The differently-diverged parasitic protist Giardia lamblia is known to have minimal machinery for vesicular transport. Yet, it has three paralogues of SNAP, a crucial component that together with NSF brings about disassembly of the cis-SNARE complex formed following vesicle fusion to target membranes. Given that most opisthokont hosts of this gut parasite express only one α-SNAP, this study was undertaken to determine whether these giardial SNAP proteins have undergone functional divergence.
All three SNAP paralogues are expressed in trophozoites, encysting trophozoites and cysts. Even though one of them clusters with γ-SNAP sequences in a phylogenetic tree, functional complementation analysis in yeast indicates that all the three proteins are functionally orthologous to α-SNAP. Localization studies showed a mostly non-overlapping distribution of these α-SNAPs in trophozoites, encysting cells and cysts. In addition, two of the paralogues exhibit substantial subcellular redistribution during encystation, which was also seen following exposure to oxidative stress. However, the expression of the three genes remained unchanged during this redistribution process. There is also a difference in the affinity of each of these α-SNAP paralogues for GlNSF.
None of the genes encoding the three α-SNAPs are pseudogenes and the encoded proteins are likely to discharge non-redundant functions in the different morphological states of G. lamblia. Based on the difference in the interaction of individual α-SNAPs with GlNSF and their non-overlapping pattern of subcellular redistribution during encystation and under stress conditions, it may be concluded that the three giardial α-SNAP paralogues have undergone functional divergence. Presence of one of the giardial α-SNAPs at the PDRs of flagella, where neither GlNSF nor any of the SNAREs localize, indicates that this α-SNAP discharges a SNARE-independent role in this gut pathogen.
Giardia lamblia is a gut pathogen that causes the diarrheal disease giardiasis. In addition, this protist serves as an excellent model to study how eukaryotic evolution has proceeded along different paths . Studies show that the molecular machinery for multiple cellular processes of this protist are highly diverged compared to that present in most well-studied model eukaryotes . Whether the differently-diverged cellular pathways of present-day Giardia results from early divergence from the main line of eukaryotic evolution or is a consequence of reductive evolution is still debatable. This divergence is particularly evident in the machinery for maintaining its endomembrane system, which is composed of fewer compartments compared to that present in most eukaryotes . Besides the endoplasmic reticulum (ER), the only other identifiable endomembrane compartments are the small peripheral vesicles (PVs) that discharge both endosomal and lysosomal functions . Thus, this parasite is an interesting model to study how vesicular trafficking proceeds in the backdrop of reduced endomembrane compartment diversity and uncovering the molecular machinery that supports this system is important to understand how life has evolved to use different approaches to address the same challenge.
The exchange of material between different endomembrane compartments takes place either through direct contact between the organellar membranes or via transport vesicles . Such exchanges are very precise and sustained by a complex machinery whose molecular components include both proteins and lipids . Vesicle-mediated exchange requires membrane deformation and vesicle budding from donor membrane, transport of these vesicles on the cytoskeletal network, followed by docking at the appropriate target membrane, and finally its fusion to the target membrane . Proteins that ensure the fidelity of this process include: (i) the adapter protein (AP) complex and members of the Sar/Arf GTPases that selectively load appropriate cargo molecules into the budding vesicle and recruit the coat proteins at the donor compartment; (ii) the coat proteins (clathrin, COPI and COPII) that deform the membrane to form the vesicle; and (iii) the Rab GTPases, tether proteins and the SNAREs, which ensure that the vesicles only fuse to the appropriate acceptor compartment . While tether proteins extend out from the acceptor membrane to "scout" for the correct vesicle and bring it closer, the SNAREs operate over a shorter distance and the formation of the trans-SNARE complex brings about the fusion between the membranes of the vesicle and acceptor compartment.
Different members of the SNARE proteins decorate the surfaces of various vesicles and also the target compartments. A trans-SNARE complex is formed when the SNARE on the incoming vesicle precisely pairs-up with the cognate SNAREs on the target membrane. This pairing enables the vesicle and target membranes to come close together such that they can fuse . Following membrane fusion, the helical bundle of the newly-formed cis-SNARE complex is disassembled so that the SNAREs arriving on the incoming vesicle may be recycled back to the donor compartment. The strong intermolecular interactions within the cis-SNARE complexes are broken with the help of a protein complex, consisting of the AAA ATPase N-ethylmaleimide sensitive factor (NSF) and soluble NSF-attachment protein (SNAP), as the energy derived from ATP hydrolysis drives cis-SNARE uncoupling .
As previously mentioned, the machinery supporting the endomembrane system of Giardia is composed of fewer components. For example, instead of four, only two AP complexes have been identified in Giardia; in the tethering complexes, three components of both HOPS and TRAPP1, and two components of DSL1 were found to be missing [10, 11]. Only eight Rab GTPases have been identified in this protist, in contrast to the large repertoire of Rab members in other parasitic protists such as Trichomonas and Entamoeba [12–15]. Even the ESCRT machinery for endosomal sorting is composed of fewer components, with either entire complexes, such as ESCRT-I, being absent, or complexes being composed of fewer subunits, as in the case of ESCRT-II and ESCRT-III [16, 17].
Interestingly, there appear to be exceptions to this observed reduction of cellular machinery, as in the case of SNAPs. Many organisms, including mammals, have multiple paralogues of SNAPs, termed α-, β- and γ-SNAPs . In mammals, while α- and β-SNAPs share a high degree of homology (> 80% identity), γ-SNAP shares only 20% identity with α-SNAP. Also, while both α- and γ-SNAPs are expressed in most tissues, expression of β-SNAP is restricted to the brain . In addition, expression of α-SNAP commences in the developing embryo and continues into adulthood, but β-SNAP is expressed only after birth . These SNAPs also discharge different cellular functions. α-SNAP is required for transport from ER to the Golgi, within the Golgi, homotypic vesicle fusion, store-operated calcium release and ER/endosome membrane fusion [20–22]. β-SNAP is functionally similar, except it has a different affinity for a binding partner, PICK1 . γ-SNAP can stimulate the Ca2+-dependent exocytosis but is unable to function in ER to Golgi transport [24, 25]. While most eukaryotes characterized thus far have only one copy of α-SNAP, two α-SNAPs have been reported in Giardia . Here we report the presence of not two, but three SNAP genes in the Giardia genome. All the three genes were expressed in trophozoites, encysting trophozoites and cysts. The predicted secondary and tertiary structures of all the three proteins are similar to the yeast α-SNAP, Sec17, and all three giardial genes can rescue the growth defect of the sec17-1 temperature-sensitive yeast mutant. Interestingly, there is a distinct difference in the subcellular distribution of all three paralogues. Apart from the anticipated localization at the cell periphery where many membranous compartments are present, two of the paralogues exhibit a dramatic change in subcellular distribution both during encystation and oxidative stress. This observed difference in subcellular distribution is indicative of the paralogues performing distinct functions in this protist.
Given that SNAPs play pivotal roles in vesicle-mediated trafficking, we wanted to characterize the previously-reported putative giardial orthologues encoded by the ORFs GL50803_17224 and GL50803_16521 . This report also identified the ORF GL50803_10856 as a putative NSF. However, domain analysis in Pfam indicates that like the first two ORFs, the protein encoded by the last also contains a SNAP domain and consistently, GiardiaDB annotates it as an α-SNAP. To determine if the proteins encoded by these ORFs have the potential to function as SNAPs, we analyzed their sequence to determine their probable secondary structures. All characterized SNAP orthologues are small α-helical proteins, including the 292 residue-long S. cerevisiae orthologue, Sec17 . The three putative giardial SNAPs are comparable in size; while GL50803_17224 encodes 298 amino acids, GL50803_16521 and GL50803_10856 are composed of 292 and 294 amino acids, respectively. However, the three ORFs do not share extensive sequence homology; while the sequence of GL50803_17224 is 30.6 and 22.6% identical to GL50803_16521 and GL50803_10856, respectively, the remaining two ORFs share 21.6% identity. Secondary structure predictions indicate that all the three putative proteins are composed of α-helices and loops (Fig. 1a). This conclusion is independently supported by circular dichroism spectroscopy, which indicates that the percentage helicity of the three proteins is comparable to Sec17 (Additional file 1: Figure S1). Consistent with the crystal structure of Sec17, the three giardial proteins are predicted to have fourteen α-helices each (Fig. 1a) . One notable difference is that while in the Sec17 crystal structure, the membrane-interacting hydrophobic patch, GFMKLF, adopts a short α turn (α-1'), the hydrophobic residues in the corresponding region of all the three putative giardial orthologues are located in an unstructured loop between helices α1 and α2 . Thus, all the three giardial proteins are like Sec17 both in terms of their size and the secondary structure they are likely to adopt.
A previous report had identified residues on the SNARE-binding surface of the Bos taurus α-SNAP, of which all but one (Y200) were charged . Perusal of the crystal structure of Sec17 showed that almost all the analogous positions are also occupied by similar residues, including W196 occupying a position comparable to Y200 (Fig. 1b). Given the low sequence identity of the three giardial proteins with known SNAP orthologues (Additional file 2: Tables S1 and S2), we wanted to determine their possible tertiary structures to see if similarly-charged amino acid residues occupy analogous positions on their surface as well. The possible tertiary structures of the three giardial proteins were determined using protein threading. While most positions on the modeled structures of the giardial SNAPs were occupied by residues analogous to those of the bovine α-SNAP, there were few exceptions: (i) in GL50803_17224, a histidine (H158) was present in place of a canonical arginine or lysine; (ii) there was an interchange of position between an aromatic (F198) and a positively charged residues (K194) in GL50803_16521; and (iii) two positively charged residues (K159 and R160) were shifted more towards the edge of the concave face in GL50803_10856 (dotted circles in Fig. 1b; Additional file 2: Table S3). Barring these minor variations, the pattern of distribution of the positively-charged residues was mostly conserved for all the three giardial proteins and Sec17, indicating that they are likely to be functionally analogous.
Existing literature documents that two of the three α-SNAP proteins, α-SNAP17224 and α-SNAP16521, are expressed in trophozoites and up to 14 h of encystation . Reverse transcription PCR indicates that all the three identified genes are transcribed in trophozoites, encysting trophozoites (8 and 16 h following induction of encystation), and cysts (Additional file 1: Figure S2). Thus, the identified ORFs are not pseudogenes and the activity encoded by each is required in all stages of this parasite's life-cycle. Perusal of the genomes of the other Giardia isolates included in the GiardiaDB (assemblage A2 isolate DH, assemblage B isolate GS, assemblage B isolate GS_B and assemblage E isolate P15) indicates that each of these also encodes three putative α-SNAPs (data not shown). The presence of multiple α-SNAP paralogues in the genome of a unicellular protist that lacks diversity of endomembrane compartments is unusual given that most eukaryotic genomes encode only one orthologue of this protein; exceptions include Arabidopsis thaliana, which has two (Additional file 2: Tables S1 and S2) . To determine if the identified sequences are α- or γ-SNAPs, we reconstructed a phylogenetic tree with known α- and γ-SNAP sequences from diverse taxonomic groups. Sequences of putative SNAP paralogues from the closely-related diplomonad, S. salmonicida and Trepomonas sp., were also included. From the tree topology, we observed that while two sets of sequences, one with GL50803_17224 and another with GL50803_16521 of the reference strain (assemblage A, isolate WB), cluster together with the α-SNAPs, the third set, with GL50803_10856, clusters closer to the γ-SNAPs (Fig. 2). Trepomonas, a close relative of Giardia, also contains three SNAP paralogues, of which two cluster with the γ-SNAPs and the third is within the α-SNAP cluster. Unlike Giardia and Trepomonas, S. salmonicida encodes only two putative SNAPs, of which one clusters with the α-SNAPs while the other with the γ-SNAPs. Based on this analysis, it appears that while two of the identified giardial sequences are likely to be α-SNAPs, the third may be a γ-SNAP.
It is known that α-, but not β- or γ-SNAP can substitute for Sec17 . We used this criterion to evaluate if the identified giardial proteins are α- or γ-SNAP orthologues. Towards this, we used a mutant yeast strain having the temperature-sensitive sec17-1 allele, which is functional at 30 °C but not at 37 °C . We used functional complementation to assess if any of three giardial genes can functionally substitute for the sec17-1 hypomorph. All three giardial genes were expressed in the sec17-1 mutant under the control of a constitutive promoter. Expression of yeast's own SEC17 gene from the same vector served as a positive control while yeast transformants containing just the vector backbone served as negative control (Fig. 3). All the five transformants exhibited equivalent growth at the permissive temperature of 30 °C. At the non-permissive temperature of 37 °C, mutants expressing the plasmid-borne copy of SEC17 exhibited robust growth while those transformants harboring just the vector backbone failed to grow at this non-permissive temperature (Fig. 3). Growth of yeast cells expressing any one of the three giardial genes was comparable to the positive control. Thus, the survival of the temperature sensitive sec17-1 mutant expressing any of the three giardial genes at 37 °C indicates that even though one of the three SNAP sequences from Giardia clusters with γ-SNAPs, all these genes encode proteins that are functionally equivalent to Sec17 of yeast, which is an α-SNAP. Based on the results of this complementation analysis, we conclude that all the three SNAP proteins of Giardia are orthologous to α- and not γ-SNAP.
The presence of three putative α-SNAPs that are expressed in multiple stages of the parasite's life-cycle raises the possibility that these may have undergone functional divergence during the course of evolution. Thus, while one of them may be under strong selection pressure to discharge the essential functions of an α-SNAP, the other(s) may have been adapted to perform alternative functions. To investigate if such functional divergence has taken place, we wanted to determine the subcellular distribution of the three α-SNAPs. Towards this, we raised polyclonal antibodies against α-SNAP17224 and α-SNAP16521 in rabbit and against α-SNAP10856 in mouse. Each of the polyclonal antibodies specifically detected only the corresponding protein that had been purified from E. coli as a band of ~34 kDa; none of them detected any of the other two α-SNAPs (Additional file 1: Figure S3a). Each antibody also detected a single band in G. lamblia trophozoite extract whose size corresponded to that predicted for the α-SNAPs of Giardia (Additional file 1: Figure S3a). Thus, in the absence of any observed cross-reactivity, it can be concluded that each antibody recognizes its target α-SNAP with a high degree of specificity. This also indicates substantial structural differences amongst these three proteins.
The polyclonal antibodies were used for immunolocalization of the three α-SNAPs in all the different stages of the parasite life-cycle in which their expression had been detected previously (Additional file 1: Figure S2). Given that all three genes can functionally complement the sec17-1 temperature-sensitive allele (Fig. 3), it is expected that these proteins will be associated with vesicles and/or membrane compartments such as the ER. Consistently, a previous study has already reported the localization of α-SNAP16521 to the PVs . We observed that along with α-SNAP16521, α-SNAP10856 also localizes to the PVs as the signal for this protein colocalizes with that of the fluorescent dye Lucifer yellow, which is endocytosed and delivered to acidic compartments (Fig. 4a; Additional file 1: Figure S4a). α-SNAP10856 also colocalizes with the PX domain-containing protein encoded by the ORF GL50803_16548, which is known to localize to the PVs (Additional file 1: Figure S4b) [34, 35]. Unlike the other two, α-SNAP17224 localized to the anterior part of the cell, mostly around the two nuclei; another pool of the protein appeared to be cytoplasmic (Fig. 4a). This difference in the subcellular localization between α-SNAP17224 and the two other α-SNAP paralogues indicates that the former performs a specific function that is different from that of the other two. Thus, there appears to be a functional divergence for at least one of the three α-SNAPs.
We observed additional evidence of such functional divergence in trophozoites undergoing encystation. While the pattern of localization of α-SNAP10856 in trophozoites and encysting cells remained the same, that of the other two paralogues changed significantly (Fig. 4a). At 8 and 16 h after induction of encystation, α-SNAP17224 still localized at the perinuclear regions, but its cytoplasmic distribution was no longer evident. Such a distribution persisted even after 30 h of induction (Additional file 1: Figure S5a). However, at 48 h post-induction, it was exclusively located at the PVs, with no signal at the perinuclear region (Fig. 4a; Additional file 3: Figure S9 and Additional file 2: Table S6). α-SNAP16521 also underwent a change in cellular distribution during encystation, but this change was much more rapid compared to α-SNAP17224. At 8 and 16 h post-induction, α-SNAP16521 was present exclusively at the paraflagellar dense rods (PDRs), which are electron-dense structures that are associated with the anterior, caudal and posteriolateral flagella (Fig. 4a; Additional file 1: Figure S5b and c, Additional file 3: Figure S9 and Additional file 2: Table S6) . This transition from the PVs to the PDRs starts as early as 1.5 h after the start of encystation as such cells exhibit both peripheral as well as distribution to the PDRs of the anterior and caudal flagella; complete redistribution of the signal appears to be completed by 4 h (Additional file 1: Figure S5b). The signal was more prominent at the PDRs of the anterior flagella, compared to that of the posteriolateral or caudal flagella. At the 48 h time-point, besides the signal at the PDRs, α-SNAP16521 was also observed at the periphery of the ventral disc (Fig. 4a and Additional file 1: Figure S5d). This α-SNAP continued to associate with flagellar structures even in the tetranucleated cysts and also localized to cytoplasmic puncta (Fig. 4a). α-SNAP17224 and α-SNAP10856 also exhibited a similar punctate distribution in cysts, without any association with the flagella. These puncta are likely to be vesicles as many of these are also positive for the lipid-binding PX domain-containing protein mentioned above (Additional file 1: Figure S6). Although the change in the pattern of subcellular distribution during encystation is unique to each α-SNAP, the pattern of expression of the corresponding genes was very similar during this period. Real-time PCR indicated that the expression of all the three genes remained largely unchanged during encystation, with significant downregulation observed only in cysts (Fig. 4b, lower panel). The expression of the gene encoding CWP1 served as a positive control (Fig. 4b, upper panel) . Such subcellular redistribution of proteins, without any change at the level of transcription, has been previously observed for other giardial proteins, such as β'COP subunit, Rab11, YiP, heavy chain of clathrin, DRP, ESCP and Rpn10 [13, 38–40]. Thus, many regulatory changes in this protist appear to be dependent on protein relocation, rather than synthesis of new proteins.
There are two situations in which two of the α-SNAPs exhibit a similar peripheral distribution in trophozoites or encysting trophozoites: (i) α-SNAP16521 and α-SNAP10856 in trophozoites and (ii) α-SNAP17224 and α-SNAP10856, 48 h post-induction of encystation (Fig. 4a). To determine if the two above-mentioned protein pairs colocalize, we carried out quantitative colocalization analysis for each pair under the conditions in which they exhibit similar cellular distribution. Scattergrams of multiple images of both the protein pairs did not indicate any substantial colocalization as the distributions of the green and red-colored pixels did not overlap significantly (Fig. 4c). Both the scattergram and the intensity plot of the two different fluorophores indicate that while there was some overlap for α-SNAP16521 and α-SNAP10856 in trophozoites, the same was not observed for α-SNAP17224 and α-SNAP10856, in encysting trophozoites (Fig. 4c). This is supported by colocalization analysis of multiple images wherein both the Pearson's correlation (Pr) and overlap coefficient (OC) values are below 0.5 for each protein pair (Fig. 4d). Incidentally, SNAP16521 and α-SNAP17224 never localize to the same subcellular region under any of the conditions tested (trophozoites, encysting trophozoites and cysts) (Fig. 4a). Given this lack of colocalization of the three α-SNAP paralogues, it may be concluded that there are functional differences among these three proteins.
Even though all the three paralogues function as α-SNAP in yeast, they exhibit heterogeneity in terms of their localization in Giardia. This scenario may arise if one or more of these paralogues do not function as an α-SNAP. Since α-SNAP and NSF assemble into the functional 20S complex that brings about SNARE unwinding, we examined if the three paralogues colocalize with the giardial NSF (GlNSF), which is encoded by the ORF 50803_114776. Immunolocalization of GlNSF in trophozoites was performed with an antibody (raised in rat) that specifically recognized this protein in giardial protein extract (Additional file 1: Figure S3b). It was observed that GlNSF localized to the PVs and the "brush border" structures associated with the part of the anterior flagella that is proximal to the basal bodies (Fig. 5a) . This distribution persisted in 48 h encysting cells, with the enhanced signal at the brush borders. While none of the α-SNAPs localized to the brush borders, all of them exhibit a peripheral distribution in either trophozoites or encysting trophozoites (Fig. 4a). Hence for colocalization of the α-SNAPs with GlNSF, conditions were chosen where each of the α-SNAPs exhibit a predominantly peripheral distribution, 48 h encysting trophozoites for α-SNAP17224, trophozoites for α-SNAP16521, and both trophozoites and encysting trophozoites (48 h) for α-SNAP10856 (Fig. 5a). Under each of the conditions tested, all the three α-SNAPs showed significant colocalization with the GlNSF at the cell periphery, but not at the brush borders of the anterior flagella (Fig. 5a, Additional file 1: Figure S7). Both the Pr and the OC values indicate significant colocalization in multiple cells (Fig. 5b). Since all three α-SNAPs colocalize with GlNSF and each of them functionally complements the sec17-1 temperature-sensitive allele (Fig. 3), it is likely that they function as α-SNAP in Giardia. Interestingly, barring α-SNAP10856, the two other α-SNAPs and GlNSF localize to regions where there is no overlap of the SNAP and NSF signals. These include the perinuclear regions for α-SNAP17224, the PDR for α-SNAP16521, and the brush borders for GlNSF. Thus, it seems likely that in addition to functioning in the 20S complex, each of these proteins also discharges additional cellular function(s).
Given that the three α-SNAP paralogues colocalize with GlNSF, we wanted to test whether each of these three proteins is capable of directly interacting with the latter. We used yeast two-hybrid to assess this binary interaction (Fig. 5c). It is known that yeast Sec17 physically interacts with its own NSF (Sec18) . Consequently, these two proteins exhibit strong interaction in our two-hybrid assay as cells co-expressing Sec17 from the bait vector (pGBT9 with TRP1 selection marker) and Sec18 from the prey vector (pGAD424 with LEU2 selection marker), exhibited robust growth, not only on plates lacking histidine (LTH 3-AT), but also on plates lacking adenine (LTA); in comparison to the former, the later selection medium imposes greater stringency as only cells harboring strongly interacting bait-prey pairs can grow in the absence of exogenously-added adenine . Results of the spot assay indicated that the interaction between GlNSF and α-SNAP10856 was comparable to that between Sec17 and Sec18 as yeast transformants expressing this giardial protein pair were able to grow on both LTH 3-AT and LTA plates (Fig 5c). However, estimation of the activity of the LacZ reporter gene, which provides a quantitative estimation of binary interaction, indicates that the interaction between the giardial proteins was weaker compared to that between the yeast proteins (Fig. 5d). Transformants expressing GlNSF and α-SNAP17224 exhibited growth on LTH 3-AT, but not on LTA plates (Fig. 5c). Even the β-galactosidase activity indicates that this giardial α-SNAP's interaction with GlNSF was weaker compared to that between GlNSF and α-SNAP10856 (Fig. 5d). However, there does not appear to be any interaction between GlNSF and α-SNAP16521 as there is no growth on both LTA and LTH 3-AT plates. Even the color of the spot growing on plates lacking leucine and tryptophan (LT) is comparable to those of the negative control (Fig. 5c), as is the β-galactosidase activity of this transformant (Fig. 5d). Based on these observations there appears to be a lack of binary interaction between α-SNAP16521 and GlNSF; however, we cannot rule out an interaction between these two proteins in vivo where additional cellular factors may stabilize this interacting pair. Taken together, the results of the yeast two-hybrid assay indicate that GlNSF interacts differentially with the three α-SNAPs, with the strongest interaction taking place with α-SNAP10856, followed by that with α-SNAP17224, and very little or no interaction with α-SNAP16521. This observed difference in the interaction of the three paralogous proteins with GlNSF further underscores the fact that the three giardial α-SNAPs are likely to have undergone functional divergence in this protist.
Based on the observed relocalization of α-SNAP17224 and α-SNAP16521 during encystation, we hypothesize that change in subcellular distribution of these two α-SNAPs may be part of Giardia's response to changes in the external environment. If this hypothesis is true, then similar changes may occur when cells encounter oxidative stress. We chose oxidative stress in particular because existing literature indicates that Giardia is likely to have a unique mechanism for handling this stress. Not only is Giardia unable to tolerate elevated oxygen levels, it lacks several key components that are used by most eukaryotes to detoxify cellular reactive oxygen species . Thus, while Giardia lacks enzymes such as catalase, glutathione peroxidase, and superoxide dismutase, its defense against oxidative stress includes noncanonical enzymes like an NADH oxidase, an NADH peroxidase, and a flavodiiron protein, to name a few . To investigate if the presence of multiple paralogues of α-SNAP may be a part of this organism's atypical mechanism to combat oxidative stress, we monitored the cellular distribution of these three proteins after inducing oxidative stress in trophozoites with two different agents, H2O2 (150 μM) and metronidazole (1 μg/ml) [45, 46]. Treatment with both reagents for a period of 1 h was sufficient to induce intracellular oxidative stress, as monitored by the conversion of DCFDA to the fluorescent DCF (Additional file 1: Figure S8). Following induction of oxidative stress, the relocalization of α-SNAP17224 and α-SNAP16521 was similar to that previously observed during the late stages of encystation (Compare Fig. 4a and Fig. 6; Additional file 3: Figure S10a and b, Additional file 2: Table S7). Thus, while α-SNAP16521 relocalized from the cell periphery to the PDRs, the signal for α-SNAP17224 moved from the perinuclear region to the cell periphery. However, unlike the change observed during encystation, a punctate cytoplasmic signal was also observed in both cases, with a cluster of puncta at the perinuclear region especially in case of α-SNAP17224 (Fig. 6). Consistent with the observed lack of change in subcellular distribution during encystation (Fig. 4a), α-SNAP10856 remained at the cell periphery even during oxidative stress (Fig. 6). However, like the other two α-SNAPs, cytoplasmic puncta were also observed in this case. Thus, in contrast to α-SNAP10856, the change in the distribution of α-SNAP17224 and α-SNAP16521 during both encystation and oxidative stress indicates that these two paralogues may have acquired additional functions during the course of evolution.
Although most eukaryotes encode only one α-SNAP, here we report that there are three paralogues in Giardia. These three genes are expressed in multiple morphological states of the parasite, thereby indicating that these three proteins are unlikely to discharge redundant functions in these different stages. Although α-SNAP10856 clusters with γ-SNAP sequences in the reconstructed phylogenetic tree, the results of the complementation analysis clearly indicate that it is functionally orthologous to α-SNAP (Fig. 3). The presence of three functional α-SNAP paralogues in a unicellular organism appears to be in stark contrast to most multicellular eukaryotes having only one homolog. Interestingly, the presence of multiple SNAPs has been documented in 47 protist genomes, including those of Trichomonas, Entamoeba, Naegleria, Trypanosoma and Leishmania and in silico analyses indicate that some of these putative proteins may be γ-SNAPs  (Dirk Fasshauer, personal communication). We have identified two SNAP paralogues in S. salmonicida and three in Trepomonas (Fig. 2). Thus, the presence of multiple SNAPs seems to be the norm for protists. Given that Protista represents a paraphyletic group, it is not possible to ascertain if a single gene duplication event gave rise to the expansion of the SNAP family, or if there were multiple independent such events. However, the fact that there is a difference in the number of SNAPs in closely-related diplomonad species, two in S. salmonicida and three in both Giardia and Trepomonas, indicates that later duplication events have also contributed to the expansion of this family of proteins in Protista.
While the results of the complementation assay indicate that each of the three paralogues are functionally analogous to Sec17 of yeast, the distinct subcellular localization of each protein in Giardia indicates that they do not perform redundant functions. In addition, results of the two-hybrid assay indicate that GlNSF had varying affinity for the three α-SNAPs. Given that GlNSF binds to α-SNAP10856 with the highest affinity, it may be hypothesized that the bulk of giardial cis-SNARE unpairing may be carried out by this paralogue. Its localization to the PV, where most of the SNAREs are present, lends support to this hypothesis . Notably, this distribution does not change even during encystation, and after exposure to oxidative stress. Conversely, GlNSF has little or no interaction with α-SNAP16521 and this paralogue relocalizes to PDR region where neither GlNSF nor the SNAREs are documented to be present, suggesting that NSF attachment may not be necessary for the function(s) this protein discharges at the PDR. However, since α-SNAP16521 localizes to the PVs in trophozoites, its participation in SNARE complex disassembly cannot be ruled out.
One reason for the presence of such complexity in this unicellular protist may be the necessity to maintain Giardia's asymmetric cell morphology. Unlike amoeboid protists, Giardia has a very unique tear-drop shape. The maintenance of this distinct asymmetric shape is likely to entail polarized vesicular trafficking to selective regions of the cell surface. However, the morphologically-simple endomembrane system of Giardia appears to lack key sorting stations, such as the Golgi. In the absence of readily-identifiable sorting compartments, Giardia may have evolved another system of determinants that allow selective targeting of vesicles to specific regions of the plasma membrane so that the shape of the cell is maintained. α-SNAP is already known to participate in such selective targeting in metazoans; it has been documented to enable polarized sorting to the apical surface of neuroepithelium as a single mutation causes missorting of apical proteins such as E-cadherin, β-catenin and F-actin . The factors that play a role in giardial cell shape maintenance are difficult to trace as mutant hunts cannot be undertaken in this polyploid organism. Thus, it would be interesting to determine if there are any morphological changes following selective ablation of any one of these α-SNAPs.
The presence of α-SNAP17224 and α-SNAP16521 at cellular locations where NSF is not detectable is intriguing (Figs. 4a, 5a). One possibility is that α-SNAP may be performing NSF-independent functions at these locations. Existing literature indicates that α-SNAP has various NSF-independent roles . An example of this is α-SNAP's involvement in regulating calcium uptake via calcium release activated calcium (CRAC) channels . Hexameric assemblies of the Orai1 protein at the PM forms the CRAC channel pores and this assembly process is regulated by α-SNAP as its deficiency results in loss of calcium selectivity of the CRAC channels, leading to dysregulated entry of sodium into the cell . In another instance, α-SNAP interacts directly with the Bcl-2 family member, BNIP1, which is known to regulate the morphology of the ER . Incidentally, we have also observed enhanced α-SNAP17224 signal at perinuclear regions, which are known to be occupied by the ER (Fig. 4a). The distribution of α-SNAP16521 at the PDRs is also indicative of a noncanonical role for this paralogue. Other proteins of Giardia with similar localization at the PDR include four proteins belonging to the Nek family of kinases (ORFs 5375, 92498, 16279 and 101534) and the catalytic subunits of protein phosphatase 2A (PP2Ac) and protein kinase A (PKAc) [53–55]. Nek proteins are documented to regulate ciliary function and assembly . They also coordinate ciliary function with the cell cycle as they regulate the function of the centrioles, which serve both as basal bodies and microtubule-organizing centers . Consistently, a study encompassing a large number of unikont and bikont organisms indicated that there is a direct correlation between the number of Nek genes encoded by a given genome and the presence of diving ciliated cell(s) in that particular organism . Giardia has 56 active Neks, although its genome encodes 198; no other genome encodes such a large number of Neks and it is hypothesized that the expansion of this family of kinases is necessary to solve the challenges of coordinating the division of eight flagella with the cell cycle [58, 59]. Both PKAc and PP2Ac localize to the PDR in trophozoites [53, 54]. Since the signal for both proteins disappear simultaneously from the PDR of the anterior flagella early in encystation, it is postulated that they function in the same pathway in Giardia [53, 54]. PKAc is a known regulator of flagellar motility through its phosphorylation of dynein . Thus, it is possible that since several other proteins that regulate flagellar function also localize to the PDRs, α-SNAP16521 may play a similar role. The difference in the α-SNAP16521 signal intensity at various PDRs may be because of differences in motility of each flagellar pair . It may be noted that we have not detected the α-SNAPs at all the subcellular location where SNAREs of Giardia are known to localize . For example, while gQb3 localizes to mitosomes, we have not observed similar distribution for any of the α-SNAPs. However, we cannot rule out the presence of a minor, and therefore undetectable, pool of any of the three α-SNAPs at other locations within the cell.
The present study has uncovered the presence of three paralogues of α-SNAPs in G. lamblia. Expression of these three genes during multiple morphological states of the protist indicates that the function of each of these three α-SNAPs is required by the cell. Thus, these must be performing non-redundant functions. Antibodies raised against each of these three proteins were used to determine their subcellular distribution during different stages of the parasite's life-cycle, and also upon exposure of the trophozoites to oxidative stress. These immunofluorescence experiments indicated a non-overlapping pattern of subcellular redistribution, without any accompanying change in the expression of the corresponding genes. In addition, two-hybrid assay established that these three paralogues have varying affinity for GlNSF. Taken together, it may be concluded that the three α-SNAP paralogues have undergone functional divergence in this protist. It was observed that α-SNAP16521 localizes to the PDRs associated with the anterior, posterolateral and caudal flagellar pairs. Given that nither GlNSF nor any of the SNAREs localize to the PDRs, it is possible that this paralogue performs an NSF-independent function. Thus, following duplication of the α-SNAP genes of Giardia, there may have been neofunctionalization of some of the paralogues.
The protein sequences of Giardia SNAPs were curated from GiardiaDB and secondary structure predictions were carried out using iterative threading assembly refinement (I-TASSER) and Protein Homology/analogY Recognition Engine (Phyre2) servers [62, 63]. By default, both approaches used the crystal structure of S. cerevisiae Sec17 (PDB ID: 1QQE) as a template . Based on these predictions, the secondary structural elements were marked on the multiple-sequence alignment that was generated with ClustalW, with editing in Jalview [64, 65]. Three-dimensional automatic threading models were generated in I-TASSER server, with the crystal structure of Sec17 as a template. The conserved positively charged residues were marked in Pymol .
Sequences of both α- and γ-SNAPs from organisms were curated using either NCBI (https://www.ncbi.nlm.nih.gov), UniProt (https://www.uniprot.org) or Eukaryotic Pathogen Database Resources (https://eupathdb.org) [67–69]. Domain analysis was performed with Pfam to ensure that the identified sequences contained a SNAP domain . The curated sequences were used to reconstruct a maximum likelihood tree using MEGA7, with 100 bootstrap replicates .
Trophozoites of Assemblage A isolate Portland-1 were grown in slanted 15 ml culture tubes containing Diamond TYI-S-33 medium (pH 6.8) and the encystation was carried out as previously described [72, 73]. In order to obtain a pure preparation of cysts, 72 h post-induction of encystation, cells were harvested and incubated in distilled water at 4 °C, for 24 h for selective lysis of trophozoites that did not undergo encystation.
The temperature-sensitive strain RSY269 (MATα ura3-52 his4-619 sec17-1) was used for functional complementation . SEC17 and all the three Giardia α-SNAPs were PCR amplified with primers are listed in Additional file 2: Table S4. The PCR products were cloned under the control of a constitutive yeast promoter, in a 2 μm vector having URA3 as a selectable marker (Additional file 2: Table S5). Each construct was individually transformed into RSY269. Resulting transformants were grown overnight in liquid YCM lacking uracil; serial dilutions of these cultures were spotted onto YCM plates lacking uracil and incubated at 30 and 37 °C.
Each of the three giardial α-SNAPs was expressed and purified from BL21 (DE3) as previously described, except 0.2 mM IPTG was used . The N-terminal region of GlNSF was also induced with the same concentration of IPTG but was purified from the pellet fraction, as previously described . The primers used for cloning in pET32a are listed in Additional file 2: Table S4. The purified proteins were used to raise antibodies against α-SNAP17224 and α-SNAP16521 in rabbit, against α-SNAP10856 in mouse and against GlNSF in rat. All animal experiments adhered to the guidelines approved by the Institutional Animal Ethical Committee of Bose Institute (IAEC/BI/37/2015).
Immunofluorescence was performed in trophozoites, encysting trophozoites (time of encystation indicated in respective figures) and cysts as previously described . Briefly, cells were harvested by chilling the culture tubes on ice, followed by centrifugation at 1000× g for 10 min. After washing with 1× PBS, the cells were fixed with 4% formaldehyde for 20 min at room temperature (RT). Following fixation, cells were harvested by centrifugation and treated with 0.1 M glycine for 5 min at RT. Subsequently, trophozoites and encysting trophozoites were permeabilized with 0.1% Triton X-100 in 1× PBS (v/v) for 15 min, followed by blocking with 2% BSA for 2 h at RT. Cysts were permeabilized with 0.2% Triton X-100 and 0.1% SDS for 15 min, before blocking with 2% BSA. For labeling of all the four giardial proteins, the respective primary antisera were used at 1:50 dilution in 0.2% BSA and incubated for overnight at 4 °C, with shaking. The following day, cells were washed thrice with 1× PBS and incubated with 1:400 dilution of any combination of the following secondary antibodies, as per requirement: Alexa Fluor 488-conjugated goat anti-rabbit, Alexa Fluor 488-conjugated goat anti-mouse, Alexa Fluor 594-conjugated goat anti-mouse, Alexa Fluor 594-conjugated goat anti-rabbit and Alexa Fluor 488-conjugated goat anti-rat. All secondary antibodies were procured from Abcam (Cambridge, UK). Before washing away secondary antibodies, cells incubated with DAPI at 1 μg/ml concentration for 15 min. Finally, cells washed three times with 1× PBS and resuspended in antifade medium (0.1% p-phenylenediamine in 90% glycerol). Samples were imaged with the 63× objective of a confocal laser scanning microscope (Leica TCS SP8, Wetzlar, Germany). 3D deconvolution and colocalization analyses were performed with Leica Application Suit X and images were assembled with Adobe Photoshop CS3 and Adobe Illustrator CS3.
Statistical analyses for all the colocalization studies were performed with Leica Application Suit X (LAS X) software. The correlation coefficients, Pearson (Pr) and overlap (OC), values for each colocalization experiment were calculated on the basis of pixel-wise correlation between the signals emitted by the two fluorophores in each layer of six independent Z-stack. The Pr and OC values were plotted with GraphPad Prism 5.
cDNA preparation and real-time PCR was performed as previously described, with primers designed against unique regions of giardial α-SNAPs (Additional file 2: Table S4) [35, 40]. PCR condition was as follows: initial denaturation at 95 °C for 5 min, second denaturation at 95 °C for 30 s, and annealing for 20 s at 56, 64.5 or 65.8 °C for SNAP17224, SNAP16521 and SNAP10856, respectively. The CT values obtained for the three α-SNAP genes were normalized against the expression of ribosomal protein S5 (GL50803_12981), while the expression of the CWP1 gene served as positive control for encystation [37, 46]. Each experiment was performed in triplicate, with three technical replicates for each and data validation was done using two-tailed, paired t-test analysis in GraphPad Prism 5.
Yeast two-hybrid assay was performed using full length NSF and α-SNAPs of both yeast and Giardia, which were cloned in pGAD424 (prey vector having LEU2 marker) and pGBT9 (bait vector having TRP1 marker), respectively (Clonetech Laboratories, Mountain View, USA); the resulting fusion proteins had either the Gal4 activation domain (AD) or its DNA binding domain (BD), respectively (Additional file 2: Table S5) . As per requirement, different pairs of the AD and BD constructs were co-transformed into the yeast strain PJ69-4A and the growth of each transformant, on YCM plates either lacking leucine, tryptophan and adenine (LTA) or lacking leucine, tryptophan and histidine, but having 2.5 mM 3-AT (LTH 3-AT), was monitored following incubation at 30 °C for 2 to 3 days . The binary interaction between the various NSF and α-SNAP orthologues were also measured quantitatively by assessing the β-galactosidase activity by determining the nmol of o-nitrophenol formed from the hydrolysis of ONPG per min per mg of protein . For this assay, experiments were performed in triplicate with two technical replicates for each sample. Results were statistically validated using a two-tailed, paired t-test in GraphPad Prism 5 software.
Trophozoites were grown to confluency. The old medium was replaced with freshly-prepared medium and cells were allowed to grow for another 2 h, prior to treatment with oxidative stress-inducing agents, either 150 μM H2O2 or 1 μg/ml metronidazole, for a period of 1 h, at 37 °C [45, 46]. To confirm intracellular ROS generation, cells were first harvested by chilling the tubes on ice, harvested by centrifugation at 1000× g for 10 min, washed thrice with warm PBS and treated with 2',7'-dichlorodihydrofluoresceine diacetate (H2DCFDA) (Sigma D6883, St. Louis, USA) at a concentration of 1.5 μM, for 15 mins at 37 °C . Finally, cells were fixed with 2% paraformaldehyde, washed thrice with PBS and observed under a confocal microscope.
We acknowledge Dr A. K. Sil for his valuable suggestions during the course of this study and also for critically reading the manuscript. We thank Professor Randy Schekman and Professor Peter Novick for providing yeast strains. We acknowledge the Central Instrumentation Facility of Bose Institute, Kolkata, India, for DNA sequencing and confocal imaging and thank its staff members A. Poddar and S. Ghosh-Chakraborty for imaging and S. Biswas for CD spectroscopy. We are thankful to Dr A. Ghosh for helping with database searches and Dr A. Banerjee of Leica Microsystems for providing technical assistance during image processing. We acknowledge the assistance of D. Samanta and P. P. Dey of the Centre for Translational Animal Research, Bose Institute. We thank Professor P. Chakrabarti and Dr A. K. Sil for allowing us to use their laboratory facilities. We also thank members of the Sarkar Laboratory, Bose Institute, Kolkata, India, for proofreading the manuscript and providing valuable comments.
This study was supported by funds provided by the Bose Institute, Kolkata, India. SPD received fellowship support from the INSPIRE programme of the Department of Science and Technology, Government of India (IF120153).
SPD and SS conceived the study. SPD and AM carried out the experiment. KJ raised the antibodies. SG participated in the study design, provided critical inputs and helped draft the manuscript. SPD and SS analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.
All animal experiments were carried out under the approval of the Institutional Animal Ethics Committee, Bose Institute, Kolkata (IAEC/BI/37/2015).
Kienley N. Phylogenetic studies of the vesicular fusion machinery. PhD Thesis, Georg-August-Universität, Göttingen; 2010. https://d-nb.info/1042305226/34.
DeLano WL. The PyMOL Molecular Graphics System. DeLano Scientific LLC, Palo Alto, CA, USA. 2002. https://www.pymol.org. Accessed 21 Feb 2018.


A U.S. Coast Guard helicopter spotted the body of an Illinois man Monday morning off of Porter Beach after he apparently drowned while kayaking Sunday, according to a release from the Indiana Department of Natural Resources' Division of Law Enforcement.
Porter County Coroner Chuck Harris identified the man as Alex Lopez, 21, of Plainfield, Ill. Lopez's body was recovered around 7 a.m. Monday in 10 feet of water by the Porter Volunteer Fire Department.
Lopez, who was kayaking with friends Sunday afternoon and fell out of a kayak, was not wearing a life vest at the time of recovery, Harris said, adding the investigation is ongoing and toxicology tests are pending.
An investigation revealed Lopez and two friends were kayaking approximately 150 yards offshore from Porter Beach in the Indiana Dunes National Lakeshore when their kayaks capsized shortly after 5 p.m. Sunday, according to the release.
Lopez went under the surface of the water and did not return. His two friends were wearing life jackets and were able to stay afloat and summon help, according to the release.
Emergency responders from several agencies responded to the scene and initiated a search for Lopez that lasted several hours. After dark, the search was postponed until early Monday morning.
A U.S. Coast Guard helicopter located a body floating in the water near the area around 7 a.m. Monday and directed members of the Porter Volunteer Fire Department to the area to assist with recovering the body, which was positively identified as Lopez.
Conservation officers are investigating the incident as a boating accident and the cause of death is suspected accidental drowning. Alcohol may have been a contributing factor in the incident, according to the release.
Conservation officers were assisted by Indiana Dunes National Lakeshore park rangers; Porter Police; U.S. Coast Guard; Lake County Sheriff's Office; and the Burns Harbor, Chesterton, Portage and Porter fire departments.


We all knew it was coming, we just didn't know in what form. Today in New York City, TAG Heuer CEO Jean-Claude Biver introduced the brand's very first connected wristwatch. The TAG Heuer Connected is a 46 mm watch that looks very much like the new mechanical Carrera with Caliber 01 inside, but instead of a self-winding, Swiss-made movement, we have a Google-powered operating system.
The watch takes the shape of a modern TAG Carrera, with a 46 mm case made of grade 2 titanium, complete with titanium deployant buckle. The stock black rubber strap may be swapped to a host of different colors, and the watch looks and feels very much like any TAG. However, there are no chronograph pushers here, just one crown that acts as the control center for the connected timepiece.
The watch features three different TAG Heuer faces – a true chronograph, a three hander, and a GMT – and the chronograph works by tapping the screen once to start and stop, and twice for resetting. It is important to note that this is not a hybrid at all, but rather a true smartwatch. There is nothing mechanical about it.
The price of the Android powered TAG Heuer Connected smartwatch will be $1,500 and it will be available for purchase in the U.S. at TAG stores and online today. The cleverest thing about the entire watch is that after the two-year plan on the TAG Heuer Connected watch is up, any owner may trade it in to a TAG retailer, and for an additional $1,500, receive a mechanical Carrera in its place. An interesting feeder program if there ever was one.
Oh, and to answer your #watchnerd question: the watch cannot be "Swiss Made" because the movement is digital, but the case is indeed Swiss made – a nice touch, certainly.
Full specs below, and to watch our video on the TAG Connected watch, click here. You can read more about it at TAG Heuer online.


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Previously there was fragmentation with opportunity to take part in the event throughout the district, each area ran on slightly different formats / rules. Whilst the rules were clearly distributed, they were not always endorsed which led to lots of issues when selecting a team for the Level 3 event.
There were issues around quality of young leaders umpiring the High5 Netball.
We consulted with all Secondary School colleagues and it became clear that space to host the events and sufficient young leaders to run the event were a major issue.
We organised An All England Netball High5 Workshop, to be delivered locally, at no cost to participants, as a twilight after school session. We targeted the Secondary Schools to send young leaders and invited partner Primary Schools to send teachers / staff. Although, we only had a '10' day turn around, as we felt is was critical to hold the workshop before our cluster competitions started, we managed to attract sufficient candidates (16) to run the course.
Due to huge numbers of schools (21 schools) taking part in the competition, we had to identify '4' different locations across the district to accommodate the event, and identify sufficient volunteers to run the event. We contacted the local Netball Club who did provide qualified volunteers, we contacted Newton Rigg College who not only provided one of the sites, they also identified '8' young leaders to help run the event.
We dramatically improved participation rates.
Very many positive comments on the day from teachers and parents about the management of the event. One Headteacher said, 'I knew it would be a great event when you were organising the day'.
All schools are very clear about the rules and the criteria to enable them to go through to the Partnership final, which will then select a team for the School Games Final.
Success factors include consultation with lots of partners in Eden.
Making a great contact with Newton Rigg College which will hopefully grow and have mutual benefit.
We hadn't planned for such huge numbers entering the event, so next time we will plan and have sufficient venues in place from the start.


text:
Q: Swift - UIScrollView Scrolls Partially My problem is slightly different from other's 'Swift UIScrollView' problems when using auto layout:
Problem:
Unlike others, when I run my app, it scrolls. My problem is that the scroll cuts off the bottom 20-30% of the content. I can pull to see the buttons did build and are down there, but when I let it go the scroll snaps back to a false bottom which cuts off my content!!! I've been trying, for days, to fix it to scroll the entire height but it continues to cut off!!
Description of app:
I used auto layout to layout 6 buttons and labels. Each button a rectangular image, with a label directly beneath it. (sorry, the site won't let me post pictures!)
I have my views arranged like this:
MainView > ScrollView > ContentView > Buttons & Labels
I have my contentView pinned to my ScrollView and my ScrollView pinned to my MainView. My buttons and labels all have constraints that are building correctly, to create a list that looks like:
Rectangular button
Label beneath it
Spacing
Rectangular button
Label beneath it
Spacing
Etc.
Can anyone tell me why I can't scroll the full length of the view?

A: Your description of how your items are constrained is vague, so I'm going to list all of the constraints you need to make this work.  Then you can compare what you have to what you need and adjust accordingly.

*

*Your ScrollView should be pinned on all 4 sides to the MainView.  (This isn't absolutely necessary.  You can constrain your ScrollView however you want, but make sure it can grow as the device and/or orientation changes).

*Your ContentView should be pinned on all 4 sides to the ScrollView with offsets of 0.

*Since you want your ScrollView to scroll vertically only, constrain the width of the ContentView to the width of the ScrollView using an Equal Widths constraint.  To do this, in the Document Outline view, Control-drag from your ContentView to your ScrollView and select Equal Widths from the pop up.

*The height of ContentView will be set by the sum total height of everything in it.  In order for this to work, your topmost button needs to be pinned to the top of the ContentView, all of your buttons and labels should be pinned to their nearest neighbors, and the bottommost label should be pinned to the bottom of the ContentView.  In addition, all of your buttons and labels should have constraints for their widths and heights.  I would suggest setting an explicit width constraint and explicit height constraint for your buttons and centering them horizontally in the ContentView.  For your labels, set an explicit height constraint and pin the left and right edges to the ContentView.
If you have these constraints and no other ones, your ContentView will be properly sized.

A: Using contentView, like you said, usually fixes the issue. So chances are you need to take a second look at your contraints. Maybe try this solution in a clean/new project to see that it works. (it does work). My guess would be that some of your constraints conflict each other.
Otherwise I think it would be a good idea to setContentSize of your scrollView in your viewDidLoad.
Another hack would be to place 2 UI objects with their alphas set to zero on the right top corner and left bottom corner. This would hint scrollView to set its contentSize.

redpajama_set_name: RedPajamaStackExchange


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First Vice President of the Pan-African Parliament (PAP) Stephen Masele underscored the importance of Egypt's chairmanship of the African Union under the leadership of President Abdel Fattah El Sisi.
The PAP and the African Union (AU) will support Cairo and closely coordinate with it in the coming stage, Masele told MENA on Saturday ahead of the AU Summit, slated for Sunday and Monday.
The Tanzanian MP lauded President Sisi's efforts and his keenness to support the youth and provide them with opportunities to take part in the decision-making process. These efforts are a model to follow in African states, he added.
He also commended Sisi's efforts to confront terrorism, noting that the Egyptian leader has great experience in dealing with this phenomenon.
President El-Sisi has successfully won the war against terrorism in Egypt amid security problems suffered by neighboring countries, such as Libya, in addition to a state of instability sweeping the region, Masele said.
On that score, the PAP vice president praised Cairo's efforts to help achieve peace and stability in Libya, which he said is a key issue for Egypt and for all African countries.


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text:
Jeb's time warp
Is he ready for a rapid-response, Twitter-driven modern campaign?
By DARREN SAMUELSOHN and MAGGIE HABERMAN
Jeb Bush is trying to show he's ready to run a modern-day presidential campaign: When he declared last week that he's "actively" exploring a presidential run, the former Florida governor went straight to Facebook and Twitter instead of the more traditional route of a speech or news release.
But Bush will have a lot more work to do than just showing off his social media proficiency to prove he's not full of cobwebs. It's been 12 years since he last ran for anything. And while GOP donors are excited about another Bush making a presidential run surrounded by top-notch staff and all the high-tech gadgets he can buy, it's another thing to campaign in the real-time Web, where a delayed reaction or a thin-skinned response can quickly dominate a news cycle.
In the 48 hours after Bush made his announcement, Bush was outflanked by Sen. Marco Rubio on the Cuba debate. Then Sen. Rand Paul beat Bush to the punch on one of the fundamentals of an online campaign: controlling the space atop Google search results for his name. Paul's RandPAC bought ad space tied to searches for Bush and used the prime real estate to poke the former governor's politics.
( Also on POLITICO: George H.W. Bush hospitalized)
"I think this is one of the biggest challenges, the fact he hasn't run for office since 2002," said Craig Stevens, a former George W. Bush administration spokesman. "That's why I wouldn't have been surprised if he didn't decide to run, because it's such a different game."
"There's almost nothing in common between the campaign environment of 12 years ago and the campaign environment today. So much has changed. A new entrant like this is going to have to adjust to that," added Kurt Luidhardt, vice president and co-founder of the Prosper Group, a GOP digital media firm with ties to two other potential 2016 candidates, New Jersey Gov. Chris Christie and Indiana Gov. Mike Pence.
Bush isn't the only potential candidate facing challenges in the rapid-response Internet era. Hillary Clinton was outgunned on technology by the Obama campaign in 2008 and has made sparing use of Twitter, employing it essentially as a press release outlet. She has been praised for how her team at the State Department used social media in global policy. But campaigns are a different story, and her task getting up to speed isn't that different from Bush's.
Bush will no doubt have the money to assemble a strong media and digital campaign. But he's also rusty, and that opens the presumptive GOP front-runner to the kinds of early campaign trail flubs that his advisers will be working hard to avoid.
( Also on POLITICO: Let the good times poll)
"He's going to be out of practice. No matter what you do, whether it's playing piano, shooting basketball or doing campaign work, you've got to get back into practice," said Stevens, who also worked on the George W. Bush 2000 and Romney 2008 campaigns and now lives in New Hampshire.
Mindy Finn, a digital GOP operative who has worked for George W. Bush and Mitt Romney, said Jeb Bush is entering a dangerous media environment in dealing with "how quickly a small issue can become a big issue and kind of take over a campaign." Bush's status as a favorite of the establishment wing of the party also makes him an inviting target for lower-tier candidates to take on through social media.
"It's a David vs. Goliath dynamic that I think any candidate that underestimates that does that at their own peril," Finn said.
In a contrast to Bush, fellow Floridian Rubio was out ahead early on the news about the U.S. normalizing relations with Cuba. Rubio, who over the past year has been building up his operation ahead of a potential campaign, appeared in front of reporters in the Capitol about 15 minutes after Obama ended his remarks from the White House and also had a statement out hours ahead of Bush. By contrast, both Bush and Clinton issued statements electronically, and in Clinton's case, late in the day.
( Also on POLITICO: Sen. Elizabeth Warren blasts FDA proposal on gay sex)
Paul also struck fast against Bush via Google, demonstrating with an inexpensive online search results ad just how much the campaign world has changed. The Kentucky Republican's PAC — which already has a digital director on hand, former Mitch McConnell and Ted Cruz campaign adviser Vincent Harris — secured the paid advertising slot on all "Jeb Bush" Google search results in the Washington, D.C., area. The two ads were short, touted Paul and went directly at Bush's conservative bona fides.
"Join a movement working to shrink government. Not grow it," one ad read.
Republicans say Bush's failure to fire back in real time is a troubling early sign that stems from his limited campaign team. "He is behind right now," Stevens said.
Bush was also burned by the abbreviated news cycle in early 2013, when he described illegal immigration as an "act of love." Conservatives pounced, and that headline dominated news for days until his next public appearance, when he tried to clarify. Clinton was similarly seared during the 2014 midterms when an unusual statement about corporations not creating jobs went viral over a weekend, and she didn't address it until she'd already held several other appearances.
If he takes the next step of formally launching a campaign, Bush would presumably staff up quickly with a large team that's expected to include operatives from his brother's presidential teams, as well as longtime allies going back to his Florida governor days. At a closed-door strategy session of GOP tech operatives earlier this month, first reported by POLITICO, media consultant Mike Murphy served as Bush's informal representative.
But some other top digital talent is very likely spoken for already. Targeted Victory, one of the largest GOP digital shops, has ties with two other potential 2016 contenders: Mitt Romney and Paul Ryan. And other Republican tech firms and individual consultants say they are hoping to maintain their connections with potential 2016 candidates, from Rubio to Paul to Cruz, Christie and Bobby Jindal.
Bush is no stranger to technology, and, when he was last politically active, saw it as a point of pride.
He regularly shared his email address – [email protected] — during speeches around Florida and kept up regular conversations with middle school children, reporters and pretty much anyone else who wanted to write him. His official Florida governor portrait showed his BlackBerry in the background.
"I was digital before digital was cool, I guess, now it's like commonplace," Bush told WPLG-10 earlier this month where he also said he planned to publish an e-book as well as release 250,000 emails from his time in Tallahassee.
Apart from his presidential announcement, Bush's recent Facebook posts are a mix of press statements, defensive swipes at Democrats and up-close photos: Jeb during a Chanukah menorah lighting and hugging his son George P. Bush after he was elected Texas land commissioner.
On the day of his announcement that he would establish a leadership PAC, Bush's Facebook likes jumped by 10 percent: to a little more than 122,000. And while followers are hardly seen as the best metric for online success, it remains one key indication of how he's doing. And in that respect, Bush is miles behind Romney's 11.3 million likes; Ryan's 5 million; Paul's 1.8 million; Rubio's 709,000; or even Christie's 175,000.
In a 2003 Washington Post article, Bush responded via email to a series of questions from reporter Mark Leibovich about his online habits that dated back at that point about a decade. His account [email protected] received about 200 to 300 messages a day. "I use it now to keep me connected to friends and constituents," he said.
But email is a contained, specific medium. Twitter is essentially an electronic town hall. It also remains to be seen how Bush will make his emails public — whether they're easily searchable online or not.
Some Democratic operatives believe Bush will be eaten alive on social media by his antagonists on the right. But allies of the former governor say he'll be fine, especially with the help of a resurgent party infrastructure that demonstrated in 2014 that it is just as capable of using all the data, digital advertising and get-out-the-vote microtargeting that Obama used to great effect.
"If he does decide to run, we'll make a commitment to digital technology at the center of our campaign at a level never seen before in U.S. politics," said a source close to Bush. "We're working with the best people in the business."
Steven Moore, a manager director at the GOP digital firm CampaignGrid, said Bush's tech background demonstrates he'll make the right moves in a presidential campaign. "He's not been living in a cave for the last 12 years either," said Moore, whose firm worked on Christie's 2013 gubernatorial campaign.
"He's a very bright man and clearly is an innovator and has been an innovator. I'd expect that innovator ethos would carry forward," Moore added. "There's no reason to believe he's suddenly going to switch to an IBM Selectric typewriter and carry a BlackBerry, with all due respect to the BlackBerry people."

redpajama_set_name: RedPajamaCommonCrawl


During the next seven days receive 35% off all New Society print and ebooks (excludes New Catalyst titles) when you order online at www.newsociety.com. Just enter the code SUMMER18 at check out and hit redeem. And while you wait for your books to arrive you can check out our Summer Reading Sale blog where we will feature books that inspire you to grow, make, build and change!


So, it's no secret that I love pretty bikes. Literally a pretty frame colour and I am sold.
So, it's no secret that I love pretty bikes. Literally a pretty frame colour and I am sold. So, imagine how I felt when Liv Cycling released a gorgeous looking TT bike (Liv Avow) this year.
I didn't buy it on impulse when it was first released, I kept considering it for quite some time. When I still wanted it a few months later I decided if I would train for and ride Ironman Italy on it then that was justified. So, I purchased it from Giant in Chelmsford. There were some modifications I wanted to do before riding it though, I wanted to change the gearing to Shimano Di2 (electronic) and to change the tri bar set up to one I knew to be comfortable. I also wanted some new wheels. So, this meant that whilst I have owned the bike for a while my first outing on it was just 3 days before the Cotswold Classic Triathlon.
I took it out on the Thursday evening before the race and it was a bit disastrous, the base bar wasn't correctly tightened so within 2 miles my tri bars were pointing to the floor. Not a great start, stopped and sorted them out and did a few miles on the hoods, just getting used to the feel of the bike, it handles differently to my road bikes and the brakes felt quite different. After a few miles I was a little more confident and tried the tri bars, I soon worked out I needed to tweak the position a bit as my knees were meeting my elbows and the angle my arms were at was putting too much pressure up through my shoulders. My other half Adam was out with me and was very patient each time we needed to stop and make some adjustments. It was literally a case of ride 2 miles, stop, tweak, ride another 2, stop tweak again. But eventually I was reasonably comfortable, and we headed home.
The next day I took the bike out for a little 12-mile loop close to home, just to check I felt ok on it, then it was time to get everything ready for the triathlon as we were leaving Saturday morning to get to the Cotswolds and get registered etc. ready for me to race on Sunday.
I haven't done a triathlon since Dubai 70.3 in February. A shoulder injury just before Dubai meant I struggled in Dubai, then the recovery from that, coupled with other events meant that I have done very little swimming this year, and no real triathlon focused training. I entered this event as a test race before Ironman Italy as I really wasn't sure how I would cope.
Morning of the event, usual 4am alarm and a struggle to make myself eat (I eventually managed half a bagel) then we headed to the start, racking was quite easy with each competitor's area clearly numbered, so I set up my transition and headed towards the swim start.
I am always crazily nervous before the swim start, I really don't know why, I'm not a bad swimmer, nothing terrible has ever happened to me in a mass swim start but I really don't like it. The water temperature was just over 22 degrees and I had planned to swim non-wetsuit, but the air temperature felt cold, so I had a last-minute change of plan and put my wetsuit on. I really did not want to start, and right up until the countdown I was considering changing my mind and just spending the day watching by the lake. I didn't want to race. Adam was super supportive, knew I was feeling awful, helped me get my wetsuit on, went to buy me a bottle of water and made sure I got in to the water.
The swim was fine, after about 200mtrs I was regretting the wetsuit decision, but too late to do anything about it. All of my wave started off super-fast, so I had plenty of space for about the first half a mile, and I caught the majority of the pack about half way through the swim. 45 minutes later the swim was over. Now time to test out the new bike!
Running out of transition somehow my race belt had loosened, so there was a moment of flapping just over the mount line while I had to stop to adjust it! Then off and on to the bike leg. The route was 2 x 28-mile laps, reasonably flat, a 'hill' of sorts about half way into the lap, followed by a few undulations, then back to being reasonably flat again. The first lap took me 1hr 32 mins, and the 2nd lap 1hr 36 mins (a little slower but the hills felt bigger a 2nd time round and I also had to stop at a couple of junctions) so 3hrs 8 minutes later I was back in transition. Not a ground-breaking bike leg, but a good solid 17.5mph and on a bike I was unfamiliar with I was quite pleased. I probably spent 50% of the time on the bars and 50% in the hoods, but I don't think that's a bad ratio considering I'd only ridden the bike twice.
Then there was just the run left to go. The run was predominantly trail, it runs around the back of the swim lake, then out on to a road for a short while before heading into a country park. It was pretty, I hadn't researched the event very well so the fact it was off road threw me a bit, but also gave me something to focus on and made me concentrate on picking my feet up. It was 3 loops of 4.4 miles and I was doing ok until halfway. 10km into the run I lost the motivation to move and had a few walks along the road section in the sun. It was 32 degrees, so a lot of people were feeling the heat quite a bit. My run became a fast walk/jog in sections, but I was ok with this, better to be able to finish than to push too hard in the heat and struggle.
6 hours and 29 minutes later I finished. 2nd triathlon of the year done, reasonably successfully in extreme heat with a new bike. No PB's but a good solid day. I also finished in position 666... Make of that what you will.
I really enjoyed the event, it was just the right size, small enough to feel quite personal, but big enough that there were always other competitors around. The marshals were super friendly and enthusiastic, great little crowd of dedicated supporters as you came through the bike and run laps. The location was also incredible, the most picturesque setting. I will almost certainly be back.


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Human Rights Law Review1
HRLR Online2
A Jailhouse Lawyer's Manual3
1The flagship journal, each volume published in three issues every academic year.
2An online forum for rigorous discussion and analysis of current issues in human rights law.
3A handbook of legal rights and procedures designed for use by people in prison.
JLM Fundraiser
The Time Trap: Addressing the Stereotypes that Undermine Tribal Sovereignty
Issue 53.1
Adam Crepelle, Assistant Professor, Antonin Scalia Law School, George Mason University; Director, Tribal Law & Economics Program, at the Law & Economics Center; Associate Professor and Managing Fellow, Native American Law and Policy Institute, Southern University Law Center; Campbell Fellow, The Hoover Institution at Stanford University; Associate Justice, Pascua Yaqui Tribe.
History is deeply embedded in federal Indian law. According to jurisprudence, Indians were nonagricultural "savages" prior to 1492. Indians' supposed lack of sophistication played a vital role in foundational cases determining Indian rights and the extent of tribal sovereignty. The process of stare decisis has resulted in repetition of the principles formulated on the belief in Indian simplicity; consequently, historic ideas of Indians continue to impact present-day Indian rights—often for the worse. This is the time trap.
The time trap is the popular belief that Indian cultures were simple, non-commercial, hunter-gatherers prior to European arrival. Encapsulated within this belief is the idea that indigenous cultures are static and erode as they merge with mainstream society. However, this perception is incorrect: the indigenous peoples of North America had complex societies prior to 1492, including agriculture and expansive trade networks. Indian tribes organically incorporated previously unknown items from Europe, such as the horse and gun, into their cultures. This Article asserts that reexamining how society and the law view Indian history is the key to unlocking the time trap.
More H.R.L.R.
There Is No Such Thing As A "Legal Name"
Austin A. Baker and J. Remy Green
Adam Crepelle
Hard Drive Heritage: Digital Cultural Property in the Law of Armed Conflict
Richard Ong


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New salvage food store opens in Sioux Falls
Posted: Apr 9, 2021 / 6:12 PM CDT
/ Update: Apr 9, 2021 / 6:13 PM CDT
SIOUX FALLS, SD (KELO) – There is a new option to fill your cart in Sioux Falls. It's called Fair Market, but it's not a supermarket. It's a salvage food store.
Kristin Johnson and her husband Greg opened the business last month along Carolyn Avenue in Sioux Falls.
"We have regulars here, some people come 2-3 times a week," said Kristin Johnson, co-owner of Fair Market.
The products in this store come from salvaged food warehouses and can end up there for a variety of reasons.
"Food isn't sold because it's damaged, sometimes it's expired," Johnson said.
When a shipment of collected non-perishable food arrives at Fair Market, it is sorted and anything that can be sold is repaired and put on the shelf.
"Re-glue a box where the glue came off or wipe down a box because something else exploded next to it," Johnson said.
Kristin says the food in her store comes at a very low price.
Client Esther Barber says it's working really well for her growing family.
"We like the low cost items that we can store our shelves with," said customer Esther Barber.
But that's not the only reason Kristin says her store is important.
"For me I have two daughters, they are of school age that I look at and I think I give them a planet and if it went to the landfill it's that many cubic feet of landfill space that were used by something that could have fed a person. In terms of "feeding the person," we are talking about inequalities in our system and I feel like it starts a lot with food. your stomach is empty, how are you going to learn? "said Johnson.
She hopes her new store can help make a difference.
You can also find housewares at Fair Market.
Bowral arrests: NSW police clash with anti-mask organic food store owners
MasterChef judge visits Edinburgh zero waste grocery store to cook up a winning dish
Global Grocers opens international food store for multicultural taste in Penticton – Penticton News
Amazon opens first non-food store in UK
Colorado is home to the world's largest comic book store
Tattered Cover opens children's bookstore at Stanley Marketplace


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← US civil rights group to sue CIA over secret prisons
Rice 'to talk tough on CIA claim' →
KBR workers in Iraq paid 50 cents an hour
Posted on December 4, 2005 by Team VCS
While the United States spends billions on troop support in Iraq, the people serving the meals, scooping the ice cream, and washing the dishes make as little as 50 cents an hour.
The U.S. military has paid Halliburton subsidiary KBR about $12 billion so far for so-called logistics support to U.S. military personnel in Iraq, the largest contract of its kind ever. Around 80,000 troops are served meals at dining facilities every day under the contract — the other 60,000 or so fend for themselves in field kitchens or by eating military issue "Meals Ready to Eat."
KBR in turn hires that work out entirely to subcontractors whose job it is to recruit, transport, house, feed and pay "third-country" nationals to stock, prepare, serve and clean up at the dining facilities at 43 bases across Iraq.
Those workers are recruited from countries with already low wages, where jobs are scarce. And as pressure to keep the logistics contract cost down has increased, subcontractors have moved from country to country in search of cheaper labor markets.
That is what brought around 770 workers from Sierra Leone, Africa, to Iraq in July to work for ESS Support Services Worldwide, A British-based food service company specializing, according to its Web site, in "remote site, defense and off-shore locations."
Most of the workers are deemed unskilled and work seven days a week for 12 hours a day, according to their contracts, one of which was obtained by United Press International. In practice, workers said in interviews, most only work six days a week.
There is no provision for sick leave. Any employee who threatens a strike or attempts to organize is subject to immediate dismissal and the employee required to pay for his return plane ticket.
For this they are paid $150 a month, roughly 45 cents an hour.
Salaries are deposited in bank accounts in Africa so the money is available to the workers' families.
The workers also get a $40 a month cash allowance on top of that, but the contract states the money is a gift, and the amount discretionary and may be eliminated. Their housing — three to a standard size trailer — laundry, food and uniforms are provided free.
Employees are prohibited from discussing the contract and "ESS internal issues or complaints" with anyone outside the company, including the military and media. A copy of the contract was provided to UPI by an ESS employee via e-mail, with the assistance of a U.S. military officer. The worker quit Iraq and has now returned to Sierra Leone.
Neither the U.S. military nor prime contractor KBR sets a minimum wage standard for workers employed by any sub-contractor under the Logistics Civilian Augmentation Program — known as LOGCAP. Jana Weston, the deputy program director of LOGCAP, told UPI in September she was unaware that some workers were being paid $150 a month.
"KBR's subcontractors are required to comply with all applicable labor laws and provisions in the country in which they work. KBR's non-U.S. subcontractors operate within industries and geographic regions that are highly competitive in regard to recruiting and retaining employees. As such, the subcontractors are driven to pay market wages and benefits," said Melissa Norcross, a spokeswoman for Halliburton in Houston, Texas.
The dining services subcontracts, like LOGCAP overall, are cost-plus arrangements, meaning the contractor is reimbursed for its costs and then paid a percentage on top of that for profit.
As a result, the sub-contractors' costs are closely guarded propriety information and no details of wages and contract arrangements for other LOGCAP sub-contractors could be obtained to compare them to those paid by ESS.
Previous to the Sierra Leone contract, ESS employed workers from Sri Lanka who were paid about $400 a month for the same work, according to a U.S. military officer who oversees the logistics contract at one of the bases where ESS provides dining services.
Paul Kelly, ESS group corporate affairs director, acknowledged previous workers were paid more in an interview in October with UPI. He tagged the higher wages to less competition for the LOGCAP subcontract. When more companies entered the fray, ESS found cheaper labor to improve its pricing to the government.
"Initially wage rates were higher because there were fewer companies bidding (on the subcontract)," said Kelly, declining to confirm the old wage. "As more companies competed for contracts — labor is one of those areas companies have been targeting" to drive down the bids.
The switch to cheaper labor came as Gen. George Casey, the commander of U.S. forces in Iraq, launched an effort to drive down the costs of LOGCAP.
"Increasing expenditures in theater … jeopardize our ability to maintain public support as the costs associated with our operations continue to rise," he wrote in a memo issued to commanders this summer, and exclusively reported by UPI. "Our spending in theater not only affects us directly, it has a ripple effect throughout all of the services."
"When we started there were a handful of companies competing. Now there are probably 20 or 30, which does drive price down, and of course that's what LOGCAP wants," said Kelly.
Kelly said the monthly wage offered to the Sierra Leoneans is far higher than what they would earn at home. According to the Sierra Leone worker, some 7,000 candidates showed up to apply for the 750 jobs ESS offered in June 2005.
"The important thing to bear in mind here is that it is a little bit too simplistic to compare pay scales as we might look at them from a U.S. or U.K. perspective," Kelly said. "You have to look at job type and how does this compare with what an employee would earn it in its own country."
"What we don't do is pay them less than what they earn in their own country," Kelly said. "We take employee welfare very seriously."
Sierra Leone is an extremely poor country, with a market-based economy and a per capita income of less than $100 per year. For the last decade a violent insurgency has destroyed the local economy. The government approved a minimum wage of about $4 a week for a 40-hour work week, according to the State Department's 2004 human rights report.
Kelly added that even mid-level government officials in the country earn only about $40 a month, although that would be a for a 40 hour week.
Kelly said, however, that ESS' recruiting of workers in Sierra Leone does not only have to do with competition under LOGCAP and low labor costs. Geo-politics has played a role as well.
"The catalyst for having to go to Sierra Leone to recruit in the first was that the respective governments of India, Pakistan, Sri Lanka, Thailand and the Philippines have all put an official ban on their nationals working or traveling in Iraq," states an e-mail sent to Kelly from the Sierra Leone recruiter.
According to Kelly, the Iraq LOGCAP subcontract is not profitable enough to warrant the risk and difficulty of fulfilling it, especially given the fact the company must insure its workers in a very hazardous war zone.
"We've already flagged that we are very heavily scaling down our presence in the Middle East simply because the margin we can earn does not justify the logistical complexities and the risk," Kelly said. "The question is do you want to put employees into a position where it's still very dangerous out there and logistically very complicated?"
Acknowledging the Sierra Leonean workers agreed to work for the advertised pay, military personnel interviewed in Iraq still expressed surprise and discomfort at the wages paid to those feeding and cleaning up after them every day, with whom many say they are quite friendly. Two Army officers alerted UPI to the issue and arranged the first meeting with the Sierra Leone worker.
Workers' living conditions on bases with dining facilities in Iraq are inherently hazardous, owing to the violent insurgency, but materially good — most have hot showers, electricity and air-conditioned sleeping quarters.


This beautiful wedding venue is located in Paphos Cyprus. Vasilias Nikoklis Inn, an old stone built inn makes a perfect setting for private and intimate weddings and events. The venue can take up to 80 guests both for ceremony and reception and is exclusively hired for the day. Accommodation is also available, 8 charming double rooms can be hired for the day perfect for bridal preparations and also for an overnight stay.
The venue is operated by a local family and has been hosting weddings since 2008, all members of the family are focused in making couples and their families and friends feel like home and have unforgettable memories of their special day in Cyprus. Together with the fantastic and romantic environment of Vasilias Nikoklis Inn, great knowledge and support is provided by the weddings team so the whole procedure from booking day till the wedding day is stress free. All couples booking direct with Vasilias Nikoklis enjoy the benefits of an online wedding planning platform where couples can access 24/7, built up their plans and control their budget. So if you are dreaming of dancing as Mr and Mrs under a sky full of stars at the yard of a rustic private venue in Cyprus please get in touch!
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Sitting on sofas and relaxing in our favorite coffee shops do not bring transformation. Although rest is necessary and an essential joy, they do not define who we are. We become what our trials and enemies attempt to silence.
It may not be what we look forward to, but the Lord will regularly bring you to battlefields that launch us into the next phase of living. These confrontations always mark seasonal shifts. Without them, you cannot go to any new level or see any expansion of God's miraculous influence.
So, on this day, encourage yourself! Come to your fight "in the name of the Lord of hosts" – by the One who reigns over your life. And let every enemy know that they have defied the armies of the living God. Stand your ground. Wield the weapon God has provided. And let your victory redefine you.
I love the preparation of David before getting to this place. He had many trials while protecting the family's sheep! Through this, he had come to KNOW the person and power of his delivering God! I mean, Who fights with a tiger or a bear and survives? Trials, had in David, produced extraordinary strength and staying power! Becoming skilled with the tools needed for the task, his slingshot, and having gained an unwavering courage and faith knowing that he can't beat a lion or a bear but he knew who could. So he says to Goliath, I'm gonna fight you with my slingshot that is not a weapon of war just to show everyone here the battle is the Lords! Trouble, if it turns us to the Lord , produces character! Woo hoo!
That is so awesome! Every trial gives us the necessary tools for the greater tests!
Oh how this stirs my heart greatly; not to mention it is one of my absolute favorite encounters of David!
I would not be where I am today had I tucked tail and ran from what I had been called to face (daily, weekly, seasonally). AND this I am sure of, what I go through today (KNOWING the LORD of hosts is with me) will shape my tomorrow's to have generational (physical & spiritual) implications.
Amen, Becky! To know that everything we go through, both in battle and victory, is for the generations, is truly a powerful encouragement!
AMEN!! Even with all the battles I have had to face medically and emotionaly I would not change a single thing. Even though in the middle of them I thought much differently at times, I am who I am and where I am because of them today. I also have a much higher level of faith because of them! GOD IS SO GOOD ALL THE TIME!!
It's been a huge joy to watch the Lord's transformation of your life through the years!


NEJLT is the journal of NEALT, the Northern European Association of Language Technology, an organization covering the geographical area of the Nordic countries (Denmark including the Faroe Islands and Greenland, Finland, Norway, Iceland, Sweden), the Baltic countries (Estonia, Latvia, Lithuania) and Northwest Russia. NEJLT is established to be a channel for the dissemination of research results in all areas of language technology and is not restricted to the geographical area of NEALT. The official language of NEJLT is English.
Language technology (LT) is the science and technology of processing natural language by computers. NEJLT primarily serves the R&D community in this area. Presentations and discussions of well founded and innovative theoretical and empirical approaches, as well as relevant evaluations and real life applications, are all suitable topics for articles in the journal.
NEALT also has three other publication series: monographs, proceedings and reports, from 2012 also published by LiU Epress. Before 2012 they were published at DSpace at Tartu University Library.


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Le competizioni di lotta ai VII Giochi della Francofonia si sono svolti dall'8 al 10 settembre 2013 a Nizza, in Francia. Si sono svolti 16 tornei, 8 maschili ed 8 femminili, tutti di lotta libera.

Podi

Uomini

Donne

Medagliere

Collegamenti esterni
 Libro dei risultati

Sport ai VII Giochi della Francofonia
Lotta ai Giochi della Francofonia
Lotta a Nizza
Competizioni di lotta in Francia
Lotta nel 2013

redpajama_set_name: RedPajamaWikipedia