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Congress Is on a Roll, But Will It Last?
Congress has recently passed several consequential pieces of legislation including a highway spending bill and an authorization to raise the federal debt ceiling. However, WSJ's Jerry Seib questions if the collaboration will last into 2016. Photo:AP
This transcript has been automatically generated and may not be 100% accurate.
... Congress finally seems to be getting some things done these days in the last few weeks alone is passed a bill to raise the federal debt ceiling as highway bill defense spending bill it's on the verge of passing a bill that would fund the government for the rest of the fiscal year ... and would in the process lift a decades old embargo on oil exports ... all this raises the obvious question of when was this mean that Congress is gonna roll into twenty sixteen prepared to get some things that ... they can force me the answer is probably not for a variety of reasons ... reason number one is a Paul Ryan the new house speaker who's been helping accomplish some these recent things ... is getting a bit of a pass from the Conservatives in his caucus right now they're letting go on some things that give them some successes that come in October continued to do that and to the new year ... be well Nancy Poloz see that Democratic leaders getting some pushback from her members ... they think that for example in the state budget deal which they agreed ... to lifting the embargo on oil exports isn't enough in return so she's getting to push back as well ... third I don't think the White House is interested in having a big agenda in Congress next year the president alone the number of things you'd like to do in most of those who prefer to do on his own driving going through the Congressional meat grinder ... and finally it's a political year next year everybody in the house ... thirty the Senate in addition to the presidential candidates will be running for reelection ... that's not a good environment for getting things done ... so even the trade bill the Pacific trade bill which a lot of people thought might be the one big accomplishment of twenty sixteen ... Republican leaders in Congress are now saying ... Allah says way to do was to wait till after the twenty sixteen election to approve that ... so I think the progress for saying now may turn out to be a bit of a Mirage ...


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The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii.
IMPORTANCE The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma.
Apicomplexans are a phylum of obligate intracellular parasites that cause a significant disease burden in humans and animals. The most successful of these is Toxoplasma gondii, which infects approximately one-third of the human population globally and is a threat to immunocompromised individuals and neonates (1). The most notorious apicomplexan parasite is Plasmodium falciparum, which is the causative agent of malaria, which claims ~1 million lives annually (2). A hallmark of apicomplexans is the conservation of a number of unique organelles that serve critical roles in parasite biology and thus are attractive targets for the development of novel therapeutic interventions.
One of these unique organelles is the inner membrane complex (IMC), which consists of two distinct elements: a series of flattened membrane sacs called alveoli underlying the parasite plasma membrane and a supporting rigid cytoskeletal network of intermediate filaments (3, 4). In Toxoplasma species, the alveolar sacs of the IMC are arranged as three rows of fused rectangular membrane plates, sutured together like a quilt, and capped by a single large alveolar plate at the apical end of the parasite (5). While Plasmodium merozoites appear to possess only a single alveolar sac, distinct segmented plates that are sutured together are visible in Plasmodium gametocytes (6, 7). In apicomplexans, the alveolar sacs and underlying cytoskeleton are layered on top of subpellicular microtubules emanating from an apical microtubule-organizing center (8).
The IMC has important functions in parasite motility, host cell invasion, and intracellular replication. The outer leaflet of the IMC membrane acts as the anchor for the actin-myosin motor that powers parasite gliding and invasion (9, 10). In addition, the IMC serves as the structural scaffold for the formation of daughter cells within the mother during asexual reproduction. In Toxoplasma, two daughter buds arise from the maternal parasite, a process called endodyogeny, while in Plasmodium, multiple daughters are formed, termed merogony (11). The nascent IMC appears at the onset of bud formation, and replicated organelles are packaged within this compartment as the buds elongate. The daughter buds complete their maturation by adopting the maternal plasma membrane.
Despite the crucial roles of the IMC, the protein composition of this cellular compartment is still largely unknown. The majority of IMC proteins that have been identified to date belong to a family of articulin-like proteins called alveolins, which are characterized by conserved repeat motifs flanked by variable sequences (12, 13). These proteins are one of the distinguishing hallmarks of the Alveolata infrakingdom that includes apicomplexans, dinoflagellates, and ciliates. In apicomplexans, the alveolins form part of the cytoskeletal network that provides structural stability to the IMC (8).
Even fewer proteins have been localized to the alveolar subcompartment of the IMC. Some of these include components of the parasite actin-myosin motor, such as Gap40, Gap45, and Gap50, which form a complex that recruits the myosin machinery, including MyoA and MLC1, to the IMC (14–16). Another subset of proteins in the membrane sacs is the IMC subcompartment proteins (ISP1, -2, -3, and -4), which are tethered to the membrane bilayers via posttranslational acylations (17, 18). In T. gondii, each ISP localizes to distinct subcompartments of the IMC, which may be delineated by specific subsets of alveoli (17, 18). For example, ISP1 is found exclusively in the apical cap, while ISP2 and -4 are present in the center of the IMC but not the cap or base of the parasite. ISP3, on the other hand, localizes to the center and the base of the IMC. Although their precise functions are unknown, the ISPs appear to be involved in daughter bud formation, as disruption of ISP2 leads to a dysregulation of endodyogeny (17).
The paucity of characterized IMC proteins led us to identify new protein constituents of this organelle. We utilized a technique named BioID that has been developed to uncover novel proteins and elucidate complex networks of protein-protein interactions (19). The BioID approach relies on the proximity-dependent biotinylation of interacting partners and proximal proteins by a promiscuous biotin ligase, BirA*, that is fused to the protein of interest. BirA* contains a mutation that abolishes substrate specificity, allowing it to label proteins interacting with or proximal to the ligase-target protein fusion within its native environment. The biotinylated partners or near neighbors can then be affinity purified for identification by mass spectrometry. The method has proved particularly valuable for cytoskeletal or membrane-bound bait proteins, which are solubilized only under harsh detergent conditions that often disrupt protein-protein interactions in standard immunoprecipitation assays. A further advantage of BioID is the ability to capture transient or weak interactions that could be missed by other techniques. This approach has been successfully used to uncover new components of the nuclear lamina, nuclear pore, and centrioles in mammalian cells, as well as novel bilobe proteins in Trypanosoma brucei (19–22).
We adapted the BioID technique for T. gondii and, as a proof of principle, used the IMC membrane protein ISP3 as bait to identify novel IMC proteins. We demonstrate that the ISP3-BirA* fusion localizes properly to the IMC, is catalytically active, and labels previously described IMC proteins as well as novel targets. A number of these targets were verified by epitope tagging, thereby validating our approach. We further characterized two of these proteins by gene disruption and show that one of them appears to be essential in Toxoplasma. To evaluate labeling in a different suborganellar compartment of the IMC, we then applied the BioID approach to a novel IMC cytoskeletal network protein that is restricted to the apical cap. An array of known and novel targets was again identified, of which a selection of new targets were verified by localization. Of particular interest, we uncovered a group of proteins that localize to the transverse and longitudinal sutures of the rectangular alveolar plates and demonstrate that these novel constituents associate with both the membrane and cytoskeletal elements of the IMC. Together, these studies establish a methodology for identifying novel proteins in Toxoplasma subcellular compartments, substantially increase the repertoire of known IMC proteins, and provide a foundation for evaluating protein-protein interactions in the IMC.
ISP3-BirA* biotinylates proteins in the IMC.To identify novel IMC components by BioID, we generated a construct containing ISP3 fused to BirA* with a C-terminal hemagglutinin (HA) tag, driven from the ISP3 promoter (Fig. 1A). The ISP3-BirA* fusion was integrated into the genome of RHΔhpt (parental) parasites, and localization of the fusion was assessed by immunofluorescence assay (IFA). As expected, we observed staining in the central and posterior subcompartments of the IMC but not in the apical cap (Fig. 1B), a pattern consistent with endogenous ISP3 (17). Correct IMC localization was confirmed by colocalization with IMC3 (see Fig. S1A in the supplemental material).
Figure S1
Copyright © 2015 Chen et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
To determine if ISP3-BirA* could label proximal proteins in the IMC, we grew parasites in medium supplemented with biotin and stained for biotinylated proteins using fluorophore-conjugated streptavidin. Wild-type T. gondii parasites have endogenously biotinylated proteins in the apicoplast (and to a lesser degree in the mitochondria) that are labeled by streptavidin, thus representing the substantial background of this method in Toxoplasma compared with other cell types (Fig. 1B, top) (23). In ISP3-BirA*-expressing parasites, we also detected robust streptavidin staining in the periphery that colocalized with ISP3-BirA*, indicating that the biotin ligase fusion was active and labeled proteins in the IMC (Fig. 1B, middle). Surprisingly, while ISP3-BirA* was enriched in daughter buds during endodyogeny (see Fig. S1A), lower but detectable amounts of biotinylation were seen in daughter buds (Fig. 1B, bottom).
We then assessed the labeling of T. gondii proteins by ISP3-BirA* in whole-cell lysates by Western blotting with a streptavidin-horseradish peroxidase (HRP) probe. Although background biotinylated proteins were detectable in parental lysates when the blot was overexposed (see Fig. S1B), there was a significant increase in biotinylated proteins in lysates of ISP3-BirA* parasites, and only when biotin was present in the growth medium (Fig. 1C). These labeled proteins were unique to ISP3-BirA* parasites and did not appear to correspond to background proteins from the parental line. Collectively, these data demonstrate that ISP3-BirA* is active and biotinylates proteins at the parasite periphery.
Identification of novel IMC proteins by BioID.To identify targets labeled by ISP3-BirA*, we affinity purified biotinylated proteins from parasite lysates for MuDPIT mass spectrometric analysis. Biotinylated proteins from both parental and ISP3-BirA* lysates were significantly enriched using streptavidin-conjugated magnetic beads (Fig. 2A). Proteins identified by mass spectrometry that were unique to or significantly enriched in ISP3-BirA* samples were scored as hits based on the number of identified spectra and unique peptides (see Table S1 in the supplemental material). The highest-scoring protein in our data set was the ISP3 bait, which is in agreement with previous BioID studies in which the bait-BirA* fusion biotinylated itself and was the top hit (19–22). Importantly, some of the highest-scoring hits in our data set included proteins known to associate with IMC membranes: MLC1, GAP45, GAP50, HSP20, Rab11b, and ISP1 (14–17, 24, 25). In addition, proteins in the IMC cytoskeletal network, such as IMCs 1, 10, and 14, were enriched in our data set (4, 12). These data indicate that ISP3-BirA* biotinylates proteins in both the membrane sacs and the underlying network of the IMC.
Table S1
Copyright © 2015 Chen et al.
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Next, we utilized gene expression data to filter hits from our BioID data set for further investigation (summarized in Table 1). IMC proteins are synthesized de novo during endodyogeny, with their gene expression peaking during each round of daughter bud formation at the S and/or M phase (26). We selected a group of hypothetical proteins with this signature cyclical expression pattern similar to known IMC proteins (see Fig. S2A in the supplemental material) and determined their localization in parasites via epitope tagging at their C termini.
Figure S2
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This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Characterization of novel IMC proteins identified by BioID.Among our ISP3-BirA* hits, we epitope-tagged six proteins that shared the same localization in parasites as ISP3: TgGT1_286580 (here referred to as IMC17), TgGT1_295360 (IMC18), TgGT_217510 (IMC19), TgGT1_271930 (IMC20), TgGT1_232030 (IMC21), and TgGT1_313380 (Table 1). One of these proteins, TgGT1_313380, had been independently identified from a purification of the MORN1 basal complex, designated ILP1, and characterized elsewhere (27). These proteins have no known functions (Table 1), although IMC17 was annotated as a putative lysophospholipase based on an α/β hydrolase fold predicted by BLAST analysis (28). We localized these hits to the central and basal subcompartments of the IMC but not in the apical cap (Fig. 2B), consistent with proximal biotinylation by ISP3-BirA*. In agreement with the preferential labeling of the maternal IMC by the ISP3-BirA fusion (Fig. 1B, bottom), IMCs 17/18/20 appear to be deposited onto the maternal IMC after endodyogeny and were not detected in daughter buds (see Fig. S2B in the supplemental material). In contrast, IMCs 19/21 were detected in both the maternal IMC and nascent daughter buds, as shown by colocalization with IMC3 during endodyogeny (see Fig. S2C).
Next, we used detergent extraction assays to determine whether these IMC proteins are associated with the membrane sacs or the underlying cytoskeleton (Fig. 2C). Proteins embedded in the IMC network (such as the alveolins) are resistant to extraction by the nonionic detergent Triton X-100, whereas proteins attached to the membrane sacs (such as the ISPs) are solubilized under these conditions (4). IMCs 18/19/20 were entirely resistant to extraction by Triton X-100, indicating these proteins are anchored into the network of the IMC. IMC17 was partially soluble in these extraction assays, suggesting an association with the underlying cytoskeleton that is not as firm as these other novel IMC proteins or alveolins. In contrast, IMC21 appears to localize to the membrane sacs, as it was solubilized in detergent. This result is supported by the presence of a predicted palmitoylation site (a cysteine residue at position 2) that may mediate tethering to the IMC membranes. The identification of hits from both the membrane sacs and the underlying cytoskeleton indicates that ISP3-BirA* labels proteins in both of these IMC suborganellar compartments.
In addition to these proteins in the central/posterior regions of the IMC, we also localized two proteins to the apical cap of the parasite: TgGT1_311840 and TgGT1_250820. TgGT1_311840 was one of the highest-scoring hits on our data set. TgGT1_250820, on the other hand, had a much lower rank but was chosen for analysis based on its IMC-like cyclical expression pattern (see Fig. S2A). Staining of TgGT1_311840 and TgGT1_250820 overlapped perfectly with that of ISP1 in both mature and dividing parasites (Fig. 2D and Fig. S2C in the supplemental material, respectively), demonstrating these proteins also localize to the nascent IMC during replication. We subsequently designated TgGT1_311840 and TgGT1_250820 as apical cap protein 1 (AC1) and AC2. Consistent with extraction data from other ISP3 BioID hits, we found these two apical cap proteins reside in separate subcompartments of the IMC: AC1 associates with the membrane sacs (consistent with a predicted palmitoylation site at Cys-8), while AC2 is anchored into the cytoskeletal network (Fig. 2E). Two additional proteins from the ISP3 BioID data set were localized to a unique compartment of the IMC and are discussed below. As expected, not all proteins chosen for endogenous tagging localized to the IMC: three proteins, TgGT1_240570, TgGT1_320740, and TgGT1_232340, stained punctate vesicular structures throughout the parasite (see Table S1 and Fig. S2D in the supplemental material).
Of these new IMC proteins that we localized, we chose to functionally characterize IMC18 and ILP1 by gene disruption using a combinatorial epitope-tagging/Cre-lox strategy (see the approach in Fig. S3A in the supplemental material). This strategy is a modification of the Cre-lox gene excision system previously established for Toxoplasma (29). We endogenously tagged both the target gene and the upstream gene with an epitope tag followed downstream by an loxP site, thereby flanking the target gene with loxP sites. We performed this genetic engineering in DiCre-ku80::KillerRedfloxYFP parasites that express inducible Cre recombinase and also activate yellow fluorescent protein (YFP) expression upon induction with rapamycin (30). In both target strains, we observed the loss of the 3× HA-tagged target protein at the IMC as well as concomitant expression of YFP upon induction of Cre activity (Fig. 3A and C). Disruption of IMC18 did not have any observable gross effects on parasite morphology, as assessed by IFA (Fig. 3A, bottom), or parasite growth, as measured by plaque assays (data not shown). In contrast, Δilp1 vacuoles were highly disordered and contained morphologically abnormal parasites that appeared enlarged and/or malformed (Fig. 3C), defects that were reminiscent of ILP1-overexpressing parasites (27). In addition, we observed a general mislocalization of organelles, such as mitochondria and apicoplasts, within parasites lacking ILP1 (Fig. 3D). We were unable to successfully clone an ILP1-null, YFP-positive parasite after repeated rounds of limiting dilution from knockout-positive pools, suggesting ablation of this gene is lethal.
Figure S3
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This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
AC2-BirA* biotinylates proteins in the apical cap of T. gondii.We also wanted to explore whether the BioID approach was applicable to the cytoskeletal network of the IMC. We simultaneously chose to focus on proteins in the apical cap, and thus we fused BirA* to the cytoskeletal apical cap protein AC2 (Fig. 4A). In this case, we engineered the BirA* sequence into a vector for endogenous tagging and integrated it into the AC2 locus by homologous recombination. The resulting AC2-BirA* fusion localized correctly to the apical cap and also labeled proteins in the apex of mature and dividing parasites upon addition of biotin, as detected by streptavidin staining that colocalized with the bait protein (Fig. 4A).
To identify the proteins labeled by AC2-BirA*, we again purified biotinylated proteins from parasites and analyzed them by MuDPIT mass spectrometry (see Table S2 in the supplemental material). Our top hits were enriched for the alveolins IMC1, -3, -4, -6, -7, -10, -12, -13, and -14 (12), indicating that AC2-BirA* preferentially labels proteins in the IMC cytoskeletal network. In addition, several IMC proteins prominent in the apical cap, including ISP1, PhIL1, and IMC11, scored highly in our experiment (12, 17, 31). These hits suggest that the BirA* ligase, when fused to the appropriate bait protein, can be used to selectively biotinylate proteins in specific suborganellar compartments of the parasite.
Table S2
Copyright © 2015 Chen et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Identification of cytoskeletal proteins in the apical cap of T. gondii by BioID.To determine if AC2-BirA* labeled novel targets in the apical cap, we localized several hypothetical proteins with IMC-like cyclical expression patterns from our hits (see Fig. S4A in the supplemental material). By endogenous tagging, we identified 5 novel proteins in the apical cap: TgGT1_308860 (designated AC3), TgGT1_214880 (AC4), TgGT1_235380 (AC5), TgGT1_251850 (AC6), and TgGT1_225690 (AC7). All of these proteins colocalized with the apical cap marker ISP1 in both parental and daughter parasites (Fig. 4B; see also Fig. S4B). In addition, we localized 3 proteins from this data set to the central and posterior regions of the IMC: TgGT1_316340 (IMC22), TgGT1_304670 (IMC23), and TgGT1_258470 (IMC24), all of which colocalized with IMC3 (Fig. 4C). Importantly, we determined using detergent extraction assays that 7 of these 8 proteins reside in the IMC cytoskeleton (see Fig. S5 in the supplemental material), validating BioID as an approach to identify cytoskeletal components in T. gondii parasites. Consistent with our previous BioID experiment, we also tagged two proteins that are not in the IMC: TgGT1_249870 labeled cytoplasmic puncta in the parasite while TgGT1_232130 localized to the subpellicular microtubules (see Table S2 and Fig. S4C). During the course of these studies, AC5 and TgGT1_232130 were detected by mass spectrometric analysis of immunoprecipitations of the subpellicular microtubule-associated protein TrxL1, although their localization was not determined (32).
Figure S4
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This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S5
Copyright © 2015 Chen et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Identification of proteins that localize to the alveolar sutures of the IMC.The IMC alveoli are organized as a patchwork of rectangular plates plus the single cone-shaped plate termed the apical cap (Fig. 5A). These plates are sutured together by unknown components to form the IMC. Recently, a protein called CBAP/SIP was identified that appears to label the transverse junctions of the IMC plates, as well as more prominent rings at the apical and basal ends of the parasite (33, 34). One of the most remarkable findings of our IMC BioID approach was the discovery of three proteins (TgGT1_235340 and TgGT1_219170 from the ISP3 BioID data set and TgGT1_220930 from AC2 BioID) that localize to the precise pattern expected for the IMC sutures. This suture staining pattern labels both longitudinal and transverse structures extending along the center and posterior of the IMC (Fig. 5B) but not the apical cap or subpellicular microtubules (see Fig. S6A in the supplemental material) (32). This staining agrees well with the approximate number, size, and shape of the IMC plates shown in freeze fracture electron microscopy studies (5). In independent gene-tagging studies in our lab, we also discovered a fourth IMC suture protein that we also report here (TgGT1_305930). Since TgGT1_235340, TgGT1_219170, TgGT1_220930, and TgGT1_305930 are the first markers that delineate the entirety (e.g., both the longitudinal and transverse) of the sutures between the membrane plates, we designated these proteins IMC suture components (ISCs) 1 to 4, respectively.
Figure S6
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This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
While ISCs 1, 2, and 4 do not contain any apparent functional domains by BLAST analyses, ISC3 contains similarity to choline transporter-like proteins, which may suggest a role in IMC membrane biogenesis (35). Each of the ISCs can be detected on the nascent IMC during endodyogeny, although ISC3 is much more prominent in the budding daughters compared to the other ISCs (see Fig. S6B). ISC3 is also unique in that it contains 8 predicted transmembrane domains that presumably span the alveolar membranes. In agreement with this, ISC3 is released with other IMC membrane proteins upon detergent extraction (see Fig. S6C). ISC2 is also soluble in these assays, although it lacks predicted transmembrane domains or acylation sites, suggesting indirect attachment to the IMC membranes and/or a loose interaction with the cytoskeleton. Surprisingly, ISCs 1/4 appear to be firmly embedded into the IMC cytoskeleton but also have predicted palmitoylation sites that may tether them to the membranes (Table 1). Taken together, these data indicate that there are both membrane and cytoskeletal components of the IMC sutures.
We further examined the ISCs with superresolution microscopy and in relation to other IMC proteins. We visualized the brightest-staining ISC (ISC3) using both confocal and superresolution (STED) microscopy and found that the longitudinal and transverse IMC sutures resolved as puncta rather than continuous lines at higher resolutions (Fig. 5C). We also costained ISC2 with CBAP/SIP, which showed colocalization at the transverse sutures (Fig. 5D), in agreement with their initial discoveries (33, 34). Finally, we had previously hypothesized that the posterior boundary of ISP2, which localizes to the central subcompartment of the IMC, is delineated by discrete alveolar plates (17). To examine this possibility, we colocalized ISP2 with ISC2 and found that the ISP2 subcompartment does indeed terminate at an alveolar junction, as the edges of the ISP2 boundary aligned perfectly with a transverse suture (Fig. 5E). These data provide support for a model in which the IMC subcompartments are defined by specific subsets of alveoli (17).
The Toxoplasma IMC consists of distinct membrane and cytoskeletal elements that can be problematic for identifying protein-protein interactions through conventional methods, particularly transient or weaker interactions. In addition, only a small number of IMC proteins (~30) have been identified, and the IMC total protein composition is largely unknown. To circumvent these problems, we adapted the BioID technique for T. gondii and demonstrate that BirA* fusions can biotinylate a robust number of proteins in the IMC. These proteins were identified via MuDPIT despite the inherent background of endogenous biotinylated proteins present in the apicoplast (23). Determining whether these proteins are bona fide interacting partners or merely proximal proteins will need to be addressed using other approaches, such as coimmunoprecipitations, two-hybrid assays, or split green fluorescent protein (GFP). However, we anticipate that the highest-scoring mass spectrometry hits are more likely to represent true binding partners. Additional experimental replicates and reverse BioID experiments with prey proteins will also help to verify interactions and may reveal important interaction networks within the IMC.
We chose the IMC membrane protein ISP3 as bait for the initial BirA* fusion, because ISP3 can be fused to a large tag, such as YFP, and traffic correctly to the IMC (17). Because ISP3 is inserted into the IMC membranes by coordinated acylations (17), we hypothesized that ISP3-BirA* should label proteins in the alveoli and potentially some in the adjacent IMC cytoskeleton. Indeed, our ISP3 BioID data set contains known and novel proteins from both suborganellar compartments (see Table S1 in the supplemental material). Surprisingly, while the ISP3-BirA* fusion targets properly and is enriched in daughter buds like endogenous ISP3 (17), its biotinylation activity appears to be mostly confined to the maternal IMC (Fig. 1B, bottom). This suggests that biotinylation activity of the fusion is altered during endodyogeny or modulated by the surrounding compartment. Alternatively, the lower signal in the nascent IMC could be attributed to the fact that daughter buds have less time to accumulate the biotinylation signal than the maternal IMC. In agreement with this activity, the novel proteins identified by ISP3 BioID are present in, and in some cases restricted to, the maternal IMC. The biotinylation of ISCs by ISP3-BirA* is not surprising, as the sutures appear to be in direct contact with the alveolar membrane sacs. The identification of two new apical cap proteins via ISP3 BioID is less expected but could be attributed to labeling at the junctions between the cap and its neighboring alveolar plates or during early stages of daughter bud formation. It is also clear that ISP3-BirA* does not label the entire subcompartment, as our data set did not include the ISP family members ISP2 and ISP4, whose localizations partially overlap ISP3 in the IMC (17, 18). We anticipate that the extent of biotinylation by BirA* fusions will be dependent on the activity of the particular bait used as well as its proximity to other proteins. It is also important to note that filtering our hits from both data sets described here based on IMC-like gene expression profiles yielded proteins that do not localize to the IMC. Four of these proteins stained punctate vesicular structures in the parasite and one labeled the subpellicular microtubules (see Table S1 and S2 and Fig. S2D and S4C in the supplemental material). Although they were enriched above background proteins from parental parasites, it is not known whether these five hits represent true interactors/near neighbors of our bait proteins.
To begin functional analyses of these novel IMC proteins, we used a modification of the Cre-lox system developed previously to disrupt the genes encoding IMC18 and ILP1 (29). This modified system requires that both the target gene and the upstream gene are arranged in the same orientation in the genome and amenable to endogenous tagging, which results in loxP sites flanking the gene of interest (developed by the M. W. White lab; see Fig. S3A in the supplemental material). This approach has the added benefit of localizing the upstream gene product, if it is unknown (see Fig. S3B and D). Δimc18 parasites displayed no apparent phenotype in vitro, although this result should be taken with caution, as significant compensation has recently been reported in other gene knockouts in T. gondii (36–38). IMC18 does not appear to have paralogs that are detectable by BLAST analysis (data not shown), but we cannot rule out the possibility of other IMC proteins providing functional redundancy. In contrast, disruption of ILP1 resulted in gross morphological defects (Fig. 3C and D) and appears to be lethal to the parasite. These data are in agreement with overexpression studies of ILP1 in Toxoplasma, which resulted in aberrant nuclei and IMC cytoskeletons (27). Similarly, ablation of the ILP1 homolog in Plasmodium berghei produced enlarged, swollen parasites with microtubule and motility defects (39). While the ILP1 homolog is not essential in P. berghei, we were unable to obtain a viable T. gondii Δilp1 mutant in spite of extensive rounds of cloning Cre-induced knockout parasites. It is possible that Plasmodium contains compensatory factors and that their homologs in Toxoplasma are either not present or have lost their ability to suffice in the absence of ILP1. While the tagging/knockout approach used here is rapid and inducible with rapamycin only a subset of the genes in our ISP3 BioID data set have the correct gene orientation for this approach. The development of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene disruption systems in T. gondii will facilitate the functional analysis of large groups of novel genes, such as those identified in this study (40, 41).
The results from our second BioID experiment with AC2 demonstrate the utility of the technique for exploring cytoskeletal networks in T. gondii and for the targeted enrichment of proteins from specific subcompartments of the parasite. In this case, we observed robust labeling of known and novel cytoskeletal proteins in the IMC, many of which are restricted to the apical cap (see Table S2). Interestingly, we found that the 15 novel IMC proteins identified here segregated into two groups of proteins: those that localize to the central and basal subcompartments of the IMC and those that reside in the apical cap (as opposed to IMC1, which stains throughout the IMC). Our results, taken together with other known IMC proteins (12, 17), suggest that the IMC is a highly compartmentalized organelle containing many compartment-specific proteins. As has been previously described for alveolins (12), we observed that some apical cap proteins were detected earlier or later in daughter buds relative to ISP1 (e.g., AC1 is visible on buds before ISP1, while AC4 is detected after [summarized in Table 1]). Analyzing precisely when each of these markers is detected on the nascent IMC will help provide a spatial and temporal understanding of daughter bud formation during endodyogeny.
Perhaps the most intriguing group of proteins uncovered in this study is the ISCs, which appear to specifically label the sutures of the IMC. These proteins localize to both transverse and longitudinal sutures, clearly demarking the rectangular alveolar sacs of the IMC (Fig. 5B). By labeling the entirety of the sutures, the ISCs support the localization of CBAP/SIP to the transverse sutures and indicate that there are differences in protein composition between transverse and longitudinal sutures (33, 34). The staining pattern of MAL13P1.228, a P. falciparum-specific IMC protein reported to localize primarily to rectangular transverse sutures in gametocytes, appears to be more reminiscent of CBAP/SIP than the ISCs (6). Importantly, the ISCs also provide evidence that the IMC subcompartments, delineated by individual ISPs, represent distinct subsets of alveolar plates (17). Although the precise mechanisms targeting ISPs to their subcompartments are unknown, it is possible that these subsets of alveoli express specific enzymes (such as palmitoyl acyltransferases) or receptors that facilitate proper ISP localization.
Interestingly, the ISCs appear to associate with both the membrane and cytoskeletal elements of the IMC. Of the ISCs identified here, ISC3 most closely fits what might be predicted of a membrane suture protein in that it contains 8 predicted transmembrane domains that are presumably anchored in the alveolar sacs and released upon detergent extraction (see Fig. S6C). ISC3 is also similar to choline transporter-like proteins, which import choline for the synthesis of phospholipids for cellular membranes (35). This may suggest a role for ISC3 in choline acquisition for building IMC membranes. In contrast, ISCs 1/4 are embedded in the cytoskeleton and may provide a molecular bridge between the sutured alveolar sacs and the underlying network. ISC2 is released by detergent extraction but lacks apparent transmembrane domains or acylation sites that could tether it to the IMC membranes. It is tempting to speculate that these four ISCs form a complex at the sutures, with ISC2 linking the membrane-bound ISC3 to the ISCs anchored in the cytoskeleton. However, additional studies will be required to elucidate these interactions and determine the function of the ISCs at the sutures. The ISCs also provide new candidate BioID bait proteins that will undoubtedly reveal more constituents of the alveolar sutures and provide clues on how these ISCs interact to form this critical structure within the parasite.
Toxoplasma and host cell culture.The T. gondii RHΔhpt, RHΔku80Δhpt, and modified strains were grown on confluent monolayers of human foreskin fibroblast (HFF) host cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, as previously described (42).
Antibodies.The following previously described primary antibodies were used in immunofluorescence (IFA) or Western blot assays: anti-IMC1 (monoclonal antibody [MAb] 45.15) (43), anti-ISP1 (MAb 7E8) (17), mouse anti-ISP3 (17), anti-F1 ATP synthase β subunit (MAb 5F4) (44), anti-ATrx1 (MAb 11G8) (45), anti-ROP7 (MAb 1B10) (46), rabbit anti-ROP13 (47), and rat anti-IMC3 (12). The hemagglutinin (HA) epitope was detected with mouse anti-HA (MAb HA.11) (Covance) or rabbit anti-HA (Invitrogen). The c-Myc epitope was detected with mouse anti-Myc (MAb 9E10) (NeoMarkers) or rabbit anti-Myc (Sigma).
IFA and Western blotting.For IFA, HFFs were grown to confluence on coverslips and infected with T. gondii parasites. After 18 to 36 h, the coverslips were fixed and processed for indirect immunofluorescence as previously described (48). Primary antibodies were detected by species-specific secondary antibodies conjugated to Alexa 594/488/350. The coverslips were mounted in Vectashield (Vector Labs) and viewed with an Axio Imager.Z1 fluorescence microscope (Zeiss) as previously described (17).
For Western blotting, parasites were lysed in Laemmli sample buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 1% 2-mercaptoethanol, 0.1% bromophenol blue), and lysates were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. Blots were probed with the indicated primary antibodies, followed by secondary antibodies conjugated to horseradish peroxidase (HRP). Target proteins were visualized by chemiluminescence.
Second-copy expression of ISP3-BirA*.To generate the ISP3-BirA* fusion, the BirA* sequence (19) was PCR amplified (P1/P2; see Table S3 in the supplemental material) and inserted into pISP3-HA-HPT in frame (17), between the 3′ end of ISP3 and the HA epitope tag, to generate pISP3-BirA*-HA-HPT. One hundred micrograms of the construct was linearized with HindIII and transfected into RHΔhpt parasites. The parasites were selected with medium containing 50 µg/ml mycophenolic acid-xanthine, cloned by limiting dilution, and screened by Western blotting and IFA against the HA tag. A clone expressing the correct fusion protein was selected and designated ISP3-BirA*.
Table S3
Copyright © 2015 Chen et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Epitope tagging of BioID hits.For endogenous tagging of proteins, we used the plasmids p3×HA-LIC-DHFR, p3×HA-LIC-HPT, or p3×HA-LIC-CAT (18). For this study, we generated pBirA*-3×HA-LIC-DHFR by inserting the BirA* sequence into p3×HA-LIC-DHFR, in frame between the LIC sites and 3× HA tag. A 3′ portion of each gene was PCR amplified using the designated primers (see Table S3) and inserted into the respective plasmid using a ligation-independent cloning approach to generate a 3× HA epitope tag fusion prior to the stop codon of each gene. One hundred micrograms of each construct was linearized within the amplicon and transfected into RHΔku80Δhpt parasites. Transgenic parasites were selected in the appropriate drug medium (containing either 1 µM pyrimethamine, 50 µg/ml mycophenolic acid-xanthine, or 1 µM chloramphenicol) and cloned by limiting dilution. Clones that had undergone the intended recombination event were screened by IFA and Western blotting against the 3× HA tag. For Cre-lox knockout experiments, we used the plasmids p3×HA-LIC-CAT-loxP and p3×Myc-LIC-HPT-loxP (which contain loxP sites downstream of the drug marker; developed by the M. W. White lab, University of South Florida) for endogenously tagging the target gene and its upstream gene, respectively, in DiCre-ku80::KillerRedfloxYFP parasites (30). Cre-mediated gene excision was performed as previously described (29).
Detergent extractions.Extracellular parasites were washed in phosphate-buffered saline (PBS), pelleted, and lysed in 1 ml of 1% Triton X-100 lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl) supplemented with Complete protease inhibitor cocktail (Roche) for 30 min on ice. Lysates were centrifuged for 15 min at 14,000 × g. Equivalent amounts of total, supernatant, and pellet fractions were separated by SDS-PAGE and analyzed by Western blotting.
Affinity capture of biotinylated proteins.HFF monolayers infected with parasites expressing BirA* fusions or the respective parental line were grown in medium containing 150 µM biotin for 24 h prior to parasite egress. A total of 150 µM of biotin yielded more robust BirA* labeling within the IMC (which was optimal by 24 h [data not shown]) compared to 50 µM used previously for mammalian cells (19). Extracellular parasites were collected, washed in PBS, and lysed in RIPA buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40) supplemented with Complete protease inhibitor cocktail (Roche) for 30 min on ice. Lysates were centrifuged for 15 min at 14,000 × g to pellet insoluble debris and then incubated with PureProteome streptavidin magnetic beads at room temperature for 4 h under gentle agitation. Beads were collected using magnets and washed five times in RIPA buffer, followed by three washes in 8 M urea buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl). Ten percent of each sample was boiled in Laemmli sample buffer, and eluted proteins were analyzed by Western blotting by streptavidin-HRP prior to mass spectrometry.
Mass spectrometry of biotinylated proteins.Purified proteins bound to streptavidin beads were reduced, alkylated, and digested by sequential addition of Lys-C and trypsin proteases (49, 50). The peptide mixture was desalted using C18 tips and fractionated online using a 75-µm inner diameter fritted fused silica capillary column with a 5-µm pulled electrospray tip and packed in house with 15 cm of Luna C18(2) 3 µm reversed-phase particles. The gradient was delivered by an easy-nLC 1000 ultrahigh-pressure liquid chromatography (UHPLC) system (Thermo Scientific). Tandem mass spectrometry (MS/MS) spectra were collected on a Q-Exactive mass spectrometer (Thermo Scientific) (51, 52). Data analysis was performed using the ProLuCID and DTASelect2 implemented in the Integrated Proteomics pipeline IP2 (Integrated Proteomics Applications, Inc., San Diego, CA) (53–55). Protein and peptide identifications were filtered using DTASelect and required a minimum of two unique peptides per protein and a peptide-level false-positive rate of less than 5%, as estimated by a decoy database strategy (56). Normalized spectral abundance factor (NSAF) values were calculated as described (57).
Immunocytochemistry and confocal and superresolution imaging.Extracellular parasites were fixed, laid onto coverslips, and processed for indirect immunofluorescence as previously described (48). The coverslips were mounted in ProLong Gold (Molecular Probes) and imaged using a TCS SP5 MP-STED confocal microscope (Leica Microsystems) as described (58). In brief, the ATTO 647N-labeled parasites were imaged on an avalanche photodiode (APD; PerkinElmer) using an oil immersion objective (HCX PL APO CS ×100/1.40-numerical-apterature STED), a 640-nm pulsed diode laser (PicoQuant) for excitation, and a 750-nm pulse (Mai Tai Broadband; Spectra-Physics) for STED depletion.
We thank the following for generously providing reagents: Gary Ward (University of Vermont) for anti-IMC1 antibodies and the pCBAP-3×Myc-LIC-DHFR plasmid, Michael W. White (University of South Florida) for the p3×HA-LIC-CAT-loxP and p3×Myc-LIC-HPT-loxP plasmids, Marc-Jan Gubbels (Boston College) for anti-IMC3 antibodies, Markus Meissner (University Of Glasgow) for providing DiCre-ku80::KillerRedfloxYFP parasites, and Ke Hu (Indiana University) for the pTub-mEmeraldFP-TrxL2 plasmid.
Confocal and superresolution imaging was performed at the California NanoSystems Institute (CNSI) Advanced Light Microscopy/Spectroscopy Shared Resource Facility at UCLA, supported in part by funding from an NSF Major Research Instrumentation grant (CHE-0722519). This work was supported by NIH grant no. AI064616 to P.J.B. and no. GM089778 to J.A.W.
- Received 20 November 2014
- Accepted 28 December 2014
- Published 17 February 2015
- Copyright © 2015 Chen et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

subset: commoncrawl


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For additional information, read the Nomination Form PDF
NRHP Listing Date 08/27/2009
NRHP Reference Number 09000663
The South East Street Historic District is in a portion of the Town of Culpeper that grew rapidly during the 1850s with the expansion of the railroad into the area. The district figured heavily in the Civil War through its association with various Union and Confederate military campaigns and encampments. In fact, substantial Union casualties in the area resulted in the establishment in 1867 of the district's Culpeper National Cemetery (previously listed on the state and national registers) following the Civil War. Today the district features a representative collection of residential architectural styles popular between 1835 and 1955.
VLR: Virginia Landmarks Register
NPS: National Park Service
NRHP: National Register of Historic Places
NHL: National Historic Landmark
Updated April 4, 2018


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You know what mama always said: "make sure you take your anti-oxidants!" Not really. Mom may have reminded you to take your vitamins and minerals, but she may not have known about the damage free radicals can do or how to prevent that damage.
Free radicals cause "oxidation deterioration." In short, they cause damage at a cellular level. This damage can come from stress, pollutants in the environment, food that is not good for you, or various diseases and illnesses. This damage can cause anything from aging (perhaps earlier than you should) or other problems like cancers and Parkinson's disease.
A free radical takes an electron from another molecule, which makes it unstable. That molecule, in turn, will take an electron from another molecule. Eventually, the whole cell will become damaged. This process may lead to broken cell membranes, which will alter what enters and exits the cell. The damaged molecules could also mutate and grow tumors or change the DNA code.
You can prevent this damage by taking or eating antioxidants. Antioxidants give electrons, while free radicals seek to take an electron away.
Antioxidants neutralize that free radical from doing damage. Many people know that colorful fruits and vegetables are full of antioxidants, but there are essential oils that also contain these life-enhancing molecules.
Essential Oils You Need
I always make sure my essential oils are high-quality, so recently I've been using SenseLab essential oils. The best part of these oils is that they are food safe, which makes me feel good about using them when I make dinner. I rather use Oregano essential oil than regular oregano seasoning from the grocery store. Using oregano essential oils helps protect you from free radicals. It's also a natural antibiotic!
High Antioxidant Essential Oils
Some of my favorite antioxidant essential oils have a high ORAC score (that means that they are high in antioxidants).
Two of them I make sure I always have on hand are Myrrh and Frankincense. Both of these were actually gifts to our Lord Jesus at his birth so you know they must have been important! The Magi knew how prized these herbs were, even back then. Not only are Myrrh and Frankincense excellent antioxidant essential oils, but they are also antiseptic and great to diffuse during prayer.
Another great essential oil is clove, which is both high in antioxidants and great for sore teeth. It's also a great one to mix with other oils like cinnamon and orange in your diffuser during the fall and winter season. Smells like Christmas!
While most people will stick with more "well-known" essential oils like peppermint and lavender essential oils, which are both great for allergy season, I really think Vetiver should be in everyone's health arsenal. SenseLab reported that "significantly inhibited DNA damage, clastogenic effects and cell cycle arrest in the bone marrow cells of mice that were given cisplatin (Vetiver) for five consecutive days." That's impressive!
SenseLab knows how important antioxidants are. For your convenience, they made an entire page of their website just for antioxidant essential oils. Check that out here.
For a more complete list of oils with exact ORAC scores, please check out the list from the blog, There's an EO for That.
Remember to keep your health your number 1 priority in 2019.


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This article is a stub, an article too short to provide more than rudimentary information about a subject. You can help Pro Wrestling Wiki by expanding it.
Kōji Kanemoto, (October 31, 1966), is a Japanese professional wrestler.
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- IWGP Junior Heavyweight Tag Team Championship (4 times) - with Minoru Tanaka (1), Jushin Liger (1), Wataru Inoue (1), and El Samurai (1)
- UWA World Junior Light Heavyweight Championship (1 time)
- Best of the Super Juniors Tournament Winner (1998 & 2009)
- GHC Junior Heavyweight Tag Team Championship (1 time) - with Tiger Mask IV
- 5 Star Match (1997) vs. El Samurai on June 5
- Most Outstanding Wrestler (1998)
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Memoriam." (Dedicated by Permission to the Poet-Laureate.) Fcap. 8vo, 2s. The Education of the Human Race. Translated from the German of Gotthold Ephraim Lessing. Fcap.
The Great Frozen Sea: A Personal Narrative of the Voyage of the "Alert ... - Página 24
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text: Introducing Paleontology New dinosaur sported a curious set of chompers Fossils discovered in France linked to previously unknown Cretaceous species ALL TEETH While researchers have only the dinosaur's upper jaw bone and some teeth, this reconstruction takes a stab at a portrait of Matheronodon provincialis, a new species. Lukas Panzarin Share this:FacebookTwitterPocketRedditPrintEmail By Helen Thompson October 26, 2017 at 4:00 pm This article is only available to Science News subscribers. Subscribers, enter your e-mail address to access our archives. Client key* E-mail* Log in Not a subscriber? Science News is a nonprofit. Support us by subscribing now. Subscribe


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2015 65 JAGUAR XF 2.2 D R-SPORT BLACK 4d 200 BHP
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- Insurance group43
Performance & Economy
- Fuel consumption (urban)46.2 mpg
- Fuel consumption (extra urban)65.0 mpg
- Fuel consumption (combined)52.0 mpg
- 0 - 60 mph8.5 seconds
- Top speed140.0 mph
- Cylinders4
- Engine power200 bhp
- Engine torque332 lbs/ft
- Height1460mm
- Height inclusive of roof railsmm
- Length4961mm
- Wheelbase2909mm
- Width1877mm
- Width including mirrorsmm
Interior / Exterior
- 12V socket in rear of vehicle
- 18 way electric adjustable front seats with driver memory
- 2 X front 12v power sockets
- 3 rear seat head restraints
- 3 spoke leather steering wheel
- Auxiliary heater
- Auxiliary input socket
- Bluetooth audio streaming
- Bond Grain leather instrument panel and door top rolls
- Bond Grain leather upholstery
- Bright metal foot pedals
- Coat hooks
- DAB digital radio module
- Drivers footrest
- Dual zone climate control
- Folding rear centre armest
- Footwell lights
- Front armrest
- Front centre console storage box
- Front seatback pockets
- Interior mood lighting
- Isofix child seat preparation
- Jaguar sense
- JaguarDrive selector
- Media interface
- Meridian 770W surround sound system with 12 speakers
- Reach/rake electric adjustable steering column + entry/exit tilt away
- Rear centre head restraint
- Steering wheel mounted audio/cruise controls
- Sunvisors with illuminated vanity mirrors
- Twin front cupholders
- Twin rear cupholders
- 20" Black Kalimnos alloy wheels
- Alloy space saver spare wheel
- Approach lamps
- Automatic headlights
- Bi-Xenon headlights with LED day running lights
- Black electric adjustable heated door mirrors
- Black window surrounds
- Black/body colour bumpers
- Body coloured boot lid finisher
- Door mirror memory
- Headlight washers
- Heated front windscreen and timer
- Heated rear window with timer
- Knurled Aluminium IP Finisher
- Laminated windscreen
- Rain sensor windscreen wipers
- Rear spoiler
ComfortNo features available.
Safety / Other
- Alarm/immobiliser
- Anti-whiplash front headrests
- Bluetooth telephone connectivity
- Brake pad warning light
- Cornering brake control
- Deadlocks
- Drive away door locking
- Driver information centre
- Driver/Front Passenger airbags
- DSC - Dynamic Stability Control
- Electronic parking brake
- Front Parking Aid
- Front/rear seatbelt pre-tensioners
- Keyless Start
- Navigation system with HDD mapping, RDS/TMC + DVD player
- Rear parking sensor
- Remote central locking
- Reversing camera
- Service interval indicator
- Servotronic PAS
- Side airbags
- Side curtain airbags
- Three 3 point rear seatbelts
- Trip computer with dual function
- Tyre pressure monitoring system
OtherNo features available.
Factory Fitted Extras
For more info on this vehicle call our showroom on 0288774 8918
Every effort has been made to ensure the accuracy of the above information but errors may occur. Please check with a salesperson.
- Registered: 2015
- Mileage: 25000 miles
- Engine Size: 2.2 L
- Fuel Type: DieselGearbox: AutomaticBody Style: SaloonColour: WhiteOwners: TBC
For more info on this vehicle call our showroom on 0288774 8918
JAGUAR XF R-SPORT BLACK 200BHP AUTOMATIC/PADDLE SHIFT LATE 2015 Finance available Call for a quick quote or apply via our website www.ajmsalesltd.com 25k, just serviced, 20" factory alloys, upgraded meridian surround sound entertainment system, full black heated memory electric seats, Windsor clad leather, gloss veneer/aluminium trim, beautiful special order pearlescent white paint, full electric pack, sat nav, Bluetooth, xenon headlights, spare wheel, reverse camera, multifunction steering wheel, cruise control, front and rear park distance control. This Jaguar XF offers a compelling mix of comfort technology and driver kicks, phone for full details 07881614000/ 02887748918 £15,495
Every effort has been made to ensure the accuracy of the above information but errors may occur. Please check with a salesperson.
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|60 mths
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Additional Information
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*The Acceptance & Option fees that may apply can vary from time to time and from lender to lender, fees are already accounted for within the payments displayed and are also included within the total amount payable.
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Home Entertainment
April 20, 2011
"Never Apologize": Malcolm McDowell Stars In The Spirited Documentary About Controversial British Director Lindsay Anderson To Debut On DVD May 31
(April 20, 2011 – Burbank, CA) – "Never Apologize," the fascinating documentary of Malcolm McDowell's celebration of the provocative Lindsay Anderson and their famous colleagues, will be released on May 31 for the first time on DVD in the U.S. A unique blend of film, theatre and storytelling and a Cannes Film Festival selection (2007), the documentary weaves a mesmerizing mosaic about Anderson, his friend, mentor and director, who brought him to international prominence through his debut role in "If...," which led Stanley Kubrick, after years of searching, to cast McDowell in "A Clockwork Orange." Alan Bates, Bette Davis, John Ford, John Gielgud, Lillian Gish, Richard Harris, Laurence Olivier, Rachel Roberts and Princess Diana spring to life in McDowell's hilarious, chilling and moving accounts, drawn from his memories and through writings by and about Anderson, one of the most significant forces in British culture. Directed by Mike Kaplan, who worked extensively with both Kubrick and Anderson, "Never Apologize" evolved from productions at the Edinburgh Festival and London's National Theatre. A wealth of archival material surrounds McDowell's compelling, inspired and wide-ranging performance, emerging as a genuine cinema experience ("a surprising coup that grips consistently for every one of its 111 minutes"- David Robinson, Sight and Sound). McDowell stars in the upcoming film "Vamps," directed by Amy Heckerling, opening later this year. And "Franklin & Bash," the new TNT comedy-drama TV series debuting this summer features McDowell as the head of a buttoned-down law firm joined by two young, fly-by-the-seat-of-their-pants street lawyers who cause a seismic culture clash within the firm. He will also be featured in the upcoming film "Silent Hill Revelation," the sequel to the 2006 hit "Silent Hill." About Warner Home Video: With operations in 90 international territories, Warner Home Video, a division of Warner Bros. Home Entertainment Inc., commands the largest home entertainment distribution infrastructure in the global video marketplace. Warner Home Video's film library is the largest of any studio, offering top quality new and vintage titles from the repertoires of Warner Bros. Pictures, Turner Entertainment, Castle Rock Entertainment, HBO Video and New Line Cinema.


text: Sony just broke out it's latest and greatest Android device, the Neo. The device is able to plug into your HDTV via HDMI and can be controlled with a standard remote. The 3.7-inch screen on the device sports a healthy resolution of 854 x 480 and uses Sony's Bravia graphics engine. The phone has a 1GHz Snapdragon CPU, an 8 megapixel camera with LED flash (and a 2 megapixel front-facing camera), and will be available in an array of colors (silver, red, and blue). We're getting more info right now, so stay tuned!


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Thanks to the innovative formulas in K-beauty, toners are no longer a negligible step in most of our routines. Gone are the days when toners were full of alcohol and did nothing but dry our skin out. Now, toners improve how the rest of our skin care products perform and even have some benefits of their own! So join the toner train ASAP by peeping this year's standouts below:
IMAGE Etude House
ETUDE HOUSE Soon Jung pH 5.5 Relief Toner, SM Mall of Asia
After cleansing, this alcohol-free product helps lower your skin's pH level to 5.5, which is the ideal pH for a healthy moisture barrier.
IMAGE K-Beauty Cafe
DEAR, KLAIRS Supple Preparation Facial Toner, P880, K-Beauty Cafe
Make sure those essences and serums absorb into your skin better with this pH-balancing toner.
IMAGE K-Beauty Cafe
PYUNKANG YUL Essence Toner, P925, K-Beauty Cafe
A waterless (you read that right) toner that's as nourishing as an essence and a moisturizer.
IMAGE Mizon
MIZON Vita Lemon Sparkling Toner, P523, Beautymnl
In need of a quick skin refresh? This will brighten your skin and your day.
IMAGE Banila Co
BANILA CO VV Vitalizing Toner, P1150, SM North Edsa
A firming toner with V-collagen, niacinamide, and peptides for that age-defying glow.
IMAGE Beauty Bar
YADAH Anti-T Toner, P750, Beauty Bar
Packed with skin-healing plant extracts like black raspberry and cactus, this protects your skin from breakouts without that drying, burning sensation.
IMAGE Huxley
HUXLEY Secret of Sahara Toner: Extract It, P1450, Beautymnl
The Sahara prickly pear seed oil, which is this hydrating toner's star ingredient, is extracted from a plant that survives in the desert's extreme heat from drying out.
IMAGE The Face Shop
THE FACE SHOP Yehwadam Revitalizing Toner, P1495, Freestate.ph
Tone your way to radiance and out of dullness with this revitalizing toner packed with ginseng extract.
IMAGE Nature Republic
NATURE REPUBLIC Aloe Vera 92% Soothing Gel Mist, P260, Althea
The brand that brought us the best-selling aloe gel also has a soothing mist you can use as a toner. Bye, redness.
IMAGE Innisfree
INNISFREE Jeju Volcanic Pore Toner, P800, Beautymnl
To remove excess sebum before you moisturize, swipe this toner all over the skin.


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The phrases "complementary" and "various" are generally used to discuss with non-conventional strategies of diagnosing, stopping, or treating cancer or its symptoms. The techniques used in CAM are principally less invasive than standard medical practices, which means that they don't rely on surgical procedure or standard medications. " Between Heaven and Earth " is a guide that can assist you understand why historic Chinese medication can still be priceless as we speak and how it may be used with extra modern Western practices.
Specialists have not researched many CAM dietary supplements and practices sufficient to inform how effective they are as treatments. Despite their deep wells of medical expertise, many top hospitals are providing to help treat serious medical issues with reiki — a follow based on the idea that lightly touching patients can unleash a cosmic power circulation that may heal them naturally.
But the Facilities for Medicare and Medicaid Services will not pay for acupuncture, dismissing the scientific evidence as inadequate. Relating to treating ache and persistent illness, many doctors are turning to therapies like acupuncture and meditation—however using them as part of a bigger, integrative strategy to health. Most complementary health approaches fall into certainly one of two subgroups—natural merchandise or thoughts and physique practices.
The mission of NCCIH is to define, by way of rigorous scientific investigation, the usefulness and security of complementary and integrative well being interventions and their roles in improving well being and well being care. I believe we've clearly established right here that it is honest to say CAM practitioners typically will use treatments anyplace alongside the scientific spectrum with little or even no regard for logic or evidence.
If you are looking for a CAM practitioner, converse along with your major health care supplier(s) or somebody you consider to be knowledgeable about CAM regarding the therapy in which you are interested. Standard medication is based on scientific knowledge of the body and uses treatments that have been proven effective through scientific research.


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We offer the finest ingredients
Our goal is to help you with your health and beauty needs. We are available anytime to consult or offer suggestions.
We offer a varitey of Hemp products we cannot sell through website, but email us for more information at email@example.com
We now offer your choice of ziploc baggies or FSC Certified Unbleached Totally Chlorine-Free (TCF) paper packaging. For a small up-charge we can package your butters or soaps in totally organic paper instead of BPA-Free Plastic.
Have an issue you're reluctant to talk about? Don't! We are here to help and guide in anyway we can!
We offer articles on a variety of issues and products for your health, beauty and DIY needs.
If you are not 100% satisfied with your purchase, you can return the product and get a full refund or exchange; return the product within 14 days from the date you purchased it.
As with any organic product always test for a potential allergic reaction.
Check out our Events Page!
Currently MOST April Events are cancelled.
Check back frequently as we will keep you updated!
September 3-7 PA Labor Day Markeplace Irwin, PA


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Boys basketball: East Haven drops Shelton in opener
The Shelton High boys basketball team fell to visiting East Haven High, 77-65, on Wednesday night in their season opener at Murray Gymnasium.
Malachi Campbell scored 19 points to power the Yellow Jackets, while Nick Durso and Zach Vineyard chipped in with 16 points apiece.
For Shelton, Casey Belade had 19 points, Jeff Stockmal scored 15 and Matt Terrasi 13.
For the full game story, check out next week's Herald.


text: Wide Curtain Fabric Rolls – 235cm (92 inch) – Made in USA – Great Designs – 1.29 Kg USDJuly 28, 2016 by Dan Knutzen Leave a CommentWHATSAPP ME 001-214-288-5003 USAIs this email not displaying correctly? View it in your browser.:start Curtain FabricMade in USA Description: – Assorted Designs – 235cm (92 inch) width – Average Roll 40 yards – 15,000 kgs available – Price: $1.29/kg USD EXW Ready to ship! For more info: Simon 1-214-288-5003 Whatsapp [email protected] [email protected] Leave a Reply Cancel replyYour email address will not be published. Required fields are marked *CommentName * Email * Website


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Are you in the market for a new Volkswagen model? If you're on the hunt for a family-sized vehicle with exciting features, you should check out the 2019 VW Golf at Alexandria Volkswagen. We'll look at how the 2019 Golf compares to the 2019 Golf GTI and determine which model fits your budget and lifestyle the best. We're here to help you find the right vehicle that will make your Arlington commute more enjoyable.
Starting Price
If high performance is at the top of your list, you will want to experience the Golf GTI! Its upgraded engine gives you more power and speed while driving on the streets in Washington DC, but will come at a higher price. While the Golf offers slightly less power than the GTI, the price is more approachable. Here is a quick look at what you can expect with the newest Golf and Golf GTI.
Volkswagen Golf:
Volkswagen Golf GTI:
The interior of both models offers an abundance of high-class technology to keep your commutes to Springfield exciting. Both models come standard with the VW Car-Net App Connect, SiriusXM satellite radio, and the chic leather-wrapped steering wheel. What makes the Golf GTI stand out are features like:
Comparing the Volkswagen Golf vs. GTI, you will notice quite a bit of difference. The GTI was made for drivers who have a fast-paced lifestyle and want that in a vehicle, while the Golf offers strong performance, but at a more affordable price point. The best way to make your final decision is to visit Alexandria Volkswagen and hit the road for a test drive and see firsthand what each model has to offer. Contact us today for questions! Whether you're looking for a used Volkswagen Golf GTI, or for details on a Volkswagen Golf lease, we're here to help. Don't forget to ask about our current vehicle specials and check out the available Volkswagen Golf GTI accessories.
* Ask your dealer if this vehicle meets the emissions requirements of the state in which you are registering the vehicle. Starting MSRP excludes destination, taxes, title, other options, and dealer charges. Dealer sets actual price. Specifications, equipment, options, and prices are subject to change without notice. Some items may be unavailable when vehicle is built. Ask your dealer about availability of options and verify that the vehicle includes the equipment you ordered.
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844-385-8469Call Now
107 W Glebe Rd • Alexandria, VA 22305Get Directions
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The Good, The Bad, and The Smugly: In a regular series, our writer picks out 3 fantasy picks with contrasting fortunes so far this season.
It's been two weeks since the last article, so how has everyone been doing?
The Good
Pierre-Emerick Aubameyang. This week's 'Good' Award has to go to Auba. Arsenal were already convincingly beating Fulham before this man came on and left his mark on the game. Two goals and an assist in 28 minutes? Outstanding performance, and surely the best definition of the term 'super sub'.
Arsenal gave Fulham a leathering last weekend. The Cottagers now have the worst defensive record in the Premier League, conceding 21 goals in 8 games. Swiss cheese. On the other hand, The Gunners are flying it. Putting five past poor ol' Bettinelli last weekend ensured them 4th place, just pipping Spurs to this spot. This was with a comparatively weak lineup, with big players like Auba and Ozil starting on the bench. Though, Arsenal definitely seem to have their silky mojo back (Looking at you, Ramsey).
Pierre was a class act when he came on. He didn't take too long to jump into proceedings. Indeed, it took him all of five minutes to assist Ramsey in that previously mentioned sumptuous goal. He then showed he still has an eye for the back of the net, by scoring a lovely volley with his lesser foot. Finally, he put one more passed an already downtrodden Bettinelli to secure the three bonus points for himself. I kinda feel bad for Lacazette.
If you have Auba, hang on with both hands. He has taken slightly longer to perform this season, but I reckon this is the beginning of something beautiful. He is now putting away chances that he was missing in the first few games. Currently sitting at £10.7 and 8.9% ownership, he could even be a tasty differential.
The Bad
Harry Kane. Scoring a measly point, there's no doubt in my mind who gets the 'Bad' award this weekend. It was both a poor personal and team performance. Harry looked lethargic against Cardiff, having lost the ball the most times of any player in the match (4).
For a game which people thought that Spurs would win by a few goals, it didn't exactly turn out that way. Tottenham were quite lucky to walk away with three points on Saturday, in all honesty. Everybody was misfiring, not only Kane, as Moura and Son should have had a goal to their names as well. Alas, it was left for the aptly-named Dier to score one of the easiest goals I'll ever see. I'm actually surprised he got the ball in between the sticks, I've seen this lad take some shots from outside the box, and they're not pretty.
Back to Kane, however, and it was a very disappointing performance from a man who scored 13 points in gameweek 7. He also lost the most duels of any player in the match which, to me, clearly shows the man is tired. He's been a workhorse for Spurs in recent times. He has gone from last season to the World Cup, straight back into this season and midweek Champions League.
I mean, I don't want to see him benched, but the stats don't lie. I'd like to see him get a rest this weekend, but that's probably unlikely with upcoming international games against Croatia and Spain. Lord knows this man needs a break.
In my opinion, I would stick with Kane if you already own him. I don't see a point in getting rid, seeing as this boy always turns up in the long run. His form so far is definitely and, uncharacteristically, streaky. I wouldn't, however, say it's cause for giving him the boot just yet (not the golden boot either, for that matter).
The Smugly
Matt Doherty. As an Irishman, I'm pleased to give this week's 'Smugly' award to a fellow Paddy. When I look at what Doherty has given to Wolves, I wonder how Cyrus Christie has made the cut for Ireland's RB spot. Maybe I'm still sour after the latter handed that first goal to Christensen, ultimately tossing us out of the World Cup before it even began in that fateful 5-1 hammering from the Danes. Whoops, bit of a segue. I think this article was supposed to be about Fantasy Football, or something.
Doherty has scored an excellent 15 points this gameweek. In a defensively solid side who have only conceded six goals (fourth best in the league!), Matt is bombing up and down that right flank. This is proving a lucrative choice, as he has now opened his goalscoring account for the 18/19 season with a delightful one-two before rifling a shot past Hennessey.
With his fourth clean sheet of the season, Doherty looks to be part of a Burnley-esque budget defence which does offer attacking value in the shape of its wing-backs. The best part? He's currently priced at £4.7 and is owned by 9.6% of players. Not bad for an inexpensive defender who has now reached double digits for a third week thus far.
If you don't have Matt or any Wolves defender, I'd definitely consider him. Wolverhampton face Watford and Brighton in that order over the next two gameweeks. Those two haven't exactly been banging in goals of late. Maybe, this is an opportune time for this tough Wolves defence.
Join us after the international break for a chance to see who will be next gameweek's Good, Bad and, most importantly, the 'smugly'.


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Around the World in a Connected Helicopter
Here's the story behind the technology that is fueling this record-setting flight
July 19, 2017
Dubbed the C150 Global Odyssey (C150GO), the flight celebrates the 150th anniversary of Canada's confederation. Throughout the planned 37-day mission, they've stopped in Quebec, Iceland and London.
Bob and Steven Dengler take the C150 Global Odyssey (C150GO), the flight for their 37 day mission around the world.
"No one has more expertise than Honeywell when it comes to getting critical data on and off an aircraft and making flights safer and more efficient," said Warren Nechtman, vice president, Connected Aircraft Systems, Honeywell Aerospace. "This is why we're happy to sponsor the C150 Global Odyssey."
Honeywell's Aspire 200 Satellite Communications system provides Wi-Fi speeds of up to 350 Kbps, enabling access to updated weather information throughout the journey.
Helicopter connectivity is a challenging feat because rotor blades interrupt the satellite signal to and from the aircraft. Honeywell has solved this problem with our Aspire 200 Satellite Communications system. The Aspire 200 and GoDirect Cabin Connectivity , plus highly reliable data connectivity through Inmarsat's global satellite constellation , let the team experience Wi-Fi speeds of up to 350 Kbps. These speeds enable real-time uploads to social media (including Twitter), videoconferencing and access to updated weather information throughout their journey.
The C150GO is the brainchild of Bob and Steven Dengler, a father-son team with a passion for aviation.
"The connectivity capabilities of Honeywell's Aspire 200 allows us to share photos and videos of our adventure from the air, but it also provides real-time data to manage mission risk as we fly. This greatly enhances our safety no matter where we are in the world," said Bob Dengler. "Our biggest need for this journey is reliable, global service that provides consistent coverage anywhere in the world. Honeywell's offering does that and more."
Read more about the mission and follow the team's progress at C150 Global Odyssey. Find out where the crew is on the world map real-time.


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Operation Reef Defence: Airlie Beach
A new report from the Climate Council released this week reveals the urgent need for Australia to kick its coal habit to protect the Great Barrier Reef.
The report highlights the chilling finding that even if global warming is limited to 1.5°, coral reefs could lose a further 70-90% of cover.
This would effectively eliminate the Great Barrier Reef as we know it.
Adani is hellbent on building one of the world's biggest and most destructive coalmines, which would effectively sign the death warrant for our Great Barrier Reef. Operation Reef Defence heard Australia's voice: we will not trade the Great Barrier Reef for one of the world's most destructive coalmines.
WATCH NOW to revisit our journey along the east coast of Australia – from Sydney to Abbot Point and many points in between: https://www.seashepherdglobal.org/our-campaigns/reef-defence/
Indigenous elder Ken Dodds paddled out to meet the Steve Irwin to welcome supporters in their fight to #StopAdani on Operation Reef Defence.
Video Credit: Jax Oliver / Sea Shepherd.


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Mohammed Azharuddin's last-minute backing out rendered Sunday's Badminton Association of India (BAI) elections in Chennai a one-horse race and the former cricket captain said he decided to skip it after failing to get a stay order on the contest.
The cricketer-turned-politician created a stir with his announcement to contest BAI elections but it turned out to be a damp squib as he left the ring allowing long-standing president VK Verma, who has been at the federation's helm for 12 years, another term.
"I decided against contesting after we did not get the stay order," Azharuddin said from Hyderabad.
"I wanted to fight but it was going to be difficult after that," he said after Verma was elected unopposed in Chennai.
Even though he was reluctant to elaborate, one of his associates said they needed more time to prepare.
"You cannot overnight overthrow someone like Verma, who has been here for such a long time. We needed some more time to prepare and hence sought a stay order. Azhar Bhai also had to go to Europe as part of a delegation and even though he returned midway, we did not have enough time," he said.
Azharuddin, meanwhile, insisted Verma's re-election is illegal since it violates the recent Sports Ministry guidelines that fix a federation president's term to a maximum of 12 years.
"I was not there but I'm, told the election was conducted in a manner which was not free and fair. It was stage-managed and Verma won in complete violation of the Sports Ministry guideline," Azharuddin said.
Asked what would be his course of action now, Azharuddin said, "I will talk to people and see what can be done. We will wait and see."
Azharuddin also doubted Verma's claim of being unanimously elected in Chennai.
"I did not keep a tab but I'm told it was neither free nor fair. I'm not convinced about this claim of unanimous election either.
"They elected three secretaries which baffles me. I don't know how can they explain that. You generally have a one secretary and two joint secretaries. He in fact had the coach [Pullela Gopichand] in the AGM as well, now that is strange," Azharuddin said.
"See, I have nothing personal against anyone. I just wanted to bring more professionalism and accountability in the running of the game and the federation. I'll talk to people now and then decide what to do," he added.


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Sunderland's lead at the top of the National League Northern Division was cut to six points after their game at Tranmere Rovers was the only fixture postponed yesterday.
Middlesbrough recorded their fourth league win when a 52nd-minute Laura Thompson goal earned victory over Oldham.
Newcastle have now conceded 10 goals in two games after they lost 4-1 at Doncaster in the Northern Combination.
They were trailing by four goals when Stacey Crooks grabbed a second-half consolation.
Chester-le-Street Town completed a double over Hutton Crunswick with a 7-0 win at Chester Moor.
Striker Natalie Gutteridge scored a hat-trick and Laura Gibbon two with Cherie MacDougall and Sam Hill also on target. Nissan reached the semi-finals of the Bill Godward Cup with a 3-1 win over Lumley.
Nissan meet Blyth Town after a 4-2 win over Darlington RA.
Blyth Spartans face South Durham Royals in the other semi-final. They beat Killingworth 2-1 through two Joanne Dixon goals.
South Durham Royals' beat Crook Town 6-1 .


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Question Description
I need an explanation for this Business question to help me study.
Assume that the average firm in your company's industry is expected to grow at a constant rate of 4% and that its dividend yield is 6%. Your company is about as risky as the average firm in the industry, but it has just successfully completed some R&D work that leads you to expect that its earnings and dividends will grow at a rate of 50% [D1 = D0(1 + g) = D0(1.50)] this year and 25% the following year, after which growth should return to the 4% industry average. If the last dividend paid (D0) was $1.75, what is the value per share of your firm's stock? Round your answer to the nearest cent. Do not round your intermediate computations.
Final Answer
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