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About Development
About the Node
Localized JAK/STAT signaling is required for oriented cell rearrangement in a tubular epithelium
Katherine A. Johansen, D. David Iwaki, Judith A. Lengyel
Development 2003 130: 135-145; doi: 10.1242/dev.00202
Katherine A. Johansen
D. David Iwaki
Judith A. Lengyel
Rearrangement of cells constrained within an epithelium is a key process that contributes to tubular morphogenesis. We show that activation in a gradient of the highly conserved JAK/STAT pathway is essential for orienting the cell rearrangement that drives elongation of a genetically tractable model. Using loss-of-function and gain-of-function experiments, we show that the components of the pathway from ligand to the activated transcriptional regulator STAT are required for cell rearrangement in the Drosophila embryonic hindgut. The difference in effect between localized expression of ligand (Unpaired) and dominant active JAK (Hopscotch) demonstrates that the ligand plays a cell non-autonomous role in hindgut cell rearrangement. Taken together with the appearance of STAT92E in a gradient in the hindgut epithelium, these results support a model in which an anteroposterior gradient of ligand results in a gradient of activated STAT. These results provide the first example in which JAK/STAT signaling plays a required role in orienting cell rearrangement that elongates an epithelium.
JAK/STAT pathway
Cell rearrangement
Tubulogenesis
The generation of complex shapes, both of the whole embryo and its constituent tissues and organs, depends on the coordination of multiple cellular processes; these include cell shape change, proliferation, cell migration and epithelial-mesenchymal transitions. An essential contributor to the form-generating processes of gastrulation and epithelial morphogenesis is oriented cell rearrangement. During gastrulation in frogs and fish, cells intercalate in the mediolateral axis, and converge towards the midline; this process has been termed convergent extension and results in dramatic elongation of the embryonic anteroposterior axis (reviewed by Keller et al., 2000; Wallingford et al., 2002). Although these cell rearrangements during gastrulation occur primarily among mesenchymal cells, cell rearrangement can also occur among cells that are bound together in an epithelium. Elongation of a number of epithelial sheets, such as the Drosophila germband and the C. elegans dorsal epidermis, has been shown to occur by mediolateral intercalation relative to the midline (Irvine and Wieschaus, 1994; Heid et al., 2001). Elongation of epithelial tubes such as the sea urchin archenteron, C. elegans intestine, insect renal tubule and Drosophila posterior spiracles and hindgut is also driven by cell rearrangement; in these cases, cell intercalation is oriented circumferentially (perpendicular to the tube proximodistal axis) rather than towards the midline (Fig. 1B) (Ettensohn, 1985; Leung et al., 1999; Skaer, 1993; Brown and Castelli-Gair Hombria, 2000; Iwaki et al., 2001) (reviewed by Wallingford et al., 2002).
Signaling molecules expressed in the hindgut. The hindgut epithelium (pink) elongates during stages 11 to 16, changing from a short, wide tube to a long, narrow tube (A); the cell rearrangement that is a major contributor to this process is shown in B. The three domains of the hindgut (SI, small intestine; LI, large intestine; RE, rectum), and the genes encoding signaling molecules expressed therein, are indicated in (C); anterior is towards the left, dorsal is upwards and the gray region indicates boundary cells. The points of attachment of the renal tubules are indicated near the anterior of the SI by `V'. Hindgut morphology, outlined by apical staining with anti-Crb (red) and nuclear staining with anti-β-Gal (green), is shown in stage 16 wild-type (D), drm (E), bowl (F) and (G) lin embryos, all carrying the bynapro enhancer trap that drives expression of lacZ in the nuclei of the hindgut. (H-K) upd expression, detected by in situ hybridization in stage 13 embryos, is seen only in the anterior hindgut (small intestine) of wild-type (H, black arrowhead), is missing from drm (I) and bowl (J) embryos (white arrowheads), and is expanded throughout most of the hindgut in lin embryos (K, black arrowhead).
An unsolved problem of great interest is the source of the polarizing information that causes rearranging cells to intercalate in one axis, and not in another. Although convergent extension in both frogs and fish has been shown to require the planar cell polarity pathway initiated by non-canonical Wnt signaling, the nature of the positional cues that orient the rearranging cells has not been defined (reviewed by Wallingford et al., 2002). Similarly, known genes required for oriented cell rearrangement in the C. elegans dorsal epidermis and Drosophila posterior spiracles encode putative transcription factors that are thought to confer morphogenetic capacity, rather than regulate expression of cue-providing molecules (Heid et al., 2001; Brown and Castelli-Gair Hombria, 2000). As localized, secreted signals have been demonstrated to guide various types of cell migration and epithelial outgrowth in both Drosophila and mammals (Duchek et al., 2001) (reviewed by Metzger and Krasnow, 1999; Hogan and Kolodziej, 2002; Moore, 2001; Rollins, 1997), an appealing hypothesis is that rearranging cells orient with respect to spatially localized cell signaling molecules.
The conserved JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is widely used; it has been shown to play a required role in a variety of processes including hematopoiesis, sex determination, lymphocyte migration, and border cell migration (reviewed by Ward et al., 2000; Luo and Dearolf, 2001; Sefton et al., 2000; Moore, 2001; Vila-Coro et al., 1999; Silver and Montell, 2001; Beccari et al., 2002). A suggestion that JAK/STAT signaling might be involved in epithelial cell rearrangement comes from the observation that the ligand for the Drosophila JAK/STAT pathway, Unpaired (Upd; Os — FlyBase) is expressed in a highly localized position at the anterior of the embryonic hindgut epithelium (Iwaki et al., 2001). Mutants that block the elongation of the hindgut, which occurs largely by cell rearrangement, also alter the localized pattern of upd expression (Iwaki et al., 2001).
Exploiting the genetic simplicity of Drosophila, we present evidence that the JAK/STAT pathway orients cell rearrangement in the hindgut, a simple epithelial tubule. In contrast to the situation in mammals where there are four different JAKs and seven different STATs (Imada and Leonard, 2000), the Drosophila genome encodes only one known ligand (upd), one receptor (the cytokine-like domeless, dome, also known as master of marelle), one JAK (hopscotch, hop) and one STAT (Stat92E, also known as marelle) (reviewed by Castelli-Gair Hombria and Brown, 2002). For examining cell movement during tubulogenesis, the hindgut is a particularly useful model as it elongates by cell rearrangement without either cell proliferation or apoptosis (Iwaki et al., 2001) (reviewed by Lengyel and Iwaki, 2002). Analysis of loss-of-function mutants shows that the key components of the Drosophila JAK/STAT pathway are required to achieve a fully elongated hindgut; gain-of-function (overexpression) studies show that uniform high level activation of the pathway is not sufficient, while localized production of ligand is necessary to promote oriented cell rearrangement. Our results support a model in which an anteroposterior gradient of ligand activates STAT activity in a similar gradient, leading to orientation of cell rearrangement. This is the first example of a required role for JAK/STAT signaling in orienting cell rearrangement that drives elongation of an epithelium.
Drosophila stocks
Mutant alleles used (the strongest available) were: updos1A (a deletion) (Ferrus et al., 1990); hopC111 (Perrimon and Mahowald, 1986); Stat92E06346 (Hou et al., 1996); drm3, drm6 and drmP1 (a deletion) (Green et al., 2002); bynapro (Murakami et al., 1995); bowl1 (Wang and Coulter, 1996); lin2 (Hatini et al., 2000); wg7L74 (Tearle and Nusslein-Volhard, 1987); and twi1 (Nusslein-Volhard et al., 1984). For temperature shift experiments, the temperature-sensitive allele wgIL114 (Nusslein-Volhard et al., 1984) was used. Three GAL4 lines that drive expression uniformly throughout the hindgut were used: bynGAL4 drives strong expression starting at stage 7, while fkhGAL4 and 455.2GAL4 drive progressively weaker expression starting at stages 8 and 9, respectively (San Martin and Bate, 2001; Fuss and Hoch, 2002; Iwaki and Lengyel, 2002) (D. D. I., unpublished). drmGAL4 drives expression only in the small intestine domain of the hindgut (Green et al., 2002). UAS lines used were UAS-upd (Zeidler et al., 1999), UAS-hopTML (Harrison et al., 1995), UAS-Stat92E (from Steven Hou, unpublished) and UAS-domeΔCYT3.2 (deletion of the Dome cytoplasmic domain) (Brown et al., 2001).
Generation of germline clones
Germline clones of hop and Stat92E were made using the FLP-DFS technique (Chou et al., 1993). For hop, larvae of the genotype hopC111 FRT101/ovoD1 FRT101; hsFLP38 were heat shocked at 37°C for 2 hours on each of days 4 and 5 after egg deposition. The eclosed females of this genotype were then mated to FM7, act-lacZ males to obtain progeny, half of which lacked both maternal and zygotic contributions of hop (referred to as hopm-z-). For Stat92E, larvae of the genotype hsFLP122;FRT82B ovoD1/FRT82B Stat92EP1681 were similarly heat shocked and mated to Stat92E06346/TM3 ftz-lacZ males to obtain Stat92Em-z-progeny.
Whole-mount in situ hybridization was carried out as described (Pignoni and Zipursky, 1997; Tautz and Pfeifle, 1989). Digoxigenin-labeled RNA probes (Roche Molecular Biochemicals) were made from cDNA templates of upd (Harrison et al., 1998), dome (Brown et al., 2001), hop (Binari and Perrimon, 1994), Stat92E (Hou et al., 1996; Yan et al., 1996), drm (Green et al., 2002), Ser (Thomas et al., 1991), hh (Lee et al., 1992) and dri (Gregory et al., 1996). Antibody staining of embryos was performed using standard techniques (Ashburner, 1989). Antibodies (and dilutions) used were α-Crb (1:100), α-Wg (1:100) and α-En (1:5), all available from the Developmental Studies Hybridoma Bank at the University of Iowa Department of Biological Sciences; α -Stat92E (Chen et al., 2002) (1:1000); α-phosphorylated histone H3 (Upstate Biotechnology, 1:500); α-Con (Meadows et al., 1994) (1:30); and α-β-Galactosidase (Promega, 1:1000, Cappel, 1:500). For transverse sections, embryos were fixed in either 2% glutaraldehyde plus 1% osmium tetroxide, or in 4% formaldehyde, embedded in Epon and sectioned at 2 μ m. Sections were stained with 0.025% Methylene Blue and 0.01% Toluidine Blue in 0.025% sodium tetraborate buffer. Light microscopy was carried out using a Zeiss Axiophot microscope, and images were captured with a Sony DKC-5000 digital camera. Confocal microscopy was carried out using a Carl Zeiss LSM 310 with a 40× objective and 1.5× digital zoom; images were acquired and processed using Zeiss LSM software. Hindgut lengths were measured as previously described (Iwaki et al., 2001) using images acquired with a Hamamatsu camera and Axiovision software. Total hindgut cell number in embryos of different mutant genotypes was determined by counting nuclei (identified by anti-β-Galactosidase staining) that were expressing lacZ due to the presence of either bynapro or bynGAL4,UAS-lacZ.nls, following previously published procedures (Iwaki et al., 2001). The number of cells in the hindgut circumference was determined by counting cells in eight to ten serial transverse sections through the large intestine of three to four different embryos. Embryos were staged according to Campos-Ortega and Hartenstein (Campos-Ortega and Hartenstein, 1997).
Candidate cues for hindgut elongation
The Drosophila hindgut epithelium elongates and narrows during a 10-hour period of embryogenesis (Fig. 1A), driven largely by cell rearrangement (Fig. 1B) (reviewed by Lengyel and Iwaki, 2002). This cell rearrangement must be oriented by some type of polarity, which probably arises from locally expressed signaling molecules. The developing hindgut is patterned into three morphologically distinct regions along its anteroposterior axis: the small intestine at the most anterior (to which the renal tubules connect), the large intestine and the rectum at the most posterior. Genes encoding at least seven cell signaling molecules are expressed differentially in these three regions (Fig. 1C). The putative transcriptional regulator encoding genes drumstick (drm), bowel (bowl) and lines (lin) control patterned expression of these signaling molecules (in particular upd), and are concomitantly required for cell rearrangement and elongation of the hindgut (Fig. 1C-K). The signal-encoding genes Ser, Dl, hh, dpp and spi, however, are not required for hindgut cell rearrangement (Iwaki and Lengyel, 2002; Fuss and Hoch, 2002; Hoch and Pankratz, 1996; Takashima and Murakami, 2001) (data not shown).
wg is expressed throughout the hindgut primordium starting at stage 5; by late stage 10 it is expressed at the most anterior and most posterior of the hindgut (Fig. 1C). To assess the required role of wg expression at these different stages, we carried out temperature shift experiments with a temperature-sensitive wg mutant (see supplementary figure S1 at http://dev.biologists.org/supplemental/). While early lack of wg function results in a dramatically smaller hindgut, the elimination of wg function during stage 10 (prior to the period of major elongation of the hindgut) or later allows essentially normal hindgut elongation. Thus, localized expression of Ser, hh, spi, dpp, Dl and wg does not appear to play a required role in hindgut cell rearrangement.
Localized expression of upd, dome and Stat92E in the developing hindgut
upd, encoding the ligand for the Drosophila JAK/STAT pathway, is only expressed in the small intestine (Fig. 1C,H) and is regulated by genes controlling hindgut cell rearrangement. In drm and bowl mutants, expression of upd is missing from the small intestine (Fig. 1I,J), while in lin mutants, upd expression is expanded throughout much of the hindgut (Fig. 1K). These results raise the possibility that localized Upd might provide an orienting cue for rearranging hindgut cells.
If it plays a role in hindgut cell rearrangement, upd must be expressed before and during the period of major hindgut elongation, i.e. between stages 11 and 16 (Fig. 1A); genes encoding the other known components of the Drosophila JAK/STAT signaling pathway, summarized in Fig. 2A, should also be expressed at the same stages, both within and adjacent to upd-expressing cells. We used in situ hybridization to characterize the expression of upd, dome, hop and Stat92E during stages just prior to and during hindgut elongation; characterization of Stat92E protein expression is presented in a subsequent section.
upd and JAK/STAT pathway components are expressed at the anterior of the elongating hindgut. The components of the Drosophila JAK/STAT signaling pathway (ligand Upd, receptor Dome, JAK Hopscotch and STAT) are shown in A and discussed in text. Beginning at stage 9, upd expression is detected in a narrow ring of cells at the anterior of the developing hindgut (at junction with posterior midgut) (B), while expression of dome and Stat92E in this domain is weaker and more diffuse (E,H). By stage 11, expression of upd in the prospective small intestine is strong (C), and increased expression of dome and Stat92E is observed in domains overlapping with, but more extensive than that of upd (F,I, respectively). These expression patterns for upd, dome and Stat92E are maintained throughout embryogenesis (D,GJ, respectively; stage 14). Expression in the anterior hindgut, detected by in situ hybridization, is indicated by brackets (B,C,E,F,H,I) and arrowheads (D,G,J).
Expression of upd in the hindgut is first detected at stage 9 in a narrow ring of cells that will become the small intestine (Fig. 2B). Expression in the prospective small intestine is maintained during stages 10 and 11 (Fig. 2C), where it can be seen just posterior to the everting renal tubules (note that in the hindgut at these germband-extended stages, `posterior' is toward the head). During stages 12-14, when the hindgut undergoes a major part of its elongation, upd expression is seen throughout the now distinct small intestine (Fig. 2D). Expression of upd is maintained throughout the small intestine during the remainder of embryogenesis.
In addition to upd, we examined expression in the hindgut of genes encoding the JAK/STAT receptor (dome), JAK (hop) and STAT (Stat92E). hop is expressed uniformly throughout the embryo, including the hindgut as it elongates (data not shown) (Binari and Perrimon, 1994). Expression of both dome and Stat92E is detected weakly at the anterior of the hindgut beginning at stage 9, becomes significantly stronger by stage 11, and is maintained through stage 14 (Fig. 2E-J). For both the receptor- and STAT-encoding genes, expression domains in the hindgut epithelium overlap with and extend beyond the narrow domain of upd expression (Fig. 2, brackets). Most significantly, expression of dome and Stat92E extends to a more posterior position in the hindgut epithelium than does expression of upd (Fig. 2, compare C,F,I, brackets). Thus, the mRNA expression of the ligand, receptor and STAT components in the hindgut prior to and during its elongation is consistent with a role for JAK/STAT signaling in hindgut cell rearrangement.
Required role of upd and JAK/STAT pathway components in hindgut elongation
To assess the requirement for upd and JAK/STAT signaling in hindgut elongation, we examined hindgut morphology, length and circumference in wholemounts and transverse sections of embryos deficient for upd, dome, hop or Stat92E. In embryos lacking zygotic upd function, the hindgut reaches only about half its normal length and is somewhat wider (Fig. 3B). Hindguts are also incompletely elongated and wider in embryos lacking both maternal and zygotic activity of either hop or Stat92E (Fig. 3C,D). Embryos lacking zygotic dome function have hindguts only slightly shorter than wild type (data not shown), presumably because of the maternal contribution of dome (Brown et al., 2001). However, when a dominant negative form of Dome (UAS-domeΔCYT3.2) is expressed uniformly using bynGAL4, hindguts are significantly shorter and wider (Fig. 3I). Length measurements reveal that upd-, hop- and Stat92E-deficient, as well as DomeDN- expressing hindguts, while not as short and wide as those of drm, bowl and lin embryos (compare Fig. 1B-D), are nevertheless 40-50% shorter than those of wild-type (summarized in Fig. 3Q). Consistent with their wider appearance in whole-mount embryos, upd, dome, hop and Stat92E-deficient hindguts have a greater number of cells in their circumference (19-27) than do wild-type hindguts (12) (Fig. 3F-H,M,Q). Overall, the shorter and wider appearance of the hindgut is roughly similar among embryos lacking the different components of the JAK/STAT pathway.
Spatially localized JAK/STAT signaling is required for hindgut elongation and cell rearrangement. Compared with wild type (A), the hindgut is shorter and wider in updos1A (B), hopm-z- (C) and Stat92Em-z- (D) embryos. As seen in transverse section, compared with wild-type (E), there are more cells in the hindgut circumference of updos1A (F), hopm-z- (G) and Stat92Em-z- (H) embryos. The hindgut is also shorter and wider when bynGAL4 is used to drive uniform hindgut expression of UAS constructs that activate JAK/STAT signaling, namely UAS-upd (J) and UAS-hopTML (K); the defect is more severe when both UAS-hopTML and UAS-Stat92E are driven together (L). Inhibition of JAK/STAT signaling by bynGAL4 driven expression of the dominant negative UAS-domeΔCYT3.2 also results in a shorter, wider hindgut (I); transverse sections of embryos of these genotypes similarly reveal a greater than normal number of cells in the hindgut circumference (M-P). All embryos shown are at stage 16; wholemounts are stained with anti-Crb and sections are of the large intestine. Averages of total number of hindgut cells, cells in hindgut circumference, and hindgut lengths observed in the loss-of-function mutants and in bynGAL4 embryos expressing various JAK/STAT components are summarized in(Q.
The shorter hindgut length is not due to a deficiency of cells, nor is the excess number of cells in the circumference due to overproliferation, as the total number of hindgut epithelial cells is 96, 89 and 85% of wild-type for upd, hop and Stat92E mutant embryos, respectively (Fig. 3Q). Further, staining of upd embryos with anti-phosphorylated histone H3, a marker of mitosis, did not detect more cell division than observed in wild type (data not shown). The number of cells in the wild-type hindgut at stage 11 is ∼50; by stage 16, this number has been reduced dramatically (to ∼12) by cell rearrangement (Iwaki et al., 2001). Because by stage 16 the number of cells in the circumference of upd, hop and Stat92E embryos is reduced only partially (to roughly 20-30), while total cell number is essentially the same as seen in wild-type (Fig. 3Q), it must be concluded that, in the JAK/STAT loss-of-function mutants tested, there is a defect in hindgut cell rearrangement.
Requirement for localized JAK/STAT signaling
If localized JAK/STAT signaling provides an orienting cue for cell rearrangement, then ectopic JAK/STAT signaling throughout the hindgut would be expected to disrupt this process. To test this, we used bynGAL4 to drive various UAS constructs uniformly in the hindgut epithelium. Uniform expression of upd results in hindguts that elongate to only about 65% of the wild-type length and have about 50% more than the normal number of cells in their circumference (Fig. 3J,N,Q). In the testis, eye and hemocytes, ectopic expression of upd or activated JAK causes increased cell proliferation (Kiger et al., 2001; Tulina and Matunis, 2001; Chen et al., 2002; Luo et al., 1997). However, total cell number in hindguts ectopically expressing upd is only 76% of normal (Fig. 3Q); consistent with this, staining with anti-phosphorylated histone H3 did not detect excess cell proliferation (data not shown). As uniform expression of upd in the hindgut does not result in an increase (but rather a reduction) in hindgut cell number, the excess number of circumferential cells seen in upd overexpressing hindguts must arise from a defect in cell rearrangement.
To ask whether localized activation of other components of the JAK/STAT pathway is required for hindgut elongation, we expressed uniformly an activated form of the Drosophila JAK (UAS-hopTML). Similar to what was seen for uniform expression of upd, total cell number did not differ significantly from wild type, but the resulting hindguts were shorter and had more circumferential cells (Fig. 3K,O,Q). When both UAS-hopTML and UAS-Stat92E are driven by bynGAL4 (Fig. 3L,P), the hindgut elongation defect is more severe than UAS-hopTML alone.
The ectopic expression studies presented here demonstrate that, while components of JAK/STAT signaling are required, activation of the pathway at uniformly high levels throughout the hindgut is not compatible with normal cell rearrangement. Experiments to be presented in a subsequent section further support the idea that spatially restricted JAK/STAT signaling is necessary for hindgut cell rearrangement.
Disruption of JAK/STAT signaling does not affect hindgut patterning
As proper cell rearrangement is correlated with correct hindgut patterning (Iwaki et al., 2001), it could be argued that, rather than affecting the orientation of rearranging cells directly, upd and JAK/STAT signaling control cell rearrangement by affecting patterning. To test this idea, we assessed gene expression characteristic of the different hindgut regions (Fig. 4A) in upd mutants and in embryos ectopically expressing upd throughout the hindgut. Examination of all markers tested (except wg, see below) supports the conclusion that all three domains — small intestine, large intestine, and rectum — as well as boundary cell rows and rings are present and correctly patterned in upd mutant hindguts (Fig. 4B; updos1A row). Similarly, all three hindgut domains and boundary cells are present when upd is uniformly expressed in the hindgut (Fig. 4B; bynGAL4; UAS-upd row). The only domain missing from hindguts lacking upd is a small, wg-expressing region at the extreme anterior of the small intestine that is established during stages 10/11 (Fig. 4A,B). As our temperature-shift experiments showed that activity of wg is not required for hindgut elongation after stage 10, this defect in wg expression cannot be the basis for the effect of upd loss-of-function on hindgut elongation.
upd has only minimal effect on hindgut patterning. Spatially localized patterns of gene expression in distinct domains of the stage 14 hindgut and posterior midgut are summarized in A; points of renal tubule attachment are indicated by `V'. Gene expression patterns in A are shown in wholemounts of wild-type (wt), upd loss-of-function (updos1A) and upd gain-of-function (byn-GAL4; UAS-upd) embryos (B). In the absence of upd activity (updos1A), expression of Wg at the most anterior of the small intestine is reduced (white arrowhead), while expression patterns of hh and Ser in small intestine, En in large intestine, dri in boundary cells and hh, Ser and Wg in rectum are not affected. When upd is expressed uniformly throughout the hindgut (byn-GAL4; UAS-upd), expression of Wg in the anterior small intestine is slightly upregulated (black arrowhead), while all other spatially localized gene expression patterns examined appear normal. Expression of hh, Ser and dri was detected by in situ hybridization, that of Wg and En by antibody staining.
In summary, all domains of the hindgut, with the exception of a small number of cells at its anteriormost tip, are correctly patterned in hindguts either lacking or uniformly expressing upd. We therefore conclude that, rather than affecting patterning primarily and morphogenesis secondarily, upd and JAK/STAT signaling directly affect the cell rearrangement that elongates the hindgut.
Spatially restricted upd is required for hindgut cell rearrangement
If localized expression of upd at the anterior of the hindgut epithelium is required for hindgut cell rearrangement, then expression of upd in this domain should rescue the elongation defect in embryos lacking upd. We therefore used drmGAL4 to drive expression of UAS-upd only at the anterior of the hindgut in embryos lacking upd function. The resulting hindguts appeared morphologically normal, with an average length ∼93% that of wild type (Fig. 5C,D,I). Thus, anteriorly localized (in the small intestine) expression of upd in an upd mutant background is sufficient for elongation.
Anteriorly localized upd expression is required for hindgut elongation. When upd is expressed throughout the hindgut (using bynGAL4) in an updos1A mutant background, the hindgut is shorter and more deformed (A) and has as many or more cells in transverse section (B) than that seen in updos1A embryos (compare with Fig. 3B,F). When upd is expressed only in the small intestine (using drmGAL4) in an updos1A mutant background, the length of the hindgut and number of cells in transverse section is almost indistinguishable from that seen in wild-type (C,D; compare with Fig. 3A,E). The drm3/drm6 hindgut is short and wide (E) and has an excessive number of cells in its circumference (F). When upd is expressed only in the small intestine (using drmGAL4) in a drm3/drm6 mutant background, the hindgut is more elongated (G), and has fewer cells in its circumference (H) than seen in drm3/drm6. This phenotype is similar to the rescue of the drm3/drm6 hindgut that is seen when drmGAL4 is used to drive UAS-drm (R. B. Green, PhD thesis, UCLA, 2002). Hindgut apical surfaces are outlined by anti-Crb staining; embryos are stage 16; GAL4-driven expression patterns are shown on the right of each pair of panels. Length measurements and circumference counts are summarized in I.
Is restriction of upd expression only to the hindgut anterior necessary for the cell rearrangement that drives elongation? To answer this, we used the uniformly expressed bynGAL4 to drive upd in an upd mutant background. Rather than rescuing (like the drmGAL4 driver), this resulted in severely defective, short and wide hindguts (Fig. 5A,B). It could be argued that the early and high level of expression driven by bynGAL4 causes a level of activation of JAK/STAT pathway that inhibits cell rearrangement. We therefore performed the same experiment with fkhGAL4 and 455.2GAL4, which (respectively) drive expression uniformly in the hindgut at progressively lower levels and later times (Iwaki and Lengyel, 2002; Fuss et al., 2000; San Martin and Bate, 2001) (D. D. I., unpublished). We observed neither rescue of the upd hindgut phenotype nor a phenotype more defective than that of upd alone (in contrast to the result with bynGAL4; data not shown). Thus, anteriorly localized upd expression appears to be both necessary and sufficient for cell rearrangement in the large intestine.
An important issue is whether upd plays a permissive role in the hindgut (giving cells the ability to rearrange), or an instructive role (orienting cells as they rearrange). As none of three different levels of uniform upd expression rescued the upd loss-of-function phenotype, it seems unlikely that the function of JAK/STAT signaling in the hindgut is simply to promote the capacity of cells to rearrange. Rather, the data support the notion that the required role of localized expression of upd is to provide an instructive cue that orients cell rearrangement.
upd signaling mediates drm function in hindgut
Although upd cannot entirely mediate the effect of drm on hindgut cell rearrangement (as drm hindguts are shorter and wider than those of upd; Fig. 5E,F), we asked whether, and to what extent, expression of UAS-upd under control of drmGAL4 could rescue the drm loss-of-function phenotype. Strikingly, expression of upd at the anterior of the drm mutant hindgut is sufficient to bring about significant rescue of the drm hindgut phenotype, as assessed in both wholemounts and transverse sections (Fig. 5G,H). Compared with drm hindguts, the partially rescued hindguts are 45% longer, and have 35% fewer cells in their circumference (Fig. 5I). The rescue of the drm hindgut phenotype by anteriorly expressed upd thus demonstrates that upd is a key mediator of drm function in the hindgut.
Upd signal is received by cells of the large intestine
Given the small number of cells in its circumference by stage 16 (Fig. 3E) (Iwaki et al., 2001), the large intestine must undergo significant cell rearrangement as it elongates. Yet upd, which we have shown controls cell rearrangement throughout much of the hindgut, is expressed only in the small intestine, and not in the large intestine. Because action of Upd over a distance has been described in the eye disc (Zeidler et al., 1999), it seemed possible that Upd produced in the small intestine might control cell rearrangement by activating JAK/STAT signaling in the large intestine (Fig. 1C,H). During stages 11 and 12, as cell rearrangement is elongating the hindgut, the prospective large intestine is 50 to 60 μm in length (Fig. 6A,B); this is the distance over which Upd would have to diffuse to affect the entire large intestine.
upd activates the JAK/STAT pathway in cells posterior to the small intestine. Sagittal sections through the hindgut at stages 11 (A) and 12 (B) show outlines of the everting renal tubules (black dashes), small intestine primordium (red dots), large intestine primordium (yellow dots) and rectum primordium (orange dots). Estimates of the lengths of the small intestine (SI) and the large intestine (LI) are shown based on analysis of the sets of serial sections to which A and B belong. Scale bar: 50 μm. Levels of Stat92E protein and mRNA are barely detectable in updos1A embryos (C and E, respectively), but dramatically upregulated in bynGAL4; UAS-upd embryos (D and F, respectively); Stat92E protein is also upregulated in bynGAL4, UAS-hopTML embryos (L). Stat92E protein is detected along a significant region of the length of the anterior hindgut, as observed in both whole-mount (G,I) and sagittally sectioned embryos (H,J) at stages 11 (G,H) and 12 (I,J). The hindgut is outlined by black dots (H,J), and red dots indicate the observed anteroposterior gradient of Stat92E protein (H,J). Consistent with this, double staining for both upd mRNA (in situ hybridization) and Stat92E protein (antibody staining) shows expression of Stat92E in an anterior-to-posterior gradient (brown, posterior extent marked with open arrowheads) that extends posterior to the domain of upd expression (blue, posterior extent marked with black arrowheads) (K). Expression of UAS-upd with drmGAL4 in an upd mutant background rescues the upd hindgut elongation defect (M), while expression of UAS-hopTML with drmGAL4 in an upd mutant background fails to rescue (N); α Crb is used to outline hindgut morphology. In C-G,I, the lumen of the hindgut is indicated with dots; the anterior limit of the small intestine is indicated with a larger dot.
In addition to activating phosphorylation of Stat92E (Luo and Dearolf, 2001), expression of upd has also been shown to upregulate Stat92E protein levels during embryogenesis (Chen et al., 2002). We therefore investigated expression of Stat92E mRNA and protein as reporters for receipt of the Upd signal. Antibody staining shows that, in the stage 11 hindgut, Stat92E protein is greatly reduced in the absence of upd function, and dramatically upregulated when upd is uniformly expressed (Fig. 6C,D). This regulation appears to occur at the transcriptional level, as Stat92E mRNA is similarly reduced in the absence of upd function, and upregulated when upd is uniformly expressed in the hindgut (Fig. 6E,F). We conclude that staining with anti-Stat92E identifies cells receiving Upd signal.
During stages 11 to 12, Stat92E protein is detected in a domain that extends more posteriorly than the upd expression domain at the hindgut anterior (compare Fig. 2C and Fig. 6G,I); this can be seen most clearly when both upd mRNA and Stat92E protein are labeled (Fig. 6K). Sagittal sections reveal that Stat92E is present in the nuclei of the hindgut epithelium at stages 11 and 12, (Fig. 6H,J); therefore, during these stages, at least some of the large intestine epithelial cells are receiving Upd signal. Significantly, Stat92E appears to be present in the hindgut as a gradient, with the most anterior cells (at the point of renal tubule evagination) most strongly labeled; during stages 11 to 12, this gradient extends to at least 20% to 45% of the length of the large intestine primordium (Fig. 6G-J). Because of limits in dynamic range of the anti-Stat92E assay, we cannot presently determine whether, at some point, this gradient extends over the entire large intestine.
Stat92E is present not only in the nuclei of the hindgut epithelium, but also in the nuclei of the hindgut visceral mesoderm (Fig. 6J; see supplementary figure S2 at http://dev.biologists.org/supplemental/). However, as determined by staining with anti-Connectin, the hindgut visceral mesoderm is normal in upd mutant embryos (see supplementary figure S2); furthermore, the hindguts of twi mutants, which lack all mesoderm, are able to undergo a significant amount of elongation (see supplementary figure S2) (San Martin and Bate, 2001). Thus, even though cells of the visceral mesoderm receive the Upd signal, they do not appear to play a significant role in the rearrangement of cells within the hindgut epithelium.
The expression of Stat92E in the hindgut epithelium, while suggestive, does not reveal whether receipt of Upd signal by prospective large intestine cells is required for their rearrangement. Because in an upd mutant embryo expression of upd at the anterior of the hindgut rescues elongation (Fig. 5C), we asked whether expression of dominant active Hop (hopTML) might also rescue. As a control, we expressed hopTML uniformly in the hindgut; similar to what we observed for upd, this results in a dramatic upregulation of Stat92E (Fig. 6, compare D with L). A key finding is that, in contrast to the essentially complete rescue of the upd hindgut phenotype when upd is expressed anteriorly (Fig. 6M), anterior expression of hopTML in an upd background does not rescue hindgut elongation (Fig. 6N). These results demonstrate that activation of the JAK/STAT pathway only in cells of the prospective small intestine (i.e. the domain that expresses upd) is not sufficient for normal hindgut cell rearrangement. Thus, upd is required in a non-cell autonomous fashion for hindgut cell rearrangement.
The observed upregulation of Stat92E in cells of the hindgut posterior to the small intestine is consistent with the cell non-autonomous function of upd in the hindgut. The fact that cell rearrangement is severely abnormal when uniform levels of Stat92E and HopTML are driven together (Fig. 3L,P) further supports the idea that Stat92E must be distributed non-uniformly, i.e. as a gradient, in order for cells to rearrange. The observed gradient of Stat92E, reaching to at least 20% of the length of the large intestine during stage 11, and to at least 45% during stage 12 (Fig. 6H,J), suggests that there is a corresponding gradient of the activating ligand Upd in the hindgut. Both the distance (roughly 20 to 40 μ m, see Fig. 6A,B) and time (stages 9 through 12, roughly 4-6 hours) over which this postulated Upd gradient is established are consistent with parameters described for formation of gradients of Upd, Dpp and Wg in imaginal disc tissues (Zeidler et al., 1999; Entchev et al., 2000; Teleman and Cohen, 2000; Strigini and Cohen, 2000), as described below.
We have shown that elongation of the Drosophila hindgut by cell rearrangement requires the Upd ligand and the JAK/STAT pathway components Dome (receptor), Hop (JAK) and Stat92E. As elongation does not occur when expression of ligand or activation of the pathway is uniform, but only when the source of ligand is localized to the hindgut anterior, the requirement for localized JAK/STAT signaling in hindgut elongation can be characterized as instructive, rather than permissive. As patterning is normal in hindguts both lacking and uniformly expressing upd, the required role of JAK/STAT signaling in hindgut morphogenesis is likely via direct effects on cell movement.
Upd is required non-autonomously for hindgut cell rearrangement
The rescue of the upd phenotype by anteriorly localized expression in the hindgut of upd, but not of activated JAK (Hopscotch), demonstrates that there is a requirement for upd function that is not cell autonomous. In other words, upd is required in cells (those of the large intestine that undergo the greatest rearrangement) different from cells that produce it (those of the small intestine). A number of examples have been described in which localized expression of a signaling molecule (including Upd) is required non-autonomously for cell rearrangement, morphogenesis or motility. In the Drosophila eye imaginal disc, expression of Upd at the midline is required to establish a dorsoventral polarity that orients ommatidial rotation (Zeidler et al., 1999). In both Drosophila tracheae and the vertebrate lung, branching morphogenesis of the epithelium depends on localized expression of FGF in adjacent mesenchyme (reviewed by Metzger and Krasnow, 1999; Hogan and Kolodziej, 2002).
Localized activation of JAK/STAT signaling has been shown to play a role in cell motility in a number of contexts. In Drosophila, localized expression of Upd in the anterior polar cells of the egg chamber acts to coordinate the migration of the adjacent border cells (Silver and Montell, 2001; Beccari et al., 2002). In mammals, cytokines expressed in target tissues act to attract both migrating lymphocytes and tumor (reviewed by Moore, 2001; Muller et al., 2001; Murphy, 2001; Vila-Coro et al., 1999). Our finding that localized (only in the small intestine) expression of upd is both necessary and sufficient for rearrangement of cells in the large intestine indicates that Upd must have an organizational, action-at-a-distance function in controlling cell rearrangement during tubular morphogenesis.
A predicted gradient of Upd in the hindgut
The rescue experiments discussed above establish that there is a cell non-autonomous requirement for upd in hindgut elongation. Consistent with this, there is evidence that Upd is present and required in an anteroposterior gradient in the hindgut. Prior to and during hindgut elongation, both Stat92E mRNA and Stat92E protein are detected not only in the small intestine epithelium (and the visceral mesoderm surrounding the small intestine), but also in the epithelium posterior to the small intestine; this expression of Stat92E appears to be in a gradient. In the Drosophila eye imaginal disc, a gradient of Upd is required to orient the rotation of ommatidial cell clusters (Zeidler et al., 1999); in addition, there is evidence for a gradient of Upd and Stat92E in patterning of the follicular epithelium of the Drosophila egg chamber (R. Xi, J. R. McGregor and D. A. Harrison, unpublished). As expression of Stat92E depends on upd (this work) (Chen et al., 2002), it is likely that Upd protein is present in the hindgut epithelium as an anteroposterior gradient, with its highest level in the upd-expressing cells of the small intestine, and lowest level in posterior, upd non-expressing cells of the large intestine. Expression of SOCS36E (suppressor of cytokine signaling at 36E), which is regulated by upd, overlaps with and extends significantly beyond the domain of upd expression (Karsten et al., 2002), further supporting the idea that there is a gradient of Upd in the hindgut. A model that summarizes the observed localized expression of upd mRNA, a gradient of Stat92E protein, and cell rearrangement leading to elongation is shown in Fig. 7.
Model for hindgut elongation by Upd/JAK/STAT signaling. Expression of upd in the small intestine (SI) under the control of Drm and Lin results in the formation of a gradient of Upd protein in cells posterior to the small intestine. In response to signaling by Upd, a gradient of Stat92E is established; this leads to the orientation of cell rearrangement in the hindgut.
In the Drosophila eye imaginal disc, anti-Upd staining and the behavior of clones of mutant cells that have lost components of the JAK/STAT pathway indicate that Upd is present in a gradient that extends at least 50 μ m beyond its midline mRNA expression domain (Zeidler et al., 1999). In the Drosophila hindgut, we have shown that Stat92E is a reliable reporter for the presence of Upd. Two to four hours after upd is first expressed at the anterior of the hindgut (stage 9), Stat92E can be detected at least 30-40 μm from the site of upd expression (stages 11 and 12). These time and distance parameters are similar to those observed during generation of the Upd gradient in the eye, and the Dpp and Wg gradients in wing imaginal discs, which form over distances of roughly 40-80 μm in 1-8 hours (Zeidler et al., 1999; Entchev et al., 2000; Teleman and Cohen, 2000; Strigini and Cohen, 2000). Thus, it is reasonable to imagine that a gradient of Upd is established in the developing hindgut in a short enough time frame to affect cell rearrangement.
The essential consequence of JAK/STAT signaling is activation of the STAT protein, which leads to altered transcriptional programs (Darnell, 1997; Horvath, 2000). STAT has been shown in a number of contexts to be required for cell motility (Sano et al., 1999; Yamashita et al., 2002; Silver and Montell, 2001; Beccari et al., 2002), and therefore probably regulates expression of genes controlling cytoskeletal assembly and cell adhesion. In these contexts, however, activation of STAT does not appear to be required to orient cell movement, but rather to facilitate or promote it. As Stat92E is required for hindgut elongation, and its protein product appears to be present in a gradient along the anteroposterior axis, this raises the intriguing question of how a gradient of a transcription factor might orient cell rearrangement.
Our results demonstrate a new role for Upd and the JAK/STAT pathway, namely the control of cell rearrangement that elongates a tubular epithelium. Given the widespread occurrence of JAK/STAT signaling, further analysis of the mechanism by which JAK/STAT signaling controls hindgut cell rearrangement in Drosophila is likely to provide insights into the control of cell motility during many processes, including organogenesis, wound healing and cancer metastasis.
We thank D. Harrison for sharing information prior to publication, J. Axelrod, U. Banerjee, D. Harrison, K. Lyons and members of the Lengyel laboratory for helpful comments on the manuscript, and S. Yu for excellent technical assistance. K. A. J. and D. D. I. were supported by USPHS National Research Service Awards GM07185 and GM07104, respectively. This work was supported by NIH grant HD09948 to J. A. L.
Supplementary figures available on-line
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'I hope to empower': Meet music education lecturer LaSaundra Booth
Photo by Samantha Lewis | The Daily Tar Heel
Dr. LaSaundra Booth plays piano in the piano lab at Kenan Music Building on Tuesday, Mar. 8, 2022. Dr. Booth was recently featured as the NAfME 2021 All-National Honors Orchestra Conductor.
By Kelly Kendall
March 20, 2022 | 9:18pm EDT
On her first day of orchestra in the fourth grade, LaSaundra Booth arrived late.
She wanted to play the violin, but it had already been assigned. She was given a cello instead.
But when she sat down with the cello, Booth said she quickly fell in love with the string instrument.
That cello would be the beginning of Booth's seasoned career in music and music education — a career that would lead her to being named the National Association for Music Education 2021 All-National Symphony Orchestra's conductor.
Booth has been a lecturer of music education in the UNC Department of Music since last fall. Originally from Greensboro, Booth said she has performed and taught in areas across the state.
Booth's music career began as a student. Throughout grade school, she honed her skills as a cellist and eventually was invited to play with the Raleigh Symphony Orchestra as a senior in high school.
Booth said this experience inspired her to pursue a degree in music performance at N.C. Central University.
In 2003, Booth said she began teaching music in the public school system, and has since taught at the elementary, middle, high and collegiate levels. Throughout this time, Booth said she witnessed cuts to fine arts education funding in schools.
Determined to continue making music accessible for students of all backgrounds and financial statuses, Booth founded the Wake Forest Community Youth Orchestra, a nonprofit organization that provides instruments and musical training to over 350 children across three counties.
Without public schools funding music education, she said, many students like herself would miss out on the opportunity to play instruments.
"When there's a child in need, who's there to help them?" Booth said. "If public schools won't do anything about it, I'll just start my own, and that's what we did. We don't turn students away because of a lack of an instrument or inability to pay, so we kind of fill the void that is needed in our community."
Impact at UNC
Now at UNC, Booth teaches courses in music education.
Evan Feldman, associate chair professor of performance, composition and music education in the Department of Music, said Booth's legacy will be one that continues for generations.
"I think it's an extension of who she is," Feldman said. "She's molding teachers now that are in some way, or going to be, a reflection of her and are going to have some of her DNA in them."
Senior music major Joe Figliolo said he has worked with Booth this past year, and plans to enter the field of music education. He said Booth has built a sense of confidence in him.
"She came in very passionate about making sure that we go into the field and as we go into our interviews, as we go into our internships and whatever it may be, we just always feel confident in ourselves," Figliolo said. "She's like when you walk into a room there's only one Joe Figliolo in this world."
For Booth's accomplishments as a performer and instructor, the National Association for Music Education selected her to conduct the 2021 All-National Symphony Orchestra, which brings talented students from across the country together to perform.
This year's ensemble was virtual, but Booth still got to connect with students and hold virtual performances. She chose pieces from two Black female composers — Mary D. Watkins and Florence Price — for the symphony to perform.
Booth said these women served as inspirations for her.
"For me, conducting is more than just doing a piece technically accurate or precise," she said. "I really try to build a connection with the students. I want to know about the composer and their background. One of the great things about being able to get invited to them is that they allow me to select what I want to perform, and I did not hesitate to program works by two women of color."
Whether at the elementary or collegiate level, Booth has worked to transform music education. And moving forward, she said she will continue to strive to positively impact her students.
"I hope to empower," Booth said. "If there's one word I'm taking into 2022, it's 'empower.' I want to empower the next generation of teacher leaders."
@kelly2cats_
university@dailytarheel.com | elevate@dailytarheel.com
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redpajama_set_name: RedPajamaCommonCrawl


text:
Q: Numeric text list to parent/children elements I have text file that looks like this:
1. Apple
1.1. Apricot
1.2. Avocado
1.2.1. Banana
1.2.1.2. Blackberry
1.2.2. Boysenberry
1.3. Cherry
1.4. Dragonfruit
1.5. Plantain
1.5.1. Pear
1.5.1.1. Pomelo

And so on.
I need hierarchy array with children / parents elements from this simple text input.
Iam stuck with this, need help.

A: You can represent the hierarchy as a list of dictionaries in python. The list would like like this:
[{'id': '1', 'parent': '0', 'name': 'Apple'},
{'id': '1.1', 'parent': '1', 'name': 'Apricot'},
{'id': '1.2', 'parent': '1', 'name': 'Avocado'},
{'id': '1.2.1', 'parent': '1.2', 'name': 'Banana'},
{'id': '1.2.1.2', 'parent': '1.2.1', 'name': 'Blackberry'},
{'id': '1.2.2', 'parent': '1.2', 'name': 'Boysenberry'},
{'id': '1.3', 'parent': '1', 'name': 'Cherry'},
{'id': '1.4', 'parent': '1', 'name': 'Dragonfruit'},
{'id': '1.5', 'parent': '1', 'name': 'Plantain'},
{'id': '1.5.1', 'parent': '1.5', 'name': 'Pear'},
{'id': '1.5.1.1', 'parent': '1.5.1', 'name': 'Pomelo'}]

Here, each dictionary inside the list represents each row in the text file. The row contains the id, its parent and the associated name.
Code for reproducing the list of dictionaries (Stack.txt contains the text file in the question):
res = []

with open("Stack.txt","r") as fin:
    for line in fin:
        var = str(line)
        var = var.rstrip('\n')
        var = var.split(' ')
        dic = {}
        if var[0] == "1.":
            dic["parent"] = "0"
            dic["id"] = "1"
            dic["name"] = var[1]
            res.append(dic)
        else:
            temp = var[0][:-1]
            id =  temp[-1]
            parent = temp[:-2]
            id = parent + "." + id
            dic["parent"] = parent
            dic["id"] = id
            dic["name"] = var[1]
            res.append(dic)

fin.close()
for x in res:
    print x

redpajama_set_name: RedPajamaStackExchange


Dress mannequins on black.Fabric will be cut to a metric fat quarter unless otherwise requested.&nbs..
Dress mannequins on purple.Fabric will be cut to a metric fat quarter unless otherwise requested.&nb..
Sweet little multi-coloured bows on pink.Fabric will be cut to a metric fat quarter unless otherwise..
Sweet little multi-coloured bows on white.Fabric will be cut to a metric fat quarter unless otherwis..
Fat quarter bundle in corals from Sunnyside Up and Sundrops by Corey Yoder for Moda.The price is bas..
Fat quarter bundle in greys from Sunnyside Up, Flower Mill and Sundrops by Corey Yoder for Moda.The ..
Fat quarter bundle in whites from Sunnyside Up, Flower Mill and Sundrops by Corey Yoder for Moda.The..


CPU heat sinks and other cooling systems are responsible for cooling electronic components and circuits. They use an impeller to ensure quick heat dissipation. Among other applications, they are used in computers and EDP systems for cooling microprocessors.


For those wanting to escape the towns and cities and enjoy a more quiet way of life and enjoy mother nature, then Grenada and Tobago is the perfect twin-centre holiday for you. In Grenada, step away from the beach to enjoy hikes across the green hills and stunning vistas of Grand Etang National Park, or head underwater and uncover the Underwater Sculpture Park a great visit for diving enthusiasts. In Tobago the adventures can continue, take a swim at Argyll Falls, the island's signature waterfall. Jump on a boat and head to Buccoo Reef, boasting a colourful array of underwater life. Don't forget to visit the Nylon Pool, situated between the reef and the shore, an offshore sandbank that has formed a pool in the middle of the ocean.
Spice Island Beach Resort lies on the stunning white sands of Grand Anse Beach offering a wide range of luxury services and amenities. Enjoy a pampering at the spa, indulge in fine dining at one of the resort's restaurants or just lay on the beach and watch the world go by. Great for couples looking for a romantic escape or a family looking for quality time - this resort has it all.
Situated on the Tobago Plantations lies the Magdalena Grand Beach and Golf Resort. Surrounded by an 18-hole golf course and close to nearby attractions such as Scarborough and Pigeon Point Nature Park. Choose from five restaurants, enjoy complimentary bicycle hire, five star PADI Dive Centre and plenty of activities for younger explorers.


text:
Q: How to get a Checkbox value in Velocity for a customfield In JIRA I have a custom field which is a Checkbox. In Velocity I'm trying to get the value of the checkbox, but I just seem to get the Name of customfield returned instead of the value:
#set($Exclude = $customFieldManager.getCustomFieldObject("customfield_10108").getValue($issue))

This is returned for all records, regardless of whether the checkbox is ticked or not:

[Exclude]

(this is the label of the checkbox)
I also tried the following with no luck:
 #set($Exclude = $customFieldManager.getValueFromIssue("customfield_10108", $issue))

I don't really care what comes back (0,1 or True/False).
I've also tried this with a Radio Button rather than a Checkbox, but I get the same issue.

A: This took me much more time than it should, but finally found it:
#set($Exclude = $customFieldManager.getCustomFieldObject("customfield_10108"))
#if(!$Exclude.hasValue($issue))
<whatever you want to do when the checbox is not checked>
#end


text:
People | 20 QUESTIONS
Professional cyclist Mayuko Hagiwara: 'Challenge yourself as much as possible'
Name: Mayuko Hagiwara
Nationality: Japanese
Occupation: Professional cyclist
1. What do the kanji that comprise your first name — 麻由子 — mean? Would you say they match your personality? I don't exactly know what it means but my parents decided on it based on the number of kanji strokes that it comprises. They wanted me to be liked by a lot of people, and that's why they chose this name for me.
2. Where do you go to escape Japan? Okinawa, because an important friend lives there. It's also the perfect place for training.
3. Whom in Japan do you most admire? (Artist) Taro Okamoto, because my father really liked his art and respected his ideas. Okamoto was a fantastic creator who was able to challenge people's way of thinking. He also demonstrated tremendous passion.
4. When I think of Japan, I think of … beauty and an abundance of convenience.
5. What do you miss most about Japan when you are overseas? I mostly miss my best friend (in Okinawa), my other friends and meals that have been cooked by my mother.
6. What's your favorite Japanese word or phrase? 根性 (konjyo, which means willpower or guts). My grandmother showed me how to live this way.
7. What's your favorite phrase in any language? Grintare, which means "blessing" in Italian. I was surprised to discover that a Western language includes such a word. This notion is part of our Japanese spirit.
8. What inspired you to become a racing cyclist? Delight.
9. What do you hope to accomplish in the sport? To compete against myself as much as I can in order to accomplish my own goals.
10. What's the best way of training for cycling? Any tips? Go out for a ride with my friends and then celebrate finishing with a cold beer.
11. We've heard that you initially found European racing difficult. What's the biggest difference between racing in Japan and racing in Europe? They're totally different. It almost feels like racing in Japan and racing in Europe are different sports altogether.
12. You became the first Japanese to win a stage in a grand tour, winning stage 6 of the Giro d'Italia Femminile in Italy in 2015. Care to share any strategies you employed during the race? The most important thing for me is to work hard to do whatever I can for myself and my team. Always.
13. How important is teamwork to racing? Teamwork is everything in cycling. If everyone in the team is individually strong but have different objectives during a race, it's impossible to ever be victorious.
14. What is one thing in your life you can't replace? My bicycle. My life would be incomplete without it.
15. If you could have dinner with anyone from history, who would it be? I would like to have dinner with members of my family or close friends.
16. If someone wrote a biography about you and your life, what do you think the title should be? "Tora, Tora, Tora."
17. How do you relax when you aren't working? I listen to my favorite music, take a walk in nature, or catch up with my family and friends.
18. Who would win a fight between a lion and tiger? The tiger, because I was born in the Year of the Tiger in the Chinese zodiac.
19. What do you want to be when you grow up? I never know.
20. Do you have any words of advice for young people? Challenge yourself as much as possible and work hard to achieve whatever you can. Don't be afraid to make a mistake.
Mayuko Hagiwara currently rides for Wiggle.co.uk team Wiggle High5. For more information, visit www.wigglehighfive.com/mayuko-hagiwara-jpn.
LATEST PEOPLE STORIES
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There aren't many female filmmakers in Japan, making even fewer female film editors. Aika Miyake is an exception. "Somewhere in my mind is an optimistic me, hoping that my example will inspire o...
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Name: Kenro Taura Age: 62 Nationality: Japanese Occupation: Executive director of Kiko Network
Potential in symbiotic ties, marketing yet to be tapped
Name: Sanjeev Sinha Title: President, India Japan Partnership Fund and Group; Founding President, IIT Alumni Association Japan URL:
cycling, Taro Okamoto, Mayuko Hagiwara


It's a boring wet day on Hazard River until Jack Wilde and his friends find a note on an abandoned boat. The message is damaged but they can all read the words HELP ME.
The gang must find out who needs help and why. But as they get closer to the answer will they be the ones who need help?


text:
Danyło Iwanysziw – ukraiński działacz społeczny, poseł do Sejmu Krajowego Galicji III kadencji (1870-1876), włościanin z Hruszowa.

Wybrany w IV kurii obwodu Sambor, z okręgu wyborczego nr 23 Łąka-Medenice.

Bibliografia

 Wykaz Członków Sejmu krajowego królestwa Galicyi i Lodomeryi, tudzież Wielkiego Księstwa Krakowskiego w roku 1870, Lwów 1870.

Posłowie Sejmu Krajowego Galicji III kadencji
Ukraińscy działacze ludowi w Galicji

redpajama_set_name: RedPajamaWikipedia


Objective: Artix has sent you to patrol a local graveyard. Something is causing the undead to overflow onto the road and attack travelers.
Objective completed: You have made sure that the undead that had escaped the graveyard won't be harming anyone else, especially the fearsome superslime SQUISHNOX!
**Please note that only characters levels 1-19 will receive weapon drops in this quest due to the Universal Loot Table.
-- alexefb, Andy8, Basils, ChaoticFlame, coolyellow, cof_oro, Evilbooyaa, flameveal, foxbox6666, frolock, Gandlo de Sandra, Lianue for rewards.
-- Megaket Genevus, Poisonxx, Punkedelmo, roc phoenix flame, ShiitakeWarrior, Scakk, WhiteDragon master, Windy, zzat for rewards.


Only "after a wee bit o' the coin"? If only it were so innocent as a good old-fashioned scam. After seeing the way she looked at Audrey's sketchbook (not to mention the fact that she kept it), I can't help but suspect something a little more sinister.
Oh, and panel five: what is that man eating, and where can I buy one?
Geez, it's just another deep-fried ostrich leg. Any half-decent state fair south of Ohio and east of New Mexico has them.
And maybe she'll notice her sketchbook is missing at the same time.


text:
Peabody Award (dosł. Nagroda Peabody'ego), pełna nazwa: George Foster Peabody Award (dosł. Nagroda George'a Fostera Peabody'ego) – amerykańska nagroda medialna, wręczana za wybitne dokonania w przemyśle radiowym, telewizyjnym i internetowym. Historycznym ośrodkiem i miejscem jej wyłaniania jest Uniwersytet Georgii, pod dyrekcją dziekana wydziału dziennikarstwa i mediów masowych, w Athens.

Historia
Nagroda została powołana w 1939 r. przez amerykańskie Narodowe Stowarzyszenie Nadawców (National Association of Broadcasters), jako wyróżnienie dla wybitnych audycji radiowych. Po wybraniu przez stowarzyszenie miejsca wyłaniania nagrody, patronem (przydzielonym w 1940 r.) został przedsiębiorca i filantrop George Peabody, darczyńca uniwersytetu. Od 1948 r. nagroda obejmuje telewizję, od 1989 roku sieci kablowe, a od 2003 także szeroko rozumiane nowe media - w 2012 roku nagroda została przydzielona blogowi internetowemu.

Ogólna charakterystyka
Odbiorcą Nagrody są osoby bądź organizacje odpowiedzialne za powstanie lub dystrybucję danego dzieła, a w szczególnych przypadkach podmioty odpowiedzialne za więcej niż jedno dzieło w ocenianym roku. Wyróżniane bywają również instytucje bądź sieci medialne, jak np. Radio BBC. Nagroda nie wyklucza nominacji dzieł nadawanych poza Stanami Zjednoczonymi, jednak w swojej historii liczba nagród przyznanych twórcom zagranicznym jest niewielka.

Nagroda Peabody'ego, w zamyśle twórców radiowy odpowiednik Nagrody Pulitzera, jest ścisłe powiązana z uniwersytetem i z tego powodu cieszy się dużym prestiżem w amerykańskim przemyśle medialnym. Zdaniem byłego członka komitetu nagrody, Fraziera Moore'a, wyróżnia się ona od np. Nagrody Emmy międzynarodową perspektywą oceniania, jurorzy zaś są restrykcyjni i reprezentują różnorodne środowiska artystyczne i różne doświadczenie. Jak podkreśla z kolei pełniący od 2001 do 2013 roku funkcję dyrektora komisji nagrody, profesor Horace Newcomb, spośród ponad tysiąca nominacji każdego roku każda współzawodniczy ze sobą na równi, a wśród oceniających panuje opinia, że posiadają "jedno kryterium, a jest nim doskonałość".

Ocenianie
Wczesnego lutego każdego roku około 30 komisji rozpoczyna badanie przydzielonej im części spośród ponad 1000 wszystkich zgłoszeń. Skład tych komisji tworzą wykładowcy, pracownicy oraz wybrani studenci Uniwersytetu Georgii. Po analizie składają one pisemną rekomendację dla każdego ze swoich faworytów.  Nominację może złożyć dowolna osoba, jeżeli zrobiła to przed ustalonym przez regulamin terminem oraz uiściła wyznaczoną składkę (na rok 2015 wynosiła ona 225$ dla radia i wytworów audio, 350$ dla telewizji i pozostałych mediów). Zawężone listy trafiają do kilkunastoosobowego (obecnie liczącego 17 osób), głównego ciała komisji Nagrody Peabody'ego, do którego należą przedstawiciele amerykańskich środowisk naukowych, krytyków artystycznych, dziennikarzy i specjalistów przemysłu medialnego. Jurorzy omawiają między sobą kandydatury na zasadzie wolnej dyskusji, na terenie kampusu uczelni. Ostatnie ze spotkań odbywa się pod koniec marca. By zwyciężyć, dana nominacja musi zdobyć jednogłośną aprobatę każdego z członków jury. Liczba nagród nie jest z góry określona, a zwycięzcy nie są w żaden sposób stopniowani bądź kategoryzowani. Uroczystość wręczania nagród odbywa się w Nowym Jorku późną wiosną.

Przypisy

Nagrody telewizyjne
Nagrody radiowe
Radio w Stanach Zjednoczonych
Telewizja w Stanach Zjednoczonych
Nagrody internetowe
Amerykańskie nagrody


Donald McKay exemplifies an active senior and citizen in the community of Decatur. Whether he is on the dance floor, swimming at the local fitness center or reading the New York Times, Don is completely engaged. Mr. McKay and his late wife Susan, moved into Decatur Christian Towers in 2001.
A writer and editor of articles on business, economic, and political matters, Mr. McKay was named one of the "Outstanding Young Men of America" in 1971 for his work as editor of Boston's Senior News. Later, living in the Atlanta metro Mr. McKay received a "Certificate of Appreciation in Recognition of Outstanding Service" at the Mediation Unit of the U.S Equal Employment Opportunity Commission, Atlanta District Office. Recently, he served as a Program Assistant at the Literacy Volunteers of Atlanta, a position funded by AmeriCorps VISTA, a domestic version of the Peace Corps.


text:
Menard County is the name of two counties in the United States:

 Menard County, Illinois
 Menard County, Texas


text:
CLINICAL TRIALS PROFILE FOR FURAZOLIDONE
All Clinical Trials for Furazolidone
Trial ID
NCT00520949 Quadruple Therapy for Triple Therapy Resistant Helicobacter Pylori Infection Completed Aga Khan University N/A 2006-10-01 Triple therapy, a combination of proton pump inhibitor with two antibiotics, is the gold standard for anti-Helicobacter pylori treatment. Usual antibiotics are clarithromycin, and either amoxicillin or one of the nitroimidazoles (metronidazole). However, there is an increasing evidence of H. pylori resistance to classical triple therapy. Another reason for this failure being low patient compliance with treatment. A regimen useful in one geographical area may not be effective or practical in another area. The aim of this study was to eradicate H. pylori infection resistant to triple therapy, establish the efficacy and safety of a 14-day therapeutic regimen to eradicate of H. pylori in patients who have failed with the classical triple therapy (omeprazole, clarithromycin and amoxicillin) given for 14 days.
NCT00741052 Ciprofloxacin Multiple Dose for Adult Cholera Completed International Centre for Diarrhoeal Disease Research, Bangladesh Phase 3 2007-07-01 Cholera is an important diarrhoeal disease and an important cause of death, particularly during epidemic outbreaks, in Bangladesh and many other developing countries. Used as an adjunct to management of dehydration, antimicrobial therapy using an appropriate agent reduces diarrhoea duration and stool volume in severe cholera by about half. The usefulness of antimicrobials has, however, been greatly eroded by the increasing prevalence of resistant strains of V. cholerae O1. From October 2004 at the Matlab Hospital and from December 2004 at the Dhaka Hospital of ICDDR, B, V. cholerae strains became increasingly resistant to tetracycline and erythromycin- two drugs used in the treatment of severe cholera in adults and children respectively. Because of this high prevalence of resistance we resorted in early 2005 to using ciprofloxacin for treatment against multi drug resistant V. cholerae. Although all isolates were susceptible to ciprofloxacin when standard thresholds for disc-diffusion or E-test were used, but majority of the strains demonstrated a MIC value of 0.250 µg/ml, over hundred-folds greater than the V. cholerae strains tested in earlier years, which generally had a MIC of <0.003 µg/ml. In this randomized, double blind, controlled trial we will assess clinical and bacteriological response to 12 hourly oral dose of ciprofloxacin for 3 days in which the first two doses will be 1 g each and the later 4 doses will be 500 mg each, and compare them with a single 1 g oral dose of azithromycin. We are using azithromycin as the comparator drug because current circulating V. cholerae isolates are susceptible (MIC ≤ 0.125 µg/ml) to this azithromycin, and single-dose azithromycin has been evaluated earlier to be effective in the treatment of cholera.
NCT01668927 Empirical Rescue Therapies of Helicobacter Pylori Infection Completed Shanghai Jiao Tong University School of Medicine Phase 4 2012-07-01 The increase of antibiotic resistance to H. pylori causes failure of treatment. Furazolidone, amoxicillin and tetracycline are good candidates for rescue therapy since resistance to these three antimicrobials was rare. It is necessary to assess the efficacy and safety of these four bismuth-containing quadruple regimens with above antibiotics as empirical rescue therapies for H. pylori eradication.
NCT01805934 Quadruple Therapy Versus Dual Therapy as Rescue Regimens for Helicobacter Pylori Infection Completed Shanghai Jiao Tong University School of Medicine Phase 2 2012-05-01 The eradication rates of first-line treatment for Helicobacter pylori(Hp) infection are not satisfactory. The study aims to compare the efficacy and safety between quadruple therapy with furazolidone and dual therapy with high doses of amoxicillin as rescue regimens for Hp.
NCT02894268 Doxycycline- and Furazolidone-containing Quadruple Regimen is Superior of Tailored Therapy Recruiting Sir Run Run Shaw Hospital Phase 4 2016-05-01 Doxycycline- and Furazolidone-containing Quadruple Regimen can be a successful rescue treatment for Helicobacter pylori Infection patients after Failure of several therapy. It is superior of tailored therapy as rescue treatment for helicobacter pylori Infection after failure of several therapy.
NCT03139253 Antimicrobial Susceptibility Testing Guided Triple Therapy in Salvage Helicobacter Pylori Treatment Not yet recruiting Yanqing Li Phase 4 2017-05-30 Helicobacter pylori (H. pylori), which infects about 50% of the global population, has been recognized as a main risk factor of multiple gastric pathologies, especially non-cardiac gastric cancer. Strongly evidence supports that H. pylori eradication is an effective approach to reduce the incidence of those pathologies.
>Trial ID
>Title
>Status
>Sponsor
>Phase
>Start Date
>Summary
Clinical Trial Conditions for Furazolidone
Condition Name
Condition Name for Furazolidone
Intervention Trials
Helicobacter Pylori Infection 3
Scarred Peptic Ulcer 1
Functional Dyspepsia 1
Rescue Therapy for Helicobacter Pylori 1
[disabled in preview] 0
This preview shows a limited data set
Subscribe for full access, or try a Trial
Export unavailable in trial.
Subscribe for complete access.
Condition MeSH
Condition MeSH for Furazolidone
Helicobacter Infections 4
Disease Susceptibility 1
Stomach Ulcer 1
Clinical Trial Locations for Furazolidone
Trials by Country
Trials by Country for Furazolidone
Location Trials
Clinical Trial Progress for Furazolidone
Clinical Trial Phase
Clinical Trial Phase for Furazolidone
Clinical Trial Phase Trials
Phase 4 7
Clinical Trial Status
Clinical Trial Status for Furazolidone
Completed 4
Not yet recruiting 4
Clinical Trial Sponsors for Furazolidone
Sponsor Name
Sponsor Name for Furazolidone
Sponsor Trials
Shandong University 2
Shanghai Jiao Tong University School of Medicine 2
Livzon Pharmaceutical Group Inc. 1
Sponsor Type
Sponsor Type for Furazolidone
Identify first generic entrants


We're excited to announce that Emma has a brand new integration with Salesforce!
The integration features seamless 2-way contact syncing, response tracking directly in Salesforce, automation based on field changes, and so much more that helps you put your customers at the heart of your marketing.
Here's Senior Product Manager (and Salesforce enthusiast) Matt Thackston to tell you all about it.
Emma for Salesforce keeps your Emma Audience and your Salesforce account in sync both ways. Say goodbye to the days of importing and exporting .xls files to keep your data current.
Your mailing results display directly in Salesforce so you can quickly see which of your leads are opening, clicking, and converting. You'll know what's working (and what's not), so your follow-up strategy becomes crystal clear.
Our integration also gives you the power to create a series of automated emails that fire based on a field change to a contact's record in Salesforce. When paired with our Advanced Automation suite, your customer data becomes the powerhouse at the core of every campaign you create. It's smart, strategic email marketing at its best.
There's even more where that came from, so head over to the Salesforce AppExchange to see the full list of features.
Ready to check it out? Our Salesforce integration is available for all Emma Plus, Premium, and Enterprise customers. If you're not in one of those packages (or not sure) and want to take it for a spin, just let us know.


The shopper satisfaction is our primary focus on. We uphold a consistent level of professionalism, quality, credibility and repair for Steel Pipe Fittings Malleable Elbow , Steel Pipe Fittings , Water Pipe Fitting Male Tee , Are you still wanting for a quality product that is in accordance along with your excellent organization image while expanding your solution range? Consider our quality merchandise. Your choice will prove to get intelligent!
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text:
Sectors >
Antwerp to host heavy lift freight forwarding network - Sectors
Antwerp to host heavy lift freight forwarding network
September 15 - The over-dimensional specialists network, the Project Professionals Group, will host its annual conference from June 30 to July 3, 2010 in the Port of Antwerp.
PPG general manager Kevin Stephens said Antwerp was the successful nomination following this year's conference hosted by the Port of Corpus Christi in Texas.
Stephens said: "PPG is looking forward to meeting in Belgium and at one of Europe's major heavy lift project cargo ports."
Some 150 freight movers, ship owners and related industry companies from over 50 countries are expected to attend the 2010 conference.
Centauro crosses the Andes
July 18 - Centauro Argentina has coordinated the delivery of turbines for a hydroelectric energy project in Mendoza.
Gustav Seeland adds to fleet
July 18 - Hamburg-based Gustav Seeland is expanding its fleet with an MK 88 Plus mobile crane.


Boston, MA - As you may know, BPS district is in the process of finding and replacing the current interim superintendent Laura Perille after Tommy Chang's sudden departure. Bianca Vázquez Toness writes about the incompetencies that Chang possessed and overall experience when he first took on the job. As a result, BPS is looking to fill the role with a candidate who is both well -versed about the challenges that the district faces and equipped to pick up from where Chang left off. During this search a number of activists have found discrepancies in the approach and Catherine Mcgloin further expounds on this issue in the The Bay State Banner. Some of these activist groups include Boston Network for Black Student Achievement and Citizens for Public Schools and the Boston Branch of the NAACP.
All of the organizations together insist on BPS district hiring process to be transparent, inclusive and strategic by specifically choosing someone who is an extremely competent leader in education.In fact, At-large City Councilor Annissa Essaibi-George believes that the position should be given to a teacher. In addition, many of the activists believe that the school committee must clearly reject the hiring of the interim superintendent so that competitive candidates may feel comfortable applying. Hopefully, the position will be filled by or before the up coming school year so that the candidate will have time to prepare for all that the job entails.
Mayor Walsh, on the other hand, is not concerned about filling the position and plans to start recruiting in October. In order to be transparent in the process, the mayor has opted to creating a search committee for the superintendent role. Activists are determined to have a say concerning the individuals who will comprise of this committee and are looking to recieve a response from the school board concerning their request by next Wednesday.


It is a federal crime to knowingly create and submit false campaign finance reports to the Federal Election Commission.
Typically, the Government charges the filing of a false campaign disclosure form as being a violation of 18 U.S.C. § 1001, which is a general statute that prohibits filing materially false statements within the jurisdiction of any federal agency. Although 18 U.S.C. § 1001 is a charge that can be filed in connection with knowingly filing false campaign disclosure forms with the Federal Election Commission, the typical charge that is brought in these cases is an alleged violation of 2 U.S.C. § 441(a), the Federal Election Statute. This statute makes it a crime to knowingly and willfully accept and/or receive contributions in excess of the limits of the Election Act. If two or more persons conspire to violate this law, another prosecutor's preferred charge is to allege a violation of 18 U.S.C. § 371, the general conspiracy statute.
Our firm has been involved in federal prosecutions for violation of state campaign disclosure laws where federal monies or federal grants were implicated. There are a variety of tools at the disposal of a prosecutor in such cases but the main creative tool employed here is the money laundering statute. This statute, found at Title 18 U.S.C. § 1957, prohibits spending in excess of $10,000.00 where the source of the funds cannot be determined from the face of the financial transactions. The creative use of money orders or even cash deposits designed to conceal the identity of the owner of the funds can likely be used as a creative prosecutor's tool in campaign finance prosecutions.
Most cases involving campaign financial disclosure laws are cases brought where there has been a conspiracy to conceal the amount of donations and/or the identity of the donors, particularly where large amounts of monies in excess of spending limits are involved. Thus, most federal prosecutions in this area are predicated upon the general conspiracy provision found at 18 U.S.C. § 371, the false statements provisions found at 18 U.S.C. § 1001 and the specific illegal campaign excessive spending violations found at 2 U.S.C. § 1446.
Finch McCranie LLP has experience representing persons involved in federal criminal investigations for alleged campaign disclosure violations. Such cases typically are serious matters involving significant financial penalties and possible jail time in the event of a conviction. Please contact us today for a free initial consultation.