text:
- Research
- Open Access
The potassium channel Ether à go-go is a novel prognostic factor with functional relevance in acute myeloid leukemia
- Jasmin R Agarwal1,
- Frank Griesinger2,
- Walter Stühmer1 and
- Luis A Pardo1Email author
© Agarwal et al; licensee BioMed Central Ltd. 2010
- Received: 9 October 2009
- Accepted: 27 January 2010
- Published: 27 January 2010
The voltage-gated potassium channel hEag1 (KV10.1) has been related to cancer biology. The physiological expression of the human channel is restricted to the brain but it is frequently and abundantly expressed in many solid tumors, thereby making it a promising target for a specific diagnosis and therapy. Because chronic lymphatic leukemia has been described not to express hEag1, it has been assumed that the channel is not expressed in hematopoietic neoplasms in general.
Here we show that this assumption is not correct, because the channel is up-regulated in myelodysplastic syndromes, chronic myeloid leukemia and almost half of the tested acute myeloid leukemias in a subtype-dependent fashion. Most interestingly, channel expression strongly correlated with increasing age, higher relapse rates and a significantly shorter overall survival. Multivariate Cox regression analysis revealed hEag1 expression levels in AML as an independent predictive factor for reduced disease-free and overall survival; such an association had not been reported before. As a functional correlate, specific hEag1 blockade inhibited the proliferation and migration of several AML cell lines and primary cultured AML cells in vitro.
Our observations implicate hEag1 as novel target for diagnostic, prognostic and/or therapeutic approaches in AML.
- Acute Myeloid Leukemia
- Chronic Myeloid Leukemia
- K562 Cell
- Acute Myeloid Leukemia Patient
- Astemizole
Acute myeloid leukemia (AML), characterized by strong proliferation of undifferentiated hematopoietic progenitor cells, is the most common type of acute leukemia in adults. AML subtypes are very heterogeneous, with different chromosomal aberrations, therapeutic response and outcome. Age and certain chromosomal aberrations are markers for a good or bad prognosis, but 45% of AML have a normal karyotype and an unclear intermediate prognosis with a five-year survival rate of only 40% [1]. Prognosis factors are crucial for therapy decisions like bone marrow transplantations, which can cure the disease but are not devoid of severe side effects. An increasing number of genetic parameters (mainly gene mutations implicated in hematopoietic differentiation or transcription regulation) are being identified as predictive factors. Examples are FLT3-ITD (Internal Tandem Duplication of Receptor Tyrosine Kinase) or partial tandem duplications of the MLL gene (Mixed Lineage Leukemia), which are associated with a poor prognosis [2, 3]. Therapies against certain subtypes with distinct biological features offer noticeably improved outcome, as established for AML M3 with 90% survival, highlighting the need for specific therapeutic regimes [4].
Increasing evidence relates ion channels to cancer pathogenesis and prompts their use in diagnosis and therapy [5]. Voltage-gated K+-channels show the highest variability among ion channels with over 70 genes. The channels expressed in a given cell are specific not only for the cell type but also for its physiological status. These channels represent a suitable distinctive element for both healthy and tumor cells and have aroused significant interest in cancer research [5–7]. Our group is specifically interested in the human voltage-gated potassium ion channel ether à go-go 1 (hEag1) because of its pathological expression in tumor cells and its potentially oncogenic properties [8–13]. The physiological expression of hEag1 is largely restricted to the brain where its role is still unknown [14], although transient hEag1 expression induces myoblast fusion and exit from the cell cycle during myoblast differentiation [15]. hEag1 expression and functional characteristics are modulated in neuronal cells throughout the cell cycle [16] and by many factors like the cytoskeleton or calmodulin [8, 17]. During neuroblastoma differentiation, hEag1 expression is strongly down-regulated [18].
Non-neural cells aberrantly overexpressing hEag1 acquire phenotypical characteristics of malignancy and induce aggressive tumor growth in immunodeficient SCID mice [12]. hEag1 is significantly overexpressed in many tumor cell lines and more than 75% primary solid tumors from different histological origins like breast, colon or cervix carcinomas [10, 19] and sarcomas [11]. Importantly, the channel cannot be detected in the originating normal tissues. hEag1 inhibition by the antihistamine astemizole, the tricyclic antidepressant imipramine or hEag1 specific monoclonal antibodies reduces tumor cell proliferation in vitro and in vivo [9, 11, 20–23]. In summary, the expression of hEag1 in many solid tumors is clearly established though its functional role in carcinogenesis or tumor maintenance is still under investigation.
In chronic lymphatic leukemias the related channel HERG (human Eag-related gene, KV11.1) could be identified, but no hEag1 expression was detected [24]; it was subsequently assumed that hEag1 had no relevant role in leukemias.
Leukemias and lymphomas are an important and frequent group of tumors with very distinct pathophysiological features in contrast to solid tumors. The goal of the current study was to determine if different types of leukemias share the biological feature of solid tumors to express hEag1. Additionally, we wanted to determine if any potential expression might have functional correlates or even prognostic value. Within leukemias we focused on AML and analyzed hEag1 expression by quantitative real-time PCR (qPCR) and correlated it to several patient characteristics. A possible role of hEag1 in leukemia cell proliferation could be shown by growth and migration inhibition of the hEag1-expressing cell lines PLB-985, K562, UT-7 and HEL and primary clinical samples by the potassium channel blockers astemizole, imipramine, the hEag1 specific monoclonal antibody mAb56 and siRNA knockdown [9]. No involvement of hEag1 expression during HL-60 cell differentiation was detected. Cell cycle related changes and apoptosis induction were analyzed to determine possible inhibitor effects useful for any hEag1-based therapy.
hEag1 is frequently expressed in primary myeloid leukemias and leukemia cell lines
As the expression of the hEag1 channel in leukemias is unknown our aim was to study the prevalence of hEag1 in different kinds of leukemias.
Patient characteristics
Study population
hEag1 positive
No (%)
47 (40)
Gender, no. of males (%)
23 (50)
Age, years median (range)
60 (1-86)
31 (1-89)
Age groups, N (%)
child (1-18 years)
5 (13)
adult (19-59 years)
18 (47)
elder (60-90 years)
23 (53)
FAB classification, N (%)
2 (28)
9 (45)
14 (56)
10 (53)
3 (21)
3 (60)
1 (33)
5 (45)
Cytogenetic analysis, N (%)
25 (66)
t (8;21)
3 (43)
inv (16)
t (15;17)
11q23 abnormalities
1 (10)
4 (80)
chromosome 5 or 7 abnormalities
7 (44)
Trisomy 8 alone
2 (33)
other single abnormality
1 (17)
multiple abnormalities
9 (41)
Cytogenetic classification, no. (%)
3 (19)
25 (51)
17 (36)
Stem cell transplantation
5 (23)
No stem cell transplantation
19 (34)
CR1 achieved, no. (%)
13 (34)
not achieved
9 (33)
Relapse, no. (%)
early relapse (within 6 months)
5 (50)
late relapse
11 (34)
no relapse within 3 years
3 (12)
Death, no. (%)
early death (within 6 months)
13 (50)
late death
14 (44)
Disease free survival (patient no.)
median, months
disease free at 3 years, no. (%)
5 (13)
23 (37)
Overall survival (patient no.)
median, months
alive at 3 years, no. (%)
6 (16)
29 (47)
alive at 5 years, no. (%)
2 (5)
19 (31)
Therapy protocol
Peripheral blood blasts, % median (range)
65 (0-100)
42 (0-95)
66 (2-100)
Bone marrow blasts, % median (range)
85 (0-100)
90 (25-98)
85 (0-100)
WBC, ×10 9 /l median (range)
33.7 (0.9-400)
50.8 (2.6-222
32 (0.9-400)
Acute lymphoblastic leukemia (ALL) (n = 8) and biphenotypic acute leukemia (n = 3) were hEag1-negative in all samples analyzed.
hEag1 was detected in 5 of 13 cases (38%) of chronic myeloid leukemia (CML), and interestingly, 58% of the samples with myelodysplastic syndrome (MDS; n = 12) expressed hEag1. Many MDS progress into AML, but inducing factors and time until progression are still unknown. One hEag1 positive patient with MDS progressed quickly after two months into an AML M2, another patient died after five months. Two other patients with hEag1 positive MDS were followed for more than one year and two patients for more than two years without any progression. We further followed one patient with AML M4 that was hEag1 positive at first diagnosis. After 14 months, a relapse occurred and the amount of hEag1 expression was strongly increased.
40% of the analyzed AML samples expressed hEag1 (47 of 118 samples). According to the FAB (French-American-British) classification, a subtype-dependent expression pattern was observed: AML FAB M3 (n = 8) and M4Eo (n = 6) were consistently hEag1-negative. Within the other subgroups, hEag1 was expressed in 2 of 7 M0 (28%), 9 of 20 M1 (45%), 3 of 14 M5 (21%), 3 of 5 M6 (60%) and 1 of 3 M7 cases (33%). The most common subtypes M2 and M4 frequently expressed hEag1 (14 of 25 cases (56%) and 10 of 19 cases (53%), respectively) (Fig. 1).
In order to establish specificity of the hEag1 inhibitor effects during proliferation assays, we also determined the expression of HERG (KV11.1) in the hEag1 positive cell lines. UT-7 cells expressed equal and K562 cells higher levels of HERG channels compared to hEag1, whereas we did not detect HERG in PLB-985, HL-60 or HEL cells. All four patient samples were negative for HERG expression.
Protein expression was confirmed by flow cytometry using an anti-hEag1 specific monoclonal antibody (mAb49) directed against an extracellular loop of the channel. The results are summarized in Fig. 2B. Some background fluorescence was detected in all cell lines, including hEag1-negative patient samples. For this reason, we subtracted the magnitude of the mean fluorescence intensity (MFI) shift observed in Patient 2 cells (hEag1 negative in PCR) from all measurements. Under these conditions, hEag1 knockdown by siRNA in PLB-985 and K562 cells completely abolished the increase in fluorescence intensity (Fig. 2C), indicating that the MFI shift is due to hEag1 protein expression.
The hEag1 channel is indeed expressed in several cell lines and primary clinical specimens of newly diagnosed myeloid leukemias.
hEag1 expression in AML is subtype-dependent and correlates with increasing age
Broad statistical analyses were performed for AML because of the relatively high number of samples available. There was no significant correlation between hEag1 expression and initial laboratory parameters such as platelet counts (median, 58 vs. 54 × 109/l; p = 0.62), LDH (511 vs. 451 U/L; p = 0.24), hemoglobin (9.5 vs. 8.8 g/dL; p = 0.36), peripheral blood blasts (42 vs. 66%; p = 0.39) and bone marrow blasts (90 vs. 85%; p = 0.94). White blood cell counts were higher in hEag1 expressing samples without reaching statistical significance (median, 50.8 vs.32 × 109/L; p = 0.76). There was also no difference in gender distribution between hEag1 positive and hEag1 negative patients (p = 1.0).
Three prognostic groups can be defined in AML according to cytogenetic aberrations. The favorable group is defined by t(8;21), t(15;17), inv(16) or young age. The intermediate group consists of patients with normal cytogenetics, +8, +21, +22 or -9q, while a complex karyotype, -7, -5, MLL anomalies or FLT3-ITD (31) correlate with poor prognosis. hEag1 expression was often associated with an intermediate prognosis in 25 of 49 patients (51%). In 17 of 47 patients it correlated with an unfavorable prognosis (36%, p = 0.66), while only 3 of 16 patients (19%) with a favorable prognosis profile expressed hEag1. Chromosome 5 or 7 abnormalities (n = 16) or multiple aberrations (n = 22) were often associated with hEag1 expression in 44% and 41%, respectively. Interestingly, 4 of 5 patients (80%) with FLT3-ITD were hEag1 positive.
The highest prevalence of hEag1 was reached in the most frequent subtypes AML M2 and M4 with expression in more than half of the cases. Channel expression significantly correlated with increasing age.
The hEag1 expression level in primary AML is a novel independent predictive factor for a poor outcome
The frequent expression of hEag1 in AML suggests an involvement of the channel in the pathophysiology of this disease, which might be linked to patient outcome. To test this hypothesis, statistical quantifications were performed.
To determine if hEag1 expression adds predictive value to age or karyotype alone, we used Cox regression multivariate analysis. hEag1 expression levels, age and karyotype are independent predictive parameters for the overall survival of AML patients (p = 0.044, 0.058 and 0.057, respectively). In a positive/negative thresholded analysis, without taking into account expression level in the positive patients, channel expression did not add predictive value over age alone in the elderly and mid-age patients (older than 65 and 17.28 to 65 years; p = 0.59 and 0.44), but was still highly significant as a bad prognostic factor in young patients (p = 0.0022).
In summary, we conclude that the expression level of hEag1 strongly correlates with shorter survival expectancy in AML and can be used as a novel independent predictive factor, which eventually could be introduced into routine prognosis analysis for advanced grouping and therapy planning of AML patients.
Functional effects of hEag1 inhibition in vitro
Previous studies [26] described impairment of leukemia cell proliferation by HERG inhibition. HERG is abundantly expressed in K562 cells and in low amounts in UT-7 cells. It is conceivable that part of the observed effect is due to HERG blockade. This argument is not applicable to PLB-985 or primary cultured cells, where we did not detect any expression of HERG. To clarify the extent of hEag1 implication in the regulation of proliferation of these cell lines, we used the monoclonal antibody mAb56, a very specific inhibitor of hEag1 that does not modify HERG and even not hEag2 activity. 10 or 20 μg/mL mAb56 inhibited PLB-985 by 70%, UT-7 by 80% and K562 proliferation by 98% after 4 days (p < 0.05). The mAb56 abolished the proliferation of P4 primary cells.
Finally, knockdown of hEag1 expression by siRNA in PLB-985 and K562 cells also resulted in up to 80% diminished proliferation over 5 days. (hEag1 knockdown by siRNA was not possible in UT-7 cells.) Representative proliferation curves are shown in Fig. 5. Effective hEag1 knockdown by siRNA was confirmed by flow cytometric detection of cells stained with anti-hEag1 mAb49 (see Fig. 2C). Altogether, our data suggest that hEag1 is implicated in the regulation of leukemia cell proliferation.
Apoptosis induction is a desirable therapeutic effect. For K562 cells, an intense induction of apoptosis was observed after treatment with imipramine and astemizole up to 81% after 1 day (Fig. 6B). Increased apoptosis was not detected in other cell lines, nor was it induced by siRNA or mAb56, indicating that the increased apoptosis is not solely due to hEag1 blockade. In primary AML cells from one patient (P4) apoptosis and necrosis was 2 times increased by 10 μg/mL mAb56 and 20 μM imipramine (Fig. 6B).
As transient hEag1 expression is important during differentiation of other cell types like myoblasts, we tested its possible involvement in hematopoietic differentiation. We used the model cell line HL-60, which is arrested at the promyelocytic stage, but can be terminally differentiated over 6 days into granulocytes by retinoic acid. Cell differentiation was shown by Giemsa stain of cell nuclei and an increase of CD38 expression in flow cytometry measurements. We determined hEag1 expression every 2 h during differentiation but did not detect any hEag1 expression at any time during the entire differentiation process (data not shown).
Our functional analyses of AML cell lines and primary AML specimens implicate a role of hEag1 in migration and cell proliferation, without a clear cell cycle arrest. Because hEag1 inhibitors are able to block proliferation and migration they might be useful for therapeutic approaches.
The involvement of hEag1 potassium channels in leukemias had not been systematically studied, although its relevance for many solid tumors is well established. We analyzed the prevalence and prognostic impact of hEag1 expression in 154 patients with newly diagnosed leukemia with special focus on 118 AML patients. In solid tumors, hEag1 is expressed in over 70% of carcinomas and sarcomas [10, 11]. Smith et al. [24] did not detect hEag1 in CLL, and therefore we did not study CLL samples. In ALL we did not detect any hEag1 expression; although the sample number was low. This might indicate that hEag1 expression is not relevant in lymphatic leukemias.
A completely different scenario applied for myeloid leukemias. Although we detected hEag1 expression in one third of the patients with CML, we did not perform further statistical analyses due to the limited availability of samples. We therefore concentrated on AML, where we found an interesting subtype-dependent hEag1 expression, since one half of the cases of the most common subtypes M2 and M4 expressed hEag1, and this correlated with increasing age, higher relapse rates and a significantly shorter overall survival.
Interestingly, 50% of MDS expressed hEag1. This could represent a similar situation to the report by Farias et al. [19] in cervix carcinoma, where they detected hEag1 expression in cervix carcinomas and cervical hyperplasias that are often pre-forms of carcinomas. If this is also true for AML, hEag1 expression outside the brain might be useful as an early marker for malignant cell transformation, although such a far-reaching conclusion would require the support of prospective studies in a large sample population.
66% of the patients with normal cytogenetic AML expressed hEag1. These patients with a normal karyotype represent a very heterogeneous group with regard to prognosis and therapy response. Several molecular markers like FLT3-ITD, MLL, CEBPA (CCAAT/enhancer binding protein) or BAALC (brain and acute leukemia, cytoplasmic) further characterize some of these patients but there is still highly unmet medical need for other factors to establish a clear prognostic profile in every individual patient [28, 29]. It was therefore important to determine if hEag1 expression in AML has prognostic relevance. In other studies, hEag1 expression was associated with an unfavorable outcome in sarcomas, but no multivariate Cox regression analyses were performed, so it is not know if hEag1 expression has any predictive potential in sarcoma [11]. In our study, we observed a clearly decreased survival in hEag1 expressing patients. This could be due to higher expression frequencies in elder patients, since children with AML respond better to therapy than adults. However, multivariate Cox regression analysis determined an independent predictive value for hEag1 expression (p = 0.044), karyotype (p = 0.049), age (p = 0.058) and cytology (p = 0.057), while gender was not predictive (p = 0.20). The median overall survival of 10 months of hEag1 overexpressing patients was strongly reduced compared to hEag1 non-expressing patients with 52 months OS (p = 0.0019). The patient's prognosis is important for therapeutic decisions like bone marrow transplantation, which can prolong the survival or even cure the disease. But it is accompanied with risky side effects that might not be acceptable for patients who anyway have a good prognosis with chemotherapeutic treatment. hEag1 could be used as an additional prognostic factor as it significantly correlates with a bad prognosis. These findings could justify a prospective study to evaluate the use of hEag1 as predictive factor during routine clinical profiling of newly diagnosed AML patients.
Finally, hEag1 expression has been proposed as the basis of a potential new therapeutic target, due to its restricted expression outside the central nervous system and the effects of hEag1 inhibition on the proliferation of several tumor cell types in vitro [11, 23] and in vivo [9, 20]. We observed that hEag1 inhibition reduced the proliferation of several AML cell lines. Most available blockers of hEag1 are also effective inhibitors of other channels and enzymes, some of which could be present in the model cells used in this study. However, the observation that the specific anti-hEag1 antibody mAb56 and siRNA show similar strong inhibitory effects as the tested drugs argues for an implication of hEag1 in the proliferation of leukemia cells. The limited number of cell lines expressing hEag1 precluded establishing a reliable correlation between expression levels and impact of channel inhibition on cell proliferation. We could not perform experiments with hEag1 overexpression because we did not achieve reliable transfection efficiencies on leukemia cell lines, either by lipofection or nucleofection, even if control vectors could be successfully transfected, which might indicate deleterious effects of hEag1 overexpression on the cells
One of the primary AML cell preparations with the highest hEag1 content showed comparable behavior to established cell lines. The inhibition achieved through hEag1 inhibitors was even faster and more intense than in cell lines. These results prompt further experiments to establish if leukemias with a particular expression pattern might be suitable targets for anti- Eag1 strategies.
Other members of the Eag K+-channel subfamily have been implicated in the pathophysiology of leukemias. HERG channels have been described to be up-regulated in a wide spectrum of hematopoietic malignancies like AML (~70%), CML, ALL or lymphomas, while it is not expressed during physiological hematopoietic development or in normal peripheral blood cells [30]. HERG channels regulate leukemia blast proliferation, improve AML cell migration and invasiveness and correlate with higher relapse and shorter survival [26, 31]. The inhibition of HERG reduces leukemia cell growth and arrests cells at the G1/S transition phase [27]. Therefore, HERG channels could have broader therapeutic applications in leukemia than hEag1, but its ubiquitous expression throughout the body and its crucial role in cardiac repolarization are challenging problems for a HERG-based therapy because severe side effects like fatal arrhythmias can occur [32].
In K562 cells, which express high levels of both hEag1 and HERG, we observed a dramatic induction of apoptosis induced by both imipramine and astemizole. It is tempting to speculate that simultaneous inhibition of hEag1 and HERG in those cell types expressing both channels could be an efficacious therapeutic approach.
In combination with routinely used induction therapeutics, the hEag1 blockers astemizole and mAb56 were able to increase the apoptotic response in PLB-985 cells. This could be advantageous by increasing leukemia responsiveness during the critically important induction therapy. However, this effect was cell type specific, indicating that accurate profiling of the AML cells, together with complete toxicity profiles of such drug combinations will be required and have to be critically evaluated in primary cells and animal models.
An increased migration capability is one important sign of malignancy. hEag1 expressing leukemia cell lines show higher migration rates than non-expressing ones. hEag1 inhibitors were able to reduce cell migration. In the context of a therapeutic usage those inhibitors might be important that are able to prevent several features of malignancy like proliferation, migration or functional properties of these transformed cells.
The potential of hEag1 inhibitors for a usage in leukemia treatment has to be carefully further analyzed in primary cells in vitro and mouse models in vivo.
We showed that hEag1 is indeed frequently expressed in myeloid leukemia and MDS. In AML it is expressed with high frequency in the most common subtypes and it correlates with increasing age, higher relapse rates and a significantly shorter overall survival. Statistical analyses revealed hEag1 as an independent predictive factor for a worse outcome. Functional studies showed the potential of hEag1 inhibitors to reduce proliferation and migration of AML cell lines and primary cells. Our data suggest a potential for hEag1 as tumor marker for early tumor screening of hematopoietic disorders, as prognostic factor in certain AML types and as therapeutic target of specific inhibitors in the treatment of AML. Antibody-labeled aggressive therapy with radionuclides or toxic molecules could act locally and highly efficiently. More detailed analysis will be performed to establish hEag1 in the diagnosis and treatment of leukemias.
Patients and samples
RNA or frozen cells isolated from the peripheral blood or bone marrow from patients with newly diagnosed AML were obtained from the hematology department of the University Medicine Göttingen. Molecular genetic analyses of patient's blood samples were performed with their informed consent and upon approval by the local ethics committee. Patient details are summarized in Table 1. The median follow up was 4.2 years (range 0.75-7.2 years). Treatment regimes are summarized in Additional File 1.
Definition of response, relapse and end points
Complete remission (CR) was defined by ≤ 5% bone marrow blasts, neutrophile counts ≥ 1000/μL, platelets ≥ 100,000/μL and lack of extramedullar disease. Cytogenetic remission was defined by normal cytogenetics, and molecular remission by negative molecular studies. Disease-free survival (DFS) was measured from CR to relapse. Overall survival (OS) was defined as time from diagnosis to death irrespective of the cause. Living patients were censored at the date of last follow-up.
RNA isolation, cDNA synthesis and real time PCR
Mononuclear cells were isolated from peripheral blood or bone marrow by density centrifugation over a Ficoll gradient and RNA was isolated from the buffy coat [33]. Either lysates from frozen cells or total RNA was obtained. Total RNA was isolated from 5 × 106 cells using the RNeasy mini isolation kit for animal cells (Qiagen, Hilden, Germany) with additional DNAse treatment according to the manufacturer's protocol. cDNA synthesis and real time PCR were performed as previously described [10]. Quantification was performed using the ΔΔCT method (cell lines) or after Pfaffl [34].
Cell culture
The human myeloid leukemia cell lines HL-60 (AML M2, DSMZ ACC 3, Braunschweig, Germany), K562 (CML in blast crisis, ACC 10), PLB-985 (AML M4, ACC 139; this is a subclone of HL-60 cells), HEL (AML M6, ACC 11), CMK (AML M7, ACC 392) and KASUMI-1 (AML M2, ACC 220) were cultured in RPMI1640 medium with 10% fetal calf serum (Invitrogen, Karlsruhe, Germany). UT-7 cells (AML M7, ACC 137) were cultured in alpha-MEM medium with 20% FCS and 5 ng/mL GM-CSF. Cells were maintained at 37°C and 5% CO2 in a humidified atmosphere.
Peripheral blood from 4 patients was obtained at the time of initial diagnosis prior to therapy. Primary leukemic blasts were isolated by density centrifugation for 30 min at 800 × g (Lymphoprep, Axis-Shield, Oslo, Norway) Cells from the buffy coat were washed three times with PBS and cultured in RPMI1640 medium supplemented with 20% FCS, 1% pyruvate (Sigma-Aldrich, Steinheim, Germany), 1% insulin-transferrin-selenium (ScienCell, Carlsbad, CA) and 1% penicillin/streptomycin (Invitrogen).
Differentiation of HL-60 cells into granulocytes was induced with 2 μM retinoic acid over 6 days [35, 36]. During differentiation, samples were obtained every 2 h and analyzed for hEag1 expression up to day 6. Differentiation was monitored by successive Giemsa stains, and confirmed after 6 days by flow cytometry, through up-regulation of CD38 [37].
To measure apoptosis under induction drugs, 2 × 105 PLB-985, K562 and primary P4 cells in 24-well plates were incubated with 2 μM idarubicin, 20 μM etoposide, 6 μM cytarabine or 4 μM doxorubicin either alone or in combination with 4 μM astemizole or 10 μg/mL mAb56 for 24 h. Apoptosis was assessed with the Caspase-Glo 3/7 Assay system (Promega) measuring luciferase-induced luminescence in a 96-well plate reader according to the manufacturer's instructions.
Proliferation assays
PLB-985, UT-7, K562, HEL or primary clinical cells were seeded in 96-well plates (2,000 cells/well or 10,000 primary cells/well) and cultured with different concentrations of astemizole (0.5-4 μM), imipramine (5-20 μM) or mAb56 (5-20 μg/mL) for up to 5 days. These inhibitor concentrations are below non-specific cytotoxic doses (5 μM astemizole [21] and 50 μM imipramine [22]). The functional monoclonal anti-hEag1 antibody mAb56 (10 μg/mL) was used as hEag1-specific blocker because it does not affect HERG or hEag2 [9]. Cell proliferation was measured every 24 h by Alamar Blue (Invitrogen) fluorescence after 2 h incubation with the dye in a 1420 Victor2 Multilabel Counter (Ex 544, Em 590 nm; Wallac-Perkin-Elmer, Waltham, MA). The relative proliferation was normalized to control cell growth without inhibitor.
hEag1 silencing was performed using 30 nM siRNA (sense: r(CAG CCA UCU UGG UCC CUU A)dTdT, antisense: r(UAA GGG ACC AAG AUG GCU G)dTdA). Transfection was performed by nucleofection (Amaxa, Lonza, Cologne, Germany) according to the manufactures recommendations with 2 × 106 K562 or PLB-985. No transfection was achieved in UT-7 cells. Commercial non-targeting scrambled siRNA (Ambion, Darmstadt, Germany) and anti-GAPDH siRNA were used as negative and positive control, respectively. RNA and protein knockdown after 2 days was confirmed by real-time PCR and flow cytometry.
Migration analysis
3 × 105 cells were seeded in the upper reservoir of 24-well Boyden chambers in serum-free medium. The lower compartment contained medium with 20% FCS. After 24 h incubation at 37°C, cells in the lower chamber were centrifuged, resuspended in 10 μL medium and counted in a Neubauer chamber.
Flow cytometry
For hEag1 protein analysis, cells were fixed for 10 min in 10% formalin, quenched with 100 mM glycine, permeabilized for 5 min in 0.1% Triton-X100 and incubated overnight in blocking buffer (0.2% gelatin in PBS). The primary antihEag1 antibody (mAb49) was used at a concentration of 1.3 μg/mL for 1.5 h. Cells were washed three times with blocking buffer and incubated in 1 μg/mL AlexaFluor 488-labeled secondary antibody for 2 h. The whole procedure was performed at 4°C. Finally, cells were washed four times, resuspended in PBS and measured in a BD FACSAria flow cytometer (Becton Dickinson, Heidelberg, Germany). Only single viable cells (as determined by forward-sideward scatter) were gated and further analyzed.
Apoptosis analysis was performed using Annexin V-AlexaFluor 488 or 680 (Vybrant Apoptosis Assay Kit #2, Invitrogen) and 100 μg/mL propidium iodide according to the manufacturer's instructions.
For cell cycle determination, cells were washed once with PBS and incubated for 15 min in 50 μg/mL propidium iodide, 0.3% saponine and 100 U/mL RNAse A in PBS at 4°C. Flow cytometry measurements were performed within 1 hour, measuring less than 200 cells/second. Data were analyzed with FlowJo software (TreeStar, Ashland, OR).
Differentiated HL-60 cells were stained with 25 μg/mL anti-CD38 antibody per 106 cells in 100 μL final volume. After 20 min incubation at room temperature, cells were washed and resuspended in PBS.
Statistical analysis
Estimated probabilities of OS and DFS and time-to-event curves were constructed according to the Kaplan-Meier method and were compared with the log-rank χ2 test. Group data are expressed as mean ± SD. Student's t-test was used for single comparison at 95% confidence. Prognostic factors were examined by multivariate Cox proportional Hazards Survival Regression analysis [38]. Patients with CML or MDS were not statistically analyzed because of small sample numbers.
We thank U. Kutzke and B. Heidrich for technical assistance, Dr. K. Jung (Department of Medical Statistics, Göttingen University) for advice on statistics, Sabrina Becker (University Medicine, Göttingen) for leukemia samples, Stefanie Brems (University Medicine, Mannheim) for several cell lines, Prof. Büchner (University Münster) and Prof. Reinhardt (Hannover Medical School) for clinical information and M. Djannatian and A. Agarwal for suggestions on the manuscript.
Authors' Affiliations
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- Gavrilova-Ruch O, Schönherr K, Gessner G, Schönherr R, Klapperstuck T, Wohlrab W, Heinemann SH: Effects of imipramine on ion channels and proliferation of IGR1 melanoma cells. J Membr Biol. 2002, 188: 137-149. 10.1007/s00232-001-0181-3View ArticlePubMedGoogle Scholar
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Despite presenting a character clear-up earlier this week, readers were still questioning the information on Sean Slater's appearance in EastEnders.
I can 100% assure you that the character WILL be walking the streets of Walford and joining his sister, Stacey (Lacey Turner), along with the rest of the Slater clan.
A spokesman confirmed to DS: "Sean Slater will be joining the Square later this year. No decision has been made as yet as to who will play the character. He's going to enter the Square with a bang and will be at the centre of some of the biggest storylines of the year."

subset: commoncrawl


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Little Giant Automatic Stock Waterer - Black Epoxy Finish
Product Number: C18073
In Stock (Ships within 1-2 business days)
A constant water level in this unit is maintained by an improved adjustable float that is molded from high-grade nylon for a lifetime of trouble-free operation. Rigidly formed of heavy-gauge prime draw-quality steel, the fast, easy-cleaning bowl has a large, deep drinking area. Outlasts and outperforms other watering systems, and it's backed by a one-year warranty. The Little Giant waterer fits 1/2 in. pipes and is easily installed at any height with U-bolts or lag bolts. Can be connected left or right. Unit is 4-1/2- H, bowl width is 8 in. Front to back dia. is 8 in. Front to back of bowl is 7-1/4 in. Nonsiphoning.
of 0 Reviews
Your guide to an exceptional shopping experience.


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Yesterday we spent our time talking about social justice. The question we were asked is "What is my/our role as bishops in a hurting and broken world?"
I was humbled by the conversations. Bishops in other parts of the world simply face more external hurt and more societal brokenness that we can imagine. I spoke to bishops whose people fear for their lives because of the government. One told me that his house is filled with students who have no place to live because they have no parents. Another said that the churches had been torn down so now they worship under trees.
What is common is a desire for the church to be the Good News that gives hope and promise to people. The purpose of this conference is less to legislate a new structure for the Communion and more to energize our collective effort to be the Church God needs us to be for our day and time.
I also reflected that in face of such pain it is easy to become paralyzed. "What can I do?" we ask. But we begin by seeing more clearly how things are and then by asking God to give us the first step. If we cannot help the people across the globe directly, what can we do right now?
Today we go to London to visit the Lambeth Palace and Buckingham Palace.
Keep all of us in your prayers as I keep you in mine.


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We have all done admire to friends and acquaintances having tattoos and especially having strange tattoos on various inspirations . Tattoo but on science, seen? Undoubtedly, most of us don't aware science tattoos. We are living the 21st century, where people are very curious beauty, fashion, personality and son on. Similarly, tattoos are playing a big role in the fashion industry. Today, the tattoo is not only a small sign on the body. Currently, folk covers the whole body to it. As the market is full of a wide range of tattoos, here CinemaJAGAT is providing the name of some unique, attractive science tattoos name and their image. Hope you will like………….
Below are 20 of the most wonderful science tattoos you've ever seen!
1) A modified version of the phylogenetic tree of life
2) The spiral of Earth's orbit in space
3) The Bishop theorem
4) The number one chico design for tree of life
5) The planetary symbols of the solar system
6) The wisdom of Carl Sagan
7) A molecule serotonin
8) An amino acid sequence
9) The human DNA in combination with the Tree of Life
10) The pyramidal neuron of the hippocampus
11) An animal cell
12) a crystallographic x-ray image of a DNA molecule
13) the molecular structure of Vemurafenib, a hand drug for the treatment of cancer
14) A molecule ATP
15) The device of the International Linear Accelerator Program
16 ) the Drake equation used to estimate the number of active our extraterrestrial civilizations in the Milky Way
17) The Carl Sagan Pulsar map
18) O Charles Darwin
19) From space to Earth
20) Einstein and Darwin
Also, read the post……>>>>>> Top 10 Tattoos| Best Tattoos| Top 10 Best Tattoos
View One More post…… >>>>>> What Pitfalls Hiding OPEN-Office ? Implications Of Open Offices
Read the post…..>>>>> Ultimately Depression Identity ? Which Age More Affected
Also, read one more post…..>>>>> How Do Interior Designers Beautify Your Homes?


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The Health Advantages Of Using A Seat Pad
In recent times most jobs require people to sit for long hours in a day. It is a past trend to walk to work or to board a bus for a few minutes drive. Due to the high cost of living in the urban areas have made people move to the rural areas. They also move from the urban area due to the problems of pollution and congestion. Living far from the workplaces people have no otherwise other than traveling to their offices. They spend the whole day sitting in their offices then spend more sitting hours as they go back home.
Sting for long hours can lead to future health issues. Those who travel for many hours they should consider getting the seat pads. According to the research the body of a human being experiences discomfort as soon as you start driving. The seat increases the temperature, and the body becomes more uncomfortable. It shows that the chair you use determines how well the body will be comfortable and determines your health. When you lack proper support in the chairs we sit on then there are chances that we experience back pains.
The manufactures who made the office chairs did not emphasize on the back support since the chairs are simple and cheap. There are other types of the seat with extra support but are expensive. The seat pads improve the support structure of an uncomfortable seat and give you an upright position. Using a seat pad gives the spine its normal upright structure. It avoids the back muscle strains and can make a huge difference as time goes by. The seat pads suits every type of chairs and sitting circumstances.
The call center employees, the receptionist and those who work in offices are the most affected people in using the seat pad. They can be used either at home or in the car. The seat pads are made from a light material making them portable and people can use one seat pad at home and at the office. People use a lot of money in search for relaxation. It is economical to buy a seat pad massage. Check here and get a clear picture of the advantages of the seat pads. This story give their explanation about the seat pads.
The massage seat pad has different types. All you need to do is to position the massage seat pad where you experience pain. The second type of massage seat pad is the plug model. They provide a different setting warming though they are a bit complex. Parts like the neck and the shoulder muscle gets the full message since the seat pad moves on specific spots in the massage process. Massage seat pads have many benefits including the improvement of blood circulation into the body.


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"It's hard to imagine a time when a political party has been so willfully dysfunctional. The Republican Congress of the last decade has been profoundly uninterested in what might be generously called "governing." Instead, they impulsively chase every passing ball into the street, oblivious to danger. Unsurprisingly, it's coming back to haunt them. Even Republican voters seem disturbed with how little the GOP manages to get done in Washington. For reasons that may seem obvious but somehow confound Republicans, taxpayers aren't thrilled when they hear stories like this one from a former Benghazi committee staffer-turned-whistleblower:"


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The Reserve Visitor Centre at Kranji Way is a great place to start your exploration.
Archerfishes often hang around under the bridge especially when the tide is turning.
crocodiles in Singapore.
shorebirds in Singapore.
tree climbing crabs as they climb up the mangrove trees to avoid swimming predators that come in with the water. Mudskippers climb up too! More about mudskippers in Singapore.
Marvelous Mangrove activities!
The Reserve has many regular activities, all are free but registration is required due to limited places.
At Sungei Buloh only, join a fascinating demonstration of traditional prawn farming and harvesting. The kids will have a great time watching the different kinds of marine life that turn up when harvesting a traditional prawn farm! The next demo is on 24 Mar (Sat).
Guided tours in Mandarin (Chinese) are also regularly conducted at the Reserve. Join the upcoming ones on 18 Feb (Sun) and 24 Mar (Sat).
This weekend, lots of mangrove fun at the Reserve!
Links to more information
- 3 Feb (Sat): World Wetlands Day - Sungei Buloh Wetland Family Adventure
- 3 Feb (Sat): What's in my mangroves?
- 3-4 Feb (Sun): World Wetlands Day activities by NParks
Links to more information
- More about World Wetlands Day celebrations in Singapore. At Sungei Buloh and elsewhere.
- Sungei Buloh Wetland Reserve by NParks
- 9 reasons to visit Sungei Buloh by the Cheekiemonkies, the family adventures of Kelvin, daddy blogger.
- Sungei Buloh Wetland Reserve with kids by Bumble Bee Mum.
- Surveys and trips to Sungei Buloh on wild shores of singapore.


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The Thousand Autumns of Jacob de Zoet
A Novel
David Mitchell
Random House: 484 pp., $26
David Mitchell's new work, "The Thousand Autumns of Jacob de Zoet," is conventional in more ways than one. Not only is the novel, set in Japan at the end of the 18th century, the least experimental book the British novelist has ever written — in fact, it cleanly passes as "historical fiction" — but with each passing book, he embraces a new genre, an innovative approach to fiction that has become the two-time Booker finalist's quirky signature. Throughout his barely decade-long career, the 41-year-old has moved limberly from one literary form to another. After the high-wire sampling of disparate genres in his formally daring Calvino- and Murakami-soaked "Cloud Atlas" (2004), who could have foreseen the intimate 2006 "Black Swan Green," a semi-autobiographical coming-of-age story of a stuttering 13-year-old in Thatcher's England?
Like "Black Swan Green," Mitchell's new book is more verbal calisthenics than structural gymnastics. It almost completely forgoes the first-person voice that Mitchell mastered in his prior work and limits itself to a short span of years (a nanosecond compared to the centuries-jumping "Cloud Atlas") and a few locales in and off Japan.
It is 1799 and the eponymous hero is a pious clerk from Holland during the waning days of the Dutch East Indies Co.'s trade monopoly with shogun-era Japan. The country's terra firma has yet to re-welcome the footprints of foreigners, so most of "Thousand Autumns" takes place on Dejima, the tiny island in Nagasaki's harbor where perforce exchanges of all sorts (mercantile, military, linguistic) take place. This little patch is a strange floating world of its own, a cramped universe containing the swindling Dutch company officials and their warehouses, gouty sailors speaking in salty doggerel, Asian magistrates and chamberlains, Japanese interpreters and a small contingency of East Asian slaves. There is also a half-civilized ape, called William Pitt, that first appears in the novel holding a bloody, freshly amputated leg courtesy of Dejima's resident Dutch surgeon, Marinus.
The clerk Jacob de Zoet has embarked on his career in the East to earn the guilders and esteem that will allow him to marry a sweetheart back home, and his superiors exploit his gullibility by asking him to work on a chronicle of petty corruption (a trick that exonerates the company's senior figures). His naiveté leads to a stunning inability to read the allegiances and motivations of those around him, setting in course their betrayal of him and his exile in Dejima. "The Orient is all about signals," one character muses, a theme running through "Thousand Autumns." The novel is a fiasco of missed signals and worse misunderstandings. Some are comical, as the Japanese go-betweens struggle to comprehend and translate Dutch phrases such as "impotent" and "proof positive," but many have grave consequences.
Mitchell milks this sense of tragedy in the second section of the novel, when a feudal warlord enslaves Orito, the Japanese midwife whom De Zoet has fallen for, in a bizarre mountaintop nunnery where ritual baby-harvesting (euphemistically called "The Engiftment") and murder are practiced. De Zoet undertakes to rescue her with the help of his closest Japanese interpreter friend, who organizes a commando-style mission undone by yet another act of betrayal and subterfuge. It's hard to know what Mitchell had in mind with this melodramatic detour into a comic-book Japonica of Shinto exoticism and Asian despotism, but it points to the author's unexpectedly weak handling of a crowded cast and twisting plot. The narrative is pockmarked with too many meanwhile-back-at-the-temple leaps, and the thread shows too often when Mitchell tries to stitch together the book's set pieces and character studies. In his earlier books, the disconnect of stories across time and space were fascinatingly and proddingly jarring. Here, they're frequently just jarring.
Which isn't to say that "Thousand Autumns" is a flop — far from it. When not tripping over the intricacies of its plot line, the novel features some of Mitchell's most luscious writing yet. For all the baroque movement of the story, the language is an exercise in extravagant control. Little flashes of haiku illuminate the text ("a night-soil man's buckets, swinging on his pole, stain the air"), and Mitchell interrupts his characters' thoughts with his own aperçus that have the feel of tiny punches. They have a cumulative effect. Walking through Dejima, De Zoet "smells steamed rice, sewage, incense, lemons, sawdust, yeast and rotting seaweed." In one beautiful passage, Mitchell laconically notes: "The incandescent sun is caged by a glowing bay tree." Later and more ominously we're told that "the yeasty moon is caged in his half-Japanese, half-Dutch window." And in a bravura moment in which Mitchell switches to first-person narration, a Sumatran slave philosophizes on what it means to have a name and on what part of himself a person can actually be said to possess. Having had everything else taken from him, the slave consoles himself with the sovereign pleasure of owning his own thoughts: "So I created a mind like an island … protected by deep blue sea. On my mind-island, there are no bad-smelling Dutchmen, or sneering Malay servants or Japanese men." Islands — and thinking — become their own forms of defiance.
But the island of Dejima can never bask in a protective sea. It's a zone of cultural contact where change is violently foreshadowed. The midwife Orito's teacher is the surgeon Marinus, also a learned philospher and an irascible naturalist who could have jumped ship from Richard Holmes' "The Age of Wonder." The scientific progress he represents is accompanied by surgical blood and gore (gleefully operating on an unanesthetized sailor, Marinus triumphantly holds up his booty, a gallstone "as big as an acorn and the yellow of a diseased tooth"). Dejima is cast as a showdown between Enlightenment and Engiftment. When an English ship arrives to wrest the island from Dutch control, its captain growls about blasting the Japanese into the 19th century. Unknown to him but already quite clear to Orito and De Zoet and the 21st century reader, upheaval doesn't always require gunboats and cannon shot.
Banks is the Brooklyn, N.Y.-based former editor of Bookforum.


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Some Medical practitioners remain in opposition to the use of marijuana still trapped with all of the negatives that are affiliated with THC, it can be time and energy to glimpse previous all These Unwanted side effects because the health and fitness benefits connected to CBD are undoubtedly about to outweigh every one of the negatives.
Steve I would want to talk to you immediately or messages I ponder if all this is accurate I'm using Those people tiny amount of him I broke my neck a several years again and they'd to wire it back again and keep me in position it's been a tricky 1 but I'm executing nicely, it's just so high-priced is is yours expensive
Cannabidiol, improved known as CBD, is becoming Just about the most properly-investigated and sought-just after cannabinoids in the cannabis plant – specially just after several high-profile media reports in the likes of CNN and ABC News caught the attention of most U.
Every single individual has an Endocannabinoid technique or even the ECS for short. The function of This technique is to manage quite a few normal and biological processes like appetite, mood, sleep, and pain.
I have pretty critical back pain. I discovered that CBD allows reduce the pain and also will help with my anxiety. I had been really skeptical, however I am happy that I stumbled on CBD oils.
The results of CBD oil are highly powerful On the subject of mental-connected ailments. But that's not where the benefits close. CBD oil is likewise regarded an exceedingly Secure remedy solution that the human body responds perfectly.
These receptors are existing during our physique but they are most considerable in locations like our immune procedure cbd oil for anxiety and Mind. The body is chargeable for making its own cannabinoids that share a similar chemical framework with cannabinoids which might be depending on plants – for instance CBD.
CBD benefits are authentic simply because CBD does make solid medicinal and therapeutic effects for even the most common ailments.
Though other cannabinoids (namely THC) are acknowledged to encourage CB1 receptors within the central anxious process to produce brain-altering outcomes, CBD has been proven in animal styles to operate in more of a "backdoor" way that has a naturally-happening enzyme often known as FAAH. When inhibited, this enzyme causes a Establish-up of anandamide from the brain, which is regarded as being the human body's most important By natural means-developed endocannabinoid (usually known as "the bliss molecule").
Another thing really should be quite obvious that CBD and THC are two elements precisely opposite of 1-A different. Which means if THC has the Homes to make you high, CBD does not. Hence, it is commonly not regarded as damaging as THC.
Both Desprez and McAllister placed impacted cells in a very petri dish coupled with cannabidiol. The results had been relatively astonishing because the Make contact with of cannabidiol with afflicted cells did not just cease behaving mad, but will also began to go back to their authentic state.
I wonder too. I can't smoke "weed" or choose thc oil this way both (I did like a teenager). I'm very serious about cannabis butter. I would like I could get health care for my anxiety and inflammation from an undiagnosed autoimmune problem. My c1 to skull gets inflamed for the reason that my head basically has become continually poping out of put For some time which also brings about pain and swelling along with autoimmune artery inflammation in my neck (go figure) which also results the base of my cranium simultaneously. I also get what I'd describe as epileptic seizures during the night time. It wakes me up by having an electrical burning sensation with the best of my cranium towards the roof of my mouth to toungue.
The truth is, studies recommend that in excess of four hundred million individuals world wide are diagnosed with the health-related affliction, resulting in over five million deaths inside of a year. The worst portion is usually that diabetes has started to become additional widespread than previously – due to our life and our latest eating behavior.
The observance in the test also discovered the main rationale driving the successful effects. Following coming in connection with cannabidiol, the above-expression of your effected cells stops.


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Castillon Côtes-de-Bordeaux AC 2016
Further details
- Winemaker
- Château Beynat
- Location
- Saint-Magne de Castillon, 33350
- Size of domaine
- 6.50 hectares
- Grape
- 70% Merlot, 30% Cabernet Franc on Riparia, 3309 and 10114 rootstock. Massale selection. 25 year old vines average. 5.000 vines per hectare. Vines trained guyot bordelaise.
- Terroir
- Clay and limestone at an elevation of 80 metres and facing south and south/east. 10° to 20° slopes. Depth of the soil is 50cm.
- Viticulture
- Organic viticulture (certified by Ecocert©). De budding and green harvesting only if needed. Mechanical management of grass and mowing. Soil is ploughed during Spring. Leave grass to grow throughout the vineyard. Sulphur and copper are used against oidium and mildew. Organic fertilizers are used during Spring. Machine harvested and table de trie used to check every individual grape. 42 hectos per hectare yields.
- Vinification
- Grapes are all destemmed. No prefermentive cold maceration. Grapes are not crushed before fermentation. Alcoholic fermentation in concrete vats for 10 to 14 days (at 25°C; max 27°C) using natural, indigenous yeasts. No chaptalisation. Vats are thermoregulated. 2 pumping over and 1 cap punching per day during fermentation. 20 days cuvaison. Pressed with a vertical hydraulic press. Press wine is blended with free run juice. Aged for 18 to 24 months (on average) in concrete vats with no racking. No bâtonnage, light filtration and no collage. Two bottlings during the year (one between January and March and another one between October and December). 35.000 bottles produced on average.
- Related links
- None available.


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Department of Mechanical Engineering, Northwestern University, Evanston, IL 60208
A comprehensive approach for computing the lubricant flow as functions of the roughness orientation is presented. By using a mapping function, a periodic, anisotropic surface is transformed into an isotropic one. The transformed isotropic roughness has only one characteristic wavelength, so that a double scale analysis based on this wavelength of roughness can be developed to calculate the flow factors introduced in the average Reynolds equation. In addition, the Bhushan number instead of the conventional Peklenik number is used to describe the surface property more clearly. The new analysis provides an accurate evaluation of the roughness effects with only a second order approximation. The agreement is excellent even for incipient surface contact. An explicit relationship between the nondiagonal flow factors, which are associated with the pumping flux, and the diagonal ones have been established. It has been found that the maximum pumping action takes place when the asperities are oriented at 45 degrees to the direction of pressure gradient or sliding motion. The pumping effect caused by the relative sliding is about 50 percent greater than the effect caused by the pressure gradient. Strictly speaking, the asperities oriented at 45 degrees to the sliding direction will give different flow factors for different Bhushan numbers. This differs from some prior studies for periodic or area-distributed random roughness. Marked difference in flow factors due to the difference of asperity orientation is also observed. It suggests that caution should be used when the average Reynolds equation is applied, such as in the case of axisymmetric punching where various asperity orientations exist.
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Are you one of those people who are always suffering setbacks? Does little ever seem to go right for you? Do you sometimes feel that the universe is out to get you? Do you wonder:
Why do I have bad luck? Is bad luck real?
Let me let you into a secret:
Your luck is no worse—and no better—than anyone else's. It just feels that way. Better still, there are two simple things you can do which will reverse your feelings of being unlucky and change your luck.
1. Stop believing that what happens in your life is down to the vagaries of luck, destiny, supernatural forces, malevolent other people, or anything else outside yourself.
Psychologists call this "external locus of control." It's a kind of fatalism, where people believe that they can do little or nothing personally to change their lives.
Because of this, they either merely hope for the best, focus on trying to change their luck by various kinds of superstition, or submit passively to whatever comes—while complaining that it doesn't match their hopes.
Most successful people take the opposite view. They have "internal locus of control." They believe that what happens in their life is nearly all down to them; and that even when chance events occur, what is important is not the event itself, but how you respond to it.
This makes them pro-active, engaged, ready to try new things, and keen to find the means to change whatever in their lives they don't like.
They aren't fatalistic and they don't blame bad luck for what isn't right in their world. They look for a way to make things better.
Are they luckier than the others? Of course not.
Luck is random—that's what chance means—so they are just as likely to suffer setbacks as anyone else.
What's different is their response. When things go wrong, they quickly look for ways to put them right. They don't whine, pity themselves, or complain about "bad luck." They try to learn from what happened to avoid or correct it next time and get on with living their life as best they can. They have this Motivation Engine, which most people lack, to keep them going.
No one is habitually luckier or unluckier than anyone else. It may seem so, over the short term (Random events often come in groups, just as random numbers often lie close together for several instances—which is why gamblers tend to see patterns where none exist).
When you take a longer perspective, random chance is just . . . random. Yet those who feel that they are less lucky, typically pay far more attention to short-term instances of bad luck, convincing themselves of the correctness of their belief.
Your locus of control isn't genetic. You learned it somehow. If it isn't working for you, change it.
2. Remember that whatever you pay attention to grows in your mind.
If you focus on what's going wrong in your life—especially if you see it as "bad luck" you can do nothing about—it will seem blacker and more malevolent.
In a short time, you'll become so convinced that everything is against you that you'll notice more and more instances where this appears to be true. As a result, you will drown yourself in negative energy and almost certainly stop trying, convinced that nothing you can do will improve your prospects.
Fatalism feeds on itself until people become passive "victims" of life's blows. The "losers" in life are those who are convinced they will fail before they start anything; sure that their "bad luck" will ruin any prospects of success.
They rarely notice that the true reasons for their failure are ignorance, laziness, lack of skill, lack of forethought, or just plain foolishness—all of which they could do something to correct, if only they would stop blaming other people or "bad luck" for their personal deficiencies.
Your attention is under your control. Send it where you want it to go. Starve the negative thoughts until they die.
To improve your fortune and have "good luck", first decide that what happens is nearly always down to you; then try focusing on what works and what turns out well, not the bad stuff.
Your "fate" really does depend on the choices that you make. When random events happen, as they always will, do you choose to try to turn them to your advantage or just complain about them?
If you think you're "suffering from bad luck", you can really change things up and start life over. It may even be a lot easier than you thought:


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Welcome version 3 of ITIL®! The Information Technology Infrastructure Library has become the growing standard for managing any infrastructure of information technology. The standard consists of several books that detail the best practices for management, development, and monitoring the infrastructure.
For the readers not familiar with past iterations of ITIL®, the collection of books fell into two primary sets of disciplines: Service Delivery and Service Support. Service Delivery focuses on those processes required to proactively provide IT services to the business. Most processes, like Service Level Management, are planning processes from which the context to deliver IT services are formed. Service Support concentrates on the services themselves, ensuring that the user has access and ensuring that the services remain in an operational mode. Processes like problem management and change management are found in Service Support.
Since May 2007, ITIL® has restructured the collection of books to better reflect the life cycle of IT Service Management. Five key volumes now make up the ITIL® framework.
- Service Strategy
- Service Design
- Service Transition
- Service Operation
- Continual Service Improvement
Service Strategy consists of processes to build a long term strategy for the IT environment. It focuses on the creating the direction of IT Services. To support the strategies developed, the architecture of the IT infrastructure must be design. The architecture takes into account the physical and software assets, processes, documentation, and staffing. The majority of processes found in Service Delivery can be found in the new volume of Service Design.
In order to fulfill on the design opportunities of the architecture, it must be built. Service Transition moves the business from design into operational mode. In most situations, transition requires supporting the IT environment as before while adopting new design components to the environment. Once the architecture is implemented, Service Operation provides the structure for maintaining the performance requirements of the architecture. Many of the Service Support processes can be found in Service Operations.
The final volume of Continual Service Improvement provides the best practices for handling changing business needs and implementing improvements to service delivery.
To find out more about ITIL® v3, go to The Art of Service website, theartofservice.com.


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Accredited College with Bachelor's Degree & Graduate Programs Online & On-Campus | National University
By submitting this form, I confirm that the information provided on this form is accurate and complete and I agree to receive email communications, phone calls, and SMS/Text Messages about Educational opportunities from National University System and/or it's affiliates. I understand that I may withdraw my consent at any time. I understand that if I am submitting my personal data from outside of the United States, I am consenting to the transfer of my personal data to, and it's storage in, the United States, and I understand that my personal data will be subject to processing in accordance with U.S. laws. Please review our privacy policy or contact us for more details at email@example.com.
NUVHS offers a completely online dual diploma program that provides international students the opportunity to earn an accredited United States high school diploma while concurrently earning a diploma in their home country.
Through this program, global students considering attending an American university have the opportunity to enroll in U.S. college-prep courses to prepare for a rigorous American college education. Students will improve their English language skills and interact with American students through engaging online courses led by highly qualified high school instructors.
International students will have access to:
After meeting English Language Proficiency (ELP) requirements, international students typically transfer a large number of their local high school credits and require as few as 12 additional online courses over four years to earn their NUVHS diploma.
For more information, simply fill out the form and an advisor will contact you soon.


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Bour Basehit Beats Boston [VIDEO]
The Boston Red Sox bullpen failed its first test since finding out closer Koji Uehara would be lost for the season as they blew a 4-run lead and lose to Miami 5-4 in 10 innings.The Marlins cut the lead in half in the bottom of the 6th inning off Sox starter Steven Wright which led to the bullpen taking over.
Ichiro Suzuki came up with a sacrifice fly in the 7th inning off Alexi Ogando. With no Uehara Boston called upon Junichi Tazawa to close out the game but allowed a game tying sacrifice fly to Adeiny Hechavarria.
In the bottom of the 10th Craig Breslow entered the game and served up a leadoff triple to Dee Gordon followed by an intentional walk to Martin Prado setting up the walk-off single to left center field by Justin Bour.
Mookie Betts led the Sox in the loss with 3 hits and 2 rbi while Jackie Bradley Jr. added 2 hits, 2 rbi and a spectacular catch in left field.
Eduardo Rodriguez will take the mound in the series finale tonight.


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Fishing is turning out to be the 'fisherman's tale' of Brexit. Full of drama and daring in the telling, but little of substance on dry land.
By telling fishy tales, Brexiteers have rewarded the Scottish Conservative MPs for saving Theresa May's political life by pushing them overboard.
Fishing has mythical importance to Brexiteers, who exploited its powerful betrayal narrative. Brussels' flawed Common Fisheries Policy (CFP) forced British fishermen to scrap boats, throw back catches, and open up British waters to foreign competitors. If Brexit was a vote to take control of the UK's laws, borders and money, it was also a vote to take control of its seas.
Fishing represents one of the few areas where the UK has a clear advantage in talks with the EU. It is a tiny part of the UK economy, but has much greater value to the EU side, something so clear to Whitehall that it was plotted on a graph in the government's Brexit analysis. One of Germany's biggest fish-processing plants is in Angela Merkel's constituency.
This meant the UK had a great bargaining position, but the Government decided to use this to make a deal. Michael Gove promised a speedy exit from the CFP, but the UK wanted too many other things from the EU to fight for it. The announcement that the UK will stay bound by CFP quotas through a post-Brexit transition was always coming in on the tide.
May needs fishing votes as much as Merkel, but the constituencies where they count are in Ruth Davidson's backyard. Scottish Tories are furious about the transition terms on fishing, but there is little they can do.
Davidson and industry leaders want guarantees that fishermen won't be let down again in the final Brexit deal, but a vote on that could come before talks on future EU fishing access. When Davidson and Gove co-authored an article on fishing two weeks ago, it was seen as a power play. In fact, it was probably softening up the ground for yesterday's announcement. The lesson is: don't believe stories about the one that got away.


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At People Water it doesn't matter to us whether we are building a new well or fixing a broken one. The only thing that matters is that PEOPLE are getting clean water and with that clean water a chance to live a better life. That's why we do what we do, and that's why we drink what we drink.
Through our Drop for Drop initiative, we develop or restore new sources of clean water, either by drilling a new well, repairing an existing well, establishing a water purification system, or implementing new water-providing technologies.
When properly maintained, a well can last a very long time. However, they require regular maintenance, right now it is estimated that there are over 800,000 broken wells throughout the world.
Many wells breakdown simply because maintenance is not being performed or was simply not planned into the original project. People Water's model is built on the idea of sustainability. Because our Wells are supported through a for-profit business model, we are able to plan, repair and maintain wells consistently. Think of it as circular, something we like to call Good Karma!
Have a drink with us and together we will change the World one Drop at a time.
your water impacts their water
An average well when properly maintained can deliver up to approximately 3,000 liters of clean water a day.
Many people suffer from diseases associated with lack of access to safe drinking water, and drinking dirty water accounts for a high number of hospital patients.
Investment in drinking water and sanitation will result in 272,000,000 more days of school each year.
Women and children often have to make long exhausting walks to get water miles away.
When a well is put into a community it provides positive change. No more drinking dirty water, no more long exhausting walks for women and children, and most importantly, the people of the village can be healthier.
By providing PEOPLE clean water, we are able to help individuals in many areas of their lives. Once clean water is made available, children are much healthier and are able to go to school and stay in school. Communities as a whole do better financially because people are staying with their jobs or helping their families and not losing time fetching water or taking care of a sick child. The effect of clean water to a community is literally life changing.
Sign up to hear from us about news, specials, and events.


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Kansas State University
Kansas State beats No. 25 Oklahoma 72-66
Just when it looked like the game was slipping away, Kansas State took control.
That's the kind of poise, resolve and toughness the Wildcats showed during a 72-66 victory over Oklahoma on Tuesday at Bramlage Coliseum.
Not only did the come-from-behind win come three days after a humbling blowout loss at Kansas, it came in dramatic fashion.
For much of the night, it appeared Oklahoma was simply too strong for the Wildcats. Case in point: The Sooners held a 62-56 lead with 5 minutes, 37 seconds remaining despite making 33.3 percent of their shots, getting two points from leading scorer Cameron Clark and allowing K-State to make three-pointers at an uncharacteristically high rate.
Despite those key factors working against them, they had confidence and a two-possession lead.
But it was K-State who dominated down the stretch.
Less than three minutes later, the Wildcats were on top 66-63 and on their way to their third win over a ranked foe.
"They made a run, we made a run," Weber said. "We were just fortunate at the end that we were able to make the run that gave us the victory. That's what you hope you can do at home, find a way to win. It's great. That's a good win."
The game-changing swing began in unexpected fashion when freshman forward Wesley Iwundu, who had made one three in his first 16 games, nailed a three-pointer from the corner. Then freshman guard Marcus Foster, who led K-State with 18 points, made a jumper, Thomas Gipson hit a layup and Foster hit another three. Just like that, K-State went from trailing by six to leading by three with 2:07 to go.
Oklahoma made several attempts to retake the lead, but K-State repeatedly turned it away. Gipson and Iwundu neutralized Sooners big man Ryan Spangler — who tormented the Wildcats with 21 points and 14 rebounds — on the glass. Shane Southwell, who had 16 points and eight rebounds, stripped the ball away from Clark as he drove to the basket. And Nino Williams, who had 10 points and nine rebounds, helped the Wildcats close things out at the foul line.
"I hit a big shot in the corner," said Iwundu, who had 11 points and six rebounds. "I was wide open. I really had no choice but to make it. That and rebounding (was important late). That was something I really wanted to be good at this game."
With the win, K-State (13-4, 3-1 Big 12) regained some of the momentum it lost over the weekend. It has won 11 of its last 12 games.
"That was big for us," Foster said. "Coaches were talking about how last year they lost to Kansas and they couldn't recover right and lost to Iowa State. That was our emphasis coming into this game, we've got to bounce back from that Kansas loss and get it out of our head. We did a good job of not thinking about it. Our focus was on OU."
No. 25 Oklahoma (13-4, 2-2) lost its first game after breaking into the top 25.
"It was a tough game," Oklahoma coach Lon Kruger said. "I thought down the stretch K-State did some really good things. They made shots when they needed to, got loose balls and got second-chance opportunities. We did not convert on our end.
"When you are in a position to win on the road you would like to go ahead and do that. We're not quite ready, I guess."
Not against K-State's defense, anyway. It held Oklahoma well below its average of 87 points, limiting Clark to two points on nine shots. Clark entered the game as the Big 12's leading scorer, but he struggled to find good looks on Tuesday.
Combined with some hot shooting in the first half, which featured threes from Foster, Iwundu and Will Spradling on five straight possessions, and the Wildcats had found a way to frustrate the Sooners.
"That was a big boost," Southwell said. "You saw the energy in the building. I think it got them a little bit rattled, especially on offense. When we came down and made a three they came down and just shot the ball instead of running their sets, which they are very good at.
"When we hit threes and get momentum like that it is very big on both sides of the ball."
K-State's hot start allowed it to take a nine-point lead midway through the first half and head into halftime with a 39-38 advantage.
It relied on something more essential late.
"That was a character game and a gut-check game," Weber said. "Those are the games you have to win at home."


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More painting was done so a bath and hair wash was required before the pumpkin suite went on.
Pumpkin carving
Really chuffed with how the pumpkins and the lantern turned out - Rye was fascinated.
Story telling. A really lovely book that goes through different sounds and different animals etc that can be found each season. Beautiful illustrations too.
I took Rye out too, not to trick and treat, but I thought a walk to see the other kiddies in costumes would be fun - except we didn't see anyone. Not sure if I was too early, or if no one tricks or treats around here. I only had one group come to the door - very suprised considering the number of children around here.
We had lovely pitta bread with our pumpkin soup:
Later once Rye was settled I lit the candles on my alter, plus extra for my passed Mum, Dad and brother and made a few wishes. I sat and contempated and watched the flames and then snuggled down to sweet dreams.


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West Bengal and Sikkim (14 days)
Kolkata-Darjeeling-Pelling-Yuksom-Ravangla-Gangtok-Kalimpong-Gorumara-Bagdogra- Kolkata.
A two week expedition of West Bengal and the remote kingdom of Sikkim - home to fabulous scenery, amazing religious monuments and lively cultures - offering outstanding experiences which will live long in your memory.
Day 1: Arrival Kolkata
Upon your arrival at Kolkata airport, you will be met on arrival and transferred to your hotel for overnight stay.
Day 2: Kolkata (B)
After breakfast, we take a sightseeing tour of the city accompanied by a local guide who will take you to all the famous monuments, temples and bazaars, including a visit to the imposing Victoria Memorial. Opened by the Prince of Wales in 1921, this vision in marble is often called the Taj of the British Raj. We also visit the New Market a bazaar on Chowringhee Road, the stately Indian Museum founded in 1814 and the oldest and largest in India, St Pauls Cathedral and the extraordinary Marble Palace which houses paintings by Rubens, Titian and Reynolds.
In the evening transfer to railway station from where you board the Darjeeling Mail train to New Jalpaiguri. Overnight onboard the train.
Day 3: Darjeeling (D)
Arrive New Jaipalguri around 0800hrs. Upon reaching New Jaipalguri, drive on by road to Darjeeling (90 km from NJP Railway Station). Check-in at the hotel. Evening at leisure to explore the hill town. Overnight at hotel.
Day 4: Darjeeling (B/D)
Wake-up early in the morning and reach Tiger Hill to enjoy the magnificent view of sunrise on the Mt. Kanchanzonga. Stop midway to visit the Batasia Loop and Ghoom Monastery. Back to the hotel for breakfast then start a sightseeing tour covering the Himalayan Mountaineering Institute, the Zoological Gardens, the Tibetan Refugee Self Help Centre and a visit to a tea estate. Later, transfer to Darjeeling Himalayan Railway, affectionately known as the Toy Train for a Joy Ride to Ghoom, surely the most romantic way to approach the majestic and mysterious Himalayas. In the evening, take a guided walk through the Mall area, full of food stalls, markets and shops. Afterward, head back to your hotel for dinner and overnight.
Day 5: Darjeeling-Pelling (B/D)
After an early breakfast, drive 130km to Pelling. The journey affords opportunities to stop and admire magnificent scenery, including misty pine forests, tea gardens, river valleys, waterfalls, and of course the snow capped mountains of Khangchendzonga. The pleasant little town of Pelling is the base from where trekkers set out for treks into West Sikkim.
Day 6: Pelling (B/D)
This morning visit two of Sikkims most revered monasteries. The oldest, Sangachoeling Monastery, was founded around 1697 (walk approx 40 mins). The second oldest, the awe-inspiring Pemayangtse (Perfect Sublime Lotus) Monastery, founded in 1705, houses a good collection of wall paintings and sculptures. Also visit the Rabdentse Ruins.
Day 7: Pelling-Yuksom (B/L/D)
Drive 52km from Pelling to Yuksom, the first capital of Sikkim en-route the sacred Khecheopalri Lake, upon which leaf lamps are floated during evening prayers. Situated at an altitude of 1780m, it is the starting point for mountaineering expeditions and treks to higher Khangchendzonga ranges. You may like to visit the Dhubdi Monastery, located on a hilltop about an hours trek from Yuksom. Built in the 17th century, its name means the retreat.
Day 8: Yuksum-Ravangla (B/L/D)
Ravangla is popular for its breathtaking views of the Himalayas and for its tranquility. You may like to simply relax and enjoy the scenery here, or you may prefer to trek through the surrounding forests of oak, pine, chestnut, magnolia and rhododendron trees.
Day 9: Ravangla-Gangtok (B/D)
This morning drive 65 km to Gangtok stopping off on the way to visit Singtam and its major attraction, the Bermoik Monastery. Gangtok, the capital of Sikkim, is known for its extravagant scenic beauty and is famous for its monasteries. Check into your hotel with free time in the afternoon to explore the fascinating bazaar on MG Road.
Day 10: Gangtok (B/D)
Rise early to view sunrise over the Kanchenjunga range from Tashi View Point. After breakfast take in the sights around Gangtok, including Ganesh Tok, Hanuman Tok, and the Institute of Tibetology. Later visit the serene monastery at Rumtek. The contrast between the bustle of busy Gangtok and the quiet of the monasteries and gompas at Rumtek, is striking.
Day 11: Gangtok (B/D)
An excursion today to Tsomgo Lake, set in an area of spectacular scenic beauty. This is the closest point that westerners can access to the Tibetan border, some 20km further on. While there you can walk or ride a yak around the lake (direct Payment). Yaks may not be the most comfortable animals to ride but this is an unmissable opportunity!
Day 12: Gangtok-Kalimpong (B/D)
Leaving Sikkim, make the 85 km journey to Kalimpong, known for its horticulture (particularly its wide array of orchids and gladioli) and spectacular views. In the afternoon take in some of the highlights of this picturesque town including the Dello Hills, Dr Grahams Childrens Home, a small factory making paper by traditional methods, and flower nurseries.
Day 13: Kalimpong-Gorumara. (B/L/D)
Today drive to Gurumara National Park. Located in the Terai region of the Himalayan foothills, this medium-sized wildlife sanctuary of grasslands and forests is primarily known for its population of Indian Rhinoceros. The best way to view wildlife is from the numerous well-located watchtowers. Afternoon enjoy Jeep Safari at Gorumara National Park- Overnight at Chalsa/Gorumara.
Day 14: Gorumara-Bagdogra- Kolkata (B)
After breakfast, drive to Bagdogra airport (80kms) to board your flight to Kolkata, departs at 1510hrs and arrives Kolkata at 1610hrs. On arrival, transfer to your hotel where a day-use room is available for your comfort. The remains of the day is at leisure followed by evening transfer to airport.
Please note that, whilst every effort will be made to carry out the programme shown above, small changes may sometimes be necessary.
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The Jean Marcel watch - the fine line between expense and investment.
The collections of Jean Marcel and celebrated master watch maker Vincent Calabrese have always been innovative, if not revolutionary, and have been distinguished with the highest international prizes - The Gold Medal in 1977 and the Silver Medal in 1989 from the Geneva Inventors Salon. Marcel's model Tourbillon Volant was admitted to the collection of the International Horological Museum at La Chaux-de-Fonds, Switzerland.
There is the Mystery Collection, which uses a special reconstructed self-winding movement to shows the hours, minutes and seconds without the use of hands, and there is the Cisitalia Grand Prix 360, which indicates hours, minutes and seconds in a three-register format, also using a special reconstructed self-winding movement. The added care and features in the finish of the watches is evident in the sapphire crystal each watch has. By gently breathing on the sapphire glass, the name Mystery appears, and then vanishes again. Production of these exquisite timepieces is limited to 100 pieces per month.
The Jean Marcel watch stands at the top of international watch concepts. The finest quality materials and innovation in movements and design have kept Jean Marcel at the forefront of Swiss watchmaking.
|Watch Brands
Auguste Reymond Baume et Mercier Bell and Ross Breitling Cartier Concord Dubey and Schaldenbrand Ebel Givenchy Gucci IWC Krieger Longines MOMO Movado Omega Rolex TAG Heuer XEMEX Zenith


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Through Angel's Eyes is about to explode onto the teen fiction marketplace. Reserve your copy now and start seeing life Through Angel's Eyes.
Alabama, 1963 - a world is about to explode . . .
Angel Dunbar's life is about to be engulfed by the flames.
She'll be humiliated, spat upon, beaten and left for dead.
And she'll fall in love . . . with a dangerous twist.
Want to know what really happened when Birmingham exploded?
Then you've just got to live it . . . Through Angel's Eyes.
Read it first with your free chapter download here
About The Author
Steve Theunissen is a 47 year old middle school teacher who lives in Tauranga, New Zealand. Through Angel's Eyes, Steve's first novel, draws upon his life-long study of the Civil Rights movement and the inspiration that he has received from Martin Luther King and from the Birmingham, Alabama child marchers.
22 Marire Drive Ohauiti, Tauranga | 64 07 5446161
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For 75 years, Carolina Casualty has been a premier insurance brand in the trucking industry. We specialize in writing liability, physical damage and motor truck cargo insurance for local, intermediate and long-haul truckers, as well as private carriers.
Originally incorporated in North Carolina in 1943, the company moved to Jacksonville, Florida in 1958 after an affiliation with the Insurance Company of the South, followed by a merger between the two companies in 1966. In January 1985, W. R. Berkley Corporation purchased Carolina Casualty.
Carolina Casualty is headquartered in Jacksonville, Florida, with underwriting team members in Knoxville, Tennessee and Boise, Idaho. Additionally, our risk management team members are strategically located across the country. With specialized expertise in underwriting, claims and risk management, our nationwide team of professionals considers risks for which other companies are not prepared.
Carolina Casualty is a member company of W. R. Berkley Corporation, an insurance holding company that is among the largest commercial lines writers in the United States.
W. R. Berkley Insurance Group Ratings:
A+ (Superior) Financial Size Category XV by A.M. Best
A+ (Strong) by Standard & Poor's