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Cytochrome P450scc metabolizes vitamin D3 to 20-hydroxyvitamin D3 (20(OH)D3) and 20,23(OH)2D3, as well as 1-hydroxyvitamin D3 to 1α,20-dihydroxyvitamin D3 (1,20(OH)2D3). It also cleaves the side chain of 7-dehydrocholesterol producing 7-dehydropregnenolone (7DHP), which can be transformed to 20(OH)7DHP. UVB induces transformation of the steroidal 5,7-dienes to pregnacalciferol (pD) and a lumisterol-like compounds (pL).
Methods and Findings
To define the biological significance of these P450scc-initiated pathways, we tested the effects of their 5,7-diene precursors and secosteroidal products on leukemia cell differentiation and proliferation in comparison to 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). These secosteroids inhibited proliferation and induced erythroid differentiation of K562 human chronic myeloid and MEL mouse leukemia cells with 20(OH)D3 and 20,23(OH)2D3 being either equipotent or slightly less potent than 1,25(OH)2D3, while 1,20(OH)2D3, pD and pL compounds were slightly or moderately less potent. The compounds also inhibited proliferation and induced monocytic differentiation of HL-60 promyelocytic and U937 promonocytic human leukemia cells. Among them 1,25(OH)2D3 was the most potent, 20(OH)D3, 20,23(OH)2D3 and 1,20(OH)2D3 were less active, and pD and pL compounds were the least potent. Since it had been previously proven that secosteroids without the side chain (pD) have no effect on systemic calcium levels we performed additional testing in rats and found that 20(OH)D3 had no calcemic activity at concentration as high as 1 µg/kg, whereas, 1,20(OH)2D3 was slightly to moderately calcemic and 1,25(OH)2D3 had strong calcemic activity.
Citation: Slominski AT, Janjetovic Z, Fuller BE, Zmijewski MA, Tuckey RC, Nguyen MN, et al. (2010) Products of Vitamin D3 or 7-Dehydrocholesterol Metabolism by Cytochrome P450scc Show Anti-Leukemia Effects, Having Low or Absent Calcemic Activity. PLoS ONE 5(3): e9907. https://doi.org/10.1371/journal.pone.0009907
Editor: Jürgen Schauber, Ludwig-Maximilians-University, Germany
Received: November 16, 2009; Accepted: February 26, 2010; Published: March 26, 2010
Copyright: © 2010 Slominski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: NIH grant R01A052190 to AS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
In vivo, vitamin D3 (D3) is generated in the epidermis through photochemical transformation of 7-dehydrocholesterol (7DHC) after absorption of ultraviolet radiation B (UVB; wavelength 280–320 nm) energy –. The active form of vitamin D3, 1,25(OH)2D3, is generated systemically through sequential 25- and 1α– hydroxylation in the liver and kidney, respectively , . It is also produced locally in different organs including skin , . 1,25(OH)2D3, in addition to being a major regulator of body calcium homeostasis, also inhibits cell proliferation, stimulates differentiation and/or apoptosis, has tumorostatic and anticarcinogenic properties, and protects DNA against oxidative damage , –. Importantly, 1,25(OH)2D3 and its derivatives also display potent anti-leukemic activities –. Different leukemia lines serve as excellent models for testing vitamin D analogs. Unfortunately, the toxicity (hypercalcemia) of high levels of vitamin D largely prevents the use of pharmacological doses of 1,25(OH)2D3 for either the prevention or treatment of cancer. Therefore, there is a continuing search to find vitamin D analogues (for example, Gemini analogs) that retain antiproliferative activity but that are non-calcemic, acting as partial receptor agonists for the VDR –. In addition, more than 30 years ago Holick et al showed that shortening the side chain of vitamin D or eliminating it (producing 20-hydroxypregnacalciferol (20(OH)pD), attenuates or eliminates the calcemic effect .
Cytochrome P450scc hydroxylates vitamin D3 in a sequential manner, predominantly to 20-hydroxyvitamin D3 (20(OH)D3). Although further hydroxylation producing 20,23(OH)2D3 and other metabolites also occurs, these hydroxy-derivatives represent a minority of reaction products –. 20(OH)D3 can also be produced by P450scc in purified adrenal mitochondria  and acts as a potent inducer of keratinocytes differentiation  and inhibitor of NF-κΒ activity . P450scc also hydroxylates 1-hydroxyvitamin D3 to 1,20-dihydroxyvitamin D3 (1,20(OH)2D3) a product that shows antiproliferative activity towards human keratinocytes . Finally, P450scc in vitro or ex-vivo in adrenal glands, cleaves the side chain of 7-dehydrocholesterol (7DHC) producing 7-dehydropregnenolone (7DHP), which can undergo further oxidations by steroidogenic enzymes , . 7DHP and it hydroxyderivatives, can undergo UVB-induced transformation to corresponding pregnacalciferol (pD) and lumisterol-like (pL) compounds –. These secosteroids with only a two-carbon side chain (pD and 20(OH)pD) have been shown to lack calcemic activity in vivo .
Thus, having a large panel of novel secosteroidal compounds that can be biologically active but do not have a side chain or have a side chain modified at C20 (similar to the Gemini type analogues), we decided to test whether any of these novel compounds could act as relatively non-toxic anti-leukemic agents.
1. Structures and origin of novel secosteroids (see Figure S1)
Vitamin D3, produced through photolytic transformation of 7-DHC, is hydroxylated to 1,25(OH)2D3 (calcitriol) by the sequential actions of 25- and 1α-hydroxylases . Vitamin D3 is also hydroxylated by P450scc to 20(OH)D3 and 20,23(OH)2D3 , , while 1,20(OH)2D3 is generated through hydroxylation of 1(OH)D3 by P450scc  or by the action of 1α-hydroxylase on 20(OH)D3 . 20(OH)D3 can also derive from UVB induced photolytic transformation of 20(OH)7DHC that is produced by hydroxylation of 7DHC by P450scc . 20(OH)7DHC is further transformed to 7DHP by P450scc ,  then after absorption of UVB photons forms pD or pL depending on the UV energy . 7DHP is potentially reduced by 20-hydroxysteroid dehydrogenase to 20(OH)7DHP  which undergoes UVB induced transformation to 20(OH)pD .
2. Novel secosteroids have low calcemic effect
One of us (MFH) has documented that elimination of all but two carbons of the side chain in vitamin D3, leading to production of 20(OH)pD and pD, eliminates the calcemic effect of the secosteroid . Since P450scc hydroxylates the side chain of D3 and 1(OH)D3 at C20, we tested the calcemic effects of the resulting products in rats at a doses 0.1 µg/kg–3 µg/kg of body weight (Fig. 1). 20(OH)D3 at dose as high as 3.0 µg/kg had no calcemic activity (calcium = 10.4±1.5 vs 9.3±1.3 mg/dL for control), whereas 1,25(OH)2D3 at the same dose had the expected strong calcemic effect raising calcium to 16.0±1.2 mg/dL. Interestingly, addition of a 1α-hydroxyl group to 20(OH)D3 promoted calcemic activity raising calcium to 13.9±0.8 mg/dL. Thus for 20(OH)D3, the lack of 1α-hydroxylation underlies its inability to influence serum calcium. 1,20(OH)2D3 displays a calcemic effect, however less than that of 1,25(OH)2D3. This is consistent with a fundamental role of the hydroxyl group at the 1 alpha-position of vitamin D for the regulation of serum calcium levels , .
3. Novel secosteroidal products show anti-leukemic activity
Anti-leukemic effects of 20(OH)D3, 20,23(OH)2D3, 1,20(OH)2D3, pD, 20OHpD and pL in comparison to 1,25(OH)2D3 and to precursor steroidal 5,7-dienes (for structures see Figure S1), were tested in K562 human chronic myeloid and MEL mouse erytholeukemia and human HL-60 promyelocytic and U937 promonocytic leukemia cells. The choice of 10−7 M as the concentration of drugs to use in the majority of experiments was based on initial experiments (not shown) as well as on already published data , , , , demonstrating that this concentration is optimal for comparative studies.
The secosteroids inhibited cell proliferation, however, with different potencies in different cell lines. Specifically, in mouse (Mel) erythroleukemia the strongest inhibitory effects were seen for both 1,25(OH)2D3 and 20,23(OH)2D3 with slightly lower (p<0.05) but strong effects for 20(OH)D3 (p<0.01) (Fig. 2A) (Table S1A). 1,20(OH)2D3 was less potent than 20(OH)D3 (p<0.05), however, more potent than pD, pL, and 7DHP (p<0.05), while 20(OH)pD and 20(OH)7DHP had no effect (Table S1A). The latter compounds were less efficient than 1,25(OH)2D3, 20,23(OH)2D3 and 20(OH)D3 (p<0.05 or 0.01) (Table S1A).
The anti-proliferative effects of novel secosteroidal and steroidal 5,7-diene compounds on Mel mouse erythroleukemia (A), human K562 myeloid (B), HL-60 human promyelocytic (C), U937 promonocytic (D) leukemias. After treatment for 7 days with different compounds (10−7 M), cells were stained with trypan blue and the number of viable cells counted. Statistically significant effects are marked as follow *p<0.05; **p<0.01; ***p<0.001.
1,25(OH)2D3 and 20,23(OH)2D3 were the most effective in inhibiting proliferation of K562 human myeloid leukemia while slightly lower (p<0.05) but highly significant inhibition was seen for 20(OH)D3 (p<0.01) (Fig. 2B) (Table S1B). 1,20(OH)2D3 and pL were least potent, and 7DHP, 20(OH)7DHP and 20(OH)pD had no effect on these cells (Table S1B). Interestingly pD had a significant inhibitory effect on proliferation, which was not statistically different from the effects of 1,25(OH)2D3, 20,23(OH)2D3, 20(OH)D3 and 1,20(OH)2D3.
In human HL-60 promyelocytic and U937 myeloid leukemia all of the secosteroids and steroidal 5.7-dienes showed statistically significant inhibitory effects, however, their relative potency were different for both cell lines (Fig. 2C, D). The relative potency for HL-60 cells was as follows: 1,25(OH)2D3>20(OH)D3 = 20,23(OH)2D3 = 1,20(OH)2D3 = 20(OH)pD>pD = pL( = 7DHP) >20(OH)7DHP( = 7DHP), while for K562 cells it was 1,25(OH)2D3 >20(OH)D3 = 1,20(OH)2D3>20,23(OH)2D3 = pL = 7DHP = 20(OH)7DHP>pD = 20(OH)pD, respectively (Fig. 2C, D) (Table S1C, D).
The induction of erythroid differentiation was evident by the pink-red cell pellet of K562 cells after incubation for 7 days in the presence of secosteroids and steroidal 5.7-dienes (Fig. S2A). The benzidine staining shows that hemoglobin synthesis begins at day 2 (data not shown) and approximately 60%–80% of the cells treated with drugs for 7 days stained positively for benzidine (Fig. 3C; Fig. S2B). 1,25(OH)2D3, 20(OH)D3, 20,23(OH)2D3, 1,20(OH)2D3, pD and pL had similar potency for induction of differentiation as determined from the number of benzidine positive cells (Fig. 3; Table S2A). 20(OH)pD, 7DHP and 20(OH)7DHP were significantly less potent than 1,25(OH)2D3, 20(OH)D3 and 20,23(OH)2D3, but of similar in potency to other compounds tested with the exception of 1,20(OH)2D3 which was more potent than 7DHP (Table S2A).
Induction of erythroid differentiation determined was from the the concentration of hemoglobin in an equal number of cells. Cells were treated for 7 days with different compounds (10−7 M), stained with benzidine solution and the amount of hemoglobin as hemin in Mel (A) or K562 (B) leukemia cells was determined by measuring the absorbance at 600 nm after lysis of the cells. ***p<0.001. C.The number of benzidine positive K562 cells after 7 days of treatment with different compounds (10−7 M). Cells were stained with benzidine solution and the number of benzidine positive (blue) cells was determined by counting 200 cells per microscopic field. The results are present as % of benzidine positive cells in comparison to total cell number. *p<0.05; **p<0.01; ***p<0.001.
When the level of differentiation was determined from the relative concentration of hemoglobin as hemin measured spectrophotometrically in an equal number of cells, all compounds tested induced significant cell differentiation. 1,25(OH)2D3 was more potent than other compounds with the sequence of potency being: 1,25(OH)2D3>20,23(OH)2D3> 20(OH)D3>pL>pD>1,20(OH)2D3>7DHP>20(OH)pD>20(OH)7DHP for K562 cells and 1,25(OH)2D3>20,23(OH)2D3>20(OH)D3>1,20(OH)2D3>pL = 20(OH)pD>7DHP = 20(OH)7DHP>pD in Mel cells (Fig. 3A and B; Table S2B, C).
The induction of differentiation of HL-60 and U937 cells towards monocyte-like morphology was evaluated by NBT reduction and cell morphology. The first signs of morphological differentiation were observed at 72 h for all compounds tested with the differentiation being most pronounced for 1,25(OH)2D3 (data not shown). At day 5, the monocytic maturation pattern was similar for all compounds (Fig. S3) and by day 7 of treatment 82–88% of cells were NBT positive. In HL-60 cells 1,25(OH)2D3, 20,23(OH)2D3, 20(OH)D3, 1,20(OH)2D3, pL and pD showed similar potency in stimulation the formation of NBT positive cells. 20(OH)pD and 5,7-dienes were slightly less potent than vitamin D3 hydroxyderivatives on day 5 of treatment (Table S2D; Fig. S3). On Day 7, 1,25(OH)2D3, was equipotent to 20,23(OH)2D3 but was more efficient in induction of morphological differentiation than the other compounds tested (Fig. 4A; Table S3E). 20,23(OH)2D3 showed similar potency to 20(OH)D3, 1,20(OH)2D3, pL, pD and 20(OH)7DHP, while 20(OH)pD and 7DHP were less active (Fig. 4A; Table S2E). This was further confirmed when the absorbance of the supernatants from lysates was measured at 715 nm (Fig. 4C; Table S2F, G).
A and B. Cells were treated for 7 days with different compounds (10−7 M), stained with NBT and the number of NBT positive (blue) cells was determined counting 200 cells per field, and expressed as a % of the total cells. Induction of differentiation measured by the absorbance at 715 nm after lysis of the equal number of NBT stained cells is in C and D. *p<0.05; **p<0.01; ***p<0.001.
In U937 cells, all compounds tested induced cell differentiation by day 5 of treatment with 20,23(OH)2D3 being more potent than 1,20(OH)2D3, 7DHP, pL, pD and 20(OH)pD (figure not shown; Table S2H). On day 7, 1,25(OH)2D3 was the most potent compound, with 20,23(OH)2D3 being more potent than 7DHP, pL and pD and 20(OH)D3 being more potent than 7DHP (Fig. 4B, Table S2I). When the level of differentiation was measured spectrophotometrically to asses the relative effect on an equal number of cells, on day 5 of treatment all compounds induced differentiation with the exception of 7DHP and pL. 1,25(OH)2D3, 20(OH)D3 and pD were the most potent with 20(OH)D3 being significantly more potent than 1,20(OH)2D3, 7DHP, pL and 20(OH)7DHP (Table S2J). On day 7, the strongest induction of monocyte-like differentiation was observed for 1,25(OH)2D3, 20(OH)D3 and pL with other compounds being less potent, and 7DHP and 20(OH)pD having no effect (Fig. 4D, Table S2K).
4. Real time PCR and flow cytometry studies on HL-60 cells
Secosteroids with a full side chain (1,25(OH)2D3, 20,23(OH)2D3, 20(OH)D3, 1,20(OH)2D3) and those with just a two carbon side chain (pD and 20(OH)pD) were chosen for further studies on their antileukemic effects on HL-60 cells. Expression of CD11b and transferin genes was induced significantly and in a dose dependent manner only for secosteroids with a full-length side chain (1,25(OH)2D3, 20,23(OH)2D3, 20(OH)D3 and 1,20(OH)2D3) (Fig. 5), while pD and 20(OH)pD3 had no or minimal effects (not shown). These effects on CD11b were confirmed at the protein level by flow cytometry where 1,25(OH)2D3, 20,23(OH)2D3 and 20(OH)D3 were the strongest inducers of CD11b expression with 1,20(OH)2D3 being less potent and pD and 20(OH)pD3 having no effect (Fig. 6). Interestingly, among tested compounds only 1,25(OH)2D3 and 1,20(OH)2D3 stimulated CYP24 gene expression, with 1,20(OH)2D3 showing comparatively low potency (not shown). For example, at concentration of 10−8M 1,25(OH)2D3 stimulated CYP24 expression 700 times, while 1,20(OH)2D3 stimulated it only 8 times (not shown).
Real time PCR analysis of the expression of CD11b and transferrin genes in HL-60 cells. Cells were treated for 3 days with graded concentrations of 1,25(OH)2D3 (A), 20(OH)D3 (B), 20,23(OH)2D3 (C) and 1,20(OH)2D3 (D), mRNA was isolated and the gene expression analyzed. *p<0.05; **p<0.01; ***p<0.001 determined with one way ANOVA.
Flow cytometry analysis of CD11b protein expression in HL-60 cells. Cells were treated for 5 days with 10−6 M 1,25(OH)2D3 (A), 20(OH)D3 (B), 20,23(OH)2D3 (C), 1,20(OH)2D3 (D), pD (E) and 20(OH)pD (F), mRNA was isolated and the % of cells expressing CD11b protein determined. *p<0.05; **p<0.01; ***p<0.001.
Cell cycle analysis showed that secosteroids with a full-length side chain (1,25(OH)2D3, 20,23(OH)2D3, 20(OH)D3, 1,20(OH)2D3) induced highly significant G1/G0 phase arrest, while pD and 20(OH)pD were less potent (Fig. 7). Among the former, 20,23(OH)2D3 was the strongest inducer of arrest at G1/G0 and the strongest inhibitor of S and G2/M phases (p<0.05)(Fig. 7).
Cell cycle analysis of the HL-60 human promyelocytic human leukemia shows arrest in G1/G0 phase (A). Statistically significant inhibition of S (B) and G2/M (C) phases is also evident for 1,25(OH)2D3, 20(OH)D3, 20,23(OH)2D3, 1,20(OH)2D3 but absent for pD and 20(OH)pD. Representative flow cytometry analysis for 20,23(OH)2D3 is in D. *p<0.05; **p<0.01; ***p<0.001.
5. Molecular modeling studies
To understand the potential binding mode of 20S(OH)D3 in VDR, we selected the crystal structure of the VDR complex with calcitriol (PDB code: 1DB1)and performed molecular modeling studies using Schrodinger Suite 2009 (Schrodinger Inc., New York, NY). The docking program successfully reproduced the original binding pose of the native ligand with an excellent docking score (−13.5, Supplemental Fig. S4). All of the six hydrogen bonding interactions between the ligand and the residues in VDR are reproduced in this validation: 3-OH to Ser274/Tyr143; 1-OH to Ser233/Arg270; and 25-OH to His393/HIP301.
Next we examined the potential binding of 20S(OH)D3 to the VDR. The 20S(OH) isomer (the metabolite generated by P450scc enzymes) overlaps with the native ligand very well with a docking score −11.4. We also performed docking calculations for a variety of D3 metabolites shown in Figure S1. The results are listed in Table S3. Overall, all the metabolites have good docking score, suggesting they may bind well to the VDR. In general, metabolites having 1 alfa-OH have better docking scores than those that do not have. Shorter chain analogs (pD3 analogs) generally have worse docking scores, suggesting that the removal of the side chain may be detrimental to the VDR binding. While we are fully aware of the limitations with molecular modeling, these docking studies suggest that there are high binding affinities of these compounds for the VDR, and the biological activity of these metabolites may still be generated via their interactions with the VDR.
Recently we have provided evidence that the mammalian P450scc system is capable of sequential hydroxylation of the side chain of vitamin D3 at position 20 and 23 , ,  to metabolites that are biologically active in skin cells , , . P450scc (in vitro and ex-vivo) can also transform 7DHC to 7DHP which can be further hydroxylated by classical steroidogenic enzymes to different steroidal 5,7-dienal hydroxy-products , . These, if exposed to UVB, can change to secosteroidal products with only a 2-carbon side chain – some of which are biologically active in skin cells , . In this study we show that novel secosteroids and 5,7-dienes, including 20,23(OH)2D3, 20(OH)D3, 1,20(OH)2D3, pD, pL, 20(OH)pD, 7DHP and 20(OH)7DHP, can inhibit proliferation and induce differentiation of MEL mouse erytholeukemia, human K562 chronic myeloid, HL-60 promyelocytic and U937 promonocytic leukemia cells, which is dependent on the cell type and the structure of the compound tested. These results are consistent with our separate studies showing that 20(OH)D3, 20,23(OH)2D3 and 1,20(OH)2D3 act in very similar manner to 1,25(OH)2D3 by inhibiting proliferation and inducing differentiation of cultured human keratinocytes , , .
In vivo data shown in Fig. 1 indicate that the calcemic effect of vitamin D3 derivatives can largely be separated from their other properties (inhibition of proliferation and stimulation of differentiation) as clearly demonstrated for 20(OH)D3. Interestingly, removal of the key hydroxyl group at C-1 whose presence is known to play a role in calcium regulation , , , has only a slight or no influence on the induction of differentiation of monocytes and leukemia cells (20,23(OH)2D3, 20(OH)D3, 1,20(OH)2D3 act in very similar manner (see figures 3–7). Interestingly, all of the secosteroids we tested had no significant effect on the induction of CYP24 expression, or a very small effect in the case of 1,20(OH)2D3, compared to the large stimulation by 1,25(OH)2D3. Again, this observation is in full agreement with our studies on human keratinocytes, where 20(OH)D3 and 20,23(OH)2D3 have shown poor induction of CYP24, while having equipotent activity with 1,25(OH)2D3 in the regulation of cellular phenotype , . Importantly, gene silencing technology used on human keratinocytes has clearly demonstrated that the phenotypic action of 20(OH)D3 and 20,23(OH)2D3 are mediated through activation of VDR , , . Therefore, because of this intriguing dissociation between phenotypic effects seen in leukemia cells (present studies) and keratinocytes (parallel studies) we have examined the potential binding of 20(OH)D3 to the VDR (figure S4). We have found that the 20S(OH) isomer (the metabolite generated by P450scc , ) overlaps with the native ligand (calcitriol) very well having comparable docking score (figure S4). This opens a new and exciting area to study on the correlation between structure and activity of P450scc and CYP27A1 and CYP27B1 generated active vitamin D3 hydroxyderivatives Since these compounds also differ in the presence or absence of a hydroxyl group at C-25, we suggest that a C-25 hydroxyl group, in addition to C-1 (see above), is necessary for strong induction of CYP24 gene expression. Future studies on this subject are mandatory, since CYP24 is the major enzyme causing inactivation of vitamin D, and respectively slower rates of inactivation could be predicted for the P450scc-derived secosteroids, which should be of advantage for therapeutic use of these compounds.
The observed activities of the various secosteroids and steroidal 5–7dienes we tested were influenced by the genetic background of the cell lines used, suggesting the existence of cell-type specific factors that can modulate activities. Such factors might include the cocktail of transcription factors present, and the concentration of vitamin D metabolizing enzymes in the cells such as CYP24, CYP27A1 and CYP27B1. Despite this variability between cell types, the non-calcemic 20(OH)D3 and its hydroxyl-derivatives generally exerted effects on cell proliferation and differentiation comparable to those of 1,25(OH)2D3. Furthermore we observed functional similarity with the effects induced by 20(OH)D3 and 20,23(OH)2D3 in epidermal keratinocytes, e.g., G1/G0 arrest connected with induction of their differentiation program , . The shortening of the side chain resulted in decreased activity, which is in agreement with lower docking score in comparison to 1,25(OH)2D3 (Table S3). Nevertheless, these compounds, pD3 and 20-OH pD3, do induce leukemia cell differentiation, similarly as in melanoma cells , . This suggests that the mechanism of action of the tested compounds is conserved among different cell types including leukemias (cell of hematopoietic origin), normal skin keratinocytes ,  and malignant melanoma cells , .
Acute myeloid leukemias (AML) are high grade malignancies originating from blood cell precursors in bone marrow. Depending on the lineage of leukemic cells they can be divided into myeloid, monocytic, erythroid and megakaryocytic. AML is characterized by accumulation of immature forms (blasts) in bone marrow and subsequently in peripheral blood and are rapidly and universally fatal if not treated. Developed over the last three decades, chemotherapeutic regimens to treat AML markedly improved the rate of remission and survival in AML patients. However, many leukemias show primary resistance to current therapy and many patients suffers from AML relapse following initial remission. Relapsed disease often becomes resistant to currently available chemotherapeutic drugs and short of bone marrow transplant such relapsed patients become incurable and die. These findings necessitate a continuing search for new drugs or adjuvant therapeutic approaches to treat AML.It is well established that hematopoietic cells and their malignant counterparts (leukemic blasts) express VDR. Moreover, in vitro studies of myeloid leukemia cells show that there is strong response to the active form of vitamin D ranging from induction of differentiation to induction of apoptosis or autophagy, with resulting attenuation of proliferation , –, . Unfortunately in vivo use of these promising drugs is limited by their toxic (calcemic) activity. In this study we report that newly identified and characterized derivatives of vitamin D that have low or no calcemic activity still retain their potent anti-leukemic effects. Furthermore, the above compounds have the potential to be normally generated in skin (from their 5,7-dienes precursors) exposed to solar radiation –, . Potentially these derivatives can be synthesized in other tissues in the body expressing P450scc, for example the adrenal gland , , , , . Interestingly the number of tissues in which expression of P450scc has been identified is growing and includes bone, thymus, skin and brain as examples , –, raising the possibility of local production of the ligands identified in this study in the bone marrow environment. Therefore, we believe that we have identified a family of non-toxic and physiological compounds that can be used in therapy of acute leukemias , , –.
In summary, novel secosteroidal products of P450scc metabolism of vitamin D3 and pro-vitamin D3, show excellent anti-leukemia activity in vitro with 20(OH)D3 deserving special attention because of its relatively high potency and lack of calcemic activity. Moreover, since these compounds potentially can be synthesized in vivo in adrenal gland or generated by skin and theoretically may be produced by many other tissues including bone, further studies are required to define their potential physiological and pathophysiological role in bone marrow homeostasis and leukemiagenesis.
Materials and Methods
1. Steroids and secosteroids
Chemical structures of the compounds tested are shown in Figure S1. 1,25(OH)2D3 was purchased from (Fluka). 20(OH)D3 and 20,23(OH)2D3 were biochemically synthesized as described previously using an in vitro reconstituted P450scc system with vitamin D3 (Sigma) as substrate , . 1,20(OH)2D3 was biochemically synthesized from 1α-hydroxyvitamin D3 (Sigma) as described previously .The products were purified by TLC followed by RP-HPLC and identities were confirmed based on mass and UV spectra, as well as on retention times in comparison to standards previously characterized by NMR , , . The compounds were aliquoted, dried and stored at −80°C until use.
7DHP and 20(OH)7DHP were synthesized and purified as described previously , . After NMR confirmation of their structures, the steroids were purified by HPLC, aliquoted and stored for further use or subjected to photochemical transformation using a Biorad UV Transilluminator 2000 (Biorad, Hercules, CA). Spectral characteristics of the UVB (280–320 nm) source were published previously ,  and its strength (4.8±0.2 mW cm−2) was routinely measured with a digital UVB Meter Model 6.0 (Solartech Inc., Harrison Twp, MI). Irradiation was followed by incubation for 14 h at room temperature then selected products were purified by RP-HPLC chromatography as described , . The major products were identified on the basis of their retention time and characteristic UV absorption. Initial identification was confirmed by means of MS and NMR measurements , . The resulting secosteroids including pregnacalciferol (pD), pregnalumisterol (pL) and 20-hydroxy-pregnacalciferol (20(OH)pD) were RP-HPLC purified, aliquoted and stored at −80°C , .
2. Testing on leukemia cell lines
MEL mouse erythroleukemia , human K562 chronic myeloid leukemia (purchased from American Tissue Culture Collection, USA), and human HL-60 human promyelocytic  and U937 promonocytic leukemia  cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS) (Atlanta Biologics)) and 1% penicillin/streptomycin/amphotericin antibiotic solution (Sigma) at 37°C in 5% CO2. Test compounds were dissolved in ethanol and added to the cultures to reach final concentrations as listed. Ethanol at a concentration of 0.1% was used as a vehicle (negative) control, while 2% DMSO or 32 nM TPA served as positive controls for studies on cell differentiation. U937 and MEL cell lines were generous gift from Dr S. Hanissian (University of Tennessee HSC) and HL-60 from Dr M. Radic (University of Tennessee HSC).
Cells at concentrations of 2×106 cells/ml (K56 and MEL lines) and 107 cells/ml (HL-60 and U937 lines) were inoculated into T25 flasks (TPP, Midwest Scientific) containing 10 ml of RPMI supplemented with charcoal treated 10% FBS. The test compounds were added at concentrations of 10−7 M every day with media being changed every 72 hours. After 7 days days the cells were stained in 0.4% trypan blue (Sigma) and the viable cells counted with a hemacytometer.
Cell cycle analysis.
Cell cycle analysis was performed by flow cytometry following standard protocols used in our laboratories , . Briefly, HL-60 cells were cultured in RPMI 1640 plus charcoal treated 10% FBS (HyClone). Test compounds were added to a final concentration 10−6 M in 0.1% ethanol every day with media being changed every third day. On day five the cells were washed in PBS, fixed in 70% cold ethanol and stained with propidium iodine (Sigma). DNA content analysis was performed with a FACS Calibur flow cytometer.
To estimate erythroid differentiation (production of hemoglobin), first we evaluated the number of benzidine positive cells after 5 and 7 days in culture. Cells were centrifuged and washed four times with PBS and resuspended in 1 ml of fresh PBS. For hemoglobin determination, a benzidine staining solution was freshly prepared by mixing one part of 30% hydrogen peroxide, one part of base stock solution of 3% benzidine in 90% acetic acid, and 5 parts of water. The solution was diluted 1∶10 with the cell suspension and 250 µl aliquots added to 4 wells of a 24-well plate. After 10 min of incubation at room temperature, benzidine-positive cells were counted under the microscope with a minimum of 200 cells scored. Second, to define the relative content of hemoglobin spectrophotometrically , equal number of cells (7×106) were washed with cold PBS and lysed for 20 min in 100 µl of lysis buffer (0.2% Triton X-100 in 100 mM potassium phosphate buffer, pH 7.8). The lysates were centrifuged for 15 min at 1500 r.p.m. and 100 µl of the supernatant was incubated with 2 ml of benzidine solution (5 mg/ml in glacial acetic acid) and 2 ml 30% H2O2 for 10 min at room temperature in the dark. Absorption was measured at 600 nm. Data are shown as fold increases in comparison to the level of hemoglobin in vehicle-treated cells.
Differentiation of HL-60 or U937 cells toward monocytes-like morphology and NBT-reduction was assessed after 5 and 7 days. Cells (2×106) were washed with PBS four times and resuspended in 200 µl of NBT solution (4 mg/ml). After the addition of 200 µl of TPA solution (2 µg/ml in PBS) cells were incubated at 37°C for 60 min in 24-well plates. Cell differentiation was assessed by intracellular blue formazan deposits. The NBT positive and negative cells were scored under light microscopy examination (20×) with a minimum of 200 cells scored. . For spectrophotometric analysis the cells were washed twice with buffer containing cold bovine serum albumin solution (17 mg/ml BSA, 137 mM NaCl, 5 mM KCl, 0.8 mM MgSO4, 10 mM, HEPES, pH 7.4) to remove unreacted NBT, and the insoluble formazan deposits in the resulting pellets were solubilized in 1 ml of 90% DMSO, 0.1% SDS and 0.01 mM NaOH by vigorous vortexing. The samples were centrifuged 5 min at 1,500 x g to remove the cellular debris, and then the absorbance of supernatants measured at 715 nm. Data are expressed as change in A715/106 cells .
Staining of the CD11b cell differentiation marker was carried out according to the manufacturer’s protocol as follows. Cultured cells were resuspended in cold wash buffer (PBS supplemented with 1% FBS and 1% human serum to block non-specific Fc-mediated binding) at a concentration of 2×106/ml. After centrifugation, the cell pellet was resuspended in 100 µl wash buffer. Aliquots (50 µl) of this were incubated with 50 µl of diluted antibody, either phycoerythrin-conjugated mouse anti-human CD11b antibody or phycoerythrin-conjugated mouse IgG isotype as control (both from BD Pharmingen, San Diego, California). After 30 min the cells were centrifuged at at 400 x g and resuspended in 1 ml of wash buffer. This step was repeated with cells finally being resuspended in 200 µl of wash buffer for flow cytometry .
Real-time RT PCR.
The RNA from HL-60 cells treated as described above was isolated using the Absolutely RNA Miniprep Kit (Stratagene, La Jolla, CA). Reverse transcription (1 µg RNA/reaction) was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany). Real-time PCR was performed using undiluted cDNA and a TaqMan PCR Master Mix. Reactions (in triplicate) were performed at 50°C for 2 min, 95°C for 10 min and then 50 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. The primers and probes were designed with the universal probe library (Roche). Data were collected on a Roche Light Cycler 480. The amount of amplified product for each gene was compared to that for β actin using a comparative CT method. A list of the primers used for RT-PCR DNA amplification is shown in Table S4.
3. Testing of calcemic effect
The calcemic effects of 20(OH)D3 and 1,20(OH)2D3 were compared with 25(OH)D3 and 1,25(OH)2D3. Briefly, weanling rats were obtained from Holtzman and fed a vitamin D deficient diet for 3 months before they were divided into 13 groups (6 animals per group) and injected with either vehicle (propylene glycol) or 3 concentrations (0.3 µg/kg, 1.0 µg/kg, 3.0 µg/kg) of 25(OH)D3, 1,25(OH)2D3, 20(OH)D3 or 1,20(OH)2D3 dissolved in propylene glycol for 7 consecutive days. A day after the final dosing, blood was obtained by heart puncture and serum was prepared. Serum calcium was determined by using a kit (DICA-500 QuantiChrom™) from BioAssay Systems (Hayward, CA). Data were analyzed using a student's 1-tailed independent t-test with a p value indicating significance at p<0.05.
Structures and origin of novel secosteroids. Arrows show direction of enzymatic reactions or photochemical transformation. 20HSD: 20-hydroxysteroid dehydrogenase; P40scc: CYP11A1; 1-alpha hydroxylase: CYP27B1.
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Morphological aspects of erythroid differentiation in K562 human leukemia after 7 days of treatment with 10−7M of listed compounds. A. Representative cell pellets after 7 days of treatment with 1,25(OH)2D3 and 20,23(OH)2D3. B. Representative microscopic fields showing benzidine positive K562 cells. The cells were stained with benzidine solution and photographed in light microscopy, 20× magnification.
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Morphological aspects of monocytic differentiation in HL-60 (A) and U932 (B) human leukemia cells after 5 days of treatment with 10−7M of listed compounds. The cells were stained with NBT solution (blue) and photographed in light microscopy, 20× magnification.
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Superimposition of 20S(OH)D3 (dark green, docking score −11.4) and the native ligands in VDR (docking score −13.5). Yellow dotted lines shows that the docking program well reproduced the six hydrogen bonding interactions between the native ligand and VDR that are presented in the crystal structures.
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Student t test analysis of inhibitory effect of tested compounds on leukemia cells proliferation
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Student t test analysis of stimulatory effect of tested compounds on leukemia differentiation.
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Docking scores of the biding of tested ligands to the VDR.
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Conceived and designed the experiments: ATS ZJ TC MH. Performed the experiments: ZJ BEF MAZ RCT MNN TS WL TC. Analyzed the data: ATS ZJ MAZ RCT TS WL JKZ DM TC GL MH. Contributed reagents/materials/analysis tools: ATS MAZ RCT MNN WL JKZ DM TC GL MH. Wrote the paper: ATS ZJ RCT GL.
- 1. Holick MF, Clark MB (1978) The photobiogenesis and metabolism of vitamin D. Fed Proc 37: 2567–74.
- 2. Bikle DD, Nemanic MK, Gee E, Elias P (1986) 1,25-Dihydroxyvitamin D3 production by human keratinocytes. Kinetics and regulation. J Clin Invest 78: 557–66.
- 3. Bar M, Domaschke D, Meye A, Lehmann B, Meurer M (2007) Wavelength-dependent induction of CYP24A1-mRNA after UVB-triggered calcitriol synthesis in cultured human keratinocytes. J Invest Dermatol 127: 206–13.
- 4. DeLuca HF (2004) Overview of general physiologic features and functions of vitamin D. Am J Clin Nutr 80: 1689S–96S.
- 5. Holick MF (2007) Vitamin D deficiency. N Engl J Med 357: 266–81.
- 6. Bikle DD (2007) What is new in vitamin D: 2006-2007. Curr Opin Rheumatol 19: 383–8.
- 7. Kamradt J, Rafi L, Mitschele T, Meineke V, Gartner BC, et al. (2003) Analysis of the vitamin D system in cutaneous malignancies. Recent Results Cancer Res 164: 259–69.
- 8. Holick MF (2003) Vitamin D: A millenium perspective. J Cell Biochem 88: 296–307.
- 9. Schwartz GG, Blot WJ (2006) Vitamin D status and cancer incidence and mortality: something new under the sun. J Natl Cancer Inst 98: 428–30.
- 10. Nagpal S, Na S, Rathnachalam R (2005) Noncalcemic actions of vitamin D receptor ligands. Endocr Rev 26: 662–87.
- 11. Meyskens FL Jr, Kopecky KJ, Appelbaum FR, Balcerzak SP, Samlowski W, et al. (1995) Effects of vitamin A on survival in patients with chronic myelogenous leukemia: a SWOG randomized trial. Leuk Res 19: 605–12.
- 12. Wu-Wong JR, Nakane M, Ma J, Dixon D, Gagne G (2006) Vitamin D receptor (VDR) localization in human promyelocytic leukemia cells. Leuk Lymphoma 47: 727–32.
- 13. Srivastava MD, Ambrus JL (2004) Effect of 1,25(OH)2 Vitamin D3 analogs on differentiation induction and cytokine modulation in blasts from acute myeloid leukemia patients. Leuk Lymphoma 45: 2119–26.
- 14. Sicinski RR, Prahl JM, Smith CM, DeLuca HF (1998) New 1alpha,25-dihydroxy-19-norvitamin D3 compounds of high biological activity: synthesis and biological evaluation of 2-hydroxymethyl, 2-methyl, and 2-methylene analogues. J Med Chem 41: 4662–74.
- 15. James SY, Williams MA, Newland AC, Colston KW (1999) Leukemia cell differentiation: cellular and molecular interactions of retinoids and vitamin D. Gen Pharmacol 32: 143–54.
- 16. Munker R, Kobayashi T, Elstner E, Norman AW, Uskokovic M, et al. (1996) A new series of vitamin D analogs is highly active for clonal inhibition, differentiation, and induction of WAF1 in myeloid leukemia. Blood 88: 2201–9.
- 17. Nakagawa K, Sasaki Y, Kato S, Kubodera N, Okano T (2005) 22-Oxa-1alpha,25-dihydroxyvitamin D3 inhibits metastasis and angiogenesis in lung cancer. Carcinogenesis 26: 1044–54.
- 18. Spina CS, Tangpricha V, Uskokovic M, Adorinic L, Maehr H, et al. (2006) Vitamin D and cancer. Anticancer Res 26: 2515–24.
- 19. Spina CS, Ton L, Yao M, Maehr H, Wolfe MM, et al. (2007) Selective vitamin D receptor modulators and their effects on colorectal tumor growth. J Steroid Biochem Mol Biol 103: 757–62.
- 20. Masuda S, Jones G (2006) Promise of vitamin D analogues in the treatment of hyperproliferative conditions. Mol Cancer Ther 5: 797–808.
- 21. Holick MF, Garabedian M, Schnoes HK, DeLuca HF (1975) Relationship of 25-hydroxyvitamin D3 side chain structure to biological activity. J Biol Chem 250: 226–30.
- 22. Slominski A, Semak I, Zjawiony J, Wortsman J, Li W, Szczesniewski A, et al. (2005) The cytochrome P450scc system opens an alternate pathway of vitamin D3 metabolism. Febs J 272: 4080–90.
- 23. Tuckey RC, Li W, Zjawiony JK, Zmijewski MA, Nguyen MN, et al. (2008) Pathways and products for the metabolism of vitamin D3 by cytochrome P450scc. Febs J 275: 2585–96.
- 24. Tuckey RC, Nguyen MN, Slominski A (2008) Kinetics of vitamin D3 metabolism by cytochrome P450scc (CYP11A1) in phospholipid vesicles and cyclodextrin. Int J Biochem Cell Biol.
- 25. Guryev O, Carvalho RA, Usanov S, Gilep A, Estabrook RW (2003) A pathway for the metabolism of vitamin D3: unique hydroxylated metabolites formed during catalysis with cytochrome P450scc (CYP11A1). Proc Natl Acad Sci U S A 100: 14754–9.
- 26. Zbytek B, Janjetovic Z, Tuckey RC, Zmijewski MA, Sweatman TW, J, et al. (2008) 20-Hydroxyvitamin D3, a product of vitamin D3 hydroxylation by cytochrome P450scc, stimulates keratinocyte differentiation. J Invest Dermatol 128: 2271–80.
- 27. Janjetovic Z, Zmijewski MA, Tuckey RC, DeLeon DA, Nguyen MN, et al. (2009) 20-Hydroxycholecalciferol, product of vitamin D3 hydroxylation by P450scc, decreases NF-kappaB activity by increasing IkappaB alpha levels in human keratinocytes. PLoS One 4: e5988.
- 28. Tuckey RC, Janjetovic Z, Li W, Nguyen MN, Zmijewski MA, et al. (2008) Metabolism of 1alpha-hydroxyvitamin D3 by cytochrome P450scc to biologically active 1alpha,20-dihydroxyvitamin D3. J Steroid Biochem Mol Biol.
- 29. Slominski AT, Zmijewski MA, Semak I, Sweatman T, Janjetovic Z, et al. (2009) Sequential metabolism of 7-dehydrocholesterol to steroidal 5,7-dienes in adrenal glands and its biological implication in the skin. PLoS One 4: e4309.
- 30. Slominski A, Zjawiony J, Wortsman J, Semak I, Stewart J, et al. (2004) A novel pathway for sequential transformation of 7-dehydrocholesterol and expression of the P450scc system in mammalian skin. Eur J Biochem 271: 4178–88.
- 31. Zmijewski MA, Li W, Zjawiony JK, Sweatman TW, Chen J, et al. (2009) Photo-conversion of two epimers (20R and 20S) of pregna-5,7-diene-3beta, 17alpha, 20-triol and their bioactivity in melanoma cells. Steroids 74: 218–28.
- 32. Zmijewski MA, Li W, Zjawiony JK, Sweatman TW, Chen J, et al. (2008) Synthesis and photo-conversion of androsta- and pregna-5,7-dienes to vitamin D3-like derivatives. Photochem Photobiol Sci 7: 1570–6.
- 33. Tang E, Voo K, Nguyen M, Tuckey R (2010) Metabolism of substrates Incorporated into phospholipid vesicles by mouse 25-Hydroxyvitamin D3 1α-hydroxylase (CYP27B1). J Steroid Biochem Mol Biol in press.
- 34. Janjetovic Z, Tuckey RC, Nguyen MN, Thorpe EM Jr, Slominski AT (2010) 20,23-dihydroxyvitamin D3, novel P450scc product, stimulates differentiation and inhibits proliferation and NF-kappaB activity in human keratinocytes. J Cell Physiol 223: 36–48.
- 35. Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D (2000) The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5: 173–9.
- 36. Holick MF (2004) Sunlight and vitamin D for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease. Am J Clin Nutr 80: 1678S–88S.
- 37. Slominski A, Wortsman J, Paus R, Elias PM, Tobin DJ, et al. (2008) Skin as an endocrine organ: implications for its function. Drug Discov Today Dis Mech 5: 137–144.
- 38. Slominski A, Semak I, Zjawiony J, Wortsman J, Gandy MN, et al. (2005) Enzymatic metabolism of ergosterol by cytochrome p450scc to biologically active 17alpha,24-dihydroxyergosterol. Chem Biol 12: 931–9.
- 39. Qiao S, Okret S, Jondal M (2009) Thymocyte-synthesized glucocorticoids play a role in thymocyte homeostasis and are down-regulated by adrenocorticotropic hormone. Endocrinology 150: 4163–9.
- 40. Teplyuk NM, Haupt LM, Ling L, Dombrowski C, Mun FK, et al. (2009) The osteogenic transcription factor Runx2 regulates components of the fibroblast growth factor/proteoglycan signaling axis in osteoblasts. J Cell Biochem 107: 144–54.
- 41. Mukai H, Tsurugizawa T, Ogiue-Ikeda M, Murakami G, Hojo Y, et al. (2006) Local neurosteroid production in the hippocampus: influence on synaptic plasticity of memory. Neuroendocrinology 84: 255–63.
- 42. Tiala I, Suomela S, Huuhtanen J, Wakkinen J, Holtta-Vuori M, et al. (2007) The CCHCR1 (HCR) gene is relevant for skin steroidogenesis and downregulated in cultured psoriatic keratinocytes. J Mol Med 85: 589–601.
- 43. Thiboutot D, Jabara S, McAllister JM, Sivarajah A, Gilliland K, et al. (2003) Human skin is a steroidogenic tissue: steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1). J Invest Dermatol 120: 905–14.
- 44. Slominski A, Ermak G, Mihm M (1996) ACTH receptor, CYP11A1, CYP17 and CYP21A2 genes are expressed in skin. J Clin Endocrinol Metab 81: 2746–9.
- 45. Fischer TW, Sweatman TW, Semak I, Sayre RM, Wortsman J, et al. (2006) Constitutive and UV-induced metabolism of melatonin in keratinocytes and cell-free systems. Faseb J 20: 1564–6.
- 46. Pisarchik AV, Dolmatovich TV, Kartel NA (2001) [Modulation of the cat gene expression in E. coli cells by simple (AC)20 repeats]. Mol Gen Mikrobiol Virusol 15–8.
- 47. Reddy PM, Shen CK (1993) Erythroid differentiation of mouse erythroleukemia cells results in reorganization of protein-DNA complexes in the mouse beta maj globin promoter but not its distal enhancer. Mol Cell Biol 13: 1093–103.
- 48. Gallagher R, Collins S, Trujillo J, McCredie K, Ahearn M, T, et al. (1979) Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood 54: 713–33.
- 49. Nilsson K, Sundstrom C (1974) Establishment and characteristics of two unique cell lines from patients with lymphosarcoma. Int J Cancer 13: 808–23.
- 50. Slominski A, Zbytek B, Pisarchik A, Slominski RM, Zmijewski MA, et al. (2006) CRH functions as a growth factor/cytokine in the skin. J Cell Physiol 206: 780–91.
- 51. Zhang D, Cho E, Wong J (2007) A critical role for the co-repressor N-CoR in erythroid differentiation and heme synthesis. Cell Res 17: 804–14.
- 52. Mahdavi M, Yazdanparast R (2007) Gnidilatimonoein from Daphne mucronata induces differentiation and apoptosis in leukemia cell lines. Arch Pharm Res 30: 177–81.
- 53. Fabiani R, De Bartolomeo A, Rosignoli P, Servili M, Selvaggini R, et al. (2006) Virgin olive oil phenols inhibit proliferation of human promyelocytic leukemia cells (HL60) by inducing apoptosis and differentiation. J Nutr 136: 614–9.
- 54. Ali AS, Chopra R, Robertson J, Testa NG (2000) Detection of hTERT protein by flow cytometry. Leukemia 14: 2176–81.


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text:
The popularity of UFC is at an all-time high. People of all...
Barfly: Dana White and Ed O’Neill Discuss Heavyweights, Mayweather and PED’s
UFC Pres Dana White and “Al Bundy” Ed O’Neill sit down on FOXSport’s Barfly to talk about a host of topics including UFC 145, Jon Jones vs. Dan Henderson, Heavyweights, Mayweather and performance enhancing drugs.
Dana talks about having to make the decision of what to do with Alistair Overeem, Chuck Liddell getting that itch to fight again and the Nevada State Athletic Commission’s rule allowing Floyd Mayweather put off his 90 days in jail to fight on May 5th.

domain: sports


text:
Tests continue on U.S. 378 bridge over Thurmond Lake
Engineers might need another six weeks to complete their inquiry into problems with underwater support columns at a new bridge over Thurmond Lake along U.S. Hwy. 378.
Construction on the 1,680-foot span that will link Lincolnton, Ga., with McCormick, S.C., began in April 2010 and scheduled to finish last July.
However, after identifying problems related to possible movement in the bridge supports, known as "bents," engineers launched a series of tests that may further delay a new completion date later this month.
"We are in the process of testing the concrete, which will take some time, and we anticipate that testing process will be completed in the next six weeks," said Cissy McNure, a spokeswoman for the Georgia Department of Transportation, in an e-mailed response to questions.
The support columns are anchored at the lake's bottom in water as much as 80 feet deep. Engineers believe the deep, cold water - and differences in temperature - may have affected the curing of the concrete and caused stresses in the seal it was designed to create.
The contractor for the $22.4 million project is Alabama-based Scott Bridge Co., which also managed the $192 million construction of the Interstate 20/Bobby Jones Expressway juncture in Augusta.
Once completed, the 44-foot-wide span will have two travel lanes and two emergency lanes and - at its highest point - will be 108 feet above the lake at full pool.

domain: news


text:
The Chemainus Business Improvement Association is obviously onto something.
In the spirit of the season, it was decided to hold a five-kilometre Family Fun Pumpkin Spice Run on Saturday, Nov. 10 at 10 a.m. The public responded in a big way, with 233 participants registered.
“We’re thrilled to have so many participants in this fun run,” noted CBIA coordinator Shelley Johnstone. “This far exceeded our expectations. It’s truly inspiring when the community comes together for some active fun in support of Cowichan Neighbourhood House. We’ve had remarkable business support as well, with sponsorships, in-kind donations and people-power help. Chemainus is a truly special community.”
There is no race day registration so those who didn’t act in time are out of luck until next year. But there’s still ways to participate.
“We’d love to have community members come out and cheer everyone on, anywhere along the course,” Johnstone enthused. “And everyone is welcome to the post-race Party in the Park.”
Registered participants must sign-in and pick-up their race bib and pumpkin prize pack between 8:45 a.m. and 9:30 a.m. on Saturday at Waterwheel Park, on Waterwheel Crescent.
Johnstone noted all participants will gather in Waterwheel Park for an important pre-race announcement at 9:45 a.m. From there, everyone walks together to the start line on the TransCanada Trail.
The route goes along the TransCanada Trail on Chemainus Road before winding back through the downtown core and finishing at Waterwheel Park for the post-race party.
Strollers are welcome on the course, but dogs are not allowed.
Participants who will be walking and participants with strollers are asked to start at the back of the pack.
With part of the course being an out-and-back, it means people will be travelling in both directions on the trail at the same time.
There are no water stations along the route, but plenty will be available at the finish and during the post-race party.
There will be treats, music and prize giveaways during the after-party.
See route map, above left, showing the Trans Canada Trail course and finishing downtown.


NASA Crewed Soyuz Mission to International Space Station from Baikonur Cosmodrome to Launch Dec. 8
By Space Coast Daily // December 5, 2021
nasa & space news
(NASA) – A veteran Russian cosmonaut and two Japanese private citizens are set to launch to the International Space Station on Wednesday, Dec. 8, and return to Earth on Sunday, Dec. 19.
Roscosmos cosmonaut Alexander Misurkin will join spaceflight participants Yusaku Maezawa and Yozo Hirano on the Soyuz MS-20 spacecraft launching from the Baikonur Cosmodrome in Kazakhstan at 2:38 a.m. EST Wednesday, Dec. 8 (12:38 p.m. Baikonur time).
Launch, docking, and return activities will air live on NASA Television, the NASA app, and the agency’s website.
After a four-orbit, six-hour journey, the Soyuz will dock to the station’s Poisk module at 8:41 a.m. About two hours after docking, hatches between the Soyuz and the station will open and the crew members will greet each other.
Once on station, the trio will join Expedition 66 Commander Anton Shkaplerov and cosmonaut Pyotr Dubrov of Roscosmos, as well as NASA astronauts Mark Vande Hei, Raja Chari, Tom Marshburn, and Kayla Barron, and ESA astronaut Matthias Maurer, for approximately 12 days on the orbital laboratory.
On Sunday, Dec. 19, Misurkin, Maezawa, and Hirano will complete their mission, undocking the Soyuz from the Poisk module before heading for a parachute-assisted landing on the steppe of Kazakhstan at 10:18 p.m. EST (9:18 a.m. Monday, Dec. 20, Kazakhstan time).
CLICK HERE FOR BREVARD COUNTY NEWS


text:
In this revealing documentary, veteran correspondent John Merrow takes you behind the ivy-covered walls of our colleges and universities to see if they are delivering on their promise.
Debunking commonly held notions about the rite of passage known as the college experience, this PBS documentary follows 30 students and their teachers along the path of higher education, from admission to graduation, and exposes the disappointment, disorientation and deflation many students feel -- in both public and private schools. This revealing study also addresses the quality and readiness of America's future work force.
Films For Action empowers citizens with the information they need to help create a more just, sustainable, and democratic society.
We receive no government or corporate funding and rely on our supporters to keep us going. Donate today.

domain: sociology


Jaguar XF is Getting an Enhanced Performance-look with R-Sport Trim
There are several options for those who are looking forward to getting a stylish sport sedan, however with any added expense of springing for a full-on performance model. The majority of the European automakers can readily give options with a customized uplifting package. BMW is offering their M-Sport aero kits, Audi got S-Line on offer, and Mercedes has released AMG Sport packs for the customers to do the upgrading. As the latest, Jaguar is also getting on to action by launching their all-new R-Sport line, offering the Sporty trim to the brand models.Photo 1: JAGUAR
Expected to get unveiled in the Geneva Motor Show, the new R-Sport trim has got some similarity to that of the XFR and XFR-S, but featuring a more economical engine as well as a relatively accessible entry price. The XF R-Sport is notable for a more sportier front bumper, rear spoiler, side sills, and 17-inch wheels and also a unique style badging, which will set it apart from the causal Jaguar XF look. Not just the outside, but there is interior upgrades as well and also there is an optional blackish styling pack to offer a more stingy touch.
Jaguar will be featuring the XF R-Sport in both sedan as well as wagon models with 2.2-liter turbo diesel, which can produce 161 HP and about 295 pound-feet torque, which is fitted with a spot start system. It is capable of making XF R-Sport far more frugal than 5.0-liter V8 when it comes to the real deal. As announced by Jaguar spokesperson, this version may not be getting released to US as you can get it other places; “however, it is surely capable of producing a similar aesthetics in the North American market too” he informed.Photo 2: Jaguar XF is Getting an Enhanced Performance-look with R-Sport Trim
However, we will get a closer look of the deal in the upcoming Geneva Motor Show next week, which will a better idea about which all markets it is going to seize and how the performance will be graded.


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Published on July 4th, 2016 | by gareth0
Resident Evil 7 Demo Breaks Records
The demo was a very nice teaser to the game and with the great showing at E3, Capcom is poised to deliver a great new game in the series. The demo has set records and here is the official word from Capcom.
About Resident Evil 7 Teaser: Beginning Hour
The Resident Evil 7 Teaser: Beginning Hour demo portrays events leading up to the main game and ties directly into the impactful PlayStation VR “KITCHEN” tech demo first shown at E3 2015. In the new teaser demo, the player awakens inside a dilapidated cottage on an ominous plantation mansion. Can they make it out alive? PlayStation Plus subscribers can now download the standalone teaser demo for PlayStation 4 and will have first access before it is available on other platforms at a later date.
About Resident Evil 7 biohazard
Resident Evil 7 biohazard is the next major entry in the renowned Resident Evil series and sets a new course for the franchise as it leverages its roots and opens the door to a truly terrifying horror experience. A dramatic new shift for the series to first person view in a photorealistic style powered by Capcom’s new RE Engine, Resident Evil 7 delivers an unprecedented level of immersion that brings the thrilling horror up close and personal. The intense horror experience will be further amplified when playing the game in the included PlayStation VR Mode option on PS4.
Set in modern day rural America and taking place after the dramatic events of Resident Evil® 6, players experience the terror directly from the first person perspective. Resident Evil 7 embodies the series’ signature gameplay elements of exploration and tense atmosphere that first coined “survival horror” some twenty years ago; meanwhile, a complete refresh of gameplay systems simultaneously propels the survival horror experience to the next level.

domain: entertainment


text:
- Verify Your Rep
- About Intelife
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The DVR is a cost-effective, digital video recorder with 4-, 8- or 16-channel capabilities.
The NVR is a high-performance network video recorder designed to support video streams from IP video cameras.

domain: medical


text:
Several faculty and students from the department of geography, geology and planning recently attended the North-Central/South-Central Joint meeting of the Geological Society of America (GSA) March 19-21 in Pittsburgh, PA.
In attendance were Drs. Kevin Mickus, Kevin Evans, Charles Rovey and Doug Gouzie with graduate students Mo’amen Almaz, Grant Spoering, Wes Weichert and Ben … [Read more...]
Two CNAS students presented at the Geological Society of America’s annual meeting held in Denver, Colorado Sept. 24-28. Each student presented a poster on their research and had the opportunity to listen to lectures by experts in the field and network with other professionals.
Grant Spoering, a geospatial science graduate student, presented his research titled “Detrial … [Read more...]

domain: geography


Its DJ Big N who brings out this afrobeat tune titled “The Trilogy”. He compiles short verses of each of the artiste featured and turns it into one big tune. He featured Reekado Banks, Iyanya and Ycee. Download and Listen Up!
DJ Big N feat. Reekado Banks, Iyanya & Ycee – The Trilogy [Download]


text:
CHARLESTON, SC (WCSC) - Authorities with the Charleston County Sheriff's Office say they are investigating three people, who were injured in a car wreck last week, in connection to a series of burglaries in a West Ashley neighborhood.
On May 24, two juveniles and 18-year-old Abraham Jenkins were taken to the hospital after a car wreck involving a stolen Crown Victoria in Hollywood following an attempted traffic stop.
Jenkins was charged with possession of a stolen motor vehicle and failure to stop for blue lights.
Major Jim Brady announced on Wednesday that the sheriff's office and the Charleston Police Department are working together to investigate whether the car and the people who were in the vehicle were involved in several burglaries in the Shadow Moss subdivision.
Authorities said deputies attempted to stop a 2006 Ford Crown Victoria on Bees Ferry Road near the Shadow Moss subdivision on May 24. The sheriff's Office says that during the pursuit, Jenkins was driving extremely reckless "without caution or regard for public safety and disobeyed all traffic laws."
Deputies report that the driver reached speeds of 90 to 100 miles per hour while driving through several busy intersections and crossed over double lines in the roadway forcing other drivers off the roadway.
The pursuit ended on Wiltown Road when the driver lost control of the car, hit a tree and overturned. The two juveniles in the car were ejected and Jenkins had to be extricated from the car. They were taken to local hospitals for treatment of non-life-threatening injuries.
The sheriff's office says that the license tag on the Crown Victoria was reported stolen through Charleston City Police Department and the vehicle itself reported stolen through the Charleston County Sheriff's Office.


text:
The National Working Committee (NWC) of the factional National Chairman of the Labour Party ( LP), Lamidi Apapa, has rejoiced with the party’s flag bearer, Mr Peter Gregory Obi, on his 62nd birthday.
The Apapa-led NWC of the party, as much as they jubilate with Obi, said they regret fielding him as the party’s presidential candidate for the 2023 presidential election, Naija News gathered.
According to the faction, Obi, rather than representing varied interests in the party, chose the interest of a faction.
The faction, in a statement issued on Wednesday night by Suspended National Publicity Secretary of the Labour Party, Comrade Abayomi Arabambi, said as he celebrates his 62 birthday, he should reflect on being a statesman rather than a regional bigot.
Arabambi said, “The National Working Committee (NWC) of the Labour Party (LP) felicitates with its popular presidential candidate, Mr. Peter Gregory Obi, on his 62nd anniversary.
“The Party wishes that the occasion should provide the celebrant a period of deep reflection and ponder on the citizens-conferred status of a statesman who represents interests beyond his ethnic leaning
“The Lamidi Apapa-led National Working Committee of the Party hereby charges the celebrant to, on this occasion of thanksgiving to God, to be reflective of the Nigerian interest, which he seeks to serve.
“While we in the Labour party join him to thank God and wish him a good health and prosperity we want to also remind him that his popularity emanates from the love of the people for the Labour party and galvanized by his representation of the people.
“In wishing him well, we want him to state that we regret fielding him on the platform of the party, in the light of recent development particularly actions that undermined the discipline the Labour party has been known for.
“It is indeed worrisome that even though he went to the Presidential Electoral Tribunal as a non-member of the Labour Party and lied severally that there was a time they issued subpoena to INEC for all those document and maintained that they, they were unable to pay all the documents they seek to tender, kicks regret in our party stakeholders.
“We are further disturbed that our presidential candidate would rely on a “non party agent’ who came to court to defend the alleged stolen mandate,” this is regrettable as it is laughable.
“We wish him well for his 62nd year birthday. We made a mistake to have fielded him to, which we will never repeat again.
“We had thought that this birthday will offer him an opportunity to have a reflection that would make him a better Nigerian and not an Ethnic bigot.”


Ontario’s overdue property reassessment is on hold until at least 2024, leaving many commercial ratepayers with a further wait to realize tax reductions from pandemic-related value erosion.
New CMHC data explores the prevelance and implications of development-led evictions and other trends impacting Toronto's rental housing landscape.
One of many the industries impacted by COVID-19, the short-term rental market will likely see some recover in 2021, according to a new report from Beyond Pricing.
Aging residential towers are in dire need of investment, something the pandemic has underscored given the scores of frontline workers who’ve been disproportionately affected.
As affordable rental housing continues to be a pressing need in the GTA, some housing experts believe infill development is the solution we've been looking for.
As urban planners re-evaluate the way our cities were designed through the lens of today’s health and wellness needs, the concept of 'the 15-minute city' is making a comeback.
The City of Winnipeg has invited commercial building owners and institutional facility managers to affix their Energy Star Portfolio Manager results on a publicly accessible map.
As rents continue to climb in Canada’s major metropolitan areas, “creative” and “collaborative” solutions will be needed to tackle the challenges of urban growth.
Housing Now, an initiative to activate City-owned lands for the development of affordable housing, has officially begun phase one of the program.
The study assesses and scores 110 prominent international cities on 22 indicators deemed to support and/or influence the age cohort born between 1997 and 2012.
Rules surrounding short-term rentals have been the subject of much debate in cities all across Canada. What will the long-term effects of the new rules be on Toronto's multi-res apartment owners?
A spokesperson for the Ministry of Municipal Affairs and Housing says the underlying principle of improving accessibility will be maintained, and connects the revisions to the government's commitment to cut red tape that is slowing down the production of new housing.
Much of the current negative sentiment relates to the emergence of informal rooming houses in predominantly single-family residential areas near suburban post-secondary campuses.
The new policy would give developers a basis to challenge any local government's veto of non-employment land uses in suburban office parks.
Commercial property owners who host clothing donation bins could run afoul of their province's Occupiers Liability Act or Occupational Health and Safety Act should an incident occur.
As 2019 begins, Canadians continue to face a well-chronicled slate of challenges when it comes to finding affordable rental housing. Here, housing experts share their insights...and even a few solutions.
Hamilton-area landlords are speaking out after city councillors voted to move ahead on a rental housing licensing pilot affecting properties with six or fewer units. If approved, landlords will be required to obtain a rental housing license for $200.


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Telltale Games has responded to allegations that several of its employees posted perfect ten user scores on Metacritic for a game they worked on – Jurassic Park.
"Telltale Games do not censor or muzzle its employees in what they post on the internet," the company said in an official statement. "However, it is being communicated internally that anyone who posts in an industry forum will acknowledge that they are a Telltale employee. In this instance, two people who were proud of the game they worked on, posted positively on Metacritic under recognizable online forum and XBLA account names."
GameSpot was the first news outlet to notice unusual user scores for Jurassic Park on Friday, when the game received four perfect ten scores.
Metacritic users have responded by scoring the game with "revenge zeros," which brought the game down to an average user score of 2.9, based on 79 ratings.

domain: game


How many new police will we get from latest class?
261 NEW police will graduate from the academy today, and two will join the ranks of Coffs/Clarence police.
16 recruits will join the Northern Region of policing, with figures showing the two will be allocated to our district.
Our surrounding districts will only receive one extra officer in the allocation, with the Mid North Coast command gaining one, while Richmond to the north not receiving.
This new allocation follows another two officers coming into the area from the February class, with one allocated to Richmond.
Across the state, 'Class 337' includes 176 policemen and 85 policewomen who will complete 12 months on-the-job training and study of the Associate Degree in Policing Practice by distance education with Charles Sturt University before being confirmed to the rank of constable.
NSW Police Commissioner Mick Fuller APM will be joined by Governor of NSW, Her Excellency, The Honourable Margaret Beasley AO GC, NSW Premier, The Honourable Gladys Berejiklian, and Minister for Police and Emergency Services, The Honourable David Elliot MP, to inspect the new recruits on the parade ground.
Commissioner Fuller said the newly-attested officers will hit the ground running on Monday when they start at their new postings at police stations across the state.


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My cousin got married this weekend. Congrats Becca and Joe!
I made this for them as a wedding gift. It makes a great centerpiece and memory.
Things You Will Need:
A clear bottle
A color printer
Card stock paper
Get together some text. I used first Corinthians, lyrics from their wedding song, the location of the wedding, their names, etc. use words that will mean something to them.
Print out your text. Using a ruler and exacto knife, cut between the text so you have many lines. I wrote over important text with a pink pen for emphasis. You can use a color printer to color the text if you wish.
Wrap the line of text around the pencil in a swirling fashion.
Place all of your swirly text into your glass bottle and shake it up.
Ta Daaa! A lovely memory. You could go all out and write lovey dovey sentiments all over the bottle and add pretty ribbons if you would like. I prefer the bottle as it is. Enjoy!

domain: hobby


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Inside Smart Cities is by, for and about smart city leaders. Send your city news and opinion pieces for inclusion in the Inside Smart Cities newsletter.
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Lenovo Smart Tab M10 TB-X605F
Secondary Camera: 2 MPix
Average of 3 scores (from 10 reviews)
Reviews for the Lenovo Smart Tab M10 TB-X605F
There are much better Android tablets to be had, but the Lenovo Smart Tab M10 HD is good enough for casual media consumption for those who also want a device that can double as a smart display.
Single Review, online available, Medium, Date: 12/08/2020
Rating: Total score: 70%
Source: Gear Diary
Having a Smart Display in your living space makes for a great central hub where you and other family members can check schedules, play music or Youtube video, make video calls, control your Google or Alexa integrated home, and display favorite photos. Having that display also function as a family tablet when it’s not docked adds extra convenience and value. The Lenovo Smart Tab M8 and Smart Tab M10 are 2-in-1 devices that can serve as smart displays or as stand-alone tablets.
Comparison, online available, Very Long, Date: 10/21/2020
Source: Laptop Mag
The Lenovo Smart Tab M10 2nd Gen is an exciting product, and we can't wait to get our hands on time with it on the show floor. Stay tuned for that as well as our full review and benchmarks for the device later in the year. For more news on laptops, tablets and more, check out our CES 2020 hub page.
Single Review, online available, Medium, Date: 01/07/2020
Source: WinFuture DE→EN Archive.org version
Single Review, online available, Short, Date: 10/20/2019
Source: Computerhoy ES→EN Archive.org version
Single Review, online available, Short, Date: 08/18/2019
Rating: Total score: 85%
Source: Ofertaman ES→EN Archive.org version
Positive: Great display; good price; decent processor; nice speakers; FM radio; USB-C. Negative: Plastic case; only 16GB ROM; short battery life; poor cameras.
Single Review, online available, Very Short, Date: 12/24/2019
Source: Ofertaman ES→EN Archive.org version
Positive: Nice display; powerful processor; decent speakers; USB-C. Negative: Plastic case; only 16GB ROM; short battery life; poor cameras.
Single Review, online available, Very Short, Date: 10/22/2019
Source: Tabletowo PL→EN Archive.org version
Single Review, online available, Long, Date: 11/24/2019
Rating: Total score: 65%
Source: Komputerswiat PL→EN
Positive: Decent hardware. Negative: Average performance; poor display.
Single Review, online available, Short, Date: 10/23/2020
Source: Komputerswiat PL→EN
Positive: Nice display; impressive design; decent speakers; long battery life.
Single Review, online available, Medium, Date: 01/27/2020
While most tablets are only used as an oversized Android device for general browsing and playing videos, Lenovo has decided to change that assumption all together with its latest Lenovo Smart Tab M10 TB-X605F. Being a smart tablet, it’s taking the best combination of smart technology and Android technology to a whole new level. Well, having been nominated as a finalist for Best of CES in 2019, this tab definitely needs to be noted, because it can function as a typical Android tablet while it can also be placed within a companion Bluetooth speaker dock to act as an Alexa-powered smart display, quite similar to Amazon’s Echo Show. The Bluetooth speaker not only features a Bluetooth connection for user convenience and comfort, it also utilizes physical volume control. The speaker can be used entirely on its own, as well, by connecting it to other devices via Bluetooth to act as a speaker. There is obviously some level of versatility with this device that can really delight users.
This functionality of the Lenovo Smart Tab also provides a better user experience, since, as far as general tablet usage is concerned, most of the time users tend to skip recharging the device when it is not in use, leading to a complete drainage of the battery when it really needs to be used. However, with this new smart invention, this issue can be handled in a much smarter way, as there is definitely a use for the tablet as a smart display, even when it is not used as a typical Android tablet. Such an awesome thought process and creation by Lenovo definitely deserves accolades.
As far as the display is concerned, the Smart Tab M10 has a Full-HD 10.1-inch screen for a greater viewing pleasure. The texts and photos displayed are really sharp and account for an awesome overall user experience. Despite having a great level of brightness for inside and outside usage in the shadows, this tablet is obviously quite dark to be used in direct sunlight. In terms of overall display brightness, the Lenovo Smart Tab P10 can be said to be better than the M10. This might be a slight drawback for this product, but the wide viewing angles of the IPS panel incorporated within the Smart Tab M10 should not be disregarded.
On the whole, in comparison to the P10, the M10 is definitely cheaper in terms of price, but it does fall short when it comes to specifications. While the P10 delivers 4 GB of RAM with 64 GB of storage, the M10 only delivers a maximum of 3 GB of RAM along with 32 GB of storage space. The P10 also comes with a fingerprint reader, which is excluded from the M10. So, the M10 definitely delivers a mid-range smart tablet experience for its price, but it obviously is not the best one out there.
Hands-on article by Jagadisa Rajarathnam
Qualcomm Adreno 506: Integrated graphics card in the Snapdragon 625 SoC and based on the new 500-series architecture with support for Vulkan, OpenGL ES 3.1 + AE (3.2) and UBWC.
Only some 3D games with very low demands are playable with these cards.
SD 450: Lower mainstream octa-core ARM Cortex-A53 SoC clocked at up to 1.8 GHz and the Adreno 506 GPU, a LPDDR3 single channel memory controller as well as an X9 LTE (Cat. 7 300 / 150 Mbps) modem.» Further information can be found in our Comparison of Mobile Processsors.
This is a standard display format for tablet computers or small convertibles. You see more on the screen than on a smartphone but you can't use big resolutions well. On the other hand, mobility is not a problem.» To find out how fine a display is, see our DPI List.
Lenovo: Lenovo ("Le" from English legend, novo (Latin) for new) was founded in 1984 as a Chinese computer trading company. As of 2004, the company was the largest laptop manufacturer in China and, after acquiring IBM's PC division in 2005, the fourth largest in the world. In addition to desktops and notebooks, the company manufactures monitors, projectors, servers, etc, and specializes in developing, manufacturing and marketing consumer electronics, personal computers, software, enterprise solutions and related services.
In 2016, the company ranked first in the world in computer sales. It still held it in 2023 with about 23% global market share. Important product lines are Thinkpad, Legion and Ideapad.
In 2011, it acquired a majority stake in Medion AG, a European computer hardware manufacturer. In 2014, Motorola Mobility was purchased, which gave Lenovo a boost in the smartphone market.
73.33%: This rating is poor. More than three quarters of the models are rated better. That is rather not a purchase recommendation. Even if verbal ratings in this area do not sound that bad ("sufficient" or "satisfactory"), they are usually euphemisms that disguise a classification as a below-average laptop.
» Further information can be found in our Notebook Purchase Guide.


St. Andrew’s Choir under the direction of Cheryl Weeks consists of approximately 14 singers who practice on Wednesday evenings at 6:30 p.m. from September to June. In addition to leading the congregational singing each Sunday, the choir prepares a wide variety of anthems for most Sundays. The Annual Carol Service is an important part of our musical year. Dr. Kenneth Cameron is the church organist. New members are gladly welcomed.
The Kilted Choir is a group of men which sings Scottish tunes and sacred music at some services and other events, accompanied by Chris McDonald on the piano. A highlight of the year is the St. Andrew’s service and Ceilidh in late November. New members are welcome and a kilt is required!


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Titanium sheet and plate are commonly used in manufacturing today with the most popular grades being 2 and 5. Grade 2 Titanium Grade 2 is the commercially pure titanium used in most of the chemical processing plants and is cold formable.
The Bulletsafe Bulletproof Handgun Shield is the perfect product when you need protection beyond your vest. Bulletproof shields are useful when the threat of being shot is highest or when you need to protect more than just yourself. Officers can use a ballistic shield to knock on bad guys doors or to shield bystanders from a dangerous situation.
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LYNX T is a lighter antiriot version of the titaniummade bulletproof quotSpherequot helmet used by OMON. This helmet is built from multilayer fibreglass with detachable 2mm thick steel visor with ballistic plastic glass sighting window. Builtin radio headset is the original SHG7 K113 set supplied by OKB quotOctavaquot.
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All of our shields use ballistic metals which are superior to resinfixed ballistic fibers for rigid applications. All products have a ballistic steel core and one model supplements that with a titanium strike face for even greater ballistic protection
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Our Titanium/Steel combination Hard Armor Plates provide all the protection you will find in Threat Level III steel plates but at about 30 less the weight. Standard Features NIJ threat level III Single curve Multihit capability Defeats 7.62 mm NATO 148 gr. .308 caliber FMJ at 2780 fps Front / Back placement
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Tesla Adds Titanium Underbody Shield and Aluminum Deflector Plates to Model S Elon Musk Chairman Product Architect amp CEO March 28 2014 These incidents unfortunately received more national headlines than the other 200000 gasoline car fires that happened last year in North America alone.
Jerry Miculek took it upon himself to find out by crafting a titanium shield that is exactly to spec aside from the metal its made out of. It39s the right size and weighs eight pounds just like ...
Trauma Plates Galls is here to keep you safe in highrisk situations that expose you to the threat of blunt trauma or bullet impact. Trauma plates come in several options including steel construction for a hard shock plate steelpolymer combination or titanium with a rubberized coating.
I am shooting Titanium grade 5 plates with pistol calibers. What Body Armor Should You Run Flat Curved Curved Buildup and Square AR500 Armor Duration 517. AR500 Armor 46233 views
Pro shooter tests bulletproof Captain America shield. Watch quotGreatest Shooter of All Timequot Jerry Miculek test a homemade solid titanium version of Captain America39s famous round shield.
For the study researchers manufactured a hard armor system consisting of a ceramic faceplate a CMF core and a thin back plate made of aluminum. The armor was tested using .50 caliber ball and armorpiercing rounds. The armor was tested with the rounds being fired at impact velocities from 500 meters per second up to 885 meters per second.
titanium that can be specified under the MILDTL46077G. While all four classes have the same strength and ballistic requirements the objective has been to increase the oxygen content to a maximum of 0.30 allowing the use of lowercost processing technologies such as Electron Beam or Plasma Melting for both Class 3 and 4. Armor grade titanium ...
Find great deals on eBay for titanium body armor and titanium armor plate. Shop with confidence. ... AA SHIELD Bullet Proof Panel Body Armor Inserts Plate Lvl IIIA 3A ...
This armor is NOT the one tested in the video but is an example of alloy plates available for armor systems. Im not sure that wearing around an inch and a quarter thick plate of titanium is an improvement over the thinner steel based on bulkiness but it sure does stop the rounds even a black tip .50BMG which almost made it through.
titanium diboride overview. Titanium Diboride TiB 2 is an extremely hard ceramic used for example in specialized ballistic armor applications.Additionally unlike most ceramic compositions it is electrically conductive.
Body armor or bullet proof vests can also be legally purchased at a gun show or in a brick and mortar store. You dont have to show any particular type of ID to do so and youre free to walk away from the purchase carrying or wearing that body armor or bullet proof vest.
TITANIUM ARMOR HISTORY In general the armor alloy with the best ballistic properties is the one that is the hardest yet does not exhibit brittleness. When titanium armor MILA46077 was being developed in the late 1950s it was difficult to keep test plates from cracking under ballistic attack.
There is a wide range of shield products available online on the platform of DHgate from Pendant Necklaces Necklaces amp Pendants Jewelry of different brands. Browse through our categorydefining titanium shield products in a simple secure and faster way.
For a movie accurate bullet proof titanium shield I would be very interested in purchasing that even considering your high cost of production. Grade 5 is the same as 6Al 4V. In order to get the best properties out of it you need to heat treat it though. Grade 2 is a pure titanium not an...
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Mechanical and ballistic properties of selected titanium alloys used in modern armor systems Ti6Al4V is an alphabeta titanium alloy comprised of 90 Ti 6 Al and 4 V. It has a density of 4.43g/cm3 a hardness of 334HB a yield strength of 880MPa tensile strength of 950MPa charpy impact at 17 J and elongation at 14 in 2.
This ballistic also comes in a number of other sizes including 12x16 for smaller childrens backs or laptop bags and 12x18 for larger backpacks. The Tuffypacks 11x14 ballistic panel is a great flexible alternative to our Integrated Bulletproof Backpacks. See our Guide on Bulletproof Backpacks and Backpack Armor for more information.


text:
I only have limited time using this hardness tester, but so far it seems to be pretty accurate once calibrated. I will order more and recommend this hardness tester to my friends!
Sarah|3/5/2023 10:10 PM
It's worth five stars
Great buy. This portable Shore A hardness tester is well-made and feels durable to the touch. I'm very satisfied!
John|3/3/2023 12:28 AM
Reliable Shore A hardness tester
I have used this Shore A hardness tester in my manufacturing business and it has been an invaluable tester. Accurate and reliable, and the digital display makes it easy to read.
Brooks|2/22/2023 7:34 PM
Highly recommended for this quality hardness tester
The sisco hardness tester saves us a lot of time and money by allowing us to test the hardness of materials quickly and accurately. The user interface is very intuitive and the machine itself is built to last.
The digital Shore hardness durometer, type B, features hardness values 90 A to 20 D, measurement range 10~90 H, accuracy ≤ ± 1H and resolution 0.1H, suitable materials moderately hard rubber, thermoplastic elastomers, paper products, and fibrous materials. Portable hardness tester with R0.1 cone indenter supports USB data transmission.
Scale C Shore hardness durometer for sale online. It is also a digital hardness tester that has a hardness value of 90 B~20 D, testing range of 10~90 H, precision ≤ ± 1H, and resolution of 0.1H, measuring materials include medium-hard rubber, thermoplastic elastomers, medium-hard plastics and thermoplastics, etc. Portable Shore hardness tester supports USB data transmission and matches the indenter 0.79 truncated (frustum) cone.
Digital hardness tester is available with hardness value above 90 A, measurement range 10~90 H, accuracy ≤ ± 1H, resolution 0.1H, indenter R0.1 cone. Portable Shore hardness tester is suitable for measuring materials of the type D shore hardness durometer are hard rubber, thermoplastic elastomers, harder plastics, rigid thermoplastics and so on.
Type DO digital Shore hardness tester for sale online, designed for testing moderately hard rubber, thermoplastic elastomers, and very dense textile windings. Portable hardness tester provides hardness values 90 C to 20 D, testing range 10~90 H, accuracy ≤ ± 1H, resolution 0.1H, indenter R1.2 spherical radius.
Shop on sisco.com for all of your industrial test and measurement equipment & accessories including portable detectors, meters, testers, scales, etc.

domain: chemistry


Prater Shannon posted an update 4 days, 21 hours ago
The "SAP Licensed Improvement Associate – SAP HANA 2. SPS05" certification examination confirms how the prospect provides the understanding to build up indigenous application programs jogging in the SAP HANA System. This certification develops on the standard information received through relevant SAP HANA training. The courses addresses the improvement languages and tools, utilized to recognize the various parts of a software application in SAP HANA.
SAP C_HANADEV_17 Certification Notices
To make certain accomplishment, SAP suggests mixing training classes and hands-on experience to put together to your C_HANADEV_17 certification test as concerns will try out your capability to utilize the skills you possess obtained in education.
You are unacceptable to use any reference point resources throughout the certification test (no access to on the internet documentation or any SAP system).
SAP C_HANADEV_17 Certification Topic Areas
Please visit below the list of subjects that may be protected in this particular SAP C_HANADEV_17 certification and the courses that cover them. Its precision does not comprise a real claim; SAP stocks the legal right to upgrade the test content items and topics, weighting) at any time.
– Exposing Data as OData Services> 12%
– SQLScript Development> 12%
– Using Calculation Views> 12%
– Developing a Multi-Target Application> 12%
– Creating the Persistence Data Model8% – 12%
– Creating and Integrating the UI< 8%
– Managing Source Code Using Git< 8%
– Using Database Security< 8%
– Defining Application Security< 8%
SAP C_HANADEV_17 Certification Standard Details
SAP C_HANADEV_17 Certification Examination Prep
All SAP advisor certification can be purchased as Cloud Certifications in the Certification Hub and may be scheduled with merchandise code CER006. With CER006 – SAP Certification from the Cloud, you may occupy to 6 assessments attempts that you pick in a 12 months – from where ever and when it fits you! Examination dates might be booked and chosen independently.
Every specific certification includes its unique list of planning techniques. We establish them as "Subject matter Areas" and they may be found on each and every exam description. You will discover the amount of questions, the time period of the test, what places you will end up examined on, and suggested program content and work it is possible to reference.
SAP C_HANADEV_17 Certification exams may have unscored things that are being tested for forthcoming releases of your exam. These unscored items are randomly handed out over the C_HANADEV_17 certification subject areas and so are not measured for the last score. The whole amount of items of an exam as advertised from the Coaching Retail outlet is rarely exceeded when unscored products are employed.
SAP C_HANADEV_17 Exam Details:-
– Exam Code:- C_HANADEV_17
– Exam Name:- SAP Certified Development Associate – SAP HANA 2.0 SPS05
– Delivery Methods:- Certification
– Level:- Associate
– Exam:- 80 questions
– Sample Questions:- View more
– Cut Score:- 64%
– Duration:- 180 mins
– Languages:- English
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Last week, the Federal Highway Administration conducted a two-day demonstration on partially automated truck platooning in the Washington, D.C. metro area using a three-truck platoon. A previous demonstration of this nature was conducted near Los Angeles last March.
For the past four years, the FHWA’s Exploratory Advanced Research team has been working on this project with the California Department of Transportation in order to showcase the capabilities and potential of utilizing three-truck platoons with Vehicle-to-Vehicle Connectivity (V2V) and Cooperative Adaptive Cruise Control (CACC). This technology was developed under the EAR project, in conjunction with the University of California Berkeley Partners for Advanced Transportation Technology, and allows trucks to operate more smoothly as a unit, reducing and controlling the gaps between vehicles. Here is an informative video of how truck platooning works - bear in mind that this particular video is not affiliated with this project, although it outlines the same principles at use.
So why is this important? There are actually a number of reasons why this advance is pretty exciting, especially in the United States.
- The controlled speed and drag of platooning will decrease emissions AND reduce traffic congestion.
- Early results of testing predict up to a 15% fuel savings for following trucks.
- Improved road efficiency, travel time, and consequently, reduced delivery time. A boost to truckload on-time performance is always a plus.
- Reduced operating costs for fleet operators.
- Expected to create higher driver retention AND reduce driving workload and fatigue to drivers. Because this system is partially automated, drivers are still very critical for steering the vehicle and remaining vigilant to take over full control of the vehicle at any time for safe driving.
- This type of technological transportation advance helps to make the United States more competitive on a global scale as well.
The Federal Highway Administration expects truck platooning to increase our highway capacity by making freight transportation more efficient, meaning that we can get more out of the highway system we currently have, in addition to relieving traffic congestion and reducing costs to the freight industry. This advancement has the potential to have a direct and positive impact on the economy of the United States. With the successes of these demonstrations, truck manufacturers and their suppliers are working to commercialize the system by late 2018. Increasing logistics efficiency appears to be on a continuous upward track.
For more information on the FHWA's truck platooning program, click here.


10th November 2006
Texan rockers BLUE OCTOBER have cancelled upcoming live performances in North America after frontman JUSTIN FURSTENFELD re-injured his leg. Furstenfeld had surgery on his leg earlier this year (06), and he has been taken to...
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