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A nutrient deprivation-induced locus in Sinorhizobium meliloti strain 1021 was identified by use of a Tn5-luxAB reporter gene transposon. The tagged locus is comprised of two open reading frames (ORFs) designatedndiA and ndiB for nutrient deprivation-induced genes A and B. Comparison of the deduced amino acid sequences of bothndiA and ndiB to the protein databases failed to reveal similarity to any known genes. The expression of thendi locus was found to be induced by carbon and nitrogen deprivation, osmotic stress, and oxygen limitation and during entry into stationary phase. To identify regulatory components involved in the control of ndi gene expression, a second round of mutagenesis was performed on the primaryndiB::Tn5-luxAB-tagged strain (C22) with transposon Tn1721. A double-mutant strain was obtained that lacked ndi locus transcriptional activity under all of the inducing conditions tested. The Tn1721-tagged gene showed a high degree of similarity to tryptophan-rich sensory protein TspO from Rhodobacter sphaeroides, as well as to mitochondrial benzodiazepine receptor pK18 from mammals. Induction of thendi::Tn5-luxAB reporter gene fusion was restored under all inducing conditions by introducing thetspO coding region, from either S. meliloti orR. sphaeroides, in trans. Furthermore, it was found that, in addition to tspO,fixL, which encodes the sensor protein of an oxygen-sensing two-component system, is required for full expression of thendi locus, but only under low oxygen tension.
In nature, bacterial growth is restricted by a wide variety of environmental factors. One factor of particular importance is the lack of essential nutrients. In most natural settings, at least some essential nutrients are limiting; therefore, negligible growth or dormancy is the more typical physiological state of bacteria (24). Understanding how bacteria are able to monitor, sense, and respond to their environment in this nutrient-deprived state is fundamental to our understanding of microbial biology and ecology. Some bacteria, such asBacillus and Myxococcus spp., sporulate when challenged with environmental stresses (11, 14). However, the majority of bacteria do not appear to differentiate into these stress-resistant forms. Research on primarily Escherichia coli, Salmonella typhimurium, and Vibriospp. has shown that under starvation conditions, these nonsporulating bacteria enter into a specific genetic program which results in the generation of a survival state supporting the persistence of the species until conditions improve (17, 19, 28, 35).
Research on the starvation survival of bacteria whose natural habitat is soil, such as Pseudomonas or Rhizobium spp., has been relatively limited thus far (see Discussion). Soil is generally a harsh, oligotrophic environment (31, 40, 44). Nutrient deprivation and oxygen limitation may represent the most prevailing stress conditions for bacteria that persist in this environment. Although a small amount of organic matter is present in most soils, the bulk of it is in a recalcitrant form, such as lignin or humus (1). Furthermore, in soils where utilizable carbon is available, the lack of inorganic nutrients such as phosphorus or nitrogen may limit growth. Rhizosphere soil has been reported to be a somewhat less oligotrophic environment than bulk soil, due to the presence of plant root exudates as readily utilizable nutrients. However, even in the rhizosphere, bacterial growth and activity are generally limited to the short periods when these exudates are available (18). It is likely that bacteria, which have evolved within the soil and rhizosphere, have developed distinct mechanisms for persisting in a nutrient-limited soil environment.
We have been investigating gene expression during nutrient deprivation in an indigenous soil bacterium, Sinorhizobium meliloti. This bacterium is a capable of establishing a symbiosis with the legume alfalfa (Medicago sativa) during which a new specialized organ is formed, the nitrogen-fixing root nodule. These nodules provide the proper physiological conditions for the bacteria to survive in the absence of competing microflora and to reduce atmospheric dinitrogen to ammonia, which is then assimilated by the plant (reviewed in references 34 and41). The symbiotic properties of rhizobia have been thoroughly investigated (34), yet little is known about how these bacteria are able to persist in their free-living state in the soil and rhizosphere. Previously, we reported the isolation of 33S. meliloti strains with Tn5-luxAB reporter gene fusions induced by deprivation of carbon, nitrogen, or both (23). Here we report further characterization of one of these strains (C22) that harbors a transcriptional fusion induced by oxygen, nitrogen, or carbon deprivation; by osmotic stress; and during entry into postexponential stationary phase. The locus containing this fusion has been designated ndi for nutrient deprivation induced. In addition, we describe the identification of anS. meliloti gene encoding a homologue of the tryptophan-rich sensory protein TspO from Rhodobacter sphaeroides which is involved in regulating the expression of thendi locus.
Media and growth conditions. S. meliloti strains were grown on or in rich TY medium (4) or defined minimal GTS medium (23) at 28°C, as indicated. GTS-N is GTS medium lacking nitrogen. GTS-C is GTS medium lacking carbon. GTS+NaCl is GTS containing NaCl (400 mM). GTS+sucrose is GTS containing sucrose (30%).E. coli strains were grown on rich medium (Luria-Bertani medium) at 37°C. Antibiotics were added at various concentrations.S. meliloti received streptomycin at 250 μg ml−1, kanamycin at 200 μg ml−1, tetracycline at 10 μg ml−1, and spectinomycin at 50 μg ml−1. E. coli received ampicillin at 100 μg ml−1, kanamycin at 50 μg ml−1, spectinomycin at 100 μg ml−1, and tetracycline at 5 μg ml−1.
Survival during stationary phase.Strains 1021, C22, 12,C-4, and R-C22 were grown in GTS medium containing limiting amounts of carbon (0.025% glucose). Triplicate cultures (25 ml) were grown in 125-ml flasks with shaking at 28°C for 21 days. Viable cell counts of each culture were determined at regular intervals by plating a dilution series (five replicates) on TY medium containing the appropriate antibiotics. The data were recorded as CFU per milliliter.
Nodulation experiments. S. meliloti mutant strains C22 and 12,C-4 were screened for the symbiotic phenotype by inoculation on alfalfa (M. sativa) seedling roots and for the ability to fix nitrogen as described previously (22).
Substrate utilization experiments.To test strains for the ability to utilize or oxidize a variety of different carbon sources, GN Biolog Microplates from BiOLOG (Hayward, Calif.) were used as described by the manufacturer.
DNA manipulations and plasmid constructions.The strains and plasmids used in this study are described in Table1. Plasmid DNA for restriction analysis and DNA sequencing was prepared using Wizard minipreps from Promega (Madison, Wis.). Chromosomal DNA was isolated from S. meliloti strains as described by de Bruijn et al. (6). All enzymes for DNA manipulations were purchased from Boehringer Mannheim (Indianapolis, Ind.) or New England Biolabs (Beverly, Mass.). Restriction enzyme digests and ligations were carried out as described by Sambrook et al. (32). Probes were labeled with [α-32P]dATP using a random primer kit (Boehringer Mannheim) and following the manufacturer's instructions. Plasmids were introduced into E. coli hosts by electroporation and intoS. meliloti strains via triparental conjugation (5).
Two plasmids (pTtspO and pRstspO) were constructed for complementation studies with double-mutant strain 12,C-4. To construct plasmid pTtspO, we replaced the gfp coding region in plasmid pTB93F (8) with an S. meliloti tspO-like open reading frame (ORF) containing a PpuMI-SalI fragment derived from plasmid pLtspO. Expression of the S. meliloti tspO-like ORF in this construct is controlled by the constitutivetrp promoter of pTB93F. To construct plasmid pRstspO, we replaced the gfp coding region in plasmid pTB93F (8) with a BamHI-KpnI fragment containing the R. sphaeroides tspO gene from plasmid pUI1126 (48). Expression of R. sphaeroides tspO in this construct is also under the control of the constitutive trppromoter of pTB93F.
Transposon mutagenesis.Tn1721 transposon mutants were generated by triparental conjugation (5; A. Milcamps, P. Struffi, and F. J. de Bruijn, submitted for publication) using E. coli strain DH5α carrying plasmid pJOE105 (donor; Tcr) or plasmid pRK2013 (helper; Kmr) and the S. meliloti insertion mutant C22 (recipient). Three thousand transposon mutants were isolated, and single colonies were purified, grown in liquid TY medium, and stored in microtiter plates at −80°C. The resulting double-mutant strains were screened for luciferase activity on agar plates under the inducing conditions specified using a Hamamatsu Photonic System model C1966-20 (Photonic Microscopy) as described by Milcamps et al. (23).
Isolation of the Tn1721-tagged locus and genetic techniques.The Tn1721-tagged locus from double-mutant strain 12,C-4 was isolated by creating a partial genomic library in pBluescript II KS, using 12,C-4 genomic HindIII fragments between 4.0 and 5.0 kb, and screening this library via colony hybridization using a probe corresponding to the first 350 bp of the 5′ end of Tn1721. This clone was designated pBSR. In order to obtain additional flanking sequence information, a pLAFR1 genomic library of S. meliloti 1021 (6) was probed with pBSR to isolate the wild-type genomic clone of the S. meliloti tspO locus. The obtained pLAFR1 genomic clone was designated pLtspO.
Phage φM12 was used for general transduction as described by Finan et al. (7) to reconstructtspO::Tn1721 as a single mutation. This strain is designated R-C22.
Induction and quantitative measurement of luciferase activity.To evaluate expression under various stress conditions, strain C22 or strains harboring pC22lux were grown in TY broth with the appropriate antibiotics for 36 h, subcultured in triplicate into GTS or TY broth (1:100 dilution), and grown overnight to early exponential growth phase (optical density between 0.1 and 0.3). The cultures were maintained under aeration for experiments evaluatingluxAB gene expression during stationary phase or constantly bubbled with a mixture of 1% oxygen in nitrogen to test the effect of microaerophilic conditions on expression. Alternatively, the cells were centrifuged at room temperature and the pellets were resuspended in regular or modified GTS medium for examination of expression under nutrient deprivation conditions or osmotic stress. All cultures were incubated in a rotory shaker at 28°C, and luciferase activity and cell density were measured at the designated time points.
Luciferase activity was determined with a luminometer (model TD-20e; Turner Designs, Sunnyvale, Calif.) by mixing 140 μl of bacterial culture with 10 μl of n-decyl aldehyde (Sigma, St. Louis, Mo.) and immediately starting the analysis. The aldehyde solution was prepared in water (0.1%, vol/vol) and vortexed for 10 min to form an emulsion. Photons were counted for 20 s, and data was recorded as light units (LU). The assay was performed in triplicate on each culture. Calibration of the luminometer by the method of Hastings and Weber (10) was used to determine that 1 LU equaled approximately 1.4 × 107 photons.
To quantitatively determine the luminescence of carbon-limited and therefore energy-starved cells, a 500-μl aliquot of culture was mixed with an equal portion of complete GTS medium and vortexed briefly. This mixture was incubated at room temperature for 15 min, and a 140-μl aliquot was used for luciferase activity testing as described above.
Since oxygen is required for luciferase activity, the cells that were exposed to low oxygen tension (1%) were aerated briefly before testing by pelleting a 750-μl aliquot of culture and resuspending the pellet in an equal amount of fresh GTS medium. This sample was subsequently immediately tested for luciferase activity as described above.
DNA sequence analysis.Sequence analysis of DNA fragments carried in Bluescript vectors was performed using standard primers and primer walking strategies. The sequencing was carried out at the DNA sequencing facility at Michigan State University. Initial DNA sequence analysis was carried out using the Sequencher software package (Gene Code Corporation, Ann Arbor, Mich.). ORFs were identified by analyzing the DNA sequence with a codon preference program based on codon usage for S. meliloti (C. Halling, University of Chicago, Chicago, Ill.) and by examining the DNA sequence for start codons and Shine-Dalgarno motifs around translational start sites indicated by the codon usage program. Possible promoter regions were identified by searching for characteristic motifs in the DNA sequence using the Predict Promoter for Prokaryotes program (29a, 29b). Database searches were conducted through the National Center for Biotechnology Information (NCBI) web page using the Gapped BLAST program (2). Alignments of deduced amino acid sequences were obtained using the pileup program of the GCG software package (Genetics Computer Group, Madison, Wis.).
S. meliloti strain C22 carries a Tn5-luxAB insertion in a novel locus.Tn5-luxAB insertion mutant C22 was isolated in a previous screen for S. meliloti strains carrying luxABreporter gene fusions induced under nutrient deprivation conditions (23). The tagged locus in strain C22 was cloned by excision from the genome as an EcoRI fragment (EcoRI does not cleave within Tn5-luxAB), self-ligated, and electroporated into E. coli (DH5α). The resulting plasmid (pC22) is self-replicating due to the presence of oriVwithin the Tn5-luxAB transposon (46). The sequence of the DNA flanking the Tn5-luxAB insertion site was determined using unique outward reading primers corresponding to the left and right ends of the transposon, as described previously (23). Additional sequence in the region was determined by primer walking. DNA sequence analysis of the region flanking the Tn5-luxAB insertion (4.7 kb) predicted four ORFs (Fig.1). The site of insertion in strain C22 was found to be located near the middle of an ORF that, upon comparison of the deduced amino acid sequence to the NCBI protein databases, failed to reveal significant similarity to any known protein. This ORF has been designated ndiB for nutrient deprivation-induced gene B. The predicted translational stop codon (TGA) of the ORF just upstream of ndiB, which has been designatedndiA, and the predicted start (ATG) site of ndiB were found to overlap (GAAGGAGAGACTGCAATG AA). Therefore, it is possible that these two genes are translationally coupled and constitute an operon. In addition, comparison of the deduced amino acid sequence of ndiA also showed no similarity to any sequence in the NCBI protein databases; therefore, both ndiA and ndiB appear to be novel.
Two strategies were used to delimit the predicted promoter region of the ndiAB locus. First, plasmid pC22 was digested withEcoRI and the entire fragment was cloned into broad-host-range vector pTB93 (pC22lux) and subsequently introduced into S. meliloti strain 1021. Induction of luciferase activity during oxygen, nitrogen, or carbon deprivation; osmotic stress; and stationary phase was observed in the strain containing plasmid pC22lux (data not shown), indicating that the completendi promoter region is contained within the 4.7-kb fragment. In addition, the DNA sequence analysis program Promoter Predictions for Prokaryotes (http://www.fruitfly.org /seq_tools/promoter.html) predicts a putative promoter region (score, 0.91) approximately 275 bp upstream of ndiA (Fig. 1).
An ORF encoding a protein with a high degree of similarity (31% identity, 46% similarity) to various repressor proteins, such as PurR and LacI (33), was found adjacent to and downstream (194 bp) of the ndiAB genes, along with a putative promoter region (score, 0.98) identified by the Predict Promoter Program (Fig. 1). It is highly similar to the consensus (−35, −10) promoter element ofE. coli (9). It should be noted that within the 4.7-kb sequence analyzed, the promoter region upstream ofndiA and this promoter region were the only two promoters predicted by the program.
In addition, a divergent ORF was predicted at the 5′ end of the putative ndiAB operon, containing a region with significant similarity (41% identity, 60% similarity) to the geneosmY from E. coli, which encodes an osmotically induced periplasmic protein whose function is unknown (51,52).
The ndiB::Tn5-luxAB gene fusion in strain C22 is induced by O2, N, and C deprivation; by osmotic stress; and during stationary phase.The expression of the chromosomal ndiB::Tn5-luxAB fusion harbored by strain C22 was quantitatively examined by measuring luminescence (relative LU [RLU]) under a variety of growth conditions. Thendi promoter was found to be active at low levels throughout exponential growth (≅3 RLU) and induced 12- to 27-fold after 4 h of carbon deprivation, nitrogen deprivation, or osmotic stress and at reduced oxygen tension (Fig. 2). In addition, an eightfold induction of thendiB::Tn5-luxAB fusion was observed as the cells of strain C22 entered stationary phase in complex TY medium (Fig.3). This expression profile was also observed in stationary-phase cells grown in defined GTS medium (data not shown). The observed luxAB reporter gene expression pattern persisted for approximately 8 h after entry into stationary phase. However, since luciferase activity is dependent on the energy status of the cell due to the requirement for reducing equivalents (reduced flavin mononucleotide) to catalyze the bioluminescence reaction (21), the persistence of expression during later stages of stationary phase (after 24 h) could not be quantitatively evaluated.
A homologue of tspO regulates expression of thendi locus.In order to identify genes involved in the regulation of the ndi locus, strain C22 (ndiB::Tn5-luxAB) was mutagenized with a second transposon (Tn1721) as described in Materials and Methods and a library of 3,000 double mutants was screened for altered patterns of ndiB::Tn5-luxAB expression. One double mutant, designated strain 12,C-4, was found in which thendiB::Tn5-luxAB reporter gene fusion was found to be no longer expressed under any of the previously determined inducing conditions. Southern hybridization analysis of strain 12,C-4 confirmed that this strain contained a single Tn1721insertion and that the insertion site did not fall within the initial Tn5-luxAB-tagged locus (data not shown).
The corresponding Tn1721-tagged locus from double-mutant strain 12,C-4 was isolated as described in Materials and Methods. The structure and DNA sequence of the region surrounding the transposon were determined (Fig. 4). The site of insertion was found to be in a gene encoding a protein with a high degree of similarity to the 18-kDa outer membrane component (pK18) of the mammalian mitochondrial benzodiazepine receptor (≅45% identity, 65% similarity) (20), as well as to an outer membrane oxygen sensor protein (TspO) of R. sphaeroides (42% identity, 67% similarity) (48-50).
Upstream (53 bp) of the tspO-like gene, a 225-bp ORF (ORF2) was found with a high degree of similarity (55% identity) to the protein encoded by the yuzA gene from Bacillus subtilis (accession no. Z99120). This protein, whose function is unknown, is approximately the same size as the predicted protein in ORF2 (78 amino acids; Fig. 4). Upstream of ORF2 (29 bp) was found another ORF (ORF1), in the same transcriptional direction, which shares a high degree of similarity with a gene encoding a transforming growth factor-induced protein from Synechocystis sp. (46% identity) (15) and a major secreted protein MBP70 precursor from Mycobacterium bovis (39% identity; accession no.A37195). Both of these genes encode secreted proteins that affect eukaryotic gene expression.
A putative promoter region was identified by computer analysis just upstream of ORF1 (score, 0.94), and a divergent ORF (ORF4) upstream of this putative promoter region was identified with similarity to a gene encoding an epoxidase from Methanobacterium thermoautotrophicum (32% identity and 50% similarity) (accession no. AAB85164). In addition, downstream of thetspO-like gene, a divergent ORF encoding a protein with low similarity (31% identity and 46% similarity) to a Tra protein from Streptomyces lividans plasmid pIJ101 (accession no. P22409) was found (data not shown). Thus, the sequence analysis predicts that three ORFs (ORF1, ORF2, and ORF3, which encodes TspO) constitute a Tn1721-tagged operon.
To determine if the dark phenotype of double-mutant strain 12,C-4 was exclusively due to the mutation in the tspO-like gene, a plasmid (pTtspO) which constitutively expresses the coding region of only the tspO-like ORF was introduced into strain 12,C-4 via conjugation for complementation studies. This region was found to restore induction of the ndi locus under all of the previously tested induction conditions (Fig.5). In addition, the tspO gene from R. sphaeroides was also found to restore proper induction patterns when provided in trans (pRstspO) and expressed under the control of the same constitutive promoter (Fig. 5).
FixL is involved in regulating expression of the ndilocus.As described above, expression of the Tn5-luxABfusion in strain C22 is induced by nitrogen and oxygen deprivation (Fig. 2). Two regulatory pathways have been described in S. meliloti that are involved in the control of nitrogen deprivation and microaerobically induced genes, namely, the ntr system, consisting of the ntrA (30) and ntrBC(37) genes, and the fixLJ system (3), respectively. In order to evaluate the involvement of these signaling pathways in the control of ndiB expression, plasmid pC22lux was introduced into fixL::Tn5-233 (J. Trzebiatowki, unpublished data) and ntrC::Tn5(37) mutant strains of S. meliloti 1021 and luciferase activity was monitored over time under inducing conditions. A similar reporter gene expression profile was found in the wild-type and ntrC mutant backgrounds during nitrogen deprivation (data not shown), indicating that ntrC is not required forndiB expression during nitrogen deprivation. However, a much lower level of luciferase expression was found under oxygen-limiting conditions in the fixL mutant strain than in the wild-type background (Table 2). To further evaluate this observation, a double chromosomal (S. meliloti 1021fixL::Tn5-233 ndiB::Tn5-luxAB) mutant was created by transducing thendi::Tn5-luxAB insertion into S. meliloti 1021 fixL::Tn5-233. This strain (fixL::Tn5-233 ndiB::Tn5-luxAB) was examined for luciferase activity under oxygen-limiting conditions. A decreased level ofndiB::Tn5-luxAB expression during oxygen deprivation was observed, compared to that found in the wild-type background (Table 2), confirming the data obtained with strains carrying the plasmid-borne fusion. Therefore, it appears thatfixL is required for full induction ofndiB::Tn5-luxAB reporter gene expression under low oxygen tensions. Furthermore, ndiB-luxAB reporter gene expression patterns were found to be unaltered in thefixL mutant under all of the other inducing conditions tested (carbon and nitrogen deprivation and osmotic stress) (data not shown). Therefore, fixL involvement in the regulation of thendi locus appears to be specific to sensing and/or responding to low oxygen tension.
Luciferase activity is dependent on the energy status of the cell. Therefore, it was possible that the observed decrease inndiB::Tn5-luxAB reporter gene expression in the fixL and tspO mutant backgrounds could be the result of a general effect of the physiology of the cells on luciferase activity. To examine this question, a control strain (CV2) harboring a constitutively expressed Tn5-luxAB fusion in a gene whose function is unknown (23) was used. Double-mutant strains CV2:tspO::Tn1721 and CV2:fixL::Tn5-233 were created by φM12 transduction and examined for luciferase activity under low oxygen tension. Inactivation of the fixL or tspO gene did not affect the luciferase expression of the Tn5-luxAB reporter gene (data not shown). Therefore, expression of the ndi locus, rather than luciferase activity per se, appears to be dependent on fixL and tspO.
Phenotypic analysis of mutant strains.The growth rate and survival characteristics of strains C22, 12,C-4, and R-C22 were examined. These strains, as well as wild-type strain 1021, were grown in minimal GTS medium, and viable cell counts of each culture were determined at regular intervals for 21 days. All three strains were found to have growth rates similar to that of the wild-type strain. Furthermore, a decrease in viable counts was not observed during stationary phase, indicating that these mutants are not impaired in the ability to persist under starvation conditions (data not shown). In addition, the ability to utilize or oxidize a wide variety of different carbon sources was screened using the GN Biolog Microplates. All three strains had utilization patterns similar to that demonstrated by the wild-type strain (data not shown). Therefore, these mutants do not appear to be deficient in carbon utilization.
The abilities of the C22 and 12,C-4 mutants to nodulate and to fix nitrogen symbiotically were examined in a plant infection test. Both strains were able to nodulate the plant with equivalent numbers of nodules, and the nodules demonstrated levels of nitrogenase activity comparable to that of plants infected with the wild-type strain. Therefore, ndiB and tspO do not appear to be absolutely required for the formation and function of the symbiotic association.
Rhizobia provide a unique model system with which to investigate environmental control of gene expression in a bacterium indigenous to soil. These bacteria persist in the free-living state in bulk soil or in an endosymbiotic state in plants. In order to do so, rhizobia must survive under stressful conditions in soil, competitively sense and utilize signaling molecules and growth-promoting nutrients excreted from the plant into the rhizosphere, and adapt to the homeostatic environment within the host plant tissue. It is evident that these three distinct modes of existence require a high degree of physiological adaptability and mechanisms to sense changes in environmental parameters.
The nutrient deprivation responses in rhizobia are just beginning to be investigated. Recently, evidence for a general starvation response inRhizobium leguminosarum similar to that found in E. coli and Vibrio sp. has been reported (38) and Uhde et al. (39) have identified S. melilotimutants that are affected in stationary-phase survival (39). In addition, phosphate stress-induced genes in S. meliloti(36), as well genes expressed during carbon or nitrogen deprivation, have been reported (23) and starvation-induced changes in chemotaxis, motility, and flagellation have also been reported (42).
Although S. meliloti starvation or stress response research has only just begun, there is great potential for rapid progress in this well-studied organism. Many of the environmental parameters bacteria encounter in soil, such as low oxygen tensions, nitrogen and carbon limitation, and osmotic changes, are also experienced in planta, either during the infection process or after symbiosis is established. Consequently, a number of genes have been identified during studies on symbiosis that are involved in both physiological adaptations in the free-living state and legume infection (26). These include functions required for the synthesis of cytochrome complexes, amino acids, nucleotides, and chaperonins (GroEL), as well as utilization of different carbon and energy sources (26). It is likely that these genes play a role in the starvation or stress response of rhizobia; however, further studies are required to determine this.
As indicated earlier, upstream of ndiA is a divergent ORF with similarity to an osmY-encoded protein whose function is unknown. Interestingly, this gene has also been found to be induced by carbon starvation (43) and during stationary phase (13,16), in addition to osmotic stress (52), in E. coli. It should be noted, however, that the similarity is restricted to the last 71 amino acids of the protein encoded byosmY. The first 130 amino acids of osmY do not show similarity to the ORF. In addition, an ORF was identified adjacent to and downstream of the ndi locus which is predicted to encode a protein with striking similarity to transcriptional repressor proteins and it is possible that this protein is involved in controlling expression of the ndi locus. The high background levels of luciferase activity exhibited by cells carrying plasmid pC22lux, even in a tspO mutant background (Table 2), may indicate titration of repressor molecules resulting in a higher level of expression. However, additional experiments are required to determine if this putative regulatory protein is involved in controlling expression of the ndi locus.
To our knowledge, this is the first report that a tspO-like gene in S. meliloti is involved in signal transduction. Recently, however, this same locus was identified by Oke and Long (27) in a screen for S. meliloti genes expressed predominantly in the nodule during the intermediate stages of nodule development. As stated earlier, the environmental parameters used to evaluate gene expression in this study are likely to be found in both the free-living state and in planta, so it is not surprising that the same locus should be identified under these distinctly different screens. Their studies found that plants infected with cells containing a disruption in nex-18 (this gene correlates with ORF1 in Fig. 4) resulted in an altered symbiotic phenotype in that a mixture of Fix+ and Fix− nodules were elicited by the plants infected with this mutant strain. It is therefore evident that this putative operon is expressed during the intermediate stages of nodule development and that ORF1 of this putative operon is important for symbiosis (27). However, further studies are required to determine the function and role of this locus in the development of the symbiotic association.
The TspO outer membrane receptor does not appear to be ubiquitous in nature. Homologues of pK18/TspO have been found in vertebrates, invertebrates, plants, and some yeasts; however, they have not been observed in the majority of the genomes of prokaryotes whose DNA sequencing has been completed. Since a member of the alpha subdivision of purple bacteria is the likely source of the endosymbiont that gave rise to the mammalian mitochondrion (45), the finding of a pK18 orthologue in R. sphaeroides, a member of this subdivision, has been of great interest. Moreover, Yeliseev et al. (50) have demonstrated that the rat pK18 gene could complement a TspO-deficient strain of R. sphaeroides, indicating functional homology. The finding of a pK18/TspO homologue inS. meliloti is also intriguing. Members of the genusSinorhizobium also belong to the alpha subdivision of purple bacteria; therefore, it is not surprising that similar proteins are retained in these organisms. However, since only a few prokaryotes contain this protein, it may be evolutionarily significant that mitochondria, members of the alpha subdivision of purple bacteria that are the likely source of the endosymbiont that gave rise to the mammalian mitochondrion, and S. meliloti, an endosymbiont, contain this protein.
In Rhodobacter spp., TspO is located in the outer membrane and is associated with the major outer membrane porins. In the mitochondrion, the data indicate that pK18 is also localized to the outer membrane and is associated with the voltage-dependent anion channel (20). Moreover, both the TspO and pk18 complexes bind and transport dicarboxylic tetrapyrrole intermediates of the heme biosynthetic pathway (20, 48). This has been proposed as the likely mechanism by which TspO regulates gene expression inRhodobacter (47, 49). Although it seems counterintuitive that synthesis of porphyrins would occur during nutrient deprivation, it has been reported that under a variety of conditions, such as iron deficiency, starvation for heme, or oxygen limitation, bacterial cells produce and excrete porphyrins (12,25, 29). We propose that heme itself or an intermediate of the heme biosynthetic pathway acts as an effector molecule or triggers the synthesis of another effector molecule that binds to a repressor, which then inhibits transcription of the ndi locus. When the concentration of porphyrins or putative effector molecules is decreased, the repressor can no longer inhibit transcription and expression of the ndi locus is restored. It is possible that in the TspO mutant, the concentrations of porphyrins remains high under physiologically stressful conditions; therefore, there is constant repression of the ndi locus.
In addition, FixL appears to be required for full induction of thendi locus under low oxygen tension. This control may be imposed directly through the response regulator FixJ, or the control may be an indirect effect; that is, FixL may control porphyrin biosynthesis in response to oxygen tensions and thereby indirectly regulate expression of the ndi locus when the oxygen tension is low. However, TspO appears to be epistatic to FixL, since no expression is observed in the tspO mutant, even though FixL is still present.
The mechanism by which TspO in Rhodobacter controls the transport of porphyrins and the nature of the signal that TspO senses remains to be determined. In addition, further investigations are necessary to determine if these two related TspO homologues in S. meliloti and Rhodobacter spp. actually function in a similar manner and respond to the same signals. Nonetheless, these two proteins are involved in regulating gene expression and are likely to provide a new and important way to think about signal transduction in prokaryotes.
This work was supported by NSF STC grant DEB9120006 from the Center of Microbial Ecology and grant DE-FG02-91ER20021 from the Department of Energy.
We thank Jodi Trzebiatowski for providing us withfixL::Tn5-233 and for many helpful discussions and Sam Kaplan for generously providing us with a variety of tspO constructs. We also thank Julie Hines for technical assistance.
- Received 14 August 2000.
- Accepted 5 October 2000.
↵* Corresponding author. Present address: Dartmouth Medical School Department of Microbiology, Vail Building, Rm. 202, Hanover, NH 03755-3842. Phone: (603) 650-1247. Fax: (603) 650-1318. E-mail: medavey{at}dartmouth.edu.
- Copyright © 2000 American Society for Microbiology


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subset: commoncrawl


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Netflix's The Witcher Producer Discusses the Show's Potential
The series will be full of surprises with plenty to talk about.
By Chris Morse
We have heard plenty about the development of the upcoming The Witcher Netflix series from writer, executive producer, and showrunner Lauren S. Hissrich, who frequently shares insight into the show's production via social media, but today we heard from another one of its producers with his own insight to share about the television adaptation.
Casting for the series still underway, but in the meantime, illustrator, director, and animator Tomasz Bagiński recently chimed in on the ongoing development with Polish website Wirtualne Media, as reported by Poland in English. Bagiński is going as far as comparing The Witcher to Amazon's in-development Lord of the Rings TV series, but notes that his team has a bit of a head start.
This is one of only a few series that is evoking so many emotions. We can only compare it to the noise surrounding "The Lord of the Rings" series being produced by Amazon. They have not created a single line of the script yet; we are much further.
The approach to developing this series will more heavily emphasize the Eastern European roots of author Andrzej Sapkowski's source material, as opposed to "Americanizing" it. As such, filming will take place there and is set to begin this winter. "We do not want it to be a typical American series," Bagiński explained. "It will be a great proposal not only of fans of The Witcher but also to viewers all over the world. There will be a lot of surprises, the series will evoke discussions for sure."
Given the nature of the story, its politics, and the social issues present throughout the narrative, having the series take those ideas head-on to get people talking would definitely be the right approach for a series with such robust world-building. We can't wait to see those aspects of The Witcher come to life.
The producer also revealed that the author Sapkowski is indeed involved in the process, but did not go into any specifics other than the fact that he is being kept up to date on the progress and will see all of the scripts. Since the series is based directly on the books rather than the popular video game adaptations, having input from the writer himself will go a long way towards getting the show right.
There is no official release date just yet, but previous reports have suggested that we may have to wait until 2020 to see The Witcher arrive on Netflix. We here at Dead Entertainment will keep you posted with all of the latest as it develops.
The Witcher Netflix Series Showrunner Offers Insight into Writing Process
Here's why they have to figure out how the season will begin and end before anything else.
Read More


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I checked out The Expendables 2 yesterday and couldn't have had a better time at a Saturday afternoon movie screening. First off, my local AMC now has reserved seating with servers catering to your every whim, but more importantly, TE2 is just a damn good action flick. It's sad to think that the younger generations have never really truly watched an action film due to the pussification of Hollywood studios, and their penchant for making as much money as humanly possible by putting out only PG-13 rated movies. Luckily, some of the greatest action stars from the 70's, 80's, and 90's are still alive today, and thanks to Sly Stallone they can still deliver a movie that's full of guns, blood, and corny one liners with perfection.
The Expendables 2
EB 9 out of 10 Buddhas
The Expendables 2 opens with a high-octane punch to the face, and it never slows down until the final credits. I was ready to proclaim it as the greatest action movie of the 21st century after the 10 minute intro was through, and the Skull and bullet shells Expendables logo appeared to signify that the movie had officially started. What can I say? I'm a guy who grew up watching Arnold and Stallone in movies like Commando and Rambo. Back then there was no such thing as a plot. These movies were more about kill counts and catchphrases, and that's exactly what The Expendables 2 has recreated.
Seriously one of the greatest opening scenes of all-time
There is a plot in TE2, if you can call it that, but like I said it mattered not. This movie is pure man fun, and the ladies will love it too. Watching all of these geezers gathered together on a single screen is something special, and their chemistry is perfect. I can't tell you how many times my entire theater burst out laughing while watching this movie, and the giggles weren't just for cheesy lines. Some of the action is so over-the-top and brutal, that it warrants laughter. Just keep that in mind when you hear Stallone's, "Rest in Pieces" line towards the end of the movie. It's easily one of the greatest kill scenes of all-time, and it's definitely an homage to the overly violent action movies from the 80's.
In addition to TE2 just being a fun and entertaining shoot 'em up, it also serves up some serious nostalgia for action movie fans. Arnold, Sly, Van Damme (he played a great villain), Chuck, Willis, and more, all in the same movie? You can't beat that with a M-60! It honestly just felt awesome to watch these guys do what they do best again. F*ck political careers and serious movies! These dudes were put on this Earth to eat steroids, look ripped, and shoot off more ammo than an entire 3rd world country even possesses. That's exactly what they were tasked with doing in TE2, and they all pulled it off with flying colors.
The Muscles from Brussels did a fine job as the film's antagonist
On top of having a retardly high kill count and gruesome deaths, action movies also have to be full of lame one-liners, and TE2 is packed with them. I think each of the main 80's action stars all said their iconic catchphrases at least once, or had another cast member say it in a mocking form essentially making fun of themselves and the genre. It was great! Just wait until Chuck Norris shows up. The writers didn't even try to hide the jokes and obvious references to all of the Chuck Norris sayings we've seen in email chains, and on websites dedicated to the legend of Norris. I thought his inclusion may have ruined this film, but I was dead wrong. He was used perfectly, and his scenes were some of the funniest in the film.
I honestly can't find a single fault with this movie. The creators knew exactly what they were doing, and they pulled it off perfectly. More movies like this need to get made, so I hope the likes of Stallone and Arnold don't die off yet from abuse, because there isn't a single young male actor out there that can touch these guy's jock straps when it comes to making action movies. They may not move and look like they used to, but they know how to shoot guns with unlimited ammo and have a good time. I guess if I had to come up with one complaint I'd say that some of the kill shots produced so much blood and giblets it almost looked too fake, but I'd be a p*ssy for saying so. There's no such thing as too much fake blood and giblets when it comes to a throw back action flick period!
I could watch old guys shooting machine guns all day long
The Expendables 2 is hands down the greatest action movie of the 21st century. It's better than the original because of its ability to shamelessly make fun of itself and its actors, and the kill count has been increased dramatically. If you grew up watching these guys in the 80's then you owe it to yourself to watch this movie. It transported me all the way back to my youth when I used to grab sticks in the back yard with no shirt on pretending to shoot hundreds of faceless enemies as if I were Rambo. I'd wager that it'll make you feel the same way. For you younger folk you should go see it just to realize how awesome action films used to be before the US became overly sensitive and wussified.
For all of its upside I give The Expendables 2 an EB 9 out of 10 Buddhas. It's well worth a screening, so get your puny a*s to the theater today! You've been wanting to see this movie on a repetitive loop…
[#ff entbuddha] "Making you a better geek, one post at a time!"


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Hey yall
Im driving to Reno from SF tomorrow for my intro exam, and I was wondering if anyone has any podcast suggestions to help with studying for the exam. Also if anyone has any topics or regions that you think I should pay extra attention, it would be greatly appreciated.
Well, how did you do!?
Passed! Our intro class had a 100% pass rate, and was a great experience!
Good job man. Have fun with your continued studies and let me know when you pass that certified!


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Good Afternoon Friends,
Today I'd like to talk about this really wonderful tea I tried for the first time today. As you may or may not know, I am not usually a fan of green tea, but once again, my expectations have been blown away by another one of DAVIDsTEA's.
Toasted Walnut-Green Tea-Low Caffeine
Steeping: 3 minutes at 170ºF (76ºC) – .18oz (5g) tea- 16oz ( 475ml) mug
Aroma: light fresh grass, coconut, tropical fruit, maple walnut
Flavor: light, earthy, and vegetal with a perfect blend of coconut, pineapple, and nuttiness
Liquor: light golden honey
If I had to describe this tea, I find this tea to be perfect for the transition of seasons: from it's light, sweet tropical fruit taste of Summer that moves into a welcoming of the warmth and hearty nut flavor that's reminiscent of Autumn. I mainly enjoy and prefer black, pu'erh, and lapsang teas because of their boldness and earthiness. This tea felt to me that it possessed that earthiness but without that bold intensity that black and earthy teas have, which is a nice change. All around, I found this tea to be very enjoyable, and I definitely plan to order this in the future!
One thing, of many things, that I like about DAVIDsTEA's is their sample sizes and variety of said samples. When you spend a certain amount on their tea, you're able to choose up to 3 samples. They're a small .18oz(5g) package of tea with it's name, description, ingredients, and brewing instructions. To me, I find this very thoughtful and helpful for those whom either have allergies/sensitivities, or people like me who want to know how to get the most out of their cuppa with some recommended instructions. The only thing I would change about the samples is the brewing temperature indicator. For example, on the toasted walnut tea sample, it just states that the water should be hot without a direct temperature. I feel that adding an actual temperature suggestion would be more efficient for proper brewing, especially for someone new to tea. All around, I was really impressed with this tea, and would rate for a green tea, an 8/10.
I have a few more sample packs to try and review, so please look forward to more tea reviews soon!
Here is a link to the tea for those interested as well as a link to the sample pack that it comes in.
Toasted Walnut Tea
David's Top 12
Thank you for stopping by to read my review!


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Elaboração e análise de indicadores baseados em dados do Sistema Nacional de Pós-Graduação, presentes nos Cadernos de Indicadores Capes
Fioroni, Joyce
MetadataShow full item record
The Post-Graduation system in Brazil is a strategical factor to the socioeconomic and cultural development process of brazilian society, marched by a strict evaluation process and recognition of the Post-Graduation Program (PGP) by the national and international scientific community. The evaluation process stands in this system managed by "Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)" and besides the initiatives of CAPES in endow the country with an efficient database about the situation and evolution of Post-Graduation, there is a underutilization of information by the factors involved on National Post-Graduation System (NPGS) due to form and volume of available data. The general object of this own paper work is the elaboration and review of indicators based on data of NPGS present on Indicators Notebooks of Courses Recommended and Recognized by CAPES, in area of Engineering II. The method used was the exploratory case study and the case unity was the knowledge area from CAPES Engineering II: subarea Materials Engineering and Metallurgic. Collected the data from Notebooks of Bibliographic Production of PGP on area as a documental strategy. Also, was used bibliometrics as technique of information analysis to the elaboration of Bibliometrics Indicators with the support of Software VantagePoint. As results was elaborated and reviewed the indicators of scientific production from PGP: collaboration between the PGP, the evolution of publications, the representativeness of PGP, ranking of periodic and main areas of knowledge. It concludes that the Indicators Notebooks by CAPES consolidate a national source of information, public and with easy access of scientific production national that enables the construction of suitable indicators to decision making process ordering improvement of PGP and the public politics of C&T.


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Tarun scratched his head.
He found the sum too difficult to solve.
This was the second time he tried.
He felt a bit frustrated.
Shadow was still barking.
He had started barking about ten minutes ago.
It was a big distraction.
Still, Tarun did not want to get up to see why Shadow was barking.
Finally he got up.
"What's wrong with you, Shadow?" he asked aloud, whilst walking towards the kennel.
Shadow wagged his tail once or twice.
Then he turned the other way and started barking loud as ever.
Tarun saw the reason only then.
An iguana was making its way around the garden.
On seeing Tarun, it disappeared through an opening in the parapet wall.
"Now, keep quiet. I want to do my sums in peace" Tarun told Shadow.
Shadow wagged his tail again.
Just then Tarun saw a parrot at the bird feed.
He watched the beautiful bird for a while before entering the house.
He sat at the desk and started working on the sum again.
To his surprise he could solve it this time.
Tarun was overjoyed.
He took Shadow out of the kennel and played with him for some time.
Later in the evening he told Thaththa about his breakthrough.
"Sometimes, all what our mind needs is a small break!" he replied.
Tarun completely agreed with him.
Why was Tarun frustrated?
What did the iguana do after seeing Tarun?
According to father what does mind need at times?


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JAY-Z Teases New Video for "Family Feud"
Published Dec 28, 2017JAY-Z has already released a number of videos for tracks off of his 4:44 LP, and he'll share another before 2017 comes to a close with the arrival of a clip for "Family Feud."
In a teaser posted to Twitter, the clip finds Jay and Beyoncé in a confession booth. Their daughter, Blue Ivy, also makes an appearance. You can watch the teaser in the player below.
The video for "Family Feud" arrives tomorrow (December 29) and will almost certainly be a Tidal exclusive.
JAY-Z had previously released visuals for 4:44 tracks "MaNyfaCedGod," "Moonlight," "Adnis," "Kill Jay Z," "BAM," "The Story of O.J.," "Legacy," "Smile," "Marcy Me" and the title cut.
We named 4:44 one of our Top 10 Hip-Hop Albums of 2017. You can order it on CD via Umusic.
.@S_C_'s "Family Feud" x 12/29 x #TIDAL: https://t.co/We5OlPBrLl pic.twitter.com/sNoRbI8R56— TIDAL (@TIDAL) December 28, 2017


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As a local Realtor for the past 16 years and a homeowner myself, I am very familiar with O'ahu's local market conditions and real estate trends. Whether you are new to the market, thinking of moving here, or you are an experienced investor, I have the expertise and resources to help you meet your real estate goals.
I moved to O'ahu 22 years ago from San Diego and fell in love with this paradise, where my family has made our home. When we bought our house here, we did not know anything about the real estate market, and I wished we had an expert to help and guide us. Shortly thereafter, I decided to pursue a real estate career to provide this service to others.
I provide comprehensive marketing solutions for sellers, and I am experienced and skilled at finding properties to best suit my client's needs. I studied for several years at a French-language university, and I have worked with a broad range of international clients from Canada, Australia, Europe, Asia, and the United States.
I feel blessed to live here. Hawai'i provides a unique opportunity to enjoy a well-rounded living experience, with the best climate, famous beaches, and golf courses, along with the friendly aloha spirit of the islands. Hawai'i property is a good investment and an excellent choice for your retirement years.
Throughout my practice, I've been nominated for Aloha 'Āina REALTOR® Awards. In my business, I strive to go beyond expectations for all my clients. My career is based on a foundation of dedication, honesty and integrity.
I specialize in single-family homes, condos and helping foreign buyers find their perfect O'ahu, Hawai'i real estate.
Please reach out to me at 808-389-3237, or via email at email@example.com for assistance.
{{::listing.address.line_1}}, {{::listing.address.city}}
My family and I wouldn't have had our dream house if it was not for Sudi's effort, follow through, professionalism, and creativity. Our house was exactly what we had been looking for and more within the budget. She was always very thorough and accessible. Always friendly and informative, clear and organized. She made the transaction easy with her coordination. Sudi's help extended well beyond a real estate agent and we are proud to say that we have a great friend. Beyond her hard work, her honesty, humility, integrity, and kindness in looking after her clients above and beyond her duty of care.


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In another universe I am not the one writing this. In another universe I am still who I am, but wholly different–the personification of another facet of myself. In another universe I am the same man but different, a secondary, tertiary–an nth degree version of myself wholly unknown yet wholly undifferentiated.
That's a good way of saying it.
Where all these universes meet–a place in time or space, removed from either or both–there is an integral self that exists beyond all possibilities.
The act of reality forming from thought differentiates these forms, causing universal constants to slip behind subliminal ideals, each variable taking upon itself a new manifestation based upon those factors that surround it.
Today I feel undifferentiated. It's an integral part of my identity.
In another universe I discovered not writing, but art. Instead of abandoning my doodling for the precision of words, I took to drawing with paramount skill. I stumbled upon Photoshop–possibly illegally, I cannot say–instead of the free alternatives I use here, and there I allowed my passion for design to grow.
In college I dabbled in interior decorating, but my interests coalesced into a lavish graphic design program. I interned with local businesses on a rebranding project and decided to take a minor in marketing. After graduation I got a job with a national nonprofit designing media. My work was hailed as critical in the acquisition of marriage equality in three states and adoption rights in four others.
After more than a dozen years in the business of nonprofit, I settled down and took to illustrating children's books, even venturing to write a few of my own. I dabbled in a few young adult novels, but the burgeoning mythology inside me became nothing more than a pastime as I took to drawing the various gods and goddesses I imagined. Eventually I'd have an open gallery dedicated to each of them, but their story remained untold.
I died at 73, peacefully slipping away in my sleep.
In another universe I took physics first and my senior interests became invested not in understanding the world around me, but the world inside me–a simple course in biology made me want to take a second course in biology. I entered college not knowing precisely what I wanted to do, but after a bout of undergraduate research, I pursued my Ph.D and entered the realm of professional research.
Although I did not receive the Nobel Prize myself, my work in stem cells contributed to the work of those who did. I was not resentful, however, since I had seen the fruit of my work branching off into possible cures for diseases like diabetes and a dozen others. Throughout my career I pursued various committee and board positions alongside my research, assisting in the creation of policy that furthered the health of our nation.
In my mid thirties I met a man and we wished to have children–children biologically fathered by both of us. With my background in stem cells, which had always been focused upon the embryonic variety, I was able to gain grants and research partners to investigate possible ways of making this dream a reality. After eleven tedious years of research, we were able to move forward with a full-scale trial.
Our first child matured into adulthood and became an honor student, hailed worldwide as a scientific miracle, but was killed in a car accident. Our research continued.
In another universe there was a break-in. I was shot at seventeen.
In another universe I took the advice I'd been given and pursued a degree in history and followed it as such with an MFA in creative writing. I became a little-known local author for many years, writing mysteries and science-fiction thrillers, but only achieved critical acclaim when I began writing an advice column for a state newspaper. The column was syndicated nationally and I was even offered a TV spot, but kindly declined. I preferred working behind a screen, not on the other side of it.
Keeping mostly to myself, I never married.
In another universe I ditched mathematics altogether and ran full-force into politics. I took an internship after graduation, pursued a master's degree in public administration, and then ran for local office. After serving ten years in the state senate, I ran for federal office–and lost. I took two more years to build a base, connecting with my intended constituents, and then ran again to win.
I held this office for twelve years, passing laws to reform our nation's welfare program and to promote a stronger public education system that not only provided a higher quality product but was also more efficient and lower in cost. I was asked to be the presidential candidate's running mate, and agreeing largely with her platform, we joined forces. We won the popular vote, and thanks to electoral college reformation, we won the race.
After serving two terms as vice president, I ran a successful campaign for presidency in the following election. Unfortunately, after two years, I was assassinated.
In another universe I could not tell you my childhood or my friends or my family, but in another universe I could tell you my ambitions, my passions, my accomplishments–the whole of my life and my death.
It's a simple feat, staring across these primordial strings into unknown realms and witnessing creation occurring alongside and far ahead our own universe. It's an easy sight–simply it's all my own imaginings, the things I foresee in my own future, the things I foresee if I adjust my past accordingly.
I sometimes–no, I often–wonder how it might be if for just a moment I could meet myself, if for a moment we could clasp hands and let flow between us all the things we know–how much more effective a leader, a student, a man I would be if this were so! The knowledge of a dozen universes in an instant I would know.
In another universe, my life is ordered. Complete.
In this universe, I can detail my past and elaborate my present, but ask me of my future and suddenly the slate is blank. I can enunciate my current plans, my present engagements, the many and myriad ways my attention is torn and toggled, but ask me of tomorrow, and my tongue is tied.
In this universe I haven't a clue where I'm headed. I can speak ages of my visions and my passions, but when they fall in place together, it's a conglomerate mess, muddled and confused. I lack a plan longer than my outstretched arm, any order past the clutter strewn across my desk.
In this universe I can discern neither my future nor my fate.
It's a dismal universe to live in.


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Monday, December 18, 2006
Most Post Its-Feature Rhythms
I've been drawing from the feature rhythms and finding my caricatures are coming easier and surer, as well as the out of the head kind of stuff I'm doing on these post its. I'm going to combine feature rhythm plus the simple musculature notes from Scott Williams and see where that leads.
Posted by Mr Goodson at 9:31 PM
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Nintendo's UK Store Appears To Have Confirmed Mortal Kombat 11 Switch Delay In Europe
Mortal Kombat 11 was expected to arrive worldwide on the Switch in April until several European retailers updated their product listings for the Switch version of the game, changing the launch date to 10th May.
At the time, these retailers included Amazon and even the Warner Bros. store (the company responsible for publishing the game). If this delay wasn't already set in stone, the official Nintendo UK store is now listing the game's arrival for 10th May 2019 as well. This pretty-much confirms the latest entry won't arrive on the Switch in this region the same day as other platforms.
On the plus side, if you do decide to pre-order the game from the UK Nintendo store, there's a choice between the standalone game or a special bundle. The bundle priced at £59.99 contains a copy of the game and a Mortal Kombat Pixel Pals Lamp. The lamps to choose from include Raiden, Sub-Zero and Scorpion. Take a look below:
The Switch release of Mortal Kombat 11 in North America appears to be on track, with sites like GameStop and the local Nintendo store still showing the original launch date of 23rd April 2019.
This European delay isn't the only Mortal Kombat 11 setback, after it was confirmed earlier in the month Switch owners would miss out on the Collector's Edition. Fortunately, pre-ordering any version of the game will still unlock the special character, Shao Kahn.
Would a small delay like this encourage you to purchase another version of the game? Will you still be buying the game when it does release on the Switch? Tell us below.
Source : https://www.nintendolife.com/news/2019/01/nintendos_uk_store_appears_to_have_confirmed_mortal_kombat_11_switch_delay_in_europe
Bender Bending Rodríguez
Latest posts by Bender Bending Rodríguez (see all)
- Shovel Knight Showdown – The Enchantress character highlight - December 6, 2019
- Local multiplayer action game Demons with Shotguns coming to PS4 on December 17, Xbox One on December 24 - December 6, 2019
- Football Manager 2020 Touch Kicks Off On Switch Next Week - December 6, 2019


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AHAVA Starter Kit
Get the best of AHAVA at an even better price. Try 4 of AHAVA's bestselling products in convenient travel sizes. Perfect for frequent travelers or for those new to AHAVA!
Purifying Mud Mask A unique blend of purifying Dead Sea Mud, minerals and natural extracts, this mask deeply cleans and detoxifies pores for a fresh, even complexion. Absorbs excess oils as it smoothes and hydrates for a radiant complexion. Horsetail extract helps to soothe skin with its antiseptic and anti-inflammatory properties. Jojoba oil helps to nourish and moisturize skin.
Essential Day Moisturizer Targeted for most skin types, this best-selling daily moisturizer infuses skin with lasting hydration, leaving skin feeling supple and soft all day long. Protects against environmental aggressors with Vitamin E and soothes with Aloe Vera
Mineral Hand Cream Give your hands an extra infusion of smoothness with this light hand cream in value size. It's rich in nourishing Dead Sea minerals that hydrate and soften skin for increased all day comfort and safeguard effectively against environmental damage. Witch Hazel extracts smoothes skin, providing a refreshing, revitalizing effect.
Mineral Botanic Velvet Cream Wash (Hibiscus & Fig) A luxurious cream body wash formulated with the power of Dead Sea minerals and enriched with Hibiscus, Fig, vitamins and antioxidants. Pair with Hibiscus & Fig Body Lotion to nourish and soften skin. Hibiscus helps to smooth and moisturize while promoting the healing of dry skin with natural anti-bacterial properties. Fig Extract contains anti-inflammatory properties as well as natural moisturizers.
Starter Kit includes:
Purifying Mud Mask 0.9 oz. / 25 gr
Essential Day Moisturizer 0.51 oz. / 15 ml
Mineral Hand Cream 0.68 oz. / 20 ml
Mineral Botanic Velvet Cream Wash (Hibiscus & Fig) 3 oz. / 85 ml
AHAVA's Mesh Travel Bag
Purifying Mud Mask Kaolin, Silt (Dead Sea Mud), Aqua (Mineral Spring Water), Cocamide MEA & DMDM Hydantoin & Sodium Lauroamphoacetate & Sodium Trideceth Sulfate, Propanediol (Corn derived Glycol), Glyceryl Stearate, Ethylhexyl Palmitate, Aloe Barbadensis Leaf Extract & Propylene Glycol, Glycerin, Simmondsia Chinensis (Jojoba) Seed Oil, Ceteareth-30 & Cetearyl Alcohol, Maris Sal (Dead Sea Water), Sodium Lactate, Zinc Oxide, Caprylyl Glycol & Chlorphenesin & Phenoxyethanol, Panthenol (Pro Vitamin B5) , Chamomilla Recutita (Matricaria) Flower Oil, Stearyl Alcohol, Magnesium Aluminum Silicate, Hydroxyethylcellulose, Peg-40 Stearate, Allantoin, Lactic Acid, Dimethicone, Sorbitan Tristearate, Xanthan Gum, PEG-5 Cocoate & PEG-8 Dicocoate & PEG-9 & Iodopropynyl Butylcarbamate, Triclosan, Methylisothiazolinone, BHA, Tocopherol (Vitamin E).
Essential Day Moisturizer Aqua (Mineral Spring Water), Ethylhexyl Palmitate, Isopropylmyristate, Glyceryl Stearate, Cetyl Alcohol, Propanediol(Corn derived Glycol), Alanine & Creatine & Glycerin & Glycine & Magnesium Aspartate & Saccharide Hydrolysate & Urea, Ethylhexyl Methoxycinnamate, Peg-40 Stearate, Sorbitan Tristearate, Caprylyl Glycol & Chlorphenesin & Phenoxyethanol, Maris Sal (Dead Sea Water), Aloe Barbadensis Leaf Juice, Dimethicone, Allantoin, Parfum (Fragrance), Tocopherol (Vitamin E), Butylphenyl Methylpropional, Coumarin, Hexyl Cinnamal, Hydroxyisohexyl 3-Cyclohexene Carboxaldehyde, Linalool
Mineral Hand Cream Aqua (Mineral Spring Water), Ethylhexyl Palmitate, Ceteareth-30 & Cetearyl Alcohol, Cetyl Alcohol, Glycerin, Sodium Cetearyl Sulfate, Hamamelis Virginiana (Witch Hazel) Flower Water, Propanediol(Corn derived Glycol), Phenoxyethanol & Ethylhexylglycerin, Maris Sal (Dead Sea Water), Dimethicone, Parfum (Fragrance), Allantoin, Linalool, Hexyl Cinnamal, Hydroxyisohexyl 3-Cyclohexene Carboxaldehyde, Butylphenyl Methylpropional, Coumarin, Alpha- Isomethyl Ionone, Citronellol, Geraniol, Isoeugenol, Benzyl Alcohol, Benzyl Benzoate, Benzyl Salicylate.
Mineral Botanic Velvet Cream WashAqua (Mineral Spring Water), Glycerin, Ceteareth-30 & Cetearyl Alcohol, Isohexadecane, Cyclomethicone, Cetyl Alcohol, Sodium Cetearyl Sulfate, Ethylhexyl Palmitate, Caprylyl Glycol & Chlorphenesin & Phenoxyethanol, Butyrospermum Parkii (Shea Butter), Maris Aqua (Dead Sea Water), Parfum (Fragrance), Aloe Barbadensis Leaf Juice, Xanthan Gum, Nelumbo Nucifera (Lotus) Flower Extract & Propylene Glycol Dicaprylate\Dicaprate & Aqua (Water), Castanea Sativa (Chestnut) Seed Extract, Lactic Acid, Alpha Isomethyl Ionone, Butylphenyl Methylpropional, Citronellol, Hexyl Cinnamal, Limonene, Linalool.


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High blood pressure in cats
High blood pressure is not something you immidiately associate with cats, so it is a bit of a shock when the vet diagnosis it and takes it very seriously. This is because high blood pressure in cats (hypertension) is rarely a primary condition in cats. It is rather the result of underlying condition, the most common being chronic kidney failure or hyperthyroidism. This is often referred to as secondary hypertension. There are other causes of high blood pressure in cats including obesity and Cushing's disease - a tumour in the pituitary or adrenal gland which causes excessive production of corticosteroids. Hypertension can have a very serious consequences including blindness, neurological disorders or even congestive heart failure.
Conditions associated with hypertension:
- Bleeding into the eye from burst blood vessel may result in detachment of the retina and swelling which often leads to the permanent blindness in cats.
- Stroke
- Heart failure. When the blood pressure is too high, the heart has to work doubly hard to pump the blood. Overtime that may cause thickening of the walls of the heart chambers which when untreated can lead to the congestive heart failure.
- High blood pressure is also a contributing factor to kidney damage and may in the case of renal failure, make the condition much worse.
One of the major problems in early diagnosis of hypertension in cats is luck of obvious symptoms. As a result, very often the first thing which the owner notices is a sudden onset of blindness due to bleeding into the eye and damage to the retina. Unfortunately, at this stage it is often too late to save the cat's eyesight. Because the high blood pressure is often a secondary condition, any potential signs of hypertension such as breathlessness or lethargy are likely to me masked by symptoms of primary condition.
And yet the diagnosis of high blood pressure is very simple, actually as simple as in humans since The equipment used is often similar to that used routinely in people, with an inflatable cuff placed around one of the front legs or the tail. Measuring blood pressure only takes a few minutes, is completely pain-free and is extremely well tolerated by most cats. So the diagnosis can be made within minutes. It is now recommended that blood pressure should me measured in older cats as part of the routine veterinary examination. This would serve two main function, prevention of serious complication arising from the hypertension, but also perhaps an early signs of an underlying condition which causes the high blood pressure.
Management of hypertension involves administration of drugs which lower the blood pressure. Although, in human there are a number of agents which are very successful in controlling blood pressure, a good anti-hypertensive drug is still largely lacking. Although a number of agents are available for treatment of hypertension these are not specifically licensed for this use in cats. Examples of drugs commonly used are amlodipine (trade name Istin) which is a calcium channel blocker and benazepril (trade name Fortekor) - an ACE inhibitor. In general amlodipine is more effective in cats although in some cats a combination of both of these drugs may be necessary for adequate control of the blood pressure.
The prognosis
Cat with primary hypertension can be managed successfully with anti-hypertensive drugs and as long as there is no permanent health imperment before the diagnosis of high blood pressure, there is no reason for the cat to lead a long and normal life. With secondary hypertension, the prognosis very much depend upon the primary illness, its nature and severity. So for example, if the hypertension is caused by hyperthyroidism, the condition is treatable and following successful treatment of hyperthyroidism, the cat's blood pressure should stabilised as well. In the case of high blood pressure caused by chronic renal failure, the prognosis are considerably poorer. However, in all cases it is important to control the blood pressure since the reduction of blood pressure will be beneficial. For example, in humans it is well recognised that lowering blood pressure slows down the degeneration of kidney, and it becomes more and more apparent that the same may be true for cats.


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A trip to Deli Farm Charcuterie
On Friday 16th December, the apprentices set off on a trip to Delabole to visit the team at Deli Farm Charcuterie.
We arrived at 10:30 and were greeted by Jean, Martin and Becky. We split into two groups and while one group had the charcuterie tour, the other group went up the road to the wind turbines.
First for us in Group A was the tour, but before we could enter the building we were shown into a porta-cabin where we covered up with white overalls, a hair net and plastic shoe covers.
First stop on our tour was the preparation room where Jean talked to us all about what was involved in producing air-dried meats. There are several different elements that are vital to the preparation and air drying such as humidity levels, temperature and contamination control.
We were then guided into the drying room, which was a lot colder than the previous one! Jean explained it was much emptier than usual due to a busy lead up to Christmas. Depending on the product, the meats can dry for up to 7 months and grow a white mould around the skin which is completely safe to eat.
After looking around the drying room, we made our way into the packaging room. Jean explained that at busy times there can be up to four of them in there as everything is packaged to order.
As we finished our tour, the apprentices had lots of interesting questions and we met up with Group B to swap activities. We all got in the van and drove 5 minutes up the road to another part of the farm where the 10 wind turbines stand.
Once we'd walked to the base of one of the turbines we were all surprised to learn that we were going in! Once inside, Martin answered all of our questions and explained how the turbines work. Only in a hurricane will the turbines go into survival mode and shut down.
Once back at the farm, we were all shown into Martin and Jean's house where we learnt about their solar panels and finished the afternoon with a quiz, a tasting session and hot dogs.
We were all loathed to go back out into the cold to drive back, but all agreed it had been a great morning.
Thank you to everybody at Deli Farm Charcuterie!


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Most companies especially in the IT service domain looking for prospective clients do so by setting up a B2B appointment. This business process helps to connect with the member of another company looking for business. The B2B appointment setting is mostly made with the movers and shakers of the potential client.
There are many appointment setting services that help to set up a B2B appointment. These appointment setting services help to tag, identify and generate sales leads and fix business appointments. This helps to save time spent on cold calls and more time in lead generation ideas and preparing strong sales pitch to the client.
It is always easier to sell to a prospective client if you have a pre-set appointment. Even this is made easy by appointment setters.
B2B Appointment Setting Services have appointment setters who have a contact sheet of all potential leads and set appointments with only interested parties and do away with leads which might not be revenue generator in the near future.
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MARComm is a worldwide marketing solution provider that helps to achieve, excel and deliver the optimal sales and marketing initiative. They are keen observers' and have multitudinous experts studying the market and follow close to 5000 companies globally. They are highly qualified in lead and demand generation. Inventive-SMi core strength is B2B technology vendors and lead generation ideas. Software companies can achieve and meet revenue and marketing targets with MARComm.
We provide lead generation & appointment garnering to many technology companies all over the world. For pricing and solutions,
Get in touch with us today!


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Who am I? Do you have a crush on me? Are you the police? … both?
My name is Andrew and as I write this I am a 27 year old masters student studying horn performance. I love anything and everything to do with the art of practicing, performing, and teaching! When I am not busy reading or playing, you can find me working as the brand manager for an online poker company or, on the very rare occasion, playing some Starcraft.
I decided to start this blog to share my interests and as an effort to make connections with people who share those interests. However, I also believe that we are at a very interesting and exciting time for the industry. What we do, whether it be teaching or performing, is all about communication. Think about how drastically communication has changed over the past 10 years! As a student, it also seems that the industry is changing, that the traditional careers many of our teachers had aren't really options for a vast majority of us – but this does not mean that there are not new opportunities! I hope that through my posts and conversations with you lovely people we can help to find and shape the face of the industry in the years to come.
I could ramble on for days, but that is not nearly as interesting as hearing what you have to say! Ideally this blog would not be run by just me, so if you share my interests and want to get into blogging, shoot me an email at firstname.lastname@example.com
Happy practicing!
Sarah Pulfer permalinkJanuary 5, 2012 6:26 pm
Andrew! Blog looks good! I am glad you reminded everyone of the SMART system of goal-setting. Happy New Year!


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VANCOUVER, CANADA — (Marketwired – October 27, 2016) Moovly Media Inc. (TSX VENTURE: MVY) (FRANKFURT: OPV2) ("Moovly" or the "Company") announces a partnership with stock media provider VideoBlocks, adding over half a million multimedia content assets, valued at over 10 million USD, to the Moovly platform. This partnership propels Moovly into a clear content leadership position, with an order of magnitude of more content than its nearest rival.
VideoBlocks has won numerous awards including top #11 media company and was among the top 4 fastest growing media companies in back to back years by Inc.com. Through this partnership, Moovly will make VideoBlocks' professional quality digital assets including 115,000 HD video clips, 350,000 images, photos and vectors plus 100,000 music tracks, sounds effects and loops available to its users.
Geert Coppens, CTO of Moovly: "This partnership significantly increases the quality, variety and number of library items available to our users. The comprehensive VideoBlocks libraries will enable our users to create truly magnificent multimedia content using the objects of thousands of illustrators, video and sound artists, motion graphics specialists and professional photographers."
TJ Leonard, CEO of VideoBlocks: "VideoBlocks is pleased to partner with Moovly who shares our mission of making professional-quality creative media available to everyone."
The addition of the VideoBlocks digital assets to the Moovly platform is further evidence of Moovly's vision and technology leadership in the space, made possible by the second generation advanced platform launched in May 2016. The Moovly platform with its powerful APIs enables flexible access, 3rd party integration and utilization of Moovly's technology, examples of which are the recently launched mobile app and now this content integration.
About Moovly
Moovly is a cloud-based multimedia platform that enables everyone to create engaging multimedia content by making it affordable, intuitive and simple. Users choose from extensive content libraries in various styles and can add their own images, movies and sounds to create captivating videos and presentations. Finished work is published on major social channels like Facebook and YouTube, shared for viewing or editing in the online Moovly User Gallery or downloaded with the click of a button.
Today, Moovly is used in education, businesses of any size and government, non-government and non-profit organizations. Moovly is also adopted by private consumers and broadcasters to create videos that explain, promote, engage, inspire, educate and entertain. Videos can be played on mobile devices, TV screens, digital billboards and more.
Moovly makes video content reusable, easy to edit, fast to update and translatable, all in-house. Platform flexibility and powerful API mean Moovly can offer, custom styles and branded libraries, white-labelled and custom skinned interfaces, single sign-on high-level security, quality of service priority queuing, premium support and commercial other services.
For additional information regarding Moovly, please refer to its website at www.moovly.com.
For further information please contact:


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Press Information Bureau, Hyderabad
Government of India
Karimnagar, March 1, 2016:
Every citizen must concentrate on digital payments
MLA and Mayor inaugurate Digi-dhan Mela at Karimnagar
Digi-dhan Mela was conducted at Ambedkar Stadium in Karimnagar today. The MLA Sri Gangula Kamalakar alongwith the Mayor Sri Ravindar Singh, Collector Sri Sarfraz Ahmad inaugurated the mela. On this occasion the MLA and the Mayor said that both the central and state governments are of the view that all payments should be through the cashless and digital route only. If money is transferred online/debit card/ credit card/PoS the money flow is safe and there would be no misuse. Post-demonetisation, the Government at the Centre has decided to organise cashless transactions throughout the country and for this 100 days- 100 cities initiative has been adopted. Many banks, fertiliser companies and Mobile payment companies set up stalls to educate people about digital payments. People are advised to enroll for Aadhar cards during the Mela. Changes and modifications in Aadhar cards were made. Arrangements for opening New bank accounts also were put in place. Cultural shows were arranged. 300 applications were received for Aadhar cards, Deputy Director Shri Anand Reddy said. The event was supervised by State Government representative Shri Sailesh Reddy, Joint Collector Shri Badri Srinivas, DRO Aayesha Masrat Khalum.


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Thank you for attending Open House! What a great evening. It was so fun to see families socializing and getting to know teachers.
We handed out only a few CMAS test scores, so we will be preparing them to send home with your students. We will send out some notifications when those go out.
Thanks for your support!


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Joint Youth Ministry
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Donations & Payments
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Almaden Hills UMC
Cambrian Park UMC
Willow Glen UMC
High School Mission Trip
Pine Ridge, South Dakota
June 14th-June 23rd
Next Step RSVP Here
Trip details -education nights/payments
Where: Staying at churches/hotels on the way up and down in various cities/states but spending our days in Pine Ridge, South Dakota at the facility Next Step lives at for the summer.
Cost- $1050- This includes transportation, food, rental cars, gas, mission trip payment to Next Step, activities, shirts and much more.
The JYM can pay up to half for students trip if needed for each student attending. Every hour a student and parent volunteers at upcoming fundraising events counts as $10 off your trip. If you need more help financially with the trip we can work that out also. We have never needed to turn someone away for lack of finances and we don't want to do that for this trip either. Please talk with us. We want this opportunity for your student and for our JYM family.
Deposit of $300 due March 3rd.
Students joining us on the trip are required to attend two of the four education nights offered to better prepare us for our time in South Dakota. Additionally all families need to help at the Evening at the Country Fair on March 24th in some capacity.
Junior High Summer Camp
Hartland Christian Camp
July 7th - July 12th
Camp Sign UP
Current Junior High Students - Join us for an amazing week! We will travel to Hartland Christian Camp located in Badger, CA (Near King's Canyon National Park). Hartland has amazing opportunities for students, such as high ropes activities, paint ball, swimming pool, canoes, crafts and crazy games! They also have a strong focus on Jesus and student faith development., Hartland is also a great place for students to create friendships with fellow JYM students, and learn to trust their cabin leaders, as well as Lisa & Kat. We hope you will join us!
Who: Current 6th - 8th grade students
When: July 7th - 12th
Hartland Christian Camp
Cost: $575 - financial aid is available!
First Deposit of $25 is due at sign up to hold your spot
Second deposit of $250 is due April 28th
Final deposit of $300 is due June 9th. You can make payments online, by check (made out to WGUMC-JYM) or cash
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Learn the newest information about residential and industrial real property in the Seattle area and Pacific Northwest. Did You Know? In the first 10 days of the North Bay fireplace, we posted 390 tales about the hearth. And so they were shared nearly 137,000 times. About Website – I'm an utilized researcher with a PhD in Sociology. I have a coverage background in equity and diversity, race and ethnicity, gender and sexuality, and interdisciplinary leadership. This page is related to my research weblog.I write about social justice to increase understanding of social difference (together with, race, gender and sexuality).
Intuitively we know costs have gone up. Statistically, the number of gross sales in Key West as a whole are down by 22{eade898a9d02c76e81358dea3a25c7837930eccf03f6e0d93a37b89984f1578c} in comparison with 2015. The demand for houses has remained intense for the twenty-two years I have been selling homes right here. In 1996 and 1997 a buyer could buy a cigar maker cottage for someplace around $250,000. Now a cigar maker cottage will value near a million dollars. Back in 1996 the best priced houses bought around a million dollars. In 2017 there were no sales beneath a million in the Casa Marina but there have been 14 over a million – three over $three,000,000. The demand for nice homes in good places is there. The inventory of accessible homes on the market in the cheaper price vary in Previous Town and Casa Marina is lacking.
India's rank in the International House Worth Index has jumped 13 spots to reach the ninth position amongst 55 international markets, on the again of accelerating costs in mainstream residential sector. Manhattan actual estate gross sales and prices took a fall in the fourth quarter, and so they're prone to slide even additional. Actual Estate Categorical surpassed my expectations in each … Read More


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ATLANTA — Bill Haas had a sinking feeling when he heard the gallery groan, first indication that his shot had tumbled down the slope and into the lake. When he saw the ball only half-submerged in water, Haas figured he still had the slightest chance.
To somehow save par.
Against all odds, to stay alive in his sudden-death playoff with Hunter Mahan at the Tour Championship, the richest playoff in golf history with FedEx Cup and its $10 million bonus riding on the outcome.
"It was an all or nothing shot," Haas said.
As he did through the final, frenzied hour at East Lake on Sunday, it worked to near perfection. Haas splashed the ball out onto the green to 3 feet for par, then won the Tour Championship — and the FedEx Cup — on the third playoff hole.
Haas won a combined $11.44 million, including $10 million for capturing the FedEx Cup. Mahan, who failed to save par from a bunker on the third playoff hole, had to settle for $864,000 as the runner-up and $700,000 for finishing seventh in the FedEx Cup.
Haas won for the first time this year, and the payoff could be more than just a massive bank deposit. Fred Couples makes his final captain's pick on Tuesday for the Presidents Cup, and Haas put on quite a show.
"I did what I could do," Haas said.
Even if Couples wasn't watching, his assistant captain had a great view: Jay Haas, Bill's dad, was in the gallery at East Lake, and raised his arms as his 29-year-old son delivered the riveting conclusion of the event.
"I'm proud of him the way he came back," Jay Haas said.
Only a week ago, Haas was poised to make the Presidents Cup on his own until a 42 on the back nine at Cog Hill.
He was atop the leaderboard Saturday afternoon at East Lake until a bogey-double bogey finish.
And he almost let it get away from him again. Haas had a three-shot lead when he walked off the 15th green, only to make bogey from the trees on the 16th and bogey from the gallery on the 18th for a 2-under 68.
Mahan had to make par on the 232-yard closing hole. He hit a clutch chip — the biggest weakness in his game — and holed a 5-foot par putt for a 71 to join Haas in the high-stakes playoff.
They returned to the par-3 18th and the pressure was obvious with so much money at stake. Haas hit his shot well right, into the gallery for the third straight time, then hit a tough chip to 10 feet. Mahan went in the bunker and blasted out to 6 feet. Haas appeared to be down to his last shot, but holed the par putt to stay alive.
What followed was an amazing turnaround.
From the right bunker, his approach to the 17th on the second playoff hole tumbled down the hill and barely into the lake, the top half of the ball still showing. Mahan hit a pitching wedge to 15 feet, and must have thought the $11.44 million was his.
Mahan was in that same lake on Thursday when he removed his socks and shoes and played back to the fairway, so when he saw what Haas had done, he knew it wasn't over. He just didn't think — no one did, really — that Haas could hit such a spectacular shot.
"I thought I had won on the second playoff hole, and then he hits it out of the water to 2 feet," Mahan said. "So it seemed like he was destined to win this week."
Water splashed upward, and the ball settled 3 feet away. Mahan's birdie putt just missed on the high side of the cup.
Back to the 18th for the third time in less than an hour, Haas avoided the gallery this time and went left of the green. Mahan again found the bunker, only this time he blasted out some 15 feet by the hole and missed. Haas chipped to 3 feet and holed it for the biggest putt of his young career.
He calmly pumped his fist toward the green and let out a huge sigh.
"I'm very fortunate," Haas said. "This is pretty unbelievable."


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Allie Troeckler may eventually fall short of taking down the Civic Memorial Eagles girls basketball career scoring record set by Katie Broadway at 2,079 points.
But Troeckler did break Broadway's single-game scoring record Saturday night in the Eagles' 73-63 victory over the Taylorville Tornadoes in Bethalto. CM, a winner in five straight games, improves to 7-2 one season after graduating five starters from a 31-3 team.
A 5-foot-10 sophomore, Troeckler scored 44 points on 14-for-17 shooting from the field and 16-for-22 from the free-throw line. Troeckler, who is averaging 21.9 points per game, also finished with 10 rebounds, five steals and three blocked shots.
Broadway, who went on to a standout Division I collegiate career at Illinois State after graduating from CM in 2008, held the previous mark at 39 points in a game during her junior season.
The Eagles led the Tornadoes 41-22 at halftime, but Taylorville rallied with a 28-point third quarter to draw within 54-50. Katelyn Turbyfill had 10 points, six rebounds and five assists for the Eagles, who got eight points from Alaira Tyus.
Quincy Notre Dame 68, Alton 42 – The host Raiders led 24-10 after one quarter and 46-20 by halftime and beat the Redbirds at The Pit in Quincy.
"This was a good experience for us despite the loss," said Alton coach Bob Rickman, whose Redbirds fall to 2-6. "Traveling and playing a team that puts pressure on us will be an experience I hope we can learn from and better prepare us for the tough teams in our league."
Alton, which returns to Southwestern Conference play Thursday at home against Collinsville, committed 34 turnovers and shot 43 percent (18 of 42) from the field against QND. The 6-3 Raiders shot 53 percent (31 of 58) from the field, was 3 of 11 from the foul line and made 24 turnovers.
Alton junior LaJarvia Brown scored 19 points on 9 of 14 shooting from the field. Jewel Wagner had eight points and six rebounds for the Redbirds, who also got six rebounds each from Megan Paulda and Cheriena Mitchell.
"Unfortunately for us, we have too many ups and downs in our play," Rickman said. "We make a great play and then turn it back over. We have great defense, and one cut, late reaction on rotating and they get an easy basket. It is something we just have to keep trying to fix."
Edwardsville 77, Normal 38 – The Tigers overpowered the Ironmen at Lucco-Jackson Gym in Edwardsville to improve to 7-0. The Tigers, who led 39-20 at halftime, shot 55 percent (30 of 54) from the field and made 17 of 25 free throws.
Freshman Rachel Pranger finished with 27 points and 10 rebounds while making 10 of 14 shots from the field and 7 of 8 from the foul line. Sophomore Criste'on Waters added 19 points and freshman Kate Martin had nine points and seven rebounds for Edwardsville, which got eight steals from Aaliyah Box.
Southwestern 49, Breese Mater Dei 37 – The Piasa Birds won their sixth straight while sending their record to 10-1 with the road victory in Breese. Mater Dei is 2-5. Maddy Greeling scored 16 points, Kelsey Rhaodes had 12, Erin Laubscher had 10 and Payton Heyen chipped in seven for the Birds, who led 22-11 at the half.
Carrollton 58, Brussels 47 – Carrollton was down one point at halftime before coming back to beat the Raiders in Brussels. The Hawks are 6-1. Maddie Struble scored 19 points, Madison Mountain had 13 and Mackenzie Ruyle and Rachel Williams each added eight for Carrollton. Brussels, up 29-28 at the half, got 13 points from Alexa Pikesley and 11 from Baylee Kiel.
Bunker Hill 39, Metro East Lutheran 32 – The host Minutemaids broke an 18-18 halftime tie to get the win at Hlafka Hall in Bunker Hill. Taylor Oxford scored 11 points and Heidi Ostendorf scored 10 for the 3-6 Minutemaids, who added seven points from Ava Ostendorf. Metro (4-5) got 12 points from Abby Yurchuck and 11 from Karly Schley.
Pawnee 38, Staunton 36 – The Bulldogs rallied from a 26-17 halftime deficit but could not complete the comeback on their home court in Staunton. Jenna Parmentier made 6 of 11 shots and led Staunton with 14 points and eight rebounds. Karissa Pesavento had seven steals and Devin Barrett had seven rebounds for the Dogs, who fell to 0-8.


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Atkins has gained a world design competition to design a 5-megastar lodge resort set within a gorgeous water-stuffed quarry within the Songjiang district with reference to Shanghai in China. Its beautiful thought designs impressed by using the pure water and panorama options of the quarry captured the creativeness of judges to quash competitors from two different world firms. The modern design of the four hundred-bed resort lodge stands two ranges better than the rock face of the a hundred metre deep quarry and includes underwater public areas and guestrooms.
it is going to contain convention facilities for up to 1,000 people, a banqueting centre, restaurants, cafés and sports services. Sustainability is fundamental to the design starting from using green roofing for the buildings above the bottom stage to geothermal energy extraction. An aquatic theme runs in the course of the design both visually and functionally. Two underwater ranges will house a restaurant and guestrooms going through a ten-metre deep aquarium. the lowest stage of the hotel will incorporate a leisure complex with a swimming pool and water-based sports activities. An extreme sports activities centre for actions corresponding to rock mountaineering and bungee leaping can be cantilevered over the quarry and accessed by special lifts from the water level of the hotel.
learn more at Songjiang resort, Atkins, world structure news, structure jobs.


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Method to Harmony the Hoverboard
- Comment : 0
Although taking a look at for that first time in the Hoverboard, people typically get in touch with us. Any person began at some stage, though to the begun, these look like silly concerns. The troubles really the truth is the honesty how the Hoverboard might drift on top of the surface area without the need of urgent it. What we are talking about it is a definitely healthy technique that will change when suggested to advancement. Inside the season, take pleasure in or otherwise it these found comes to be normally referred to as Hoverboard on the internet. The manner in which through which you employ by soothing regarding it one certain is equivalent to you have adhere heading catastrophe, to smooth floor far from each other. With your extra fat likewise well balanced between toes and also your pumping systems, the device does not move, also when you are produced from by a person. The toes should guidance notably basic-alongside to make sure that you can manage forwards.
It generally does not work with a series of pressure, and you will understand that you have to toned fairly in order to protect against the swegway from continue without the demand of you. It requires from the 2nd for this mind to discover of to tone to acquire basis strain the dimensions to have effective inside it, and an offered quantity of regarding 5 mins. Complete this considering that before they understand it runs they try to motion in regards to the mobile, lots of individuals that go down. You-could not activity worrying this like walking up a stairway given that turning the electric motors is prompted with the gear. Of using the golf ball in the foundation concerning the methods at the same time as you do, you need to invest some time to motion on and off with ft. For those that hop-off of utilizing the rounds of even feet or exchange frontward, you are confirmed to get a crazy and also brief trip.
The really first next you us it, we suggest you continue to be around a wall framework, desk, or friend for aid by putting one solitary basic delicately concerning the cellular taking advantage of the additional schedule even so securely on the flooring, and boost amongst the gyros. Try switching your foundation coupled and obtain before putting full extra fat relating to this a. Motion up with your more amounts of time acquiring interest structure up or likewise not to exchange guidance along after you have recognized the typical region. It is therefore organic and all-natural you will certainly never ever bear in mind you experienced in the starting every single time you have it.