella Ma started pitching investors on their San Francisco-based startup, Little Passports , both had young children and Norman was pregnant. The overwhelming majority of the investors they met with were men who wanted to know "if we were running this as a 'lifestyle company,'" Ma recalls. Investors passed and word got around Silicon Valley that "there's no way women like this could grow a company fast enough" to satisfy venture capitalists, Norman says. Yet grow it did, to $5 million in revenue five years later. Norman and Ma eventually raised nearly $2 million for their education business, which sells monthly subscription packages to help kids learn about geography. Much of the funding came from a female investor group that saw in the idea potential that eluded many men.
With 'brogrammer' culture spreading through the male-dominated world of tech, Little Passports' experience reflects a contrasting trend: Women are running an increasing number of America's startups, and they make up a growing share of the angel investors funding them. Today women make up about 20 percent of both the entrepreneurs and investors involved in angel deals, up from single-digits a decade ago, according to the University of New Hampshire's Center for Venture Research (PDF). Women made up 23 percent of all entrepreneurs seeking angel capital in 2013, up from 9 percent in 2005. There were fewer than 20,000 female angel investors in 2005, but that number increased to nearly 58,000 by last year.
The group that funded Little Passports, Golden Seeds , was founded nine years ago, making it an early female investor group. Managing director Jo Ann Corkran says the group has 300 investors, 72 percent of them women. They are committed to investing in companies that have in top executive positions at least one woman who holds significant equity and decision-making power. All together, members from chapters in New York, Boston, Silicon Valley, and Texas have invested $50 million in 61 companies, Corkran says. The group also holds regular open office hours, giving company founders a chance to meet informally with experienced investors.
Recent survey of 863 board members of publicly traded companies in the U.S.
There's no mystery about why women are underrepresented in the investor community, Corkran says. "If you make 77¢ on the dollar and you compound that over a lifetime, you end up with women having a lot less free cash flow." Corkran is aware of the perception that women aren't as comfortable with risk, but she doesn't buy it. "They are as thoughtful and as willing to take a knowledgeable, considered risk as anybody else," she says. Natalia Oberti Noguera, founder and chief executive of Pipeline Fellowship , an angel investing boot camp for women, agrees. Since its April 2011 launch, her program has trained more than 80 women, who have committed over $400,000 in investment. Having started in New York, the group has expanded to Atlanta, Austin, Boston, Chicago, Los Angeles, Miami, San Francisco, Seattle and Washington.
The idea came about after Oberti Noguera attended a gathering of nearly two dozen investors as one of two women in the room. "These people were deciding whether or not to invest and they went around the room saying, 'Well, my wife and her friends say this,' or 'My girlfriend says that.' I realized then and there that women do not have a seat at the table." A Silicon Valley pitch-fest at which her all-female team presented to an all-male investor panel provided a further vivid lesson. "We were told, 'The fact that you're an all-woman team is too distracting.' I came out of that realizing that we weren't taken seriously," she says. If women are becoming increasingly influential as angel investors, however, they still have a way to go in the venture capital world. Alyse Killeen is an associate at March Capital Partners in Los Angeles, specializing in the fields of health and life science. Last year, she founded a networking and professional development group called Women In Venture . The group has about 18 members, but only two—herself and one other woman—currently hold jobs at venture capital firms. The group's goal is to provide encouragement and professional development that will help keep women in the field and advance them. Women now make up just 4 percent of venture capital partners. "We want to make it at least 20 percent women," Killeen says. She feels that along with her efforts, entrepreneurs themselves are pushing advancement for women. Both male and female founders are actively looking for diversity in their investment teams and on their boards and management teams, she says. "We have had a few competitive deals come in because entrepreneurs who could have chosen from between 20 to 30 firms chose us because they wanted to work with a woman investor. It's like, 'Listen, all of our engineers look essentially the same, but we believe you can help us recruit women, and that will give us an edge,'" Kileen says. Oberti Noguera is also hopeful, but says she won't back off any time soon. "I tell people, don't complain," she says. "Just raise awareness of the issues and disrupt within the system, while creating our own systems. That's the way we're going to make progress."
YOU FIGHT LIKE a GIRL
Better Identification of Viking Corpses Reveals: Half of the Warriors Were Female
Shieldmaidens are not a myth. A recent archaeological discovery has shattered the stereotype of exclusively male Viking warriors sailing out to war while their long-suffering wives wait at home with baby Vikings. Plus, some other findings are challenging that whole "rape and pillage" thing, too. Researchers at the University of Western Australia decided to revamp the way they studied Viking remains. Previously, researchers had misidentified skeletons as male simply because they were buried with their swords and shields. (Female remains were identified by their oval brooches, and not much else.) By studying osteological signs of gender within the bones themselves, researchers discovered that approximately half of the remains were actually female warriors, given a proper burial with their weapons.
It's been so difficult for people to envision women's historical contributions as solely getting married and dying in childbirth, but you can't argue with numbers—and fifty/fifty is pretty damn good. The presence of female warriors also has researchers now wondering just how accurate the stereotypes of raping and pillaging actually are:"Women may have accompanied male Vikings in those early invasions of England, in much greater numbers than scholars earlier supposed, (Researcher) McLeod concludes. Rather than the ravaging rovers of legend, the Vikings arrived as marriage-minded colonists." In many ways, this discovery is well-timed with the recent uproar over Thor becoming a title for both sexes instead of an exclusively male name. Fingers crossed this means that pop culture could start including more female warriors than just Sif and Lagertha (from The History Channel's Vikings, above). Just so long as they're not wearing boob plate armor. Because, as we're always re-learning, women have always fought. [UPDATE: Commenter Andrew W. gives a wonderfully precise and informative look at the findings in this comment below, adding some context to the idea of female Viking warriors.]
PREVIOUSLY : GREAT SEX STRIKES thru HISTORY – RETHINKING the LIPSTICK INDEX
"Part of a girl's puberty ritual among the Khoisan, for example, is a dance called the Eland Bull Dance, where the girl plays the part of the bull, sending out a "wrong sex, wrong species" signal. Meanwhile, the women of the camp dance around her as if mating with the bull, taunting the local men with their complete lack of interest in them. "The message to the males is absolutely clear – you go off, you hunt some eland, and then we'll see. It's a sex strike in all but name," says Power. The Hadza have a similar ritual called "epeme", linked to symbolic menstruation and the new moon, associated with a mythical heroine who hunts down male zebra and wears their penises.
In a recently completed study, Power found that in a range of traditional societies puberty rituals link menstrual taboos with hunting, lunar phase and "wrong sex" signals in ways that meet the predictions of the sham menstruation and sex strike theory. "It's wildly improbable that any of that is going to be there by accident," she says. And there is more evidence in the archaeological record, says Watts. His study of 74 sites in southern Africa dating from more than 20,000 years ago reveals an explosion in the use of red ochre and other red pigments between about 100,000 and 120,000 years ago."


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The Yankees have recalled RHP Tommy Kahnle from Triple-A Scranton/Wilkes-Barre, while sending down RHP Luis Cessa, the team announced on Friday.
Kahnle has had a rough 2018 in the big leagues as injury and bad outings forced him down to Triple-A. First, it was shoulder and bicep tendinitis that kept him out early on in the season. Upon his return, Kahnle didn't appear to be the same pitcher as last season, with his velocity down a bit and erratic pitchers.
Over eight apperances (nine innings), Kahnle owns a 7.00 ERA and 1.78 WHIP with the Yankees on the year.
In Triple-A, though, he seems to have figured it out. He is pitching to a much-better 2.49 ERA and his WHIP is close to one at 1.06. He will look to re-establish himself as a solid arm out of the bullpen.
Cessa, on the other hand, was called up ahead of the Yankees' series with the Red Sox. But things went bad for him on Thursday night when he allowed five runs on seven hits over 3.2 innings.


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Why Hit And Run Internet Marketers Will Never Be Rich
So many affiliate marketers and internet marketers out there today think the secret to success online is simple:
Ok are you ready for the secret internet marketing formula for not getting rich? Here it is!
Super Internet Marketing Get Rich Quick Formula
1.Pick a "Hot" niche just ready to be tapped!
2. Put up a squeeze page or "money site"
3. Send traffic
4. Watch the dough roll in
5. Move on to the next hot niche and repeat steps 1-4…
Why It Doesn't Work
Despite thousands of internet marketers out there trying to get rich quick chasing the hottest niche or the newest fad, it just doesn't create long-term success. Why?
Because it's just short-term thinking. If you think you're just going to put up a squeeze page, create some shitty product or free offer, build a list and sell them stuff, that's fine. But you'll never build long-term relationships that way. And when it comes to business this is the REAL secret formula for getting rich:
Long Term Relationships = Long Term Wealth
The only way to get rich and keep it in business, especially in the internet marketing business, is to create long-term relationships. The truth is it DOES NOT MATTER what niche you pick when it comes to getting rich. You can get rich teaching people how to train their dogs, or how to pick up women, or how to make money blogging, or how to clean their house… you can get rich in any business and in any industry.
How do I know it's possible to get rich IN ANY NICHE…?
Because someone else already has!
But when you look at people who have gotten rich and stayed rich in any niche or industry, it's never just some fly-by-night operation with shitty products and no focus on customers. It's always a person or a company that focused on serving people, adding more value to their market and kept at it… sometimes for years, sometimes for decades!
When it comes to internet marketing, you shouldn't just think about what's going to make you the most money this month or this week – you should think about what business do you want to be in for the next 10, 20 or 30 years? That's how you build A REAL BUSINESS online.
So next time you get a new idea to go sell toys online because it's "hot" right now – think about your long-term success. Is that really going to help you create a lasting business that you can sell or pass on to your children? Is it going to leave a legacy for you and your family? Or is it just another one of those get-rich-quick ideas?
P.S. Want to sell more books online? Not sure how to market yourself and build your author platform?
If so, click here to grab my free report on 10 Ways to Sell More Books Online right now.
How To Double Your Online Sales
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[…] trying to get rich quick or make money on auto-pilot without actually doing anything sounds good – but it's not […]


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Services and facilities
URH Apartments Vila de Tossa offers fully equipped holiday apartments in Tossa de Mar, so you can enjoy your holidays with complete comfort at your own pace. You will also have access to the facilities and services of our hotel URH Tossa de Mar, located just a few yards away. Everything you need for your stay in Tossa de Mar… and much more!
Check-in is carried out at Hotel URH Vila de Tossa (200 yds. away from the apartments). At the hotel reception desk you can pick up your apartment keys and pay your deposit.
Facilities in our hotels
At our hotel URH Tossa de Mar you will have access to the following services: swimming pool with solarium, social room, board games, pool table, table football, luggage storage and snack bar.
Pets welcome
Come with your pet on your next holiday! At URH Apartments Vila de Tossa you can stay with your pet by paying a small surcharge, subject to prior request and confirmation from the hotel.
Other services
Reception desk 24/7 (Hotel URH Vila de Tossa)
Partner car park 200 yds. away (15-20€/day)
Massage service (surcharge)
Laundry service (surcharge)


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High street closures at five-year low
New research from PricewaterhouseCoopers (PwC) and the Local Data Company (LDC), shows coffee shops led the rise in new store openings last year.
According to the report, the number of coffee shops increased by 4% (or 80 units) last year. British high streets lost 14 stores a day in 2015. And around 5,138 shops closed down in town centres in 2015, compared to 5,839 high street closures in 2014.
This was offset by 4,640 new openings last year, taking the net number of store losses to 498, compared with 987 in 2014. PwC and LDC said this was the lowest closure rate in five years, since the peak in 2012, when 20 stores were closing on average each day.
Mike Jervis, partner at PwC, said: "The lower rate of closures in 2015 reflects optimism among retailers, and indeed most consumer confidence indices support this. In addition, retail insolvencies are at an historical low."
He added: "The openings are concentrated on experience-type outlets, especially food and beverage, and I'd also expect to see more growth in discount store openings this year."
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LOUISVILLE, Ky. (AP) – A Kentucky jury has recommended a 60-year prison sentence for a man who killed a Pakistani immigrant in a road rage shooting in 2015.
Media reports say Christopher McCullum faced life in prison after being found guilty Tuesday of killing 41-year-old Mukhtar Ahmad as they drove down Interstate 71.
Testifying Wednesday in Louisville in the sentencing phase of trial, a tearful McCullum told jurors he was not in his right mind when he fired the shots in the stranger's car.
Defense attorney Josh Hartman asked jurors to recommend a 20-year sentence, saying his client deserved punishment but was a non-violent man lost to drug and alcohol abuse.
But Assistant Commonwealth's Attorney Doug Meisel asked for a life sentence.
McCullum was convicted of murder, wanton endangerment and tampering with evidence.
Source: www.washingtontimes.com stories: Politics comments powered by HyperComments


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Singing Hills Draws Retail and Housing to Hwy 46
Presidian Hotels & Resorts, a local hotel owner/operator and developer, broke ground recently on a Hampton Inn by Hilton and adjoining convention center in far North Central San Antonio.
The project is taking place 13 miles north of Loop 1604's intersection with U.S. 281, right at 281 and Highway 46. The hotel will have 87 rooms while the convention facility will measure 5,300 square feet, including office and meeting space. The hotel is designed to appeal to short-distance travelers. It is slated for completion in summer 2017.
The Hampton Inn by Hilton is part of the Singing Hills development, a master-planned community that when built out will have more than 246,150 square feet of retail space, more than 350 single-family residential units, a multifamily site, an entertainment complex, 25 additional spots for various uses, and possibly office space. Singing Hills is located not far from Bulverde, Spring Branch, Stone Oak and Boerne. It's a short drive from New Braunfels, too. It's in the middle of continuous residential and commercial growth in the Hill Country. Some real estate industry outlets call it one of the fastest growing areas in the nation.
Measuring 250 acres, FLY OVER VIDEO HERE, Singing Hills now includes a 200,000-square-foot Wal-Mart supercenter, a Security Service Federal Credit Union branch, and a blend of fast-service eateries. REOC San Antonio is developing Singing Hills, and taking care of leasing and sales while Middleman Construction Co. is handling overall surrounding construction. Mdn Architects is the firm overseeing designs for the community. The concept for Singing Hills is to offer an array of services and products, from general merchandise and urgent health care, to meeting professional and entertainment needs in an expanding community. REOC is installing more than $50 million in site infrastructure.


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Boys can be a puzzle for parents, particularly mothers. As we strive to remain connected with our adolescent sons, it can be difficult to understand why this can sometimes be a battle. According to psychologist and author, Steve Biddulph, boys experience three developmental phases of boyhood that are key to understanding and raising boys:
- 0-6 years: boys will bond with their mothers first and learn to grow in emotional stability
- 6-14 years: boys will bond with their fathers as they navigate masculinity and how to be male
- 14+ years: boys look beyond their parents for mentorship as they experience a huge testosterone surge.
Biddulph gained notoriety in 1997, with his book, Raising Boys, which explores the development of boys from birth to manhood and discusses the role of schools in raising boys. A follow-up edition has now been released, looking at the challenges of the 21st century.
While much has changed in the 21 years since Biddulph first released Raising boys, his core message remains the same: boys need you. They require love, affection and your time. In that regard, the future looks promising. According to Biddulph, fathers have trebled the amount of time they spend with their children in just one generation.
We uncover five key themes of the Raising boys latest release:
1. Boys need to take risks
Risk-taking is natural for boys and forms an essential part of their growing up. Boys need to learn to take risks safely while still allowing for new and challenging experiences, particularly during adolescence. Read our tips on how to successfully manage risk-taking in boys.
2. Set strict screen time limitations
With the rise of gaming and digital devices boys are at risk of becoming isolated and underdeveloping social skills. While digital proficiency is essential in today's world, left to themselves, boys' screen time can easily get out of control, so parents need to set firm limits and expectations. This can help your son to maintain relationships with friends, remain connected with family, and stay physically active. Discover our 7 strategies for limiting screen time.
3. Boys need to cry
Biddulph believes there is still widespread suppression of emotion in boys. It is thought that if boys don't cry, their emotions will present as anger or violence. He encourages parents to allow boys to cry and recognise sad feelings as a natural occurrence. Support your son through these times, by encouraging him to 'let it out', and resist the urge to hasten him in getting past it.
4. Actively teach boys to be gentle and sensitive
This is particularly important in teaching boys how to have respectful relationships with women. Both mother and father need to model positive male/female relationships by treating each other with respect, compassion and empathy. Parents should behave in the same way they would like their son to behave.
5. Male role models are important
The importance of a strong male role model is key. From age six, boys will look to their fathers. At adolescence, male role models separate from the father figure will become more significant as he enters into manhood. Male teachers, sporting coaches and family friends will all have a huge role to play in raising balanced men. Ensure your son is surrounded with positive role models with different backgrounds.
Much of Biddulph's findings are based upon what he believes to be fundamental differences in the ways boys and girls develop. We continue to learn more and more about the ways that boys' and girls' brains function.
However, teaching boys is second nature at Trinity. While researchers are still debating whether boys learn differently to girls, at Trinity we have educated boys in mind, body and spirit for over a century. Our mission is to provide a thoroughly Christian education for boys from Pre-Kindergarten to Year 12, imparting knowledge and understanding of the world we live in, and recognising the importance of spiritual qualities in every sphere of learning.
Fuelled by a pastorally aware culture with exceptionally high levels of individual student attention, we aim to know, understand and nurture each student to help him realise his potential, passion and purpose in life, ensuring that every Trinity boy is challenged, inspired and guided to become a successful, compassionate, internationally-minded man.
To learn more about the Trinity difference and to discover why we're one of Sydney's leading schools for boys, download our prospectus.


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