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The ability to modify cellular properties such as adhesion is of interest in the design and performance of biotechnology-related processes. The current study was undertaken in order to evaluate the effectiveness of modulating cellular adhesion in HeLa cells from a genomics perspective. Using DNA microarrays, differences in gene expression between two phenotypically distinct, anchorage-dependent and anchorage-independent, HeLa cell lines were identified. With the aid of several statistical methods and an extensive literature search, two genes were selected as potential targets for further study: siat7e and lama4.
Subsequently, experiments were carried out to investigate the effects of siat7e and lama4 separately, on adhesion in HeLa cells by altering their expression in vivo. Decreasing the expression of siat7e, a type II membrane glycosylating sialyltransferase, in anchorage-independent HeLa cells using short interfering RNA (siRNA) resulted in greater aggregation (i.e. clumping) and morphological changes as compared to untreated anchorage-independent HeLa cells. Similar effects were seen in anchorage-independent HeLa cells when the expression of lama4 which encodes laminin α4, a member of the laminin family of glycoproteins, was enhanced as compared to untreated anchorage-independent HeLa cells. Using a shear flow chamber, an attachment assay was developed; illustrating either increased expression of siat7e or decreased expression of lama4 in anchorage-dependent HeLa cells reduced cellular adhesion.
Collectively, the results of this study are consistent with the roles siat7e and lama4 play in adhesion processes in vivo and indicate modifying the expression of either gene can influence adhesion in HeLa cells. The strategy of applying bioinformatics techniques to characterize and manipulate phenotypic behaviors is a powerful tool for altering the properties of various cell lines for desired biotechnology objectives.
In the design of biological processes for the production of therapeutic and/or diagnostic compounds from mammalian cells, it is vital to take into account the properties of the cell line being used (Chu et al., 2005). Details pertaining to a cell line in terms of its: growth pattern, nutritional requirements, glycosylation capabilities, and response to stimuli are important parameters to consider in order to properly design specific processes (Lum et al., 2004).
An important cellular property in biotechnology applications is adhesion–a cell's ability to attach to a surface in order to grow (Zhu et al., 2002). Depending on the exact application, a cell line that does not adhere to a surface, anchorage-independent, may be strongly preferred over a cell line that adheres to a surface, anchorage-dependent (Lum et al., 2004; Karu et al., 2001). For a different application, an anchorage-dependent cell line may be preferable over an anchorage-independent cell line. Being able to manipulate the cellular feature of adhesion would, therefore, benefit biotechnology applications and is the basis of the current study (Lum et al., 2004; Zhu et al., 2002).
A variety of studies have been conducted to evaluate the importance of cellular properties for the production of specific products. Researchers have also identified possible pathways to modify cellular properties by employing specific selection methods. In relation to adhesion, most studies have focused on either quantifying observations on a genetic level or exploring the effects of specific compounds (Springer et al., 1976). For instance, selenite, a hydrous calcium sulfate, has been shown to reduce the ability of HeLa cells to attach to fibronectin (Zhu et al., 1998). Researchers also showed that blocking the expression of pten, a tumor suppressor gene, in 293T cells using siRNA resulted in a loss of adhesion as well as a change in morphology (Mise-Omata et al, 2005; Crowther, 2002). Additional studies have highlighted a number of genes thought to be involved in mediating adhesion such as rhoA, rac1, and cdc42; although the exact mechanisms have not been fully elucidated (Mise-Omata et al, 2005; Hatzimanikatis and Lee, 1999; Tavazoie et al., 1999).
In the present study DNA microarrays were used to identify genomic differences between anchorage-independent and anchorage-dependent HeLa cells. Data generated from these arrays were normalized and then screened using a variety of imaging tools. Based on the results of several clustering algorithms, a review of previous studies, and their relative expression levels, two genes were selected for further investigation. The expression levels of these two genes, siat7e and lama4, were verified using reverse transcription-polymerase chain reaction (RT-PCR). Once verified, the expression level of either gene was manipulated and the adhesion features of these 'altered' cells were characterized first using a particle counter and then a shear flow chamber.
The two cell lines, anchorage-dependent and anchorage-independent HeLa cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA) (Catalog Nos. CCL-2 and CCL-2.2, respectively). Both cell lines were grown in BioFlo 3000 bioreactors (New Brunswick Scientific Co., Edison, NJ) with a working volume of 1.5L. Runs were conducted for up to 7 days after inoculation with constant sampling to characterize growth parameters. At least two different runs were carried out for each cell line. The media used was DMEM (Biosource International, Camarillo, CA) with 10% FBS (Biosource International, Camarillo, CA). The anchorage-dependent HeLa cells were grown on Cytodex 3 (Amersham Biosciences, Piscataway, NJ) microcarriers. Each reactor was seeded with 5.0 × 105 cells/mL (Bleckwenn et al., 2005; Masters, 2002).
Samples from the bioreactors were taken at regular intervals to test media composition (i.e. pH, Glucose, Lactate), cell viability, and cell density. Cells used to seed the bioreactors were synchronized using serum deprivation for 24 hours (Simon et al., 2002). Samples were collected in 2mL RNase/DNase-free micro tubes (Marsh Biomediacal Products, Rochester, NY), combined with TRIzol reagent (Invitrogen, Carlsbad, CA) and stored at −80°C.
Total RNA was isolated from samples using an Invitrogen kit (Micro-to-Midi Total RNA Purification System). Purified RNA was quantified using a spectrophotometer, GeneQuant Pro (Biochrom Ltd, Cambridge, UK). The absorbance values at 260nm and 280nm were measured and the quality of RNA was determined by the ratio of these numbers (A260/280). Only samples with an A260/280 of at least 1.8 were used for microarray analysis (Butte, 2002). Each sample, consisting of approximately 10μg of total RNA, was reverse transcribed, labeled, and prepared for microarray hybridization using a protocol from The Institute for Genomic Research (TIGR) in Rockville, MD: "Aminoallyl labeling of RNA for Microarrays" (SOP # M004, Rev. 2) with an effective date of 3/4/2002.
High quality microscope slides (Corning, Corning, NY) were printed with a set of 32,448 spots corresponding to approximately 14,000 unique ESTs (genes) by TIGR (Rockville, MD) using an array fabricator (Intelligent Automation, Rockville, MD). These complementary DNA (cDNA) microarrays were prepared and hybridized using a protocol "Microarray labeled probe hybridization" (SOP # M005, Rev. 3) available from TIGR with an effective date of 9/11/2002. After washing and drying, each slide was ready for scanning and image analysis using a GenePix 4000B (Molecular Devices Corporation, Sunnyvale, CA). To ensure proper labeling efficiency the dyes for 2 arrays were swapped (Lee and Quackenbush, 2000).
Acuity software (Molecular Devices Corporation, Sunnyvale, CA) was used to normalize, filter, and cluster the data. Due to similarities between the two cell lines, total intensity normalization was deemed most appropriate and therefore applied to the data (Xiang et al., 2003; Burke, 2000). Next, the data were filtered as part of a quality control step to remove spots with poor signal quality, defined as having low total signal, non-circular shapes, or a signal below local background (Saeed et al., 2003; Conway and Schoolnik, 2003; Hegde, et al., 2000). In addition, genes with highly variable (i.e. inconsistent) expression ratios were removed.
In order to explore the data further, the following clustering algorithms were applied: self-organizing maps (SOMs), principle component analysis (PCA), and hierarchical clustering (Saeed et al., 2003, Quackenbush, 2001). PCA was used to estimate the number of distinct clusters likely to form in the data (Quackenbush, 2001). This algorithm revealed 8 or 13 distinct clusters would best segregate the data, based on the inherent structure of the data. SOMs and hierarchical clustering were then applied in order to construct groups of genes with potentially similar functions or roles based on expression patterns. The parameters typically specified for either of these algorithms such as scaling, the similarity metric, or the linkage method were continually modified to best separate the data into meaningful groups. Once a final value for each of these parameters was selected, the resulting groups were probed to identify subsets of genes with relevance to adhesion. This was done by searching for genes closely associated with other genes known to be involved in adhesion such as integrins and cadherins. Of these genes, the most differentially expressed were then identified and evaluated based on their known or proposed functionalities as indicated in the literature. Using this approach, two genes were selected for further investigation; siat7e and lama4.
To enhance statistical significance associated with gene-specific expression levels, four oligonucleotide microarrays were also hybridized. The arrays used were Human Genome U133 Plus 2.0 (Affymetrix, Santa Clara, CA) prepared with over 54,000 spots corresponding to roughly 38,000 genes. Two of these arrays were hybridized with RNA extracted from anchorage-dependent HeLa cells while the remaining two arrays were hybridized with RNA extracted from anchorage-independent HeLa cells. The GeneChip arrays were assayed using the Affymetrix protocol "Eukaryotic sample and array processing" (701027, Rev. 5).
Although, the differences and similarities between cDNA and oligonucleotide microarrays are numerous, for the purposes of this text, only two differences are of particular interest; sample hybridization and resulting data. The cDNA microarrays utilized in the present study were dual-channel whereas the oligonucleotide microarrays were single-channel. When an array is hybridized with two samples, each labeled with a different dye, the array is referred to as being a dual-channel array. Conversely, when only one sample is hybridized onto an array, the array is referred to as being a single-channel array. As a result of this difference, data generated from the dual-channel arrays were in terms of relative intensity values (i.e. ratio form) whereas data from the single-channel arrays were in terms of absolute intensity values (Zhu et al., 2002; Nakanishi et al., 2001).
In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to verify the microarray results. The protocols used were provided by Applied Biosystems (4333458, Rev. B & 4335626, Rev. C, both dated 05/2004). Each gene was assayed four times through two separate experiments to establish greater statistical significance.
Sequencing information for both siat7e and lama4 were obtained using two public online databases; Harvester (http://harvester.embl.de/, European Molecular Biology Laboratory, Heidelberg, Germany) and GenBank (http://www.ncbi.nlm.nih.gov/, National Institutes of Health, Bethesda, MD). For each gene, full-length DNA constructs (GeneCopoeia, Germantown, MD) and siRNA were purchased (Ambion, Austin, TX). The siRNA sequences: siRNA ID # 11236 (lama4) and siRNA ID # 112233 (siat7e) were used.
For the anchorage-dependent HeLa cells, the transfecting agent used was Lipofectamine 2000 (Invitrogen, Carlsbad, CA), the transfecting agent used for the anchorage-independent HeLa cells was Lipofectamine LTX (Invitrogen, Carlsbad, CA). First, a frozen vial of the cell line to be transfected was thawed and then used to seed a 25cm2 cell culture flask (Corning, Corning, NY). Within 24 hours, spent media was replaced with fresh media, DMEM (Biosource International, Camarillo, CA) with 10% FBS (Biosource International, Camarillo, CA). Upon reaching confluency, the cells were expanded; first into a 75cm2 cell culture flask and then into a 162cm2 cell culture flask. When the 162cm2 flask was approximately 90% confluent, cells were passaged into two 6-well plates (Corning, Corning, NY) with fresh media. Once the cells were at least 80% confluent, transfection was performed. Samples for subsequent analysis were taken after 48 hours for verification of successful transfection using RT-PCR.
Physiological changes, post-transfection, were observed with a DM IRB microscope and attached camera (Leica Camera, Allendale, NJ). Quantification of observable changes was performed using a Counter Z (Beckman Coulter, Fullerton, CA). In order to assay the level of adhesion for a specific manipulation, the formation and distribution of cellular aggregates in both control and treated cells were analyzed. Two separate samples were prepared by diluting each in an electrolyte solution at a ratio of 1:2 and run through the Beckman Coulter counter for varying sizes. In addition, three different 6-well plates were seeded and analyzed.
To account for variability introduced during the transfection process the following controls were established: (i) untreated cells, (ii) cells treated only with the transfecting agent, Lipofectamine 2000 (Invitrogen, Carlsbad, CA), (iii) cells transfected with nonsense sequence siRNA, Silencer Negative siRNA Control # 1 (Ambion, Austin, TX), and (iv) cells transfected with Silencer GAPDH siRNA (Ambion, Austin, TX). Transfection with nonsense siRNA served as the negative control whereas transfection with siRNA specific for GAPDH served as the positive control (used to optimize conditions for transfection). Subsequently, the mRNA levels were monitored using RT-PCR. The data labeled 'control' in Figures 3 & 4 were calculated by averaging the values from all four controls previously mentioned, due to very similar distribution patterns.
Based on previous work with the anchorage-independent HeLa cells the following ranges (in μm) were selected for investigation: <5, 5-15, 16-30, 31-60, 61-80, 81-100, and >100 (Masters, 2002). These ranges were not meant to be definitive but rather suggestive of different possible groupings for HeLa cells with an average diameter of 12-15μm. In addition, when taken together these ranges are free of gaps and therefore present a complete picture of the aggregation taking place. The first range (<5) represented debris and cellular fragments and therefore was considered background. The next two ranges (5-15 and 16-30) represented single cells, dividing cells, and pairs of cells. The 31-60 range represented small-to-medium sized aggregates of 3-6 cells. Finally, the last three ranges represented medium-to-large aggregates comprised of 6 or more cells.
The expression levels of either siat7e or lama4 were altered by transfecting plasmid DNA conferring resistance to the compound geneticin (neomycin gene). This resistance allowed for selection of clones expressing the plasmid. To enhance the expression of either gene, a vector containing the full-length gene sequence was used. On the other hand, to reduce the expression of either gene, a vector containing the appropriate siRNA sequence was used. The stability of the cell lines with DNA plasmid inserts was tested using western blot analysis (lama4) and RT-PCR (siat7e). After every fourth passage, a western blot or RT-PCR was performed to verify continued repression or enhancement of the specific gene being targeted. Prior to transfection, a kill curve was constructed to optimize the concentration of geneticin incubated with the cells resulting in 100% cell death (750μg/mL).
In order to distinguish these clones from one another and from the original cell lines, the following designations were used: anchorage-dependent [lama4 −], anchorage-dependent [siat7e +], anchorage-independent [lama4 +], and anchorage-independent [siat7e −]. The first two cell lines were prepared using anchorage-dependent HeLa cells. In the case of the anchorage-dependent [lama4 −] cells, the expression of lama4 was reduced by inserting a vector embedded with siRNA specific for lama4. Anchorage-dependent [siat7e +] cells were prepared by inserting an expression vector that contained both the full-length sequence of the gene siat7e and a CMV promoter (GeneCopoeia, Germantown, MD). Similarly, anchorage-independent [lama4 +] cells were constructed from anchorage-independent HeLa cells by introducing an expression vector that contained the full-length lama4 gene. The anchorage-independent [siat7e −] cells were also prepared from anchorage-independent HeLa cells using a vector that contained siRNA specific for siat7e.
To quantify adhesion properties in these modified cell lines, a shear flow chamber was utilized. The apparatus was placed onto the stage of an inverted microscope with a camera capable of generating real-time photos and video (Abulencia et al., 2003). By growing cells on a 35mm × 10mm cell culture dish (Corning, Corning, NY) cells could be placed directly inside the shear chamber. As such, varying levels of shear stress were introduced using Hanks' based, enzyme-free cell dissociation buffer (Invitrogen, Carlsbad, CA) past the adherent cells on the culture dish. Preliminary experiments established the range of shear stresses applicable to the cell lines used in the present study (data not included). Subsequent experiments quantified each cell line's adhesion property based on the number of cells that would detach for a given period of time and a given shear stress. The number of cells detaching during the course of a single experiment were converted into percentages using the initial number of cells present in the viewable region. The following shear stress values were used 19.2, 24.0, and 28.8 dyn/cm2; each for one minute in succession for a single cell culture dish. At least three separate dishes were assayed for a given cell line to establish statistical parameters.
Growth characterization of anchorage-dependent and anchorage-independent HeLa cells was performed in bioreactors under the same set of conditions. Samples for microarray analysis were taken at the same time point, when both cell lines were in exponential growth. Both cell lines were experiencing similar environments as determined by measuring metabolic concentrations, cell density, pH, and viability. For example, both cell lines had a glucose concentration of approximately 3 g/L and a lactate concentration of approximately 1 g/L. In addition, both cells lines had a viability of at least 94%.
Total RNA samples from anchorage-dependent and anchorage-independent HeLa cell cultures were purified, reverse transcribed, and analyzed using cDNA microarrays. Initial data analysis used GenePix Pro to visualize the hybridization on each of the 4 hybridized microarray slides. Over 8,400 of the approximately 14,000 genes spotted on the arrays were of sufficient quality to be included in the normalization procedure using the total intensity model. Following normalization, the data were filtered to identify genes with the highest and lowest expression ratios per array, defined as follows:
In the present study, anchorage-independent HeLa cells were selected as the test sample whereas anchorage-dependent HeLa cells were selected as the control sample. Since there were 4 hybridized cDNA microarrays, there were 4 expression ratios per gene. An expression ratio of unity indicates equal hybridization between the two samples being assayed. Therefore, expression ratios above 1.0 are often referred to as being upregulated whereas expression ratios below 1.0 are often referred to as being downregulated.
An initial round of screening yielded approximately 3,500 genes based on consistency between slides in terms of how close the expression ratio from any one slide was to the median expression ratio calculated from all four slides. Next, the data were filtered to identify genes with expression ratios from the slides in the same direction (i.e. either upregulated or downregulated across all four slides). As a result, the number of genes selected was reduced to 667. These genes were then organized using the following clustering algorithms: self-organizing maps (SOMs), principle component analysis (PCA), and hierarchical clustering (Saeed et al., 2003, Quackenbush, 2001). PCA indicated either groups of 8 or 13 clusters were likely to form. Using this information, SOMs and hierarchical clustering were then applied to the data. By changing the parameters typically specified for either of these algorithms, new configurations were constructed and evaluated. The resulting groups were then probed to identify subsets of genes with relevance to adhesion by searching for genes closely associated with other genes known to be involved in adhesion such as integrins and cadherins. Of these genes, the most differentially expressed were then identified, as shown in Table 1. Next, these genes were evaluated based on the known or proposed functionalities of these genes, as indicated in the literature. In the end, two genes were selected for further analysis based on their proposed functionalities, expression levels, and clustering outcomes; siat7e and lama4.
The expression of siat7e was higher in anchorage-independent HeLa cells than in anchorage-dependent HeLa cells, while the expression of lama4 was lower in anchorage-independent HeLa cells than in anchorage-dependent HeLa cells. Both Figure 1 and Table 2 illustrate these expression ratios. It should be noted that although, it is common to find highly differentially expressed genes in typical microarray-related studies, in many cases it is simply a matter of what is or is not excluded. In the present study large differences in expression levels were not seen because: the two cell lines studied were very similar, care was taken to ensure the quality and quantity of RNA used for microarray analysis, and borderline spots were not considered in the subsequent analysis.
As can be seen in Figure 1, the expression ratios of siat7e were consistently above 1 while the expression ratios for lama4 were consistently below 1. However, the inherent variability between these slides in terms of expression ratios for the two selected genes established the need for verification. To validate the results from the cDNA microarrays, two pairs of oligonuceotide microarrays as well as a series of RT-PCR experiments were conducted. The results of these experiments are shown in Table 2 which demonstrates that the median expression levels across all three platforms are consistent with one another for several genes, including two control genes, gapd and pgk1.
Given the transcriptional differences of siat7e and lama4 between anchorage-independent and anchorage-dependent HeLa cells, as well as the results of various clustering algorithms, the expression of these genes could play a role in the adhesion and/or growth characteristics of HeLa cells. If so, then enhancing or inhibiting their expression should affect cell physiology. To evaluate the effect of siat7e on cell behavior, the expression of siat7e in anchorage-dependent HeLa cells was enhanced via transient transfection of the full-length gene. As shown in Figure 2 by comparing the image of anchorage-dependent HeLa cells (A) with the image of anchorage-dependent HeLa cells with enhanced expression of siat7e (B), enhancing the expression of siat7e caused a decrease in the percentage of viable cells attached to the surface as well as a change in cell shape for some cells from elongated, cylindrical cells to compact, spherical cells. In addition, it was observed that for a given period of time under the same growth conditions (i.e. media, temperature, etc.) the anchorage-independent HeLa cells grew to higher cell densities. (data not shown).
Reducing the transcription of lama4 in anchorage-dependent HeLa cells by transiently transfecting the cells with gene-specific siRNA also resulted in a change illustrated in Figure 2 when comparing the image of anchorage-dependent HeLa cells (A) with the image of anchorage-dependent HeLa cells with reduced expression of lama4 (C). In this case, the change was a 10% reduction in the percentage of attached cells. Simultaneous modifications to the expression levels of siat7e and lama4 in anchorage-dependent HeLa cells did not augment these observed changes (data not shown).
Reducing the expression level of siat7e or enhancing the expression of lama4 on anchorage-independent HeLa cells via transient transfections were also studied. The effect of reducing the expression of siat7e using gene-specific siRNA can be seen in Figures 2 and and3.3. Comparing the image of anchorage-independent HeLa cells (D) to the image of anchorage-independent HeLa cells with reduced expression of siat7e (E) in Figure 2 reveals the effects of reducing the expression of siat7e in anchorage-independent HeLa cells. This reduction results in an increase in the percentage of cells adhering to the surface, an increase in the percentage of cells adhering to one another (i.e. clumping), and a decrease in the percentage of cells detached from the surface. In addition, a greater number of cells appeared to elongate and take on shapes that were non-spherical, especially when attached to the surface. In order to investigate further the behavior of anchorage independent HeLa cells following downregulation of siat7e, an assay was devised to measure size distribution of cells and cells associated in aggregates in culture.
As seen for the control populations in Figure 3, one-half of the anchorage-independent HeLa cells existed individually (5 – 15 μm) because an individual HeLa cell is roughly 12 μm in diameter (for the narrowest portion of the cell). However, for siat7e-specific siRNA treated cells, the percentage of individually existing cells was reduced to one-third. In addition, there was an increase in the percentage of cells existing in larger clusters following inhibition of siat7e. Differences between siat7e-specific siRNA treated cells and control cells were statistically significant for three ranges, 5-15 μm, 61-80 μm, and 81-100 μm using a one-tailed Student's t-test with at least a 10% significance level. Indeed, the anchorage independent [siat7e-] cells exhibited greater clustering at all three size groupings above 60 μm.
The results of enhancing the expression of lama4 in anchorage-independent HeLa can be seen in Figures 2 and and4.4. By comparing the image of anchorage-independent HeLa cells (D) and the image of anchorage-independent HeLa cells with enhanced expression of lama4 (F) in Figure 2, it can be seen that upregulating lama4 in anchorage-independent HeLa cells resulted in an increase in the percentage of cells adhering to the surface or clumping together, and a morphological change from spherical to more elongated shapes for those cells adhering to the surface. Shown in Figure 4 are the results of the cell size distribution assay comparing anchorage-independent HeLa and anchorage-independent Hela with enhanced expression of lama4. Enhancing the expression of lama4 in anchorage-independent HeLa cells resulted in a reduction in the percentage of cells existing individually (55% to 41%) and an increase in the percentage of cells existing in clusters; a phenomenon similar to that observed when reducing the expression of siat7e in anchorage-independent HeLa cells. Differences between anchorage-independent HeLa cells with inserts of lama4 and control cells were statistically significant for two ranges; 5-15 μm and >60 μm, using a one-tailed Student's t-test with at least a 10% significance level. The clusters greater than 60 um were not separated into individual categories since there were fewer large clusters in the anchorage independent HeLa with enhanced expression of lama4 compared with anchorage independent HeLa with reduced expression of siat7e.
Next, the expression levels of either siat7e or lama4 were altered by transfecting plasmid DNA. Each plasmid used contained the neomycin gene, conferring resistance to the compound geneticin. This resistance allowed for selection of clones expressing the plasmid. To enhance the expression of either gene, a vector containing the full-length gene sequence was used. On the other hand, to reduce the expression of either gene, a vector containing the appropriate siRNA sequence was used. Each cell line expressed either the siat7e or the lama4 gene or gene-specific siRNA, constitutively. To verify the adhesion properties of these plasmid-expressing cell lines, a shear flow chamber previously used for characterizing cellular properties was utilized. A range of shear stresses over which a clear distinction could be made between the control cell lines, anchorage-dependent HeLa and anchorage-independent HeLa was established.
Figure 5 illustrates the percentages of cells dissociating due to different shear stresses. In particular, Figure 5A shows these percentages for anchorage-dependent HeLa cells that were either unmodified, contained enhanced siat7e expression, or contained reduced lama4 expression. The rate of dissociation and the percentage of cells dissociating with increasing shear stress were greater for both of these modified cell lines than for the unmodified anchorage-dependent cells, possibly indicating weakened adhesion. Similarly, Figure 5B illustrates the percentages of anchorage-independent HeLa cells dissociating. Three cell lines are shown in Figure 5B, anchorage independent HeLa cells that were either unmodified, contained reduced siat7e expression, or contained enhanced lama4 expression. The percentage of cells dissociated was lower for the cells with reduced siat7e expression compared to the unmodified anchorage-independent HeLa cells and same cells with increasing lama4 expression. In general, all of the anchorage-independent cell lines were less adherent than the anchorage-dependent cell lines.
By averaging multiple runs for each cell line and converting the number of dissociated cells into percentages it was possible to illustrate differences in adhesion between the different cell lines. When the expression of lama4 was reduced in anchorage-dependent HeLa cells, the percentage of cells dissociating increased from 12% ± 5% to 23% ± 5%. When the expression of siat7e was reduced in anchorage-independent HeLa cells, the percentage of cells dissociating decreased from an average of 72% ± 6% to 53% ± 5%. The opposite pattern of modified adhesion ability was obtained for the reciprocal cases. When the expression of lama4 was enhanced in anchorage-independent HeLa cells, the percentage of cells dissociating decreased from 72% ± 6% to 61% ± 4%. When the expression of siat7e was enhanced in anchorage-dependent HeLa cells, the percentage of cells dissociating increased from 12% ± 5% to 32% ± 5%.
In the present study, DNA microarrays along with other genomics tools were used to identify the genes siat7e and lama4 as influential to the adhesion of HeLa cells. The selection of these two genes from a broader list of differentially expressed genes was based on: proposed or known functionalities detailed in previous studies, expression levels, and clustering outcomes. Anchorage-independent HeLa cells exhibited higher expression of siat7e and lower expression of lama4 relative to anchorage-dependent HeLa cells. The expression levels of these two genes were then modified in vivo and the resulting changes were quantified using: a microscope, a particle counter, and an attachment assay. By either enhancing the expression of siat7e or reducing the expression of lama4 in anchorage-dependent HeLa cells, a reduction in cellular adhesion was observed. Conversely, by either reducing the expression of siat7e or enhancing the expression of lama4 in anchorage-independent HeLa cells, enhanced aggregation and cellular adhesion were observed.
These findings on the impact of siat7e and lama4 on cellular adhesion are consistent with prior studies on the functions of these genes. The gene lama4 encodes laminin α4, a secreted protein consisting of 1,816 amino acids and detected in various tissues including the skin, placenta, heart, lung, skeletal muscle, and pancreas (Richards et al., 1996). Laminins, a family of secreted glycoproteins, are a major component of the basal lamina; a layer found beneath epithelial cells and surrounding connective tissue with functional relevance to a variety of cellular properties such as morphology, polarity, adhesion, and migration (Haralson and Hassell, 1995; Gonzalez et al., 2002; Kalmykova et al., 2002; Colognato and Yurchenco, 2000). Each laminin molecule consists of three distinct subunits (α-, β-, and γ-chains) making it a heterotrimer (Colognato and Yurchenco, 2000). Laminin α4 acts as a ligand for several integrin heterodimers including αvβ3 and α3β1, both of which are involved in angiogenesis and endothelial cell interaction (Gonzalez et al., 2002; Reddy and Kalraiya, 2006).
Previous studies have explored the functionality of different laminins in a variety of settings with specific interactions in mind. In the context of wound healing, migration, and adhesion, laminin 2 and laminin 4 were deemed good substrates for human keratinocytes; cells found in the skin, hair, and nails that synthesize keratin (Kalmykova et al., 2002). In addition, both laminins support epidermal growth factor-mediated migration and in relation to other laminins, have a higher number of cell surface receptors (Kalmykova et al., 2002). Similarly, coating various surfaces with peptide motifs shared by laminins such as Ile-Lys-Val-Ala-Val (IKVAV) increased cellular adhesion to those surfaces (Lin et al., 2006). Laminin also appears to support the growth of mouse embryonic stem cells by contributing to the adsorption of serum-derived proteins and enhancing the stability of resulting cellular assemblies (Brynda et al., 2005). Thus, the increase in expression of laminin in anchorage-dependent HeLa cells is entirely consistent with the role this protein plays in cellular adhesion processes.
In contrast, the expression level of siat7e in anchorage-independent HeLa cells was more than twice the expression level in anchorage-dependent HeLa cells. Siat7e encodes a type II membrane protein, α-N-acetylgalactosaminide α2,6 sialyltransferase V (ST6GalNAc V), found in the Golgi apparatus. This protein is responsible for the synthesis of GD1α (disialoganglioside) from GM1b (monosialoganglioside) which involves the addition of a N-acetylneuraminic acid (NeuAc) onto the N-acetylgalactosamine (GalNAc) residue of the acceptor molecule, NeuAcα2,3Galβ1,3GalNAcβ1,4Galβ1,4Glcβ1-Ceramide where Gal represents Galactose and Glc represents Glucose (Ikehara et al., 1999; Okajima et al., 1999; Taki et al., 1997; Yu et al., 2004; Yanagisawa et al., 2004). As a result, the protein ST6GalNAc V is also referred to as GD1α synthase (Sawada et al., 1999; Taki et al., 1997; Ito et al., 2003). A ganglioside consists of a glycosphingolipid (glycolipids containing sphingosine) with one or more sialic acids; predominantly NeuAc (Wiegandt, 1985). Gangliosides are known to be critical to numerous cellular functions including cell-cell interactions, tissue differentiation, and cell adhesion (Liu et al., 2004; Okajima et al., 1999; Tsuchida et al., 2003; Hakomori, 1981). The presence of sialic acids and the nature of their linkages on a ganglioside can be especially relevant to biological functions (Okajima et al., 1999).
More recently it was uncovered that ST6GalNAc V along with ST6GalNAc VI have another sialyltransferase function; the addition of a second NeuAc onto the N-acetylglucosamine (GlcNAc) residue of monosialyl lactotetraosylceramide (Lc4) with an α2,6 linkage (Tsuchida et al., 2003). Monosialyl Lc4 (NeuAcα2,3Galβ1,3GlcNAcβ1,3Galβ1,4Glcβ1-Ceramide) serves as a precursor for the synthesis of either monosialyl or disialyl Lewis a (Lea). Both monosialyl Lea and disialyl Lea are thought to play important roles in a variety of cellular processes including oncogenesis (Magnani, 2004; Itai et al., 1991; Julien et al., 2001).
Consistent with these studies, the gene expression of ST6GalNAc VI was found to be downregulated in a number of colon carcinoma cell lines (Miyazaki et al., 2004; Suzuki et al., 2006). Furthermore, transfection of several cell lines with ST6GalNAc VI revealed an increase in disialyl Lea expression along with a reduction in monosialyl Lea expression (Tsuchida et al., 2003; Miyazaki et al., 2004; Suetake et al., 2003). Furthermore, the transfection altered the adhesive properties of the cells by lowering their adhesion to E-selectin while increasing their binding to sialic acid binding Ig-like lectin 7 (Siglec-7) (Miyazaki et al., 2004). Therefore, altering the sialic acid content on a glycolipid by modifying the expression of this family of sialylatransferases can confer significant changes in cellular adhesion. Although, both ST6GalNAc V and ST6GalNAc VI share a number of features including sequence motifs, overall structure, and function, only ST6GalNAc V was found to be upregulated in the current study for anchorage-independent HeLa cell lines (Patel and Balaji, 2006). Nonetheless, a change in expression of siat7e in the attachment-independent cell lines is consistent with the role it plays in cellular adhesion processes. It would be interesting to determine the expression levels of binding partners to these glycolipids on the surface of HeLa cell lines.
Thus, changes of the expression levels of lama4 and siat7e are consistent with the evolution of different adhesion properties for the two HeLa cell lines evaluated in the current study. Furthermore, it has been suggested that sialylatransferase genes can be subject to methylation silencing (Sakakura et al., 2005; Miyazaki et al., 2004; Reddy and Kalraiya, 2006; Brynda et al., 2005). As such, it is conceivable a similar epigenetic transformation may be occurring in order for HeLa cells to adapt to suspension conditions. Finally, there is the potential for synergistic effects between laminins and sialylatransferases, although the current study did not reveal any significant alteration in the adhesion properties of the HeLa cells when the two genes were modified in concert (Wang et al., 2006; Suzuki et al., 2002).
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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According to our records, Peter Schumann is possibly single.
Help us build our profile of Peter Schumann!
to add information, pictures and relationships, join in discussions and get credit for your contributions.
Peter Schumann (born 1934) is the founder and director of the Bread & Puppet Theater. Born in Silesia, he was a sculptor and dancer in Germany before moving to the United States in 1961. In 1963 he founded Bread & Puppet in New York City, and in 1970 moved to the Northeast Kingdom of Vermont, eventually settling in Glover, Vermont where the company still performs. Schumann's best known work is the Domestic Resurrection Circus, performed annually by the Bread and Puppet Theater until 1998.
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Need a new look? Come to Juma African Hair Braiding Styling Salon & Fashion. Our stylists will take time to hear what you want and give you a style you'll love. From braiding to weaving, we've got you covered; our salon will relax you while we do all the pampering. You'll leave feeling fabulous, looking stylish and loving your updated appearance! Call our salon in Bronx, New York, to make an appointment with one of our professional stylists.
"We Do Excellent Work on Your Hair"
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Call today to make an appointment with one of our hairstylists. We can accommodate even the busiest schedules, so pick up the phone today.
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(718) 231-9876 | (347) 458-9162
Business Hours
Mon - Sun: 09:30 AM - 10:00 PM
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Juma African Hair Braiding Styling Salon & Fashion
Additional Phone: (347) 458-9162
Email: email@example.com
4064 White Plains RoadPhone: (718) 231-9876
Bronx, NY 10466
Bronx, NY 10466
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Spicy prawn soup
Nutrition and extra info
- Easily halved
Nutrition: per serving
- kcal327
- fat17g
- saturates10g
- carbs32g
- sugars4g
- fibre4g
- protein16g
- salt0.97g
- 1 tbsp sunflower oil
Sunflower oil
A variety of oils can be used for baking. Sunflower is the one we use most often at Good Food as…
- 300g bag crunchy stir-fry vegetables
- 140g shiitake mushroom, sliced
- 2 tbsp Thai green curry paste
- 400g can reduced-fat coconut milk
- 200ml vegetable or fish stock
- 300g medium straight-to-wok noodle
- 200g bag large, raw prawn
There are thousands of different species of prawn, but tiger, king and North Atlantic are the…
Heat a wok, add the oil, then stir-fry the veg and mushrooms for 2-3 mins. Take out and set aside, then tip the curry paste into the pan and fry for 1 min.
Pour in the coconut milk and stock. Bring to the boil, drop in the noodles and prawns, then reduce the heat and simmer for 4 mins until the prawns are cooked through. Stir in the veg, then serve.
If you can't find a can of coconut milk, use a block of creamed coconut or a carton of coconut cream, diluted as instructed.
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Comments, questions and tips
Comments (62)
Yummy. Try frying a little garlic and fresh chilli in oil for no more than 30 seconds (without browning the garlic) before adding the veges. Then, as others have suggested, a little fish sauce and fresh lime juice after it's taken off the heat before serving. Also, a small handful of fresh bean sprouts, sliced spring onion and/or fresh chopped chilli as a garnish works well too.
Tried this last night - simply amazing! The taste was great, very easy to make. Even my husband is raving about it. Only thing was the liquid soaked up quickly - so I added more fish stock. Also added chilli and ginger for extra 'kick'. Will definately be making it again - and again. Can't wait to share it with my sister.
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Publisher's Description+
What's new in this version:
* Updated graphics to better handle all the different variety of handsets, tablets, and themes.* Tablet menu updated to use the Action Bar intelligently.* Updated for Android 3.2. If your tablet is still running Android 3.0 or 3.1, you are highly encouraged to update to 3.2.* Better memory management by way of recycling images that are not in view quicker.* Several bugs fixed.


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Welcome to the Appalachian State University Women's Golf scholarship and program info page. Here you will get specifics on the college and information on their Women's Golf program like who to contact about recruitment, names of past alumni, what scholarship opportunities can be had and ways to start the recruiting process.
Appalachian State University Women's golf Program Coaching & Recruiting Personnel
If you're hoping to get recruited it is critical to understand who to build a relationship with - and NCSA has the information you need. The Head Coach of the Appalachian State University Women's Golf program is Heather Brown. Additional essential staff include:
Main campus of 410 acres with 890 other acres in Boone (population: 14,000), 90 miles from Winston-Salem. Major airports Charlotte and Greensboro (both 100 miles); bus between Greensboro. University-owned bus service.
Noah Hannon
Class of 2017
Roman Galaska
Class of 2017
Men's Soccer
Michael Montalvo
Class of 2016
Men's Soccer
Jonathan McDaid
Class of 2016
D'Ante Smith
Class of 2016


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Description: Teeveen international ventures is located in Alhaji Isa Williams Street, Lagos Island, Lagos. Our c
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DENVER, Colo., Feb. 10, 2014 (SEND2PRESS NEWSWIRE) -- Therapy Partner, a national leader in providing online, streamlined and clean technology for the needs of clinicians in private practice (psychologists, psychiatrists, and therapists), has launched 3.0 a progressive, single device application, for complete practice management. Therapy Partner 3.0 has a user interface that is intuitive and is driven by "Book-Bill-Document" proprietary technology; streamlining the daily tasks that clinicians are faced with in business.
Therapy Partner 3.0 boasts an impressive suite of features from a new intuitive and easy to use interface including: Progress Notes, Integrated Calendaring/Patient Scheduling, Comprehensive Billing, Automated Statements, Credit Card Processing, and more. The new proprietary "Book-Bill-Document" feature allows clinicians to easily book clients for appointments, bill multiple sessions across multiple days with one click using the "Quick-Bill" feature, and allows providers to quickly add a progress note. Therapy Partner 3.0 is available for use from your smart phone, tablet, and computer. Comprehensive state of the art storage of patient and financial information are included in the newest version of Therapy Partner 3.0.
Dr. Jessica Dolgan, company CEO and licensed psychologist, states that Therapy Partner 3.0 "was designed to be task driven, in that the primary tools clinicians access through Therapy Partner 3.0 are based on their most frequent tasks and activities in their clinical day. Our goal was to develop an application that is fast, clean, intuitive, and easy, allowing our clinicians to focus on clinical care." 3.0 sales support Dr. Dolgan's vision, with an immediate and continued spike in sales noted since the products' release.
Therapy Partner continues to prove itself as a national leader in the development of technology for clinicians in private practice. 30 percent of Therapy Partner 3.0's new annual sales continue to come from current customer referrals of colleagues. In addition, clinicians using the product consistently show an increase in revenue of 20 percent in the first year of using Therapy Partner 3.0. Representatives at Therapy Partner note that the clinicians increase in revenue is "directly related to having a single-device technology application that allows them to easily bill patients directly for service, consultation, and late cancels/no-shows."
Therapy Partner offers an annual subscription based service with plans beginning as low as $54 per month. Therapy Partner also provides flat credit card transaction rates, and they broker the relationship with their partner bank, allowing clinicians to only interface with a dedicated Account Manager at Therapy Partner.
The psychological industry has been requesting this type of well-considered, intuitive technology to address their practice management needs, technology that allows clinicians to focus on providing quality clinical care. Therapy Partner 3.0 brings to clinicians a cost effective, efficient, single-device complete practice management solution that allows them to focus on what they do best, provide quality clinical care to their patients.
More information: http://www.TherapyPartner.com/.
About Therapy Partner:
Based in Denver, Colorado, Therapy Partner continues to be a progressive technology company focused on developing advanced, single device technology for streamlining scheduling, billing and progress notes as well as a complete host of practice management tools. Therapy Partner, owned by Dr. Jessica Dolgan, a licensed psychologist, is a privately held company, operating since 2005. The tech firm is known for customized support and dedicated account managers, as well as diligently striving to continue to enhance and develop progressive technology for Psychologists, Psychiatrists, and Therapists.
*(LOGO 72dpi: http://www.therapypartner.com/PublicWebsiteMedia/Therapy-Partner-Practice-Management-PRLogo.gif )
*(VIDEO YouTube: http://youtu.be/zjsVq_97U6s )
Therapy Partner, Announces Launch of 3.0, Including Integrated Scheduling, Billing, and Progress Notes
DENVER, Colo., Feb. 10, 2014 (SEND2PRESS NEWSWIRE) -- Therapy Partner, a national leader in providing online, streamlined and clean technology for the needs of clinicians in private practice (psychologists, psychiatrists, and therapists), has launched 3.0 a progressive, single device application, for complete practice management. Therapy Partner 3.0 has a user interface that is intuitive and is driven by "Book-Bill-Document" proprietary technology; streamlining the daily tasks that clinicians are faced with in business.
For more information regarding this press release, please visit:
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After just meeting one another the two have fallen for each other.
And decide to now get married after it has only been a day of knowing one another.
"I'll lay all my fortunes at your feet and follow you, my lord, all over the world."
"Sleep dwell upon thine eyes, peace in thy breast. Would I were sleep and peace, so sweet to rest. Hence will I to my ghostly friar's close cell, His help to crave and my dear hap to tell."
Romeo rushes to Friar's cell to ask for his advice.
Friar then tells Romeo that he will marry the two in secret since there love is forbidden.
Friar justifes that the two houses children's romance could end the feud.
"Oh, let us hence. I stand on sudden haste."
"But come, young waverer, come, go with me, In one respect I'll thy assistant be, For this alliance may so happy prove To turn your households' rancor to pure love."


text:
Widely acclaimed for its outstanding step-by-step illustrations, comprehensive coverage, and reader-friendly format, Textbook of Pediatric Emergency Procedures is now in its thoroughly updated Second Edition. In 137 chapters, the book provides clear, complete instructions on every emergency medicine procedure performed on infants, children, and adolescents. More than 1, 000 detailed drawings, most in two-color, show how to perform each procedure. This edition includes a new chapter on new technologies and techniques for managing the difficult airway. Other new topics include use of tissue adhesives in laceration repair, focused abdominal sonography for trauma (FAST), management of priapism, reimplanting an avulsed permanent tooth, use of automatic external defibrillators, and procedures related to bioterrorism.Percutaneous transtracheal ventilation for emergency dental appliance removal. Anesthesiology. I985 ... Manual translaryngeal jet ventilation. Emerg Med Clin North Am. ... Konrad B, Siedlecki K, Bosch S, et al. Asphyxiation by laryngealanbsp;...
|Textbook of Pediatric Emergency Procedures
|Christopher King, Fred M. Henretig
|Lippincott Williams & Wilkins - 2008


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Sky News presenter Kay Burley isn't normally one to be lost for words, but one royal baby onlooker had the veteran journalist stumped.
While out asking crowds of well-wishers for their reaction to the news the Kate Middleton, the Duchess of Cambridge, had given birth to a little boy, one member of the crowd said "I hear it's a black boy".
A brief moment of stunned silence followed, as Kay moved swiftly on..
Watch the full clip above.
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Seen a scammer? Been caught out? Let the world know so they don't get burned. Add information if you see one that's already been logged - scammers move quickly and change details often to avoid detection. Details you log here will be in major search engines within an hour or two.Please Like, Share, Tweet etc. to spread the word.
|No Name
2013-09-21 13:58:59
Memphis, TN, United States
| Mrs. Catherine Davidson
Wills/Probate Scam
|Mrs. Catherine Davidson ⇒
|Email Address
|law/chamber/solicitors/je/ws/WILL/9834520012 ⇒
|Scam Website
|email@example.com ⇒
|447024015890 ⇒ Reverse Phone Lookup (by Spokeo


text:
Quick Look
Property brokered by
Get more listing details on brokerage site
Presented By:
Reece Coleman
02 9302 9555
Email Agent
Reece Coleman
02 9302 9555
Email Agent
Property DetailsTaking pride of place in the Redcliffe Peninsula, the Newport waterside community will provide a wide range of waterside and waterfront homes suitable for different lifestyle needs. Set amongst the flourishing Redcliffe Peninsula, Newport will feature a proposed retail and dining precinct in the town centre, a proposed child care centre, a retirement village, foreshore parks and a unique 23 hectare non-tidal lake offering high mast boat access to Moreton Bay.A perfect blend of amenity and the lifestyle opportunities provided by proximity to the water, Newport typifies all that is desirable about living by the water. From a peaceful walk to a bike run, kayaking or weekend sailing, it's all here at Newport.Whether it's fish and chips at Redcliffe, indulging in retail therapy at Westfield North Lakes, catching a flick at the cinemas, playing a round of golf at the North Lakes Resort Golf Club, or relaxing on the long soft sand beaches of Redcliffe- there's plenty of places to while away the afternoon. Regardless of the season, your waterside lifestyle will allow you to take a break from the everyday hustle and bustle of city life.This quality home by award winning Dwyer Quality Homes will suit a range of new home buyers looking for the perfect waterside location close to employment, education, retail, entertainment, lifestyle and health services and opportunities only 35 minutes to the Brisbane CBD.- Brand new 184m2 4 bedroom, 2 bathroom home with double lockup garage- Free flowing living rooms with separate home media room- Stone kitchen, quality European appliances, large pantry- 35min Brisbane CBD, 26 minutes Brisbane Airport, 3 min to Kippa-Ring railway- Vibrant retail, entertainment, recreation and high quality education facilities close by- Prime investment opportunity in high growth area with strong rental demand- Dual land and building contract for Australian residents; minimises stamp duty- Brand new house & land package, foreign investor compliantLand Size: Approx 350m2Lease this home out for $500pw, no management fees or vacancy for 3-yearsDisclaimer: The Agency gathers all information contained herein from sources we believe reliable. We have no reason to doubt its accuracy, however we cannot guarantee it. All interested parties should make & rely upon their own inquiries whether or not this information is in fact accurate.
Area Detailed Information
Area Weather
Area Cost Index
Area Property Prices
Additional Property InformationThis residential listing located at Lot 505 Newport Estate, Newport has four bedrooms and two bathrooms. The property is currently selling at $385,932.
This property is listed by Reece Coleman of The Agency | Australia headquartered in Australia.


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Autumn Leaves (Fall leaves)
>> Friday, November 18, 2011
If you live in the Northern Hemisphere, about now you can be sure of one thing. Your garden is full of fallen leaves. While many people see these as an inconvenience merely to be swept up and disposed of, in reality they are a great resource for the gardener
Given enough time, fallen leaves turn into one of the most wonderful products for your garden- leaf mold. A dark brown crumbly soil conditioner that will enhance the growing power of your soil.
All you need to get from leaves to leaf mold, is time and a small amount of water and patience. If you have a large enough garden, the easiest thing to do is to construct a pen for your leaves- four wooden posts stuck firmly into the soil with something between the posts to hold the leaves in. Chicken wire is traditionally used, and cheap.
Alternatively you could build a bin made of wooden delivery pallets or whatever spare wood you have around the garden. A leaf bin is much easier to construct than a compost heap in which you want to compost foodstuffs,as it does not need to be vermin proof. It does not need to be of a minimum size to allow for hot composting, all it needs is to hold the leaves in an not let them blow back around your garden, allow some air circulation and allow the odd shower of rain to moisten the leaves.
If you do not have enough room for a dedicated leaf mold bin, you could just place leaves in a black plastic sack, punch several holes in the sack and leave for a year or two. Not the most pretty option, but effective. There are also a number of decomposable leaf sacks on the market that you can pack you leaves in. The advantage of these is that if you forget about them, you won't have black plastic lying around your garden, and they look alot prettier than a pile of black rubbish sacks.


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State completes damage assessment survey
August 15, 2014
The following resources are out assisting the public and working through-out different areas in Douglas County:
Douglas County Maintenance Department
2 Grader Fish Springs Area
2 Loaders Johnson Lane Area
1 Broom Truck Johnson Lane Area
2 Bottom Dumps Johnson Lane Area
1 Back Hoe Johnson Lane Area
A&A Construction
1 Loaders Johnson Lane Area
1 Dump Trucks Johnson Lane Area
1 Grader Johnson Lane Area
Crocket Engineering
1 Grader Johnson Lane Area
1 Loader Johnson Lane Area
1 End Dumps Johnson Lane Area
2 Loader Fish Springs
2 End Dumps Fish Springs
Curtis &Sons Construction
1 End Dumps Fish Springs Area
1 Loader Fish Springs Area
2 Grader Fish Springs Area
Gardnerville Town
1 Street Sweeper Johnson Lane
Town of Minden
1 Street Sweeper Johnson Lane
If you have significant flood damage on your street please call Public Works
(775)783-6480 and they will respond to your inquiry in order of severity.
The Douglas County Emergency Reverse 911 system is back up and running, and the however the National Weather Service issues appropriate warnings from their radar system and the Emergency Broadcast System. If Douglas County needed to issue evacuation notices the Emergency Alert System would be activated.
A public Douglas County flash flood hotline (775)782-9993 and email address email@example.com have been set up for residents to report damages for the state assessment. Please leave a detailed message with your name, contact information and address for damage assessment. This Hotline and email will close on August 15, 2014.
A night-time Aug. 6 flash flood that filled Pine Nut Creek and sent water and mud across Fish Springs may have been the second 100-year event in a month.
A 100-year flood means there's only a 1 percent chance of a certain sized event in any given year.
Interim County Manager Larry Werner said there's a 63 percent chance that there will be two of those sized floods in a year.
Those weren't hard odds to beat with an unprecedented monsoon season, which brought moisture to Western Nevada and prompted four floods resulting in damage to public and private property.
The night of the Fish Springs flood, National Weather Service reported Doppler radar indicated an inch of rain had fallen at the top of Pine Nut Creek in 45 minutes.
According to the weather service's tables that would come close to being a 200-year flood, which would see 1.11 inches of rain over 30 minutes.
Werner said the weather pattern that resulted in flooding across the county is remarkable.
"None of us have ever seen this before," he said. "We were hit by an extraordinary storm. There's no way we could afford to design structures to withstand that."
Exacerbating the flooding was the large amount of sediment included in the July 20 flood.
"The mud flow was a different sort of animal," he said. "Every time it hit a culvert it would plug it up and then jump over the road and do it again the next time."
A state damage assessment team visited 75 properties on Wednesday, as they toured Fish Springs and Johnson Lane to gather information about the level of damage from flooding in Douglas County.
Team members also contacted residents who used the county's flood hotline or email, according to county spokeswoman Melissa Blosser.
"We made it personal yesterday with those impacted and I am proud of that effort," said District Fire Chief Tod Carlini, who is also county emergency manager. "We went to the people yesterday, and we want to thank everyone who called in and reported damage. It is important that we document damages as we attempt to qualify for state funding."
On July 20, the base of Hot Springs Mountain received 1.21 inches of rain in just over an hour, sending water and mud into northern Carson Valley neighborhoods.
Another storm near Mount Siegel in the Pine Nut Mountains on Aug. 6 sent water roaring down Pine Nut Creek, cutting off the community of Fish Springs. The declaration came with a fourth flood on Monday that rushed into upper Stephanie Way overwhelming already filled culverts.
Once the assessment team finishes its report, it will be reviewed by the state and then sent to the governor, who will determine whether the damage qualifies the county for a state declaration.
Should the governor declare a disaster in Douglas County, residents would be eligible for low-interest loans to restore their property.
Blosser said all of the information received from emails and calls has been collected and will be used to produce a location specific flood damage map to further access damages and review areas of concern.
County workers and contractors are cleaning up debris left from the flood and clearing ditches and culverts.
"We can assure you we have everything we have access to out and on the ground, cleaning ditches and clearing debris," said Chris Oakden, road maintenance operations superintendent. "As a public entity there are some things we just can't do, for instance cleaning up private property. Our main priority is cleaning out the ditches, clearing debris from public roadways and not letting things get backed up while maintaining the safety of our employees and our residents."
Werner said the county is working on mapping the north Valley floods in the same way it did Fish Springs.
More than 200 properties were damaged including those who lost their yards to flood waters.
"Some people were angry we wouldn't put the dirt that had washed out of their yards back," he said.


text:
Bluebonnets by Ed Owens
- March 17, 2013
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Doing the Faukner thing . . .we'll leave the Porch Light on.
Ed – I think we need to clarify something. Blythe WAS my favorite character until Miki's Mom (the EM EYE ELL EFF !!!!) came along. That woman is hot. As much as I'm intrigued by Blythe and the graves of her 13 ex-husbands, the seldom seen but provocatively drawn Mrs. Amasawa has leapfrogged Blythe and skyrocketed to the top of my list.
As for my "character in the middle," I have a problem: Roy, Buck, and the pachyderm would be close to the top, and Grandpa, Gordon, and the Major nearer to Smokey-level status. That would leave Miki and Alice in between. Thus, my dilemma.
If I say Alice is in the middle, she'll be high-pissed she's not Numero Uno. If I don't mention her at all, she may come after me with one of Blythe's butcher knives.
You see my dilemma? Can I plead the Fifth?
P.S. Speaking of Miki's mom leapfrogging Blythe, even though this is a family strip I wouldn't mind seeing THAT sometime. Do you do requests? Mrs. Amasawa and Blythe cavorting in a gym in exercise outfits could be a great "one-off" April Fool's Day panel!
P.P.S. I turned 60 last Wednesday, so I guess my status as a dirty old man is now official.
Hey, I don't blame you. Go enjoy the weather.
66! Hell! I thought you said you were old!
Oh, and Happy birthday.
Happy Birthday to my Cyber Big Sis!
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The African feathered headdress Juju Hat (Tyn hat) originates from Cameroon and was worn by royalty and medicine men, who used them during dance ceremonies.This Vintage Tyn (JuJu) is made of feathers tightly sewn onto a raffia base and the finished product folds neatly into a cylindrical shape. The opened hat can be displayed on the wall as a decoration to adorn any corner or display area. They have become a very popular design accessory for the home.
Measures 31" wide across
Measures 31" wide across
Meet the owner of africanhouseLearn more about the shop and process
Juju (Tyn) Hat...White
Only 1 available
- Vintage item
- Materials: feathers, twine, wood, cloth
- Ships worldwide from United States
- Feedback: 69 reviews
- Favorited by: 19 people
- Listed on Aug 22, 2016
- 502 views
- 19 favorites


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The Human Resources Department is charged with ensuring the city has a competent and diverse workforce dedicated to meeting the city's goals. To fulfill this mission, the department is guided by the following directives:
The City of Elgin is an Equal Opportunity/Reasonable Accommodation Employer. Applicants are considered solely on the basis of their qualifications as required for the position they seek, and no discrimination is exercised because of their political or religious opinions or affiliations, or because of their race, religion, color, sex, sexual orientation, national origin, age, disability, veteran status or marital status.
Apply for a job.
You may be eligible for the following benefits working for the city of Elgin:
Leaves & Holidays


text:
Sept. 21 Men's Health event at RMH to focus on 'Diabetic Foot Health'
| Date Posted: 9/9/2010
ROCKINGHAM – You may not know it, but your feet are at high risk for infection and worse if you have uncontrolled diabetes.
Dr. MirFattah Mirian
Diabetes affects circulation (blood flow), which can make your feet less able to fight infection and to heal. Uncontrolled diabetes can damage the nerves in your feet and legs, causing a condition called diabetic neuropathy that makes it difficult for you to feel heat, cold or pain.
Severe foot problems can lead to even more serious complications, including amputation. In fact, according to the American Diabetes Association, people with diabetes are far more likely to have a foot or leg amputated than other people.
On Tuesday, Sept. 21, Dr. MirFattah Mirian, a Richmond County podiatrist, and Melissa Herman, a certified diabetes educator with FirstHealth's Diabetes Self-Management Program, will be featured in a Men's Health Event on "Diabetic Foot Health" to be held at FirstHealth Richmond Memorial Hospital.
Scheduled to be held from 6 to 8 p.m. in the hospital's Third-Floor Auditorium, the program will focus on the prevention and complications of diabetes, especially as they apply to foot health.
"Anyone can have foot problems," says Dr. Mirian, "but even something as usually uncomplicated as a blister can lead to very serious problems for a person with diabetes. During our program, Melissa Herman and I will discuss the importance of foot health and provide information on daily foot care and diet, both of which can help lessen the chance of diabetes complications."
The Men's Health Night program on "Diabetic Foot Health" is sponsored by the FirstHealth Richmond Memorial Hospital Foundation, which supports FirstHealth services for the residents of Richmond County.
"Diabetes is a very serious problem in Richmond County, which has a diabetes rate that is more than twice that of the entire state of North Carolina," says Ken Goodman, chair of the RMH Foundation's Board of Trustees. "In their Sept. 21 program, Dr. Mirian and Ms. Herman plan to share information about diabetes prevention and control that both men and women should find helpful, and the Foundation is happy to have a role in this important event."
The Sept. 21 Men's Health Night program is free, but space is limited, and registration is required. Please call (910) 417-3772 or (800) 213-3284 for more information or to register.
Participants in the "Diabetic Foot Health" program will receive a $5-off coupon for a Peripheral Vascular Disease screening conducted during the RMH Wellness Screening Day on Oct. 9.
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Surprise cockfags!
Right before classes started, my college has a long orientation, that's full of parties, placement exams, and general lightheartedness before school finally starts. It culminates with a big, free concert on the quad, which is where my story begins.
Tenten went to my high school, and we'd been friends, though we'd never really been close. We'd hang out occasionally in the same group, and we thought it was kind of neat when we ended up going to the same college but never really thought much about it. When I arrived at the concert on the quad, though, I saw her and immediately went over to her, for lack of any other familiar faces.
She's absolutely gorgeous, by the way. She's incredibly voluptuous. She's one of those girls with ridiculous curves, her tits are very nice, but it's her ass that's perfect. It's big, firm, and always looks beautifully inviting through her pants. To top it off, she's always happy and smiling. She's got a cute face, and she's always cuter when smiling.
That evening, she was wearing a tight pink tank top and a short, matching skirt. She was barefoot, and I immediately noticed her long, pale legs in the warm summer twilight. When she saw me, she ran over, excited.
"Hey!" she yelled a little too loud and hugged me. She'd obviously been drinking a bit, but so had I. When she hugged me, she crushed her lovely tits into my chest. "Did you just get here?"
I told her I had, and we chatted about our summers, what classes we would be taking, and other, trivial matters when the music started.
Now, Tenten loves to dance. She's a fantastic dancer, and won all sorts of awards and what not in high school for it, and even did some professional dancing. As a result, she's in incredible shape, she can do the splits without warming up at all, just to give you an idea.
Naturally, she started dancing once the band started and since I was closest to her, she elected me to be her partner. I was totally cool with that; a sexy girl, gyrating against me on a hot summer evening? How could I refuse? She wrapped her arms around my, moving her hips to the music, getting closer to me, and then drawing away, only to get closer again. I did my best to keep up since my dance experiences leans more towards swing and lindy hop because I'm old fashioned like that, but I think I must've done a good job. I honestly can't tell you if I busted out any special moves or if it were just the atmosphere of hundreds of other hot teenage bodies around us, but pretty soon, she was pressed up against me, our hips grinding together. I remember at the time imagining how we'd have been reprimanded in high school for doing that, and out of habit, I looked around to make sure no chaperones were about to descend on us. Of course as you are reading this there were none, just tons of other kids, doing the same thing.
Tenten turned around and wiggled her luscious ass against my crotch. I held her by the hips as she ground against me, making exaggerated moans, and trying to sing along to the music through her laughter. A few guys tried to dance with her too, but I held onto her out of instinct, not letting any other guy come close. She grinned at me.
"You're so cute," she said in a loud whisper right in my ear. She nipped at my ear lobe and went back to dancing. I was hard now, and I was sure she could feel my cock through my pants. We were facing one another again, and she straddled my leg, rubbing down on it in rhythm to the music. I gripped her ass pressed her down hard on my leg, and she moaned for real. I groped her ass, trying to squeeze as much of her firm, perfect butt as I could. She loved this and alternated between pressing down on my leg, and pushing her ass into my hands.
I reached under her skirt, and found myself holding her bare ass cheeks. All she wore underneath was a thong, so I rubbed and groped her flesh, massaging her. A few people noticed, and watched but neither of us cared. I heard some girls exclaiming how hot it was, and since it was night by now, and it had cooled down dramatically, they must have been referring to us.
I worked my hands under her thong, and ran my finger down her ass crack, over her tight asshole, to feel her pussy. And my god, she was wet. She was soaking beyond belief; juice had actually run down her legs, mingling with the sweat already clinging to her. There was even a growing wet spot on my pants where she had been rubbing herself.
Now I began to rub her pussy. It was totally shaved, and slicked from so much juice. Tenten through her head back and moaned when I found her clit, still dancing. Now, she was grinding her hips onto my hand. I was drunk with lust and my cock was begging to get free. It was dark now, and the quad was just bathed in the light of the street lamps, so I undid my pants, pulling out my cock, and pressing it against her. She gasped, but pressed back.
I wanted more, though, and I had already decided what I was going to do. I pulled her thong aside, held her close, lifting her up by the hips a bit, and pressed my cock inside of her in one, swift move. I went in all the way immediately; her wet, greased cunt offered no resistance. She gasped, and her faced showed surprise, but then she moaned, and began moving her hips against my, all in time with the music. Only the people nearest us could see what was happening and a small ring of people formed around us, watching us fuck. It was dark enough where we were that they probably couldn't see our faces, but they could definitely figure out what was going on. None of them dared to try and stop us.
"They must be so drunk," one girl said.
"Fuck, this is getting me hot," commented another. In a second, she was making out with a guy next to her.
I was thrusting into Tenten as hard and fast as I could, afraid someone would try to stop our fun. She did what she could to grind down on me but gave up and, throwing her arms around my neck, she wrapped her legs around my waist, so that I was supporting both of us. I held her by her ass, and kept fucking her. She was so wet, it was easy to pulse my dick in and out of her tight cunt as hard and fast as I could. Plus, her body weight always drove her down onto my cock harder and deeper than before. She latched her lips onto my neck, sucking hard, and then biting hard, trying to hold in her moans. I gripped her tight, and massaging her ass while holding her. She loved this too, and she moaned loud and long when I pressed a finger into her asshole while I fucked her. Her cunt tightened around me, and I couldn't take it--I started to cum. I slammed her down against me, so that her clit was smashed against my crotch, and this drove her over the edge. I felt her pussy walls tightening and loosening around me, making my orgasm even better.
When we finished cumming, she put her legs down and just leaned against me, panting. I ran my hand up and down her back, suddenly feeling great affection for her. I tilted her head up and kissed her for the first time, no tongue, sweet. The people around us began to disperse when they saw the show was over. Tenten and I were kissing harder and faster, though, exploring each other's mouths almost frantically, trying to make up for lost time. I reached under her tank top, running my hands over her smooth, firm belly, slicked with sweat, and up to her tits. I rubbed them through her bra, and she bit my lower lip, and purred.
Finally, I took her by the hand and led her away from the crowds. We went to a far corner of the quad that looked deserted, and tumbled to the ground. I covered her face with kisses, and she nipped back at me. At some point, I can't remember when, our shirts came off and so did her bra. I balanced my hands on her bare tits, her large, hard pink nipples poking into my palms, while I rained down sloppy wet kisses onto her face. I kissed down her to her neck, and gave her a long, slow lick across her throat, culminating in a playful little bite. I bit her hot flesh over and over, moving down to her breast, and took her nipple into my mouth. They were large, easily the biggest I've seen, and stood proudly on her firm mounds. I chewed on it, taking care not to bit too hard, and then just sucked. I suckled her for several minutes while she ran her hand through my hair. Compared to the passion and intensity of our first fuck, this was very relaxing. I abandoned suckling to lick and nip at the rest of her breast before repeating all of that on her other hard, swollen nipple.
While I worked on her other tit, I stroked her belly gently, letting my fingers play over her sweat slicked skin. It must have tickled a bit, because she squirmed a bit, but she purred like a pleased little kitten anyway. I slid my hand down to her crotch, pulled her thong down and tossed it away, and cupped her pussy, rubbing my palm gently against her hot box. This made her purr even louder. I kissed my way down her stomach, leaving a trail of marks from hard, suckle kisses, until I got to her pussy. Her scent found my nose before I saw her wet twat, which radiated heat everywhere. In the dim light, I could see my semen dribbling out so I spread her lips and pressed my tongue inside, giving her a long, firm lick from the inside and out, up to her clit. I licked back down, pushing my tongue inside, and started all over. M grabbed my head, and pressed my face into her pussy, and then, to top it all over, she wrapped her legs around my head. I figured I'd be there till she came, so I picked up the pace, swirling my tongue up and down, over her pussy. I made a low, humming noise to make my tongue and lips vibrate, and this elicited more loud moans from my sexy girl. After several minutes, I abandoned licking to focus on sucking. I got her whole pussy in my mouth--lips, clit, and all--and began to suck hard, while diddling her with my tongue. I found what she really liked. She started shaking and kept on trembling for several minutes before she started to thrash and cum. Hot juice dribbled into my mouth, and her legs tightened around my head, crushing my face into her pussy. I could barely breath but fortunately, after a few seconds, she went limp and released me.
"S-sorry..." she said, staring up at the starry sky. "That was really good."
I decided after that, I deserved something in return so I spread her legs again, pressed my cock against her and in I was. She gave a weak moan, but was content to let me do the work. I pound my cock in and out of her drenched pussy faster than before, since she was so wet. It felt like I wasn't even touching her cunt walls--that's how wet she was. I was pounding her so hard, that the rubbing started to build up this delicious heat in our genitals, that spread throughout out bodies. She felt it finally, moaned, and wrapped her arms and legs around me. It didn't take me long to cum again, and I sprayed even more of my seed into her cunt.
"Oh, God, yes!" she moaned, squeezing me tight, her pussy milking my cock. I kissed her hard for a minute before pulling out. I straddled her chest, and she, grinning, took my limp, sloppy cock into her mouth. She slurped with great enthusiasm at my dick, and I felt her tongue swirl around my cock head, and slide up and down my shaft. I thrust a bit into her mouth, and she liked this, so I picked up the speed, fucking her face. We must have gotten too into it, though, because I was going pretty hard soon and she started to gag. She pulled off my cock and stuck it between her tits. She didn't even miss a beat--she crushed them around my cock and, lubricated my both of our cum and her spit, I started to fuck her generous tits. I flicked her swollen nipples back and forth while I did it and soon, we were both moaning.
I wanted more, though.
"Get on your hands and knees," I ordered. She obeyed and I spread her gorgeous ass cheeks, fondling her well used pussy. I penetrated her again, and she moaned. I thrust a few times, and I'm sure she figured she was about to get fucked doggy style. Instead, I began to tease her asshole. "Can I fuck you here?" I asked. I expected her to say no, but I figured it was worth a shot.
"Yeah, just don't go too hard," she replied. I was surprised but I didn't need anything else. I pulled out and began to press my cock in between her gorgeous ass cheeks. I felt her tight little hole open up and begin to consume me. She let out a long, loud noise, halfway between a moan and a groan as her ass swallowed up my cock. I pressed in as deep as I could, and she was moaning one long, continuous moan. Her ass felt so fantastic around my cock. It was infinitely tighter than her cunt and I began to move inside of her.
"God, yes!" Tenten moaned. "Oh my God, I never thought this would feel so good!" She pressed her ass against me, trying to take more of my length in her tight hole. I was thrusting at a steady pace but that wasn't enough for her.
"Fuck, pound me!" she demanded. I picked up speed, slamming my cock in and out of her ass. She loved this and practically screamed. It was then that I noticed other people standing not too far away from us, watching. I listened carefully and I heard them talking:
"Oh my God, are they the same ones who were fucking before?"
"They're really into it."
"Hey, is he fucking her in the ass?"
Tenten must have heard. "Yes!" she screamed. "He's fucking my ass!" I had thought she'd sobered up by now, but apparently not. Her scream brought some laughs and even cheers from the people watching. They got a little closer and a few of them dispersed, probably off to do the same thing themselves.
I was getting close so I decided to experiment. I pulled out all the way and Tenten whimpered in disappoint. She gave a loud scream, though, when I slammed back into her in one stroke burying my cock in her asshole. I did this again and again and she was squirming and almost spazing in pleasure and pain. Then, I pulled out, dug my cock into her pussy, gave a few thrusts, and then jammed it back into her ass. I did this over and over, alternately between fucking her pussy and her ass. She was going wild and started to cum; she had been rubbing her clit but my cock sent her over the edge. I decided I wanted to cum too, so I embedded my cock as deep in her ass as I could and let loose, filling up her tight butt with cum. As we both came, I gripped her hot tits. They were hanging down and had been swinging back and forth as we fucked, like tempting pendulums.
Afterwards, we lay there, making out and snuggling. We fell asleep in the warm summer air and woke up an hour later. We found out clothes, dressed, and I pressed her up against a tree. I dug my cock into her pussy again and we fucked once more. It was hard, fast, dirty sex, she wasn't wet, so it hurt both of us, but we didn't care. We wanted all the fucking we could get. I came hard into her almost dry pussy and she almost collapsed on top of me.
She got a boyfriend right away, some guy...Hyuga I think, so we haven't had sex since then, but I think the first time was enough. We're closer friends than we were before and she gets excited every time she sees me on campus, runs over to me, even dragging her boy along in tow. I sort of wonder how I measure up to him, but I haven't had the balls to ask her. I do hope I get to have her again sometime in the future, but I'm not about to try and break them up or anything mean like that. Still, she's always giving me these wicked little grins, especially when she's with her boyfriend...
Admit it, you thought I'd give you guys Kyuubi, right? Did you learn nothing from Chapter Four? I'm a pathological Liar!


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Paul Smith Men's Green Zig-zag Cotton T-shirt
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Featuring an all-over zig-zag pattern, this eye-catching t-shirt from Paul Smith Jeans will form a stylish base for your casual attire this season. The regular-fit silhouette features a crew neck, short sleeves and a brand label at the side seam, ensuring a classic finish. Paul Smith Jeans cotton-jersey t-shirt. Pulls on. Crew neck, ribbed neckline, short sleeves, all-over zig-zag pattern, brand tab at side seam, notched hem. 100% cotton. Color: green
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DETROIT — Relentless rain has swamped southeastern Michigan, turning roads into deep canals, ruining daily commutes and threatening communities with floods.
Forecasters say the rain that started Thursday is expected to stretch into the weekend.
Parts of Detroit received more than 5 inches of rain. Flooding closed sections of Interstate 75 at times, and northbound lanes of the Lodge Freeway under Cobo Center were shut down due to water and sewage.
Lt. Mike Shaw of the state police says, "This water rises very quickly."
News media posted online pictures of submerged vehicles. One man was up to his neck in water. Non-emergency surgeries were canceled due to flooding at Harper Hospital in Detroit. More than 275 street drains were cleared in Detroit.
The National Weather Service says some areas could get another inch of rain Friday.


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As a Children's Miracle Network hospital, there are many local events and promotions happening each day that support Children's Hospital of Wisconsin.
Be sure to visit your local Speedway store on Fridays this summer and round up your purchase at the register to support Children's Hospital of Wisconsin!
Know a RE/MAX agent or live near a RE/MAX office? Chances are they donate to Children's Hospital of Wisconsin by giving a dollar amount or percentage from each of their transactions! Find a RE/MAX agent or office near you here.
This month, when you visit Ace Hardware locations cashiers will ask if you want to round your purchase's total up to the nearest dollar amount to benefit Children's Hospital of Wisconsin. Be sure to stop by your local Ace and support Children's Hospital of Wisconsin.
At the end of the month, Walmart and Sam's Clubs around the state will begin their annual fundraising campaign for CMN hospitals like Children's Hospital of Wisconsin! Associates will ask you to make a donation to Children's Hospital of Wisconsin at checkout. Last year, 80 Walmart and Sam's Club locations raised over $850,000 for Children's Hospital. This campaign will run from August 29th to October 9th!
Visiting the Fond Du Lac or Oak Creek areas this summer? If so, be sure to stop by Love's Travel Stops and support Children's Hospital of Wisconsin through a $1 Miracle Balloon donation! This campaign will run August 26th through September 30th!


text: The hotel offers a wide range of services and facilities to ensure a comfortable stay, making it the perfect choice for business travellers and holidaymakers alike. Wireless internet access in public areas allows guests to stay connected. The accommodation features rooms with a living room, a kitchen and a bathroom. The rooms have a double bed and a sofa bed. Extra beds can be requested. A fridge, a microwave and a tea/coffee station are provided as standard. Internet access, a TV, a radio, a DVD player, an alarm clock and WiFi ensure optimal comfort. The bathrooms are fitted with a shower and a bathtub. A hairdryer and bathrobes are provided for everyday use. The establishment offers non-smoking rooms. The hotel offers attractions including sport and entertainment opportunities. The hot tub is the perfect place to relax. The accommodation offers massage treatments (for a fee) to travellers. Copyright GIATA 2004 - 2016. Breakfast can be booked.


text:
The NTU CMOS Emerging Technology Group kicked off in October 2009 and has grown substantially over the years. We have talented researchers from all over the world. Our research focus is to develop CMOS Emerging Technologies for Big-data Collecting and Computing System, which mainly include following research perspectives:
(1) 3D/2.5D Data Server Research Platform for Big-data Computation
(2) THz Radio Research Platform for Big-data Communication
(3) Integrated Sensor Training Platform for Big-data Collection.
Our objective is to provide both academia and industry with a platform of innovations and services on CMOS based emerging technologies. Our group has close collaboration inside NTU, A*STAR, Intel, Huawei, HP, and universities in USA/China. Our projects are currently sponsored by NTU, NRF, MOE and MINDEF.


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Schoeps CCM2S Diffuse field omni
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Schoeps CCM 2S is an omnidirectional pattern microphone with the frequency response characteristic of the CCM 2S falls between that of the CCM 2 and the CCM 3, making this microphone suitable for a wide range of applications.
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|Schoeps CCM2S Diffuse field omni
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Got a question about this product? We have the answer. CALL US: 020 84452446
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PreSonus Eris E8 (Pair)Inc. VAT: £398.00 Ex. VAT: £331.67


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Reopened my shop on ETSY again ;) new prints available for sale;) + Polaroids. Please go take a look at www.etsy.com/shop/satelinasg
I'm not proud of it, but I just recently discovered all the awesomeness on Etsy. Will add your shop to my favorites :)
I'm sure your beauty will shine through in whatever you do! I haven't been on Etsy before, but have heard of it recently.
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i did broadcasts with Suicidegirls persicope account for a while and its really fun.
but on periscope its have to be "safe for work" and no boobies allowed =( so I've deicide to try real web modeling and just made my profile on jasmin.
anyone got experience with webcam modeling ?
here is the link if u want to join and seem me strip, playing with...
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I love the idea, but you are never online :( 4 days since last time and I didnt find you there either
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Fosterville Jobs
Looking for jobs in Fosterville? Snagajob has great opportunities with the area's leading companies. And applying for Fosterville jobs has never been easier. Look at the job listings below, click on a job title that interests you and hit the "apply now" button. Part-time jobs, teen jobs, student jobs and summer jobs are now just a click away. Start your job search now.
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Hotel Doubletree JFK Airport
135-30 140th Street, 11436 Jamaica - Long Island, United States |Show on the map
from €69
Doubletree Hotel JFK Airport provides excellent prices without compromising your comfort, making it the ideal accommodation
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Tripadvisor Score Score based on 824 reviews
Services and terms: Hotel Doubletree JFK Airport
Standard Room Services
- Air Conditioning
- Coffee & Tea Making Facilities
- Safe
- Satellite TV
- Pay TV
- Radio
- Hairdryer
- Telephone
Accommodation facilities
- Lift
- Bar/Café
- Business Centre
- Laundry
- People with reduced mobility access
- Non-smoker Rooms
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- 24 hr Room Service
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Useful information: Hotel Doubletree JFK Airport
Other names for Hotel Doubletree JFK Airport:
Radisson Hotel Jfk Airport
The Radisson Hotel Jamaica Ny
Location: Hotel Doubletree JFK Airport
The closest major airports to Doubletree Hotel JFK Airport are:New York, NY (JFK-John F. Kennedy Intl.) - 3 km / 1.8 miNew York, NY (LGA-LaGuardia) - 13.4 km / 8.3 miThe preferred airport for Doubletree Hotel JFK Airport is New York, NY (JFK-John F. Kennedy Intl.). Distances are calculated in a straight line from the property's location to the point of interest or airport and may not reflect actual travel distance. Distances are displayed to the nearest 0.1 mile and kilometre.
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