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Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity.
The discoidin domain receptors (DDRs), DDR1 and DDR2, comprise a family of receptor tyrosine kinases (RTKs) that function as collagen receptors (1,2). The DDRs are the only RTKs that are activated by a component of the extracellular matrix. Several collagen types, both fibrillar and non-fibrillar types, activate the DDRs, with the two receptors displaying different specificities for certain collagen types (1–4). Structurally, the DDRs are characterized in their extracellular regions by the presence of a collagen-binding discoidin homology domain and a domain unique to the DDRs (stalk region) (Fig. 1). A transmembrane region is followed by a large cytoplasmic juxtamembrane domain, and, finally a C-terminal tyrosine kinase domain. Both DDRs form ligand-independent dimers on the cell surface (5). We have recently defined the mode of collagen recognition by the discoidin domain of DDR2 using X-ray crystallography (6).
The DDRs control fundamental cellular processes including cell proliferation, adhesion and migration (7,8). In addition, the DDRs regulate extracellular matrix remodeling by controlling matrix metalloproteinase expression and activity. Both receptors are associated with human diseases, including fibrotic disorders of the lung (DDR1), liver (DDR2) and kidney (DDR1); atherosclerosis (DDR1); osteoarthritis (DDR2); rheumatoid arthritis (DDR2); and several types of malignancies (7).
DDR2, like DDR1, is widely expressed in different tissues during development and in postnatal tissues (7). While DDR1 is mostly confined to epithelial cells and leukocytes, DDR2 is found on cells of mesenchymal origin. Mice lacking DDR2 exhibit dwarfism (9,10), indicating that DDR2 plays an important role in bone growth. Labrador et al. (9) showed that targeted deletion of Ddr2 in mice led to shortened long bones due to reduced chondrocyte proliferation. In another study, Kano et al. (10) characterized a spontaneously occurring, autosomal-recessive mutation in their mouse colony that led to a 150 kb deletion encompassing the first 17 exons of the Ddr2 gene. These latter mice, termed slie mutant mice, were infertile in addition to being dwarfed (10).
Recently, DDR2 function has been implicated in human skeletal growth. Three DDR2 gene missense mutations and one splice site mutation were each shown to cause spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications [SMED-SL (MIM 271665)] (11). SMED-SL is a rare, autosomal recessive, human growth disorder. Patients exhibit normal intelligence, disproportionate short stature, platyspondyly, abnormal epiphyses and metaphyses, narrow chest, shortening of the lower and upper limbs, short broad fingers and premature calcifications (12,13).
DDR2 is a member of the subfamily of RTKs that also include ROR2. Mutations in the ROR2 gene have been shown to be the underlying cause of the recessive form of Robinow Syndrome, a skeletal dysplasia characterized by short stature, limb shortening, genital hypoplasia and craniofacial abnormalities (14). We have recently shown that all the missense pathogenic mutations in ROR2 are retained in the endoplasmic reticulum (ER) and are substrates for the ER quality control system, demonstrating that ER retention is the underlying cellular mechanism of this condition (15,16) (Ali et al., unpublished data). Given the phylogenetic relatedness of DDR2 to ROR2, we reasoned that most SMED-SL causing missense mutations in DDR2 are likely to result in the retention of DDR2 protein in the ER. In addition, patients with SMED-SL phenotype have been reported in the United Arab Emirates (UAE), but the molecular defects responsible for their symptoms have not been established (17) (Al-Gazali et al., unpublished data). In this article, we report the evaluation of two families from the UAE with SMED-SL and the identification of two mutations including a novel missense mutation in DDR2. In addition, we report the elucidation of the cellular and biochemical mechanisms of all the missense mutations so far found in patients with this condition. While the previously established SMED-SL DDR2 mutations indeed caused ER retention of mutated DDR2, we demonstrate that the novel SMED-SL mutation (p.E113K) resulted in a ligand-binding defect. These latter data fit well with our recent structural data on the ligand-binding site of DDR2 (6). Furthermore, our data demonstrate further the widespread involvement of the ER quality control systems in the cellular mechanisms of numerous secretroy pathway targeted proteins (15,18–20).
Two consanguineous families with SMED-SL from the UAE have been evaluated (see Supplementary Material, Fig. S1 for the pedigrees). The first is a Pakistani family with two affected children. The clinical details of the affected children are described in detail below. The second family is of Egyptian background with two affected children and was described in detail before (17). The family was found to have the same mutation, c.2254C>T (p.R752C), as found in the Palestinian families described by Bargal et al. (11). The severity of the clinical features in this family, particularly the degree of chondral calcifications, was also similar to the clinical features of the families described by Bargal et al. (11). Both children in this family died, one at the age of 13 years due to respiratory complications and the other one died suddenly at 8 years of age due to cord compression.
This male child was the product of normal pregnancy. Delivery at term was by lower section Cesarean section. Prenatal ultrasound showed short limbs and short stubby hands. His birth weight was 2700 gm (5%) and length was 47 cm (10%). At birth, he was noted to have in addition to the short limbs and short hands, bowing of the lower limbs. The diagnosis of achondroplasia was suggested. During the first year of life and the early childhood years, he had repeated chest infections which were not severe enough to require hospital admissions. These attacks got better with time and stopped when he was 6 years old. His early developmental milestones were normal. He was operated on both legs to correct the bowing. He was evaluated by us at the age of 9.5 years. His weight was 27.5 kg (10%) and height was 99 cm ( < 3%, −6 SD). He had dysmorphic features and shortening of all limbs with very short puffy hands (Table 1). He was attending normal school at the appropriate grade and was top of the class. Neurological examination was normal. Skeletal survey revealed changes typical of SMED-SL including short and broad long bones with irregular flared metaphyses of the proximal and distal ends and genu varum (Table 1). There was a small thoracic diameter with short and broad ribs with very few calcifications at the costo-chondral margin (Fig. 2A).
Brother of case 1, the product of normal pregnancy and term delivery by LCS. His weight was 2.9 kg (10%) and length was 49 cm (25%). Short limbs and short hands were diagnosed prenatally. He was found to have bowing of limbs in addition to the shortening. He had, similar to his brother, repeated respiratory infections not requiring hospital admissions. He was evaluated by us at 8 years of age. His weight was 17.6 kg (<5%) and height was 94 cm (<3%, −6 SD). He had similar dysmorphic features as his brother but his chest appeared much narrower than his brother's. He was attending normal school at the appropriate grade and was top of the class. Neurological examination was normal. Skeletal survey revealed similar changes to his brother (Fig. 2B and Table 1).
The genomic DNA from SMED-SL affected individuals and their parents (Supplementary Material, Fig. S1) was isolated and all the exons and flanking regions of the DDR2 gene were amplified by PCR as described in the Materials and Methods. Direct DNA sequencing revealed two homozygous missense mutations: c.337G>A (p.E113K) for family 1 and c.2254C>T (p.R752C) for family 2 (Supplementary Material, Fig. S2). The parents in each case were heterozygous for the mutation found in their affected offspring. The nucleotide change c.337G>A has not been found in 100 ethnically matched controls. In addition, the p.E113K mutation is novel and is located in the ligand-binding domain of the DDR2 protein (Fig. 1). The amino acid change is physiochemically significant (a change of the negatively charged glutamic acid to the positively charged lysine) and affects an important residue within the discoidin domain, the DDR2 ligand-binding domain (6). Furthermore, this residue is conserved in the DDR2 protein from different species including zebrafish, mouse and human (Supplementary Material, Fig. S3). These pieces of data together with the functional assays described below confirm the pathogenicity of this amino acid substitution. On the other hand, the mutation (p.R752C) found in the family of Egyptian origin is a recurring mutation and has been recently found as a founder mutation in five Palestinian families from Jerusalem (11). This mutation is located in the DDR2 intracellular domain, as shown in Figure 1.
We hypothesized that most of the missense mutations in DDR2 (Fig. 1) will result in the retention of the mutated protein in the ER. This assumption was based on the relatedness of DDR2 to ROR2 and our findings that all the pathogenic missense mutations in ROR2 have been shown to be retained in the ER (15,16). Therefore, we generated all the missense mutations found in SMED-SL patients including those reported by Bargal et al. (11) in a mammalian expression system using site-directed mutagenesis and expressed the proteins in HeLa cells as described in the Materials and Methods. Confocal microscopy imaging revealed that the C-terminally HA-tagged wild-type DDR2 protein was primarily localized to the cell surface and colocalized with the plasma membrane marker GFP-h-Ras (Fig. 3A–C). We also observed some intracellular staining of the wild-type protein which presumably represents the newly synthesized protein that is in transit to the cell surface. Similarly, the p.E113K mutant was located to the periphery of the cell and colocalized with GFP-h-Ras (Fig. 3D–F). In contrast, confocal imaging of p.T713I, p.I726R and p.R752C mutants revealed an intracellular and reticular localization of the proteins, which is clearly different from that of the wild-type protein, with no colocalization with the plasma membrane marker (Fig. 3G–O). This intracellular staining is reminiscent of ER staining and we therefore co-stained cells expressing the wild-type and mutant proteins with a well established ER marker (calnexin). As shown in Figure 4A–F, the wild-type protein and p.E113K mutant showed distinct and different localizations from that of calnexin, confirming plasma membrane locations. On the other hand, the three intracellular missense mutants (p.T713I, p.I726R and p.R752C) showed a high degree of colocalization with the ER marker (Fig. 4G–O) indicating ER localization. Further biochemical studies (the following sections) confirmed the ER localization of the three proteins.
The first step in collagen-induced DDR2 transmembrane signaling is activation of DDR2, which manifests itself in autophosphorylation of cytoplasmic tyrosine residues (1,2). To analyze whether SMED-SL disease mutants could be activated by binding to collagen, we transiently expressed the mutants and wild-type DDR2 in HEK293 cells. HEK293 cells were chosen for these experiments, as DDR autophosphorylation in these cells can be easily monitored by western blotting of cell lysates (1,21). In transfected HEK293 cells, wild-type DDR2 is usually observed as a mixture of three (sometimes four) molecular weight forms between ~125 and 130 kDa (5,21,22), with the upper forms representing complex glycosylated mature forms. Unlike wild-type DDR2, however, for which the expected molecular weight forms were detected (Fig. 5A), the SMED-SL mutants p.T713I, p.I726R and p.R752C were detected predominantly as the lowest molecular weight species (Fig. 5A), with mutants p.T713I and p.I726R displaying a small amount of the middle forms. None of these mutants displayed the highest molecular weight form of DDR2. In addition, Endo H treatment of cell lysates expressing wild-type or mutant DDR2 showed that the predominant forms of T713I-DDR2, I726R-DDR2 and R752C-DDR2 were sensitive to Endo H digestion, as was the lowest molecular weight form of wild-type DDR2 (Fig. 5A), suggesting that these molecular weight species represent immature biosynthetic precursors of DDR2. This is in agreement with the protein localization data in HeLa cells, presented in Figures 3 and and4.4. The predominant forms of wild-type DDR2, however, were the upper molecular weight species, which were largely absent in the mutant receptors. These forms were Endo H resistant and thus represent the complex glycosylated mature forms of DDR2.
As expected, a strong collagen-induced phosphorylation signal was observed for the upper forms of wild-type DDR2 (Fig. 5B, upper blot lanes 1 and 2). In keeping with the lack of plasma membrane localization of T713I-DDR2, I726R-DDR2 and R752C-DDR2, no collagen I-induced phosphorylation could be detected for any of these mutant receptors (Fig. 5B, upper blot lanes 3–7).
In contrast to mutants T713I-DDR2, I726R-DDR2 and R752C-DDR2, which displayed predominant ER localization, E113K-DDR2 was found on the plasma membrane in HeLa cells (Figs 3 and and4).4). Transient expression of E113K-DDR2 in HEK293 cells resulted in the appearance of the same molecular weight forms as observed for wild-type DDR2, with only the lowest molecular weight form showing sensitivity to Endo H treatment (Fig. 6A). The upper species of E113K-DDR2 are thus complex glycosylated and likely represent mature forms of the receptor. Unlike wild-type DDR2, however, E113K-DDR2 could not be activated by collagen I. Even at high concentrations of collagen I (50 µg/ml), no collagen I-induced phosphorylation signal was detected (Fig. 6B), indicating that plasma-membrane localized E113K-DDR2 is defective in transmembrane signaling.
We recently identified glutamate 113 as one of the major collagen-binding residues in DDR2 (6). Glu113 forms a salt bridge with Arg105 in the DDR2 discoidin domain. Substitution of the glutamate by lysine would disrupt the salt bridge and is expected to negatively impact on ligand binding by E113K-DDR2. To analyze the effect of p.E113K substitution on DDR2 collagen binding, we created a soluble ectodomain construct with the p.E113K mutation and expressed it in HEK293-EBNA cells. This construct was secreted at similar levels to the corresponding wild-type protein, indicating that the mutation did not result in gross abnormalities of protein folding. The mutant protein failed to bind to collagens types I and II in an established solid-phase binding assay, displaying similar low-level binding to a DDR2 variant harboring the p.W52A mutation (Fig. 7B and C). We and others recently showed that Trp52, like Glu113, plays a critical role in collagen recognition by DDR2, and that substitution of Trp52 by Ala abolishes collagen I and collagen II binding (6,23). We conclude that substitution of Glu113 by Lys leads to loss of ligand binding, which in turn is responsible for lack of collagen-induced signaling of full-length E113K-DDR2.
Here we report the clinical features of two siblings diagnosed with SMED-SL and a novel DDR2 missense mutation identified in these patients. This mutation (p.E113K) is located in the extracellular discoidin domain and affects a surface-exposed residue that was previously identified as an important amino acid in the collagen-binding site of DDR2 (6,23). Additionally, we identified another mutation (p.R752C) in an Egyptian family with SMED-SL. This DDR2 mutation, localized in exon 17 of the DDR2 gene, which encodes part of the intracellular tyrosine kinase domain, is a recurring mutation previously found in five Arab families from Jerusalem affected with SMED-SL (11).
Here we demonstrate the effects of all known SMED-SL causing missense mutations on DDR2. Our cellular and biochemical analysis of these four DDR2 mutations showed that these fall into two classes: all previously identified missense mutations cause a trafficking defect with the affected proteins mis-localizing to the ER, while the novel p.E113K-DDR2 mutation is localized in the cell similar to wild-type DDR2 but is defective in transmembrane signaling due to a ligand-binding defect. A previous report identified mutations in the DDR2 gene as causative genetic defects for SMED-SL (11), but did not illuminate the mechanism of disease. Bargal et al. (11) speculated on the possible biochemical consequences of the disease mutations p.T713I, p.I726R and p.R752C. Because the affected amino acids are located in the active site of the DDR2 kinase, it was proposed that the substitutions may influence the stability of the activation loop, ATP binding affinity or substrate specificity, respectively (11). The possibility that protein folding may be affected was not considered. DDR2 is a member of the RTK family of plasma membrane receptors and is phylogenetically related to ROR2 (24). Missense mutations in the ROR2 gene were recently shown to cause retention of the mutated proteins in the ER as a result of the protein folding quality control within this organelle named ER-Associated Protein Degradation (ERAD) (15,16). ERAD ensures that only properly folded proteins and fully assembled complexes are allowed to exit the ER to their final destinations. Proteins that fail to attain the required functional native conformations due to mutations or failure to acquire the needed posttranslational modifications, such as proteolytic cleavage of signal peptides, glycosylation and GPI-anchoring, are retained in the ER for a short period of time but then get targeted for re-translocation to the cytoplasm and degradation by the ubiquitin/proteasome systems (25–29).
In this report, we show that three out of four missense mutations (p.T713I, p.I726R and p.R752C) found in SMED-SL patients result in the retention of the affected proteins in the ER. ER retention of the three mutants is strongly supported by the confocal images presented in Figures 3 and and44 and the biochemical findings on the sensitivity to Endo H treatment presented in Figure 5. We noticed lower levels of expression of the ER-retained mutant proteins in HEK293 cells, compared with wild-type DDR2, as can be seen in Figure 5B. These low steady-state levels of the mutant proteins may be due to ERAD degradation. Additionally, our findings add another genetic condition to the 60 or so known to be caused by ERAD (15,18,19,30).
Unlike the ER-retained mutant DDR2 receptors, E113K-DDR2 was found at the cell surface, similar to wild-type DDR2 (Fig. 3A–F), indicating a different disease-causing mechanism. Glu113 is located in the DDR2 extracellular discoidin domain. The DDR2 discoidin domain adopts a β-barrel structure consisting of eight β-strands (23). At the top of the barrel, five protruding loops create a trench that forms the collagen-binding site (6,23). Glu113, which forms a salt bridge with Arg105, is one of several key residues that contact collagen (6). Figure 8, which shows a cartoon drawing of the DDR2 discoidin domain bound to a collagen peptide, illustrates how Glu113 is involved in the DDR2-collagen interaction. Data presented in Figure 7 demonstrate that E113K-DDR2 had lost most of the collagen-binding activity. This result agrees well with the previous observation that mutation of E113Q reduced collagen-binding to ~10% of wild-type DDR2 (23). Thus, Glu113 is one of the main DDR2 residues contributing to collagen-binding. It is also noted that Glu113 and Arg105 are conserved in DDR1, further underscoring the importance of the Glu113-Arg105 salt bridge in the DDR-collagen interaction. This is the first report on the clinical relevance of substitutions at this site.
We noted some variations in the phenotype between the two families we are reporting here. For example, the clinical phenotype and the degree of chondral calcification of the second family (mutation p.R752C) were very severe (17). Both children had extensive calcifications which appeared by 1 years of age and were very progressive and widespread involving almost all cartilages. In addition, both children had repeated respiratory infections requiring hospital admissions and leading to death in the older child. The younger sib had in addition symptoms of cord compression, which led to his sudden death. The phenotype in the affected children from the first family (mutation p.E113K), on the other hand, was less severe with very few mild respiratory infections not requiring hospital admissions. In addition, the chondral calcification was limited and much less severe than in the p.R752C family. This variability might be due to retention of some residual activity in the p.E113K mutant compared with the p.R752C mutant. Alternatively, cells expressing the ER-retained mutated protein (p. R752C) may experience ER stress, which would not be experienced by cells expressing the correctly targeted p.E113K protein. However, the clinical significance of these presumed differences in ER stress responses are unknown at this stage.
How does DDR2 control bone growth? Very little is known about the role of DDR2 in bone growth. DDR2 is expressed on chondrocytes and its elimination in the mouse leads to suppression of chondrocyte cell proliferation (9). It is assumed that the interaction of DDR2 with collagen II in the proliferative zone of the growth plate is responsible for controlling chondrocyte proliferation. DDR2 is also a receptor for another collagen type with relevance to bone growth, collagen X (4). While collagen II is found in all types of cartilage, collagen X expression is restricted to the hypertrophic zone of the growth plate. We show here that E113K-DDR2 is unable to interact with collagen II (Fig. 7C). Further studies are required to establish whether the mutant protein is also defective in binding to collagen X.
In mice with targeted DDR2 deletion, the growth plates were observed to be shortened (9). In the only available histological study of a chostrochondral junction from a SMED-SL patient, extremely abnormal cartilage was found (12). The growth plate was found to lack proliferating chondrocyte columns. Chondrocytes were small and sparse in the resting zone, often surrounded by a 'ring-like' amorphous matrix, which in turn could be surrounded by a more densely packed structure of fibrillar collagen. Clustering of chondrocytes within lacunae was also observed. Taken together with our data, it can be speculated that in SMED-SL patients, lack of DDR2 interaction with collagen II results in impaired DDR2 signaling, leading to severe suppression of chondrocyte proliferation, which may be responsible for poor linear bone growth. Further morphological studies on patient samples with known DDR2 mutations would be required to confirm this hypothesis.
In summary, we report a novel mutation in the DDR2 gene in patients affected with SMED-SL and elucidate the cellular and biochemical mechanisms underlying all reported SMED-SL disease mutations. Two different mechanisms, failure to reach the plasma membrane and impaired ligand binding, were found to result in the growth disorder. This study supports the notion that DDR2 signaling is important in human development and that the interaction of DDR2 with collagen is essential for proper skeletal growth.
The research was approved by Al-Ain Medical District Human Research Ethics Committee and informed consent was obtained from the parents. Patients were evaluated by a clinical geneticist and their radiography imaging were also evaluated.
Bovine serum albumin was obtained from Fisher Scientific (Loughborough, UK). Collagen I (acid-soluble from rat tail; C-7661) was purchased from Sigma (Poole, UK). Bovine collagen II, ELISA grade, was from Chondrex Inc. (Redmond, WA, USA). Endoglycosidase H (Endo Hf) was purchased from New England Biolabs (Hertfordshire, UK). The antibodies and their sources were as follows: antibodies for immunofluorescence: mouse anti-HA-Tag monoclonal antibody (dilution 1:200; Cell Signaling Technology), rabbit anti-calnexin polyclonal antibody (dilution 1:500; StressGen Biotechnologies), Alexa Fluor 568-goat anti-mouse IgG (dilution 1:200; Molecular Probes), Alexa Fluor 488-goat anti-rabbit IgG (dilution 1:200; Molecular Probes). Antibodies for western blotting: goat-anti-DDR2 (AF2538) was from R&D Systems (Abingdon, UK); mouse anti-phosphotyrosine, clone 4G10, from Upstate Biotechnology (Lake Placid, NY, USA); peroxidase-conjugated goat anti-human Fc from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Secondary antibodies for western blotting were as follows: rabbit-anti-goat Ig-horseradish peroxidase (Zymed Laboratories Inc., San Francisco, CA, USA); sheep-anti-mouse Ig-horseradish peroxidase (Amersham Biosciences UK, Chalfont St Giles, UK).
Genomic DNA was isolated from the peripheral blood leukocytes using Flexigene DNA Kit (Qiagen, GmbH) according to the manufacturer's instructions. DNA was dissolved in TE (Tris-EDTA) buffer and quantitated by spectrophotometry.
Exon-specific primers for DDR2 gene (Supplementary Material, Table S1) were designed using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/) and used to amplify the coding exons 3–18 and their flanking regions. Amplification was carried out using 1U of Taq polymerase (Qiagen, GmbH), 0.2 mm of each of the dNTPs, 1× PCR reaction buffer, 1.5 mm MgCl2, 5 pmol of each forward and reverse primers and 50 ng of genomic DNA template in 25 µl reactions. The PCR was performed by an initial 5 min denaturation at 95°C followed by 40 cycles of 95°C for 30 s, 58°C–62°C for 35 s and 72°C for 45 s with a final elongation at 72°C for 10 min.
Sequencing was performed using the dideoxy method by fluorescent automated sequencing on the ABI 3130xl genetic analyzer (Applied Biosystems, Foster City, CA, USA). The sequence data were analysed using Sequencing analysis software v5.3 (ABI, Foster City, CA, USA). Mutations are designated on the coding DDR2 sequence with reference to GenBank accession number NM_001014796.1 with the A of the initiation codon ATG as number +1.
The HA-tagged version of the wild-type DDR2 was generated by sequential site-directed mutagenesis to introduce the nine amino acids of the HA tag (YPYDVPDYA) in two cycles using the pcDNA3.1-DDR2 plasmid (21) as a template. In the first cycle, we introduced five amino acids (YPYDV) of the HA tag prior to the stop codon of the DDR2 gene using the following mutagenesis primers.
HA1-F: CCACAAGGCGACGAGTACCCATACGATGTTTGATGCTGTCAGTGCC and HA1-R: GGCACTGACAGCATCAAACATCGTATGGGTACTCGTCGCCTTGTGG. In the second cycle, the above construct was used as a template and the remaining four amino acids (PDYA) of the HA tag were introduced using the following mutagenesis primers. HA2-F: TACCCATACGATGTTCCAGATTACGCTTGATGCTGTCAGTGCC and HA2-R: GGCACTGACAGCATCAAGCGTAATCTGGAACATCGTATGGGTA.
The missense mutants were introduced by QuickChange site-directed mutagenesis using Turbo Pfu polymerase (Stratagene) and using the untagged pcDNA3.1-DDR2 (21) and the C-terminally HA-tagged pcDNA3.1DDR2 as templates. The primers for the site-directed mutagenesis to introduce the missense mutations were as follows. DDR2-E113K-F: GAGGTCATGGCATCAAGTTTGCCCCCATG; DDR2-E113K-R: CATGGGGGCAAACTTGATGCCATGACCTC; DDR2-T713I-F: CGAGATCTGGCCATTCGAAACTGTTTAG; DDR2-T713I-R: CTAAACAGTTTCGAATGGCCAGATCTCG; DDR2-I726-F: TACACAATCAAGAGAGCTGACTTTGGA; DDR2-I726R-R: TCCAAAGTCAGCTCTCTTGATTGTGTA; DDR2-R752 C-F: GTGCTCCCTATCTGCTGGATGTCTTGG; DDR2-R752C-R: CCAAGACATCCAGCAGATAGGGAGCAC.
cDNA encoding E113K-DDR2 ectodomain (Lys22-Thr398) was obtained by PCR amplification and cloned into a modified pCEP-Pu vector containing a human Fc sequence, as described for the respective wild-type and W52A-DDR2 constructs (6).
All constructs were confirmed by direct DNA sequencing of the purified plasmids.
HeLa cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum, 2 mm l-glutamine and 100 U/ml penicillin/streptomycin at 37°C with 10% CO2. For transfection, cells were grown in 24 well tissue culture plates. Transfection was performed using the liposomal transfection reagent FuGENE 6 (Roche Biochemicals), according to manufacturer's instructions. A mixture of 1 µg of DDR2 wild-type or mutant plasmid DNA and 4 µl of FuGENE 6 in 94 µl of OPTIMEM I medium was applied to each well of the cultured cells. Twenty-four hours after transfection, the cells were fixed and processed for microscopy.
Human embryonic kidney (HEK) 293 cells (ATCC, Manassas, VA, USA) and HEK293-EBNA cells (Invitrogen Ltd., Paisley, UK) were cultured in Dulbecco's modified Eagle's medium/F12 medium (Invitrogen) supplemented with 10% fetal bovine serum, penicillin (10 U/ml) and streptomycin (100 µg/ml) at 37°C with 5% CO2. For transfection, cells were grown in 24 well tissue culture plates (autophosphorylation assays) or 12 well tissue culture plates (deglycosylation assays) and were transfected with 1 or 2 µg, respectively, of DDR2 wild-type or mutant plasmid DNA using calcium phosphate precipitation.
For immunofluorescence, cover slip-grown HeLa cells were washed with phosphate-buffered saline (PBS), fixed by methanol at −20°C for 4 min. Fixed cells were washed in PBS three times then incubated in blocking solution (1% BSA in PBS) for 30 min at room temperature. The fixed cells were then incubated at room temperature for 1 h with either mouse monoclonal anti-HA antibody alone or co-stained with both mouse monoclonal anti-HA antibody and rabbit polyclonal anti-calnexin antibody. After washing with PBS, the cells were incubated with the appropriate secondary antibodies for 1 h at room temperature, washed several times with PBS and mounted in immuno fluor medium (ICN Biomedicals). Data were acquired using Leica DM-IRBE or Nikon confocal microscopes. For presentation, images were pseudocolored as either red (DDR2) or green (calnexin and EGFP-H-Ras), contrast enhanced and overlayed using Adobe Photoshop® (Adobe Inc.). All images presented are single sections in the z-plane.
Forty hours after transfection, HEK293 cells were lysed in 1% Nonidet P-40, 150 mm NaCl, 50 mm Tris, pH 7.4, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin. Twenty microliter of each cell lysate was denatured in 1X glycoprotein denaturing buffer (0.5% SDS and 1% β-mercaptoethanol) for 5 min at 100°C. The denatured lysates were then split into two equal aliquots which were incubated for 3 h at 37°C in the presence or absence of 10 U of Endoglycosidase H. Samples were then resolved on 7.5% SDS–PAGE followed by blotting onto nitrocellulose membranes and probing with anti-DDR2 antibodies, as described below.
The assay was performed as described previously in detail (21). Twenty-four hours after transfection, HEK293 cells were incubated with serum-free medium for 16 h. Cells were then stimulated with collagen I (2–50 µg/ml) for 90 min at 37°C. Cells were lysed in 1% Nonidet P-40, 150 mm NaCl, 50 mm Tris, pH 7.4, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 1 mm sodium orthovanadate and 5 mm NaF. Aliquots of the lysates were analyzed by SDS–PAGE followed by blotting onto nitrocellulose membranes. The duplicate blots were probed with either anti-phosphotyrosine monoclonal antibody or anti-DDR2 antibodies followed by corresponding horseradish peroxidise-conjugated secondary antibodies. Detection was performed using Enhanced Chemiluminescence Plus (Amersham Biosciences) and an Ettan DIGE Imager (GE Healthcare Biosciences).
The production and purification of recombinant DDR2 proteins was essentially as previously described (6,21). The recombinant Fc-tagged wild-type and E113K-DDR2 extracellular domain proteins were isolated from episomally transfected HEK293-EBNA cells and purified by affinity chromatography on a HiTrap rProtein A column (17-5079-01, GE Healthcare Biosciences, Uppsala, SW, USA) using an ÄKTA™ Purifier (GE Healthcare Biosciences). Both proteins were obtained in similar yields (4.0 mg per liter of conditioned medium for wild-type DDR2, 3.5 mg per liter of conditioned medium for E113K-DDR2).
The assay procedure has been described previously in detail (21). Briefly, rat tail collagen I or bovine collagen II was diluted in 50 mm Tris pH 7.5, 100 mm NaCl to 10 µg/ml and coated onto Maxisorp 96-well plates (Fisher Scientific) overnight at room temperature. Wells were then blocked for 1 h at room temperature with 1 mg/ml bovine serum albumin in phosphate-buffered saline plus 0.05% Tween-20. Recombinant Fc-tagged wild-type or mutant DDR2 extracellular domain proteins, diluted in incubation buffer (0.5 mg/ml bovine serum albumin in phosphate-buffered saline plus 0.05% Tween 20), were added for 3 h at room temperature. Wells were then washed six times with incubation buffer. Bound Fc-tagged DDR2 proteins were then detected with goat anti-human Fc coupled to horseradish peroxidase (1:3333 dilution), added for 1 h at room temperature followed by six washes with incubation buffer. A color reaction was subsequently performed using o-phenylenediamine dihydrochloride (Sigma), added for 3–5 min. The reaction was stopped with 3 M H2SO4, and plates were read at 492 nm on a 96-well plate reader.
This work was supported by the Dubai Harvard Foundation for Medical Research (grant number 2008-04 to B.R.A. and L.A.); and the UK Medical Research Council (G0701121 to B.L.). Funding to pay the Open Access publication charges for this article was provided by MRC grant G0701121.
We would like to thank the patients and their families for taking part in this study. We are especially grateful to Prof Erhard Hohenester for providing Figure 8. We also like to thank Dr Alistair Hume for his help and comments and Mr Saeed Tariq for his help with confocal imaging.
Conflict of Interest statement. The authors declare that there are no conflicts of interest in this study.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Updated by CasinosAvenue on 03/11/2016See Infos & Directions See the games
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"The "Florins Church" on Florinsmarkt Square" Top 5 Page for this destination Florinskirche Tip by wadekorzan
Florinskirche, Koblenz: 4 reviews and 13 photos
Just around the corner from the Deutsche Kaiser, you will find the Florinsmarkt Square, 1 of the many many beautiful squares in Koblenz. This square was not damaged by the bombing of World War II, but was rebuilt after 1689 when Louis XIV of France invaded and destroyed the city.
The Florins Church was originally built in the 12th century. It was rebuilt after being partially desroyed by the French under Louis XIV, the "Sun King", and when napolean took over the Rhineland, he turned the church into a stable for his horses. After being a stable for several years, he decided to give it back to the city as a present. being a bit smelly and dirty, the Catholics (Koblenz and the Rhineland are predominantly Catholic) decided they did not want the church back and they gave it to the Protestants. it has been a Protestant church ever since! It is 1 of the 3 main churches in the Old town, but is the only Protestant church of the three..
Website: http://www.stiftung-florinskirche.de
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super sixes
Who doesn't love a list? These are our pick of the best events from beginner triathlons to the toughest 10k's.
Run it, jog it, or walk this new endurance challenge along Britain's longest waterway. It's 100k, 50k, or 25k on the Grand Union Canal towpath, a 'hidden green corridor' that winds its way from the heart of London out towards the Chilterns. The scenery will surprise you â it's the Capital's best kept secret; the Challenge will inspire you – it's London's great escape!
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36 Trowbridge Lane,Clementon NJ, 08021
Sold Price
3 beds , 2 Full/1 Half baths
1,408 sq. ft
Status: Sold
Berkshire Hathaway HomeServices Fox & Roach, REALTORS
(866) 486-5636
Last Updated: 2/24/2017 10:07 PM
Description for 36 Trowbridge Lane Clementon, NJ 08021
Two Story townhouse, located in Trowbridge community, with three bedrooms and two and a half bathrooms. First floor offers living room with fireplace, kitchen with ample cabinets space, dining area, and powder room. Second floor features master bedroom with full bathroom, two additional bedroom and full bathroom. Also has a full basement for all your storage needs. Go see it today!! Sold as is buyer responsible for any/all certifications and or repairs, township or lender. This property was built prior to 1978 and lead-based paint potentially exists.
Listing Information
Electric Water Heater, Public Water Service
Public Sewer
School Information
Room Information
First Floor Laundry
Interior Features
Central Air
Full, Unfinished Basement
Exterior / Lot Features
Assigned Parking Space
Sidewalks, Street Lights, Front Yard, Back Yard
Financial Considerations
Inside AccessTM
Current market conditions for 36 Trowbridge Lane
Active Market Trends
Source:MLS DATA
Median list price$99,450
Days on market175
Properties for sale94
Sold Market Trends
Source:MLS DATA
Median sold price$57,500
Sales to list price9,180.0%
Properties sold49
Median sq ft price$39
Arrows indicate 90 day trend
Listing Price History for 36 Trowbridge Lane
Clementon Real Estate
Clementon is located in New Jersey. Clementon, New Jersey 08021 has a population of 5,008.
The median household income in Clementon, New Jersey 08021 is $45,667. The median household income for the surrounding county is $62,320 compared to the national median of $53,046. The median age of people living in Clementon 08021 is 40.1 years.
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Listing Courtesy of Re/Max of Barrington
Listing information
- Beds: 5
- Total Rooms: 10
- Parking Type Description: Attached 3 cars
- Total Square Footage: 4,000 ($174.98 per sq. foot)
- Estimated Annual Taxes: $2,130
- High School: Crystal Lake Central High School
- Style: 2-Story
- Baths: 4 (Full: 3 3/4: 0 1/2: 1 Other: 0)
- Stories: 0
- New Construction: Yes
- Lot Area: 0.79
- Association Fee: $250
- Middle School: Richard F Bernotas Middle School
- Year Built: 2016
- Status: Active
- Fireplaces: 1
- Foreclosure: No
- Lot Size: 255X200X75X225
- County: Mc Henry
- Elementary School: West Elementary School
Additional Rooms 5th Bedroom, Breakfast Room, Mud Room, Office
Age NEW Proposed Construction
Air Conditioning Central Air
Appliances All Stainless Steel Kitchen Appliances, Dishwasher, High End Refrigerator, Microwave, Oven-Double
Area Crystal Lake / Lakewood / Prairie Grove
Area Amenities Pond/Lake
Assessment Includes Common Insurance
Basement Full
Basement Bathrooms (Y/N) No
Basement Description Bathroom Rough-In, Unfinished
Bath Amenities Double Sink, Separate Shower, Whirlpool
Built Before 1978 (Y/N) No
Dining Room Separate
Disability Access and/or Equipped Yes
Disability Access/Equipment Details 2+ Access Exits J, 32 inch or more wide doors, 36 inch or more wide halls, Doors w/Lever Handles, Doors-Pocket, Doors-Swing-In, Entry Slope less than 1 foot
Driveway Asphalt
Electricity 200+ Amp Service
Energy/Green Building Rating Source Other
Equipment Ceiling Fan, CO Detectors, Humidifier, Radon Mitigation System(s), Sump Pump
Exterior Building Type Brick, Cedar, Other
Exterior Property Features Patio
Fireplace Details Gas Logs, Gas Starter, Wood Burning
Fireplace Location Family Room
Foundation Concrete
Garage Details 7 Foot or more high garage door, Garage Door Opener(s)
Garage On-Site Yes
Garage Ownership Owned
Garage Type Attached
General Information Commuter Train, School Bus Service
Heat/Fuel Forced Air, Gas
Interior Property Features 2nd Floor Laundry, Hardwood Floors
Is Parking Included in Price Yes
Kitchen Type Eating Area-Table Space, Island, Pantry-Walk-in
Lot Size .50-.99 Acre
Master Association Fee No
Master Bedroom Bath Full
Other Bedrooms (Below Grade) - 0, Model - The "Heather" Expanded, Special Service Area - T, Tax Year - 2014, Total Full/Half Baths - 3.1, Township - Grafton
Ownership Fee Simple w/ HO Assn.
Parking Garage
Possession Specific Date
Property Type Detached Single
Recent Rehab (Y/N) No
Roof Type Asphalt/Glass (Shingles)
Sewer Septic-Private
Special Assessments U
Style Of House Traditional
Tax Exemptions None
Type Detached 2 Stories
Virtual Tour None
Water Well-Private
Waterfront (Y/N) No
5th Bdrm Main Level 13X13
Breakfast Rm Main Level 12X12
Dining Room Main Level 15X13
Family Room Main Level 17X17
Kitchen Main Level 19X17
Mud Main Level 10X7
Office Main Level 14X13
Bedroom 2 2nd Level 14X13
Bedroom 3 2nd Level 13X13
Bedroom 4 2nd Level 13X13
Laundry Room 2nd Level 10X8
Master Bedroom 2nd Level 17X17
Listing Courtesy of Re/Max of Barrington


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Friday night. Station Bar & Lounge. I'll be relaxed...read more
Page 2 of 15
SHOWS, yeah I got em, in South Eastern Queensland. In two weeks time I am performing at The Bison Bar in Nambour, solo and intimate. That means that I get undressed while I play. I am trying not to do too many solo shows at the moment, but for the Sunshine Coast I make special […]..read more
Thursday 18th February – Union Hotel Newtown, two sets with band + Wifey About to head to the airport to pick up the band who are flying in from Brisbane and Melbourne because I am independently wealthy and can afford such indulgences. We're gonna have a quick rehearsal at Troy Horse and then head to […]..read more
Moore's Law in full effect, things have changed out of sight since my last post. Everything is different. I'm just not feeling it as far as writing long newsletters lately. Could be the heat, could be that I'm too busy doing things, could be the PS4. Anyway I have a little tour of Tasmania with […]..read more


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no power to pcm port
tried to read codes but have no power to pcm port i cant find a fuse for it had some one tell me iy uses the cig lighter fuse which is blown is this correct
9 Answers
As you can see (click on it to enlarge) pcm is upper, second from right, CIG is elsewhere, lower in middle
is there another pcm port to read codes besides the one under steering wheel
But when you say pcm port, that's not where you plug scanner in to retrieve codes. You use the OBD port, a completely different port. Is that what you meant? PCM is Power Control Module
No, that would be it. The OBD port under the steering wheel, this is not your car but is very similar
Actually Powertrain Control Module ...and one time I tried to scan a car and thought the same thing, no power to OBD port but the batteries in my scanner were dead
that what i mean the obd port is there another one?
Nope, only one
was driving on hyway car lost acceleration sounded like a backfire but not threw exhaust died will not start will turn over got spark after cranking gets a strong fuel smell tryed to read codes no power to obd port
Yikes, That makes me think a timing issue
Your Answer
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Is The Collektion Hotels & Resorts incorrect or missing key business information? Update your Hotfrog listing today.
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TheCollektion represents some of the world's finest hotels & resorts,offering the utmost unique travel experiences aimed to capture theimagination of discerning travelers by constantly pushing theboundaries of luxury and surpassing expectation.
As a hotel sales & marketing company, The Collektion is designedspecifically to drive and strengthen each individual hotel partners'market share from our office in London and Singapore.
The dynamic team atThe Collektion brings along a wealth of experience in key sales roleson property and globally within The Ritz-Carlton Hotel Company, FourSeasons Hotels & Resorts Worldwide, Hilton, InterContinentalHotels, Preferred Hotel Group and St Regis amongst other hotels.
Is The Collektion Hotels & Resorts in 16 Raffles Quay, 33-03 Hong Leong Building, Singapore Singapore 048581 your business? Claim your listing and attract more leads by adding more content, photos and other business details.
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10 Anson Rd #27-08 International Plaza Singapore 079903
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Valley Art Association
Valley Art, established in 1966, celebrated its 50th anniversary in 2016. Valley Art is located in the heart of historic downtown Forest Grove on Main Street, just one block west of Pacific University.
As one of the oldest nonprofit, all-volunteer art associations in the Pacific Northwest, we feature over 200 established and emerging local artists. We offer an eclectic assortment of paintings, fine pottery, jewelry, wood carvings, glass art, metal sculpture, textiles and garden art, to name a few. We pride ourselves in finding unique items at exceptional values. There are also art classes in pottery, watercolor, oil painting, mixed media, quilting and there are several classes for children – truly something for everyone.
POTTERY CLASSES – Fall and Winter
Saturday Pottery Classes (age 16 and up) with Bob Hackney – Saturdays, December 10-31 (no class on Christmas Eve) & January 7-28. Cost for each month of classes is $140 for Valley Art members, $150 for nonmembers.
Classes are on Saturdays from 10 a.m. to 2 p.m. for beginners at all skill levels. No production work allowed. Students will throw on the wheel and hand-build. Clay is $13-$15 per bag and tool kits are $15. Bring an old towel for clean up, wear old clothing and closed toe shoes.
There are also some Tuesday classes. Contact Bob at 971-331-3094 or firstname.lastname@example.com to register.
Christine Hurayt will teach pottery classes on Saturday again beginning in February. Stay tuned for more information.
featuring the band
Saturday, February 18th
– 7:30 – 11:00 pm
Italian masquerade fundraiser for Valley Art Gallery – music, food and masked adventure! Photo booth and one glass of wine free at the no-host bar.
Your ticket is also your chance to win a framed original oil painting by Roderick Smith, a $1,500 value.
Admission: $25
Come to Valley Art (2022 Main St., Forest Grove 97116) between Feb. 1-4, fill out an entry form and purchase an "art box" for $10. Yes, it looks like an ordinary cigar box but it's actually your entry in our Smokin' Hot Art Show.
Take the box home and transform it. Make it into anything you like – a jewelry chest, a bird nest, a purse, a book. Everyone starts with similar cigar boxes. Then, bring it back to the gallery by March 4th.
All art boxes will become the property of Valley Art and be displayed and marked for sale for $50 each. The proceeds from the boxes will go to Helvi Smith, a Forest Grove artist we all want to support.
If you have questions call 503-341-9701.
Valley Art Supporters:
This is an exciting time for Valley Art, as we celebrate 50 years in Forest Grove and begin our 51st. The gallery was started in 1966 by 12 artists. Two years later it became a legitimate business.
Today it is a thriving art gallery, pottery studio and educational nonprofit organization, run entirely by volunteers. We have over 325 members, about 100 artists and nearly 50 active volunteers.
Currently we are working on raising money to help with the refurbishing of our building which needs some expensive updating. The storefront of Valley Art requires stabilization of the 1930s era infrastructure. We welcome your help. Donations have arrived ranging from $25,000, $1,000 to $250 and $10.
Donations are welcome anytime, but remember: When the year 2017 comes to an end, we ask you consider Valley Art in your year-end giving. Your contribution is tax deductible under Section 501 (c)3 of the IRS tax code. Stop by the gallery or send your donation to VAA, P.O. Box 333, Forest Grove, OR 97116.
IN ADDITION to our beautiful gallery space we offer two separate classroom spaces, one with wonderful natural lighting for workshops and club meetings as well as a 600 sq. ft. pottery studio for both adult and children's classes.


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Celebrate a special graduate and her accomplishments with this classic blue photo graduation card! Antique brocade and a classic blue & ivory color scheme showcase your photo, which we can leave in original color or convert to vintage sepia or black & white. To suit your own needs, we can change all the bachelor university graduation celebration party wording for an announcement or different graduation level and change the deep blue & ivory colors to your own school colors.
See more ideas & personalize this design here: Classic Photo Graduation Invitations (simply share your color and design requests in the personalization form as needed).


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DeeAnn Lee Douglass, 53 of Bruce, passed away Friday, Dec. 30, 2016, at Baptist Memorial Hospital in Oxford. Born February 2, 1963 in Alameda, Ca., she was the daughter of Robert Christensen and Donna Swift Milkvoich. She served in the Navy and was a former accountant. She was a member of Old Town Baptist Church. [Read more...]
DeeAnn Douglas
Amber Hathcock
Amber Dawn Hathcock, 40, went to her heavenly home Sunday, Dec. 25, 2016 at her residence in Calhoun City. She was born August 3, 1976 in Texas City, Tx., the daughter of Roger Schneckenburger and Christine McKinney Deubner. [Read more...]
Malline Parker
Malline Marshall Parker, 75, went to her heavenly home Saturday, Dec. 31, 2016 at Baptist Memorial Hospital in Oxford. She was born August 2, 1941 in Yoakum, TX, the daughter of Hulan and Juetta McCord Marshall. She was a member of New Providence Baptist Church. [Read more...]
Mary Alice Flanagan
Mary Alice Herring Flanagan "Miss Myrt," 101, formerly of Gore Springs, passed away December 13, 2016 in Mobile, Al. She was born Oct.03, 1915 to Granville and Mary McCleskey Herring in Vardaman. She was preceded in death by her parents; husband, Wiley Flanagan; granddaughter Theresa Ambron, and son-in-law Rex Brewer. She is survived by her daughter Mary Flanagan Brewer, granddaughter Danette (Kevin) Woods, great granddaughters Kylee Carr (Thomas) Gaston and Kara Carr, and great great grandsons Paxton and Parker Boykin, and Carson Gaston.Azalea City Funeral Home in Charge of Services. A graveside service will be held at Pittsboro Cemetery at a later date.
Ladell Alexander
Ladell S. Alexander, 96, of Vardaman, died Tuesday, Dec. 27, 2016, at Baptist Health Services
Nursing Home in Calhoun City. Born June 25, 1920 in Vardaman, she was the daughter of John Christopher and Cassie
Pettit Stewart. She was the widow of the late Bro. Steven Bane Alexander. She was a homemaker and a member of Poplar Springs Baptist Church. [Read more...]
Lynda Smith
Lynda Jane Smith, 65, of Pittsboro, died Monday, Dec. 26, 2016, at Pontotoc Health Services Hospital.
Born June 18, 1951 in Vardaman, her parents were the late William Rex and Jonnie Lee Deaton Dye.
She was a former finance loan officer and manager, and a member of Old Town Baptist Church. [Read more...]
William Harrison
William Earl Harrison, 83, went to his heavenly home Sunday, Dec. 25, 2016 at Sanctuary Hospice House in Tupelo. He was born July 21, 1933 in Banner, the son of Charlie C. and Tishie Stricklin Harrison. He was retired from Motts, Inc. in Water Valley and a member of Philadelphia Church of God. [Read more...]
Amy Harrelson
Amy Lynn Harrelson, 52, of Ridgeland, passed away Wednesday, Dec. 21, 2016, at The University of Mississippi Medical Center. Amy was born in Greenwood on May 14, 1964. She attended Mississippi Baptist High School from grades 1-12. She received a Bachelor's Degree from Mississippi State University, and a Master's Degree in Elementary Education from Mississippi College. [Read more...]
Carlton Dover
Carlton "Jabe" Dover, 73, of Vardaman, died Monday, Dec. 12, 2016, at North Mississippi Medical Center in Tupelo, following a sudden illness. He was born November 9, 1943, in Randolph to Elton and Bessie Dover. He was a self-employed mechanic, and attended Thorn Church of Christ. [Read more...]
Larry Rowsey Sr.
Larry Gresham Rowsey, Sr., 75, of Hernando passed away Wednesday, Dec. 21, 2016. He enjoyed the outdoors, his farm, riding his Kubota and spending time with his friends and family. A successful businessman, he was the owner of Rowsey Furniture Company and Rowsey Farms. He was very involved in the Baptist Church and loved to sing and listen to hymns. He adored his grandchild, Gracyn, whom he called "Tadpole", and enjoyed being a grandfather. [Read more...]


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Council to Help Company's 1,100 Credit Unions Identify Marketing Strategies to Generate Opportunities for Expansion and Continued Loan Growth
Ontario, CA, April 1, 2014 – CU Direct (www.cudirect.com), the nation's leading provider of lending, automotive and strategic solutions to the credit union industry, has announced the formation of its North American Automotive Marketing Council.
CU Direct's new marketing council will take a leadership role in identifying strategies that meet the needs of a rapidly changing automotive environment and identifying opportunities for credit unions to expand market presence and increase auto lending. The volunteer council includes marketing executives and leaders from a dozen CU Direct partner credit unions from across the U.S. and Canada.
"The mission of the new council is to foster the creation of proactive solutions that meet the evolving needs of consumers, credit unions and the automotive industry, as well as to enhance support of CU Direct's credit union partner base, and generate increased loan growth for member credit unions," stated Marci Francisco, senior director, automotive marketing & business development at CU Direct.
"One of the great strengths of credit unions is the synergy we gain by working together for the greater good of our members," said LJ Tarman, vice president, marketing and public relations, NuVision Federal Credit Union. "The collaboration of this council will help us provide even more benefits for all credit union members by developing new opportunities within the ever- changing lending landscape."
The council will also take a lead role in promoting CU Direct's automotive products and services to the credit union marketplace, and act as a liaison with CU Direct's executive team, providing thought leadership on marketing methods and strategies for enhancing the credit union community's success in the greater automotive space. The inaugural meeting of the newly formed council is slated for May.
"I feel privileged to join this dynamic team of credit union professionals, as we work together in helping our peers develop products and services that help them generate more loans, while effectively managing the indirect channels," said Andrea Pruna, vice president, marketing at Northeast Credit Union.
"The marketing council will ensure that our CU Direct programs are implemented full-circle within our credit unions. I'm honored to be a part of the council" noted Glenda Burkett, vice president, marketing at Credit Union of America.
Members of CU Direct's North American Credit Union Marketing Council include: Stephen Black, vice president, marketing, at Seattle, Washington-based BECU; Glenda Burkett, vice president, marketing, at Wichita, Kansas-based Credit Union of America; Tammy Gallegos, vice president, marketing, communications and service quality, at Riverdale, Utah-based America First Credit Union; Matt Kaudy, vice president, marketing and business development, at Upper Marlboro, Maryland's NASA FCU; Marc L'Ecuyer, market manager, consumer products, at Levis, Quebec, Cananda-based Desjardins Group; Andrea Pruna, vice president, marketing, at Portsmouth, New Hampshire's Northeast Credit Union; Cathy Rios, vice president, marketing, and Stacy Fifield, CLO at Vacaville, California's Travis Credit Union; Mike Schnably, senior vice president, sales & marketing, at Exton, Pennsylvania-based Citadel FCU; Greg Stroud, vice president, sales & marketing, at San Antonio, Texas-based Security Service FCU; Heather Summers, senior vice president, marketing, at Westminster, California's LBS Financial Credit Union; L.J. Tarman, vice president, marketing & public relations, at Huntington Beach, California-based NuVision FCU; and Keith Troup, vice president of lending/CLO at Olympia Washington's Washington State Employees Credit Union.
## #
Established in 1994, CU Direct has helped the credit union industry fund over $138 billion in loans by being the nation's leading lending and automotive solutions provider for credit unions. Representing more than 1,070 credit unions, CU Direct specializes in solutions that help credit unions generate loans, manage risk and provide value to members. CU Direct offers a diverse, extensive library of products and services designed to help credit unions advance their lending programs and achieve overall portfolio success. Solutions include CUDL, Lending Insights, Lending 360, OnSpot Financing, Vero, and its suite of auto buying products. For more information about CU Direct visit www.cudirect.com.


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Posted: Friday, October 28, 2016 1:18 PM
PLEASE READ!!! Assured leasing solutions is a scam! 877-833-5876 this is the number however you have to dial an extension to speak with anyone. Mr Larry Stevens claims to be the executive assistant of the company. He says he will get you approved for an apartment and once he does that you have to pay $600 what he doesn't tell you is that the his so-called company does not sign the lease until after they receive the $600. The $600 can only pay be paid online not through money order nor any other means. When mr. Stevens said the application had been approved I started asking him questions about his company trying to get a little bit of a better understanding as to where my money is going and he got very irritated with me and told me he can't understand why I'm asking so many questions. I was a bit turned off after that conversation so I did a little research of my own and checked into website Landover.WS and found the same one 877 number. Each time I called the number I would dial a different extension and seem to always get in contact with mr. Stevens I was looking to speak with someone else to legitimize this company however every extension went to mr. Stevens. When I asked mr. Stevens who his supervisor was he told me it was a man named mr. Mitchell and I stated to him that I had dialed mr. Mitchell's extension and still have gotten in contact with mr. Stevens he told me that he was the executive assistant to mr. Mitchell and no one could speak to mr. Mitchell he also denied me the ability to speak with anyone else in his office and got very frustrated with me when I insisted that I wanted to speak with someone else because I did not believe his company was legit. I also spoke with the leasing agent who was trying to assist me in getting an apartment along with the help with mr. Stevens and she informed me without my prompting that he seemed a bit pushy and skeptical. I spoke with mr. Stevens after this and told him I would not be continuing with my application with his company and he was very frustrated he argued with me and text me for at least about 15 minutes arguing with me about whether or not his company is legit. Im sorry but that in itself was a red flag that the son so called executive assistant has to time text argue with me about my decision to move forward with his company. Im glad i did not continue with this scam and i hope no one else falls for it. The only other reviews i found for this company ASSURED LEASING SOLUTIONS stated that mr stevens took her money and he did not deliver on an apartment nor refund her money.
• Location: Atlanta, usa
• Post ID: 43093948 georgia


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This is a view of Tate Modern, London, at night. The silver birch trees alongside the Thames are floodlit at night and this creates an ethereal feeling. The silvery trunks almost glow in the electric light. You can see the lights of the museum through the trees.
Oil painting by Camilla Gray
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Radio V 99 is a wonderful radio station for Jazz in addition to world music fans. A large number of music fans on the globe who loves Jazz kinds of music and also there are a few fans who loves to know some great bunch of World music. Mais, there are so few radio stations on the globe which provides them both. Radio V 99 is that type of radio station that can handle both Jazz and world music.
Radio V 99 adresse officielle du site est www.radiov99.com
Radio V 99 adresse officielle du site est www.radiov99.com
S'il vous plaît attendre 10-20 Radio secondes chargement ...
Populaire Bulgarie Radio:
Radio Veronika
Danse FolkBG
bTV Radio
Alpha Radio
Radio FolkBG


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House Sitter in San Leandro
My names Angela,.i live in Sanleando. Im in desperetely in need for a job. I do housecleaning, wsrehouse work and even worked.for UPS
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Ottomans & Storage Cubes
+5 more Lounge II Petite Slipcovered Ottoman and a... Clearance EUR 539.51our reg. price. EUR 599.56
Ottomans are the ultimate accent for both style and functionality in the home. Use them as casual extra living room seating, or roll them up to the sofa or sectional as a footrest. Push two or more together to create a unique DIY coffee table or side table near the sofa. Storage ottomans are a chic and clever way to hide and organize extra throw blankets, remote controls, magazines and any other clutter. For big families, consider a storage cube in the playroom for added toy, game and stuffed animal organization.


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It's almost impossible to measure this accurately when breastfeeding, so use frequency and duration of feeds as a guide instead. In The New Contented Little Baby Book, Gina Ford suggests a daily guide:
- 2-6 weeks: 7 feeds
- 6-12 weeks: 5-6 feeds
- 3-6 months: 4-5 feeds
A helpful checklistIf your baby doesn't drink the exact amount, don't worry - no one told him the rules! Here's a rough checklist that should show if he's getting enough nutrition in his milk:
- He has yellowy, seedy poo five times a day to begin with, slowing to an average of one a day from 3 months onwards.
- His nappy is wet and/or heavy. Hold a fresh one in the other hand and compare the weight of the two if you're unsure.
- His wee is clear. If it's yellow he could be dehydrated and not getting enough to drink.
- A bottle has the advantage of giving an accurate measure of how much he's taken.
- At the breast he should be making gulping and swallowing noises.
- He's happy after a feed and doesn't cry for more.
- He's following his height and weight chart to the satisfaction of your doctor or health visitor. And remember, it's normal to go a few weeks where there's no gain, sometimes even a small weight loss, usually followed by a growth spurt.
The Department of Health recommends that you feed nothing but milk for the first 6 months. However, some health professionals will advise that you wean as early as 4 months if your baby's particularly hungry and seems ready. Don't automatically assume that slow weight gain means it's time to wean. Early solids are less about calories and more about texture, and the calories your baby needs to gain weight well will still come from milk. You should discuss this option with your health visitor before you begin.


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Lee Honish - Short Sale Coach
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Honish had a 20+ year career as a foreclosure asset manager/loss mitigator working for large mortgage lenders. Early in 2007, he knew that agents needed to know what the banks know in order to close short sales in record time. He developed a program that has a 99% close ratio, which is both astonishing and truly making a difference. "It is satisfying to provide useful education to a group of professionals in our nation that desperately need to work together. Real estate professionals just need to put the right strategy to work for them, so they can be a part of the solution, not a part of the problem."
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There are many factors that influence the addictiveness of a drug. The route of administration is a major determinant of the speed of drug entry into the body and to receptors in the central nervous system. Clinical studies are in progress which determine the importance of different routes of administration and the relationship of blood concentrations of drug and active metabolites to concomitant drug effects. Studies of heroin, cocaine, methamphetamine, codeine and cannabis administration by the intravenous, smoked, intranasal and oral routes are underway or in the data analysis stage. Pharmacodynamic effects include subjective, cognitive and psychomotor performance, physiological measures and specific brain activity as assessed by fMRI. Plasma, urine, oral fluid, sweat, hair and skin are analyzed for drug and metabolites by solid phase extraction-gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. The intravenous and smoked routes produce a rapid onset of pharmacological effects together with an early appearance of drug and metabolites in blood. Evidence of delays in distribution to effector sites are observed following cannabis smoking, but drug effects following heroin and cocaine administration coincided more closely with concurrent blood concentrations. Onset of effects is delayed following intranasal and especially after oral administration. The emerging face of drug abuse is the plethora of new designer drugs. Two main classes of designer drugs are the synthetic cannabinoids and synthetic cathinones. The pharmacodynamics and pharmacokinetics of these new drugs are mostly unknown. We are endeavoring to conduct a controlled synthetic cannabinoid human administration study, but until this is possible, we are using human hepatocyte cultures and high resolution accurate mass spectrometry to characterize the metabolic profiles of these new designer drugs.
|Barnes, Allan J; Young, Sheena; Spinelli, Eliani et al. (2014) Evaluation of a homogenous enzyme immunoassay for the detection of synthetic cannabinoids in urine. Forensic Sci Int 241:27-34
|Koola, Maju Mathew; Gorelick, David A; McMahon, Robert P et al. (2014) Psychiatric symptom differences in people with schizophrenia associated with substantial lifetime substance use but no current substance use disorder. Schizophr Res 152:315-6
|Anizan, Sebastien; Ellefsen, Kayla; Concheiro, Marta et al. (2014) 3,4-Methylenedioxypyrovalerone (MDPV) and metabolites quantification in human and rat plasma by liquid chromatography-high resolution mass spectrometry. Anal Chim Acta 827:54-63
|Ellefsen, Kayla N; Concheiro, Marta; Beck, Olof et al. (2014) Quantification of cocaine and metabolites in exhaled breath by liquid chromatography-high-resolution mass spectrometry following controlled administration of intravenous cocaine. Anal Bioanal Chem :
|Lee, Dayong; Huestis, Marilyn A (2014) Current knowledge on cannabinoids in oral fluid. Drug Test Anal 6:88-111
|Newmeyer, Matthew N; Desrosiers, Nathalie A; Lee, Dayong et al. (2014) Cannabinoid disposition in oral fluid after controlled cannabis smoking in frequent and occasional smokers. Drug Test Anal 6:1002-10
|Milman, Garry; Bergamaschi, Mateus M; Lee, Dayong et al. (2014) Plasma cannabinoid concentrations during dronabinol pharmacotherapy for cannabis dependence. Ther Drug Monit 36:218-24
|Bergamaschi, Mateus M; Queiroz, Regina H C; Chagas, Marcos H N et al. (2014) Rimonabant effects on anxiety induced by simulated public speaking in healthy humans: a preliminary report. Hum Psychopharmacol 29:94-9
|Gandhi, Adarsh S; Zhu, Mingshe; Pang, Shaokun et al. (2013) First Characterization of AKB-48 Metabolism, a Novel Synthetic Cannabinoid, Using Human Hepatocytes and High-Resolution Mass Spectrometry. AAPS J :
|Walsh, J Michael; Verstraete, Alain G; Huestis, Marilyn A et al. (2008) Guidelines for research on drugged driving. Addiction 103:1258-68


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Hot Links: Pastor Tasered, Jewish Site Vandalized, and Frogs
The governing board of the Gilbert Unified School District approved an increase in athletic fees for Gilbert high schools Tuesday night. Parents will now pay almost double for their kids to participate in sports -- up to $100 per sport from the current fee of $55...A Valley pastor named Steve Anderson says he was tasered by DPS officers during a traffic stop in April, and he's got the YouTube video to prove it. DPS had no immediate comment, except to say an investigation is under way...Someone painted swatiskas and white power graffiti on the site of the future Chabad Center for Jewish Life in Chandler. Police are investigating, and could consider this a hate crime...Mesa Police Chief George Gascon may have been offered the top job in the San Franscisco Police Department. A source told CBS 5 News that Gascon is a finalist for the Chief of Police position there. Gascon and the Mesa Police Department would neither confirm nor deny the report...The Phoenix Zoo is working to save more than 1,300 Chiricahua Leopard Frog tadpoles. The frogs are an endangered species, with only 80 habitats remaining from the 400 locations where it used to thrive...A woman drove into a gas pump at Circle K near 83rd and Glendale avenues early this morning, sparking a fire. Police say a child on a bicycle rode over and hit the emergency shutoff valve. No injuries were reported, except maybe a bruised ego on the part of the inattentive driver.
Gilbert district OKs higher sports fees
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Ann Murray, Former School Board Member Busted for Using N-Word, Running for Reelection
Former Broward School Board transportation department supervisor Ann Murray is running for reelection for the Broward School Board in August. But before voters decide between her and or her opponent -- West Park City Commissioner Felicia Brunson -- there's the little matter of Murray's past use of the "N word."
Back in 2007, a year before her election to the School Board, Murray's allegedly used the racial epithet while describing the cheap seats at Sun Life Stadium.
Her use of the word became public in 2011, and Murray was called to resign by the vice president of the Democratic Black Caucus of Broward County.
See also: Activist Calls For Ann Murray To Resign
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For her part, Murray, 71, apologized for the comment, though she rejected calls for her resignation from the four-year position she had won in a special election in 2008.
But in 2011, her remarks became public when fellow supervisor Lisa Spince reported in a written statement to the board that Murray had made the derogatory comment.
It was while Murray and Spince were making plans for the Super Bowl that year that Murray recalled a Miami Dolphins game they attended with others from the transportation department.
"Do you remember when a group of us from transportation came down to watch a Bills game?" Murray said, according to Spince's written statement. "Yeah, they had us up in nigger heaven."
Spince, who is white, said she commented to Murray that the term was offensive. There also happened to be an African-American bus driver nearby who overheard the conversation.
When the remarks became public, the chairwoman of the Democratic Black Caucus of Broward County, Freda Stevens, called Murray to clear the air and get a public apology.
Murray, Stevens said, went on a rant trying to explain why she used the term and went on to say she had "black blood" in her family.
"You all do not know if I have black blood in my family," Stevens said Murray told her over the phone. "I cannot make up for what happened 400 years ago. I know that some of you all are just sensitive, but I have been called names, whitey and honky. I just brushed it off."
Murray is now running against Brunson in District 1.
According to the Sun Sentinel, Brunson challenged Murray on her comments during a meeting with the paper's editorial board.
"If you can make a remark like that about African-Americans, you can make it about any other minority group," Brunson said. "It was totally insensitive and something that should not be tolerated."
Murray defended herself, telling the board that she doesn't use the N-word, except for in this one case, and that it was likely because of her poor upbringing in her native Boston.
Murray explained that the nosebleed sections at the Boston Garden in her day were known as the "N-word" seats.
Voters from the district, which encompasses Dania Beach, Hallandale Beach, Miramar, Pembroke Park, Pembroke Pines, and West Park, will be hitting up the voting booths on August 26.
Send your story tips to the author, Chris Joseph. Follow Chris Joseph on Twitter
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2 Circle Drive,Monmouth Beach NJ, 07750
4 beds , 3 Full/1 Half baths
Status: Active
Berkshire Hathaway HomeServices New Jersey Properties
(800) 548-3466
Last Updated: 2/22/2017 5:53 AM
Description for 2 Circle Drive Monmouth Beach, NJ 07750
This fabulous WATERFRONT home is a first time summer rental! Enjoy summer at this beautifully decorated home in pristine condition located on a cul de sac with magnificent waterfront. Open,neutral floor plan plus 4bedrooms plus bonus room with 3 beds; 3 1/2 baths; family room has pool table plus bar for your enjoyment; upgraded gourmet eat in kitchen. Enjoy dock for boat and water sports plus use of 2 kayaks. Take in amazing waterfront views from the balconies of your bedrooms plus rooms on first level. Porch spans the width of home to enjoy the waterfront with various seating arrangements. Available July and August for $30,000 per month or both months. Please no pets or smokers. Close to beach, shopping, restaurants, and fast ferry service, bus and train.
Listing Information
Gas Water Heater, Public Water Service
Public Sewer
School Information
Room Information
Den/Study, Family Room, Laundry Room, Living Room
Eat-In Kitchen
Dual Sinks, Tub Only, Full Bath
Interior Features
Central Vacuum, Dishwasher, Dryer, Microwave, Refrigerator, Stove, Washer
Carpet Wall to Wall, Wood
Central Air, A/C - Zone, Ceiling Fan(s)
Gas Heat, Forced Air, Zone
Bay Window(s)
Furnished, Beamed, Walk-in Closets
Exterior / Lot Features
2 Garage Spaces, Driveway
River, Water
River, Water Front, Seawall/Bulkhead, Outdoor Lights, Sliding Glass Door, River Front
Financial Considerations
Inside AccessTM
Current market conditions for 2 Circle Drive
Active Market Trends
Source:MLS DATA
Median list price$697,000
Days on market112
Properties for sale48
Sold Market Trends
Source:MLS DATA
Median sold price$335,250
Sales to list price9,372.0%
Properties sold16
Median sq ft price$277
Arrows indicate 90 day trend
Listing Price History for 2 Circle Drive
Monmouth Beach Real Estate
Monmouth Beach is located in New Jersey. Monmouth Beach, New Jersey 07750 has a population of 3,298.
The median household income in Monmouth Beach, New Jersey 07750 is $91,023. The median household income for the surrounding county is $84,746 compared to the national median of $53,046. The median age of people living in Monmouth Beach 07750 is 48.4 years.
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Register for FREE to watch the full 17:45 video!
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You only watched the first 20 seconds of this video.
Download: Low Quality Video - High Quality Video - Original Video
Yummy girlfriend swallowing my load
By: anonymous2
Added: Jan 27, 2010
Time: 17:45
Votes: 3
Favorited: 8
Views: 6952
- Posted by secofstate on 14 Oct 10
Seventeen minutes of blowing with a final cum in the mouth from a very nice looking gal
- Posted by pierrepoint on 28 Jan 10
Lazy guy...and hard working blowing GF !!


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Researchers in Japan coaxed human induced pluripotent stem cells into becoming a liver bud—an early structure that is seen when livers form—when they recreated cellular interactions that normally occur during bud development (Takebe T et al. Nature. doi:10.1038/nature12271 [published online July 3, 2013]).
The approach involved mixing hepatic progenitors derived from the stem cells with endothelial and mesenchymal cells. Staining and gene-expression analyses revealed a resemblance between the stem cell–derived buds and naturally occurring liver buds.
Hampton T. Researchers Use Human Stem Cells to Build a Functional Liver. JAMA. 2013;310(5):470. doi:10.1001/jama.2013.178396
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Buy 5 and get 1 free in this mixed case special to sample a range of different white wine styles. This wine case features hand-selected wines from some of Australia's best boutique wineries and contains a range of styles including Riesling, Pinot Gris, Chardonnay, Semillion Sauvignon Blanc and Pinot Grigio. This 5 + 1 Wine Case contains; 1 x Sidewood Estate 2012 Cremello Chardonnay, 1 x Thompson Estate 2013 Estate Range - Semillon Sauvignon Blanc, 1 x Brockenchack Wines 2015 Mackenzie William Riesling, 1 x Mr Mick 2015 Riesling, 1 x Bervini 2014 Pinot Grigio Veneto, Italy, and 1 x Hickinbotham 2015 Pinot Gris.
We deliver Australia wide and all items will generally ship next business day. Estimated delivery times and total charges are calculated and displayed on checkout.
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Craft beer's all about exploring a diverse range of styles and flavours, just like wine. Our hand-selected mixed cases offer the perfect way to sample a range of our favourite craft beers and different varieties.Discover more about BoozeBud.
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An increase in illicit drug use in Northern Ireland may well have links to the resolution of political conflict, which started in the mid 1990s. Social issues, heretofore hidden, have emerged into the limelight and may be worsened by paramilitary involvement. Registered addicts in the four Health Board areas have shown an increase from 1997 with the… (More)
The aim of this study was to examine the pattern of attendance of patients requesting Emergency Hormonal Contraception (EHC) at an accident and emergency department before and after a government driven change in legislation, which allowed EHC to be sold over-the-counter by trained pharmacists, to women aged 16 years and above. We employed retrospective… (More)
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Brocade provides network solutions that help the organizations transition smoothly to a virtualized world where applications and information can reside anywhere. These solutions deliver the unique capabilities for a more flexible IT infrastructure with unmatched simplicity, non-stop networking, application optimization, and investment protection.
Technology Data Group's Network Software mailing lists are proven to increase response rates and are the industry standard for email list software. We manage all of your opt-in email lists, email campaigns and email marketing, our exclusively tailored mailing lists are one-stop source of specialty and compiled for every requirement.Get the count of product users across different regions, format given below: Key Benefits


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Thanksgiving Craft Idea - Felt Door Sign
Thanksgiving Door Sign
This craft Idea goes along with the felt door sign theme. I found a coloring page that I liked to use in my door sign, then assembled all the needed supplies, to complete the project. For this project you will need:
- 12" x 18" Stiff Easy felt panel whatever colors you choose.
- and 9" x 12" soft felt panel in various colors in accordance to your choice of colors.
- Embroidery thread (optional) for sewing method
- felt glue (optional) for no sew method
This is a very easy project to make using the no sew method but can also be made using a sewing method with embroidery stitches, I used the embroider stitches to decorate and embellish the turkey but used a no sew method on the rest of the door sign. However you can choose completely no sew, or to sew it all as in previous door sign Ideas which are posted at the bottom of this page. Just before the comments section.
Turkey Coloring Page
Completing the Thanksgiving Felt Door Sign
I first found a coloring page with the image I wanted to use. Going with the thanksgiving theme, I found a turkey image that would work well. You can find many coloring pages online for these projects.
I then made a few copies so I would have extra when I go to cut out the pieces if need.
I assigned colors to all the pieces of the turkey design and made sure to get soft felt panels of all the colors. I then used light head and bond product and traced all the pieces on to the light heat and bond with will make the felt pieces easier to cut out.
Light heat and bond is a product that is used to bond fabrics together using and iron on method. It comes in sheets of paper and can usually be found at any craft store. Once the pieces are traced onto the heat and bond paper you can then make a rough cut around the pieces and bond to the respective colors of felt. Then cut all the pieces out precisely the size of the piece.
Once all the pieces are cut out you will then be able to assemble the pieces like a puzzle to form your image, onto the 9" x 12" color of soft felt of your choice.
In this example I then decided on a design to embellish the turkey by using embroidery thread and stitches to make different designs on the feathers and around the turkey outline. However this is not necessary if you prefer the no sew method you may also achieve a similar effect using felt tip markers.
Once this was complete I then applied the turkey panel to the larger stiff easy felt panel and added welcome lettering at the bottom. You may want to add your own lettering ideas, such as, Happy Thanksgiving, Happy Turkey Day, etc.
To apply them I chose to use the light heat and bond again for a quit method to assemble the project, but this can also be sewn by hand as I have done with other felt door signs.
The last step is to apply loops at the top to hang on a dowel rod or you may add hooks for hanging as well. Then nice thing about this project is you there are options to make it as easy or a difficult depending on the crafters choice. No sew methods make this a very easy craft for young children to complete with adult supervision.
Pictures of some of the steps are added below.
More Craft Ideas - Felt Door Signs
- Dog Days of Summer Craft Idea - Felt Door Sign
This door sign is meant to be displayed in the month of August, Since there are no other American holidays in the month of August I wanted to do something fun and different. So I chose to do a Dog Days of...
- Halloween Craft Idea - Haunted House Felt Door Sign
One of the greatest ways to dress up the front of your house for any holiday is with door signs. However many door signs you might find out on the market are just the same stuff you have seen year after year....
- Christmas Craft Idea - Felt Santa Door Sign
This Christmas craft idea, is very easy to make and can be adapted to different skill levels. It is very inexpensive to make and can be adapted to any picture you would like to use. I chose a Santa face,...
More by this Author
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An article about great ways to make money by making and selling your quilts.
- 10
One of the easiest kinds of quilts to make are baby quilts. They are much smaller that large bed size quilts and fun to make. They can be made my machine or made by hand. Of course making a quilt by...
- 32
A step by step guide to making a Denim Rag Quilt. With full size pictures. This particular quilt is 60"x70" in size. And will fit a twin size bed easily, But my husband saw what I was doing and has now claimed the quilt...
Comments 2 comments
Love this!! Ugh, I have to find more time in my life to be able to accomplish this... What a wonderful idea...


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Does any of you know someone who is interested in sharing an apartment in Amsterdam or who has a room that I can rent for most weekends and perhaps Protected content per week starting Jan/Feb 09?
I am looking to return to the Netherlands after 12 yrs abroad. Since I am still working abroad I might need a base for a few months to get organised.
I would appreciate to receive and ideas and recommendation you might have.
Many thanks, Ingrid


text: FRANKFURT (MarketWatch) -- BHP Billiton plc BHP, -1.73% BHP, +0.98% Rio Tinto Ltd. RIO, -1.37% RIO, +2.00% and ThyssenKrupp AG TKA, -0.10% were all downgraded to hold from buy on Tuesday by Societe Generale. Regarding mining giants BHP Billiton and Rio Tinto, the broker said that momentum from iron-ore price talks may fade, while both groups' valuations are now less compelling. Antofagasta plc ANTO, +0.12% a Chile-based copper mining group, was also downgraded to sell from hold, because lots of good news is already priced in, Societe Generale said. As for Germany's ThyssenKrupp, the broker said that there is a risk of margin squeeze and subdued demand in OECD.


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