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Telomerase transgenics are an important tool to assess the role of telomerase in cancer, as well as to evaluate the potential use of telomerase for gene therapy of age‐associated diseases. Here, we have targeted the expression of the catalytic component of mouse telomerase, mTERT, to basal keratinocytes using the bovine keratin 5 promoter. These telomerase‐transgenic mice are viable and show histologically normal stratified epithelia with high levels of telomerase activity and normal telomere length. Interestingly, the epidermis of these mice is highly responsive to the mitogenic effects of phorbol esters, and it is more susceptible than that of wild‐type littermates to the development skin tumors upon chemical carcinogenesis. The epidermis of telomerase‐transgenic mice also shows an increased wound‐healing rate compared with wild‐type littermates. These results suggest that, contrary to the general assumption, telomerase actively promotes proliferation in cells that have sufficiently long telomeres and unravel potential risks of gene therapy for age‐associated diseases based on telomerase upregulation.
Telomeres consist of tandem DNA repeats and specific proteins and protect the chromosome ends from degradation, recombination and DNA repair activities (reviewed in Blackburn, 1991; Greider, 1996). In this regard, loss of telomeric function, either by loss of telomeric repeats or by mutation of telomeric proteins, is associated with increased chromosomal instability and loss of cell viability (Counter et al., 1992; Blasco et al., 1997; van Steensel et al., 1998; Samper et al., 2000).
Telomerase is the cellular reverse transcriptase that synthesizes telomeric repeats de novo (Greider and Blackburn, 1985; reviewed in Nugent and Lundblad, 1998). It is composed of a catalytic subunit known as TERT (telomerase reverse transcriptase) (Harrington et al., 1997a; Kilian et al., 1997; Lingner et al., 1997; Meyerson et al., 1997; Nakamura et al., 1997; Greenberg et al., 1998; Martín‐Rivera et al., 1998), an RNA molecule or TERC (telomerase RNA component), which is used as template for the addition of new telomeric repeats (Greider and Blackburn, 1989; Singer and Gottschling, 1994; Blasco et al., 1995; Feng et al., 1995), and associated proteins (Collins et al., 1995; Harrington et al., 1997b; Nakayama et al., 1997; Gandhi and Collins, 1998; Mitchell et al., 1999).
Telomerase activity is upregulated in the vast majority of human tumors as compared with normal somatic tissues. It has been shown that expression of the catalytic subunit of telomerase, TERT, in cultured human primary cells reconstitutes telomerase activity and allows immortal growth (Bodnar et al., 1998; Kiyono et al., 1998; Jiang et al., 1999; Morales et al., 1999). Furthermore, TERT‐mediated telomerase activation is able to cooperate with oncogenes in transforming cultured primary human cells into neoplastic cells (Hahn et al., 1999). In addition, it has been shown recently that TERT‐driven cell proliferation results in activation of the c‐myc oncogene (Wang et al., 2000). These findings in cultured cells have opened up the possibility that telomerase upregulation, which occurs in >90% of all human tumors (reviewed in Shay and Bacchetti, 1997), may contribute actively to tumor growth (reviewed in Weitzman and Yaniv, 1999). On the other hand, telomerase activity reconstitution in adult somatic cells or tissues is envisaged as a potential approach for gene therapy of age‐related diseases. To address directly if tissue‐specific telomerase overexpression has an impact on tumor susceptibility or normal tissue biology, we targeted expression of the catalytic component of mouse telomerase, mTERT (Greenberg et al., 1998; Martín‐Rivera et al., 1998), to basal keratinocytes of stratified epithelia. Here, we describe the construction and characterization of these mice.
Results and discussion
In vivo reconstitution of telomerase activity in transgenic K5‐mTERT mice
We used the 5′‐regulatory region of the bovine keratin K5 gene (Ramírez et al., 1994; Murillas et al., 1995) to target mTERT expression to basal keratinocytes (see Figure 1A for details of the bovine K5 promoter region used to express mTERT) (Materials and methods). Eight different founder K5‐mTERT transgenic mice were identified (T1–T8) that showed increased telomerase activity in the skin of the tail compared with wild‐type littermates (Figure 1B). Two of these founders, T1 = 8043 and T8 = 7664 (highlighted in red in Figure 1B) showed the highest in vivo reconstitution of telomerase activity in the tail skin. All the results described here were obtained using the K5‐mTERT mice derived from the T1 founder, K5‐mTERT (T1) (asterisk in Figure 1B), and were confirmed, where indicated, with the T8 founder‐derived K5‐mTERT transgenics, K5‐mTERT (T8), to rule out possible non‐specific effects due to transgene insertion.
In agreement with the known expression pattern of keratin K5 (Ramírez et al., 1994; Murillas et al., 1995), the stratified epithelia of K5‐mTERT (T1) mice tested, including uterus, trachea and skin, showed significant upregulation of telomerase activity as compared with the corresponding littermate wild‐type tissues (Figure 1B and C). This telomerase upregulation coincided with increased mTERT mRNA levels in K5‐mTERT transgenic skin (between 25‐ and 50‐fold) compared with wild‐type controls (data not shown). Other tissues of the K5‐mTERT (T1) mice where the transgene is not expressed (i.e. liver, brain, lung, ovary and whole thymus) did not show upregulation of telomerase activity as compared with wild‐type littermates (Figure 1C).
Telomere length in wild‐type and K5‐mTERT transgenic basal keratinocytes
Previous studies using cultured human cells showed that TERT‐mediated telomerase activity reconstitution resulted in extension or maintenance of telomere length (Bodnar et al., 1998; Kiyono et al., 1998; Hahn et al., 1999). We measured telomere length in K5‐mTERT (T1) skin keratinocytes by quantitative fluorescence in situ hybridization (Q‐FISH) on skin sections as previously described (González‐Suárez et al., 2000). Figure 2A shows Q‐FISH images of interphase nuclei in the skin of age‐matched (8‐week‐old) littermate wild‐type and K5‐mTERT (T1) mice; the basal layer of skin keratynocytes is indicated with a yellow arrow. Quantification of the fluorescence intensity of telomere dots showed that telomeres in K5‐mTERT (T1) epidermis are similar in length to those of age‐matched littermate wild‐type mice (Figure 2B; black bars). Primary keratinocyte cultures derived from newborn wild‐type and K5‐mTERT (T1) mice also showed similar telomere length using Flow‐FISH, an independent quantitative FISH technique to measure telomere length that is based on flow cytometry (see Materials and methods) (Rufer et al., 1998) (Figure 2C). All together, these results suggest that TERT‐driven telomerase upregulation in basal skin keratinocytes does not result in a significant extension of telomeres in the skin as compared with age‐matched wild‐type mice.
K5‐mTERT stratified epithelia are histologically normal
K5‐mTERT (T1 and T8) mice, ranging from newborn to 19 months old, were phenotypically indistinguishable from the corresponding age‐matched wild‐type littermates and showed no pathologies, no spontaneous development of epithelial tumors and no loss of viability with age (not shown). In agreement with this, histopathological analysis of stratified epithelia (skin, oral cavity, esophagus, forestomach and vagina) from 15‐ to 19‐month‐old K5‐mTERT (T1 and T8, as indicated) and the corresponding age‐matched littermate wild‐type controls revealed no macroscopic or histological differences between genotypes, indicating that transgenic telomerase expression per se does not alter the stratified epithelium structure (Figure 3). These results suggest that potential telomerase reconstitution for gene therapy of epithelial aging‐associated diseases would not have any deleterious effects on the structure of stratified epithelia.
Increased tumor susceptibility and mortality of K5‐mTERT transgenics produced by chemical carcinogens
To study further the effects of telomerase overexpression on the normal biology and function of stratified epithelia, we first addressed whether transgenic telomerase expression impacts on epithelial tumor formation upon exposure to chemical carcinogens. The stages of initiation, promotion and tumor progression in the classical skin chemical carcinogenesis model are well characterized (Heckner et al., 1982). In wild‐type mice, initiation using 7,12‐ dimethylbenz[a]anthracene (DMBA) and subsequent promotion with 12‐o‐tetradecanoylphorbol 13‐acetate (TPA) leads to papillomas that are hyperplastic, well‐differentiated, skin lesions. H‐ras activation, loss of the p53 tumor suppressor and telomerase upregulation are reported in the majority of DMBA‐initiated papillomas (Balmain et al., 1984, 1992; Quintanilla et al., 1986; Bednarek et al., 1997). Furthermore, when telomeres are critically short, as in late generation mice deficient for the RNA component of mouse telomerase, Terc (Blasco et al., 1997; Lee et al., 1998), the absence of telomerase activity has a dramatic inhibitory impact on skin tumorigenesis (González‐Suárez et al., 2000), indicating that tumor formation in skin requires telomere maintenance above a threshold length.
Age‐matched (8‐ to 12‐week‐old) wild‐type and K5‐mTERT (T1) littermate mice received a single DMBA treatment followed by TPA–acetone treatment twice a week for 15 weeks (Materials and methods). Six weeks after the start of DMBA + TPA treatment, papillomas appeared in both wild‐type and K5‐mTERT (T1) mice, and these papillomas continued to grow in size throughout treatment (Figure 4A). The number of papillomas in K5‐mTERT (T1) mice was significantly increased compared with wild‐type littermates (Figure 4A and B). In particular, at week 15 (the end of TPA treatment), a total of 222 and 109 papillomas for K5‐mTERT (T1) and wild‐type mice, respectively, were counted (Figure 4A); this corresponds to 20.0 ± 9.8 and 9.9 ± 6.3 papillomas per mouse for K5‐mTERT (T1) and wild‐type mice, respectively (Figure 4B; Student's t‐test P = 0.0091). After termination of TPA treatment at week 15 (asterisk in Figure 4A), many of the papillomas regressed in both genotypes, and a similar percentage of them progressed to larger lesions, maintaining the difference in total number of papillomas between wild‐type and K5‐mTERT (T1) littermates. In particular, 6.4 and 7.2% of the papillomas at week 15 progressed to lesions >1 cm at week 30, in wild‐type and K5‐mTERT (T1) mice, respectively (Figure 4A). Despite the difference in total number of papillomas, no histological differences were found between wild‐type and K5‐mTERT (T1) papillomas (Figure 6A; papilloma in wild‐type and K5‐mTERT T1 mice).
The increased susceptibility of transgenic K5‐mTERT (T1) skin to develop papillomas due to chemical carcinogenesis was confirmed using K5‐mTERT mice derived from a different founder, T8 (Figure 1B). K5‐mTERT (T8) transgenics also showed an increased number of papillomas upon DMBA + TPA treatment as compared with the corresponding wild‐type littermates (Figure 5).
Strikingly, 80% of the DMBA + TPA‐treated K5‐mTERT (T1) mice (eight out of 10) died before week 60 after the start of treatment, whereas 90% of the similarly treated wild‐type cohorts survived during this time (nine out of 10; Figure 4C). Death of the DMBA + TPA‐treated K5‐mTERT (T1) mice was associated with several pathologies (see Table I for quantifications) including the following. (i) Severe gastric hemorrhages resulting from an abnormal forestomach epithelium, which showed a marked hyperplasia and hyperkeratosis (Figure 6A; compare the normal forestomach epithelium in DMBA + TPA‐treated wild‐type mice with the abnormal forestomach in DMBA + TPA‐treated K5‐mTERT mice). This observation suggests that other K5‐mTERT (T1) stratified epithelia besides the skin are prone to tumorigenesis upon DMBA + TPA treatment. (ii) Benign epidermal lessions: numerous keratoacanthomas and blue nevi (melanocyte accumulations in the skin). (iii) Pre‐malignant epidermal lesions: severe dysplastic papillomas. (iv) Malignant epidermal tumors: basal cell carcinomas, malignant keratoacanthomas. (v) Tumors of non‐epithelial origin: subcutaneous fibrosarcoma, uterus histiocytic sarcoma, liver histiocytic sarcoma, cecum lymphoma, as well as infiltrations of tumoral lymphoid cells in various tissues (multicentric lymphomas).
Hence, the increased mortality of K5‐mTERT mice upon DMBA + TPA carcinogenesis coincides with a higher susceptibility of these mice to develop neoplasias.
Telomeric Q‐FISH analysis on skin sections from DMBA + TPA‐treated mice showed that telomeres were similar or slightly elongated in K5‐mTERT (T1) mice as compared with wild‐type controls (Figure 2B; gray bars).
K5‐mTERT skins are more sensitive to the mitogenic effects of phorbol esters
Curiously, whereas wild‐type mice showed areas of histo logically normal skin surrounding papillomas 7 weeks after termination of DMBA + TPA treatment (Figure 6A; normal skin), the DMBA + TPA‐treated K5‐mTERT (T1) mice maintained a marked hyperplasia of the skin, with regions showing up to 10 layers of keratinocytes (Figure 6A; hyperplastic skin). These observations suggested an increased proliferative response of K5‐mTERT (T1) basal keratinocytes to repetitive TPA treatment. To study this further, wild‐type and K5‐mTERT (T1) mice were treated weekly with TPA (5 μg of TPA in 200 μl of acetone) in the absence of previous treatment with the carcinogen DMBA. After 3 weeks of repetitive TPA exposure, K5‐mTERT (T1) mice showed a more marked hyperplasia of the skin than similarly treated wild‐type mice (Figure 6B; hyperplastic skin at week 3 of TPA treatment). Strikingly, after prolonged (3 and 7 weeks) repetitive TPA exposure, the K5‐mTERT (T1) mice showed incipient papilloma lesions in the skin, which were never detected in similarly treated wild‐type mice (Figure 6B; incipient papillomas at weeks 3 and 7 of TPA treatment). Some of these incipient papilloma lesions in the K5‐mTERT (T1) transgenics progressed to macroscopic papillomas upon further TPA treatment; again, this was never observed in the similarly treated wild‐type littermates (not shown). Furthermore, probably due to licking of the skin‐treated region, TPA‐treated K5‐mTERT mice also showed areas of focal hyperplasia in the esophagus and forestomach epithelia, which were absent in similarly treated wild‐type mice (Figure 6B; hyperplastic esophagus at week 7 of TPA treatment).
Taken together, these results suggest that constitutive telomerase activity in stratified epithelia results in increased proliferation of basal keratinocytes in response to TPA treatment. This could be a way in which constitutive telomerase expression helps to promote tumor formation and progression in the K5‐mTERT transgenic mice subjected to chemical carcinogenesis of the skin.
Increased wound healing in K5‐mTERT epidermis as compared with wild‐type epidermis
As an independent way to assay the functionality of K5‐mTERT (T1) trangenic epithelia, we carried out wound‐healing experiments in the skin of 2‐ to 4‐month‐old littermate wild‐type and K5‐mTERT (T1) transgenics. Wound healing is a complex process involving cellular proliferation, growth factor production and an immune system reaction. Two consecutive 13 mm2 (4 mm diameter) circular punch biopsies were performed on the back skin of six wild‐type and six K5‐mTERT (T1) littermate mice. The rate of wound healing was monitored as the percentage of the initial wound area left open with time after the punch was made (see Materials and methods). One wound was made 2 days after the other and the animals were killed 7 days after the first wound was made. K5‐mTERT (T1) transgenics showed faster wound healing compared with wild‐type littermates (Figure 7A–C). Figure 7A shows the percentage of the initial wound area open in wild‐type and K5‐mTERT (T1) mice (six mice in each group) 3 and 4 days after each wound was created. The average wound areas at day 3 were 82.85 ± 12.38 and 46.95 ± 8.3% for wild‐type and K5‐mTERT (T1) mice, respectively (Student's t‐tests P = 0.001); and 75.45 ± 17.11 and 30.8 ± 15.5% for wild‐type and K5‐mTERT (T1) mice, respectively, at day 4 (Student's t‐tests P = 0.0031) (Figure 7A). These differences in wound healing between genotypes were highly significant, as indicated by the Student's t‐test values (P <0.01). As examples, Figure 7B and C shows the rate of healing of the two wounds (wound 1 and 2) in two representative pairs of wild‐type and K5‐mTERT (T1) littermates. Figure 7D shows representative images of wild‐type and K5‐mTERT (T1) wounds 4 days after the first wound was made (1 and 2 in Figure 7D correspond to the first and second wound, respectively); the two wild‐type wounds remained opened whereas the two K5‐mTERT (T1) wounds were already closed. As a standard for wound size, fresh punch biopsies were also made prior to sacrifice of the mice (marked with a 3 in Figure 7D). Histology of the wounds confirmed a faster rate of re‐epithelilization of the K5‐mTERT (T1) wounds as compared with wild‐type littermates (not shown).
Taken together, these results indicate that the skin of K5‐mTERT (T1) mice has a faster wound‐healing rate than that of the corresponding wild‐type littermates. This may reflect a proliferative advantage of telomerase‐transgenic skins as compared with controls in response to proliferative signals associated with wound healing, in agreement with the greater hyperplasia seen in K5‐mTERT epithelia as compared with wild‐type littermates upon treatment with the mitogen TPA.
Normal p53, Ras and c‐Myc levels in K5‐mTERT skin
p53 loss of function and H‐ras upregulation are described as occurring in multistage chemical carcinogenesis of the skin, and are thought to favor the appearance of tumors (Balmain et al., 1984, 1992; Quintanilla et al., 1986). To determine whether constitutive telomerase expression induces changes associated with transformation in the skin, we studied several tumor markers such as p53, Ras and c‐Myc in the skin of wild‐type and littermate K5‐mTERT (T1) mice. To analyze p53 status in skin keratinocytes, we performed immunohistochemistry of the skin with anti‐p53 antibody, as previously described (González‐Suárez et al., 2000) (see Materials and methods). There is no significant variation in p53 levels between wild‐type and K5‐mTERT (T1) untreated skin or DMBA + TPA‐induced papillomas (Figure 8A), suggesting that transgenic telomerase expression does not affect normal p53 levels; as a control, we show the skin of a p53‐deficient mouse (Figure 8A). Furthermore, there was no significant difference in Ras protein levels in nuclear extracts prepared from wild‐type and K5‐mTERT (T1) primary keratinocyte cultures, as determined by western blot (Figure 8B). Detection of Ku70 nuclear protein was used as a loading control (Figure 8B).
It has been shown recently that TERT‐driven cell proliferation in cultured cells can result in the activation of the c‐myc oncogene (Wang et al., 2000). We studied c‐myc mRNA levels in the skin from several K5‐mTERT (T1 and T8, as indicated) mice and corresponding wild‐type littermate controls. No significant differences in the c‐myc mRNA levels were observed between genotypes (see Materials and methods; Figure 8C and Table II for quantitation of c‐Myc levels using a phosphoimager), suggesting that transgenic mTERT expression in the skin does not result in c‐myc upregulation.
We show here that constitutive high levels of telomerase activity in stratified epithelia do not alter the normal epithelium structure and are not associated with changes in p53, Ras or c‐Myc levels, in agreement with previous reports showing that TERT immortalized human cultured cells do not show changes associated with transformation (Jiang et al., 1999; Morales et al., 1999). However, following DMBA + TPA treatment of the skin, K5‐mTERT (T1 and T8) mice are significantly more susceptible to the development of neoplasias than the corresponding wild‐type littermates. This increased tumor susceptibility of K5‐mTERT transgenic skin is accompanied by a greater proliferation of basal keratinocytes upon TPA treatment, as well as by an increased would‐healing rate of the K5‐mTERT skin compared with wild‐type mice. All these observations are in agreement with a direct or indirect role for telomerase in signaling proliferation under mitogenic conditions. These results agree with previous observations that indicated that first generation Terc−/− mice, which lack telomerase activity but still have long telomeres, show a lower incidence of papillomas than wild‐type mice (González‐Suárez et al., 2000). These findings unravel the potential risks of gene therapy for age‐associated diseases based on telomerase upregulation, and indicate that telomerase upregulation, which occurs in the vast majority of mouse and human tumors, actively promotes tumor growth even in the presence of long telomeres. Since the average length of telomeres in wild‐type and K5‐mTERT skin was similar, it is tempting to speculate that telomere conformation must be different whether or not telomerase activity is present in the cell, and that telomeres that are being 'maintained' by telomerase could be signaling proliferation and/or survival under mitogenic conditions.
Finally, K5‐mTERT mice constitute a new experimental model to study the direct impact of transgenic telomerase expression in both skin tumorigenesis and aging in vivo. Tissue‐specific telomerase‐transgenic mice are essential to study the impact of potential use of telomerase for gene therapy of age‐related diseases. K5‐mTERT mice also constitute a new animal model system to study the effectiveness of anti‐telomerase therapies in cancer.
Materials and methods
Generation and genotyping of transgenic K5‐mTERT mice
An EcoRI fragment containing the full‐length mTERT cDNA and 3′‐untranslated sequences was cloned 3′ downstream of 5 kb of bovine keratin K5 regulatory sequences (see Figure 1A) (Ramírez et al., 1994; Martín‐Rivera et al., 1998). The transgene was digested with NotI, purified using Genclean (Bio 101 Inc.), adjusted to a final concentration of ∼2 μg/ml and microinjected into (C57BL/6 × DBA/2) mouse embryos, as described (Hogan et al., 1986). Eight different founder mice were identified by Southern blot analysis of tail DNA, using the 5 kb fragment corresponding to the bovine keratin K5 promoter as a probe (not shown). The experiments described here were carried out with founder T1 and were reproduced, as indicated, with founder T8, which shows a similar upregulation of telomerase activity (T1 = 8043 and T8 = 7664; highlighted in red in Figure 1B). The other founders showing lower reconstitution of telomerase activity in the skin were not included in this study.
Handling of mice
Mice were housed in our barrier area, where pathogen‐free procedures are employed in all mouse rooms. Quarterly health monitoring reports have been negative for all pathogens in accordance with FELASA recommendations (Federation of European Laboratory Animal Science Associations).
Telomerase assays
Telomerase activity was measured with a modified telomeric repeat amplification protocol (TRAP), as described (Blasco et al., 1997).
Tumor induction experiments
Ten and 12 age‐matched (8‐ to 12‐week‐old) mice of each genotype, wild‐type and K5‐mTERT (T1), respectively, were shaved and treated with a single dose of DMBA (0.1 μg/μl in acetone; Sigma). Nine age‐matched (8‐week‐old) mice of each genotype, wild‐type and K5‐mTERT (T8), were shaved and treated with a single dose of DMBA (0.1 μg/μl in acetone; Sigma). In all cases, subsequently mice were treated twice weekly with TPA (12.5 μg in 200 μl acetone each treatment; Sigma), for 15 weeks. Four control mice of each genotype were treated with acetone only. In samples analyzed after weekly cumulative TPA doses, the TPA concentration used was 12.3 μg in 200 μl of acetone.
Histopathological analyses and immunochemistry
Papilloma, skin, esophagus, forestomach, oral mucosa and vagina sections from 15‐ to 19‐month‐old wild‐type and K5‐mTERT (T1 or T8, as indicated) mice were fixed in 10% buffered formalin and stained with hematoxylin–eosin. Images were captured with an Olympus‐Vanox microscope at a 20× magnification.
For the DMBA + TPA‐treated mice that died, necropsy of animals was performed and all organs were examined macroscopically. Tumoral and normal tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections of 4 μm were stained with hematoxylin–eosin. Images were captured with an Olympus‐Vanox microscope at a 20× magnification.
Immunohistochemistry for p53 was performed on deparaffinated sections (Vectabond slides) after pressure cooker processing. For p53 detection, we used a streptavidin–biotin complex technique, with a polyclonal rabbit anti‐mouse p53 antibody, CM5 (Novocastra Laboratory Ltd; 1:1500 dilution, overnight, 4°C), and a biotinylated anti‐rabbit IgG (Vector Laboratory; 1:400 dilution, 30 min, room temperature), followed by streptavidin peroxidase (Zymed Laboratory, Inc.; 1:20 dilution, 30 min, room temperature); the chromogen was developed with diaminobenzidine, and slides were counterstained with hematoxylin. Control slides were obtained by replacing the primary antibody with TBS (not shown) (see also González‐Suárez et al., 2000).
Wound‐healing experiments
Two to three full‐thickness punch biopsies extending through the epidermis and dermis (punch diameter 4 mm; PFM, Köln, Germany) were performed in six wild‐type and six K5‐mTERT mice (2–4 months of age) after depilation. Mice were anesthesized prior to wound creation. The wound‐healing rate was calculated as the percentage of initial wound area with time. Wound areas were calculated with the formula (area = πr2; where r is the ratio of the wound).
Telomere length measurements
For Q‐FISH, paraffin‐embedded skin sections (untreated or treated with DMBA + TPA) were hybridized with a PNA‐tel probe, and telomere length was determined as described (Zijlmans et al., 1997; González‐Suárez et al., 2000). Slides were deparaffinated in three xylene washes (3 min each), then treated for 3 min with a 100, 95 and 70% ethanol series. More than 50 keratinocyte nuclei from each genotype were captured at a 100× magnification and the telomere fluorescence was integrated using spot IOD analysis in the TFL‐TELO program. For Flow‐FISH, primary mouse keratinocytes (105) were prepared (Hennings, 1994) and hybridized using the Flow‐FISH protocol (Rufer et al., 1998). Telomere fluorescence of 2000–3000 nuclei gated at the G1−G0 cell cycle stage was measured using a Coulter EPICS XL flow cytometer with SYSTEM 2 software. The same cells were hybridized in parallel without the PNA–fluorescein isothiocyanate (FITC) probe to obtain background fluorescence values, which were subtracted from the fluorescence intensity of each sample. We used three wild‐type and six K5‐mTERT (T1) primary keratinocyte cultures.
Western blots
Nuclear extracts were prepared from three primary wild‐type and four K5‐mTERT (T1) keratinocyte cultures (Hennings, 1994), and western blots were performed as previously described (González‐Suárez et al., 2000). The protein concentration per well was 20 μg. For Ras detection, pan‐ras antibody (Ab‐3; Calbiochem) at a 1:1000 dilution was used. For Ku86 detection, anti‐human p70 (Hamiya Biomedical Company, 1:000 dilution) was used. The percentage of the acrylamide gel was 15%.
Northern blots
Total RNA was extracted from the skin of a total of four wild‐type and four K5‐mTERT (T1 or T8, as indicated) littermate mice essentially as described (Chomczynski and Sacchi, 1987). A 50 μg aliquot of RNA was used per lane in the northern blot. A 1500 nucleotide fragment of the human c‐myc gene was used as probe for murine c‐myc mRNA detection; as a positive control, total RNA from murine lymphocyte cell line Ba/FO3 upon interleukin‐2 stimulation was used (Hatakeyama et al., 1989). As control for loading, actin was also detected in the blots.
We are indebted to F.Larcher for help in isolating primary keratinocytes. We thank E.Santos and Rosa Serrano for mouse care and genotyping, P.Jurado for her contribution to the construction of the K5‐mTERT transgene, and M.Serrano and C.Mark for critical reading of the manuscript. E.G.‐S. is a predoctoral fellow from the Fondo de Investigaciones Sanitarias. E.S. is a predoctoral fellow from the Regional Government of Madrid (CAM). Research at the laboratory of M.A.B. is funded by Swiss Bridge Award 2000, by grants PM97‐0133 from the Ministry of Science and Technology (MCT), Spain, 08.1/0030/98 from the CAM, FIGH‐CT1999‐00009, FIGH‐CT‐1999‐00002 and QLG1‐1999‐01341 from the European Union, and by the Department of Immunology and Oncology (DIO). Research at the laboratory of J.L.J. was funded by grant PM98‐0039 from the MCT. The DIO was founded and is supported by the Spanish Research Council (CSIC) and by the Pharmacia Corporation.
- Copyright © 2001 European Molecular Biology Organization


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Hike for June 3 - Champagne-sur-Seine - Vulaines-sur-Seine/Samoreau - Fontainebleau
The hike will start in Champagne-sur-Seine (right bank) and will end at Fontainebleau-Avon train station. 17k, leave or take.
We will walk through the forest to Vulaines-sur-Seine/Samoreau. After lunch there will be a bail-out point as we will be near the Vulaines-sur-Seine/
Samoreau train station (9k). However, getting on the train at this station may be tricky as the step (footboard) can be very high and very narrow and you have to stand on it to push the door handle up, no problem if several people bail out and can help each other. Also in Vulaines-sur-Seine, a museum dedicated to French poet Stéphane Mallarmé. http://www.musee-mallarme.fr/ root
Then we will cross the river to the forest.
There will be a few ups and downs, a short steep ascent at the very start, with steps.
If mosquitoes like you, come prepared, there may be a few in the morning. Wear good footwear, poles are a good idea. Carry enough water. Lunch should be around 12:15/30pm.
All hikers need personal accident and liability insurance.
-Take train GAMU from Gare de Lyon direction/to Montargis at 9.19, arriving at Melun at 9.44, then change for train KOHO to Montereau at 9.50 arriving in Champagne-sur-Seine at 10.15 ; Zone 5. Departure should be from Hall 1-lettered platforms- but check on arrival. I will meet you at Gare de Lyon, we will sit in the last car or one before last if the last car is crowded.
-Return trains from Fontainebleau-Avon every half hour. 16.00 (arr:16.41) - 16:30 (arr:17.15)
There is a brasserie next to the train station.
-Return trains from Vulaines-sur-Seine/Samoreau Via Melun, every hour at .47: 13.47-14.06/14.13 arr Gare de Lyon 14.41…
Unless you have a "Passe Navigo" or benefit from discounted fares, buy a "Mobilis 5 zones" (16.60€)
See you on Wednesday!
ps: Sorry, but I don't have a cell phone.

subset: commoncrawl


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The Origins of Guided Self Healing
Guided Self Healing draws from the four historical traditions in psychology plus an emerging fifth tradition. It pulls from
Cognitive Behavioral Therapy
that people have beliefs that on a deeper level they know are limiting and untrue, and that transforming these beliefs, and the behavior that results from them, transforms people's lives. Like cognitive behavioral therapy and
Positive Psychology,
it recognizes our personal sense of agency, with all of the responsibility and accountability this implies.
From the
world, it draws the idea that the inability to resolve conflicting forces leads to difficulty and that the uncovering of the roots of the conflict leads to greater psychological wellbeing. It draws from
Psychoanalysis, Depth Psychology
and the
that people experience life through the lens of very rich and comprehensive archetypal stories and when one becomes aware of and in different relationship with these stories, people are freer. From
Post Modern Narrative Therapies
, it draws the idea that we are embedded in these stories, and if we could externalize the problems inherent in them and experience them as opportunities to resolve problems, as opposed to them being statements of who we are, shifts can occur.
From the
world, it opens us to the realization that we are evolving souls, and there is greater meaning to what happens to us as human beings. From
Humanistic Psychology,
it deeply acknowledges the ascendency of each person's existential, subjective experience. From
Transpersonal Psychology,
it recognizes the existence of a multidimensional world. From
Buddhist Psychology
, we gain the concept of mindfulness, awareness, allowing and self acceptance in the service of the ultimate transmutation of self. From many traditions, we realize there are three fundamental problems for human beings: craving, fear and overwhelm. The resolution of these problems opens us to the possibilities of loving relationship, the wisdom of understanding, and action based in true presence.
Systemic Theory,
it recognizes the importance of context and the mutually influencing interconnectedness of all of life. Problems are ultimately only solvable when you consider all that could possibly impact them, both directly and indirectly. The problem's impact also needs to be considered.
There is an emerging tradition that incorporates recent advances in trauma and body-centered therapies and energy psychology
From the advances in
Trauma Therapy
, it acknowledges the powerful ideas of post traumatic stress and the profound anxiety and stuckness that occur when we are unable to integrate experience. From
Body-centered Psychotherapy
, it opens to the realization that it is far more powerful to experience life in its immediacy instead of just reflecting on and talking about it. From
Energy Psychology
, we garner the provocative idea that acupressure can release traumatic blocks, as can crossing midlines (think EMDR) and that working with chakras and fields can improve our sense of presence and wellbeing.
We can see that Guided Self Healing incorporates and synthesizes concepts and contributions from many different approaches. What distinguishes it from other therapeutic modalities is that it integrates these into an inclusive holistic framework where the whole is greater than the sum of its parts.
"While there are anecdotal reports supporting the effectiveness of Guided Self-Healing ("GSH"), there is no current scientific research supporting its effectiveness in the absence of other techniques. Dr. Hahn also includes standard psychological methods in assessment and psychotherapy."
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Our Services
Space provides a vital source of support to Aylesbury in a time of much need in our town. (past Mayor of Aylesbury, Cllr. Ranjula Takodra)
The Space project does very valuable work within the local community, providing support to many vulnerable people going through hard times for a variety of reasons. The one to one counselling sessions, weekly group drop ins and workshops run by Space help to give their users back a sense of self belief and the ability to work through their problems. (David Lidington, MP for Aylesbury)
Space is an excellent service, very valuable and much needed. It helped to have someone caring to talk to.
It provides me with a lot of company.
A place to go when you are feeling alone.
I used to come to Space as a member, seeking support when I was going through a very dark and lonely time. Now life is a lot easier to cope with for me and I enjoy working as a volunteer giving something back and helping others see some light too.
It feels like an air-pocket, a light-house, an oasis. It's the only space where i feel being loved unconditionally, where my mind and soul are nourished, where i get to know my own humility and others. It is my most effective 'anti-depressant' without the harmful side effects. Life without Space? It would feel like being 'homeless'!


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2018 Port Operating Handbook
The Port Waters of Geelong Operating Handbook provides advice and instructions for ships masters, agents and owners responsible for entering the port waters of Geelong.
Port Waters of Geelong - Operating Handbook
Click Here to view the Port Operating Handbook - (Updated 1/10/18)
Geelong Port Harbour Masters Directions
Click here to view the Geelong Harbour Masters Direction
Port Water of Hastings - Operating Handbook
2017 Port of Hastings Operating Handbook
Port of Hastings Harbour Masters Directions
Click Here to view the Hastings Harbour Masters Directions - 1/7/2017
Update Notices
Click Here to go to the Notices to Mariners
Marine Notices are issued by AMSA and contain matters of National relevance.
Click Here to view the Federal Marine Notices
The current Port Operating Handbook is available from the Victorian Regional Channels Authority or alternatively it can be downloaded from the VRCA website.


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For a very important date.
A due date, that is, which has come and gone.
I've never been late before, and it's very much driving me nuts. Logically, I realize a woman can't be pregnant forever. This baby must come out. But how can she still be sitting in there, so tight and cozy, when her date has come and gone? I feel like she's becoming an eternal part of me and will never leave.
Before everyone starts giving me advice for encouraging her exit, I've already heard and probably already tried it. Multiple times, because even if she wasn't ready three days ago, she might be now, right?
People keep saying wonderfully observant things like, "You're still pregnant!" Oh, is that what the problem is? I wasn't sure. Or this one: "Are you having contractions?" Yes, and I have been for three weeks. Would you like to know my sleep patterns as well? But this one's the worst: "How are you feeling?" Take a guess. Take a wild guess. I'm overweight with a painful contracting watermelon in my belly, I don't sleep well, my feet, hands, and face are swollen, I can't bend over, everything aches--how do you think I'm feeling?
About the only thing you can say to me is, "I'm so sorry." And then move on. Let's turn the conversation another direction.
I try to keep myself distracted by filling my calendar with events, but as each day approaches, I secretly hope I'll have to cancel because I have a new baby. As much as I love my writer's group, I'm not thrilled that I'll be making it to our meeting tonight. As close as the airport is, I'm not excited that I'm available to give my mom a ride. Even though I planned out our dinner menu for the week, I wish I wasn't cooking it. It's great to get these things accomplished, but I wish I couldn't.
For you on the outside looking in, you know with an even clearer head than I that it's only a matter of days. She will come. And I'll be glad I got so much done before she got here. So what's the big deal?
Well... if you've ever been late, I'm sure you know.
Since I'm still pregnant, my goal today is to tally up the blog tour points and get those posted! Oh, and Inevitable goes free for ONE DAY ONLY tomorrow! I'll give you links tomorrow!


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Who I am and why I write
In my real life, off of the Internets, I am a sociologist...this means I am qualified to look at a situation, state the obvious about it and add statistics to back up said statements e.g., Balls bounce down 50% of the time and up 50% of the time. For the most part, I resist my training and attempt to navigate the depths of life issues and resolve them from the root...which is probably the reason I didn't get along with a single professor in college.
Update on the Cookie Series
First I want to thank those who are coming from the old website. This is now where you will find the latest news about my work.
Speaking of news, I am pleased to announce that Parts II and III of Cookie are out for editing and will be available soon. I'll announce their release dates as soon as I get them.
As a reminder, Part I is available here on the site from the "Bookstore" page. For anyone having trust issues with the PayPal link on this site, the book is also available on Amazon.com at a $2.00 price difference. Buy from me and save.
In addition, all purchases of Part I directly from me by June 30, 2014 will receive Part II free of charge on it's release!
January 07th, 2014
Hello Readers, and welcome to my little page. I thought I would take the time to introduce myself and answer what most people are probably wondering....why is this chick calling herself Lady Jaye? No, I'm not royalty LOL! It's pretty simple. I'm just me and although I love the Internet, I also love my privacy. This way I can share the best of my stories on the D/L.
About Cookie
Cookie is designed to be at least a 4 part short story series. I haven't yet made a final determination on length. I wrote it this way to allow for digestion of the story and it's pretty intense emotional content. I do not recommend it for readers under the age of 18 without parental consent.
I sincerely hope you enjoy and will leave me comments as you get started with the story or send me email! I love email!
Thank you for stopping by!
January 07th, 2014
I am truly excited about the initial responses from you guys! Please keep bearing with me and the manual ordering process. I want to eventually automate delivery but it's going to take a little time.
*Note to first time Mac ebook readers* I have just been made aware that Mac/iPad/iPod users need to install software prior to getting their epub formats to work but once it's installed, it never has to be again. I am working on getting a good list of free apps for the iPad and iPod for you! I am happy to help get your different versions up and running so that you can enjoy Cookie!


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Cardiff Metropolitan University is holding a Creative Writing Symposium 'Writing Between the Lines' on Saturday 3rd September.
The event will be led by Cardiff School of Education Academic Associates Dr Lucy Windridge and Selina Philpin and supported by academic staff Dr Kate North and Prof Jeff Wallace.
The symposium places a focus on how creative writing combines the philosophical enquiry of how we understand ourselves and the world we live in, with practice and self-reflection as a method of research.
The event is set to bring together postgraduate candidates, teachers and practitioners to examine the relationship between theoretical study and creative practice with the aim of improving and upholding the sustainability of this expanding discipline at Masters and Doctoral level.
The symposium is open to contributors from all subject areas and disciplines, though it is anticipated that it will be of principal appeal to those interested in creative writing as a research methodology within the following fields: literary studies; visual arts; philosophy; creative writing; film production; education; performance; cultural geography; critical and cultural theory.
Dr Lucy Windrige said: "Creative writing is an ever-changing, ever-shifting concern in the business of examining the nature of being. Through the groupings of new assemblages of ideas, words and critical thinking, the practice of writing generates original thought.
Selina Philpin continued: "Although it is clear creative writing is a discipline which can involve a high degree of intellectual input, it is still difficult to pin down and justify it as a specific research methodology, something which we hope to address via this event."
For more information and to register, please contact: firstname.lastname@example.com or visit the website: www.cardiffmet.ac.uk/BetweentheLines


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July 1, 2014
COLUMBUS, Ohio – The Big Ten Conference has revealed the schedule of league games for 2015, the inaugural season of men's lacrosse as a B1G-sponsored sport. Ohio State will host Johns Hopkins and Maryland in 2015, while traveling for games at Penn State, Michigan and Rutgers.
"It is exciting for our program to now have the chance to compete for Big Ten championships like many other Buckeye teams," Nick Myers, Ohio State head coach, said. "We understand the history and tradition of the Big Ten Conference and are looking forward to the opportunity to contribute to Ohio State's success in the league."
Ohio State's first B1G match will be at Penn State the weekend of March 27-29 in State College, Pa. The Buckeyes and Nittany Lions have met each season since 2003 and 18 times overall, with Ohio State 5-3 in the last eight meetings between the teams.
The weekend of April 3-5, the Buckeyes will host Johns Hopkins, which was added to the B1G as an affiliate member for men's lacrosse only. The teams faced off in Baltimore in 2014, with the Blue Jays coming away with a 10-9 win in triple overtime. The 2015 matchup will be the sixth between the teams, with the series dating to 1999.
The Buckeyes and Wolverines will face off as Big Ten foes for the first time the weekend of April 10-12 in Ann Arbor, Mich. Michigan moved to Division I for the 2012 season, with Ohio State winning all three matchups between the teams since that time. In 2014, the Buckeyes downed the Wolverines, 15-6, in the Showdown in the Shoe in Ohio Stadium, and Ohio State won the last meeting in Ann Arbor, 17-8, in 2013. Overall, Ohio State and Michigan have met 15 times (dating to 1966), with Ohio State 12-3 in those games.
Up next for the Buckeyes will be a home game vs. new B1G conference member Maryland the weekend of April 17-19. The game will be the fifth in the all-time series between the teams and the first since an NCAA first round game in 2003 in College Park, Md. The series between the Buckeyes and Terrapins began in 1990 and the 2015 game will be the first between the teams in Columbus.
The Buckeyes' B1G schedule wraps up with a game at new conference school Rutgers the weekend of April 24-26. The series between Ohio State and the Scarlet Knights began in 1953, with the last matchup a win by the Buckeyes in 1999 in Piscataway.
The first Big Ten Tournament will be hosted by Maryland in College Park, Md. The tournament will featured a four-team field, with two semifinal matches April 30 and the championship May 2.
Specific dates for each B1G game, as well as the Buckeyes' complete non-conference schedule, will be announced at a later date.


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Thank you to everyone who has posted on this blog! I had no idea how many people would be posting here!
I've learned so much from you, and hope I've provided you with some good information as well.
I'd love your input as to how I can make this blog even better. Wii fit running and Wii fit yoga are popular topics on this blog. As we continue to talk about those two fun features of the Wii fit, what else would you like to see featured on this Wii blog? I'd love to hear your thoughts!
Thank you again for posting on my "WiiScoop" blog! I started this blog as a hobby, and am just so happy to see there are others here that like the Wii just as much as I do!
I hope everyone is having a great weekend!
Happy Gaming,


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Debt. It's a word that gives all of us the same sensation, and a burden which, in varying capacities, can weigh us down. But these are challenging times, and it's fair to say that the stigma for many surrounding debt has changed from one of borderline shame to begrudging reality. But it's quietly becoming a big problem for our country, with 2.5 million Brits now dependent upon credit cards for day-to-day living, and total consumer debt rising by 10 per cent in the last 12 months.
We've all had to dip our toes in the credit card waters occasionally – not least of all when it comes to our children's birthday celebrations or Christmas. But no one wants to get into a debt spiral and set foot on that slippery slope. The good news is that the road to debt freedom doesn't need to be a long and arduous one. Just a few tweaks here and there can set you on your way so that you can not only get your head above water but establish long-term financial security for you and your family.
A budget facelift
Chances are, you won't need to make any dramatic changes to your spending, and it's always easier than you think to free up space in your monthly budget without making major sacrifices. But a good starting point is to write down all the things you spend your money on each day. Keep a record of it; even if it's just for a week. At the end of it, ask yourself: "Where can I cut down on costs?" Do you buy food out instead of a packed lunch? Could you look at buying clothes and other good second hand? Could you shop in bulk and save? Or consider free activities for your children, instead of pricey ones? All these things make a big difference when added together, and there are good tools to help you get to make these savings.
Emergency savings
It's easy to put a monthly figure in your mind as to what you need in order to get by. But every month is different. Think Christmas, birthdays, holidays, or big one-off purchases (eg: furniture). To avoid the shock factor when these events come around, why not set up multiple accounts, and squirrel away a few pounds into them during 'normal' months. That way, you'll have your bases covered when it comes to those irregular, unexpected purchases.
Get debt savvy
Take note: interest matters. And credit cards in particular charge a lot! So if you have multiple lines of credit, channel all your energies into dealing with the most expensive debts first, even if it means you're just making minimum repayments on your cheaper debts. This top-down approach will mean you start saving from day one. It may even be worth looking at getting a personal loan to consolidate your debts, thereby cutting the head off the monster.
Make Summer 2019 AWESOME!
Kids not old enough for summer camp?
Hold your own at home!
Make this summer with your kids awesome!
Balance transfers
Another effective option if you have high credit card debt is to look at a balance transfer. Here you simply transfer your existing credit card debt onto a card with a new provider, who in turn gives you a window (sometimes up to three years) in which they charge no interest or fees. That way you can chip away without fear of the debt getting away from you. Careful though – if you don't pay it all off within the window, the charges can be hefty!
Debt management plans
For those with more severe problems with debt, there is the debt management plan option. In this case, you make payments to a company, who then shares this money around with your various creditors in order to pay them off. Companies such as Payplan do this without charging any fees, thus making it a perfectly viable option if you would like to avoid the burden of deciding the optimal amounts to allocate to each debt.
Of course, not all debt is bad. Something like a mortgage is actually an achievement for our generation, and there are many ways in which debt can help to set us up for the future. But of course, the end goal is always going to be financial independence and a clean slate from any such obligations. But as the above shows, you can achieve this sooner than you think, and without having to give up too much in the way of time, effort or pleasure. So don't delay, and get moving on a plan that suits you today.
This is a sponsored post on behalf of Lending Works


text: Contact the Studio You can contact the studio directly through the form on this page. If you'd like to phone or send a traditional email please use the links on the bottom of the page. Either way, we'd love to hear from you. Your Name* First Last Email* PhoneThe best way to contact youEmailPhoneWhat are you interested in studying? Piano Guitar Voice Message*How did you hear about West Seattle Music Studio? Internet Search West Seattle Blog City Scene B-Town Blog Current Student Friends/Family Join our Mailing ListYesNo This iframe contains the logic required to handle Ajax powered Gravity Forms.


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Guinot Franchise
Stand out from competitors with our globally recognised brand name!
Established for over 50 years, Guinot remains a pioneering skincare brand. It is the number one professional brand in France. Driven by results, and famous for its machine-based and manual treatment technologies, Guinot is continuously investing in research and development to remain at the forefront of the industry.
You will find Guinot treatments and products in over 10,000 salons in 70 countries worldwide. It is only available through appointed salons and spas, and most recently, through its growing network of franchise salons. The success of the franchise model in France has lead Guinot to bring the exciting salon franchise opportunity to the UK and Ireland.
We are currently seeking individuals to be part of our UK expansion. If you are hard working, focused and willing to follow a proven business system, we would love to hear from you.
The Guinot franchise opportunity
Guinot has a recognised and established brand image which vastly increases the chances of making your salon business a huge success in such a competitive market. Our iconic interior design is instantly recognisable to help with brand promotion, and as members of the British Franchise Association (bfa), a Guinot franchisee can be assured of ethical business practices and fair franchising.
"Opening a business is always a risk, but with Guinot's strong brand and highly experienced team, we would recommend this opportunity to other first time salon owners looking to enter the beauty salon industry, or to existing salon businesses that want to take their business in a new direction. I would highly recommend working with Guinot... If you have the passion and perseverance to make a business in the beauty industry work, then I would recommend Guinot every time!"
Tina Rai, Guinot franchisee, Gerrards Cross
The role of the franchisee:
Franchisee responsibilities will include tasks such as:
- Managing the daily operation of the business
- Managing payroll and admin for your employees
- Active networking and promotion of the franchise business within your local community
- Recruitment, motivation and management of a team
- Monitoring of individual therapist performance and salon business performance indicators
- Implementing marketing initiatives
Two ways to franchise with Guinot:
1. Conversion
This opportunity is ideal for you if you already own a salon and want to get a step ahead of the competition and take your salon to the next level. Or, if you own a salon and are thinking of retiring in 5-10 years, this is a good opportunity for you to build a valuable business that you could sell on in retirement.
Statistically, Guinot salons in France that converted to a Guinot franchise experienced an average turnover increase of 20%.
2. New
This is the perfect opportunity for those with a passion for the beauty industry who do not have an existing salon, have little or no industry experience or for those looking to expand their business portfolio.
How much does the franchise cost?
The typical start-up cost for a NEW salon business is £90,000 - £130,000 (€100,000 - €145,000). This is inclusive of franchise fee, all set-up costs and operating expenses. Each project must be looked at individually, but these figures give a good indication of the potential outlays.
For an EXISTING salon looking to convert to a Guinot franchise, the start-up costs can be as low as £35,000 (€40,000), but again, each project must be considered individually.
Please note that due to bank lending, your personal contribution may only need to be 30-50% of the start-up cost. We can put you in contact with a franchise professional from a leading high street bank who will be able to assist you in securing a loan for the rest of the cost (subject to status and fulfilling certain criteria).
How we train and support you
- Local market analysis
- Support creating business plan – 3 years P&L, Cash Flow Forecast & Balance Sheet
- Site selection
- Salon design
- Assistance in staff recruitment
- Initial training
- Pre-launch marketing activity
- Merchandising
- Operating procedures
- Analysis of figures and regular review of KPIs
- Continuous training
- Ongoing marketing support
- Regional Seminars and National Conference
- Branded Software
- Website set-up and management
- Franchise-only Facebook forum
- Expansion
Once your salon has launched, you will receive marketing support throughout the year. Every 6 weeks you are provided with franchise-exclusive promotions to implement.
All supporting online and offline digital collateral is provided, along with easy to follow implementation guides and how-to tutorials to make sure you are up to date with the latest marketing channels and can use them effectively to promote your business.
Who we are looking for...
There are many attributes we look for in our franchisees, and although previous experience in the beauty industry is not necessary, possessing the following qualities really does help!
- Being an inspiring leader
- Business acumen
- Enthusiasm
- Dedication
- Determination
- Self-motivation
- Energy
- A passion for beauty
- A commitment to customer care
"I knew that I wanted a new challenge and that I wanted to be my own boss, and a Guinot franchise seemed like a great option for me. Although I had the confidence to run my own beauty salon, I didn't have the confidence to do it on my own. Teaming up with Guinot has proved successful and rewarding and I'm looking forward to the future"
Justyna Rostek, Guinot franchisee, Earlsfield


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save your Nokia phone's battery lifeJanuary 8th, 2013 by admin | No Comments | Filed in MOBILE
Sharetop ten tips to save your Nokia battery life. 1. Switch off your phone and restart your phone again, then you can restore your phone settings. stop your running applications. 2. Stop and remove your screen saver for saving your battery life. 3. set a simple theme for your phone. 4. you donot recharge your battery while your battery goes very low label 5. minimize your phone volume to save your battery lif
How to remove battery from laptop.January 7th, 2013 by admin | No Comments | Filed in HARDWARE
Share He your laptop battery does not work properly or damage your battery then you can remove your battery from your laptop. To remove your laptop battery please follows the following step carefully.Step1: first you shutdown your computer and disconnect your charges cable from your laptop and also disconnect other accessories (example: speaker, DSL cable, USB cable etc.) step2: you close your LID (laptop s


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NEW ZEALAND No 2 OUTPOST CEMETERY
No. 1 and 2 Outposts were respectively South and North of the Fisherman's Hut. It was near these points that the 7th and 12th Australian Infantry Battalions landed on 25th April 1915; and on the 30th the Posts were made by the Nelson Company of the Canterbury Infantry Battalion NZEF, who buried there some of the Australian dead. No. 2 Post was the scene of heavy fighting at the end of May; and it was one of the starting-points for the Battle of Sari Bair. It contained the best well in Anzac, and the 16th Casualty Clearing Station and the New Zealand Dental Corps clinic were established near to it.
New Zealand No. 2 Outpost Cemetery was named from the burials carried out by the Nelson Company. It is, in fact, one long grave, on the East side of the Anzac-Suvla road, between No. 2 Outpost and Embarkation Pier Cemeteries. It was made in September 1915. The cemetery covers an area of 469 square yards, and it contains the graves of 2 named soldiers from the United Kingdom and 150 whose names and units are alike unknown. Special memorials are erected to 13 soldiers from New Zealand, 10 from the United Kingdom and 8 from Australia, known or believed to be buried in the cemetery. On the North and South sides of the cemetery are thick belts of shrubs, and on the East side a belt of trees thirty feet wide.
New Zealand No.2 Outpost Cemetery is a small cemetery within 100 metres of No.2 Outpost Cemetery, on the east side of the Anzac-Suvla road near Embarkation Pier Cemetery.
Colonel Neville Manders Australian Army Medical Corps AIF
- Killed in action 9th August 1915, aged 55.
- Deputy Director of Medical Services (DDMS) at ANZAC - one of the senior medical officers.
- Was killed by a stray bullet while having breakfast at No 3 Outpost.
Captain C.G.Lutyens 6th Bn East Lancashire Regiment
- Killed in action 9th August 1915, aged 28.
- From Hascombe, Surrey.
- Relative of the architect Edwin Lutyens.


text: Options Full Datasheet Printer Friendly Datasheet Prepare for Printing Full Datasheet Printer Friendly Datasheet Prepare for Printing Have Questions? Ask Us! Request A Quote Today! Add To Compare List anti-ADAMTS4 antibody :: Mouse anti-Human A Disintegrin And Metalloproteinase With Thrombospondin 4 (ADAMTS4) Monoclonal Antibody Catalog # MBS2110708 Unit / Price 0.1 mg / $220 +1 FREE 8GB USB 0.2 mg / $290 +1 FREE 8GB USB 1 mg / $690 +1 FREE 8GB USB Product Name Product Info Accession #s Product Desc Diseases/Tissues/Pathways Applications References Product Name A Disintegrin And Metalloproteinase With Thrombospondin 4 (ADAMTS4), Monoclonal Antibody Full Product Name HRP-Linked Monoclonal Antibody to A Disintegrin And Metalloproteinase With Thrombospondin 4 (ADAMTS4) Product Synonym Names ADMP-1; Aggrecanase-1 Product Gene Name anti-ADAMTS4 antibody[Similar Products] Matching Pairs Unconjugated Antibody: A Disintegrin And Metalloproteinase With Thrombospondin 4 (ADAMTS4) Clone #1# (MBS2001335)HRP Conjugated Antibody: A Disintegrin And Metalloproteinase With Thrombospondin 4 (ADAMTS4) Clone #D4 (MBS2110708) Matching Pairs HRP Conjugated Antibody: A Disintegrin And Metalloproteinase With Thrombospondin 4 (ADAMTS4) Clone #D4 (MBS2110708)Immunogen: A Disintegrin And Metalloproteinase With Thrombospondin 4 (ADAMTS4) (MBS2009500) Research Use Only For Research Use Only. Not for use in diagnostic procedures. TOP Immunogen Sequence Thr437~Pro 575 OMIM 603876 3D Structure ModBase 3D Structure for O75173 Clonality Monoclonal Isotype IgG Clone Number D4 Host Mouse Species Reactivity Human Specificity The antibody is a Mouse monoclonal antibody raised against ADAMTS4. It has been selected for its ability to recognize ADAMTS4 in immunohistochemical staining and western blotting. Purity/Purification Antigen-specific affinity chromatography followed by Protein A affinity chromatography Form/Format Liquid Concentration 500ug/ml (lot specific) Conjugation HRP Preparation and Storage Store at 4 degree C for frequent use. Aliquot and store at -20 degree C for 12 months.Avoid repeated freeze/thaw cycles.The thermal stability is described by the loss rate. The loss rate was determined by accelerated thermal degradation test, that is, incubate the protein at 37 degree C for 48h, and no obvious degradation and precipitation were observed. The loss rate is less than 5% within the expiration date under appropriate storage condition. ISO Certification Manufactured in an ISO 9001:2008 and ISO 13485:2003 Certified Laboratory. Supply Chain Verification Manufactured in a lab with traceable raw materials. Bulk orders can typically be prepared to the customerâs specifications, please inquire. Other Notes Small volumes of anti-ADAMTS4 antibody vial(s) may occasionally become entrapped in the seal of the product vial during shipment and storage. If necessary, briefly centrifuge the vial on a tabletop centrifuge to dislodge any liquid in the container`s cap. Certain products may require to ship with dry ice and additional dry ice fee may apply. TOP Applications Tested/Suitable for anti-ADAMTS4 antibody Western Blot (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC), Immunoprecipitation (IP) TOP NCBI/Uniprot data below describe general gene information for ADAMTS4. It may not necessarily be applicable to this product. NCBI GI # 157427675 NCBI GeneID 9507 NCBI Accession # NP_005090.3 [Other Products] NCBI GenBank Nucleotide # NM_005099.6 [Other Products] UniProt Primary Accession # O75173 [Other Products] UniProt Related Accession # O75173[Other Products] TOP NCBI Official Full Name A disintegrin and metalloproteinase with thrombospondin motifs 4 isoform 1 preproprotein NCBI Official Synonym Full Names ADAM metallopeptidase with thrombospondin type 1 motif 4 NCBI Official Symbol ADAMTS4 [Similar Products] NCBI Official Synonym Symbols ADMP-1; ADAMTS-2; ADAMTS-4 [Similar Products] NCBI Protein Information A disintegrin and metalloproteinase with thrombospondin motifs 4 UniProt Protein Name A disintegrin and metalloproteinase with thrombospondin motifs 4 UniProt Synonym Protein Names ADMP-1; Aggrecanase-1 Protein Family A disintegrin and metalloproteinase with thrombospondin motifs UniProt Gene Name ADAMTS4 [Similar Products] UniProt Synonym Gene Names KIAA0688; ADAM-TS 4; ADAM-TS4; ADAMTS-4 [Similar Products] UniProt Entry Name ATS4_HUMAN TOP NCBI Summary for ADAMTS4 This gene encodes a member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) protein family. Members of this family share several distinct protein modules, including a propeptide region, a metalloproteinase domain, a disintegrin-like domain, and a thrombospondin type 1 (TS) motif. Individual members of this family differ in the number of C-terminal TS motifs, and some have unique C-terminal domains. The enzyme encoded by this gene lacks a C-terminal TS motif. The encoded preproprotein is proteolytically processed to generate the mature protease. This protease is responsible for the degradation of aggrecan, a major proteoglycan of cartilage, and brevican, a brain-specific extracellular matrix protein. The expression of this gene is upregulated in arthritic disease and this may contribute to disease progression through the degradation of aggrecan. Alternative splicing results in multiple transcript variants, at least one of which encodes an isoform that is proteolytically processed. [provided by RefSeq, Feb 2016] TOP UniProt Comments for ADAMTS4 ADAMTS4: Cleaves aggrecan, a cartilage proteoglycan, and may be involved in its turnover. May play an important role in the destruction of aggrecan in arthritic diseases. Could also be a critical factor in the exacerbation of neurodegeneration in Alzheimer disease. Cleaves aggrecan at the '392-Glu-|-Ala-393' site.Protein type: EC 18.104.22.168; Secreted, signal peptide; Protease; Motility/polarity/chemotaxis; SecretedChromosomal Location of Human Ortholog: 1q21-q23Cellular Component: extracellular matrix; proteinaceous extracellular matrix; extracellular space; extracellular regionMolecular Function: peptidase activity; protein binding; protease binding; metallopeptidase activity; zinc ion binding; metalloendopeptidase activityBiological Process: extracellular matrix disassembly; extracellular matrix organization and biogenesis; defense response to bacterium; proteolysis; skeletal development Research Articles on ADAMTS4 1. The lower levels of ADAMTS4 in the primary ovarian insufficiency group, when compared with the idiopathic hypogonadotropic hypogonadism patients pointed out that even limited hormone secretion and ovulation in the POI group, may have protective effect on cardiovascular system. The higher levels of ADAMTS4 and lipid accumulation in the IHH group demonstrated the increased risk of these patients for cardiovascular disease. TOP Precautions All of MyBioSource's Products are for scientific laboratory research purposes and are not for diagnostic, therapeutics, prophylactic or in vivo use. Through your purchase, you expressly represent and warrant to MyBioSource that you will properly test and use any Products purchased from MyBioSource in accordance with industry standards. MyBioSource and its authorized distributors reserve the right to refuse to process any order where we reasonably believe that the intended use will fall outside of our acceptable guidelines. Disclaimer While every efforts were made to ensure the accuracy of the information provided in this datasheet, MyBioSource will not be liable for any omissions or errors contained herein. 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TOP Products associated with anti-ADAMTS4 antibody Reference Product PubMed Publications ACAN antibody >6 publications with ADAMTS4 and ACAN BCAN antibody >3 publications with ADAMTS4 and BCAN TGFB1 antibody >3 publications with ADAMTS4 and TGFB1 FURIN antibody >3 publications with ADAMTS4 and FURIN OSM antibody >2 publications with ADAMTS4 and OSM IL1B antibody >2 publications with ADAMTS4 and IL1B IL17A antibody >1 publications with ADAMTS4 and IL17A SRPX2 antibody >1 publications with ADAMTS4 and SRPX2 Pathways associated with anti-ADAMTS4 antibody Products by Pathway Pathway Diagram Endochondral Ossification Pathway antibodies Endochondral Ossification Pathway Diagram Diseases associated with anti-ADAMTS4 antibody Disease Name Pubmed Publications Inflammation Antibodies >16 publications with ADAMTS4 and Inflammation Neoplasms Antibodies >14 publications with ADAMTS4 and Neoplasms Necrosis Antibodies >14 publications with ADAMTS4 and Necrosis Arthritis, Rheumatoid Antibodies >7 publications with ADAMTS4 and Arthritis, Rheumatoid Nervous System Diseases Antibodies >5 publications with ADAMTS4 and Nervous System Diseases Atherosclerosis Antibodies >5 publications with ADAMTS4 and Atherosclerosis Brain Diseases Antibodies >5 publications with ADAMTS4 and Brain Diseases Heart Diseases Antibodies >4 publications with ADAMTS4 and Heart Diseases Myocardial Infarction Antibodies >4 publications with ADAMTS4 and Myocardial Infarction Carcinoma, Squamous Cell Antibodies >2 publications with ADAMTS4 and Carcinoma, Squamous Cell Organs/Tissues associated with anti-ADAMTS4 antibody Organ/Tissue Name Pubmed Publications Brain Antibodies >13 publications with ADAMTS4 and Brain Muscle Antibodies >4 publications with ADAMTS4 and Muscle Embryonic Tissue Antibodies >3 publications with ADAMTS4 and Embryonic Tissue Lung Antibodies >1 publications with ADAMTS4 and Lung TOP © 2006-18 MyBioSource.com. 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Favorite restaurants on Maui, Hawaii Island and Kauai
Must try: Holuakoa Gardens and Cafe (Hawaii Island), Honu (Maui), The Banyan Tree (Maui), Josselin's Tapas Bar and Grill (Kauai.)
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The portions are hearty, large enough to fill up a farm boy. But delicate touches present themselves throughout, reflecting this farm-boy chef's French Culinary Institute training in New York: a careful brunoise of carrots, gnocchi paired with a turmeric cream sauce threaded with preserved lemon peel for pops of brightness (though the gnocchi are more like flour dumplings than potato clouds).
It seems so improbable that such a restaurant exists, "in the middle of nowhere," which, more precisely, is Holualoa, 20 minutes up the mountain above Kona. It has a population of about 6,000 and the main drag of galleries would have you believe most of the residents are artists. In San Francisco, Holuakoa Café would be at home among the other locavore restaurants; in Honolulu, it would be the poster child for the farm-to-table movement. And yet, perhaps it can exist nowhere else. Read says "the people here are similar-minded, trying to express a healthy food system, a healthy community conspired by love." His local sourcing is enabled by the Big Island, which has the most and cheapest agricultural land in the state. This restaurant, a five-year-old project by a 49-year-old chef channeling Alice Waters, is exactly where it belongs.
Maui: Honu
1295 Front St., Lahaina, (808) 667-9390, honumaui.com.
Price range: Appetizers $3.50 to $27; entrées $15 to $69
A lot of the seafood in Hawaii is imported. Honu, Mark Ellman's latest venture, is one of the few establishments that owns up to this simple, sometimes inconvenient truth, by embracing American seafood in an open-air setting on Lahaina shores.
The menu is East Coast and West Coast seafood shack rolled into one, with Dungeness crab cocktails, fried Ipswich clams and Maine lobster rolls, plus a scattering of locally caught ahi dishes and catch-of-the-day preparations. It stirs up memories of coastal cities where I have lived. The Dungeness crab tank at the entrance of Honu reminds me of steam kettles of crab at San Francisco's Fisherman's Wharf. (Sadly, however, the inhabitants of Honu's tank lack the spunk of Fisherman's Wharf crabs; at least one of them is belly up. I can only hope the dead ones aren't served.)
Sweet chunks of Maine lobster coated lightly in mayonnaise overflow from a properly top-split, toasted Portuguese sweetbread roll. A bite takes me back to Boston college days; while others raided fraternity houses for free beer, we'd sneak lobsters from their walk-in refrigerators.
If Honu could just throw a beach clambake, my seafood nostalgia would be complete, and I'd be happy as, well, a clam.
But new memories are to be made here at Honu, sitting across from my date (my marriage to whom has signaled the end of fraternity-house raiding). It could hardly be more romantic, with the waves lapping at our feet . . . except we have just ordered the fried pig ears. Maybe the Pacific oysters on the half shell would have been a better aphrodisiac. The pig ears are actually addictive, if unromantic (even the whisper of lavender in the batter doesn't change this); they're like a porky, fatty version of fried calamari. They beg to be chased with beer, to wash away some of the grease.
More charming is the Dore-style monchong, dipped in flour and egg, sautéed, and finished with a lemon caper butter sauce. The fish is moist and meaty, propped up on a bed of nutty quinoa punctuated with pomegranate seeds. What really elevates this dish, though, is the dusting of dill pollen, which delivers a fresh, grassy punch with more intensity than dill alone.
Ellman's restaurants have occupied both ends of the casual/formal spectrum; he owns Mala Ocean Tavern, next door, and started (and sold) Maui Tacos. Honu doesn't exist as much in between as it does at each endpoint, with $39-and-up entrées and $16 pizzas, a concept that seems to appeal to families. There are many: a family to our left with a child folding slices of pizza into his mouth, another on the other side with a boy expertly cracking a salt-and-pepper crab (he would make a Chinese mother proud). I'm not a huge fan of the pizzas. While the pizza toppings are fresh and flavorful, like a spicy Italian sausage with arugula atop a bright San Marzano sauce, the crust is more California Pizza Kitchen—sweet and dense—than airy, Neopolitan-style.
Maui: The Banyan Tree at The Ritz Carlton
1 Ritz Carlton Drive, Kapalua, (808) 669-6200
Price: $80 for a four-course menu
Next to us, a man, sitting at the head of a large party, gestures too wildly and sends a bottle of wine flying into glasses. A team of servers descends and cleans up the spilled wine and broken glasses while the man stands to the side. ("Such a good wine," he laments.) In less than five minutes, the whole episode is over, a new bottle of wine is opened, joviality resumes and all is right again in the bubble of The Ritz Carlton. It seems there are few things The Ritz Carlton cannot fix.


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Getting a first hand report on emergency room health care the hard way
As some of you who follow me on Facebook know, I spent a good chunk of the day in the emergency room at Waukesha Memorial. I'm okay, and I'm taking it easy today at home with some pain killers and antibiotics. I'm not sure of the street value of this stuff.
It was not a heart attack (the ticker is fine) but a severe pain just below the ribs made me turn my car around and drive to the emergency room. Yes, I drove. Spare me the lecture. If I just stopped the car and called 911, God knows where the ambulance would have taken me. As long as I was capable of driving I was going to drive.
It's funny what you think of in those moments. My thought really was I needed to go to Waukesha Memorial because my wife would find me there.
The too-busy receptionist asked me what was wrong then gave some forms to fill out. She wouldn't even answer my questions. I doubt she heard me sarcastically yell at her, "Thanks for helping!" After that, I have to say the rest of the staff was very helpful.
I had to laugh at the technician or nurse that stuck my hand for the IV. She gave a version of the standard, "Now this might sting." Compared to what? The severe pain that was causing me to barely breathe? "Death, where is thy sting?" She got the IV tube in me and then they offered the morphine.
To give you an idea of the pain, the first blast (first ever) of morphine didn't help. I was even remembering bits of poetry by Coleridge. (I was the only one to get the joke.) However, when they upped the dosage – that was pretty good. Except staring straight ahead was like being in a glass elevator going down, only to suddenly start over and then back down.
By this time Doreen was there. The hospital wanted a urine sample. The nurse really wanted me to use the bedpan but, since he was not totally opposed, I opted for walking to the men's room with Doreen's help. "I've made it to plenty of bathrooms in bars," I assured them.
The EKG and the chest x-ray ruled out the heart attack. The staff was convinced it was a kidney stone except the pain wouldn't cooperate by being in the right place. No back pain, the pain wasn't moving, etc. The CT scan couldn't kind any kidney stones. Doreen and I thought it was my gall bladder but that's on the wrong side.
Doreen and I ran through my diet the last couple of days in case my recent healthier eating habits were trying to kill me.
Finally the doctor decided it must be an infection, as I have a nice little pocket of fluid just above the diaphragm. We're not 100% sure that's what it was, but given that I've been fighting fluid in my inner ears for the last week, it seems possible.
So I'm taking it easy today. For some reason nobody wants me to drive on these pain killers. But the good news is I'm okay, just a little shaken by the whole experience, and a little foggy from the Oxycodone. And I can't get the stupid probes off.
The things I do just to get out of cutting the grass.


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It's that time of year when we are rushing to holiday parties every weekend, sometimes we even do a little hosting too. So you know what parties means…appetizers! Whether your friends are stopping by quickly for a small gift or ornament exchange, or you're hosting a fun holiday bash, you'll likely need plenty of show stopping and easy appetizers to nibble on.
I've compiled a list of appetizers that are worthy of this festive season, both in looks and in flavor! Some of these appetizers are light and others are a little heavier so you can skip dinner. I won't say which ones are which 🙂
Smoked Salmon Dip is a holiday no brainer since it will save you all of the steps that are usually required by assembling a smoked salmon platter. It's ready in just a few minutes and will be a crowd pleaser.
This Cranberry Pistachio Baked Brie is downright gorgeous. Every party needs a Baked Brie (<– new party rule. You like?).
Mushroom Polenta Bites are the perfect addition to your holiday party spread, especially if you're looking for something a little heartier. This combo is a stunner.
Fully Loaded Guacamole isn't your average guacamole, which means that it is perfect for your holiday spread.
Need a bar snack? These crunchy roasted chickpeas are addicting, crunchy, and slightly salty…they're the ultimate bar snack for holiday parties.
Spanakopita is one of my favorite things in the world and these little bit sized Spanakopita Bites are totally party worthy. Plus, veggies. Right?!
Herb and Cheese Puff Pastry Straws are another great option for a bar snack. These are easy to make, but they will wow your guests.
And, one last one…Mini Taco Cups! Need I say more?!
Happy Weekend Friends! I hope you enjoy some marvelous snacks and appetizers.


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What hurts more: Vodka Or Whiskey?
Learn how certain types of alcohol can either worsen your hangover symptoms or possibly help to prevent a hangover.
Detoxicated is a natural hangover supplement formulated by a medical doctor. Using high-quality, natural ingredients that support the body's ability to metabolize alcohol, Detoxicated helps with hangovers by minimizing the toxic byproducts that cause them. This means that you can wake up feeling refreshed and ready to work, work-out, or just feel great. Combat your hangover naturally. Your body and liver will thank you.
Always drink responsibly. Never drink and drive.
Curious about how prickly pear helps with your hangovers? Learn about how these scientists discovered prickly pear as a potential natural hangover prevention.keep reading >
A potential hangover cure!? We hope so. Learn about why these leaves might become the next best natural hangover remedy.keep reading >


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The competitions included on the European calendar :
EUROPEAN CHAMPIONSHIPS (Lead – Bouldering – Speed)
Held every two years, the next European Championships will take place in 2019. Lead and Speed Championships will be held at the Edinburgh International Climbing Arena (UK) from 4-6th October. Bouldering dates and venue are yet to be confirmed.
Lead, Boulder and Speed Combined will be in April 2020, Moscow.
European Youth Championships (Lead – Bouldering – Speed)
The European Youth Championships are held every year.
European Youth Championships 2018 Overall Results
European Cup (Lead – Bouldering – Speed)
The European Cups (adults) started in 2013.
- These are the main guidelines:
- The rules will be the same as the European Youth Cups.
- Prize money is not mandatory. When prize money is provided, it must be provided to the top three climbers at a minimum (more to the first, then second, then third,...)
- The registration fee is an individual competitor fee divided in two: 15 euros paid to the IFSC Europe (via an invoice to the national federation) and 15 euros maximum paid to the organizer (at the registration desk of the competition)
- The calendar fee is 500 euros per event
European Youth Cup (Lead – Bouldering – Speed)
The results of the 2018 European Youth cup can be accessed via the following links.
Regional European competitions
- Adults or Youth
- These competitions are recognized by the ECSC in order to help the development of sport climbing. They are not included in any European ranking.
- National federations can group together in one event and may organize their national championships or competitions on this event.
- No specific rules are mandatory but event rules must be sent to the regional representative and validated by the Executive Board.
- International licenses are not required, but national licenses are mandatory with the individual insurance required in the hosting country.
- Open to all the relevant competitors of the European region as defined in the ECSC regulations, and only these competitors.
European promotional events
Promotional events, festivals, outdoor meetings, … are held under the same conditions as the IFSC promotional Events
New events
New events with new formats could arise and be tested in Europe.
Click here to download the European Rules 2019. (pdf File: 644.91 KB)
Click here to download the European Paraclimbing Rules 2013. (pdf File: 93 KB)


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Friday, February 28, 2014
sign of spring?
Looking for signs of Spring... I saw a robin yesterday and the kids and I stopped to watch it hop along the part of the yard where the snow has melted. More snow is on it's way in a few days and I've seen no flowers yet.
Time has seemed to stand still this week as I battle a cold and fever. Lots of knitting and lighting candles and soup.
I knit a pair of robin wrist warmers over 2 days from a Tiny Owl knits pattern. I'd like to re-do the left one at some point as the tension is off.
I'm knitting with rainbow yarn today and plotting out some ideas for a piece of black silk I bought and plan to make a dress with.
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