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Lysyl oxidase propeptide (LOX-PP) ectopic overexpression inhibits the growth of cancer xenografts. Here the ability and mode of action of purified recombinant LOX-PP (rLOX-PP) protein to inhibit the growth of pre-existing xenografts was determined. Experimental approaches employed were direct intratumoral injection (i.t.) of rLOX-PP protein into murine breast cancer NF639 xenografts, and application of a slow release formulation of rLOX-PP implanted adjacent to tumors in NCR nu/nu mice (n = 10). Tumors were monitored for growth, and after sacrifice were subjected to immunohistochemical and Western blot analyses for several markers of proliferation, apoptosis, and for rLOX-PP itself. Direct i.t. injection of rLOX-PP significantly reduced tumor volume on days 20, 22 and 25 and tumor weight at harvest on day 25 by 30% compared to control. Implantation of beads preloaded with 35 micrograms rLOX-PP (n = 10) in vivo reduced tumor volume and weight at sacrifice when compared to empty beads (p<0.05). A 30% reduction of tumor volume on days 22 and 25 (p<0.05) and final tumor weight on day 25 (p<0.05) were observed with a reduced tumor growth rate of 60% after implantation. rLOX-PP significantly reduced the expression of proliferation markers and Erk1/2 MAP kinase activation, while prominent increases in apoptosis markers were observed. rLOX-PP was detected by immunohistochemistry in harvested rLOX-PP tumors, but not in controls. Data provide pre-clinical findings that support proof of principle for the therapeutic anti-cancer potential of rLOX-PP protein formulations.
Citation: Bais MV, Nugent MA, Stephens DN, Sume SS, Kirsch KH, Sonenshein GE, et al. (2012) Recombinant Lysyl Oxidase Propeptide Protein Inhibits Growth and Promotes Apoptosis of Pre-Existing Murine Breast Cancer Xenografts. PLoS ONE 7(2): e31188. https://doi.org/10.1371/journal.pone.0031188
Editor: Surinder K. Batra, University of Nebraska Medical Center, United States of America
Received: July 15, 2011; Accepted: January 3, 2012; Published: February 8, 2012
Copyright: © 2012 Bais 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: This research was supported by NIH grants: R01 DE14066, RO1 CA82742, and R01 HL088572, and Department of Defense Idea Award W81XWH-08-1-0349 PC073646. The funders had no role in the 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.
Lysyl oxidase (LOX) is a copper-dependent, extracellular matrix enzyme required for the normal biosynthesis of mature and functional collagens and elastin [1]. LOX is made as a 50 kDa proenzyme and processed extracellularly to a ∼30 kDa mature LOX enzyme and the ∼18 kDa propeptide (LOX-PP) by procollagen C-proteinases [2], encoded by the bone morphogenetic protein-1 and the tolloid-like-1 and -2 genes [3], [4]. The LOX gene has been shown to have "RAS rescission" activity, and this tumor suppressor activity of the LOX gene has been mapped to its propeptide region [5]. Natural and recombinant LOX-PP has N- and O-linked carbohydrates and a very high isoelectric point. The characterization of purified recombinant LOX-PP (rLOX-PP) showed that it is a highly disordered protein and is, therefore, predicted to have multiple binding partners and more than one mechanism of action [6].
LOX enzyme activity has been reported to promote tumor invasiveness [7], [8]. LOX enzyme and LOX-PP are derived from the same mRNA, as summarized above. Interestingly, estrogen receptor positive breast cancer tumors are less invasive than estrogen receptor negative tumors, and express lower levels of LOX mRNA [9]. Taken together, data suggest a model in which abnormally low levels of LOX-PP may permit tumor growth, whereas abnormally high LOX enzyme may promote tumor invasion. Our interest here is to investigate the potential of rLOX-PP protein to inhibit tumor growth in vivo.
LOX-PP inhibits RAS-dependent transformation of NIH 3T3 fibroblasts assessed by inhibition of cell proliferation, growth in soft agar and Akt- and Erk1/2 dependent induction of NF-kappaB in vitro [5]. In breast cancer, LOX-PP was shown to reverse the invasive phenotype of Her2/neu driven breast cancer cells, inhibit epithelial to mesenchymal transition, and inhibit migration and branching colony formation. Retrovirus mediated ectopic overexpression of LOX-PP in NF639 cells suppresses xenograft tumor formation in nude mice [10]. In human lung and pancreatic cancer cells, LOX-PP reduces migration, Erk1/2 and Akt signaling, and growth in soft agar and represses BCL-2 levels [11]. Doxorubicin is a chemotherapeutic drug which induces apoptosis in cancer cells. Pre-treatment of pancreatic and breast cancer cells with rLOX-PP sensitizes the cancer cells to doxorubicin-induced apoptosis in vitro, whereas LOX-PP alone did not appear to induce apoptosis [12]. LOX-PP inhibits different signaling pathways. LOX-PP reduces tyrosine phosphorylated FAK and p130cas protein, and reduces haptotaxis of NF639, MDA-MB-231 and Hs578T breast cancer cells and thus attenuates fibronectin mediated integrin signaling [13]. rLOX-PP inhibits FGF-2 induced DNA synthesis, Erk1/2 and Akt signaling and FRS2alpha activation in DU145 prostate cancer cells and in the phenotypically normal MC3T3-E1 osteoblastic cell line [14], [15]. Interestingly, the single nucleotide Arg158G polymorphism in a carboxy-terminal conserved region results in loss of some of the LOX-PP tumor suppressor properties [9], while some intracellular mechanisms of action depend on the amino-terminal end [16]. Taken together, the various studies have shown that LOX-PP has a tumor suppressor property, inhibits RAS, FGF-2, and FAK signaling, and that different conserved regions mediate its activity.
These studies strongly suggest that LOX-PP could have potential as a cancer therapeutic. The major goal of this study was to evaluate the ability of rLOX-PP protein to inhibit or reverse the growth of pre-existing breast cancer xenografts, first by direct intratumoral injection (i.t.); and second, by a slow release formulation in order to establish proof of principle that rLOX-PP protein formulations can have the potential as an in vivo cancer therapeutic. In order to gain insights into the mode of action of rLOX-PP protein in vivo, the effect on breast cancer xenograft cell proliferation and apoptosis was determined. Moreover, data suggest that LOX-PP protein persists in tumors particularly when administered by a slow release formulation in vivo. These findings support the notion that formulations of rLOX-PP may ultimately prove to have therapeutic utility in cancer therapy.
Materials and Methods
Cell line and Mouse xenograft model
The mouse mammary tumor virus (MMTV)-Her-2/neu NF639 cell line kindly provided by Dr. P. Leder [17] was grown in DMEM supplemented with 10% FBS. All experiments were performed as approved by Boston University Medical Center IACUC. In both experiments, the single cell suspensions in DMEM were prepared. NF639 cells (4×106 cells) were injected subcutaneously into the midline dorsa (n = 10/condition) of NCR nu/nu mice (Charles River Laboratories), and tumors allowed to grow to 100 mm3. Caliper measurements were made at intervals to monitor the volume of all tumors.
Intratumoral injection
Purified rLOX-PP (10 micrograms) expressed by HEK293 cells [6] in PBS or PBS alone (vehicle) was injected once per day i.t. for 5 days starting on day 17 when the average tumor volume was 100 mm3. Mice were monitored and maintained until day 21 at which time the PBS injected control tumors reached 1000 mm3.
rLOX-PP slow release formulation in alginate beads
Alginate beads containing rLOX-PP and empty control beads were made according to a standard protocol [18] as follows. rLOX-PP (1 mg/ml) [6] and 18 mg/ml sodium alginate were mixed at a ratio of 1∶2 and the slurry was passed drop-wise through a 21 gauge needle into a solution of CaCl2 (1.5% w/v) with gentle mixing. Mixing was continued for five minutes, and then beads were allowed to settle for 10 minutes. Beads were washed three times in sterile water and stored in 150 mM NaCl, 1 mM CaCl2 at 4°C until used. LOX-PP incorporation within the beads was calculated based on a 70% incorporation efficiency measured previously with other growth factors of similar size and molecular properties [19], [20]. Empty beads were made without addition of rLOX-PP. Bead size was measured with a digital caliper (Fisher Scientific) and an average diameter of 1.50±0.12 mm (n = 20) was noted. Animal studies were conducted with a single preparation of beads to avoid batch to batch variability.
rLOX-PP in vitro release kinetics from LOX-PP/alginate beads
rLOX-PP release kinetics from calcium alginate beads were determined in vitro over a 14 day period. Three beads containing 1 µg rLOX-PP each were incubated in 1 ml release buffer (150 mM NaCl, 1 mM CaCl2, 0.05% gelatin) with n = 4 at 37°C with constant shaking. The supernatant was collected on days 0, 1, 3, 7, 10 and 14. At each time point, the entire supernatant was removed and replenished with 1 ml fresh release buffer. Samples were boiled in 1X SDS PAGE sample buffer and aliquots were subjected to Western blot analysis, and compared to standard curves of rLOX-PP determined from the same blots after densitometric analyses and linear regression with the aid of a Versadoc densitometer and Quantity One software (BioRad). Total cumulative rLOX-PP released was then calculated.
In vivo implantation of rLOX-PP alginate beads
NF639 cells (4×106) were injected once in the dorsum of each mouse and tumors were allowed to grow to 300 mm3 (n = 10/group). Then, 35 micrograms rLOX-PP in beads, or empty alginate beads (n = 10) were surgically implanted adjacent to each experimental and control tumor, respectively, on lateral sides of tumor borders and subsequently sutured with vicryl 4.0 biodegradable sutures. Tumor volumes were monitored by caliper measurements made at regular intervals, and tumor weights were determined at sacrifice.
Histology and immunohistochemistry
All tumors were harvested at sacrifice and then fixed in 4% paraformaldehyde for three hours, and then placed in 30% sucrose overnight at 4°C. Frozen sections were made and subjected to immunohistochemistry and quantitative histomorphometric analyses [21]. Four tumors, 3 to 4 sections per tumor, were used for immunohistochemistry staining analysis for the proliferation marker, Ki-67 (Abcam Inc.; ab15580), phospho-histone H3 (Millipore; 06-570), the apoptosis marker active caspase 3 (Trevigen, Gaithersburg, MD, USA; 2305-PC-020), and LOX-PP itself (Novus, NB110-41568). Apoptosis was further evaluated using a kit for the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay according to the manufacturer's instructions (Trevigen, Gaithersburg, MD, USA; 4815-30-K) [22]. Quantitation of micrographs was performed independently by two experienced researches MB and SSS) in a blinded fashion according to previous protocols performed by this laboratory [21], [23], [24]. Five independent 0.09 mm2 grids on three sections per animal were measured; a minimum of three mice per group was analyzed as indicated for each assay.
Western blotting
Snap frozen tumors were ground to a fine powder in liquid nitrogen and then extracted into SDS PAGE sample buffer (0.1 mM Tris-HCl, 4% SDS, 10% glycerol, 5% β-mercaptoethanol). Protein concentrations were determined using Nano Orange assay kits (Molecular Probes, Eugene, OR, USA). Approximately 10 micrograms of protein from 3 tumors per experimental group were then subjected to 10% SDS PAGE and Western blotting with primary antibodies for mitosis marker phospho-histone H3 (Millipore, Billerica, MA, USA; 06-570), the downstream targets of Ras signaling phospho-Erk1/2, total Erk1/2, and normalization control beta-actin (Cell Signaling, Danvers, MA, USA; antibodies 9101S, 9102S and 4970L, respectively). Anti-rabbit secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA; 7074S). The quantifications were performed by a digital densitometry system and Quantity One software (Versadoc; BioRad, Eugene, OR, USA).
Intratumoral injection of rLOX-PP inhibits pre-existing breast cancer xenografts
Earlier studies have shown that the ectopic expression of LOX-PP inhibits breast and pancreatic cancer xenograft growth, and proliferation of prostate cancer cells in vitro [10], [12], [14]. To evaluate whether rLOX-PP protein can inhibit the growth of preexisting tumors in vivo, we used a breast cancer xenograft model. NF639 breast cancer cells (4×106 cells) were injected subcutaneously into the dorsal region of nude mice and tumors allowed to grow to a volume of 100 mm3 (day 17). Mice were then divided randomly into two groups with 10 mice each. Mice were injected once per day beginning on day 17 for five consecutive days with 10 micrograms rLOX-PP or vehicle (PBS) in a volume of 100 microliters. Caliper measurements were continued through day 25 at which time tumors injected with vehicle (controls) reached a volume of 1000 mm3. Data in Figure 1A show that on day 25 the average tumor volume in the rLOX-PP i.t. injection group was 743±101 mm3 compared with PBS control (1043±199 mm3) showing significantly inhibited tumor growth in LOX-PP treated tumors (p≤0.05). The growth rate was plotted from the tumor volume measurements beginning on day 18. The growth rate in the vehicle group was 125 mm3 per day compared to 85 mm3 per day in rLOX-PP treated tumors (Figure 1B). Consistent with the above findings, the tumor weights determined at sacrifice were smaller in the rLOX-PP injected group (Figure 1C) but not significantly different from control (p = 0.41). Thus, the data show that i.t. injection of rLOX-PP significantly inhibits the tumor volume growth by 28% on day 25 and growth rate by 32% after day 18 compared to PBS, and a trend toward diminished tumor weight was found.
(A) Subcutaneous xenografts of NF639 breast cancer cells were injected once daily with 10 micrograms rLOX-PP for 5 days beginning on day 17 (arrows). Tumor volumes were monitored by caliper measurements (n = 10; *, p<0.05); (B) The tumor growth rates were determined from measurements made beginning on day 18 and were derived from linear regression analyses of the data. (C) Tumor weights at sacrifice on day 25 (p = 0.4, n = 10).
Sustained release of rLOX-PP
To evaluate whether rLOX-PP could be stabilized for slow and sustained release and provide enhanced stability and possibly increased biological activity in vivo, we incorporated rLOX-PP into alginate beads. Alginate is a naturally occurring aquatic plant polysaccharide polymer and alginate beads have been used previously for successful slow release of FGF-2 and other proteins with high isoelectric points without loss of biological activity [18], [25]. rLOX-PP is also a highly basic protein with a high isoelectric point [6]. In order to determine whether rLOX-PP can be released from alginate beads in a time-dependent manner, in vitro release kinetics were determined as outlined in Materials and Methods. Data in Figure 2 indicate that rLOX-PP is released continuously over the experimental period of 14 days (p≤0.001) with apparent linear release between day 1 and day 7 (Figure 2B), slowing after day 7 (Figure 2C). In total, 50% of rLOX-PP encapsulated in alginate beads was released into buffer by day 14 (Figure 2C) with the remainder still bound to alginate. In vivo, tightly bound LOX-PP would potentially be released because alginate is progressively resorbed [26].
(A) Western blot of known amounts of rLOX-PP ranging from 0.1 to 10 ng of rLOX-PP; (B) Western blot of rLOX-PP-bead supernatants collected at different intervals; (C) calculated cumulative release of rLOX-PP; (n = 4; *, p<0.001).
To determine the activity of rLOX-PP/alginate beads to effectively inhibit breast cancer xenograft growth we first established NF639 tumors as described in Materials and Methods. Empty alginate beads or beads containing a total of 35 micrograms rLOX-PP were then implanted adjacent to the xenografts through a small incision made on each of two sides of the tumor. rLOX-PP/alginate beads significantly inhibited tumor growth compared to empty beads on day 21 and 25 (Figure 3A). Tumor size in animals implanted with rLOX-PP were 7%, 36% and 38% lower on days 18, 21, and 25, respectively, compared to control mice. The tumor growth rate was determined by plotting tumor volumes beginning on day 18 and revealed a rate of 110 mm3/day for control tumors (empty beads) compared to 42 mm3/day for the rLOX-PP treated tumors (rLOX-PP/alginate beads), demonstrating a 61% reduction in growth rate after rLOX-PP bead implantation adjacent to tumor xenografts (Figure 3B). The final average tumor weight at sacrifice was 30% lower after rLOX-PP/alginate bead implantation (Figure 3C). In summary, data indicate that rLOX-PP/alginate bead implantation resulted in lower final tumor volume by 38% on day 25, 30% lower final tumor weight, and a 61% lower tumor growth. Taken together, findings show that both i.t. injection of rLOX-PP and slow release formulations of rLOX-PP effectively inhibit NF639 xenograft growth. In addition, it is apparent that a lower amount of rLOX-PP (35 micrograms) when administered in a slow release formulation is more effective at inhibiting tumor growth than i.t. injection of a higher total amount of rLOX-PP (a total of 50 micrograms: 10 micrograms per day as described above).
(A) Subcutaneous 100 mm3 xenografts of NF639 breast cancer cells were established in mice. rLOX-PP/alginate or empty beads were implanted on day 15 (arrow). Tumors were monitored by caliper measurements (n = 10; *, p<0.05, ***, p<0.01). (B) The tumor growth rates were determined from measurements made beginning on day 18 and were derived from linear regression analyses of the data; (C) tumor weights determined at sacrifice on day 25 (n = 10; *, p<0.05).
rLOX-PP inhibits tumor growth by reducing proliferation and increasing apoptosis
In order to gain insights into mechanisms by which LOX-PP inhibits tumor growth in vivo, we first evaluated relative expression levels of Ki-67 by immunohistochemistry in harvested tumors. Ki-67 is a marker for cell proliferation, and is expressed in all phases of the cell cycle except G0, and is a predictive and prognostic marker for breast cancer [27], [28], [29], [30], [31]. A higher number of Ki-67 immunopositive cells were observed in sections injected with PBS or implanted with empty beads compared to rLOX-PP injection or rLOX-PP/alginate beads implantation, as seen in representative sections (Figure 4A and 4B). The proportion of Ki-67 immunopositive cells was reduced by both direct i.t. injection of rLOX-PP and rLOX-PP/alginate beads compared to their respective controls (Figure 4C; p<0.001). In addition, histone H3 is phosphorylated specifically during mitosis and is also frequently used as a proliferation marker in cancer. Both Ki-67 and phosphorylated histone H3 have been used for identification of aggressive human breast and ovarian cancer and positively correlate with the tumor grade [32], [33]. Data in Figure 5A and 5B suggest the presence of a reduced number of phospho-histone H3 stained cells in both direct i.t. injection of rLOX-PP and rLOX-PP/alginate bead treated tumors compared to their respective controls. The quantitation of phospho-histone H3 from tumor tissue protein extracts by Western blotting showed an average reduction of 45–50% in both i.t. injection of LOX-PP (Figure 5C; p<0.05) and rLOX-PP/alginate beads (Figure 5D; p<0.05). Thus, direct i.t. injection of rLOX-PP and rLOX-PP/alginate beads each respectively reduced Ki-67 and phospho-histone H3 by approximately 50% in this breast cancer xenograft model.
(A) Immunostaining with Ki-67 antibody or non-immune IgG control tumors after (A) direct i.t. injection of rLOX-PP compared to PBS and (B) after implantation of empty and rLOX-PP alginate beads. Black arrows mark some positive-stained cells; the white arrows point to an inset containing an enlarged image of a stained cell. (C) The corresponding quantifications are shown (***, p<0.001; n = 3). Scale bar = 0.005 mm.
(A) Immunostaining with phospho- histone H3 antibody or nonimmune IgG control tumors after (A) direct i.t. injection of rLOX-PP (L) compared to PBS (P) and (B) after implantation of empty (EB) and rLOX-PP (LB) alginate beads. Black arrows mark some positive-stained cells; the white arrows point to an inset containing an enlarged image of a stained cell. Scale bars correspond to 0.005 mm for 400× and 0.002 mm for 1000×. Arrows mark some positively stained cells. (C) Western blot analyses of 17 kDa phospho- histone H3, 36 kDa beta-actin and the corresponding quantifications of protein extracts from tumors after (C) direct i.t. injections of rLOX-PP compared to PBS and (D) after implantation of empty and rLOX-PP alginate beads (*, p<0.01; n = 3).
Apoptosis is induced by at least two different pathways: death receptor (extrinsic) and mitochondrial (intrinsic) pathways. Both pathways converge at caspase 3 activation that can be detected by immunohistochemistry. Representative micrographs (Figures 6A and 6B) and quantitative scoring of all data (Figure 6C) suggest that both i.t. injection and slow release of rLOX-PP increased the number of cells staining for active cleaved caspase-3 in breast cancer xenografts.
(A) Immunostaining of tumors with active caspase 3 antibody, or non-immune IgG control. (A) i.t. rLOX-PP- or PBS injected tumors, and (B) tumors treated with surgically implanted empty alginate beads or rLOX-PP/alginate beads. Black arrows mark some positive-stained cells; the white arrows point to an inset containing an enlarged image of a stained cell. Scale bar = 0.005 mm for 400× and 0.002 mm for 1000×; (C) the corresponding quantifications (***, p<0.001; n = 3).
To determine more directly whether apoptosis and DNA fragmentation were enhanced by rLOX-PP, we performed TUNEL assays as indicated in Materials and Methods. TUNEL positive cells (Figure 7A and B) were found to be significantly increased to about 10% of cells in sections with rLOX-PP i.t. injection or rLOX-PP/alginate beads, compared to about 2% for controls (Figure 7C; p<0.0001). Taken together with activated caspase 3 measurements shown in Figure 6, the data indicate that rLOX-PP inhibits proliferation and stimulates apoptosis in breast cancer xenografts.
(A) TUNEL staining of tumors treated i.t. with rLOX-PP, or tumors injected with negative control PBS; (B) tumors treated with surgically implanted empty alginate beads or rLOX-PP alginate beads. Black arrows mark some positive-stained cells; the white arrows point to an inset containing an enlarged image of a stained cell. Scale bar = 0.005 mm for 400× and 0.002 mm for 1000×; (C) the corresponding quantifications (***, p<0.001; n = 3). Arrows mark some positive-stained cells.
We next investigated whether LOX-PP itself could be detected in harvested tumors using a specific anti-LOX-PP antibody in immunohistochemistry assays [34]. As seen in Figure 8, data indicate that rLOX-PP is easily detected at elevated levels in tumors from mice implanted with rLOX-PP alginate beads compared to empty beads. By contrast, although the same trend is observed in tumors derived from direct i.t. injection experiments, the level of detectable rLOX-PP appears to be less than for rLOX-PP/alginate.
Immunostaining of tumors after sacrifice with LOX-PP antibody or non-immune IgG control from mice after (A) direct i.t. injection of rLOX-PP compared to PBS or (B) after implantation of empty or rLOX-PP-containing alginate beads. Scale bars = 0.005 mm. Arrows mark some positive-stained cells.
We next evaluated a downstream target of Ras signaling to ask whether the mechanism of rLOX-PP protein in vivo is likely to occur by mechanisms that we have described in vitro and in ectopic expression studies both in vitro and in vivo. Figure 9 shows that Erk1/2 phosphorylation was inhibited by more than 70% after rLOX-PP i.t. injections (Figure 9A) and rLOX-PP/alginate slow release (Figure 9B) compared to respective controls. Thus, data are consistent with findings that rLOX-PP protein functions by inhibiting Ras signaling in a pre-clinical breast cancer xenograft model.
Western blot analysis of 42 and 44 kDa phospho-Erk, total Erk1/2 and 36 kDa beta-actin from tumor extracts after (A) direct i.t. injections of rLOX-PP (L) compared to PBS (P); (B) after implantation of empty (EB) and rLOX-PP (LB) alginate beads; (C) the corresponding quantifications are shown (*, p<0.05; n = 3).
Here we show for the first time that rLOX-PP protein inhibits pre-existing tumor growth after direct i.t. injection or when applied in a slow release formulation. Intratumoral injection studies have been classically done in initial studies to evaluate the ability of proteins to inhibit tumor growth. Direct i.t. injection of tumor necrosis factor-alpha and interferons, or interferons alone, were shown to inhibit xenograft growth in mice [35], [36], [37]. In human patients, i.t. injection of recombinant IL-2 induced tumor killing, tumor necrosis and lymphocytic infiltration [38]. Thus, consistent with other pre-clinical and clinical studies, it is here shown that direct i.t. injection of rLOX-PP inhibited the growth of a pre-existing breast cancer xenograft. Interestingly, the slow release formulation appeared to be more effective than i.t. injection of naked rLOX-PP in the inhibition of NF639 xenograft growth. Although, LOX-PP has been shown to have tumor suppressor properties, the effectiveness of rLOX-PP protein to inhibit pre-existing tumor growth has not been previously evaluated in vivo. Such studies are important in the evaluation of therapeutic potential.
Alginate beads have been used previously for successful slow release of recombinant proteins with a high isoelectric point without loss of biological activity [18], [25]. In human clinical studies, alginate beads containing human mature allogenic chondrocytes were found to be an effective treatment for cartilage defects in the knee showing significant improvement within six months which shows that alginate is safe and feasible for application in vivo in patients [39]. Alginate formulations, therefore, could have potential for use in pre-clinical human studies, although other carriers such as polylactide/polyglycolide polymers may be more widely used [40]. Encapsulated proteins in alginate beads are released by diffusion from and degradation of alginate beads for in vivo applications [41], and the present study is significant in that it provides proof of principle that rLOX-PP is effective in vivo, especially in a slow release formulation. At sacrifice, it was apparent that most of the bead material had been resorbed in all implanted mice, as expected. Only 1–2 beads out of the twelve beads implanted per mouse could be found at sacrifice (data not shown) suggesting that more than 80% of rLOX-PP was released in vivo.
The single implantation of alginate incorporated rLOX-PP is more efficient than five i.t. injections of rLOX-PP. The total amount of rLOX-PP incorporated in alginate beads was 35 micrograms and resulted in a more significant reduction in tumor xenograft volume, growth rate and tumor weight compared to 50 micrograms rLOX-PP direct i.t. injections over five days. Similarly, other studies have shown that alginate encapsulation of recombinant proteins are highly active in vivo. For example, similar to our data with rLOX-PP, in vivo subcutaneous implantation of a single dose of alginate beads containing IL-17R was more effective in reducing inflammation when compared to multiple subcutaneous injections of this anti-inflammatory protein [41]. FGF-2 plays an important role in tissue repair and has a very short half-life when administered parenterally. Surgical implantation of FGF-2 bound to heparin-Sepharose beads encapsulated in calcium alginate microcapsules in an arterial injury model was more efficient in delivering FGF-2 within the arterial wall when compared to intravenous administration [18]. Thus, consistent with other studies, rLOX-PP inhibits tumor xenograft by direct i.t. injection or implantation of alginate encapsulated rLOX-PP, yet the latter shows a stronger effect with an apparently increased persistence of rLOX-PP in tumors as seen in Figure 6. Although there are many possibilities to improve the current formulation of rLOX-PP for in vivo application, the current formulation of alginate beads with encapsulated rLOX-PP inhibited tumor growth rate by more than 61% and provides proof of principle that rLOX-PP protein in some form has therapeutic potential. It now becomes of considerable interest to determine the pharmacokinetics, pharmacodynamics, and stability and effectiveness of systemically applied naked rLOX-PP and of protected formulations of rLOX-PP that would be acceptable for human trials in order to develop rLOX-PP as an anti-cancer drug candidate.
In order to investigate mechanisms of inhibition, we evaluated effects of rLOX-PP on cell proliferation and apoptosis. rLOX-PP inhibits cell proliferation determined by reduced expression of two independent proliferation markers: Ki-67 and phospho-histone H3. Ki-67 has been used to evaluate the responsiveness of chemotherapeutics to breast cancer patients and evaluate the risk of tumor incidence after treatments [42], [43]. Ki-67 has diagnostic importance in distinguishing between benign and malignant tumors [44], [45], [46], and is a prognostic factor for clinical breast cancer patients [47], [48], [49]. Phospho-histone H3 has been similarly employed to evaluate tumor grade and aggressiveness [32], [33]. Phospho-histone H3 is a prognostic proliferation marker in triple negative invasive lymph node-negative breast cancer [50].
Data further show that rLOX-PP induces apoptosis in breast cancer xenografts which was evaluated by activated caspase 3 immunostaining and TUNEL assays. Apoptosis is induced by different pathways which converge at caspase 3 activation, a pro-apoptotic marker [51]. Recently, it has been shown that rLOX-PP sensitizes pancreatic and breast cancer cells to doxorubicin induced apoptosis in vitro [12], but whether rLOX-PP alone could induce apoptosis in breast cancer xenografts in vivo was not known. The mechanism by which rLOX-PP induces apoptosis of cancer cells in vivo in the absence of other chemotherapeutic agents as seen in the present study requires further evaluations. The NCR nu/nu mouse model employed in the present study is T-cell deficient, but contains natural killer (NK) cells and B cells. It is, therefore, possible to speculate that the increased apoptosis observed in rLOX-PP treated tumors in vivo could be driven by enhanced sensitivity to NK cell-derived TRAIL, or FasL stimulated signaling [52], [53]. A greater understanding of molecular mechanisms of action of LOX-PP may permit the design of combinatorial therapeutic approaches that would be more effective than individual chemotherapeutic approaches. As noted, it is of interest that rLOX-PP inhibits tumor growth in vivo characterized by inhibition of cell proliferation and induction of apoptosis, whereas in vitro rLOX-PP alone inhibits proliferation but has not been seen to promote apoptosis [12].
Earlier in vitro studies done with ectopic rLOX-PP expression in cancer cell lines indicate that LOX-PP inhibits Ras-mediated activation of Erk1/2 by inhibiting FGF-2 to FGFR1 receptor binding and activation, and by direct interaction with Hsp70 and c-Raf [14], [15], [16]. The current study extends these observations to rLOX-PP protein administered in vivo in which we show highly significant inhibition of Erk1/2 phosphorylation. It is likely that mechanisms of LOX-PP identified primarily in vitro are operative in vivo, but further study of individual molecular interactions in vivo with wild type and mutant forms of rLOX-PP will be required to investigate the relationship between specific molecular interactions and actual inhibition of tumor growth. The observation of similarities in the magnitudes of rLOX-PP administration on altering levels of both proliferation and apoptosis markers, whereas tumor inhibition is more pronounced in the slow release model is likely to be coincidental. Without more continuous assays of changes in the expression of these markers throughout the entire experimental period, it is difficult to comment further. Importantly, the current study establishes experimental systems by which a variety of questions can now be addressed in vivo.
In conclusion, rLOX-PP protein is effective in inhibiting mouse xenograft growth. The data show that alginate bound rLOX-PP is more efficient in inhibiting breast cancer xenograft compared to direct i.t injections. Further understanding of the mechanisms by which rLOX-PP inhibits breast cancer xenograft growth will enhance the ability to design potentially more effective combinations of anti-cancer regimens.
Author Contributions
Conceived and designed the experiments: PCT MVB MAN. Performed the experiments: MVB SSS DNS MAN. Analyzed the data: MVB PCT GES KHK. Contributed reagents/materials/analysis tools: MVB SSS DNS MAN KHK. Wrote the paper: MVB PCT. All authors critically reviewed and approved the manuscript.
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text:
|Home > Zyna
♀ Zyna
What does Zyna mean?
Zyna [zy-na] as a girls' name is a Greek name, and the name Zyna means "guest, stranger". Zyna is an alternate form of Xenia (Greek).
ASSOCIATED WITH greek
VARIANTS Zina, Zenia, Zena, Zeena, Zaina, Xena▼, Xeenia
OTHER FORM VIA XENIA XiaCREATIVE FORMS
(female) Zeana, Zynai, ..
(male) Zynac, Zyno, .. MIDDLE NAME PAIRINGS
Zyna Gitty (Z.G.), ..
How popular is Zyna?
Zyna is a rare first name for females. Zyna is an equivalently rare surname too for both adults and children. (2000 U.S. DEMOGRAPHICS)
Which version is better?with its source form and related girl names. Prominent alternative forms of Zyna (NOT IN TOP 2000) listed in the Top 2000 are Xena, Xenia, Zaina (#1862 FROM RECENT DATA), Zena and Zina. Other forms, like Xeenia, are uncommon. These forms of Zyna were favored by parents 19 years ago (MEDIAN #1618) and have become much less popular since, with the version Xena becoming less fashionable. (2015 BIRTH STATISTICS)
Similar Names
Zyna is alike in pronunciation to Ciana, Cyanea, Sana, Sanna, Seana, Sena▼, Siana, Siena▲, Signa, Sonia▼, Sonya▼, Xeni, Zahna, Zanna, Zaynah, Zeenya, Zenna, Zinia, Zinnia, Zinnya, Zinya, Zoanna, Zona▼, Zonia and Zuni. Other recommended names are Anna▼, Bina, Buna, Byna, Chyna▼, Dayna▼, Dena▼, Duna, Dyana, Dyna, Erna▼, Etna, Fyona, Gena▼, Gina▼, Iona▼, Iryna, Jana▼, Kena, Leyna, Lona▼, Lyna, Mona▼, Moyna, Myrna▼, Nena, Nyna, Orna, Rana, Reyna▲, Ryana, Teyna, Tina▼, Tyana, Una▼, Vana, Vina▼, Vyna, Yana, Zayra, Zenda, Zeta, Zia, Zin, Zita, Zoia, Zoyya (see Zoya), Zuba, Zuma and Zyta. These names tend to be more commonly used than Zyna.See names in meaning and etymology.

subset: commoncrawl


text:
Murrells Inlet Post Office
654 Bellamy Ave, Murrells Inlet, SC 29576
Phone: 843-651-2422
Fax: 843-357-9084
Toll-Free: 1-800-Ask-USPS® (275-8777)
Monday-Friday 10:00am - 4:00pm
Saturday 10:00am - 12:00pm
Sunday Closed
Monday-Saturday 12:01am - 11:59pm
Sunday Closed
Monday-Saturday 12:01am - 11:59pm
Sunday Closed
Monday-Friday 11:30am
Saturday-Sunday Closed
Monday-Friday 4:45pm
Saturday 2:15pm
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Monday-Friday 9:30am - 4:00pm
Saturday-Sunday Closed
Monday-Friday 9:30am - 4:30pm
Saturday 10:00am - 12:00pm
Sunday Closed
*This facility does not process US Passports applications or renewals. Check nearby locations below.
- Burial Flags
- Business Reply Mail Account Balance
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- Duck Stamps
- General Delivery
- Global Express Guaranteed®
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- Pickup Accountable Mail
- Pickup Hold Mail
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Visit our Links Page for Change of Address, Hold Mail/Stop Delivery, PO Boxes™ rentals and fees, Rates on Postage Stamps and Freight/Shipping, Holidays, Bulk Mail, Money Orders, and Available Jobs.
*Please call to verify information. Is anything incorrect? Let us know here
Nearby Post Offices:
Surfside Beach
420 Highway 17 N
3.5 miles away
7140 Highway 707
4.5 miles away
Pawleys Island
10993 Ocean Hwy
5.5 miles away
Myrtle Beach
505 N Kings Hwy
8.3 miles away
Georgetown County
Reviews*Comments below are not read by postal employees. Please do not post personal information.
I was delivering furniture to a house down off Seacoast Dr. and wasn't able to lift the HEAVY dresser & chest....This gentleman stopped, got out of his truck, and helped me carry both pieces up a tall flight of stairs. ...THANKS!!! I only know his first name..Rod..Thanks again Rod!..
Paul Choberka


text:
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IBM survey says ads are the biggest reason people consider canceling their streaming video services
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What this year's M&A activity tells us about media and entertainment
With Dalian Wanda Group's $3.5 billion acquisition of Legendary Entertainment in January, this year's media and entertainment M&A activity kicked off with a bang that hasn't slowed down. Comcast's $3.8 billion acquisition of DreamWorks Animation just three months later continued the trend of content consolidation and IP aggregation. The common denominator is access… Read More
Five companies considered buying LinkedIn
A new SEC filing posted Friday gives more clarity about what led up to Microsoft acquiring LinkedIn for more than $26 billion. And it proves that Microsoft wasn't the only prospective buyer. It shows that the talks between LinkedIn CEO Jeff Weiner and Microsoft CEO Satya Nadella began on February 16 of this year, just 12 days after the earnings debacle where LinkedIn shares plummeted. Read More
IBM and Cisco team up on enterprise collaboration to stave off rivals like Slack and Microsoft
Earlier this month, IBM and Cisco announced they would work together to integrate IBM's Watson artificial intelligence technology into edge routers from Cisco, and today the two IT giants are deepening their partnership again, as they aim for a bigger piece of the enterprise collaboration market being chased by the likes of fast-growing, popular upstarts like Slack, large… Read More
IBM demos an employee hack for gathering live video data via drone
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As Symantec flexes its muscle in security, who will step up to the challenge?
As the security market shakes up in more ways than one, who wins, who struggles and who will get acquired next? Read More
IBM's Watson makes a move into self-driving cars with Olli, a minibus from Local Motors
IBM today took the wraps off its first big foray into the world of self-driving cars, not as the driver of them, but as the brain behind making your self-driving journey a little more interesting. IBM Watson, the company's AI platform, is powering services in Olli — an electric-powered vehicle that can carry up to 12 people designed by Local Motors, a car maker based out of… Read More
IBM expands its cloud with SugarCRM platform integration and VMware Horizon Air deal
IBM has been trying to carve out a place for itself as a go-to cloud repository for all enterprise software services — not just those IBM builds itself. And today, two of its earliest partners in that realm announced new services that take their relationships to the next phase. VMware, a big player in desktop virtualization, will now offer its Horizon Air cloud-hosted desktops and… Read More
IBM and Cisco ink IoT deal integrating Watson AI into Cisco edge routers
IBM has been giving a push both to its Watson artificial intelligence and Internet of Things businesses as part of its bigger strategy to grow new streams of revenue to offset declines in older ones. Today came the latest development on that front: IBM has inked a deal with Cisco to bring Watson IoT technology into Cisco's architecture, specifically aimed at providing… Read More
A big (over $4.6 billion) week in enterprise M&A highlights the business market's critical needs
It's been a blistering start to summer if you're an investor interested in business software. With the U.S. Memorial Day holiday in the rearview mirror and temperatures beginning to climb across the country, the heat index for dealmaking among companies focused on selling technology to businesses seems to have gone up as well. But beneath the big deals for Demandware (bought… Read More
Want to know who to vote for? IBM's Watson Elections will make an emotional decision for you
Sad, mad, glad, confused about the presidential race so far? Well, if you are really lost on who to vote for, IBM may have a solution depending on your mood. Watson Elections, one of the surprisingly few political applications for IBM's supercomputer, debuted on stage this morning at TechCrunch's Disrupt Hackathon in Brooklyn, New York to help us decided the best candidate fitting… Read More
IBM launches quantum computing as a cloud service
Quantum computing is still very much in the early research stage, but IBM is hoping to accelerate the progress around it by making a quantum computer available to researchers as a cloud service. It is a bold and ambitious idea, although still very much a small step in trying to understand quantum computing processing. IBM is allowing interested parties to access a 5 qubit quantum… Read More
IBM raises its blockchain game with secure cloud services and Docker integration
Bitcoin may still be the most famous application of blockchain technology, but the distributed, encrypted database architecture is now being applied to a range of other services, from different kinds of (non Bitcoin) financial transactions to anything else that requires secure tracking. And today, IBM laid out its claim for some of that new business: the IT giant announced a new set of… Read More
Brought to you by the letters A and I: Sesame Workshop, IBM developing edtech for pre-schoolers
If you think Tickle Me Elmo is freaky already, wait until those talking monsters can beat you at Jeopardy!. Sesame Workshop, the educational nonprofit behind Sesame Street and its iconic characters, this week announced a partnership with IBM Watson to develop edtech for pre-school children. Sesame Workshop is no newcomer to tech. The organization previously set up a venture fund in… Read More


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Arts, Literature, Education, Educational TechnologyFollow
Listen in with me while I read classic books from the public domain. I am not reading anything for the podcast at the moment. Previous books I read for the podcast include Pride and Prejudice by Jane Austen, The Adventures of Huckleberry Finn by Mark Twain, and Anne of Green Gables by L. M. Montgomery.
November 5th, 2006
Episode 91 of 118 episodes
Anne's Impressions of Sunday School
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In a time where we're all threatened by a rhetoric of hate from the people in power; A Gay And A NonGay challenges many of our differences head on and promises that no matter who you are, or what you're into (Bruce Springsteen or Britney), love is love and gay and nongays can be friends.

An independent podcast from James Barr (@imjamesbarr) and Dan Hudson (@DanHudson). Contact us on Twitter, IG or Facebook @gaynongay


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Answers Home
» Health & Wellness
» Medications
I's gots a cold!
I'm 99% sure Yoru has a URI. I've had cats with URI with the same symptoms in the past. Wheezing, sniffling, coughing, runny eyes/nose. I know he'd need antibiotics but he's too skiddish and timid to get him into a carrier and take him to a vet. He won't even go near it and completly spazzes out if. He doesn't even like coming inside the house for more than a few minutes, much less a tiny carrier or the car. Do you think the vet might give me the antibiotics without seeing him or maybe give me a quick acting mild sedative to knock Yoru out till the vet visit is over?
Asked by
Yoru "YoYo" Puffs
on Jan 3rd 2011
in Medications
- Cast your vote for which answer you think is best!
URIs can be caused by viruses or by bacteria. Antibiotics will only work if he has a bacterial infection. Sometimes, a viral infection will lead to a bacterial one. In general, viral infections involve clear or brownish discharge, and bacterial ones will produce yellow or green. Only a vet can tell for sure what's wrong with Yoru. I doubt that any vet would prescribe medications without seeing your cat first. As for sedatives, I've given cats Valium and Xanax (vet prescribed), which make them woozy but don't knock them out. Again, it's unlikely that a vet would prescribe any med strong enough to knock out a cat without administering it him/herself. Call your vet for advice. My cats don't have problems with carriers or traveling; maybe someone else can give you advice about how to get Yoru into a carrier. Good luck.
Chibi answered on 1/3/11. Helpful? / 0
If Yoru is as feral as you say he is, then getting him to a vet will be difficult, but not impossible. Unfortunately, no vet I know would prescribe meds w/o seeing an animal first. I believe the only way to get Yoru to a vet would be to trap him using a humane trap. You will not be able to "catch" him and get him into a carrier. Most humane traps do fit in the back of a car. You can contact a local rescue or even your animal control officer thru your police department for them to loan you one. Bait it with the smelliest wet food you can find since his sense of smell is probably compromised. Do not try to remove him from the trap, you'll be attacked. You can try asking for sedative, but with a respiratory infection, I doubt if the vet would give you one. And make sure whatever vet you go to, they are accustomed to handling ferals.
Thanks for caring about this poor guy--and let us know how he's doing.
Izadore (Izzie) answered on 1/4/11. Helpful? / 0


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TRIPOLI: Libyan leader Muammar Gaddafi's regime has offered rebels controlling the port of Misrata an amnesty if they stop fighting.
A Justice Ministry statement called on ''all armed groups in Misrata to lay down their weapons in exchange for an amnesty''.
The offer was good until Tuesday, a government spokesman said.
On Friday NATO warships stopped Colonel Gaddafi's forces from laying anti-ship mines in Misrata's harbour, the alliance said, accusing the regime of trying to disrupt the flow of aid to the city, which has been besieged for two months.
The government has also said that ''armed gangs are trying to make mischief between Tunisia and Libya and to create a humanitarian crisis in the region to justify an intervention by NATO''.
On Thursday Tunisia accused Libya of violating its territorial integrity after rebels and Gaddafi forces clashed at a border crossing.
Agence France-Presse


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Christa R. Haggai, Attorney-at-Law
The wrist joint is made up of several bones, the radius and ulna, which are the bones in the forearm. The radius is named so because the radius (bone) acts like the radius (of a circle). The ulna acts as the center point to the circle because when the arm is rotated the ulna does not move. The radius (bone) acts like the radius (of a circle) because it rotates around the ulna. The carpus is the cluster of bones between the radius and ulna. Falls and high impact car accidents are some of the more common causes for wrist fractures in Los Angeles.
After experiencing a slip and fall accident or a high impact motor vehicle accident, the following symptoms may indicate that you have a wrist fracture:
Like any other fracture of a bone, wrist fractures are diagnosed by imaging, such as x-ray, CT scan or MRI. If the broken ends of the bone are not aligned properly, your doctor will need to manipulate the pieces back into their proper positions — a process called fracture reduction. Depending on the amount of pain and swelling you have, you may need a muscle relaxant, a sedative or even a general anesthetic before this procedure. After the bones are properly aligned they will need to be immobilized for proper healing. While many fractures may heal with realignment and immobilization, some require surgery, which could include placement or devices, rods, screws and pins. Like with leg fractures, surgery may include internal fixation or open reduction with internal fixation.
After either splinting or surgery, physical therapy will be needed in order to regain mobility, reduce stiffness and soreness in the arm, wrist and hand. Rehabilitation may take several months depending up the type of fracture and severity of the injury.


text: Coronation Street: Jennie McAlpine on Tyrone's plan to flee - "It's a bad idea. He will get caught" The actress reveals how Fiz will find life as a fugitive from the law: "She's under no illusion that they can get away with it" Comments Written By David Brown 12:01 AM, 05 February 2013 Next week's episodes of Coronation Street will see Tyrone (Alan Halsall) and Fiz hiding out in Wales, having fled Weatherfield with Hope and Ruby in tow. But with the police stepping up their search and Kirsty (Natalie Gumede) agreeing to do a public appeal, Fiz soon starts to fret about the futility of their plan. Here, actress Jennie McAlpine talks about Fiz's worries for Tyrone and what she'd do should the man she loves be sent to prison: How does Fiz feel about the way Kirsty has framed Tyrone? Fiz loves Tyrone. He's her soulmate. And she's not surprised that Kirsty has done this as she knows she's capable of anything. But even so, it is unbelievable and she's stunned at how good Kirsty is at lying. Kirsty has almost started to believe the lies herself, that's how good she is. And the fact that she has got other people on her side shows how good she is. Even people who've known Tyrone for so long have been taken in hook, line and sinker. What does Fiz think about Ty's plan to snatch Ruby and do a runner?Fiz thinks the plan is a bad idea from the start and she does say that. Tyrone knows it is wrong too and he's secretive about the details with her. It is a bad idea - he will get caught. He might have Ruby for a short time but there is no way this is a long-term plan. But he's determined and because Fiz loves him she agrees to go along with it. How does Fiz feel about leaving everyone behind and going on the run?Fiz is a very sensible person with her own child, so she feels terrible deceiving her family. So she does it quickly, like pulling a plaster off. She just goes home packs and says they have to do it that night. She tells Chesney she's just moving into Tina's flat with Tyrone. But she's under no illusion that they can get away with it – yet she trusts Tyrone and would go anywhere with him. As a mother herself, does Fiz feel it's right to take Ruby away from Kirsty? Yes, she has no qualms about that. She's a good mum herself, she adores Hope and - let's face it - she knows all about crap mums with Cilla as a parent! So she knows that Kirsty is not a good mum - Fiz realises that just because you give birth to a baby doesn't automatically mean you are going to be a good mum. Could you describe the sequence of events for us?Well, they head for the ferry terminal, but you will have to wait and see how they get on - it doesn't go to plan from the outset. Chesney learns that they've gone missing as it is all over the press. Fiz phones him and he shouts like a parent does at a kid that's gone missing, but he is partly pleased to have heard from her. I love their relationship - even though she's done what she's done, she rings him and knows that he's her little brother and that he'll help her if he can. When the police catch up with them, Tyrone says that Fiz had nothing to with the abduction. How does she feel about that?She feels sad because she did agree to go along with it, Tyrone was going to do it anyway, but she could have said no. So, really, she is responsible too. But that is Tyrone through and through - he is selfless. What's her biggest fear about what could happen to Tyrone?She's really worried and, when she is back in the street, she uses every opportunity to tell all of Kirsty's mates and supporters the truth - that Tyrone is the good guy. But running off didn't do them any favours and made him look guilty. It's just played into Kirsty's hands - she is really calm and measured and Fiz is anything but. Every time someone sees them, Fiz is the one screaming and shouting. If Tyrone is sent to prison, will Fiz stand by him and wait for him to come home? She spends a lot of time prison visiting doesn't she, poor Fiz! At least she doesn't have the wear the yellow bib this time. But yes, she will stand by him. She knows that he hasn't done anything wrong and it 's one of those great stories where the viewer also knows he is innocent. People love being in on a secret. Maybe there will be a free Tyrone campaign! Like this? Share it. Facebook Twitter Google Tumblr Pinterest Blogger Reddit VKontakt Print Email


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A report on Bloomberg report claims search giant Google is looking to ditch Intel as a chip supplier and make its own CPUs, using a custom processor developed with an ARM license. Assuming that even is true, don't hold your breath on that one happening any time soon.
The amount of work to be done would be massive, expensive, time consuming and probably have little to no return for years if not decades. In the process, Google might just weaken itself, although it has no real competitors to be much of a threat.
Google has massive data centers around the world, many of them larger than a football stadium. To cut out costs, the company stopped buying servers from the big three vendors – IBM, HP and Dell – and began making its own stripped down, bare bones server designs. It redesigned the standard rack motherboard, eliminating things like the video port, excessive USB ports or other ports and certain chips on the motherboard.
Because it outsources motherboards, it has to buy CPUs directly from Intel. Bloomberg said Google is Intel's fifth-largest customer, accounting for 4.3% of Intel's total sales.
Now, if Google went the ARM route, it would save a whole lot of money. ARM licensees pay a fee on every chip sold. Well, Google won't be selling the chips, it would be for its own consumption. Google might even negotiate for no fee, just a one-shot license purchase, since it's not selling a product. I don't know how the license fee would work out but it would have to be a lot better than the hundreds of dollars for a Xeon E3000 series processor.
Second, it would save Google probably in the millions of dollars in electricity fees, since ARM processors typically consume under 10 watts of electricity, while the Xeon is around 75 to 90, unless you get the low-power version, which is 35 watts but is also underpowered when compared to the mainstream Xeon. Multiply that over the millions of chips in data centers and the savings accumulates rapidly.
Now for the down sides. Google needs a 64-bit chip, and there isn't one available. Oh, some have tried, like Apple and Qualcomm, but those are not for sale nor are they fully 64-bit, and they don't have many of the extras that a server processor demands, like error correcting code (ECC) memory or reliability and availability features such as failover and load balancing. A server processor is a lot more than just an instruction processing device, it has to keep the server up and running no matter what.
ARM is working on a 64-bit design of its own but won't ship it until late 2014 or early 2015. Then comes the qualifying it with motherboards, and all of the internal gear used in data centers, and then the software, and then the services… you get the idea. It will be a very, very long and expensive process.
Then there's the issue of who will make it in the volumes Google demands. TSMC is overloaded as it is. Globalfoundries might not have the capacity. And Intel certainly won't make them.
There are already ARM-based servers in the works, like HP's Moonshot and Dell's Copper. These are in the early stages and are used primarily as front-end systems to back-end services. You don't need a powerful Xeon processor just to present an HTML page and take some input. However, we don't have any sales figures yet for these servers, so there's no way of knowing how well they are doing.
I don't doubt that Google has the engineering skill to pull something like this off, and it will undoubtedly do its own custom silicon for the motherboards, perhaps to improve parallelism and inter-server communication. And since Google owns the Motorola smartphone business, I can see it doing chip design there as well. But making its own CPU that's a worthy alternative to Intel? The investment is just too great.


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Whats on... With Jess
June 22, 2013 , 7:58 AM by Joanne Woods
We often hear that 6.10am is too early to find out what's happening around Victoria this weekend. So here is the audio to that segment with Jess Mcguire! Now you don't need to set your alarm.
You can follow this conversation by subscribing to the comment feed for this post.
The comments to this entry are closed.


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As a redshirt freshman at Arizona State, he showed his immense potential with touchdown receptions of 62 and 37 yards.
Then came a seemingly limitless run of bad luck. Smith caught 10 passes during his Arizona State career, which was derailed by an ankle injury that cost him all of 2007, a torn hamstring that limited him to one game in 2008 and a torn ACL that cost him all of 2009. Given a sixth year of eligibility, Smith caught just one pass as a senior.
Invited to participate in the inaugural Eastham Energy All-Star Game, which was played at Arizona State's Sun Devil Stadium, Smith finally put it all together. In an all-star game so small of scale that official statistics were not kept, Smith topped 100 receiving yards, had a 54-yard reception and a 15-yard touchdown catch, and was named the offensive MVP.
Smith (6-foot-1, 202 pounds) parlayed that strong performance into an outstanding pro day workout, with a 4.39 in the 40-yard dash, a 40-inch vertical jump and 19 reps on the bench.
He went undrafted in 2011 but signed with Carolina, only to never get a shot because of an eye infection. Midway through training camp, he signed with Seattle but didn't make the final roster.
Smith was out of football for a year, then returned to Arizona State for its pro day in 2012. While there, according to agent Steve Weinberg, Smith struck up a conversation with Seahawks coach Pete Carroll, who tried to bring Smith to USC to play cornerback. Carroll suggested Smith return to his high school roots and play cornerback. It was sage advice coming from Carroll. Arguably the top cornerback in the league is Seattle's Richard Sherman, who was a star receiver at Stanford before a knee injury and subsequent move to corner.
Smith spent 2012 making the position switch, working out at Fischer Sports in Phoenix with Will Sullivan, who is one of Darrelle Revis' trainers. He participated in the NFL's regional combine in Seattle, and did well enough there to get an invite to the super regional combine in Dallas.
Scouts were impressed but, with no college film of Smith playing cornerback, not impressed enough to offer a contract. The Packers brought in Smith as one of 27 tryout players at last weekend's rookie orientation camp. An imposing figure at cornerback with his size and an intriguing prospect with his athleticism, the Packers signed Smith to the 90-man roster this week.
Coincidentally, one of the players Smith will be battling for a roster spot is Loyce Means. It was Means who stole the show at that all-star game, scoring touchdowns on an interception, fumble return and blocked field goal. Means' story is even more remarkable, as his signing stemmed from bumping into Packers scout Alonzo Highsmith at an airport in Memphis.
As a cornerback, Smith is a developmental prospect, to be sure, but his size and speed make him an interesting candidate to play immediately on special teams, whether it's covering kicks or as a possible return threat.
Agree or disagree?: Discuss hot Packers topics in our, free forums. Leave Bill a question in the subscribers-only Packers Pro Club forum.
Bill Huber is publisher of Packer Report magazine and PackerReport.com and has written for Packer Report since 1997. E-mail him at firstname.lastname@example.com, or leave him a question in Packer Report's subscribers-only Packers Pro Club forum. Find Bill on Twitter at twitter.com/PackerReport.


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I really liked the template – Businessus | Portfolio + Blog | HTML Template But I want to use it for my wordpress blog. Could you please tell me how can I set it up for wordpress. Do you have the same template for wordpress? If yes please do let me know where I can find it. I would really like to buy it.
Thanks and Regards, Minal
I can fix it to work like wordpress theme. Contact me if you want.
I recommend that you contact the author direct from their portfolio page – the contact form on that page – they already have one template in wordpress so I would recommend using the same author.
Hi jonathan01,
Thankyou for your reply. I did write to the author from the portfolio page but haven't recieved any response. Incidently there are many comments on the template page asking for the same, but there is no response. Please help me find the WP template for the same theme.
Regards, Minal
If you have attempted to contact the author and had no reply after a few days I would then post a topic on these forums for a wordpress dev to convert for you – you should receive multiple offers and you will be able to see previous wordpress templates they have created to make sure they come up to your standard.
Hope that helps!


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This series of posters illustrates the universality of physical laws by connecting the concepts of rotation, speed, distance, pressure, time, mass, density and acceleration to both Olympic competitions and cosmic phenomena. Each poster includes both... (View More) a definition of the physics term and its units of measurement, and features an image of an Olympic/Paralympic athlete, a cosmic event, and a common everyday item as examples of the application of that physics concept. The posters, offered in English, Portuguese, and/or Spanish, are available to download and post in after-school facilities, community centers, libraries, science centers as well as classrooms. (View Less)
Using new and recycled materials, learners construct a rocket car that moves by the use of a balloon and a straw system. Following trial runs, learners engineer revisions to the car design to improve the distance that it travels. Templates,... (View More) pictures, worksheets and a lengthy list of suggested construction materials are provided. This activity was designed for use in a library program. (View Less)
Materials Cost: $1 - $5 per group of students
This online repository includes captioned images designed to tell a story about the science of light. Developed for the United Nation's International Year of Light 2015 (IYL2015), the images showcase the incredible variety of light-based science... (View More) being researched today across the electromagnetic spectrum, across scientific disciplines, and across technological platforms. (View Less)
In this activity, children use common craft materials and ultraviolet (UV)-sensitive beads to construct a person (or dog or imaginary creature). They use sunscreen, foil, paper, and more to test materials that might protect UV Kid from being exposed... (View More) to too much UV radiation. Includes background for facilitators. This activity is part of the "Explore!" series of activities designed to engage children in space and planetary science in libraries and informal learning environments. (View Less)
Materials Cost: $5 - $10 per group of students
This multimedia ibook introduces the physical concepts related to plasma globes commonly seen in science museums. The characters Camilla Corona and Colours O'IRIS discuss the concepts of plasma, electric fields, and atom electron loss and recapture... (View More) in simple terms without requiring extensive vocabulary. The Sun is used as an example of plasma, with similarities and differences between it and the plasma globes highlighted. For those who wish to go farther, a glossary is provided that expands upon the concepts in the comic. NASA resources on the Sun and related topics are also provided. This comic is part of the series Tales from Stanford Solar. (View Less)
Students are tasked with virtually designing a spacecraft to withstand the harsh environment of the Van Allen Radiation belts- the location of many communication, GPS and weather satellites. The details of the challenge, along with videos on... (View More) radiation, a materials list (including descriptions, densities, costs, and testing), and subsystems information are included. (View Less)
This Science On a Sphere video and docent show (script and playlist), explores factors that render Earth habitable and influence Earth's energy budget. The video gives an overview of NASA's Search for "Goldilocks Planets" - planets that are not too... (View More) hot or too cold for liquid water. (View Less)
This game helps students understand how multiple remote sensing wave bands can be combined to reveal new information about the weather. SciJinks is a joint NASA/NOAA educational website targeting middle school-aged children and their educators. It... (View More) explores weather and Earth science through articles, videos, images, and games. (View Less)
The bending and scattering of sunlight to create blue skies and red sunsets is explained and illustrated in this article. SciJinks is a joint NASA/NOAA educational website targeting middle school-aged children and their educators. It explores... (View More) weather and Earth science through articles, videos, images, and games. (View Less)
This comic addresses the question "What is color?" Using the Sun as an example, the comic discusses how visible light (white light) contains all the colors of the rainbow. It goes on to describe why our Sun is white, our sky is blue, and why sunsets... (View More) are red/orange. The discussion ends with a thought-question and provides further information on NASA missions and websites that address issues related to the Sun. The comic is illustrated mostly with NASA imagery and is part of the series Tales from Stanford Solar, featuring Camilla Corona and Colours O'Iris. The topic "What is Color?" was inspired by the 2014 Alan Alda Flame Challenge, an international competition asking scientists to communicate complex science in ways that would interest and enlighten an 11-year-old. (View Less)


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Lacrosse news straight to your inbox.
Monday July 23rd, 2012 4:35pm
Remember the first day of school every year? A new teacher, a new class, and a bunch of new faces you have never seen before all come together in an awkward meeting to become a class for the year. Individually, it is always harder to make new friends, most likely because it is hard to establish a common ground. Yet, if there is already a common ground shared — like lacrosse for a youth team from New York and Texas — the union could become a long lasting friendship.
The Long Island Express Tenacious Turtles are considered one of the best young lacrosse programs in the country and compete in tournaments on a regular basis. The young Turtles compete at the 6th grade level and are a difficult foe to play against. The Turtles have always been eager to play new competition, and when Joe DeSimone got a call one night asking if the Turtles would be interested in playing a wayward team from Texas in a scrimmage oneFriday night, he didn't hesitate to set up the game.
What happened after the game went beyond the field became close camaraderie, resulting in the teams meeting again this weekend at the Konica Minolta Big City Classic.
"It's a simple answer," says Joe, when asked how the friendship between the two teams blossomed. "their passion for the game."
This team from Texas illustrates the growth of a great game from what was once a sport played by feuding tribes into a sport of fellowship, honor and respect. In the same respect, the Tenacious Turtles show that there is a desire to assist in growing the sport as well. As Joe put it, "some people just have opinions and don't focus on the passion of the game," a trait he feels is necessary in growing the sport.
Over the course of the weekend, the two teams followed each other to and from their games during a tournament. It wasn't hard to spot the Mustangs though, as they drove around on the Dallas Cowboys team bus, courtesy of Jerry Jones the owner of the Cowboys. "We were fortunate enough to have one of Jerry's grandsons on the team and he loaned us the bus" says Roby Mize, a parent of one of the Mustang players.
"The game was going to be a great learning experience for us," Mize says of the scrimmage with the Turtles. Texas is seen as a state where football reigns supreme. To many sports enthusiasts, it is considered to be The Mecca of High School Football. Yet, Mize is quick to point out that football is a fall sport while lacrosse is played in the spring. "(It's a) great crossover sport for skill players," he says, "quarterbacks, running backs, they love lacrosse."
Roby and Joe exchanged notes and tips from a coaching perspective that weekend, and have stayed in contact over the past year. When the Turtles won a tournament recently that would allow them to play at halftime of the Duke vs. Syracuse game during Sunday's Big City Classic, Joe knew just who to invite to play against them.
The two teams will play against one another on Friday night, then meet again on Sunday. They will also be doing an on field T-shirt exchange, an impromptu tradition that started last year. "One of the kids asked for a (Turtles) T-shirt, and the kids exchanged them right off their backs."
The two teams hope to meet beforehand where they will tailgate and reconnect once again. They also look to keep the tradition going more in the years to come. A tradition befitting of the game of lacrosse.


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Can someone give me a rough Dyno estimate?
I just purchased an 85 silverado just wondering a rough estimate for horsepower as best a guess as someone can give me i will give you all the info i have also any suggestions for my truck would be appreciated. heres the specs 350, unknown pistons,heads 882's stock from what ive been told, 280 comp cam magnum im guessing from what the previous owner told me he didnt remember edelbrock performer rpm intake and edelbrock 600 cfm carb he says the gears are 392's i dont have the truck to look at right now because i am deployed to iraq and i got a good deal through one of my wifes friends any suggestion would be appreciated
... with no headers and stock compression you're probably not making more than 250-300. A 9.5 motor with decent exhaust would probalby make 350.
oops my bad i left out the long tube headers and flowmasters
oh, those are pretty good heads, I would say not less than 325, maybe even 350, again, depending on CR.
How much more power would i get if i switched to a 750 CFM carb or is that overkill?
moderator please move this post to engines
It won't be overkill but I doubt you will notice any gain with the carb change.
how much horsepower is too much for the stock bottom end?
when the rods start knocking.:thumbup:
500-600 HP, not more than 5500-6000. R's are more what kills it, the rods stretch and break.
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Leif – a new Australian-made skincare range from Sydney design studio Container
You gotta respect a company who know their niche and stick to it. Sydney based design studio Container have established a stellar reputation for one thing and one thing only – picture perfect packaging. This boutique studio designs and manufactures the most elegant containers for some of Australia's best boutique skincare, haircare and cosmetics labels – brands such as Kevin Murphy and O&M. It is such truly beautiful, innovative, yet understated work – and perfectly exemplifies why bespoke packaging is so important in the beauty industry.
Late last year Container launched a very special personal project. After years consulting to high profile beauty brands, the Container crew were inspired to create their very own product range. 'Leif' originally began as a design study – but eventually grew into a fully fledged skincare range, which finally launched in November last year. One of Leif's founders, Jonnie Vigar, says their goal with this range was to create a simple, natural product with a distinctly Australian flavour. Drawing upon botanical extracts including Lemon Myrtle, Wild Rosella, Tea Tree and Eucalyptus, the range is free from artificial colours, parabens, synthetic fragrances and all those other nasties. The packaging is 100% recyclable.
As is often the case with Australian tall poppies, it seems *Wallpaper magazine has outed Leif before us locals have even caught on! Doh – why does that always happen!? The Leif range was recently shortlisted for 'Best New Grooming Product' in the *Wallpaper's Design Awards 2011. Very impressive and well deserved. I have a feeling we'll be seeing a lot more of these guys…!
For now you can buy Leif products only in Sydney! Outrageous. I challenge Melbourne retailers to get cracking and snap up this product before someone else does! On your marks, get set – EMAIL!