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During the development of the Drosophila central nervous system the process of midline crossing is orchestrated by a number of guidance receptors and ligands. Many key axon guidance molecules have been identified in both invertebrates and vertebrates, but the transcriptional regulation of growth cone guidance remains largely unknown. It is established that translational regulation plays a role in midline crossing, and there are indications that transcriptional regulation is also involved. To investigate this issue, we conducted a genome-wide study of transcription in Drosophila embryos using wild type and a number of well-characterized Drosophila guidance mutants and transgenics. We also analyzed a previously published microarray time course of Drosophila embryonic development with an axon guidance focus.
Using hopach, a novel clustering method which is well suited to microarray data analysis, we identified groups of genes with similar expression patterns across guidance mutants and transgenics. We then systematically characterized the resulting clusters with respect to their relevance to axon guidance using two complementary controlled vocabularies: the Gene Ontology (GO) and anatomical annotations of the Atlas of Pattern of Gene Expression (APoGE) in situ hybridization database. The analysis indicates that regulation of gene expression does play a role in the process of axon guidance in Drosophila. We also find a strong link between axon guidance and hemocyte migration, a result that agrees with mounting evidence that axon guidance molecules are co-opted in vertebrate vascularization. Cell cyclin activity in the context of axon guidance is also suggested from our array data. RNA and protein expression patterns of cell cyclins in axon guidance mutants and transgenics support this possible link.
This study provides important insights into the regulation of axon guidance in vivo.
The process by which axons cross the midline during development of the Central Nervous System (CNS) in Drosophila is of great interest and has been the focus of much scientific research [1,2]. After neuroblast delamination and cell fate decisions, axons undergo a journey that will eventually wire them to the appropriate targets. This journey includes a series of steps, one of which is the decision to cross (or not cross) the midline. Four key regulators of midline crossing are known: slit, robo, robo2 and comm. Growth cones in which the robo and robo2 receptors are present at the membrane sense the presence of the slit ligand and do not cross the midline [3,4]. Conversely, growth cones without robo and robo2 at the membrane are not able to respond to the repulsive cue of slit and do cross the midline. robo levels at the growth cone membrane are regulated by comm.
A major question in midline crossing is how regulation occurs through gene expression, i.e., to what extent is transcription relevant in the control of growth cone pathways. It is well established that rapid local translation does occur [5,6]. Some literature also indicates that transcription is relevant [7-9]. We designed this study of transcription during CNS development to i) find relevant genes in the axon guidance process, ii) elucidate the role of transcription regulation during midline crossing in Drosophila, and iii) determine whether any upstream changes are visible in axon guidance mutants. Our analysis uses two whole-genome microarray data sets: (1) axon guidance – Drosophila embryos, stage 15, of axon guidance Gain Of Function (GOF) and Loss Of Function (LOF) mutants, and (2) time course – a developmental wild type time course, consisting of 12 non-overlapping 1 hour collection points (previously published [10]). The axon guidance (AG) data was collected as part of this study and is published here for the first time. We have thoroughly reanalyzed the previously published time course (TC) data.
To identify clusters of co-expressed genes in each data set, we employ the hopach clustering methodology [11]. Clustering algorithms have been successfully applied to microarray studies, but the systematic analysis of clustering output remains a major challenge. A significant obstacle is the lack of reproducibility, or even assessment of variability, of clustering results. Clustering output can vary dramatically from one repetition of an experiment to another, particularly with the small sample sizes typical in microarray experiments. The R statistical package hopach [11] uses a bootstrap resampling method to assess cluster variability, producing a measure of membership of each gene in every cluster. For this study, we adapted hopach to use these cluster membership values to assign genes to clusters, while allowing genes to belong to more than one cluster – a type of fuzzy clustering. This modification improves reproducibility and results in cluster output that is suitable for functional characterization. hopach is freely available and is easily used by biologists, providing a statistically sound framework for analysis of microarray data.
We characterize gene clusters with two complementary sources of annotation, the Gene Ontology (GO) [12], and the Atlas of Pattern of Gene Expression (APoGE), a component of the Berkeley Drosophila Genome Project [10]. APoGE provides pictures of the expression pattern of each gene in the Drosophila embryo and a description of the anatomical structures in which the gene is expressed. A controlled vocabulary is used by individual curators (Amy Beaton and Volker Hartenstein), experts who assure consistency in each call. The goals and scope of APoGE are described in [10]. Its use for characterization of microarray data has never been explored. Together, GO and APoGE provide a rich, multi-dimensional vocabulary for annotating hopach clusters. In this study, we analyze clusters in which genes with terms related to axon guidance are significantly over represented. Querying the APoGE in situ database for genes expressed with specific spatial and temporal patterns provides a means to further explore the axon guidance clusters.
Applying this approach, we identify striking changes in gene expression across GOF/LOF, as well as groups of axon guidance genes co-expressed during wild type development. These findings suggest that regulation of gene expression does in fact play a role in midline crossing. In addition, our analysis indicates that axon guidance genes may be involved in the process of hemocyte migration, and that cell cyclins are active during midline crossing. These observations are confirmed through independent RNA and protein expression studies.
Results and discussion
Transcriptional Control in Axon Guidance Mutants
Our hopach analysis of gene expression profiles from 17 Drosophila axon guidance mutants identified 517 clusters of median size 27 (range 1 to 821). Figure 1 shows the profiles of these clusters ordered by the hopach algorithm so that clusters close to each other contain genes with similar expression profiles. If regulation during the course of axon guidance occurs mainly through protein translation, the changes observed in a microarray study will not yield coherent groups of genes with known function in axon guidance. Yet, we found several clusters significantly enriched for axon guidance genes.
Figure 1. Medoid Heat Maps of Axon Guidance and Time Course. Heat map of the medoids identified in (1a) axon guidance and (1b) developmental time course experiments. The medoid is a single gene from each cluster, selected by hopach as the cluster profile because its pattern of expression is the most representative of that cluster. Medoid plots provide a global overview of the clustering results. The order of clusters in these plots is uniquely determined by the hopach algorithm. Expression relative to the control is shown on a red to yellow color scale, with red indicating suppressed and yellow indicating induced expression. The number of genes represented by each profile (i.e., row) is variable from cluster to cluster. (1a) Medoids for the axon guidance data. There are distinct regions of clusters, for instance the initial 37 clusters are similar with stronger expression for the gain of function FRM and UR.mef. A distinct region follows, in which there is a significant decrease in the mutants robo, robo2, slit.robo and the transgenic UC2x. (1b) Medoids for the developmental time course. The initial clusters show only late expression with the subsequent clusters showing earlier and earlier expression. Note: the size of each cluster is not represented in this plot. For instance Cluster TC139 has 6626 genes -almost half of all microarray probes are represented by one single row.
We ranked clusters based on their association with relevant terms from GO ("axon guidance") and APoGE ("ventral nerve cord", "embryonic brain", and "ventral midline") as described in the Methods. The top four clusters for each term are shown in Table 1. Cluster AG132 ranks first for "axon guidance", "embryonic brain" and "ventral midline". It is the cluster with the highest ranks in both the GO and the APoGE vocabularies. This cluster contains a number of genes well known from the axon guidance literature, such as dock, plexB, Cam, CadN, caps, spen, Fs(2)Ket, Con, RhoGAP19D, Rac2, and shot. AG164 is another cluster that also contains relevant genes in axon guidance mechanisms, such as trio, WASp, Nedd4, robo3, MICAL, RhoGAPp190, rhoGAP88C, RhoGAP18B, lola, side, and Fmr1 [2]. A full list of genes in all of the top ranking clusters and a more detailed exploration of each cluster are available in the Supplementary Materials(Additional files 1234).
Table 1. Significant Clusters for Relevant Controlled Vocabulary Terms in Axon Guidance Data
Querying APoGE, we identified a large subset of genes in Cluster AG132 which are expressed in the commissures and midline (Figure 2). Several genes of unknown function (CG6448, CG6930, nrv3, CG11347, CG11798, CG13624, and CG31666) share this distinctive pattern of expression with the known genes dock, gBeta, brat and Cam. This similarity provides some insight into the functions of the less well characterized genes and suggests their involvement in axon guidance.
Figure 2. Genes Present in APoGE for Cluster AG132. Genes present in APoGE for Cluster AG132 show a similar pattern in embryonic brain and ventral nerve cord. All embryos are viewed ventrally. (2a) dock has a key role in axon guidance [47]. (2b) Cam has been implicated in photoreceptor light termination, muscle synapses and midline crossing [48]. (2c) Gbeta13F mutants have neuroblasts defects [49]. (2d) Brat is reported as involved in the regulation of cellular rRNA. Genes (2f) to (2m) are genes of unknown function with a similar pattern of expression.
Hemocyte Migration
Querying APoGE for Cluster AG164 reveals genes with hemocyte expression, including Dgk, pyd, 103E12, CG8780, CG11100, CG18549 and CG32354. Hemocytes are Drosophila blood cells, which migrate during embryonic development [13], with the direct involvement of the Vascular Endothelial Growth Factor (VEGF) pathway [14]. Hemocytes are relevant in CNS development [15], and it is known that mutants for Pvr, a key molecule in the VEGF pathway, show a longitudinal phenotype and CNS condensation defects [16].
The microarray data indicates that genes expressed in hemocytes are significantly upregulated in slit and robo12, but not in slit.robo. This pattern is intriguing, because the axon guidance phenotype (all axons collapsed at the midline) is similar in the three mutants. Motivated by this observation, we investigated whether hemocytes show any spatial defects in mutants for the slit ligand (slit), the double mutant for slit's receptors robo1 and robo2 (robo12), and the double mutant for slit and robo (slit.robo). Figure 3 is representative of the data we observe using two diffent probes (fray and CG25C); the CNS scaffold is visible by staining with the antibody BP102. The robo12, slit and slit.robo embryos have the same midline CNS phenotype, with all axons collapsed at the midline (highlighted with the green arrow). Hemocyte expression, however, differs between the mutants. The hemocytes are absent from the middle segments in slit and slit.robo embryos (3c, 3d, 3g and 3h), whereas the defect is not observed in the robo12 embryos (3e and 3f). This raises the speculation that slit might be part of some repellent mechanism for hemocytes, with robo assuming a different role than in axon guidance. From the in situ hybridization data it is not possible to determine whether genes expressed in hemocytes are up regulated in slit and slit.robo mutants. The hemocytes are absent in the middle segments, but expression in hemocytes in other segments could be higher, leading to a higher overall expression in whole embryos. Nevertheless, we can conclude that the slit and slit.robo have the same pattern, different from robo12, as the microarray data suggested.
Figure 3. Hemocyte Location in Axon Guidance Mutants. In blue, RNA in situ hybridization showing expression for CG25C, a hemocyte probe. The CNS scaffold (brown) is stained with the BP102 antibody, and indicates the CNS phenotype. The ladder-like WT CNS is visible in WT; the collapsed phenotype is observed in slit, robo12 and slit.robo embryos. (3a), (3c), (3e) and (3g) are ventral views; (3b), (3d), (3g) and (3h) are lateral views. Green arrows indicates the middle segments where in some mutants the hemocytes are absent. (3a) and (3b) are WT embryos where the hemocytes are present ventrally along the entire anterior-posterior axis. (3c) and (3d) are homozygous slit embryos where the hemocytes are not present in the middle section of the embryo. (3e) and (3f) are double mutants for robo and robo2 (robo12). The collapsed midline phenotype is similar to slit and slit.robo mutants, and yet, contrary to slit and slit.robo, the hemocytes are present throughout the whole embryo. (3g) and (3h) are embryos double mutant for robo and slit. They are similar to the slit homozygous mutants in that the hemocytes are not present in the middle section of the embryo.
Transcriptional Control in Time Course
Applying hopach to the developmental data set first published in [10] results in 253 clusters. One of these (cluster TC139) contains 6626 non-differentially expressed genes (with a similar level of expression in all developmental stages). This result illustrates the ability of the hopach algorithm to place low-variability genes in a single large cluster, while forming distinct (much smaller) clusters for other patterns.
The key axon guidance genes slit, comm, comm2 and robo2 belong to clusters significantly enriched for the GO term "axon guidance" (Table 2). The genes comm, comm2 and robo2 are all present in Cluster TC91, which is a small cluster (15 members) with a pattern of expression characterized by a peak around stages 4 to 9. Comm is known to be transiently expressed in the commissural axons and midline glia, simultaneously with robo [17,18]. However, the similarity in expression for comm, comm2, and robo2 throughout the whole 18 hours of development has never been reported. Given the exceptional similarity between the expression of these three genes it would be interesting to explore if they are controlled by the same upstream mechanism, or whether they control a common downstream mechanism. Included in the adjacent clusters (TC90 to TC97), hence with a very similar pattern of expression, there are other important axon guidance genes (RhoGEF4, neur, and RhoGEF3) and many genes involved in Notch signaling (E(spl), neur, Brd, m4, HLHm5, HLHmgamma, Dl, bib and malpha). Notch involvement in axon guidance has been previously reported [19]. The genes spi, hh, argos, gsb, Dl, slp2, siz, and ems are also in these clusters and have documented phenotypes in axon guidance or the CNS scaffold.
Table 2. Significant Clusters for Relevant Controlled Vocabulary Terms in Time Course Data
A query of APoGE for genes in Clusters TC90 to TC97 with midline expression similar to comm, comm2 and robo2, yielded argos. The gene argos has been shown to be relevant in axon guidance in the visual system [20], which further strengthens the possibility of a role for argos in midline axon guidance. Sulfated (Sulf1) is expressed in cells that appear to be adjacent to comm2 and robo2 expressing cells. Sulf1 has not been previously implicated in axon guidance. It is our future project to investigate whether either argos or Sulf1 has a genetic interaction with comm, comm2 and robo2.
Cell Cyclins
Time course clusters TC140, TC141 and TC142 are the most relevant clusters for the APoGE terms ("embryonic brain", "ventral nerve cord", "ventral midline"). They do not contain axon guidance genes, but they have many cyclins, including CycA, polo, cdc2, CycB3 CycE and ago. Cell cyclins are involved in cell division and in CNS development, playing an important role in neuroblast division. However, a role for cyclins has not been reported in post-mitotic neurons, the type of neurons involved in axon guidance. It is known that CycA and CycE mutants have severe defects in the commissures and longitudinal axon tracts of the CNS [21,22], and Cdk5 controls some aspects of axon patterning in vivo [23]. The cyclins CycA, cdc2 and CycB3 genetically interact with each other [24,25] and are important in the earlier stages of intense cell division [26]. Agreeing with this role, the TC data shows high expression early in development, but suggests other functions at later stages because i) there is sustained expression well past the intense initial cell divisions, and ii) the APoGE images show a similar pattern of expression for all cyclins for later stages.
Co-expression of Cell Cyclins in Axon Guidance and Time Course Data
Interestingly, we also identify expression changes in cyclins in the AG data set. Clusters AG1 to AG93 are a group of cell cyclin rich clusters that contain the cyclins smg, polo, CycB3, swa, pim, cdc2, twe, CycB, and fzy. Table 3 shows the percentage of genes present in APoGE with cell cyclin-like expression in each data set separately and simultaneously in both of them. We reason that genes belonging simultaneously to the two independent data sets' cell cyclin rich cluster space have a higher probability of being a cell cyclin or cell cyclin related gene. Figure 4 depicts the genes from this common cluster space with cyclin-like expression. There are three types of genes in this set: i) genes with a role as cyclins (CybB3, cdc2, CycA, fzy, mei-S332, G-ialpha65A, and Set), ii) genes with no known role as cyclins (lola, Df31, Set, UTPase, Sd, msf11, Chrac16, Su(var)2–10, and Uch-L3), and iii) genes with little known information (CG15141, CG5175, CG8478, and CG31639). We plan to research the proposed role of the latter two groups of genes in the context of axon guidance.
Table 3. Cell Cyclin Expression in Axon Guidance and Time Course
Figure 4. RNA Expression in APoGE for Common Genes in Axon Guidance and Time Course Relevant Clusters. (4a), (4c), (4d), (4g), (4h), (4i), (4j), (4k), (4q), (4r), ventral view. (4b), (4f), (4l), (4m), (4n), (4o), (4p), (4s) and (4t), lateral view. (4a) CycA is a cell cyclin whose mutants have severe defects in the commissures and longitudinal axon tracts of the CNS [21]. (4b) CycB3 is a cell cyclin, required for spindle organization. (4c) cdc2 is a cell cyclin; cdc2 mutants have CNS defects [50]. (4d) fizzy is involved in cell cycle and mutants result in degeneration of CNS and absence of PNS. (4e) mei-S332 is a gene involved in meiosis and mitosis. (4f) G-ialpha65A is involved in mitosis and is also involved in CNS development. (4g) lola is a transcription factor required for axon guidance. (4h) Df31 is involved in chromatin remodelling. (4i) Set is involved in DNA replication with reported cyclin binding characteristics. (4j) dUTPase is involved in nucleic acid metabolism. (4k) scalloped (sd) is involved in wing development and in CNS development. (4l) Chrac-16 is a member of the chromatin accessibility complex. (4m) Su(var)2–10 is involved in chromosome organization and biogenesis. (4n) Uch-L3 has been reported as a member of the regulatory complex of the 26 S Proteasome. (4o) BCL7-like and (4p) msb1l are genes for which little is known. (4q) CG15141, (4r) CG5175, (4s) CG8478, and (4t) CG31639 are genes with no functional information available but with the cell cyclin pattern of expression.
Cell Cyclin Protein and RNA Expression in Axon Guidance Mutants and Transgenics
The cell cyclin clusters show changes in transcript levels of several cyclins (CycA, CycB, cdc2, CycB3, fzy) in both mutant and transgenic embryos. Most notably, the microarray data indicates that for all the robo GOF, robo overexpression is always accompanied by an increase in the transcription of cyclins. Hence we decided to verify if indeed cyclin RNA and protein are increased in robo GOF embryos. We assessed the precise location of these expression changes through in situ hybridization. To verify that changes in cyclin transcript levels correlate with changes in protein levels, we employed immunocytochemistry with various antibodies against cell cyclins. We analyzed the RNA and protein patterns for cyclins (Figure 5) in three robo GOF: (a) UR.mef – robo GOF is overexpressed in all muscles (mef2 driver), (b) UR.yf – a stronger robo than wild-type (robo-yf) is overexpressed in the CNS (elav driver), and (c) FRM – the cytoplasmic domain of robo is overexpressed in the CNS (elav driver). In agreement with the microarray data, robo overexpression in post-mitotic neurons (driver elav) is accompanied with a large increase in cyclin expression both at the level of RNA and protein (black arrows in Figure 5c,5d, and 5e). The increase in cyclin expression is especially clear when overexpressing robo in the muscles (driver mef2), making it visible in the pleural external oblique muscles (5f) and the dorsal external muscles (5g). These findings support the hypothesized link between cell cyclins and axon guidance.
Figure 5. RNA and Protein Cell cyclin Expression in the Axon Guidance Mutants and Transgenics. All embryos are shown ventrally. (5a), (5c), (5e), (5f), (5g), (5i), and (5l) are mRNA expression patterns. (5b), (5d), (5h), (5j), and (5k) are protein expression patterns. (5a) CycA RNA expression (blue) in WT embryos, stage 13. The scaffold of the CNS is stained with antibody BP102 (brown). Cells expressing cyclins are visible on the ventral surface in the middle of the segmental commissures of the VNC. (5b) CycA protein staining in WT embryo, showing a similar pattern to the CycA RNA expression. (5c) CycB RNA expression in UR.yf embryos. There is a large increase in cyclin expression and cyclin-expressing cells are away from the midline (black arrow). There are no cyclin expressing cells at the midline. (5d) CycB protein staining in UR.yf embryos (black arrow). The pattern is similar to the RNA expression. (5e). CycB3 RNA expression in FRM embryos. There is an increase of cyclin expression. (5f) and (5g) CycB RNA expression for embryos with robo overexpressed in the muscles (UR.mef); an increase is seen in the muscles (red arrow), with the pleural external oblique muscles clearly visible in (5f). (5h) Cdc2 protein staining in UR.mef embryos. The staining is very clear in the muscles (red arrow) showing cyclin expression on the muscles, whereas in the WT embryos there is no such expression. (5i) CycA RNA expression in a slit embryo. The cyclin expressing cells are present on a narrow region outside of the VNC, compared to their central and lateral location in the WT (compare regions highlighted with the green arrow); staining at the midline cells does not change. (5j) The midline cells express CycB. In a wt embryo, additional cyclin-expressing cells are also visible in the area abutting the midline. However, in the slit embryo, cyclin-expressing cells are not visible in this area, instead appearing pushed outside the VNC. Also the more lateral cells are absent in the slit embryo. (5k) CycB protein staining of a robo12 embryo. As in the slit embryo (5j), cyclin expressing cells are absent from the area abutting the midline. (5l) CycA RNA expression in robo embryos. The cells expressing cyclins are located as in WT.
The microarray data does not indicate a significant change in cell cyclin expression in axon guidance mutants. But in view of the robo GOF results, we decided to analyze the cyclin expression in the axon guidance mutants slit, robo, and robo12. Consistent with the microarray data, there are no visible quantitative cyclin expression changes in these mutants. Yet several major anatomical rearrangements in the cyclin expressing cells are evident. In wild type embryos, cyclin staining is visible from the dorsal side up to and at the midline. In the mutants for slit (5j) and robo12 (5k), there is a distinct gap in cyclin staining around the midline, and no dorsal cyclin-expressing cells are visible. At the midline, slit and robo12 mutants show WT-like cyclin staining. In robo mutants (5l) no apparent change is observed.
Midline crossing in Drosophila is a model of choice for axon guidance, and yet we are only beginning to understand the role of transcription in the control of axon guidance. Control does occur through protein translation. In such a spatially and temporally fine-tuned process, it is intuitive that transcription would also be involved. Previous work in flies and mice points in this direction; the role of transcription factors is also emerging as very relevant, as reviewed in [27]. To investigate this topic we have undertaken a whole-genome expression study of mutants and transgenics of the key axon guidance genes. If control of midline crossing proceeds through transcription, the genetic perturbations induced in the mutants and transgenics should reveal clusters enriched for genes known to be involved in axon guidance. We also investigated the expression patterns of known axon guidance genes during normal development in a wild type microarray time course, using a previously published microarray data set.
We have found that the hopach package provides a robust, yet flexible, approach to clustering gene expression data. hopach is one of several hybrid algorithms [28] which are gaining popularity in the microarray community. The extension of hopach proposed here has the advantage that all genes can be assigned to a cluster, even when all pair-wise distances cannot be stored in computer memory. Also, the assignment of genes to clusters are based on bootstrap membership values, accounting directly for variability and making results reproducible [11].
To characterize clusters, we recommend integrating gene annotations from multiple complementary controlled vocabularies. In this study we used GO and APoGE, which combine the bulk of literature knowledge with nascent efforts towards a complete genetic expression characterization in Drosophila. When co-expression in microarray data is coupled with a shared RNA expression pattern and/or shared terms in the GO vocabulary, gene annotation via the "guilt by association" paradigm utilized so often in functional genomics becomes a much less tenuous method. For a specific cluster space, we used two independent data sets – a mutant/transgenic data set and a time course data set – and focused on genes which are co-expressed on both data sets. This approach is conceptually similar to techniques where two different axes are used, such as 2-D electrophoresis protein separation.
Our analysis revealed clear co-expression of axon guidance genes in both the GOF/LOF data and the developmental time course. This finding indicates that transcription does in fact play a role in control of midline crossing. We identified several clusters containing many genes known to be relevant in midline crossing, but also numerous less well characterized genes. Cluster AG132 is one of the most interesting clusters; genes such as dock, plexB, Cam, CadN, caps, spen, Fs(2)Ket, Con, RhoGAP19D, Rac2, and shot belong to this cluster. By querying APoGE for spatial/temporal expression patterns of interest, we were able to immediately identify several dozen candidates for further studies. We have selected a pool of candidates meeting both of the following criteria: 1) membership in a selected axon guidance cluster (as discussed in this paper), and 2) a known pattern of expression in the wild-type CNS, according to APoGE. In short, these genes are present in the right time and place to be involved in axon guidance, and further, their expression patterns are perturbed when the guidance process is disrupted. This candidate pool is thus a very attractive target for genetic analysis, the more so because many candidate genes are already represented by mutant stocks in publically available collections. Sensitized genetic backgrounds, such as the slit and robo heterozygotes, have been successfully used in the past to identify additional genes involved in pathfinding [29]. We suggest using trans-heterozygote analysis to search for interactions between (1) candidate genes within the same cluster (such as AG132, AG164, and TC91), and (2) candidate genes and key axon guidance genes (slit, robo, etc.)
Our analysis suggests that known axon guidance genes may also be involved in hemocyte migration. Cluster AG164 contains a significant number of genes expressed in hemocytes, the Drosophila blood cells. Hemocytes migrate during development, with the VEGF pathway involved in the guidance process. We observed through RNA expression analysis that hemocyte migration does not proceed normally in slit and slit.robo – hemocytes are absent in the central segments of the embryo. In robo12, a double mutant for genes robo and robo2 (the known receptors for slit), hemocyte migration is normal, even though the CNS phenotype is the same as in slit and slit.robo. Hence, we speculate that slit may function independently of its robo receptors in this context. Blood vessel migration has been linked in mammals to the same molecular processes as axon guidance during recent studies [30]. Both slit and robo have been implicated in several ways in the vascularization system of vertebrates [31,32]. Since vascularization is a more recent evolutionary development than axon guidance [33,30], it appears that some of the molecules involved in axon guidance may have been co-opted by vascularization mechanisms fairly early during evolution. Prompted by the observation that numerous cell cyclin genes are present in a few clusters in our microarray data, we analyzed RNA and protein patterns of cyclins in axon guidance mutants and transgenics. We examined several robo GOF (UR.mef, UR.yf, FRM), because they show the strongest overexpression in the cyclin cluster of the axon guidance microarray data. We also studied the LOF mutants of each of the key axon guidance genes slit, robo and robo12. The overexpression of robo is accompanied with an increase in RNA and protein levels of several cyclins. We also observe that cyclin expressing cells are dislocated in slit and in the double robo12 mutants. The cyclin expressing cells are visible away from the midline, exactly the opposite direction of the axons that are stalled at the midline in these two mutants. Our results suggest that coordinated expression of cyclins may play a role in timing the midline crossing process. The role of cell cyclins might be to adequately time the developmental state of the neurons and to assure that all the multiple signaling pathways for axon guidance are working in the proper time and location, similar to the role cyclins play in the checkpoints in cell division [23].
In conclusion, this study has shown that axon guidance is under transcriptional control. We have also observed that genes transcriptionally regulated in axon guidance mutants and transgenics are expressed in hemocytes, indicating that blood vessel migration may employ a mechanism involving axon guidance molecules. Furthermore, our results suggest that coordinated expression of cell cyclins may play a role in timing the midline crossing process. Together, these results provide new insights into the roles of axon guidance genes in vivo and demonstrate the validity of our whole genome approach as a method to study transcriptional networks in other biological systems.
Drosophila Stocks
We used 17 mutants and transgenic animals in this experiment. Flies were placed in cages at room temperature and plates were changed every hour. We collected embryos at late stage 15, and visually assessed before RNA extraction. For lethal mutations, the CyO-Kruppel GFP balancer was used [34] and the homozygous mutants were selected against fluorescence under a UV microscope. All mutants are available from Bloomington.
WT flies are Canton-S flies. comm embryos result from crossing CommΔe39[35] with commP [36]. commΔe39 is a null allele. commP is a 900 bp deletion of the 5' UTR and the transcription start site. comm/WT was obtained by crossing commΔe39 with WT flies. The robo LOF is robo4 and is a point mutation [35]. robo.WT was obtained by crossing robo4with WT flies. robo2x123 results from an excision of EP 2582 [4]. robo12 is the robo, robo2 double mutant resulting from the recombination of robo4 with robo2x123 [4]. slit2 comes from an EMS screen and is a point mutation [37]. slit.WT results from crossing slit2 with WT flies. slit.robo was obtained by recombination of slit1 with robo4[29]. UC is the comm GOF, and corresponds to a single copy of the UAS-comm construct crossed with the driver elavGal4 [3]. UC2x is a stronger comm GOF and was obtained using two copies of UAS-comm crossed with the driver elavGal4 (this work). UR is a robo GOF that results from crossing UAS-robo flies with the postmitotic neural driver elavGal4 flies [38]. UR.yf is a phenotypically stronger robo GOF obtained from the cross of UAS-robo-yf flies with the postmitotic driver elavGal4 [39]. UR.mef designates embryos with robo overexpression in the muscles, produced by crossing UAS-robo-yf flies with the mesodermal driver mef2Gal4 [40]. UR2 is a robo2 GOF obtained by crossing the construct UAS-robo2 with the elavGal4 driver [4]. FRM is a robo GOF, obtained by crossing the driver elavGal4 with a UAS chimeric construct, which combines the robo cytoplasmic domain with the frazzled extracellular domain [41]. UNC5 is the unc-5 GOF, obtained by crossing, a GS-element insert located upstream of unc-5, with the elavGal4 (GSunc5 kindly provided by John Thomas).
Developmental Time Course Stocks
The time course data set was published and details reported in [10]. There are 12 samples of 3 replicates each of non-overlapping 1 hour collection, starting from 30 to 90 minutes and ending at 11.5 to 12.5 hours post egg laying.
DNA Microarrays and Target Preparation
RNA was extracted from the embryos using QIAGEN columns according to manufacturer recommendations. Embryonic RNA was hybridized to Affymetrix DrosGenome1 microarrays according to the standard Affymetrix protocols.
All analysis was performed in the R statistical programming language (v2.0.0), available at [42], with the Bioconductor (release 1.5) add-on packages rma, siggenes, and hopach, available at [43].
Data Processing
Expression measures were calculated using the RMA algorithm in the package rma, applied as indicated in [44], with the recommended quantile-quantile normalization procedure. The siggenes package was used to rank all genes based on differential expression (log2 ratio) relative to a control, which was RNA from WT flies. The top ranked most differentially expressed genes in each condition (mutant or transgenic) were selected until their number totaled 2000 for each experiment. These provided the initial seed data set for hierarchical clustering.
Clustering with hopach
hopach is a hybrid hierarchical clustering method specifically designed for analysis of microarray data. The algorithm builds a non-binary, hierarchical tree, but also assigns genes to clusters [11]. The final level of the tree provides a meaningful ordered list in which nearby genes are similarly expressed. The first level of the tree with maximal average cluster homogeneity is identified and cluster labels are assigned based on the partitioning of genes in this level. Then, each gene's membership in every cluster is assessed using a non-parametric bootstrap method that fixes the cluster profiles, and for each of many resampled data sets reassigns every gene to the cluster whose profile is closest. The proportion in which a gene appears in each cluster is a measure of its cluster membership. Thus, the variability of the estimated cluster labels can be directly assessed from a single data set through this resampling approach.
We adapted the hopach methodology as follows:
1. Initial clusters
Apply the standard hopach algorithm with an appropriate choice of distance to a set of several thousand pre-selected genes. This produces initial clusters, each represented by a medoid gene.
2. Bootstrap extended clusters
Run the non-parametric bootstrap with the fixed medoids (from the previous step) as described in [11] to obtain a percentage membership value (between 0 and 1, with 1 meaning that a gene only belongs to that cluster) for each gene in every cluster. Reassign genes to clusters based on an appropriate threshold for bootstrap membership. Possible values include > 0.8 (genes belong to only one cluster, very homogenous clusters), > 0.5 (genes belong to only one cluster, less homogeneous) and > 0.3 (genes can be present in more than one cluster – fuzzy clustering). Note that this step is performed using all genes in the data set (not just selected genes), so that every gene is now assigned to a cluster.
3. Final clusters
Optionally, apply hopach again to every extended cluster to produce a new set of sub-clusters. In general, large clusters will be further divided at this step, whereas smaller ones will not. When the membership threshold in Step 2 is large enough that genes belong to only one cluster, this second application of hopach can be used to create a full hierarchical tree and a unique, final ordering of all genes. If this step is skipped, the bootstrap extended clusters are the final clusters.
4. Order within clusters
Reorder the genes in each cluster based on distance to the medoid – the medoid for each cluster is thus ranked one, genes with low ranks form the core of the cluster, and genes with high ranks are more peripheral.
This modified hopach method was applied to each data set separately, beginning with 2000 selected genes and including all genes on the Affymetrix DrosGenome1 microarray in Step 2. We used cosine-angle (uncentered correlation) distance (Step 1) and a membership threshold of 0.3 (Step 2).
Controlled Vocabularies
The vocabularies for GO were downloaded from [45]. APoGE vocabulary can be obtained upon request. We selected terms relevant to axon guidance: "axon guidance" (GO:0007411) in GO; "ventral nerve cord", "embryonic brain" and "ventral midline" in APoGE. Over representation in each cluster was evaluated using the hypergeometric distribution. Clusters of size 10 or larger were ordered based on hypergeometric p-value, which gives the probability of finding by chance alone the observed number of genes or more in that cluster annotated with the term (conditional on the total number of genes, the size of the cluster, and the frequency of the annotation over all genes). For each term, this produces one p-value for each cluster. For the most interesting clusters, we queried all the available pictures in APoGE, searching for patterns consistent with relevance to axon guidance mechanisms.
In Situ Hybridization and Antibody Staining
In situ hybridization were done to assess RNA expression of several specific genes. The experiment was conducted in 96 well plates and followed the protocol published in [10]. Antibody staining of whole mount embryos followed the standard procedure outlined in [46,29]. BP102 (a gift from Corey Goodman) is a specific antibody for the Drosophila embryo central nervous system. B102 was used at concentrations of 1:10. The antibodies against the cyclins CycA, CycB, CycB3 and Cdc2 were kindly provided by Patrick O'Farrel and were used at concentrations of 1:5, 1:5, 1:5 and 1:4. The HRP-conjugated secondary antibodies [Jackson Labs] were used at a 1:250 dilution.
Authors' contributions
TRM designed the experiment, carried out the microarray hybridizations and analysis, and performed the in situ and protein hybridizations. JP participated in experiment design and in the in situ and protein hybridizations. PT participated in the design of the study and is the responsible for the APoGE database. KSP contributed to design of the study, microarray analysis and writing the manuscript. All authors read and approved the final manuscript.
Additional file 1. Axon Guidance Cluster 132. Annotations and In situ images for genes in Cluster AG132.
Format: PDF Size: 4.9MB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 2. Axon Guidance Cluster 164. Annotations and In situ images for genes in Cluster AG164.
Format: PDF Size: 7.1MB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 3. Time Course Cluster 91. Annotations and In situ images for genes in Cluster TC91.
Format: PDF Size: 4.2MB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 4. Hopach Clustering Results. Tables of gene cluster assignments for axon guidance and time course microarray data sets.
Format: XLS Size: 3.3MB Download file
This file can be viewed with: Microsoft Excel Viewer
We thank Corey S. Goodman, Beth Blankemeier, Eileen Li, António Coutinho, and Carlos Duarte. Tiago R. Magalhães was supported by a fellowship from the Fundação para a Ciência e a Tecnologia.
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An older gentleman asked me to sell some saddles for him. They have been stored in his tack room for many years. This one is older...I cannot find a brand name on it. It is stamped 1500 on the bottom and I think some words,but I can't make them out. I am not a western rider at all! So I am hoping I measured the seat correctly. If I did it is a 15". I don't know about gullet,bars,etc.?
**It is darkish brown NOT reddish. In most of the pics I had my flash on....in the last few I didn't,but it is the correct color in the last ones. Dark brown.
This saddle is in great shape,sound tree,pretty braided stirrups,comes with lagtigo, I don't see any obvious huge flaws on it,but I know it is an older one so it isn't brand-new.
He only wanted $200 including shipping on it so I told him I would list it in case anyone on here was interested.

subset: commoncrawl


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So many briefings, interviews and exhibits, so little time. Completely absorbing the fire hose of data coming at you every day is sometimes a futile struggle. I've filed about eight news stories today for the Flight Daily News, but only scratched the surface of my notebook. Here's a few random bits from today's news. (I snapped the photo above at daybreak on Sunday morning.)
1. Sukhoi is suddenly coy about the prospects for the first flight of the T-50 demonstrator by end-year. It was only a few months ago that Sukhoi CEO Mikhail Pogosyan confidently predicted the PAK-FA demonstrator would fly before 2010. Asked about that prediction today, he told reporters that Sukhoi would have more information about first flight at the Farnborough air show next July.
2. In a press briefing about Northrop Grumman's unmanned systems, a completely new aircraft design was pictured on one of the slides. I asked what it was. The Northrop executive identified the UAV as the "WildThing", a fan-in-wing-powered design aimed at the naval cargo market. That was interesting. A few hours later, Northrop notified reporters that it wasn't called "WildThing" after all. It is instead called the MUVR. Okay, so then what is WildThing? Northrop replies: "WildThing is a proprietary vertical-lift concept for fleet self-defense in small-deck shipboard operations".
3. Northrop Grumman is expecting a contract award for KC-X in March. So much for Secretary of Defense's Bob Gates' objective to sign a deal by the end of the year.
4. The next 90 days could be very interesting in the F-35 Joint Strike Fighter flight test program. First, BF-1 and BF-2 return to flight. The first optimized conventional-takeoff-landing prototype, AF-1, will also fly within that timeframe. If Lockheed Martin can pull it off, the planned burst of activity would end a nearly 9-month-old lull in multi-aircraft flight test operations.
5. Two different companies — Northrop Grumman and Raytheon — are eyeing the concept of dramatically scaling-up same basic airframe design for the MQ-X role. Northrop calls it the Bat and Raytheon calls it the KillerBee, but both come from the same design by Swift Engineering. Raytheon's legal right to make this pursuit is a matter of dispute between the two companies.
Random 5: Paris Air Show Day 1
By Stephen Trimble on 15 June, 2009 in Uncategorised
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RIDGELAND – Coach Jamie Mitchell came to Starkville with the plan to get the program back in a position to where it's winning state championships.
The 24th-ranked Mississippi State Bulldogs are getting contributions from unexpected sources when it's needed the most.
The Starkville Yellowjackets have come too far to turn back now.
There will be a Starkville High School football reunion among coaches tonight in Quitman.
Mississippi State has had success winning its last four games which as earned a No. 24 ranking.
The Ridgeland Titans are about to find out if their legs can carry them all the way to the Class 5A state championship game.
The Starkville Yellowjackets have already had a great football season.
The Egg Bowl is only two days away and Mississippi State is going for its third consecutive series win.
Porsha Porter provided the backbone to give the Mississippi State Lady Bulldogs a lift on Wednesday.
By DANNY P. SMITH


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Lord help us if the Phillies sneak in and grab the last wildcard spot. They will be an eXtemely tough out for anyone, especially the Reds.
Lord help us if the Phillies sneak in and grab the last wildcard spot. They will be an eXtemely tough out for anyone, especially the Reds.
agreed, but unfortunately we cant control how bad the dodgers, pirates and cards have been playing
Brewers as well. Very dangerous 2 teams
Philadelphia has a 4% playoff chance right now its amazing how many people think they have a real shot at jumping so many teams and getting in.
only 3 games back with about 3 weeks to go
If the Cardinals choke the Phillies will still need the Dodgers, D-Backs, Pirates and Brewers all to not keep pace as well. But I would love to see the Cardinals blow it to one of them preferably the Dodgers or Pirates.
Now the Phillies are 3 back, they better pull for the Diamondbacks tonight since if the Dodgers win they will not be able to pick up ground regardless of win the Cards vs Dodgers game tomorrow since they would be tied.
At this point I'd much rather play the Cards than the Phillies. Chances are they will not be our problem until the second round unless Washington starts losing games which they have shown no signs of doing.
I'm not buying the have only a 4% chance of going 15-4 while the Cards go 11-8 or worse, Dodgers 12-8 or worse, Pirates 13-7 or worse....and Brewers 14-5 or worse.
Not buying the math....the most difficult is the Phillies end of it.
This is going to be exciting....you post way more about this than anyone. I'm checking the scores often on every game that involves the Braves, Cardinals, Phillies, Brewers, Pirates and Dbacks. If you already know it just can't be the Brewers and Phillies then don't update us on them.
Enjoy it....don't assume the Braves and Cards are locks.
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FRIDAY, July 30
July 21 at 5 a.m. -- non-injury, Ky 1054 South, Ricky Johnson, 19, Berry, driving a 2003 Ford F150.
July 18 at 1:15 p.m. -- non-injury, US 27 North, an unidentrified driver, driving a 1994 Dodge Power Ram. The driver left the scene of the accident.
July 20 at 11:17 p.m. -- non-injury, US 62 East, Ashley A. Vickers, 18, Cynthiana, driving a 2005 Ford Focus.
July 17 at 9 p.m. -- non-injury, Ky 353, April Claypool, 22, Cynthiana, driving a 2007 Chevrolet 4x4.
• Madison Jo Milner was born to Emily Huffman and Brandon Milner on May 29, 2010 at Central Baptist Hospital. She weighed 4 lbs.
• Mallory Maree Houg was born to Susie Crump and Jonathan Houg at Harrison Memorial Hospital on June 14, 2010. She weighed 7 lbs. 2 oz.
• Tre'quan Roger-Allen Commodore was born to Jessica and Roger Commodore of Cynthiana on July 19, 2010 at Harrison Memorial Hospital. He weighed 7 lbs. 9 oz.
Coach Mark Carroll is in his second week of practice with the 2010 Harrison County Thorobreds football team and is optimistic about the chances for the season.
The Thorobreds return six senior and two junior starters from last year's defense and have six other players who contributed last year with more expected from them this year.
We should use more crape myrtles in Kentuckiana, they are not just for more warm, temperate climates; in fact, there are a great many that go unbothered by an average winter in our parts.
There is often a lull in the garden at this time, our early summer blooms are fading and our late summer garden has yet to pop; but the crape myrtle can provide a colorful bridge between the two. This summer blooming shrub is in full glory by the end of July through August, the months of high summer, as I like to call it.
High school graduates to age 25 are invited for Rev2.0 night. They meet twice a month on the second and fourth Thursday at Rohs Opera House. Will meet Thursday, July 22 from 6:30-8 p.m. There will be games, prizes, food and fun. Childcare is provided on site. All this is free.
For more information contact Carri Haakinson at 588-6816.
The Revolution Youth Group
• Suzanne Shepherd and Glenn Joseph Wilson to marry on Aug. 14, 2010 at 6:30 p.m. A reception will follow the ceremony. Invitations were sent.
• Erin Lindsey Galasso and James Matthew Cooper plan later summer wedding at The Church of the Good Shepherd in Lexington, Ky.
Harrison County High School golf coach Jason Crisp is in his second year of coaching the boys' golf team and will be coaching the girls' team for the first time this season.
The teams are as different as night and day. The girls have virtually no varsity experience while the boys return four of their top five from last year. Crisp had tryouts for the boys' team while there are only four girls so there was no need for a tryout.
10 years ago . . .
Cynthiana Summer Showdown at
River Road Park in August
There will be an open fast pitch softball tournament at River Road Park Aug. 13-15 for teams 8 and under through 16 and under. Some games will also be played at Shirley Field at the high school.
The tournament will be played under high school rules and will feature games of seven innings or 75 minutes. Entry is $275 and will close Aug. 8.
Team signup will be on a first come first served basis.
Mudcats win coach-pitch tournament
Copyright © 2015
The Cynthiana Democrat is your source for local news, sports, events, and information in Cynthiana, KY and the surrounding area.


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Family: Wowed by wigs
Posted: April 8, 2014 at 11:30 a.m.
Rob Pickens admits he gets a bit attached to the wigs.
That's obvious during a late March afternoon, which finds him in the basement of the Arkansas Repertory Theatre, surrounded by some of about 50 wigs from its recent production of Les Miserables, writes Helaine Williams in Family.
Lovingly handling the wigs — mostly synthetic, with touches of human hair — worn by such principal characters as Fantine, Madame Thenardier, Cosette and Jean Valjean, he refers to the wigs by the characters' names and human pronouns.
This is "just because … they have so much character to them," Pickens says. "You have to get into character a little bit when you're styling the wigs. Like how Madame Thenardier would put up her hair in her dingy mirror every morning — pop some dead flowers in it," and go on about her day.
"It's amazing the transformation power wigs have," Pickens says.
See Wednesday's Arkansas Democrat-Gazette for more.
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You can now invite anyone to meet at your favorite Starbucks.
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34 year old man | Mounds View, MN, USA | Seeking women 26-36 within 50 miles of Saint Paul, MN
Former HipHop instructor turned Real Estate agent.
If you're interested in someone who likes to workout, have fun, enjoys business, and eating food, than that makes two of us...! A little bit of jokester, otherwise typically direct and to the point. I am a real estate agent so I do work around the clock for my clients BUT I also get to set my own schedule which I use to typically sleep in since I hate leaving my bed in morning...
Coffee and conversation , Business networking , Cooking , Dining out , Movies/Videos , Performing arts , Playing cards , Playing sports , Video games , Watching sports
Aerobics, Bowling, Dancing, Other types of exercise, Running, Weights / Machines, Yoga
I exercise 3-4 times per week.
I like Cats, Dogs
Some other viewpoint
I like to try new restaurants and bars whenever possible, but I do have a couple of favorites. NE Social, Champps in NB, Wild Bills in Blaine
I love movies that make me laugh or ponder life in a new perspective, or a great 2 hours of computer animated visuals will do. I am a huge nerd for Netflix documentaries!! I like competition TV like top shot, top chef, faceoff,
For fun I exercise, hang out with friends and family, make food, read, watch movies, whatever will help me improve or relax.
I read tons of books on sales, marketing, self improvement, history, strategy, body language, the mind/brain, psychology, and leadership. Then I have read a few fantasy series (Harry Potter) but not that often.
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|5'10" (177cms)
|5'0" (152cms) to 5'11" (180cms)
|Body type:
|Athletic and toned
|Slender, About average, Curvy, Athletic and toned
|No preference
|Dark blonde
|No preference
|No preference
|Social Drinker
|Social Drinker, Moderately
|Financial / Accounting / Real Estate
|No preference
Real Estate Agent with Keller Williams, former hiphop instructor and choreographer
|$35,001 to $50,000
|Less Than $25,000, $25,001 to $35,000, $35,001 to $50,000, $50,001 to $75,000
|Never Married
|Never Married
|Have kids:
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|English, Spanish
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|Some college
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text:
As of Saturday, May 4, 2013
© Copyright 2015 Albany Herald
ATLANTA -- The Indiana Pacers brought the energy and the inside game to Game 6, beating the Atlanta Hawks 81-73 Friday night at Philips Arena to close out their first-round playoff series four games to two.
The Pacers now advance to the Eastern Conference semifinals to play the New York Knicks, who eliminated the Boston Celtics on Friday night.
Roy Hibbert broke through the mental barrier of a Pacers' winless streak in Atlanta that had reached 13 games until Friday night. Hibbert scored 17 points and grabbing 11 rebounds.
Indiana's George Hill had 21 points and seven rebounds, and David West added 21 points -- 10 in the third quarter -- and eight rebounds. Lance Stephenson compiled eight points, 11 rebounds and six assists.
The Hawks' co-captains, Al Horford and Josh Smith, each had only four points at halftime. Horford finished with 15 points and seven rebounds while Smith ended with 14 points and nine rebounds. Devin Harris scored 14 points and Kyle Korver added 12 points.
Hawks coach Larry Drew met separately with Jeff Teague Thursday, impressing upon him that scoring was only part of his role. Drew urged Teague to be aggressive, but Teague was unable to produce, finishing with 10 points and only two assists.
In a sluggish second quarter, the Pacers mustered 16 points on 7-for-22 shooting. The Hawks scored only nine points on 1-for-15. The crowd was booing as the Hawks missed open shot after open shot.
The Pacers went to the locker room at halftime up 37-29. The eight-point lead was the largest either team had had at halftime as the visiting team.
The Pacers had to have been pleased to be up 21-20 after the first quarter. Earlier in the series, the Hawks had made runs at the start of the second quarter in Games 3 and 4.
But on Friday, the Hawks couldn't buy a basket. They went scoreless from 10:52 to 5:08 in the period. Much had to do with the Pacers, who kept the Hawks' transition game in check by easily beating Atlanta back on defense.
The Hawks also missed their first two shots of the second half, finally scoring on a goaltending call at 9:38 in the third quarter.
The Pacers were on a 19-4 run halfway through the quarter. Drew was imploring his players. "This is gut-check time!"
But the Hawks had nothing and the Pacers built a 19-point lead.
To their credit the Hawks didn't throw in the towel, creeping back to trail 69-58 at 9:10 in the fourth quarter. Suddenly, they awoke and narrowed the gap to 73-68 on a 16-4 run with 4:02 left in the game.
The Hawks got as close as 78-73 with 47 seconds left.
NOTES: Drew said before the game that the adjustments the Pacers made in Game 5, which Indiana won 106-83 on their home court, didn't affect his team. "We just have to be better," Drew said, "at some of the things we need to do." … Drew said he had emphasized to his players not to put too much pressure on themselves, not to look ahead to a Game 7 on the road and give all of their attention to Game 6. "Game 7 is not important right now," he said. Drew couldn't recall ever having been part of a series where the home-court advantage was so pronounced. "That's why home court is so important," he said. "You play with a little more juice, I think." … Pacers coach Frank Vogel had no adjustments to announce before the game. "In terms of things the series hasn't seen yet," he said, "we're probably beyond that." What he was happiest about, he said, is that "I think our guys are finally understanding how to guard them. It took a couple games to get there, obviously had to make some adjustments when they made their adjustments. Now it's about doing it." … Vogel packed three suits for the trip and told his players to pack for six days. "You asked me if there was a psychological move to tell the team to pack for six days," he said. "It's practical."
Knicks finish off Celtics
BOSTON - Despite a valiant comeback try by the Boston Celtics, both in the series and the game, the New York Knicks are in the second round of the NBA playoffs for the first time since 2000.
The Celtics, who rallied from an 0-3 deficit and created designs on becoming the first NBA team ever to come back to win after being three games down, ran off 20 straight points in the fourth quarter and were within four with 3:34 left. But Carmelo Anthony took over in the closing minutes and the Knicks left TD Garden with an 88-80 Game 6 victory on Friday to win the series 4-2.
New York moves on to face Indiana in the second round.
Anthony, who finished with 21 points, seven rebounds and five assists (he had six assists in the first five games), scored seven points in the closing minutes to help hold off the Boston charge, which started with the Celtics trailing by 26 points with less than 10 minutes left in the game.
The game might mark more than the end of the season for the Celtics, who did all they could without the injured Rajon Rondo. It could also mark the end of the Boston career of Paul Pierce. He has a year left on his contract at close to $15 million, but the team can buy him out for $5 million.
Last year, with the Celtics trying to stay alive against Miami in the conference final, Pierce went 4-for-18 from the floor and scored nine points. Friday night, he was 4-for-15, including 1-for-9 from 3-point range, and had 14 points, seven rebounds, five assists and five turnovers.
Iman Shumpert had 17 points and Pablo Prigioni 14 (going 4-for-6 on 3-pointers) in a balanced New York attack.
Jeff Green led the Celtics with 21 points before fouling out, while Kevin Garnett had 15 points and 10 rebounds and Jason Terry 14 points.
With Pierce going 1-for-10 from the floor, the Knicks probably should have led by 25 or more at the half. Instead, New York saw half of an 18-point lead evaporate before an old-fashioned 3-point play by Anthony with 4.5 seconds left in the half made it 39-27 at the break.
The Celtics missed their first nine 3-pointers before Green and Terry connected during a 10-0 run in the second quarter. They ended the half with 11 turnovers.
The Knicks then came out and had the lead back up to 18 3:04 into the second half. But Boston again answered, cutting it to 10 on a Terry three with 4:38 left in the third quarter.
Then came a 12-2 New York burst, with Shumpert nailing a pair of threes, and doing the Terry "Jet" celebration down the court. The burst gave the Knicks a 20-point lead after three and had boos coming from the crowd. Some started leaving.
They should have stayed. Things got interesting after the Knicks built a 26-point lead early in the fourth quarter.
NOTES: Knicks 3-point specialist Steve Novak was missing because of back spasms. "I just think it went out, locked up on him (in Game 5)," coach Mike Woodson said. "He couldn't come back after the halftime, so we've got to move on." … Celtics coach Doc Rivers, ending his pregame media chat, said, "Let's hope we're talking about basketball after the game." … No. 1 Knicks fan Spike Lee, resplendent in Knicks orange, arrived about an hour before tipoff. When Celtics radio analyst Cedric Maxwell approached, Lee waved him away. They then had a chat. … Injured Knick Amar'e Stoudemire, who underwent right knee surgery on March 11 and hasn't played since, was on the floor working out before the teams came out. He isn't expected to play Sunday (regardless of which round) but could return later in a second-round series. "I don't think he's at that stage yet of playing in a game," Woodson said. … Recently retired former Knick and Celtic Rasheed Wallace was also out shooting around. … Rivers on the unpleasantness between the teams: "There's nothing wrong with a little bit of healthy dislike." … The Elias Sports Bureau says the Celtics became the first team in NBA playoff history to be perfect from the foul line in two straight games: 19 of 19 and 17 of 17. … Only the 1951 Knicks, the 1994 Nuggets and 2003 Trail Blazers rallied to force a Game 7 before the Celtics and Rockets both attempted to do it Friday night. The other 100 3-0 leaders entering this season won their series and did it in less than seven games. In fact, according to Elias, Boston and Houston are only the 11th and 12th teams to force a Game 6.
Report: LeBron to be named NBA MVP for fourth time, make history
The NBA will name Miami Heat star LeBron James as league MVP, ESPN reported Friday, citing league sources.
It will be James' fourth MVP honor. He also won it last year.
Only Michael Jordan, Bill Russell, Kareem Abdul-Jabbar and Wilt Chamberlain have also won the award at least four times.
Other candidates included Oklahoma City Thunder's Kevin Durant and Los Angeles Clippers point guard Chris Paul.
League commissioner David Stern will present the MVP trophy to James in Miami on Sunday, a day before the Heat begin their second-round playoff series against either the Brooklyn Nets or Chicago Bulls.
Miami swept the Milwaukee Bucks in the first round.
James told reporters Friday that he is just focused on winning another championship, but coach Erik Spoelstra spoke about the MVP honor.
"He's earned it," Spoelstra said. "He's having an even more historic season than he's had the last two years. Most people probably didn't think he could go to a higher level after last season, yet he reinvented himself to show that he could. Our pinch-ourselves moment was when we signed him. But since we've had him, we don't take him for granted."
James averaged 26.8 points, 8.0 rebounds, 7.3 assists, 1.7 steals and one block in 76 regular season games. He also shot a career-best 56.6 percent from the field and 40.6 percent from 3-point range.
The Heat went a franchise-best 66-16 this season, including a 27-game winning streak — second-longest in NBA history behind the Los Angeles Lakers' 33-game streak in 1971-72.
Kobe, mom headed for ugly legal battle
Los Angeles Lakers star Kobe Bryant is trying to stop his mother from selling off memorabilia from earlier in his career.
After Bryant's lawyers ordered New Jersey-based Goldin Auctions to cease-and-desist selling off the items, the online auction house filed a lawsuit against Bryant, according to CSN Philly.
Pamela Bryant sold the items to Goldin Auctions recently.
Kobe Bryant claims that his mother does not have the right to sell the items, but she contends in the lawsuit that he told her years ago that he does not want them.
The items include several of his high school uniforms, NBA championship rings and many other collectibles.
Pamela Bryant wants to auction off the items to buy a house, according to the lawsuit.
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More like this story
- NBA PLAYOFFS ROUNDUP: Pacers put Hawks on brink of elimination with Game 5 win ( May 1, 2013 )
- NBA PLAYOFFS ROUNDUP: Knicks go up 3-0 on Celtics with 90-76 win ( April 26, 2013 )
- Boston rests Big 4; Hawks still barely win ( April 21, 2012 )
- NBA PLAYOFFS ROUNDUP: Celtics clip Hawks in OT in Game 3 ( May 5, 2012 )
- Hawks beat Celtics in playoff opener ( April 29, 2012 )


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WorkForce? More like workhorse. This feature-packed, office-friendly multifunction printer has a list price of $299.99. Best feature? Double-sided printing!


text:
Senior Member
15 Forum Posts
G'day all,
I'm about to finalise the design of my rover using the CYC5588AXI-060. It's replacing a Zilog eZ80F91 module, which only required 2 debug/programming pins, TCK and TDI (and ground, obviously!).
However, I'm unclear about exactly which pins are required for the SWD mode debug/programming interface. I'd appreciate it if someone could confirm exactly which pins need to be connected to the header for non-JTAG programming. I'd also like to understand if I need to make any changes to the programming routine if only the SWD pins are used. The family and device datasheets mention switching to SWD from the JTAG connection, but not details of which of the common pins need to be connected first. Any help would be appreciated!


text:
Seibertron.com - The Ultimate Transformers Fansite
Rhinox23 wrote:
Does anyone else's UM have this issue? Does mine just have loose/weak joints? Is there anyway to remedy this?
wilcosu35 wrote:mine's also really loose in the joints. and he also came out of the box missing one of his leg wheels
i'm really on the fence about him. qc-wise he's bad, he looks dull and his transformation is just plain simple and boring
Henry921 wrote:You can always be counted on to listen to reason, Pryme.
Dead Metal wrote:Have you ever, and i mean ever seen/read/heard something that is completely original and does not copy/homage/pay tribute to something else? Here's a hint: Nope. You never have and you never will.
Return to Transformers Toys Discussion
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text:
4 User Responses
The office staff is the worst in Greenville, the only person that went out of their way to help you doesn't even work there anymore. They always act like they are doing you a favor to wait on you, especially when I bring my children in, which they despise. Their model is on the 2nd floor, how are handicap people supposed to look at it? Not that Snoopy would care because she would never show apartments, just scream at you if your rent is five minutes late and the only reason you were late is because your father just died. Trust me, put your money into a house which is how much it cost to live here!
To me it seems like livedhereforyears and justmystyle are part of the office staff. Wherever there is a complaint, there is a livedhereforyears and justmystyle hero to save the day for Carlyle. What they don't get is that people tell about THEIR experiences, not about livedhereforyears's and justmystyle's.
I think it's funny how the office staff secretly puts nice plugs about themselves in as if they are true tenants. I think it's hilarious how snobbish they are considering they work in the front office of an apartment complex. APARTMENTS! I mean, how snobbish can you be, none of these people are fit to own. Get off your high horse.
I think you worked here for a while
I agree. The office staff is not very nice. In fact, they are want to be snobs and like they are better than everyone else. News flash, they're not! They need to focus on the tenants and not themselves.
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text:
Oilers complete third-period comeback in shootout win over Devils
Oct 8, 2013, 12:46 AM EDT
The Edmonton Oilers began the third period of Monday's game against the New Jersey Devils trailing by three goals and in danger of losing their third straight to begin the new season.
And that's when the comeback started. The Oilers scored four times in the third period to actually take a one-goal lead and then won the game 5-4 in a shootout.
"Like I've said before, all it takes is one shift, one period to get the season going. That's all we needed," said Oilers' forward Taylor Hall, as per the team's Twitter account.
The Oilers had lost their first two games to begin the 2013-14 season and had looked bad doing so. They had been outscored 11-6 coming into Monday, and their performance caused another public tongue lashing from new coach Dallas Eakins.
"The city and the fans should be angry, as angry as we are," Eakins said following the Oilers' loss in Vancouver Saturday. "It's great that they care. Our team is upset too."
It didn't look like those words had much influence on his team Monday.
The Devils had a 2-0 lead after the first period and a 3-0 cushion after 40 minutes. Jaromir Jagr also scored his first as a member of the Devils in the opening period.
It didn't look good.
But the Oilers managed to come alive in the second period, scoring four times in the span of seven minutes and 47 seconds.
Oilers' goalie Jason Labarbera got credit for the win, while Ryan Nugent-Hopkins scored in his return to the Edmonton lineup after recovering from shoulder surgery in April.
- peterjohnjoseph - Oct 8, 2013 at 1:04 AM
Glad to see Ferrence get on the board, and lead this team to a win. Hopefully this gives them some confidence.
- 950003cups - Oct 8, 2013 at 1:05 AM
This team is slowly becoming a big joke. They dropped a 3 spot in the 3rd vs Labarbara????
- mpg44 - Oct 8, 2013 at 6:08 AM
I agree except for the slowly part.
- valoisvipers - Oct 8, 2013 at 2:33 PM
Atleast they are putting pucks in the net. The rest will work it's self out and you will still have the Flyers to troll on.
- mp1131211 - Oct 8, 2013 at 1:16 AM
That third period was just bad. First the Devils blew it and then the Oilers blew it. Neither of them could play with the lead tonight.
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text:
Brookport, Illinois
|United States
|37°7′30″N 88°37′38″W / 37.12500°N 88.62722°WCoordinates: 37°7′30″N 88°37′38″W / 37.12500°N 88.62722°W
|0.66 sq mi (2 km2)
|- land
|0.65 sq mi (2 km2)
|- water
|0.01 sq mi (0 km2)
|1,054 (2000)
|1,321.8 / sq mi (510 / km2)
|- summer (DST)
|Postal code
|Area code
|Wikimedia Commons: Brookport, Illinois
Brookport is a city in Massac County, Illinois, United States. The population was 1,054 at the 2000 census. It is part of the Paducah, KY-IL Micropolitan Statistical Area.
Brookport is located at 37°7′30″N 88°37′38″W / 37.12500°N 88.62722°W (37.125056, -88.627218).[1]
According to the 2010 census, the city has a total area of 0.66 square miles (1.7 km2), of which 0.65 square miles (1.7 km2) (or 98.48%) is land and 0.01 square miles (0.026 km2) (or 1.52%) is water.[2]
Brookport is located on the north bank of the Ohio River, opposite Paducah, Kentucky.
The city is located on US Route 45.
There is a bridge across the Ohio River at Brookport, that connects Kentucky and Illinois. This bridge runs between Paducah and Brookport. The bridge is named for Irvin S. Cobb, who was born in Paducah; the bridge was built by the WPA. Lock and dam 52 is also located in Brookport and is run by the Army Corps of engineers.
As of the census[3] of 2000, there were 1,054 people, 450 households, and 283 families residing in the city. The population density was 1,321.8 people per square mile (508.7/km²). There were 519 housing units at an average density of 650.9/sq mi (250.5/km²). The racial makeup of the city was 90.61% White, 7.12% African American, 0.47% Native American, 0.28% Asian, 0.47% from other races, and 1.04% from two or more races. Hispanic or Latino of any race were 1.90% of the population.
There were 450 households out of which 30.0% had children under the age of 18 living with them, 44.2% were married couples living together, 14.7% had a female householder with no husband present, and 37.1% were non-families. 33.8% of all households were made up of individuals and 15.1% had someone living alone who was 65 years of age or older. The average household size was 2.34 and the average family size was 2.97.
In the city the population was spread out with 28.1% under the age of 18, 8.3% from 18 to 24, 26.7% from 25 to 44, 21.4% from 45 to 64, and 15.6% who were 65 years of age or older. The median age was 35 years. For every 100 females there were 94.5 males. For every 100 females age 18 and over, there were 90.9 males.
The median income for a household in the city was $24,438, and the median income for a family was $30,000. Males had a median income of $31,250 versus $19,188 for females. The per capita income for the city was $11,751. About 16.2% of families and 19.8% of the population were below the poverty line, including 28.5% of those under age 18 and 17.5% of those age 65 or over.
See also[edit]
- ^ "US Gazetteer files: 2010, 2000, and 1990". United States Census Bureau. 2011-02-12. Retrieved 2011-04-23.
- ^ "Places: Illinois". 2010 Census Gazetteer Files. United States Census Bureau. Retrieved 2012-05-03.
- ^ "American FactFinder". United States Census Bureau. Retrieved 2008-01-31.


text: The Rescue: The Embassy Mission wiki last edited by Jagged85 on 10/09/13 11:33PM View full history Rescue: The Embassy Mission is a short NES game, involving 4 different types of gameplay segments as the player attempts to rescue hostages and eliminate the terrorist threat. In the opening cutscene, terrorists take an embassy hostage. The player controls 6 SWAT team members infiltrating the embassy to rescue the hostages within. The player sets two separate difficulties (dubbed Rank and Mission), determining variables such as enemy reaction time and the presence of innocents who can be injured by the actions of the player. Regardless of the settings, the goals of the story and geometry of the levels remain the same. The player starts controlling the three snipers on the team. In the first segment, the player must maneuver the individual snipers to the entrances of the buildings surrounding the embassy to take up their sniper posts. The first sniper must make it to the closest sniper post, the second the middle point and the third to the farthest point. If a sniper is killed during this segment, the next sniper attempts to make it to his sniper post, with the last sniper point becoming unavailable to the player. To reach the points, the player must avoid the terrorist's searchlights. The player can roll using the a and b buttons, go prone and crawl with the down arrow and take cover in doorways, windows and other objects using the up arrow to avoid being spotted. Once the snipers are in position, the player can take control of the snipers again. The perspective switches from side scrolling to through the sniper's scope. The player can eliminate terrorists who happen to be near the windows. Reducing the number of terrorists inside the Embassy, makes further gameplay segments easier. The directional pad moves the aims the rifle, a and b fire. Each of the three sniper points have a vantage point on a different side of the embassy. In the third gameplay segment the player takes control of the assault team, who have been dropped off on the roof by a helicopter. Individually the three assault team members must rappel down the side of the embassy and enter through a window. The player can enter on any of the three floors. The player uses the up and down buttons to carefully descend. Pressing down causes the player to descend, but if the player descends to far without catching the rope, the team member will fall and die. The up button catches the rope and climbs upward. If the player encounters a silhouette in the window and crosses that window, the rope will be cut by that terrorist. These silhouettes are the terrorists that can be eliminated by the sniper, the player can switch to the sniper at any time to eliminate a suddenly appearing enemy before crossing that window. When the player reaches the desired floor, the a and b buttons are used to break through the window. The final gameplay segment is in the first person. Individually the assault team members make their way through the three floors of the building, eliminating the terrorists. Movement is handled through screens. The player can better understand the character's position through a minimap. Up on the direction pad always moves one screen forward (including through doors and using stairs), left and right turn left and right one screen. The a and b buttons fire the weapon. When a fire button is continuously fired, the direction pad can be used to aim the gun as the gun continuously fires. In certain difficulty settings, the hostages will appear in the map and can be shot by the assault team. At lower difficulties, everyone (terrorists and hostages) appear on the minimap. The game ends when all terrorists are eliminated. On most difficulty settings, the player views an ending cutscene rating the player's performance in the mission. The number of hostages and team members who survived are tallied and a newspaper article detailing the casualties is displayed.