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Polarizing cells extensively restructure cellular components in a spatially and temporally coupled manner along the major axis of cellular extension. Budding yeast are a useful model of polarized growth, helping to define many molecular components of this conserved process. Besides budding, yeast cells also differentiate upon treatment with pheromone from the opposite mating type, forming a mating projection (the 'shmoo') by directional restructuring of the cytoskeleton, localized vesicular transport and overall reorganization of the cytosol. To characterize the proteomic localization changes accompanying polarized growth, we developed and implemented a novel cell microarray-based imaging assay for measuring the spatial redistribution of a large fraction of the yeast proteome, and applied this assay to identify proteins localized along the mating projection following pheromone treatment. We further trained a machine learning algorithm to refine the cell imaging screen, identifying additional shmoo-localized proteins. In all, we identified 74 proteins that specifically localize to the mating projection, including previously uncharacterized proteins (Ycr043c, Ydr348c, Yer071c, Ymr295c, and Yor304c-a) and known polarization complexes such as the exocyst. Functional analysis of these proteins, coupled with quantitative analysis of individual organelle movements during shmoo formation, suggests a model in which the basic machinery for cell polarization is generally conserved between processes forming the bud and the shmoo, with a distinct subset of proteins used only for shmoo formation. The net effect is a defined ordering of major organelles along the polarization axis, with specific proteins implicated at the proximal growth tip.
Polarizing cells undergo widespread molecular rearrangements in a spatially and temporally concerted manner, reorganizing cellular components along the primary cellular polarized growth axis. Polarization mechanisms are important for both basic cell division and specialized growth processes, such as formation of neuronal processes,(1) cell motility(2) and asymmetric stem cell differentiation.(3) The resultant coordinated asymmetry affects major developmental, cell division, and metabolic programs in the cell. The budding yeast, Saccharomyces cerevisiae, has proved an excellent model system for studying polarized growth, as it restructures the cell differentially in vegetative growth, mating and filamentous growth.(4) Each of these developmental fates is characterized by a distinct morphology, and the stimulus and outcomes of each differentiation pathway are unique. For example, internal bud cues from the previous bud site drive the cycle of bud emergence and cytokinesis. External cues such as mating pheromone and nutrient starvation trigger signal transduction pathways that induce formation of a mating projection or the filamentous 'foraging' phenotype, respectively. These changes in cellular morphology are accompanied by dynamic changes in the cellular proteome that function in setting up the polarization axis and orchestrating specific activities required for the appropriate growth response.
Wild-type yeast cells, upon treatment with pheromone from the opposite mating type, undergo a differentiation program typified by the formation of a mating projection (the 'shmoo') accompanied by cell cycle arrest in the G1 phase (reviewed in refs (4 and 5)). Many proteins are redistributed to different subcellular locations culminating in the formation of a highly structured polarization axis oriented toward the shmoo tip. Although similar polarization mechanisms dominate the formation of a bud and a mating projection, there are obvious differences underlying the two processes. While budding is preordained by location of the previous budding landmark, mating projection formation appears to be a dynamic process that relies in the wild on a directional pheromone gradient. Mating-competent cells can therefore continuously sense pheromone gradients and alter the site of projection formation accordingly, and in some instances, even sequentially form multiple 'shmoos'.(4) Cells subjected in the laboratory to uniform pheromone concentrations form randomly oriented mating projections. The shape and structure of mating projections also differ from buds, with the conspicuous constriction that marks buds being largely absent at the base of the 'shmoos'. This region contains the septins, chitin, and pheromone induced proteins such as Afr1p that interact with the septins. It is therefore likely that an altered set of protein interactions during the mating process determines the shape and morphology of the projection and distinguishes it from bud formation during vegetative growth. This is more evident when one analyzes finer details such as the growth dynamics of a bud relative to a shmoo. Bud growth becomes isotropic after the apical growth phase, while a shmoo displays more unidirectional growth dynamics, possibly because of differential rates of recruitment of proteins that deposit cell wall constituents. These differences arise due to the distinct functional goals that each process seeks to achieve—nuclear segregation to the daughter and cytokinesis during vegetative growth, as opposed to cell fusion and karyogamy during mating.(4) Systematic identification of the proteins associated with each process will therefore begin to shed light on their mechanistic differences.
Significant strides have been made in technologies for increasing the throughput of imaging protein spatial localization in mammalian cells (e.g., see refs (6 and 7)), but the power of yeast genetics has already resulted in proteome-wide imaging and epitope tagging strategies being successfully employed in this model organism. Cellular imaging of protein localization has been conducted on transposon-tagged(8) as well as recombination-based tagged open reading frame (ORF) libraries.(9) Immunofluorescence and live-cell imaging of the tagged strains in S. cerevisiae has identified the subcellular localization of most of the proteome under standard laboratory conditions and these data are now accessible through the TRIPLES, GFP/UCSF, and other databases.
We previously reported the development of spotted cell microarrays(10) (cell chips) for measuring cell morphology and morphology defects across collections of thousands of yeast strains, more recently applied to measure a bacterial protein's localization in thousands of differing genetic backgrounds.(11) Briefly, spotted cell microarrays allow for cells of different genetic backgrounds to be robotically arrayed onto coated glass slides at high density, then each strain imaged in turn using automated microscopy. The cell microarray approach is readily adapted to measure eukaryotic protein subcellular localization by taking advantage of the availability of epitope-tagged strain collections, such as the green fluorescent protein (GFP)-tagged strain collection.(9) In this strain set, each of the ~4200 S. cerevisiae strains carries a genomic copy of the Aequoria victoria GFP (S65T) gene fused to the carboxy-terminus of a different open reading frame. Arraying this strain set on spotted cell microarrays and imaging the entire set of strains thus measures the subcellular localizations of ~4200 proteins in parallel, providing a measure of each tagged protein's localization under the assayed conditions. This approach might logically be combined with immunofluorescence experiments, as a major advantage of the cell chips is the minimal use of expensive reagents on the chips, achieved by limiting the use of antibodies and dyes to single microscope slides. Imaging entire libraries on chips also results in reduced imaging times in comparison to, for example, imaging the 50 96-well plates required for the complete GFP tagged collection.
In this study, we have attempted to map the changes in localization of the yeast proteome upon formation of a mating projection. Although individual proteins that localize to the shmoo tip have been characterized (e.g., the shmoo tip marker Fus1(12)), proteome-wide screens have not been performed to measure such localization changes due to their expensive and cumbersome nature. We developed and implemented a cell microarray-based imaging assay for measuring the spatial redistribution of a large fraction of the yeast proteome, and applied this assay to identify proteins localized along the mating projection following pheromone treatment. By further incorporating information about known yeast gene associations and about protein localization during vegetative growth, we trained a machine learning algorithm to refine the cell imaging screen, resulting in a total of 74 proteins identified that specifically localize to the mating projection. Functional analysis of these proteins, coupled with analyses of individual organelle movements during shmoo formation, suggests a model in which the basic machinery for cell polarization is generally conserved between processes forming the bud and the shmoo, with a distinct subset of proteins used only for shmoo formation. The net effect is a defined ordering of major organelles along the polarization axis, with specific proteins implicated at the proximal growth tip.
Spotted cell microarrays were manufactured from the S. cerevisiae GFP tagged clone collection (Invitrogen), in which each of ~4200 individual strains with genetic background EY0986 (ATCC 201388: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 (S288C)) was chromosomally tagged with the coding sequence of A. victoria GFP (S65T) at the carboxy-terminal end of an open reading frame.(9) This collection was copied in 96 well plates using a Biomek FX liquid handling robot, inoculating each strain into 200 μL of YPD containing 17% glycerol, growing at 30 °C for ~2 days without shaking, mixed on a plate shaker, and sealed and frozen at −80 °C.
GFP-tagged strains were treated with alpha factor in the following manner: A copy of the GFP clone collection was thawed from −80 °C and a 1% inoculum used to seed a fresh copy in YPD medium. After growth to near saturation for ~36 h at 30 °C, this intermediate copy was used at 1% to inoculate another such one in YPD. This copy was grown overnight at 30 °C and was washed three times to inactivate secreted extracellular Bar1p protease that degrades alpha factor. Subsequently, alpha factor was added to each sample well in the collection at 75 μg/mL final concentration and allowed to incubate for ~3 h at 30 °C. The samples were fixed using freshly prepared 2% formaldehyde for 1 h at 30 °C and the excess fixative was removed by three successive washing rounds of YPD containing 17% glycerol before resuspension in 100 μL of YPD plus 17% glycerol. These sample plates were stored at −80 C or directly printed.
Cell microarrays were printed onto freshly treated poly-l-lysine coated slides via contact deposition of a suspension of yeast cells from the arrayed yeast collection using a DNA microarray spotting robot. Locations of cell spots were identified by scanning the freshly printed slides using a GenePix microarray scanner, then cells in each spot were imaged using an automated Nikon E800 fluorescence microscope and Photometrix Coolsnap CCD camera as in Narayanaswamy et al.,(10) collecting DIC images and fluorescent images in the DAPI and GFP channels from each spot, on two replicate slides, for a total of ~16000 microscope images per slide. Images were stored and analyzed using the Cellma cell microarray image database software.(10) Two independent graders manually inspected the images, selecting strains in which the GFP-fusion protein showed punctate localization proximal to the shmoo tip. Manual follow-up assays were then performed in the absence of fixative two or more times on independent live cultures (biological replicates), requiring reproducibility of shmoo localization in all assays. Additional cell microarrays were printed and imaged from the GFP-ORF strain collection in the absence of pheromone, serving as a negative control for the localization. This set also allowed comparison of cell microarray-based localization measurements with those observed in the original analysis of the strain set.(9) For all shmoo-tip localized proteins identified, our untreated controls matched the published localizations, but the proteins localized to the shmoo in the presence of pheromone in all (≥3) replicate analyses.
Doubly fluorescent strains for colocalization were constructed by mating and tetrad dissection. Red fluorescent protein (RFP) tagged marker strains (EY0987; ATCC 201389: MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 (S288C)) for nine organelles and cellular structures (gift of the laboratory of Erin O'Shea) were mated with the FUS1-GFP strain (EY0986), and diploids selected on Lys(−) Met(−) media. After sporulation, tetrads were dissected and doubly fluorescent 'a' type yeast propagated.
Quantitative image analysis was performed using custom MATLAB image processing software. Primary software features include background noise removal, cell region identification, cell boundary segmentation, and subcellular feature description. All fluorescent images underwent background noise removal which was accomplished through background image subtraction and median filtering to remove speckle noise. Yeast cells were identified by implementation of the Kittler and Illingworth automated thresholding method(13) on composite images for each field of view. These composite images were created by stacking all fluorescent channels as well as a histogram-modified DIC image. Cell boundary segmentation was accomplished using a seeded watershed algorithm in which the cell seed was generated using a fluorescent image of the DAPI stained nuclei. When DAPI stain was not available, cell segmentation was performed manually through image annotation in ImageJ.(14) To remove improperly segmented cells, automatic cell segmentation results were required to meet predefined minimum and maximum cell size restrictions. Subcellular feature identification was accomplished using cell-by-cell automated thresholding on each of the fluorescent channels.
Still images of the RFP/GFP/DAPI labeled yeast following alpha factor exposure were analyzed as above. Yeast cells identified in the images were computationally reoriented along the axis between the center of fluorescence of the DAPI stained nuclei and the GFP labeled shmoo tip. These rotated cells were then stretched or compressed so that the distance between these two points was uniform for all cells. After these transformations, each cell in the still images was overlaid so that RFP labeled organelle localization could be visualized.
To minimize false negative observations from the high-throughput screen, we trained a naïve Bayesian classifier(15) to identify additional shmoo-tip localized proteins. For the training set, we employed the proteins identified in the high-throughput screen. Classifier features were aggregated from data provided by the UCSF GFP screen(9) and the functional gene network of Lee et al.16,17 The features collected for each gene were the sum of the gene's network log likelihood scores (LLS) to the set of training genes, the ratio of the sum of the gene's LLS scores to the training set genes divided by the sum of LLS scores to all other genes, protein abundance (molecules per cell), and cell location (using primary cellular location). All features were numeric values except location which was treated as a discrete estimator (e.g., binary flags for nucleus, bud neck, etc.). The classifier was implemented in Weka and applied to the set of 5804 yeast genes, either using all training genes as the training/test set or using 10-fold cross-validation (both gave similar results). The classifier proved reasonably predictive, with an area under a 10-fold cross-validated receiver-operator characteristic (ROC) curve of 0.843. At the score threshold selected for experimental validation, shmoo-localized proteins were predicted with a cross-validated true positive rate of 0.51 and a false positive rate of 0.027. This corresponds to recovering 20 of the 37 shmoo genes (cross-validation: 19). An additional 151 (cross-validation: 153) genes not identified in the initial screen were also classified as shmoo genes using a 0.5 probability cutoff. Of the 151 genes, 118 were present in the GFP library and were rescreened manually; 37 of these proved to be shmoo localized.
It has been previously observed that organelles move during the pheromone-induced, polarized growth of yeast.(18) To establish conditions for screening for genes involved in polarized growth, we used fluorescence microscopy to identify reproducible trends in organellar movements. We systematically measured the subcellular localization of 9 major organelles via organelle-specific fluorescent protein fusion markers. For each organelle, we constructed a yeast strain expressing both a red fluorescent protein (RFP)-tagged organelle marker, and an orientation marker, a green fluorescent protein (GFP)-tagged shmoo tip marker, Fus1. The positions of the two fluorescent protein markers and the nucleus (detected by staining with DAPI dye) were determined before and after treatment with the mating pheromone alpha factor. Image analysis was used to quantify each organelle's spatial distribution across >100 individual cells in an image (Figure (Figure1).1). Organellar positions were calculated after reorienting and scaling each individual cell in the image to a common reference frame (the axis from the nucleus to the shmoo tip).
Protocol for quantifying organellar localization following pheromone treatment. Synthetic alpha factor was added to vegetatively growing cells containing GFP tagged Fus1 and one of nine different RFP tagged organellar marker proteins. Computational image ...
Each organelle marker strain (Figure (Figure2)2) strongly revealed the formation of an axis of polarity, presumably toward the pheromone gradient in anticipation of the mating process and subsequent cellular fusion. Several common features about organellar arrangement were evident: The nucleated actin filaments that form the actin cables were polarized toward the shmoo tip (as evidenced by Sac6-RFP fusions, the cross-linking fimbrin protein used as the marker). This was consistent with actin cables acting as 'conduits' for transporting organelles during mating, just as they do during vegetative growth.(4) The spindle pole body core component, Spc42, was prominent in its movement toward the shmoo tip, indicating that the tethered spindle microtubules, the nuclear envelope and the chromosomes were also reoriented.18,19 In consequence, the entire nucleus was also reoriented toward the shmoo tip, with the nucleolus being preferentially located at the opposite side of the nucleus relative to the shmoo tip. Another consequence was extensive vesicular trafficking of cargo intended for membrane deposition and new cell wall formation at the site of the shmoo projection (as evidenced by localization of clathrin protein Chc1 with Fus1-GFP). No bias toward the polarization tip was observed for the endoplasmic reticulum marker Sec13, a component of the Nup84 nuclear pore complex and COPII complex. As expected, there was a broad distribution of a Golgi cisternal marker, Anp1, both toward and away from the shmoo tip. While polarization was along the shmoo tip axis, transport may occur in both directions. The endosome marker, Snf7 tended to accumulate away from the shmoo tip, possibly implicating the involvement of the endocytic pathway in trafficking components between the membrane and the vacuole (for example, the pheromone receptor Ste2 which upon pheromone binding and ubiquitination, is internalized and endocytosed into vesicles).(20) Analysis of yeast expressing the GFP-tagged mitochondrial marker Ilv3 (data not shown) revealed mitochondria throughout the shmooing cells, occasionally shmoo-tip localized but also generally found widely distributed.
Organelles are differentially distributed along the polarization axis. Results of rotating and overlaying thresholded fluorescent intensity distributions for all shmooing cells from multiple still images are shown for each of nine organellar markers. ...
Overall, our results are consistent with the establishment of an alpha factor-induced polarization axis which leads to the spatial reorganization of multiple organelles in a roughly ordered fashion from proximal to the shmoo tip to the distal end of the cell.
Having established a reproducible cell-by-cell organellar reorganization along the polarization axis, we attempted to identify the numerous proteins and protein complexes that mediated these cell-wide changes in morphology and distribution. We therefore undertook a systematic, high-throughput analysis of alpha factor-induced changes in protein localization of GFP-tagged yeast strains. It should be noted that a comprehensive survey of proteins localized to the shmoo tip has not previously been carried out despite many studies21−24 that have implicated key roles for individual proteins in this dynamic cellular compartment. Building on our previous success in constructing and screening yeast cell microarrays,(10) we screened the collection of GFP-tagged yeast strains for pheromone-induced changes in protein localization.
An overview of the cell microarray assay is presented in Figure Figure3.3. Briefly, after treating the library of GFP-tagged fusion protein expression strains with alpha factor, we fixed and robotically printed(9) the strains onto poly lysine-coated microscope slides(10) and imaged the fixed strains using automated microscopy. We screened for proteins showing a clear localization proximal to the shmoo tip. In all, 187 proteins were chosen from the initial screen, based on a lenient criterion that included even marginal examples. These 187 strains were then manually retested in the absence of fixative, which improved signal-to-noise value because of reduced background fluorescence. The follow-up screen yielded 37 strains in which GFP fusion proteins were consistently localized to the shmoo tip (Table (Table11).
Schematic of the work flow for performing the proteome-wide localization screen. The GFP tagged yeast library was treated with mating pheromone, the cells were fixed, and then microarrayed onto a microscope slide. High-throughput microscopy of the arrayed ...
Manually Verified Shmoo Tip Localized Genes Identified by the Cell Chip
Statistical analysis of the functions present(25) showed a significant enrichment for several pathways and processes, including genes involved in secretion/exocytosis, budding/cell polarity, cytoskeletal organization, and pheromone response/mating specificity. Representative examples are shown in Figure Figure44.
Representative components of cellular systems found localized to the shmoo tip. Each panel shows a few cells from one yeast strain expressing a GFP-tagged protein localized to the shmoo tip following pheromone response. Components of the exocyst were ...
In contrast, of the 150 proteins not confirmed by the manual follow-up imaging assay, roughly 65% were either mitochondrial proteins or proteins that were localized at the shmoo neck or at the base of the shmoo tip. These classes were obviously difficult to distinguish from true proximal shmoo localization, leading to their inclusion in our initial lenient screen. The remaining 35% of the excluded proteins exhibited punctate structures not reproducibly associated with the shmoo tip (e.g., peroxisomal proteins). The high-throughput survey recovered about 15% of the SGD-annotated, shmoo-localized proteins (Figure (Figure5,5, Table Table1).1). This high false negative rate is likely due to fixation induced autofluorescence, which masks low-abundance proteins.
Venn diagram summarizing the proteins recovered. (A) Intersection of the cell chip and classifier identified proteins with the known set of shmoo tip localized proteins as annotated in the Saccharomyces Genome Database. (B) Of the 306 strains manually ...
To reduce the high false negative rate, we took advantage of near genome-wide data sets of protein localization in vegetative growing yeast cells9,26 and an integrated probabilistic gene network16,17,27 of functional genomic and proteomics data in order to develop a machine learning algorithm for predicting additional proteins localized to the shmoo tip (for example, proteins that had been missed because of low expression or penetrance). As detailed in , our initial set of 37 proteins obtained from the cell chip screen was used to train a naïve Bayesian classifier. Application of the classifier identified 151 proteins exceeding a 50% probability score threshold. An advantage of this approach is that the delimited set of candidate genes could then be individually assayed in the absence of fixative, a task that was greatly simplified because 118 of the 151 proteins were already present in the extant GFP library. We manually retested each of these 118 GFP fusion strains for protein localization to the shmoo tip. From this set, 37 additional proteins (~31%) were confirmed to be shmoo-localized (Table (Table22).
Manually Verified Shmoo Tip Localized Proteins Identified by the Classifier
The classifier-guided retesting strategy doubled the coverage of the screen, and any remaining false negatives could be rationalized based upon low protein abundance. Of genes in the GFP library, we could identify 63% of the known shmoo tip-localized proteins that were present at >2500 molecules/cell, but less than 21% of known proteins with <2500 molecules/cell. On average, the shmoo tip proteins identified via the classifier method were less abundant than those recovered via the cell chip method lending credence to using a network guided approach to expand an initial list of seed genes. Although we were interested in specifically identifying proteins localized to the shmoo tip and therefore picked the highest confidence linkages, it must be mentioned that this process might be iterated to potentially reveal more genes that are related to the pheromone response pathway.
The 74 shmoo-tip localized proteins (37 from the cell microarray screen, 37 from computational prediction and screening) showed a marked enrichment for Gene Ontology functional categories related to polarized growth (with p < 10−6 being the threshold of probability calculated using a hypergeometric distribution(28) that the intersection of given list with any functional category occurs by chance), with the strongest enrichment observed for the GO Biological Process annotation establishment of cell polarity (p < 10−35), followed by annotations including anatomical structure morphogenesis (p < 10−32), cellular bud site selection (p < 10−29), cytokinetic process (p < 10−28), vesicle-mediated transport (p < 10−22), reproduction (p < 10−20), endocytosis (p < 10−18), actin filament orgnanization (p < 10−13), exocytosis (p < 10−12), and conjugation (p < 10−6). More specifically, we note that many of the proteins localized to the shmoo tip belong to protein complexes that are also involved in mediating polarized growth at the bud tip during vegetative growth (Figure (Figure5).5). Thus, there appears to be broad adaptive reuse of the polarization machinery between these two processes. In addition, 41 of the 74 proteins have human orthologs (Tables (Tables11 and and2),2), indicating the general conservation of these processes across eukaryotes.
The exocyst is an evolutionarily conserved octameric protein complex thought to be targeted to the plasma membrane through the interaction of Exo70 with phospholipids(29) and stabilized by Sec3.(30) One of the major functions of the exocyst is to direct docking of myosin motor driven vesicles at the bud site during vegetative growth; the peripheral exocyst component Sec3 has also been previously implicated in pheromone-induced polarization.(31) We show here that all 8 subunits of the exocyst localize to the shmoo tip (Figures (Figures44 and and6),6), strongly supporting a conserved role in the polarization machinery of the two polarized growth processes.
Spatially distinct localization of different vesicle transport processes is apparent along the shmoo tip. Several representatives of each process are shown. Proteins involved in exocytosis, including components of the exocyst, are tightly localized to ...
The pentameric septin ring, a collar-like structure that is one of the first organizing components at the site of polarized growth, may also be localized at the shmoo neck,4,32 given that a number of its component proteins (Cdc10, Cdc11 and Shs1) were recovered in our screens (Figure (Figure4).4). Cdc12, another septin component, displayed filamentous structure and orientation toward the polarization axis. However, Cdc3, the fifth component, was absent from the GFP library, and thus could not be identified by either screen.
On a larger scale, there is clearly remodeling of the actin cytoskeleton and vesicular transport (to enable membrane and cell wall deposition) during shmoo formation,(33) and these proteins were heavily represented in our screens (Figures (Figures44 and and6;6; Tables Tables11 and and2).2). Sac6, a fimbrin protein involved in bundling actin monomers to filaments, was localized at the shmoo tip, as was the twinfilin Twf1, a protein that assists in actin monomer localization to sites of rapid filament formation.(34) Twinfilin localization is assisted by the cap binding proteins Cap1 and Cap2,(34) which were also recovered in the screen. The dynamic nature of actin reorganization at the shmoo tip was indicated by the identification of Aip1, which is known to regulate cofilin mediated actin depolymerization.(35)
The actin regulating kinases Ark1 and Prk1 were found and are known to modulate actin cortical patch components and endocytic pathways through phosphorylation cycles.36,37 Substrates of Ark1 and Prk1 that were recovered included Sla1, Sla2, Pan1, Ent1, and Ent2 suggesting the involvement of the Pan1-End3-Sla1p complex that is known to be required not only for actin cytoskeleton organization but also for normal cell wall morphogenesis.(38) Srv2, another tip-localized protein, binds adenylyl cyclase and ADP-actin monomers and may facilitate regulation of actin dynamics and cell morphogenesis.(39)
Consistent with the idea that Sla1 couples proteins in the actin cortical patch with the endocytic machinery,40,41 we observed cortical patch proteins such as Las17 (homologue of human Wiskott Aldrich syndrome protein), Vrp1 (homologue of human wasp interacting protein), Abp1 and Abp140 as well as proteins involved in endosomal vesicle trafficking, endocytosis and exocytosis at the shmoo tip. For instance, proteins such as Sfb3, Sec31 and Shr342,43 mediate sorting of COPII vesicles between the ER and Golgi, and Sec18,(44) which is involved in post-Golgi vesicular transport. Other endosomal pathway proteins such as Bsp1 that functions as an adaptor linking the synaptojanin protein Inp52 implicated in TGN-to-early endosome sorting to the cortical actin cytoskeleton(45) were also identified. The type V myosin, Myo2 along with its interacting partners and cargo peroxisomes compose the observed vesicular transport phenomena directed at the shmoo tip. Myo2, for example, interacts with Chs5 to deliver chitin synthase enzyme Chs3 and with Smy1 to transport vesicles, and organelles such as vacuole(46) toward the tip. The final step involves docking and tethering of the endosomal vesicles to the site of polarized growth. Sec2,(47) a protein that functions as the Guanine nucleotide exchange factor for the rab GTPase Sec4, mediates vesicle tethering to the exocyst and localizes at the shmoo tip. Proteins involved in cellular processes such as maintenance of cell wall integrity (Rgd1, Slg1, Wsc2, Wsc3) and in cell fusion (Kel1, Kel2 and Fus1) are tip localized as well as tying polarization events and pheromone specific functions at a systemic level.
An interesting observation made from the shmoo tip localization screen was the relative positioning of the proteins that were involved in various phases of vesicle mediated transport in our images (Figure (Figure6).6). Proteins that were involved in exocytosis, such as the components of the exocyst, were spatially situated tightly at the shmoo tip as compared to the rest of the proteins from the screen. These proteins are involved in the final stages of protein transport such as protein docking and secretion and presumably indicate regions of membrane-vesicle interactions. In contrast, proteins of endocytosis localized more broadly around the shmoo tip, often with a punctate distribution possibly defining hot spots of endocytosis, and including proteins such as Sla1, Las17 and others. Formation of such hot spots is still an active area of research with recent discoveries being made in yeast of 'eisosomes' that appear to mark endocytic sites.(48) The actin cytoskeleton and cortical proteins play important roles in such processes with Rvs161 (also identified in this screen) interacting with eisosomal marker proteins.(27) Lastly, proteins involved in ER-Golgi and trans-golgi to plasma membrane vesicle trafficking showed a definite bias of GFP signal toward the shmoo tip, presumably reflecting vesicular transport, but were even more broadly distributed around the tip than the exocytic or endocytic proteins.
Several uncharacterized proteins recovered in our screen, such as Ymr295c,(49) Ydr348c, and Yor304c-a, localize to both the bud(9) and shmoo tips suggesting a role in the general polarization machinery. Examination of these proteins' functional associations in the yeast functional gene network(27) provides some suggestions for their connection to particular aspects of polarized growth (Figure (Figure7A):7A): Yor304c-a is most tightly functionally associated with Bud6, a central protein in signaling polarization (also recovered in the screen) and Duo1, a cytoskeletal protein. Ymr295c and Ydr348c link to each other, with the former also strongly associated with glycolytic transcription factor Gcr1, and the latter associated with cell cycle progression genes Clb2 and Cdc28. This may suggest a role for these genes connecting polarization with processes such as cell cycle state and metabolism.
For uncharacterized proteins identified in the screen, some insights into their roles in polarized growth can be obtained from their functional associations in gene networks. The gene neighbors (yellow circles) of five uncharacterized proteins (red circles) ...
In addition to proteins playing a role in polarization during vegetative growth and mating, we identified a set of proteins that may be unique to one or the other process. Among the 81 false positive GFP-tagged strains predicted to be shmoo tip-localized by the classifier, several were involved in vesicular transport with 18 localizing to the bud tip (Figure (Figure5).5). In other words, even though the computational method did not always directly predict localization to the mating tip, it predicted proteins involved in processes very similar to and possibly mechanistically overlapping with shmoo formation. For example, Boi2 and Bnr1 were conspicuously absent from the shmoo tip but were present at the bud tip and may reflect differential organization and positioning of the cytoskeletal architecture during pheromone induced mating as compared to vegetative buds.(50) In contrast, the formins Bni1 and Boi1 were found proximal to the shmoo tip consistent with their role in remodeling this dynamic locale.(51)
Additional proteins were found, emphasizing the differential sets of interactions driving the processes of mating and cell fusion. The shmoo tip marker Fus1(12) was recovered, as was Bck1, a cytosolic protein kinase in vegetative cells that appears to concentrate at the polarized shmoo tip. This is presumably due to its role in regulating cell wall integrity, a process closely connected to the mating response pathway.(52) Similarly, Mid2, a cell wall sensor normally distributed uniformly around the cell wall(9) shows a 3-fold increase in expression levels upon pheromone stimulation.(53) Its biased distribution around the shmoo tip is indicative of the increased occurrence of cell wall deposition at the site of polarized growth.(54)
Yer071c, a protein annotated as having a punctate composite cytosolic localization, and Ycr043c, a Golgi annotated protein,(9) were also recovered and have not been previously implicated in polarization. Some suggestions for more precise function can again be gained from the yeast functional network (Figure (Figure7B),7B), which connects Yer071c with actin cap binding proteins Cap1 and Cap2, while Ycr043c links to Rvs161, a lipid raft protein, all of which we observed at the shmoo tip. In addition, Car1, an arginase that when deleted causes cells to undergo G1 cell cycle arrest upon starvation(55) and Lsg1, a protein involved in 60S ribosome biogenesis are two other proteins we identified in this screen, but that were not obviously mating related. Our screen therefore adds evidence and provides putative hypotheses for the involvement of such uncharacterized proteins in the mating polarization pathway.
We report a large-scale imaging-based screen for proteins showing spatial localization to the growing tip of the yeast mating projection following pheromone response. A summary of our findings is presented in Figure Figure8.8. Our screen has enabled us to align the proteome down to the level of individual proteins identified along the polarization axis induced by pheromone stimulation. Upon the basis of this systematic assay, we demonstrate that the bulk of shmoo-tip localized proteins are conserved with those localized to the bud tip during vegetative growth. These data therefore support a model for the adaptive reuse of polarized growth machinery between different polarized growth processes. For example, Huh et al.(9) had shown that actin related genes are structured along the polarization axis during budding. We show that this observation is true during mating as well. This process is not automatic since the budding and mating projection sites are distinct and therefore the point of origin of cellular asymmetry is likely also different. Moreover, the 67 proteins shared between the mating and budding polarization processes (Figure (Figure5)5) are significantly more conserved between human and yeast than expected by chance (39 of the 67 yeast genes have human orthologs,(56)p < 0.023, chi-square test), indicating that the functions of these core polarization components are likely to be preserved across eukaryotes. Although the process of establishment of cell polarity by proteins such as Cdc42, Cdc24 and Far1 are well-studied, the exact molecular mechanisms toward predictive cellular models are still wanting.(57) Our efforts are a first step toward screening proteome-wide dynamics to understand precisely how biological systems spatially coordinate protein interactions and regulate their dynamics.
Our screen recovered 37 proteins from the cell chip (out of 188 strains manually retested) and 37 from the classifier (out of 118 manually tested). This set had an overall intersection of 13 strains with the known set from SGD (Figure (Figure5).5). A majority of known genes that this screen missed were low-abundance signaling proteins with signals too weak to be detected by GFP alone. In the 118 strains tested for the classifier, there were 81 false positives that did not localize to the shmoo tip. Of these 81, 18 were annotated to be bud localized and included bud specific formins such as Bnr1 and Boi2. Of the proteins recovered in this screen, 7 were unique to shmoo tip. These findings, summarized in Figure Figure8,8, emphasize the involvement of a differential set of protein interactions driving each process toward their separate goals after the initial shared polarization machinery has been put in place.
Finally, this screen uncovers novel players in the polarization pathway. For example, proteins such as Ymr295c, Yor304c-a, and Ydr348c showed marked localization to the polarized growth tip in both budding and shmooing and thus likely represent conserved components of the general polarized growth pathway.
We thank Vishy Iyer for critical comments and help with array printing, Alice Zhao for help with imaging, and Erin O'Shea for the RFP-tagged organellar marker yeast strains. This work was supported by grants from the NIH (GM06779-01, GM076536-01), Welch Foundation (F1515, F1654), and a Packard Fellowship (E.M.M.).
National Institutes of Health, United States
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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- English
- français
Pathology of Monin-Obukhov similarity in the stable boundary layer
The performance of Monin-Obukhov similarity (MOS) theory has been investigated for four types of events of transient physical processes in the stable nocturnal boundary layer during the Cooperative Atmosphere-Surface Exchange Study-99 (CASES-99). These four types were internal gravity waves, Kelvin-Helmholtz (K-H) shear instability, a density current, and a low-level jet. It was found that the MOS theory generally holds whenever a new equilibrium is reached or recovered during or after these events but fails in the period of transition or the developing stage of the events with newly generated turbulence. Specifically, MOS was found to hold during the entire process of the nonbreaking internal gravity waves and during the well-developed stages both of the density current and of the low-level jet. It was found not valid during the process of the K-H instability and the developing stages of the density current. Characteristic features of these four types of events are also presented and analyzed.
- View record in Web of Science
Keywords: LOW-LEVEL JET; PROFILE RELATIONSHIPS; TURBULENCE STRUCTURE; STRONG STABILITY; DENSITY-CURRENT; GREAT-PLAINS; HEAT; WAVE; TEMPERATURE; CASES-99
Record created on 2005-09-08, modified on 2017-02-23

subset: commoncrawl


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It's been 13 years since Underworld hit theaters, bringing us a never ending war between vampires and lycans (a.k.a. werewolves) with Kate Beckinsale caught in the middle. The flick was a surprise hit, spawning three sequels filled with death, destruction and leather outfits. And if you thought 2012's Underworld: Awakening was the end of it all, you'd be wrong, because there's a brand new sequel set to hit theaters early next year.
The first trailer for Underworld: Blood Wars was released today, and it looks like we'll be getting even more of the franchise's edgy action. Kate Beckinsale returns as Selene, an immortal vampire warrior forced to fight in the battle between the blood-sucking vampires and their furry lycan enemies. This time out she's ready to end the war for good, but it won't be easy, as she's on the run from both lycans and the vampire clan that betrayed her.
If you haven't kept up with the Underworld franchise, don't worry. All you really need to know is that vampires and werewolves are at war and they'll use whatever it takes to kill each other, from guns and swords to fists and fangs. Also, everyone's a leather-clad, combat-ready badass. If you weren't convinced, check out our breakdown of the most killer moments in the first Underworld: Blood Wars trailer.
1. That's A Vampire Riding A Horse
The trailer jumps back in time early on to give us a look at the beginnings of the war between vampires and lycans, complete with a armored vampire warrior outrunning two werewolves on horseback. He's also got a sweet sword that pops out some spiky metal disks perfect for wolf hunting. Here's to hoping they fly backwards and kill those hairy terrors.
2. Werewolves At War
The Underworld movies may deal with werewolves and vampires, but they lean more into the action genre than horror, as seen in this epic lycan army assault. The brute strength of the lycans is on full display during their assault on the ancient vampires, promising some sweet medieval flashbacks to complement the modern story.
3. Selene Dodges A Falling Helicopter
Selene's vampire powers give her some pretty incredible strength, speed and reflexes, seen firsthand as she dives away from this helicopter crash. Being a vampire would probably have helped her survive the crash anyway, but at least she won't get bruised up so bad.
This one's a bit of a cheat since it's actually a clip from the 2003's Underworld, but c'mon this is a defining moment from the series. It epitomizes the franchise's dedication to creative action, even if it comes across as a bit self-serious 13 years later.
Are you a vampire fan? Check these out for more:
- Why Buffy The Vampire Slayer Is One Of The Most Influential Shows Of All Time
- 22 Years Later And The Cast Of Interview With The Vampire Are Still Killing It
- 12 Facts You Didn't Know About the Twilight Franchise
5. Holy Crap, It's Tywin Lannister
Thankfully, Selene has some help on her mission from Thomas, the leader of one of the world's last remaining vampire clans. Game of Thrones actor Charles Dance returns as the character, who was first introduced in Underworld: Awakening, and we expect to see a performance that would make Tywin Lannister proud.
6. Blood Tastes Extremely Good
In case you forgot this movie has vampires in it, the trailer dedicates some time to some good 'ol fashioned blood drinking. But this is classy, so the vamp's drinking from a beautiful glass chalice instead of straight from a victim's veins.
7. Showing Off Some Fangs
Thomas isn't Selene's only ally, as she also has some help from his son David. Actor Theo James took some time off the Divergent series to return as the character, and probably had a lot of fun going crazy for this scene.
8. Look, It's The Title
Remember those spiky disk weapons from earlier in the trailer? Well, they're back, and this time Selene's tossing them out to create the title graphics for the movie. Does that mean she knows the centuries-long war she's been fighting is actually a film designed for our amusement? Woah.
9. Selene's Disappearing Act
We get an extra look at the action after the titles appear as Selene shows off her hand-to-hand combat skills. Luckily she has the ability to simply disappear if the fighting gets to be a little too tough.
Underworld: Blood Wars will rage war on audiences when it hits theaters on January 6, 2017. Are you excited for Kate Beckinsale's next outing as the vampiric Selene? Let us know in the comments below.


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CEVDCUS06Contemporary English Version
6 # Is 55.1,2; Si 24.21. God blesses those people
who want to obey him#5.6 who want to obey him: Or "who want to do right" or "who want everyone to be treated right."
more than to eat or drink.
They will be given
what they want!
Contemporary English Version® © 1995 American Bible Society. All rights reserved. Bible text from the Contemporary English Version (CEV) is not to be reproduced in copies or otherwise by any means except as permitted in writing by American Bible Society, 1865 Broadway, New York, NY 10023 (www.americanbible.org).


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The Andes Mountain Range:
The longest Mountain Range in the World
Over 4,400 Miles Long!
The longest Mountain Range in the World
Over 4,400 Miles Long!
Located along the entire western coast of South America, the Andes mountain range is about 4,500 miles long. The mountain range spans seven countries — Venezuela, Colombia, Ecuador, Peru, Bolivia, Chile and Argentina — and can be found between 10 degrees north and 57 degrees south latitudes and 70 degrees to 80 degrees west longitude. It is up to 500 miles wide.
The origin of the name Andes has not been definitively determined; most agree that it comes from the Quechua word anti, which translates to "high crest." A minority argue that it comes from Anti Suyu, which is one of the four regions of the Incas who once inhabited the range.
Want to know what it's like flying over the Andes Mountains? Watch this video ---------------------------------->
The film below is a great documentary done by the BBC about the Andes


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Carnivorous Plant with 'Water Fresh' pot
Will keep pests under control naturally
Amazing 'Water-Fresh' pot - Reservoir insures your plants have enough water
Uniquely attractive looks - All individual in character.
Like having your own pet - Excellent gift for Children and Adults alike.
Please note: Only one plant will be supplied and may not be exactly as shown.
Adults and children will be fascinated by this mix of carnivorous plants. These plants get most of their nutrients from trapping animals, and are uniquely attractive. These plants will eat small insects around your office, home or classroom, and are a natural way to keep pests under control. They are not difficult to grow, especially with a 'Water-Fresh' pot. This pot has a reservoir built in, meaning your plant's needs are taken care of. Simply fill again when empty. It's like having your very own pet, without the total commitment.
Pot size 6cm. Height (inc pot) 20 cm approximately.


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Finances Accounting plays an important role in any business. Why do you think it is important for a business to keep track of its expenses?. Lesson Objective Complete a payroll register. Content Vocabulary. payroll register
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Accounting plays an important role in any business.
Why do you think it is important for a business to keep track of its expenses?
Complete a payroll register.
Content Vocabulary
payroll register
A record of the gross income, deductions, and net income of a company's employees.
Natural Foods Center pays its employees weekly.
Mary Clark prepares the payroll register for the center's five employees from the following information.
Clark has prepared the payroll register for the week of March 2.
Step: Find the net pay for the week.
Ajani Samuel is a printer employed by Kwik Copy. He is single and claims no allowances. Determine his net pay for a week when his gross pay was $285. Medicare is 1.45 percent of gross, Social Security is 6.2 percent of gross, and city tax is 1.5 percent of gross. Use the tables on pages 788-791 for federal income tax (FIT).
Step: Find the net pay.
Medicare: (0.0145 × $285) = $ 4.13
Social Security: (0.062 × $285) = 17.67
City tax: (0.015 × $285) = 4.28
$285.00 – ($4.13 + $17.67 + $4.28 + $29.00) =
$285.00 – $54.08 = $230.92
Find the total net pay.
Gross pay: $399.70
Federal income tax: $10
State income tax: $6.40
Social security: 6.2 percent of gross
Medicare: 1.45 percent of gross
Health insurance: $65
Find the net pay.
Total gross pay: $24,600
Total deductions: $9,930


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Diploma Two Semesters & One Summer Term Fall semester entry only F__a_ll_ S_e_m__e_s_t_e_r ________________________________________1_8_ Course # Title Credits CON160-C Principles of Carpentry I 5 CON176-C Carpentry Lab I 10 Elective Math* 3 _S_p_ri_n_g_ _S_e_m__e_s_te_r______________________________________2_2_ CON161-C Principles of Carpentry II 5 CON178-C Carpentry Lab II 10 CON164-C Design & Cost Estimating 4 Elective Communications* 3 _S_u_m_m__e_r_ T_e_r_m__________________________________________7_ CON169-C Principles of Carpentry III 2 _C_O_N_1_9_3_-_C______C__a_rp_e_n_t_ry_ _L_a_b_ I_II__________________________5_ * You may select any math or communications course with A, C or E suffix. Students should check specific course requirements under the Construction Technology program if they are considering completing the AAS Degree option. CARPENTRY As a carpentry student, you will get hands-on experience in the shop as well as field experience through the frameto finish construction of an energy efficient two or three bedroom home. This will include job site safety; floor, wall and roof framing, door and window installation/finish, drywall installation and finishing, cabinet and countertop installation, exterior finish such as roofing, siding and soffits, along with classroom time to discuss the theory behind these concepts. An emphasis is placed on the care and use of hand and power tools, proper use and function of equipment and materials, as well as an emphasis on the 'House as a System' concept. 'Green' building concepts are discussed and utilized. Building design/estimating and CAD drawing will be taught as well as math and communication skills. Successful students become employed in an industry with the increasing need for skilled carpenters. For more information about our graduation rates, the median debt of students who completed the program, and other important information, please visit our website at www.nwicc.edu/compliance. 84 Northwest Iowa Community College Catalog 2017–2018
To see the actual publication please follow the link above


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This Frame it All Two Stepper Raised Garden Bed is easily assembled without any tools. The Classic White Composite Timbers are scratch-proof, hose down or wipe clean. They are connected at each corner with secure snap-on, screw-less Stacking Joints in a striking, dense black contrast. This premium garden combines functionality and elegance and is extendable in every direction by adding timbers and joints; to showcase your produce or flowers.
- Screw-less stacking joints
- Adjusts easily to a variety of heights, shapes and sizes
- Eco-friendly made from recycled plastic
- Five year warranty
- Flowers and mulch not included


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From time to time we hear that Brazil has been a great success in becoming more energy self-sufficient especially through an aggressive (meaning heavily subsidized) sugar-ethanol program. But note this story in today's New York Times: Brazil is going to nearly double its investment in offshore oil and gas exploration, to $174 billion over the next five years. This at a time of low oil and gas prices.
So by all means, let's use Brazil as an example of sound energy policy.


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MOVIE SYNOPSIS [ General Audiences ] With the main performance of Bug Hall, this film directed by Mathew Miller was premiered in cinema or TV in 2015. The film "Subterranea" had an original production of USA. You can watch Subterranea online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2015.
Rating: 5 by 12 users./
Runtime: 99 minutes
Bug Hall, William Katt, Nicholas Turturro, Lily Gladstone, Tashia Gates, Ken White, Derrick Shawn, Amber Mason, Russ Gay, Dan Molloy, Joseph Grady, Katie Kohler, Kyndra Gilvarry, Ali Tabibnejad, David Mills-Low and Ann Peacock.
99-minute film (full movie) directed by Mathew Miller.
The original audio of Subterranea has dialogues in Dolby Digital, it was produced in USA.
It premiered in cinemas or television in 2015.
Screenplay written by Peter Nicholls and Mathew Miller.
Brandon Woodard.
Keith Miller, Eric D. Wilkinson, Andrew Collier, Peter Huth and Mathew Miller.
Composed by Michael Holmes.
Without commercial breaks, the full movie Subterranea has a duration of 99 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, Subterranea in FULLTV Movies.


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Deficit Trailer (2007)
"When you have it all....you don't have anything" 21 May 2007 Drama, Foreign 79 mins
Déficit follows one day of crisis in the life of Cristobal (Gael Garcia Bernal), a spoilt, rich kid throwing a party for his friends at his parents' luxurious villa. A big fence and a live-in staff of servants are there to shield Cristobal from the harsher realities of life, while his younger sister Elisa (Camila Sodi), there with her own crowd, uses drugs as her chosen means of escape. There are, however, some realities that cannot be kept at bay forever, like the reason behind their parents' prolonged absence, the gradual breakdown in the villa's amenities, and Cristobal's dwindling university prospects.
Watch the official Deficit 2007 trailer in HD below or find more Deficit videos on Vidimovie.


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The conflict against ISIS in both Iraq and Syria has provided a fertile ground for delivering a wide range of air-launched weapons.
Nations from France, USA, UK, and of course Russia have come to the conflict with their own inventory of weapons, from completely unguided to precision, but consistently attracting headlines depending on the writer's stand-point.
Islamic State has been unable to acquire or develop air-defence systems capable of effectively countering US Coalition or Russian aircraft .
A loss of 34% has been recorded in Islamic State terrority since mid 2015, with occupied ISIS terrirtory now representing 60,000sq km, - an area eqivilant in size to Latvia.
US Coalition airstrikes in 2016
Uncontested airspace has allowed the widest variety of attack air platforms to be employed in Iraq and Syria, seeing significant testing of new sensors and weapons.
Attack aircraft being used in Syria and Iraq
A full array of weaponry has been used against Islamic State with a diverse set of targets being engaged. Air strikes have intensified as the coalition seek to dislodge Islamic State from Mosul and Raqqa. The large number of deployed platforms have represented the self-interests of each contributing country, rather than a collective military campaign.
Jane's Aerospace, Defence & Security Editorial Staff


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Forums > Photography Talk > Most famous model..
Who was the most famous model you have ever photographed??
How was your experience???
Jul 06 08 06:08 pm Link
Posts: 1779
Denver, Colorado, US
Most famous for me was Miss Conduct #371. It rocked beyond words
Jul 06 08 06:10 pm Link
Posts: 3844
Peoria, Illinois, US
Brooke Lynne MM# 435770 (my avatar)
I don't know if other people consider her famous, but her port is extremely accomplished, and she's worked with some of the best. I was afraid that I would be nowhere near up to par, but she had her avatar as one of my images for a while, and I can't begin to explain how good that made me feel.
She's highly impressive, and I would recommend her to anybody.
Jul 06 08 06:17 pm Link
Posts: 525
Aiea, Hawaii, US
Francine Dee is the latest one - also on my avi.
vince PTP
Jul 06 08 06:21 pm Link
Posts: 77
Laguna Beach, California, US
ICE-T and his wife Coco-on my port. Awesome people very down to earth and friendly!
Jul 06 08 06:25 pm Link
I photographed once Raquel Zimmerman in one backstage in NY.
She posed for me during 10 min... Hahaha
Jul 06 08 06:31 pm Link
Posts: 3037
New York, New York, US
Caroline Murphy when she first got to NYC--professional and friendly.
Mischa Barton and Lindsey Lohan (as child models)--both great child models.
Vanessa Paradis (aka Mrs Johnny Depp, Chanel model) when she was promoting her singing--brief shoot and I didn't really know who she was.
David Duchovney (who a was briefly a Calvin Klein model)--very nice, funny.
Jul 06 08 06:55 pm Link
Posts: 458
Portland, Oregon, US
Aria Giovanni a couple years ago. It was strange but a good time.
Jul 06 08 06:57 pm Link
you have a big list of celebrities in your portfolio!
I saw your album, amazing work! Congratulations!
Jul 06 08 08:22 pm Link
Posts: 5
Costa Mesa, California, US
kathy ireland...anna nicole smith
Jul 06 08 08:28 pm Link
Posts: 7657
Los Angeles, California, US
Playmate Shauna Sand. Good shoot. She was very down to earth.
Jul 06 08 08:52 pm Link
James Loy Photography wrote:
are they in your portfolio?
daninredondo wrote:
I'm curious to see your playmate! Did you photograph her for playboy??
Posts: 2524
Allentown, Pennsylvania, US
i count Candace Nirvana amongst my friends these days. she seems kinda well known in the circles i hang with.
she also brought Carlotta Champagne to my place once and i had a chance to work with her as well.
Jul 07 08 11:04 am Link
Posts: 4114
Los Angeles, California, US
I guess Betcee May Mayhem #8001. She's steadily becoming more well known.
My experiences with her have been fantastic. She is a great model and a great person. Professional, creative, hard working, Very very intelligent, helpful, dedicated to her craft and passionate about her art.
Wenona, Mayhem #25021 is also very well known. Very similar experience as that with Betcee May.
Jul 07 08 11:51 am Link
Posts: 1571
Atlanta, Georgia, US
Chanel Iman, Seung-Hyun, Agyness Deyn, Lily Donaldson, just to name a few
Jul 07 08 12:00 pm Link
Posts: 1464
Edison, New Jersey, US
My first shooting was with a fitness model Julian Fantechi (www.julian-fantechi.com), who triggered me to learn photography.
I never realized he was acting so well and fully understood my instructions, even if my photography was totally different from his usual styleâ¦
Jul 07 08 12:24 pm Link
Posts: 7804
Boston, Massachusetts, US
Never shot a famous model.
How does one go about doing such a thing?
Jul 07 08 12:26 pm Link
Leeann Tweeden from Fredericks of Hollywood for a swimwear catalog, Karen McDougal PMOY 98 for a swimwear catalog, Amber Marie Goetz the second ever PB Cyber Girl, Veronica Trollerud (Avi), Renee Jaquith from Perfect 10 and Sheri Stowe.
Jul 07 08 12:29 pm Link
Posts: 3621
New York, New York, US
I have worked for Naomi Campbell, Helena Christensen, Giselle Bundchen, Karlina Kurkova, Natalia Vodianova and Caroline Murphy...... Unfortunatly that was as a limo driver not a photographer. (For all you MUA's I also have worked for Pat McGrath as well.)
Jul 07 08 12:43 pm Link
Posts: 213
Miami, Florida, US
Worked with Nadia Auerman, Christi Turlington, Claudia Shiffer, Kristen McNenemy, Stephanie Seymor, Sylverster Stalone, Elton John, ⦠while assisting with Richard Avedon in New York.
Would have loved to photograph any of them.
... catching the light!
Jul 07 08 12:46 pm Link
Posts: 122
Berlin, Berlin, Germany
Jul 07 08 12:47 pm Link
Posts: 259
Alexandria, Virginia, US
dfhpHOTOGRAPHY wrote:
Snap! How do you know them? And, I love Law & Order haha
Jul 07 08 12:57 pm Link
As an assistant I've worked with Jenna Jamieson, Julie Meadows, Roselyn Sanchez, Brooke Burke, Jaime Bergman, Gretchen Stockdale, Cindy Margolis, Cindy Rothrock and a number of others I can't seem to remember All were great poeple to work with, especially Cindy Margolis and Gretchen Stockdale
Jul 07 08 12:59 pm Link
tony federico wrote:
Yes...did she throw anything at you?
Jul 07 08 01:12 pm Link
Posts: 122
Berlin, Berlin, Germany
Brian Baybo wrote:
Jul 07 08 01:25 pm Link
Posts: 4369
Austin, Texas, US
No one all that famous.
Probably Holley Dorrough (MM #8932) a couple of times. She was very nice, very professional both times.
Jul 07 08 01:29 pm Link
Posts: 4281
Raleigh, North Carolina, US
I find myself avoiding higher profile models. I don't know if I can articulate why that is (besides the fact that I can't afford them and they often won't do TFCD with someone at my level), except to say that the path of least resistance in creating images often involves using someone who is less well-known and more willing to take chances doing something risky to make something beautiful.
Though someday before she gets too old, I'd love to do an album cover for Bjork sometime.
Jul 07 08 01:55 pm Link
tony federico wrote:
Disable your cap lock It is considered rude to post in all caps
Jul 07 08 01:57 pm Link
Posts: 48041
Hollywood, Florida, US
Sheila Bleck, one of the top heavyweight female bodybuilders.
Others, but can't list names due to the nature of their fame.
Jul 07 08 02:02 pm Link
Posts: 2369
San Ramon, California, US
As for models, headshots for Angie Harmon back in the mid 90's and Tawny Kitaen in the late 90âs
Jul 07 08 02:25 pm Link
Posts: 123
Westmont, Illinois, US
Cindy Crawford.
I was working with her to start her career when she was 15. I met her on a newspaper assignment and she stood out from the rest right from the start! Beautiful, talented and smart, she took to it easily and was a pleasure to work with. I watched her meteoric rise to the top and felt glad that I had helped her get there. There are several pictures in my profile from those early shoots. Enjoy!
Roger Legel
Posts: 2369
San Ramon, California, US
tony federico wrote:
Sorry, I don't recognize her.
tony federico wrote:
So is Whatâs Up Magazine a hard bound magazine or Webzine? I love your work but canât find the magazine on any racks or news stands.
Posts: 29259
Toronto, Ontario, Canada
A few years ago I shot Katrina.
She stared in the first season of Webdreams:
Jul 07 08 02:40 pm Link


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referencia : DADCG01+FCDD051
Intermediate model of the Active range, the ACTIVE XPRESS is a little more punchy than the Xpress Easy model. Made for female skiers of all levels who want to have fun on the track in full quietude. With very progressive edges and rocker combined with...DYNASTAR
Intermediate model of the Active range, the ACTIVE XPRESS is a little more punchy than the Xpress Easy model. Made for female skiers of all levels who want to have fun on the track in full quietude. With very progressive edges and rocker combined with a light weight, it rotates with ease, while remaining very stable and gripping. Easy to manage and scalable, this ski has the particularity of forgiving everything.
Specially developed to meet the needs of good female skiers, the XPRESS 11 W binding combines excellent performance and unparalleled lightness without the slightest concession regarding the safety of its user. The fitting and adjustment of these bindings is very simply and easily done without any tool. The XPRESS system has been awarded with the ISPO Eco-responsibility Award in 2012 in Munich.
- Middle Sidewalls construction: Sandwich construction in the middle of the ski, the sidewalls under the foot of the skier provide performance and accuracy. The CAP construction at the ends lightens the skis and allows easier pivoting.
- Tip Rocker : Adapted rocker at tip only. For easier pivoting on flat skis with a smooth and forgiving feel in the turns. The rise is moderated so that once the ski is on its edge, the ski works in the turn providing full-length edge contact.
- Multicell Core : High density Multicell Core that can be combined with multiple materials such as glass fibre or titanal. The benefits: Very good lateral torsion / optimal flex distribution / significantly improved sensations on the ski
- Guaranteed Made in France : The "Guaranteed Made in France" label is issued by an independent organisation which guarantees that products are French-made, based on two criteria that the company must satisfy at the same time: the main specifications of the production process are French and at least 50% of the manufacturing cost is of French origin.
- DIN value: 3.5/11
- Lateral Elasticity- toe piece: 45 mm
AFD plate : AFC
- Heel piece: XPress
- Heel piece: XPress
- Vertical elasticity- heel piece: 12 mm
- Brake: 2P 70/85 mm
- Adjustment of length: 36 mm
- Height/width adjustment: 36 mm
|Espátula (mm)
|Patin (mm)
|Talón (mm)
|Radio (m)
|Peso (g)
Desde 1963: Establecido desde 1963 en el valle de Chamonix, cerca de Mont Blanc, Dynastar reune a hombres y mujeres que comparten, a traves de valores comunes y la cultura comun, una pasion por el esqui y el rendimiento. Dynastar pone, desde hace 48 anos, sus conocimientos y experiencia al servicio de los esquiadores apasionados implicados y que les proporcione tanto el equipo tecnico y de diseno, de alta calidad.
Dynastar innova constantemente para ofrecer los amantes del esqui lo mejor en terminos de esquiabilidad. Con la aparicion del Rocker, todos los esquis de la gama estan equipados con esta nueva tecnologia que se adapta a cualquier nivel de la practica, la pista, fuera pista, con cada uno de los beneficios especificos.


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Biker in GTA 5 style chase out-runs POLICE chopper in thrilling clip
A DRAMATIC clip has surfaced showing the heart-racing moment a crazed biker out-races a police helicopter in scenes that could be straight out of GTA 5.
The adrenaline-fuelled footage – captured on the driver's helmet camera – documents the driver's epic bid to escape from the cops as they chase him in a helicopter.
The above video – which is just under 90 seconds long – shows a man notice a police helicopter monitoring him while he goes to get a cold drink from a petrol garage.
Suddenly the man pulls out of the petrol stop and speeds off down the highway.
What ensues is a pulsating high-speed chase through the streets of Philadelphia, as the biker uses his nerves-of-steel to outmanoeuvre the cops.
The race came to an end when the man evaded the helicopter by stopping under an overpass on the motorway and pulling to the side of the road.
The helicopter at first stopped to survey the underpass but after finding nothing it moved on to search for the renegade biker elsewhere.
A description at the start of the video alleges that the footage was sent by a friend to YouTube user Carbon RR Pilot who then uploaded it.
Whether or not that's true, the stunning footage looks like a scene right out of PlayStation game Grand Theft Auto.
The clip has since gone viral being viewed almost 400,000 times.
One viewer jokingly commented: "As a fellow rider please let me know when he expires."
Another YouTuber wondered why the video was posted, adding: "And here come the Philly police department with a warrant.
"Are you dumb or what buddy? This guy's getting caught now."
Another high-speed chase made the headlines last week when a criminal stole the police car he was put in after being arrested – sparking an hour-long chase.
Some bikers prefer a more "free" feeling when they're riding – like this lad who triggered a mad police chase after he ripped through a quiet suburban town wearing nothing but his birthday suit.


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Carlos Ruiz Zafon Quotes About FeelingsQuotes about: Feelings
I couldn't help thinking that if I, by pure chance, had found a whole universe in a single unknown book, buried in that endless necropolis, tens of thousands more would remain unexplored, forgotten forever. I felt myself surrounded by millions of abandoned pages, by worlds and souls without an owner sinking in an ocean of darkness, while the world that throbbed outside the library seemed to be losing its memory, day after day, unknowingly, feeling all the wiser the more it forgot.
He was a very private person, and sometimes it seemed to me that he was no longer interested in the world or in other people... I got the feeling that Julián was living in the past, locked in his memories. Julián lived within himself, for his books and inside them - a comfortable prison of his own design." "You say this as if you envied him." "There are worse prisons than words.
It's hard work, writing, you know. Honestly, a fight every day against your own limitations. You have to squeeze books out of your brain, you're constantly trying to solve challenges. I think most writers enjoy the feeling of having written something, rather than the process of writing it.
Carlos Ruiz Zafon
- Born: September 25, 1964
- Occupation: Novelist
- Cite this Page: Citation


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Two Friends Smocked Rag Doll Dress 3T
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I am crazy about Raggedy Ann! This dress is just too cute! Size 3T and is 23.5" long. It is in excellent condition. Sorry,bow not included. .
I am listing several items and will be keeping my descriptions short. Please check out my other auctions. All clothes are washed in cold water and are hung to dry. We are a pet free/smoke free home. Most all items are worn only once. Please contact me if you have any questions prior to bidding.Please note that I do my best to describe each item as honestly as possible. If for some reason you are not satisfied please contact me prior to leaving feedback. I will do my best to resolve the problem. Shipping will be $3.50 per item and by the USPS. Payment is expected within 3 days of auctions end. Paypal is preferred. Thank you for looking.


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This week I used the "Queen's Herald" stamp. I knew the moment I saw it that it would make a very fun and unique Easter card. After a few attempts at a card idea that wasn't working out, I pulled out a piece of vellum and tried stamping on it. Then I had the idea of embossing it with copper embossing powder. These stamps are so detailed that I am astonished at how well it came out. (I definitely recommend using a detail embossing powder if you try this yourself. You will get the best image.)
The embossed copper looks cool and has excellent detail but still does not match the beauty of the original stamp image. Next week I will feature another card using this stamp so you can see the inked image as well.
Queen's Herald Stamp ($9.50)
Nature's Blessings Fine Rubber Art Stamps on Etsy
I'd like to thank Nature's Blessings again for providing me with some of their lovely stamps. If you like my creations, please leave a comment below. Thank you!


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We've all seen movies or heard of stories of sex games gone wrong. Some kinky affair where you strangle your partner a little too hard or too long and suddenly, a passionate affair turns into a nightmare. No one wants to have this happen to them (much less their partner) so as per usual, we're here to help.
Don't be a drunken fool
While it's always great to have some alcohol to lubricate any situation, being overly intoxicated during any kinky game is a sure fire way to disaster. It's always important to remain in control especially when it involves games that require tying up, strangulation, cutting, wax, etc.
The same goes for drugs
What you do with your private time is none of our business, but if you have the life of someone else on your hand, try not to be too doped up, especially on drugs that dull your senses. You might not be able to tell if you're going too far.
Safe words
During role-playing or Dom games many couples also use Safe Words so they know what the limit is during play. So it goes without saying that if you're using anything to obstruct the mouth of your partner, make sure there is enough room for them to speak out the safe word. Or else they could be trying to tell you to stop but you won't be able to hear or understand them.
Make sure you can get them out quickly
If you're tying your partner up, or wrapping, or whatever you do, make sure you can get them out of the get up quickly in case anything goes wrong. Sometimes all it takes it a few extra seconds for something good to go bad.
And most importantly, if you partner calls out the safe word, stop immediately.
We know, we know, we don't want to take the fun out of your games, but it's always better to be safe and have boundaries then to have regrets! So have fun my kinky people, but remember my tips and all shall be well in your dirty, naughty world!
What other measures do you take to safeguard your play? Let me know in the comments!


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My Experience Working on Evidence-Informed Policymaking in Kenya
In the first in a series of guest blogs from Evidence Works 2016 delegates, Hon. Susan Musyoka shares some of the challenges faced by the Parliamentary Caucus on Evidence-Informed Decision-Making which she chairs, and the strategies being used to overcome them. In this blog, she also highlights the good work of the Caucus that includes delivering sensitization workshops on evidence use for MPs, holding open policy 'cafes', capacity-building endeavours, and broadening access to research and information.


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It's one week until I leave for Portland and yes, I am counting the days. I could use a few more days in the week, but at some point I just have to say I'm done. I'm trying to finish all my last minute things, making lists and making them again. My husband still has to fireproof all the drapes with this awful liquid stuff, and make a rod to hang my cute little candle chandelier. No easy task for sure but that's why we have husbands! Right?
Remember those little pillows I showed you awhile back? Well, I'm desperately trying to photograph them myself because I can't get my photographer here in time. So I took this pretty picture, uploaded it to my computer and then realized "Oh No!" The pillow actually says weet reams instead of Sweet Dreams. Would you buy a pillow that says weet reams? Come on, just for me? That way I don't have to redo the photo. LOL! Ok, I'll redo it, but it's your fault if I'm not ready for market!
Now look who wants a first class seat on the airplane? It's Bitty Kitty! She's been a trouble maker for sure. It took five times to get her "just right". That poor little body has gone from skinny to plump. Then she wouldn't let me put pins in her so I had to add a mouse. Finally I have a Bitty Kitty Pincushion to go with Bitty Bird and Bitty Bunny. She sits in a saucer, just wishing for some milk. (:
So now it's back to the photo shoot. Wish me luck. Weet reams everyone!


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Labour's economic blueprint set out by shadow chancellor John McDonnell
A Labour government would balance Britain's books "fairly", avoiding the cuts to benefits and services resulting from Conservative austerity, shadow chancellor John McDonnell has said.
In his first major speech in the post, Mr McDonnell set the scene for tax hikes on the rich, saying that when a Labour government needs to raise money it will do so by "fairer, more progressive taxation" which does not impose a burden on middle and low-earners.
He promised an "aggressive" drive to force multinational corporations like Starbucks, Vodafone, Amazon and Google to pay "their fair share of taxes" and to cut "subsidies" enjoyed by business. And he said Labour would boost growth through an "active monetary policy" - effectively printing money to stimulate the economy.
Mr McDonnell's blueprint won a wary reaction from business, with the CBI warning that an active monetary policy could fuel inflation and drive up interest rates, while the British Chambers of Commerce said Labour "must not confuse supporting growth with state control over the economy".
And Vodafone said there was "no truth" in the shadow chancellor's claim it was dodging tax, insisting it has "always paid its taxes" and pointing to a £360 million bill in direct UK taxes for 2014/15, when its profits in the country amounted to just £41 million.
In his keynote address to the Labour annual conference in Brighton, the veteran left-winger accused Conservatives of making middle-earners and the poor bear the burden of eliminating Britain's deficit, while protecting the richest from the consequences of the economic crisis.
He said Labour would "halt Conservative tax cuts to the wealthy paid for by cuts to families' income", end the "brutal" treatment of disabled people subjected to fitness-to-work assessments and deliver a "real living wage", higher than the £7.20 "national living wage" offered by George Osborne.
"Austerity is not an economic necessity, it's a political choice," said Mr McDonnell.
"The leadership of the Conservative Party made a conscious decision six years ago that the very richest would be protected and it wouldn't be those who caused the economic crisis who would pay for it. Although they said they were One Nation Tories, they've demonstrated time and time again, they don't represent one nation, they represent the 1%."
Insisting that Labour would not be "deficit deniers" Mr McDonnell said: "I tell you straight from here on in Labour will always ensure that this country lives within its means. We will tackle the deficit."
But he added: "T his is the dividing line between Labour and Conservative. Unlike them, we will not tackle the deficit on the backs of middle and low-earners and especially by attacking the poorest in our society.
"We will tackle the deficit fairly and we can do it."
He acknowledged that Labour "need to prove to the British people we can run the economy better than the rich elite that runs it now".
But he said the party's strategy would be based on growing the economy by strategically investing in key industries and sectors.
And he promised, to loud applause: "Labour's plan to balance the books will be aggressive.
"We will force people like Starbucks, Vodafone, Amazon and Google and all the others to pay their fair share of taxes."
He went on: "There will be cuts to tackle the deficit but our cuts will not be the number of police officers on our streets or nurses in our hospitals or teachers in our classrooms.
"They will be cuts to the corporate welfare system. There will be cuts to subsidies paid to companies that take the money and fail to provide the jobs."
If Labour inherits a deficit in 2020, it will pay it down "at a speed that does not put into jeopardy sustainable economic growth", he promised.
Mr McDonnell said Labour would launch a wholesale review of its economic policies, guided by an advisory panel featuring a number of internationally renowned economists.
Labour will review the nation's major economic institutions, including the Treasury, Bank of England and HM Revenue and Customs, to ensure they are "fit for purpose", he said. Former civil service head Lord Kerslake will lead the review of Treasury operations.
While the Bank will retain its independence, Labour will look at extending its mandate to require it to target employment, growth and earnings as well as inflation.
Mr McDonnell urged Labour moderates who have refused to serve under new leader Jeremy Corbyn to "come back and help us succeed".
He acknowledged that fierce debates over Labour's future direction lie ahead, but insisted that these should not be interpreted as splits in the party.
"We are in an era of new politics," said the shadow chancellor. " People will be encouraged to express their views in constructive debate.
"Don't mistake debate for division. Don't mistake democracy for disunity."
Responding for the Conservatives, Treasury minister David Gauke said: "Labour's tax rises would hurt hard-working people, threatening every family's security.
"And as (Bank of England governor) Mark Carney has said, Labour's policy to end the Bank of England's independence and print money would drive up the cost of living.
"That's why Labour are a serious risk to Britain's economic security."
But unions welcomed Mr McDonnell's speech, which was hailed as "inspirational and historic" by Public and Commercial Services union general secretary Mark Serwotka, while Unison general secretary Dave Prentis said it provided "hope for the future".
Asked how he would make multinationals pay their share of taxes, Mr McDonnell told Channel 4 News: "I'm going to meet all these companies and have a conversation about their responsibilities within our community.
"Hopefully, some of them will pay voluntarily. But if it requires taxation legislation to be changed, we will do it."
Mr McDonnell said Labour's current policy is to raise the top rate of income tax to 50%, but added: "We want to go out there and consult people about what the proper tax rate should be. I think that 50% at the moment is the right level, because I don't think income tax is the big issue. The big issue is making sure businesses and corporations pay their way."
Speaking at an anti-austerity fringe meeting, Mr McDonnell called on delegates to "automatically support" workers when they want to take action and resist.
He reiterated his desire for "peaceful, non-violent direct action" as a way of showing more people how they can get involved in opposing the Government.
Mr McDonnell, addressing how to "combat" austerity in communities, said: "Each of us now has a responsibility on our shoulders to recognise that we have a role to play in this.
"And that means when we go back to our constituencies and our communities, we start gathering people together to organise our own meetings to talk about how we battle against austerity in our own communities, not just nationally.
"And the way we do it is the traditional methods in all we do - if it is austerity as a result of cuts in wages or threats to jobs or privatisation or whatever, you know as well as I do now that there's momentum building within the unions to resist.
"So our job therefore is when individual groups of workers want to take action, we automatically support.
"There is no standing on the sidelines any more because that's the tactic the establishment uses - divide and rule."


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Desperate bid to avoid meltdown
Britons are being advised to consider leaving Tokyo as workers continue their desperate attempts to prevent meltdown at Japan's stricken nuclear plant.
Military helicopters sprayed seawater onto damaged reactors at Fukushima Dai-ichi, which has been rocked by a series of explosions and fires since Friday's 9.0-magnitude earthquake and tsunami.
The efforts to cool reactors came as US officials warned of a shortage of water in a storage pool meant to stop nuclear fuel rods from overheating.
The Foreign Office (FCO) issued advice to any British nationals in - or to the north of - Tokyo to consider leaving the area because of the "evolving situation" in Fukushima and potential disruptions to the supply of goods, transport, communications and power.
Early on Thursday morning, the FCO updated its advice, urging Britons to remain outside an 80km radius of the plant "as an additional precautionary measure". It said the call was in line with the US government's advice to its citizens in Japan.
Six days after the disaster, the official death toll reached more than 4,300, but experts believe the figure will climb to more than 10,000.
Police say more 452,000 people are staying in schools and other shelters, as supplies of fuel, medicine and other necessities run short.
Meanwhile, an International Rescue Corps (IRC) team which wanted to help in the rescue effort said it was prevented from travelling to the disaster zone because the UK embassy in Tokyo refused to issue the necessary documentation. Willie McMartin said: "It is sheer disbelief that we got to Tokyo, that we were told that we would get the document provided we get a one-sheet letter from the embassy, and they refused it."
But Foreign Secretary William Hague pinned the blame for the aborted mission on the team's own failure to be properly equipped.
In a rare address to the nation, Emperor Akihito, 77, said: "It is important that each of us shares the difficult days that lie ahead. I pray that we will all take care of each other and overcome this tragedy."


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Category Archives: DIVA founder Stanley Kay
2017 Stanley Kay Scholarship winner Maria Marmarou
Maria Marmarou, 17, is a junior at Kutztown Area Senior High School. She studies drum set, timpani, and mallets with Dr. Willis Rapp. She has participated in many jazz festivals with Kutztown's jazz band and has won various awards. She … Continue reading
Apply for the 2017 Stanley Kay Scholarship
Applications are now OPEN for the 2017 Stanley Kay Scholarship. The application deadline is May 15. This year's scholarship winner will attend COTA Camp Jazz. Click here to read about the scholarship and how to apply.
Now Available! Special Kay
Special Kay, our first "all-original" recording, featuring the hard-swinging, joyful, and poignant compositions of our founder and creative force, Stanley Kay, is now available. Visit the CD page to order!
2016 Stanley Kay Scholarship winner Nick Kepron
The winner of the 2016 Stanley Kay Scholarship is Nick Kepron. Nick will be attending the Daniel Glass New York Jazz Intensive Drummers Collective workshop. Congratulations, Nick!
2016 SK Scholarship summer camp options
Know a promising young drummer? Are YOU a promising young drummer? The Stanley Kay Scholarship offers drummers between the ages of 15-22 an opportunity to attend COTA CampJazz or the Daniel Glass New York Jazz Intensive. Visit the Scholarship page … Continue reading
Apply for the 2016 Stanley Kay Scholarship
Applications are now open for the 2016 Stanley Kay Scholarship. DIVA honors the musical legacy, integrity, creativity and excellence of Stanley Kay through a scholarship, offering an opportunity for a seriously swingin' drummer between the ages of 15—22 to attend … Continue reading
2015 Stanley Kay Scholarship co-winner Nick Kepron
Nick Kepron is thrilled to be a recipient of the 2015 Stanley Kay Scholarship. Nick, 16, is a drummer, abstract artist, composer and bandleader. An Honors student entering his Junior year at Cherry Hill High School East in Cherry Hill, New Jersey, he has been the drummer, co-leader and … Continue reading
2015 Stanley Kay Scholarship co-winner Maya Stepansky
Maya Stepansky is greatly honored to receive the 2015 Stanley Kay Scholarship and attend the NYSMF. A 10th grade student at Montclair High School, Maya's passion for drumming took off at age 9 when she was selected as drum majorette … Continue reading
2015 Stanley Kay Scholarship winners
Congratulations to Maya Stepansky and Nick Kepron, winners of this year's Stanley Kay Scholarship. Check back for photos and bios of these two talented young artists. Visit the Stanley Kay Scholarship page for information on the program, how to donate, and … Continue reading
Mixing Special Kay
Sherrie and master engineer Kent Heckman mixing our upcoming CD Special Kay at Red Rock Recording in Saylorsburg, PA. Recorded live in June, 2014 at Tannery Pond Community Center, it features the compositions of our founder Stanley Kay – our first … Continue reading


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In Stock
The Gaslow UK Bayonet Adapter adapts foreign Euro style fill points to fit UK fill nozzles. It is not required on a Gaslow Filler Kit.
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© 2017 Hamilton Gas Products Ltd | All Rights Reserved


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Serious yet dynamic, Borkum Riff Bourbon Whiskey has a unique, captivating taste and aroma. Infused with eight-year old Kentucky Bourbon, it combines the natural flavour of the tobacco with a distinctive aroma that is both traditional and discreetly masculine.
$24.99 Retail: 29.99 Save $5.00
- Disclaimer
- The statements, views and opinions presented in customer reviews are those of the author and are not endorsed by, nor do they necessarily reflect, the opinions of BnB Enterprise, product manufacturers, employees, or any other organization.
- BnB Enterprise does not warranty or guarantee the correctness of the information provided or its fitness for any purpose.
Borkum Riff Bourbon Pipe Tobacco
I have enjoyed smoking this tobacco for many years. I hope BNB Tobacco continues to offer it. They have given me great service!
Like the larger, looser pack cans. Much less spillage when transferring to pouch.
Borkum Riff review
Only problem is that about one out of three orders I find the product too dry. I have been smoking this brand for many years and continue to enjoy it.
Good Smoking
Borkum Riff Bourbon blend has a nice aroma, light flovor and is cool burning, my favorite after over 50 years of pipe smoking.
Delivered early, fresh product!!!!!!!!!
Borkum Riff Bourbon (the original B-R)
Still the best commercially produced pipe tobacco on the market.
Good Product Delivered Poorly
BnB Tobacco processed and shipped expected product in timely fashion, but USPS mangled carton --- inside, 3 of 8 "cans" had paper sides separated from ends.
39.99 $29.99
39.99 $33.99
39.99 $29.99
39.99 $29.99
29.99 $24.99
39.99 $33.99


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The 24-70mm f/2.8 Carl Zeiss T* Alpha A-Mount Standard Zoom Lens from Sony is a bright zoom lens, specifically designed by Carl Zeiss for the Sony Alpha-series of DSLR cameras.
Unique to this lens is the Carl Zeiss T* (T-star) coating that helps reduce lens flare, while increasing contrast. When used with cropped APS-C sized DSLR cameras, the equivalent focal length for this lens is 36-105mm. It is a compact all-in-one lens, and is ideal for snapshots, portraits, interiors or outdoor scenery.
The lens' maximum aperture is a fast f/2.8 and can be maintained throughout the zoom range. Its minimum aperture is f/22. It also has 9 circular aperture blades that work to produce smooth, out of focus background effects.


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Use this form to contact us about Expand2Web Theme support and questions.
We want you to be successful, so don't hesitate to reach and and let us know if you have any questions. Or if you just want to share your masterpiece with us, we'd like to hear about that too!
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Who are we?
We are an independent network of people who have suffered sexualized violence, people who love people who have suffered sexualized violence, and people who are just plain fed up with the harm that sexualized violence does in our communities and around the world. We are people who are concerned, specifically, about the spiritual dimensions of sexualized violence and the presence of sexualized violence in communities of faith. Many of us have a connection to the Mennonite church in North America (MC USA and MC Canada), whether we are participating members, former members or are connected in some other way. Those with no Mennonite connection whatsoever are also welcome in this space and in the work we do together.
OSU is all of the people who connect here. Our daily operations are run by Hilary J. Scarsella. If you would like to get involved, let us know or contact Hilary directly.
What do we do?
We support each other, advocate for people who have suffered sexualized violence, and organize to end sexualized violence in our communities – lovingly, fiercely, imperfectly, and as best we can. Here are a few of the specific ways we go about our work:
- We do our best to make the OSU website a public space where people who have suffered sexualized violence can share their stories, be heard, and be received with support and respect. Speaking up and refusing to carry the burden of keeping the life-destroying secret of sexualized violence can be a powerful part of survivors' processes of rebuilding ourselves and our lives in the aftermath of violence. Because sexualized violence thrives in secrecy, breaking the silence – one story at a time – is also an act that cracks away at systemic perpetuation of sexualized violence in our communities. Because it is our priority that OSU be an environment in which people who share testimony of sexualized violence can feel supported in doing so, comments made on personal testimony are carefully moderated.
- We offer those who want to share their testimony on OSU support in the writing process. Have an experience you want to share and don't know how to begin? You are most welcome to send us a message.
- We create space for conversation around sexualized violence and its prevalence in communities of faith. If our communities are going to become places of hospitality, safety and justice for survivors of sexualized violence and those who are vulnerable to such violence in the future, we need space to talk about what's wrong and how we can make it right. The articles posted on the OSU blog aim to prompt that conversation from as many different angles as possible. We encourage all of our readers to engage OSU posts with an attitude of respect and a desire to both learn and contribute.
- As we are able, we offer educational and liturgical resources for individuals and communities wanting to dig deep in their commitment to resist sexualized violence and stand in loving solidarity with victims and survivors. Check our resources page (which will be under development through the summer of 2016) for a full description of what we offer and links to materials put together by people and organizations we think are great.
- In all of our work, we are committed to addressing sexualized violence through an intersectional lens that considers the ways that race, culture, class, age, ability, gender, sexuality and more impact our understanding of sexualized violence and what needs to be done to stop it.
**Our Stories Untold partners closely with SNAPmenno, the Mennonite chapter of the international Survivors Network of those Abused by Priests.


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Product Description
The two-piece plastic shell snaps together, making installation easy, and the built-in screen protector will guard against smudges and fingerprints. This Otterbox Defender for iPhone 4/4S case comes with a 1450 mAh lithium-ion battery.
OtterBox Defender with ION Intelligence for iPhone 4/4S:
- For iPhone 4/4S
- Rubber plugs protect main openings of device
- Access all features of the device through the case
- Graphite OtterBox case for iPhone 4 intelligently knows when to charge the phone automatically
- 10 LEDs visually indicate remaining case charge
- 2-piece plastic shell snaps together around device
- Otterbox Defender case for iPhone 4 with ION intelligence has built-in screen protector
- Built-in 1450mAh lithium-ion battery
- Color: Graphite
- Includes micro-USB cable