Abstract model organism Drosophila melanogaster. We showed that ferritin H and L transcripts are coexpressed during embryogenesis and that both subunits are essential for embryonic development. Ferritin overexpression impaired the survival of iron-deprived flies. In vivo expression of GFP-tagged holoferritin confirmed that iron-loaded ferritin molecules traffic through the Golgi organelle and are secreted into hemolymph. A constant ratio of ferritin H and L subunits, secured via tight post-transcriptional regulation, is characteristic of the secreted ferritin in flies. Differential cellular expression, conserved post-transcriptional regulation via the iron regulatory element, and distinct subcellular localization of the ferritin subunits prior to the assembly of holoferritin are all important steps mediating iron homeostasis. Our study revealed both conserved features and insect-specific adaptations of ferritin nanocages and provides novel imaging possibilities for their in vivo characterization.
IRON is an essential element of aerobic life. Cells have evolved highly regulated molecular pathways that ensure iron incorporation into heme (Ponka 1997) or formation of iron-sulfur clusters (Rouault and Tong 2005). Cellular and systemic iron levels are tightly regulated to ensure bioavailability and protect from the hazards of iron overload (Hentze et al. 2004). Ferritin, a heteropolymer composed of H and L subunits, acts as the primary iron-storage molecule (Harrison and Arosio 1996). The H subunit contains a ferroxidase center, which enables the mature heteropolymer to oxidize soluble ferrous iron, whereas the L chain provides the nucleation centers for deposition of the ferrihydrite mineral (Santambrogio et al. 1996).
In Drosophila, Ferritin 1 heavy chain homolog (Fer1HCH) and Ferritin 2 light chain homolog (Fer2LCH) encode the ferritin subunits that compose the major, secreted form of ferritin (Charlesworth et al. 1997; Georgieva et al. 1999, 2002). The crystal structure of secreted ferritin from Trichoplusia ni revealed a symmetrical arrangement of H and L chains (Hamburger et al. 2005). Inter- and intrasubunit disulfide bonds were shown to be important for the folding/assembly of T. ni ferritin, and the respective cysteine residues mediating these bonds were also conserved in Drosophila melanogaster, suggesting that the ferritins of the two species share the same mode of assembly (Hamburger et al. 2005). The Fer1HCH amino acid residues that are required for ferroxidase activity in mammals were conserved in the insect ferritin structure, and a predicted Fer2LCH ferrihydrite nucleation site formed by the L-chains was also found (Hamburger et al. 2005).
As in vertebrates, the IRE/IRP system functions in Drosophila (Rothenberger et al. 1990; Missirlis et al. 2003). A functional IRE is present in the 5′-UTR of the Fer1HCH mRNA, but only in certain splice variants that are preferentially encoded under iron-limiting conditions (Lind et al. 1998; Georgieva et al. 1999). In contrast, no IRE is present in Fer2LCH mRNA (Georgieva et al. 2002). IRP homologs are expressed in the fly (Muckenthaler et al. 1998), and one homolog (IRP-1A) has been shown to bind to IREs from both Drosophila and mammals (Lind et al. 2006).
This article shows that mutational inactivation of either Fer1HCH or Fer2LCH in Drosophila, as well as the disruption of the ferroxidase center of Fer1HCH, results in developmental arrest and fly embryonic lethality. We characterize a novel fly strain expressing GFP-tagged Fer1HCH and show that GFP-Fer1HCH is incorporated into endogenous functional ferritin. We use this strain to study induction and trafficking of ferritin in the fly midgut, the major iron-storing organ in the insect.
Fly strain Fer1HCHG188/TM3 was sent to us by Lynn Cooley and is available at (Kelso et al. 2004). This line was generated from a screen utilizing a mobile exon encoding GFP carried in a P-transposable element (Morin et al. 2001), which landed in the second intron of the Fer1HCH gene (Figure 2A). Fer1HCH451/TM3, Sb, ry and Fer2LCH35/TM3, Sb, ry were generated during a large-scale mutagenesis screen (Spradling et al. 1999) (nos.11497 and 11483, respectively). To generate UAS-Fer1HCH and UAS-Fer2LCH flies, expressed sequence tags LD03437 and LD01936 pUAST. Clone LD03437 contains the Fer1HCH IRE, but we have omitted it from the transgenic construct using a downstream XhoI restriction site present within the 5′-UTR for cloning. To generate ferroxidase-inactive UAS-Fer1HCH* we have mutated E87 to K87 and H90 to G90 (Cozzi et al. 2000) and injected into embryos using conventional techniques (Rubin and Spradling 1982). Overexpression was accomplished by means of the UAS/Gal4 binary expression system (Brand and Perrimon 1993). All phenotypes reported were confirmed with independent transgene insertions and we generated recombinant chromosomes (on the X and the third) carrying both UAS-Fer1HCH and UAS-Fer2LCH. Levels of ferritin expression were assessed by Western blotting and were found increasingly elevated in the following genotypes: (1) induction from the third chromosome recombinant +/+; Actin-Gal4/+; UAS-Fer1HCH, UAS-Fer2LCH/+; (2) induction from the X chromosome recombinant UAS-Fer1HCH, UAS-Fer2LCH/+; Actin-Gal4/+; +/+; and (3) induction from the X and third chromosomes UAS-Fer1HCH, UAS-Fer2LCH/+; Actin-Gal4/+; UAS-Fer1HCH, UAS-Fer2LCH/+.
Prussian blue staining and immunohistochemistry:
Midguts from larvae were dissected in PBS and quickly transferred to 4% formaldehyde in PBS for 30 min. Following washes with PBS, the tissue was permeabilized by treating with 1% Tween in PBS for 15 min. For detection of ferric iron, the samples were incubated in the dark with Prussian blue stain [2% K3Fe(CN)6, 2% HCl] for 45 min. Five washes with water followed and the preparations were mounted in water on glass slides (mounting in PBS will result in loss of the blue stain due to the change in pH) and imaged on a Nikon microscope (see below). For immunohistochemistry, we used the primary antibodies mouse anti-GFP (1:1000) from Molecular Probes (Eugene, OR) (A-11120) in combination with rabbit anti-Lava lamp (1:250) (a gift from John Sisson; Sisson et al. 2000) or rabbit anti-Fer2LCH (1:500) (Missirlis et al. 2006), followed by secondary fluorescent1000) from Molecular Probes (A-11029(1:1000) from Molecular Probes (A-11035). Preparatiopreparation for microscopy.
Images of GFP-ferritin in freshly dissected intestines mounted in PBS were captured using a Nikon ECLIPSE E600 microscope linked to a Nikon digital camera DXM1200F and were further processed by ACT-1, the application program for the DXM1200F digital camera and Adobe Photoshop. GFP was visualized using a Nikon mercury lamp and the B-2E/C FITC filter. Confocal microscope images of dissected tissues were captured with a 40×/1.2 NA water objective on a Zeiss 510 Meta invertedwith a 505-550 emission filter for GFP and the 543-nm helium-neon laser line with a 560–615 emission filter for Alexa Fluor 546-conjugated antibodies.
To study post-transcriptional regulation of Fer1HCH and Fer2LCH in Drosophila, we utilized flies with P-element insertions in these genes. We focused on three P elements: Fer1HCH451 and Fer2LCH35, which result in a loss-of-function mutant for each of the respective ferritin chains, and Fer1HCHG188, which adds GFP to the H subunit (Figure 2A). Fer2LCH35 is predicted to cause a genetic null mutation because the transposable element disrupts the open reading frame, whereas in Fer1HCH451 the P element resides in intronic sequences and the mechanism by which it disrupts gene function is unknown (Figure 2A). When homozygous, these insertions are embryonic or first instar larval lethal. Each insertion present over a deficiency chromosome lacking both genes also resulted in embryonic lethality, indicating that both ferritin-subunit chains perform essential functions in Drosophila.
Characterization of Drosophila strains containing transposable elements in Fer1HCH/Fer2LCH. (A) Fer1HCH and Fer2LCH share the same genetic locus at polytene position 99F on the third chromosome. Open and shaded boxes represent UTRs and the ORFs, respectively. The genomic insertions of P elements are shown as triangles and described in the text. (B) Western blot analysis of whole fly extracts from wild-type flies or the heterozygous Fer1HCH451/+ and Fer2LCH35/+ flies. Antibodies used are indicated at the bottom of the images. Levels of both ferritin subunits are reduced irrespective of which gene is affected by the transposon insertion. (C) Transgene-dependent rescue of the embryonic lethality associated with Fer1HCH451/451 and Fer2LCH35/35 and Fer1HCHG188/451 demonstrates the specific functions provided by the two genes. UAS-Fer1HCH* has no ferroxidase activity and cannot support ferritin function in vivo. (D) Predicted transcripts generated from the chromosome bearing the Fer1HCHG188 P element are depicted, indicating that both IRE- and non-IRE-containing transcripts (Lind et al. 1998; Georgieva et al. 1999) will encode GFP-tagged versions of the Fer1HCH subunit. Note that the predicted cleavage site for generation of the mature polypeptide is contained in the second exon of the Fer1HCH gene, prior to the GFP insertion. (E) Protein extracts from wild-type and Fer1HCHG188/+ adult flies were subjected to reducing SDS–PAGE; Western blots were probed with Fer2LCH and Fer1HCH peptide antibodies. Fer2LCH levels remain unchanged between the two samples, whereas there is less Fer1HCH and a higher-molecular-weight species appearing in Fer1HCHG188/+, migrating at the predicted size for a GFP-Fer1HCH fusion protein. (F) Nonreducing SDS–PAGE separation of lysates from mouse liver and wild-type, Fer1HCH451/+, and Fer1HCHG188/+ Drosophila strains. Flies were allowed to feed for 24 hr on food containing 55FeCl3-NTA. (Left) Western blots; (right) autoradiograph of a portion of the gel run in parallel. All detectable iron in extracts from Fer1HCHG188/+ flies coincides with the ferritin heteropolymers specific to this strain. Higher-molecular-weight species bands most probably reflect different ratios of native and GFP-tagged Fer1HCH subunits within ferritin heteropolymers.
To investigate possible homeostatic interactions between the two proteins, their expression in heterozygous adult flies was examined by Western blot analysis (Figure 2B). As expected, Fer1HCH levels were markedly reduced in Fer1HCH451/+ heterozygotes and the same was true for Fer2LCH levels in Fer2LCH35/+ flies. Unexpectedly, endogenous levels of Fer2LCH were also low in Fer1HCH451/+, and Fer1HCH levels were low in Fer2LCH35/+ flies. Because trans-heterozygous (Fer1HCH451/Fer2LCH35) flies are fully viable, we hypothesized that neither of these insertions woboth genes simultaneously. To address the potential role of the P-element insertions on transcription at the locus, we ubiquitously overexpressed Fer1HCH in Fer1HCH451/451 homozygous flies and restored adult viability (Figure 2C; see also materials and methods). When Fer1HCH was overexpressed in homozygous Fer2LCH35/35 flies, viability was not restored. The converse was true for overexpression of Fer2LCH (Figure 2C). These results indicate that the respective P elements interfere specifically with Fer1HCH and Fer2LCH expression and that reduction in levels of the alternate chain in each mutant is most likely due to post-transcriptional regulation.
We also generated flies that express Fer1HCH with a mutation that inactivates the ferroxidase activity, on the basis of analogy to the ferroxidase-null human allele (Cozzi et al. 2000). Expression of the mutated transgene was not able to functionally substitute for Fer1HCH and rescue the lethality of Fer1HCH451/451 flies. Thus, the ferroxidase activity of Fer1HCH provides an essential function in vivo and is likely required for iron loading of the ferritin shell.
A GFP-containing P element integrated into the second intron of Fer1HCH was used to study ferritin expression (Figure 2A) (Morin et al. 2001). Intron/exon donor and acceptor splice sites flank the GFP sequence on the P element; consequently, all Fer1HCHG188 mRNA types (IRE +/−) are predicted to contain the GFP exon, which should encode GFP in frame with the Fer1HCH protein (Figure 2D). Therefore, post-transcriptional regulation of GFP-Fer1HCH mediated by the IRP-1A (Lind et al. 2006) would remain unaffected by the GFP exon. GFP is inserted two amino acids downstream of the predicted cleavage site for the Fer1HCH signal peptide (Charlesworth et al. 1997), which is encoded from the second exon of the gene (Figure 2D). Thus, the full-length mature polypeptide is predicted to translocate to the endoplasmic reticulum and contain the GFP attached to the N terminus, after the signal peptide is cleaved.
To address whether the expressed GFP-Fer1HCH polypeptide contributed to the formation of the holoferritin in the transgenic insect, we separated the same extracts used in Figure 2E by SDS–PAGE electrophoresis under nonreducpreserve higher-molecular-weight ferritin heteropolymers (Missirlis et al. 2006). In extracts from wild-type flies we observed a single ferritin species that migrated close to mouse liver ferritin (Figure 2F, left blots). As expected from the lower levels of both ferritin subunits in Fer1HCH451/+ heterozygotes, these flies expressed less total ferritin. Conversely, in extracts from heterozygous Fer1HCHG188/+ flies that expressed GFP-tagged ferritin, several higher-molecular-weight bands could be detected. Thus, ferritin polymers are formed in these flies and the different molecular-weight species most likely reflect varying ratios of Fer1HCH and GFP-Fer1HCH chains in polymers. Importantly, no ferritin composed solely of native Fer1HCH and Fer2LCH was detected in the heterozygous Fer1HCHG188/+ flies, suggesting that the GFP-tagged ferritins are the functional ferritins of these animals. However, homozygous Fer1HCHG188/G188 and heteroallelic Fer1HCHG188/451 embryos failed to develop into larvae, indicating that the fly cannot survive when all Fer1HCH subunits are tagged with GFP. We wondered if GFP tagging of all Fer1HCH subunits hindered ferritin assembly or whether it obstructed access of an iron carrier to the assembled ferritin. If failure to assemble ferritin with 12 GFP-Fer1HCH subunits was the cause of lethality, we expected to rescue the lethality of Fer1HCHG188/451 flies by overexpression of the mutant ferroxidase construct UAS-Fer1HCH*. This expectation arose from the fact that GFP-Fer1HCH should provide intact ferroxidase centers to the holomer, and mutant Fer1HCH* should allow normal assembly. However, overexpression of wild-type UAS-Fer1HCH rescued the lethality of Fer1HCHG188/451 flies, but UAS-Fer1HCH* did not (Figure 2C), suggesting that a more likely explanation for the lethality of embryos expressing only GFP-Fer1HCH subunits is that the GFP tag blocks Fer1HCH-interacting proteins from accessing the ferroxidase centers of holoferritin.
GFP-ferritin is iron loaded:
We sought to demonstrate that the GFP-tagged ferritin in the Fer1HCHG188/+ heterozygous flies was indeed able to sequester iron and substitute for nontagged holoferritin. To this end, we fed adult flies with radioactive iron in the form of 55FeCl3. Nitrilotriacetate was added to maintain 55Fe3+ in solution at the neutral pH of food, and whole-fly homogenates were prepared after the insects were allowed to feed for 24 hr. Autoradiographs of the nondenaturing SDS–PAGE gels clearly indicated that iron was incorporated in ferritin from wild-type or Fer1HCH451/+ flies (Figure 2F, right, lanes 1 and 2) but also in GFP-tagged ferritin (Figure 2F, right, lane 3).
The observation that the same amount of iron was associated with half the amount of ferritin protein in the heterozygous Fer1HCH451/+ strain (Figure 2F, middle lanes) suggests that the iron load per ferritin holomer can increase when total ferritin decreases. Also, the absence of 55Fe incorporation in the heterozygous Fer1HCHG188/+ strain at the same molecular weight where putative residual normal ferritins would run suggests that these flies survive on ferritin containing both GFP-tagged and nontagged Fer1HCH subunits. Our speculation that GFP may partially inhibit iron loading is consistent with our findings that assembled ferritin molecules with fewer GFP tags (distinguished by their faster migration on Figure 2F) are more heavily iron loaded than assembled ferritin molecules with many GFP-Fer1HCH subunits (migrating more slowly on Figure 2F). Thus, although the GFP-Fer1HCH in solo is not fully functional, it coassembles in vivo with Fer1HCH to form functional fluorescent ferritin.
Since iron overload is known to induce ferritin expression in vertebrates and flies alike, we sought to test the consequences of overexpression of either ferritin H or L genes under normal or low iron levels. Immunoblotting of lysates prepared from whole fly extracts revealed that ubiquitous overexpression of either of the two single chains individually was not sufficient to significantly alter total ferritin amounts or the relative ratio between the two chains (Figure 3A). In contrast, when both UAS-Fer1HCH and UAS-Fer2LCH were simultaneously activated with the same Actin-Gal4 driver, robust overexpression was achieved in both sexes. Expression of the ferroxidase inactive UAS-Fer1HCH* was also demonstrated when coexpressed with UAS-Fer2LCH (Figure 3A, right). Overexpression of ferritin was also revealed when the proteins were separated in nonreducing gels (Figure 3C, left). We confirmed successful expression from the transgenes by performing RT–PCR using primers that were specific to the transgenic mRNA (Figure 3B).
Phenotypes associated with ubiquitous overexpression of ferritin. To achieve ferritin overexpression, we crossed homozygous female flies for the indicated transgene to males from the Actin-Gal4/Cyo stock. The progeny carrying the Actin-Gal4 driver was selected. (A) Western blot analysis from whole fly extracts indicates that overexpression of the single-ferritin chains fails to alter their relative ratio. If a Fer1HCH or a Fer1HCH* subunit is overexpressed in parallel with Fer2LCH, significant overexpression is achieved. (B) To verify that transgenic message is expressed, RT–PCR was performed with primers specific to the UAS-derived transcripts. (Ccontrol, mild, and stronger ferritin overexpression strains, and the Fer1HCH*, Fer2LCH overexpression strain (genotypes are indicated in materials and methods) probed with anti-Fer2LCH. (Right) AnInterestingly, similar amounts of total iron are contained in the different size pools of total ferritin. Ferritin containing a mixture of functional endogenous Fer1HCH and nonfunctional transgene-derived Fer1HCH* subunits is still able to bind iron. (D) Survival on 10 mm paraquat is enhanced in female, but not male flies overexpressing ferritin. The solid line represents the control +/+; Actin-Gal4/+flies, the three lightly shaded lines represent flies in which the single Fer1HCH, Fer2LCH, and Fer1HCH* are overexpressed (but not stabilized), while the two darkly shaded lines represent ferritin overexpressors (1H, 2L and 1H*, 2L). (E) Parental crosses were set up in vials containing 180 μm Bathophenanthroline disulfate to cause iron deficiency, regular food or 5 mm ferric ammonium citrate to cause iron overload. Male progeny of flies overexpressing ferritin (i.e., carrying the Actin-Gal4 driver) surviving to adulthood are compared to flies where ferritin overexpression is not induced from the transgene (i.e., carrying the Cyo balancer chromosome). The number of flies counted containing the Cyo balancer chromosome is indicated at the top of each bar. Different levels of ferritin overexpression were achieved by using independent recombinant chromosomes and their combination. Iron deficiency combined with ferritin overexpression can be lethal, whereas ferritin overexpression is well tolerated under conditions of iron overload.
Ferritin overexpression in mammalian cells causes functional iron deficiency due to iron chelation (Cozzi et al. 2000; Wilkinson et al. 2006). However, no significant alteration of total radioactive iron associated with overexpressed ferritin was detected after 24 hr of feeding (Figure 3C, right). If the feeding period was extended for 5 days, a slight increase was seen in sequestration of radiolabeled iron into the ferritin of overexpressors (data not shown), but overall (see also Figure 2F) the results show that, at least in Drosophila, ferritin protein levels are not the determining factor controlling iron storage into ferritin. Overexpression of ferroxidase-inactive UAS-Fer1HCH* together with UAS-Fer2LCH in the presence of functional endogenous Fer1HCH subunits results in ferritin heteropolymers that are still potent iron-storage complexes.
In contrast to Fer3HCH overexpression, which did not affect development, flies overexpressing Fer1HCH and Fer2LCH are at a developmental disadvantage compared to their siblings (Figure 3E). We scored the progeny from the cross Actin-Gal4/Cyo × UAS-Fer1HCH, UAS-Fer2LCH/UAS-Fer1HCH, UAS-Fer2LCH by gender and by presence or absence of the Actin-Gal4 driver. We used two independent sets of recombinant chromosomes (one on the X and one on the third) for UAS-Fer1HCH and UAS-Fer2LCH and also flies that carried both recombinant chromosomes, allowing for testing if the phenotype was dosage sensitive. We show the results in male flies (Figure 3E), where the competitive disadvantage of ferritin overexpression is more pronounced than in females. Importantly, the effects of ferritin overexpression are more dramatic under iron-limiting conditions induced by addition of the iron chelator Bathophenanthroline disulfate in the food, whereas the lethal effects can be rescued by dietary iron supplementation. Therefore, our results indicate that ubiquitous ferritin overexpression in the absence of iron overload can be deleterious, by a mechanism that implicates the iron-chelating properties of ferritin.
Ferritin transcripts hav:
The genomic proximity between Fer1HCH and Fer2LCH could facilitate similar expression patterns to coordinate biosynthesis of the ferritin heteropolymer. In situ hybridizations of antisense RNA probes against the two genes in whole mount preparations of embryos at different stages of development were performed. We reasoned that if the predominant sites of Fer1HCH and Fer2LCH expression were different, it would be unlikely that they shared common regulatory enhancers, as previously suggested (Dunkov and Georgieva 1999, 2006). Nevertheless, we found that the different transcripts were expressed in highly specific, yet similar, patterns during embryogenesis (Figure 4). Fer1HCH and Fer2LCH were expressed during oogenesis and the maternal transcripts became evenly distributed in the egg and early embryo (data not shown), where they could be detected in the blastoderm (Figure 4, A and B). Tissue-specific transcripts were first detected during germ-band elongation in cells of the mesoderm (Figure 4, C and D), which are specified to give rise to the fat bodies and amnioserosa (Figure 4, E and F). Staining was also seen in cells destined to become macrophages in the anterior head region of embryos (Figure 4, C and D). During germ-band retraction and dorsal closure, the amnioserosa retained ferritin mRNAs (Figures 4, G and H). At late stages of embryogenesis, cells in the developing midgut initiated ferritin transcription (Figure 4, I and J). The similar embryonic expression patterns of Fer1HCH and Fer2LCH suggest that both ferritin subunits are expressed in each cell type where ferritin is required.
Intestinal sites of constitutive and inducible ferritin expression differ. (A and B) Dissected larval midgut from the Fer1HCHG188/+ line. Fluorescence identifies the iron region in the middle midgut (arrows). (C and D) Dissected midgut from larvae of the same genotype raised on food containing 1 mm ferric ammonium citrate. Note that ferritin protein accumulates in the anterior midgut. (E–H) Prussian blue staining of midguts from control or iron-fed larvae: (E) anterior midgut, control diet; (F) iron region, control diet; (G) anterior midgut, iron diet; (H) iron region, iron diet. (I and J) Fer1HCHG188/+ larvae were raised on food containing 1 mm CuSO4 and midguts were imaged using UV excitation and the DAPI filter. (I) Cu-metallothionein fluorescence (orange, asterisks) is prominent in the posterior midgut. (J) Cu-metallothionein fluorescence is also found in a cluster of cells anterior to the iron region, where constitutive metallothionein expression has been reported (Mcnulty et al. 2001). (K and L) Time course of iron-dependent ferritin induction in (K) anterior midgut and (L) middle midgut dissected from third instar larvae. Extracts wereFerritin induction in the iron region is much less pronounced.
To assess the time frame of ferritin induction during iron feeding and to further demonstrate that a specific subset of cells in the anterior midgut shows a strong response to iron levels, we determined endogenous ferritin levels in a time course following iron feeding. For this experiment we used wild-type third instar larvae (100 hr old at 25°) and dissected their anterior and middle midguts for Western blot analysis. The results showed a clear induction of the ferritin heteropolymers 3 hr postfeeding in the anterior midgut (Figure 5K). Consistent with our imaging and iron-staining results, the induction of ferritin was largely restricted to the anterior midgut and was much less pronounced in the iron region (Figure 5L).
Another essential transition metal and dietary nutrient for Drosophila is copper (Zhou et al. 2003; Selvaraj et al. 2005). Copper-containing (cuprophilic) cells function in the acidification of the midgut and are present at different sites than the iron region (Poulson and Bowen 1952; Hoppler and Bienz 1994; Dubreuil et al. 2001). Copper-metallothionein complexes in these cells fluoresce orange–red upon ultraviolet illumination (Mcnulty et al. 2001). We imaged simultaneously the copper-metallothionein fluorescence and GFP-ferritin in midguts from larvae fed a diet containing 1 mm Cu2+. The results showed that ferritin-expressing cells indeed form a distinct cellular population from cells that contain copper (Figure 5, I and J; see also discussion).
Ferritin induction at subcellular resolution:
To investigate which cellular compartment accumulates iron-loaded ferritin in midgut cells, we costained GFP-Fer1HCH-expressing cells with an antibody raised against the Golgi-associated protein Lava lamp (Sisson et al. 2000). Confocal images from a single cell in the iron region clearly showed that all GFP-ferritin localized within the Golgi compartment (Figure 6, A–C), consistent with the images obtained by electron microscopy (Locke and Leung 1984; Nichol and Law 1990). A few Golgi bodies in cells from the iron region were devoid of ferritin (Figure 6C, red). We also imaged the same cells stained with antibodies against the Fer2LCH subunits. As expected from their tight association revealed by structural studies, biochemical analysis, and electron microscopy, there was complete colocalization with GFP-Fer1HCH in the Golgi complex of these specialized cells (Figure 6, D–F). Finally, we focused on the cells of the anterior midgut that do not normally express ferritin, but potently do so in the presence of high iron levels (see Figure 5, A–D). The cells were stained for both ferritin chains during the time course of ferritin induction (Figure 6, G–I). Prior to iron feeding, GFP-Fer1HCH was not expressed and low levels of Fer2LCH were detected (Figure 6G). At 1 hr postinduction GFP-Fer1HCH was detected m, but we could also detect Fer2LCH-positive Golgi that were devoid of GFP-Fer1HCH subunits (Figure 6H). In contrast, at 4 hr postinduction the two subunits were strongly induced and were seen only in complex with one another within the Golgi (Figure 6I).
Ferritin resides in the Golgi apparatus of midgut cells. (A) Section of a single cell in the iron region of a dissected midgut from Fer1HCHG188/+ larvae imaged by confocal microscopy. Note the punctate subcellular localization, consistent with membrane-enclosed ferritin compartments seen by EM in midgut preparations from another insect species (Locke and Leung 1984). (B) The same cell was stained with anti-Lava lamp, a Golgi-associated protein. (C) Yellow identifies the Golgi complex as the primary location of ferritin in cells of the iron region. (D–F) A single cell expressing GFP-Fer1HCH in the iron region was stained with anti-Fer2LCH; colocalization of the ferritin chains was seen in the Golgi. (G–I) Time course of ferritin induction in single cells of the anterior midgut. Merged images of GFP-Fer1HCH in green and Fer2LCH in red are shown. (G) Prior to iron feeding, low levels of the ferritin subunits are expressed. (H) One hour postfeeding, GFP-Fer1HCH is in the endoplasmic reticulum, while Fer2LCH is detected only in Golgi bodies. (I) Four hours postfeeding, the two chains colocalize in the Golgi apparatus.
Collectively, our results have identified different specialized intestinal sites for iron and copper metabolism and showed that ferritin synthesis is differentially regulated along the intestine. Significantly, our results also validate the use of the Fer1HCHG188 line as a faithful reporter of endogenous ferritin expression.
In contrast to results from mammalian cell or animal models (Thompson et al. 2003; Wilkinson et al. 2006), but consistent with what is known from human patients with hereditary hyperferritinemia cataract syndrome (Cazzola 2002), experimental reduction or increase of ferritin levels through genetic manipulation in Drosophila caused only very mild alterations in the insect's iron homeostasis. These results point toward an independent regulatory system that controls iron sequestration into ferritin. The nature of this system is currently unknown, but could involve a putative iron chaperone that delivers iron to ferritin (Napier et al. 2005). However, the hypothesized chaperone's function does not completely override the need for ferritin regulation, as shown by the phenotype of ferritin overexpressing flies that was lethal under low iron conditions, but was rescued with iron supplementation (Figure 3E). Alternatively, localization of ferritin in the Golgi apparatus of insect cells may prevent it from contact with the cytosolic and mitochondrial iron pools. It is currently not known how iron is delivered to the ferritin that resides in the secretory pathway of cells.
We provided evidence that the ferritin genes are coexpressed during embryogenesis. We wondered whether their genetic proximity is conserved in other Drosophila species with fully sequenced genomes. To this end, we used the EvoPrinter, a new multigenomic DNA sequence analysis tool that facilitates the rapid ithe context of a single species (Odenwald et al. 2005). In silico analysis of the Fer1HCH and Fer2LCH genomic locus by the EvoPrinter produced an output of the combined mutational histories of six Drosophila species, superimposed on a reference sequence from D. melanogaster (supplemental Figure 1 at). The results not only showed that clustering of the two genes is conserved, but also identified potentially shared regulatory regions. From the 17,670 bp spanning the two genes that were analyzed, only 914 (or 5.2%) were conserved in an identical position in all six species tested. One-third of these conserved sequences (306) were contained within the open reading frames. Surprisingly, over half of them (491) were not contained in the cDNA sequence, but rather were clustered in two regions: downstream of the Fer2LCH-transcribed region or in the second intron of Fer1HCH (supplemental Figure 1). A particular stretch of 10 bp, TTTGCACACG, was found three times in the second intron of Fer1HCH and could represent a binding site for an unidentified factor. The remaining conserved base pairs (117) were mostly concentrated in the 5′-UTR of Fer1HCH, including the IRE itself. The conservation of the IRE over ∼e underscores the functional significance of the IRE/IRP control system in all Drosophila species.
Note added in proof: The conserved stretch of 10 bp, TTTGCACACG, identified here by EvoPrinterhas been independently described by others (H. Yepiskoposyan, D. Egli, T. Fergestad, A. Selvaraj, C. Treiber et al., 2006, Transcriptome response to heavy metal stress in Drosophila reveals a newNucleic Acids Res. 34: 4866–4977). These authors have previously characterized the binding of metal transcription factor 1 to this sequence.


This beautiful planet known as the Earth offers everything that she has with arms wide open. As inhabitants of this place, we have every right to make the best of this life in the path that we choose. In order to make this life a success, there are a few parameters one should follow. A good amount of planning is required to generate a result that is positive in nature. Every day requires us to make decisions, no matter how small. With this, we exercise the power of freewill and accordingly carve out the road which we will take. Your every thought, and every choice will lead you down a different path. It is helpful to realise where you are headed, or at least know the direction in which you wish to travel. Then, also ask yourself this question. Are you aligned with your goals?
To materialise any goal, it is first important to be aligned. Spend some time in introspection to find out how you want to see yourself. One very crucial element in this process is that we need to take the right steps at the right time. When you see an opportunity opening up, take it. Is there something that you really want?It could be a job, an audition, or even a trip?
A useful tip is that you could learn from your mistakes, we all make them and they are valuable in teaching us lessons. But is it possible that learn from other people's mistakes as well?


The content of these updates can be found in the release notes below.


Reducing energy costs with smart metering integrations
Amidst other factors, the increase in energy costs has played a significant role. Energy prices have been on the rise since 2021, having more than doubled since.
And while the Energy Price Guarantee will help reduce costs for domestic consumption, energy prices for businesses may increase by more than 500%.
“Put simply, business owners can’t manage their energy bills effectively if they have no way of determining where and how exactly their energy is being used, and this is where smart meters come in.
“Having access to reliable and in-depth energy consumption data enables businesses to make the changes they need to effectively reduce consumption and costs.”
Many still rely on outdated or ineffective metering, which might provide an accurate picture of how much energy a business is using, but no data regarding the ways in which it’s being used, rendering them almost entirely futile.
And often, existing metering systems are altogether not fit for purpose, whether installed incorrectly or connected to the wrong energy management platforms and communications systems.
Smart metering is particularly useful when paired with aM&T (automatic Monitoring and Targeting) equipment, which offers practical ways to read meters’ data, and in turn help pinpoint sources of energy wastage.
“If used properly, aM&T technology can generate cost savings of 10-15%. In the current climate, this can be incredibly beneficial for businesses,” adds David.
aM&T software can help identify whether a business’ daily energy usage lines up with what’s expected and from there, identify opportunities to reduce consumption.
This technology can provide businesses with a clear view of what their peak hours or days are for energy usage, whether there are energy spikes at particular times, and where exactly most energy is being used.
Tags Engineering Slider Smart manufacturing
Dispensing robots: what’s the risk?
ABB to divest UK technical engineering consultancy business to TÜV Rheinland


The gist of the personal attacks made against me were that I was a lazy debater, made awful arguments, and sounded like a donkey.  These are sentiments I heard when I debated.  But why does a person's choice of argument open them up for attack?Why has Towson's choice of argument opened them up for personal attack?
Why don't you want to be held accountable for your words?And to the community, why does this person think it's safe to say it here, but not safe to say it out there?Is there a lesson there?
There is a second part of this discussion as well and it involves debating race in debate rounds.  I think this topic is so delicate and I personally struggle with it.  One note, which I think is important, is that I don't think that Louisville and certainly not Towson called people racist because they were white.  I know this personally because I had to defend against them and I am black.  It is about a way of debating.  The more I engage in this discussion the more I understand why Louisville debated me how they did and it's because I debated them the way you did.  I applaud them for it now, but I also understand why many in the community are angry in the moment because in many ways Louisville's argument doesn't account for the people they are debating, many of which are very good people with very good hearts.  So, you must think to yourself you don't know me?You don't know that I have done X, Y, and Z.  I am not racist!
I think a good starting place for all of this is respect.  It's lacking here.  It starts with listening and trying to understand these very difficult issues. It's going to require some thinking outside the box.  And, it's going to require that some people who would rather imagine a world where they can rearrange our troop to imagine a world in which they participate in the oppression of others...


co’
Alexander accepted those recommendations and added three more including immediate expulsion of students found responsible for hazing and the right of university officials to randomly search fraternity and sorority houses.
“” Claitor responded.
However, Claitor said he felt that if universities and national fraternal organizations set out specific rules and policies that clearly discouraged and presented penalties for hazing, as most currently do, then this would not be considered means for punitive damages.
Donahue also argued that harsher criminal punishment would be appropriate and effective in preventing hazing, but punitive damages would not.
Claitor disagreed. “Ie asked Claitor why he did not include injury resulting from hazing along with death.bill. </s> Photo-documenting several occasions can present you with experience, particularly if you continue to be a budding event professional photographer. You might not receive payment with this deed however the experience you will get through this is immeasurable and can greatly help you prepare to as being a professional event professional photographer. Setting up your blog or perhaps an online journal of some kind, provides you with the chance to showcase your pictures to anybody who visits your website.
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Argentine president Mauricio Macri arrived on Monday evening to Moscow the first leg of his latest tour of European countries, and the first of 2018, with the purpose of promoting Argentina and attracting investments. In Moscow president Macri and First Lady Juliana Awada are scheduled to meet with president Vladimir Putin at the Kremlin.
Russia is considered an excellent market for Argentine food exports, and to increase bilateral trade, but Russian energy companies, government and private, are also among the most dynamic, and the idea of president Macri is to interest them to invest in Argentina and its potential development in wind, hydroelectric, oil and gas sectors.
From Moscow, president Macri and his delegation will hich convenes the main business and political leaders between January 23/26. On this occasion for the first time in years, US president Donald Trump has announced his attendance. The last time was when ex president Bill Clinton.
It will be a great chance for the Argentine government to watch global tendencies, political and economic. Davos will be a


HPD officer promoted
Hutchinson Police Officer Josh Jones was promoted to the rank of sergeant on March 2.
Jones, the son of a former Tucson, Ariz., police officer, started  HPD.
from 2004 until 2012, deploying twice to Iraq and once to Afghanistan, for a combined three years overseas. Jones was a shift supervisor for the tactical operations center of an aviation battalion, supporting combat operations on the ground.
After he left active duty, he joined the Kansas National Guard, where he was promoted to sergeant as a team leader and squad leader. He was recently promoted to staff sergeant and he is excited for the added responsibility and challenges he will face.
Since being hired at the HPE, he has been an adviser with the Reno County Cadet program for six years and Field Training Officer for three years, mentoring young people what it takes to be in law enforcement, asthis department. He also serves as a Force on Force instructor.
In his free time, Jones volunteers at his church, where he is a member of the worship team and the youth pastor, along with his wifeReno County.
(Editor’s note: This brief ran Sunday, March 8, with the wrong photo. The News apologizes for the error.)


Must have '(Recommended)' as the last element in the Search result title line.
'(Recommended)' must have a text size less than or equal to the Search result title text size (within accessibility guidelines).
Must not have a heading (e.g. Featured results:).
'Spelling suggestions' appearing on 'featured search' results pages must be displayed as per Checkpoint 18—Spelling suggestions.
'Featured search' results must be displayed below the Search box group and above the Result set description, refer to figure 26.


Warm temperatures and high angle spring sunshine contributed to these natural wet avalanches in Bullen Basin.
We noted a few fresh naturals, this one in the South Fork of High Creek Canyon.
Thanks Amy and Eric Flygare for sending in this shot of a triggered wind slab under the large cornice on Cornice Ridge. The heavily tracked slope at around 9500' in elevation faces east northeast.


(1) Spider Pig wearing Lipstick
(3) A boy
(4) Barack Obama (I’m dark skinned so maybe I can pull it off)
(7) 80s rocker chick
Obama lol politcal costumes are halarious
or u can be Michella Obama
she has great styl1.


table – still, I want to be a good group member! I ended up with 5 full minutes to spare for some edits.
She remembers the day he looked out at his prized roses – her husband had just trimmed then – and said, “Well, they’ll bloom nice next year . . . but I won’t see them.” One day he wanted to drive across the line – in their family the American border was always called, the line. He wasn’t really supposed to drive but she wouldn’t stop him. Behind the wheel of his truck he looked at her and said, “What a great feeling to get out on the road – I could go anywhere.” They both knew he couldn’t go anywhere – his morphine was back at the house and she didn’t see that he had packed extra pads to deal with the issue of incontinence. Dying with a tumor in your bladder is a messy business.
This blog is a bit like cleaning off my desk – just what has been going on in my life this week?
I remember how sad I was when Derrida died and the way in which he was so vilified in a New York Times obituary. When an outpouring of negative reaction to the Times piece came out, I learned that Derrida was particularly remembered for his generosity toward students – when he held a 1/3 teaching position at Irvine, he taught more than was required of him and the time he spent with students was unparalleled. Derrida would attend conferences on deconstructionism and give serious attention to all presentations. He would take careful notes and ask thoughtful, respectful questions of each presenter – be they lowly student or peer. As a graduate student myself at the time, this only endeared him more to me.
Have the characters in a book, movie, or TV series ever really got to you? Let me know. I’d like to close this post by drawing the diverse threads of these gleanings together but am at a loss as to how. I suppose it is all about living and thinking and being – what more is there?
A friend recently commented that she was hanging laundry on a clothesline for the first time in her life. I shared something with her that I came across while pursuing my PhD. (A graduate student in a certain frame of mind – desperately seeking dissertation topic – tunes into the oddest things.) I had heard of a woman in Newfoundland who did an entire PhD dissertation on how people hang out their laundry and what it says about them – personally, socially, and culturally. She included an array of clothesline photos to illustrate her points.
I believe in a neat clothesline – something you can look on with pride. There should be symmetry – what the hell – artistry, to what you have just caused to fly free over the landscape. Laundry should be hung so each piece is connected to the piece in front (this allows things to really whip in the breeze and not bunch up). As you hang out the clothes, little metal gadgets that clip and hold the two lines together should definitely be employed – these compensate for the weight of the wet laundry and keep the line at a proper height. I don’t have any hard and fast rules about the order of the hanging. Mostly I try not to overstress my clothes pins by clipping thick items together with the same pin.
I feel offended when confronted with laundry hung on a clothesline in a haphazard or sloppy fashion. This is why I never allow my husband, Bruce, to hang laundry. It isn’t really an issue – he hates doing this task and I really like it. Each piece of laundry has to be shaken out before hanging – you don’t just toss a folded over or squished up piece of fabric on the line – it will dry permanently set in that state. Geez – anyone knows that.
Hanging laundry to dry outside makes me philosophical – it is


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Separatist leader Count Dooku approved Passel Argente's plan to attack Kamino, and Argente decided to employ Merai as the on-location commander of the assault forces. It was a poorly planned and coordinated attack, with the loosely defined objective of destroying the main reactor of Tipoca City, thereby hampering the Republic's ability to create clones. With such limited resources and intelligence, Merai charged headlong into the assault.
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Defenses to Theft/Larceny Charges
- Age (Although being a minor is not a complete defense, it may serve to lessen the penalty.)
Under Texas theft / larceny law, offenders may receive charges varying from a "Class C" misdemeanor for the most minor of thefts, all the way up to a first degree felony for the most serious. The following guidelines are generally set for varying degrees of theft based on the amount or value of property taken:
- "Class C" misdemeanor: $50 or less
See Theft Pen


An event that saw one in five New Zealanders stop and celebrate te reo Māori together has been named a finalist in the prestigious, global Reed Awards.
Te Taura Whiri i te Reo Māori and digital strategy company, For Purpose were announced as finalists in several categories by award organisers, Campaigns and Elections in Washington DC this week.
The Reed Awards celebrate the very best in grassroots advocacy and social campaigns from across the world.


Smoke Free Horry County
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To find non-smoking rooms in the Myrtle Beach area, visit the VisitMyrtleBeach.com advanced lodging search.
For more information on Smoke Free Horry County, visit SmokeFreeHorry.com.


Non-Profit Leader Receives HV Beautiful Business Award
She notes The Queens Galley also offers a food-service and hospitality training program, and teaches families about healthy cooking, nutrition, and how to best budget for food shopping.
The was given to Diane Reeder during an event in Milton.


< 100% Effort ?
By TheFez
July 1, 2012 in Coursework, Advising, and Exams
I am just getting ready to enter my PhD program. I met with the director to talk about 1st year courses. He made an interesting comment (that I later found out he made to another student when he was entering the program).
The gist of it was this....
Most students have an "all or nothing" approach to coursework. They don't know how to put in any less than 100% effort. They try to be perfect. Some students can "modulate" their effort. To see what is required and just do the right amount. They are then able to work on more stuff.
It surprised me. It sounded like the first time I was being given advice by a professor to "satisfice" and not maximize (in econ terms). Giving this some serious thought since until now I have been one of those 100%+ers.
There is more to do in graduate school than can reasonably be done by a mere mortal (read: me. Probably you too). The advice you got is a very good one and you should give it some serious thought. Once you start school, you are going to have many simultaneous demands on your time: reading and writing for several courses; teaching requirements; research requirements; possibly, some service requirements (e.g. small organizational duties within your department). At some point - as soon as possible- you have to learn to prioritize. You can't possibly do all of the required reading for all your courses, for example. It's also sometimes a strategic choice to submit final papers that are "good enough", not "publishable as is". I have friends who don't want their professors to see anything less than perfect work and guess what - they have taken a number of incompletes and are behind on their requirements. I have chosen to submit papers that will earn me my desired grade but nothing more. If it's good/interesting work, I can follow up on it after the deadlines, but there is no need to get myself all worked up to meet some made-up deadline. Same goes for teaching duties. You can invest as much as you possibly can and still be lacking in terms of additional study aids/office hours/advice you could give your students.
You need to learn not to let any one aspect of your work take over all your work time. For that matter, you also need to learn not to let your work take over your life. It's important to learn to schedule time in your calendar for off-work activities(!), or else other duties will just take over that free time. It's also important to take time off to rest - and schedule it too, if you must. Just block off time for reading, writing, teaching, hobbies, rest, anything else you want. You may have noticed that this sounds like a heavy load -- it is. This is where prioritizing and doing less-than-perfect work in some areas becomes crucial. I'm not advocating for doing average work in all aspects and all the time, but rather learning where it's important to show your strong suits and where you can get by with doing less. Courses are often a place where ok-but-not-perfect work is really all you need.
ZeChocMoose, Alyanumbers, booksnlooks and 1 other
I also got the same advice, twice! When I started my MSc and when I was visiting PhD schools. I heard it from both profs and current graduate students. In my current MSc experience, it's definitely true and good advice. I also learned that it is MUCH easier to say "I'm going to prioritize" than to actually sacrifice effort. But I think I've learned to do it by now
Sometimes the advice comes with further details, like -- grad courses can sometimes be taught by profs that don't give a damn about the course. They just want it over and done with and you're not going to get anything out of it. So just get that minimum pass. Similarly, if you are taking a course that is just to fulfill a requirement but doesn't help your research or career, then just get that minimum pass. Save your course-related efforts for subjects that are interesting to you, or for courses that your adviser is teaching.
In addition, your grad school grades won't count for very much. Most post-doc applications won't really look at them, but some fellowships/scholarships might. In undergrad, the point of school is to take courses, and learn. In graduate school, many people (profs and students) view courses as a formality, something to get out of the way. Like a driving exam, first aid certification, safety training, etc, the goal is to demonstrate that you are proficient. (i.e. courses are still important, but for different reasons than in undergrad).
A related piece of advice is knowing when to ask for help. Sometimes students want to impress their supervisor by trying to solve the problem all on their own and spend weeks doing so. Sometimes the problem is something that the student couldn't have possibly known (or would have taken a long time). Knowing when to balance asking for help to save time vs. still being independent and learning on your own is something I'm still trying to figure out too.
And finally, grad school is really a place where "you get out what you put in". You can easily devote all your time to grad school. You will probably be rewarded too, with high grades, good research etc. But the cost is high! You need to know what you want to get out, and how much time you're willing to spend on school. If you don't draw the line, you will end up spending all your time doing work. A lot of students find it useful to log their hours and note what they were doing (just in general, e..g. research, courses, teaching, etc.) and then review it to see if they are spending time the way they want to. For me, a ~50-60 hour work week is what I've been aiming for.
@TheFez--
You might benefit from spending time thinking about why you were surprised by the professor's guidance and why you're an overachiever.
For example, if you find that the reasons for both center around a desire to do right by others or to please those whom you admire, that insight could be very useful when you run into a professor who simply cannot be pleased or satisfied and/or changes constantly the criteria of evaluation.
Also, you might realize that you have assumptions about the rules that some of the rules are much different. This insight may allow you to handle with increased skill situations in which one finds others appearing to a different game..
Finally, you may find an insight that leads you to realize that you're holding others to the same expectations you hold yourself. Such an insight can be especially useful when working with undergraduates who don't share your interests and/or have other priorities.
To be clear, I am not making assumptions about you. I'm offering suggestions so that you can vet your own assumptions and make changes as you see fit.
Ampere and pinkrobot
Program:Social Psychology
To echo the above, put just enough effort into your coursework so you don't look like an idiot. Too much time on coursework means you're not researching enough. Research gets jobs, grades don't. Same advice for TA work.
ETA: Forgot that grades often matter for scholarships. At my university you'd want good grades in the first year or two, until you get a good fellowship. Once you're funded they don't matter and most people take them pass/fail from that point on.
Edited July 12, 2012 by lewin00


Department co-head José Manuel Argüello is noted by market onlookers for his relevant capabilities within the IP sphere. One client enthuses: "He is very clear when explaining all the aspects of IP."


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 </s> PILOT BUTTE, Sask. – A grass fire that burned two to three acres in Pilot Butte, Sask. marked an early start to the grass fire season, the Pilot Butte Fire Department said Tuesday.
At approximately 1 p.m., firefighters from Pilot Butte, Balgonie and White City were called to a grass fire northeast end of the Regina Wildfire Federation gun range. The fire also extended into the White Butte trails.
“It’s rough terrain in there,” Kevin Dell, the Pilot Butte fire chief, said.
Dell said the wind, which gusted around 30 kilometres per hour, also helped the fire to spread around the dry grass.
Dell said this is the first large grass fire in the area this year and weather conditions have increased the risk in the area.
“There’s very little moisture laying around this spring,” Dell said.
“The snow melted and went pretty quick. Everything is drying up quite well. Grass fire season is a little earlier than normal, almost two weeks early.
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Edinburgh bookshops: The 11Omicron variant takes heat out of Currys as sales cool ahead of Christmas
Electrical goods retailer Currys has warned of cooling sales in the crucial run-up to Christmas.
15th Dec 2021, 9:21am - 3 min read
Updated 15th Dec 2021, 9:21am
However, underlying profits in the UK and Ireland rose from £25 million to £33m following cost-cutting measures across the business.
Chief executive Alex Baldock told investors: "Our market , but the stronger business we've built can ride out both the industry-wide disruption to supply chains and bumpy demand."
At a group level, including its international businesses in Greece and the Nordic region, sales were down 2 per cent to £4.8bn, with pre-tax profits rising 6.7 per cent to £48m.
Despite its cautious outlook, the company, which has gone through a series of name changes, said it would spend £75m buying up shares from January and would also pay out a 1p-a-share dividend to shareholders.
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Meanwhile, pressure has come from supply chains, with Currys saying it has seen challenging conditions.
Baldock added: "Since the start of the Covid pandemic in early 2020 there has been a shortage of high-demand tech products or products where manufacturing has been limited due to pandemic restrictions."
urry...
CurrysIrelandOmicron


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Iperformance, so despite having hit a few more red carpets than usual lately (side note: I decline at least four event invitations each week, so it could be a lot worse), the opening night of Kooza was one launch I simply couldn't pass up! The show was incredible, pairing a hauntingly beautiful narrative with a variety of acts that took my breath away - and even had me white knuckling the seat in front of me, in the case of the famous Wheel of Death!
wow so exciting and this look is just out of the world.


HTC Nexus 9 specs and launch details leaked
As far as accessories are concerned, the tablet is said to have a strong and beautiful Magic Cover leather that comes in a various of colors (mainly natural and black), Magic Cover TPU (with colors like coral, mint indigo, amethyst, limestone, and black) and will also include a Keyboard Folio.
Other features previously leaked about the tablet include a 1.6main camera. The device will also offer NFC, 4GB RAM, GPS and MDM9x25 modem (the LTE version).
https://www.inferse.com/17023/att-htc-one-m8-android-4-4-3-update-now-available/
HTC tablet ref docs hint, Android L or LMP stands for “Lemon Meringue Pie”
Even though NVIDIA did not give the specifics about the devices that will get the 64-bit Tegra K1 processor, it said that some of its partners will definitely benefit from this processor as well as the Android L, which is already being developed. With HTC and Google being among the main partners of NVDIA, the Nexus 9 is touted as one of the devices that will have the “”, which are features that are synonymous with the 64-bit Tegra K1 chip, according to NVIDIA.
www.inferse.com/16901/htc-one-m8-vs-lumia-930/
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Coach Griggs Focuses on More than Xs and Os
“I think the kids here are great,” Coach Griggs says after his first few weeks with the team. “Right now, I’m trying to get to know the players on an individual basis and let them know what I’m seeing as a coach with fresh eyes. I’m really just making them aware that I see what they are doing. I want engaged and focused to be able to execute a play, and how aware they are of the importance of being in the right position, even before the ball is hiked. Mental focus and awareness are huge parts of the game and can get you through challenges when things get tough.”
Coach Griggs joins the team of high school staff coaches that includes John Alerich, Vincent Gualtieri, Jim Hastings, Zach Metkler, John Moritz, Brandon Rios, Mark Shemanski, and Bob Sporio.
What Got Griggs to the NFL
Coach Griggs’ own journey to the NFL is an unlikely, yet inspirational story itself. It begins in New Jersey, where he grew up, and was a member of his high school’s football team. He played during his sophomore and junior years, but the school district went on strike in his senior year, so the team only played about four games that season. Apparently, that was enough to draw the attention of scouts from Villanova University. But after his junior year of college, the university cut its football program. But destiny seemed to have a mind of its own as The Ohio State University recruited him to play for the Buckeyes.
“I didn’t even start until the end of the year,” recalls Coach Griggs of his time with the Buckeyes. “I thought, ‘Great, that was nice and now it’s over,’ and was prepared to move on.”
But then, in the offseason, his teammate mentioned that Pro Day that was being held on The Ohio State campus.
“The first coach I saw when I opened the door was one of ours, and he asked me what I was doing there,” recalls Coach Griggs. “He told me, Sorry, this is just for All-Americans and kids the scouts have asked to see.’ But another one of our coaches looked at me and said I could run (the timed trials) and put my name on the list. No one was really looking that closely at me, but a couple of scouts did ask if I had film. I told them I didn’t have any film. Then, they asked if I had an agent. Of course, I didn’t!
When my roommate told me a few days later that someone named “Dick Vernon,” or something like that, had called, I said, “Do you mean Dick Vermeil (former coach of the Eagles)?”
It was. For the next four years Coach Griggs played for Philadelphia and, in 1985, was selected as the team’s Man of the Year for his work in the community. He spent another three years with the Cleveland Browns before retiring.
On to Pittsburgh…
Director of Athletics Mike O’Brien says of Coach Griggs: “He is a tremendous guy annts to know who you are as a person and he relates exceptionally well to everyone. He’s just a very positive person to be around.”
Coach Loughran adds, “Before you can become a good coach, you have to be able to make personal connections and relate to the kids. I’m happy that he’s been able to do that in the short time he’s been with us.”


rewarded by her career choice.She greatly enjoys the wide variety of cases she sees in general practice, as well ao well.
More about Dr. Kirsten Moore, D.V.M.
Dr.Moore has lived in SE Michigan her whole life. She grew up with dogs, a cat, hamsters and a bunny. She worked at a pet store and was very involved in 4-H and canine obedience training. She had many friends with horses and was finally able to enjoy becoming a horse owner when her daughter was growing up.
Dr.Moore and her husband have two grown children. Their son and daughter-in-law live in Georgia. Dr. Moore is fortunate enough to share a beautiful home with her daughter, son-in-law and grandson Ethan, born in late 2015. Dr. Moore and her family enjoy playing with Ethan, as well as watching their surfing the internet, bowling, ping pong, board games and of course they enjoy playing with their numerous cats and walking their two dogs.
Over the years, Dr. Moore has enjoyed many different mixed breed cats, as well as various breeds of dog including; Dachshunds, retriever and shepherd mixes, an English Pointer, a Finnish Spitz and three Papillons. She currently has two lovely tortie cats (mom and daughter), but also enjoys her daughter’s English Setter, Golden Retriever and two more household cats. Dr. Moore doesn’t think she could ever choose a favorite pet, because each one is special.
Jessica started at the clinic in 2016. She came with previous veterinary and customer service experience. She owns and operates a small hobby farm, which includes, 3 dogs, 2 cats, 3 horses, 20 chickens, 2 ducks and one rabbit. She hopes to add more soon. Her husband works with horses as a farrier/blacksmith. Jess loves all things animals, is a huge foodie and enjoys traveling – sheshe visited family.
Vicky worked at the clinic for eleven years as a receptionist until 2014 when she became the Practice Manager. After earnshe worked in resort management, until deciding to follow her true love of working with animals and their people. Besides being a great receptionist, Vicky also does most of the clinic’s payroll and bookkeeping. She has a soft spot for shelter and rescue pets, owning four, two dogs Rocky and Louie, and two cats Siamon and Jax. Vicky is an excellent cook,nd touring the countryside by motorcycle. </s>
DBx: Again, I am increasingly struck and distressed by how much of modern economics – economics since the time of J.M. Keynes (1883-1946) – is an elaborate practice of assuring the man-in-the-street that his economically untutored sense of the way economies work is, in fact, spot-on correct. The man-in-the-street, seeing only what Deirdre McCloskey calls the “first act” of economic phenomena, concludes that the first act is all that there is.
If modern astronomy were akin to modern economics, a great deal of modern astronomy (although by no means all of it) would be devoted to explaining – with the use of fantastic mathematics and elaborate empirical demonstrations – that the man-in-the-street has been correct all along in believing that the suna stationary earOffice Phone Number 704-635-8273. </s> News: New Left 4 Dead prequel, Splinter Cell: Blacklist and Pikman 2 announced, Vivendi selling their shares in Activision Blizzard, control your xbox with your smart phone, Zombie U announced for Wii U, analysts’ say 360 still has a few years, 38 studios files for bankruptcy.
We finished up with some E3 prediction recaps. </s> Christmas is a joyous time of year full of baubles, handmade ornaments, family traditions and good food. My sisters and I had matching handknit Christmas Stockings made by Mrs. Katterman. Hanging the stocking on the mantelpiece was a special


Young ICU nurse Whitney Kirkpatrick’s lifdoor looking for her boyfriend of nine months, Gregory Thomas. He is not the wealthy, successful oil executive he claims to be. Instead, Gregory has spun an elaborate web of lies — and he’s been funding his extravagant lifestyle with Whitney’s money. With the support of family and friends, Whitney finds the strength to move on and to educate others about identity theft and white-collar crime.


Barcelona Confirm Pedri Injury Blow
The 18-year-old went off injured during the 2-0 win in La Liga at the weekend
Barca have now revealed the full extent of Pedri's injury, with a statement on their tored."
Test confirm that @Pedri is out after pulling the soleus muscle in his left leg pic.twitter.com/WLtE5Zwe29
Barcelona’s upcoming fixtures
They have hauled themselves back into the Liga title race and now trail leaders Atletico Madrid by only two points, albeit having played two games more.
LaLiga would appear to be Barcecond leg in the French capital is on March 10, meaning Pedri would have a little over a week to shake off the problem if he is to face Mauricio Pochettino’s side.
Copa del Rey Barcelona La Liga Pedri


Asirelli P. Horn clauses form of logic: algebraic static semantics of programs. Internal note IEI-B82-23, 1982.
(English) We give an algebraic static semantics far logic programs in the Horn Clauses Form of Logic. The semantics is expressed by means of set expressions derived from the static analysis of the clausal definition of programs. Static semantics is derived from a case analysis of the semantics of clauses as defined according to fixpoint semantics and the operational semantics associated to hyperresolution and instantiation rules, so that consistency between the various semantics is ensured. For each predicate a set expression is generated (pattern) which can be, in the end, computed by computing the union and intersection of sets in the set expression. A notation is introduced to represent sets of data, built by means of function, i.e data constructors. We have considered a logic language with monadic predicates and functions symbols, as a first approach to the study of static semantics for logic programs in general.


Noosa Coastguard will tow you in no problem from 50 miles out to sea in a gale .....$50 a year......only joking you have to have 2 engines in South Africa to go offshore...
Still two mouths to feed, not bad if you can afford it...
I second, go with one power unit. redundancy is great and makes total sense in certain applications or where major amounts of power is needed. Look at single prop airplanes for instance, there are thousands flying around without worry and if they fails the consequences are much greater than in a boat!
You can get off your high horse about me putting some "poor sucker" at risk rescuing me, it's never happened. Further more, I am the "poor sucker" who drives the rescue boat...
poor ol Jack Savage would not be happy with the mess you plan on doing to his nice boat...just restore it as it was made, they are an excellent boat as it is...
She is a nice boat, well worth looking after and restoring. I am sure that you will get a nice engine at the right price too with a bit of "scouting"...


Beckenham can be considered Bromley'n't much development going on in Beckenham.
While looking on the map, Beckenham might look a bit isolated from Central London. There is however the fact that the district is connected to Central London to as many as nine railway stations and a number of major roads, which makes the area a rather attractive business location. Notable landmarks in the area include the famous Chinese Garage, as well as the celebrated Kesley Park, which is part of the beautiful Kesley Estate.


Designed to perfection, these shoes are appropriate for daily wear with any outfit of your choice. Pair it with a nice, crisp shirt & formal trousers to achieve a desirable look.


Reviews Routers & Bridges Belkin F5D8632-4 - wireless router - DSL - 802.11b/g/n (draft) - desktop : "Belting Belkin Router!
Belkin's N1 Vision Wireless Router with integrated Modem shows relevant network information on its interactive network display. You can quickly view y...
Review of ""
"Belting Belkin Router!"
Th…
It also means that my dad can use my connection from his house next door if his net ever goes down (although he'd need me to give him the security password which I shall talk about below) as our network shows up on his laptop when he's reconnecting to his home network haha. This also reinforces the security aspect that I will address in a few moments as you don't know who else can pick up your network on a wireless search within your area!d…
• 64-bit WEP. This is similar to 128-bit WEP, but not as powerful-bit WEP."
Nice detailed review. I gave up on Belkin stuff after a couple of terrible wireless access points and network cards, but looks like they may have got better!


It's nearly 41 years ago to the day, Malaysia witnessed a landmark event in its history. The event is known as the May the 13th Incident or the Sino-Malay Sectarian violence of Kuala Lumpur. Malaysia after getting its independence in 1963, Kenyans keep note of that year, saw increasing economic inequality between the Chinese and the indigenous Malay people. The newly formed Malaysia included Malaya (the Malaysian Peninsular), Singapore, North Borneo and Sarawak. However in 1964, race riots brought about by the perceived inequality between the wealthy Chinese and the poorer Malay. Most Malay were angered by the fact that the government was willing to look the other side placating Chinese interests at the expense of the indigenous Malay. These issues persisted up till the elections of 1969.
The government that took over from here saw that they could no longer placate Chinese interest at the expense of the Malays and they responded by implementing affirmative action policies that were referred to as the New Economic Policy (NEP) of 1969. Some saw this measure to be draconian as it further entrenched very strong Malay privileges into the constitution. Years on the affirmative action measures have failed to bear any tangible benefits for the Malay as the inequality still exists. It is so bad that the national statisitcs office does not release official income statistics due to their sensitivity. This has lead to the government lead by the prime minister Najib Razak unveiling a New Economic Model.
In Kenya, we face the similar situation. We have the choice of staying on the current path and facing similar tensions in 2012 or we have the chance at least to take the monumental leap and create a new constitution. However, there is a great deal of noise over this process and as each day passes the chances of getting a new contitution gets slimmer and slimmer as the noise gets louder and louder.
I am really angered by the Church's position on both the Kadhi courts and the abortion debate. They completely lack perspective and someone can serioulsy question their collective memory and compassion for the country. In terms of Kadhi courts, I really don't get how for people who begin their National anthem by 'Oh God of all creation" can refer to the kadhi situation as something that will favour Islam over Christianity.  Clearly the anthem already shows a national acceptance of biblical creationism and yet the church conveniently overlooks this fact when arguing against the kadhi courts issue. On the note of abortion, the constitution clearly states that abortion would only be legal, if a medical practitioner deems it to be necessary if the mothers life is in danger. I really don't understand what is wrong with this, would they rather a wife, sister, mother or daughter lose their life in order to uphold biblical teachings? If their relatives were in the same situation, would they rather lose their relatives and maybe also lose the child just to uphold the bible?.
It has been about two weeks since the last article on inflation measurement in Kenya. Since then I have arrived in the land that inspires the blog and gotten more views on the matter. The content of the article is clearly vindicated by the general consensus from Bankers on their unwillingness to reduce their interest rates. It was expected that with lower inflation figures, there would have been downward pressure on bank lending rates as their real interest rates fall.
Peering further on this issue, there will be double pressure to disregard inflation and make economic growth the key issue for the Central Bank. Nowhere in the draft does it mandate the Central Bank to actively control inflation. In the current Central Bank Act, it clearly states that "The principal object of the bank shall be to formulate and implement monetary policy directed to achieving and maintaining stability in the general level of prices". In the current draft, the Central Bank is mandated to control amongst others the value of the currency of the republic, issue notes and coins, act as a banker and financial adviser of the government and conduct monetary policy aimed at achieving growth. The focus here then shifts from controlling inflation to stimulating growth.
The Central Bank as well as the National Assembly will thus have a focus on growth instead of having a system of balances where the Central Bank focuses on inflation and the National Assembly focuses on growth. Growth with unbridled inflation is merely cosmetic as in the long run, real growth will be stagnant. This debate is rife in South Africa where some sections of the African National Congress (ANC) contend against the Reserve Banks stance on inflation targeting. The results are clear as South Africa has managed to steer away from a meltdown of their financial system and has avoided the disastrous unemployment that their peers in the middle income countries category have suffered.


Joan C. Gaudette, 83, of Manahawkin, NJ passed on Sunday, December 2, 2018. Born in Williamstown, PA to her late parents, George and Ethel Adams, she is a long time resident of Manahawkin, NJ. Surviving are her beloved husband, David Gaudette.


In the gospels, Jesus told his disciples, ", NKJV). Christ's warning to them and us is "Without hope in me, multitudes of people are literally going to die of fright!"
Jesus provided the ultimate example of this kind"…Father, into Your hands I commit My spirit…
You may wonder, "But didn't Jesus say he had the power?" (See John 10:18.) Since he had the power to "take up his life again," why did he resign it into God's hand to be preserved?The answer is obvious. Jesus did it to set an example for all of his sheep to follow!
If we are being asked to trust our lives to someone, then we have to know that this someone has the power to keep us from all danger, threats and violence. We must have the confidence of the apostle Paul as he writes, "…


Carnage in Catalunya
In Racing News 0 comment
The MotoGP weekend in Catalunya gave us a lot of chaos and carnage. 29 riders failed to finish the race across all 3 classes with 11 MotoGP riders crashing out and only 13 taking the chequered flag. This was an unprecedented level of attrition and it will go down in history as being one of the race weekends with the least finishers.
Moto2 'only' had 6 non-finishers which was relatively low compared to Moto3's 12 and MotoGP's 11. Remy Gardener was caught up in a first lap incident with Lorenzo Baldassarri, who he accused of forcing him into a crash. Whether these claims were substantiated or not, championship leader Baldassarri later crashed out and lost his lead in the championship. The king of crashes, Sam Lowes managed to stay on his bike for a change and see the chequered flag.
The Moto3 race was extremely chaotic. 3 different riders abandoned the race whilst leading for starters. There was a very clumsy move from Can Oncu, missing his breaking into turn 4 and bowling 4 other riders out of the race. The infamous turn 10 also gave us some incidents, most notably on the first lap when Tatsuki Suzuki crashed while challenging for the lead. Later on Rodrigo took Binder out and later on Toba was also taken out after being clipped. A bizarre incident saw Filip Salac retire after both his visor and screen simultaneously fell off while heading down into turn 10. Marcos Ramirez held on to win after a race full of jostling and action.
It truly was one of the most bizarre MotoGP races for a long time. Lap one saw wildcard Bradley Smith take out his team-mate Aleix Espargaro heading into turn 10. Espargaro appeared to be severely hurt and had to be carried off his bike. Karel Abraham also crashed on the opening lap in an unrelated incident. The most talked about move of the race was when Lorenzo, who was finally finding some speed on the Honda, made a late move breaking into turn 10 , lost the front and hit Dovisioso, which set off a chain reaction of crashes culminating in Yamaha team-mates Vinales & Rossi also retiring after that incident. That move pretty much ended the race as it allowed Marc Marquez plenty of breathing room to ride away unchallenged after his nearest 4 rivals were out.
Rins had a bucking bronco moment on his bike while defending 2nd place into turn 1. He managed to stay on and recover to a very respectable 4th after what looked like a sure crash. Bagnaia continued his miserable run of form with a 4th consecutive DNF whilst Morbidelli suffered his second big crash of the weekend whilst running in the top 10.
On a slightly different note, it must be noted that Marc won all 3 races! How is this so? Well, Marcos Ramirez won in Moto3. Alex Marquez won in Moto2 with brother Marc taking the victory in MotoGP. Mark my words, this won't be the last time that the Marquez brothers do the double in a race weekend. It was younger brother Alex's 3rd victory in a row which moved him top of the championship standings. Marquez tripled his lead in front after his main rivals crashed and Canet also managed to extend his lead in the Moto3 championship with his 2nd place and retirements from those close to him.
We now have another 2 weeks to catch our breath before the action is back in 2 weeks time at the classic Assen track.
Motogp, Motogp barcelona, Motogp catalunya
FIM Helmet Homologation in MotoGP Causes Confusion
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Stage 6 Summary July 12, 2019 June 15, 2019


The chair takes its name from the design concept in which the unnecessary branches are removed, leaving the usable frame for support of the backrest and seat. Making the pure structure transparent.


Saudi Immigration Officials extended amnesty period till 15th November 2017, Earlier it was told to illegals to benefit before 15th October 2017, Now it is confirmed that it is extended again for one month.
- Indian Embassy in Riyadh also announced "Saudi Government has granted another precious chance to remaining undocumented Indian nationals in KSA to avail of the Amnesty 2017. This is the last chance, valid till mid November 2017, for eligible persons desirous of returning to India, without paying any fine or facing ban on return to KSA in future".
- These amnesty is available for all nationals for required help call your Embassy. This procedure let you take safely final exit with out any penalty or fine or ban from re entering in Saudi Arabia.


Home News Mortgage Advisor vs. Property Agent: What’s the Difference?
Buying a home is an exciting time, however it’s undeniable hen it feels a little overwhelming.
A Mortgage advisor is there to help you navigate the financial aspect of buying a home.
With so many loan productscan be tough (hint: it ). At HPartners, we assist our clients to find the right mortgage product, considering the terms of the loan and assessing whether the loan is flexible enough to adapt to life changes.
What are the benefits of a Mortgage Advisor?
Need help finding the right loan? That’s where we can help you, get in touch with one of our Mortgage Advisors today.
A Buying Agent will help you with the entire buying process, apart from finding the right mortgage. From searching for the right property, attending inspections on your behalf, liaising with real estate agents and arranging pre-settlement processes (such as building and pest inspections), a Buying Agent will take the stress out of buying a home by handling the run-around for you.
Buy your home at the right price with the assistance of a skilled negotiatorWhat does a Property Selling Agent do?
If you are looking to sell your house, a Property Selling Agent will help you to manage the selling process. From assessing how much your property is worth, advising on how to add value to your home (within your budget) and pairing you with a real estate agent who is likely to get you the best price, a Selling Agent will be with you at every step of the way.
At HPartners, we recommend Property Chase to our clients who are looking for additional home buying or selling assistance.
Who are Property Chase? They are a boutique buyers agency that has specialised in buying property throughout Brisbane, Toowoomba and surrounding areas for over 14 years. Find out more about what Property Chase does here.


From these, we can calculate a set of n ‘pseudo-values’:
Partial estimates and pseudo-values for the tiger data are shown in the table below:
If the bias is proportional to 1/n, the bias will cancel out, and the mean of the pseudo-values (35 for the tiger data) will be an unbiased estimator of S.
You may have noticed that the species or animals which drop out are those which were detected on only one occasion, called "singletons". Burnham and Overton (1979) showed that the jackknife estimator can be calculated from the number of singletons, \( f_1 \):
\[ S^*=S_{obs}+\frac{n-1}{n} f_1 \]
The tiger data has 10 animals caught only once, \(f_1 = 10\), so \(S^* = 35 \).
The ‘drop-one-out’ method is a first-order jackknife; we can drop out more than one occasion.
If we drop out k occasions at a time, we can calculate a kth-order jackknife estimate. The details can get quite complex, but Burnham and Overton (1979) provide equations making use of frequency of capture. For example, the second-order jackknife estimate is
\[ S_2^*=S_{obs}+\frac{2n-3}{n} f_1 - \frac{(n-2)^2}{n(n-1)} f_2 \]
where \(f_2\) is the number of animals or species recorded twice; \(f_2\) = 6 for the tiger data, so we have
\[S_2^*=26+\frac{2×10-3}{10} 10 - \frac{(10-2)^2}{10(10-1)} 6=26+17-4.2667=38.733\]
Since a whole series of jackknife estimates are available, which should we use?
Higher-order jackknives have smaller bias, but they also have larger standard errors, so there is a trade-off between bias and precision. Burnham and Overton (1979) provide a stopping rule which compares the difference between successive jackknives with the standard error, which is implemented in Program CAPTURE and the R code in the wiqid package.
CAPTURE and wiqid  add a further refinement: they allow for interpolation between the two best jackknife estimates. So their estimate of the number of tigers in the Kanha Reserve is 33.32, between the values from the first-order jackknife and "zero-order" jackknife, which is just \(S_{obs} \).
Karanth, K. U., Nichols, J. D., Kumar, N. S., Link, W. A., & Hines, J. E. (2004) Tigers and their prey: Predicting carnivore densities from prey abundance. Proceedings of the National Academy of Sciences, 101, 4854-4858.