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Small extracellular vesicles (sEVs) generated from the endolysosomal system, often referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics, as they carry valuable biological information and reflect their cells of origin. Herein, we propose a simple and inexpensive electrical method for label-free detection and profiling of sEVs in the size range of exosomes. The detection method is based on the electrokinetic principle, where the change in the streaming current is monitored as the surface markers of the sEVs interact with the affinity reagents immobilized on the inner surface of a silica microcapillary. As a proof-of-concept, we detected sEVs derived from the non-small-cell lung cancer (NSCLC) cell line H1975 for a set of representative surface markers, such as epidermal growth factor receptor (EGFR), CD9, and CD63. The detection sensitivity was estimated to be similar to 175000 sEVs, which represents a sensor surface coverage of only 0.04%. We further validated the ability of the sensor to measure the expression level of a membrane protein by using sEVs displaying artificially altered expressions of EGFR and CD63, which were derived from NSCLC and human embryonic kidney (HEK) 293T cells, respectively. The analysis revealed that the changes in EGFR and CD63 expressions in sEVs can be detected with a sensitivity in the order of 10% and 3%, respectively, of their parental cell expressions. The method can be easily parallelized and combined with existing microfluidic-based EV isolation technologies, allowing for rapid detection and monitoring of sEVs for cancer diagnosis.
Silk is considered to be a potential biomaterial for a wide number of biomedical applications. Silk fibroin (SF) can be retrieved in sufficient quantities from the cocoons produced by silkworms. While it is easy to formulate into scaffolds with favorable mechanical properties, the natural SF does not contain bioactive functions. Spider silk proteins, on the contrary, can be produced in fusion with bioactive protein domains, but the recombinant procedures are expensive, and large-scale production is challenging. We combine the two types of silk to fabricate affordable, functional tissue-engineered constructs for wound-healing applications. Nanofibrous mats and microporous scaffolds made of natural silkworm SF are used as a bulk material that are top-coated with the recombinant spider silk protein (4RepCT) in fusion with a cell-binding motif, antimicrobial peptides, and a growth factor. For this, the inherent silk properties are utilized to form interactions between the two silk types by self-assembly. The intended function, that is, improved cell adhesion, antimicrobial activity, and growth factor stimulation, could be demonstrated for the obtained functionalized silk mats. As a skin prototype, SF scaffolds coated with functionalized silk are cocultured with multiple cell types to demonstrate formation of a bilayered tissue construct with a keratinized epidermal layer under in vitro conditions. The encouraging results support this strategy of fabrication of an affordable bioactive SF-spider silk-based biomaterial for wound dressings and skin substitutes.
Affibody molecules are small affinity-engineered scaffold proteins which can be engineered to bind to desired targets. The therapeutic potential of using an affibody molecule targeting HER2, fused to an albumin-binding domain (ABD) and conjugated with the cytotoxic maytansine derivate MC-DM1 (AffiDC), has been validated. Biodistribution studies in mice revealed an elevated hepatic uptake of the AffiDC, but histopathological examination of livers showed no major signs of toxicity. However, previous clinical experience with antibody drug conjugates have revealed a moderateto high-grade hepatotoxicity in treated patients, which merits efforts to also minimize hepatic uptake of the AffiDCs. In this study, the aim was to reduce the hepatic uptake of AffiDCs and optimize their in vivo targeting properties. We have investigated if incorporation of hydrophilic glutamate-based spacers adjacent to MC-DM1 in the AffiDC, (Z(HER2:2891))(2) -ABD-MC-DM1, would counteract the hydrophobic nature of MC-DM1 and, hence, reduce hepatic uptake. Two new AffiDCs including either a triglutamate-spacer-, (Z(HER2:2891))(2)-ABD-E-3-MC-DM1, or a hexaglutamate-spacer-, (Z(HER2:2891))(2)-ABD-E-6-MC-DM1 next to the site of MC-DM1 conjugation were designed. We radiolabeled the hydrophilized AffiDCs and compared them, both in vitro and in vivo, with the previously investigated (Z(HER2:2891))(2)-ABD-MC-DM1 drug conjugate containing no glutamate spacer. All three AffiDCs demonstrated specific binding to HER2 and comparable in vitro cytotoxicity. A comparative biodistribution study of the three radiolabeled AffiDCs showed that the addition of glutamates reduced drug accumulation in the liver while preserving the tumor uptake. These results confirmed the relation between DM1 hydrophobicity and liver accumulation. We believe that the drug development approach described here may also be useful for other affinity protein-based drug conjugates to further improve their in vivo properties and facilitate their clinical translatability.
Carbonic anhydrase IX (CAIX) is a cancer-associated molecular target for several classes of therapeutics. CAIX is overexpressed in a large fraction of renal cell carcinomas (RCC). Radionuclide molecular imaging of CAIX-expression might offer a non-invasive methodology for stratification of patients with disseminated RCC for CAIX-targeting therapeutics. Radiolabeled monoclonal antibodies and their fragments are actively investigated for imaging of CAIX expression. Promising alternatives are small non-immunoglobulin scaffold proteins, such as affibody molecules. A CAIX-targeting affibody ZCAIX:2 was re-designed with the aim to decrease off-target interactions and increase imaging contrast. The new tracer, DOTA-HE3-ZCAIX:2, was labeled with In-111 and characterized in vitro. Tumor-targeting properties of [In-111]In-DOTA-HE3-ZCAIX:2 were compared head-to-head with properties of the parental variant, [(99)mTc]Tc(CO)(3)-HE3-ZCAIX:2, and the most promising antibody fragment-based tracer, [In-111]In-DTPA-G250(Fab')(2), in the same batch of nude mice bearing CAIX-expressing RCC xenografts. Compared to the (99)mTc-labeled parental variant, [In-111]In-DOTA-HE3-ZCAIX:2 provides significantly higher tumor-to-lung, tumor-to-bone and tumor-to-liver ratios, which is essential for imaging of CAIX expression in the major metastatic sites of RCC. [In-111]In-DOTA-HE3-ZCAIX:2 offers significantly higher tumor-to-organ ratios compared with [In-111]In-G250(Fab']2. In conclusion, [In-111]In-DOTA-HE3-ZCAIX:2 can be considered as a highly promising tracer for imaging of CAIX expression in RCC metastases based on our results and literature data.
A unique strategy for effective, versatile, and facile surface biofunctionalization employing a recombinant spider silk protein genetically functionalized with the antibody-binding Z domain (Z-4RepCT) is reported. It is demonstrated that Z-silk can be applied to a variety of materials and platform designs as a truly one-step and chemical-free surface modification that site specifically captures antibodies while simultaneously reducing nonspecific adsorption. As a model surface, SiO2 is used to optimize and characterize Z-silk performance compared to the Z domain immobilized by a standard silanization method. First, Z-silk adsorption is investigated and verified its biofunctionality in a long-term stability experiment. To assess the binding capacity and protein-protein interaction stability of Z-silk, the coating is used to capture human antibodies in various assay formats. An eightfold higher binding capacity and 40-fold lower detection limit are obtained in the immunofluorescence assay, and the complex stability of captured antibodies is shown to be improved by a factor of 20. Applicability of Z-silk to functionalize microfluidic devices is demonstrated by antibody detection in an electrokinetic microcapillary biosensor. To test Z-silk for biomarker applications, real-time detection and quantification of human immunoglobulin G are performed in a plasma sample and C1q capture from human serum using an anti-C1q antibody.
Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the ZCa–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification
Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1-10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1-10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1-10 in analyses. The pre-maturated GFP 1-10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1-10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1-10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.
Epidermal growth factor receptor (EGFR) is overexpressed in a number of cancers and is the molecular target for several anti-cancer therapeutics. Radionuclide molecular imaging of EGFR expression should enable personalization of anti-cancer treatment. Affibody molecule is a promising type of high-affinity imaging probes based on a non-immunoglobulin scaffold. A series of derivatives of the anti-EGFR affibody molecule ZEGFR:2377, having peptide-based cysteine-containing chelators for conjugation of Tc-99m, was designed and evaluated. It was found that glutamate-containing chelators Gly-Gly-Glu-Cys (GGEC), Gly-Glu-Glu-Cys (GEEC) and Glu-Glu-Glu-Cys (EEEC) provide the best labeling stability. The glutamate containing conjugates bound to EGFR-expressing cells specifically and with high affinity. Specific targeting of EGFR-expressing xenografts in mice was demonstrated. The number of glutamate residues in the chelator had strong influence on biodistribution of radiolabeled affibody molecules. Increase of glutamate content was associated with lower uptake in normal tissues. The Tc-99m-labeled variant containing the EEEC chelator provided the highest tumor-to-organ ratios. In conclusion, optimizing the composition of peptide-based chelators enhances contrast of imaging of EGFR-expression using affibody molecules.
Mucoadhesion is defined as the adhesion of a material to the mucus gel covering the mucous membranes. The mechanisms controlling mucoadhesion include nonspecific electrostatic interactions and specific interactions between the materials and the mucins, the heavily glycosylated proteins that form the mucus gel. Mucoadhesive materials can be used to develop mucosal wound dressings and noninvasive transmucosal drug delivery systems. Spider silk, which is strong, biocompatible, biodegradable, nontoxic, and lightweight would serve as an excellent base for the development of such materials. Here, we investigated two variants of the partial spider silk protein 4RepCT genetically engineered in order to functionalize them with mucoadhesive properties. The pLys-4RepCT variant was functionalized with six cationically charged lysines, aiming to provide nonspecific adhesion from electrostatic interactions with the anionically charged mucins, while the hGal3-4RepCT variant was genetically fused with the Human Galectin-3 Carbohydrate Recognition Domain which specifically binds the mucin glycans Gal beta 1-3GlcNAc and Gal beta 1-4GlcNAc. First, we demonstrated that coatings, fibers, meshes, and foams can be readily made from both silk variants. Measured by the adsorption of both bovine submaxillary mucin and pig gastric mucin, the newly produced silk materials showed enhanced mucin binding properties compared with materials of wild-type (4RepCT) silk. Moreover, we showed that pLys-4RepCT silk coatings bind mucins through electrostatic interactions, while hGal3-4RepCT silk coatings bind mucins through specific glycan-protein interactions. We envision that the two new mucoadhesive silk variants pLys-4RepCT and hGal3-4RepCT, alone or combined with other biofunctional silk proteins, constitute useful new building blocks for a range of silk protein-based materials for mucosal treatments.
Human epidermal growth factor receptor type 3 (HER3) plays a crucial role in the progression of many cancer types. In vivo radionuclide imaging could be a reliable method for repetitive detection of HER3-expression in tumors. The main challenge of HER3-imaging is the low expression in tumors together with endogenous receptor expression in normal tissues, particularly the liver. A HER3-targeting affibody molecule labeled with radiocobalt via a NOTA chelator [Co-57]Co-NOTA-Z(08699) has demonstrated the most favorable biodistribution profile with the lowest unspecific hepatic uptake and high activity uptake in tumors. We hypothesized that specific uptake of labeled affibody monomer might be selectively blocked in the liver but not in tumors by a co-injection of non-labeled corresponding trivalent affibody (Z(08699))(3). Biodistribution of [Co-57]Co-NOTA-Z(08699) and [In-111]ln-DOTA-(Z(08699))(3) was studied in BxPC-3 xenografted mice. [Co-57]Co-NOTA-Z(08699) was co-injected with unlabeled trivalent affibody DOTA-(Z(08699))(3) at different monomer:trimer molar ratios. HER3-expression in xenografts was imaged using [Co-57]Co-NOTA-Z(08699) and [Co-57]Co-NOTA-Z(08699): DOTA-(Z(08699))(3). Hepatic activity uptake of [Co-57] Co-NOTA-Z(08699): DOTA-(Z(08699))(3) decreased with increasing monomer:trimer molar ratio. The tumor activity uptake and tumor-to-liver ratios were the highest for the 1:3 ratio. SPECT/CT images confirmed the biodistribution data. Imaging of HER3 expression can be improved by co-injection of a radiolabeled monomeric affi body-based imaging probe together with a trivalent affibody.
Electrokinetic principles such as streaming current and streaming potential are extensively used for surface characterization. Recently, they have also been used in biosensors, resulting in enhanced sensitivity and simpler device architecture. Theoretical models regarding streaming current/potential studies of particle-covered surfaces have identified features such as the particle size, shape and surface charge to influence the electrokinetic signals and consequently, the sensitivity and effective operational regime of the biosensor. By using a set of well-characterized proteins with varying size and net surface charge, this article experimentally verifies the theoretical predictions about their influence on the sensor signal. Increasing protein size was shown to enhance the signal when their net surface charge was either opposite to that of the sensor surface, or close to zero, in agreement with the theoretical predictions. However, the effect gradually saturated as the protein size exceeded the coulomb screening length of the electrolyte. In contrast, the proteins containing the same type of charge as the surface showed little or no difference, except that the signal inverted. The magnitude of the surface charge was also shown to influence the signal. The sensitivity of the technique for protein detection varied over two orders of magnitude, depending upon the size and surface charge. Furthermore, the capacity of the electrokinetic method for direct electrical detection of various proteins, including those carrying little or no net electric charges, is demonstrated.
Antibody DNA conjugates are powerful tools for DNA-assisted protein analysis. Growing usage of these methods demands efficient production of high-quality conjugates. We developed an easy and fast synthesis route yielding covalent antibody-DNA conjugates with a defined conjugation site and low batch-to-batch variability. We utilize the Z domain from protein A, containing the unnatural amino acid 4-benzoylphenylalanine (BPA) for photoaffinity labeling of the antibodies' Fc region. Z(xBPA) domains are C-terminally modified with triple-glycine (G(3))-modified DNA-oligonucleotides enzymatic Sortase A coupling. We reliable modification of the most commonly used IgG's. To prove our conjugates' functionality, we detected antibody-antigen binding events in an assay called Droplet Barcode Sequencing for Protein analysis (DBS-Pro). It confirms not only retained functionality for both conjugate parts but also the potential of using DBS-Pro for quantifying protein abundances. As intermediates are easily storable and our approach is modular, it offers a convenient strategy for screening various antibody-DNA conjugates using the same starting material.
Affibody molecules are engineered scaffold proteins, which demonstrated excellent binding to selected tumor-associated molecular abnormalities in vivo and highly sensitive and specific radionuclide imaging of Her2-expressing tumors in clinics. Recently, we have shown that peptide nucleic acid (PNA)-mediated affibody-based pretargeted radionuclide therapy using beta-emitting radionuclide 177Lu extended significantly survival of mice bearing human Her2-expressing tumor xenografts. In this study, we evaluated two approaches to use positron emission tomography (PET) for stratification of patients for affibody-based pretargeting therapy. The primary targeting probe ZHER2:342-SR-HP1 and the secondary probe HP2 (both conjugated with DOTA chelator) were labeled with the positron-emitting radionuclide 68Ga. Biodistribution of both probes was measured in BALB/C nu/nu mice bearing either SKOV-3 xenografts with high Her2 expression or DU-145 xenografts with low Her2 expression. 68Ga-HP2 was evaluated in the pretargeting setting. Tumor uptake of both probes was compared with the uptake of pretargeted 177Lu-HP2. The uptake of both 68Ga-ZHER2:342-SR-HP1 and 68Ga-HP2 depended on Her2-expression level providing clear discrimination of between tumors with high and low Her2 expression. Tumor uptake of 68Ga-HP2 correlated better with the uptake of 177Lu-HP2 than the uptake of 68Ga-ZHER2:342-SR-HP1. The use of 68Ga-HP2 as a theranostics counterpart would be preferable approach for clinical translation.
Radionuclide molecular imaging of HER2 expression in disseminated cancer enables stratification of patients for HER2-targeted therapies. DARP in G3, a small (14 kDa) engineered scaffold protein, is a promising probe for imaging of HER2. We hypothesized that position (C- or N-terminus) and composition (hexahistidine or (HE)(3)) of histidine-containing tags would influence the biodistribution of [Tc-99m]Tc(CO)(3)-labeled DARP in G3. To test the hypothesis, G3 variants containing tags at N-terminus (H-6-G3 and (HE)(3)-G3) or at C-terminus (G3-H-6 and G3-(HE)(3)) were labeled with [Tc-99m]Tc(CO)(3). Labeling yield, label stability, specificity and affinity of the binding to HER2, biodistribution and tumor targeting properties of these variants were compared side-by-side. There was no substantial influence of position and composition of the tags on binding of [Tc-99m]Tc(CO)(3)-labeled variants to HER2. The specificity of HER2 targeting in vivo was confirmed. The tumor uptake in BALB/c nu/nu mice bearing SKOV3 xenografts was similar for all variants. On the opposite, there was a strong influence of the tags on uptake in normal tissues. The tumor-to-liver ratio for [Tc-99m]Tc(CO)(3)-(HE)(3)-G3 was three-fold higher compared to the hexahistidine-tag containing variants. Overall, [Tc-99m]Tc(CO)(3)-(HE)(3)-G3 variant provided the highest tumor-to-lung, tumor-to-liver, tumor-to-bone and tumor-to-muscle ratios, which should improve sensitivity of HER2 imaging in these common metastatic sites.
Evaluation of human epidermal growth factor receptor 2 (HER2) expression levels in breast and gastroesophageal cancer is used for the stratification of patients for HER2-targeting therapies. The use of radionuclide molecular imaging may facilitate such evaluation in a non-invasive way. Designed ankyrin repeat proteins (DARPins) are engineered scaffold proteins with high potential as probes for radionuclide molecular imaging. DARPin G3 binds with high affinity to HER2 and may be used to visualize this important therapeutic target. Studies on other engineered scaffold proteins have demonstrated that selection of the optimal labeling approach improves the sensitivity and specificity of radionuclide imaging. The present study compared two methods of labeling G3, direct and indirect radioiodination, to select an approach providing the best imaging contrast. G3-H6 was labeled with iodine-124, iodine-125 and iodine-131 using a direct method. A novel construct bearing a C-terminal cysteine, G3-GGGC, was site-specifically labeled using [125I]I-iodo-[(4-hydroxyphenyl)ethyl]maleimide (HPEM). The two radiolabeled G3 variants preserved binding specificity and high affinity to HER2-expressing cells. The specificity of tumor targeting in vivo was demonstrated. Biodistribution comparison of [131I]I-G3-H6 and [125I]I-HPEM-G3-GGGC in mice, bearing HER2-expressing SKOV3 xenografts, demonstrated an appreciable contribution of hepatobiliary excretion to the clearance of [125I]I-HPEM-G3-GGGC and a decreased tumor uptake compared to [131I]I-G3-H6. The direct label provided higher tumor-to-blood and tumor-to-organ ratios compared with the indirect label at 4 h post-injection. The feasibility of high contrast PET/CT imaging of HER2 expression in SKOV3 xenografts in mice using [124I]I-G3-H6 was demonstrated. In conclusion, direct radioiodination is the preferable approach for labeling DARPin G3 with iodine-123 and iodine-124 for clinical single photon emission computed tomography and positron emission tomography imaging.
Affibody molecules are small proteins engineered using a nonanti-body scaffold. Radiolabeled Affibody molecules are excellent imaging probes, but their application to radionuclide therapy has been prevented by high renal reabsorption. The aim of this study was to test the hypothesis that Affibody-based peptide nucleic acid (PNA)-mediated pretargeted therapy of human epidermal growth factor receptor 2 (HER2)-expressing cancer extends survival without accompanying renal toxicity. Methods: A HER2-targeting Affibody molecule ligated with an AGTCGTGATGTAGTC PNA hybridization probe (Z(HER2:342)-SR-HP1) was used as the primary pretargeting agent. A complementary AGTCGTGATGTAGTC PNA conjugated to the chelator DOTA and labeled with the radionuclide Lu-177 (Lu-177-HP2) was used as the secondary agent. The influence of different factors on pretargeting was investigated. Experimental radionuclide therapy in mice bearing SKOV-3 xenografts was performed in 6 cycles separated by 7 d. Results: Optimal tumor targeting was achieved when 16 MBq/3.5 mu g (0.65 nmol) of Lu-177-HP2 was injected 16 h after injection of 100 mu g (7.7 nmol) of Z(HER2:342)-SR-HP1. The calculated absorbed dose to tumors was 1,075 mGy/MBq, whereas the absorbed dose to kidneys was 206 mGy/MBq and the absorbed dose to blood (surrogate of bone marrow) was 4 mGy/MBq. Survival of mice was significantly longer (P < 0.05) in the treatment group (66 d) than in the control groups treated with the same amount of Z(HER2:342)-SR-HP1 only (37 d), the same amount and activity of Lu-177-HP2 only (32 d), or phosphate-buffered saline (37 d). Conclusion: The studied pretargeting system can deliver an absorbed dose to tumors appreciably exceeding absorbed doses to critical organs, making Affibody-based PNA-mediated pretargeted radionuclide therapy highly attractive.
Protein fragment complementation assays (PCA) rely on a proximity-driven reconstitution of a split reporter protein activity, typically via interaction between bait and prey units separately fused to the reporter protein halves. The PCA principle can also be formatted for use in immunossays for analyte detection, e.g., via the use of small immunoglobulin binding proteins (IgBp) as fusion partners to split-reporter protein fragments for conversion of pairs of antibodies into split-protein half-probes. However, the non-covalent binding between IgBp and antibodies is not ideal for development of robust assays. Here, the authors describe how split-enzyme reporter halves can be both site-specifically and covalently photoconjugated at antibody Fc-parts for use in homogeneous dual-antibody in vitro immunoassays based on analyte-dependent split-enzyme fragment complementation. The half-probes consist of parts of a beta-lactamase split-protein reporter fused to an immunoglobulin Fc binding domain equipped with a unique cysteine residue at which a photoactivable maleimide benzophenone group (MBP) is attached. Using such antibody conjugates the authors obtain an analyte-driven complementation of the reporter enzyme fragments monitored via conversion of a chromogenic substrate. Results from detection of human interferon-gamma and the extracellular domain of HER2 is shown. The described principles for site-specific conjugation of proteins to antibodies should be broadly applicable.
Protein engineering provides powerful tools to create useful proteins with desired properties. In this thesis, rational design principles have been used for development of fusion proteins that can interact with the neonatal Fc receptor (FcRn) for potential medical applications.
FcRn is widely expressed in the human body. The natural ligands of FcRn are immunoglobulin G (IgG) and serum albumin (SA). FcRn can bind to both proteins in a pH dependent manner and endow them with an unusually long half-life in vivo. Protein building blocks interacting directly or indirectly with FcRn may potentially be used to either piggy-back on the FcRn-system for extension of the in vivo half-life or to saturate the system to decrease the in vivo half-life of the natural ligands. In this thesis, I have explored an FcRn binding affibody molecule (ZFcRn) and/or an albumin binding domain (ABD) for these purposes.
In study I and II, the prolactin receptor was found to often be expressed in glioblastoma multiforme tumors from patients as well as in glioblastoma multiforme cells lines. We investigated a novel antagonist of the prolactin receptor in vitro and found that it could block signaling through the receptor as well as cellular invasiveness. An antagonist of prolactin receptor could thus potentially become a drug for treatment of glioblastoma multiforme. However, the antagonist will likely have a short plasma half-life due to its small molecular size, which limits its usability. Therefore, it was expressed as a fusion to ABD, which interacts indirectly with FcRn. The produced fusion protein was found to be able to block signaling through the prolactin receptor in vitro and also had a prolonged plasma half-life in vivo.
The goal of study III was to investigate the properties of human growth hormone (hGH) when it was expressed as a protein fusion with ZFcRn, interacting directly with FcRn, and/or ABD. The fusion proteins, ZFcRn-hGH, ABD-hGH, and ZFcRn-ABD-hGH could be recombinantly expressed and successfully purified to homogeneity. They had the expected binding abilities to FcRn, SA and hGH receptor. They were all found to be able to induce signaling over the plasma membrane in a model cell line.
Patients suffering from many autoimmune diseases produce particular IgG molecules, which are responsible for the disease symptoms. A potential treatment could be to increase the catabolism of these IgGs to relieve disease symptoms. In study IV, an FcRn interacting affibody molecule was investigated for IgG depletion by blocking the IgG/FcRn interaction. In vitro, we first found that the affibody molecule shares a common binding site with IgG on FcRn, which indicates that the affibody should be able to block IgG from binding to FcRn. In vivo, we injected large amounts of the affibody molecules in different formats in mice and found up to 39% reduction of total endogenous IgG. In a clinical setting, reduction of total IgG level would also reduce the disease causing IgGs, and potentially ameliorate the symptoms of IgG-driven autoimmune diseases.
Taken together, I have in this thesis explored application of FcRn interacting molecules for extension of biological half-lives of therapeutically relevant proteins and reduction of total IgG level by FcRn blocking.
Growth hormone replacement therapy has been used to treat children and adults with
growth hormone deficiency for more than three decades. Growth hormone has a short
biological half-life, requiring daily subcutaneous injections, and long acting versions are
therefore desirable. In this study we have analyzed three fusion proteins, ZFcRn-hGH,
ABD-hGH and ZFcRn-ABD-hGH, consisting of the human growth hormone (hGH) and an
affibody molecule binding the neonatal Fc receptor (ZFcRn) and/or an albumin binding
domain (ABD). Both ZFcRn and the ABD may have the ability to endow hGH with an
extended plasma half-life. The fusion proteins could be recombinantly expressed in the
periplasmic space of Escherichia coli and easily purified to homogeneity. All three fusion
proteins appeared to have a strong interaction with the growth hormone receptor. ABDhGH
and ZFcRn-ABD-hGH had a strong affinity for HSA (KD 0.006 and 0.02 nM,
respectively). ZFcRn-hGH and ZFcRn-ABD-hGH had moderately strong affinity for mouse
neonatal Fc receptor at pH 6.0 (KD 200 and 100 nM, respectively). The fusion proteins
thus retained the expected binding abilities of the individual domains. Further
characterization showed that the fusion proteins could induce phosphorylation of signal
transducer and activator of transcription 5 (STAT5) in the model cell line U251-MG,
further showing that the hGH-part of the fusion proteins was functional.


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373 Eagle Drive, Jupiter, FL 33477
Bedrooms: 5
Bathrooms: 6 | 1
Sq. Ft.: 6,916
Type: Single Family Home
Listing #RX-10408887
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Masterfully re-modeled in 2015/2016 Designer Traditional 5 bedroom, 51/2 baths home with separate Guesthouse.Grand Foyer Entrance with mahogany doors and venetian plaster walls throughout into living room. Dramatic curving staircase with newer hand wrought iron railings. Newer 8" wide board solid walnut floors throughout. Full led ceiling lighting. Designer kitchen with high end appliances , and butler pantry. Beautiful white counter tops and Italian tile backsplash/ separate wet bar for entertaining. Spectacular newer master suite & his/hers baths with artisan custom tile work and french doors leading to private terrace. Impact doors & windows. Also movie room/Custom design chandeliers and fixtures.Extra large laundry room- New pebble tec finished heated pool, A Must See!
Property Features
Location Information
County: Palm Beach
MLS Area: 5100
Latitude: 26.898804
Longitude: -80.081383
Geo Area: PB31
Subdivision: ADMIRALS COVE
Directions: Alt A1A north of Donald Ross or PGA Blvd to entrance on left. Manned Gate
Development Name: Admirals Cove
Subdivision Info: Basketball, Bike – Jog, Boating, Business Center, Clubhouse, Community Room, Elevator, Exercise Room, Golf Course, Lobby, Manager on Site,
Interior Features
Rooms: Cabana Bath, Den/Office, Family, Laundry-Util/Closet, Media
Interior: Cook Island, Foyer, Pantry, Roman Tub, Upstairs Living Area, Volume Ceiling, Walk-in Closet
1/2 Baths: 1
Has Dining Room: yes
Dining Room Description: Breakfast Area, Dining-Living, Eat-In Kitchen, Formal
Family Room Description: 26X15
Den: 15X14
Living Room Description: 36X19
Kitchen Description: 18X15
Heating: Central, Zoned
Cooling: Central
Floors: Marble, Wood Floor
Additional Equipment: Auto Garage Open, Central Vacuum, Dishwasher, Disposal, Dryer, Ice Maker, Microwave, Range – Electric, Refrigerator, Storm Shutters, Washer
Exterior Features
Stories: 2
Construction: CBS
Exterior: Auto Sprinkler, Built-in Grill, Covered Patio, Fence, Summer Kitchen, Zoned Sprinkler
Roof: Concrete Tile
Possession: At Closing
Utilities: Underground
Security System: Burglar Alarm, Gate – Manned, Security Patrol
Parking Description: 2+ Spaces, Driveway, Garage – Attached
Has Garage: yes
Garage Spaces: 3
Has a Pool: yes
Pool Description: Freeform, Heated, Inground, Salt Chlorination
Guest House: 1 Bath, 1 Bedrooms, Kitchen
Has Golf Course: yes
Lot Description: 1/2 to < 1 Acre
Zoning: Res
Building Total Area (Sq. Ft.): 8, 786
Waterfront Description: Interior Canal, Navigable, No Fixed Bridges, Ocean Access, Seawall
Has Waterfront: yes
View Description: Canal, Pool
Additional Information
Status: Active
Property Type: SFR
Property SubType: Single Family Detached
Year Built: 1992
Is Short Sale: No
HOA Fee: 742.9
Restrictions: Buyer Approval, Tenant Approval
Pets: Yes
Boat Service: Attended, Electric Available, Fuel, Full Service, Lift, Marina, Private Dock, Restroom, Up to 80 Ft Boat, Yacht Club
Design: Traditional
Membership Equity: Yes; $175, 000
Taxes: City/County
Tax Year: 2017
Tax Amount: 68, 929
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Seller's Representative:
NV Realty Group LLC/PB
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Perth weather: Record-breaking heatwave forecast
Perth is set to swelter though another record-breaking heatwave, forecast to kick off on Wednesday with a toasty 38C.
The Bureau of Meteorology is predicting a four day run of temperatures above 39C from Thursday — something that hasn't been seen in December since records began at the new meteorological site in 1993.
It comes after Perth experienced the hottest first week in December on record with temperatures averaging 36C.
Thursday has been forecast to be 39C, followed by three days of 40C.
And, the overnight temperatures are unlikely to provide much relief with it unlikely to drop below 20C.
Duty forecaster Jim Richardson said a ridge in the south of the State and a trough developing down the west coast over the next few days were to blame for the hot conditions.
"It's a pretty classic set up for Perth ... the trough down the west coast and you have easterlies or north easterlies just dragging this hotter air from further inland and you either get late sea breezes or weak sea breezes."
Wednesday: 20C-38C. Very hot and sunny.
Thursday: 21C-39C. Very hot and sunny.
Friday: 25C-40C. Very hot and sunny.
Saturday: 24C-40C. Very hot and sunny.
Sunday: 23C-40C. Very hot and sunny.
Monday: 21C-32C. Very hot and sunny.
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Layers of mouthwatering flavours; dried fruits, peanut brittle, cream caramel, coffee, cedar and a beautifully luscious texture with freshness, lift and a long, rich finish. *Gold Medal Decanter World Wine Awards 2018*
The NV Apostoles VORS is a blend of Palomino made into Palo Cortado and 10% Pedro Ximenez, assembled after their first five years of aging after which they completed their 30 years in barrel together. It shows a mahogany color and a predominant note of raisins among the hazelnuts, candied fruit and sweet vanilla. The off-dry palate has intense flavors of dates, dry figs and raisins, with great persistence and length. The blending makes it very approachable and good to combine with cured cheeses. A unique, off-dry Palo Cortado. (LG)
A flagship wine of González Byass, Apóstoles is a blend of 18- to 20-year-old wines, including 13 percent from sweet Pedro Ximénez. That's a high percentage for a Palo Cortado, but the strength of the Apóstoles base wine is such that it can absorb the intense sweetness, allowing it to add some gentleness to the wine. The salty flavors are wrapped in a layer of hazelnut tones, but the intense minerality is what predominates.
A unique and individual style of Sherry, even among palo cortados. It's dark amber in color, with a pungent mix of aromas that include molasses, raw brown sugar, chocolate and campfire. Feels fresh on the tongue, with coffee, dark chocolate and core peach and citrus flavors. (MS)
Rather friendly for a Sherry, with ginger, almond paste, toffee and roasted peanut notes forming the core. A light bitter almond edge keeps the finish honest. Purists may decry the lack of austerity here, but if you want to ease someone into Sherry, this might be the way. (JM)
VORS means "Very Old and Rare Sherry." The solera from which the sherry is sourced must be an average age of 30 years, and only the very best older soleras are able to put the VORS label on their wine. Gonzales Byass is one of the dwindling number of shippers with soleras of this age and quality. To be more precise, this solera of Palo Cortado (the rarest style of sherry in production) began in 1862, in honor of a vineyard visit by Queen Isabel II. That is an old solera! A bit of Pedro Ximenez is blended in here which adds some sweetness to this very old Palo Cortado. It's a singular bottling from one of the best-known shippers in Jerez.
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LAWRENCE, KANSAS – Coming back from Rio, four former Jayhawk track and field athletes spoke on Friday at Rock Chalk Park about competing in the 2016 Olympics. One of the four competitors, sprinter Kyle Clemons, left Rio with some extra weight around his neck.
Clemons won a gold medal for Team USA in the 4×400 meter relay. Clemons ran the third-leg in the preliminary round as the group placed second with a time of 2:58.38. Clemons did not run for the team in the Finals, where a time of 2:57.30 edged out second place Jamaica.
Clemons, a volunteer assistant for the KU track team, continues to accommodate fans that want to see his gold medal, though he does not always wear it.
"I've had it pretty much in my glove box in my car," Clemons said. "Wherever I go somebody may want to see it and I don't want to go all the way back home."
"After it wears down, settles down, everything like that I'll probably just put it in a case."
Clemons, discus thrower Mason Finley and triple jumper Andrea Geubelle were all part of the same 2009 recruiting class.
"Six years ago were being recruited together to be here at the University and we kind of marked our ground here," Geubelle said. "Now we've taken that next step."
With Olympic experience under their belt, Geubelle and Finley still have aspirations to join Clemons as an Olympic medalist.
"It's cool to see the success of my teammate," Finley said. "It definitely lights a fire under my butt."
Finley finished 11th in the discus with a mark of 62.05 meters. His performance was the best Olympic discus finish by an American since 2004.
Geubelle finished 21st in the triple jump with a mark of 13.93 meters, a tremendous feat after she left the sport in 2013 for over a year following a torn patellar tendon in the right knee.
The fourth former Jayhawk, Daina Levy, competed for the Jamaican national team. Levy was the first Jamaican female to compete in the Olympic hammer throw. Her mark of 60.35 meters landed her a 30th place finish.
Levy's time with the Jamaican team has allowed her to interact with superstar sprinter Usain Bolt, but she is not starstruck by her interactions with him.
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Romania's economy grew by 2.8 per cent in 2014 compared to 2013, surpassing the 2.2 per cent growth level on which the budget was based and the European Commission's 2.1 per cent forecast published in the autumn of 2013, a report published on the Finance Ministry's website shows.
The development was based on the higher-than-expect growth in private consumption (4.5 per cent compared to initial forecasts of 1.6 per cent) and public consumption (5.3 per cent compared to initial forecasts of 1.7 per cent). On the other hand, an unpleasant surprise came from investment expenditures, which dropped 3.5 per cent year-on-year while the budget was based on a growth forecast of 4 per cent and the EC had forecast a growth of 3.5 per cent.
"This was determined by high uncertainties among investors, foreign investors in particular, as well as the government investments' low contribution. It is worth noting that the drop in investments eased in Q3, and there was even a re-launch of growth in Q4," the report reads.
Thus, in 2014 Romania registered the fourth-highest economic growth level in the EU.
When it comes to economic sectors, industry and constructions had a weaker-than-expected evolution. In industry however, the difference was moderate, the sector registering 0.8 per cent growth compared to a 0.9 per cent growth forecast. Constructions however stagnated in 2014, despite the fact that a growth of 0.5 per cent had been forecast. The agriculture sector's growth exceeded expectations, standing at 0.1 per cent compared to a forecast of -0.2 per cent. The services sector grew by 1.3 per cent, compared to an estimate of 0.7 per cent, while net tax revenues grew by 0.5 per cent compared to the 0.2 per cent forecast on which the budget was based.


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Massage elk river Guildford
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Welcome to Radiant Body Massage!
Sheraton Hotels
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text: Lasik Self Evaluation Test Name* First Last PhoneEmail*Do you have trouble seeing far away or up close?*Up closeFar awayHow interested are you in being able to play sports without glasses or contacts?*It's very important to me NOT to wear glasses for activities such as sports.It's not important to me. I do not mind wearing glasses.What is your age range?*Under 2121 - 4040 - 6970+Are you interested in seeing well up close (reading) without glasses?*It's very important to me NOT to wear reading glasses.It's not important to me. I do not mind wearing reading glasses to see things up close.Do you wear glasses or contact lenses?*GlassesContact lensesBothNeitherWould your career or business activities improve if you were to become less dependent on glasses or contacts?*YesNoMaybeOver 98% of LASIK patients see 20/40 or better after surgery. The results of LASIK laser vision correction have been tremendous for literally millions of people. Despite the amazing safety and results of this procedure there are associated risks. Are you willing to discuss these risks with our LASIK coordinator?*YesNo


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Breakdown Co-ordinator *French, Spanish, Italian speaking*
Uptime Solutions is the breakdown co-ordination service for Volvo and Renault Trucks. In our Rugby office we are responsible for the UK, France, Spain and Italy.
*This role is a temporary contract via agency with the potential to go permanent.*
You will be the point of contact for each case, receiving calls from Drivers and asessing where they are located, noting down key details into the case management system. You will then contact the nearest dealership to request assistance from a Technician, process payments and warranty checks. You will also liase with the Uptime team in Gent, Belgium for assistance where required.
Shift pattern
We operate between the hours of 6am and 11pm and work on a 4 week rotation of early or afternoon shifts, Mon-Fri only.
Week 1 6:00-14:30
Week 2 14:30-23:00
Week 3 7:00-15:30
Week 4 14:00-22:30
On each shift for the Rugby South team there are up to 11 of us covering 3 different countries, France, Spain and Italy. We speak a mixture of languages and come from all across the world including Europe, South America and Africa. We like celebrating our diversity and regularly share our favourite foods at Fika (Swedish for coffee and cake time!) We have an open plan office with desks that allow you to stand or sit while you work.
Take over active cases at the beginning of a shift
Assure proactive follow up of open cases
Take incoming calls as a priority
Communicate effectively and proactively will all parties to co-ordinate the breakdown service
Secure the financial arrangements related to a breakdown according to warranty, soft products and financial routines – standards
Escalate cases according to the escalation process
Ask customers and dealers questions repairing dealer in order to provide the right information to the relevant parties
Correct when possible or report inaccurate/incomplete information when discovered within databases or systems
You will feel confident communicating over the phone in at least 2 languages including, French, Spanish or Italian. You will have a good understanding of computer systems and have experience in customer service either in an office environment , hospitality or similar.
In this role you will receive 4 weeks of training including classroom sessions and shadowing other team members to make sure you are confident in the role.
We will help you develop the skills you need to progress at Volvo Group and with access to an international business who knows where that could take you!
We offer a competitive salary and lots of benefits including 6% shift bonus, cycle to work, health care, pension, annual bonus, discount on Volvo and Renault cars and 25 days holiday.
We speak your language! Come and join the #VolvoFamily, apply today and if sucessful we will invite you to an initial telephone asessment.
Om oss
The Volvo Group is one of the world's leading manufacturers of trucks, buses, construction equipment and marine and industrial engines under the leading brands Volvo, Renault Trucks, Mack, UD Trucks, Eicher, SDLG, Terex Trucks, Prevost, Nova Bus, UD Bus, Sunwin Bus and Volvo Penta.
The Volvo Trucks brand has been built up over decades and enjoys a solid position worldwide. It is one of the world's best known and respected brands within the commercial vehicle industry. It is associated with the core values quality, safety and environmental care. With Volvo Trucks you will be part of a global and diverse team. We work with passion, we trust each other and we embrace change to stay ahead. We make our customers win.
Vi vill lära känna dig
Ett bekräftelsemail skickas till dig efter att du färdigställt din ansökan. Du kan uppdatera din profil även efter inskickad ansökan.
Om du blir kallad till en intervju informerar vi dig kring ansökningsprocessens samtliga delmoment såsom andra intervju, tester och referenstagning.
Samtliga kandidater blir informerade när den sökta tjänsten är tillsatt. Du kan välja ifall du önskar behålla ditt CV i vår databas för att få förslag på andra jobb som matchar din profil.
Vid anställningsstart genomgår du en introduktion som syftar till att på kort tid få dig att bli en del av ditt nya team samt börja kunna genomföra dina nya arbetsuppgifter på bästa möjliga sätt.


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Mumbai International Emigration and Luxury Property Expo
Mumbai International Emigration and Luxury Property Expo: the trade show
Mumbai International Luxury Property and Residency Conference
Date 15-16 January 2020
Venue The St. Regis Mumbai (462, Senapati Bapat Marg, Worli, 400013), India.
Website: https://ilpexpo.com/exhibitions/mumbai/2020/jan
An exhibition on investment immigration and international high-end real estate
Mumbai International Luxury Property and Residency Conference is a large-scale
conference and exhibition.
It combines a conference on the first day (15 January), with an exhibition, workshops,
and networking opportunities on the second day (16 January).
Exhibitors and attendees
The event brings together high-end real estate and immigration market professionals. Our visitors include private individuals interested in immigration and acquisition of upscale properties,
international entrepreneurs, home buyers, and prospective immigrants.
Regular exhibitors of the Mumbai IELP Expo include:
- Government agencies from 30 countries, including the USA, Canada, the EU (Spain,
Cyprus, Malta etc.), the Caribbean (Antigua & Barbuda, Dominica, St Kitts and Nevis),
and more;
-International associations, including FIABCI and the Investment Migration Council;
- Prominent immigration companies, including Henley& Partners and CS Global
- Luxury real estate market leaders, including Sotheby's Real Estate, Christie's, and
Knight Frank;
-Major personal financial consultancies, including EY, KPMG, PwC, and Deloitte; and
- Other leaders in the related fields.
Why Mumbai?
- As of 2018, total HNWI (High Net Worth Individuals) wealth worldwide amounted to US
$70 trillion. India has 218,600 HNWIs, who collectively control US $877 billion.
-As of 2017, more than 17 million Indian immigrants were residing abroad.
- Indians favor the UAE as an immigration destination. More than 3.5 million Indian
nationals reside in the Emirates.
- 2 million Indian immigrants reside in the USA.
-In 2013-2014, Indians invested more than US $5.8 billion in residential properties in the
-Indians prefer property investments in Singapore, New York, Dubai, and London.
- In London, of buyers of properties worth US $1 million or more, 3% are Indian
The conference covers a range of topics, including immigration, acquisition of upmarket real
estate overseas, and personal financial consulting. Attendees had the opportunity to get their
questions answered by our professional partners and receive bespoke advice from
acknowledged and reputable industry experts.
To join the event, please apply on the official website: https://ilpexpo.com/exhibitions/mumbai/2020/jan
Event profile Mumbai International Emigration and Luxury Property Expo
- Industries:
- Frequency: Biennial
- Scope: International
Next edition Mumbai International Emigration and Luxury Property Expo
- Edition finished
- Add to Calendar
Mumbai International Emigration and Luxury Property Expo editions
|Mumbai International Emigration and Luxury Property Expo 2019
|From to
|Mumbai, India
|The St. Regis Mumbai
Mumbai International Emigration and Luxury Property Expo organizer
- London (United Kingdom)


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For those of you who are reading this, I am Luke Golden. I have held the position of Education and Stewardship Coordinator at Save Our Canyons for the past year and a half. It has been my pleasure to lead the Wilderness Stewardship Project as well as our other volunteer opportunities and SOCkids. It is my belief that active involvement with our SLC community is critical in order to preserve and protect our beautiful Wasatch Mountains. Growing up in a small mountain town in Northern New Hampshire has helped to shape my views on how to protect and use wild areas that are home to so much flora and fauna.
Another year of our volunteer projects and Wilderness Stewardship Project has come to an end. This year, we turned our time and dedication towards restoration in Lone Peak Wilderness, installation of new wilderness signs within four different wilderness areas, and five trash clean up days in Big Cottonwood Canyon. For everyone who is not familiar with the Wilderness Stewardship Project (WSP), this is a project that is aimed at improving wilderness areas in the Central Wasatch through trail maintenance and restoration, while focusing on educating the public about these areas. In 2018, Save Our Canyons, with help from the David Kelby Johnson Memorial Foundation, launched an effort to promote volunteerism, educate and engage students, and improve stewardship within the designated wilderness areas in the Central Wasatch Mountains.
We started off the 2019 season with a bang! Our first WSP volunteer events of the year started in late May, when we installed six signs in four different wilderness areas. Over the winter, Save Our Canyons received a grant from the National Wilderness Stewardship Alliance that gave us the funding to purchase six wilderness portal signs for the Salt Lake Ranger District to be installed in Lone Peak, Twin Peaks, Mount Olympus, and Deseret Peak Wilderness Areas. We installed these wilderness portal signs from May 28-31st with the help of Nice InContact volunteers for each volunteer sign installation day. Installing these wilderness portal signs was no day in the park by any sense. Each sign that we installed required four volunteers to carry the eight foot long and six inch wide post up the trail to the old portal sign. Once we carried all the materials to the old portal sign, we dug out the old one and installed the new one.
After replacing a couple of the signs, it became clear that the crew who installed these old signs 20 years prior did not follow the forest service guidelines for installing trail signs. This was evident because some posts were only one foot in the ground, some maybe two feet, others with no nails in the bottom of the post to keep it secure (deadmaning) and others were not properly buried. The Forest Service Requires sign posts to be dug three feet deep into the ground, to be deadmaned, and to be buried in a particular way that requires the layering of rocks and gravel with the hopes that these signs can direct hikers for 20 years or so. We successfully removed and installed every wilderness sign. These wilderness sign installation days were well worth it when the newly purchased signs were installed and the old falling apart signs retired from their duty of informing hikers about these unique areas. Many thanks go out to all of those who volunteered.
We know that the majority of the trash in the Cottonwood Canyons is not in the wilderness areas, but along the roadside. To improve the water and environment of the Central Wasatch Mountains we set up five volunteer days to pick up trash in Big Cottonwood Canyon. We would like to thank CHG Healthcare for participating in four out of five volunteer days and The Grauer School of Encinitas, California for contacting SOC to set up the fifth. We were able to cover seven miles of Big Cottonwood Canyon while picking up 21 garbage bags full of trash. Each day as we picked up trash in BCC, it became clear that different areas of the canyon accumulated more trash than other spots. Not surprisingly, the areas with the highest amount of trash are at the base of the canyon, Butler to Mineral Fork, Mill D North/Reynolds Flat area, outside Spruces Campground area, and outside Solitude Ski Area — which see a lot of use. There are other areas in the canyon that could use a clean-up as well, but we expect to get to those next summer. We accomplished a lot but were unable to clean the entire Big Cottonwood Canyon Highway due to the all the other volunteer projects that we had going this summer. Also Save Our Canyons is a small five person operation. We are all busy with different projects and campaigns that we believe are worthwhile and can bring positive change to the Wasatch. If we are able to increase our funding towards projects like WSP or trash picks, we would be able to accomplish even more.
It was a pleasure working with the students from The Grauer School because I was able to provide their first experience in the Wasatch Mountains. Many of them had questions about the snow, animals, environment, and why the water that flows through these mountains is important. I was pleased to hear from some of the students that they perform trash pick-ups at the local beaches near their homes. They noticed that a lot of the trash we were finding was similar to what they find on the beaches, but that there was much more on the coast they reported that each day new trash lines the beaches. One of the teachers said that the beach in California could use it every day and that we in a way were lucky because the trash does not replace itself at the same rate that it does in the ocean. Even though our mountains might receive a lesser amount of trash, any is too much. It is of the utmost importance that all who enter and enjoy these mountains are thoughtful and considerate and contribute to keeping them pristine. These areas are not our homes, but the homes of the native plants and animals. When we are in the mountains, we are merely visitors.
As summer came into full swing, our traditional Wilderness Stewardship Project began on June 15th with the focus on the Lone Peak Wilderness. Bells Canyon Trail was our first overused and unappreciated trail of this season. For this event, we cleared five water drainages, cut brush, and closed seven user made trails. As we were hiking up the trail, we noticed one thing that stood out quickly; the group size. We saw numerous groups hiking within the Lone Peak Wilderness that exceeded 20 plus people. Designated wilderness areas have group size limits to reduce the impact on the area and the group size on the Salt Lake Ranger District of Lone Peak Wilderness is 10 people. I strongly encourage anyone who hikes to get informed in the rules of the area the area that they choose. The rules exist for good reasons. Save Our Canyons has created a variety of opportunities to learn about the wilderness rules. We developed a wilderness education plan to educate the valley about wilderness areas in hopes that people recreating in these areas have a minimal impact. This education plan consists of web pages that are linked to our website, brochures that present the importance of these areas, their rules and how you can recreate in ways that are not detrimental to these areas. If you are interested in improving your knowledge or sharing the information about wilderness areas, you are more than welcome to stop by the office and grab some brochures or check it out on the wilderness tab on our website. Brochures are also available at our business partners which include Patagonia, Fisher Brewery, Momentum Gym, Wasatch Touring, Cotopaxi, IME and a couple others around the city.
During the remaining four WSP days, we spent our time focusing our efforts on the Red Pine Lake Trail due to the high use it experiences and also that it was being rerouted this past summer. During these four volunteer days we cleared 20 drainages, cut brush, closed nine user made trails, removed 10 illegal fire pits, and rerouted .2 miles of the Red Ped Pine Lake Trail. We were happy to participate in the lower reroute of the two reroutes on the Red Pine Lake Trail to make it easier for hikers and to improve water drainage off the trail to lessen the impacts of erosion. The Salt Lake Ranger District, with the help of other local districts, The Cottonwood Canyons Foundation, and the community came together to accomplish this. The reroute was beautifully done and I suggest that everyone take a stroll up this trail to see all the work that was done by people who care for these areas. While we were rerouting the trail, many hikers commented on the new trail, mentioning that it is much easier and that the reroute was beautiful and was well done.
One of my favorite parts about the Wilderness Stewardship Project is meeting the volunteers and listening to the stories they have about the Wasatch and how it has changed. I enjoy hearing about their experiences hiking throughout the world and in Utah, and why they were inspired to volunteer with Save Our Canyons. One common theme is that every volunteer shares an appreciation of the Wasatch and has a darn good story to share as well. Our experiences in the mountains, whether in the Wasatch or another range, shape us to be outdoor enthusiasts and engaged activists. Mountains bring people together and shape the beliefs of communities. When we get together, it's obvious that we share common goals for the future of these mountains and wilderness areas.
As the WSP season came to a wrap, we had our last volunteer day on October 5th with plans to remove illegal fire pits from Red Pine Lake. As the morning of the 5th arrived, I drove to the Little Cottonwood Park and Ride to meet the volunteers who had contacted me. I was expecting a usual turn out of 5-7 volunteers who were ready to dedicate their Saturday to improving our wilderness areas. But as I waited outside my car on this chilly fall morning, volunteers started to arrive. A few volunteers arrived, but to my surprise, the stream continued until my car was surrounded with people of all ages ready to conquer the Red Pine Lake Trail one more time. An astonishing 18 volunteers came out to show their appreciation and dedication on our last day of the year.
Previously, our highest turn out over the 12 WSP volunteer days over two years was 8 volunteers. It warmed my heart to see the age range of those who came to help out. We had students from local high schools as well as adults in their retirement. High volunteer turn out is an important factor for this project because we are able to cover more ground and spread out the workload. With 18 volunteers we were able to have two groups of nine split up to cover both sides of the lake. Due to it being late in the season, the lake was very low, which uncovered most of the trash that was in the area. It ws sad to see the accumulation of broken glass that was found around and in the lake. It was also discouraging to see how many illegal fire pits were in the area. The Forest Service does not want dead wood to be burned because it provides homes to birds and insects as well provides nutrients to the soil so that plants can grow. The fire rings also scar the earth leaving black stains in our wilderness areas. It is my hope that more and more people each year will educate themselves and others about the Wasatch and how to recreate properly in this area so that we can keep these mountains healthy. I strongly encourage anyone who is interested to stop by our office to pick up informational materials or contact a member at SOC for further education. Over the past two seasons of running this project, I have noticed that it can be challenging to get people to show up to volunteer on their weekends, so it was thrilling to see the support over the course of the summer and especially on our last day. As a result of the volunteers' efforts, we were able to make a larger restorative impact on the area. I would happily spend any weekend with these dedicated and passionate individuals working on improving the Wasatch.
Over the course of the summer we had 14 volunteer days where we worked with 130 volunteers. Thanks to our passionate group of outdoor enthusiasts, we performed 463 hours of volunteer work in the Wasatch Mountains through trash pick-ups, wilderness trail maintenance/restoration, and wilderness sign installations. Many agencies such as the Forest Service rely on volunteers to help them with projects such as trail maintenance or highway trash pick ups. This summer we saved the Forest Service and UDOT a combine $11,774.09 though our service hours to clean up and maintain the Central Wasatch Mountains.
I would like to thank everyone involved in helping to restore our Wasatch Mountains to what they should be. I encourage everyone to share their stories with friends and family about your time volunteering with Save Our Canyons because it is our hope that we can attract even more volunteers next year. It is our mission to keep these areas clean and with your help we believe we can continue to keep the Wasatch pristine.


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Secrets at the Sunshine Hotel: the murder of Vern Holbrook
February 13, 2015 Crime, Crime Files, Dateline, News 6 Comments
Tonight's Dateline, "Secrets at the Sunshine Hotel" delves into the grizzly 2013 attack of 78-year-old Yakima, Washington real estate broker Vern Holbrook. The man was lured to a vacant home by a couple posing as home buyers. Luis Gomez-Monges and Adriana Mendez left their three children behind in the car when they followed Holbrook into his home, and the struck him from behind. He was found badly beaten, and with his throat cut, but was still alive. Holbrook passed away several months later, but it became clear that there were other people involved in his murder, including family.


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SEC's Insider Trading Probe May Nab 44 Investment FirmsMani
The Securities and Exchange Commission said that nearly four dozen funds may have traded on insider information coming out of a congressional committee. This could make the SEC's congressional insider trading probe one of the largest in history.
SEC's probe
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Recent research has revealed that a person or team wearing red is more likely to win a physical contest than a person or team wearing another color. In the present research, we examined whether red influences perceptions of relative dominance and threat in an imagined same-sex competitive context, and did so attending to the distinction between wearing red oneself and viewing red on an opponent. Results revealed a bidirectional effect: wearing red enhanced perceptions of one's relative dominance and threat, and viewing an opponent in red enhanced perceptions of the opponent's relative dominance and threat. These effects were observed across sex, and participants seemed unaware of the influence of red on their responses. Our findings lead to practical suggestions regarding the use of colored attire in sport contexts, and add to an emerging, provocative literature indicating that red has a subtle but important influence on psychological functioning.
Roger Feltman and Andrew J. Elliot
Dennis Dreiskaemper, Bernd Strauss, Norbert Hagemann and Dirk Büsch
Hill and Barton (2005) showed that fighters in tae kwon do, boxing, and wrestling who wore red jerseys during the 2004 Olympic Games won more often than those wearing blue jerseys. Regarding these results, this study investigated the effects of jersey color during a combat situation on fighters' physical parameters of strength and heart rate. An artificial, experimental combat situation was created in which the color of sport attire was assigned randomly. Fourteen pairs of male athletes matched for weight, height, and age had to fight each other: once in a red jersey and once in a blue. Heart rate (before, during, and after the fight) and strength (before the fight) were tested wearing the blue and the red jerseys. Participants wearing red jerseys had significantly higher heart rates and significantly higher pre-contest values on the strength test. Results showed that participants' body functions are influenced by wearing red equipment.
Justin H. Rigby and Austin M. Hagan
dependent on the photobiomodulation therapy's wavelength, which determines the therapy's specific color of light. 2 Different wavelengths in the visible, from blue to red, and infrared spectrums have been used in treating musculoskeletal conditions in physical medicine and rehabilitation. Blue light has
Bjoern Krenn
In the current study we questioned the impact of uniform color in boxing, taekwondo and wrestling. On 18 photos showing two athletes competing, the hue of each uniform was modified to blue, green or red. For each photo, six color conditions were generated (blue-red, blue-green, green-red and vice versa). In three experiments these 108 photos were randomly presented. Participants (N = 210) had to select the athlete that seemed to be more aggressive, fairer or more likely to win the fight. Results revealed that athletes wearing red in boxing and wrestling were judged more aggressive and more likely to win than athletes wearing blue or green uniforms. In addition, athletes wearing green were judged fairer in boxing and wrestling than athletes wearing red. In taekwondo we did not find any significant impact of uniform color. Results suggest that uniform color in combat sports carries specific meanings that affect others' judgments.
Shelley Lucas
Originally named the Red Zinger Classic, the Coors International Bicycle Classic was contested for fourteen years, 1975-1988, primarily in Colorado, but expanding to neighboring states and even Hawaii in its later years. As a stage race, this multi-day event featured several individual stages (or
Margo Mountjoy, Jorunn Sundgot-Borgen, Louise Burke, Kathryn E. Ackerman, Cheri Blauwet, Naama Constantini, Constance Lebrun, Bronwen Lundy, Anna Melin, Nanna Meyer, Roberta Sherman, Adam S. Tenforde, Monica Klungland Torstveit and Richard Budgett
In 2014, the International Olympic Committee (IOC) published a consensus statement entitled "Beyond the Female Athlete Triad: Relative Energy Deficiency in Sport (RED-S)". The syndrome of RED-S refers to: "impaired physiological functioning caused by relative energy deficiency, and includes but is
Courtney Szto
Product (RED) was launched in 2006 as an initiative to activate the corporate sector in the fight against HIV/AIDS in Africa. In 2009, Nike joined Product (RED)'s list of corporate partners with its "Lace Up, Save Lives" campaign. Nike (RED) directs 100% of its profits toward HIV/AIDS treatment and prevention through the Global Fund to Fight AIDS, Tuberculosis and Malaria and grassroots soccer programs in Africa. This case study questions the symbolism projected by Nike (RED) and its implications by applying Jean Baudrillard's theories on consumption and hyperreality. The manner in which Nike (RED) represents Africa, HIV/AIDS, soccer, and sport for development and peace are all discussed as mediated simulations that position Nike as the producer of knowledge. Data analysis observes that Nike (RED) laces produce a hyperreality, whereby the origin of truth becomes, according to Baudrillard, indecipherable and soccer becomes more important than HIV/AIDS.
Scott C. Forbes, Darren G. Candow, Jonathan P. Little, Charlene Magnus and Philip D. Chilibeck
The purpose of this study was to determine the effects of Red Bull energy drink on Wingate cycle performance and muscle endurance. Healthy young adults (N = 15, 11 men, 4 women, 21 ± 5 y old) participated in a crossover study in which they were randomized to supplement with Red Bull (2 mg/kg body mass of caffeine) or isoenergetic, isovolumetric, noncaffeinated placebo, separated by 7 d. Muscle endurance (bench press) was assessed by the maximum number of repetitions over 3 sets (separated by 1-min rest intervals) at an intensity corresponding to 70% of baseline 1-repetition maximum. Three 30-s Wingate cycling tests (load = 0.075 kp/kg body mass), with 2 min recovery between tests, were used to assess peak and average power output. Red Bull energy drink significantly increased total bench-press repetitions over 3 sets (Red Bull = 34 ± 9 vs. placebo = 32 ± 8, P < 0.05) but had no effect on Wingate peak or average power (Red Bull = 701 ± 124 W vs. placebo = 700 ± 132 W, Red Bull = 479 ± 74 W vs. placebo = 471 ± 74 W, respectively). Red Bull energy drink significantly increased upper body muscle endurance but had no effect on anaerobic peak or average power during repeated Wingate cycling tests in young healthy adults.
Alan Klein
This study examines racial tolerance through the intersection of the media, fans, and the Boston Red Sox. Through the 1998 season Red Sox home games in which Dominican Pedro Martinez pitched attracted large numbers of Latinos. This marked the first time that large numbers of people of color regularly attended Fenway Park. Media reports simultaneously promoted both an awareness of this cultural phenomenon and portrayed it as widely applauded. In presenting a story of Boston's "embracing the ace," the media reports also wound up pushing a view of widespread approval of the new Latino presence both in Fenway and society at large. This study sought to compare the impressions of widespread exuberance for Martinez and the Dominicans at the Park with actual interviews of those Anglos at the Park. It also sought to examine what motivated the Dominicans to attend in such large numbers and to so publicly celebrate their identity. The results showed that Anglos held a fractured view about Dominicans: a very positive view of Pedro Martinez as a Dominican but a fairly evenly split view of Dominicans in general. For their part, Dominicans were unconcerned with what Anglos thought and came to the game only to lend support to their Latino hero, as well as bask in his reflected glow. One methodological conclusion arrived at is that media content analysis must be cross checked against some sort of data and must not be assumed to accurately reflect social reality.
George Wilson, Dan Martin, James P. Morton and Graeme L. Close
The relative energy deficiency in sport (RED-S) syndrome was recently developed in recognition that male athletes display evidence of impaired physiological function that may be related to low energy availability ( Mountjoy et al., 2014 ). Jockeys are unique among professional athletes in that they


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Why Life Doesn't Stop After Retirement
This page may contain affiliate links, which means if you open such a link and purchase the item, I will receive a commission.
Many of us who are lucky enough to retire from work at the right age, will need to think about our options. For example, do you stop working full-time and head into part-time work? It's difficult for many of us to stop working altogether, so we carry on in some capacity. You can't blame those that do as they have probably dedicated their lives to their industry and profession.
Maybe you could move to a warmer climate or abroad. Just pick up your savings, your suitcases and go. Many people choose to do this because they want to live in paradise for the last leg of life. You can't blame them because it's about time you enjoyed some relaxing time in the sun.
Another option when you retire is to move to a community that is centered around other people that are at a similar time in their lives. In some of these communities, you get looked after by healthcare professionals who tend to all your medical needs and life is kept upbeat with lots of different activities.
Mentoring young professionals
So often we think of retirement as for old people. Well, retirement these days is at the age of around 70. But with modern diets, medicine and exercise routines, 70 isn't that old. There are politicians, and business owners around the world that are around this age and they still work a full-time schedule. So if you would like to continue working in your industry in some form, you can always help the younger generation.
Mentoring professionals are becoming more and more popular in every aspect. You get to teach the brash and bold youngsters about the industry, show them how certain things are done and or approached and help them become a success in their own lives. If this interests you, look to join up to an industry mentoring program. You might also be able to do this within your own company so check before you go elsewhere.
A shared retirement
Perhaps you would rather be surrounded by others in your situation as well. In that, a company with a great reputation such as Yukana is one of your top options. They have a private residence, giving you privacy and space for yourself. You'll be in a newly renovated unit and apartment, which is set inside beautiful gardens. There's always the freshest food which is made daily and you can call upon the staff to make you whatever kind of snack you need during the day. The whole concept is simulating your life outside of the retirement home so it's as normal as possible.
Travel the world
Now perhaps more than ever, it's your time to explore the world. You've worked hard all your life, saved up and invested wisely so now you can take it easy and go globetrotting. There are discounts for retirees all the time in all kinds of holiday agencies. Cruise holidays are particularly open to giving seniors discounts on their voyages and package plans.
Retirement doesn't mean retirement from life. You can continue to work in some kind of manner in your industry by mentoring the young that are coming through and help them to become successful. You could choose to live alongside and share time with other retirees. Or maybe it's finally time to travel the world now that you will have plenty of free time in retirement.
Have you thought about what you might want to do when you retire? I'm interested in hearing about it, so leave me a note in the comments.
31 Comments
No, it surely doesn't. I am already making my retirement plans and I want to see more of this world.
I think I will continue to travel even when I get old. Life is not over when you reach your retirement!
Exactly this! People typically go one of two ways in retirement, either they stay active and healthy, or they stay home day after day and regress. When we retire, we absolutely want to be doing more. Going on walks, travelling and more! WHy work all your life just to stop and not to anything when you have all the free time in the world?
Sarah M
We should embrace each stages of life. I am glad that you are enjoying your time after retirement.
There's still life after retirement, or maybe that's the start of life if you have worked so hard all your life before retiring. 🙂
Pool Operator Talk
I like the idea of mentorship. it is so rewarding to help a person on there way to what they wish to become and then to watch them soar from a distance, all the while knowing that you helped them to discover their own potential. That, and the traveling, Got to travel as well.
We want to travel a lot after retirement
Neil Alvin Nicerio
Same here. I'd love to travel the world too.
Life doesn't stop when you retired instead do the things that make your life happy 🙂 Nice post!
Mr. MFC | Morning Fresh Cent
I have been investing ever since I started my first job and savings even way before then. I want to retire earlier than the expected age so I can travel and learn how to play golf.
Martin - MPG TRAVEL TOURS
Life should not stop after retirement.. I just wish we could retire sooner to enjoy more of the more mobile years!
Polly Amora
Hi Jenn, I hope you're well. I've been investing on my retirement since I was in my 20s though I have no plans of retiring yet (I'm still in my 30's). 😀 I do a bit of mentoring myself and I plan to continue this after I retire. Traveling is definitely one of my plans too!
Vanessa Cast (@MomInBusiness_)
Life doesn't stop when you retired, it's just the start of another amazing stage in your life and with all the accumulated experience you can help others to figure it out. Keep these awesome articles coming!
Jaana McEntee
My plan is just to follow my dreams..Hope my bones are still good for this at this age 🙂
blair villanueva
Many of my colleagues are afraid of retirement, however I am excited for mine 😀
That's why I invest for my health so that I cant achieve that stage.
I love this article. I will definitely be passing alone to my grandparents.
This was a great post. I am still young, so I have many years ahead of me. I am sure when I retire, there will be different options. This is a great article to share with my Mother though. I can see her getting some great advice from reading this, thanks.
Annita Nikole
I'm glad you are discussing this. For some reason, some people I know see retirement as time to check out or basically be done with life. Then they just sit on the sofa, watch bad daytime TV and do nothing. Not what I want to do!
i would to share your to ma grandpa!! AND I know he loved to read this post !! thank you dear
Neil Alvin Nicerio
I agree with you. My mom and dad just retired and they are more busy now with their farm in the province.
I am still young havent thought of retirement yet, lol.
Exactly what we plan to do, mentor the young while traveling the world!
You, Me and Benny
this is wonderful!! thank you for sharing with us!
Life definitely does not have to stop after retirement. This is great proof of it, I wish to travel the world after retirement as well. — Lili
Definitely live somewhere peaceful closer to family, and lots of traveling and cruising.
CARMEN | Wellington World Travels
Actually I thought about mentoring/coaching/speaking in my retirement (or close to retirement). trying to figure out if I should take a course first, and on what topic.
Nursery Rhymes Girl
Hi HoppyMomma,
I always love to share your posts with my mom! Thank you! 🙂 I agree!
Sounds like you're having a great retirement! My dad retired years ago and now he's a professor.
Sanjota Purohit
Yes, retirement is not end of the life. Life just gets into different phase and one should have plans on how to spend it.
Great post
So we've been trying to find the best retirement plan! And talking what what do after retirement Thanks for sharing this!


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On 10th October 2020 the Lodge will be based near Stratford upon Avon.
The meeting will take place at
Alderson House
23 High Street
The subject of the meeting will be a demonstration of the examination of a candidate and his initiation as practised by the National Grand Lodge of Romania.
Much to the relief of the majority of our membership the demonstration will be in English
There willl be a practice at 11:00 a.m. A light buffet will be available at Noon and lodge will tyle at 1.00 p.m.
There will be a full weekend of activities
Click here for for full details and details of the superb hotel accomodation


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