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Get in touch with Journey The Way
Get in touch directly with the pastors at Journey The Way using the form hosted below. We look forward to hearing from you!
Love God. Love People. Turn the World Upside Down.
147 S Hillside
Wichita, KS 67211
Copyright 2019, Journey The Way. All Rights Reserved.


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Intel enters VR arena with Project Alloy all-in-one headset
Chipmaker cuts the cord on virtual reality
SAN FRANCISCO: INTEL HAS made its first moves into the world of virtual reality (VR) with the launch of Project Alloy, the first "completely contained" head-mounted display (HMD). Project Alloy was...
Huawei files applications to trademark its mobile OS 'Hongmeng' in several countries
Huawei is reportedly seeking to use its new operating system in a variety of gadgets
Toshiba Europe to rebrand as Dynabook
Goodbye Toshiba, hello Dynabook?!?
Google unveils Stadia gaming platform
Google claims its new service will free players from the limitations of traditional consoles and PCs
Hands-on review: Microsoft HoloLens 2
Microsoft's HoloLens 2 shows that augmented reality headsets could be a valuable collaboration and work tool - but at a price
Apple: Slowing iPhone sales behind first profits warning since 2002
Apple loses $250bn in value since becoming $1trn company in August
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subset: commoncrawl


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Mar 10, 2016
Really interesting course. Good introduction to the dynamics of group behavior and collaboration. Nice overview of advantages of crowdsourcing, but no mention of any ethical or economic concerns.
Apr 08, 2017
Great course, very practical and a great pack of skills which will help me interpret problems better, especially ones involving multiple people (which is basically all problems.)
von Kevin M•
Dec 27, 2018
I don't think introduction needed to be stretched so long but it was good :)
von Giny C•
Mar 31, 2019
Interesting course :)
von Will H•
Feb 26, 2019
von Mikhail S•
Feb 04, 2018
Good stuff!
von Devin H•
Aug 30, 2016
I loved learning more about social computation and the power of contributions by many people to reach a common design solution.
von Michael L•
Mar 10, 2016
Really interesting course. Good introduction to the dynamics of group behavior and collaboration. Nice overview of advantages of crowdsourcing, but no mention of any ethical or economic concerns.
von Nivedita N•
Jan 08, 2016
Very informational . I personally learnt a lot.
von Vaibhav M B•
Feb 13, 2017
The course has very good material especially in terms of crowdsourcing. Gave me really good insights in social thinking and how to relate it to your product
von diego a•
Oct 11, 2015
Excellent Course. I really like the explanation and the topics. I 've learned a lot
von Wallace L•
Aug 28, 2016
I learned a lot! :) thanks coursera and mentors!
von Sergi B•
Oct 26, 2017
Everything about the course is great - the concepts, the presentation of the concepts, the assignments and even the quiz.
von Oana D•
Mar 17, 2016
relevant and useful
von Ingrida M•
Sep 17, 2015
Amazing course with a lot of secrets about social computing from USA to other countries :)
von Zunaira E•
Feb 15, 2017
This was an excellent primer on how social collaboration and crowdsourcing works. Gave a good understanding of the concepts involved when creating such platforms.
von Anastasia S•
Mar 16, 2016
Great course. Do not forget to submit your work on time, otherwise you will not be graded.
von ctvv3010•
Jun 24, 2018
very cool projects to exercise
von Adriana C•
Mar 13, 2016
Very interesting topic!
von Renee•
Jan 10, 2018
Opening a new field of computing to me!
von Juliana S•
Nov 10, 2016
This course was very usable and important to know.
von Jean W•
Mar 15, 2017
I enjoyed this course the most in the specialization so far. I think it could be awesome as a Part 1 and Part 2, or maybe you all could offer an Advanced Social Computing course. This is such an interesting topic, and it's a huge part of how new technologies are being incorporated into the way we collaborate in many areas of social and professional life.
von Aram Y•
May 02, 2017
I had awesome experience while studying a t this course. Must-attend in case you are interested in society psychology
von Emily A•
Mar 15, 2016
I really enjoyed this class a lot! The assignments directly tested the weekly material and I thought they were useful activities. The end quiz was good to re-think the entire course and reflect on all of the material we learned.
von Sicong C•
Jan 16, 2017
It is a great class. But I would like to see more examples and related learning material.
von Anuj M•
May 07, 2017
This course is very interesting, informative and same time tough (not easy).
von Pablo P G•
Mar 29, 2017
Excellent course, provides tools needed to design applications based on social networks


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I did it again. I spent all day cheerfully snapping pictures of my food and then realized that I'd left my camera cord in Seattle this morning. Bad blogger. I apologize for the bland post but here is "What I Ate Wednesday" with pictures to come!
The Noms:
32 oz ginger tea with 2 frozen wheatgrass cubes and stevia
32 oz carrot, kale, romaine juice with with stevia and cinnamon
Deceptively Chocolatey Mint Green Smoothie:
1 head romaine
1 cup spring water
4 tbsp carob powder
1/4 cup fresh mint
1/2 an avocado
1 dropper chocolate stevia
1 dropper vanilla stevia
pinch sea salt
Blend until smooth and creamy.
Dinner was a big bowl of kelp noodles, 2 free-range scrambled eggs, steamed spinach, shredded cabbage, dulse, coconut vinegar, and stevia.
Dessert was a couple ounces of Trader Joe's organic 72% dark chocolate. 'Twas delish.
I've been immensely enjoying having smoothies during the day which are simultaneously light and filling. I find that I feel better with less overall density in my day and I haven't had the desire to overeat at dinner with a good, hydrating smoothie mid-afternoon (usually Pumpkin Pie in a Bowl). I'm feeling very free with this lifestyle right now, I used to think that "juicing 'til dinner" was a sort of be-all-end-all and I'm excited to notice that I'm a lot more flexible than that. I know when I can bend and compromise and how. Some days juicing until dinner feels awesome, sometimes (especially when I'm really active) I love some fruit. Sometimes I have lunch and keep the day as a whole really light. Learning to listen to my body has been such a process but I've never felt more in tune with it than I do right now.


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Talin is a cytoskeletal protein that binds to integrin β cytoplasmic tails and regulates integrin activation. Talin1 ablation in mice disrupts gastrulation and causes embryonic lethality. However, the role of talin in mammalian epithelial morphogenesis is poorly understood. Here we demonstrate that embryoid bodies (EBs) differentiated from talin1-null embryonic stem cells are defective in integrin adhesion complex assembly, epiblast elongation, and lineage differentiation. These defects are accompanied by a significant reduction in integrin β1 protein levels due to accelerated degradation through an MG-132-sensitive proteasomal pathway. Overexpression of integrin β1 or MG-132 treatment in mutant EBs largely rescues the phenotype. In addition, epiblast cells isolated from talin1-null EBs exhibit impaired cell spreading and focal adhesion formation. Transfection of the mutant cells with green fluorescent protein (GFP)-tagged wild-type but not mutant talin1 that is defective in integrin binding normalizes integrin β1 protein levels and restores focal adhesion formation. Significantly, cell adhesion and spreading are also improved by overexpression of integrin β1. All together, these results suggest that talin1 binding to integrin promotes epiblast adhesion and morphogenesis in part by preventing integrin β1 degradation.
Talin is a 270-kDa adaptor protein that is localized predominantly in macromolecular complexes formed at the junctions between cells and the extracellular matrix (ECM) where it has been shown to connect the integrin family of cell adhesion molecules to the actin cytoskeleton (4, 9, 20, 43, 50). In addition, talin is thought to be a key regulator of integrin activation (1, 45), and small interfering RNA (siRNA)-mediated knockdown of talin in various cell lines reduced the affinity of β1 and β3 integrins for their ligands (47). In vivo, conditional deletion of the talin1 gene in mouse platelet precursors inhibited ligand-induced αIIbβ3 integrin activation and platelet aggregation, leading to impaired hemostasis in the whole animal (13, 36, 40). Similarly, in B-lymphocytes, talin1 is required for α4β1 (VLA-4) and αLβ2 (LFA-1) integrin activation and thereby for homing to lymph nodes and bone marrow (31).
At the organismal level, talin is required for embryonic development and tissue morphogenesis. In Caenorhabditis elegans, RNA interference-induced talin suppression impaired distal tip cell migration and disrupted the muscle actin cytoskeleton, leading to gonad malformation and paralysis (8). In Drosophila melanogaster, talin is concentrated at the muscle attachment site in an integrin-dependent manner. Talin-null mutants die during embryogenesis, displaying a muscle detachment phenotype similar to that conferred by integrin-null mutations (3). In these invertebrates, talin functions mainly to link ECM-bound integrins to the actin cytoskeleton during tissue morphogenesis and maintenance (15). The mutant phenotype is evident when the actin cytoskeleton is under stress conditions, such as muscle contraction and cell migration. Unlike C. elegans and Drosophila, which have only one talin gene, Dictyostelium discoideum has two talin genes which play distinct roles in the single-cell and the multicellular slug phases (48). Similarly, vertebrates, including the mouse and human, have two talin genes, talin1 and talin2 (10, 33, 44). In adult mice, talin1 is expressed ubiquitously, while talin2, though widely expressed, is most abundant in the testis, brain, and muscles (10, 33, 44). Ablation of talin1 arrested embryonic development at gastrulation, and the mutant embryos were smaller in size, abnormally organized, and deficient in extraembryonic tissues. The ectoderm layer was able to form a columnar epithelium but contained fewer cells (34). In contrast, talin2 knockout mice were viable and fertile (5, 7). These data indicate that talin1 is essential for embryogenesis and show that talin2 does not compensate for loss of talin1, although whether talin2 is expressed during the early stages of embryogenesis was not established. By comparison, mice lacking integrin β1 die at the peri-implantation stage and fail to form organized germ layers (11, 46).
In this study we utilized mouse embryonic stem (ES) cell-derived embryoid bodies (EB), epithelial cysts that are structurally similar to the peri-implantation embryo, to study the mechanisms by which talin1 regulates epithelial morphogenesis and lineage differentiation. We show that talin1 ablation diminishes the assembly of integrin-based adhesion and signaling complexes and impairs epiblast elongation and lineage differentiation. These defects are likely due to the enhanced degradation of integrin β1 through an MG-132-sensitive proteasomal pathway because in the absence of talin1 (i) levels of integrin β1 protein (but not its activation) are significantly reduced, (ii) the protein half-life of integrin β1 is shortened by ∼50%, and (iii) overexpression of integrin β1 or treatment with the proteasome inhibitor MG-132 promotes the assembly of integrin adhesion complexes and epiblast epithelialization. These results uncover a new mechanism by which talin1 promotes epithelial morphogenesis by preventing integrin β1 degradation.
Culturing of ES cell and embryoid bodies.The ES cell lines used for this study were wild-type R1 and HM1 ES cells, talin1+/− (C39) and talin1−/− (J26 and A28) ES cells (41), and integrin β1−/− (G201) ES cells (11). Wild-type and talin1+/− and talin1−/− ES cells were cultured on mitomycin C-treated STO cells. Integrin β1-null ES cells were grown directly on plastic culture dishes (27). EB differentiation was initiated from ES cell aggregates in suspension culture as described previously (28). Integrin β1−/− EBs were cultured in poly-hydroxyethyl methacrylate (HEMA)-coated bacteriological petri dishes (29).
Reagents.Paxillin, focal adhesion kinase (FAK), FAK pY397, integrin-linked kinase (ILK), neural cell adhesion molecule (NCAM), E-cadherin, and integrin β1 (clones 9EG7 and HMβ1-1) monoclonal antibodies (MAbs) were from BD Biosciences. Vinculin (clones hVIN1 and VIN-11-5), talin (clone 8d4) and β-tubulin MAbs were from Sigma. Mouse monoclonal antibodies specific for talin1 (97H6) and talin2 (68E7) were raised against residues 482 to 911 of each isoform. Human integrin β1 (clone HUTS-4) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MAbs were from Chemicon. β-Dystroglycan MAb was from Novocastra. Polyclonal antibodies (pAb) to laminin α1 rG70 and nidogen were gifts from Peter Yurchenco (Robert Wood Johnson Medical School). Flk-1 pAb and interleukin-2R (IL-2R) MAbs were from Santa Cruz Biotechnologies. IL-2R pAb was from Cell Signaling Technology. α-Fetoprotein (α-FP) pAb was from ICN. Fluorescein isothiocyanate (FITC)-, Cy3-, Cy5-, and horseradish peroxidase (HRP)-conjugated secondary antibodies were from Jackson Immunochemicals. Green fluorescent protein (GFP) pAb and phycoerythrin (PE)- and Alexa 488-conjugated secondary antibodies were from Invitrogen.
Immunofluorescence.E6.5 mouse embryos and EBs were fixed with 3% paraformaldehyde, embedded in OCT compound, and sectioned with a Leica cryostat. The experimental procedures and animal care were approved by the Institutional Animal Care and Use Committee. EB processing and immunostaining were carried out as described previously (28). The nucleus was counterstained with DAPI (4′,6-diamidino-2-phenylindole). Slides were examined with a Nikon inverted fluorescence microscope (Eclipse TE 2000), and digital images were acquired with a cooled charge-coupled-device (CCD) camera (Hamamatsu) controlled by IP Lab 4.0 software (Scanalytics). To measure the epiblast height, the phase image of an EB cross section was evenly divided into 4 parts by two perpendicular lines. The epiblast height along the lines was measured using Adobe Photoshop and averaged. Data were analyzed by the Student t test or one-way analysis of variance with Holm-Sidak comparisons with SigmaStat (version 3.5; Systat) and expressed as the mean ± the standard deviation (SD).
Immunoprecipitation and immunoblotting.EBs were collected, washed once in phosphate-buffered saline, and lysed in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate) containing protease and phosphatase inhibitor cocktails. Protein concentrations were determined using bicinchoninic acid (BCA) reagents (Pierce). For immunoprecipitation, cells lysates were precleared with protein G agarose and then incubated with primary antibodies and protein G beads overnight. Immunoprecipitates were washed and analyzed by SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes which were blocked with 5% nonfat dry milk. After incubation with primary antibodies, specific signals were detected with HRP-conjugated secondary antibodies and ECL reagents.
RT-PCR.Total RNA was isolated with the TRIzol reagent (Invitrogen) and reverse transcribed to cDNA using AMV reverse transcriptase (RT) (Stratagene). PCR primers used for detecting integrin β1A mRNA in talin1+/− and talin1−/− EBs were as follows: 5′-TAGCAAATTGCCAGACGGAG (forward) and 5′-TCTCTTTCCTGCAGTAAGCGTC (reverse). The primers for detecting human and mouse integrin β1A in EBs expressing human integrin β1 were 5′-TTGTGAAGCCAGCCACGGAC (forward) and 5′-CAACCACACCAGCTACAATTGG (reverse) and 5′-ATGTCTAGCGTCAAATGGTC (forward) and 5′-CAACCACGCCTGCTACAATTGG (reverse). PCR conditions for α-FP, neurofilament protein 68 (NF68), ζ-globulin, and 18S were described previously (26, 35).
Transfection of ES cells and epiblast cells.ES cells were transfected with pCXN2-hβ1-GFP or pCXN2 using Lipofectamine 2000 (Invitrogen). Stable cell clones were established based on GFP expression. For transient transfection of epiblast cells, endoderm was selectively removed from 5-day EBs by brief incubation in deionized water for 20 s, and the addition of 10 volumes of fresh medium followed. The remaining EBs containing basement membranes (BMs) and epiblast cells were dissociated with trypsin-EDTA. Isolated epiblast cells were cultured on laminin-111 (10-μg/ml)-coated culture dishes or glass coverslips for 12 h. The cells were transfected with phβ1-GFP (kindly provided by Martin Humphries of University of Manchester) (38), pEGFP-talin1, pEGFP-talin1 W359A, pEGFP, or pTac-β1 (kindly provided by Susan LaFlamme of Albany Medical College) (22) using Lipofectamine 2000. Twenty-four or forty-eight hours after transfection, the cells were used for immunostaining, an integrin degradation assay, or fluorescence-activated cell sorter (FACS) analysis.
Protein degradation assay.Three-day EBs and epiblast cells transfected with Tac-β1 were treated with cycloheximide (100 μg/ml) to block new protein synthesis. Medium was changed every 4 h with fresh cycloheximide added. Cell lysates were collected at the indicated time points and analyzed by immunoblotting with integrin β1 (HMβ1-1), IL-2R, and GAPDH antibodies. The results of 3 independent experiments were plotted, and the integrin β1 (Tac-β1)/GAPDH ratio at the 0-hour time point was designated 1.
FACS.Epiblast cells were surface labeled with either the HMβ1-1 or 9EG7 integrin β1 monoclonal antibody in Hank's solution containing 2 mg/ml bovine serum albumin for 1 h at 4°C. Isotype-matched hamster or rat IgG was used as a control. The cells were washed and fixed in 3% paraformaldehyde for 10 min. The primary antibody bound to the cell surface was detected with FITC- or PE-conjugated secondary antibodies. To assess the ligand binding of α5β1 integrin, the glutathione S-transferase (GST)-tagged fibronectin fragment FN9-11 (the expression vector was kindly provided by Mark Ginsberg of University of California, San Diego) was produced by bacterial expression and biotinylated using an EZ-link biotinylation kit (Pierce). The cells were incubated with biotinylated FN9-11 in the presence or absence of 0.5 mM MnCl2 or 5 mM EDTA. After being washed, the bound ligand was detected with FITC-conjugated streptavidin. The mean fluorescent intensity of the specific labeling was analyzed on a BD FACSCalibur flow cytometer (BD Biosciences).
Talin1 ablation impairs epiblast elongation and lineage differentiation.To determine the role of talin1 in embryonic epithelial morphogenesis, we analyzed EBs cultured from wild-type and talin1+/− (clone C39) and talin1−/− (clones J26 and A28) ES cells. EBs differentiated from J26 and A28 ES cells displayed similar phenotypes, and therefore, only the results obtained with J26 ES cells are reported. Talin1+/− EBs differentiated normally and began to form endoderm and underlying BM at day 2 in suspension culture, and by day 3, a distinct epiblast epithelium was observed by live phase microscopy (Fig. 1A). By day 5, the EBs were fully cavitated and had developed into epithelial cysts. Although talin1−/− EBs did form endoderm and BM, epiblast epithelial morphogenesis was delayed and was less efficient compared to that for talin1+/− EBs (Fig. 1B). Particularly, the epiblast height of 5-day talin1−/− EBs was only 50% that of the heterozygous controls (Fig. 1C and D). These results suggest that the loss of talin1 selectively affects epiblast epithelial morphogenesis.
The epiblast is the precursor of the three definitive germ layers that give rise to all the cell types of the body. To examine the consequence of defective epiblast morphogenesis on lineage differentiation, we performed RT-PCR for transcripts of the visceral endoderm marker α-FP, the neuronal marker NF68, and the mesoderm marker ζ-globin. The α-FP mRNA level was not altered in the absence of talin1, which is in agreement with the morphological evidence of endoderm differentiation. However, the transcripts of NF68 and ζ-globin were decreased in 5-day talin1−/− EBs (Fig. 1E and F). We also carried out immunoblot analysis for the expression of the neuronal marker NCAM and the cardiovascular lineage marker Flk-1 in 8-day EBs. As shown in Fig. 1G, NCAM expression was significantly reduced in talin1−/− EBs, while Flk-1 was detected only in talin1+/− EBs. These results suggest that talin1 ablation inhibits the differentiation of ectoderm and mesoderm lineages.
Both talin1 and talin2 locate to the epithelium-BM adhesion site depending on integrin.Under standard cell culture conditions, talin is localized primarily to integrin-containing focal adhesions (FAs). To gain insight into the mechanisms by which talin1 regulates embryonic epithelial morphogenesis, we studied the spatial expression of talin1 and talin2 in both E6.5 mouse embryos and EBs by immunofluorescence microscopy. We utilized isotype-specific monoclonal antibodies raised against mouse talin1 (97H6) or talin2 (68E7) (Fig. 2A). Both antibodies stained the basal side of the endoderm/epiblast adjacent to the embryonic BM as well as the apex of the endoderm (Fig. 2B). In the BM zone, talin1 and talin2 colocalized with integrin β1. In the early stages of EB differentiation before endoderm and BM formation, talin1 and talin2 were distributed mainly in the cytoplasm (Fig. 2C and data not shown). BM formation led to the recruitment of both talin1 and talin2 to the endoderm/epiblast-BM adhesion site, where they colocalized with integrin β1, and both talin1 and talin2 coimmunoprecipitated with integrin β1 (Fig. 2D). Interestingly, in β1-null EBs, talin1 expression was increased, while talin2 was markedly decreased, suggesting that the two talins are differentially regulated by integrins (Fig. 2D). We further showed that talin1 and talin2 were absent from the cell-BM adhesion site in integrin β1−/− EBs (Fig. 2E). Taken together, these results indicate that both talin1 and talin2 interact with β1 integrins at epithelium-BM adhesions during embryonic epithelial morphogenesis.
Talin1 maintains integrin β1 protein expression and integrin-based epithelium-BM adhesion complexes.Next, we tested whether talin, as in Drosophila, might regulate the assembly of integrin-based cell-BM adhesion complexes (3). Five-day talin1+/− and talin1−/− EBs were immunostained for integrin β1 and classic FA proteins, including talin, FAK, paxillin, vinculin, and ILK. In talin1+/− EBs, both integrin β1 and the FA proteins colocalized at cell-BM adhesions in a continuous fashion (Fig. 3A and data not shown). Furthermore, activated phospho-FAK was localized in the BM zone. These results suggest the formation of integrin-based adhesion and signaling complexes at the epithelium-BM interface. In contrast, talin1−/− EBs showed a marked reduction of integrin β1 in the BM zone as detected using both the 9EG7 antibody that is specific for activated integrin β1 and the HMβ1-1 antibody that recognizes both the active and inactive forms (24, 37). There was an accompanying reduction of FA protein staining at cell-BM adhesions, although interestingly, talin2 appeared relatively unaffected. These results suggest that talin1 is required for the assembly of integrin-based epithelium-BM adhesion complexes and that talin2 does not compensate for the loss of talin1 in this respect. Despite the diminished assembly of integrin-based cell-BM adhesion complexes in talin1−/− EBs, we did not observe any detachment of endoderm or epiblast cells from the BM. However, the endoderm cells appeared much more rounded in the null EBs, suggesting weakened integrin-BM adhesions (Fig. 1C).
To test whether the diminished assembly of adhesion complexes might be caused by the reduced expression of integrins and FA proteins, we performed immunoblot analysis on 5-day EBs (Fig. 3B). We confirmed that talin1 was not expressed in talin1−/− EBs and that talin2 was expressed in both talin1+/− and talin1−/− EBs and was not upregulated in the absence of talin1. Significantly, integrin β1 expression was reduced by ∼70% as detected using the HMβ1-1 antibody. Moreover, time course studies showed that although talin1+/− EBs expressed integrin β1 at all stages of the differentiation process, it was completely absent from talin1−/− EBs at day 1, although it was present at day 2 (Fig. 3C). Integrin α5 and α6, the two major α subunits that pair with the β1 subunit in EBs (29), were also significantly downregulated in talin1−/− EBs (Fig. 3B). In contrast, β-dystroglycan, a major BM receptor that is upregulated in integrin β1−/− EBs (27), showed a significant increase in talin1−/− EBs, while the expression of the cell-cell adhesion protein E-cadherin was unchanged. These results suggest that talin1 is required for the maintenance of the normal integrin level and that the increased dystroglycan expression might be a compensatory response to decreased integrin protein levels. We also analyzed the expression and phosphorylation of other FA proteins in talin1−/− EBs by immunoblotting (Fig. 3B). FAK activation, as assessed by phosphorylation at Tyr397, was decreased in parallel with reduced FAK protein expression. ILK expression was also reduced by ∼50% in talin1−/− EBs (data not shown). Paxillin phosphorylation at Tyr118, which has been shown to be catalyzed by activated FAK (2), was inhibited in the absence of talin1, although the expression of paxillin was slightly upregulated. All together, these results suggest that talin1 promotes the assembly of epithelium-BM adhesion and signaling complexes likely by regulating the expression of integrin β1, FAK, and ILK at the protein level.
To assess the effect of talin1 ablation on integrin β1 activation, we performed FACS analysis on epiblast cells isolated from 5-day EBs. Activated β1 and total integrin β1 on the cell surface were labeled with the monoclonal antibody 9EG7 and HMβ1-1, respectively. Isotype-matched preimmune IgG was used as a nonspecific binding control. As shown in Fig. 3D, the total integrin β1 was reduced by 57% in talin1−/− epiblast cells, and this was accompanied by a proportional reduction in activated β1 by 55%. These results suggest that the loss of talin1 reduces integrin β1 expression but not activation. To confirm the above finding, we assessed the ligand binding activity of α5β1 integrin using the fibronectin fragment FN9-11. Surface binding of FN9-11 was reduced by 54% in talin1−/− epiblast cells compared to control talin1+/− cells (Fig. 3E). The specificity of the assay is indicated by the fact that EDTA treatment abolished much of the FN9-11 binding. In the presence of MnCl2, which activates integrins independent of cellular activators, such as talin, FN9-11 binding was only slightly increased in both talin1+/− and talin1−/− epiblast cells. Again, this indicates that the bulk of integrin β1 expressed on talin1−/− cells is already in the activated form. Collectively, these results suggest that talin1 ablation reduces integrin β1 expression but not its activation in epiblast cells.
Integrin β1 protein degradation is accelerated in the absence of talin1.To test whether the reduced integrin β1 protein in talin1−/− EBs might be due to decreased gene transcription, we performed RT-PCR using primers specific for mouse integrin β1. We found that the integrin β1 mRNA level was slightly increased in talin1−/− EBs, suggesting that the decrease of integrin β1 protein must be the result of posttranscriptional events (Fig. 4A and B). Next, we investigated whether talin1 might regulate the stability of the integrin β1 protein. Three-day talin1+/− and talin1−/− EBs were treated with 100 μg/ml cycloheximide to block new protein synthesis, and EB extracts were collected at 4, 8, 12, and 24 h after cycloheximide treatment. As shown in Fig. 4C and D, the rate of integrin β1 protein degradation was significantly accelerated in talin1−/− EBs compared to the talin+/− control. Based on this assay, we estimate the integrin β1 protein half-life to be 23.1 ± 5.0 h in talin1+/− EBs and 10.6 ± 0.5 h in talin1−/− EBs (P < 0.01, n = 3). These results suggest that the interaction of talin1 with β1 integrins prevents integrin β1 protein degradation.
Proteasome inhibition prevents integrin β1 degradation and promotes epiblast morphogenesis in talin1−/− EBs.To elucidate the proteolytic pathways that might be involved in the degradation of the integrin β1 protein, we incubated 3-day talin1+/− and talin1−/− EBs for 16 h with the proteasome inhibitor MG-132, the calpain inhibitor ALLN, or the lysosome inhibitor chloroquine. Immunoblot analysis revealed that in the solvent control, the integrin β1 protein level of talin1−/− EBs was only 35.8% of that of talin1+/− EBs when normalized to actin. However, treatment of talin1−/− EBs with MG-132 restored the integrin β1 protein to the talin1+/− level (Fig. 5A). In contrast, in ALLN- and chloroquine-treated talin1−/− EBs the integrin β1 protein levels remained 34.6 and 28.4% of the talin1+/− control level. These results suggest that talin1 ablation increases the turnover rate of integrin β1 mainly through an MG-132-sensitive degradation pathway, although the fact that chloroquine treatment slightly increased the integrin β1 protein in both talin1+/− and talin1−/− EBs suggests that lysosomes may also be involved in integrin turnover. The restoration of integrin β1 protein levels in MG-132-treated talin1−/− EBs resulted in increased accumulation of integrin β1, paxillin, and phospho-FAK Tyr397 in the BM zone (Fig. 5B). The efficiency of epiblast epithelialization was also improved by MG-132 treatment (Fig. 5C). Furthermore, the epiblast cells of the treated EBs appeared more elongated compared to those of the solvent control. To confirm this, we quantified the epiblast height with IP Lab image analysis software and showed that inhibition of integrin β1 degradation by MG-132 increased the epiblast height by 60% in talin1−/− EBs (Fig. 5D). Similarly, lineage differentiation was restored by MG-132 treatment as indicated by increases in the mRNAs for NF68 and ζ-globin (Fig. 5E and F). All together, these results suggest that talin1 regulates adhesion assembly and epiblast morphogenesis in part by preventing integrin β1 degradation through an MG-132-sensitive proteasomal pathway.
Talin1-integrin interaction is required for the maintenance of integrin stability and focal adhesion formation.To determine whether talin1 binding to integrin β1 is required for the maintenance of normal integrin β1 levels, we transfected talin1+/− and talin1−/− epiblast cells with GFP-tagged talin1 or a mutant talin1 that is defective in integrin binding (talin1 W359A) (12, 14). Compared with talin1+/− epiblast cells which spread nicely on the laminin-111 substrate and formed prominent FAs, talin−/− cells spread poorly and did not assemble FAs (Fig. 6A). However, transfection with wild-type GFP-talin1 completely rescued cell spreading and FA formation in talin1−/− cells. GFP-talin1 colocalized with paxillin in FAs. In contrast, transfection of the talin1−/− cells with GFP-talin1 W359A did not improve cell spreading or FA formation, and the integrin-binding talin1 mutant remained largely in the cytoplasm. Interestingly, the GFP-talin1 W359A mutant was incorporated into the FAs assembled by talin1+/− epiblast cells. These results suggest that the talin1-integrin interaction is required for epiblast spreading and FA formation but is dispensable for talin1 recruitment to preexisting FAs.
We next examined the surface expression of integrin β1 in the transfected cells by FACS analysis. Forty-eight hours after transfection, cells were fixed and immunostained with the HMβ1-1 antibody followed by a phycoerythrin-conjugated secondary antibody. Analysis of the transfected, GFP-positive cells revealed that integrin β1 on the cell surface was reduced by 58% in talin1−/− epiblast cells compared with that of talin1+/− controls (Fig. 6B). Transfection of talin1−/− cells with wild-type talin1 largely restored integrin β1 expression, whereas transfection with talin1 W359A had no effect. We also performed immunoblot analysis of the transfected cells and obtained a similar result (data not shown). These data establish that a talin1-integrin interaction is required for the maintenance of normal integrin β1 levels in epiblast cells.
Overexpression of integrin β1 in talin1−/− EBs improves epithelial morphogenesis.To test whether the reduced integrin β1 expression in talin1−/− EBs contributes to the defect in the assembly of cell adhesion complexes and epiblast morphogenesis, we stably transfected talin1−/− EBs with GFP-tagged human integrin β1 (hβ1-GFP), which has been shown to support cell attachment and FA assembly when expressed in integrin β1−/− fibroblasts (38). RT-PCR analyses of 5-day EBs using primers for a nonhomologous region of the human and mouse integrin β1 mRNA revealed that the transgene was expressed at twice the level of the endogenous mouse gene (Fig. 7A). Immunoblotting and immunofluorescence confirmed the expression and appropriate localization of the hβ1-GFP fusion protein (Fig. 7B and C). As a consequence of hβ1-GFP overexpression, the recruitment of paxillin and phospho-FAK to epithelium-BM adhesions was significantly increased in talin1−/− EBs compared to the vector control, indicating enhanced cell-BM adhesion assembly and signaling (Fig. 7D). Furthermore, hβ1-GFP overexpression in talin1−/− EBs increased both the efficiency of epiblast epithelialization and the epiblast height (Fig. 7E and F). We also determined the stability of hβ1-GFP in talin1−/− EBs and found its half-life to be 10 h, slightly shorter than endogenous integrin β1. These results suggest that the reduction in integrin β1 expression in talin1−/− EBs contributes to impaired epiblast morphogenesis and that the talin1−/− phenotype can be partially rescued by overexpressing integrin β1 to compensate for its increased degradation.
To further assess whether the reduced integrin expression after talin1 ablation causes impaired epiblast-matrix interactions, we transiently transfected EB-derived epiblast cells with either hβ1-GFP or the control vector. After transfection, cells were plated onto the laminin-111 substrate for 24 h and immunostained for human integrin β1 and paxillin. Normal epiblast cells spread nicely on laminin-111 and formed FAs (Fig. 7G). In contrast, talin1−/− epiblast cells transfected with GFP alone attached but failed to spread or form FAs (Fig. 7H). However, forced expression of hβ1-GFP in the mutant cells partially rescued cell spreading and FA formation compared to those of the untransfected or GFP-transfected cells. The exogenous integrin β1-GFP could be detected at the FAs with the monoclonal antibody HUTS-4 which reacts with the activated human integrin β1 (30). These results confirm that the phenotype of talin1−/− epiblast cells is due at least in part to reduced integrin β1 expression.
Next, we assessed whether the ligand binding site in the integrin β1 ectodomain is required for the rescue effect. To this end, we transiently transfected epiblast cells with a construct that consists of the extracellular and the transmembrane domains of IL-2 receptor and the intracellular domain of integrin β1 (Tac-β1) (22). Expression of Tac-β1 failed to rescue cell spreading and FA formation in talin1−/− cells (Fig. 7I). However, Tac-β1 could be detected at FAs when expressed at low or intermediate levels in talin1+/− cells. High-level transgene expression caused cells to round up. These results suggest that reduced integrin β1 protein levels at least partially account for the defective assembly of epiblast-BM adhesions and that an intact ligand binding domain is required for integrin β1 to rescue the talin1−/− epiblast phenotype.
To test whether the interaction between talin1 and integrin β1 tails is sufficient to regulate integrin turnover, we treated Tac-β1-transfected epiblast cells with cycloheximide to block new protein synthesis and analyzed Tac-β1 protein levels over time by immunoblotting. In contrast to the accelerated degradation of endogenous integrin β1 and transfected hβ1-GFP in talin1−/− EBs, the degradation rate of Tac-β1 in talin1−/− epiblast cells was not increased compared to that in the talin1+/− control (Fig. 7J and K). This result shows that the integrin extracellular domain plays a key role in determining the rate of integrin turnover in the absence of talin1.
In the present study, we report that talin1 ablation in EBs impairs epiblast epithelial formation and lineage differentiation. These defects correlate with a substantial reduction in integrin β1 expression and in the absence of talin1, integrin β1 was rapidly degraded through an MG-132-sensitive pathway. Rescue experiments show that talin1 binding to integrin is required for the maintenance of normal integrin β1 levels and FA formation. Moreover, overexpression of a human integrin β1-GFP fusion protein partially rescued the talin1-null phenotype. These results uncover a new mechanism by which talin1 regulates integrin function in epithelial cells and thereby epithelial morphogenesis.
Previous studies in Drosophila have showed that talin colocalizes with integrin at FA-like structures formed at the basal surface of the wing imaginal disc epithelia (3). Talin mutations resulted in the loss of integrin from these structures and vice versa. This is in agreement with our findings in talin1−/− and integrin β1−/− EBs, which suggests that talin and integrin are interdependent for the assembly of these FA-like structures. Loss of talin in fly wing epithelia causes the separation of the dorsal and ventral epithelial layers and leads to a wing blister phenotype (3, 42). In fly tracheal epithelia, talin mutations do not affect terminal bud formation and lumen morphogenesis early in development but result in convoluted lumens and branch degeneration during larval life, indicating that talin is required for tracheal epithelial maintenance (25). Our analyses of talin1−/− EBs extend these findings and demonstrate that talin1 is required for epiblast elongation and efficient epiblast epithelialization. Taken together, these results establish a critical role for talin1 in the formation and maintenance of epithelial tissues.
Conditional deletion of the talin1 gene in mouse megakaryocytes inhibited platelet adhesion and aggregation and led to severe defects in hemostasis (13, 36, 40). This phenotype was attributed to the low affinity of platelet integrins in the absence of talin1 (there is no talin2 in platelets), and they could not be activated in response to platelet agonists. In contrast, β1 integrin activation in differentiating EBs is unaffected in the absence of talin1 when assessed by FACS analysis using the 9EG7 antibody which recognizes only activated integrin β1 and using the α5β1 integrin ligand FN9-11 fragment. This could be due to the compensation by talin2, which is expressed in normal and talin1−/− EBs and continues to localize to the epithelium-BM adhesion. Indeed, cell spreading and focal adhesion assembly in talin1−/− fibroblasts is dependent on upregulation of talin2 (50). Moreover, defects in cell spreading and FA that result from talin1 knockdown in endothelial cells (which express only talin1), can be rescued by expressing talin2 (21). However, integrin β1 activation measured both with the 9EG7 antibody and via adhesion assays was also reported to be unaffected in primary cultures of fetal myoblasts in which both talin1 and talin2 were deleted, although myoblast fusion was severely compromised (7). The kindlin family of FERM domain proteins has been shown to synergize with talin to activate integrins, and ILK has also been shown to support integrin activation (16, 23). Therefore, it is possible that talin2 synergizes with other pathways to activate integrin β1 in the talin1−/− epiblast.
In contrast to platelets, skeletal muscle, and fibroblasts where integrin β1 expression was not affected by deleting the talin1 gene (6, 40, 50), talin1−/− EBs had a significant reduction of integrin β1 at the protein level. A similar finding has been reported for undifferentiated talin1−/− ES cells, but not for differentiated ES cells (41). While the reason for this difference is unclear, it is unlikely to be due simply to compensation by talin2 since fibroblasts express much less talin2 than EBs and platelets lack talin2 altogether (data not shown). One possible explanation is that integrins are highly dynamic and have faster turnover rates in rapidly expanding epiblast and undifferentiated ES cells. We found that the half-life of the integrin β1 protein is 23 h in EBs versus 96 h in confluent embryonic fibroblasts. In these undifferentiated cell types, talin1 may actively participate in the regulation of integrin turnover. In this study, we further show that the enhanced degradation of the integrin β1 protein is sensitive to the proteasome inhibitor MG-132 and that MG-132 partially reversed the epithelial defects observed with talin1−/− EBs. There is growing evidence that E3 ligases play an important role in regulating cell adhesion and migration (17), and focal adhesion proteins, including talin and paxillin, have been shown to be ubiquitinated (18, 19). In addition, ILK has been shown to regulate ubiquitination of Notch1. However, we were unable to detect ubiquitination of integrin β1, α5, or α6 using anti-hemagglutinin (HA) antibody in epiblast cells transiently transfected with HA-tagged ubiquitin (data not shown). This suggests that α5β1 and α6β1 integrin may not be the direct targets for ubiquitination in EBs. Nonetheless, transfection of the talin1−/− epiblast cells or EBs with human integrin β1 largely rescued cell spreading, FA adhesion assembly, and epiblast epithelial morphogenesis. Our data suggest that talin1 promotes epiblast cell adhesion and embryonic epithelial formation at least in part by regulating integrin turnover.
The data presented in this study may also impact the interpretation of the mouse talin1 knockout phenotype which is characterized by defects in the organization and size of the pregastrulation (E6.5) embryo (34). While the phenotype is slightly less severe than that of the integrin β1 knockout (11, 46), it was sufficiently early to lead to the conclusion that talin2 was probably not expressed in these early embryos and was therefore unable to compensate for loss of talin1. However, the current study demonstrates that both talin1 and talin2 are expressed in the same locations in cells of the epiblast at around this time (Fig. 2B). Therefore, it now seems possible that the talin1−/− embryonic phenotype is due, at least in part, to a reduction of integrin β1 levels in the epiblast as a result of increased integrin degradation. The presence of some β1 integrins in the talin1−/− epiblast (as in the EBs) could account for the decreased severity of the talin1−/− phenotype compared to the integrin β1−/− phenotype. We are currently investigating this possibility.
How talin1 regulates integrin stability is not yet known. One possibility is that talin1 may stabilize integrin clusters at the epithelium-BM adhesions by linking ligand-bound integrins to the actin cytoskeleton. Indeed, our immunofluorescence results showed that the clustering of integrin β1 at these FA-like structures was significantly reduced in talin1−/− EBs. In the absence of talin1, unbound integrins on the cell surface may be internalized and degraded, causing a reduction of integrin levels (39). Second, talin has been suggested to facilitate the exit of integrin from a subplasma membrane compartment to the cell surface. Antisense-induced suppression of talin in HeLa cells led to retention of integrin in this compartment (32). In this case, the immature integrin accumulated inside the cell may undergo proteasome-mediated degradation (49). Our immunostaining results do not support this hypothesis because no such intracellular pool of integrin β1 was found in either differentiated EBs or isolated epiblast cells. Lastly, we found that microtubules on the basal side of epiblast cells were disoriented in the mutant EBs. Since microtubules are required for membrane protein recycling (39), changes in the microtubule cytoarchitecture may compromise integrin trafficking and lead to a reduction in integrin levels. This intriguing possibility warrants further investigation.
This work was supported by a grant from the National Institutes of Health (R01GM081674) to S.L. and by grants from the Wellcome Trust (077532/Z/05/Z) and Cancer Research UK to D.R.C.
We thank Ashwini Kumar for his assistance in the FACS analysis and Uta Praekelt for the generation of the isoform-specific talin1 and talin2 monoclonal antibodies.
- Received 9 December 2010.
- Returned for modification 29 January 2011.
- Accepted 21 May 2011.
- Accepted manuscript posted online 13 June 2011.
- Copyright © 2011, American Society for Microbiology. All Rights Reserved.


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About Lubo
LUBO is a platform for the entrepreneurial feminine in the creative industry. The collective is here to help us grow our network and know-how through monthly Meet-Ups, Workshops and events.
Behind LUbo
CHARLOTTE ORR is a textile artist and designer who works with botanical dyes and organic fibers. Her passion to work with ecological principles through local land-based sources (and foraging most of her dye materials from the Santa Cruz / SF Bay Area) is the driving force behind her sustainable home goods business, Stand West.
"LUBO was inspired by a growing desire to surround myself with like minded creatives. After speaking to fellow feminine driven entrepreneurs in the Santa Cruz area about the idea of LUBO, I quickly realized that creating a networking platform to learn from one another and utilize each other's skill sets was a collective desire. My hope is to offer a well curated, highly informative space where we not only come together to find community and support but have the opportunity to learn more about what it means to run a successful business from a feminine perspective."


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writer's block
a usually temporary condition in which a writer finds it impossible to proceed with the writing of a novel, play, or other work.
13 Quotes All Writers Will Relate ToWriting is beautiful...and it's painful. Even these skilled writers have faced writer's block, and they have the words for it.
Nearby words
write-in candidate, write-off, write-protect, write-up, writer, writer's block, writer's cramp, writerly, writhe, writhen, writing
Origin of writer's block
First recorded in 1945–50
Dictionary.com Unabridged Based on the Random House Unabridged Dictionary, © Random House, Inc. 2019


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Tuesday, May 28, 2019
SoCal here we come!
14 comments:
Have a safe trip!ReplyDeleteReplies
Thanks Billy...unfortunately the German care packages come to an end...:-(Delete
Have a safe trip... and let me know if you stop by the Bay Area. I'll show you around the local flea markets. Plus I have some stuff to give you.ReplyDeleteReplies
Hey Fuji, will do. I am sure we'll be up there on occasion...and my job will probably have me up there frequently too.Delete
Moving to another foreign country, eh? :)ReplyDelete
Wishing you and the family the safest of trips!Replies
Ha, coming back to America always feels foreign everytime I've returned! Thanks and hopefully I'll be back up and blogging sooner than later.Delete
Good luck with the move!! I look forward to seeing more posts/videos from California!ReplyDeleteReplies
Thanks Sean. Glad to read your blog about meeting up with Dave in Japan. So cool. Hopefully, I'll be able to get some unique California posts up every once in a while.Delete
safe travels, SMM! Hope the move goes smoothly!ReplyDeleteReplies
Thanks! It will be a challenge for sure!Delete
Good luck with the move my friend! If you ever find yourself back in Dayton, let me know!ReplyDeleteReplies
Adam, thanks a bunch. I am sure we'll be back in Dayton at least once a year. Hopefully we can hook up! Scope out some local card shops!Delete
Wishing you a smooth move and enjoy California dreamin'ReplyDeleteReplies
You bet! Will work on my tan!Delete


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NEW – Spirit Warriors
Modeled after the popular Ninja Warriors, come join us for strength training, obstacles and ninja warrior courses – including our very own warped wall!
Welcome to our Spirit Gymnastics Sports Academy Family! It is our privilege to share our love for Gymnastics, Cheer, Dance and Sports with your child.
Our mission is to build and encourage true character in all ages as they learn the sports of gymnastics, cheer, dance and more. We value the qualities of discipline, structure and excellence. We offer a wholesome environment where our students and staff develop strength, discipline and self-esteem as they strive to reach their goals through hard work and the reward of success not measured in trophies but in lifelong lessons.
Modeled after the popular Ninja Warriors, come join us for strength training, obstacles and ninja warrior courses – including our very own warped wall!
Our competitive gymnastics team include girls of all competitive levels. They train with Teresa, Debra, Meghan, Susan and Hunter on all four events and conditioning. Our girls also learn good sportsmanship, team work and how to support and encourage each other throughout the season. Our coaches support their goals whether that's to have fun or […]
The program consists of training fun designed for athletes with Special Needs and preparing them to participate in gymnastics meets with HUGS divisions. Parents are welcome to stay and participate or observe the program. Instruction is provided by certified Spirit Gymnastics coaches and volunteers trained to work with special needs children. Lakeland Regional Rehabilitation staff […]
The FREE program consists of training fun designed for athletes with Special Needs. Parents are welcome to stay and participate or observe the program. Instruction is provided by certified Spirit Gymnastics coaches and volunteers trained to work with special needs children. Lakeland Regional Rehabilitation staff partners with us, providing occupational therapist as part of the […]
This invite only class for gymnasts ages seven to ten is part of USA Gymnastics TOPs program. TOPs (Talent Opportunity Program), is a talent search and educational program for female gymnasts ages 7-10 and their coaches. During the months of June and July gymnasts age 7-10 are evaluated on physical abilities at the state or […]
The perfect opportunity for parents to enjoy a night out! Drop the kids off at Spirit for an action-packed night of gymnastics, free play, the bounce house and pizza! Ask
This invite-only class is designed to prep our youngest talent for the gymnastics competitive team. Athletes learn foundational skills and start working on learning early team routines so they're ready for team tryouts and their first competitive season.
For our more advanced tumblers, this class focuses exclusively on more advanced tumbling skills such as tucks and passes.
A class focused exclusively on tumbling skills – athletes learn handsprings and more!
Our advanced intermediate class focuses on all four events. Students learn more advanced skills such as back and front tumbling passes.
One step up from beginner, our athletes work through all four events: floor, bars, beam and vault.
Students spend the first half of class learning the basics of dance such as foot positions. The remainder of the class is spent learning the fundamentals of gymnastics.
A time for homeschool students to come together, make friends and learn the basics of gymnastics.
Open to the public to allow anyone to come and enjoy the gymnastics experience for $5 on Tuesday and Thursday mornings and $10 on Friday evenings. The time consists of free play with our trained coaches here to supervise. Highlights include a bounce house, trampoline and rope climbs. Participants must have a signed waiver on […]
A class for children from walking to three years old. Mom or Dad can come on the floor with their student. We review colors, numbers and shapes while incorporating early, foundational gymnastics skills. The class also incorporates parachute games, trampoline jumps and the swing.
A safe and fun introduction to rolling, bouncing, twisting and all around movement awaits every child that enters our gym! From warming up their muscles to going upside down around the uneven bars, they will experience it all! We focus first on knowledge of the equipment and their comfort level with each event they visit. […]
Rolling and cartwheels and handstands and more! All designed for our youngest tumblers ages three to four.
Our Spirit vans will pick our students up from school and bring them back to the gym for an afternoon of gymnastics rotation, snack time, games, homework, team work and fun!
Basketball, soccer, baseball, football, tumbling and more! Boys will get a fun workout, be introduced to a variety of sports and learn teamwork and sportsmanship along the way.
Young athletes will be introduced to all male gymnastics equipment, tumbling skills, strength building routines and basics that will help them across any sport. They'll learn sportsmanship, teamwork and perseverance along the way!
Young athletes will be introduced to all male gymnastics equipment, tumbling skills, strength building routines and basics that will help them across any sport.
Boys will get a fun workout, be introduced to a variety of sports and learn teamwork and sportsmanship along the way.
A safe and fun introduction to rolling, bouncing, twisting and all around movement awaits every child that enters our gym!
The fourth annual Spirit Classic will take place March 8-9, 2019 in Lakeland, FL.
Join team from across the state and beyond in beautiful Central Florida for this premiere, growing event!
"I am first and foremost very proud to be a gym owner with the opportunity to train young athletes as they work hard to achieve their goals year after year. I know you take the same pride in your young athletes and have the same positive impact on their development as I do. My same philosophy carries over to the Spirit Classic and I will work very hard to see everyone leaves with a smile on their face, feeling good about the experience."
~Teresa Proctor
Owner & Head Coach
Spirit Gymnastics Youth Association, a 501c3 nonprofit, is proud to offer free modified gymnastics for special needs athletes.Learn More
Susan is a team coach who can be found putting the girls through their workout of the day, coaching new skills on bars or pushing our TOPS girls to new heights.
Anthony works with our after school kids and helps coach our boys classes.
Ticia works with our after school program and our morning homeschool classes. She also coaches several of our preschool classes, beginner and tumbling classes.
Madison works with our after school kids. She also coaches some of our tumbling classes and our cheer prep class.
AbiGayle has been a part of the Spirit family since 2016. She is a coach for HUGS, our special needs program.
Emily is the fearless leader of our Spirit All Stars cheer team. She can also be found coaching tumbling skills or coaching our rec kids.
Jayden works with our SPLAT after school program. She also works with our tiny tumble students and beginner rec classes.
Angel works with our gymnastics pre-team girls. She oversees parents night out and camps. Angel also works with our mom, pop and tot, beginner and intermediate rec classes.
Abi coaches our pre-team gymnastics team. She also works with our after school program, our youngest beginner rec classes and our intermediate rec classes.
Hunter is one of our gymnastics team coaches. He also coaches our total tumble and advanced rec classes.
Abi works in our SPLAT kids after school program, camp programs and parent's night out. She also coaches beginner and intermediate rec classes.
Chae works with our after school kids. She also works with our tiny tumblers and beginner gymnasts.
Sarah works in our after school program and our HUGS special needs program. Sarah also works with our tumbling classes and our beginner to intermediate rec classes. You may also find Sarah working with our gymnastics pre-team girls.
Keith heads up our Boys in Action and Boys in Action Sports programs, bringing his passion for sports and passion for mentoring young men to the gym. When he's not teaching a class, you can likely find him maintaining the facility or building our next great addition.
Debra coaches team and a few rec classes. Her daughter is a gymnast who has competed on Spirit's competitive team for years, and you may have seen her son (also known a "The Spirit Baby") tagging along in the gym!
Teresa is our fearless leader – the owner of the gym. With more than 20 years of experience as a gymnastics coach, she mentors our coaches, molds our girls in to awesome young ladies and gymnasts, and treats all families who walk through the doors as if they're her own family.


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Hong Kong's powerful family-run conglomerates are rushing to reassure investors of their succession plans, spurred by a scandal involving the city's biggest property developer that has thrown the spotlight on plans to hand down businesses.
Lui Chee-woo, the 82-year-old founder of developer K. Wah International Holdings and Galaxy Entertainment Group, which runs casinos in Macau, outlined on Thursday how management of the two companies would pass to his children.
Three other Hong Kong firms with extensive property holdings - The Wharf (Holdings), Henderson Land Development and New World Development - also gave details of the roles younger family members would play.
Some of Asia's largest listed companies are family run, and succession plans are tightly held secrets, often only revealed through wills at the death of their founders.
The move to give details of the plans comes amid a high-profile corruption investigation involving Sun Hung Kai Properties, which has seen its shares plunge 22 percent since co-chairmen Thomas and Raymond Kwok were arrested in March.
Institutional investors have pressed for details of the company's succession plan and while Sun Hung Kai says it has one, it has not been announced.
"It definitely shows that an unclear game plan for the future of a company creates a lot of problems," said Nicole Wong, property analyst at brokerage CLSA Asia-Pacific Markets.
"It's shock therapy."
With many tycoons behind Hong Kong's most powerful companies in their 80s, the issue of succession is at the forefront of investors' minds.
Asia's richest man, Li Ka-shing, last week said that his eldest son, Victor, would take over at Cheung Kong (Holdings) and Hutchison Whampoa, the real estate to telecoms empire. The 83-year-old founder also said he would bankroll younger son Richard's deals.
Hong Kong tycoons have typically rebuffed questions about succession while they were still at the helm.
Analysts said the fact that companies were drawing up such plans meant that leadership should pass from entrepreneurs used to making tough business decisions to managers running mature businesses.
"It's nothing to do with policy, it's nothing to do with the government or the economy, it's just about time," said Alfred Lau, property analyst at Bocom International, of the transition process companies were getting to grips with.
At K. Wah, founder Lui said that while he was not retiring, he had divided his empire.
"They are already in their positions," a spokeswoman for K. Wah told Reuters on Thursday. "He did not exactly use the word succession plan, but it does have similar meaning."
Eldest son Francis Lui will take charge of Galaxy and run the Macau business, the spokeswoman said.
The second son, Lawrence, will take care of the family's business in the United States, while younger son Alexander Lui will take over the property business in Hong Kong, she said.
Daughter Paddy Lui will run its hotel business in Hong Kong, while younger daughter Lui Wai-ling heads group administration.
Wharf, a conglomerate that spans ports, communications and property, confirmed to Reuters on Thursday how it has divided up the company's units. Chairman Peter Woo's daughter, Jennifer, is handling luxury retailer Lane Crawford, while son Douglas is managing director of its development arm, Wheelock Properties.
A spokeswoman at Henderson Land confirmed that chairman Lee Shau Kee's eldest son, Peter Lee, is responsible for the group's business in mainland China, while younger son, Martin Lee, looks after the business in Hong Kong.
"You can say it's a succession plan," the Henderson spokeswoman said. "They are both vice chairmen of the group but they have different core focus."


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You are here
National Wages Council seeks public feedback on wage, employment-related issues
SINGAPORE'S National Wages Council is asking for public feedback on wages and employment-related issues which it will consider as it develops annual guidelines on these matters.
The council - which comprises employers, worker and government representatives - will meet in April and May to develop these guidelines. Feedback should be shared by April 15 for it to be considered.
The National Wages Council is chaired by DBS chairman Peter Seah, who has been at the helm since 2015.
In its deliberations on wage recommendations, the council considers factors such as Singapore's economic performance, as well as the domestic and global economic outlook, said the Manpower Ministry in a media release.
Singapore's economic competitiveness, labour market conditions, inflation and productivity growth are also taken into account, it said.
Last year, the council raised the basic monthly pay threshold for workers it gives quantitative guidelines for to S$1,300, up from S$1,200, and recommended that this group be given built-in wage increments of between S$50 and S$70.
It also proposed - for the first time - that companies with productivity gains in the previous year give them a one-off bonus of between S$300 and S$600, either in a lump sum or over several payments.
For other workers, the council did not specify any amount or proportion for the bonus.
The guidelines came after healthy growth in the Singapore economy of 3.6 per cent in 2017. Labour productivity, measured as real value added per worker, also rose 4.1 per cent in that year.
Last year, Singapore's economy grew 3.2 per cent, and labour productivity grew 2.5 per cent.
Feedback can be sent by e-mail to the council's secretariat at firstname.lastname@example.com
It can also be mailed to:
National Wages Council
c/o Manpower Planning and Policy Division
18 Havelock Road #07-01
Singapore 059764
The fax number is 6534 4840.


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For the last month or so cash gold produced about 7 wild spikes to the upside, which readers can all ignore. Anomalies like this do happen in futures forcing me to double check by the switch to line mode to see if the spike is real.
As wild as this bullish phase has been it's pretty normal for diagonal wave structures. The 5th wave is another zigzag with one 5th wave being part of a "C" wave bullish phase.
The May/June zigzag is not even close to having an even "C" wave but I look for zigzags anyways. I allow much more extreme zigzag lengths like in 1930-1932 stock market decline. When a market dishes out a long "C" wave then I allow it and incorporate it into my version of the EWP.
The Gold price can crash again as this potential 4th wave correction has not finished.
Hits: 14


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For many owners of residential beach homes in Massachusetts, their ocean view is an important and enjoyable part of their property. In order to make certain changes to the home, therefore, local zoning laws generally require a special permit or variance. In a July 12, 2019 Massachusetts real estate case, the owner of a beach house opposed the building permits granted to her neighbors for a tear-down and rebuild of a new residence. The matter came before the Land Court on appeal by the plaintiff, following the decision of the local zoning board.
Both of the parties' properties were on a peninsula near the ocean. The plaintiff resided in a single-story home with a deck. The defendants reportedly tore down an existing, single-story, non-conforming residence and built a new two-story home in its place. Although the plaintiff's view of the ocean from her deck became partially obstructed by the second story of the defendants' new house, the height of the house fully complied with current zoning. Other aspects of the house, however, were not.
On appeal, the plaintiff argued that the defendants' new home increased the nonconformity of the floor area, open space, and setbacks beyond the limits allowed under the town zoning bylaw. While the defendants had obtained a building permit to construct the house, the plaintiff asserted that the defendants were required to obtain a special permit or variance for the new build.


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Feedback welcomed on council's budget
A reminder that there are a few days left for submissions and comments on the Wudinna District Council's annual business plan for 2019-20.
The 2019-20 annual business plan is available at the council's office and can also be viewed online at the council's website at wudinna.sa.gov.au.
Written submissions regarding the budget will be received until 5pm on Monday, July 15.
If you are not into reading a full annual business plan take a look at the great slide presentation developed by staff on council's website or Facebook page - it gives a great overview.
Our council's 2019-20 average increase in rates is recommended at 3.4 per cent.
The state government's forced Solid Waste Levy increases on our district of 24.6 per cent will take the waste levy cost to $32,349 for the 2019-20 year.
It is planned to absorb this increase into the existing budget estimates this year.
Prior to the council's meeting on Tuesday, July 16 an hour between 11am and noon is allocated for submissions and representations to be made by interested people.
Council staff can assist if anyone requires more details on the process.
The end of June saw Ben Wagner farewelled as he had retired after some 20 years in the gardening position. He was congratulated and thanked for his work at his farewell.
Also a welcome to new gardener Jay Bunney and his family to the district.
Do not forget we are celebrating National Tree Day on Sunday, July 28 at Wudinna.
Following the tree planting which starts at 10am there will be a community walk starting at 11am and a community barbecue at Apex Park in Wudinna at noon.
More details are available from the council office and all are welcome.
Council and contracted grading services have been busy on the roads across the district, making the most of ideal conditions following much welcome rain.
A thank you to clubs and committees, builders and contractors who have worked with council on the Drought Communities Program projects.
A full report will be provided for the community and the federal government, demonstrating the added value such projects can bring to our rural regions.
Wudinna District Council mayor


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Fleet Advantage established a record amount of lease originations exceeding $200 million for the period January 1 through June 30, 2019. read more
JUL 16, 2019 - 6:30 am
CEO of Fleet Advantage, John Flynn, was included in Supply & Demand Chain Executive magazine's list of 2019 Pros to Know in the supply chain industry. read more
FEB 26, 2019 - 6:35 am
Fleet Advantage reached a significant company milestone, securing $1 billion in total lease originations, with more than $200 million in originations in 2017 alone. read more
JAN 18, 2018 - 5:31 am
Fleet Advantage reported results of its Q3/17 operating cycle, with more than $80 million secured in loan originations during the quarter. read more
NOV 15, 2017 - 6:21 am
Fleet Advantage expanded the role of Jim Griffin, who will now serve in the capacity of both chief technology officer and chief operating officer. read more
AUG 16, 2016 - 7:21 am
Fleet Advantage was awarded the Technology Leadership Award for Fleet TCO Optimization Solutions in North America by the global industry research and consulting firm Frost & Sullivan. read more
JUN 1, 2016 - 7:19 am


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Gated community with lots of charm! When you walk into the home, you're immediately welcomed with original hardwood flooring and take a moment to take in the 18 feet of soaring ceiling!! Master bedroom is downstairs along with flex room in which can also be used as a bedroom, study, or den. Kitchen is loaded with plenty of cabinet space, stainless steel appliances, and large island. Master suite includes spacious garden tub, separate dual vanities and large walk in closet. Home has open concept and features assortment of bay windows. Check out the unique and exciting backyard basketball setup in which contains a premium, custom engineered outdoor shock absorbing court!
Attached, Garage, Garage Door Opener
Bay Windows, Cable TV Available, High Speed Internet Available
Built-in Microwave, Dishwasher, Disposal, Oven - Gas, Vent Mechanism, Water Line to Refrigerator
Central Heat-Elec, Central Heat-Gas
Other, Sprinkler System, Sport Court
Users may not reproduce or redistribute the data found on this site. The data is for viewing purposes only. Data is deemed reliable, but is not guaranteed accurate by the MLS or NTREIS.
Listing information last updated on October 22nd, 2019 at 3:45am CDT.


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928 DICKINSON WAY, French Creek, British Columbia, V9P1Z7
$1,699,000 - For sale
**Rare Opportunity with Stunning Walk on Waterfront*** At over .6 of an acre this sprawling brand new home built by Departure Bay Construction offers stunning views from all angles. Enjoy the sunsets from your gourmet kitchen, or take the path down to your private sandy beach, and very own custom built pergola. This property has it all, including an 912 sq ft detached over height shop for your RV or boat, in addition to the attached double garage and RV parking. You'll be popular with a home like this, and there's plenty of room with a massive bonus room with ensuite and wet bar above the garage, 3 bedrooms and 2 more baths on the main floor. GST applicable. (id:2471)
- Property Address:928 DICKINSON WAY, French Creek, British Columbia, V9P1Z7
- Property Type:Single Family
- Title:Freehold
- Lot Size:0.63
- Acreage:0.63 ac
- Access:Easy access
- Driveway:Garage (2 spaces)
- Waterfront Type:Waterfront on ocean
- Structure Style:House
- Building Age:2018
- Bedrooms:3
- Bathrooms:3
- Square Footage:2862 sqft
- Style:Detached
- Amenities Nearby:Recreation
|Main level
|8 ft ,8 in x 11 ft ,5 in
|Main level
|10 ft x 14 ft
|Living room
|Main level
|14 ft ,8 in x 18 ft
|Dining room
|Main level
|7 ft x 16 ft
|Main level
|15 ft ,7 in x 9 ft
|Main level
|11 ft ,10 in x 10 ft
|Main level
|16 ft ,5 in x 9 ft ,10 in
|Laundry room
|Main level
|10 ft x 14 ft
|Master bedroom
|Main level
|14 ft x 17 ft
|Main level
|24 ft ,4 in x 24 ft
|3pc Bathroom
|Main level
|5pc Ensuite bath
|Main level
|Recreation room
|Upper Level
|14 ft ,10 in x 13 ft ,8 in
|3pc Bathroom
|Upper Level
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- Map 928 DICKINSON WAY, French Creek, British Columbia, V9P1Z7
- Neighbourhood Information
928 DICKINSON WAY, French Creek, British Columbia, V9P1Z7
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Information and discussion on all aspects of British photographic history
The Nicholas Brothers & A. T. W. Penn: photographers of South India 1855–1885 examines the successful studios established by John and James Perratt Nicholas and Albert Penn in Madras and Ootacamund. The text is illustrated with over 100 plates and 150 figures, the majority of which are published here for the first time. The book further reproduces a catalogue of Nicholas & Co.'s photographs from 1881, which will serve as an invaluable tool for researchers and collectors.
In the 1850s and 60s, Madras was an important centre for the rapidly developing art of photography. Dr Alexander Hunter founded the Madras School of Arts in 1850 and the Madras Photographic Society in 1857, where John Nicholas served on the committee. Pioneering photographers Linnaeus Tripe, John Parting, Edmund David Lyon, Willoughby Wallace Hooper and Samuel Bourne all contributed to the rapid advance of photography in the region. James Perratt Nicholas and A. T. W. Penn continued their work to the end of the nineteenth century.
This publication marks the end of a 12-year research project for the author, who scrupulously documents three decades of work by James Perratt Nicholas and A. T. W. Penn. It begins with the early years of the Nicholas studios in Madras and Ootacamund, explains how the business achieved success in the 1870s and 1880s and concludes with the introduction of the Kodak, the rise of the amateur photographer, and the inevitable decline in the studios' profitability that followed.
The Nicholas Brothers & A. T. W. Penn: photographers of South India 1855–1885 is being published by Quaritch in Summer 2014. If you would like to be contacted when it is available for purchase at the special prepublication price, please contact: Alice Ford-Smith: firstname.lastname@example.com
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I've just finished reading this. An excellent book; well researched and produced; with a lot of fine illustrations; many of which I hadn't ever seen before. An absolute must for anyone interested in early photography in India.
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I know how to install Bootstrap manually or with composer. But I want to know that which is best practice to use it. If I install through composer then how to include bootstrap files. Looking forward for best answer that can teach me best way for it.
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Gabriel BassPosted at 16:52h, 05 March
What would Melanie May say? Melanie May may say many things… Great to see your fun and delicious blog up and running… On my end, I ended up settling in Colombia, currently in Bogotá with a 4 month old son and trying to get the music career off the ground… life's pretty sweet…. Anyway, whenever you want to head back to one of your favorite countries, we'll be here waiting for you with open arms. Abrazos grandes!


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The Hospice offers incredibly rewarding work, where staff can make a very real difference to patients' lives, with a patient staff ratio that supports meaningful care. We are offering hours to suit you. The casual positions will work shortfalls on a rotating roster Monday to Sunday. Experience in caring for people with a life limiting illness would be advantageous. Ongoing training and education will be offered to you to achieve our goal of meeting the needs of every palliative care patient in our region.
Applicants must have current Annual Practising Certificate and be eligible to work in New Zealand.
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Werewolves On The Moon [WWOM]
"What Would Happen?"
Loyalty, Power, Curiosity, and an Overwhelming Drive for Success. These are the defining traits of the Werewolves On The Moon.
What would happen? The ultimate of hypotheticals. This statement turned creed empowers us, pushes us to make the impossible a reality, standing up to whatever the darkness throws our way.
Atheon, Crota, Oryx, Aksis, Ghaul, all crushed in the thrill of the hunt by an unrelenting desire to further the pack. You hear whispers of our conquest, like Bigfoot and the Boogyman we exist as a myth that has to be seen to be believed.
For we are the Werewolves On The Moon, and we are impossibility personified.
Invite OnlyThis clan is invite only.
82 Members
Created October 29, 2014


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Authored by two experienced clinicians, this insightful guide provides a pragmatic roadmap for treating adult survivors of complex psychological trauma. Christine Courtois and Julian Ford present their effective, research-based approach for helping clients move through three clearly defined phases of posttraumatic recovery. Two detailed case examples run throughout the book, illustrating how to plan and implement strengths-based interventions that use a secure therapeutic alliance as a catalyst for change. Essential topics include managing crises, treating severe affect dysregulation and dissociation, and dealing with the emotional impact of this type of work. The companion Web page offers downloadable reflection questions for clinicians and extensive listings of professional and self-help resources.See also Drs. Courtois and Ford's edited volume, Treating Complex Traumatic Stress Disorders, which presents research on the nature of complex trauma and reviews a range of evidence-based treatment models for adults.
Reviews and Endorsements:
"The state of the art of treating complex trauma, both the basics and the nuances, as summarized by two wise and sophisticated experts."--Judith Lewis Herman, MD, Department of Psychiatry, Harvard Medical School
"Courtois and Ford, two internationally acknowledged experts, have written a magnificent book on the nature and phase-oriented treatment of complex trauma. Destined to become an instant classic, the book is illustrated throughout with instructive case material. It is at once an excellent guide for the uninitiated and an outstanding resource for experienced clinicians in the field. As most forms of complex trauma occur in interpersonal relationships, the book's emphasis on the therapeutic relationship as the vehicle for healing contributes to its importance."--Onno van der Hart, PhD, Emeritus Professor of Psychopathology of Chronic Traumatization, Utrecht University, The Netherlands
"Finally! We have long needed a clinical guidebook that takes treatment providers through the process of therapy for complex posttraumatic stress syndromes, which are among the greatest challenges that professionals encounter. Courtois and Ford's broad and deep clinical experience shines through in their discussion of assessment, engagement, and treatment--as does their optimism, passion, and hope."--Sandra L. Bloom, MD, founder, The Sanctuary Model
"Courtois and Ford provide detailed guidelines for conducting their sequenced treatment approach, from preparation, assessment, and planning, to each of the phases of therapy. The book covers the 'meat and potatoes' of trauma therapy: ensuring client safety, cultivating the therapeutic relationship, and processing trauma memories. It includes strategies from different theoretical approaches and treatment modalities and addresses more complex processes, such as psychic emptiness and dissociation. Written with exceptional clarity, compassion, and respect, and richly illustrated with clinical material, this is an invaluable resource for clinicians."--Sandra C. Paivio, PhD, CPsych, Department of Psychology, University of Windsor, Ontario, Canada
"Both authors have worked extensively with survivors of complex trauma, and share their wisdom and experience in this book. At a time when this clinical population is becoming more visible to clinicians, this thoughtful, extensively documented work is a real treasure. The emphasis on therapist self-care and the ways in which the authors underscore the central importance of therapist self-awareness and self-regulation are particularly valuable. This book will be required reading for all of my interns."--Laura S. Brown, PhD, ABPP, Director, Fremont Community Therapy Project, Seattle, Washington
Caversham Booksellers98 Harbord St, Toronto, ON M5S 1G6 Canada
(click for map and directions)All prices in $cdn
Copyright 2019
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Attractive 4 bedroom detached bungalow conveniently located in a popular area within a 10 minute drive of Waterford City centre and amenities. Set back from the road with well attended gardens the bungalow enjoys privacy and scenic overland views. Beautifully presented and tastefully decorated with bright cheerful colours the accommodation comprises of entrance porch, entrance hall, sittingroom, shower room, kitchen, sunroom, utility room, 4 bedrooms and main bathroom.
The property is located 2.6km from Ferrybank and its amenities which include schools, grocery stores, specialist shops, chemist, butchers, hairdressers, restaurant, licensed premises, library, doctors and dentists surgeries and sports and leisure facilites. M9 Waterford to Dublin Motorway 4.4km.
Entrance Porch
Entrance Hall - 4.1m x 1.5m
uPVC front door. Tiled floor.
Sittingroom - 4.2m x 3.7m
Antique reproduction fireplace with solid fuel stove. Maple hardwood floor.
Shower room
With w.h.b, w.c, shower cabinet and electric shower. Tiled walls and floor.
Archway to inner hallway - 7.9m x 1.0m
Airing cupboard with folding double doors. Tiled floor.
Bedroom 1 - 4.2m x 3.0m
Built in wardrobe and dressing table.
Bedroom 2 - 3.7m x 3.1m
Main Bathroom -
W.c, w.h.b with pine surround, wall light fitting.
Bedroom 3 - 3.1m x 3.1m
Bedroom 4 - 5.5m x 3.8m
Antique reproduction fireplace with solid fuel stove. Antique over mantle. Built in display cabinet with cupboards underneath.
Kitchen - 4.4m x 3.17m
Circular stainless steel sink unit. Built in wall and floor units and worktops. Integrated Belling electric cooker. Extractor. Microwave. American Fridge. Built in shelves. Tiled floor. Wall mounted china cabinet.
Sunroom - 2.8m x 2.4m
Pine shelving. uPVC double glazed patio doors to railed decking overlooking the back garden with picturesque overland views.
Utility room - 2.0m x 1.8m
With circular s.s.s unit. Plumbed for washing machine and dishwasher. Pine shelving. Tiled floor.
Splayed masonry wall entrance and gate piers. Tarmacadam driveway to large parking area. Well attended lawns with Laurel hedging boundaries. Attractive double wrought iron gates to side of parking area. Water filter unit.
Enclosed back garden with lawn and raised deck. Shrub flower beds and rockeries. Detached workshop 28 sq.m.
Excellent condition throughout
Walls and roof insulated to a high specification
Stairs to attic partly floored, suitable for conversion subject to planning consent
uPVC double glazed windows
Oil fired central heating
Fibre broadband available
Local amenities in close proximity include creche, Strangsmills school and Waterford Golf Club.
Phonewatch alarm
Regulated by RICS
Monday to Friday 9.30am - 5pm.
Closed 1pm - 2pm.
REA O'Shea O'Toole,
11 Gladstone Street,
Waterford City,
Co. Waterford,
00353 51 876 757