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A novel thioredoxin-like protein encoded by the C. elegans dpy-11 gene is required for body and sensory organ morphogenesis
Frankie C. F. Ko, King L. Chow
Development 2002 129: 1185-1194;
Frankie C. F. Ko
King L. Chow
Sensory ray morphogenesis in C. elegans requires active cellular interaction regulated by multiple genetic activities. We report here the cloning of one of these genes, dpy-11, which encodes a membrane-associated thioredoxin-like protein. The DPY-11 protein is made exclusively in the hypodermis and resides in the cytoplasmic compartment. Whereas the TRX domain of DPY-11 displays a catalytic activity in vitro, mapping of lesions in different mutant alleles and functional analysis of deletion transgenes reveal that both this enzymatic activity and transmembrane topology are essential for determining body shape and ray morphology. Based on the abnormal features in both the expressing and non-expressing ray cells, we propose that the DPY-11 is required in the hypodermis for modification of its substrates. In turn, ray cell interaction and the whole morphogenetic process can be modulated by these substrate molecules.
Ray and body morphogenesis
dpy-11
Morphogenesis is an important developmental process that eventually determines the form and shape of an organ or body structure. This process usually involves multiple regulated steps with cell shape changes, cell movement and cell-cell communication as the major events. Provision of a competent extracellular environment and proper display of cell surface proteins are both essential to ensure that normal cell migration and cellular communication occur.
Molecules in the extracellular matrix are known to be critical for establishing the morphology of C. elegans (Kramer, 1994). Collagens encoded by a number of dpy loci, when mutated, give a dumpy phenotype (von Mende et al., 1988; Levy et al., 1993). These molecules that are essential for establishing the shape and integrity of animal body are often extensively modified posttranslationally (Norman and Moerman, 2000; Friedman et al., 2000). Therefore, molecular modification of the extracellular matrix (ECM) can be used to modulate and facilitate both cellular differentiation and morphogenetic processes (Vitale et al., 1997). In addition to the matrix proteins, ligand molecules and cell surface receptors are also targets for modification. For example, proteolytic modification is essential for the synthesis of a biologically active ligand in the Hedgehog signaling pathway that controls segment polarity (Wodarz and Nusse, 1998; McMahon, 2000). Glycosylation of Notch by Fringe can regulate the downstream signaling activity in Drosophila (Bruckner et al., 2000; Fortini, 2000; Moloney et al., 2000). Covalent addition of polysialic acid to NCAM or integrin molecules can alter cellular adhesion properties and tissue differentiation (Hoffman and Edelman et al., 1984; Pretzlaff et al., 2000). In C. elegans, the dpy-18 gene encoding a prolyl-4-hydroxylase has been shown to hydroxylate its target substrate for protein disulfide isomerase (PDI) modification and maintenance of the body shape (Friedman et al., 2000; Hill et al., 2000; Winter and Page, 2000). Failure to modify the substrate molecules can also result in incorrect protein folding or assembly leading to retention of the precursor molecules intracellularly and possibly cellular swelling (Wallis et al., 1990; Walmsley et al., 1999). In addition, many proteins and signaling molecules involved in cell-cell communication are cysteine-rich and can form disulfide bonds, e.g., Frizzled and its related proteins, fibronectin, collagen and cell adhesion molecules (CAMs) (Wodarz and Nusse, 1998; Balzar et al., 2001; Ichii et al., 2001). Intra- and intermolecular disulfide bond formation in these proteins can determine their molecular folding as well as biological activity.
Thioredoxin is an essential cellular redox cofactor for a variety of biochemical reactions and cellular functions. Most thioredoxins are soluble proteins of about a hundred amino acids. It facilitates the thioredoxin reductase-mediated electrons flow from NADPH to FAD, acts as a hydrogen donor for the ribonucleotide reductase in the process of deoxyribonucleotide synthesis and also functions as 'protein disulfide oxido-reductase' to reduce the disulfide bonds in proteins like insulin and NFκB transcription factor (Holmgren, 1985; Qin et al., 1995). With such diverse functional roles, thioredoxin gene-knockout animals exhibit embryonic lethality, although the primary cause of this phenotype was not identified (Matsui et al., 1996). Thus, while the three-dimensional structure of thioredoxin has been resolved, the developmental function of thioredoxin-like molecule remains a mystery (Eklund et al., 1991; Holmgren, 1995; Martin, 1995). In the past decade, two N-terminal membrane associated thioredoxin-like molecules were isolated from the soybean plant and the symbiotic bacteria inhabiting its root nodules (Loferer et al., 1993; Shi and Bhattacharyya, 1996). Their protein structure has led to the hypothesis that these thioredoxin variants may have an additional function of facilitating communication between the host and its symbionts. More recently, a human thioredoxin-like molecule bearing a transmembrane domain was reported to be the product of a TFGβ inducible gene (Matsuo et al., 2001). Although it resides predominantly on the endoplasmic reticulum and displays an apoptosis-suppressing effect in HEK293 cells treated with low doses of brefeldin, its functional role in normal developmental process was not addressed.
We report here that the C. elegans dpy-11 gene encodes yet another variant of the thioredoxin-like proteins with both a signal peptide and a transmembrane domain. While dpy-11 is distinctly different from the related gene found in human, we show that its expression in hypodermal cells is essential for establishing normal body shape in larval and adult nematodes. The function of its gene product in the context of body and sensory ray morphogenesis will be discussed.
Worm cultures
Worm strains were cultured and maintained as described by Brenner (Brenner, 1974) at 20°C unless stated otherwise. All dpy-11 alleles were either obtained from the Caenorhabditis Genetics Center, Minnesota, or kindly provided by the Hodgkin laboratory and the Baillie laboratory.
Phenotypic analysis of dpy-11 alleles
All dpy-11 mutants were characterized by their respective body and male tail phenotypes under Nomarski microscopy. Except the reference allele e224 that was coupled with him-5(e1490)V, all alleles were in him-8(e1489)IV background. The average body length was obtained by measuring 30 males with a micrometer. The ray phenotypes were recorded using a Ram Index defined by the degree of sensory ray swelling, with 0 indicating wild type (Fig. 1G) and 5 representing the most severe phenotype (Fig. 1L). Thirty males of each genotype were scored and the value was averaged.
Body and tail phenotype of (A,G) wild-type worms, and mutants carrying various dpy-11 alleles: (B) e504, (C) s360, (D,I) e224, (H) e431, (J) s261. (E,K) Mutant worm rescued by p72WCP and (F,L) wild-type worm with dpy-11 RNAi. Scale bar: 100 μm (A-F); 20 μm (G-L).
Mapping the dpy-11 and lesions in mutant alleles
dpy-11 was mapped between gad-1 and egl-46. Cosmids spanning this region were injected individually into dpy-11(e224) animals with dpy-13::gfp, a reporter driven by the dpy-13 promoter, at a concentration of 100-150 ng/μl (Stinchcomb et al., 1985; Mello et al., 1991). A positive rescuing cosmid W06G7 was identified. An Acc65I-SphI rescuing fragment from W06G7 had been identified and subcloned into pSP72 as p72WAS. Further deletion derivatives of p72WAS were generated by restriction enzyme cleavage and self-ligation.
The genomic DNA of 15 dpy-11 alleles was extracted as described previously (Sulston and Hodgkin, 1988). About 400-600 ng of genomic DNA was used for PCR amplification with Taq polymerase (Gibco) and dpy-11 gene-specific primers. The product DNA fragment was sequenced with the BigDye Terminal Sequencing Kit (Perkin Elmer) using the same primer sets.
RNA interference of dpy-11 gene and characterization of cellular defects
RNAi experiments were performed as described by Fire et al. (Fire et al., 1998). Sense and antisense RNA were synthesized from the EST cDNA clone, yk109h7, with T3 and T7 polymerase respectively (Promega in vitro Transcription Kit). F1 progeny from injected individuals were examined under Nomarski microscopy.
gfp reporter driven by one of the different cell-type specific promoters, dpy-11 or dpy-13 (hypodermal cell, this study), ram-5 (structural cell) (Yu et al., 2000) and sek-1 (neuronal cell) (Yu et al., 2000) were introduced into dpy-11 mutant animals. The transformed animals were examined by fluorescence microscopy. RNAi was also performed in these marker strains to reveal the severe cellular defects.
Engineering of reporter, expression and deletion constructs
A HincII-BsmI 1.6 kb fragment carrying the dpy-11 promoter region from p72WAS.3 was linked to the worm cDNA yk109h7 and human cDNA DKFZp564E1962 to generate pd11cDNA and pd11hcDNA respectively. For ectopic expression of dpy-11, a SphI site was first introduced in front of the start codon in dpy-11 cDNA by site-directed mutagenesis with primer KC293:5′-CCAGCATGCTGCTCCG-3′ (Barik, 1993). A 3 kb SacI/SphI fragment carrying the ram-5 promoter was cloned in front of this modified dpy-11 cDNA to generate pr5cDNΑ (Yu et al., 2000).
A 2.7 kb 5′ flanking region of dpy-11 was subcloned into pPD95.70 to construct a gfp reporter transgene with a nucleus localization signal (NLS), pd11GFPN. A non-nucleus localized version of reporter, pd11GFP-N, was generated by deleting the NLS from pd11GFPN. These constructs were injected with markers pRF4 (rol-6d) into wild-type animals or with pd11cDNA into dpy-11 mutants for expression pattern examination.
The DPY-11 localization and rescue efficiency were examined by transforming dpy-11 animals with the testing constructs. The wild-type pattern was first revealed with the full length fusion transgene, pd11D11GFP, which had the gfp fragment from pPD95.69 ligated to a HindIII site introduced at the stop codon of dpy-11 cDNA by site-directed mutagenesis with KC 296: 5′-CGAAGAAGACCAAGCTTTAAATTCTATGCAACTTCC-3′ and KC297: 5′-GGAAGTTGCATAGAATTTAAAGCTTGGTCTTCTTCG-3′. The plasmid encoding a tagged and C terminus-truncated version of DPY-11, pd11ΔCTGFP, was made by deleting an internal AvaII/HindIII fragment in pd11D11GFP followed by self-ligation. A 3 kb ram-5 promoter fragment was inserted into pd11D11GFP to replace the dpy-11 promoter to generate pr5D11GFP for examining the subcellular localization of DPY-11 in a different cell type. A HincII site was introduced in front of the CT-domain in the dpy-11 cDNA by site-directed mutagenesis with primers KC273: 5′-CGTTATCATCATCGTCGACCAAGTATTCCC-3′ and KC274: 5′-GGGAATACTTGGTCGACGATGATGATAACG-3′. Subsequently, the HincII cut CT fragment was cloned into the 3′ end of the GFP gene in pD119.16 with HincII-SmaI. This GFP with CT domain at the 3′ end was used to replace the GFP in pd11ΔCTGFP and was designated pd11AGFPC.
Starting with the functional dpy-11 genomic cDNA fusion fragment, pd11cDNA, pd11ΔSP was generated with the signal peptide (aa 2-34) removed by BsmI and BglII digestion followed by self-ligation. pd11ΔS was made to remove the spacer fragment from aa 135-177 by joining the SmaI site (introduced at aa134/135 by mutagenesis with a primer KC299: 5′-GTACCCGGGAACTGGGTCGATCAC-3′) and an EheI site (5′ to the transmembrane domain generated with primers KC271: 5′-GGGCCAGCTACGGCGCCCTCTTCGCCGGAG-3′ and KC272: 5′-CTCCGGCGAAGAGGGCGCCGTAGCTGGCCC-3′). A HincII site was introduced at position aa 198/199 with primers KC273 and KC274 (mentioned above). The fragment with the TM domain from aa178-198 was removed by EheI and HincII digestion to generate pd11ΔTM. pd11ΔCT was engineered by introducing a frameshift mutation at the AvaII site (aa 205), which resulted in truncation of the C-terminal tail. To replace the transmembrane domain of DPY-11, a BspEI (blunted)/ApaI fragment containing a pat-3 TM gfp fusion from pPD122.39 was used to substitute the DPY-11 TM at the EheI to ApaI of the cDNA to generate pd11TMGFP.
For protein expression, the thioredoxin-like domain of DPY-11 (PpuMI to XhoI from cDNA, yk109h7) was subcloned into PpuMI and XhoI site of pPD95.75. After a SmaI site was introduced after the TRX domain by site-directed mutagenesis with primer KC299 as described above, the SmaI fragment encompassing aa 19-133 was subcloned into the pGEX-2T vector to generate pG2TD11T (wild type). A G76E mutant version of the thioredoxin-like domain with glycine 76 changed to glutamic acid was engineered by mutagenesis with primer KC369 (5′-GGAATCAAGGTTGAAGAAGTCGATG-3′) and KC370 (5′-CATCGACTTCTTCAACCTTGATTCC-3′) and was designated pG2TG76E.
Protein production and purification
A 2 l culture of BL21(DE3) transformed with pG2TD11T was grown to log phase followed by induction with 1 mM IPTG at 30°C for 4-5 hours. The culture was lysed in PBS by French Press (SLM Instruments), sonicated for 15 minutes on ice and passed through a glutathione sepharose 4B column (Amersham Pharmacia Biotech, cat. no.17-0756-01). The GST-tagged TRX-like domain was eluted with 20 mM reduced glutathione in 50 mM Tris-HCl (pH 8.0) and quantified by the Bradford assay. After overnight dialysis in PBS, the protein was either temporarily stored in 4°C or frozen at –70°C. The G76E mutant protein and GST control protein were expressed using the respective expression plasmids. The purified protein samples of wild type (pG2TD11T), G76E mutant (pG2TG76E) and GST (pGEX2T) were separated using 12% SDS-PAGE.
Insulin reduction assay
Insulin stock solution (10 mg/ml; Sigma, cat. no. I5500) was prepared according to the method of Holmgren (Holmgren, 1979). The E. coli recombinant thioredoxin (Sigma cat. no. T0910) and GST were used as positive and negative control respectively. 70 μl of insulin stock solution was added to the cuvettes with 630 μl assay solutions containing 1 mM dithiothreitol (DTT) and 200 μl enzyme at different concentrations (0 μM, 1 μM, 2 μM and 3 μM) to start the reaction. The final reaction mixture contained 1× PBS, 2 mM EDTA, 1 mg/ml insulin and 1 mM DTT. The absorbency was measured in a Beckman 650 spectrophotometer at wavelength of 650 nm at 25°C. The data was recorded at 5 minutes intervals for a period of 30 minutes.
Characterization of dpy-11 mutant phenotypes
dpy-11 mutant was first isolated based on its dumpy body phenotype observed in all larval and adult stages (Brenner, 1974). Its swollen ray (Ram, ray morphology abnormal) phenotype as noted by Baird and Emmons (Baird and Emmons, 1990) indicates that dpy-11 is also required for sensory organ morphogenesis. We examined the available dpy-11 alleles on these two diagnostic features, and the results are summarized in Table 1. The Ram Index (RI) is a measure of the severity of swollen rays: mutants score the highest of 5 (Fig. 1L) and wild-type animals score 0 (Fig. 1G,H). The dpy-11 reference allele e224 together with e390, e455 and e733 had mildly swollen rays with a RI of about 2 (Fig. 1I). These mutants were weakly dumpy with the body length ranging from 0.60 mm to 0.66 mm (Fig. 1D). Strong Dpy and Ram phenotypes were found in e33, e207, e504, e752, e794, e1180 and s261 alleles (Fig. 1B,J). Their body length ranged from 0.38 mm to 0.46 mm, and their respective RI from 3.5 to 4.8 (Fig. 1J). In general, there was a good correlation of the severity of dumpiness and ray swelling.
However, there were exceptions. s10 and s360 mutants had a strong Dpy phenotype (0.43 mm to 0.49 mm) but exhibited almost wild-type rays (Fig. 1C). e395 and e431 alleles had weak Dpy body (from 0.63 mm to 0.65 mm) with normal rays (Fig. 1H). When these four alleles were tested for temperature sensitivity, only s10 mutants presented a swollen ray tip phenotype with greater than 90% penetrance at 25°C. The other three mutants had wild-type male tails, independent of the testing temperature.
When heterozygous animals of these 15 alleles were examined, they all showed wild-type body length and rays. This observation suggests that these mutations are loss-of-function mutations. In addition, double heterozygous worms of e207 (putative null) and e224 alleles (weak dumpy) had a strong Dpy phenotype with intermediate Ram rays of RI of 2.7 (Table 1). Hence, the body morphology, in comparison with ray development, appears to be more sensitive to the reduction of dpy-11 gene activity.
Cellular defects of dpy-11 mutant sensory ray
To characterize the sensory ray abnormality in dpy-11 mutants, cell-specific marker gfp transgenes driven by hypodermal, structural and neuronal cell-specific promoters were introduced into animals carrying the e224 allele. The hypodermis and structural cell processes of the mutant animals had abnormal swelling (Fig. 2D,F) in contrast to those of the wild-type worms (Fig. 2C,E). The neuronal processes in this reference allele appeared less abnormal although the processes were rough and irregular in shape with occasional nodule-like swelling. These neuronal nodules were obvious in RNAi-treated males (Fig. 2H, arrowhead), which also exhibited more severe swelling of hypodermis and structural cell processes (data not shown).
Cellular features of wild type (A,C,E,G) and dpy-11 (e224) (B,D,F) or dpy-11(RNAi) (H) male tails as visualized by (A,B) Nomarski optics; (C-H) cell-type specific markers to identify: hypodermal cells (C,D; marked by pd11GFP-N), structural cell (E,F; marked by ram-5::gfp) and neuronal processes (G and H; marked by sek-1::gfp). Scale bars: 20 μm.
Definition of the dpy-11 gene locus
dpy-11 was genetically mapped between egl-46 and gad-1. Cosmids spanning this region were injected into e224 mutants to test for rescue activity. Positive rescuing cosmids, W06G7 and F46E10, were identified (Fig. 3A). The physical location of dpy-11 was simultaneously confirmed with another overlapping cosmid T04F11 (J. Pak and A. Spence, personal communication). Serial deletion constructs derived from W06G7 were made to localize the rescuing activity (Fig. 3A). The p72WCP fragment contained the F46E10.9 gene and a truncated F46E10.8 ORF. Exhibition of wild-type dpy-11 activity by this fragment suggests that F46E10.9 is dpy-11. The identity was subsequently confirmed by two approaches: (1) a transgene with a F46E10.9-derived cDNA, yk109h7, linked to the F46E10.9 5′ flanking sequence rescued the mutant phenotype efficiently; (2) RNAi with this cDNA phenocopied the dpy-11 Ram and Dpy phenotypes in wild-type animals (Fig. 1F,L). This RNAi result confirms again that both phenotypes seen in all our mutants are due to loss-of-function of dpy-11.
The dpy-11 locus and the predicted protein structure. (A) Cosmids and their deletion derivatives used for defining the dpy-11 locus. (B) Genomic organization of dpy-11 and the lesions in mutant alleles. (C) Predicted structure of the DPY-11 protein. The GenBank accession number of the dpy-11 gene is AF250045.
dpy-11 is exclusively expressed in the hypodermis
The spatial and temporal expression patterns of dpy-11 were examined with a reporter assay. The green fluorescent protein gene (gfp) under the control of a 2.7 kb dpy-11 5′ flanking sequence, which could rescue mutants when coupled to the cDNA, was used to generate transgenic worms in both wild-type and mutant backgrounds. The reporter constructs, with or without the nuclear localization signal (NLS), revealed a similar pattern of signals independent of the rol-6d or pd11cDNA markers used for transgenesis. The expression profile is therefore reflective of the authentic dpy-11 transcriptional activity and related to the body and ray morphogenesis (Fig. 4A,B). Fluorescence was detected in the hypodermal cells as early as the comma stage (data not shown) and throughout all the larval stages in the body hypodermis (Fig. 4A,B) with the exclusion of seam cells (Fig. 4B, arrowheads). At the late L3 to L4 stages when ray cell differentiation took place, no dpy-11 promoter driven GFP signal was detected in the ray precursor (Rn) cells. Neither was there any signal detected in cells of the Rn.a branch differentiating into future neuronal derivatives. GFP signal was observed only in the Rn.p cells after they were born, all of which eventually fused to form the tail seam or fused with the body hypodermal syncytium (data not shown).
Expression patterns of dpy-11 and subcellular localization of its product. (C,E,G) Nomarski images of D,F,H, respectively. (A) dpy-11 promoter-driven gfp reporter with a nuclear localization signal in L3 larva. (B) Cytosolic gfp reporter in L4 larva showed no seam cell expression (arrowhead). (C,D) The DPY-11 tagged with GFP at the C-terminus (pd11ΔCTGFP) presented a heterogeneous cytosolic expression pattern in the hypodermis around the vulva. (E,F) The fusion transgene encoding full-length DPY-11 with a GFP tagged between the transmembrane domain and the C-terminus (pd11AGFPC) presented a heterogeneous cytosolic expression pattern in the body hypodermis. (G,H) In gastrulating embryos, cytosolic expression (pd11ΔCTGFP) was detected in unfused hypodermal cells (arrows) with membrane association detected in some dying cells (arrowheads). Scale bars: 100 μm (A,B); 10 μm (C-H).
To examine the subcellular localization of the DPY-11 protein, the fusion transgene encoding full-length DPY-11 with a GFP tagged between the transmembrane domain and the C terminus was constructed (pd11AGFPC, Fig. 8, construct 10). This fusion transgene was almost as active as a wild-type transgene in complementation rescue assay. The fusion products presented a heterogeneous signal with a membranous organization in the expressing hypodermis (Fig. 4C,D). A similar pattern was noted when a C terminus-tagged GFP fusion DPY-11 (pd11ΔCTGFP, Fig. 8, construct 8) was examined (Fig. 4E,F). The pattern is distinctly different from that of cytosolic GFP (Fig. 4B). This distribution pattern suggests that the DPY-11 protein is associated with subcellular membranous organelles. In addition, in some dying transgenic embryos where cells started to disintegrate, membrane-associated GFP signal could be observed (Fig. 4G,H, arrowheads) while non-dying cells displayed the same heterogeneous profile (Fig. 4G,H, arrows). These results suggest that DPY-11 molecules are residing on a membranous structure, possibly endoplasmic reticulum or Golgi apparatus.
Structural organization of DPY-11
DPY-11 has 246 amino acids as predicted from the ORF present in the cDNA. Based on the secondary structure and sequence analyses, it has a putative signal peptide at the N-terminus (SP), a thioredoxin-like domain (TRX), a 50 amino acid spacer (S), a putative transmembrane domain (TM) and a 46 amino acid C-terminal region (CT) (Fig. 3C) (Schultz et al., 2000). The thioredoxin-like domain shared 48-53% similarity with thioredoxin molecules from bacteria and eukaryotes (Fig. 5A). DPY-11 also had a potential ER retention signal sequence, KKTK, at its C terminus (Hong, 1996).
Amino acid sequence alignment of DPY-11. (A) Thioredoxin domains in different organisms: C. elegans (Q09433), C. nephridii (X14630), E. coli (K02845), H. sapiens (J04026). (B) DPY-11 homologs in human (AL080080) and fruitfly (AAF47072). The numbers in brackets are GenBank accession numbers.
Full protein sequence alignment has identified two proteins, from human and Drosophila respectively, sharing structural similarity to DPY-11 (Fig. 5B). These DPY-11-like proteins have an N-terminal signal peptide, a thioredoxin-like (TRX) domain, a spacer sequence, a transmembrane domain and a C terminus of different lengths. In their respective TRX domains, they all had a putative catalytic site with CPAC sequences, in contrast to the conserved CGPC sequence found in most thioredoxins. Such conservation suggests that these DPY-11-like proteins may indeed be functionally related. However, when a construct with the human dpy-11-like cDNA driven by the worm dpy-11 promoter was introduced into dpy-11 mutants, no rescue activity was observed, possibly because of the divergence in their sequences and thus biological specificity (Fig. 8, construct 2).
DPY-11 TRX domain has reducing activity
To verify that the TRX-like domain indeed has catalytic activity, we over-produced, in bacteria, a truncated DPY-11 fragment with aa 19-133 encompassing the TRX domain to test for its reducing activity. When the wild-type and the G76E mutant fusion proteins were produced and purified, a 40 kDa band were detected with SDS-PAGE in WT and G76E lanes respectively (Fig. 6). A band of about 26 kDa, possibly representing the GST protein, was also noted in GST lane. This result is consistent with the predicted size of 14 kDa for the recombinant truncated DPY-11 fragment. After these proteins were purified, thrombin treatment was performed to release the DPY-11 TRX domain. However, insoluble precipitates with no enzymatic activity were obtained (data not shown), so that GST fusion proteins were used subsequently for all the enzymatic assays.
Expression and purification of the GST fusion construct. WT represents the GST fusion product of the TRX-domain from amino acids 19-133 of DPY-11. G76E is a similar fusion protein with the glycine at position 76 changed to glutamic acid. GST is the expression of an empty pGEX-2T GST vector, which produced only the GST tag.
The insulin reduction assay monitors the break down of the disulfide bond between insulin chains A and B in the presence of DTT (Holmgren, 1985). In such an assay, wild-type fusion protein displayed higher reducing activity than the G76E mutant version and the two negative controls (Fig. 7A). By 30 minutes into the reaction, this wild-type TRX domain displayed an activity comparable to the level of the E. coli thioredoxin control. However, it is interesting to note that while thioredoxin could reduce the insulin soon after the reaction was started, GST-DPY11 exhibited a 10 minutes delay.
Insulin reduction assay of GST-DPY-11 protein. (A) A time course of the enzymatic activity assayed at a fixed concentration (3 μM) of the tested proteins. (B) A comparison of the catalytic activities of the fusion protein and thioredoxin at different concentrations and a fixed reaction time of 20 minutes. Black squares represent the E. coli recombinant thioredoxin; white triangles represent the GST fusion protein of wild-type TRX domain; black triangles represent the mutant fusion protein with glycine 76 mutated to glutamic acid; white circles represent the GST alone and black circles, the buffer alone.
When 20 minutes was chosen as the reaction time for examining the concentration effect of these recombinant enzymes, the wild-type fusion protein again showed higher activity than the mutant version and negative control in all concentrations tested (Fig. 7B). The wild-type TRX-like domain was less active than the positive control, but clearly displayed a reducing activity. Hence, the intrinsic property of this TRX-like domain is probably relevant to the biological function of DPY-11 in body and ray morphogenesis.
The DPY-11 function within the hypodermal cell is dependent on the signal peptide, TM domain and its C terminal tail
DPY-11 protein has a unique domain organization. Its enzymatic property may not account for the full spectrum of its biological activity. Deletion constructs were made and tested for the structural requirement of the domains of this molecule (Fig. 8). Product from the pd11TMGFP transgene (construct 12) had the authentic TM and C terminus eliminated and could present a fusion product with only the DPY-11 N-terminal region on the hypodermal cell surface (data not shown). This molecule exhibited no mutant rescue activity at all. The same was true when the TM domain alone was deleted (construct 6). Similarly, deletion of either the signal peptide or the spacer also completely abolished the mutant rescue activity (construct 4 and 5). These results argue that the topology and the membrane anchorage of the protein appear to be important for its biological function.
A schematic representation of different mutant constructs made for the detection of rescue activity and subcellular localization. The percentage of transformed mutant animals reverted to wild type is indicated and the number of animals scored is in brackets. Localization of the tagged mutant DPY-11 proteins within the expressing cells is shown on the right column. The GFP part is not to scale.
In contrast, deletion of 41 amino acids of the C-terminal tail, as in the product encoded by pd11ΔCT or pd11ΔCTGFP (construct 7 and 8), did not abolish the rescue activity completely. About 20% of its wild-type activity was retained. The results show that these 41 amino acids at the C terminus are critically required though it is not essential for DPY-11 function. When the complete CT region was included in the transgene, such as in pd11D11GFP (construct 9), rescue efficiency was much improved. Placing the CT domain on the very C terminus, as in pd11AGFPC (construct 10), could increase the rescue efficiency to above 90%, i.e., only a mild compromise of the wild-type transgene activity.
As the expression pattern of dpy-11 was confined to the hypodermal tissue and the topology of the encoded product appeared critical, we tested if the expression of dpy-11 in other ray cells could provide the same biological activity possibly mediated through protein modification outside the expressing cell. When the dpy-11 cDNA was driven by the ram-5 promoter in the structural cell of dpy-11 mutants, no reversion to the wild-type phenotype was observed (Fig. 8, constructs 3 and 11). We therefore hypothesize that DPY-11 modifies its substrate molecules within the hypodermis. The dpy-11 activity present in the structural cell using the ram-5 promoter was mis-expressed either in tissue or temporal specificity, or even both, so that wild-type ray morphogenesis process could not be restored.
Mapping of dpy-11 mutants
With a number of mutant alleles of the dpy-11 gene available, the mapping of their lesions could provide further support to the deduction from our deletion analysis. The mapping results are shown in Table 1 and Fig. 3B. Nonsense mutations were detected in e207, e504 and e752 alleles on the 5′ end of the predicted TRX domain. These mutations led to the synthesis of truncated proteins with the entire TRX domain and the sequence beyond missing. They represent putative null mutations. The s261 allele had a mutation at the 5′ donor site of intron 2. With the intron retained, a stop codon in intron 2 would be used, resulting in a truncated protein. The e33 allele had a stop codon present on the N-terminal side of the transmembrane domain resulting in a mutant protein with no membrane anchorage. This allele has a strong Dpy and Ram phenotype just like that of the null mutation suggesting that although the truncated protein synthesized had a complete TRX domain, it presumably had no biological activity. Alleles e794 and e1180 were generated by ICR191 acridine treatment known to introduce guanine into a poly-guanine sequence (T. Barnes and S. Hekimi, personal communication). The insertion of extra nucleotides to the poly-guanine track right after the predicted signal peptide region resulted in a frame shift upstream of the TRX domain and thus gave the same null phenotype.
A second group of alleles, e224, e390, e455 and e733, had mutations all mapped at the same position converting a glycine residue in the TRX domain to glutamic acid (G76E). The conversion of a non-charged residue to a charged one in this domain probably impaired the catalytic function, as demonstrated in the in vitro enzymatic assay, and therefore lead to a partial loss-of-function phenotype with mild Dpy body and Ram rays.
Mutant animals of e395, e431, s10 and s360 alleles had Dpy bodies with wild-type rays. They had the lesions clustered within the predicted transmembrane region. The s360 allele, resulting from formaldehyde mutagenesis, had nine nucleotides deleted, which led to the shortening of the encoded transmembrane domain. The s10 allele had glycine 191 changed to a positively charged arginine. Both s10 and s360 had a strong Dpy phenotype. The e395 and e431 alleles had identical mutations changing glycine 187 to glutamic acid, and mutant animals were mildly Dpy. These four mutant alleles might have weakened the membrane association of the mutant product to a different extent such that they all had a Dpy but not a Ram phenotype. The results suggest that aberrations in the TM domain do not have the major impact on DPY-11 function in the rays that they do on body morphogenesis.
DPY-11 is a modifying enzyme
We present in this study the cloning of dpy-11 gene, which encodes a novel thioredoxin-like molecule. This putative DPY-11 protein is much larger than most thioredoxins (246 aa versus 105-127 aa) and has several distinct molecular features. The DPY-11 TRX domain is highly similar to thioredoxins of prokaryotic and eukaryotic origins, although the CPGC catalytic site consensus was substituted by a CPAC sequence. Nevertheless, the non-consensus CAPC active site has been found in an active thioredoxin, C-2 in Corynebacterium nephridii (Fig. 5A) (McFarlan et al., 1989). In this study, we demonstrated biochemically that a bacterially expressed truncated DPY-11 TRX domain had a reducing activity comparable to that of a true thioredoxin from E. coli. A similar result was also reported for the TRX domain of the human DPY-11-like protein with the same CPAC sequence (Matsuo et al., 2001). Because four dpy-11 mutant alleles with a reduced gene activity, e224, e390, e455 and e733, had their lesions mapped within this TRX domain and could reduce the catalytic activity of this domain, as illustrated in the enzymatic assay, the catalytic property of this domain apparently represents a key function of DPY-11 protein.
It is interesting to note in our in vitro enzyme assay a lag phase in the first ten minutes. A similar 10 minutes lag was also observed when human DPY-11-like product was tested (Matsuo et al., 2001). The recombinant protein displayed a sigmoid curve of catalytic activity over time. It appears that DPY-11 requires an initial activation phase before enzymatic activity could be detected. Although no precedence of such a feature has been reported for thioredoxins, the existence of such property in DPY-11 may be due to the difference of substrate specificity between this DPY-11 TRX domain and other thioredoxin. Alternatively, it may be caused by the GST tag present in the fusion recombinant proteins used in this study. This possibility could not be ascertained at this point as removal of the GST tag reduced the protein solubility tremendously and prevented us from conducting the enzymatic assay.
DPY-11 modifies substrates within the hypodermal tissue
Our deletion analysis reveals that all the structural domains appear to be essential in making a functional DPY-11 protein. The signal peptide together with the single TM domain contribute to its topology. The prediction is that DPY-11 may have the TRX domain located inside endoplasmic reticulum, acting within the hypodermis to modify its substrates, which will be displayed on the cell surface or secreted to the extracellular matrix. As an alternative, DPY-11 protein may also reside on the hypodermis surface to modify substrates deposited by the same hypodermis or by its neighboring cells into the matrix. Failure to rescue the Dpy and Ram phenotypes by displaying the catalytic domain on the hyp cell surface with pd11TMGFP transgene, or expressing full-length DPY-11 protein in the structural cell with pr5cDNA are consistent with the former prediction, i.e., DPY-11 is acting within the hypodermis. Should the latter prediction be correct, these two transgenes could have provided the necessary modified function needed in the ECM within a ray, and reverted the mutant phenotypes into wild type.
Furthermore, active full-length DPY-11 protein with a GFP tagged near the C terminus was consistently detected in the cytoplasmic compartment, with a heterogeneous membranous distribution. Thus, DPY-11 may be associated with membranous organelles. This notion is supported by two additional observations: (1) myc-tagged human DPY-11-like protein was found associated with ER in mammalian cells (Matsuo et al., 2001). (2) DPY-11 has a putative ER retention signal at the C terminus (Hong, 1996). Although apparent change of the cytosolic appearance was not observed in fusion DPY-11-GFP proteins without this KKTK sequence (pd11ΔCTGFP) or in fusion protein with the KKTK sequence masked by GFP (pd11D11GFP), these fusion proteins had a much lower mutant rescue efficiency. Obviously, the C-terminal tail is not dispensable. Whether it contributes solely to the protein localization or is also involved, in association with other cellular factors, in regulatory function remains to be determined. However, the retention of DPY-11 in the cytoplasm is definitely governed by motif(s) residing in the TM region and this C-terminal tail, since recombinant protein without these two regions, encoded by pd11TMGFP, was displayed on the cell surface instead.
The results from the deletion study also illustrate the importance of DPY-11 membrane association in both body and ray morphogenesis. Mutant protein lacking its TM domain had no mutant rescue activity at all. The e33 allele with the TM domain and sequence beyond eliminated, also presented a null phenotype. Both results argue that a secreted DPY-11 enzyme is non-functional, such that its substrate is unlikely modified in situ at the extracellular matrix, but more likely is modified within the hypodermis and is deposited extracellularly afterwards. More convincingly, mutations that would hamper the membrane anchorage ability of the mutant proteins, either by amino acids deletion (s360) or by introducing a charged amino acid into the hydrophobic TM domain (e395, e431 and s10), could reduce the DPY-11 activity and resulted in Dpy animals. Although they had normal ray morphology at 20°C, the s10 animals at 25°C, exhibiting a swollen ray tip phenotype at high penetrance, suggest that this allele has reduced dpy-11 activity (Baird and Emmons, 1990; Yu and Chow, 2000). The lack of mutant phenotype at 20°C may reflect the presence of a low but sufficient level of membrane-associated DPY-11 to sustain the less demanding ray morphogenesis. After all, membrane anchorage of DPY-11 is still important for both ray and body morphogenesis.
DPY-11 may modulate cellular interaction through its matrix substrates
Based on the gene expression pattern, the catalytic property, the localization of DPY-11 within the expressing cell and the impact of its mutations on the differentiation of all three ray cell types, we propose that DPY-11 protein is made in the hypodermal syncytium to modify its substrate molecules. These substrates, which may be signaling molecules themselves, will be deposited in the ECM to facilitate interaction between the ray cells. A large number of cuticular collagen molecules, such as those encoded by dpy-2, dpy-7, dpy-10 and dpy-13 genes, are made, modified upon maturation and secreted by the hypodermal tissues (Gilleard et al., 1997; Johnstone, 2000). Mutations of these genes produce a body phenotype very similar to that of dpy-11 mutants. They may represent good candidate substrates of DPY-11.
In addition, mutations of some of these collagen genes, e.g., dpy-2 and dpy-10, can suppress mutant phenotypes of glp-1 and mup-1 (Maine and Kimble, 1989; Goh and Bogaert, 1991). While glp-1 encodes a transmembane molecule required for communication between the distal germ nuclei and the somatic gonad, mup-1 is needed for body wall muscle attachment to the hypodermis possibly through cellular communication (Kimble and White, 1981; Austin and Kimble, 1987; Goh and Bogaert, 1991). Since both glp-1 and mup-1 are shown to genetically interact with these collagen genes, it has been postulated that there are common mechanisms shared between the developmental events guided by these two genes (Nishiwaki and Miwa, 1998). The ability of dpy-11 to suppress mup-1 suggests that dpy-11 may indeed modulate the developmental or signaling process through modifying the collagen molecules in the body (Nishiwaki and Miwa, 1998). Moreover, male tail specific collagen molecules required for normal morphogenesis of sensory rays do exist (Baird and Emmons, 1990) (R. Y. Yu and K. L. Chow, unpublished data). They are deposited in the extracellular matrix around the ray cells during the tail retraction period (R. Y. Yu and K. L. Chow, unpublished data). It is highly probable that the matrix environment within a ray would be altered in the absence of dpy-11 modifying activity, which subsequently results in impaired cellular communication and abnormal ray morphogenesis.
In summary, C. elegans dpy-11 gene encodes a thioredoxin-like protein with a transmembrane domain. Although it shares conserved structural organization with other dpy-11-like molecules, the biological and developmental relevance of such related molecules has not been determined (Matsuo et al., 2001). In this present study, we showed that the membrane association and catalytic function as well as its residence within the hypodermis are important for body and ray morphogenesis. Hence, the study of dpy-11 regulation and future identification of its substrates would offer a platform for further analysis of the disulfide oxidoreductase system in matrix function and are instrumental to our investigation on regulated cellular communication during sensory ray morphogenesis.
The authors thank Dr K. C. Chow, Raymond Yu and colleagues in the lab for critical comments of this manuscript. We are grateful to Jonathan Hodgkin, David Baillie and the Caenorhabiditis Genetics Center for providing essential mutant alleles and worm strains for this study. We also thank Yuji Kohara for the cDNA. This research was partially supported by grants, HKUST 647/96M and HKUST 6110/98M, from Research Grants Council, Hong Kong.
Accepted December 7, 2001.
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You are going to email the following A novel thioredoxin-like protein encoded by the C. elegans dpy-11 gene is required for body and sensory organ morphogenesis
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What makes us feel compassion?
How do we hold on to that, in the face of fear?
In the last year, we in Australia have found ourselves once again confronted with terrorism, and all of the hatred and fear that brings.
Certainly, there has been tension, as we wait for the expected backlash, the vengeance to pour out onto the streets, on to our screens or into our conversations. But instead of retaliation, what we're seeing is a more compassionate response. There is a desire to reach out and connect with our community. To connect with those we believe share our values as we search for what we have to hold on that feels true and strong and human.
During the Sydney Seige, the hashtag #illridewithyou became viral, a form of hashtag activism. This came through again in response to the Charlie Hebdo attacks in the form of the cartoons drawn, the #jesuischarlie hashtag, the #jesuisahmed hashtag.
Although it seems odd to search for the positives when others have lost their lives to such anger, it's interesting, and heartening to witness a shift away from a mindset that fights hatred with more hatred.
But what is the value of this? Does the viral nature of these messages hold a true mirror up to a more compassionate society? When social media so often feels like a superficial and narcissistic medium, it's difficult to believe it leaves any tangible or lasting impression on our attitudes to 'real life'.
A month has passed since the siege, and we have to wonder, have we done anything positive with how far reaching this sentiment was? Has anything stuck?
Of course, terrorism is not new. But when the planes hit the Twin Towers, it well and truly came into our homes. At least in recent memory, this was the first time that our corporate and pop culture has been challenged. And in an age before social media – the idea that we could see the tragic event unfold so instantaneously from the other side of the world represented such a stark difference from how we normally perceived violent events off shore. It felt closer and we were outraged.
It's safe to say that the collective response, and the ways in which we choose to remember it – are representative of another, perhaps more negative mindset. 'Power' shifted from smoking guns, to battles of mindsets.
The current threat is different and it definitely feels different. It's on our shores and it's coming from within the folds of our society. In this, there is a realisation that what we're fighting feels difficult to grasp in the most literal, tangible sense.
And what we're fighting against is not some specific ideological challenge, but rather against a hatred of our own ideals – of our ways of living and all that we hold dear such as freedom of expression, freedom from the threat of execution, freedom to feel safe in public spaces… These are highly symbolic attacks on the values we hold.
Importantly these attacks are not on the institutions, which may or may not represent our culture, but they are on the culture itself – in our cafés, on our streets. The next target is impossible to predict. This is a terrifying thought, but it necessarily forces us to feel compassion and empathy – because it could've been me.
Our ideals have not been challenged so strongly and so visibly for a very long time. To connect with community necessarily requires introspection – what is it that connects us? And so we feel a need to do something, to reach out for what makes us human. Social media has been instrumental in the current climate by broadcasting clear messages of hope, compassion and combined strength.
Yes it seems vapid and impermanent and almost insufficient in the face of fear – but importantly, it reflects our need to share and connect with a wider audience – to know there's an army of compassionate souls out there, in a moment of helplessness.


text:
Andy Robertson urges Scots to prosper under pressure in Euro clash
Captain Andy Robertson wants Scotland to play without fear. Picture: SNS.
GAVIN MCCAFFERTY
Published: 22:30 Sunday 08 September 2019
Scotland captain Andy Robertson has called on his team-mates to thrive on the pressure of their must-win game against Belgium and show they enjoy playing for their country.
Robertson claimed on Friday that Scotland were a "bag of nerves" after losing their early
lead in a 2-1 defeat by Russia, which left them six points off second place in their Euro 2020 qualifying group.
But the Liverpool left-back is confident there is a strong enough mentality in the squad to cope with the added pressure that comes with representing a nation.
The 25-year-old said: "I believe if we don't win then this group is too much work for us. We need a result.
"We are playing against the best team in the world so it's going to be tough but we need to give our all, play with no fear and try and enjoy it – because I don't feel we look as if we are enjoying it as much as we probably should be when we represent our country.
"Put a smile on our faces, enjoy playing football and try getting the result we need.
"It's about trying to enjoy the pressure and thriving on it. We all play with pressure
at our clubs. If you look at the whole squad, last season everyone was playing for something. That's the pressure we deal with every single
week.
"But sometimes playing for your country is that added pressure. This squad is able to deal with it, there's not a doubt about that, but maybe on Friday we didn't quite learn from the game as much.
"The last ten minutes we put three long free-kicks in and the keeper came and collected
all three. We should have read when he was coming to the 18-yard line."
The former Queen's Park, Dundee United and Hull City player added: "At your club level you are maybe representing a town or a city, when you are coming up here you are representing your whole country. Everyone wants us to do well and everyone wants us to get to tournaments and support us in that way.
"But that's pressure we want. As kids, if you said we'd be in the Scotland squad, we would have been over the moon.
"All of us know how to deal with it but it's about showing it on the pitch. We have maybe not done that enough and it's about stepping up now and showing we want to play for our country, showing how proud we are to play for our country, and the result can follow."
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text:
The Top 10 Translation Fails Of Historic Proportions
These mistranslation mishaps are a blast from the past!
By Nuno Marques
Many of us who learn languages end up using those skills professionally as translators and interpreters. But humans are fallible and we tend to forget that, up until recently, most of the population of the Western world was illiterate. Learning languages was a sign of a privileged education, and translating texts was a task accomplished by a select few for a very small audience — leading to blunders and epic translation fails that are still perpetuated today. Here are a few examples.
History's 10 Worst Translation Fails
The Waitangi Treaty
Signing agreements is serious business, especially if you're signing a bad translation. When the Maori chiefs signed the Waitangi Treaty with the English, it was imperfectly translated into the Maori language, stating the indigenous population would have control over their territory but would surrender governance to the English, maintaining authority and control over their land. The English version, however, demanded a surrender of sovereignty in all of its rights and powers. So New Zealand became a colony instead of an independent land.
Pepsi Is Necromantic
In the 1960s, Pepsi's slogan, "Come Alive With the Pepsi Generation," arrived in China to very negative responses. The reason? In Chinese, the slogan translated to "Pepsi brings your relatives back from the dead."
A Buried Meaning
Nikita Khrushchev infamously concluded a Cold-War-era speech with a threat directed at America: "We will bury you!" Needless to say, this blood-thirsty statement whipped up American fear and outrage. But the true translation of his words was more like, "We will be present when you are buried," which is a common Russian phrase meaning, essentially, "We will outlast you."
Napoleon's Brother
Lodewijk Napoleon was crowned king of Holland in 1806 by his brother, Napoleon Bonaparte. Eager to please his people and to be loved by the general population, he changed his name from Ludovic to Lodewijk and declared himself Dutch rather than French. His language skills, however, were initially so poor he ended up declaring himself the "Rabbit of 'Olland" (Konijn van 'Olland) instead of "King of Holland" (Koning van Holland).
False Friends
According to interpreter John Coleman-Holmes in his book Mâcher du Coton, a Spanish delegate once apologized to the other delegates at a conference for having a cold: "Estoy constipado, perdóname!" But his Spanish was rendered into French as, "I am constipated, please excuse me!" The interpreter attempted a swift explanation, but was only met with laughter and ridicule.
The Horned One
According to the Torah, when Moses descends from Mount Sinai bearing the ten commandments, he is glowing with karan, or "radiance." But written Hebrew has no vowels, and St. Jerome, the patron saint of translators who learned Hebrew to translate the Bible into Latin, confused karan with keren and turned radiant Moses into a horned one. So with one typo the man gained horns, a representation that can be found in Michelangelo's Moses, for instance.
The Pen Is Mightier Than The Penis
Branding is serious business, and one bad translation is enough to make a product fail. When Parker Pen expanded into Mexico, its slogan read, "It won't leak in your pocket and embarrass you." But the verb "to embarrass" was confused with embarazar, a false cognate in Spanish, so the product was promoted with the translation, "It won't leak in your pocket and make you pregnant."
Jimmy Carter Seduces Poland
When U.S. President Jimmy Carter traveled to Poland in 1977 for a news conference, he was subjected to a succession of translation fails. The interpreter responsible for conveying the president's English into Polish mangled his words, using the occasional Russian term and abusing the Polish language. When the president mentioned he had left the U.S. that morning, the interpreter stated he had left the country, never to return. To add insult to injury, the president's desire to "come to learn your opinions and understand your desires for the future" was rendered erotically as a strong lust to "get to know the Poles carnally."
Things didn't get much better when the replacement interpreter sat silently while the president spoke. Apparently he could not understand Jimmy Carter's Southern accent and chose not to speak, for fear of committing further diplomatic sins.
Jimmy Strikes Again
In 1981, while giving a speech to a small Methodist college in Japan, President Carter opened with a joke. The interpreter spoke to the crowd, and the audience immediately erupted into laughter. Surprised by the joke's success, Carter asked the interpreter how he'd managed to elicit such a reaction. The interpreter reluctantly admitted that he had said, "President Carter told a funny story. Everyone must laugh."
Lazy Translation Fails
In 2009, HSBC bank scrambled to fix a $10 million rebranding campaign after their catchphrase "Assume Nothing" was mistranslated as "Do Nothing" in various countries.
Want to avoid making your own translation gaffes?
Nuno Marques
Nuno is a puppeteer and scriptwriter who spends his time twiddling his thumbs inside and outside puppets' heads. He enjoys daydreaming, biking in Berlin and opera. Some of his best friends are made of felt and foam!
Funny Mistakes By The Everyday Language Learner
Mistakes are a crucial part of language learning and nothing to feel bad about. They can sometimes be embarrassing, but they also make for some funny stories.
By Mollie Hosmer-Dillard
Why You Shouldn't Use Google Translate On Your Trip
Remove the training wheels and connect authentically. You'll be happy you did.
Brands In Translation: 9 Cringeworthy Language Fails
These marketing translation fails will make you laugh, or maybe blush.
By Dylan Lyons


Though relatively new to the game, RoMa Craft is already a heavy hitter in the world of premium cigars, offering a variety of delicious sticks crafted from the finest leaf available, and carefully blended and hand-rolled to suit a range of tastes and preferences. Unlike many other boutique brands, RoMa Craft actually owns their own tobacco factory in Esteli, Nicaragua, where their passionate team produces the popular Intemperance, CroMagnon, and Aquitaine lines under the supervision of the extremely talented Esteban Disla. Offered in a range of capas and vitolas, discover the new luxury of RoMa Craft today.


text:
Q: How to center a div in another div using flexbox? I want to make a header image with a semi circular bottom. In the middle of this image I want a title and a button. I can't manage to center the div with the title and button using just flexbox or bootstrap. mx-auto (margin x auto) works but my-auto doesn't, align-self-center and align-items-center classes doesn't work, I have to add mx-auto. To center the circular bottom I have to use margin-left: -25vw. What is the best way to center te divs?
This is my code:

.image-wrapper {
 position: relative;
 width: 100vw;
 height: 100%;
 overflow: hidden;
}

.circular-bottom {
  width: 150vw;
  height: 90vh;
  margin-left: -25vw;
  border-bottom-left-radius: 50%;
  border-bottom-right-radius: 50%;
  overflow: hidden;
}

.circular-bottom img {
  z-index: -1;
}

.title-box {
  position: absolute;
  top: 0;
  height: 100%;
  width: 100%;
}

.title-text {
  text-align: center;
  width: 40%;
 }

Title

      button text

The image-wrapper prevent the horizontal scroll. The circular-bottom makes the bottom of the image circular using overflow hidden, it's wider for the shape of the circle. This is also the reason the image isn't a background image, it have to be cut off by using overflow hidden.

A: Adding the justify-content: center property to the flex container (.title-box in this case) should work, like this:

.image-wrapper {
 position: relative;
 width: 100vw;
 height: 100%;
 overflow: hidden;
}

.circular-bottom {
  width: 150vw;
  height: 90vh;
  margin-left: -25vw;
  border-bottom-left-radius: 50%;
  border-bottom-right-radius: 50%;
  overflow: hidden;
}

.circular-bottom img {
  z-index: -1;
}

.title-box {
  justify-content: center;
  position: absolute;
  top: 0;
  height: 100%;
  width: 100%;
}

.title-text {
  text-align: center;
  width: 40%;
 }

Title

      button text

I have already tried it and It's working for me.
Link to the working codepen: https://codepen.io/anon/pen/YLOwxX

redpajama_set_name: RedPajamaStackExchange


text:
Q: Get errors from lambda as alert I am investigate approaches for get errors from my lambda functions as alert by email. I want get error with text error and lambda name in email body. My functions is working as Async and Sync mode. I was used Cloud Watch but I faced with problems.
1 variant: In CW was created one alert for all functions(more then 300 and they will changed your count, not static value). This way has one big plus - all functions in my region was added and deleted from alert dynamically. But alert we will get only with common information - that we have some eror with some function.
2 variant: Set up for each function AWS Lambda Function Dead Letter Queues, and join with aws sns. In this way I have got email with errors but, it's work only with Async lambdas. And I should set up DLQ for each functions after created.
3 variant: Paid solutions (
Maybe anybody can advice me good solutions?

A: The way you would normally do this is by adding one alarm for every function - instead of your current setup of an alarm for all of them together.
If you were to do this manually, it's a hellish job. So consider using Cloudformation/Tropsophere/Terraform. Not only is it a good idea to version control your infrastructure, that way you can write a function/method to create and add an alarm to a lambda once.
The good thing is, once you have an alarm for every lambda, AWS will take care of the rest (tracking the errors and sending emails). And your message will be specific to every lambda, unlike now.


At Sun Life Global Investments, we believe that thinking and acting from a global perspective brings to light the best opportunities for our investors. Our goal is to help them invest confidently so they can live life on their terms. In 2018, Sun Life Global Investments acquired Excel Funds – an investment fund manager specializing in emerging markets funds. The acquisition adds to our global sub-advisor network by providing access to Aditya Birla Sun Life Asset Management, Amundi Asset Management and China Asset Management Company Limited.
As a member of the Sun Life Financial group of companies, we have access to investment professionals located around the world who for years have sought out and engaged best-in-class investment managers. We are able to leverage these preferred relationships to provide Canadian investors a diverse line up of mutual funds, investment solutions and services designed to help them pursue their financial goals, both now and in the future.
We are a fund manager however we also select global managers from other firms, who share our vision and objectives, to sub-advise funds built for our product shelf. Partnering with these managers allows us to bring additional investment expertise to the Canadian investor all offered through Sun Life Global Investments.
JPMorgan Asset Management (Canada) Inc.
These teams each have a unique, disciplined, research-based approach to selecting and managing the funds in their portfolios, with a view to attaining long-term performance results. Together, they have a worldwide reach and a long and proven history of success. This expertise, combined with the best-in-class risk management practices Sun Life Financial has developed over the last 150 years, allows us to open up a whole new world of investment opportunities for Canadian investors.


text:
package org.anc.lapps.stanford;

import edu.stanford.nlp.ling.CoreAnnotations.SentencesAnnotation;
import edu.stanford.nlp.pipeline.Annotation;
import edu.stanford.nlp.pipeline.StanfordCoreNLP;
import edu.stanford.nlp.util.CoreMap;
import org.anc.lapps.stanford.util.Converter;
import org.lappsgrid.discriminator.*;
import org.lappsgrid.annotations.ServiceMetadata;
import org.lappsgrid.serialization.Data;
import org.lappsgrid.serialization.Serializer;
import org.lappsgrid.serialization.lif.*;
import org.lappsgrid.vocabulary.Annotations;
import org.slf4j.Logger;
import org.slf4j.LoggerFactory;

import java.util.HashMap;
import java.util.List;
import java.util.Map;
import java.util.Properties;

import static org.lappsgrid.discriminator.Discriminators.Uri;

/**
 * @author Keith Suderman
 */
@ServiceMetadata(
        description = "Stanford Sentence Splitter",
        produces = { "sentence" },
        requires_format = { "text", "lif", "json" }
)
public class SentenceSplitter extends AbstractStanfordService
{
   private static final Logger logger = LoggerFactory.getLogger(SentenceSplitter.class);

   private StanfordCoreNLP service;

   public SentenceSplitter()
   {
      super(SentenceSplitter.class);
      Properties properties = new Properties();
      properties.setProperty("annotators", "tokenize, ssplit");
//      pool = new ArrayBlockingQueue<StanfordCoreNLP>(POOL_SIZE);
//      for (int i = 0; i < POOL_SIZE; ++i)
//      {
//         pool.add(new StanfordCoreNLP(properties));
//      }
      service = new StanfordCoreNLP(properties);
      if (service == null)
      {
         logger.error("Unable to create the StanfordCoreNLP object.");
      }
      else
      {
         logger.info("Standford sentence splitter created.");
      }
   }

   @Override
   public String execute(String input)
   {
      logger.info("Executing Stanford sentence splitter.");

      Data data = Serializer.parse(input, Data.class);
      if (data.getDiscriminator() == null)
      {
         return createError(Messages.MISSING_DISCRIMINATOR);
      }

      String discriminator = data.getDiscriminator();
      Container container = null;
      String json = null;
      switch (discriminator)
      {
         case Uri.ERROR:
            json = input;
            break;
         case Uri.GETMETADATA:
            json = super.getMetadata();
            break;
         case Uri.TEXT:
            if (data.getPayload() == null)
            {
               json = createError(Messages.MISSING_PAYLOAD);
            }
            else
            {
               container = new Container();
               container.setText(data.getPayload().toString());
            }
            break;
         case Uri.LAPPS: // fall through
         case Uri.JSON:
         case Uri.JSON_LD:
//            container = Serializer.parse(payload, Container.class);
            if (data.getPayload() == null)
            {
               json = createError(Messages.MISSING_PAYLOAD);
            }
            else
            {
               container = new Container((Map) data.getPayload());
            }
            break;
         default:
            json = createError(Messages.UNSUPPORTED_INPUT_TYPE + discriminator);
            break;
      }
      if (json != null)
      {
         return json;
      }

      Annotation document = new Annotation(container.getText());
//      Data<Container> data = new Data<Container>();
//      StanfordCoreNLP service = null;
//      try
//      {
         //service = pool.take();
//         service = pool.poll(DELAY, UNIT);
//         if (service == null) {
//            logger.warn("The SentenceSplitter was unable to respond to a request in a timely fashion.");
//            return DataFactory.error(Messages.BUSY);
//         }

      if (service == null)
      {
         return createError("No service object has been instantiated.");
      }
      if (document == null)
      {
         return createError("Unable to create Stanford Annotation document for text.");
      }

      service.annotate(document);
      List<CoreMap> sentences = document.get(SentencesAnnotation.class);
      View view = Converter.addSentences(new View(), sentences);
      String producer = this.getClass().getName() + ":" + Version.getVersion();
//      view.addContains(Annotations.TOKEN, producer, "tokenization:stanford");
      view.addContains(Uri.SENTENCE, producer, "sentence:stanford");
      container.getViews().add(view);
      data.setDiscriminator(Uri.LAPPS);
      data.setPayload(container);
      //data = DataFactory.json(container.toJson());

//      }
//      catch (InterruptedException e)
//      {
//         data = DataFactory.error(e.getMessage());
//      }
//      finally
//      {
//         if (service != null)
//         {
//            pool.add(service);
//         }
//      }
      logger.info("Sentence splitter complete.");
      return data.asJson();
   }

//   public Data configure(Data input)
//   {
//      return DataFactory.error("Unsupported operation.");
//   }
}

redpajama_set_name: RedPajamaGithub


Have you ever played fantasy sports with your heart instead of actually using logic? During the summer of 2018, I decided to define all conventional wisdom and draft my fantasy baseball team with just players from the New York Yankees.
The Yankees finished as one of the best teams in all of baseball while breaking the home run record. They could also have a finalist for Rookie of the Year. My one team concept could work based on the calibre of the team (face it these are not the 2018 Baltimore Orioles), but in the world of fantasy sports, this was indeed a very terrible decision.
My draft strategy was pretty simple and I was able to collect every player who suited up for the Yankees except Giancarlo Stanton. He was drafted with the third overall selection which forced me to take Aaron Judge at number five. I followed with the following selections Gary Sanchez (2nd round), Luis Severino (3), Aroldis Chapman (4), Masahiro Tanaka (5), Didi Gregorius (6), Sonny Gray (7), Greg Bird (8), David Robertson (9), CC Sabathia (10), Brett Gardner (11), Dellin Betances (12), Aaron Hicks (13), Neil Walker (14), Jordan Montgomery (15), Brandon Drury (16), Tyler Wade (17), Miguel Andujar (18), Roland Torreyes (19), Austin Romaine (20), Chad Green (21), Clint Fraizer (22), and Jacoby Ellsbury (23). Most of you could admit that I put together a decent team of players considering Gleyber Torres wasn't even on my radar on draft day. Thankfully I was able to pick him as a free agent on April 21, some 19 days after the regular season started.
As the season progressed and the trade deadline(s) came and went, I missed out on acquiring J.A. Happ and Andrew McCutchen. Both players were actually on the same fantasy team and since the Yankees didn't part with any big names from my fantasy roster, I wasn't able to trade for them. I was able to pick up other mid-season acquisitions like Zach Britton off waivers while Luke Voit, Adeiny Hechavarria, Lance Lynn were all free agents.
Over the course of the season, I made 24 moves, fifth highest in my league. The Yankees called up several prospects like Justus Sheffield, Jonathan Loaisiga, Domingo German, and Kyle Higashioka while giving opportunities to veterans like Tommy Kahnle. It was really hard to fill in all the gaps, especially when people got called up one day and they were gone the next. I was only allowed four roster moves a week, so thankfully the big club didn't fall apart or I would have gone over the limit several times over.
Now let's get to the real reason why you've read this far: the numbers. The Evil Empire (my team name) finished the season a depressing 58-144-13 for a sparkling 0.305 winning percentage and 63.5 games out of first place, in an eight-team league. To put those numbers in perspective, the first overall team finished 125-84-11 (67 more wins) for a 0.593 winning percentage. The top four teams in the league made the playoffs, with the top six teams winning 100 or more categories and seventh place finished with 98 wins. Realistically I only needed 48 more category wins to even have a shot at making the playoffs, something I have achieved in other years.
Our league had ten scoring categories, five for hitters and five for pitchers. As you would imagine, we came in dead last in most of the team stats. The Evil Empire finished eighth in runs (658), eighth in home runs (202), eighth in RBIs (646), eighth in stolen bases (58), and eighth in batting average (.249). The league leaders in each category were 979 runs, 260 home runs, 870 RBIs, 151 stolen bases, and .274 average.
New York state is famous for miracles, so wouldn't you know it our pitchers were not the worse in the league. Any finish above eighth place would be considered a success, so the Evil Empire finished sixth in wins (77), tied for sixth in saves (44), sixth in strikeouts (1225), seventh in ERA (3.78), and tied for seventh in WHIP (1.22). The league leaders in each category had 105 wins, 200 saves, 1644 strikeouts, a 2.89 ERA and a 1.06 WHIP.
My Yankees pitching staff, minus Happ, really couldn't hold a candle to the all-star lineups of other teams. The real Yankees, on the other hand, won exactly 100 games (3rd best MLB), had 49 saves (5th), threw 1634 strikeouts (2nd), while boasting a 3.78 ERA (10th best), and a 1.24 WHIP (6th best). The Yankees meanwhile scored 851 runs (2nd), collected 821 RBIs (2nd), belted out 267 home runs (1st), stole 63 bases (25th) and batted a collective .249 (16th).
Now let's hypnotically say I had the full Yankees lineup all season long. Against the all-star selections of the seven other teams in the league, here's where I could have finished with the MLB Yankees numbers: fourth in runs, first in home runs, third in RBI, eighth in stolen bases, and eighth in batting average. From the mound, we would have been finished fifth in wins, sixth in saves, second in strikeouts, seventh in ERA, and eighth in the WHIP.
There were times during the season when the Yankees rode a winning streak of multiple games, but you could never tell in fantasy baseball because a 2-1, 3-2, and 1-0 win don't equal mind-blowing fantasy stats. Everyone slumps and very few players explode with enough power that lasts through an entire seven-day fantasy match up.
To make things more interesting, I calculated the statistics of the few players I never had, McCutchen, Happ, and Staton. After the new numbers emerged, The Evil Empire was still not a playoff team but managed to improve their win total by 29 and their matchup totals by five.
After an opening week tie, the Evil Empire went on to lose seven straight matchups to move in the basement of the league, where they stayed until the end. The only win of the season came in Week 9, followed by a tie in Week 10. Things appeared to be on the rise, but they lost 11 out of the final 12 matchups to finish the season a combined 58-144-18 with a series record of 1-18-3.
Staton all by himself would have altered the fantasy world with a slash line like 102 runs, 38 home runs, 100 RBIs, 5 stolen bases, a .266 batting average. McCutchen meanwhile scored 18 runs, hit 5 home runs, collected 10 RBIs, stole a base, and batted .253 in pinstripes. Happ was a blessing in disguise with a 2.69 ERA, 7 wins (0 losses), no saves, 63 strikeouts, and a 1.05 WHIP in just 11 games in New York.
When you factor in all those extra numbers, the story of the Evil Empire would have been much different. Instead of a tie in week one, they would have won the matchup 5-4-1. The team would be 3-2-0 after five weeks thanks to two losses and then two wins. A tie, win, and loss would keep things around the .500 mark before two more wins would put the fantasy team at 6-3-1. Things would go south after Week 10 as the club would manage only five ties in the last 12 matchups to finish the year at 6-10-6. In the statistical categories, the final numbers could have been 87-117-16.
These numbers only reflect the three Yankees players who played on other teams. No one else in the league played Kyle Higashoika except for me and I didn't always bring him up or have in my lineup. My final numbers could have been better if I had A.J. Cole in my line up whenever he randomly got the call from the bullpen, or if I simply paid enough attention to the pitching matchups. I was curious about the impact these particular players would have had for my lineup. There was no way for me to get McCutchen or Happ, but I did offer to pay the league fees ($40) for the guy who drafted Stanton.
I really wanted to see how I could pull off a whole season with just one baseball team. We all know by now that my team sucked and that my strategy was worse than Oakland's Moneyball one, but I actually didn't finish in the last place. The Evil Empire won the 7th/8th place matchup 5-4-1 thanks to a hot streak by the Yankees towards the end of the season, so the history books will say I didn't finish in the last.
If you are curious about my plans for next year, I may try for Judge or Torres but I doubt I would pursue any other Yankee. It is clear if you want to win 27 world fantasy champions, you need to create an all-star team that competes all season and not for one weekend in July.


Your repositories is a mess you have repositories even for different architecture so lets clean that mess.
From the "Other Software" tab you can add or remove repositories that dont work such as for ex: http://ppa.launchpad.net trusty/main armhf Packages which is for ARM (armhf) architecture.
Also delete repositories that saying 404 Not Found in your terminal.
Go to directory /home/james from your file manager and click to view your hidden folders, the file is .wine just delete that.
Install Winetricks from Ubuntu software center.
And follow the instructions to install the latest stable or release version of wine.
Now find out what you need to install from Winetricks according to what program or game you want to use.


How many wells do you want in your drain? Probably the first decision most buyers make is to identify whether they prefer a single well or a double well. These are generally the two main configurations. Double well models are also obtainable, along with specialty designs like trough sinks and integrated drain-board sinks. An individual well is great for those who frequently prepare large turkeys, lot dinner parties or choose to preserve food for the winter. A single well is also invaluable for washing small pups and bathing grubby newborns. Double well sinks serve two cook families and the ones who like to multi-task, rinsing vegetables for the salad in one well, while cleaning in the other well.


UltraTouch is made from high quality natural fibers. These fibers contain inherent qualities that provide for extremely effective sound absorption and maximum thermal performance. UltraTouch DOES NOT ITCH and is very easy to handle and work with.


Environmental and waste management including air quality, molting of layer/breeder, and insect control.
Extension responsibilities include adult education programs in Nutrient Management Planning and Agro/Bio- security seminars. Coordinator of the Deep South Poultry Conference held at the Rural Development Center, Tifton, Georgia. Involved in the preparation and judging of 4H and FFA poultry judging contests as well as contributing to programs that assist county extension personnel.
Research programs focus on environmental and waste management issues associated with the poultry industry including air quality. Other areas of focus include the effectiveness of induced molting in broiler breeder and commercial layer flocks and darkling beetle control in broiler houses.
K. D. Dunkley, C. S. Dunkley, N. L. Njongmeta, T. R. Callaway, M. E. Hume, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2007. Comparison of in vitro fermentation and molecular microbial profiles of high fiber feed substrates incubated with chicken cecal inocula. Poult. Sci. 86(5):801-10.
K. D. Dunkley, J. L. McReynolds, C. S. Dunkley, T. R. Callaway, M. E. Hume, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2007. Molting in Salmonella Enteritidis challenged laying hens fed an alfalfa crumble diet Part I: Salmonella Enteritidis colonization and virulence gene hilA response. Poult. Sci. 86(8):1633-9.
K. D. Dunkley, M. E. Hume, J. L. McReynolds, C. S. Dunkley, T. R. Callaway, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2007. Molting in Salmonella Enteritidis challenged laying hens fed an alfalfa crumble diet Part II: Fermentation and microbial ecology response. Poult Sci. 2007 Oct;86(10):2101-9.
C.S. Dunkley, J. McReynolds, K.D. Dunkley, L. N. Njongmeta, L. Kubena, D. Nisbet, and S. C. Ricke. 2007. Molting in Salmonella Enteritidis challenged laying hens fed an alfalfa diet Part III: Blood plasma metabolite response. Poult Sci. 2007 Dec;86(12):2492-501.
C.S. Dunkley, J.L. McReynolds, K.D. Dunkley, L.F. Kubena, D.J. Nisbet and S.C Ricke. 2007. Molting in Salmonella Enteritidis challenged laying hens fed an alfalfa crumble diet Part IV: Immune and stress protein response. Poult Sci. 2007 Dec;86(12):2502-8.
K. D. Dunkley, T. R. Callaway, M. M Kundinger, C. S. Dunkley, R. C. Anderson, D. J. Nisbet, and S. C. Ricke. 2008. Growth and genetic responses of Salmonella Typhimurium in an anaerobic continuous culture during shifts in pH. Anaerobe Feb;14(1):35-42.
C.S. Dunkley, W.K. Kim, W.D James, W.C Ellis, J.A. McReynolds, L. Kubena, D.J. Nisbet and S.C. Ricke. 2008. Passage rates in poultry digestion using stable isotope tracer markers. Journal of Radioanalytical and Nuclear Chemistry vol. 276(1) :35-39.
C.S. Dunkley, T.H. Friend, J.L. McReynolds, W.K. Kim, K.D. Dunkley, C.L Woodward, L.F. Kubena, D.J. Nisbet, and S.C. Ricke. 2008. Behavior of laying hens on alfalfa crumble molt diets. Poult Sci. May 87(5):815-22.
C. S. Dunkley, W. K. Kim, T. H. Friend, C. L Woodward, J. L. McReynolds , K. D. Dunkley, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2008. Behavioral response of laying hens to different alfalfa layer ration combination fed during molting. Poult Sci. 87:1005-1011.
K.D. Dunkley, T.R. Callaway, V.I. Chalova, J.L. McReynolds, M.E. Hume, C.S. Dunkley, L.F. Kubena, D.J. Nisbet and S.C. Ricke. 2008. Foodborne Salmonella ecology in the avian gastrointestinal tract. narobedoi:10.1016/j.anarobe.2008.05.007.
W. K. Kim, T. M. Herfel, C. S. Dunkley, P. Y. Hester, T. D. Crenshaw, and S. C. Ricke.2008. The Effects of Alfalfa-based Molt Diets on Skeletal Integrity of White Leghorns. Poult Sci 2008 87: 2178-2185.


Light abounds in this modern colonial on neighborhood cul de sac just a short walk to downtown Westport. Open and welcoming, 15 Pine Drive greets all visitors with flexible floor plan to benefit any buyer. Commuters will enjoy a 3 minute drive to Metro North yet a quiet location. Beach lovers can pedal two miles to Westport's crown jewel, Compo Beach or, perhaps, Longshore for golf or swimming. First floor has expansive eat-in-kitchen with island, walk-in pantry and even one of two fireplaces setting the tone. Relaxing family room, home office, living room and dining room complete the first floor and are blanketed in light. (There is a first floor bedroom option with full bath if desired.) upstairs master and two large bedrooms, one with balcony overlooking quiet yard. Finished lower level with fourth full bath and extra high ceilings includes over 1000 sq ft of play space to accommodate any hobby, delight active family and welcome friends.
SchoolsClick map icons below to view information for schools near 15 Pine Drive Westport, CT 06880.


text:
Proud Supporters of 2018 Ealing Half Marathon.
This year, Blue Swallow Group is a proud supporter of Ealing Half Marathon as a #100Club member. Ealing Half Marathon is an annual event, with over 5000 participants. It has been awarded silver in'The Running Awards' Half Marathon 5000+ category, for 2018.
On top...
Sanzhar Braves the London Marathon, for a Good Cause.
On the 22nd of April, our director, Sanzhar, has run the hottest ever Virgin Money London Marathon, on record. Under the scorching sun, he managed to complete the 42.2 km route in a respectable 04:45:36. In his true competitive spirit, he has vowed to beat his time by...
A Complete Guide to Magento 2 Benefits
In November 2015, Magento has launched an update to it's existing platform - Magento 2, much to the excitement of it's +300k active users. As with any new product, Magento 2 did not come without early-stage bugs and minor technical issues. However, even then, an overwh...
Blue Swallow and Extendware form a Strategic Partnership
Blue Swallow is delighted to announce a formation of a strategic partnership with Extendware - an award-winning Magento Technology partner, combining industry knowledge with years of experience, making it one of the most respected players in the Magento marketplace.
Ext...
Is your E-commerce store HTTPS-positive?
Customer's trust is key to improving sales and other micro and macto-conversions, for every e-commerce company. But, it is not just about earning trust for your products; customers also need to trust your e-commerce portal since they would have to share their card, wal...
Complete Guide to Magento 2 Community vs Enterprise Edition.
Choosing between Magento Community Edition (CE) and Enterprise Edition (EE) is not easy. But, thanks to Magento 2, the job is no longer as difficult as it was with Magento 1.
Magento 1 CE and EE had very few differences and you could achieve similar performance with Mag...
3 Takeaways from TechCrunch Disrupt
The long-awaited Tech Crunch Disrupt has, once again, visited the European technology capital, to amaze us with the latest developments in the world of tech, as well as give an opportunity for young, aspiring startups to showcase their unique products and services, gra...
T: 0044 2033 725 552
team@blueswallow.co
A:18 King William Street,
London, EC4N 7BP
© 2018 Blue Swallow Group


The Handbook of Clinical Anesthesia 2e is primarily an aid to those sitting the FRCA examinations. It will also become the one-stop source of reliable clinical information once FRCA accreditation has been achieved. The qualified anesthetists will recognise the same level in-depth coverage of all aspects from basic sciences, pharmacology and physiology, through to case-by-case anesthetic management plans that made the first edition such a success. The reader will find the Handbook an indispensable aide-memoire, providing all the essential practical knowledge required by the anesthetist on co-existing medical conditions, operative procedures nd techniques.


text:
FTP_COPY=0
#ftp hostname
FTP_HOST=ftpeu.epam.com
#ftp port
FTP_PORT=21
#ftp username
FTP_USER=pBLMB-MON
#ftp password
FTP_PASS=88y4pKLxpg
#ftp path to copy from
FTP_PATH=""
#TBD#
FTP_FILEMASK=""
#whether to package spark-jobs from source code
INSTALL_PACKAGES=0
#whether to drop and recreate hive and hbase schemas
INSTALL_SCHEMAS=0
#local path to temporary directory
SCR_PATH=/tmp
DIR_TEMP="${SCR_PATH}"
#hdfs path to the 'raw' area storage
RAW_AREA=/raw
DIR_OUTPUT="${RAW_AREA}"
#hdfs path to the Hive udf dir
HDFS_UDF_DIR=/user/hive/lib
#hive table name to store 'prepared' area data
PREPARED_AREA=prepared
HIVE_TABLE="${PREPARED_AREA}"
#hive database name
HIVE_SCHEMA=blmb_v2_2
#hive principal
HIVE_PRINCIPAL="hive/ecsc00104754.minsk.epam.com@EPAM.COM"
#hive connection url
HIVE_URL="jdbc:hive2://localhost:10000/;principal=${HIVE_PRINCIPAL}"
#hive connection username
HIVE_USER=hdfs
#hive connection password
HIVE_PASS=hdfs
#threshold to split hive master table data into buckets
MASTER_THRESHOLD=300000000
#microseconds limit to throw away any latency data beyond
LIMIT=1000000
#threshold to reduce all time-related data resolution inside hbase datamarts
REPORT_THRESHOLD=1000
#group prefix to separate several inputs inside single cluster
EXECUTION_GROUP=""
#path to the bloomberg source code directory
DIR_BLMB=~/git_branch/blmb-mon
#path to the datalake ftp-to-hdfs flow container
DIR_SPARK_INGEST=/source/datalake/ingest
#path to the datalake hdfs-to-hive flow container
DIR_SPARK_IMPORT=/source/datalake/import
#path to the datalake hive-to-hbase flow container
DIR_SPARK_REPORT=/source/datalake/report
#spark-on-cluster execution engine selection
SPARK_MASTER=yarn
#spark-on-cluster execution mode
SPARK_DEPLOY_MODE=client
#spark-on-cluster virtual cores to load
SPARK_DRIVER_CORES=1
SPARK_EXECUTOR_CORES=1
#spark-on-cluster ram memory to load
SPARK_DRIVER_MEMORY=1G
SPARK_EXECUTOR_MEMORY=1G
#spark credentials in kerberized environment
SPARK_KEYTAB=/etc/security/keytabs/spark.headless.keytab
SPARK_PRINCIPAL=spark-blmb_mon_hdp@EPAM.COM
#hbase-site.xml location on spark driver node
HBASE_SITE_CONFIG=/etc/spark/conf/hbase-site.xml
#hdfs url
HDFS_URL="hdfs://ecsc00104754.minsk.epam.com:8020"
#zookeeper quorum path
ZK_QUORUM="ecsc00104764.minsk.epam.com:2181:${SPARK_PRINCIPAL}:${SPARK_KEYTAB};ecsc00104765.minsk.epam.com:2181:${SPARK_PRINCIPAL}:${SPARK_KEYTAB};ecsc00104767.minsk.epam.com:2181:${SPARK_PRINCIPAL}:${SPARK_KEYTAB}"
#phoenix python interface path
PHOENIX_BIN=/usr/hdp/current/phoenix-client/bin/


and you are 30 lbs healthier.
been through much the same thing. Prison put the pounds on (starchy foods, inactivity) and it took a lot of sweat to get it back off.
so, i hope everything else is going good for you.


text:
San Alberico, Aubrey, Albericus o Albéric (Borgoña,  - Abadía de Císter, 26 de enero de 1108) fue un monje francés, cofundador de la Orden del Císter.

Biografía
No hay datos sobre su infancia. Sólo se sabe que, joven, quiso ingresar en la vida religiosa y se puso bajo la guía de Roberto de Molesmes, que había formado una comunidad benedictina cerca de Tonnerre. El lugar, sin embargo, no era adecuado para fundar un monasterio, por lo cual, en 1075 se marcharon a Molesmes, donde se fundó el monasterio. Roberto fue el primer abad, y Alberico el prior.

Pronto, sin embargo, la comunidad tuvo problemas de convivencia y algunos monjes se rebelaron; Roberto, el abad, se marchó hasta que los otros monjes que habían estado bajo el mando de Alberico llamaron al abad ya que estos se habían sublevado de tal forma contra su prior (Alberico) que hasta lo apalearon dejándolo inconsciente. San Roberto y San Alberico, sin embargo, viendo que no podrían llevar a término su ideal monástico, fueron, con algunos de los monjes, hasta Císter o Citeaux, en la diócesis de Châlons, donde en 1098 fundaron una comunidad con una forma de vida diferente al benedictino y que fue el origen de la Orden Cisterciense. Alberico, muy devoto de la Madre de Dios, puso la orden bajo la protección de la Virgen María.

Alberico fue elegido abad cuando Roberto, por orden del papa Urbano II, dejó el monasterio. Lo fue hasta el 26 de enero de 1108, cuando murió. Alberico hizo, durante su mandato, que la regla de vida de la comunidad fuera más austera. Se le atribuye el cambio de color negro de la cogulla (es el hábito que se ponen los cistercienses para ir al coro) por el de color blanco; de ahí que en la época se les comenzó a llamar a los cistercienses: "los monjes blancos" y a los benedictinos "los monjes negros" por el color de sus cogullas. Le sucedió san Esteban Harding quien llamó a su gran abad y amigo "la lanza de plata" pues esto era lo que había sido, una lanza defensora contra los ataques al corazón de Jesus.

Referencias

Enlaces externos

 "Alberic of Cîteaux", a St. Patrick's Church website
 "History of the Cistercians", en Catholic Encyclopedia

Santos católicos de Francia del siglo XI
Fundadores de una orden o congregación católica
Santos cistercienses
Santos católicos de Francia del siglo XII
Abades cistercienses
Cistercienses de Francia del siglo XI
Escritores medievales que trataron temas de mitología griega
Cistercienses de Francia del siglo XII

redpajama_set_name: RedPajamaWikipedia


How to get cheap bus tickets from Phoenix to Killeen?
Greyhound offer a total of 8 bus schedules from Phoenix to Killeen.
Bus booking in advance is the best way to find Killeen cheap tickets.
How much is bus ticket from Phoenix to Killeen?
As mentioned before, you will find on Greyhound to Killeen low-cost tickets for only .
This bus leaves at 03:20 from Phoenix bus station or stop at PHOENIX - 2115 E BUCKEYE RD.
On the contrary, the bus leaving at 03:20 have the most expensive ticket to Killeen for about .
How to get to Killeen from Phoenix?
On average you find 8 daily schedules to Killeen from Phoenix.
Greyhound bus lines operate the largest daily fleet to Killeen.
Where are Phoenix and Killeen bus stations?
For this trip to Killeen, you can get on board at PHOENIX - 2115 E BUCKEYE RD.
Moreover, get off the bus at KILLEEN - 200 E AVE C.
When is the first and last bus to Killeen?
The first bus time to Killeen is at 03:20.
How far apart is Killeen from Phoenix?
The bus traveling length from Phoenix to Killeen is around 5h 12m.
Selecting this company, you probably arrive in about 2h 45m in Killeen.
Reserve your bus seat to Killeen in advance and have a nice trip.


JEC World was a great success! Thank you to everyone who stopped by to talk with Litzler about our Prepreg Equipment and Plasma Oxidation Ovens. We were happy to have our salesman, Lin Feng Shen, from our Shanghai office join us in Paris.